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WO2025231265A1 - Combination with alpha-2 adrenergic receptor agonist - Google Patents

Combination with alpha-2 adrenergic receptor agonist

Info

Publication number
WO2025231265A1
WO2025231265A1 PCT/US2025/027331 US2025027331W WO2025231265A1 WO 2025231265 A1 WO2025231265 A1 WO 2025231265A1 US 2025027331 W US2025027331 W US 2025027331W WO 2025231265 A1 WO2025231265 A1 WO 2025231265A1
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WO
WIPO (PCT)
Prior art keywords
alkylene
compound
pain
yield
chosen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/027331
Other languages
French (fr)
Inventor
Lingyu Zhu
Zhiqiang Cheng
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alpherabio LLC
Original Assignee
Alpherabio LLC
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Filing date
Publication date
Application filed by Alpherabio LLC filed Critical Alpherabio LLC
Publication of WO2025231265A1 publication Critical patent/WO2025231265A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings

Definitions

  • ⁇ 2AR agonists for the treatment or prevention of diseases thereof, either usded alone or in combination with a second therapeutical agent.
  • CNS central nervous system
  • Clonidine was first developed to manage hypertension. Administered orally, clonidine diffuses into the CNS and activates the ⁇ 2AR in the nucleus tractus solitarii (NTS), which in turn triggers a pathway inhibiting excitatory cardiovascular neurons. This cascade effectively reduces sympathetic outflow from the CNS, leading to a clinical decrease in arterial blood pressure.
  • NTS nucleus tractus solitarii
  • clonidine was found to induce sedation by acting through the activation of central pre- and postsynaptic ⁇ 2AR in the locus coeruleus (LC), a nucleus in the medial dorsal pons, thereby inducing sedative effects.
  • LC locus coeruleus
  • clonidine has been approved for epidural use under the trade name Duraclon, marking a significant advancement in the treatment of cancer pain.
  • Duraclon has been documented to induce centrally mediated sedation, hypotension, bradycardia, and depression of its applications, which persist throughout the analgesic treatment process. Such sedation effect significantly limits the dosages that can be administered safely.
  • the present disclosure relates to methods of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist.
  • ⁇ 2AR alpha-2 adrenergic receptor
  • the peripherally selective ⁇ 2AR agonist activates at least one sub type of ⁇ 2AR, particularly ⁇ 2A AR, ⁇ 2B AR, or ⁇ 2C AR.
  • the peripherally selective ⁇ 2AR agonist has a Kp,uu,brain is lower than 0.05, 0.02, or 0.01.
  • the disease is chosen from pain, rosacea, spasticity, and aging.
  • the peripherally selective ⁇ 2AR agonist causes reduced biological effects mediated by CNS, such as sedation, hypotension, and bradycardia, than treating with a non-peripherally selective ⁇ 2AR agonist. 2 Attorney Docket No.: 071741.11025/5WO1 [0013]
  • the present disclosure provides a peripherally selective ⁇ 2AR agonist that comprises an ⁇ 2AR activation moiety covalently linked to a peripheral distribution moiety, and its uses in the treatment of a disease.
  • the present disclosure relates to methods of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peripherally selective ⁇ 2AR agonist, wherein the peripherally selective ⁇ 2AR agonist comprises an ⁇ 2AR activation moiety covalently linked to a peripheral distribution moiety.
  • the peripherally selective ⁇ 2AR agonist causes less sedation than treating with a non-peripherally selective ⁇ 2AR agonist.
  • the present disclosure relates to a compound of formula (I-D): N NH T , or a ste er, pharmaceutically acceptable salt or solvate thereof, wherein, Y 1 is CH, or N; X 1 is chosen from H, D, and halogen; RT is defined as above in formula (I-A).
  • the present disclosure relates to a compound of formula (II): R T B , or a ste r, tautomer, pharmaceutically acceptable salt or solvate thereof, wherein, A is one chosen from: S 1 ; each R 1 is independently chosen from hydrogen, halogen, haloalkyl, hydroxyl, hydroxyalkyl, alkoxy, alkyl, and -COOH; B is one chosen from: 6 Attorney Docket No.: 071741.11025/5WO1 H N N H X S N N H; oaryl, wherein the C3-12 cycloalkyl or C2-12 heterocyclyl is optionally fused with an aryl; r is 1 or 2; n2 is 0, 1, or 2; each R 2 is independently chosen from hydrogen, halogen, hydroxyl, and alkoxy; R 3 is chosen from CN, hydroxy, alkoxy, -C(O)-C 0-12 alkylene-CN, -C 0-12 alkylene-
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound as described herein or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.
  • the present disclosure relates to the use of a compound as described herein or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, for treating or preventing a disease, including pain, glaucoma, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, presbyopia, acute kidney injury, insomnia, inflammatory disease, cancer, etc. in a subject in need thereof.
  • the present disclosure relates to a pharmaceutical combination comprising a first therapeutical agent and a second therapeutical agent, wherein the first therapeutical agent is the peripherally selective ⁇ 2AR agonist described herein.
  • the pharmaceutical combination further comprises a third therapeutic agent.
  • the second therapeutic agent is chosen from opioids, antidepressant medications, antiepileptic medications, local analgesics, nonsteroidal anti- inflammatory drugs (NSAIDs), acetaminophen (paracetamol), corticosteroids, NMDA antagonists, and selective NaV1.8 inhibitors.
  • the present disclosure relates to a method of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject (i) an effective amount of a first therapeutic agent, and (ii) a second therapeutic agent, wherein the first therapeutic agent is a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist.
  • the method further comprises administering to the subject an effective amount of a third therapeutic agent.
  • the disease is chosen from glaucoma, analgesia, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, menopausal hot flashes, dysmenorrhea, presbyopia, acute kidney injury, insomnia, inflammatory disease, and cancer.
  • the second therapeutic agent is chosen from opioids, antidepressant medications, antiepileptic medications, local analgesics, nonsteroidal anti- inflammatory drugs (NSAIDs), acetaminophen (paracetamol), corticosteroids, NMDA receptor antagonists, and selective NaV1.8 inhibitors.
  • the administration of the first therapeutic agent and the second therapeutic agent provides a synergistic effect in treating the disease.
  • the administration of the first therapeutic agent and the second therapeutic agent provides an additive effect in treating the disease.
  • the present disclosure relates to a method for treating or preventing pain in a subject in need thereof, the method comprising administering to the subject an effective amount of a first analgesic agent, and an effective amount of a second analgesic agent, wherein first analgesic agent is a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist.
  • ⁇ 2AR peripherally selective alpha-2 adrenergic receptor
  • the method for treating or preventing pain further comprising administering to the subject an effective amount of a third analgesic agent.
  • the pain is neuropathic pain, nociceptive pain, nociplastic pain, or mixed pain (a mixture of nociceptive, neuropathic, and/or nociplastic pain).
  • the second analgesic agent is any analgesic agent other than a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist.
  • the third analgesic agent is any analgesic agent other than a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist, and the third analgesic agent is different from the second analgesic agent.
  • ⁇ 2AR peripherally selective alpha-2 adrenergic receptor
  • the second analgesic agent is chosen from opioids, acetaminophen (paracetamol), local analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), antiepileptic medication, antidepressant medication, topical analgesic agents, NMDA receptor antagonists, neurotoxins, cannabinoids, voltage-gated sodium channel inhibitors, adenosine agonists, transient receptor potential (TRP) channel modulators, NGF inhibitors, purinergic receptor antagonists, adenosinergic pathway modulators, sigma-1 receptor antagonists, KCC2 enhancers, glutamate transport enhancers, TrkA receptor antagonists, somatostatin receptor ligands, Human Adenylyl Cyclase Associated Protein 1 (CAP1) modulators, and angiotensin II receptor antagonists, corticosteroids, and any combinations thereof.
  • opioids opioids
  • acetaminophen paracetamol
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • the second analgesic agent is chosen from GGG tri-agonist (e.g., retatrutide), metabotropic glutamate receptors (mGluR) modulator (e.g., basimglurant), TGF beta-1 Inhibitor (e.g., Vicatertide (SB-01)), gonadotropin-releasing hormone receptor(GnRH) antagonist (e.g., linzagolix), 11 ⁇ -HSD1 inhibitor (e.g., clofutriben), 17-HSD-1 inhibitor (e.g., OG-6219), Dual ENKephalinase Inhibitor (DENKI) (e.g., PL37), CCR2 receptor antagonist (e.g., CNTX-6970), IL- 10 receptor agonist (e.g., XT-150), nAChR dual alpha 4/beta 2 subunit stimulator (e.g., ATA-104), allosteric NEK7
  • GGG tri-agonist
  • the second analgesic agent is chosen from IRX-101, MR-107A-02, 3-VM-1001, lysergide assisted therapy, SIL-1002, TRN-261, HR-1405-01, HRF-2105, TTAX-03, KP-910, LYT-503, LL-50, ZeP-3, YR-1702, YZJ-1495, FB-1003, MK-4318, PZH-2108, HEC- 137076MsOH, VVZ-2471, SYNP-101, Pudafensine, BIOS-0618, and HSK-36357.
  • the present disclosure relates to a method for treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a codrug that comprises a first therapeutic agent and a second therapeutic agent, wherein first therapeutic agent is a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist, such as the peripherally selective ⁇ 2AR agonist described herein.
  • first therapeutic agent is a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist, such as the peripherally selective ⁇ 2AR agonist described herein.
  • ⁇ 2AR peripherally selective alpha-2 adrenergic receptor
  • the method for treating or preventing the disease has a synergistic effect or additive effect.
  • the method for treating or preventing the disease has a sparing effect on the second therapeutic agent or the second analgesic agent.
  • FIGs.1A-1H PWT value of sham and spared nerve injury SNI mouse model, vehicle and drug treatment groups at 1 hour after dosing.
  • FIG.1A 3 mg/mL pregabalin p.o.
  • FIG.1B 1 mg/mL morphine s.c.
  • FIG.1C 1 mg/mL compound 1-B p.o. and 10mg/mL compound 1-B p.o.
  • FIG.1D 1 mg/mL compound 10-B p.o.
  • FIG.1E 1 mg/mL compound 44-B p.o. and 1 mg/mL compound 45-B p.o.
  • FIG.1F 1 mg/mL compound 46-B p.o.
  • FIGs.2A-2D PWT value of sham and bone cancer pain mouse (BCP) model, vehicle and drug treatment groups at 1 hour after dosing.
  • FIG.2A 3 mg/mL pregabalin p.o.
  • FIG.2B 1 mg/mL morphine s.c.
  • FIG.2C 1 mg/mL compound 44-B p.o.
  • FIG.2D 20 mg/mL compound 1-B p.o. and : 20 mg/mL compound 44-B p.o.
  • FIGs.3A-3C PWT value of sham and post-surgery pain mouse model, vehicle and drug treatment groups at 1 hour after dosing.
  • FIG.3A 10 mg/mL compound 1-B p.o.
  • FIG.3B 10 mg/mL compound 44-B p.o.
  • FIG.3C 3 mg/mL morphine s.c.
  • FIGs.4A-4O Analgesic ratio (%) following single-drug and combination administrations in SNI and BCP mouse models.
  • FIG.4A 1 mg/mL 1-B p.o., 1 mg/mL morphine s.c. and their combination in SNI
  • FIG.4B 1 mg/mL 1-B p.o., 3 mg/mL suzetrigine p.o.
  • FIG.4C 1 mg/mL 1-B p.o., 3 mg/mL pregabalin p.o. and their combination in SNI FIG.4D: 1 mg/mL 44-B p.o., 1 mg/mL morphine s.c. and their combination in SNI 12 Attorney Docket No.: 071741.11025/5WO1
  • FIG.4E 1 mg/mL 44-B p.o., 3 mg/mL pregabalin p.o. and their combination in SNI FIG.4F: 1 mg/mL 44-B p.o., 3 mg/mL suzetrigine p.o.
  • FIG.4G 1 mg/mL 44-B p.o., 7 mg/mL duloxetine p.o. and their combination in SNI
  • FIG.4H 1 mg/mL 44-B p.o., 7 mg/mL amitriptyline p.o. and their combination in SNI
  • FIG.4I 1 mg/mL 44-B p.o., 10 mg/mL ketorolac i.p. and their combination in SNI FIG.4J: 1 mg/mL 44-B p.o., 10 mg/mL ketorolac p.o.
  • FIG.4k 1 mg/mL 44-B p.o., 1 mg/mL morphine s.c. and their combination in BCP
  • FIG.4L 1 mg/mL 44-B p.o., 3 mg/mL pregabalin p.o. and their combination in BCP
  • FIG.4M 1 mg/mL 44-B p.o., 7 mg/mL amitriptyline p.o. and their combination in BCP
  • FIG.4N 1 mg/mL 44-B p.o., 7 mg/mL duloxetine p.o.
  • FIG.4O 1 mg/mL 44-B p.o., 10 mg/mL ketorolac i.p. and their combination in BCP
  • FIGs.5A-5C Body weight curve (FIG.5A), tumor volume growth curve (FIG.5B) ), and tumor volume in day 17 (FIG.5C) of mice in each group in subcutaneous colorectal cancer syngeneic model MC38, including group 1 (control group, 0mg/kg, p.o., QD*Day0-17), group 2 (clonidine, 5mg/kg, p.o., QD*Day0-3; 2mg/kg, p.o., QD* Day4-17), group 3 (compound 1-B HCl, 5mg/kg, p.o., BID*Day0-17), and group 4 (compound 1-B HCl, 10mg/kg, p.o., BID*Day0-3; 5mg/kg,
  • FIGs.6A-6C Body weight curve (FIG.6A), tumor volume growth curve (FIG.6B), and tumor volume in day 17 (FIG.6C) of mice in each group in subcutaneous colorectal cancer syngeneic model MC38, including group 1 (control group, 0mg/kg, p.o., QD*Day0-15), group 2 (Anti-mPD-1, 10mg/kg, i.p., BIW ⁇ 5 doses), group 3 (clonidine, 2mg/kg, p.o., QD*Day0-15), group 4 (44-B HCl, 2mg/kg, p.o., QD*Day0-15), group 5 (44-B HCl, 5mg/kg, p.o., QD*Day0-15), group 6 (clonidine, 2mg/kg, p.o., QD*Day0-15, and Anti-mPD-1, 10mg
  • the phrase “at least one of A, B, and C” means that only A is present, only B is present, only C is present, both A and B are present, both A and C are present, both B and C are present, or each of A, B, and C is present.
  • “at least one of” preceding a series of elements can also encompass situations in which any one or more of the elements is present in greater than one instance, e.g., “at least one of A, B, and C” can also encompass situations in which A is present in duplicate alone or further in combination with any one or more of elements B and C.
  • the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and conjuntive options.
  • a first option refers to the applicability of the first element without the second.
  • a second option refers to the applicability of the second element without the 14 Attorney Docket No.: 071741.11025/5WO1 first.
  • a third option refers to, conjunctively,the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein.
  • any numerical value such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.”
  • a numerical value typically includes ⁇ 10% of the recited value.
  • the recitation of “10-fold” includes 9-fold and 11-fold.
  • the use of a numerical range expressly includes all possible permutations and combinations of subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
  • subject means any animal, such as a mammal, particularly a human, to whom will be or has been treated by a method described herein.
  • mammal as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, and non-human primates (NHPs), such as monkeys or apes, humans, etc.
  • pharmaceutically acceptable salt(s) means those salts of a compound of interest that are safe and effective for topical use in mammals and that possess the desired biological activity.
  • Pharmaceutically acceptable salts include salts of acidic or basic groups present in the specified compounds.
  • Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, carbonate, bicarbonate, acetate, lactate, salicylate, citrate, tartrate, propionate, butyrate, pyruvate, oxalate, malonate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., 1,1′- methylene-bis-(2-hydroxy-3
  • Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, bismuth, and diethanolamine salts.
  • Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, bismuth, and diethanolamine salts.
  • alkyl means a saturated, monovalent, unbranched or branched hydrocarbon chain. An alkyl group can be unsubstituted or substituted with one or more suitable substituents.
  • alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and pentyl (e.g., n- pentyl, isopentyl, neopentyl), etc.
  • An alkyl group can have a specified number of carbon atoms. When numbers appear in a subscript after the symbol “C”, the subscript defines with more specificity the number of carbon atoms which that particular alkyl can contain.
  • C 1 to C10 alkyl or “C1-10 alkyl” is intended to include alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms.
  • C 1 to C 8 alkyl or “C 1-8 alkyl” denotes an alkyl having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.
  • alkenyl refers to an unbranched or branched hydrocarbon chain containing at least one carbon-carbon double bond. An alkenyl group can be unsubstituted or substituted with one or more suitable substituents.
  • alkenyl groups include ethenyl, propenyl, butadienyl (including 1,2-butadienyl and 1,3-butadienyl).
  • C alkenyl
  • C 2-10 alkenyl is intended to include alkenyl groups having 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms.
  • C2 to C8 alkenyl or “C2-8 alkenyl” denotes an alkenyl having 2, 3, 4, 5, 6, 7, or 8 carbon atoms.
  • alkynyl refers to an unbranched or branched hydrocarbon chain containing at least one carbon-carbon triple bond.
  • An alkynyl group can be unsubstituted or substituted with one or more suitable substituents.
  • alkynyl also includes those groups having one triple bond and one double bond. When numbers appear in a subscript after the symbol “C”, the subscript defines with more specificity the number of carbon atoms which that particular alkynyl can contain. For example, “C2 to C10 alkynyl” or “C2-10 alkynyl” is intended to include alkynyl groups having 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms.
  • C 2 to C8 alkynyl or “C2-8 alkynyl” denotes an alkynyl having 2, 3, 4, 5, 6, 7, or 8 carbon atoms.
  • cycloalkyl refers to any stable monocyclic or polycyclic saturated hydrocarbon ring system.
  • a cycloalkyl group can be unsubstituted or substituted with one or more suitable substituents.
  • a cycloalkyl group can have a specified number of carbon atoms.
  • C3 to C6 cycloalkyl or “C3-6 cycloalkyl” includes cycloalkyl groups having 3, 4, 5, 16 Attorney Docket No.: 071741.11025/5WO1 or 6 ring carbon atoms, i.e., cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • Polycyclic cycloalkyls include bridged, fused, and spiro ring structures in which all ring atoms are carbon atoms.
  • a “spiro ring” is a polycyclic ring system in which two rings share one carbon atom, referred to as the “spiro atom,” which is typically a quaternary carbon atom.
  • a “fused ring” is a polycyclic ring system in which two rings share two adjacent atoms, referred to as “bridgehead atoms,” i.e., the two rings share one covalent bond such that the bridgehead atoms are directly connected.
  • a “bridged ring” is a polycyclic ring system in which two rings share three or more atoms separating the bridgehead atoms by a bridge containing at least one atom. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.
  • aryl as used herein is a group that contains any carbon-based aromatic group including, but not limited to, phenyl, naphthyl, anthracenyl, phenanthranyl, and the like.
  • Aryl moieties are well known and described, for example, in Lewis, R. J., ed., Hawley’s Condensed Chemical Dictionary, 13th Edition, John Wiley & Sons, Inc., New York (1997).
  • An aryl group can be substituted or unsubstituted with one or more suitable substituents.
  • An aryl group can comprise a single ring structure (i.e., monocyclic) or multiple ring structures (i.e., polycyclic, e.g., bicyclic or tricyclic).
  • an aryl group can be a monocyclic aryl group, e.g., phenyl.
  • heterocyclyl includes stable monocyclic and polycyclic hydrocarbons that contain at least one heteroatom ring member, such as sulfur, oxygen, or nitrogen, wherein the ring structure is saturated or partially unsaturated, provided the ring system is not fully aromatic.
  • a heterocyclyl group can be unsubstituted, or substituted with one or more suitable substituents at any one or more of the carbon atom(s) and/or nitrogen heteroatom(s) of the heterocyclyl.
  • a heterocyclyl can comprise a single ring structure (i.e., monocyclic) or multiple ring structures (i.e., polycyclic, e.g., bicyclic).
  • Polycyclic heterocyclyls include bridged, fused, and spiro ring structures in which at least one ring atom of at least one of the rings of the polycyclic ring system is a heteroatom, for instance oxygen, nitrogen, or sulfur, wherein bridged, fused, and spiro rings are as defined above.
  • a heterocyclyl ring can be attached to the parent molecule at any suitable heteroatom (typically nitrogen) or carbon atom of the ring.
  • the term “4- to 9-membered monocyclic or bicyclic heterocyclyl” includes any four, five, six, seven, eight, or nine membered monocyclic or bicyclic ring structure containing at least one heteroatom ring member selected from oxygen, nitrogen, and sulfur, or independently selected from oxygen and nitrogen, optionally 17 Attorney Docket No.: 071741.11025/5WO1 containing one to three additional heteroatoms independently selected from oxygen, nitrogen, and sulfur, or independently selected from oxygen and nitrogen, wherein the ring structure is saturated or partially unsaturated, provided the ring structure is not fully aromatic.
  • heterocyclyl refers to 4-, 5-, 6-, or 7-membered monocyclic groups and 6-, 7-, 8-, or 9- membered bicyclic groups which have at least one heteroatom (O, S, or N) in at least one of the rings, wherein the heteroatom-containing ring(s) typically has 1, 2, or 3 heteroatoms, such as 1 or 2 heteroatoms, independently selected from O, S, and/or N, or independently selected from O and N.
  • the subscript defines with more specificity the number of carbon atoms which that particular heterocycly can contain, in addition to the heteroatoms which that particular heterocycly can contain.
  • C1 to C10 heterocycl or “C1-10 heterocycl” is intended to include heterocycl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms.
  • C 1 to C 8 heterocycly or “C 1-8 heterocycly” denotes a heterocycl having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.
  • Examples of monocyclic heterocyclyl groups include, but are not limited to azetidinyl, oxetanyl, tetrahydrofuranyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dioxolanyl, dithiolanyl, piperidinyl, piperazinyl, dioxanyl, morpholinyl, azepanyl, oxepanyl, oxazepanyl (e.g., 1,4-oxazepanyl, 1,2-oxazepanyl) and the like.
  • bicyclic heterocyclyl groups include, but are not limited to, 2-aza- bicyclo[2.2.1]heptanyl, 8-aza-bicyclo[3.2.1]octanyl, 2-aza-spiro[3.3]heptanyl, 3- azabicyclo[2.2.2]octanyl, 3-oxa-9-azabicyclo[3.3.1]nonanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl, 7- oxa-2-azaspiro[3.5]nonanyl, and 5-azaspiro[2.3]hexanyl and the like.
  • heteroaryl includes stable monocyclic and polycyclic aromatic hydrocarbons that contain at least one heteroatom ring member such as sulfur, oxygen, or nitrogen.
  • a heteroaryl group can be unsubstituted or substituted with one or more suitable substituents.
  • a heteroaryl can comprise a single ring structure (i.e., monocyclic) or multiple ring structures (i.e., polycyclic, e.g., bicyclic or tricyclic).
  • Each ring of a heteroaryl group containing a heteroatom can contain one or two oxygen or sulfur atoms and/or from one to four nitrogen atoms provided that the total number of heteroatoms in each ring is four or less and each ring has at least one carbon atom.
  • Heteroaryl groups which are polycyclic, e.g., bicyclic or tricyclic must include at least one fully aromatic ring, but the other fused ring or rings can be aromatic or non-aromatic.
  • the fused rings completing the bicyclic group can contain only carbon atoms and can be saturated, partially saturated, or unsaturated.
  • heteroaryl can be attached to the parent molecule at any available nitrogen or carbon atom of any ring of the heteroaryl group.
  • heteroaryl refers to 5- or 6-membered monocyclic groups and 9- or 10- membered bicyclic groups which have at least one heteroatom (O, S, or N) in at least one of the rings, wherein the heteroatom-containing ring typically has 1, 2, or 3 heteroatoms, such as 1 or 2 heteroatoms, selected from O, S, and/or N.
  • a heteroaryl group can be unsubstituted, or substituted with one or more suitable substituents at any one or more of the carbon atom(s) and/or nitrogen heteroatom(s) of the heteroaryl.
  • the nitrogen and sulfur heteroatom(s) of a heteroaryl can optionally be oxidized (i.e., N ⁇ O and S(O)r, wherein r is 0, 1 or 2).
  • N ⁇ O and S(O)r wherein r is 0, 1 or 2.
  • the subscript defines with more specificity the number of carbon atoms which that particular heteroaryl can contain, in addition to the heteroatoms which that particular heteraryl can contain.
  • “C 1 to C 10 heteroaryl” or “C1-10 heteroaryl” is intended to include heteroaryl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms.
  • C1 to C8 heteroaryl or “C1-8 heteroaryl” denotes a heteroaryl having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.
  • Exemplary monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thiophenyl, oxadiazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl.
  • Exemplary bicyclic heteroaryl groups include, but are not limited to, indolyl, benzothiazolyl, benzodioxolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridinyl, furopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl.
  • alkoxy refers to an –O-alkyl group, wherein alkyl is as defined above.
  • An alkoxy group is attached to the parent molecule through a bond to an oxygen atom.
  • An alkoxy group can have a specified number of carbon atoms.
  • C1 to C10 alkoxy or “C1-10 alkoxy” is intended to include alkoxy groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms.
  • C 1 to C 4 alkoxy or “C 1-4 alkoxy” denotes an alkoxy having 1, 2, 3, or 4 carbon atoms.
  • alkoxy examples include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy, isopropoxy), butoxy (e.g., n-butoxy, isobutoxy, tert-butoxy), pentyloxy 19 Attorney Docket No.: 071741.11025/5WO1 (e.g., n-pentyloxy, isopentyloxy, neopentyloxy), etc.
  • An alkoxy group can be unsubstituted or substituted with one or more suitable substituents.
  • alkylthio or “thioalkoxy” represents an alkyl group as defined above attached to the parent molecule through a bond to a sulfur atom, for example, -S-methyl, -S-ethyl, etc.
  • Representative examples of alkylthio include, but are not limited to, -SCH3, -SCH2CH3, etc.
  • halogen means fluorine, chlorine, bromine, or iodine.
  • halo means fluoro, chloro, bromo, and iodo.
  • Haloalkyl is intended to include both branched and straight-chain saturated aliphatic hydrocarbon radicals substituted with one or more halogen atoms.
  • haloalkyl examples include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2- trifluoroethyl, heptafluoropropyl, and heptachloropropyl.
  • fluoroalkyl in particular include, but are not limited to, -CF3, -CHF2, -CH2CF3, -CF2CF3, and the like.
  • alkylamino groups have one or both hydrogen atoms of an amino group replaced with an alkyl group and is attached to the parent molecule through a bond to the nitrogen atom of the alkylamino group.
  • alkylamino includes methylamino (-NHCH3), dimethylamino (-N(CH3)2), -NHCH2CH3 and the like.
  • aminoalkyl as used herein is intended to include both branched and straight- chain saturated aliphatic hydrocarbon groups substituted with one or more amino groups.
  • C1-4 aminoalkyl is intended to include alkyl groups having 1, 2, 3, or 4 carbon atoms 20 Attorney Docket No.: 071741.11025/5WO1 substituted with one or more amino groups.
  • Aminoalkyl groups are attached to the parent molecule through a bond to a carbon atom of the alkyl moiety of the aminoalkyl group.
  • Representative examples of aminoalkyl groups include, but are not limited to, -CH 2 NH 2 , -CH 2 CH 2 NH 2 , and – CH2CH(NH2)CH3.
  • amido refers to –C(O)N(R)2, wherein each R is independently an alkyl group (including both branched and straight-chain alkyl groups) or a hydrogen atom.
  • amido groups include, but are not limited to, -C(O)NH 2 , -C(O)NHCH 3 , and –C(O)N(CH 3 ) 2 .
  • hydroxyl-substituted alkyl “hydroxylalkyl” and “hydroxyalkyl” are used interchangeably, and refer to a branched or straight-chain aliphatic hydrocarbon group substituted with one or more hydroxyl groups.
  • Hydroxyalkyl groups are attached to the parent molecule through a bond to a carbon atom of the alkyl moiety of the hydroxyalkyl group.
  • a hydroxyalkyl group can have a specified number of carbon atoms.
  • “C1 to C10 hydroxyalkyl” or “C1- 10 hydroxyalkyl” is intended to include hydroxyalkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms.
  • C1 to C4 hydroxylalkyl” or “C1-4 hydroxyalkyl” denotes a hydroxyalkyl group having 1, 2, 3, or 4 carbon atoms.
  • hydroxyalkyl examples include, but are not limited to, hydroxylmethyl (-CH 2 OH), hydroxylethyl (-CH 2 CH 2 OH), etc.
  • amide refers to –N(R’)C(O)R, wherein each R and R’ is independently chosen from hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl.
  • amide groups include, but are not limited to, -NHC(O)CH 3 , -NHC(O)CH 2 CH 3 , and – N(CH 3 )C(O)CH 3 .
  • carbamide refers to –N(R’)C(O)N(R) 2 , wherein each R and R’ is independently chosen from hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl.
  • carbamide groups include, but are not limited to, -NHC(O)NH 2 , -NHC(O)NHCH 3 (methyl carbamide), and –NHC(O)NH(Ph).
  • sulfonamide refers to –N(R’)SO2-R, wherein each R and R’ is independently chosen from hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl.
  • sulfonamide groups include, but are not limited to, -NHSO 2 CH 3 (methyl sulfonamide), and –NH SO2Ph.
  • sulfonamide groups include, but are not limited to, -NHSO 2 CH 3 (methyl sulfonamide), and –NH SO2Ph.
  • substituted as used herein with respect to any organic radical (e.g., alkyl, cycloalkyl, heteroaryl, aryl, heterocyclyl, etc.) means that at least one hydrogen atom is replaced with a non-hydrogen group, provided that all normal valencies are maintained and that the substitution results in a stable compound.
  • that group can have one or more substituents, such as from one to five substituents, one to three substituents, or one to two substituents, independently selected from the list of substituents.
  • substituents independently when used in reference to substituents, means that when more than one of such substituents is possible, such substituents can be the same or different from each other.
  • suitable substituents include, but are not limited to, alkyl, halo, haloalkyl, alkoxy, amido, hydroxy, hydroxyalkyl, amino, carboxyl, ester, oxo, cyano and the like.
  • a group is shown to be substituted with 0-3 R groups, then said group can be optionally substituted with up to three R groups, and at each occurrence, R is selected independently from the definition of R.
  • R is selected independently from the definition of R.
  • the terms “optional” or “optionally” mean that the event or circumstance described can, but need not, occur, and such a description includes the situation in which the event or circumstance does or does not occur.
  • “optionally substituted heterocyclyl” means that a substituent group can be, but need not be, present, and such a description includes the situation of the heterocyclyl group being substituted by a suitable substituent and the heterocyclyl group not being substituted by any substituent.
  • Stereoisomers includes enantiomers and diastereomers. Enantiomers are stereoisomers that are non-super-imposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemate or racemic mixture.
  • Diastereomers are stereoisomers that are not 22 Attorney Docket No.: 071741.11025/5WO1 enantiomers, i.e., they are not related as mirror images, and occur when two or more stereoisomers of a compound have different configurations at one or more of the equivalent stereocenters and are not mirror images of each other.
  • Substituent groups e.g., alkyl, heterocyclyl, etc.
  • Certain examples contain chemical structures that comprise (R) or (S) terminology.
  • Stereochemically pure isomeric forms can be obtained by techniques known in the art in view of the present disclosure.
  • diastereoisomers can be separated by physical separation methods such as fractional crystallization and chromatographic techniques, and enantiomers can be separated from each other by the selective crystallization of the diastereomeric salts with optically active acids or bases or by chiral chromatography.
  • Pure stereoisomers can also be prepared synthetically from appropriate stereochemically pure starting materials, or by using stereoselective reactions.
  • tautomer refers to compounds that are interchangeable forms of a particular compound structure and that vary in the displacement of hydrogen atoms and electrons. Tautomers are constitutional isomers of chemical compounds that readily interconvert, usually resulting in relocation of a proton (hydrogen). Thus, two structures can be in equilibrium through the movement of pi electrons and an atom (usually hydrogen). All tautomeric forms and mixtures of tautomers of the compounds described herein are included with the scope of the present disclosure. [0095] Compounds described herein can exist in solvated and unsolvated forms.
  • solvate means a physical association, e.g., by hydrogen bonding, of a compound described herein with one or more solvent molecules.
  • the solvent molecules in the solvate can be present in a regular arrangement and/or a non-ordered arrangement.
  • the solvate can comprise either a stoichiometric or nonstoichiometric amount of the solvent molecules.
  • “Solvate” encompasses both solution-phase and isolable solvates.
  • Compounds described herein can form solvates with water (i.e., hydrates) or common organic solvents.
  • Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates.
  • isotopes of atoms occurring in the compounds described herein, including intermediates and final products.
  • isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include deuterium and tritium.
  • isotopes of carbon include 13 C and 14 C.
  • the present disclosure further includes isotopically-labeled compounds.
  • isotopically- labeled or “radio-labeled” compound is a compound of the present disclosure where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring).
  • isotopically- labeled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically- labeled reagent in place of the non-labeled reagent otherwise employed.
  • the name of a compound is intended to encompass all possible existing isomeric forms, including stereoisomers (e.g., enantiomers, diastereomers, racemate or racemic mixture, and any mixture thereof) of the compound.
  • the present disclosure relates to methods of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a peripherally selective ⁇ 2AR agonist.
  • the peripherally selective ⁇ 2AR agonist activates at least one sub type of ⁇ 2AR, particularly ⁇ 2A AR, ⁇ 2B AR, or ⁇ 2C AR.
  • the disease is glaucoma, pain, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, presbyopia, acute kidney injury, insomnia, inflammatory disease, cancer, etc.
  • the disease is chosen from pain, rosacea, spasticity, and aging.
  • treating with the peripherally selective ⁇ 2AR agonist causes less sedation than treating with a non-peripherally selective ⁇ 2AR agonist, such as at similar or comparable dosage.
  • the peripherally selective ⁇ 2AR agonist comprises an ⁇ 2AR activation moiety covalently linked to a peripheral distribution moiety.
  • the present disclosure provides a peripherally selective ⁇ 2AR agonist that comprises an ⁇ 2AR activation moiety covalently linked to a peripheral distribution moiety, and its uses in the treatment or prevention of a disease.
  • a disease is glaucoma, pain, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, presbyopia, acute kidney injury, insomnia, inflammatory disease, cancer, etc.
  • the disease is pain.
  • Pain Pain, as a complex and multidimensional sensory and emotional experience, poses a significant challenge to human health.
  • neuropathic pain is caused by a lesion or disease of the somatosensory nervous system. Neuropathic pain can be divided into central neuropathic pain and peripheral neuropathic pain. Central neuropathic pain includes spinal cord injury, post-stroke pain, and MS pain, while peripheral neuropathic pain includes diabetic neuropathy, postherpetic neuralgia, HIV-associated pain, chemotherapy-induced peripheral neuropathy, and post-surgical neuropathic pain.
  • first-line treatment drugs include Gabapentinoids, tricyclic antidepressants, and noradrenaline/serotonin uptake inhibitors. Although these drugs can relieve pain to some extent, the side effects of long-term use still cause a decrease in the quality of life of patients.
  • Second-line treatment drugs such as opiate receptor agonist, not only have side effects but also have a high addiction rate, which has caused many social impacts and cannot well address the demand for neuropathic pain drugs.
  • ⁇ 2AR agonists such as clonidine and dexmedetomidine, are considered an important method for treating pain in academic research and clinical applications. Scientists have found that the intraspinal administration of ⁇ 2AR agonists can effectively relieve pain.
  • ⁇ 2AR agonists are not without limitations.
  • Existing ⁇ 2AR agonists are often associated with a range of biological reactions, including sedation, hypotension, bradycardia, drowsiness, dizziness, depression, bradycardia, orthostatic hypotension, constipation, nausea, gastric upset, dry mouth (xerostomia), dry nasal mucosa, impotence, fluid retention, edema, and pupil size.
  • These other biological effects, especially sedation set limits on the dosages that can be safely administered, thereby constraining the wide-scale utility of these drugs in long-term pain management. This not only affects the quality of life for patients but also restricts the applicability of these drugs for various types and levels of pain symptoms.
  • an effective amount means an amount of a composition or compound that elicits a biological or medicinal response in a tissue system or subject that is being sought by a researcher, veterinarian, medical doctor or other professional, which can include alleviation of the symptoms of the disease, disorder, or condition being treated.
  • An effective amount can vary depending upon a variety of factors, such as the physical condition of the subject, age, weight, health, etc.; and the particular disease, disorder, or condition to be treated. An effective amount can readily be determined by one of ordinary skill in the art in view of the present disclosure.
  • an effective amount refers to the amount of a composition or compound described herein which is sufficient to activate ⁇ 2AR.
  • an effective amount refers to the amount of a composition or compound described herein which is sufficient to treat or prevent the disease or alleviate the symptoms associated with the disease.
  • the pain is neuropathic pain, nociceptive pain, nociplastic pain, or mixed pain.
  • the term “neuropathic pain” refers to pain caused by a lesion or disease affecting the somatosensory nervous system. It results from abnormal processing of pain signals due to nerve damage or dysfunction, either in the peripheral nervous system (e.g., peripheral neuropathy) or the central nervous system (e.g., spinal cord injury, multiple sclerosis).
  • nociceptive pain refers to pain that arises from actual or threatened damage to non-neural tissue and is due to the activation of nociceptors (pain-sensing nerve fibers). It is the body’s natural response to harmful stimuli such as injury, inflammation, or mechanical stress.
  • nociplastic pain refers to pain that arises from altered nociception.
  • mixed pain refers to a mixture of nociceptive, neuropathic, and/or nociplastic pain.
  • the neuropathic pain is chosen from diabetic peripheral neuropathy, postherpetic neuralgia, chemotherapy-induced peripheral neuropathy, Fabry disease– associated pain, trigeminal neuralgia, glossopharyngeal neuralgia, occipital neuralgia, HIV- 26 Attorney Docket No.: 071741.11025/5WO1 associated neuropathy, hereditary sensory and autonomic neuropathy, Charcot-Marie-Tooth disease–related pain, small fiber neuropathy, brachial plexus avulsion, phantom limb pain, post- surgical neuropathic pain, carpal tunnel syndrome, other compression or entrapment neuropathies, amyloid neuropathy, vasculitic neuropathy, leprosy-associated pain, radiation-induced neuropathy, drug-induced neuropathy, metabolic neuropathies, endocrine-related neuropathies, toxic neuropathies, immune-mediated neuropathies, traumatic peripheral nerve injury, and heritable neuropathies, as well as post-stroke pain syndrome
  • the nociceptive pain is somatic (i.e., from skin, muscles, or joints (e.g., cuts, fractures)) or visceral (i.e., from internal organs (e.g., appendicitis)).
  • the nociceptive pain is osteoarthritis-related rheumatoid pain, arthritis-associated post-traumatic pain, musculoskeletal pain, postoperative pain, tendonitis, myofascial pain, visceral inflammatory pain (e.g., appendicitis, cystitis, pancreatitis), dysmenorrhea, or inflammatory bowel disease-related pain.
  • the mixed pain is cancer-related pain (e.g., bone metastasis pain, tumor infiltration pain, perineural invasion), complex regional pain syndrome type I, failed back surgery syndrome, low back pain with radiculopathy endometriosis-associated pelvic pain, chronic post-surgical pain, chronic pancreatitis-associated pain, interstitial cystitis/bladder pain syndrome, fibromyalgia, temporomandibular disorder, vulvodynia, irritable bowel syndrome with visceral pain, and combinations thereof.
  • the pain is acute pain or chronic pain.
  • the term “chronic pain” refers to pain that is persistent or recurrent pain lasting longer than 3 months.
  • the chronic pain is chronic primary pain, chronic cancer pain, chronic postsurgical and posttraumatic pain, chronic neuropathic pain, chronic headache and orofacial pain, chronic visceral pain, chronic musculoskeletal pain.
  • the pain is cancer pain.
  • the pain is post-surgery pain. 27 Attorney Docket No.: 071741.11025/5WO1 [0126]
  • the peripherally selective ⁇ 2AR agonist comprises an ⁇ 2AR activation moiety covalently linked to a peripheral distribution moiety.
  • the present disclosure relates to methods of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a peripherally selective ⁇ 2AR agonist, wherein the peripherally selective ⁇ 2AR agonist comprises an ⁇ 2AR activation moiety covalently linked to a peripheral distribution moiety.
  • the disease is glaucoma, pain, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, presbyopia, acute kidney injury, insomnia, inflammatory disease, cancer, etc.
  • the disease is glaucoma or cancer.
  • the disease is pain.
  • the pain is neuropathic pain, nociceptive pain, nociplastic pain, or mixed pain.
  • the neuropathic pain is chosen from diabetic peripheral neuropathy, postherpetic neuralgia, chemotherapy-induced peripheral neuropathy, Fabry disease– associated pain, trigeminal neuralgia, glossopharyngeal neuralgia, occipital neuralgia, HIV- associated neuropathy, hereditary sensory and autonomic neuropathy, Charcot-Marie-Tooth disease–related pain, small fiber neuropathy, brachial plexus avulsion, phantom limb pain, post- surgical neuropathic pain, carpal tunnel syndrome, other compression or entrapment neuropathies, amyloid neuropathy, vasculitic neuropathy, leprosy-associated pain, radiation-induced neuropathy, drug-induced neuropathy, metabolic neuropathies, endocrine-related neuropathies, toxic neuropathies, immune-mediated neuropathies, traumatic peripheral nerve
  • the nociceptive pain is somatic (i.e., from skin, muscles, or joints (e.g., cuts, fractures)) or visceral (i.e., from internal organs (e.g., appendicitis)).
  • the nociceptive pain is osteoarthritis-related rheumatoid pain, arthritis-associated post-traumatic pain, musculoskeletal pain, postoperative pain, tendonitis, 28 Attorney Docket No.: 071741.11025/5WO1 myofascial pain, visceral inflammatory pain (e.g., appendicitis, cystitis, pancreatitis), dysmenorrhea, or inflammatory bowel disease-related pain.
  • the mixed pain is cancer-related pain (e.g., bone metastasis pain, tumor infiltration pain, perineural invasion), complex regional pain syndrome type I, failed back surgery syndrome, low back pain with radiculopathy endometriosis-associated pelvic pain, chronic post-surgical pain, chronic pancreatitis-associated pain, interstitial cystitis/bladder pain syndrome, fibromyalgia, temporomandibular disorder, vulvodynia, irritable bowel syndrome with visceral pain, and combinations thereof.
  • the pain is acute pain or chronic pain.
  • the term “chronic pain” refers to pain that is persistent or recurrent pain lasting longer than 3 months.
  • the chronic pain is chronic primary pain, chronic cancer pain, chronic postsurgical and posttraumatic pain, chronic neuropathic pain, chronic headache and orofacial pain, chronic visceral pain, chronic musculoskeletal pain.
  • the pain is cancer pain.
  • the pain is post-surgery pain.
  • treating with the peripherally selective ⁇ 2AR agonist causes less side effects than treating with a non-peripherally selective ⁇ 2AR agonist, such as at similar or comparable dosage.
  • treating with the peripherally selective ⁇ 2AR agonist causes no side effects.
  • the side effect is sedation, decreasing heart rate, and decreasing blood pressure, particularly the side effect is sedation.
  • a non-peripherally selective ⁇ 2AR agonist refers to a compound can be readily distributed into the CNS after being administered into a subject, binds to and activates ⁇ 2AR receptor in both the central nervous system (brain and spinal cord) and the peripheral nervous system. Examples of non-peripherally selective ⁇ 2AR agonists include, but not limited to, dexmedetomidine, and clonidine.
  • an ⁇ 2AR agonist binds to and activates ⁇ 2AR in the central nervous system, it can produce the above mentioned side effects in patients, such as sedation, decreased heart rate, blood pressure, depression, bradycardia, orthostatic hypotension, 29 Attorney Docket No.: 071741.11025/5WO1 constipation, nausea, gastric upset, dry mouth (xerostomia), dry nasal mucosa, impotence, fluid retention, edema, and pupil size.
  • a peripherally selective ⁇ 2AR agonist refers to a compound that primarily exerts its effects outside of the central nervous system (CNS), typically because it is impeded by the blood-CNS barrier.
  • Blood-CNS barrier the physical barrier between blood and the CNS, safeguards the CNS from both toxic and pathogenic agents in the blood.
  • the blood-CNS barrier comprises the blood-brain barrier, the blood-spinal cord barrier, and the blood-CSF (cerebrospinal fluid) barrier.
  • a compound may act on the rest of the body with less or no side-effects related to their effects on the brain or spinal cord.
  • peripherally selective ⁇ 2AR agonists include, but not limited to, the compounds described herein, such as compounds of formula (I-A), (I-B), (I-C), (I-D), or (II), described herein.
  • the peripherally selective ⁇ 2AR agonist primarily binds to or activates ⁇ 2AR outside CNS, thus herby producing less or no foregoing side effects, compared to the non-peripherally selective ⁇ 2AR agonists.
  • the present invention satisfies an unmet need, and has developed a series of peripherally selective ⁇ 2AR agonists.
  • the peripherally selective ⁇ 2AR agonist binds to ⁇ 2AR with a Ki ranging from 250nM to1000nM, 50nM to 250nM, 10nM to 50nM, or less than 10nM.
  • the peripherally selective ⁇ 2AR agonist activates ⁇ 2AR with an EC50 ranging from 250nM to1000nM, 50nM to 250nM, 10nM to 50nM, or less than 10nM.
  • the non-peripherally selective ⁇ 2AR agonist and the peripherally selective ⁇ 2AR agonist can be differentiated in terms of blood-brain barrier (BBB) permeability. Drugs that specifically target the central nervous system (CNS) must first traverse the BBB. In contrast, peripherally selective drugs primarily exert their effects outside of CNS, largely because they are impeded by the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • BBB blood-brain barrier
  • CNS central nervous system
  • Kp the concentration of drug in the brain divided by concentration in the blood.
  • Kp is often calculated as “logBB”, which refers to the logarithmic ratio of the concentration of a compound in the brain and in the blood.
  • Kp is a common 30 Attorney Docket No.: 071741.11025/5WO1 numeric value for describing permeability across the blood-brain barrier.
  • a compound is considered “peripherally selective” if, upon administration to a subject, its Kp is lower than 0.4, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01.
  • Kp,uu,brain is another common numeric value for describing permeability across the blood-brain barrier.
  • Kp,uu,brain or “Kp,uu”, refers to the unbound brain-to- plasma partition coefficient.
  • Kp,uu provides a more accurate measure of distribution equilibrium between unbound fractions in brain and plasma.
  • Any methods known in the field can be used to measure Kp,uu,brain.
  • AUC Area Under the Curve
  • Another example is Steady-State Concentrations, which uses the steady-state unbound concentrations of the drug in brain interstitial fluid (C_u,brain,ss) and in plasma (C_u,plasma,ss).
  • a compound is considered “peripherally selective” if, upon administration to a subject, its Kp,uu,brain is lower than 0.4, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01. In some further embodiments, a compound is considered “peripherally selective” if, upon administration to a subject, its Kp,uu,brain is lower than 0.05, 0.04, 0.03, 0.02, or 0.01. [0154] In some embodiments, the peripherally selective ⁇ 2AR agonist comprises an ⁇ 2AR activation moiety that is covalently linked to a peripheral distribution moiety.
  • the ⁇ 2AR activation moiety is a non-peripherally selective ⁇ 2AR agonist or another peripherally selective ⁇ 2AR agonist.
  • the ⁇ 2AR activation moiety is an ⁇ 2AR agonist that is chosen from (R)-3-nitrobiphenyline, A-193080, ADX-415, AGN 192836, AGN-191103, AGN-197075, AGN-201781, AGN-241622, amitraz, Apraclonidine, AR-08, Bethanidine, Brimonidine, BRL- 48962, Bromocriptine, Cirazoline, Clonidine, Detomidine, Detomidine carboxylic acid, Dexmedetomidine, Dipivefrin, DL-Methylephedrine, Droxidopa, Epinephrine, ergotamine, etilefrine, Etomidate, Fadolmidine, Guanabenz, Guanethidine, Gu
  • the functional derivative of an ⁇ 2AR agonist refers to any compound that is derived from the ⁇ 2AR agonist by a chemical reaction.
  • the derivatives include, but not limited to, acid or base salts, prodrugs, compounds containing protected functional groups such as hydroxyl, amino, carboxyl and carbonyl groups.
  • the ⁇ 2AR activation moiety is a non-peripherally selective ⁇ 2AR agonist, such as dexmedetomidine, brimonidine, and clonidine.
  • the ⁇ 2AR activation moiety is dexmedetomidine.
  • the term “a peripheral distribution moiety” refers to a moiety that can increase or improve the peripheral selectivity of an ⁇ 2AR agonist.
  • the peripheral selectivity is increased or improved so that the ⁇ 2AR agonist is a peripherally selective ⁇ 2AR agonist.
  • the peripheral distribution moiety can be the following chemical fragments: • type A fragments; those that can increase the overall molecular polarity of the compound or reduce the overall lipophilicity of the compound; • type B fragments: those that can increase the overall molecular weight or the molecular size of the compound; and • type C fragments: those that comprises a substrate element of an efflux transporter.
  • the peripheral distribution moiety is a type A fragment.
  • the type A fragment increases the total number of intermolecular hydrogen bond (H-bond) within the compound, such as H-bond donors and H-bond acceptors.
  • the type A fragment is a H-bond donor.
  • the type A fragment increases the overall molecular polarity of the compound.
  • such type A fragments can comprise a polar functional group or a charged group.
  • Examples of the polar functional group include, but not limited to, hydroxyl, amine, amide, sulfonamide, carboxyl, ether, imine, hydroxylamine, ester, aldehyde, ketone, nitro, 32 Attorney Docket No.: 071741.11025/5WO1 phosphate, thioether, and sulfone groups.
  • Examples of the charged group include, but not limited to, quaternary ammonium and organic acids such as carboxylic acids and sulfonic acids.
  • the type A fragment reduces the overall lipophilicity of the compound.
  • type A fragments include, but not limited to, alkyl or acyl that is added to a function group such as hydroxyl and amino.
  • the type A fragment is not tertiary amine or one that can help form an intramolecular H-bond.
  • the peripheral distribution moiety is a type B fragment.
  • the type B fragment is a bulky group, which can increase the overall molecular weight and the molecular size of the compound. Examples of such type B fragments include, but not limited to, long alkyl chains, polyethylene glycol (PEG), large aromatic groups, and extra cyclic or heterocyclic groups.
  • the peripheral distribution moiety is a type C fragment.
  • the type C fragment comprises a substrate element of an efflux transporter, wherein the efflux transporter is P-glycoprotein (P-gp) transporter, breast cancer resistance protein (BCRP) transporter, or multidrug resistance protein 2 (MRP2) transporter.
  • P-gp P-glycoprotein
  • BCRP breast cancer resistance protein
  • MRP2 multidrug resistance protein 2
  • a substrate element of an efflux transporter refers to a fragment that makes the compound to become a substrate of the efflux transporter.
  • the term of “a substrate element of an efflux transporter” refers to a fragment of a substrate of the efflux transporter.
  • the type C fragment comprises a substrate element of P-gp.
  • P-gp efflux is a significant limitation to BBB permeation. Any methods known in the filed can be used to determine whether a compound is a P-gp substrate. For example, the efflux ratio obtained from in vitro P-gp assay, MDCK-MDR1, can be used to identify the substrate of P-gp. A compound is considered as a P-gp substrate if its efflux ratio is greater than 2, 5, 8, 10, 50, or 100.
  • the substrate element for P-gp contains one or more of the structural modifications described above.
  • the substrate element for P-gp is chosen from: O H N O H N O O H O O NH N P S F 3 .
  • the type C fragment comprises a substrate element of MPR2 transporter.
  • the type C fragment does not comprise a substrate element of uptake transporter, such as LAT1, GLUT1, MCT1, CAT1, CNT2, OATP, PEPT1, PEPT2, and OCT.
  • a substrate element of uptake transporter such as LAT1, GLUT1, MCT1, CAT1, CNT2, OATP, PEPT1, PEPT2, and OCT.
  • the peripheral distribution moiety reduces and/or minimizes brain exposure to a peripherally selective ⁇ 2AR agonist.
  • the peripheral distribution moiety decreases passive transcellular BBB permeability by increasing topological polar surface area (TPSA), increasing molecule weight, increasing polarity, or adding hydrogen binding, especially hydrogen bond donor.
  • TPSA topological polar surface area
  • the peripheral distribution moiety introduces an acidic group to the peripherally selective ⁇ 2AR agonist.
  • the peripheral distribution moiety comprises a substrate element for P-gp, wherein the substrate element for P-gp increases P-gp efflux by increasing lipophilicity, increasing hydrogen bond acceptors, removing steric hindrance around hydrogen bind acceptors, or removing electron-withdrawing group adjacent to hydrogen bond acceptor.
  • the peripheral distribution moiety makes a compound to become a dual substrate for both P-gp and BCRP.
  • R L is a moiety that covalently connects two functional groups or moieties within a single molecule.
  • One end of RL is connected to RP and the other end of RL is connceted to Y.
  • RL can be any moiety that serves the linking function, such as the linkers used in proteolysis targeting chimeras (PROTACs) and non-cleavable linkers used in antibody-drug conjugates (ADCs).
  • R L include, but are not limited to, polyethylene glycol (PEG) and alkyl chains of varying lengths, glycols, alkynes, triazoles, saturated heterocycles such as piperazine and piperidine, thioethers, maleimidocaproyl linker.
  • R L is chosen from alkyl, polyethylene glycol, other glycol, cycloalkyl, heterocycle, aryl, and heteroaryl; wherein the cycloalky, heterocycle, aryl, or heteroaryl is optionaly substituted with at least one substituent chosen from halogen, hydroxyl, alkyl, haloalkyl, alkoxy and hydroxyalkyl.
  • RL is one selected from the followings: Z Y N m2 Y Y m2 m2 H , m , ag e s, e ag e s ca e co ec e a y o e .
  • Rp can be the following chemical moieties: • those that can increase the overall molecular weight of the compound, such as bulky functional groups and additional molecular structures, including long alkyl chains, large aromatic groups, and extra cyclic structures like cyclohexane or cyclopentane rings; • those that can increase the overall molecular polarity of the compound, such as hydroxyl, amine, amide, sulfonamide, ether, imine, hydroxylamine, ester, aldehyde, ketone, nitro, phosphate, thioether; and • those that can bring charge to the compound, such as the functional groups that ionize at physiological pH, including carboxylic acid, quaternary ammonium and quaternary phosphonium.
  • RP when RP is not substituted with RC, RP is: O H N O H N O O O H N N . 39 Attorney Docket No.: 071741.11025/5WO1 [0190] In some embodiments, when RP is substituted with RC, RP is: O H O N O H N O O H O O NH N P S S O F 3 . [0191] As u end of RP.
  • RC is chosen from -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene- C 2-12 heterocyclyl, -C 0-12 alkylene-C 1-12 heteroaryl, -NH-C 0-12 alkylene-C 3-12 cycloalkyl, -NH-C 0-12 alkylene-C2-12 heterocyclyl, -NH-C0-12 alkylene-C1-12 heteroaryl, -O-C0-12 alkylene-C3-12 cycloalkyl, -O-C0-12 alkylene-C2-12 heterocyclyl, -O-C0-12 alkylene-C1-12 heteroaryl, and alkyl substituted with trialkylammonium, wherein each of the C 3-12 cycloalkyl, C 2-12 heterocyclyl, and C 1-12 heteroaryl is optionally substituted with one or more substituents chosen from hydroxy, alkyl, oxo,
  • RC is: Z Z s s Z s Z s N H .
  • Attorney Docket No.: 071741.11025/5WO1 [0194]
  • RC is: H NH N NH NH O O O O
  • Attorney Docket No.: 071741.11025/5WO1 O NH H O O N N O O S
  • the present disclosure relates to a compound of formula (I-D): N NH T , or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, wherein, Y 1 is CH, or N; X 1 is chosen from H, D, and halogen; and R T is defined as above in formula (I-A).
  • the compound of formula (I-A) has the formula (I-A-1): R T m , or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, wherein, R 1 is chosen from H, D, and halogen; 43 Attorney Docket No.: 071741.11025/5WO1 A is a ring chosen from phenyl, pyridinyl, thienyl, furyl, pyrrolyl, 4H-pyran, or 4H- thiopyran; R 2 , n, R 3 , m, and R T are defined as above in formula (I-A).
  • the compound of formula (I-A) has the formula (I-A-2): R T , or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, wherein, A is a ring chosen from phenyl, 1,2,3,4-tetrahydro-1-naphthyl, quinoxalinyl, pyrimidinyl, and 2,1,3-benzothiadiazol; Y is CH, N, -O-CH-, or -C-NH-; when Y is -O-C-, the oxygen atom is connected to A, and the carbon atom is connected X to both ; when arbon atom is connected to both R T and A, and the nitrogen X atom is connecte ; X is NH, O, or S; and R 2 , n, and RT are defined as above in formula (I-A).
  • the present disclosure relates to a compound of formula (II): R T B , or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, wherein, A is one chosen from: S 1 ; each R 1 is independently chosen from hydrogen, halogen, haloalkyl, hydroxyl, hydroxyalkyl, alkoxy, alkyl, and -COOH; B is one chosen from: 46 Attorney Docket No.: 071741.11025/5WO1 H N N H X S N N H; oaryl, wherein the C3-12 cycloalkyl or C2-12 heterocyclyl is optionally fused with an aryl; r is 1 or 2; n2 is 0, 1, or 2; each R 2 is independently chosen from hydrogen, halogen, hydroxyl, and alkoxy; R 3 is chosen from CN, hydroxy, alkoxy, -C(O)-C 0-12 alkylene-CN, -C 0-12
  • R 1 is halogen, such as fluorine or chlorine.
  • R 1 is alkoxy, such as -OMe.
  • R 1 is hydroxyl or -COOH or -CH 2 OH.
  • R 1 is haloalkyl, such as trifluoromethyl or -CH2CH2F.
  • B i wherein X is S, O, or NH.
  • n2 [0210] In some embodiments .
  • R 8 n4 [0211] In some embodiments, RT is .
  • ring 12 heteroaryl [0213] In some embodiments, ring M is C 3-12 cycloalkyl or C 2-12 heterocyclyl, wherein the C 3-12 cycloalkyl or C2-12 heterocyclyl is optionally fused with an aryl. [0214] In some embodiments, ring M is phenyl, pyridinyl, pyrimidinyl, thiophenyl, cyclopentyl, or cyclohexyl.
  • R 2 is hydroxyl.
  • R 2 is halogen, such as fluorine or chlorine.
  • the pharmaceutically acceptable salt of the compound of formula (I) is trifluoroacetate or hydrochloride.
  • the compound of formula (II) is a compound of formula (II-A): 49 Attorney Docket No.: 071741.11025/5WO1 R 2 R 3 H , wherein R 1 , R 2 , R 3 , and n1 are defined as above in formula (II).
  • R 1 is halogen, haloalkyl, hydroxyl, alkyl, or -COOH.
  • R 1 is methyl, ethyl, hydroxyl, fluorine, chlorine, trifluoromethyl, -CH 2 CH 2 F, or -COOH.
  • n1 is 2.
  • R 2 is hydrogen, hydroxyl, or halogen.
  • R 2 is fluorine or chlorine.
  • R 3 is -C(O)-NR 4 R 4’ or -SO 2 -NR 4 R 4’ , wherein each of R 4 and R 4’ is independently hydrogen, alkyl, alkoxy, -C0-12 alkylene-N(R 6a )t, -C0-12 alkylene-C3-12 cycloalkyl, - C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-OR 6a , or hydroxyalkyl, and the hydroxyalkyl is optionally substituted with alkoxy; wherein each of the alkyl, C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R 4a , wherein R 4a , R 6a , and t are defined as above.
  • each of R 4 and R 4’ is independently hydrogen, alkyl, alkoxy, or hydroxyalkyl. O , ed CH3 O with alkoxy, such as , wherein p is 0, 1, 2, or 3, particularly p is 2.
  • R 3 is -C(O)-NR 4 R 4 or -SO2-NR 4 R 4’ , wherein R 4 and R 4’ , together with the nitrogen atom that they are attached to, form a heterocycle comprising one or more 50 Attorney Docket No.: 071741.11025/5WO1 heteroatoms chosen from O, N, and S, particularly, R 4 and R4’, together with the nitrogen atom that they are attached to, form a six-membered heterocycle.
  • O N [0231]
  • R 3 is h drox l -COOH -CH(CH 3 )-COOH -CN , N .
  • R 4 is hydrogen or alkyl
  • R 5 is amino, alkylamino, C1-12 haloalkyl, -C0-12 alkylene-OR 6a , -C0-12 alkylene-N(R 6a )t, -C0-12 alkylene- SR 6a , -C0-12 alkylene-CN, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C 1-12 heteroaryl, -C 2-12 alkenyl, or alkyl optionally substituted with cyano or amido; wherein each of the C 3-12 cycloalkyl, C 2-12 heterocyclyl, and C 1-12 heteroaryl is optionally substituted with one or more R 4a ,
  • R 5 is amino, alkylamino, alkoxy, alkyl, or -C2-12 alkenyl.
  • R 5 is alkyl substituted with cyano, such as -CH 2 CN.
  • R 5 is alkyl substituted with amido, such as -CH2CH3CONH2.
  • R 5 is alkyl substituted with alkoxy, trialkylammonium, or thiolate.
  • R 5 when R 5 is -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, or -C0-12 alkylene-C1-12 heteroaryl, the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 , . , , 2 heteroaryl is substituted with one or more R 4a , R 4a is hydroxyl, methyl, oxo, or -C(O)-Me.
  • R 3 is m , wherein m is 0, 1, 2, 3, 4, or 5, and R 6 is sulfonamide, carbamide, or alkyl stituted with cyano.
  • R is sulfonamide of formula –N(R’)SO2-R, wherein each R and R’ is independently chosen from hydrogen and alkyl, particularly R 6 is -NHSO 2 CH 3.
  • R 6 is carbamide of formula –N(R’)C(O)N(R) 2 , wherein each R N N N H and R’ is independently chosen from hydrogen, alkyl, and heteroaryl, particular .
  • R 6 is alkyl optionally substituted with cyano, alkyl optionally substituted with cyano, particularly C1-4 alkyl substituted with cyano.
  • m is 1, 2, or 3, particularly 2.
  • R 7 is hydrogen. N O O S O [0246] In certain embodiments .
  • the compound of formula (II) is a compound of formula (II-B): 52 Attorney Docket No.: 071741.11025/5WO1 R 1 R 8 ( ) n1 (CH2) n3 O n4 , wherein R 1 , R 8 , n1, n3, and n4 are defined as above in formula (II).
  • R 1 is hydrogen or alkyl, such as alkyl, particularly methyl.
  • n1 is 2. [0252] In some embodiments, n3 is 0, 1 or 3. [0253] In some embodiments, n4 is 2, 3, or 5. [0254] In some embodiments, R 8 is alkoxy, such as C1-4 alkoxy, particularly methoxy or ethoxy. [0255] In some embodiments, R 8 is amino. [0256] In some embodiments, R 8 is alkylamino, such as C 1-4 alkylamino, particularly methylamino.
  • R 8 is amide of formula –N(R’)C(O)R, wherein each R and R’ is independently chosen from hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl.
  • R 8 is amide of formula –N(R’)C(O)R, particularly R 8 is - NHCOCH3.
  • R 8 is sulfonamide of formula –N(R’)SO 2 -R, wherein each R and R’ is independently chosen from hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl.
  • R 8 is sulfonamide of formula –N(R’)SO2-R, wherein each R and R’ is independently chosen from hydrogen, -C 0-12 alkylene-C 2-12 heterocyclyl, and alkyl, particularly R 3 is -NHSO 2 CH 3.
  • R 8 is carbamide of formula –N(R’)C(O)N(R)2, wherein each R and R’ is independently chosen from hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl.
  • R 8 is carbamide of formula –N(R’)C(O)N(R) 2 , wherein each R and R’ is independently chosen from hydrogen, alkyl, and heteroaryl, particularly R 3 is - N N 53 Attorney Docket No.: 071741.11025/5WO1 [0263]
  • R 8 is -OCH3, - O O N N H O H O O N N N N N S S .
  • the compound of formula (II) is a compound of formula (II-C): R 2 R 3 H , wherein, n2 is 1 or 2; and R 1 , R 2 , R 3 , and n1 are defined as above in formula (II).
  • R 1 is alkyl, such as methyl.
  • n1 is 2.
  • n2 is 1.
  • R 2 is hydrogen or halogen.
  • R 2 is fluorine.
  • R 3 is -C(O)-NR 4 R 4’ , wherein each of R 4 and R 4’ is independently hydrogen, hydroxy, alkyl, alkoxy, -SO2-NHCH3, -SO2-NH-Ph, -CH2-COOH, -CH2-CH2-COOH, O .
  • R 3 is -SO2-NR 4 R 4’ , wherein each of R 4 and R 4’ is independently hydrogen, hydroxy, or -C0-12 alkylene-C2-12 heterocyclyl.
  • R 3 is -NH-C(O)-R 5 , -N(CH 3 )-C(O)-R 5 or -NH-SO 2 -R 5 , wherein R 5 is alkyl, -C0-12 alkylene-alkoxy, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-NH- C1-12 alkyl, -C0- 12 alkylene-NH- C2-12 heterocyclyl, or -C0-12 alkylene-C2-12 heterocyclyl. [0273] In certain embodiments, R 5 is alkyl, such as methyl.
  • R 5 is -C 0-12 alkylene-alkoxy, such as -CH 2 -OCH 3 . 54 Attorney Docket No.: 071741.11025/5WO1 [0275] In certain embodiments, R 5 is -C 0-12 alkylene-C 3-12 cycloalkyl, such . [0276] In certain embodiments, R 5 is -C0-12 alkylene-NH- C1-12 alkyl, such 5 [0277] In certain embodiments, R is-C0-12 alkylene-NH- C2-12 heterocyclyl, such as O H H N O HN N O .
  • R 3 is -SO2-alkyl, such as -SO2-CH3.
  • R 3 is -C 0-12 alkylene-COOH, such as -COOH, -CH 2 -COOH, - C(Me) 2 -COOH, -CH 2 - CH 2 -COOH.
  • R 3 is -C0-12 alkylene-C1-12 heteroaryl, su , O N HN N N N N N N N N N -R 5
  • the compound of formula (II) is a compound of formula (II-D): 55 Attorney Docket No.: 071741.11025/5WO1 R 2 R 3 H , wherein, n2 is 0 or 1; R 3 is chosen from -C(O)-NHR 4 , -SO2-NHR 4 , -NH-C(O)-R 5 , and -NH-SO2-R 5 , and -NH-R 7 ; R 4 is -C 0-12 alkylene-NHR 6a , -C 0-12 alkylene-C 3-12 cycloalkyl, -C 0-12 alkylene-C 2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-
  • R 1 is alkyl, such as methyl. [0286] In some embodiments, R 1 is alkoxy, such as -OMe. [0287] In some embodiments, n1 is 1. [0288] In some embodiments, n1 is 2. [0289] In some embodiments, n2 is 0. [0290] In some embodiments, n2 is 1. [0291] In some embodiments, R 2 is hydrogen or halogen.
  • R 3 is -C(O)-NHR 4 or -SO2-NHR 4 , wherein R 4 is -C0-12 alkylene- NHR 6a , -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C 0-12 alkylene-OR 6a , or alkyl substituted with trialkylammonium; wherein and each of the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R 4a , wherein R 4a and R 6a are defined as above.
  • R 5 is alkyl substituted with trialkylammonium.
  • R 5 is -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, or -C 0-12 alkylene-C 1-12 heteroaryl
  • the C 3-12 cycloalkyl, C 2-12 heterocyclyl, and C 1-12 H heteroaryl is substituted with one or more R 4a , R 4a is hydroxyl, methyl, oxo, or -C(O)-Me.
  • 57 Attorney Docket No.: 071741.11025/5WO1 OMe P [0299] In certain embodiments O .
  • t he compound of formula (II) is a compound of formula (II-E): R 2 R 3 N (II-E) , wherein x is 0 or 1; y is 0 or 1; X is S, O, or NH; and R 1 , R 2 , R 3 , n1, and n2 are defined as above in formula (II).
  • x is 0, y is 1, and X is S, O, or NH.
  • x is 0 or 1
  • y is 0, and X is NH.
  • each R 1 is independently chosen from hydrogen, halogen, alkoxy, and alkyl.
  • R 1 is methyl, chlorine, or methoxy.
  • n2 is 1.
  • n2 is 2.
  • R 2 is hydrogen.
  • R 3 is -C(O)-NR 4 R 4’ , wherein each of R 4 and R 4’ is independently hydrogen or alkoxy.
  • R 3 is -SO2-NR 4 R 4’ , wherein each of R 4 and R 4’ is independently hydrogen or alkyl.
  • R 3 is -NH-C(O)-R 5 or -NH-SO 2 -R 5 , wherein R 5 is alkyl or -C 0-12 alkylene-C2-12 heterocyclyl. 58 Attorney Docket No.: 071741.11025/5WO1 [0311] In certain embodiments, R 5 is alkyl, such as methyl.
  • R 5 is -C0-12 alkylene-C2-12 heterocyclyl, such a or O F): , , R 2 is adjacent to R 3 , and R 2 and R 3 , together with the carbon atoms that they are attached to, form a heterocycle optionally substituted with one or more R 4a ; and R 1 , R 4a , and n1 are defined as above in formula (II). [0314] In some embodiments, R 2 and R 3 , together with the carbon atoms that they are attached to, form a 5- or 6-membered heterocycle optionally substituted with one or more R 4a .
  • the compound of formula (II-F) is a compound of formula (II-F- 1): M1 H ( - - ) , wherein M1 is a heterocycle optionally substituted with one or more R 4a .
  • the compound of formula (II) is a compound of formula (II-G): 59 Attorney Docket No.: 071741.11025/5WO1 R 2 r R 3 H , wherein, each R 2 is independently chosen from hydroxyl and alkoxy; R 3 is chosen from hydroxy and alkoxy; and R 1 , n1 and r are defined as above in formula (II).
  • one R 2 is adjacent to R 3 .
  • each R 2 is independently chosen from hydroxyl and methoxy.
  • R 3 is chosen from hydroxyl and methoxy.
  • the compound of formula (II-G) is a compound of formula (II-G- 1) of (II-G-2): R 2 R 2 R 3 R 3 NH , .
  • the compound of formula (II) is a compound of formula (II-H): R 2 , l; e-COOH, -O-C0-12 alkylene-COOH, -C0-12 alkylene- P(O)(OH) 2 , -C(O)-NH-SO 2 -R 5 , -C(O)-NH-C 0-12 alkylene-COOH, -NH-C 0-12 alkylene-COOH, - 60 Attorney Docket No.: 071741.11025/5WO1 N N N alkylene N ; wherein the -C0-12 alkylene-COOH is optionally osen from amino and alkylamino; and R , R , and n1 are defined as above in formula (II).
  • M is phenyl.
  • M is pyridinyl.
  • Exemplary compounds of formula (I-A), (I-B), (I-C), (I-D), or (II) include, but are not limited to, the compounds described herein, and any tautomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof.
  • Exemplary RT No. structure No. structure No. structure 74 Attorney Docket No.: 071741.11025/5WO1 H N H H 4 N N O 108 O O O 203 NH O O O O 75
  • O N 124 H 216 76
  • stereoisomers e.g., enantiomers, diastereomers
  • racemic mixtures of enantiomers which can be separated from one another using art-known resolution procedures, for instance including liquid chromatography using a chiral stationary phase.
  • stereochemically pure isomeric forms of the compounds described herein can be derived from the corresponding stereochemically pure isomeric forms of the appropriate starting materials, intermediates, or reagents.
  • the compound can be synthesized by stereospecific methods of preparation, which typically employ stereochemically pure starting materials or intermediate compounds.
  • salts of compounds described herein can be synthesized from the parent compound containing an acidic or basic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate acid or base in water or in an organic solvent, or in a mixture of the two.
  • suitable organic solvents include, but are not limited to, ether, ethyl acetate (EtOAc), ethanol, isopropanol, or acetonitrile.
  • compositions comprising a compound of formula (I-A), (I-B), (I-C), (I-D), or (II) or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, as described herein.
  • Compositions can also comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is non-toxic and should not interfere with the efficacy of the active ingredient.
  • Pharmaceutically acceptable carriers can include one or more excipients such as 83 Attorney Docket No.: 071741.11025/5WO1 binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings.
  • excipients such as 83 Attorney Docket No.: 071741.11025/5WO1 binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings.
  • the precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, intradermal, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes.
  • suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like.
  • suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like.
  • the aqueous solution/suspension can comprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives.
  • compositions can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections.
  • the parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection.
  • Compositions can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal.
  • compositions The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen depend upon the condition to be treated, such as the severity of the illness, the age, weight, and sex of the patient.
  • Pharmaceutical compositions can be formulated for different modes of administration such as for topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, or subcutaneous administration.
  • a method of preparing a pharmaceutical composition comprising combining a compound of formula (I-A), (I-B), (I-C), (I-D), or (II), or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, with at least one pharmaceutically acceptable carrier.
  • compositions can be prepared by any method known in the art in view of the present disclosure, and one of ordinary skill in the art will be familiar with such techniques used to prepare pharmaceutical compositions.
  • a pharmaceutical composition according to the present disclosure can be prepared by mixing a compound of formula (I-A), (I-B), (I-C), (I-D), or (II), with one or more pharmaceutically acceptable carriers according 84 Attorney Docket No.: 071741.11025/5WO1 to conventional pharmaceutical compounding techniques, including but not limited to, conventional admixing, dissolving, granulating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the present disclosure relates to a pharmaceutical combination comprising a first therapeutical agent and a second therapeutical agent, wherein the first therapeutical agent is a peripherally selective ⁇ 2AR agonist, such as the peripherally selective ⁇ 2AR agonist described herein.
  • the pharmaceutical combination further comprises a third therapeutic agent.
  • the peripherally selective ⁇ 2AR agonist comprises an ⁇ 2AR activation moiety covalently linked to a peripheral distribution moiety.
  • the ⁇ 2AR activation moiety has formula of m , wherein Y, A, B, R2, R3, m, and n are defined as in formula [0345] In certain embodiments, the ⁇ 2AR activation moiety has formula of Y m , wherein Y, A, B, R2, R3, m, and n are defined as in formula [0346] In certain embodiments, the ⁇ 2AR activation moiety has formula of Y , wherein Y, A, B, R2, R3, m, and n are defined as in formula N [0347] In certain embodiments, the ⁇ 2AR activation moiety has formu , wherein X 1 and Y 1 are defined as in formula (I-D).
  • the ⁇ 2AR activation moiety has formula A B , wherein A and B are defined as in formula (II).
  • R T [0349]
  • the peripheral distribution moiety has formula of , wherein RT is defined as in formula (I-A).
  • R T [0350]
  • the peripheral distribution moiety has formula of , wherein R T is defined as in formula (II).
  • the peripherally selective ⁇ 2AR agonist is a compound of formula (I-A), (I-B), (I-C), (I-D) or (II).
  • the peripherally selective ⁇ 2AR agonist is a compound of formula (II-A), (II-B), (II-C), (II-D), (II-E), (II-F), (II-G), or (II-H).
  • the second therapeutic agent is any therapeutic agent other than a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist.
  • the third therapeutic agent is any therapeutic agent other than a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist, and the third therapeutic agent is different from the second therapeutic agent.
  • the second therapeutic agent is chosen from opioids, acetaminophen (paracetamol), local analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), antiepileptic medication, antidepressant medication, topical analgesic agents, NMDA receptor antagonists, neurotoxins, cannabinoids, voltage-gated sodium channel inhibitors, adenosine agonists, transient receptor potential (TRP) channel modulators, NGF inhibitors, purinergic receptor antagonists, adenosinergic pathway modulators, sigma-1 receptor antagonists, KCC2 enhancers, glutamate transport enhancers, TrkA receptor antagonists, somatostatin receptor ligands, Human Adenylyl Cyclase Associated Protein 1 (CAP1) modulators, and angiotensin II receptor antagonists, corticosteroids, and any combinations thereof.
  • opioids acetaminophen (paracetamol)
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • antiepileptic medication
  • the opioid is chosen from meperidine, tramadol, codeine, pentazocine, dihydrocodeine, tapentadol, hydrocodone, morphine, oxycodone, methadone, 86 Attorney Docket No.: 071741.11025/5WO1 oxymorphone, hydromorphone, butorphanol, buprenorphine, fentanyl, sufentanil, remifentanil, alfentanil, thebaine, diacetyl morphine, vicodin, dinorbuprenorphine, nalbuphine, and heroin.
  • the opioid is chosen from anrikefon, cebranopadol, STC-007, MR-309, desmetramadol, LPM-3480392, YZJ-4729, RFUS-144, HY-1608, NES-100, PN-6047, Cyt-1010, TRV-250, DMX-101, ADV-502, MEB-1170, TRV-734, KUR-101, 18F-FTC-146, MUM0-1, RFUS-250, NH-16003, and sunobinop.
  • the opioid is a biased opioid ligand, such as oliceridine or SHR8554.
  • the opioid is kappa-opioid agonists, such as CR845 (Difelikefalin).
  • the nonsteroidal anti-inflammatory drug is chosen from aspirin, diflunisal, ibuprofen, naproxen, indomethacin, diclofenac, meclofenamate, mefenamic acid, meloxicam, piroxicam, nabumetone, celecoxib, etoricoxib, biphenyl acetic acid, aceclofenac, ketorolac, etodolac, ketotifen, loxoprofen, tenoxicam, zaltoprofen, zaltoprofen, esflurbiprofen, valdecoxib, lumiracoxib, imidazole salicylate, dexketoprofen, nimesulide, otenaproxes
  • the antiepileptic medication is a calcium channel blockers (CCB), sodium channel blocker, or GABA modulator.
  • CB calcium channel blockers
  • the antiepileptic medication is chosen from gabapentin, pregabalin, enacarbil, mirogabalin, crisugabalin, phenibut, baclofen, 4-fluorophenibut, 4- methylpregabalin, atagabalin (PD-200,390), imagabalin, PD-217,014, tolibut, oxcarbazepine, ziconotide, lomerizine, carbamazepine, oxcarbazepine, lamotrigine, lidocaine, ropivacaine, bupivacaine, valproic acid, topiramate, and divalproex sodium.
  • the antiepileptic medication is non- ⁇ 2 ⁇ antiepileptics, such as carbamazepine or oxcarbazepine.
  • the antiepileptic medication is chosen from pregabalin naproxencarbil, XG-004, RAP-219, AFA-281, priralfinamide, laflunimus, and CPP-115.
  • the antidepressant medication is a selective serotonin and norepinephrine reuptake inhibitor (SNRI), a tricyclic antidepressant (TCA), or a monoamine oxidases inhibitor (MAOI).
  • the selective serotonin and norepinephrine reuptake inhibitor is duloxetine, venlafaxine, desvenlafaxine, bupropion, or milnacipran.
  • the tricyclic antidepressant is amitriptyline, nortriptyline, desipramine, clomipramine, or imipramine.
  • the topical analgesic agent is lidocaine (e.g., 5%) patch or capsaicin (e.g., 8%) patch.
  • the NMDA antagonist is chosen from ketamine, methadone, memantine, amantadine, dextromethorphan, and L-4-chlorokynurenine.
  • the neurotoxin is Botulinum toxin A.
  • the cannabinoid is chosen from nabilone, THC, cannabidiol (CBD), nabiximols (Sativex), AP-707, ONO-1110, CMX-020, CNTX-6016, and OCT-461201.
  • the voltage-gated sodium channel inhibitor is the inhibitor of NaV1.3, NaV1.7, NaV1.8 and / or NaV1.9.
  • the selective NaV1.8 inhibitor is chosen from suzetrigine (VX-548), ANP-230, VX-150, JMK-000623, HBW-004285, DSP-2230, VX-993, VX-973, LTG- 001, kindolor, FZ-008, HRS-4800, HRS-2129, MK-5661, and STC-004.
  • the selective NaV1.7 inhibitor is chosen from tetrodotoxin, OLP-1002, aneratrigine, CC-8464, DSP-3905, S-151128, and ST-2427.
  • the voltage-gated sodium channel inhibitor is relutrigine or ANP- 230.
  • the transient receptor potential (TRP) channel blocker is TRPV1 agonist, TRPV1 antagonist, TRPA1 inhibitor, TRPM3 inhibitor, TRPM8 inhibitor, and TRPC5 antagonist.
  • the transient receptor potential (TRP) channel blocker is chosen from capsaicin, vocacapsaicin, resiniferatoxin (RTX), ACD-440, libvatrep, tivanisiran, JNJ- 39439335, SB-705498, AJH-2947, SER-014, SRP-001, XEN-D0501, QP-5113, LD-04185, LD- 2020, BHV-2100, Elismetrep, MT-8554, RQ-00434739, and QR-060127.
  • TRP transient receptor potential
  • the NGF inhibitor is chosen from Tanezumab, Fulranumab, Fasinumab, EP-9001A, MEDI-7352, SSS-40, DS-002, luvagrobart, TNM-009, and STSA-1001.
  • the purinergic receptor antagonist is P2X purinoreceptor antagonist, such as NC-2600, or LY-3857210, P2X3 antagonists, such as Gefapixant (Merck), Sivopixant (Shionogi), or VNUT Inhibitor such as Clodronate.
  • the adenosinergic pathway modulator is a modulator for Adenosine A1, A2A, A3 receptors; CD39/73 enzymes; or Adenosine kinase.
  • the adenosinergic pathway modulator is caffeine.
  • the sigma-1 receptor antagonist is ketamine, memantine, or amantadine
  • the KCC2 enhancer is CLP257 or kenpaullone.
  • the glutamate transport enhancer is ceftriaxone.
  • the TrkA receptor antagonist is AK-1830 or BR-01T.
  • the somatostatin receptor ligand is pasireotide, LY-3556050, or FZ002-037.
  • the Human Adenylyl Cyclase Associated Protein 1 (CAP1) modulator is FM-888 or NB-001.
  • the Angiotensin II receptor antagonist is CFTX-1554, TRD-205, or WXSH-0024.
  • the corticosteroid is chosen from dexamethasone, clobetasol, triamcinolone acetonide, difluprednate, loteprednol, and fluticasone.
  • the second therapeutic agent is chosen from GGG tri-agonist (e.g., retatrutide), metabotropic glutamate receptors (mGluR) modulator (e.g., basimglurant), TGF beta-1 Inhibitor (e.g., Vicatertide (SB-01)), gonadotropin-releasing hormone receptor(GnRH) antagonist (e.g., linzagolix), 11 ⁇ -HSD1 inhibitor (e.g., clofutriben), 17-HSD-1 inhibitor (e.g., OG-6219), Dual ENKephalinase Inhibitor (DENKI) (e.g., PL37), CCR2 receptor antagonist (e.g., CNTX-6970), IL- 10 receptor agonist (e.g., XT-150), nAChR dual alpha 4/beta 2 subunit stimulator (e.g., ATA-104), allosteric NEK7/NLRP
  • GGG tri-agonist
  • the second therapeutic agent is chosen from IRX-101, MR-107A- 02, 3-VM-1001, lysergide assisted therapy, SIL-1002, TRN-261, HR-1405-01, HRF-2105, TTAX- 03, KP-910, LYT-503, LL-50, ZeP-3, YR-1702, YZJ-1495, FB-1003, MK-4318, PZH-2108, HEC- 137076MsOH, VVZ-2471, SYNP-101, Pudafensine, BIOS-0618, and HSK-36357.
  • kits for treating or preventing pain in a subject in need thereof comprising administering to the subject an effective amount of a peripherally selective ⁇ 2AR agonist, wherein treating with the peripherally selective ⁇ 2AR agonist causes less side effects than treating with a non-peripherally selective ⁇ 2AR agonist, such as at similar or comparable dosage.
  • the peripherally selective ⁇ 2AR agonist comprises an ⁇ 2AR activation moiety covalently linked to a peripheral distribution moiety.
  • kits for treating or preventing a disease in a subject in need thereof comprising administering to the subject an effective amount of a peripherally selective ⁇ 2AR agonist, wherein the peripherally selective ⁇ 2AR agonist comprises an ⁇ 2AR activation moiety covalently linked to a peripheral distribution moiety.
  • the present disclosure relates to a method of treating or preventing a disease in a subject in need thereof, the method comprising administering to the 90 Attorney Docket No.: 071741.11025/5WO1 subject an effective amount of the pharmaceutical combination described herein, or administering to the subject (i) an effective amount of a first therapeutic agent, and (ii) an effective amount of a second therapeutic agent, wherein the first therapeutic agent is a peripherally selective ⁇ 2AR agonist, such as the peripherally selective ⁇ 2AR agonist described herein.
  • the term “effective amount” regarding a pharmaceutical combination includes the effective amount of the first therapeutical agent and the effective amount of the second therapeutical agent, and these two effective amounts can be identical or different, fixed or varying dependent on upon a variety of factors as described above.
  • the disease is chosen from glaucoma, analgesia, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, menopausal hot flashes, dysmenorrhea, presbyopia, acute kidney injury, insomnia, inflammatory disease, and cancer.
  • the disease is pain.
  • the second therapeutic agent is any therapeutic agent other than a peripherally selective ⁇ 2AR agonist.
  • the present disclosure relates to a method for treating or preventing pain in a subject in need thereof, the method comprising administering to the subject an effective amount of a first analgesic agent, and an effective amount of a second analgesic agent, wherein the first analgesic agent is a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist, such as the peripherally selective ⁇ 2AR agonist described herein.
  • the method for treating or preventing pain further comprising administering to the subject an effective amount of a third analgesic agent.
  • the present disclosure relates to a method for treating or preventing pain in a subject in need thereof, the method comprising administering to the subject an effective amount of a codrug that comprises a first analgesic agent and a second analgesic agent, wherein the first analgesic agent is a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist, such as the peripherally selective ⁇ 2AR agonist described herein.
  • a codrug that comprises a first analgesic agent and a second analgesic agent
  • the first analgesic agent is a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist, such as the peripherally selective ⁇ 2AR agonist described herein.
  • ⁇ 2AR peripherally selective alpha-2 adrenergic receptor
  • the body After administration, the body (through enzymatic cleavage or hydrolysis) breaks the linkage, releasing the two active drugs at the site of action or in systemic circulation.
  • 91 Attorney Docket No.: 071741.11025/5WO1
  • the peripherally selective ⁇ 2AR agonist comprises an ⁇ 2AR activation moiety covalently linked to a peripheral distribution moiety.
  • the pain is neuropathic pain, nociceptive pain, nociplastic pain, or mixed pain.
  • the neuropathic pain is chosen from diabetic peripheral neuropathy, postherpetic neuralgia, chemotherapy-induced peripheral neuropathy, Fabry disease– associated pain, trigeminal neuralgia, glossopharyngeal neuralgia, occipital neuralgia, HIV- associated neuropathy, hereditary sensory and autonomic neuropathy, Charcot-Marie-Tooth disease–related pain, small fiber neuropathy, brachial plexus avulsion, phantom limb pain, post- surgical neuropathic pain, carpal tunnel syndrome, other compression or entrapment neuropathies, amyloid neuropathy, vasculitic neuropathy, leprosy-associated pain, radiation-induced neuropathy, drug-induced neuropathy, metabolic neuropathies, endocrine-related neuropathies, toxic neuropathies, immune-mediated neuropathies, traumatic peripheral nerve injury, and heritable neuropathies, as well as post-stroke pain syndrome, spinal cord injury–related pain, multiple sclerosis–associated pain,
  • the nociceptive pain is somatic (i.e., from skin, muscles, or joints (e.g., cuts, fractures)) or visceral (i.e., from internal organs (e.g., appendicitis)).
  • the nociceptive pain is osteoarthritis-related rheumatoid pain, arthritis-associated post-traumatic pain, musculoskeletal pain, postoperative pain, tendonitis, myofascial pain, visceral inflammatory pain (e.g., appendicitis, cystitis, pancreatitis), dysmenorrhea, or inflammatory bowel disease-related pain.
  • the mixed pain is cancer-related pain (e.g., bone metastasis pain, tumor infiltration pain, perineural invasion), complex regional pain syndrome type I, failed back surgery syndrome, low back pain with radiculopathy endometriosis-associated pelvic pain, chronic post-surgical pain, chronic pancreatitis-associated pain, interstitial cystitis/bladder pain syndrome, 92 Attorney Docket No.: 071741.11025/5WO1 fibromyalgia, temporomandibular disorder, vulvodynia, irritable bowel syndrome with visceral pain, and combinations thereof.
  • the pain is acute pain or chronic pain.
  • the term “chronic pain” refers to pain that is persistent or recurrent pain lasting longer than 3 months.
  • the chronic pain is chronic primary pain, chronic cancer pain, chronic postsurgical and posttraumatic pain, chronic neuropathic pain, chronic headache and orofacial pain, chronic visceral pain, chronic musculoskeletal pain.
  • the pain is cancer pain.
  • the pain is post-surgery pain.
  • the second therapeutic agent or the second analgesic agent is any therapeutic agent or analgesic agent other than a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist.
  • the second analgesic agent is chosen from opioids, acetaminophen (paracetamol), local analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), antiepileptic medication, antidepressant medication, topical analgesic agents, NMDA receptor antagonists, neurotoxins, cannabinoids, voltage-gated sodium channel inhibitors, adenosine agonists, transient receptor potential (TRP) channel modulators, NGF inhibitors, purinergic receptor antagonists, adenosinergic pathway modulators, sigma-1 receptor antagonists, KCC2 enhancers, glutamate transport enhancers, TrkA receptor antagonists, somatostatin receptor ligands, Human Adenylyl Cyclase Associated Protein 1 (CAP1) modulators, and angiotensin II receptor antagonists, corticosteroids, and any combinations thereof.
  • opioids opioids
  • acetaminophen paracetamol
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • the opioid is chosen from meperidine, tramadol, codeine, pentazocine, dihydrocodeine, tapentadol, hydrocodone, morphine, oxycodone, methadone, oxymorphone, hydromorphone, butorphanol, buprenorphine, fentanyl, sufentanil, remifentanil, alfentanil, thebaine, diacetyl morphine, vicodin, dinorbuprenorphine, nalbuphine, and heroin.
  • the opioid is chosen from anrikefon, cebranopadol, STC-007, MR-309, desmetramadol, LPM-3480392, YZJ-4729, RFUS-144, HY-1608, NES-100, PN-6047, Cyt-1010, TRV-250, DMX-101, ADV-502, MEB-1170, TRV-734, KUR-101, 18F-FTC-146, MUM0-1, RFUS-250, NH-16003, and sunobinop.
  • the opioid is a biased opioid ligand, such as oliceridine or SHR8554.
  • the opioid is kappa-opioid agonists, such as CR845 (Difelikefalin).
  • the nonsteroidal anti-inflammatory drug is chosen from aspirin, diflunisal, ibuprofen, naproxen, indomethacin, diclofenac, meclofenamate, mefenamic acid, meloxicam, piroxicam, nabumetone, celecoxib, etoricoxib, biphenyl acetic acid, aceclofenac, ketorolac, etodolac, ketotifen, loxoprofen, tenoxicam, zaltoprofen, zaltoprofen, esflurbiprofen, valdecoxib, lumiracoxib, imidazole salicylate, dexketopro
  • NSAID nonsteroidal anti-inflammatory drug
  • the antiepileptic medication is a calcium channel blockers (CCB), sodium channel blocker, or GABA modulator.
  • CB calcium channel blockers
  • the antiepileptic medication is chosen from gabapentin, pregabalin, enacarbil, mirogabalin, crisugabalin, phenibut, baclofen, 4-fluorophenibut, 4- methylpregabalin, atagabalin (PD-200,390), imagabalin, PD-217,014, tolibut, oxcarbazepine, ziconotide, lomerizine, carbamazepine, oxcarbazepine, lamotrigine, lidocaine, ropivacaine, bupivacaine, valproic acid, topiramate, and divalproex sodium.
  • the antiepileptic medication is non- ⁇ 2 ⁇ antiepileptics, such as carbamazepine or oxcarbazepine.
  • the antiepileptic medication is chosen from pregabalin naproxencarbil, XG-004, RAP-219, AFA-281, priralfinamide, laflunimus, and CPP-115.
  • the antidepressant medication is a selective serotonin and norepinephrine reuptake inhibitor (SNRI), a tricyclic antidepressant (TCA), or a monoamine oxidases inhibitor (MAOI).
  • the selective serotonin and norepinephrine reuptake inhibitor is duloxetine, venlafaxine, desvenlafaxine, bupropion, or milnacipran.
  • the tricyclic antidepressant is amitriptyline, nortriptyline, desipramine, clomipramine, or imipramine.
  • the topical analgesic agent is lidocaine (e.g., 5%) patch or capsaicin (e.g., 8%) patch.
  • the NMDA antagonist is chosen from ketamine, methadone, memantine, amantadine, dextromethorphan, and L-4-chlorokynurenine.
  • the neurotoxin is Botulinum toxin A. 94 Attorney Docket No.: 071741.11025/5WO1
  • the cannabinoid is chosen from nabilone, THC, cannabidiol (CBD), nabiximols (Sativex), AP-707, ONO-1110, CMX-020, CNTX-6016, and OCT-461201.
  • the voltage-gated sodium channel inhibitor is the inhibitor of NaV1.3, NaV1.7, NaV1.8 and / or NaV1.9.
  • the selective NaV1.8 inhibitor is chosen from suzetrigine (VX-548), ANP-230, VX-150, JMK-000623, HBW-004285, DSP-2230, VX-993, VX-973, LTG- 001, kindolor, FZ-008, HRS-4800, HRS-2129, MK-5661, and STC-004.
  • the selective NaV1.7 inhibitor is chosen from tetrodotoxin, OLP-1002, aneratrigine, CC-8464, DSP-3905, S-151128, and ST-2427.
  • the voltage-gated sodium channel inhibitor is relutrigine or ANP- 230.
  • the transient receptor potential (TRP) channel blocker is TRPV1 agonist, TRPV1 antagonist, TRPA1 inhibitor, TRPM3 inhibitor, TRPM8 inhibitor, and TRPC5 antagonist.
  • the transient receptor potential (TRP) channel blocker is chosen from capsaicin, vocacapsaicin, resiniferatoxin (RTX), ACD-440, libvatrep, tivanisiran, JNJ- 39439335, SB-705498, AJH-2947, SER-014, SRP-001, XEN-D0501, QP-5113, LD-04185, LD- 2020, BHV-2100, Elismetrep, MT-8554, RQ-00434739, and QR-060127.
  • TRP transient receptor potential
  • the NGF inhibitor is chosen from Tanezumab, Fulranumab, Fasinumab, EP-9001A, MEDI-7352, SSS-40, DS-002, luvagrobart, TNM-009, and STSA-1001.
  • the purinergic receptor antagonist is P2X purinoreceptor antagonist, such as NC-2600, or LY-3857210, P2X3 antagonists, such as Gefapixant (Merck), Sivopixant (Shionogi), or VNUT Inhibitor such as Clodronate.
  • the adenosinergic pathway modulator is a modulator for Adenosine A1, A2A, A3 receptors; CD39/73 enzymes; or Adenosine kinase.
  • the adenosinergic pathway modulator is caffeine.
  • the sigma-1 receptor antagonist is ketamine, memantine, or amantadine
  • the KCC2 enhancer is CLP257 or kenpaullone.
  • the glutamate transport enhancer is ceftriaxone.
  • the TrkA receptor antagonist is AK-1830 or BR-01T. 95 Attorney Docket No.: 071741.11025/5WO1 [0447]
  • the somatostatin receptor ligand is pasireotide, LY-3556050, or FZ002-037.
  • the Human Adenylyl Cyclase Associated Protein 1 (CAP1) modulator is FM-888 or NB-001.
  • the Angiotensin II receptor antagonist is CFTX-1554, TRD-205, or WXSH-0024.
  • the corticosteroid is chosen from dexamethasone, clobetasol, triamcinolone acetonide, difluprednate, loteprednol, and fluticasone.
  • the second analgesic agent is chosen from GGG tri-agonist (e.g., retatrutide), metabotropic glutamate receptors (mGluR) modulator (e.g., basimglurant), TGF beta-1 Inhibitor (e.g., Vicatertide (SB-01)), gonadotropin-releasing hormone receptor(GnRH) antagonist (e.g., linzagolix), 11 ⁇ -HSD1 inhibitor (e.g., clofutriben), 17-HSD-1 inhibitor (e.g., OG-6219), Dual ENKephalinase Inhibitor (DENKI) (e.g., PL37), CCR2 receptor antagonist (e.g., CNTX-6970), IL- 10 receptor agonist (e.g., XT-150), nAChR dual alpha 4/beta 2 subunit stimulator (e.g., ATA-104), allosteric NEK7
  • GGG tri-agonist
  • the second analgesic agent is chosen from IRX-101, MR-107A-02, 3-VM-1001, lysergide assisted therapy, SIL-1002, TRN-261, HR-1405-01, HRF-2105, TTAX-03, KP-910, LYT-503, LL-50, ZeP-3, YR-1702, YZJ-1495, FB-1003, MK-4318, PZH-2108, HEC- 137076MsOH, VVZ-2471, SYNP-101, Pudafensine, BIOS-0618, and HSK-36357.
  • the administration of the first therapeutic agent and the second therapeutic agent provides a synergistic effect in treating the disease.
  • the administration of the first analgestic agent and the second analgestic agent provides a synergistic effect in treating the pain.
  • the term "synergistic effect” refers to a pharmacological interaction between two or more agents wherein the combined effect is greater than the sum of their individual effects. In the context of analgesic or therapeutic efficacy, synergy indicates that the combination achieves enhanced results at equal or lower doses compared to each compound used separately.
  • the synergistic effect is potentiation.
  • the term “potentiation” refers to a specific type of synergy in which one agent, often inactive or weakly active alone, significantly enhances the effect of another active compound when used in combination.
  • the administration of the first therapeutic agent and the second therapeutic agent provides an additive effect in treating the disease.
  • the administration of the first analgestic agent and the second analgestic agent provides an additive effect in treating the pain.
  • additive effect refers to a combined pharmacological effect that is equal to the sum of the effects of individual agents, without enhancement or suppression.
  • Additivity is typically defined within an acceptable experimental range (e.g., CI between 0.95 and 1.1) that accounts for biological variability.
  • CI combination index
  • the synergistic affect and additive effect in combination therapy can be measured and/or determined by any methods known in the art.
  • combination index (CI) is a quantitative metric used to assess the nature of drug-drug interactions, and CI values can be calculated based on models such as Bliss independence or Loewe additivity.
  • the drug-drug interactions in combination therapy can also be characterized by terms other than synergistic affect or additive effect.
  • the term “sparing effect” is the ability of one drug in a combination to reduce the required dose of another drug while maintaining the same therapeutic effect.
  • the sparing effect is the ability of a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist in a combination to reduce the required dose of another therapeutic agent (such as a analgesic agent) while maintaining the same therapeutic effect.
  • the first therapeutic agent has a sparing effect on the second threapeutic agent.
  • the first analgestic agent has a sparing effect on the second analgesic agent.
  • second therapeutic agents and/or analgestic agents have dose-dependent side effects, which can be severe.
  • some typical dose-dependent side effects of second analgesic agents include, addiction, sedation and drowsiness, respiratory depression, constipation, nausea and vomiting, hypotension, dizziness, somnolence (sleepiness), peripheral edema (swelling), weight gain, blurred vision, difficulty with concentration/attention, dry mouth, and fatigue.
  • Such side effects are often dose-limiting side effects, and sometimes even life-threating.
  • the present disclosure provides a combination with a peripherally selective alpha-2 adrenergic receptor ( ⁇ 2AR) agonist allows the dose of second therapeutic agent/analgesic agent to be reduced dramatically, e.g., by 66.67%, while maintaining the same therapeutic effect, and thereby mnimizes adverse events such as those dose-dependent side effects listed above.
  • ⁇ 2AR peripherally selective alpha-2 adrenergic receptor
  • first therapeutic agent and the second therapeutic agent can be administrered by way of simultaneous, sequential or separate administration.
  • the following embodiments apply to the above all general aspects of methods of use.
  • the ⁇ 2AR activation moiety has formula of u la ula [0473] In some embodiments, the ⁇ 2AR activation moiety has formula of Y , wherein Y, A, B, R2, R3, m, and n are defined as in formula N [0474] In some embodiments, the ⁇ 2AR activation moiety has formu , wherein X 1 and Y 1 are defined as in formula (I-D). 99 Attorney Docket No.: 071741.11025/5WO1 [0475] In some embodiments, the ⁇ 2AR activation moiety has formu A B , wherein A and B are defined as in formula (II).
  • the peripheral distribution moiety has formula of , wherein RT is defined as in formula (I-A).
  • R T [0477] In some embodiments, the peripheral distribution moiety has formula of , wherein R T is defined as in formula (II).
  • the compounds of formula (I-A), (I-B), (I-C), (I-D), or (II) can be useful for activating ⁇ 2AR.
  • a method of treating or preventing a disease in human or in animal comprising administering to the subject a compound or composition described herein, e.g., administering an effective amount of a compound or composition described herein.
  • the disease is glaucoma, pain, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, presbyopia, acute kidney injury, insomnia, inflammatory disease, cancer, etc.
  • the disease is pain.
  • the pain is nociceptive pain, nociplatic pain, neuropathic pain such as peripheral neuropathic pain, or mixed pain. Examples of peripheral neuropathic pain include, but 100 Attorney Docket No.: 071741.11025/5WO1 not limited to diabetic neuropathy, postherpetic neuralgia, HIV-associated pain, chemotherapy- induced peripheral neuropathy, and post-surgical neuropathic pain.
  • the compounds and pharmaceutical compositions described herein cause less side effects when treating pain, such as sedation, decreasing heart rate, and decreasing blood pressure in the treated subject.
  • the compounds and pharmaceutical compositions described herein do not cause sedative response in the treated subject.
  • TAA triethylamine
  • TES triethyl silane
  • Trt trityl group or triphenylmethyl group
  • MeOH refers to methanol.
  • EtOH refers to ethanol.
  • t-BuXphos tert-butyl-Xantphos
  • TMAl trimethylaluminum
  • Xantphos refers to 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene
  • Pd(PPH3)4 refers to tetrakis(triphenylphosphine)palladium(0) [0490]
  • Example 1 Example 1
  • Step 1 400mL THF and 36g (0.18mol, 4.0eq) of 3-bromobenzoic acid were added into a 500mL reaction flask. Following cooling to -65 °C, 135mL (4mol/L, 0.428mol, 7.5eq) of n- Butyllithium was added. The mixture was stirred at -65 °C for 2 hours before 20g (0.057mol, 1.0eq) of compound 1-1 and an additional 400mL of THF were introduced.
  • Step 2 150 ml of 55% HI, 7.5g (13.3mmol, 1.0eq) of compound 1-2, and 4.1g (133mmol, 10.0eq) of red phosphorus were added into a 200 mL sealing tube The mixture was stirred at 160 °C for 16 hours until LC-MS indicated completion. Following vacuum concentration, the residue was collected to produce 7.3g of compound 1-3, achieving a 100% yield.
  • Step 4 52ml DCM, and then 1.3g (2.37mmol, 1.0eq) of compound 1-4, 594mg (7.11mmol, 3.0eq) of methoxyammonium chloride, 2.45g (18.96mmol, 8.0eq) of DIPEA, 640mg (4.74mmol, 2.0eq) of HOBT, and 999mg (5.21mmol, 2.2eq) of EDCI were added to a 100 mL reaction flask. The mixture was stirred at room temperature for 5 hours until LC-MS confirmed the reaction's completion.
  • Step 5 18mL DCM and 900mg (1.56mmol, 1.0eq) of compound 1-5, along with 9mL of TFA, were added to a 50mL reaction flask. The reaction was stirred at room temperature for 2 hours until LC-MS showed completion. Following vacuum concentration, the residue underwent column chromatography to yield 670 mg of compound 1, achieving a 98.5% yield.
  • Step 6 Compound 1 was separated by chiral HPLC to afford compound 1-A and compound 1-B.
  • the first eluting enantiomer (compound 1-A) 103 Attorney Docket No.: 071741.11025/5WO1 with a retention time of 4.18min was isolated from the eluent with an enantiomeric excess of 100% in 80% yield.
  • the naming convention for separated enantiomers is systematic. "A” denotes the first eluting product from the chromatography, and "B" indicates the second.
  • Step 2 A 250mL three-necked round bottom flask received 113ml of DCM, 11.3 g (18mmol, 1.0eq) of compound 8-2, HSiEt3 (21g, 180mmol, 10eq), and TFA (21g, 180mmol, 10eq) under nitrogen at 0°C.
  • Step 3 17 g (18mmol, 1.0eq) of compound 8-3, TrtCl (12.6g, 45mmol, 2.5eq), 170ml of DCM and Et3N (9.1 g, 90mmol, 5eq) were mixed in a 500ml three-necked round bottom flask under nitrogen. After stirring for 16 hours at 25°C, completion was confirmed by LC-MS. The product was processed similarly to previous steps to yield 7.1 g of compound 8-4 (64%).
  • Step 4 100ml of DMF, 6 g (9.8mmol, 1.0eq) of compound 8-4, Zn(CN)2 (1.26g, 10.8mmol, 1.1eq), and Pd(PPh 3 ) 4 (1.26g, 1.1mmol, 0.11eq) were added under nitrogen to a 250ml three-necked round bottom flask. After stirring at 120°C for 2 hours, LC-MS confirmed completion. Following the standard work-up, 5.1 g of compound 8-5 (93%) was obtained.
  • Step 5 60ml of EtOH, 2 g (3.6mmol, 1.0eq) of compound 8-5, and 12mL of 30% KOH were added under nitrogen into a 100ml single-mouth flask. The mixture was refluxed for 72 hours. After concentration under vacuum and subsequent work-up, 1.9 g of compound 8-6 (91%) was purified.
  • Step 6 20ml of DCM, 1g (1.73mmol, 1.0eq) of compound 8-6, EDCI (0.432g, 2.25mmol, 1.3eq), DIPEA (0.893g, 6.92mmol, 4eq), HOBt (0.234g, 1.73mmol, 1.0eq), and methoxyammonium chloride (0.174g, 2.08mmol, 1.2eq) were combined in a 100mL single-mouth flask under nitrogen. Stirred for 16 hours at 25°C and then processed as before, this yielded 0.43 g of compound 8-7 (41%).
  • Step 7 10ml of DCM, 0.430 g (0.71mmol, 1eq) of compound 8-7, and BBr3 (0.435 g, 1.775mmol, 2.5eq) were mixed in a 25ml single-mouth flask at 0°C under nitrogen. Stirring continued for 3 hours at 0°C until LC-MS indicated completion, proceeding directly to the next step.
  • Step 8 The mixture from Step 7 and 10ml of MeOH were added to a 50ml single-mouth flask under nitrogen. Heated to reflux for 16 hours, completion was verified by LC-MS.
  • Step 2 3ml of DCM, 150mg (0.25mmol, 1eq) of compound 17-2, and 1.5ml of TFA were added to a 10ml single-mouth flask under N2 at 25°C. After stirring for 2 hours and confirmation of completion by LC-MS, the reaction mixture was vacuum concentrated and subjected to purification through a fast silica gel column, yielding 33mg of compound 17, which corresponds to a 28% yield.
  • Example 4 Synthesis of Compound 22 O OH O O O O O Br N H O O O NH2 O N S O p - , g .
  • Step 2 300mg of compound 22-2, 10ml DCM, and 5ml TFA were added to a 50mL three-necked flask. This mixture was stirred at room temperature (25°C) for 12 hours. LC-MS confirmed completion; the mixture was then diluted with water, adjusted to pH 10, and extracted with DCM. The organic phase was dried over Na 2 SO 4 , vacuum concentrated, and the resulting residue was column chromatographed on silica gel to obtain 85mg of compound 22-3 as a yellow solid, with a yield of 45.9%.
  • Step 1 In a 50mL reaction flask, 10mL of THF and 860mg (3.39mmol, 1.5eq) of 1,3- dibromo-2-fluorobenzene were combined and cooled to -65°C. Next, 1.4mL (3.39mmol, 1.5eq) of n-butyllithium was added. The mixture was stirred at this temperature for 2 hours before adding 1g 107 Attorney Docket No.: 071741.11025/5WO1 (2.26mmol, 1eq) of compound 27-1 and another 10mL of THF. It was stirred for an additional 30 minutes at -65°C, then allowed to warm to room temperature over 16 hours.
  • Step 2 A 50mL three-necked flask was charged with 600mg (0.971mmol, 1eq) of compound 27-2, 1.1g (9.71mmol, 10eq) of triethylsilane, and 1.1g (9.71mmol, 10eq) of TFA. The mixture was stirred at 25°C for 1 hour.
  • Step 3 Into a 25mL reaction flask, 10ml of DMF, 170mg (0.473mmol, 1eq) of compound 27-3, 158g (0.568mmol, 1.2eq) of triphenylmethyl chloride, and 96mg (0.946mmol, 2eq) of TEA were added. The mixture was stirred at 25°C for 12 hours until LC-MS indicated the reaction had completed.
  • Step 4 A 25mL reaction flask was prepared with 10ml DMF, 170mg (0.283mmol, 1eq) of compound 27-4, 100mg (0.848mmol, 3eq) of ZnCN, and 98mg (0.0848mmol, 0.3eq) of tetrakis(triphenylphosphine)palladium. Stirring was conducted at 150°C for 30 minutes in a microwave.
  • Step 5 To a 25mL reaction flask, 10mL of DMSO and 110mg (0.201mmol, 1eq) of compound 27-5 were added and cooled to 0°C. Then, 3ml of 30% H2O2 was added, and the mixture was stirred at 0°C for 1 hour. Following LC-MS confirmation of completion, water was added, and the organic phase was separated and concentrated under vacuum. Column chromatography purification yielded 100mg of compound 27-6, an 88.1% yield.
  • Step 2 Into a 25ml reaction flask, 5ml THF, 185mg (7.6mmol, 2.1eq) of magnesium chips, and 2g (7.2mmol, 2.0eq) of compound 28-2 were added.
  • Step 3 A 100mL reaction flask was charged with 14mL DCM, 1.4g (2.18mmol, 1.0eq) of compound 28-3, and 2.53g (21.8mmol, 10eq) of TES. After cooling to 0°C, 2.48g (21.8mmol, 109 Attorney Docket No.: 071741.11025/5WO1 10eq) of TFA was added. The mixture was warmed to 25°C for 5 hours, then concentrated under vacuum after LC-MS confirmed completion. The residue was column chromatographed to yield 500mg of compound 28-4, a 68.4% yield.
  • Step 4 In a 5mL reaction flask, 2mL THF, 100mg (0.29mmol, 1.0eq) of compound 28-4, 7mg (0.06mmol, 0.2eq) of DMAP, 94mg (0.43mmol, 1.5eq) of Boc2O, and 44mg (0.43mmol, 1.5eq) of TEA were mixed. The reaction was held at 25°C for 4 hours, confirmed by LC-MS. After vacuum concentration, the residue was purified by column chromatography, yielding 120mg of compound 28-5, an 85.5% yield.
  • Step 5 A 10mL reaction flask was loaded with 1.5mL acetic acid, 0.5ml water, and 120mg (0.25mmol, 1.0eq) of compound 28-5. After chilling to 0°C, 165mg (1.24mmol, 5eq) of NCS was added. The mixture was stirred at 0°C for 2 hours until LC-MS confirmed completion, then moved to the next step without purification. The yield was recorded as 100%.
  • Step 6 To a 50mL reaction flask, 10mL of 2M NH 2 CH 3 /THF was added and cooled to 0°C before introducing the crude compound 28-6.
  • Step 2 A 50ml closed tank received 20ml of 57 wt.% HI, 2.35g (4.599mmol, 1.0eq) of compound 31-2, and 1.43g (45.99mmol, 10eq) of red phosphorus. Stirred at 160°C overnight and checked by LC-MS for completion, the mixture was cooled to room temperature and concentrated to yield 5g of crude compound 31-3 (100% yield).
  • Step 4 Into a 200mL high-pressure reactor, 174mg (0.586mmol, 1eq) of compound 31-4, 10 mL of MeOH, 296mg (2.93mmol, 5eq) of TEA, and 48mg (0.0586mmol, 0.1eq) of PdCl 2 (dppf) 111 Attorney Docket No.: 071741.11025/5WO1 were introduced. The reaction, under 5MPa of carbon monoxide at 120°C for 48 hours, left 5% of the starting material, as shown by LC-MS. After filtration and concentration, 270mg of compound 31-5 was isolated by column chromatography (100% yield).
  • Step 5 A 50mL closed tank was charged with 100mg (0.312mmol, 1eq) of compound 31-5 and 5mL of MeOH/NH3 (15M/L). The mixture was stirred at 68°C overnight, cooled to room temperature, concentrated under vacuum, and then purified to obtain 10mg of compound 31 through pre-HPLC (10% yield). [0537] Example 8.
  • Step 2 A 50mL single-mouth flask was charged with 200mg (0.35mmol, 1eq) of compound 32-2, 5mL of dichloromethane, and 1mL of trifluoroacetic acid. The mixture was stirred at room temperature for 1 hour, with TLC confirming the reaction's completion.
  • Step 2 To a 200mL high-pressure reactor, 1g (1.74mmol, 1.0eq) of 58-1, 40 mL of MeOH, 40mL of DMSO, 530mg (5.24mmol, 3.0eq) of TEA, and 148mg (0.17mmol, 0.1eq) of PdCl2 (dppf) were added. The mixture was reacted with carbon monoxide at 5MPa and 100 °C for 48 hours, with LC-MS indicating 5% remaining raw material. After concentration, the residue was purified by column chromatography to yield 53mg of compound 58-2, a 51.2% yield.
  • Step 3 A 50mL sealed tube received 25mL of 16M NH3/MeOH and 530mg (0.89mol, 1.0eq) of compound 58-2. Stirred at 30°C for 16 hours until LC-MS confirmed completion, the residue was then purified by column chromatography to yield 360mg of compound 58-3, a 67.8% yield.
  • Step 4 Into a 10mL reaction flask, 3mL DCM, 100mg (0.17mmol, 1.0eq) of compound 58-3, and 195mg (1.7mmol, 10eq) of TES were added. After cooling to 0°C, 191mg (1.7mmol, 10eq) of TFA was introduced.
  • Example 10 Synthesis of Compound 60 O O O O O O O O O OH HCl/Et2O O N + Zn/TiCl4/THF/4h DCM 2h P/HI/AcOH/16h N NH T FA of zinc were added. This mixture was cooled to -10°C under a nitrogen atmosphere while stirring.
  • Step 2 To a 25mL three-necked flask, 10mL of DCM and 0.5g (1.54mmol, 1eq) of compound 60-1, along with 10mL of HCl/Et2O, were added. The mixture was stirred at room temperature for 3 hours. LC-MS indicated the reaction was complete. Concentrating under reduced pressure yielded 400mg of compound 60-2 as crude, with a 100% yield.
  • Step 2 Into a 100mL reaction flask, 40mL THF, 2g (7.0mmol, 1.0eq) of compound 61-1, and 3.4g (7.7mmol, 1.1eq) of (2,3-dimethylphenyl)(1-trityl-4-imidazolyl)methanone were combined and cooled to 0°C before adding 2.35g (21mmol, 3.0eq) of potassium tert-butoxide.
  • Step 3 A 25mL reaction flask was charged with 10mL DCM and 500mg of compound 61-2, followed by the addition of 2.5mL TFA. The reaction mixture was stirred at 27°C for 1 hour. LC-MS indicated completion, and after concentration, the residue was purified by column chromatography to yield 240mg of compound 61-3, an 84.5% yield.
  • Step 4 In a 10mL reaction flask, 3mL THF, 240mg of compound 61-3, and 120mg (50%) of Pd/C were added.
  • Step 5 To a 10mL reaction flask, 3mL DMF, 80mg (0.25mmol, 1.0eq) of compound 61- 4, 209mg (2.5mmol, 10eq) of methoxyammonium chloride, cooled to 0°C, then 386mg (3mmol, 12eq) of DIPEA and 142mg (0.37mmol, 1.5eq) of HATU were added.
  • Step 6 A 10mL reaction flask was prepared with 1mL DCM and 160mg of compound 61-5, and 0.5mL TFA was added. Stirred at 27°C for 1 hour, completion was confirmed by LC- MS. After concentration under vacuum, the residue was purified by prep-HPLC to yield 25mg of compound 61, a 15.3% yield.Overall yield: 2.1%. [0556] Example 12.
  • Step 2 A 100mL three-necked flask received 60ml of ACN, 3.2g (7.01mmol, 1eq) of 139-1, 3.14g (14.02mmol, 2eq) of CAS 39684-80-5, and 3.42g (10.51mmol, 1.5eq) of Cs2CO3. Stirred at 60°C for 12 hours and confirmed complete by LC-MS, the reaction was worked up and purified to give 930mg of 139-2, a 22.1% yield.
  • Step 4 A 25mL three-necked flask was charged with 5ml of THF, 130mg (0.260mmol, 1eq) of 139-3, 40mg (0.390mmol, 1.5eq) of TEA, and 57mg (0.286mmol, 1.1eq) of (Tetrahydro- 2H-pyran-4-yl)methanesulfonyl chloride (CAS 264608-29-9). Stirred at 25°C for 18 hours and verified complete by LC-MS, the mixture was worked up and purified to give 80mg of 139-4 as a white solid, yielding 46.5%.
  • Step 1 A 50mL reaction flask was charged with 25mL of toluene, 5g (0.0188mmol, 1.0eq) of 3-fluoro-4-bromobenzyl bromide, and 3.44g (0.0207mmol, 1.1eq) of triethyl phosphite. Stirred at 110°C for 18 hours, completion was confirmed by LC-MS. The reaction mixture was concentrated and the residue was purified by column chromatography to yield 5.69g of compound 156-1, with a 93.4% yield.
  • Step 2 To a 50mL reaction flask, 20mL of THF, 1g (3.08mmol, 1.0eq) of compound 156-1, and 1.36g (3.08mmol, 1.0eq) of (2,3-dimethylphenyl)(1-trityl-4-imidazolyl)methanone were added and cooled to 0°C. Then, 1.04g (9.24mmol, 3.0eq) of potassium tert-butoxide was introduced.
  • Step 3 A 10mL reaction flask received 5mL of DMF, 500mg (0.817mmol, 1.0eq) of 156- 2, 192mg (1.634mmol, 2.0eq) of zinc cyanide, and 95mg (0.0817mmol, 0.1eq) of Pd(PPh3)4. Stirred at 120°C for 18 hours, TLC indicated 50% of the raw materials remained.
  • Step 4 Into a 5mL reaction flask, 1.5mL of THF, 1.5mL of MeOH, 110mg (0.197mmol, 1.0eq) of 156-3, 54mg (0.394mmol, 2.0eq) of K2CO3, and 45mg (0.394mmol, 2.0eq) of 30% H 2 O 2 were combined under nitrogen. The mixture was stirred overnight at 20°C for 18 hours.
  • Step 5 In a 5mL reaction flask, 3mL of MeOH:THF (1:1), 90mg (0.156mmol, 1.0eq) of 156-4, and 87mg of Pd(OH) 2 were stirred at 48°C overnight under a hydrogen environment. LC- MS confirmed the reaction's completion.
  • Step 2 In a 25mL three-necked flask, 598mg (2.53mmol, 2.0eq) of 182-1 was dissolved in 6mL THF and cooled to -80°C under nitrogen. n-BuLi (2.5M, 2.5mL, 6.33mmol, 5.0eq) was added, stirred for 40 minutes, then a mixture of 560mg (1.27mmol, 1.0eq) of (2,3- dimethylphenyl)(1-trityl-4-imidazolyl)methanone in 6mL THF was introduced. After stirring for 1 hour and quenching with water, the mixture was warmed and extracted with ethyl acetate.
  • Example 15 Example 15
  • Step 2 188-1 (20g, 79.27mmol, 1.0eq) was dissolved in methanol (350mL), and NaBH 4 (14.99g, 0.39mol, 5.0eq) was added at 0°C.
  • Step 3 DMSO (50mL), 188-2 (4g, 17.83mmol, 1.0eq), PySO 3 (7.1g, 44.58mmol, 2.5eq), and TEA (4.51g, 44.58mmol, 2.5eq) were added to a 250mL flask.
  • Step 4 In a 50mL flask, THF (20mL) was combined with Zn (1.1g, 16.87mmol, 7.5eq) and TiCl4 (1.58g, 8.32mmol, 3.7eq) at -70°C, followed by 188-3 (500mg, 2.25mmol, 1.0eq) and a specified compound in THF. Stirred at 80°C for 2 hours, the reaction was completed, worked up, and purified to yield 270mg of 188-4 as a yellow solid (19.1% yield).
  • Step 6 188-5 (60mg, 0.096mmol, 1.0eq) was mixed with oxalyl dichloride (36.92mg, 0.291mmol, 3.0eq) in DCM (1mL) at 0°C, stirred at 15°C for an hour, concentrated to yield 60mg of 188-6 as a white solid (100% yield), and used directly in the next step.
  • Step 7 188-6 (60mg) was dissolved in THF (0.5 mL) and treated with NH 3 ⁇ THF (8mL) at 0°C, stirred at 15°C for an hour, concentrated to yield 60mg of 188-7 as a white solid (100% yield), and used directly in the next step.
  • Example 16 Example 16
  • Step 1 In a 100mL flask, 50mL of dichloromethane and 4-Iodo-1-trityl-1H-imidazole (11.8g, 0.027mol, 1.0eq) were combined. After cooling the mixture to 0°C, iPrMgClLiCl (1.3mol/L, 20.7mL, 0.027mol, 1.0eq) was added.
  • Step 2 Into a 200mL high-pressure tube, 180mL of dichloromethane, compound 401-1 (6.1g, 12.35mmol, 1.0eq), and MnO2 (6.44g, 74.1mmol, 6.0eq) were added. The mixture was stirred at 72°C for 5 hours. Completion was verified by LC-MS, and the mixture was then filtered to yield 5.6g of compound 401-2. achieving an 83.9% yield.
  • Step 3 A 1L reaction vessel was charged with 500 mL of diethyl ether and 20g (82.67mmol, 1eq) of 3,4-Dibromothiophene (Cas: 3141-26-2). Upon cooling to -78°C, 36.37mL (90.94mmol, 1.1eq) of n-BuLi was added dropwise. The mixture was stirred at -78°C for 30 minutes before 14.02g (90.94mmol, 1.1eq) of diethyl sulfate was added dropwise. Stirring continued at 25°C for 5 hours until LC-MS confirmed the reaction's completion.
  • Step 4 Into a 50 mL reaction flask, 5mL of THF and 387.28mg (2.03mmol, 2eq) of 401- 3 were introduced. Cooled to -78°C, 0.81mL (2.03mmol, 2eq) of n-BuLi was added dropwise. After stirring at -78°C for 30 minutes, a solution of 500mg (1.01mmol, 1.0eq) of 401-2 in 5mL of THF was added.
  • Step 5 To a 25mL flask, 7mL of dioxane, 340mg (0.561mmol, 1.0eq) of 401-4, 64.08mg (0.67mmol, 1.2eq) of MsNH4, 10.28mg (0.011mmol, 0.02eq) of Pd2(dba)3, 9.54mg (0.022mmol, 0.04eq) of tBuxphos, and 365.85mg (1.12mmol, 2eq) of Cs2CO3 were added. The mixture was stirred at 100°C for 5 hours.
  • Example 18 Synthesis of Compound 502
  • Step 1 A 500mL three-necked flask was loaded with 250mL of ACN, 25g (0.15mol, leq) of 502-1, 31.5g (0.16mol, 1.05eq) of diethyl chloromalonate, and 43g (0.31mol, 2eq) of K2CO3.
  • the reaction mixture was refluxed at 80°C overnight. After completion was confirmed by LC-MS, the mixture was concentrated under vacuum and purified via silica gel column chromatography to yield 40g of 502-2. The yield was 83%.
  • Step 2 In a IL three-necked flask, 250mL of DMF and 8g (0.21mol, 1 5eq, 60%) of NaH were combined and cooled to 0°C. Then, 40g (0.125mol, leq) of 502-2 dissolved in lOOmL of DMF was added at 0°C and stirred for 1 hour. Next, 30g (0.154mol, l.leq) of 3- (Bromomethyl)benzonitrile in lOOmL of DMF was added at 0°C, and the mixture was stirred at 58°C overnight. After completion (confirmed by LC-MS), the reaction was quenched with water, extracted with EA, dried over Na2SC>4, and concentrated. Purification by silica gel column chromatography yielded 36g of 502-3 with a 64% yield.
  • Step 3 A 500mL three-necked flask received 300mL of DMSO, 36g (0.08mol, leq) of 502-3, 9g (0.15mol, 2eq) of NaCl, and 11g (0.3mol, 4eq) of H2O. The mixture was stirred at 150°C overnight. LC-MS indicated the reaction was incomplete. The mixture was worked up similarly to previous steps and purified to yield 25g of 502-4 with an 86% yield.
  • Step 4 To a 500mL three-necked flask, 200mL of DMSO, 20g (0.055mol, leq) of 502-4, 13g (0.1 Imol, 2eq) of H2O2, and 15g (0.1 Imol, 2eq) of K2CO3 were added. The mixture was stirred at room temperature overnight and purified after standard work-up to yield 8g of 502-05. The yield was 38%.
  • Step 5 A 100mL three-necked flask was charged with 40mL of EtOH, 8g (0.021mol, 1eq) of 502-5, and 12.6g (0.21mol, 10eq) of ethylenediamine.
  • Step 2 A solution of 1-bromo-2-methoxybenzene (28.99g, 155mmol) in dry THF (150mL) was cooled to -78°C, to which n-BuLi (2.5M in hexane, 62mL, 155mmol) was added dropwise. After stirring at -78°C for 30 minutes, a solution of 503-1 (20g, 77.5mmol) in dry THF 124 Attorney Docket No.: 071741.11025/5WO1 (100mL) was added dropwise. The solution was then allowed to warm to room temperature and stirred for 16 hours.
  • n-BuLi 2.5M in hexane, 62mL, 155mmol
  • Step 3 A solution of 503-2 (1.5g, 4.9mmol), PdCl2(dppf) (360mg, 0.49mmol), and sodium carbonate (1.04g, 9.8mmol) in toluene:MeOH (10mL, 1:1 ratio) was heated at 100°C for 3 days under CO atmosphere.
  • reaction was diluted with water, extracted with EtOAc, and purified via silica gel chromatography, eluting with EtOAc/PE from 1% to 10%, to yield 700mg of methyl 3-[2-(2-methoxyphenyl)-2-oxoethyl]benzoate (503-3) as a yellow oil, with a 46.94% yield.
  • Step 4 To a solution of methyl 3-[2-(2-methoxyphenyl)-2-oxoethyl]benzoate (600mg, 2.1mmol) and O-methylhydroxylamine hydrochloride (264.38mg, 3.16mmol) in toluene (8mL), LiHMDS (1M, 8.4mL, 8.441mmol) was added and stirred at 25°C for 3 hours.
  • Step 5 A solution of N-methoxy-3-[2-(2-methoxyphenyl)-2-oxoethyl]benzamide (400mg, 1.3364mmol) and NH 4 OAc (1.545g, 20.046mmol) in IPA (8.0mL) was stirred at 25°C for 30 minutes before adding NaBH3CN (335.92mg, 5.34mmol) and heated at 80°C for 3 hours.
  • Step 6 To a solution of 3-[2-amino-2-(2-methoxyphenyl)ethyl]-N-methoxybenzamide (300mg, 0.9988mmol, 1eq) in DCM:DMF (5.0mL, 10:1 ratio), 1-chloro-2-isocyanatoethane (421.59mg, 3.9952mmol) was stirred at 25°C for 6 hours. After dilution with water and extraction with DCM, the combined organic phases were washed with brine, dried over sodium sulfate, and concentrated under vacuum. Without further purification, the crude product (250mg, 46.26% yield) was obtained as a yellow oil.
  • Step 7 To a solution of 3-(2- ⁇ [(2-chloroethyl)carbamoyl]amino ⁇ -2-(2- methoxyphenyl)ethyl)-N-methoxybenzamide (200mg, 0.4927mmol) in water (5.0mL), the mixture was heated at 100°C for 3 hours.
  • Step 1 In a 250mL flask, 100mL of THF and 13.1g (0.056mol, 1.5eq) of 1,3- dibromobenzene were combined. After cooling to -78°C, 22.4mL (0.056mol, 1.5eq) of 2.5M n- BuLi was added.
  • Step 2 A 50mL flask was charged with 20mL DMF, 1g (3.45mmol, 1.0eq) of 504-1, 492mg (5.175mmol, 1.5eq) of MsNH2, 2.25g (6.9mmol, 2.0eq) of Cs2CO3, 316mg (0.345mmol, 0.1eq) of Pd2(dba)3, and 293mg (0.69mmol, 0.2eq) of tBuxphos.
  • Step 3 To a 5mL flask, 1.5mL DCM, 134mg (0.439mmol, 1.0eq) of 504-2, 76mg (0.659mmol, 1.5eq) of TMSN 3 , and 31mg (0.0878mmol, 0.2eq) of InBr 3 were added.
  • Step 4 A 5mL flask received 1.5mL THF, 0.3mL water, 144mg (0.436mmol, 1.0eq) of 504-3, and 229mg (0.87mmol, 2.0eq) of PPh3.
  • Step 5 To a 5mL flask, 1mL dioxane, 52mg (0.171mmol, 1.0eq) of 504-4, and 60mg (0.5mmol, 2.9eq) of 2-chloroethyl isothiocyanate were added. Stirred at 80°C for 16 hours, completion confirmed by LC-MS, concentrated under vacuum and purified by pre-HPLC to yield 3.3mg of compound 504, a 5% yield. Overall yield: 2.2% [0612] Example 21.
  • Step 2 A mixture of N- ⁇ 3-[2-(2-methoxyphenyl)-2- oxoethyl]phenyl ⁇ methanesulfonamide (400mg, 0.5323mmol) and NH4OAc (1.448g, 18.785mmol) in isopropanol (8.0ml) was stirred at 25°C for 0.5 hour, then NaBH 3 CN (314.80mg, 5.0096mmol) 127 Attorney Docket No.: 071741.11025/5WO1 was added and the mixture was heated at 80°C for 4.5 hours. After cooling, the mixture was filtered through celite and concentrated.
  • Step 3 A solution of N- ⁇ 3-[2-amino-2-(2- methoxyphenyl)ethyl]phenyl ⁇ methanesulfonamide (505-2, 160mg, 0.4994mmol) and 4,5-dihydro- 1H-imidazole-2-sulfonic acid (224.96mg, 1.4982mmol) in butanol:water (5:1 ratio, 3.0ml) was heated at 120°C for 2 hours in a microwave reactor. After cooling, the mixture was concentrated, diluted with water, and extracted with EtOAc.
  • Step 4 In a 25mL bottle, 10mL of THF, 497mg (2.81mmol, 1.0eq) of 510-3 were cooled to -80°C under nitrogen.
  • Step 5 A 50mL bottle received 10mL of DMF, 570mg (1.65mmol, 1.0eq) of 510-4, 1.07g (3.3mmol, 2.0eq) of Cs 2 CO 3 , 235mg (2.47mmol, 1.5eq) of methanesulfonamide, 151mg (0.165mmol, 0.1eq) of Pd2(dba)3, and 141mg (0.33mmol, 0.2eq) of t-BuXphos. Stirred at 105°C for 2 hours under nitrogen, the reaction was worked up and purified to yield 600mg of 510-5, an 85.7% yield.
  • Step 6 In a 10mL bottle, 5mL of toluene, 200mg (0.554mmol, 1.0eq) of 510-5, 166.5mg (2.77mmol, 5.0eq) of ethylenediamine, and TMAl (1.39mL, 2.77mmol, 5.0eq) were stirred at 110°C overnight. After cooling and working up, the crude was purified to yield 29mg of 510-6, a 13.5% yield. [0623] Step 7: A 5mL bottle received 1mL of toluene and 29mg (0.0745mmol, 1.0eq) of 510-6, then 57mg (0.37mmol, 5.0eq) of POCl 3 was added.
  • Example 23 Synthesis of Compound B24 Potassium ethenylt- rifluoroborate O TosMIC, t-BuOK, CN Pd(PPh3)4 , Na 2 CO 3 CN KOH, EtOH, H 2 O O MeI, K 2 CO 3 DCE, rt, 12h 1,4-Dioxane, H2 O 100o OH C, 4h DMF, rt, 12h bromoindanone (10 g, 47.38 mmol, 1.0 equiv), and TosMIC (13.88 g, 71.07 mmol, 1.5 equiv) were added.
  • Step 2 To a 100 mL single-necked flask, 60 mL of dioxane, 6 mL of water, compound B24-1 (3 g, 13.51 mmol, 1.0 equiv), potassium ethenyltrifluoroborate (2.71 g, 20.26 mmol, 1.5 equiv), Na2CO3 (2.15 g, 20.26 mmol, 1.5 equiv), and Pd(PPh3)4 (0.78 g, 0.68 mmol, 0.05 equiv) were added. The solution was stirred at 90°C under nitrogen for 12 hours. TLC analysis confirmed the reaction was complete.
  • Step 3 To a 100 mL single-necked flask, 50 mL of ethanol, compound B24-2 (5 g, 13.53 mmol, 1.0 equiv), 7.59 g of water, and KOH (7.59 g, 135.32 mmol, 10.0 equiv) were added.
  • Step 4 To a 100 mL single-necked flask, 40 mL of DMF, 40 mL of DMF, compound B24-3 (3.9 g, 13.55 mmol, 1.0 equiv), and K2CO3 (3.74 g, 27.10 mmol, 2.0 equiv) were added. Methyl iodide (3.85 g, 27.10 mmol, 2.0 equiv) was then added to the stirred solution. The mixture was stirred at ambient temperature for 12 hours. LC-MS analysis confirmed the reaction was complete. The mixture was poured into 300 mL of water and extracted with MTBE (100 mL ⁇ 2).
  • Step 5 To a 25 mL three-necked flask, 10 mL of DCM and compound B24-4 (2 g, 9.89 mmol, 1.0 equiv) were added. Ozone was bubbled into the reaction mixture for 10 minutes. TLC analysis confirmed the reaction was complete. The solution was dried over Na2SO4, filtered, and concentrated under vacuum to afford the crude oil.
  • Step 6 To a 25 mL three-necked flask, 100 mL of THF, (2,3-dimethylphenyl)(1-trityl-4- imidazolyl)methanone (300 mg, 0.68 mmol, 1.0 equiv), compound B24-5 (139 mg, 0.68 mmol, 1.0 equiv), and zinc powder (177 mg, 2.71 mmol, 4.0 equiv) were added.
  • Step 7 To a 100 mL single-necked flask, 8 mL of THF, 8 mL of methanol, compound B24-6 (150 mg, 0.40 mmol, 1.0 equiv), and Pd(OH)2/C (80 mg) were added. The solution was stirred at 40°C under a hydrogen atmosphere for 12 hours. LC-MS analysis confirmed the reaction was complete. The solution was filtered and concentrated under vacuum.
  • Step 8 To a 10 mL single-necked flask, compound B24-7 (72 mg, 0.19 mmol, 1.0 equiv), 0.1 mL of water, 1 mL of ethanol, and NaOH (23 mg, 0.58 mmol, 3.0 equiv) were added. The reaction mixture was stirred at room temperature for 3 hours. LC-MS analysis confirmed the reaction was complete. To the reaction mixture, 0.5 mL of 2 N HCl was added, and the mixture was stirred for 5 minutes.
  • Step 1 A mixture of pyrazole (60 g, 0.881 mol, 1.0 equiv) and 3-chloroperoxybenzoic acid (152.1 g, 0.881 mol, 1.0 equiv) in ethyl acetate (3 L) was stirred at room temperature for 10 days. LC-MS showed the reaction was incomplete. The reaction mixture was concentrated and quenched with saturated sodium sulfite solution.
  • Step 2 A mixture of compound 27-01 (4.5 g, 0.0536 mol, 1.0 equiv), benzyl bromide (9.2 g, 0.0541 mol, 1.01 equiv), and DIPEA (7 g, 0.0541 mol, 1.01 equiv) in DCM (54 mL) was stirred at room temperature for 16 hours. LC-MS showed the reaction was complete.
  • Step 3 To a mixture of compound 27-02 (1 g, 5.74 mmol, 1.0 equiv) in 15 mL of THF was added n-BuLi (2.5 M, 2.5 mL, 6.31 mmol, 1.1 equiv) at -78°C.
  • Step 4 A mixture of compound 27-03 (1.16 g, 1.95 mmol, 1.0 equiv), (2,3- dimethylphenyl)(1-trityl-4-imidazolyl)methanone (1 g, 2.15 mmol, 1.1 equiv), and Pd(PPh3)2Cl2 (68 mg, 0.0975 mmol, 0.05 equiv) in toluene (10 mL) was stirred at 95°C for 16 hours. LC-MS showed the reaction was complete. The reaction mixture was filtered, and the organic solution was concentrated under vacuum.
  • Step 8 To a mixture of compound B27-07 (110 mg, 0.249 mmol, 1.0 equiv) in 2.5 mL of THF, NaH (11 mg, 0.274 mmol, 1.1 equiv) was added at 0°C. The reaction was stirred at room temperature for 0.5 hours under nitrogen, then BPO (6 mg, 0.0249 mmol, 0.1 equiv) was added. The mixture was stirred at room temperature for 5 days. LC-MS showed the reaction was incomplete. The reaction mixture was quenched with ice water (3 mL) and adjusted to pH 6 with 1 M HCl.
  • Step 2 A 50 mL reaction flask containing NaH (407.61 mg, 10.19 mmol) in DMF (5 mL) was cooled to 0°C under a nitrogen atmosphere. Ethyl 2-(diethoxyphosphoryl)propanoate (2.43 g, 10.19 mmol) in DMF (7 mL) was added dropwise. The mixture was stirred at 0°C for 1 hour. A solution of B34-1 (300 mg, 1.0191 mmol) in DMF (3 mL) was added dropwise at 0°C, and the reaction mixture was stirred at 90°C for 2 hours. LC-MS confirmed the reaction was complete.
  • Step 3 A sealed tube containing a solution of B34-2 (350 mg, 0.9247 mmol) in ethanol (10 mL), Pd/C (350 mg, 3.2889 mmol), and Pd(OH)2/C (350 mg, 2.4923 mmol) was evacuated and backfilled with hydrogen three times, then charged with hydrogen (3 MPa). The reaction mixture was stirred at 50°C for 16 hours under a hydrogen atmosphere. LC-MS confirmed the reaction was complete. The mixture was filtered, and the filter cake was washed with ethanol (30 mL). The filtrate was concentrated to dryness to afford B34-3 (300 mg) as a colorless oil. Yield: 78.03%.
  • Step 4 A 10 mL reaction flask containing a solution of B34-3 (200 mg, 0.5228 mmol) in THF (3 mL), H2O (1.5 mL), and methanol (3 mL) was stirred with NaOH (41.82 mg, 1.4056 mmol) at 25°C for 12 hours.
  • LC-MS confirmed the reaction was complete.
  • the solvent was removed, and the mixture was diluted with water (100 mL) and adjusted to pH 4 with 1 M HCl.
  • the mixture was extracted with EA (30 mL), and the organic phase was dried and concentrated.
  • Example 26 Synthesis of Compound B37 O N 3 N O HO OH O Trt C, 0 g, 140.7 mmol), ethane-1,2-diol (17.47 g, 281.4 mmol), and 4-methylbenzenesulfonic acid (2.42 g, 14 mmol) in toluene (200 mL) was heated at 110°C under a nitrogen atmosphere for 4 hours.
  • Step 2 A solution of B37-1 (3.7 g, 19.9 mmol) in THF (30 mL) was cooled to 0°C, and LAH (39.8 mL, 39.8 mmol) was added dropwise.
  • Step 3 A mixture of B37-2 (1 g, 6.3 mmol) in DCM (10 mL) and Dess-Martin periodinane (3.21 g, 7.5 mmol) was stirred at 25°C for 2 hours. TLC showed the reaction was complete.
  • Step 4 A mixture of 4-[(2,3-dimethylphenyl)carbonyl]-1-(triphenylmethyl)imidazole (1700.15 mg, 3.84 mmol), B37-3 (600 mg, 3.84 mmol), and Zn (1507.69 mg, 23.05 mmol) in THF (20 mL) was cooled to 0°C. TiCl4 (4372.16 mg, 23.05 mmol) was added dropwise. The mixture was stirred for 1 hour at 0°C and then heated to 70°C for 3 hours. LC-MS showed the reaction was complete.
  • Step 5 A mixture of TosMIC (208.91 mg, 1.07 mmol) and t-BuOK (350.20 mg, 3.12 mmol) in DMSO (3 mL) was stirred at 25°C for 30 minutes. LC-MS showed the reaction was complete.
  • Step 6 A mixture of B37-5 (120 mg, 0.4118 mmol) in 5 N KOH (5 mL) was heated at 80°C for 16 hours. LC-MS showed the reaction was complete. The mixture was quenched with water (20 mL) and extracted with EtOAc (3 ⁇ 20 mL). The combined organic layers were washed with water and brine, dried with sodium sulfate, and concentrated under vacuum to afford B37-6 (90 mg, 0.2755 mmol) as a white solid. Yield: 67%.
  • Step 7 A mixture of B37-6 (90 mg, 0.29 mmol), Pd/C (92.59 mg, 0.87 mmol), and Pd(OH)2/C (122.17 mg, 0.87 mmol) in methanol (3 mL) was heated at 50°C under a hydrogen atmosphere (3 MPa) for 16 hours in a high-pressure reactor. After cooling to ambient temperature, the mixture was filtered through Celite, and the filtrate was concentrated under vacuum. The residue was purified on a Biotage Isolera One (C18 column, eluting with 10–90% MeCN/H2O containing 0.1% TFA) to provide B37 (33.01 mg, 0.1046 mmol) as a white solid. Yield: 36%.
  • Example 27 Synthesis of Compound B40-A and B40-B Pd(OAc)2,dppp O Py,Tf2O,DCM TEA,CO,DMSO O Pd(PPh3)4,dioxane O DMP,DCM OH rt,2h OTf MeOH,78°C,7h 85°C,16h rt,1H O O ep2 s O step4 O step1 st tep3 Br Br B HO O OH HCl ers1 OH Cl rs2 pyridine (11.87 g, 0.15 mol, 3.0 eq) in DCM (100 mL) was cooled to 0°C.
  • Trifluoromethanesulfonic anhydride (23.97 g, 0.085 mol, 1.7 eq) was added at 0°C, and the mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. LC-MS analysis confirmed the reaction was complete. The reaction mixture was poured into 100 mL of ice water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and concentrated under vacuum. The residue was purified by silica gel column chromatography to afford compound B40-01 (15.53 g) as a colorless liquid. Yield: 93.6%.
  • Step 2 A mixture of B40-01 (8 g, 0.024 mol, 1.0 eq), TEA (4.62 g, 0.0461 mol, 1.9 eq), Pd(OAc)2 (1.1 g, 0.00482 mmol, 0.2 eq), and dppp (1.88 g, 0.0058 mmol, 0.24 eq) in a 1:1 mixture of DMSO and MeOH (260 mL) was stirred at 78°C under a 5 MPa CO atmosphere for 7 hours. LC-MS analysis confirmed the reaction was complete. The reaction mixture was poured into 1.2 L of ice water and extracted with DCM. The organic phase was dried over Na2SO4 and concentrated under vacuum.
  • Step 3 A mixture of B40-02 (3.357 g, 0.0139 mol, 1.0 eq), (tributylstannyl)methanol (6.68 g, 0.0208 mol, 1.5 eq), and Pd(PPh3)4 (1.2 g, 0.104 mmol, 0.075 eq) in dioxane (40 mL) was stirred at 85°C for 16 hours under a nitrogen atmosphere. LC-MS analysis showed the reaction was 138 Attorney Docket No.: 071741.11025/5WO1 incomplete.
  • Step 4 A mixture of B40-03 (610 mg, 3.14 mmol, 1.0 eq) and Dess-Martin periodinane (DMP) (2 g, 4.71 mmol, 1.5 eq) in DCM (6 mL) was stirred at room temperature for 1 hour under a nitrogen atmosphere. LC-MS analysis confirmed the reaction was complete. The reaction mixture was poured into 6 mL of saturated sodium bicarbonate solution, filtered, and extracted with DCM. The organic phase was dried over Na2SO4 and concentrated under vacuum.
  • DMP Dess-Martin periodinane
  • Step 5 A mixture of B40-04 (496 mg, 2.58 mmol, 1.0 eq), (2,3-dimethylphenyl)(1-trityl- 4-imidazolyl)methanone (1.14 g, 2.58 mmol, 1.0 eq), and Zn (1.26 g, 19.35 mmol, 7.5 eq) in THF (10 mL) was cooled to 0°C, and TiCl4 (1.81 g, 9.55 mmol, 3.7 eq) was added dropwise.
  • Step 6 A mixture of B40-05 (210 mg, 1 eq) and Pd(OH)2 (210 mg, 1.0 wt) in a 1:1 mixture of MeOH and THF (6 mL) was stirred at 45°C under a hydrogen atmosphere overnight.
  • Step 7 A total of 285 mg of B40-06 was separated on a column with dimensions 30 ⁇ 250 mm packed with CHIRALPAK® IB-10 (10 ⁇ m particle size). A mixture of 70% mobile phase A (CO2) and 30% mobile phase B (0.2% ammonia in MeOH/EtOH) was used. The operational conditions were: 35°C, flow rate 3 g/min, detection at 214 nm and 254 nm.
  • Step 8 A mixture of B40-06-P1 (125 mg, 0.345 mmol, 1.0 eq) and NaOH (55 mg, 1.38 mmol, 4.0 eq) in a 2:2:1 mixture of MeOH, THF, and H2O (2 mL) was stirred at 45°C for 16 hours under a nitrogen atmosphere.
  • Step 1 To a 250 mL reaction flask was added 60 mL THF, Zn (6.4 g, 98.74 mmol, 19 eq), cooled to 0°C, slowly added TiCl4 (9.5 g, 49.89 mmol, 9.6 eq) while controlling the temperature at 0-10°C.
  • Step 2 To a 100 mL reaction flask was added 36 mL DMF, B43-03 (1.85 g, 5.03 mmol, 1.0 eq), triphenylmethyl chloride (1.68 g, 6.04 mmol, 1.2 eq), and TEA (1.02 g, 10.07 mmol, 2 eq). The reaction mixture was stirred at 25°C for 15 hours. LC-MS showed the reaction was completed. The mixture was poured into water and extracted with EA.
  • Step 3 To a 50 mL reaction flask was added 20 mL toluene, B43-04 (1 g, 1.64 mmol, 1.0 eq), DIPEA (424 mg, 3.28 mmol, 2 eq), benzyl mercaptan (224 mg, 1.80 mmol, 1.1 eq), Pd2(dba)3 (150 mg, 0.164 mmol, 0.1 eq), and xantphos (190 mg, 0.328 mmol, 0.2 eq).
  • Step 4 To a 25 mL reaction flask was added 6 mL acetic acid and 2 mL water, B43-05 (400 mg, 0.612 mmol, 1 eq). After the mixture was cooled to 0°C, NCS (409 mg, 3.06 mmol, 5 eq) was added. The reaction mixture was stirred at 20°C for 2 hours. LC-MS showed the reaction was completed.
  • Step 5 To a 25 mL three-necked flask was added 5 mL DCM, B43-06 (theoretical yield: 385 mg, 0.612 mmol, 1 eq), TEA (620 mg, 6.12 mmol, 10 eq), and methyl 3-aminopropionate hydrochloride (427 mg, 3.06 mmol, 5 eq). The reaction mixture was stirred at 25°C for 2 hours. LC-MS showed the reaction was completed. The mixture was poured into water and extracted with DCM.
  • Step 6 A mixture of B43-07 (130 mg), Pd(OH)2/C (50 mg), CH3OH (7 mL), and THF (7 mL) was stirred at 40°C for 4 days under a hydrogen atmosphere. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to afford B43-08 (50 mg). Yield: 58.7%.
  • Step 7 A mixture of B43-08 (50 mg, 0.109 mmol, 1 eq), NaOH (9 mg, 0.219 mmol, 2 eq), CH3OH (4 mL), THF (4 mL), and H2O (2 mL) was stirred at 40°C for 18 hours. LC-MS showed the reaction was completed. The mixture was concentrated under vacuum. Then 10 mL DCM and 5 mL TFA were added, and the solution was stirred for 10 minutes before evaporating the solvent. The residue was purified by preparative HPLC to give B43 (25 mg) as a white solid. Yield: 41.0%. Overall yield: 3.37%.
  • Example 29 Synthesis of Compound B50 O O ydride h eOH , , , , g, 63 mmol, 3 eq), and MeI (6 g, 42 mmol, 2 eq) in DMF (50 mL) was stirred at 30°C overnight under N2. LC-MS showed the reaction was completed. The reaction mixture was diluted with water (100 mL) and extracted with EA (100 mL ⁇ 2). The organic phase was dried over Na2SO4 and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-01 (5 g). Yield: 95.24%.
  • Step 2 A mixture of B50-01 (5 g, 20 mmol, 1 eq), 2,4-Dimethoxyaniline (3.7 g, 22 mmol, 1.1 eq), and K2CO3 (4 g, 30 mmol, 1.5 eq) in DMF (50 mL) was stirred at 110°C overnight under N2. LC-MS showed the reaction was completed. The reaction mixture was diluted with water (50 mL) and extracted with EA (50 mL ⁇ 2). The organic phase was dried over Na2SO4 and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-02 (6 g). Yield: 70%.
  • Step 3 A mixture of B50-02 (5 g, 13 mmol, 1 eq) and TFA (40 mL) in DCM (50 mL) was stirred at 30°C for 2 hours. LC-MS showed the reaction was completed. The reaction mixture was concentrated under vacuum. The mixture was diluted with saturated sodium carbonate solution and extracted with DCM (50 mL ⁇ 1). The organic phase was dried over Na2SO4 and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-03 (3.6 g). Yield: 100%.
  • Step 4 A mixture of B50-03 (3.6 g, 15 mmol, 1 eq) and acetic anhydride (2.2 g, 21 mmol, 1.5 eq) in toluene (40 mL) was stirred at 90°C for 16 hours under N2. LC-MS showed the reaction was completed. The reaction mixture was diluted with saturated sodium bicarbonate solution (20 mL) and extracted with EA (20 mL ⁇ 2). The organic phase was dried over Na2SO4 and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-04 (3.7 g). Yield: 84.7%.
  • Step 5 A mixture of B50-04 (3.7 g, 12.7 mmol, 1 eq), (tributylstannyl)methanol (6 g, 19 mmol, 1.5 eq), and Pd(PPh3)4 (733 mg, 0.635 mmol, 0.05 eq) in 1,4-dioxane (40 mL) was refluxed at 90°C overnight under N2. LC-MS showed the reaction was incomplete. The mixture was filtered and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-05 (3.8 g). Yield: 77.6%.
  • Step 6 To a solution of B50-05 (3.8 g, 15.8 mmol, 1 eq) in DCM (25 mL), Dess-Martin periodinane (8 g, 19 mmol, 1.2 eq) was added at 0°C. The reaction mixture was stirred for 2 hours at room temperature. LC-MS showed the reaction was completed. The mixture was filtered and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-06 (2.4 g). Yield: 63.6%.
  • Step 7 To a solution of B50-06 (2.2 g, 9.2 mmol, 2.4 eq), Zn (1.7 g, 26.6 mmol, 7 eq), and (2,3-Dimethylphenyl)(1-trityl-4-imidazolyl)methanone (1.7 g, 3.8 mmol, 1 eq) in THF (170 mL), titanium tetrachloride (2.5 g, 13.3 mmol, 3.5 eq) was added at 0°C. The reaction mixture was 143 Attorney Docket No.: 071741.11025/5WO1 stirred for 3 hours at 80°C. LC-MS showed the reaction was completed.
  • Step 8 A mixture of B50-07 (800 mg, 1.96 mmol, 1 eq), Pd(OH)2/C (700 mg), TFA (200 mg), and water (10 drops) in MeOH (16 mL) was stirred at 50°C for 16 hours under H2. LC-MS showed the reaction was completed. The mixture was filtered and concentrated under vacuum to afford B50-08 (848 mg) as a crude product.
  • Step 9 A mixture of B50-08 (848 mg, 2.07 mmol, 1 eq) and SOCl2 (400 mg, 4.1 mmol, 2 eq) in MeOH (16 mL) was stirred at 80°C for 16 hours under N2. LC-MS showed the reaction was completed. The reaction mixture was concentrated under vacuum. The mixture was diluted with saturated sodium bicarbonate solution and extracted with EA (20 mL ⁇ 2). The organic phase was dried over Na2SO4 and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-09 (330 mg). Yield: 41.3%.
  • Step 10 A mixture of B50-09 (200 mg, 0.54 mmol, 1 eq) in 4 M HCl (6 mL) was stirred at 45°C for 0.5 hours. NaNO2 (45 mg, 0.65 mmol, 1.2 eq) in water (0.5 mL) was added at 0°C. The reaction mixture was stirred for 1 hour at 0°C. CuCl (270 mg, 2.7 mmol, 5 eq) in water (0.5 mL) was added at 0°C. The reaction mixture was stirred for 2 hours at 30°C. LC-MS showed the reaction was completed. The mixture was diluted with saturated sodium bicarbonate solution and extracted with EA (20 mL ⁇ 2).
  • Example 30 Synthesis of Compound B53 O O O KSC(S)OEt, acetone DLP, Vinyl pivalate, Dichloroethane DLP, Dichloroethane, CSA B 0-10o C 16h S O 80o Br opiv TFA - bromophenyl)propan-1-one (10 g, 0.0343 mol, 1 eq) was cooled to 0°C. Potassium O-ethyl xanthate (6.05 g, 0.03773 mol, 1.1 eq) was added. The reaction mixture was stirred until the starting material disappeared.
  • Step 2 A 100 mL three-necked flask containing 45 mL dichloroethane, B53-01 (9 g, 0.0271 mol, 1 eq), and vinyl pivalate (6.95 g, 0.0542 mol, 2 eq) was refluxed under nitrogen.
  • Step 3 A 500 mL three-necked flask containing 140 mL dichloroethane, B53-02 (14 g, 0.0271 mol, 1 eq), and camphorsulfonic acid (629 mg, 0.00271 mol, 0.1 eq) was refluxed under nitrogen. Lauroyl peroxide (DLP) was then added to the refluxing solution followed by additional portions (2.16 g, 5.42 mmol, 0.2 eq, every 1-1.5 hours). When TLC monitoring showed that the starting material was consumed (after 1 eq of DLP), the solution was cooled to room temperature. The organic phase was evaporated.
  • DLP Lauroyl peroxide
  • Step 4 A 100 mL three-necked flask containing 19 mL toluene, B53-03 (1.1 g, 4.66 mmol, 1 eq), and p-TsOH ⁇ H2O (3.1 g, 16.3 mmol, 2.9 eq) was refluxed for 4 hours. When the starting material was totally consumed, the mixture was allowed to cool to room temperature, neutralized with saturated Na2CO3, extracted with DCM, dried, and evaporated under reduced pressure. The residue was purified by a fast silica gel column to give 1.1 g of B53-04.
  • Step 5 A 100 mL three-necked flask containing 11 mL DCM, B53-04 (1.1 g, 4.66 mmol, 1 eq), pyridine (1.11 g, 13.98 mmol, 3 eq), and trifluoromethanesulfonic anhydride (Tf2O) (2.23 g, 7.92 mmol, 1.7 eq) was stirred for 1.5 hours at room temperature. TLC showed the reaction was complete. The organic phase was washed with water, and the aqueous phase was extracted with DCM. The organic phases were combined, dried over Na2SO4, and concentrated under reduced pressure.
  • Tf2O trifluoromethanesulfonic anhydride
  • Step 6 A 200 mL autoclave containing 22 mL MeOH, 22 mL DMSO, B53-05 (1.3 g, 3.53 mmol, 1 eq), TEA (679.2 mg, 6.71 mmol, 1.9 eq), dppp (274.75 mg, 0.847 mmol, 0.24 eq), and Pd(OAc)2 (159.92 mg, 0.706 mmol, 0.2 eq) was stirred for 6 hours at 78°C under CO (5 MPa). GC-MS showed the reaction was complete.
  • Step 7 A 25 mL three-necked flask containing 7 mL 1,4-dioxane, B53-06 (683 mg, 2.46 mmol, 1 eq), tri-n-butylstannylmethanol (1.183 g, 3.69 mmol, 1.5 eq), and Pd(PPh3)4 (142.14 mg, 0.123 mmol, 0.05 eq) was stirred for 16 hours at 80°C under nitrogen.
  • Step 8 A 25 mL three-necked flask containing 7.5 mL DCM, B53-07 (370 mg, 1.61 mmol, 1 eq), and MnO2 (1.4 g, 16.1 mmol, 10 eq) was stirred for 16 hours at 45°C. The mixture was filtered and evaporated to dryness.
  • Step 9 A 50 mL three-necked flask containing 14 mL THF and powdered Zn (763 mg, 11.67 mmol, 19 eq) was cooled to 0°C under nitrogen. TiCl4 (1.12 g, 5.89 mmol, 9.6 eq) was added dropwise, and the mixture was stirred for 1 hour at 70°C. The mixture was cooled to 0°C.
  • Step 10 A 10 mL single-necked flask containing 1 mL MeOH, 4 drops of water, 4 drops of TFA, B53-09 (100 mg, 0.252 mmol, 1 eq), and 10% Pd/C (100 mg) was stirred for 6 hours at 50°C under a hydrogen atmosphere. LC-MS showed the reaction was complete. The mixture was evaporated to dryness to give 154 mg of B53-10. Yield: 100%.
  • ⁇ 2AAR FLIPR assay This experimental protocol involved cell seeding and a FLIPR assay using the ⁇ 2AAR ( ⁇ 2A-adrenergic receptor) cell line hosted in HEK293 cells.
  • the growth media used is DMEM (11965-092, Gibco) supplemented with 10% FBS (FSP500, Excell), 300 ⁇ g/mL G418 (10131-027, Gibco), and 2 ⁇ g/mL Blasticidin S HCl (BS) (A11139-03, Gibco).
  • FBS FSP500, Excell
  • BS Blasticidin S HCl
  • Cells were then treated with 0.05% EDTA-Trypsin (25300-062, Gibco), incubated at 37°C for 1-2 minutes, and monitored under an inverted microscope. The cells were detached, resuspended in growth media, and centrifuged at room temperature at 1000 rpm for 5 minutes. After discarding the supernatant, the cell pellet was resuspended in growth media to a concentration of 10 ⁇ 10 5 cells per mL. This suspension was added to 384-well plates (19-Jul-38, Greiner) at 20 ⁇ L per well and incubated overnight at 37°C in 5% CO 2 .
  • EDTA-Trypsin 25300-062, Gibco
  • the FLIPR assay began with the preparation of the assay buffer comprising 20 mM HEPES (15630-106, Invitrogen), 1 ⁇ HBSS (14025-076, Invitrogen), and 0.5% BSA (B2064, Sigma). A 250 mM Probenecid solution was prepared in this buffer.
  • the Fluo-4 DirectTM Loading Buffer was made by dissolving Fluo-4 DirectTM crystals (F10471, Invitrogen) in the FLIPR Assay Buffer and adding Probenecid. The buffer was then vortexed and allowed to stand for over 5 minutes, shielded from light.
  • testing compounds for agonist activite were serially diluted and transferred to a 384-well compound plate (25-Jan-39, Greiner). The cell plate was then treated with 2 ⁇ Fluo-4 DirectTM loading buffer and incubated for 50 minutes at 37 oC in a 5% CO2 atmosphere, followed by 10 minutes at room temperature. Subsequently, the FLIPR assay buffer was added to the compound plate, which is then centrifuged. 169 Attorney Docket No.: 071741.11025/5WO1 [0702] The cell plate was analyzed in the FLIPR Tetra+ System for fluorescence signals. For the agonist test, reference compounds were added to the cell plates, and fluorescence was measured.
  • the “Max-Min” calculation began from Read 1 to the maximum allowed. The data were analyzed using Prism software to calculate activation percentage for agonists and inhibition percentage for antagonists. The results were then fitted using specific models to determine EC50 for agonists.
  • the experimental protocol utilized various reagents and apparatus, including Penicillin/Streptomycin (100x) (SV30010, Hyclone), Poly-L-lysine hydrobromide (P1399, Sigma), and different types of 384-well plates such as the 384-Well PP 2.0 Microplate (PP-0200, LABCYTE) and 384 well Low Dead Volume Microplate (LP-0200, LABCYTE). The use of specific reference compounds like UK14304 was also integral to the assay. [0704] Example 2.
  • ⁇ 2AAR binding assay [0705] The ⁇ 2AR Binding Assay was conducted using a stable HEK293 cell line, specifically constructed by WuXi AppTec for targeting ⁇ 2AAR. This assay primarily focused on the binding activity of the radioligand [3H]-RX 821002 (PerkinElmer, NET1153250UC) to ⁇ 2AAR, with the membrane concentration set at 0.5 ⁇ g/well and the radioligand concentration at 0.5 nM.
  • Essential equipment for this assay includes Unifilter-96 GF/C filter plates (Perkin Elmer, 6005174), 96 well conical polypropylene plates (Agilent, 5042-1385), TopSeal-A sealing film (Perkin Elmer, 6050185), a MicroBeta2 reader (CNLL0153, Perkin Elemer, 1310887), and a cell harvester (UNIFILTER-96, Perkin Elemer, 1951369), all procured from Perkin Elmer. Both the assay and wash buffers consist of 50 mM Tris-HCl at a pH of 7.4 (Tris base, Sigma, T1503-1KG).
  • the plates were dried at 50 °C for one hour.
  • the next step involved sealing the bottom of the filter plate wells with Perkin Elmer Unifilter-96 backing seal tape and adding 50 ⁇ L of MicroScint-O cocktail (PerkinElmer, 6013611) to each well.
  • the top of the plates was then sealed with TopSeal-A sealing 170 Attorney Docket No.: 071741.11025/5WO1 film.
  • the trapped 3H was quantified using a Perkin Elmer MicroBeta2 Reader.
  • MDR1-MDCK Permeability Assay [0711] MDR1-MDCK II cells (obtained from Piet Borst at the Netherlands Cancer Institute) were seeded onto Polycarbonate membranes (PC) in 96-well insert systems at 3.33 x 10 5 cells/ mL until to 4-7 days for confluent cell monolayer formation. Selected ⁇ 2AR agonist from Table 3 and Table 4 were diluted with the transport buffer (HBSS with 10.0 mM Hepes, pH7.4) from DMSO stock solution to a concentration of 2 ⁇ M (DMSO ⁇ 1%) and applied to the apical or basolateral side of the cell monolayer.
  • transport buffer HBSS with 10.0 mM Hepes, pH7.4
  • Digoxin was used as a positive control for the P-glycoprotein (P-gp) substrate, while clonidine, dexmedetomidine, faldomidine and brimonidine were used as negative control.
  • Permeation of the test compounds from A to B direction and/or B to A direction was determined in duplicate.
  • Digoxin was tested at 10.0 ⁇ M from A to B direction and B to A direction in duplicate. The plate was incubated for 2.5 hours in CO2 incubator at 37.0 ⁇ 1.0°C, with 5.0% CO 2 at saturated humidity without shaking. In addition, the efflux ratio of each compound was also determined. Test and reference compounds were quantified by LC/MS/MS analysis based on the peak area ratio of analyte/IS.
  • lucifer yellow rejection assay was applied to determine the cell monolayer integrity. Buffers were removed from both apical and basolateral chambers, followed by the addition of 75 ⁇ L of 100 ⁇ M lucifer yellow in transport buffer and 250 ⁇ L transport buffer in apical and basolateral chambers, respectively. The plate was incubated for 30 minutes at 37.0°C 176 Attorney Docket No.: 071741.11025/5WO1 with 5.0% CO2 and 95.0% relative humidity without shaking. After 30 minutes incubation, 20 ⁇ L of lucifer yellow samples were taken from the apical sides, followed by the addition of 60 ⁇ L of transport Buffer.
  • the apparent permeability coefficient Papp (dCr/dt) ⁇ Vr / (A x C0) wherein dCr/dt is the cumulative concentration of compound in the receiver chamber as a function of time ( ⁇ M/s); Vr is the solution volume in the receiver chamber (0.075 mL on the apical side, 0.25 mL on the basolateral side); A is the surface area for the transport, i.e.0.143 cm2 for the area of the monolayer; and C0 is the initial concentration in the donor chamber ( ⁇ M).
  • the binding affinity of various compounds to plasma proteins was evaluated, including clonidine HCl, dexmedetomidine HCl, 1-B HCl, and 44-B HCl, with warfarin serving as a control.
  • the experiment utilized a HT-Dialysis plate (HTD 96 b) and a dialysis membrane with a molecular weight cutoff of 12-14 kDa.
  • the plasma was derived from male C57BL/6J mice, treated with EDTA-K2 as an anticoagulant.
  • the experimental procedure commenced with plasma thawing under cold tap water, followed by centrifugation at 3220 ⁇ g for 5 minutes to eliminate clots, and pH adjustment to 7.4 ⁇ 0.1.
  • Dialysis membranes were initially hydrated in ultra-pure water for about one hour and then treated in a 20:80 ethanol-water mixture for 20 minutes. These prepared membranes could be used immediately or stored at 2-8°C for up to a month. Membranes were rinsed in ultra-pure water before use.
  • Test and control compounds were prepared at a 400 ⁇ M concentration by diluting stock solutions with DMSO. Working solutions were further diluted to create 2 ⁇ M loading matrix solutions, which were thoroughly mixed. In the assay, 50 ⁇ L aliquots of these solutions were dispensed in triplicate into a Sample Collection Plate, balanced with blank PBS to a final volume of 100 ⁇ L per well.
  • a stop solution containing acetonitrile, tolbutamide, and labetalol was added, and samples were mixed and cooled at 2 to 8°C.
  • 100 ⁇ L aliquots from the loading matrix were placed in the dialysis well's donor side, matched with an equal volume of PBS on the receiver side, and incubated at 37°C for 4 hours.
  • Post-dialysis samples from both sides were collected, balanced to 100 ⁇ L with corresponding blank fluids, treated with stop solution, vortexed, and centrifuged to prepare for LC- MS/MS analysis.
  • Data analysis involved calculating the percentages of Unbound, Bound, and Recovery of the compounds post-dialysis.
  • %Unbound was calculated as the ratio of the compound's peak area 178 Attorney Docket No.: 071741.11025/5WO1 on the receiver side to its internal standard, reflecting the fraction that crossed the membrane.
  • %Bound was the complement of %Unbound, representing the fraction retained on the donor side.
  • %Recovery was determined from the peak area ratios on both sides of the membrane, assessing the dialysis efficiency in retaining the compound.
  • the initial preparation of the dialysis membrane involved thawing brain homogenate in a water bath at room temperature and subsequently heating it at 37°C for 10 minutes.
  • the dialysis setup utilized was from HT Dialysis LLC, featuring a HT-Dialysis plate (Model HTD 96 b) and a dialysis membrane with a molecular weight cutoff of 12-14 kDa.
  • the membrane underwent a comprehensive pretreatment which included hydration in ultra-pure water at room temperature for approximately one hour. This was followed by separation and immersion in a 20:80 ethanol:water solution for about 20 minutes. After this treatment, the membranes were either used immediately or stored at 2-8°C for up to one month, with a final rinse in ultra-pure water prior to experimental use.
  • test and control substances were first dissolved to create 400 ⁇ M working solutions by mixing 4 ⁇ L of stock solution with 96 ⁇ L of DMSO. These working solutions were then further diluted to 2 ⁇ M in a blank matrix by combining 3 ⁇ L of the prepared solution with 597 ⁇ L of matrix, ensuring thorough mixing.
  • 50 ⁇ L aliquots of the 2 ⁇ M compound-matrix mixture were dispensed in triplicate into a Sample Collection Plate. Each aliquot was paired with an equal volume of blank 179 Attorney Docket No.: 071741.11025/5WO1 PBS to standardize the total volume to 100 ⁇ L per well at a 1:1 matrix to PBS ratio.
  • a stop solution comprising 500 ⁇ L of acetonitrile with tolbutamide and labetalol at 250nM each was added to stabilize the samples at T0. The samples were then shaken at 800 rpm for 10 minutes and stored at 2-8°C.
  • the dialysis procedure included assembling the dialysis device according to the manufacturer's specifications, loading the matrix aliquots into the donor side of the dialysis wells, and conducting the dialysis under a humidified atmosphere with 5% CO 2 at 37°C for 4 hours.
  • Post-dialysis 50 ⁇ L samples were collected from both the receiver and donor sides into new 96-well plates. Volumes were adjusted to 100 ⁇ L by adding an equivalent amount of the opposite blank matrix or PBS.
  • %Undiluted Unbound 100 ⁇ 1/D / ((1 / (F/T) - 1) + 1/D), where D is the dilution factor (10).
  • %Undiluted Bound was derived as 100 - %Undiluted Unbound.
  • %Recovery 100 ⁇ (F + T) / T0, with F and T representing the peak area ratios of the compound to the internal standard on the receiver and donor sides respectively, after 4 hours of incubation.
  • the brain protein binding result is shown in Table 7.
  • mice were acclimated to the test facility for at least 3 days. During this period, their general health was assessed by veterinary staff or other authorized personnel. The mice were housed in groups of up to four per cage in polysulfone cages, using either certified aspen shaving bedding or corncob bedding. This bedding was regularly tested for environmental contaminants by the manufacturer. The facility's environment was carefully controlled to maintain a temperature range of 20-26°C, relative humidity between 40 to 70%, and a 12-hour light/12-hour dark cycle, although this cycle can be interrupted for study-related activities. Temperature and humidity were continuously monitored by the Vawasala ViewLinc Monitoring system.
  • Compounds such as 5 mg/kg Clonidine HCl, 5 mg/kg dexmedetomidine HCl, 5 mg/kg and 80 mg/kg compound 1-B, and 5 mg/kg and 80 mg/kg compound 44-B were administered in a 20% HP- ⁇ -CD solution in water, with sample collections scheduled at 0.5, 1, 2, and 8 hours post-dosing.
  • Blood collections were performed from the saphenous vein or another suitable site, with approximately 0.1 mL collected per time point into pre-chilled commercial EDTA-K2 tubes. The samples were kept on wet ice until centrifugation at 4°C and 3,200 g for 10 minutes.
  • the plasma was then transferred into pre-labeled 96-well plates or polypropylene tubes, quick-frozen over dry ice, and stored at -60°C or lower until LC-MS/MS analysis.
  • Table 8 The in vivo drug distribution result is shown in Table 8. [0739] Table 8: the in vivo drug distribution Brain Spinal cord Kp, uu, Compounds dosage logBB logSB /plasma, Kp /plasma brain Clonidine HCl 5 mg/kg 2.27 1.78 0.356 0.252 0.739 Dexmedetomidine HCl 5 mg/kg 1.26 1.14 0.101 0.133 1.17 5 mg/kg below detection limit* 1-B HCl 80 mg/kg 0.0598 0.0153 -1.22 -0.732 0.032 5 mg/kg below detection limit* 44-B HCl 80 mg/kg 0.0334 0.0511 -1.39 -1.29 0.013 * drug in brain and spinal cord is below the detection limit (DL).
  • DL detection limit
  • the animals were anesthetized with Zoletil 50 (50 mg/kg, 2.5 mL/kg, i.p.) and Xylazine Hydrochloride (8 mg/kg, 2.5 mL/kg, i.p.), with a toe pinch used to ensure full anesthesia before incision, and ophthalmic ointment applied to the rodents' eyes to prevent drying of the corneas.
  • Zoletil 50 50 mg/kg, 2.5 mL/kg, i.p.
  • Xylazine Hydrochloride 8 mg/kg, 2.5 mL/kg, i.p.
  • mice were individually placed in plastic enclosures with mesh bottoms, allowing full paw access. For three consecutive days, mice were acclimated for 15 minutes each day. Mechanical allodynia baseline measurements were performed on day 14. Animals not exhibiting allodynia (PWT>0.6 g) were excluded, leaving 24 qualified animals (PWT ⁇ 0.6 g) who were then randomly divided into three groups based on their baseline PWT, in addition to 6 sham mice forming a Sham group, totaling four groups with 6-8 mice each.
  • the administration route for the therapeutic intervention for compounds 1-B with a dosage from 1 mg/mL to 20 mg/mL, and 10-B, 44-B, 45-B, 46-B, 47-B, 121, 136, 118, 156 and 175 was oral (p.o.) with a dosage of 1 mg/mL, while the ones for 1 mg/kg morphine via s.c. and 3mg/kg pregabalin via p.o. as positive control, which were prepared in a 20% HP- ⁇ -CD solution.
  • mice 1-B, 10-B, 44-B 45-B, 46-B and 47-B are the active enantiomers of 1, 10, 44, 45, 46, 47, respectively, while 121, 136, 118, and 156 are racemate.
  • the solution was vortexed to ensure thorough mixing until homogeneous.
  • the dosage administered to the mice was 10 ml/kg.
  • Mechanical allodynia tests were conducted on the left hind paw of mice, which were individually placed in plastic enclosures with mesh bottoms for full paw access and acclimated for 15 minutes prior to testing.
  • the mid-plantar hind paw was probed using a series of eight Von Frey filaments with logarithmically incremental stiffness: 0.02 g (2.36), 0.04 g (2.44), 0.07 g (2.83), 0.16 g (3.22), 0.4 g (3.61), 0.6 g (3.84), 1 g (4.08), and 1.4 g (4.17).
  • the filaments were applied perpendicularly to the paw's plantar surface with enough force to slightly buckle against it, maintaining contact for 6-8 seconds. Tests were spaced by 5-second intervals to ensure clear resolution of any response to the prior stimulus, with a sharp withdrawal or flinching upon filament removal indicating a positive response.
  • EB indicates the analgesic ratio of the other analgesia drugs, such as morphine, pregabalin, suzetrigine, duloxetine, amitriptyline, and ketorolac.
  • EAB denotes the analgesic ratio resulting from a combination treatment involving compound 1-B or compound 44-B and one of the following drugs: morphine, pregabalin, suzetrigine, duloxetine, amitriptyline, and ketorolac.
  • mice Male C3H/He mice were anesthetized with a combination of Zoletil 50 (50 mg/kg) and Xylazine 185 Attorney Docket No.: 071741.11025/5WO1 Hydrochloride (8 mg/kg) administered via intraperitoneal injection, and positioned supinely. The right hind limb was shaved and sterilized. A minimal incision was made on the right hind leg to sever the patellar ligaments and expose the condyles of the distal femur. The proximal femur was perforated using a 0.3 mL syringe needle.
  • the remaining qualified animals were then randomly assigned into four groups based on their baseline PWT values.
  • the animals received a single injection of test compounds, including pregabalin 3mg/kg p.o., morphine 1mg/kg s.c., 44-B 1mg/kg p.o., as well as a group for 1-B 20mg/kg p.o. and 44-B 20mg/kg p.o. at a dose of 10 mL/kg based on body weight, and mechanical allodynia tests were performed at various time points post- administration as dictated by different experimental requirements with sham group and vehicle group.
  • the plantar aspect of the left hind paw was cleansed with three rounds of alternating Betadine and 70% ethanol applications, allowing the surface to air-dry. A 0.5-mm longitudinal incision was then made through the skin and fascia from 2 mm proximal to the heel towards the toes. The plantar muscle was longitudinally incised while preserving the origin and insertion points. Hemostasis was achieved with gentle pressure, and the skin was closed with two mattress sutures. Post-surgery, all surgical instruments were cleaned and re-sterilized using a glass bead sterilizer.
  • the objective of this study is to evaluate the in vivo efficacy study of test srticles in the Treatment of Subcutaneous Colorectal Cancer Syngeneic Model MC38 in Female C57BL6/J mice.
  • the mice are Mus musculus C57BL6/J, female, supplied by Beijing HFK Bioscience Co. LTD, with an average age of 6-8 weeks.
  • the cage is polysulfone IVC cage, with a temperature 20-26°C and humidity 40 – 70%.
  • the light cycle is 12 hours light and 12 hours dark.
  • mice is feed by a diet of standard rodent chow, irradiated, ad libitum.
  • the water is autoclaved filtered RO (reverse osmosis) softened, filtered water, ad libitum.
  • RO reverse osmosis
  • the MC38 cancer cells were maintained in vitro with DMEM medium supplemented with 10% fetal bovine serum and 50 ⁇ g/mL Hygromycin B at 37oC in an atmosphere of 5% CO 2 in air. The cells in exponential growth phase were harvested and quantitated by cell counter before tumor inoculation. Each mouse was inoculated subcutaneously at the right rear flank region with MC38 tumor cells (1x 10 6 ) in 0.1 mL of PBS mixed with PBS for tumor development. The randomization started when the mean tumor size reached approximately 121.36 mm 3 .30 mice were enrolled in the study. All animals were randomly allocated to 5 study groups, 6 mice in each group.
  • Randomization was performed based on “Matched distribution” method. The date of randomization was denoted as day 0. [0771] The treatment was initiated on the same day of randomization (day 0) per study design. After tumor cells inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss (Body weights were measured twice per week after randomization), eye/hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail.
  • Dosing as well as tumor and body weight measurements 189 Attorney Docket No.: 071741.11025/5WO1 were conducted in a Laminar Flow Cabinet. The body weights and tumor volumes were measured by using StudyDirectorTM software (version 3.1.399.19). [0772] The body weights of all animals were monitored throughout the study and animals were euthanized if they lost over 20% of their body weight relative to the weight on the day of randomization.
  • the individual mouse was euthanized if its tumor volume exceeds 3000 mm3.
  • any animal exhibiting an ulcerated or necrotic tumor were separated immediately and singly housed and monitored daily before the animal was euthanized or until tumor regression was completed.
  • the mouse was euthanized rapidly if a) tumor ulcerates, and the ulceration diameter was greater than 5 mm, or pus or necrosis observed, and b) tumor burden, including metastasis, compromises animal’s normal physiologic performances, e.g., orientation, access to food or water, etc.
  • the body weight between randomization grouping is shown in FIG.5A.
  • the tumor growth of each treatment group and control group is shown in FIG.5B.
  • TGI tumor growth inhibition
  • T/C tumor growth inhibition
  • Table 12 Pharmacodynamic analysis of each group in Subcutaneous Colorectal Cancer Syngeneic Model MC38 D17 Group Tumor volume (mm 3 ) M ) TGI (% P Value ( mean ⁇ SE ) T/C (%) (Compared with control group) G1 2478.07 ⁇ 439.10 -- -- -- G2 766.52 ⁇ 147.17 72.62% 27.38 ⁇ 0.05 G3 1009.16 ⁇ 258.31 62.32% 37.68 ⁇ 0.05 G4 996.54 ⁇ 270.05 62.86% 37.14 ⁇ 0.05 [0775] Another in vivo efficacy study of clonidine, 44-B and their combination therapy with Anti-mPD-1 in the treatment of subcutaneous colorectal cancer syngeneic model MC38 in female C57BL6/J mice was carried out with same protocol but different treatment design.10mg/kg Anti- mPD-1 i.p.
  • BIWx5 does, 2mg/kg clonidine p.o. QDx15days, and 2mg/mL 44-B HCl p.o. 190 Attorney Docket No.: 071741.11025/5WO1 QD ⁇ 15days were administrated as the single therapy, while the 10mg/kg Anti-mPD-1 i.p. BIWx5 does and 2mg/kg clonidine p.o. QD ⁇ 15days, as well as the combination of 10mg/kg Anti-mPD-1 i.p. BIWx5 does and 5mg/kg 44-B p.o. QDx15days were administrated as the combination therapy.
  • the experiment design is in Table 13. [0776] Table 13.
  • Dosing Volume ROA Dosing Frequency & mice (mg/kg) (mg/ml) ( ⁇ L/g) Duration 1 6 Vehicle -- -- * 2 6 Anti-mPD-1 10 1 3 6 Clonidine 2 0.2 p. . y 4 6 44-B HCl 2 0.2 10 p.o. QD*15days 5 6 44-B HCl 5 0.5 10 p.o. QD*15days 6 6 Clonidine 2 0.2 10 p.o. QD*15days Anti-mPD-1 10 1 10 i.p. BIW ⁇ 5 doses 7 6 44-B HCl 2 0.2 10 p.o.
  • the TGI% (1/T/C) ⁇ 100%.
  • EA represents the TGI of compound 44-B.
  • EB indicates the TGI of An notes the analgesic ratio resulting from a combined treatment involving compound 44-B and Anti-mPD-1.
  • the data is shown in Table 14.
  • the combination group of 10mg/kg Anti-mPD-1 i.p. BIW ⁇ 5 does and 5mg/kg 44-B p.o.
  • mice Spontaneous Locomotor Activity in Mice
  • the study evaluated the effects of clonidine, brimonidine tartrate, compound 1-B HCl, compound 44-B HCl on spontaneous locomotor activity in male C57BL/6 mice. Initially, mice were acclimatized to the testing environment for 8 hours the day before the experiment, followed by at least 2 hours of habituation on the day of the test. The mice were then grouped randomly based on their body weight into six per group, ensuring a balanced distribution for the administration of the drug, which was dissolved in 20% HP- ⁇ -CD in water.
  • clonidine at a dose of 1mg/kg and compound 1-B HCl at concentrations of 1mg/kg, 10mg/kg, and 20mg/kg were freshly prepared and administered orally at a volume of 10mL/kg.
  • clonidine at a dose of 1mg/kg, brimonidine tartrate at a dose of 1mg/kg and compound 44-B HCl at a dose of 1mg/kg were freshly prepared and administered orally at a volume of 10mL/kg.
  • the locomotor activity was monitored by placing the mice in the center of a test box, with a video tracking system measuring the distance traveled every 5 minutes for 60 minutes.
  • mice were randomly assigned to groups based on their body weight to ensure homogeneity across the groups in terms of weight before any treatment was administered.
  • Rotarod training occurred two days prior to the testing phase.
  • the mice On the first training day, the mice underwent three trials on the rotarod at a speed of 6 rpm, each lasting 120 seconds, with 30-minute intervals between trials. If a mouse fell off before completing 120 seconds, it was immediately placed back on the rotarod to complete the training duration.
  • the training consisted of a single trial at the same speed of 6 rpm but extended to 300 seconds. Mice that fell before the 300-second mark were similarly returned to the rotarod to ensure they reached the full training time.
  • mice On the test day, treatments were administered orally to the mice at a dosage volume of 10 mL/kg based on their body weight. The treatments included a vehicle, clonidine (1 mg/kg), and 44- B HCl at three dosages (1 mg/kg, 10 mg/kg, and 20 mg/kg). The time of compound administration was designated as time zero.
  • the rotarod test was conducted at 30, 60, and 120 minutes post-administration, with each session lasting 300 seconds at a speed of 6 rpm. The primary measure was the latency time until a mouse fell from the rotarod, which served as an indicator of the compounds' effects on motor function. [0787] Data were recorded in Microsoft Excel and subsequently analyzed using GraphPad Prism.

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Abstract

The present disclosure relates to novel alpha-2 adrenergic receptor (α2AR) agonists and uses thereof. In particular, the present disclosure relates to imidazole containing compounds, in particular, of formula (I-A), formula (I-B), formula (I-C), formula (I-D), or formula (II). These compounds can be useful as peripherally selective α2AR agonists for the treatment or prevention of disease thereof, either used alone or in combination with a second therapeutical agent.

Description

Attorney Docket No.: 071741.11025/5WO1 COMBINATION WITH ALPHA-2 ADRENERGIC RECEPTOR AGONIST CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims priority to U.S. Patent Application No.63/640,973, filed on May 1, 2024, and U.S. Patent Application No.63/797,849, filed on April 30, 2025, the disclosures of which are incorporated herein by reference in their entireties. FIELD OF THE DISCLOSURE [0002] The present disclosure describes novel alpha-2 adrenergic receptor (α2AR) agonists and uses thereof. In particular, the present disclosure describes novel imidazole containing compounds and their derivatives. These compounds can be useful as α2AR agonists for the treatment or prevention of diseases thereof, either usded alone or in combination with a second therapeutical agent. BACKGROUND OF THE DISCLOSURE [0003] The alpha-2 adrenergic receptor (α2AR) family, as part of the G-protein–coupled receptors, plays a critical role for many central nervous system (CNS) biological functions. α2ARs are key in modulating neurotransmitter release, thus influencing a spectrum of central physiological processes. Agonists targeting these receptors, such as clonidine and dexmedetomidine, have been successfully used to treat several conditions predominantly within the CNS. Related applications include treating hypertension, sedation in intensive care, and for problems like attention-deficit/hyperactivity disorder (ADHD) and agitation associated with schizophrenia or bipolar disorder. [0004] Clonidine was first developed to manage hypertension. Administered orally, clonidine diffuses into the CNS and activates the α2AR in the nucleus tractus solitarii (NTS), which in turn triggers a pathway inhibiting excitatory cardiovascular neurons. This cascade effectively reduces sympathetic outflow from the CNS, leading to a clinical decrease in arterial blood pressure. [0005] Later, clonidine was found to induce sedation by acting through the activation of central pre- and postsynaptic α2AR in the locus coeruleus (LC), a nucleus in the medial dorsal pons, thereby inducing sedative effects. The later development and approval of dexmedetomidine for sedation, particularly in initially intubated and mechanically ventilated adult patients in intensive 1 Attorney Docket No.: 071741.11025/5WO1 care settings, was attributed to its superior α2AR selectivity and pharmacokinetic properties better suited for sedation. [0006] Beyond its antihypertensive and sedation effects, clonidine has been approved for epidural use under the trade name Duraclon, marking a significant advancement in the treatment of cancer pain. However, the therapeutic application of α2AR agonists on analgesia comes with challenges, primarily due to the range of other biological adverse effects they can cause in CNS. Duraclon has been documented to induce centrally mediated sedation, hypotension, bradycardia, and depression of its applications, which persist throughout the analgesic treatment process. Such sedation effect significantly limits the dosages that can be administered safely. As a result, although α2AR agonists like clonidine and dexmedetomidine are considered important for pain treatment in both academic research and clinical settings, the sedation effect poses substantial hurdles to their widespread use in medical applications. [0007] Therefore, it is desired to develop new classes of α2AR agonists that could provide substantial therapeutic benefits in pain management such as reduced sedation effect, thereby expanding the range of therapeutic alternatives to address the prevailing unmet medical needs. Furthermore, there remains a need for pain management that reduce the dose-dependent side effects of other analgeisics without compromising therapeutic efficacy. BRIEF SUMMARY OF THE DISCLOSURE [0008] In one general aspect, the present disclosure relates to methods of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist. [0009] In some embodiments, the peripherally selective α2AR agonist activates at least one sub type of α2AR, particularly α2A AR, α2B AR, or α2C AR. [0010] In some embodiments, the peripherally selective α2AR agonist has a Kp,uu,brain is lower than 0.05, 0.02, or 0.01. [0011] In some embodiments, the disease is chosen from pain, rosacea, spasticity, and aging. [0012] In some embodiments, the peripherally selective α2AR agonist causes reduced biological effects mediated by CNS, such as sedation, hypotension, and bradycardia, than treating with a non-peripherally selective α2AR agonist. 2 Attorney Docket No.: 071741.11025/5WO1 [0013] In another general aspect, the present disclosure provides a peripherally selective α2AR agonist that comprises an α2AR activation moiety covalently linked to a peripheral distribution moiety, and its uses in the treatment of a disease. [0014] In another general aspect, the present disclosure relates to methods of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peripherally selective α2AR agonist, wherein the peripherally selective α2AR agonist comprises an α2AR activation moiety covalently linked to a peripheral distribution moiety. [0015] In some embodiments, the peripherally selective α2AR agonist causes less sedation than treating with a non-peripherally selective α2AR agonist. [0016] In another general aspect, the present disclosure relates to a compound of formula (I-A): RT m , or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, wherein, Y is C(R1), N, -O-C, -C-NH-, -CH2-C(O)-, or -CH=N-; when Y is C(R1), R1 is chosen from H, D, and halogen; when Y is -O-C-, the oxygen atom is connected to A, and the carbon atom is connected to both RT and B; and when Y is -C-NH-, the carbon atom is connected to both RT and A, and the nitrogen atom is connected to B; A is a ring chosen from phenyl, pyridinyl, thienyl, furyl, pyrrolyl, 4H-pyran, 4H-thiopyran, 1,2,3,4-tetrahydro-1-naphthyl, tetrahydrozoline, quinoxalinyl, pyrimidinyl, and 2,1,3- benzothiadiazol; NH N X a is H and 3 Attorney Docket No.: 071741.11025/5WO1 n is 0, 1, 2, or 3; each R2 is independently chosen from H, D, halogen, alkyl, alkenyl, alkynyl, alkoxyl, ester, cycloalkyl, cycloalkoxy, aryl, aryloxy, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclolalkyl, OR4, -CN, N3, NO2, N(R4)2, OR4, SR4, C(O)R4, SO2N(R4)2, CH2SR4; wherein the alkyl, alkenyl, alkynyl, alkoxyl, ester, cycloalkyl, cycloalkoxy, aryl, aryloxy, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclolalkyl is optionally substituted with one or more R5; R4 is chosen from H, D, halogen, alkyl, alkenyl, alkynyl, alkoxyl, ester, cycloalkyl, cycloalkoxy, aryl, aryloxy, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclolalkyl, and the alkyl, alkenyl, alkynyl, alkoxyl, ester, cycloalkyl, cycloalkoxy, aryl, aryloxy, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclolalkyl is optionally substituted with one or more R5; R5 is chosen from halogen, hydroxyl, -CN, -NO2, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, cycloalkyl, cycloalkoxy, aryl, aryloxy, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl; alternatively, when A is a phenyl ring and two R2 are substituted at adjacent positions of the phenyl ring, the two R2 groups, with the carbon atoms they are connected to, form a ring that is fused to ring A to form a bicyclic ring, such as quinolinyl, indolyl, benzothienyl, benzofuryl, benzofuranyl, benzodioxolyl, 2,3-dihydrobenzo[b][l,4]dioxin-6-yl, cinnolinyl, quinoxalinyl, or 1,2,4-benzotriazinyl; m is 0, 1, 2, or 3; each R3 is independently chosen from H, D, halogen, -OH, -SH, optionally substituted alkyl, optionally substituted heterocycle, and optionally substituted aryl; alternatively, R3 is a group connected to the -NH of the imidazole ring, and R3 is of the formula of: O O O OR8 6 7 6 P in: R6 is hydrogen, alkyl, cycloalkyl, or alkenyl; R7 is an amino acid residue; and R8 is alkyl or cycloalkyl; 4 Attorney Docket No.: 071741.11025/5WO1 RT is RL-RP, and RP is optionally substituted with RC, wherein: RL is a linker, wherein one end is conneceted to RP and the other end is conneceted to Y; RP is a moiety that is connected to one end of RL; and RC is a cap, which is a moiety that is connected to RP. [0017] In another general aspect, the present disclosure relates to a compound of formula (I-B): RT Y m , or a ste lly acceptable salt or solvate thereof, wherein, Y is a bond, CH(R1), NH, -O-CH-, -C-NH-, -CH2-C(O)-, or -CH=N-; when Y is C(R1), R1 is chosen from H, D, and halogen; when Y is -O-C-, the oxygen atom is connected to A, and the carbon atom is connected to B; when Y is -C-NH-, the carbon atom is connected to A, and the nitrogen atom is connected to B; and A, B, R2, n, R3, m, and RT are defined as above in formula (I-A). [0018] In another general aspect, the present disclosure relates to a compound of formula (I-C): RT m or a ste , , lly acceptable salt or solvate thereof, wherein, Y is a bond, CH(R1), NH, -O-CH-, -C-NH-, -CH2-C(O)-, or -CH=N-; when Y is C(R1), R1 is chosen from H, D, and halogen; when Y is -O-C-, the oxygen atom is connected to A, and the carbon atom is connected to B; 5 Attorney Docket No.: 071741.11025/5WO1 when Y is -C-NH-, the carbon atom is connected to A, and the nitrogen atom is connected to B; and A, B, R2, n, R3, m, and RT are defined as above in formula (I-A). [0019] In another general aspect, the present disclosure relates to a compound of formula (I-D): N NH T , or a ste er, pharmaceutically acceptable salt or solvate thereof, wherein, Y1 is CH, or N; X1 is chosen from H, D, and halogen; RT is defined as above in formula (I-A). In another general aspect, the present disclosure relates to a compound of formula (II): RT B , or a ste r, tautomer, pharmaceutically acceptable salt or solvate thereof, wherein, A is one chosen from: S 1 ; each R1 is independently chosen from hydrogen, halogen, haloalkyl, hydroxyl, hydroxyalkyl, alkoxy, alkyl, and -COOH; B is one chosen from: 6 Attorney Docket No.: 071741.11025/5WO1 H N N H X S N N H; oaryl, wherein the C3-12 cycloalkyl or C2-12 heterocyclyl is optionally fused with an aryl; r is 1 or 2; n2 is 0, 1, or 2; each R2 is independently chosen from hydrogen, halogen, hydroxyl, and alkoxy; R3 is chosen from CN, hydroxy, alkoxy, -C(O)-C0-12 alkylene-CN, -C0-12 alkylene-C2-12 heterocyclyl, -SO2-alkyl, -C(O)-NR4R4’, -SO2-NR4R4’, -C0-12 alkylene-R3’, -O-C0-12 alkylene- COOH, -C0-12 alkylene-N(R4)-C(O)-R5, -C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-O-C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-P(=O)(R4)(R4’), O 6 O O R O CH3 m -NH-R7 or n ; wherein one -CH2- group in the -C0-12 alkylene-R3’ is , the -C0-12 alkylene-R3’ is optionally substituted with one or more substitutes chosen from amino and alkylamino, and the C2-12 heterocyclyl and C1- 12 heteroaryl are each optionally substituted with one or more R4a; R3’ is chosen from -C(O)-NR4R4’, -SO2-NR4R4’, -C0-12 alkylene-COOH, -C0-12 alkylene- N(R4)-C(O)-R5, -C0-12 alkylene-N(R4)-SO2-R5, C0-12 alkylene-C1-12 heteroaryl; each R4a is independently chosen from hydroxy, alkyl, oxo, ketone, and -C2-12 heterocyclyl; each of R4 and R4’ is independently hydrogen, alkyl, alkoxy, -SO2- N(R6a)t, -C0-12 alkylene- COOH, -C0-12 alkylene-N(R6a)t, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, - C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-OR6a, or hydroxyalkyl, wherein the hydroxyalkyl is optionally substituted with alkoxy; wherein each of the alkyl, C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a; or R4 and R4’, together with the nitrogen atom that they are attached to, form a heterocycle comprising one or more heteroatoms chosen from O, N, and S; 7 Attorney Docket No.: 071741.11025/5WO1 alternatively, when one R2 is adjacent to R3, the R2 and R3, together with the atoms that they are attached to, form a ring optionally substituted with one or more R4a;; R5 is amino, alkylamino, C1-12 haloalkyl, -C0-12 alkylene-OR6a, -C0-12 alkylene-N(R6a)t, -C0- 12 alkylene-SR6a, -C0-12 alkylene-CN, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C2-12 alkenyl, or alkyl optionally substituted with cyano, amido, trialkylammonium, or thiolate; wherein each of the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a; each R6a is independently chosen from hydrogen, C1-12 alkyl, C1-12 alkoxy, -C0-12 alkylene- C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C6-12 aryl, and -C0-12 alkylene-C1- 12 heteroaryl; wherein each of the alkyl, C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl, is optionally substituted with one or more R4a; R6 is alkoxy, amino, sulfonamide, carbamide, or alkyl optionally substituted with cyano; R7 is hydrogen, alkyl, -C0-12 alkylene-COOH, optionally substituted C3-12 cycloalkyl, C2-12 aryl, C1-12 heteroaryl, -C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-P(=O)(R4) (R4’), -C0-12 alkylene- N(R4)-C(=S)-R5, -C(=S)-R5, or alkyl optionally substituted with cyano; R8 is alkoxy, amino, alkylamino, amide, sulfonamide, or carbamide; n3 is 0, 1, 2, 3, or 4; n4 is 1, 2, 3, 4, 5, or 6; t is 2 or 3; m is 0, 1, 2, 3, 4, or 5; and n is 0, 1, 2, 3, or 4. [0020] In another aspect, the present disclosure relates to a pharmaceutical composition comprising a compound as described herein or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier. [0021] In another aspect, the present disclosure relates to the use of a compound as described herein or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, for treating or preventing a disease, including pain, glaucoma, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, presbyopia, acute kidney injury, insomnia, inflammatory disease, cancer, etc. in a subject in need thereof. 8 Attorney Docket No.: 071741.11025/5WO1 [0022] In another general aspect, the present disclosure relates to a pharmaceutical combination comprising a first therapeutical agent and a second therapeutical agent, wherein the first therapeutical agent is the peripherally selective α2AR agonist described herein. [0023] In some embodiments, the pharmaceutical combination further comprises a third therapeutic agent. [0024] In some embodiments, the second therapeutic agent is chosen from opioids, antidepressant medications, antiepileptic medications, local analgesics, nonsteroidal anti- inflammatory drugs (NSAIDs), acetaminophen (paracetamol), corticosteroids, NMDA antagonists, and selective NaV1.8 inhibitors. [0025] In another general aspect, the present disclosure relates to a method of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject (i) an effective amount of a first therapeutic agent, and (ii) a second therapeutic agent, wherein the first therapeutic agent is a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist. [0026] In some embodiments, the method further comprises administering to the subject an effective amount of a third therapeutic agent. [0027] In some embodiments, the disease is chosen from glaucoma, analgesia, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, menopausal hot flashes, dysmenorrhea, presbyopia, acute kidney injury, insomnia, inflammatory disease, and cancer. [0028] In some embodiments, the second therapeutic agent is chosen from opioids, antidepressant medications, antiepileptic medications, local analgesics, nonsteroidal anti- inflammatory drugs (NSAIDs), acetaminophen (paracetamol), corticosteroids, NMDA receptor antagonists, and selective NaV1.8 inhibitors. [0029] In some embodiments, the administration of the first therapeutic agent and the second therapeutic agent provides a synergistic effect in treating the disease. [0030] In some embodiemnts, the administration of the first therapeutic agent and the second therapeutic agent provides an additive effect in treating the disease. [0031] In another general aspect, the present disclosure relates to a method for treating or preventing pain in a subject in need thereof, the method comprising administering to the subject an effective amount of a first analgesic agent, and an effective amount of a second analgesic agent, wherein first analgesic agent is a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist. 9 Attorney Docket No.: 071741.11025/5WO1 [0032] In some embodiment, the method for treating or preventing pain further comprising administering to the subject an effective amount of a third analgesic agent. [0033] In some embodiments, the pain is neuropathic pain, nociceptive pain, nociplastic pain, or mixed pain (a mixture of nociceptive, neuropathic, and/or nociplastic pain). [0034] In some embodiments, the second analgesic agent is any analgesic agent other than a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist. [0035] In some embodiments, the third analgesic agent is any analgesic agent other than a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist, and the third analgesic agent is different from the second analgesic agent. [0036] In some embodiments, the second analgesic agent is chosen from opioids, acetaminophen (paracetamol), local analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), antiepileptic medication, antidepressant medication, topical analgesic agents, NMDA receptor antagonists, neurotoxins, cannabinoids, voltage-gated sodium channel inhibitors, adenosine agonists, transient receptor potential (TRP) channel modulators, NGF inhibitors, purinergic receptor antagonists, adenosinergic pathway modulators, sigma-1 receptor antagonists, KCC2 enhancers, glutamate transport enhancers, TrkA receptor antagonists, somatostatin receptor ligands, Human Adenylyl Cyclase Associated Protein 1 (CAP1) modulators, and angiotensin II receptor antagonists, corticosteroids, and any combinations thereof. [0037] In some embodiments, the second analgesic agent is chosen from GGG tri-agonist (e.g., retatrutide), metabotropic glutamate receptors (mGluR) modulator (e.g., basimglurant), TGF beta-1 Inhibitor (e.g., Vicatertide (SB-01)), gonadotropin-releasing hormone receptor（GnRH) antagonist (e.g., linzagolix), 11β-HSD1 inhibitor (e.g., clofutriben), 17-HSD-1 inhibitor (e.g., OG-6219), Dual ENKephalinase Inhibitor (DENKI) (e.g., PL37), CCR2 receptor antagonist (e.g., CNTX-6970), IL- 10 receptor agonist (e.g., XT-150), nAChR dual alpha 4/beta 2 subunit stimulator (e.g., ATA-104), allosteric NEK7/NLRP3 inflammasome inhibitor (e.g., HT-6184), NLRP3 inflammasome inhibitor (e.g., dapansutrile), PACAP-38 inhibitor (e.g., Lu-AG09222), membrane-associated prostaglandin E synthase-1 (mPGES-1) inhibitor (e.g., NS-580), prolactin receptor antagonist (e.g., HMI-115), Protease-Activated Receptor 2 (PAR2) Antagonist (e.g., MEDI-0618), NF-kB decoy oligonucleotide, NFkB/Nrf2 modulator (e.g., AKL-4), Proepiregulin modulator (e.g., LY- 3848575), Sphingomyelin synthase 1 (SMS1) agonist (e.g., sodium idroxioleate), Lyn inhibitor (e.g., nispomeben), guanylate cyclase-c (GC-C) agonist (e.g., IW-3300), neurotrophin-3 (NT-3) 10 Attorney Docket No.: 071741.11025/5WO1 inhibitor (e.g., LEVI-04), prostaglandin receptor, EP4 antagonist (e.g., grapiprant), agonist of the ChemR23 G-protein coupled receptor (e.g., urcosimod), inhibitor of the influenza neuraminidase enzyme (e.g., peramivir), inhibitor of AP2 associated kinase 1 (AAK1) (e.g., pilavapadin), Adenosine A3 receptor antagonist (e.g., NTM-006), Antagonist of platelet activating factor (PAF) receptor (e.g., piperidone hydrochloridum), 15-lipoxygenase inhibitor (e.g., utreloxastat), Flt3 inhibitor (e.g., BDT-272), GPCR84 inhibitor (e.g., BAY-3178275), Kindolor, Trk receptor modulator (e.g., CG-001054), selective FABP5 inhibitor (e.g., ART-26.12), erythropoietin receptor agonist (e.g., cibinetide), Dopamine reuptake inhibitor (e.g., IPTN-2021), AEAr agonist (e.g., SBS-1000), Epoxide Hydrolase (sEH) inhibitor (e.g., EC-5026), SSAO/VAP-1 inhibitor (e.g., ECC-0509), sphingosine 1 phosphate receptor 1 modulator (e.g., TRV-045), and antagonist of the lysophosphatidic acid 1 receptor (LPA1) (e.g., PIPE-791). [0038] In some embodiments, the second analgesic agent is chosen from IRX-101, MR-107A-02, 3-VM-1001, lysergide assisted therapy, SIL-1002, TRN-261, HR-1405-01, HRF-2105, TTAX-03, KP-910, LYT-503, LL-50, ZeP-3, YR-1702, YZJ-1495, FB-1003, MK-4318, PZH-2108, HEC- 137076MsOH, VVZ-2471, SYNP-101, Pudafensine, BIOS-0618, and HSK-36357. [0039] In another general aspect, the present disclosure relates to a method for treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a codrug that comprises a first therapeutic agent and a second therapeutic agent, wherein first therapeutic agent is a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist, such as the peripherally selective α2AR agonist described herein. [0040] In some embodiments, the method for treating or preventing the disease has a synergistic effect or additive effect. [0041] In some embodiments, the method for treating or preventing the disease has a sparing effect on the second therapeutic agent or the second analgesic agent. [0042] Other features and advantages of the present disclosure are apparent from additional descriptions provided herein, including different examples. The provided examples illustrate different components and methodology useful in practicing the present disclosure. Such examples do not limit the claimed disclosure. Based on the present disclosure, the skilled artisan can identify and employ other components and methodology useful for practicing the present disclosure. 11 Attorney Docket No.: 071741.11025/5WO1 BRIEF DESCRIPTION OF THE DRAWINGS [0043] The foregoing and other objects, aspects, features, and advantages of exemplary embodiments will become more apparent and may be better understood by referring to the following description taken in conjunction with the accompanying drawings. [0044] FIGs.1A-1H: PWT value of sham and spared nerve injury SNI mouse model, vehicle and drug treatment groups at 1 hour after dosing. FIG.1A: 3 mg/mL pregabalin p.o.; FIG.1B: 1 mg/mL morphine s.c.; FIG.1C: 1 mg/mL compound 1-B p.o. and 10mg/mL compound 1-B p.o.; FIG.1D: 1 mg/mL compound 10-B p.o.; FIG.1E: 1 mg/mL compound 44-B p.o. and 1 mg/mL compound 45-B p.o.; FIG.1F: 1 mg/mL compound 46-B p.o. and 1 mg/mL compound 47-B p.o.; FIG.1G: 2 mg/mL compound 121 p.o. and 2 mg/mL compound 136 p.o.; and FIG.1H: 2 mg/mL compound 118 p.o. and 2 mg/mL compound 156 p.o. [0045] FIGs.2A-2D: PWT value of sham and bone cancer pain mouse (BCP) model, vehicle and drug treatment groups at 1 hour after dosing. FIG.2A: 3 mg/mL pregabalin p.o.; FIG.2B: 1 mg/mL morphine s.c.; FIG.2C: 1 mg/mL compound 44-B p.o.; and FIG.2D: 20 mg/mL compound 1-B p.o. and : 20 mg/mL compound 44-B p.o. [0046] FIGs.3A-3C: PWT value of sham and post-surgery pain mouse model, vehicle and drug treatment groups at 1 hour after dosing. FIG.3A: 10 mg/mL compound 1-B p.o.; FIG.3B: 10 mg/mL compound 44-B p.o.; and FIG.3C: 3 mg/mL morphine s.c. [0047] FIGs.4A-4O: Analgesic ratio (%) following single-drug and combination administrations in SNI and BCP mouse models. FIG.4A: 1 mg/mL 1-B p.o., 1 mg/mL morphine s.c. and their combination in SNI FIG.4B: 1 mg/mL 1-B p.o., 3 mg/mL suzetrigine p.o. and their combination in SNI FIG.4C: 1 mg/mL 1-B p.o., 3 mg/mL pregabalin p.o. and their combination in SNI FIG.4D: 1 mg/mL 44-B p.o., 1 mg/mL morphine s.c. and their combination in SNI 12 Attorney Docket No.: 071741.11025/5WO1 FIG.4E: 1 mg/mL 44-B p.o., 3 mg/mL pregabalin p.o. and their combination in SNI FIG.4F: 1 mg/mL 44-B p.o., 3 mg/mL suzetrigine p.o. and their combination in SNI FIG.4G: 1 mg/mL 44-B p.o., 7 mg/mL duloxetine p.o. and their combination in SNI FIG.4H: 1 mg/mL 44-B p.o., 7 mg/mL amitriptyline p.o. and their combination in SNI FIG.4I: 1 mg/mL 44-B p.o., 10 mg/mL ketorolac i.p. and their combination in SNI FIG.4J: 1 mg/mL 44-B p.o., 10 mg/mL ketorolac p.o. and their combination in SNI FIG.4k: 1 mg/mL 44-B p.o., 1 mg/mL morphine s.c. and their combination in BCP FIG.4L: 1 mg/mL 44-B p.o., 3 mg/mL pregabalin p.o. and their combination in BCP FIG.4M: 1 mg/mL 44-B p.o., 7 mg/mL amitriptyline p.o. and their combination in BCP FIG.4N: 1 mg/mL 44-B p.o., 7 mg/mL duloxetine p.o. and their combination in BCP FIG.4O: 1 mg/mL 44-B p.o., 10 mg/mL ketorolac i.p. and their combination in BCP [0048] FIGs.5A-5C: Body weight curve (FIG.5A), tumor volume growth curve (FIG.5B) ), and tumor volume in day 17 (FIG.5C) of mice in each group in subcutaneous colorectal cancer syngeneic model MC38, including group 1 (control group, 0mg/kg, p.o., QD*Day0-17), group 2 (clonidine, 5mg/kg, p.o., QD*Day0-3; 2mg/kg, p.o., QD* Day4-17), group 3 (compound 1-B HCl, 5mg/kg, p.o., BID*Day0-17), and group 4 (compound 1-B HCl, 10mg/kg, p.o., BID*Day0-3; 5mg/kg, p.o., QD* Day4-17). Data is expressed as "average ± standard error". [0049] FIGs.6A-6C: Body weight curve (FIG.6A), tumor volume growth curve (FIG.6B), and tumor volume in day 17 (FIG.6C) of mice in each group in subcutaneous colorectal cancer syngeneic model MC38, including group 1 (control group, 0mg/kg, p.o., QD*Day0-15), group 2 (Anti-mPD-1, 10mg/kg, i.p., BIW×5 doses), group 3 (clonidine, 2mg/kg, p.o., QD*Day0-15), group 4 (44-B HCl, 2mg/kg, p.o., QD*Day0-15), group 5 (44-B HCl, 5mg/kg, p.o., QD*Day0-15), group 6 (clonidine, 2mg/kg, p.o., QD*Day0-15, and Anti-mPD-1, 10mg/kg, i.p., BIW×5 doses), group 7 (44-B HCl, 2mg/kg, p.o., QD*Day0-15, and Anti-mPD-1, 10mg/kg, i.p., BIW×5 doses), and group 8 (44-B HCl, 5mg/kg, p.o., QD*Day0-15, and Anti-mPD-1, 10mg/kg, i.p., BIW×5 doses). [0050] FIGs.7A-7B: Total distance travelled in 0-60 min of the test (FIG.7A) for clonidine and compound 1-B HCl and the test (FIG.7B) for clonidine, brimonidine tartrate and compound 44-B HCl. Data were expressed as Mean ± SEM (n=6). ***p<0.001 compared with Vehicle group, one- way ANOVA followed by Dunnutt’s multiple comparisons. 13 Attorney Docket No.: 071741.11025/5WO1 [0051] FIGs.8A-8D. Effects of clonidine and compound 44-B HCl on rotarod test in C57BL/6 mice 30 min after administration (FIG.8A). And its latency time at 30 min (FIG.8B), 60 min (FIG.8C), and 120 min (FIG.8D). DETAILED DESCRIPTION [0052] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the disclosure. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to the disclosure. [0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the present disclosure pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification. [0054] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference (one or more) unless the context clearly dictates otherwise. [0055] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. For example, the phrase “at least A, B, and C” means that each of A, B, and C is present. The term “at least one of” preceding a series of elements is to be understood to refer to a single element in the series or any combination of two or more elements in the series. For example, the phrase “at least one of A, B, and C” means that only A is present, only B is present, only C is present, both A and B are present, both A and C are present, both B and C are present, or each of A, B, and C is present. Depending on the context, “at least one of” preceding a series of elements can also encompass situations in which any one or more of the elements is present in greater than one instance, e.g., “at least one of A, B, and C” can also encompass situations in which A is present in duplicate alone or further in combination with any one or more of elements B and C. [0056] As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and conjuntive options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the 14 Attorney Docket No.: 071741.11025/5WO1 first. A third option refers to, conjunctively,the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.” [0057] Unless otherwise stated, any numerical value, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, the recitation of “10-fold” includes 9-fold and 11-fold. As used herein, the use of a numerical range expressly includes all possible permutations and combinations of subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise. [0058] As used herein, “subject” means any animal, such as a mammal, particularly a human, to whom will be or has been treated by a method described herein. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, and non-human primates (NHPs), such as monkeys or apes, humans, etc. [0059] The phrase “pharmaceutically acceptable salt(s)” means those salts of a compound of interest that are safe and effective for topical use in mammals and that possess the desired biological activity. Pharmaceutically acceptable salts include salts of acidic or basic groups present in the specified compounds. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, carbonate, bicarbonate, acetate, lactate, salicylate, citrate, tartrate, propionate, butyrate, pyruvate, oxalate, malonate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., 1,1′- methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain compounds used in the present disclosure can form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, bismuth, and diethanolamine salts. For a review on pharmaceutically acceptable salts see Berge et al., 66 J. Pharm. Sci.1-19 (1977), incorporated herein by reference. 15 Attorney Docket No.: 071741.11025/5WO1 [0060] As used herein, the term “alkyl” means a saturated, monovalent, unbranched or branched hydrocarbon chain. An alkyl group can be unsubstituted or substituted with one or more suitable substituents. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and pentyl (e.g., n- pentyl, isopentyl, neopentyl), etc. An alkyl group can have a specified number of carbon atoms. When numbers appear in a subscript after the symbol “C”, the subscript defines with more specificity the number of carbon atoms which that particular alkyl can contain. For example, “C1 to C10 alkyl” or “C1-10 alkyl” is intended to include alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms. Additionally, for example, “C1 to C8 alkyl” or “C1-8 alkyl” denotes an alkyl having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. [0061] As used herein, the term “alkenyl” refers to an unbranched or branched hydrocarbon chain containing at least one carbon-carbon double bond. An alkenyl group can be unsubstituted or substituted with one or more suitable substituents. Examples of alkenyl groups include ethenyl, propenyl, butadienyl (including 1,2-butadienyl and 1,3-butadienyl). When numbers appear in a subscript after the symbol “C”, the subscript defines with more specificity the number of carbon atoms which that particular alkenyl can contain. For example, “C2 to C10 alkenyl” or “C2-10 alkenyl” is intended to include alkenyl groups having 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms. Additionally, for example, “C2 to C8 alkenyl” or “C2-8 alkenyl” denotes an alkenyl having 2, 3, 4, 5, 6, 7, or 8 carbon atoms. [0062] As used herein, the term “alkynyl” refers to an unbranched or branched hydrocarbon chain containing at least one carbon-carbon triple bond. An alkynyl group can be unsubstituted or substituted with one or more suitable substituents. The term “alkynyl” also includes those groups having one triple bond and one double bond. When numbers appear in a subscript after the symbol “C”, the subscript defines with more specificity the number of carbon atoms which that particular alkynyl can contain. For example, “C2 to C10 alkynyl” or “C2-10 alkynyl” is intended to include alkynyl groups having 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms. Additionally, for example, “C2 to C8 alkynyl” or “C2-8 alkynyl” denotes an alkynyl having 2, 3, 4, 5, 6, 7, or 8 carbon atoms. [0063] As used herein, the term “cycloalkyl” refers to any stable monocyclic or polycyclic saturated hydrocarbon ring system. A cycloalkyl group can be unsubstituted or substituted with one or more suitable substituents. A cycloalkyl group can have a specified number of carbon atoms. For example, “C3 to C6 cycloalkyl” or “C3-6 cycloalkyl” includes cycloalkyl groups having 3, 4, 5, 16 Attorney Docket No.: 071741.11025/5WO1 or 6 ring carbon atoms, i.e., cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Polycyclic cycloalkyls include bridged, fused, and spiro ring structures in which all ring atoms are carbon atoms. A “spiro ring” is a polycyclic ring system in which two rings share one carbon atom, referred to as the “spiro atom,” which is typically a quaternary carbon atom. A “fused ring” is a polycyclic ring system in which two rings share two adjacent atoms, referred to as “bridgehead atoms,” i.e., the two rings share one covalent bond such that the bridgehead atoms are directly connected. A “bridged ring” is a polycyclic ring system in which two rings share three or more atoms separating the bridgehead atoms by a bridge containing at least one atom. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. [0064] The term “aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, phenyl, naphthyl, anthracenyl, phenanthranyl, and the like. Aryl moieties are well known and described, for example, in Lewis, R. J., ed., Hawley’s Condensed Chemical Dictionary, 13th Edition, John Wiley & Sons, Inc., New York (1997). An aryl group can be substituted or unsubstituted with one or more suitable substituents. An aryl group can comprise a single ring structure (i.e., monocyclic) or multiple ring structures (i.e., polycyclic, e.g., bicyclic or tricyclic). For example, an aryl group can be a monocyclic aryl group, e.g., phenyl. [0065] The term “heterocyclyl” includes stable monocyclic and polycyclic hydrocarbons that contain at least one heteroatom ring member, such as sulfur, oxygen, or nitrogen, wherein the ring structure is saturated or partially unsaturated, provided the ring system is not fully aromatic. A heterocyclyl group can be unsubstituted, or substituted with one or more suitable substituents at any one or more of the carbon atom(s) and/or nitrogen heteroatom(s) of the heterocyclyl. A heterocyclyl can comprise a single ring structure (i.e., monocyclic) or multiple ring structures (i.e., polycyclic, e.g., bicyclic). Polycyclic heterocyclyls include bridged, fused, and spiro ring structures in which at least one ring atom of at least one of the rings of the polycyclic ring system is a heteroatom, for instance oxygen, nitrogen, or sulfur, wherein bridged, fused, and spiro rings are as defined above. A heterocyclyl ring can be attached to the parent molecule at any suitable heteroatom (typically nitrogen) or carbon atom of the ring. The term “4- to 9-membered monocyclic or bicyclic heterocyclyl” includes any four, five, six, seven, eight, or nine membered monocyclic or bicyclic ring structure containing at least one heteroatom ring member selected from oxygen, nitrogen, and sulfur, or independently selected from oxygen and nitrogen, optionally 17 Attorney Docket No.: 071741.11025/5WO1 containing one to three additional heteroatoms independently selected from oxygen, nitrogen, and sulfur, or independently selected from oxygen and nitrogen, wherein the ring structure is saturated or partially unsaturated, provided the ring structure is not fully aromatic. [0066] In certain embodiments, the term “heterocyclyl” refers to 4-, 5-, 6-, or 7-membered monocyclic groups and 6-, 7-, 8-, or 9- membered bicyclic groups which have at least one heteroatom (O, S, or N) in at least one of the rings, wherein the heteroatom-containing ring(s) typically has 1, 2, or 3 heteroatoms, such as 1 or 2 heteroatoms, independently selected from O, S, and/or N, or independently selected from O and N. When numbers appear in a subscript after the symbol “C”, the subscript defines with more specificity the number of carbon atoms which that particular heterocycly can contain, in addition to the heteroatoms which that particular heterocycly can contain. For example, “C1 to C10 heterocycl” or “C1-10 heterocycl” is intended to include heterocycl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms. Additionally, for example, “C1 to C8 heterocycly” or “C1-8 heterocycly” denotes a heterocycl having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. [0067] Examples of monocyclic heterocyclyl groups include, but are not limited to azetidinyl, oxetanyl, tetrahydrofuranyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dioxolanyl, dithiolanyl, piperidinyl, piperazinyl, dioxanyl, morpholinyl, azepanyl, oxepanyl, oxazepanyl (e.g., 1,4-oxazepanyl, 1,2-oxazepanyl) and the like. Examples of bicyclic heterocyclyl groups include, but are not limited to, 2-aza- bicyclo[2.2.1]heptanyl, 8-aza-bicyclo[3.2.1]octanyl, 2-aza-spiro[3.3]heptanyl, 3- azabicyclo[2.2.2]octanyl, 3-oxa-9-azabicyclo[3.3.1]nonanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl, 7- oxa-2-azaspiro[3.5]nonanyl, and 5-azaspiro[2.3]hexanyl and the like. [0068] As used herein, the term “heteroaryl" includes stable monocyclic and polycyclic aromatic hydrocarbons that contain at least one heteroatom ring member such as sulfur, oxygen, or nitrogen. A heteroaryl group can be unsubstituted or substituted with one or more suitable substituents. A heteroaryl can comprise a single ring structure (i.e., monocyclic) or multiple ring structures (i.e., polycyclic, e.g., bicyclic or tricyclic). Each ring of a heteroaryl group containing a heteroatom can contain one or two oxygen or sulfur atoms and/or from one to four nitrogen atoms provided that the total number of heteroatoms in each ring is four or less and each ring has at least one carbon atom. Heteroaryl groups which are polycyclic, e.g., bicyclic or tricyclic must include at least one fully aromatic ring, but the other fused ring or rings can be aromatic or non-aromatic. For example, for a 18 Attorney Docket No.: 071741.11025/5WO1 bicyclic heteroaryl, the fused rings completing the bicyclic group can contain only carbon atoms and can be saturated, partially saturated, or unsaturated. A heteroaryl can be attached to the parent molecule at any available nitrogen or carbon atom of any ring of the heteroaryl group. In some embodiments, the term “heteroaryl” refers to 5- or 6-membered monocyclic groups and 9- or 10- membered bicyclic groups which have at least one heteroatom (O, S, or N) in at least one of the rings, wherein the heteroatom-containing ring typically has 1, 2, or 3 heteroatoms, such as 1 or 2 heteroatoms, selected from O, S, and/or N. A heteroaryl group can be unsubstituted, or substituted with one or more suitable substituents at any one or more of the carbon atom(s) and/or nitrogen heteroatom(s) of the heteroaryl. The nitrogen and sulfur heteroatom(s) of a heteroaryl can optionally be oxidized (i.e., N→O and S(O)r, wherein r is 0, 1 or 2). [0069] When numbers appear in a subscript after the symbol “C”, the subscript defines with more specificity the number of carbon atoms which that particular heteroaryl can contain, in addition to the heteroatoms which that particular heteraryl can contain. For example, “C1 to C10 heteroaryl” or “C1-10 heteroaryl” is intended to include heteroaryl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms. Additionally, for example, “C1 to C8 heteroaryl” or “C1-8 heteroaryl” denotes a heteroaryl having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. [0070] Exemplary monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thiophenyl, oxadiazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl. Exemplary bicyclic heteroaryl groups include, but are not limited to, indolyl, benzothiazolyl, benzodioxolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridinyl, furopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl. [0071] The term “alkoxy” as used herein refers to an –O-alkyl group, wherein alkyl is as defined above. An alkoxy group is attached to the parent molecule through a bond to an oxygen atom. An alkoxy group can have a specified number of carbon atoms. For example, “C1 to C10 alkoxy” or “C1-10 alkoxy” is intended to include alkoxy groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms. Additionally, for example, “C1 to C4 alkoxy” or “C1-4 alkoxy” denotes an alkoxy having 1, 2, 3, or 4 carbon atoms. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy, isopropoxy), butoxy (e.g., n-butoxy, isobutoxy, tert-butoxy), pentyloxy 19 Attorney Docket No.: 071741.11025/5WO1 (e.g., n-pentyloxy, isopentyloxy, neopentyloxy), etc. An alkoxy group can be unsubstituted or substituted with one or more suitable substituents. Similarly, “alkylthio” or “thioalkoxy” represents an alkyl group as defined above attached to the parent molecule through a bond to a sulfur atom, for example, -S-methyl, -S-ethyl, etc. Representative examples of alkylthio include, but are not limited to, -SCH3, -SCH2CH3, etc. [0072] As used herein, the term “halogen” means fluorine, chlorine, bromine, or iodine. Correspondingly, the term “halo” means fluoro, chloro, bromo, and iodo. [0073] “Haloalkyl” is intended to include both branched and straight-chain saturated aliphatic hydrocarbon radicals substituted with one or more halogen atoms. “Fluorinated alkyl” or “fluoroalkyl” in particular refers to any alkyl group as defined above substituted with at least one fluoro atom, e.g., one to three fluoro atoms, such as one, two, or three fluoroatoms. Examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2- trifluoroethyl, heptafluoropropyl, and heptachloropropyl. Suitable examples of fluoroalkyl in particular include, but are not limited to, -CF3, -CHF2, -CH2CF3, -CF2CF3, and the like. [0074] The terms “hydroxy” and “hydroxyl” can be used interchangeably, and refer to –OH. [0075] The term “carboxy” and “carboxyl” can be used interchangeably, and refers to –COOH. [0076] The term “ester” refers to -COOR, wherein R is alkyl as defined above. [0077] The term “cyano” refers to –CN. [0078] The term “oxo” refers to a double bonded oxygen group, i.e., a substituent group of the formula =O. [0079] The term “keto” refers to -C(O)R, wherein R is alkyl as defined above. [0080] As used herein, the term “amino” refers to –NH2. One or more hydrogen atoms of an amino group can be replaced by a substituent such as an alkyl group, which is referred to as an “alkylamino.” Alkylamino groups have one or both hydrogen atoms of an amino group replaced with an alkyl group and is attached to the parent molecule through a bond to the nitrogen atom of the alkylamino group. For example, alkylamino includes methylamino (-NHCH3), dimethylamino (-N(CH3)2), -NHCH2CH3 and the like. [0081] The term “aminoalkyl” as used herein is intended to include both branched and straight- chain saturated aliphatic hydrocarbon groups substituted with one or more amino groups. For example, “C1-4 aminoalkyl” is intended to include alkyl groups having 1, 2, 3, or 4 carbon atoms 20 Attorney Docket No.: 071741.11025/5WO1 substituted with one or more amino groups. Aminoalkyl groups are attached to the parent molecule through a bond to a carbon atom of the alkyl moiety of the aminoalkyl group. Representative examples of aminoalkyl groups include, but are not limited to, -CH2NH2, -CH2CH2NH2, and – CH2CH(NH2)CH3. [0082] As used herein, “amido” refers to –C(O)N(R)2, wherein each R is independently an alkyl group (including both branched and straight-chain alkyl groups) or a hydrogen atom. Examples of amido groups include, but are not limited to, -C(O)NH2, -C(O)NHCH3, and –C(O)N(CH3)2. [0083] The terms “hydroxyl-substituted alkyl,” “hydroxylalkyl” and “hydroxyalkyl” are used interchangeably, and refer to a branched or straight-chain aliphatic hydrocarbon group substituted with one or more hydroxyl groups. Hydroxyalkyl groups are attached to the parent molecule through a bond to a carbon atom of the alkyl moiety of the hydroxyalkyl group. A hydroxyalkyl group can have a specified number of carbon atoms. For example, “C1 to C10 hydroxyalkyl” or “C1- 10 hydroxyalkyl” is intended to include hydroxyalkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms. Additionally, for example, “C1 to C4 hydroxylalkyl” or “C1-4 hydroxyalkyl” denotes a hydroxyalkyl group having 1, 2, 3, or 4 carbon atoms. Examples of hydroxyalkyl include, but are not limited to, hydroxylmethyl (-CH2OH), hydroxylethyl (-CH2CH2OH), etc. [0084] As used herein, “amide” refers to –N(R’)C(O)R, wherein each R and R’ is independently chosen from hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl. Examples of amide groups include, but are not limited to, -NHC(O)CH3, -NHC(O)CH2CH3, and – N(CH3)C(O)CH3. [0085] As used herein, “carbamide” refers to –N(R’)C(O)N(R)2, wherein each R and R’ is independently chosen from hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl. Examples of carbamide groups include, but are not limited to, -NHC(O)NH2, -NHC(O)NHCH3 (methyl carbamide), and –NHC(O)NH(Ph). [0086] As used herein, “sulfonamide” refers to –N(R’)SO2-R, wherein each R and R’ is independently chosen from hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl. Examples of sulfonamide groups include, but are not limited to, -NHSO2CH3 (methyl sulfonamide), and –NH SO2Ph. [0087] In accordance with convention used in the art: is used in structural formulas herein to depict the bond that is the point of attachment of a group, moiety or substituent to the core, backbone, or parent molecule structure. When a bond to a 21 Attorney Docket No.: 071741.11025/5WO1 substituent is shown to cross a bond connecting two atoms in a ring, then such substituent can be bonded to any atom on the ring. [0088] The term “substituted” as used herein with respect to any organic radical (e.g., alkyl, cycloalkyl, heteroaryl, aryl, heterocyclyl, etc.) means that at least one hydrogen atom is replaced with a non-hydrogen group, provided that all normal valencies are maintained and that the substitution results in a stable compound. When a particular group is “substituted,” that group can have one or more substituents, such as from one to five substituents, one to three substituents, or one to two substituents, independently selected from the list of substituents. The term “independently” when used in reference to substituents, means that when more than one of such substituents is possible, such substituents can be the same or different from each other. Examples of suitable substituents include, but are not limited to, alkyl, halo, haloalkyl, alkoxy, amido, hydroxy, hydroxyalkyl, amino, carboxyl, ester, oxo, cyano and the like. [0089] When any variable occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-3 R groups, then said group can be optionally substituted with up to three R groups, and at each occurrence, R is selected independently from the definition of R. [0090] The terms “optional” or “optionally” mean that the event or circumstance described can, but need not, occur, and such a description includes the situation in which the event or circumstance does or does not occur. For example, “optionally substituted heterocyclyl” means that a substituent group can be, but need not be, present, and such a description includes the situation of the heterocyclyl group being substituted by a suitable substituent and the heterocyclyl group not being substituted by any substituent. [0091] One skilled in the art will recognize that in certain embodiments compounds described herein can have one or more asymmetric carbon atoms in their structure. As used herein, any chemical formulas with bonds shown only as solid lines and not as solid wedged or hashed wedged bonds, or otherwise indicated as having a particular configuration (e.g., R or S) around one or more atoms, contemplates each possible stereoisomer, or mixture of two or more stereoisomers. Stereoisomers includes enantiomers and diastereomers. Enantiomers are stereoisomers that are non-super-imposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemate or racemic mixture. Diastereomers (or diastereoisomers) are stereoisomers that are not 22 Attorney Docket No.: 071741.11025/5WO1 enantiomers, i.e., they are not related as mirror images, and occur when two or more stereoisomers of a compound have different configurations at one or more of the equivalent stereocenters and are not mirror images of each other. Substituent groups (e.g., alkyl, heterocyclyl, etc.) can contain stereocenters in either the R or S configuration. [0092] Certain examples contain chemical structures that comprise (R) or (S) terminology. When (R) or (S) is used in the name of a compound or in the chemical representation of the compound, it is intended to mean that the compound is a single isomer at that stereocenter, with established absolute configuration of either (R) or (S). [0093] Stereochemically pure isomeric forms can be obtained by techniques known in the art in view of the present disclosure. For example, diastereoisomers can be separated by physical separation methods such as fractional crystallization and chromatographic techniques, and enantiomers can be separated from each other by the selective crystallization of the diastereomeric salts with optically active acids or bases or by chiral chromatography. Pure stereoisomers can also be prepared synthetically from appropriate stereochemically pure starting materials, or by using stereoselective reactions. [0094] Compounds described herein can also form tautomers. The term “tautomer” refers to compounds that are interchangeable forms of a particular compound structure and that vary in the displacement of hydrogen atoms and electrons. Tautomers are constitutional isomers of chemical compounds that readily interconvert, usually resulting in relocation of a proton (hydrogen). Thus, two structures can be in equilibrium through the movement of pi electrons and an atom (usually hydrogen). All tautomeric forms and mixtures of tautomers of the compounds described herein are included with the scope of the present disclosure. [0095] Compounds described herein can exist in solvated and unsolvated forms. The term “solvate” means a physical association, e.g., by hydrogen bonding, of a compound described herein with one or more solvent molecules. The solvent molecules in the solvate can be present in a regular arrangement and/or a non-ordered arrangement. The solvate can comprise either a stoichiometric or nonstoichiometric amount of the solvent molecules. “Solvate” encompasses both solution-phase and isolable solvates. Compounds described herein can form solvates with water (i.e., hydrates) or common organic solvents. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Methods of solvation are generally known in the art. 23 Attorney Docket No.: 071741.11025/5WO1 [0096] Also included within the scope of the present disclosure are all isotopes of atoms occurring in the compounds described herein, including intermediates and final products. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include deuterium and tritium. Isotopes of carbon include 13C and 14C. [0097] The present disclosure further includes isotopically-labeled compounds. An “isotopically- labeled” or “radio-labeled” compound is a compound of the present disclosure where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring). Isotopically- labeled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically- labeled reagent in place of the non-labeled reagent otherwise employed. [0098] As used herein, the name of a compound is intended to encompass all possible existing isomeric forms, including stereoisomers (e.g., enantiomers, diastereomers, racemate or racemic mixture, and any mixture thereof) of the compound. [0099] In one general aspect, the present disclosure relates to methods of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a peripherally selective α2AR agonist. [0100] In some embodiments, the peripherally selective α2AR agonist activates at least one sub type of α2AR, particularly α2A AR, α2B AR, or α2C AR. [0101] In some embodiments, the disease is glaucoma, pain, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, presbyopia, acute kidney injury, insomnia, inflammatory disease, cancer, etc. [0102] In certain embodiments, the disease is chosen from pain, rosacea, spasticity, and aging. [0103] In some embodiments, treating with the peripherally selective α2AR agonist causes less sedation than treating with a non-peripherally selective α2AR agonist, such as at similar or comparable dosage. [0104] In some embodiments, the peripherally selective α2AR agonist comprises an α2AR activation moiety covalently linked to a peripheral distribution moiety. [0105] In another general aspect, the present disclosure provides a peripherally selective α2AR agonist that comprises an α2AR activation moiety covalently linked to a peripheral distribution moiety, and its uses in the treatment or prevention of a disease. 24 Attorney Docket No.: 071741.11025/5WO1 [0106] In some embodiments, the disease is glaucoma, pain, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, presbyopia, acute kidney injury, insomnia, inflammatory disease, cancer, etc. [0107] In certain embodiments, the disease is pain. [0108] Pain, as a complex and multidimensional sensory and emotional experience, poses a significant challenge to human health. It is not only an important symptom of physical diseases but also a key factor affecting the quality of life, causing great physical and mental distress to patients. Crucial components of pain are neuropathic pain and nociceptive pain. Neuropathic pain are caused by a lesion or disease of the somatosensory nervous system. Neuropathic pain can be divided into central neuropathic pain and peripheral neuropathic pain. Central neuropathic pain includes spinal cord injury, post-stroke pain, and MS pain, while peripheral neuropathic pain includes diabetic neuropathy, postherpetic neuralgia, HIV-associated pain, chemotherapy-induced peripheral neuropathy, and post-surgical neuropathic pain. Currently, first-line treatment drugs include Gabapentinoids, tricyclic antidepressants, and noradrenaline/serotonin uptake inhibitors. Although these drugs can relieve pain to some extent, the side effects of long-term use still cause a decrease in the quality of life of patients. Second-line treatment drugs, such as opiate receptor agonist, not only have side effects but also have a high addiction rate, which has caused many social impacts and cannot well address the demand for neuropathic pain drugs. [0109] α2AR agonists, such as clonidine and dexmedetomidine, are considered an important method for treating pain in academic research and clinical applications. Scientists have found that the intraspinal administration of α2AR agonists can effectively relieve pain. However, the therapeutic benefits of α2AR agonists are not without limitations. Existing α2AR agonists are often associated with a range of biological reactions, including sedation, hypotension, bradycardia, drowsiness, dizziness, depression, bradycardia, orthostatic hypotension, constipation, nausea, gastric upset, dry mouth (xerostomia), dry nasal mucosa, impotence, fluid retention, edema, and pupil size. These other biological effects, especially sedation, set limits on the dosages that can be safely administered, thereby constraining the wide-scale utility of these drugs in long-term pain management. This not only affects the quality of life for patients but also restricts the applicability of these drugs for various types and levels of pain symptoms. These biological effects, especially sedation, seriously impact the application of α2AR agonists in the field of medical application. [0110] Thus, there is a need to develop novel α2AR agonist compounds for treating pain with reduced sedation. Our work seeks to make significant contributions to the field of pain 25 Attorney Docket No.: 071741.11025/5WO1 management by offering not just more effective but also safer long-term non-opioid alternatives. The compounds and methods described herein can be useful for addressing such unmet need. [0111] As used herein, “an effective amount” means an amount of a composition or compound that elicits a biological or medicinal response in a tissue system or subject that is being sought by a researcher, veterinarian, medical doctor or other professional, which can include alleviation of the symptoms of the disease, disorder, or condition being treated. An effective amount can vary depending upon a variety of factors, such as the physical condition of the subject, age, weight, health, etc.; and the particular disease, disorder, or condition to be treated. An effective amount can readily be determined by one of ordinary skill in the art in view of the present disclosure. [0112] According to particular embodiments, an effective amount refers to the amount of a composition or compound described herein which is sufficient to activate α2AR. In another particular embodiment, an effective amount refers to the amount of a composition or compound described herein which is sufficient to treat or prevent the disease or alleviate the symptoms associated with the disease. [0113] In some embodiments, the pain is neuropathic pain, nociceptive pain, nociplastic pain, or mixed pain. [0114] As used herein, the term “neuropathic pain” refers to pain caused by a lesion or disease affecting the somatosensory nervous system. It results from abnormal processing of pain signals due to nerve damage or dysfunction, either in the peripheral nervous system (e.g., peripheral neuropathy) or the central nervous system (e.g., spinal cord injury, multiple sclerosis). [0115] As used herein, the term “nociceptive pain” refers to pain that arises from actual or threatened damage to non-neural tissue and is due to the activation of nociceptors (pain-sensing nerve fibers). It is the body’s natural response to harmful stimuli such as injury, inflammation, or mechanical stress. [0116] As used herein, the term “nociplastic pain” refers to pain that arises from altered nociception. [0117] As used herein, the term “mixed pain” refers to a mixture of nociceptive, neuropathic, and/or nociplastic pain. [0118] In certain embodiments, the neuropathic pain is chosen from diabetic peripheral neuropathy, postherpetic neuralgia, chemotherapy-induced peripheral neuropathy, Fabry disease– associated pain, trigeminal neuralgia, glossopharyngeal neuralgia, occipital neuralgia, HIV- 26 Attorney Docket No.: 071741.11025/5WO1 associated neuropathy, hereditary sensory and autonomic neuropathy, Charcot-Marie-Tooth disease–related pain, small fiber neuropathy, brachial plexus avulsion, phantom limb pain, post- surgical neuropathic pain, carpal tunnel syndrome, other compression or entrapment neuropathies, amyloid neuropathy, vasculitic neuropathy, leprosy-associated pain, radiation-induced neuropathy, drug-induced neuropathy, metabolic neuropathies, endocrine-related neuropathies, toxic neuropathies, immune-mediated neuropathies, traumatic peripheral nerve injury, and heritable neuropathies, as well as post-stroke pain syndrome, spinal cord injury–related pain, multiple sclerosis–associated pain, thalamic pain syndrome, brain tumor–associated central pain, post–brain surgery neuropathic pain, central post-traumatic pain, cerebral palsy–related central pain, Parkinson’s disease–associated central pain, syringomyelia-related pain, and any combinations thereof. [0119] In certain embodiments, the nociceptive pain is somatic (i.e., from skin, muscles, or joints (e.g., cuts, fractures)) or visceral (i.e., from internal organs (e.g., appendicitis)). [0120] In certain embodiments, the nociceptive pain is osteoarthritis-related rheumatoid pain, arthritis-associated post-traumatic pain, musculoskeletal pain, postoperative pain, tendonitis, myofascial pain, visceral inflammatory pain (e.g., appendicitis, cystitis, pancreatitis), dysmenorrhea, or inflammatory bowel disease-related pain. [0121] In certain embodiments, the mixed pain is cancer-related pain (e.g., bone metastasis pain, tumor infiltration pain, perineural invasion), complex regional pain syndrome type I, failed back surgery syndrome, low back pain with radiculopathy endometriosis-associated pelvic pain, chronic post-surgical pain, chronic pancreatitis-associated pain, interstitial cystitis/bladder pain syndrome, fibromyalgia, temporomandibular disorder, vulvodynia, irritable bowel syndrome with visceral pain, and combinations thereof. [0122] In some embodiments, the pain is acute pain or chronic pain. As used herein, the term “chronic pain” refers to pain that is persistent or recurrent pain lasting longer than 3 months. [0123] In certain embodiments, the chronic pain is chronic primary pain, chronic cancer pain, chronic postsurgical and posttraumatic pain, chronic neuropathic pain, chronic headache and orofacial pain, chronic visceral pain, chronic musculoskeletal pain. [0124] In some embodiments, the pain is cancer pain. [0125] In some embodiments, the pain is post-surgery pain. 27 Attorney Docket No.: 071741.11025/5WO1 [0126] In some embodiments, the peripherally selective α2AR agonist comprises an α2AR activation moiety covalently linked to a peripheral distribution moiety. [0127] In another general aspect, the present disclosure relates to methods of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a peripherally selective α2AR agonist, wherein the peripherally selective α2AR agonist comprises an α2AR activation moiety covalently linked to a peripheral distribution moiety. [0128] In some embodiments, the disease is glaucoma, pain, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, presbyopia, acute kidney injury, insomnia, inflammatory disease, cancer, etc. [0129] In some embodiments, the disease is glaucoma or cancer. [0130] In some embodiments, the disease is pain. [0131] In some embodiments, the pain is neuropathic pain, nociceptive pain, nociplastic pain, or mixed pain. [0132] In certain embodiments, the neuropathic pain is chosen from diabetic peripheral neuropathy, postherpetic neuralgia, chemotherapy-induced peripheral neuropathy, Fabry disease– associated pain, trigeminal neuralgia, glossopharyngeal neuralgia, occipital neuralgia, HIV- associated neuropathy, hereditary sensory and autonomic neuropathy, Charcot-Marie-Tooth disease–related pain, small fiber neuropathy, brachial plexus avulsion, phantom limb pain, post- surgical neuropathic pain, carpal tunnel syndrome, other compression or entrapment neuropathies, amyloid neuropathy, vasculitic neuropathy, leprosy-associated pain, radiation-induced neuropathy, drug-induced neuropathy, metabolic neuropathies, endocrine-related neuropathies, toxic neuropathies, immune-mediated neuropathies, traumatic peripheral nerve injury, and heritable neuropathies, as well as post-stroke pain syndrome, spinal cord injury–related pain, multiple sclerosis–associated pain, thalamic pain syndrome, brain tumor–associated central pain, post–brain surgery neuropathic pain, central post-traumatic pain, cerebral palsy–related central pain, Parkinson’s disease–associated central pain, syringomyelia-related pain, and any combinations thereof. [0133] In certain embodiments, the nociceptive pain is somatic (i.e., from skin, muscles, or joints (e.g., cuts, fractures)) or visceral (i.e., from internal organs (e.g., appendicitis)). [0134] In certain embodiments, the nociceptive pain is osteoarthritis-related rheumatoid pain, arthritis-associated post-traumatic pain, musculoskeletal pain, postoperative pain, tendonitis, 28 Attorney Docket No.: 071741.11025/5WO1 myofascial pain, visceral inflammatory pain (e.g., appendicitis, cystitis, pancreatitis), dysmenorrhea, or inflammatory bowel disease-related pain. [0135] In certain embodiments, the mixed pain is cancer-related pain (e.g., bone metastasis pain, tumor infiltration pain, perineural invasion), complex regional pain syndrome type I, failed back surgery syndrome, low back pain with radiculopathy endometriosis-associated pelvic pain, chronic post-surgical pain, chronic pancreatitis-associated pain, interstitial cystitis/bladder pain syndrome, fibromyalgia, temporomandibular disorder, vulvodynia, irritable bowel syndrome with visceral pain, and combinations thereof. [0136] In some embodiments, the pain is acute pain or chronic pain. As used herein, the term “chronic pain” refers to pain that is persistent or recurrent pain lasting longer than 3 months. [0137] In certain embodiments, the chronic pain is chronic primary pain, chronic cancer pain, chronic postsurgical and posttraumatic pain, chronic neuropathic pain, chronic headache and orofacial pain, chronic visceral pain, chronic musculoskeletal pain. [0138] In some embodiments, the pain is cancer pain. [0139] In some embodiments, the pain is post-surgery pain. [0140] In some embodiments, treating with the peripherally selective α2AR agonist causes less side effects than treating with a non-peripherally selective α2AR agonist, such as at similar or comparable dosage. [0141] In some embodiments, treating with the peripherally selective α2AR agonist causes no side effects. [0142] The following embodiments apply to all general aspects described above. [0143] In some embodiments, the side effect is sedation, decreasing heart rate, and decreasing blood pressure, particularly the side effect is sedation. [0144] As used herein, the term “a non-peripherally selective α2AR agonist” refers to a compound can be readily distributed into the CNS after being administered into a subject, binds to and activates α2AR receptor in both the central nervous system (brain and spinal cord) and the peripheral nervous system. Examples of non-peripherally selective α2AR agonists include, but not limited to, dexmedetomidine, and clonidine. [0145] Without binding to the theory, if an α2AR agonist binds to and activates α2AR in the central nervous system, it can produce the above mentioned side effects in patients, such as sedation, decreased heart rate, blood pressure, depression, bradycardia, orthostatic hypotension, 29 Attorney Docket No.: 071741.11025/5WO1 constipation, nausea, gastric upset, dry mouth (xerostomia), dry nasal mucosa, impotence, fluid retention, edema, and pupil size. [0146] As used herein, the term “a peripherally selective α2AR agonist” refers to a compound that primarily exerts its effects outside of the central nervous system (CNS), typically because it is impeded by the blood-CNS barrier. Blood-CNS barrier, the physical barrier between blood and the CNS, safeguards the CNS from both toxic and pathogenic agents in the blood. The blood-CNS barrier comprises the blood-brain barrier, the blood-spinal cord barrier, and the blood-CSF (cerebrospinal fluid) barrier. By being largely impeded from entering the CNS, a compound may act on the rest of the body with less or no side-effects related to their effects on the brain or spinal cord. Examples of peripherally selective α2AR agonists include, but not limited to, the compounds described herein, such as compounds of formula (I-A), (I-B), (I-C), (I-D), or (II), described herein. [0147] The peripherally selective α2AR agonist primarily binds to or activates α2AR outside CNS, thus herby producing less or no foregoing side effects, compared to the non-peripherally selective α2AR agonists. The present invention satisfies an unmet need, and has developed a series of peripherally selective α2AR agonists. [0148] In some embodiments, the peripherally selective α2AR agonist binds to α2AR with a Ki ranging from 250nM to1000nM, 50nM to 250nM, 10nM to 50nM, or less than 10nM. In some other embodiments, the peripherally selective α2AR agonist activates α2AR with an EC50 ranging from 250nM to1000nM, 50nM to 250nM, 10nM to 50nM, or less than 10nM. [0149] The non-peripherally selective α2AR agonist and the peripherally selective α2AR agonist can be differentiated in terms of blood-brain barrier (BBB) permeability. Drugs that specifically target the central nervous system (CNS) must first traverse the BBB. In contrast, peripherally selective drugs primarily exert their effects outside of CNS, largely because they are impeded by the blood-brain barrier (BBB). The blood-brain barrier (BBB) substantially limits the entry of these drugs into the central nervous system (CNS), leading to a predominance of the drug concentration outside the CNS compared to inside. Any methods known in the field can be used to measure a compound’s BBB permeability. For example, one experimental measure of BBB permeability is Kp, which is the concentration of drug in the brain divided by concentration in the blood. [0150] As used herein, “Kp”, or “B/P ratio”, refers to the ratio of the concentration of a compound in the brain and in the blood. Kp is often calculated as “logBB”, which refers to the logarithmic ratio of the concentration of a compound in the brain and in the blood. Kp is a common 30 Attorney Docket No.: 071741.11025/5WO1 numeric value for describing permeability across the blood-brain barrier. In some embodiments, a compound is considered “peripherally selective” if, upon administration to a subject, its Kp is lower than 0.4, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01. [0151] Kp,uu,brain is another common numeric value for describing permeability across the blood-brain barrier. As used herein, “Kp,uu,brain” or “Kp,uu”, refers to the unbound brain-to- plasma partition coefficient. It represents the ability of a drug to cross the blood-brain barrier (BBB) after systemic administration. Kp,uu provides a more accurate measure of distribution equilibrium between unbound fractions in brain and plasma. [0152] Any methods known in the field can be used to measure Kp,uu,brain. One example of Area Under the Curve (AUC) method, which calculates the AUC of the unbound drug concentration-time profile in both brain and plasma after a single dose. Another example is Steady-State Concentrations, which uses the steady-state unbound concentrations of the drug in brain interstitial fluid (C_u,brain,ss) and in plasma (C_u,plasma,ss). [0153] In some embodiments, a compound is considered “peripherally selective” if, upon administration to a subject, its Kp,uu,brain is lower than 0.4, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01. In some further embodiments, a compound is considered “peripherally selective” if, upon administration to a subject, its Kp,uu,brain is lower than 0.05, 0.04, 0.03, 0.02, or 0.01. [0154] In some embodiments, the peripherally selective α2AR agonist comprises an α2AR activation moiety that is covalently linked to a peripheral distribution moiety. [0155] In certain embodiments, the α2AR activation moiety is a non-peripherally selective α2AR agonist or another peripherally selective α2AR agonist. [0156] In certain embodiments, the α2AR activation moiety is an α2AR agonist that is chosen from (R)-3-nitrobiphenyline, A-193080, ADX-415, AGN 192836, AGN-191103, AGN-197075, AGN-201781, AGN-241622, amitraz, Apraclonidine, AR-08, Bethanidine, Brimonidine, BRL- 48962, Bromocriptine, Cirazoline, Clonidine, Detomidine, Detomidine carboxylic acid, Dexmedetomidine, Dipivefrin, DL-Methylephedrine, Droxidopa, Epinephrine, ergotamine, etilefrine, Etomidate, Fadolmidine, Guanabenz, Guanethidine, Guanfacine, Guanoxabenz, indanidine, Lofexidine, Medetomidine, mephentermine, Metamfetamine, metaraminol, methoxamine, Methyldopa, Methyldopate, Methyldopate hydrochloride, Methylnorepinephrine, mivazerol, Moxonidine, naphazoline, Norepinephrine, norfenefrine, octopamine, ODM-105, 31 Attorney Docket No.: 071741.11025/5WO1 Oxymetazoline, Pergolide, phenylpropanolamine, Povafonidine, propylhexedrine, Pseudoephedrine, Racepinephrine, rezatomidine, rilmenidine, romifidine, synephrine, talipexole, tasipimidine, Tiamenidine, Tizanidine, Xylazine, Xylometazoline, and a functional derivative thereof. [0157] As used herein, the functional derivative of an α2AR agonist refers to any compound that is derived from the α2AR agonist by a chemical reaction. Examples of the derivatives include, but not limited to, acid or base salts, prodrugs, compounds containing protected functional groups such as hydroxyl, amino, carboxyl and carbonyl groups. [0158] In certain embodiments, the α2AR activation moiety is a non-peripherally selective α2AR agonist, such as dexmedetomidine, brimonidine, and clonidine. [0159] In certain embodiments, the α2AR activation moiety is dexmedetomidine. [0160] As used herein, the term “a peripheral distribution moiety” refers to a moiety that can increase or improve the peripheral selectivity of an α2AR agonist. In some embodiments, the peripheral selectivity is increased or improved so that the α2AR agonist is a peripherally selective α2AR agonist. [0161] According to the embodiments of the present disclosure, the peripheral distribution moiety can be the following chemical fragments: • type A fragments; those that can increase the overall molecular polarity of the compound or reduce the overall lipophilicity of the compound; • type B fragments: those that can increase the overall molecular weight or the molecular size of the compound; and • type C fragments: those that comprises a substrate element of an efflux transporter. [0162] In some embodiments, the peripheral distribution moiety is a type A fragment. [0163] In certain embodiments, the type A fragment increases the total number of intermolecular hydrogen bond (H-bond) within the compound, such as H-bond donors and H-bond acceptors. In a preferred embodiment, the type A fragment is a H-bond donor. [0164] In certain embodiments, the type A fragment increases the overall molecular polarity of the compound. For example, such type A fragments can comprise a polar functional group or a charged group. Examples of the polar functional group include, but not limited to, hydroxyl, amine, amide, sulfonamide, carboxyl, ether, imine, hydroxylamine, ester, aldehyde, ketone, nitro, 32 Attorney Docket No.: 071741.11025/5WO1 phosphate, thioether, and sulfone groups. Examples of the charged group include, but not limited to, quaternary ammonium and organic acids such as carboxylic acids and sulfonic acids. [0165] In certain embodiments, the type A fragment reduces the overall lipophilicity of the compound. Examples of such type A fragments include, but not limited to, alkyl or acyl that is added to a function group such as hydroxyl and amino. [0166] In certain embodiments, the type A fragment is not tertiary amine or one that can help form an intramolecular H-bond. [0167] In some embodiments, the peripheral distribution moiety is a type B fragment. [0168] In certain embodiments, the type B fragment is a bulky group, which can increase the overall molecular weight and the molecular size of the compound. Examples of such type B fragments include, but not limited to, long alkyl chains, polyethylene glycol (PEG), large aromatic groups, and extra cyclic or heterocyclic groups. [0169] In some embodiments, the peripheral distribution moiety is a type C fragment. [0170] In certain embodiments, the type C fragment comprises a substrate element of an efflux transporter, wherein the efflux transporter is P-glycoprotein (P-gp) transporter, breast cancer resistance protein (BCRP) transporter, or multidrug resistance protein 2 (MRP2) transporter. As used herein, the term of “a substrate element of an efflux transporter” refers to a fragment that makes the compound to become a substrate of the efflux transporter. In other words, the term of “a substrate element of an efflux transporter” refers to a fragment of a substrate of the efflux transporter. [0171] In certain embodiments, the type C fragment comprises a substrate element of P-gp. [0172] Without binding to the theory, P-gp efflux is a significant limitation to BBB permeation. Any methods known in the filed can be used to determine whether a compound is a P-gp substrate. For example, the efflux ratio obtained from in vitro P-gp assay, MDCK-MDR1, can be used to identify the substrate of P-gp. A compound is considered as a P-gp substrate if its efflux ratio is greater than 2, 5, 8, 10, 50, or 100. [0173] Alternatively, there are some rules for determining potential P-gp efflux substrates: • the total number of N atom and O atom (N+O) ≥ 8; • molecular weight (MW) > 400; and /or • acid with pKa > 4. 33 Attorney Docket No.: 071741.11025/5WO1 In contrast, if a compound has N+O <4, MW < 400, and/or is a base with pKa < 8, then it is a non- substrate of P-gp. [0174] Certain structural modifications can improve P-gp efflux, such as removing steric hindrance to the hydrogen bond donating atoms by attachment of a bulky group or by unmethylation the nitrogen atom, and improving hydrogen bonding potential by removal of an adjacent electron withdrawing group or by introducing the hydrogen bonding group such as amide. In certain embodiments, the substrate element for P-gp contains one or more of the structural modifications described above. [0175] In certain embodiments, the substrate element for P-gp is chosen from: O H N O H N O O H O O NH N P S F3 . [0176] In cer , transporter. [0177] In certain embodiments, the type C fragment comprises a substrate element of MPR2 transporter. [0178] In certain embodiments, the type C fragment does not comprise a substrate element of uptake transporter, such as LAT1, GLUT1, MCT1, CAT1, CNT2, OATP, PEPT1, PEPT2, and OCT. 34 Attorney Docket No.: 071741.11025/5WO1 [0179] In some embodiments, the peripheral distribution moiety reduces and/or minimizes brain exposure to a peripherally selective α2AR agonist. [0180] In certain embodiments, the peripheral distribution moiety decreases passive transcellular BBB permeability by increasing topological polar surface area (TPSA), increasing molecule weight, increasing polarity, or adding hydrogen binding, especially hydrogen bond donor. [0181] In certain embodiments, the peripheral distribution moiety introduces an acidic group to the peripherally selective α2AR agonist. [0182] In certain embodiments, the peripheral distribution moiety comprises a substrate element for P-gp, wherein the substrate element for P-gp increases P-gp efflux by increasing lipophilicity, increasing hydrogen bond acceptors, removing steric hindrance around hydrogen bind acceptors, or removing electron-withdrawing group adjacent to hydrogen bond acceptor. [0183] In certain embodiments, the peripheral distribution moiety makes a compound to become a dual substrate for both P-gp and BCRP. Compounds [0184] In a general aspect, the present disclosure relates to a compound of formula (I-A): RT m ( - ) , or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, wherein, Y is C(R1), N, -O-C, -C-NH-, -CH2-C(O)-, or -CH=N-; when Y is C(R1), R1 is chosen from H, D, and halogen; when Y is -O-C-, the oxygen atom is connected to A, and the carbon atom is connected to both RT and B; and when Y is -C-NH-, the carbon atom is connected to both RT and A, and the nitrogen atom is connected to B; 35 Attorney Docket No.: 071741.11025/5WO1 A is a ring chosen from phenyl, pyridinyl, thienyl, furyl, pyrrolyl, 4H-pyran, 4H-thiopyran, 1,2,3,4-tetrahydro-1-naphthyl, tetrahydrozoline, quinoxalinyl, pyrimidinyl, and 2,1,3- benzothiadiazol; NH N X Ra a is H and n is 0, 1, 2, or 3; each R2 is independently chosen from H, D, halogen, alkyl, alkenyl, alkynyl, alkoxyl, ester, cycloalkyl, cycloalkoxy, aryl, aryloxy, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclolalkyl, OR4, -CN, N3, NO2, N(R4)2, OR4, SR4, C(O)R4, SO2N(R4)2, CH2SR4 ; wherein the alkyl, alkenyl, alkynyl, alkoxyl, ester, cycloalkyl, cycloalkoxy, aryl, aryloxy, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclolalkyl is optionally substituted with one or more R5; R4 is chosen from H, D, halogen, alkyl, alkenyl, alkynyl, alkoxyl, ester, cycloalkyl, cycloalkoxy, aryl, aryloxy, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclolalkyl, and the alkyl, alkenyl, alkynyl, alkoxyl, ester, cycloalkyl, cycloalkoxy, aryl, aryloxy, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclolalkyl is optionally substituted with one or more R5; R5 is chosen from halogen, hydroxyl, -CN, -NO2, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, cycloalkyl, cycloalkoxy, aryl, aryloxy, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl; alternatively, when A is a phenyl ring and two R2 are substituted at adjacent positions of the phenyl ring, the two R2 groups, with the carbon atoms they are connected to, form a ring that is fused to ring A to form a bicyclic ring, such as quinolinyl, indolyl, benzothienyl, benzofuryl, benzofuranyl, benzodioxolyl, 2,3-dihydrobenzo[b][l,4]dioxin-6-yl, cinnolinyl, quinoxalinyl, or 1,2,4-benzotriazinyl; m is 0, 1, 2, or 3; each R3 is independently chosen from H, D, halogen, -OH, -SH, optionally substituted alkyl, optionally substituted heterocycle, and optionally substituted aryl; 36 Attorney Docket No.: 071741.11025/5WO1 alternatively, R3 is a group connected to the -NH of the imidazole ring, and R3 is of the formula of: O O O OR8 R6 R7 6 P R5 O R6 R5 O O R5 R O O O or R5 O OR8 , wherein: R6 is hydrogen, alkyl, cycloalkyl, or alkenyl; R7 is an amino acid residue; and R8 is alkyl or cycloalkyl; RT is RL-RP, and RP is optionally substituted with RC, wherein: RL is a linker, wherein one end is conneceted to RP and the other end is conneceted to Y; RP is a moiety that is connected to one end of RL; and RC is a cap, which is a moiety that is connected to RP. [0185] As used herein, the term RL is a moiety that covalently connects two functional groups or moieties within a single molecule. One end of RL is connected to RP and the other end of RL is connceted to Y. RL can be any moiety that serves the linking function, such as the linkers used in proteolysis targeting chimeras (PROTACs) and non-cleavable linkers used in antibody-drug conjugates (ADCs). Examples of RL include, but are not limited to, polyethylene glycol (PEG) and alkyl chains of varying lengths, glycols, alkynes, triazoles, saturated heterocycles such as piperazine and piperidine, thioethers, maleimidocaproyl linker. [0186] In some embodiments, RL is chosen from alkyl, polyethylene glycol, other glycol, cycloalkyl, heterocycle, aryl, and heteroaryl; wherein the cycloalky, heterocycle, aryl, or heteroaryl is optionaly substituted with at least one substituent chosen from halogen, hydroxyl, alkyl, haloalkyl, alkoxy and hydroxyalkyl. 37 Attorney Docket No.: 071741.11025/5WO1 [0187] In certain embodiments, RL is one selected from the followings: Z Y N m2 Y Y m2 m2 H, m , ag e s, e ag e s ca e co ec e a y o e . [0188] According to the embodiments of the present disclosure, Rp can be the following chemical moieties: • those that can increase the overall molecular weight of the compound, such as bulky functional groups and additional molecular structures, including long alkyl chains, large aromatic groups, and extra cyclic structures like cyclohexane or cyclopentane rings; • those that can increase the overall molecular polarity of the compound, such as hydroxyl, amine, amide, sulfonamide, ether, imine, hydroxylamine, ester, aldehyde, ketone, nitro, phosphate, thioether; and • those that can bring charge to the compound, such as the functional groups that ionize at physiological pH, including carboxylic acid, quaternary ammonium and quaternary phosphonium. 38 Attorney Docket No.: 071741.11025/5WO1 [0189] In some embodiments, when RP is not substituted with RC, RP is: O H N O H N O O O H N N . 39 Attorney Docket No.: 071741.11025/5WO1 [0190] In some embodiments, when RP is substituted with RC, RP is: O H O N O H N O O H O O NH N P S S O F3 . [0191] As u end of RP. [0192] In some embodiments, RC is chosen from -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene- C2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -NH-C0-12 alkylene-C3-12 cycloalkyl, -NH-C0-12 alkylene-C2-12 heterocyclyl, -NH-C0-12 alkylene-C1-12 heteroaryl, -O-C0-12 alkylene-C3-12 cycloalkyl, -O-C0-12 alkylene-C2-12 heterocyclyl, -O-C0-12 alkylene-C1-12 heteroaryl, and alkyl substituted with trialkylammonium, wherein each of the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more substituents chosen from hydroxy, alkyl, oxo, and ketone. [0193] In certain embodiments, RC is: Z Z s s Zs Zs NH . Attorney Docket No.: 071741.11025/5WO1 [0194] In certain embodiments, RC is: H NH N NH NH O O O Attorney Docket No.: 071741.11025/5WO1 O NH H O O N N O O S [0195] In another general aspect, the present disclosure relates to a compound of formula (I-B): RT m , or a ste lly acceptable salt or solvate thereof, wherein, Y is a bond, CH(R1), NH, -O-CH-, -C-NH-, -CH2-C(O)-, or -CH=N-; when Y is C(R1), R1 is chosen from H, D, and halogen; when Y is -O-C-, the oxygen atom is connected to A, and the carbon atom is connected to B; when Y is -C-NH-, the carbon atom is connected to A, and the nitrogen atom is connected to B; and A, B, R2, n, R3, m, and RT are defined as above in formula (I-A). In another general aspect, the present disclosure relates to a compound of formula (I-C): RT m or a ste , , lly acceptable salt or solvate thereof, wherein, 42 Attorney Docket No.: 071741.11025/5WO1 Y is a bond, CH(R1), NH, -O-CH-, -C-NH-, -CH2-C(O)-, or -CH=N-; when Y is C(R1), R1 is chosen from H, D, and halogen; when Y is -O-C-, the oxygen atom is connected to A, and the carbon atom is connected to B; when Y is -C-NH-, the carbon atom is connected to A, and the nitrogen atom is connected to B; and A, B, R2, n, R3, m, and RT are defined as above in formula (I-A). [0196] In another general aspect, the present disclosure relates to a compound of formula (I-D): N NH T , or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, wherein, Y1 is CH, or N; X1 is chosen from H, D, and halogen; and RT is defined as above in formula (I-A). [0197] In some embodiments, the compound of formula (I-A) has the formula (I-A-1): RT m , or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, wherein, R1 is chosen from H, D, and halogen; 43 Attorney Docket No.: 071741.11025/5WO1 A is a ring chosen from phenyl, pyridinyl, thienyl, furyl, pyrrolyl, 4H-pyran, or 4H- thiopyran; R2, n, R3, m, and RT are defined as above in formula (I-A). [0198] In some embodiments, the compound of formula (I-A) has the formula (I-A-2): RT , or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, wherein, A is a ring chosen from phenyl, 1,2,3,4-tetrahydro-1-naphthyl, quinoxalinyl, pyrimidinyl, and 2,1,3-benzothiadiazol; Y is CH, N, -O-CH-, or -C-NH-; when Y is -O-C-, the oxygen atom is connected to A, and the carbon atom is connected X to both ; when arbon atom is connected to both RT and A, and the nitrogen X atom is connecte ; X is NH, O, or S; and R2, n, and RT are defined as above in formula (I-A). [0199] In certain embodiments, when the compound of formula (I-A) has the formula (I-A-1) or R2 r in: Attorney Docket No.: 071741.11025/5WO1 ring M is C3-12 cycloalkyl, C2-12 heterocyclyl, C6-12 aryl, or C1-12 heteroaryl, wherein the C3-12 cycloalkyl or C2-12 heterocyclyl is optionally fused with an aryl; r is 1 or 2; n2 is 0, 1, or 2; each R2 is independently chosen from hydrogen, halogen, hydroxyl, and alkoxy; R3 is chosen from CN, hydroxy, alkoxy, -C(O)-C0-12 alkylene-CN, -C0-12 alkylene-C2-12 heterocyclyl, -SO2-alkyl, -C(O)-NR4R4’, -SO2-NR4R4’, -C0-12 alkylene-R3’, -O-C0-12 alkylene- COOH, -C0-12 alkylene-N(R4)-C(O)-R5, -C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-O-C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-P(=O)(R4)(R4’), O R6 O O O CH3 m , -NH-R7, or n ; wherein one -CH2- group in the -C0-12 alkylene-R3’ is optionally , the -C0-12 alkylene-R3’ is optionally substituted with one or more substitutes chosen from amino and alkylamino, and the C2-12 heterocyclyl and C1- 12 heteroaryl are each optionally substituted with one or more R4a; R3’ is chosen from -C(O)-NR4R4’, -SO2-NR4R4’, -C0-12 alkylene-COOH, -C0-12 alkylene- N(R4)-C(O)-R5, -C0-12 alkylene-N(R4)-SO2-R5, C0-12 alkylene-C1-12 heteroaryl; each R4a is independently chosen from hydroxy, alkyl, oxo, ketone, and -C2-12 heterocyclyl; each of R4 and R4’ is independently hydrogen, alkyl, alkoxy, -SO2- N(R6a)t, -C0-12 alkylene- COOH, -C0-12 alkylene-N(R6a)t, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, - C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-OR6a, or hydroxyalkyl, wherein the hydroxyalkyl is optionally substituted with alkoxy; wherein each of the alkyl, C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a; or R4 and R4’, together with the nitrogen atom that they are attached to, form a heterocycle comprising one or more heteroatoms chosen from O, N, and S; alternatively, when one R2 is adjacent to R3, the R2 and R3, together with the atoms that they are attached to, form a ring optionally substituted with one or more R4a;; R5 is amino, alkylamino, C1-12 haloalkyl, -C0-12 alkylene-OR6a, -C0-12 alkylene-N(R6a)t, -C0- 12 alkylene-SR6a, -C0-12 alkylene-CN, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C2-12 alkenyl, or alkyl optionally substituted with cyano, amido, trialkylammonium, or thiolate; wherein each of the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a; 45 Attorney Docket No.: 071741.11025/5WO1 each R6a is independently chosen from hydrogen, C1-12 alkyl, C1-12 alkoxy, -C0-12 alkylene- C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C6-12 aryl, and -C0-12 alkylene-C1- 12 heteroaryl; wherein each of the alkyl, C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl, is optionally substituted with one or more R4a; R6 is alkoxy, amino, sulfonamide, carbamide, or alkyl optionally substituted with cyano; R7 is hydrogen, alkyl, -C0-12 alkylene-COOH, optionally substituted C3-12 cycloalkyl, C2-12 aryl, C1-12 heteroaryl, -C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-P(=O)(R4) (R4’), -C0-12 alkylene- N(R4)-C(=S)-R5, -C(=S)-R5, or alkyl optionally substituted with cyano; R8 is alkoxy, amino, alkylamino, amide, sulfonamide, or carbamide; n3 is 0, 1, 2, 3, or 4; n4 is 1, 2, 3, 4, 5, or 6; t is 2 or 3; m is 0, 1, 2, 3, 4, or 5; and n is 0, 1, 2, 3, or 4. [0200] In another general aspect, the present disclosure relates to a compound of formula (II): RT B , or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, wherein, A is one chosen from: S 1 ; each R1 is independently chosen from hydrogen, halogen, haloalkyl, hydroxyl, hydroxyalkyl, alkoxy, alkyl, and -COOH; B is one chosen from: 46 Attorney Docket No.: 071741.11025/5WO1 H N N H X S N N H; oaryl, wherein the C3-12 cycloalkyl or C2-12 heterocyclyl is optionally fused with an aryl; r is 1 or 2; n2 is 0, 1, or 2; each R2 is independently chosen from hydrogen, halogen, hydroxyl, and alkoxy; R3 is chosen from CN, hydroxy, alkoxy, -C(O)-C0-12 alkylene-CN, -C0-12 alkylene-C2-12 heterocyclyl, -SO2-alkyl, -C(O)-NR4R4’, -SO2-NR4R4’, -C0-12 alkylene-R3’, -O-C0-12 alkylene- COOH, -C0-12 alkylene-N(R4)-C(O)-R5, -C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-O-C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-P(=O)(R4)(R4’), O R6 O O O CH3 m -NH-R7 or n ; wherein one -CH2- group in the -C0-12 alkylene-R3’ is , the -C0-12 alkylene-R3’ is optionally substituted with one or more substitutes chosen from amino and alkylamino, and the C2-12 heterocyclyl and C1- 12 heteroaryl are each optionally substituted with one or more R4a; R3’ is chosen from -C(O)-NR4R4’, -SO2-NR4R4’, -C0-12 alkylene-COOH, -C0-12 alkylene- N(R4)-C(O)-R5, -C0-12 alkylene-N(R4)-SO2-R5, C0-12 alkylene-C1-12 heteroaryl; each R4a is independently chosen from hydroxy, alkyl, oxo, ketone, and -C2-12 heterocyclyl; each of R4 and R4’ is independently hydrogen, alkyl, alkoxy, -SO2- N(R6a)t, -C0-12 alkylene- COOH, -C0-12 alkylene-N(R6a)t, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, - C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-OR6a, or hydroxyalkyl, wherein the hydroxyalkyl is optionally substituted with alkoxy; wherein each of the alkyl, C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a; or R4 and R4’, together with the nitrogen atom that they are attached to, form a heterocycle comprising one or more heteroatoms chosen from O, N, and S; 47 Attorney Docket No.: 071741.11025/5WO1 alternatively, when one R2 is adjacent to R3, the R2 and R3, together with the atoms that they are attached to, form a ring optionally substituted with one or more R4a;; R5 is amino, alkylamino, C1-12 haloalkyl, -C0-12 alkylene-OR6a, -C0-12 alkylene-N(R6a)t, -C0- 12 alkylene-SR6a, -C0-12 alkylene-CN, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C2-12 alkenyl, or alkyl optionally substituted with cyano, amido, trialkylammonium, or thiolate; wherein each of the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a; each R6a is independently chosen from hydrogen, C1-12 alkyl, C1-12 alkoxy, -C0-12 alkylene- C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C6-12 aryl, and -C0-12 alkylene-C1- 12 heteroaryl; wherein each of the alkyl, C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl, is optionally substituted with one or more R4a; R6 is alkoxy, amino, sulfonamide, carbamide, or alkyl optionally substituted with cyano; R7 is hydrogen, alkyl, -C0-12 alkylene-COOH, optionally substituted C3-12 cycloalkyl, C2-12 aryl, C1-12 heteroaryl, -C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-P(=O)(R4) (R4’), -C0-12 alkylene- N(R4)-C(=S)-R5, -C(=S)-R5, or alkyl optionally substituted with cyano; R8 is alkoxy, amino, alkylamino, amide, sulfonamide, or carbamide; n3 is 0, 1, 2, 3, or 4; n4 is 1, 2, 3, 4, 5, or 6; t is 2 or 3; m is 0, 1, 2, 3, 4, or 5; and n is 0, 1, 2, 3, or 4. S [0201] In some embodiments . 1 (R1)n1 [0202] In some embodiments . [0203] In some embodiments , , . [0204] In some embodiments, R1 is halogen, such as fluorine or chlorine. [0205] In some embodiments, R1 is alkoxy, such as -OMe. [0206] In some embodiments, R1 is hydroxyl or -COOH or -CH2OH. [0207] In some embodiments, R1 is haloalkyl, such as trifluoromethyl or -CH2CH2F. 48 Attorney Docket No.: 071741.11025/5WO1 H N N S [0208] In some embodiment . N [0209] In some embodiment, B i , wherein X is S, O, or NH. n2 [0210] In some embodiments . R8 n4 [0211] In some embodiments, RT is . [0212] In some embodiments, ring 12 heteroaryl. [0213] In some embodiments, ring M is C3-12 cycloalkyl or C2-12 heterocyclyl, wherein the C3-12 cycloalkyl or C2-12 heterocyclyl is optionally fused with an aryl. [0214] In some embodiments, ring M is phenyl, pyridinyl, pyrimidinyl, thiophenyl, cyclopentyl, or cyclohexyl. . [0217] In some embodiments, R2 is hydroxyl. [0218] In some embodiments, R2 is halogen, such as fluorine or chlorine. [0219] In some embodiments, the pharmaceutically acceptable salt of the compound of formula (I) is trifluoroacetate or hydrochloride. [0220] In some embodiments, the compound of formula (II) is a compound of formula (II-A): 49 Attorney Docket No.: 071741.11025/5WO1 R2 R3 H , wherein R1, R2, R3, and n1 are defined as above in formula (II). [0221] In some embodiments, R1 is halogen, haloalkyl, hydroxyl, alkyl, or -COOH. [0222] In certain embodiments, R1 is methyl, ethyl, hydroxyl, fluorine, chlorine, trifluoromethyl, -CH2CH2F, or -COOH. [0223] In some embodiments, n1 is 2. [0224] In some embodiments, R2 is hydrogen, hydroxyl, or halogen. [0225] In certain embodiments, R2 is fluorine or chlorine. [0226] In some embodiments, R3 is -C(O)-NR4R4’or -SO2-NR4R4’, wherein each of R4 and R4’ is independently hydrogen, alkyl, alkoxy, -C0-12 alkylene-N(R6a)t, -C0-12 alkylene-C3-12 cycloalkyl, - C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-OR6a, or hydroxyalkyl, and the hydroxyalkyl is optionally substituted with alkoxy; wherein each of the alkyl, C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a, wherein R4a, R6a, and t are defined as above. [0227] In certain embodiments, each of R4 and R4’ is independently hydrogen, alkyl, alkoxy, or hydroxyalkyl. O , ed CH3 O with alkoxy, such as , wherein p is 0, 1, 2, or 3, particularly p is 2. [0230] In some em bodiments, R3 is -C(O)-NR4R4 or -SO2-NR4R4’, wherein R4 and R4’, together with the nitrogen atom that they are attached to, form a heterocycle comprising one or more 50 Attorney Docket No.: 071741.11025/5WO1 heteroatoms chosen from O, N, and S, particularly, R4 and R4’, together with the nitrogen atom that they are attached to, form a six-membered heterocycle. O N [0231] In some embodiments R3 is h drox l -COOH -CH(CH3)-COOH -CN , N . -R5, or -C0-12 alkylene-O-C0-12 alkylene-N(R4)-SO2-R5, wherein R4 is hydrogen or alkyl, and R5 is amino, alkylamino, C1-12 haloalkyl, -C0-12 alkylene-OR6a, -C0-12 alkylene-N(R6a)t, -C0-12 alkylene- SR6a, -C0-12 alkylene-CN, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C2-12 alkenyl, or alkyl optionally substituted with cyano or amido; wherein each of the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a, wherein R4a, R6a, and t are defined as above. [0233] In certain embodiments, R5 is amino, alkylamino, alkoxy, alkyl, or -C2-12 alkenyl. [0234] In certain embodiments, R5 is alkyl substituted with cyano, such as -CH2CN. [0235] In certain embodiments, R5 is alkyl substituted with amido, such as -CH2CH3CONH2. [0236] In certain embodiments, R5 is alkyl substituted with alkoxy, trialkylammonium, or thiolate. [0237] In certain embodiments, when R5 is -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, or -C0-12 alkylene-C1-12 heteroaryl, the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 , . , , , 2 heteroaryl is substituted with one or more R4a, R4a is hydroxyl, methyl, oxo, or -C(O)-Me. 51 Attorney Docket No.: 071741.11025/5WO1 O R6 [0239] In some embodiments, R3 is m , wherein m is 0, 1, 2, 3, 4, or 5, and R6 is sulfonamide, carbamide, or alkyl stituted with cyano. [0240] In certain embodiments, R is sulfonamide of formula –N(R’)SO2-R, wherein each R and R’ is independently chosen from hydrogen and alkyl, particularly R6 is -NHSO2CH3. [0241] In certain embodiments, R6 is carbamide of formula –N(R’)C(O)N(R)2, wherein each R N N NH and R’ is independently chosen from hydrogen, alkyl, and heteroaryl, particular . [0242] In certain embodiments, R6 is alkyl optionally substituted with cyano, alkyl optionally substituted with cyano, particularly C1-4 alkyl substituted with cyano. [0243] In certain embodiments, m is 1, 2, or 3, particularly 2. [0244] In some embodiments, R3 is -NH-R7, wherein R7 is hydrogen, optionally substituted C3-12 cycloalkyl, C1-12 heteroaryl, -C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-P(=O)(R4) (R4’), -C0-12 alkylene-N(R4)-C(=S)-R5, -C(=S)-R5, or alkyl optionally substituted with cyano; wherein R4, R4’ and R5 are defined as above. [0245] In certain embodiments, R7 is hydrogen. N O O S O [0246] In certain embodiments . [0247] In certain embodiments lkyl optionally substituted with cyano, particularly C1-4 alkyl substituted with cyano. O O O CH3 [0248] In certain embodiments, R3 i , n is 3 or 4, particularly 4. [0249] In some embodiments, the compound of formula (II) is a compound of formula (II-B): 52 Attorney Docket No.: 071741.11025/5WO1 R1 R8 ( )n1 (CH2) n3 O n4 , wherein R1, R8, n1, n3, and n4 are defined as above in formula (II). [0250] In some embodiments, R1 is hydrogen or alkyl, such as alkyl, particularly methyl. [0251] In some embodiments, n1 is 2. [0252] In some embodiments, n3 is 0, 1 or 3. [0253] In some embodiments, n4 is 2, 3, or 5. [0254] In some embodiments, R8 is alkoxy, such as C1-4 alkoxy, particularly methoxy or ethoxy. [0255] In some embodiments, R8 is amino. [0256] In some embodiments, R8 is alkylamino, such as C1-4 alkylamino, particularly methylamino. [0257] In some embodiments, R8 is amide of formula –N(R’)C(O)R, wherein each R and R’ is independently chosen from hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl. [0258] In some embodiments, R8 is amide of formula –N(R’)C(O)R, particularly R8 is - NHCOCH3.. [0259] In some embodiments, R8 is sulfonamide of formula –N(R’)SO2-R, wherein each R and R’ is independently chosen from hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl. [0260] In certain embodiments, R8 is sulfonamide of formula –N(R’)SO2-R, wherein each R and R’ is independently chosen from hydrogen, -C0-12 alkylene-C2-12 heterocyclyl, and alkyl, particularly R3 is -NHSO2CH3. [0261] In some embodiments, R8 is carbamide of formula –N(R’)C(O)N(R)2, wherein each R and R’ is independently chosen from hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl. [0262] In certain embodiments, R8 is carbamide of formula –N(R’)C(O)N(R)2, wherein each R and R’ is independently chosen from hydrogen, alkyl, and heteroaryl, particularly R3 is - N N 53 Attorney Docket No.: 071741.11025/5WO1 [0263] In some embodiments, when the compound is a compound of formula (II), R8 is -OCH3, - O O N N H O H O O N N N N N N S S . [0264] In some embodiments, the compound of formula (II) is a compound of formula (II-C): R2 R3 H , wherein, n2 is 1 or 2; and R1, R2, R3, and n1 are defined as above in formula (II). [0265] In some embodiments, R1 is alkyl, such as methyl. [0266] In some embodiments, n1 is 2. [0267] In some embodiments, n2 is 1. [0268] In some embodiments, R2 is hydrogen or halogen. [0269] In certain embodiments, R2 is fluorine. [0270] In some embodiments, R3 is -C(O)-NR4R4’, wherein each of R4 and R4’ is independently hydrogen, hydroxy, alkyl, alkoxy, -SO2-NHCH3, -SO2-NH-Ph, -CH2-COOH, -CH2-CH2-COOH, O . some embodiments, R3 is -SO2-NR4R4’, wherein each of R4 and R4’ is independently hydrogen, hydroxy, or -C0-12 alkylene-C2-12 heterocyclyl. [0272] In some embodiments, R3 is -NH-C(O)-R5, -N(CH3)-C(O)-R5 or -NH-SO2-R5, wherein R5 is alkyl, -C0-12 alkylene-alkoxy, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-NH- C1-12 alkyl, -C0- 12 alkylene-NH- C2-12 heterocyclyl, or -C0-12 alkylene-C2-12 heterocyclyl. [0273] In certain embodiments, R5 is alkyl, such as methyl. [0274] In certain embodiments, R5 is -C0-12 alkylene-alkoxy, such as -CH2-OCH3. 54 Attorney Docket No.: 071741.11025/5WO1 [0275] In certain embodiments, R5 is -C0-12 alkylene-C3-12 cycloalkyl, such . [0276] In certain embodiments, R5 is -C0-12 alkylene-NH- C1-12 alkyl, such 5 [0277] In certain embodiments, R is-C0-12 alkylene-NH- C2-12 heterocyclyl, such as O H H N O HN N O . O [ ] n certa n em o ments, s -C0-12 alkylene-C2-12 heterocyclyl, such as , O O . ments, R3 is -SO2-alkyl, such as -SO2-CH3. [0280] In some embodiments, R3 is -C0-12 alkylene-COOH, such as -COOH, -CH2-COOH, - C(Me)2-COOH, -CH2- CH2-COOH. OH P O [0281] In some embodiments, R3 is -C0-12 alkylene-P(=O)(R4)(R4’), such as OH . HN N [0282] In some embodiments, R3 is -C0-12 alkylene-C1-12 heteroaryl, su , O N HN N N N N N N N -R5 [0284] In some embodiments, the compound of formula (II) is a compound of formula (II-D): 55 Attorney Docket No.: 071741.11025/5WO1 R2 R3 H , wherein, n2 is 0 or 1; R3 is chosen from -C(O)-NHR4, -SO2-NHR4, -NH-C(O)-R5, and -NH-SO2-R5 , and -NH-R7; R4 is -C0-12 alkylene-NHR6a, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-OR6a , or alkyl substituted with trialkylammonium; wherein and each of the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a; R5 is -C0-12 alkylene-NHR6a, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-OR6a, or alkyl substituted with trialkylammonium; wherein each of the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a; each R6a is independently chosen from -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, and -C0-12 alkylene-C1-12 heteroaryl; wherein each of the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a; R7 is C1-12 heteroaryl, -C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-P(=O)(R4) (alkoxy), or -C0-12 alkylene-N(R4)-C(=S)-R5; and R1, R2, R4a, and n1 and are defined as above in formula (II). [0285] In some embodiments, R1 is alkyl, such as methyl. [0286] In some embodiments, R1 is alkoxy, such as -OMe. [0287] In some embodiments, n1 is 1. [0288] In some embodiments, n1 is 2. [0289] In some embodiments, n2 is 0. [0290] In some embodiments, n2 is 1. [0291] In some embodiments, R2 is hydrogen or halogen. 56 Attorney Docket No.: 071741.11025/5WO1 [0292] In some embodiments, R3 is -C(O)-NHR4or -SO2-NHR4, wherein R4 is -C0-12 alkylene- NHR6a, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-OR6a , or alkyl substituted with trialkylammonium; wherein and each of the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a, wherein R4a and R6a are defined as above. O O N+ O , ene- NHR6a, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-OR6a, or alkyl substituted with trialkylammonium; wherein each of the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a, wherein R4a and R6a are defined as above. [0295] In certain embodiments, R5 is alkyl substituted with trialkylammonium. [0296] In certain embodiments, when R5 is -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, or -C0-12 alkylene-C1-12 heteroaryl, the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 H , heteroaryl is substituted with one or more R4a, R4a is hydroxyl, methyl, oxo, or -C(O)-Me. [0298] In some embodiments, R3 is -NH-R7, wherein R7 is C1-12 heteroaryl, -C0-12 alkylene- N(R4)-SO2-R5, -C0-12 alkylene-P(=O)(R4) (alkoxy), or -C0-12 alkylene-N(R4)-C(=S)-R5; wherein R4 and R5 are defined as above. 57 Attorney Docket No.: 071741.11025/5WO1 OMe P [0299] In certain embodiments O . [0300] In some embodiments, t he compound of formula (II) is a compound of formula (II-E): R2 R3 N (II-E) , wherein x is 0 or 1; y is 0 or 1; X is S, O, or NH; and R1, R2, R3, n1, and n2 are defined as above in formula (II). [0301] In some embodiments, x is 0, y is 1, and X is S, O, or NH. [0302] In some embodiments, x is 0 or 1, y is 0, and X is NH. [0303] In some embodiments, each R1 is independently chosen from hydrogen, halogen, alkoxy, and alkyl. [0304] In certain embodiments, R1 is methyl, chlorine, or methoxy. [0305] In some embodiments, n2 is 1. [0306] In some embodiments, n2 is 2. [0307] In some embodiments, R2 is hydrogen. [0308] In some embodiments, R3 is -C(O)-NR4R4’, wherein each of R4 and R4’ is independently hydrogen or alkoxy. [0309] In some embodiments, R3 is -SO2-NR4R4’, wherein each of R4 and R4’ is independently hydrogen or alkyl. [0310] In some embodiments, R3 is -NH-C(O)-R5 or -NH-SO2-R5, wherein R5 is alkyl or -C0-12 alkylene-C2-12 heterocyclyl. 58 Attorney Docket No.: 071741.11025/5WO1 [0311] In certain embodiments, R5 is alkyl, such as methyl. O [0312] In certain embodiments, R5 is -C0-12 alkylene-C2-12 heterocyclyl, such a or O F): , , R2 is adjacent to R3, and R2 and R3, together with the carbon atoms that they are attached to, form a heterocycle optionally substituted with one or more R4a; and R1, R4a, and n1 are defined as above in formula (II). [0314] In some embodiments, R2 and R3, together with the carbon atoms that they are attached to, form a 5- or 6-membered heterocycle optionally substituted with one or more R4a. [0315] In some embodiments, the compound of formula (II-F) is a compound of formula (II-F- 1): M1 H ( - - ) , wherein M1 is a heterocycle optionally substituted with one or more R4a. [0316] In some embodiments, the compound of formula (II) is a compound of formula (II-G): 59 Attorney Docket No.: 071741.11025/5WO1 R2 r R3 H , wherein, each R2 is independently chosen from hydroxyl and alkoxy; R3 is chosen from hydroxy and alkoxy; and R1, n1 and r are defined as above in formula (II). [0317] In some embodiments, one R2 is adjacent to R3. [0318] In some embodiments, each R2 is independently chosen from hydroxyl and methoxy. [0319] In some embodiments, R3 is chosen from hydroxyl and methoxy. [0320] In some embodiments, the compound of formula (II-G) is a compound of formula (II-G- 1) of (II-G-2): R2 R2 R3 R3 NH , . [0321] In some embodiments, the compound of formula (II) is a compound of formula (II-H): R2 , l; e-COOH, -O-C0-12 alkylene-COOH, -C0-12 alkylene- P(O)(OH)2, -C(O)-NH-SO2-R5, -C(O)-NH-C0-12 alkylene-COOH, -NH-C0-12 alkylene-COOH, - 60 Attorney Docket No.: 071741.11025/5WO1 N N N alkylene N ; wherein the -C0-12 alkylene-COOH is optionally osen from amino and alkylamino; and R , R , and n1 are defined as above in formula (II). [0322] In some embodiments, M is phenyl. [0323] In some embodiments, M is pyridinyl. [0324] Exemplary compounds of formula (I-A), (I-B), (I-C), (I-D), or (II) include, but are not limited to, the compounds described herein, and any tautomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof. [0325] In particular embodiments, provided is a compound selected from the compounds descrined herein, or a tautomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof. [0326] All possible combinations of the above-indicated embodiments of compounds of formula (I-A), (I-B), (I-C), (I-D), or (II) and their tautomers, stereoisomers, pharmaceutically acceptable salts and solvates are considered to be embraced within the scope of the present disclosure. [0327] Exemplary compounds of formula (I-A), (I-B), (I-C), (I-D), or (II) include, but are not limited to, the following compounds, and any tautomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof: [0328] Table 1 No. Structure No. Structure No. Structure HN HN 61 Attorney Docket No.: 071741.11025/5WO1 NH O N H OH N NH NH H Attorney Docket No.: 071741.11025/5WO1 HN N HN O N O O NH2 N Attorney Docket No.: 071741.11025/5WO1 HN N N F O OH H 64 Attorney Docket No.: 071741.11025/5WO1 HN HN O N N 82 N N 83 O H H N 84 H H H 65 Attorney Docket No.: 071741.11025/5WO1 H HN N N HN N N O H H O 66 Attorney Docket No.: 071741.11025/5WO1 HN N N OH O O O 67 Attorney Docket No.: 071741.11025/5WO1 1 1 4 1 O O H O 68 Attorney Docket No.: 071741.11025/5WO1 HN O O O N HN N N H2N H O Attorney Docket No.: 071741.11025/5WO1 N O OH O 70 Attorney Docket No.: 071741.11025/5WO1 HN H N N O H 2 H 71 Attorney Docket No.: 071741.11025/5WO1 HN HN N N N NH 02 H Cl O 4 N O 403 H N O 501 S H S N H N 72 Attorney Docket No.: 071741.11025/5WO1 O N OH N H O OH H H 73 Attorney Docket No.: 071741.11025/5WO1 Cl B40 B41 B42 H F OH O H H [0329] Exemplary RT in formula (I-A), (I-B), (I-C), (I-D), or (II) include, but are not limited to, the following: [0330] Table 1a. Exemplary RT No. structure No. structure No. structure 74 Attorney Docket No.: 071741.11025/5WO1 H N H H 4 N N O 108 O O 203 NH O O O 75 Attorney Docket No.: 071741.11025/5WO1 O H H O N N N 20 O N 124 H 216 76 Attorney Docket No.: 071741.11025/5WO1 O H O O O H 33 N N 139 O S 229 N OH O O 77 Attorney Docket No.: 071741.11025/5WO1 H N O NH O S 55 153 242 O O NH OH 78 Attorney Docket No.: 071741.11025/5WO1 O H O N N N 66 O 164 N S N 254 H O 79 Attorney Docket No.: 071741.11025/5WO1 H N O 79 NH O 175 OH 265 80 Attorney Docket No.: 071741.11025/5WO1 NH H 90 186 N N 276 OH 81 Attorney Docket No.: 071741.11025/5WO1 H N O Cl 102 O N 197 OH 287 OH p [0331] Compounds described herein can be prepared by any number of processes as described generally below and more specifically illustrated by the exemplary compounds which follow in the Examples section herein. The compounds provided herein as prepared in the processes described below can be synthesized in the form of mixtures of stereoisomers (e.g., enantiomers, diastereomers), including racemic mixtures of enantiomers, which can be separated from one another using art-known resolution procedures, for instance including liquid chromatography using a chiral stationary phase. Additionally or alternatively, stereochemically pure isomeric forms of the compounds described herein can be derived from the corresponding stereochemically pure isomeric forms of the appropriate starting materials, intermediates, or reagents. For example, if a specific stereoisomer is desired, the compound can be synthesized by stereospecific methods of preparation, which typically employ stereochemically pure starting materials or intermediate compounds. [0332] Pharmaceutically acceptable salts of compounds described herein can be synthesized from the parent compound containing an acidic or basic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate acid or base in water or in an organic solvent, or in a mixture of the two. Examples of suitable organic solvents include, but are not limited to, ether, ethyl acetate (EtOAc), ethanol, isopropanol, or acetonitrile. [0333] By way of illustration, but not as a limitation, compounds of formula (I-A), (I-B), (I-C), (I-D), or (II) described herein can be prepared according to the following general preparation procedures shown in Scheme 1 as well as the examples shown in the present disclosure. One of 82 Attorney Docket No.: 071741.11025/5WO1 ordinary skill in the art will recognize that, to obtain various compounds of formula (I-A), (I-B), (I- C), (I-D), or (II) as described herein, starting materials can be suitably selected so that the ultimately desired substituent groups will be carried through (i.e., be stable over the course of the synthesis) the reaction scheme with or without protection as appropriate to yield the desired product. Alternatively, it may be necessary or desirable to employ, in place of the ultimately desired substituent, a suitable group that may be carried through (i.e., be stable over the course of the synthesis) the reaction scheme and replaced as appropriate with the desired substituent. [0334] If no temperature or temperature range is stated, it is to be understood that the reaction is to be conducted at room temperature. [0335] When isomerically pure samples are desired, isomeric mixtures of compounds synthesized according to Scheme 1 and Scheme 2 can be separated by chiral supercritical fluid chromatography (SFC) or high performance liquid chromatography (HPLC). Scheme 1: O O O O O 2 HN R2 O OH HN R OH OH T l P i i TFA N N H Compositions [0336] In one aspect, provided is a pharmaceutical composition comprising a compound of formula (I-A), (I-B), (I-C), (I-D), or (II) or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, as described herein. [0337] Compositions can also comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is non-toxic and should not interfere with the efficacy of the active ingredient. Pharmaceutically acceptable carriers can include one or more excipients such as 83 Attorney Docket No.: 071741.11025/5WO1 binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings. The precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, intradermal, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes. For liquid injectable preparations, for example, suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like. For solid oral preparations, for example, powders, capsules, caplets, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. For nasal sprays/inhalant mixtures, the aqueous solution/suspension can comprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives. [0338] Compositions can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections. The parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection. Compositions can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal. [0339] The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen depend upon the condition to be treated, such as the severity of the illness, the age, weight, and sex of the patient. Pharmaceutical compositions can be formulated for different modes of administration such as for topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, or subcutaneous administration. [0340] In yet another aspect, provided is a method of preparing a pharmaceutical composition comprising combining a compound of formula (I-A), (I-B), (I-C), (I-D), or (II), or a stereoisomer, tautomer, pharmaceutically acceptable salt or solvate thereof, with at least one pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by any method known in the art in view of the present disclosure, and one of ordinary skill in the art will be familiar with such techniques used to prepare pharmaceutical compositions. For example, a pharmaceutical composition according to the present disclosure can be prepared by mixing a compound of formula (I-A), (I-B), (I-C), (I-D), or (II), with one or more pharmaceutically acceptable carriers according 84 Attorney Docket No.: 071741.11025/5WO1 to conventional pharmaceutical compounding techniques, including but not limited to, conventional admixing, dissolving, granulating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical Combinations [0341] In one general aspect, the present disclosure relates to a pharmaceutical combination comprising a first therapeutical agent and a second therapeutical agent, wherein the first therapeutical agent is a peripherally selective α2AR agonist, such as the peripherally selective α2AR agonist described herein. [0342] In some embodiments, the pharmaceutical combination further comprises a third therapeutic agent. [0343] In some embodiments, the peripherally selective α2AR agonist comprises an α2AR activation moiety covalently linked to a peripheral distribution moiety. [0344] In certain embodiments, the α2AR activation moiety has formula of m , wherein Y, A, B, R2, R3, m, and n are defined as in formula [0345] In certain embodiments, the α2AR activation moiety has formula of Y m , wherein Y, A, B, R2, R3, m, and n are defined as in formula [0346] In certain embodiments, the α2AR activation moiety has formula of Y , wherein Y, A, B, R2, R3, m, and n are defined as in formula N [0347] In certain embodiments, the α2AR activation moiety has formu , wherein X1 and Y1 are defined as in formula (I-D). 85 Attorney Docket No.: 071741.11025/5WO1 [0348] In certain embodiments, the α2AR activation moiety has formula A B , wherein A and B are defined as in formula (II). RT [0349] In certain embodiments, the peripheral distribution moiety has formula of , wherein RT is defined as in formula (I-A). RT [0350] In certain embodiments, the peripheral distribution moiety has formula of , wherein RT is defined as in formula (II). [0351] In some embodiments, the peripherally selective α2AR agonist is a compound of formula (I-A), (I-B), (I-C), (I-D) or (II). [0352] In certain embodiments, the peripherally selective α2AR agonist is a compound of formula (II-A), (II-B), (II-C), (II-D), (II-E), (II-F), (II-G), or (II-H). [0353] In some embodiments, the second therapeutic agent is any therapeutic agent other than a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist. [0354] In some embodiments, the third therapeutic agent is any therapeutic agent other than a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist, and the third therapeutic agent is different from the second therapeutic agent. [0355] In some embodiments, the second therapeutic agent is chosen from opioids, acetaminophen (paracetamol), local analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), antiepileptic medication, antidepressant medication, topical analgesic agents, NMDA receptor antagonists, neurotoxins, cannabinoids, voltage-gated sodium channel inhibitors, adenosine agonists, transient receptor potential (TRP) channel modulators, NGF inhibitors, purinergic receptor antagonists, adenosinergic pathway modulators, sigma-1 receptor antagonists, KCC2 enhancers, glutamate transport enhancers, TrkA receptor antagonists, somatostatin receptor ligands, Human Adenylyl Cyclase Associated Protein 1 (CAP1) modulators, and angiotensin II receptor antagonists, corticosteroids, and any combinations thereof. [0356] In certain embodiments, the opioid is chosen from meperidine, tramadol, codeine, pentazocine, dihydrocodeine, tapentadol, hydrocodone, morphine, oxycodone, methadone, 86 Attorney Docket No.: 071741.11025/5WO1 oxymorphone, hydromorphone, butorphanol, buprenorphine, fentanyl, sufentanil, remifentanil, alfentanil, thebaine, diacetyl morphine, vicodin, dinorbuprenorphine, nalbuphine, and heroin. [0357] In certain embodiments, the opioid is chosen from anrikefon, cebranopadol, STC-007, MR-309, desmetramadol, LPM-3480392, YZJ-4729, RFUS-144, HY-1608, NES-100, PN-6047, Cyt-1010, TRV-250, DMX-101, ADV-502, MEB-1170, TRV-734, KUR-101, 18F-FTC-146, MUM0-1, RFUS-250, NH-16003, and sunobinop. [0358] In certain embodiments, the opioid is a biased opioid ligand, such as oliceridine or SHR8554. [0359] In certain embodiments, the opioid is kappa-opioid agonists, such as CR845 (Difelikefalin). [0360] In certain embodiments, the nonsteroidal anti-inflammatory drug (NSAID) is chosen from aspirin, diflunisal, ibuprofen, naproxen, indomethacin, diclofenac, meclofenamate, mefenamic acid, meloxicam, piroxicam, nabumetone, celecoxib, etoricoxib, biphenyl acetic acid, aceclofenac, ketorolac, etodolac, ketotifen, loxoprofen, tenoxicam, zaltoprofen, zaltoprofen, esflurbiprofen, valdecoxib, lumiracoxib, imidazole salicylate, dexketoprofen, nimesulide, otenaproxesul, 3D-1001, AB-001, and JLP-2004. [0361] In certain embodiments, the antiepileptic medication is a calcium channel blockers (CCB), sodium channel blocker, or GABA modulator. [0362] In certain embodiments, the antiepileptic medication is chosen from gabapentin, pregabalin, enacarbil, mirogabalin, crisugabalin, phenibut, baclofen, 4-fluorophenibut, 4- methylpregabalin, atagabalin (PD-200,390), imagabalin, PD-217,014, tolibut, oxcarbazepine, ziconotide, lomerizine, carbamazepine, oxcarbazepine, lamotrigine, lidocaine, ropivacaine, bupivacaine, valproic acid, topiramate, and divalproex sodium. [0363] In certain embodiments, the antiepileptic medication is non-α2δ antiepileptics, such as carbamazepine or oxcarbazepine. [0364] In certain embodiments, the antiepileptic medication is chosen from pregabalin naproxencarbil, XG-004, RAP-219, AFA-281, priralfinamide, laflunimus, and CPP-115. [0365] In certain embodiments, the antidepressant medication is a selective serotonin and norepinephrine reuptake inhibitor (SNRI), a tricyclic antidepressant (TCA), or a monoamine oxidases inhibitor (MAOI). 87 Attorney Docket No.: 071741.11025/5WO1 [0366] In further certain embodiments, the selective serotonin and norepinephrine reuptake inhibitor (SNRI) is duloxetine, venlafaxine, desvenlafaxine, bupropion, or milnacipran. [0367] In further certain embodiments, the tricyclic antidepressant (TCA) is amitriptyline, nortriptyline, desipramine, clomipramine, or imipramine. [0368] In certain embodiments, the topical analgesic agent is lidocaine (e.g., 5%) patch or capsaicin (e.g., 8%) patch. [0369] In certain embodiments, the NMDA antagonist is chosen from ketamine, methadone, memantine, amantadine, dextromethorphan, and L-4-chlorokynurenine. [0370] In certain embodiments, the neurotoxin is Botulinum toxin A. [0371] In certain embodiments, the cannabinoid is chosen from nabilone, THC, cannabidiol (CBD), nabiximols (Sativex), AP-707, ONO-1110, CMX-020, CNTX-6016, and OCT-461201. [0372] In certain embodiments, the voltage-gated sodium channel inhibitor is the inhibitor of NaV1.3, NaV1.7, NaV1.8 and / or NaV1.9. [0373] In further certain embodiments, the selective NaV1.8 inhibitor is chosen from suzetrigine (VX-548), ANP-230, VX-150, JMK-000623, HBW-004285, DSP-2230, VX-993, VX-973, LTG- 001, kindolor, FZ-008, HRS-4800, HRS-2129, MK-5661, and STC-004. [0374] In further certain embodiments, the selective NaV1.7 inhibitor is chosen from tetrodotoxin, OLP-1002, aneratrigine, CC-8464, DSP-3905, S-151128, and ST-2427. [0375] In certain embodiments, the voltage-gated sodium channel inhibitor is relutrigine or ANP- 230. [0376] In certain embodiments, the transient receptor potential (TRP) channel blocker is TRPV1 agonist, TRPV1 antagonist, TRPA1 inhibitor, TRPM3 inhibitor, TRPM8 inhibitor, and TRPC5 antagonist. [0377] In cerain embodiments, the transient receptor potential (TRP) channel blocker is chosen from capsaicin, vocacapsaicin, resiniferatoxin (RTX), ACD-440, libvatrep, tivanisiran, JNJ- 39439335, SB-705498, AJH-2947, SER-014, SRP-001, XEN-D0501, QP-5113, LD-04185, LD- 2020, BHV-2100, Elismetrep, MT-8554, RQ-00434739, and QR-060127. [0378] In certain embodiments, the NGF inhibitor is chosen from Tanezumab, Fulranumab, Fasinumab, EP-9001A, MEDI-7352, SSS-40, DS-002, luvagrobart, TNM-009, and STSA-1001. 88 Attorney Docket No.: 071741.11025/5WO1 [0379] In certain embodiments, the purinergic receptor antagonist is P2X purinoreceptor antagonist, such as NC-2600, or LY-3857210, P2X3 antagonists, such as Gefapixant (Merck), Sivopixant (Shionogi), or VNUT Inhibitor such as Clodronate. [0380] In certain embodiments, the adenosinergic pathway modulator is a modulator for Adenosine A1, A2A, A3 receptors; CD39/73 enzymes; or Adenosine kinase. [0381] In certain embodiments, the adenosinergic pathway modulator is caffeine. [0382] In certain embodiments, the sigma-1 receptor antagonist is ketamine, memantine, or amantadine [0383] In certain embodiments, the KCC2 enhancer is CLP257 or kenpaullone. [0384] In certain embodiments, the glutamate transport enhancer is ceftriaxone. [0385] In certain embodiments, the TrkA receptor antagonist is AK-1830 or BR-01T. [0386] In certain embodiments, the somatostatin receptor ligand is pasireotide, LY-3556050, or FZ002-037. [0387] In certain embodiments, the Human Adenylyl Cyclase Associated Protein 1 (CAP1) modulator is FM-888 or NB-001. [0388] In certain embodiments, the Angiotensin II receptor antagonist is CFTX-1554, TRD-205, or WXSH-0024. [0389] In certain embodiments, the corticosteroid is chosen from dexamethasone, clobetasol, triamcinolone acetonide, difluprednate, loteprednol, and fluticasone. [0390] In some embodiments, the second therapeutic agent is chosen from GGG tri-agonist (e.g., retatrutide), metabotropic glutamate receptors (mGluR) modulator (e.g., basimglurant), TGF beta-1 Inhibitor (e.g., Vicatertide (SB-01)), gonadotropin-releasing hormone receptor（GnRH) antagonist (e.g., linzagolix), 11β-HSD1 inhibitor (e.g., clofutriben), 17-HSD-1 inhibitor (e.g., OG-6219), Dual ENKephalinase Inhibitor (DENKI) (e.g., PL37), CCR2 receptor antagonist (e.g., CNTX-6970), IL- 10 receptor agonist (e.g., XT-150), nAChR dual alpha 4/beta 2 subunit stimulator (e.g., ATA-104), allosteric NEK7/NLRP3 inflammasome inhibitor (e.g., HT-6184), NLRP3 inflammasome inhibitor (e.g., dapansutrile), PACAP-38 inhibitor (e.g., Lu-AG09222), membrane-associated prostaglandin E synthase-1 (mPGES-1) inhibitor (e.g., NS-580), prolactin receptor antagonist (e.g., HMI-115), Protease-Activated Receptor 2 (PAR2) Antagonist (e.g., MEDI-0618), NF-kB decoy oligonucleotide, NFkB/Nrf2 modulator (e.g., AKL-4), Proepiregulin modulator (e.g., LY- 3848575), Sphingomyelin synthase 1 (SMS1) agonist (e.g., sodium idroxioleate), Lyn inhibitor 89 Attorney Docket No.: 071741.11025/5WO1 (e.g., nispomeben), guanylate cyclase-c (GC-C) agonist (e.g., IW-3300), neurotrophin-3 (NT-3) inhibitor (e.g., LEVI-04), prostaglandin receptor, EP4 antagonist (e.g., grapiprant), agonist of the ChemR23 G-protein coupled receptor (e.g., urcosimod), inhibitor of the influenza neuraminidase enzyme (e.g., peramivir), inhibitor of AP2 associated kinase 1 (AAK1) (e.g., pilavapadin), Adenosine A3 receptor antagonist (e.g., NTM-006), Antagonist of platelet activating factor (PAF) receptor (e.g., piperidone hydrochloridum), 15-lipoxygenase inhibitor (e.g., utreloxastat), Flt3 inhibitor (e.g., BDT-272), GPCR84 inhibitor (e.g., BAY-3178275), Kindolor, Trk receptor modulator (e.g., CG-001054), selective FABP5 inhibitor (e.g., ART-26.12), erythropoietin receptor agonist (e.g., cibinetide), Dopamine reuptake inhibitor (e.g., IPTN-2021), AEAr agonist (e.g., SBS-1000), Epoxide Hydrolase (sEH) inhibitor (e.g., EC-5026), SSAO/VAP-1 inhibitor (e.g., ECC-0509), sphingosine 1 phosphate receptor 1 modulator (e.g., TRV-045), and antagonist of the lysophosphatidic acid 1 receptor (LPA1) (e.g., PIPE-791). [0391] In some embodiments, the second therapeutic agent is chosen from IRX-101, MR-107A- 02, 3-VM-1001, lysergide assisted therapy, SIL-1002, TRN-261, HR-1405-01, HRF-2105, TTAX- 03, KP-910, LYT-503, LL-50, ZeP-3, YR-1702, YZJ-1495, FB-1003, MK-4318, PZH-2108, HEC- 137076MsOH, VVZ-2471, SYNP-101, Pudafensine, BIOS-0618, and HSK-36357. Methods of Use [0392] In one general aspect, provided are methods of treating or preventing pain in a subject in need thereof, the method comprising administering to the subject an effective amount of a peripherally selective α2AR agonist, wherein treating with the peripherally selective α2AR agonist causes less side effects than treating with a non-peripherally selective α2AR agonist, such as at similar or comparable dosage. [0393] In some embodiments, the peripherally selective α2AR agonist comprises an α2AR activation moiety covalently linked to a peripheral distribution moiety. [0394] In another general aspect, provided are methods of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a peripherally selective α2AR agonist, wherein the peripherally selective α2AR agonist comprises an α2AR activation moiety covalently linked to a peripheral distribution moiety. [0395] In another general aspect, the present disclosure relates to a method of treating or preventing a disease in a subject in need thereof, the method comprising administering to the 90 Attorney Docket No.: 071741.11025/5WO1 subject an effective amount of the pharmaceutical combination described herein, or administering to the subject (i) an effective amount of a first therapeutic agent, and (ii) an effective amount of a second therapeutic agent, wherein the first therapeutic agent is a peripherally selective α2AR agonist, such as the peripherally selective α2AR agonist described herein. [0396] As used herein, the term “effective amount” regarding a pharmaceutical combination includes the effective amount of the first therapeutical agent and the effective amount of the second therapeutical agent, and these two effective amounts can be identical or different, fixed or varying dependent on upon a variety of factors as described above. [0397] In some embodiments, the disease is chosen from glaucoma, analgesia, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, menopausal hot flashes, dysmenorrhea, presbyopia, acute kidney injury, insomnia, inflammatory disease, and cancer. [0398] In certain embodiments, the disease is pain. [0399] In some embodiments, the second therapeutic agent is any therapeutic agent other than a peripherally selective α2AR agonist. [0400] In another general aspect, the present disclosure relates to a method for treating or preventing pain in a subject in need thereof, the method comprising administering to the subject an effective amount of a first analgesic agent, and an effective amount of a second analgesic agent, wherein the first analgesic agent is a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist, such as the peripherally selective α2AR agonist described herein. [0401] In some embodiment, the method for treating or preventing pain further comprising administering to the subject an effective amount of a third analgesic agent. [0402] In another general aspect, the present disclosure relates to a method for treating or preventing pain in a subject in need thereof, the method comprising administering to the subject an effective amount of a codrug that comprises a first analgesic agent and a second analgesic agent, wherein the first analgesic agent is a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist, such as the peripherally selective α2AR agonist described herein. [0403] As used herein, the term “codrug” or “mutual prodrug” refers to a single chemical entity composed of two pharmacologically active drug moieties. After administration, the body (through enzymatic cleavage or hydrolysis) breaks the linkage, releasing the two active drugs at the site of action or in systemic circulation. 91 Attorney Docket No.: 071741.11025/5WO1 [0404] The following embodiments apply to the above all general aspects of methods of use involving a second therapeutic agent or a second analgestic agent. [0405] In some embodiments, the peripherally selective α2AR agonist comprises an α2AR activation moiety covalently linked to a peripheral distribution moiety. [0406] In some embodiments, the pain is neuropathic pain, nociceptive pain, nociplastic pain, or mixed pain. [0407] In certain embodiments, the neuropathic pain is chosen from diabetic peripheral neuropathy, postherpetic neuralgia, chemotherapy-induced peripheral neuropathy, Fabry disease– associated pain, trigeminal neuralgia, glossopharyngeal neuralgia, occipital neuralgia, HIV- associated neuropathy, hereditary sensory and autonomic neuropathy, Charcot-Marie-Tooth disease–related pain, small fiber neuropathy, brachial plexus avulsion, phantom limb pain, post- surgical neuropathic pain, carpal tunnel syndrome, other compression or entrapment neuropathies, amyloid neuropathy, vasculitic neuropathy, leprosy-associated pain, radiation-induced neuropathy, drug-induced neuropathy, metabolic neuropathies, endocrine-related neuropathies, toxic neuropathies, immune-mediated neuropathies, traumatic peripheral nerve injury, and heritable neuropathies, as well as post-stroke pain syndrome, spinal cord injury–related pain, multiple sclerosis–associated pain, thalamic pain syndrome, brain tumor–associated central pain, post–brain surgery neuropathic pain, central post-traumatic pain, cerebral palsy–related central pain, Parkinson’s disease–associated central pain, syringomyelia-related pain, and any combinations thereof. [0408] In certain embodiments, the nociceptive pain is somatic (i.e., from skin, muscles, or joints (e.g., cuts, fractures)) or visceral (i.e., from internal organs (e.g., appendicitis)). [0409] In certain embodiments, the nociceptive pain is osteoarthritis-related rheumatoid pain, arthritis-associated post-traumatic pain, musculoskeletal pain, postoperative pain, tendonitis, myofascial pain, visceral inflammatory pain (e.g., appendicitis, cystitis, pancreatitis), dysmenorrhea, or inflammatory bowel disease-related pain. [0410] In certain embodiments, the mixed pain is cancer-related pain (e.g., bone metastasis pain, tumor infiltration pain, perineural invasion), complex regional pain syndrome type I, failed back surgery syndrome, low back pain with radiculopathy endometriosis-associated pelvic pain, chronic post-surgical pain, chronic pancreatitis-associated pain, interstitial cystitis/bladder pain syndrome, 92 Attorney Docket No.: 071741.11025/5WO1 fibromyalgia, temporomandibular disorder, vulvodynia, irritable bowel syndrome with visceral pain, and combinations thereof. [0411] In some embodiments, the pain is acute pain or chronic pain. As used herein, the term “chronic pain” refers to pain that is persistent or recurrent pain lasting longer than 3 months. [0412] In certain embodiments, the chronic pain is chronic primary pain, chronic cancer pain, chronic postsurgical and posttraumatic pain, chronic neuropathic pain, chronic headache and orofacial pain, chronic visceral pain, chronic musculoskeletal pain. [0413] In some embodiments, the pain is cancer pain. [0414] In some embodiments, the pain is post-surgery pain. [0415] In some embodiments, the second therapeutic agent or the second analgesic agent is any therapeutic agent or analgesic agent other than a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist. [0416] In some embodiments, the second analgesic agent is chosen from opioids, acetaminophen (paracetamol), local analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), antiepileptic medication, antidepressant medication, topical analgesic agents, NMDA receptor antagonists, neurotoxins, cannabinoids, voltage-gated sodium channel inhibitors, adenosine agonists, transient receptor potential (TRP) channel modulators, NGF inhibitors, purinergic receptor antagonists, adenosinergic pathway modulators, sigma-1 receptor antagonists, KCC2 enhancers, glutamate transport enhancers, TrkA receptor antagonists, somatostatin receptor ligands, Human Adenylyl Cyclase Associated Protein 1 (CAP1) modulators, and angiotensin II receptor antagonists, corticosteroids, and any combinations thereof. [0417] In certain embodiments, the opioid is chosen from meperidine, tramadol, codeine, pentazocine, dihydrocodeine, tapentadol, hydrocodone, morphine, oxycodone, methadone, oxymorphone, hydromorphone, butorphanol, buprenorphine, fentanyl, sufentanil, remifentanil, alfentanil, thebaine, diacetyl morphine, vicodin, dinorbuprenorphine, nalbuphine, and heroin. [0418] In certain embodiments, the opioid is chosen from anrikefon, cebranopadol, STC-007, MR-309, desmetramadol, LPM-3480392, YZJ-4729, RFUS-144, HY-1608, NES-100, PN-6047, Cyt-1010, TRV-250, DMX-101, ADV-502, MEB-1170, TRV-734, KUR-101, 18F-FTC-146, MUM0-1, RFUS-250, NH-16003, and sunobinop. [0419] In certain embodiments, the opioid is a biased opioid ligand, such as oliceridine or SHR8554. 93 Attorney Docket No.: 071741.11025/5WO1 [0420] In certain embodiments, the opioid is kappa-opioid agonists, such as CR845 (Difelikefalin). [0421] In certain embodiments, the nonsteroidal anti-inflammatory drug (NSAID) is chosen from aspirin, diflunisal, ibuprofen, naproxen, indomethacin, diclofenac, meclofenamate, mefenamic acid, meloxicam, piroxicam, nabumetone, celecoxib, etoricoxib, biphenyl acetic acid, aceclofenac, ketorolac, etodolac, ketotifen, loxoprofen, tenoxicam, zaltoprofen, zaltoprofen, esflurbiprofen, valdecoxib, lumiracoxib, imidazole salicylate, dexketoprofen, nimesulide, otenaproxesul, 3D-1001, AB-001, and JLP-2004. [0422] In certain embodiments, the antiepileptic medication is a calcium channel blockers (CCB), sodium channel blocker, or GABA modulator. [0423] In certain embodiments, the antiepileptic medication is chosen from gabapentin, pregabalin, enacarbil, mirogabalin, crisugabalin, phenibut, baclofen, 4-fluorophenibut, 4- methylpregabalin, atagabalin (PD-200,390), imagabalin, PD-217,014, tolibut, oxcarbazepine, ziconotide, lomerizine, carbamazepine, oxcarbazepine, lamotrigine, lidocaine, ropivacaine, bupivacaine, valproic acid, topiramate, and divalproex sodium. [0424] In certain embodiments, the antiepileptic medication is non-α2δ antiepileptics, such as carbamazepine or oxcarbazepine. [0425] In certain embodiments, the antiepileptic medication is chosen from pregabalin naproxencarbil, XG-004, RAP-219, AFA-281, priralfinamide, laflunimus, and CPP-115. [0426] In certain embodiments, the antidepressant medication is a selective serotonin and norepinephrine reuptake inhibitor (SNRI), a tricyclic antidepressant (TCA), or a monoamine oxidases inhibitor (MAOI). [0427] In further certain embodiments, the selective serotonin and norepinephrine reuptake inhibitor (SNRI) is duloxetine, venlafaxine, desvenlafaxine, bupropion, or milnacipran. [0428] In further certain embodiments, the tricyclic antidepressant (TCA) is amitriptyline, nortriptyline, desipramine, clomipramine, or imipramine. [0429] In certain embodiments, the topical analgesic agent is lidocaine (e.g., 5%) patch or capsaicin (e.g., 8%) patch. [0430] In certain embodiments, the NMDA antagonist is chosen from ketamine, methadone, memantine, amantadine, dextromethorphan, and L-4-chlorokynurenine. [0431] In certain embodiments, the neurotoxin is Botulinum toxin A. 94 Attorney Docket No.: 071741.11025/5WO1 [0432] In certain embodiments, the cannabinoid is chosen from nabilone, THC, cannabidiol (CBD), nabiximols (Sativex), AP-707, ONO-1110, CMX-020, CNTX-6016, and OCT-461201. [0433] In certain embodiments, the voltage-gated sodium channel inhibitor is the inhibitor of NaV1.3, NaV1.7, NaV1.8 and / or NaV1.9. [0434] In further certain embodiments, the selective NaV1.8 inhibitor is chosen from suzetrigine (VX-548), ANP-230, VX-150, JMK-000623, HBW-004285, DSP-2230, VX-993, VX-973, LTG- 001, kindolor, FZ-008, HRS-4800, HRS-2129, MK-5661, and STC-004. [0435] In further certain embodiments, the selective NaV1.7 inhibitor is chosen from tetrodotoxin, OLP-1002, aneratrigine, CC-8464, DSP-3905, S-151128, and ST-2427. [0436] In certain embodiments, the voltage-gated sodium channel inhibitor is relutrigine or ANP- 230. [0437] In certain embodiments, the transient receptor potential (TRP) channel blocker is TRPV1 agonist, TRPV1 antagonist, TRPA1 inhibitor, TRPM3 inhibitor, TRPM8 inhibitor, and TRPC5 antagonist. [0438] In cerain embodiments, the transient receptor potential (TRP) channel blocker is chosen from capsaicin, vocacapsaicin, resiniferatoxin (RTX), ACD-440, libvatrep, tivanisiran, JNJ- 39439335, SB-705498, AJH-2947, SER-014, SRP-001, XEN-D0501, QP-5113, LD-04185, LD- 2020, BHV-2100, Elismetrep, MT-8554, RQ-00434739, and QR-060127. [0439] In certain embodiments, the NGF inhibitor is chosen from Tanezumab, Fulranumab, Fasinumab, EP-9001A, MEDI-7352, SSS-40, DS-002, luvagrobart, TNM-009, and STSA-1001. [0440] In certain embodiments, the purinergic receptor antagonist is P2X purinoreceptor antagonist, such as NC-2600, or LY-3857210, P2X3 antagonists, such as Gefapixant (Merck), Sivopixant (Shionogi), or VNUT Inhibitor such as Clodronate. [0441] In certain embodiments, the adenosinergic pathway modulator is a modulator for Adenosine A1, A2A, A3 receptors; CD39/73 enzymes; or Adenosine kinase. [0442] In certain embodiments, the adenosinergic pathway modulator is caffeine. [0443] In certain embodiments, the sigma-1 receptor antagonist is ketamine, memantine, or amantadine [0444] In certain embodiments, the KCC2 enhancer is CLP257 or kenpaullone. [0445] In certain embodiments, the glutamate transport enhancer is ceftriaxone. [0446] In certain embodiments, the TrkA receptor antagonist is AK-1830 or BR-01T. 95 Attorney Docket No.: 071741.11025/5WO1 [0447] In certain embodiments, the somatostatin receptor ligand is pasireotide, LY-3556050, or FZ002-037. [0448] In certain embodiments, the Human Adenylyl Cyclase Associated Protein 1 (CAP1) modulator is FM-888 or NB-001. [0449] In certain embodiments, the Angiotensin II receptor antagonist is CFTX-1554, TRD-205, or WXSH-0024. [0450] In certain embodiments, the corticosteroid is chosen from dexamethasone, clobetasol, triamcinolone acetonide, difluprednate, loteprednol, and fluticasone. [0451] In some embodiments, the second analgesic agent is chosen from GGG tri-agonist (e.g., retatrutide), metabotropic glutamate receptors (mGluR) modulator (e.g., basimglurant), TGF beta-1 Inhibitor (e.g., Vicatertide (SB-01)), gonadotropin-releasing hormone receptor（GnRH) antagonist (e.g., linzagolix), 11β-HSD1 inhibitor (e.g., clofutriben), 17-HSD-1 inhibitor (e.g., OG-6219), Dual ENKephalinase Inhibitor (DENKI) (e.g., PL37), CCR2 receptor antagonist (e.g., CNTX-6970), IL- 10 receptor agonist (e.g., XT-150), nAChR dual alpha 4/beta 2 subunit stimulator (e.g., ATA-104), allosteric NEK7/NLRP3 inflammasome inhibitor (e.g., HT-6184), NLRP3 inflammasome inhibitor (e.g., dapansutrile), PACAP-38 inhibitor (e.g., Lu-AG09222), membrane-associated prostaglandin E synthase-1 (mPGES-1) inhibitor (e.g., NS-580), prolactin receptor antagonist (e.g., HMI-115), Protease-Activated Receptor 2 (PAR2) Antagonist (e.g., MEDI-0618), NF-kB decoy oligonucleotide, NFkB/Nrf2 modulator (e.g., AKL-4), Proepiregulin modulator (e.g., LY- 3848575), Sphingomyelin synthase 1 (SMS1) agonist (e.g., sodium idroxioleate), Lyn inhibitor (e.g., nispomeben), guanylate cyclase-c (GC-C) agonist (e.g., IW-3300), neurotrophin-3 (NT-3) inhibitor (e.g., LEVI-04), prostaglandin receptor, EP4 antagonist (e.g., grapiprant), agonist of the ChemR23 G-protein coupled receptor (e.g., urcosimod), inhibitor of the influenza neuraminidase enzyme (e.g., peramivir), inhibitor of AP2 associated kinase 1 (AAK1) (e.g., pilavapadin), Adenosine A3 receptor antagonist (e.g., NTM-006), Antagonist of platelet activating factor (PAF) receptor (e.g., piperidone hydrochloridum), 15-lipoxygenase inhibitor (e.g., utreloxastat), Flt3 inhibitor (e.g., BDT-272), GPCR84 inhibitor (e.g., BAY-3178275), Kindolor, Trk receptor modulator (e.g., CG-001054), selective FABP5 inhibitor (e.g., ART-26.12), erythropoietin receptor agonist (e.g., cibinetide), Dopamine reuptake inhibitor (e.g., IPTN-2021), AEAr agonist (e.g., SBS-1000), Epoxide Hydrolase (sEH) inhibitor (e.g., EC-5026), SSAO/VAP-1 inhibitor (e.g., 96 Attorney Docket No.: 071741.11025/5WO1 ECC-0509), sphingosine 1 phosphate receptor 1 modulator (e.g., TRV-045), and antagonist of the lysophosphatidic acid 1 receptor (LPA1) (e.g., PIPE-791). [0452] In some embodiments, the second analgesic agent is chosen from IRX-101, MR-107A-02, 3-VM-1001, lysergide assisted therapy, SIL-1002, TRN-261, HR-1405-01, HRF-2105, TTAX-03, KP-910, LYT-503, LL-50, ZeP-3, YR-1702, YZJ-1495, FB-1003, MK-4318, PZH-2108, HEC- 137076MsOH, VVZ-2471, SYNP-101, Pudafensine, BIOS-0618, and HSK-36357. [0453] In some embodiments, the administration of the first therapeutic agent and the second therapeutic agent provides a synergistic effect in treating the disease. [0454] In some embodiments, the administration of the first analgestic agent and the second analgestic agent provides a synergistic effect in treating the pain. [0455] As used herein, the term "synergistic effect" refers to a pharmacological interaction between two or more agents wherein the combined effect is greater than the sum of their individual effects. In the context of analgesic or therapeutic efficacy, synergy indicates that the combination achieves enhanced results at equal or lower doses compared to each compound used separately. [0456] In certain embodiments, the synergistic effect is potentiation. As used herein, the term "potentiation" refers to a specific type of synergy in which one agent, often inactive or weakly active alone, significantly enhances the effect of another active compound when used in combination. [0457] In some embodiemnts, the administration of the first therapeutic agent and the second therapeutic agent provides an additive effect in treating the disease. [0458] In some embodiments, the administration of the first analgestic agent and the second analgestic agent provides an additive effect in treating the pain. [0459] As used herein, "additive effect" refers to a combined pharmacological effect that is equal to the sum of the effects of individual agents, without enhancement or suppression. Additivity is typically defined within an acceptable experimental range (e.g., CI between 0.95 and 1.1) that accounts for biological variability. [0460] The synergistic affect and additive effect in combination therapy can be measured and/or determined by any methods known in the art. For example, combination index (CI) is a quantitative metric used to assess the nature of drug-drug interactions, and CI values can be calculated based on models such as Bliss independence or Loewe additivity. The CI values are typically derived from 97 Attorney Docket No.: 071741.11025/5WO1 experimental data comparing the observed combination effect (EAB) to the expected effect based on individual drug activities (EA and EB), using equations such as: CI = (EA + EB × (1 – EA)) / EAB (Bliss model). [0461] In general: CI < 1 indicates synergistic interaction; CI = 1 (or within 0.95–1.1) indicates an additive effect; CI > 1 indicates antagonistic interaction. [0462] The drug-drug interactions in combination therapy can also be characterized by terms other than synergistic affect or additive effect. For example, the term “sparing effect” is the ability of one drug in a combination to reduce the required dose of another drug while maintaining the same therapeutic effect. According to embodiments of the present disclosure, the sparing effect is the ability of a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist in a combination to reduce the required dose of another therapeutic agent (such as a analgesic agent) while maintaining the same therapeutic effect. [0463] In some embodiments, the first therapeutic agent has a sparing effect on the second threapeutic agent. [0464] In some embodiments, the first analgestic agent has a sparing effect on the second analgesic agent. [0465] Many second therapeutic agents and/or analgestic agents have dose-dependent side effects, which can be severe. For example, some typical dose-dependent side effects of second analgesic agents include, addiction, sedation and drowsiness, respiratory depression, constipation, nausea and vomiting, hypotension, dizziness, somnolence (sleepiness), peripheral edema (swelling), weight gain, blurred vision, difficulty with concentration/attention, dry mouth, and fatigue. Such side effects are often dose-limiting side effects, and sometimes even life-threating. [0466] The present disclosure provides a combination with a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist allows the dose of second therapeutic agent/analgesic agent to be reduced dramatically, e.g., by 66.67%, while maintaining the same therapeutic effect, and thereby mnimizes adverse events such as those dose-dependent side effects listed above. 98 Attorney Docket No.: 071741.11025/5WO1 [0467] The dose reduction of second therapeutic agent/analgesic agents associated with the combination with a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist leads to additional advantages, including a reduced incidence of dose-dependent adverse effects such as those listed above, further enhancing the therapeutic profile in a manner unforeseen by the prior art. [0468] The reduction in dosage of second therapeutic agent/analgesic agent achieved by the combination with a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist confers tangible clinical benefits, including improved patient tolerability, reduced treatment discontinuation rates, and potential for broader patient applicability. [0469] In some embodiments, the first therapeutic agent and the second therapeutic agent can be administrered by way of simultaneous, sequential or separate administration. [0470] The following embodiments apply to the above all general aspects of methods of use. [0471] In some embodiments, the α2AR activation moiety has formula of ula ula [0473] In some embodiments, the α2AR activation moiety has formula of Y , wherein Y, A, B, R2, R3, m, and n are defined as in formula N [0474] In some embodiments, the α2AR activation moiety has formu , wherein X1 and Y1 are defined as in formula (I-D). 99 Attorney Docket No.: 071741.11025/5WO1 [0475] In some embodiments, the α2AR activation moiety has formu A B , wherein A and B are defined as in formula (II). RT [0476] In some embodiments, the peripheral distribution moiety has formula of , wherein RT is defined as in formula (I-A). RT [0477] In some embodiments, the peripheral distribution moiety has formula of , wherein RT is defined as in formula (II). [0478] In another general aspect, provided are methods of activating α2AR and methods of treating or preventing a disease in a subject, using the compounds described herein or the composition containing the compounds with one or more acceptable pharmaceutical carriers, describe herein. [0479] In some embodiments, the compounds of formula (I-A), (I-B), (I-C), (I-D), or (II) can be useful for activating α2AR. [0480] In some embodiments, provided is a method of activating α2AR in a subject in need thereof, comprising administering to the subject a compound or composition described herein, e.g., administering an effective amount of a compound or composition described herein. [0481] In some embodiments, provided is a method of treating or preventing a disease in human or in animal. [0482] In some embodiments, provided is a method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject a compound or composition described herein, e.g., administering an effective amount of a compound or composition described herein. [0483] In some embodiments, the disease is glaucoma, pain, spasticity, nasal congestion, rosacea, rhinitis, anesthesia, presbyopia, acute kidney injury, insomnia, inflammatory disease, cancer, etc. [0484] In some embodiments, the disease is pain. [0485] In some embodiments, the pain is nociceptive pain, nociplatic pain, neuropathic pain such as peripheral neuropathic pain, or mixed pain. Examples of peripheral neuropathic pain include, but 100 Attorney Docket No.: 071741.11025/5WO1 not limited to diabetic neuropathy, postherpetic neuralgia, HIV-associated pain, chemotherapy- induced peripheral neuropathy, and post-surgical neuropathic pain. [0486] In some embodiments, the compounds and pharmaceutical compositions described herein cause less side effects when treating pain, such as sedation, decreasing heart rate, and decreasing blood pressure in the treated subject. [0487] In certain embodiments, the compounds and pharmaceutical compositions described herein do not cause sedative response in the treated subject. EXAMPLES [0488] The following examples are to further illustrate the nature of the present disclosure. It should be understood that the following examples do not limit the disclosure and the scope of the present disclosure is to be determined by the appended claims. Methods of Synthesis [0489] Unless indicated otherwise, the abbreviations for chemical reagents and synthesis conditions have their ordinary meaning known in the art as follows: “ACN” refers to acetonitrile; “LDA” refers to lithium diisopropyl amide; “EA” or “EtOAc” refers to ethyl acetate; “PE” refers to petroleum ether; “r.t.” and “rt” refer to room temperature; “THF” refers to tetrahydrofuran; “DIPEA” refers to diisopropylethylamine; “DCM” refers to dichloromethane; “HOBT” refers to hydroxybenzotriazole; “TLC” refers to thin layer chromatography; “TLC” refers to thin layer chromatography; “DMF” refers to dimethylformamide; “h” refers to hours; “min” refers to minutes; “EDCI” refers to 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; “DMAP” refers to 4-Dimethylaminopyridine; 101 Attorney Docket No.: 071741.11025/5WO1 “Prep-HPLC” refers to preparative high performance liquid chromatography; “DPPF” refers to 1,1'-Bis(diphenylphosphino)ferrocene; and “NCS” refers to N-chlorosuccinimide. “TEA” refers to triethylamine. “TES” refers to triethyl silane. “Trt” refers to trityl group or triphenylmethyl group. “MeOH” refers to methanol. “EtOH” refers to ethanol. “t-BuXphos” refers to tert-butyl-Xantphos “TMAl” refers to trimethylaluminum “Xantphos” refers to 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene “Pd(PPH3)4” refers to tetrakis(triphenylphosphine)palladium(0) [0490] Example 1. Synthesis of Compound 1 O O O O O OH OH OH O N TF 3-Brmombenzoic HI, P HCl T i h l h l 2 H A H N DCM T 2h p5 [0491] Step 1: 400mL THF and 36g (0.18mol, 4.0eq) of 3-bromobenzoic acid were added into a 500mL reaction flask. Following cooling to -65 ℃, 135mL (4mol/L, 0.428mol, 7.5eq) of n- Butyllithium was added. The mixture was stirred at -65 ℃ for 2 hours before 20g (0.057mol, 1.0eq) of compound 1-1 and an additional 400mL of THF were introduced. After stirring at -65℃ for 30 minutes, the mixture was allowed to warm to room temperature over 16 hours. Completion was confirmed by LC-MS, and 270mL of saturated ammonium chloride was added. The organic phase was then separated, vacuum concentrated, and the residue was column chromatographed (DCM-DCM: MeOH=92:8) to yield 13g of compound 1-2, with a 40.4% yield. 102 Attorney Docket No.: 071741.11025/5WO1 [0492] Step 2: 150 ml of 55% HI, 7.5g (13.3mmol, 1.0eq) of compound 1-2, and 4.1g (133mmol, 10.0eq) of red phosphorus were added into a 200 mL sealing tube The mixture was stirred at 160 ℃ for 16 hours until LC-MS indicated completion. Following vacuum concentration, the residue was collected to produce 7.3g of compound 1-3, achieving a 100% yield. [0493] Step 3: 240 ml of pyridine, 12.1g (39.5mmol, 1.0eq) of compound 1-3, and 55.1g (197.5mmol, 3.0eq) of triphenylmethyl chloride were added to a 50mL reaction flask. The mixture was stirred at 50℃ for 2 hours until LC-MS confirmed completion. After vacuum concentration, the residue underwent column chromatography (DCM-DCM:MeOH=92:8) to obtain 4.5g of compound 1-4, with a 20.8% yield. [0494] Step 4: 52ml DCM, and then 1.3g (2.37mmol, 1.0eq) of compound 1-4, 594mg (7.11mmol, 3.0eq) of methoxyammonium chloride, 2.45g (18.96mmol, 8.0eq) of DIPEA, 640mg (4.74mmol, 2.0eq) of HOBT, and 999mg (5.21mmol, 2.2eq) of EDCI were added to a 100 mL reaction flask. The mixture was stirred at room temperature for 5 hours until LC-MS confirmed the reaction's completion. After vacuum concentration, the residue was purified by column chromatography (DCM-DCM:MeOH=91:9) to yield 900mg of compound 1-5, with a yield of 65.8%. [0495] Step 5: 18mL DCM and 900mg (1.56mmol, 1.0eq) of compound 1-5, along with 9mL of TFA, were added to a 50mL reaction flask. The reaction was stirred at room temperature for 2 hours until LC-MS showed completion. Following vacuum concentration, the residue underwent column chromatography to yield 670 mg of compound 1, achieving a 98.5% yield. [0496] Step 6: Compound 1 was separated by chiral HPLC to afford compound 1-A and compound 1-B. A column with the dimensions 30*250 mm packed with CHIRALPAK® IG (10µm particle size) was used as the chiral stationary phase. A mixture of 60% volume mobile phase A and 40% volume mobile phase B was used as the mobile phase. *Mobile phase A: Hexane+0.2% NH3 in MeOH *Mobile phase B: EtOH+0.2% NH3 in MeOH [0497] The operation conditions were as follows: Temperature: Ambient temperature Flow rate: 25mL/min Detection: UV 214nm 500mg of compound 1 was separated on the column. The first eluting enantiomer (compound 1-A) 103 Attorney Docket No.: 071741.11025/5WO1 with a retention time of 4.18min was isolated from the eluent with an enantiomeric excess of 100% in 80% yield. The second eluting enantiomer (compound 1-B) with a retention time of 5.83min was isolated from the eluent with an enantiomeric excess of 99.2% in 81% yield. [0498] In this application, the naming convention for separated enantiomers is systematic. "A" denotes the first eluting product from the chromatography, and "B" indicates the second. For compounds where chirality leads to four distinct products, they are labeled as "A," "B," "C," and "D," based on their elution order. Consequently, if a compound is named as X, the separated products would be systematically named "X-A," "X-B," "X-C," and "X-D." [0499] Example 2. Synthesis of Compound 8 Br N Br Br O Br O N N Trt O nzene were added in a 500mL three-necked round bottom flask under nitrogen atmosphere. At 0℃, 27mL (34.1mmol, 1.5eq) of isopropylmagnesium chloride lithium chloride complex was introduced. The mixture was stirred for 6 hours at 0℃ before adding 10g (22.7mmol, 1eq) of compound 8-1, still under 0℃. Stirring continued for 16 hours at room temperature (25℃). Then, it was poured into water, washed with ethyl acetate, dried using Na2SO4, and fast silica gel column purification yielded 11.3g of compound 8-2 (79%). [0501] Step 2: A 250mL three-necked round bottom flask received 113ml of DCM, 11.3 g (18mmol, 1.0eq) of compound 8-2, HSiEt3 (21g, 180mmol, 10eq), and TFA (21g, 180mmol, 10eq) under nitrogen at 0℃. It was stirred until reaching room temperature over 16 hours. Concentration under vacuum produced compound 8-3 (17g, crude). 104 Attorney Docket No.: 071741.11025/5WO1 [0502] Step 3: 17 g (18mmol, 1.0eq) of compound 8-3, TrtCl (12.6g, 45mmol, 2.5eq), 170ml of DCM and Et3N (9.1 g, 90mmol, 5eq) were mixed in a 500ml three-necked round bottom flask under nitrogen. After stirring for 16 hours at 25℃, completion was confirmed by LC-MS. The product was processed similarly to previous steps to yield 7.1 g of compound 8-4 (64%). [0503] Step 4: 100ml of DMF, 6 g (9.8mmol, 1.0eq) of compound 8-4, Zn(CN)2 (1.26g, 10.8mmol, 1.1eq), and Pd(PPh3)4 (1.26g, 1.1mmol, 0.11eq) were added under nitrogen to a 250ml three-necked round bottom flask. After stirring at 120℃ for 2 hours, LC-MS confirmed completion. Following the standard work-up, 5.1 g of compound 8-5 (93%) was obtained. [0504] Step 5: 60ml of EtOH, 2 g (3.6mmol, 1.0eq) of compound 8-5, and 12mL of 30% KOH were added under nitrogen into a 100ml single-mouth flask. The mixture was refluxed for 72 hours. After concentration under vacuum and subsequent work-up, 1.9 g of compound 8-6 (91%) was purified. [0505] Step 6: 20ml of DCM, 1g (1.73mmol, 1.0eq) of compound 8-6, EDCI (0.432g, 2.25mmol, 1.3eq), DIPEA (0.893g, 6.92mmol, 4eq), HOBt (0.234g, 1.73mmol, 1.0eq), and methoxyammonium chloride (0.174g, 2.08mmol, 1.2eq) were combined in a 100mL single-mouth flask under nitrogen. Stirred for 16 hours at 25℃ and then processed as before, this yielded 0.43 g of compound 8-7 (41%). [0506] Step 7:10ml of DCM, 0.430 g (0.71mmol, 1eq) of compound 8-7, and BBr3 (0.435 g, 1.775mmol, 2.5eq) were mixed in a 25ml single-mouth flask at 0℃ under nitrogen. Stirring continued for 3 hours at 0℃ until LC-MS indicated completion, proceeding directly to the next step. [0507] Step 8: The mixture from Step 7 and 10ml of MeOH were added to a 50ml single-mouth flask under nitrogen. Heated to reflux for 16 hours, completion was verified by LC-MS. After concentration under vacuum and further purification steps, including the addition of 10mL saturated NaHCO3 solution and washing with ethyl acetate, drying over Na2SO4, and purification using Liquid Phase Method, 39mg of compound 8 was obtained, marking a 16% yield. 105 Attorney Docket No.: 071741.11025/5WO1 [0508] Example 3. Synthesis of Compound 17 H H H H O N O N H H O N O N O N O N H2N HN HN NH NH NH NH O O O O O and O O H DCM, 180mg (0.33mmol, 1eq) of compound 17-1, 110mg (1.00mmol, 3eq) of TEA, and 47mg (0.5mmol, 1.5eq) of methylaminoformyl chloride. The reaction mixture, after being allowed to reach 25℃, was stirred for 16 hours. Once LC-MS confirmed the reaction's completion, it was concentrated under vacuum and then purified using a fast silica gel column, resulting in 150mg of compound 17-2 at a 75% yield. [0510] Step 2: 3ml of DCM, 150mg (0.25mmol, 1eq) of compound 17-2, and 1.5ml of TFA were added to a 10ml single-mouth flask under N2 at 25℃. After stirring for 2 hours and confirmation of completion by LC-MS, the reaction mixture was vacuum concentrated and subjected to purification through a fast silica gel column, yielding 33mg of compound 17, which corresponds to a 28% yield. [0511] Step 3: 438mg of compound 17 underwent separation using a chiral column, leading to the collection of compound 17-A (133mg), which after preparative liquid chromatography with a neutral method yielded 101mg (Yield = 23.06%), and compound 17-B (133mg), which also resulted in 101mg after similar purification, with a yield of 23.06%. [0512] Example 4. Synthesis of Compound 22 O OH O O O O Br N H O O NH2 O N S O p - , g . ol, 1eq) of compound 22-1, 335mg (1.58mmol, 2eq) of tert-butyl N-(2-bromoethyl)carbamate, and 387mg (1.189mmol, 1.5eq) of Cs2CO3. The mixture was stirred at 60°C for 12 hours. Upon completion, as indicated by LC-MS, it was poured into water and extracted with EtOAc. The organic layer was 106 Attorney Docket No.: 071741.11025/5WO1 dried over Na2SO4, vacuum concentrated, and yielded 300mg of compound 22-2 as a white solid, which was carried forward without further purification, yielding 72.5%. [0514] Step 2: 300mg of compound 22-2, 10ml DCM, and 5ml TFA were added to a 50mL three-necked flask. This mixture was stirred at room temperature (25°C) for 12 hours. LC-MS confirmed completion; the mixture was then diluted with water, adjusted to pH 10, and extracted with DCM. The organic phase was dried over Na2SO4, vacuum concentrated, and the resulting residue was column chromatographed on silica gel to obtain 85mg of compound 22-3 as a yellow solid, with a yield of 45.9%. [0515] Step 3: a 10ml three-necked flask was charged with 5ml DMF, 75mg (0.233mmol, 1eq) of compound 22-3, 75mg (0.583mmol, 2.5eq) of DIPEA, and 29mg (0.257mmol, 1.1eq) of methanesulfonyl chloride. Stirring continued at 25°C for 2 hours until LC-MS analysis confirmed the reaction's completion. The mixture was then diluted with water, extracted with EA, and the organic phase was dried over Na2SO4 and vacuum concentrated. Purification by silica gel column chromatography yielded 14mg of compound 22 as a white solid, achieving a 13.2% yield. Overall yield =4.4%. [0516] Example 5. Synthesis of Compound 27 Br Br Br O Br F [0517] Step 1: In a 50mL reaction flask, 10mL of THF and 860mg (3.39mmol, 1.5eq) of 1,3- dibromo-2-fluorobenzene were combined and cooled to -65°C. Next, 1.4mL (3.39mmol, 1.5eq) of n-butyllithium was added. The mixture was stirred at this temperature for 2 hours before adding 1g 107 Attorney Docket No.: 071741.11025/5WO1 (2.26mmol, 1eq) of compound 27-1 and another 10mL of THF. It was stirred for an additional 30 minutes at -65°C, then allowed to warm to room temperature over 16 hours. Completion was verified by LC-MS, and 20mL of saturated ammonium chloride was added. The organic phase was then separated, concentrated under vacuum, and purified via column chromatography, yielding 600mg of compound 27-2 with a yield of 43%. [0518] Step 2: A 50mL three-necked flask was charged with 600mg (0.971mmol, 1eq) of compound 27-2, 1.1g (9.71mmol, 10eq) of triethylsilane, and 1.1g (9.71mmol, 10eq) of TFA. The mixture was stirred at 25°C for 1 hour. Upon completion, confirmed by LC-MS, it was poured into water, adjusted to pH=10, extracted with EA, dried over Na2SO4, and concentrated under vacuum. Purification via column chromatography on silica gel led to the isolation of 170mg of compound 27-3 as a yellow solid, yielding 48.7%. [0519] Step 3: Into a 25mL reaction flask, 10ml of DMF, 170mg (0.473mmol, 1eq) of compound 27-3, 158g (0.568mmol, 1.2eq) of triphenylmethyl chloride, and 96mg (0.946mmol, 2eq) of TEA were added. The mixture was stirred at 25°C for 12 hours until LC-MS indicated the reaction had completed. After pouring into water, extracting with EA, drying over Na2SO4, and concentrating, the crude was purified by column chromatography, yielding 220mg of compound 27-4 with a 77.3% yield. [0520] Step 4: A 25mL reaction flask was prepared with 10ml DMF, 170mg (0.283mmol, 1eq) of compound 27-4, 100mg (0.848mmol, 3eq) of ZnCN, and 98mg (0.0848mmol, 0.3eq) of tetrakis(triphenylphosphine)palladium. Stirring was conducted at 150°C for 30 minutes in a microwave. After completion, confirmed by LC-MS, the mixture was worked up and purified by column chromatography to yield 130mg of compound 27-5, an 84% yield. [0521] Step 5: To a 25mL reaction flask, 10mL of DMSO and 110mg (0.201mmol, 1eq) of compound 27-5 were added and cooled to 0℃. Then, 3ml of 30% H2O2 was added, and the mixture was stirred at 0℃ for 1 hour. Following LC-MS confirmation of completion, water was added, and the organic phase was separated and concentrated under vacuum. Column chromatography purification yielded 100mg of compound 27-6, an 88.1% yield. [0522] Step 6: In a 25mL three-port flask, 10ml of DCM and 110mg of compound 27-6 were combined, and 5ml of TFA was added at 0℃. The mixture was allowed to reach room temperature naturally and stirred for 2 hours. Completion was indicated by LC-MS. The mixture was then 108 Attorney Docket No.: 071741.11025/5WO1 concentrated under reduced pressure, and the residue was purified by TLC to obtain 25mg of compound 27 as a white solid, with a yield of 29.4%. Overall yield =3.5%. [0523] Example 6. Synthesis of Compound 28 O N S S MAP C,4h d 28-1, 26.6g (0.16mol, 1.2eq) of benzyl bromide, and 21.5g (1.2mol, 1.2eq) of K2CO3 were combined. The mixture was heated to 95°C for 16 hours. GC-MS confirmed the reaction's completion. After filtration and concentration, column chromatography purification yielded 32g of compound 28-2 with an 88.5% yield. [0525] Step 2: Into a 25ml reaction flask, 5ml THF, 185mg (7.6mmol, 2.1eq) of magnesium chips, and 2g (7.2mmol, 2.0eq) of compound 28-2 were added. The mixture was stirred at 65°C for 1 hour before cooling to room temperature for the next step. A 50mL reaction flask received 20mL THF and 1.59g (3.6mmol, 1.0eq) of (2,3-Dimethylphenyl)-[1-(trityl)-1H-imidazol-4-yl]methanone. The previously prepared Grignard reagent was added, and the reaction was heated to 80°C for 16 hours. Completion was verified by LC-MS. After quenching with 10mL water, extraction with EA, and drying with Na2SO4, the mixture was concentrated. Column chromatography purification yielded 1.6g of compound 28-3, a 69.2% yield. [0526] Step 3: A 100mL reaction flask was charged with 14mL DCM, 1.4g (2.18mmol, 1.0eq) of compound 28-3, and 2.53g (21.8mmol, 10eq) of TES. After cooling to 0°C, 2.48g (21.8mmol, 109 Attorney Docket No.: 071741.11025/5WO1 10eq) of TFA was added. The mixture was warmed to 25°C for 5 hours, then concentrated under vacuum after LC-MS confirmed completion. The residue was column chromatographed to yield 500mg of compound 28-4, a 68.4% yield. [0527] Step 4: In a 5mL reaction flask, 2mL THF, 100mg (0.29mmol, 1.0eq) of compound 28-4, 7mg (0.06mmol, 0.2eq) of DMAP, 94mg (0.43mmol, 1.5eq) of Boc2O, and 44mg (0.43mmol, 1.5eq) of TEA were mixed. The reaction was held at 25°C for 4 hours, confirmed by LC-MS. After vacuum concentration, the residue was purified by column chromatography, yielding 120mg of compound 28-5, an 85.5% yield. [0528] Step 5: A 10mL reaction flask was loaded with 1.5mL acetic acid, 0.5ml water, and 120mg (0.25mmol, 1.0eq) of compound 28-5. After chilling to 0°C, 165mg (1.24mmol, 5eq) of NCS was added. The mixture was stirred at 0°C for 2 hours until LC-MS confirmed completion, then moved to the next step without purification. The yield was recorded as 100%. [0529] Step 6: To a 50mL reaction flask, 10mL of 2M NH2CH3/THF was added and cooled to 0°C before introducing the crude compound 28-6. Stirring proceeded at 25°C for 16 hours, as evidenced by LC-MS. After concentration under vacuum, the mixture was purified by column chromatography to yield 40mg of compound 28-7, a 40% yield. [0530] Step 7: In a 5mL reaction flask, 1mL DCM and 40mg (0.088mmol, 1.0eq) of compound 28-7 were combined. The mixture was cooled to 0°C before adding 0.5ml TFA, then allowed to warm to 25°C for 2 hours, completion shown by LC-MS. The concentrated mixture was purified through prep-HPLC to obtain 14mg of compound 28, with a yield of 44.8%. Overall yield=6.4%. 110 Attorney Docket No.: 071741.11025/5WO1 [0531] Example 7. Synthesis of Compound 31 N O N OH O 4eq 4-Bromo-2-methoxypyridine OH HI P h 27.15mmol, 4.0eq) of 4-bromo-2-methoxypyridine, and 10mL of 2.5N n-butyl lithium in n-hexane (25.1mmol, 3.7eq) were added dropwise at -65℃. The solution was maintained at -65℃ for 1 hour before adding 3g (6.79mmol, 1eq) of compound 31-1. After another 0.5 hours at -65℃, the reaction was left to proceed overnight at room temperature. Completion was verified by LC-MS. The reaction was quenched with 100ml of saturated ammonium chloride, and the organic layer was separated and concentrated. The residue was mixed with 50mL DCM, stirred for 5 minutes, filtered, and dried with an infrared lamp to yield 2.88g of compound 31-2 (77% yield). [0533] Step 2: A 50ml closed tank received 20ml of 57 wt.% HI, 2.35g (4.599mmol, 1.0eq) of compound 31-2, and 1.43g (45.99mmol, 10eq) of red phosphorus. Stirred at 160℃ overnight and checked by LC-MS for completion, the mixture was cooled to room temperature and concentrated to yield 5g of crude compound 31-3 (100% yield). [0534] Step 3: Compound 31-3 (1g, crude) and 15mL of POCl3 were added to a 5mL reaction flask. After refluxing overnight and verifying completion with LC-MS, the mixture was cooled, concentrated under vacuum, and the residue was neutralized to pH=8 with saturated sodium bicarbonate. Extraction with 40mL EA (three times), drying with anhydrous sodium sulfate, and concentration provided 174mg of compound 31-4 via column chromatography (30% yield). [0535] Step 4: Into a 200mL high-pressure reactor, 174mg (0.586mmol, 1eq) of compound 31-4, 10 mL of MeOH, 296mg (2.93mmol, 5eq) of TEA, and 48mg (0.0586mmol, 0.1eq) of PdCl2(dppf) 111 Attorney Docket No.: 071741.11025/5WO1 were introduced. The reaction, under 5MPa of carbon monoxide at 120℃ for 48 hours, left 5% of the starting material, as shown by LC-MS. After filtration and concentration, 270mg of compound 31-5 was isolated by column chromatography (100% yield). [0536] Step 5: A 50mL closed tank was charged with 100mg (0.312mmol, 1eq) of compound 31-5 and 5mL of MeOH/NH3 (15M/L). The mixture was stirred at 68℃ overnight, cooled to room temperature, concentrated under vacuum, and then purified to obtain 10mg of compound 31 through pre-HPLC (10% yield). [0537] Example 8. Synthesis of Compound 32 Br H H N N N N .51mmol, 1eq) of compound 32-1, 108mg (1.54mmol, 3eq) of 2-cyanoethylamine, 213mg (1.54mmol, 3eq) of K2CO3, 47mg (0.051mmol, 0.1eq) of Pd2(dba)3, and 55mg (0.10mmol, 0.2eq) of brettphos were added. The mixture was stirred under nitrogen at 120°C for 1 hour. The completion of the reaction was indicated by TLC. The mixture was then transferred into 100mL of water and extracted three times with 50mL of ethyl acetate. After washing the organic layer with 50mL of brine, it was dried over Na2SO4 and concentrated under vacuum to yield 1g of a crude yellow oil. This was purified via column chromatography (Petroleum Ether: Ethyl Acetate = 1:0 to 1:1) to obtain 216mg of compound 32-2 as a yellow powder, resulting in a yield of 73.36%. [0539] Step 2: A 50mL single-mouth flask was charged with 200mg (0.35mmol, 1eq) of compound 32-2, 5mL of dichloromethane, and 1mL of trifluoroacetic acid. The mixture was stirred at room temperature for 1 hour, with TLC confirming the reaction's completion. The solution was then vacuum-concentrated, and the crude product was subjected to column chromatography (Dichloromethane: Methanol = 1:0 to 90:10) to produce 120mg of compound 32. Further purification by preparative HPLC yielded 93mg of compound 32 as a yellow powder, with an overall yield of 59.92%. 112 Attorney Docket No.: 071741.11025/5WO1 Overall yield=43.96%. [0540] Example 9. Synthesis of Compound 58 3.0eq TEA 2.5eq compound 2-chloro- 0.1eq PdCl 2(dppf) O O O 3-fluoro-4-iodopyridine N Cl 5MPa CO N N 10eq TES N 2.5eq iPr-MgClLiCl O NH 16M N 2 NH 10eq TFA 2 N Cl MeOH,DMSO H3/MeOH THF,80°C,16h F 100°C 48h F 30°C 16h F DCM,100°C,3.5h F H loro-3- fluoro-4-iodopyridine were added. After cooling the mixture to 0℃, 12mL (15.5mmol, 2.5eq) of iPr-MgClLiCl was introduced. The reaction was stirred at 0℃ for 3 hours, then 1.16g (2.63mmol, 1.0eq) of 2,3-dimethylphenyl)[1-(trityl)-1H-imidazol-4-yl]methanone was added and the reaction was stirred at 80℃ for 16 hours. Following completion, confirmed by LC-MS, the reaction was quenched with 40mL water and extracted with EA. The organic layer was dried over Na2SO4 and concentrated under vacuum. The residue was purified by column chromatography to yield 2.6g of compound 58-1, achieving a 29.3% yield. [0542] Step 2: To a 200mL high-pressure reactor, 1g (1.74mmol, 1.0eq) of 58-1, 40 mL of MeOH, 40mL of DMSO, 530mg (5.24mmol, 3.0eq) of TEA, and 148mg (0.17mmol, 0.1eq) of PdCl2 (dppf) were added. The mixture was reacted with carbon monoxide at 5MPa and 100 ℃ for 48 hours, with LC-MS indicating 5% remaining raw material. After concentration, the residue was purified by column chromatography to yield 53mg of compound 58-2, a 51.2% yield. [0543] Step 3: A 50mL sealed tube received 25mL of 16M NH3/MeOH and 530mg (0.89mol, 1.0eq) of compound 58-2. Stirred at 30℃ for 16 hours until LC-MS confirmed completion, the residue was then purified by column chromatography to yield 360mg of compound 58-3, a 67.8% yield. [0544] Step 4: Into a 10mL reaction flask, 3mL DCM, 100mg (0.17mmol, 1.0eq) of compound 58-3, and 195mg (1.7mmol, 10eq) of TES were added. After cooling to 0℃, 191mg (1.7mmol, 10eq) of TFA was introduced. The reaction was then warmed to 100℃ for 3.5 hours. LC-MS showed completion, and after concentration under vacuum, the residue was purified by prep-HPLC to yield 16mg of compound 58, with a 28.8% yield. 113 Attorney Docket No.: 071741.11025/5WO1 [0545] Example 10. Synthesis of Compound 60 O O O O O O O OH HCl/Et2O O N + Zn/TiCl4/THF/4h DCM 2h P/HI/AcOH/16h N NH TFA of zinc were added. This mixture was cooled to -10℃ under a nitrogen atmosphere while stirring. Then, 4.1g (21.6mmol, 3.7eq) of titanium tetrachloride was added dropwise at -10℃. The reaction mixture was stirred at 70℃ for 16 hours, followed by the addition of 950mg (6.08mol, 1.04eq) of methyl 3-oxocyclohexanecarboxylate and 2.6g (5.85mol, 1eq) of (2,3-dimethylphenyl)(1-trityl-4- imidazolyl)methanone. Stirring continued for 4 hours at 80℃. Completion was confirmed by LC- MS. The reaction was then diluted with 100mL of water and 100mL of EA, filtered, and the filtrate was extracted with EA. After washing with brine and drying over Na2SO4, the organic layers were concentrated under reduced pressure to yield 950mg of compound 60-1 as crude, with a 50% yield. [0547] Step 2: To a 25mL three-necked flask, 10mL of DCM and 0.5g (1.54mmol, 1eq) of compound 60-1, along with 10mL of HCl/Et2O, were added. The mixture was stirred at room temperature for 3 hours. LC-MS indicated the reaction was complete. Concentrating under reduced pressure yielded 400mg of compound 60-2 as crude, with a 100% yield. [0548] Step 3: A 250mL three-necked flask received 1.2mL of AcOH, 0.9mL of hydriodic acid (55%-58%), 50mg (0.15mmol, 1.0eq) of compound 60-2, and 167mg (5.4mmol, 35eq) of phosphorus. The reaction mixture was stirred at 100℃ for 16 hours. Completion was confirmed by LC-MS. The mixture was then added to water, adjusted to pH=7, and extracted with EA. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 70mg of crude. Purification by liquid chromatography yielded 6mg of compound 60, with a 10% yield. Overall yield: 5% 114 Attorney Docket No.: 071741.11025/5WO1 [0549] Example 11. Synthesis of Compound 61 O O O 1.1eq (2,3-Dimethylphenyl) O O (1-trityl-4-imidazolyl)methanone 20V DCM 3.0eq 50% Pd/C O 1.1eq Triethyl phosphite tBuOK N O 5V TFA N O ° THF 27°C,16h 1.0eq) of methyl 3-(bromomethyl)benzoate, and 3.04g (18.3mmol, 1.1eq) of triethyl phosphite. The mixture was stirred at 110°C for 16 hours. Upon completion, confirmed by LC-MS, the solution was concentrated. The residue underwent column chromatography, yielding 5.3g of compound 61-1 with a 99% yield. [0551] Step 2: Into a 100mL reaction flask, 40mL THF, 2g (7.0mmol, 1.0eq) of compound 61-1, and 3.4g (7.7mmol, 1.1eq) of (2,3-dimethylphenyl)(1-trityl-4-imidazolyl)methanone were combined and cooled to 0°C before adding 2.35g (21mmol, 3.0eq) of potassium tert-butoxide. After stirring at 27°C for 16 hours and confirmation of completion by LC-MS, the solution was concentrated and purified by column chromatography to yield 1.03g of compound 61-2, a 26.3% yield. [0552] Step 3: A 25mL reaction flask was charged with 10mL DCM and 500mg of compound 61-2, followed by the addition of 2.5mL TFA. The reaction mixture was stirred at 27°C for 1 hour. LC-MS indicated completion, and after concentration, the residue was purified by column chromatography to yield 240mg of compound 61-3, an 84.5% yield. [0553] Step 4: In a 10mL reaction flask, 3mL THF, 240mg of compound 61-3, and 120mg (50%) of Pd/C were added. Stirred at 27°C for 16 hours and confirmed by LC-MS, the solution was filtered. The organic phase was concentrated, and the residue was purified by column chromatography to yield 150mg of compound 61-4, a 62.1% yield. [0554] Step 5: To a 10mL reaction flask, 3mL DMF, 80mg (0.25mmol, 1.0eq) of compound 61- 4, 209mg (2.5mmol, 10eq) of methoxyammonium chloride, cooled to 0°C, then 386mg (3mmol, 12eq) of DIPEA and 142mg (0.37mmol, 1.5eq) of HATU were added. Stirred at 27°C for 4.5 115 Attorney Docket No.: 071741.11025/5WO1 hours, LC-MS showed 40% remaining raw material. The mixture was concentrated under vacuum to yield 160mg of compound 61-5, achieving a 100% yield. [0555] Step 6: A 10mL reaction flask was prepared with 1mL DCM and 160mg of compound 61-5, and 0.5mL TFA was added. Stirred at 27°C for 1 hour, completion was confirmed by LC- MS. After concentration under vacuum, the residue was purified by prep-HPLC to yield 25mg of compound 61, a 15.3% yield.Overall yield: 2.1%. [0556] Example 12. Synthesis of Compound 139 O HN O NH2 1.3eqNaH/1.6eqEthyl chloroacetate/THF/r t/15h N N Trt q) of (2,3- dimethylphenyl)(1-trityl-4-imidazolyl)methanone, 2.3g (18.1mmol, 1.6eq) of ethyl chloroacetate, and 1.35g (33.9mmol, 3eq, 60% wt) of NaH were combined under a nitrogen atmosphere. The mixture was stirred at 25°C for 16 hours, confirmed complete by LC-MS, and concentrated under vacuum. After adding 50ml of 10% KOH, it was stirred for another 16 hours at 100°C, then worked up and purified via a fast silica gel column to yield 3.4g of 139-1, with a 65.9% yield. [0558] Step 2: A 100mL three-necked flask received 60ml of ACN, 3.2g (7.01mmol, 1eq) of 139-1, 3.14g (14.02mmol, 2eq) of CAS 39684-80-5, and 3.42g (10.51mmol, 1.5eq) of Cs2CO3. Stirred at 60°C for 12 hours and confirmed complete by LC-MS, the reaction was worked up and purified to give 930mg of 139-2, a 22.1% yield. [0559] Step 3: To a 25mL three-necked flask was added 300mg of 139-2, 3ml of DCM, and 3ml of 4M HCl in dioxane. After stirring at 25°C for 3 hours and confirmation of completion by LC- MS, the reaction was neutralized to pH=10, extracted, and purified to yield 130mg of 139-3 as a white solid, a 52% yield. 116 Attorney Docket No.: 071741.11025/5WO1 [0560] Step 4: A 25mL three-necked flask was charged with 5ml of THF, 130mg (0.260mmol, 1eq) of 139-3, 40mg (0.390mmol, 1.5eq) of TEA, and 57mg (0.286mmol, 1.1eq) of (Tetrahydro- 2H-pyran-4-yl)methanesulfonyl chloride (CAS 264608-29-9). Stirred at 25°C for 18 hours and verified complete by LC-MS, the mixture was worked up and purified to give 80mg of 139-4 as a white solid, yielding 46.5%. [0561] Step 5: 139-4 (80mg) was combined with Pd(OH)2/C (80mg), 5mL of methanol, and 5mL of THF, stirred at 40°C for 18 hours under a hydrogen atmosphere. The catalyst was filtered off, and the filtrate was concentrated, mixed with 10mL of DCM and 5mL of TFA, stirred for ten minutes, and dried. The residue was purified by preparative HPLC to yield 30mg of 139 as a white solid, with a 46.3% yield. Overall yield=1.63%. [0562] Example 13. Synthesis of Compound 156 Br F Br Br O NH2 [0563] Step 1: A 50mL reaction flask was charged with 25mL of toluene, 5g (0.0188mmol, 1.0eq) of 3-fluoro-4-bromobenzyl bromide, and 3.44g (0.0207mmol, 1.1eq) of triethyl phosphite. Stirred at 110°C for 18 hours, completion was confirmed by LC-MS. The reaction mixture was concentrated and the residue was purified by column chromatography to yield 5.69g of compound 156-1, with a 93.4% yield. [0564] Step 2: To a 50mL reaction flask, 20mL of THF, 1g (3.08mmol, 1.0eq) of compound 156-1, and 1.36g (3.08mmol, 1.0eq) of (2,3-dimethylphenyl)(1-trityl-4-imidazolyl)methanone were added and cooled to 0°C. Then, 1.04g (9.24mmol, 3.0eq) of potassium tert-butoxide was introduced. After stirring at 10°C for 18 hours and confirmation of completion by LC-MS, the 117 Attorney Docket No.: 071741.11025/5WO1 reaction was filtered, concentrated, and the residue was purified by column chromatography to yield 1.31g of 156-2, a 69.5% yield. [0565] Step 3: A 10mL reaction flask received 5mL of DMF, 500mg (0.817mmol, 1.0eq) of 156- 2, 192mg (1.634mmol, 2.0eq) of zinc cyanide, and 95mg (0.0817mmol, 0.1eq) of Pd(PPh3)4. Stirred at 120°C for 18 hours, TLC indicated 50% of the raw materials remained. The mixture was diluted with 40mL of ice water, extracted three times with 20mL of EA, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. Purification by column chromatography yielded 220mg of 156-3, an 83.5% yield. [0566] Step 4: Into a 5mL reaction flask, 1.5mL of THF, 1.5mL of MeOH, 110mg (0.197mmol, 1.0eq) of 156-3, 54mg (0.394mmol, 2.0eq) of K2CO3, and 45mg (0.394mmol, 2.0eq) of 30% H2O2 were combined under nitrogen. The mixture was stirred overnight at 20°C for 18 hours. Following completion, confirmed by LC-MS, the mixture was poured into 10mL of water, extracted three times with 10mL of EA, dried over Na2SO4, and concentrated under vacuum. Purification by column chromatography yielded 77mg of 156-4, a 67.7% yield. [0567] Step 5: In a 5mL reaction flask, 3mL of MeOH:THF (1:1), 90mg (0.156mmol, 1.0eq) of 156-4, and 87mg of Pd(OH)2 were stirred at 48°C overnight under a hydrogen environment. LC- MS confirmed the reaction's completion. The reaction was filtered, concentrated, and the crude product was further processed with 2mL of DCM and 0.5mL of TFA, stirred for 0.5 hours, then concentrated. Purification by pre-HPLC yielded 41mg of compound 156, with a 58.3% yield. Overall yield: 21.4% [0568] Example 14. Synthesis of Compound 182 O O 1.5eq methoxyam- O O 2.0eq XX monium chloride NH NH O 5.0eq n S S 12 HAT -BuLi, THF TE TFA NH , , . , . thiophenecarboxylic acid, 2.93g (28.98mmol, 3.0eq) of TEA, and 1.21g (14.49mmol, 1.5eq) of methoxyammonium chloride were combined. After adding 4.41g (11.59mmol, 1.2eq) of HATU, the mixture was stirred at room temperature for 12 hours. Following completion, confirmed by LC- MS, the reaction was quenched with water and extracted with ethyl acetate. After drying over 118 Attorney Docket No.: 071741.11025/5WO1 Na2SO4 and filtration, the concentrate was purified by column chromatography (PE:EA=67:33) to yield 1.25g of 182-1 as a white solid, a 54.8% yield. [0570] Step 2: In a 25mL three-necked flask, 598mg (2.53mmol, 2.0eq) of 182-1 was dissolved in 6mL THF and cooled to -80°C under nitrogen. n-BuLi (2.5M, 2.5mL, 6.33mmol, 5.0eq) was added, stirred for 40 minutes, then a mixture of 560mg (1.27mmol, 1.0eq) of (2,3- dimethylphenyl)(1-trityl-4-imidazolyl)methanone in 6mL THF was introduced. After stirring for 1 hour and quenching with water, the mixture was warmed and extracted with ethyl acetate. The organic phase was dried, filtered, and concentrated, then purified by column chromatography (DCM:MeOH=90:10) and further recrystallized with PE:MTBE=2:1 to yield 67mg of 182-2 as a light yellow solid, an 8.8% yield. [0571] Step 3: A 10mL flask received 1mL DCM, 1mL TFA, 65mg (0.11mmol, 1.0eq) of 182-2, and 38mg (0.33mmol, 3.0eq) of TES. After stirring for 2 hours, confirmed complete by LC-MS, the mixture was concentrated and purified by preparative HPLC to afford 20mg of 182 as a white solid, yielding 42.1%. Overall yield=2.03%. [0572] Example 15. Synthesis of Compound 188 2.5 eq PY·SO O O O 0.1 eq SOCl2 O 5.0 eq NaBH 3 4 2.5 eq TEA N 7.5 eq Zn, 3.7 eq TiCl4 MeOH, 85°C, 12h O MeOH, 25°C, 12h O O DMSO, 25°C, 1h + N THF, 85°C, 12h NH2 TFA [0573] Step 1: Thionyl chloride (0.582mL, 8.03mol, 0.1eq) was added to a methanol solution of adamantane-1,3-dicarboxylic acid (18g, 80.27mmol, 1.0eq) at 0°C. Stirred at 85°C for 12 hours, the reaction completion was confirmed by LC-MS. The concentrated mixture was diluted with water (50mL), neutralized to pH=8 with saturated NaHCO3, extracted with ethyl acetate (30mL*3), dried, and concentrated to yield 20g of 188-1 as a white solid (100% yield). [0574] Step 2: 188-1 (20g, 79.27mmol, 1.0eq) was dissolved in methanol (350mL), and NaBH4 (14.99g, 0.39mol, 5.0eq) was added at 0°C. After stirring at 25°C for 12 hours and confirming the 119 Attorney Docket No.: 071741.11025/5WO1 completion, the reaction was concentrated, diluted with ethyl acetate, extracted with water, dried, and purified to obtain 5g of 188-2 as a white solid (11.7% yield). [0575] Step 3: DMSO (50mL), 188-2 (4g, 17.83mmol, 1.0eq), PySO3 (7.1g, 44.58mmol, 2.5eq), and TEA (4.51g, 44.58mmol, 2.5eq) were added to a 250mL flask. Stirred at 25°C for an hour and verified by LC-MS, the mixture was processed and purified to yield 2g of 188-3 as a white solid (51.3% yield). [0576] Step 4: In a 50mL flask, THF (20mL) was combined with Zn (1.1g, 16.87mmol, 7.5eq) and TiCl4 (1.58g, 8.32mmol, 3.7eq) at -70°C, followed by 188-3 (500mg, 2.25mmol, 1.0eq) and a specified compound in THF. Stirred at 80°C for 2 hours, the reaction was completed, worked up, and purified to yield 270mg of 188-4 as a yellow solid (19.1% yield). [0577] Step 5: 188-4 (200mg) was reacted with NaOH (5mL, 8M) in methanol at 20°C, heated to 115°C for 12 hours, cooled, adjusted to pH=4, extracted, and concentrated to yield 80mg of 188-5 as a yellow solid (40.8% yield). [0578] Step 6: 188-5 (60mg, 0.096mmol, 1.0eq) was mixed with oxalyl dichloride (36.92mg, 0.291mmol, 3.0eq) in DCM (1mL) at 0°C, stirred at 15°C for an hour, concentrated to yield 60mg of 188-6 as a white solid (100% yield), and used directly in the next step. [0579] Step 7: 188-6 (60mg) was dissolved in THF (0.5 mL) and treated with NH3·THF (8mL) at 0°C, stirred at 15°C for an hour, concentrated to yield 60mg of 188-7 as a white solid (100% yield), and used directly in the next step. [0580] Step 8: 188-7 (60mg) was combined with Pd(OH)2 (60mg) in MeOH:THF (16mL, 1:1), stirred at 45°C under hydrogen, concentrated, treated with DCM (1mL) and TFA (0.5mL), concentrated, and purified to yield 20mg of 188 as a white solid (43.4% yield). Overall yield=0.2%. [0581] Example 16. Synthesis of Compound 196 O O O THF O TFA [ ] tep : nc ( . g, . mo , eq) was a e to a so ut on o ( m ), followed by dropwise addition of TiCl4 (8.2g, 0.043mol, 9.6eq) at 0°C. The reaction was then heated and 120 Attorney Docket No.: 071741.11025/5WO1 refluxed at 70°C for 1 hour. After cooling to 30°C, a THF solution containing 2,3-dihydro- benzo[1,4]dioxin-6-carbaldehyde (1.8g, 0.0108mol, 2.4eq) and (2,3-Dimethylphenyl)(1-trityl-4- imidazolyl)methanone (2g, 0.0045mol, 1eq) was introduced and refluxed at 65°C for 2 hours under nitrogen. Completion was confirmed by LC-MS. The reaction was quenched with water, extracted with ethyl acetate, dried, and concentrated. Purification via column chromatography yielded 2.1g of 196-1 as a white solid (100% yield). [0583] Step 2: 196-1 (100mg, 0.37mmol, 1eq) was combined with Pd(OH)2/C (100mg) in a THF:MeOH (1:1) solution and stirred at 40°C for 16 hours under hydrogen. Following LC-MS confirmation of completion, the reaction was filtered and concentrated to give a crude product. DCM (2mL) and TFA (1mL) were added to the crude, which was then concentrated and purified by preparative HPLC to yield 12mg of 196 as a white solid (9.7% yield). Overall yield =9.7% [0584] Example 17. Synthesis of Compound 401 Br I OH O N iPrMgClLiCl,DCM N MnO2,DCM N S diethyl sulfate, nBuLI + THF, 25°C, 12h N ° N ° N TFA [0585] Step 1: In a 100mL flask, 50mL of dichloromethane and 4-Iodo-1-trityl-1H-imidazole (11.8g, 0.027mol, 1.0eq) were combined. After cooling the mixture to 0°C, iPrMgClLiCl (1.3mol/L, 20.7mL, 0.027mol, 1.0eq) was added. The mixture was stirred at 0°C for 2 hours, then 3-Bromobenzaldehyde (5g, 0.027mol, 1.0eq) was introduced. Stirring continued at 28°C for 16 hours until LC-MS confirmed the reaction's completion. After cooling back to 0°C and quenching with 44mL of saturated ammonium chloride, the organic layer was separated, concentrated, and purified via column chromatography using ethyl acetate to yield 7.1g of 401-1, achieving a 53% yield. 121 Attorney Docket No.: 071741.11025/5WO1 [0586] Step 2: Into a 200mL high-pressure tube, 180mL of dichloromethane, compound 401-1 (6.1g, 12.35mmol, 1.0eq), and MnO2 (6.44g, 74.1mmol, 6.0eq) were added. The mixture was stirred at 72°C for 5 hours. Completion was verified by LC-MS, and the mixture was then filtered to yield 5.6g of compound 401-2. achieving an 83.9% yield. [0587] Step 3: A 1L reaction vessel was charged with 500 mL of diethyl ether and 20g (82.67mmol, 1eq) of 3,4-Dibromothiophene (Cas: 3141-26-2). Upon cooling to -78°C, 36.37mL (90.94mmol, 1.1eq) of n-BuLi was added dropwise. The mixture was stirred at -78°C for 30 minutes before 14.02g (90.94mmol, 1.1eq) of diethyl sulfate was added dropwise. Stirring continued at 25°C for 5 hours until LC-MS confirmed the reaction's completion. After adding 25mL of aqueous ammonia, the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated to yield 10g of compound 401-3, achieving a 63.3% yield. [0588] Step 4: Into a 50 mL reaction flask, 5mL of THF and 387.28mg (2.03mmol, 2eq) of 401- 3 were introduced. Cooled to -78°C, 0.81mL (2.03mmol, 2eq) of n-BuLi was added dropwise. After stirring at -78°C for 30 minutes, a solution of 500mg (1.01mmol, 1.0eq) of 401-2 in 5mL of THF was added. The reaction was then stirred at 25°C for 12 hours, as indicated by LC-MS completion. The mixture was diluted with 20mL water and the aqueous phase was extracted with EA (3 times, 5mL each), the organic layers were combined, washed with brine (3 times, 5mL each), dried over anhydrous sodium sulfate, filtered, and concentrated. Column chromatography purified the crude to yield 400mg of compound 401-4, a 65.2% yield. [0589] Step 5: To a 25mL flask, 7mL of dioxane, 340mg (0.561mmol, 1.0eq) of 401-4, 64.08mg (0.67mmol, 1.2eq) of MsNH4, 10.28mg (0.011mmol, 0.02eq) of Pd2(dba)3, 9.54mg (0.022mmol, 0.04eq) of tBuxphos, and 365.85mg (1.12mmol, 2eq) of Cs2CO3 were added. The mixture was stirred at 100°C for 5 hours. After confirmation of completion by LC-MS, it was concentrated under vacuum and purified via column chromatography to obtain 100mg of compound 401-5, yielding 35.2%. [0590] Step 6: A 5mL flask was prepared with 1mL DCM, 0.1mL TFA, 0.3mL TES, and 100mg (1.0eq) of 401-5. The mixture was stirred at 25°C for 2 hours, as shown by LC-MS completion. After concentration under vacuum, pre-HPLC purification yielded 10mg of compound 401, a 17.2% yield. Overall yield =2.5% 122 [0591] Example 18. Synthesis of Compound 502
[0592] Step 1: A 500mL three-necked flask was loaded with 250mL of ACN, 25g (0.15mol, leq) of 502-1, 31.5g (0.16mol, 1.05eq) of diethyl chloromalonate, and 43g (0.31mol, 2eq) of K2CO3. The reaction mixture was refluxed at 80°C overnight. After completion was confirmed by LC-MS, the mixture was concentrated under vacuum and purified via silica gel column chromatography to yield 40g of 502-2. The yield was 83%.
[0593] Step 2: In a IL three-necked flask, 250mL of DMF and 8g (0.21mol, 1 5eq, 60%) of NaH were combined and cooled to 0°C. Then, 40g (0.125mol, leq) of 502-2 dissolved in lOOmL of DMF was added at 0°C and stirred for 1 hour. Next, 30g (0.154mol, l.leq) of 3- (Bromomethyl)benzonitrile in lOOmL of DMF was added at 0°C, and the mixture was stirred at 58°C overnight. After completion (confirmed by LC-MS), the reaction was quenched with water, extracted with EA, dried over Na2SC>4, and concentrated. Purification by silica gel column chromatography yielded 36g of 502-3 with a 64% yield.
[0594] Step 3: A 500mL three-necked flask received 300mL of DMSO, 36g (0.08mol, leq) of 502-3, 9g (0.15mol, 2eq) of NaCl, and 11g (0.3mol, 4eq) of H2O. The mixture was stirred at 150°C overnight. LC-MS indicated the reaction was incomplete. The mixture was worked up similarly to previous steps and purified to yield 25g of 502-4 with an 86% yield.
[0595] Step 4: To a 500mL three-necked flask, 200mL of DMSO, 20g (0.055mol, leq) of 502-4, 13g (0.1 Imol, 2eq) of H2O2, and 15g (0.1 Imol, 2eq) of K2CO3 were added. The mixture was stirred at room temperature overnight and purified after standard work-up to yield 8g of 502-05. The yield was 38%. Attorney Docket No.: 071741.11025/5WO1 [0596] Step 5: A 100mL three-necked flask was charged with 40mL of EtOH, 8g (0.021mol, 1eq) of 502-5, and 12.6g (0.21mol, 10eq) of ethylenediamine. The mixture was stirred at room temperature overnight and purified to yield 7g of 502-6 with an 84% yield. [0597] Step 6: To a 25mL single-necked flask, 1g (2.53mmol, 1eq) of 502-6, 7.5mL of HMDS, and 0.5mL of TMSI were added. The mixture was stirred at 130°C overnight, concentrated under vacuum, then added to 2mL DCM and 1mL TFA, stirred for 1 hour at room temperature, and concentrated. Purification yielded 10mg of 502 with a final yield of 1%. Overall yield =0.15% [0598] Example 19. Synthesis of Compound 503 O O Br N H Br HCl Br O C , , , bromophenyl)acetic acid, 31.5g (0.16mol, 1.05eq) of diethyl chloromalonate, and 43g (0.31mol, 2eq) of K2CO3 were added. The mixture was refluxed overnight at 80°C. Following LC-MS confirmation of completion, it was concentrated and purified via silica gel chromatography, eluting with EtOAc/PE from 1/20 to 1/10 to yield 20g of 2-(3-bromophenyl)-N-methoxy-N- methylacetamide (503-1) as a yellow oil, with a 73.60% yield. [0600] Step 2: A solution of 1-bromo-2-methoxybenzene (28.99g, 155mmol) in dry THF (150mL) was cooled to -78°C, to which n-BuLi (2.5M in hexane, 62mL, 155mmol) was added dropwise. After stirring at -78°C for 30 minutes, a solution of 503-1 (20g, 77.5mmol) in dry THF 124 Attorney Docket No.: 071741.11025/5WO1 (100mL) was added dropwise. The solution was then allowed to warm to room temperature and stirred for 16 hours. The reaction was quenched with saturated NH4Cl solution, extracted with EtOAc, and purified via silica gel chromatography, eluting with EtOAc/PE at 1/50 to yield 10g of 2-(3-bromophenyl)-1-(2-methoxyphenyl)ethenone (503-2) as a yellow oil, with a 40.13% yield. [0601] Step 3: A solution of 503-2 (1.5g, 4.9mmol), PdCl2(dppf) (360mg, 0.49mmol), and sodium carbonate (1.04g, 9.8mmol) in toluene:MeOH (10mL, 1:1 ratio) was heated at 100°C for 3 days under CO atmosphere. The reaction was diluted with water, extracted with EtOAc, and purified via silica gel chromatography, eluting with EtOAc/PE from 1% to 10%, to yield 700mg of methyl 3-[2-(2-methoxyphenyl)-2-oxoethyl]benzoate (503-3) as a yellow oil, with a 46.94% yield. [0602] Step 4: To a solution of methyl 3-[2-(2-methoxyphenyl)-2-oxoethyl]benzoate (600mg, 2.1mmol) and O-methylhydroxylamine hydrochloride (264.38mg, 3.16mmol) in toluene (8mL), LiHMDS (1M, 8.4mL, 8.441mmol) was added and stirred at 25°C for 3 hours. After dilution with aqueous NH4Cl and extraction with EtOAc, the product was purified via silica gel chromatography, eluting with DCM/MeOH at 1/20, to yield 400mg of N-methoxy-3-[2-(2-methoxyphenyl)-2- oxoethyl]benzamide (503-4) as a yellow oil, with a 60.45% yield. [0603] Step 5: A solution of N-methoxy-3-[2-(2-methoxyphenyl)-2-oxoethyl]benzamide (400mg, 1.3364mmol) and NH4OAc (1.545g, 20.046mmol) in IPA (8.0mL) was stirred at 25°C for 30 minutes before adding NaBH3CN (335.92mg, 5.34mmol) and heated at 80°C for 3 hours. After adjusting the pH to 8 with 2M NaOH, the mixture was extracted with DCM and purified via silica gel chromatography, eluting with MeOH/DCM at 1/10, to yield 320mg of 3-[2-amino-2-(2- methoxyphenyl)ethyl]-N-methoxybenzamide (503-5) as a white solid, with a 71.75% yield. [0604] Step 6: To a solution of 3-[2-amino-2-(2-methoxyphenyl)ethyl]-N-methoxybenzamide (300mg, 0.9988mmol, 1eq) in DCM:DMF (5.0mL, 10:1 ratio), 1-chloro-2-isocyanatoethane (421.59mg, 3.9952mmol) was stirred at 25°C for 6 hours. After dilution with water and extraction with DCM, the combined organic phases were washed with brine, dried over sodium sulfate, and concentrated under vacuum. Without further purification, the crude product (250mg, 46.26% yield) was obtained as a yellow oil. [0605] Step 7: To a solution of 3-(2-{[(2-chloroethyl)carbamoyl]amino}-2-(2- methoxyphenyl)ethyl)-N-methoxybenzamide (200mg, 0.4927mmol) in water (5.0mL), the mixture was heated at 100°C for 3 hours. After cooling to room temperature, the mixture was purified by Biotage using a C18 column, eluting with 5% to 95% MeCN/H2O containing 0.1% NH4OH, to 125 Attorney Docket No.: 071741.11025/5WO1 afford 48.25mg of 3-[2-(4,5-dihydro-1,3-oxazol-2-ylamino)-2-(2-methoxyphenyl)ethyl]-N- methoxybenzamide (503) as a white solid, with a 24.65% yield. Overall yield =11.40% [0606] Example 20. Synthesis of Compound 504 Br H O H O C N InBr3,TMSN3 N n-BuLi,THF s2CO3,Pd2(dba)3 Br S 25°C,16h MF DCM,24°C S tBuxphos,D ,2h O O O + 110°C,16h [0607] Step 1: In a 250mL flask, 100mL of THF and 13.1g (0.056mol, 1.5eq) of 1,3- dibromobenzene were combined. After cooling to -78°C, 22.4mL (0.056mol, 1.5eq) of 2.5M n- BuLi was added. Stirred at -78°C for 1 hour, then a solution of 5g (0.0373mol, 1.0eq) of 2,3- dimethylbenzaldehyde in 10mL THF was introduced. The reaction continued at -78°C for another hour before warming to room temperature overnight. Post-completion, verified by LC-MS, the reaction was quenched with 25mL saturated ammonium chloride and the organic layer was separated, dried over Na2SO4, concentrated under vacuum, and purified via column chromatography (PE:EA=85:15) to yield 8.6g of 504-1, a 77.8% yield [0608] Step 2: A 50mL flask was charged with 20mL DMF, 1g (3.45mmol, 1.0eq) of 504-1, 492mg (5.175mmol, 1.5eq) of MsNH2, 2.25g (6.9mmol, 2.0eq) of Cs2CO3, 316mg (0.345mmol, 0.1eq) of Pd2(dba)3, and 293mg (0.69mmol, 0.2eq) of tBuxphos. Stirred at 110°C for 16 hours and completion confirmed by LC-MS, the reaction was quenched with ice water, extracted with EA, and dried over Na2SO4. Purification by column chromatography (PE:EA=1:1) yielded 180mg of 504-2, a 17% yield. [0609] Step 3: To a 5mL flask, 1.5mL DCM, 134mg (0.439mmol, 1.0eq) of 504-2, 76mg (0.659mmol, 1.5eq) of TMSN3, and 31mg (0.0878mmol, 0.2eq) of InBr3 were added. Stirred at 17°C for 2 hours, completion confirmed by LC-MS, concentrated under vacuum and purified by column chromatography (PE:EA=68:32) to yield 105mg of 504-3, a 72.4% yield. 126 Attorney Docket No.: 071741.11025/5WO1 [0610] Step 4: A 5mL flask received 1.5mL THF, 0.3mL water, 144mg (0.436mmol, 1.0eq) of 504-3, and 229mg (0.87mmol, 2.0eq) of PPh3. Stirred at 50°C for 16 hours, completion confirmed by LC-MS, concentrated under vacuum and purified by TLC (DCM:MeOH=20:1) to yield 62mg of 504-4, a 46.7% yield. [0611] Step 5: To a 5mL flask, 1mL dioxane, 52mg (0.171mmol, 1.0eq) of 504-4, and 60mg (0.5mmol, 2.9eq) of 2-chloroethyl isothiocyanate were added. Stirred at 80°C for 16 hours, completion confirmed by LC-MS, concentrated under vacuum and purified by pre-HPLC to yield 3.3mg of compound 504, a 5% yield. Overall yield: 2.2% [0612] Example 21. Synthesis of Compound 505 O S H2N O O O Br S O N NH OA N BH CN one (503-2, 2.0g, 6.6mmol), methanesulfonamide (0.75g, 7.92mmol), Pd(OAc)2 (150mg, 0.663mmol), Xantphos (0.76g, 1.32mmol), and Cs2CO3 (4.30g, 13.20mmol) in dioxane (20.0ml) was heated at 100°C for 16 hours under nitrogen. After cooling, it was diluted with aqueous NH4Cl and extracted with EtOAc. The organic layers were combined, washed with brine, dried over sodium sulfate, and concentrated. Purification by silica gel chromatography (MeOH/DCM, 1% to 10%) yielded N-{3- [2-(2-methoxyphenyl)-2-oxoethyl]phenyl}methanesulfonamide (505-1, 400mg, 16.17% yield) as a yellow oil. [0614] Step 2: A mixture of N-{3-[2-(2-methoxyphenyl)-2- oxoethyl]phenyl}methanesulfonamide (400mg, 0.5323mmol) and NH4OAc (1.448g, 18.785mmol) in isopropanol (8.0ml) was stirred at 25°C for 0.5 hour, then NaBH3CN (314.80mg, 5.0096mmol) 127 Attorney Docket No.: 071741.11025/5WO1 was added and the mixture was heated at 80°C for 4.5 hours. After cooling, the mixture was filtered through celite and concentrated. It was purified by preparative TLC (EtOAc/PE, 1/3) to obtain N-{3-[2-amino-2-(2-methoxyphenyl)ethyl]phenyl}methanesulfonamide (505-2, 240mg, 53.82% yield) as a yellow oil. [0615] Step 3: A solution of N-{3-[2-amino-2-(2- methoxyphenyl)ethyl]phenyl}methanesulfonamide (505-2, 160mg, 0.4994mmol) and 4,5-dihydro- 1H-imidazole-2-sulfonic acid (224.96mg, 1.4982mmol) in butanol:water (5:1 ratio, 3.0ml) was heated at 120°C for 2 hours in a microwave reactor. After cooling, the mixture was concentrated, diluted with water, and extracted with EtOAc. The organic phases were combined, washed with brine, dried over sodium sulfate, concentrated, and purified by Biotage using a C18 column (eluting with 10% to 95% MeCN/H2O, containing 0.1% TFA) to yield N-{3-[2-(imidazolidin-2- ylideneamino)-2-(2-methoxyphenyl)ethyl]phenyl}methanesulfonamide (505, 8.38mg, 4.31% yield) as a white solid. [0616] Example 22. Synthesis of Compound 510 TMSCN TBAF EtOH MeOH O Br Br OH THF N 30%KOH SOCl2 LDA O O + THF HCl , , , , ethylbenzyl bromide, 2g (0.02mmol, 2.0eq) of TMSCN, and 20mL (0.05mmol, 2.0eq) of TBAF in THF were combined and refluxed for 1.5 hours. After LC-MS confirmation of completion, the mixture was concentrated and purified by column chromatography (PE:EA=85:15) to yield 1.42g of 510-1, a 97.9% yield. [0618] Step 2: In a 100mL flask, 28mL of ethanol and 5.7mL of 30% KOH solution were added to 1.42g (0.0098mol, 1.0eq) of 510-1. The mixture was refluxed for 18 hours, concentrated, diluted 128 Attorney Docket No.: 071741.11025/5WO1 with 20mL water, adjusted to pH=2 with 6M HCl, filtered, and dried to yield 1.35g of 510-2 as a solid, an 84.0% yield. [0619] Step 3: A 50mL flask received 15mL of methanol and 1.3g (0.008mol, 1.0eq) of 510-2, cooled to 0°C, then 1.89g (0.0159mol, 1.5eq) of SOCl2 was added. The reaction was stirred at 60°C for 18 hours, concentrated, and purified by column chromatography (PE:EA=68:32) to yield 1.37g of 510-3, a 96.2% yield. [0620] Step 4: In a 25mL bottle, 10mL of THF, 497mg (2.81mmol, 1.0eq) of 510-3 were cooled to -80°C under nitrogen. LDA (3.37mL, 3.37mmol, 1.2eq) was added, followed by 731mg (2.95mmol, 1.05eq) of 823-78-9 in 2mL THF. The mixture was warmed to room temperature overnight, quenched, and purified by column chromatography to yield 670mg of 510-4, a 68.9% yield. [0621] Step 5: A 50mL bottle received 10mL of DMF, 570mg (1.65mmol, 1.0eq) of 510-4, 1.07g (3.3mmol, 2.0eq) of Cs2CO3, 235mg (2.47mmol, 1.5eq) of methanesulfonamide, 151mg (0.165mmol, 0.1eq) of Pd2(dba)3, and 141mg (0.33mmol, 0.2eq) of t-BuXphos. Stirred at 105°C for 2 hours under nitrogen, the reaction was worked up and purified to yield 600mg of 510-5, an 85.7% yield. [0622] Step 6: In a 10mL bottle, 5mL of toluene, 200mg (0.554mmol, 1.0eq) of 510-5, 166.5mg (2.77mmol, 5.0eq) of ethylenediamine, and TMAl (1.39mL, 2.77mmol, 5.0eq) were stirred at 110°C overnight. After cooling and working up, the crude was purified to yield 29mg of 510-6, a 13.5% yield. [0623] Step 7: A 5mL bottle received 1mL of toluene and 29mg (0.0745mmol, 1.0eq) of 510-6, then 57mg (0.37mmol, 5.0eq) of POCl3 was added. Stirred at 110°C for 3 hours, the mixture was filtered and purified to yield 3.6mg of 510, an 11.9% yield. 129 Attorney Docket No.: 071741.11025/5WO1 [0624] Example 23. Synthesis of Compound B24 Potassium ethenylt- rifluoroborate O TosMIC, t-BuOK, CN Pd(PPh3)4 , Na 2 CO 3 CN KOH, EtOH, H2O O MeI, K2CO3 DCE, rt, 12h 1,4-Dioxane, H2 O 100o OH C, 4h DMF, rt, 12h bromoindanone (10 g, 47.38 mmol, 1.0 equiv), and TosMIC (13.88 g, 71.07 mmol, 1.5 equiv) were added. The solution was stirred for 5 minutes, and then t-BuOK (10.63 g, 94.76 mmol, 2.0 equiv) was added at 25°C. The solution was stirred at ambient temperature for 12 hours. LC-MS analysis confirmed the reaction was complete. The solution was poured into 500 mL of ice water and extracted with DCM (200 mL × 2). The organic phase was dried over Na₂SO₄, filtered, and concentrated under vacuum to afford a black oil. The crude product was purified by column chromatography (PE/EA = 90:10) to give 3.5 g of compound B24-1 as an orange oil. Yield: 33.26%. [0626] Step 2: To a 100 mL single-necked flask, 60 mL of dioxane, 6 mL of water, compound B24-1 (3 g, 13.51 mmol, 1.0 equiv), potassium ethenyltrifluoroborate (2.71 g, 20.26 mmol, 1.5 equiv), Na₂CO₃ (2.15 g, 20.26 mmol, 1.5 equiv), and Pd(PPh₃)₄ (0.78 g, 0.68 mmol, 0.05 equiv) were added. The solution was stirred at 90°C under nitrogen for 12 hours. TLC analysis confirmed the reaction was complete. The mixture was poured into 300 mL of water and extracted with ethyl acetate (200 mL × 2). The organic phase was dried over Na₂SO₄, filtered, and concentrated under 130 Attorney Docket No.: 071741.11025/5WO1 vacuum to afford 5 g of B24-2 as an orange oil, which was used in the next step without further purification. Yield: 100%. [0627] Step 3: To a 100 mL single-necked flask, 50 mL of ethanol, compound B24-2 (5 g, 13.53 mmol, 1.0 equiv), 7.59 g of water, and KOH (7.59 g, 135.32 mmol, 10.0 equiv) were added. The reaction mixture was stirred at 100°C for 4 hours. LC-MS analysis confirmed the reaction was complete. The solution was poured into 300 mL of water and extracted with 100 mL of ethyl acetate. The aqueous phase was adjusted to pH 1 with concentrated HCl and then extracted with DCM (150 mL × 2). The DCM phase was dried over Na₂SO₄, filtered, and concentrated under vacuum to afford 3.9 g of compound B24-3 as an orange oil, which was used in the next step without further purification. Yield: 100%. [0628] Step 4: To a 100 mL single-necked flask, 40 mL of DMF, compound B24-3 (3.9 g, 13.55 mmol, 1.0 equiv), and K₂CO₃ (3.74 g, 27.10 mmol, 2.0 equiv) were added. Methyl iodide (3.85 g, 27.10 mmol, 2.0 equiv) was then added to the stirred solution. The mixture was stirred at ambient temperature for 12 hours. LC-MS analysis confirmed the reaction was complete. The mixture was poured into 300 mL of water and extracted with MTBE (100 mL × 2). The organic phase was washed with 100 mL of brine, dried over Na₂SO₄, filtered, and concentrated under vacuum to afford the crude oil. The crude product was purified by column chromatography (PE/EA = 95:5) to give 1.8 g of compound B24-4 as a colorless liquid. Yield: 65.69%. [0629] Step 5: To a 25 mL three-necked flask, 10 mL of DCM and compound B24-4 (2 g, 9.89 mmol, 1.0 equiv) were added. Ozone was bubbled into the reaction mixture for 10 minutes. TLC analysis confirmed the reaction was complete. The solution was dried over Na₂SO₄, filtered, and concentrated under vacuum to afford the crude oil. The crude product was purified by column chromatography (PE/EA = 90:10) to give 1.1 g of compound B24-5 as a yellow oil. Yield: 54.47%. [0630] Step 6: To a 25 mL three-necked flask, 100 mL of THF, (2,3-dimethylphenyl)(1-trityl-4- imidazolyl)methanone (300 mg, 0.68 mmol, 1.0 equiv), compound B24-5 (139 mg, 0.68 mmol, 1.0 equiv), and zinc powder (177 mg, 2.71 mmol, 4.0 equiv) were added. The solution was charged with nitrogen, and TiCl₄ (257 mg, 1.36 mmol, 2.0 equiv) was added dropwise at 10°C. The reaction mixture was stirred at 80°C for 3 hours. LC-MS analysis confirmed the reaction was complete. The mixture was cooled and poured into a mixture of 20 mL of ethyl acetate and 50 mL of saturated sodium bicarbonate solution. It was stirred for 5 minutes and filtered. The aqueous phase was 131 Attorney Docket No.: 071741.11025/5WO1 extracted with ethyl acetate, and the organic phase was dried over Na₂SO₄, filtered, and concentrated under vacuum. The residue was purified by column chromatography (DCM/MeOH = 90:10) to give 152 mg of compound B24-6 as a yellow foam solid. Yield: 36.47%. [0631] Step 7: To a 100 mL single-necked flask, 8 mL of THF, 8 mL of methanol, compound B24-6 (150 mg, 0.40 mmol, 1.0 equiv), and Pd(OH)₂/C (80 mg) were added. The solution was stirred at 40°C under a hydrogen atmosphere for 12 hours. LC-MS analysis confirmed the reaction was complete. The solution was filtered and concentrated under vacuum. The residue was purified by preparative TLC (ethyl acetate) to afford 72 mg of compound B24-7 as a white solid. Yield: 47.74%. [0632] Step 8: To a 10 mL single-necked flask, compound B24-7 (72 mg, 0.19 mmol, 1.0 equiv), 0.1 mL of water, 1 mL of ethanol, and NaOH (23 mg, 0.58 mmol, 3.0 equiv) were added. The reaction mixture was stirred at room temperature for 3 hours. LC-MS analysis confirmed the reaction was complete. To the reaction mixture, 0.5 mL of 2 N HCl was added, and the mixture was stirred for 5 minutes. The solution was concentrated under vacuum, and the residue was purified by preparative HPLC to give 24 mg of compound B24 as a white solid. Yield: 31.45%. [0633] Example 24. Synthesis of Compound B27 Bn O N N FA 5 [0634] Step 1: A mixture of pyrazole (60 g, 0.881 mol, 1.0 equiv) and 3-chloroperoxybenzoic acid (152.1 g, 0.881 mol, 1.0 equiv) in ethyl acetate (3 L) was stirred at room temperature for 10 days. LC-MS showed the reaction was incomplete. The reaction mixture was concentrated and quenched with saturated sodium sulfite solution. The organic layer was washed with saturated 132 Attorney Docket No.: 071741.11025/5WO1 sodium bicarbonate solution, dried over Na₂SO₄, filtered, and concentrated to give compound B27- 01 (8 g) as a purple liquid. Yield: 10.8%. [0635] Step 2: A mixture of compound 27-01 (4.5 g, 0.0536 mol, 1.0 equiv), benzyl bromide (9.2 g, 0.0541 mol, 1.01 equiv), and DIPEA (7 g, 0.0541 mol, 1.01 equiv) in DCM (54 mL) was stirred at room temperature for 16 hours. LC-MS showed the reaction was complete. The reaction mixture was concentrated under vacuum, and the crude product was purified by silica gel column chromatography (PE/EA = 96:4) to give compound B27-02 (7.57 g) as a colorless liquid. Yield: 81.1%. [0636] Step 3: To a mixture of compound 27-02 (1 g, 5.74 mmol, 1.0 equiv) in 15 mL of THF was added n-BuLi (2.5 M, 2.5 mL, 6.31 mmol, 1.1 equiv) at -78°C. The reaction was stirred at - 78°C for 2 hours under nitrogen, then tributyltin chloride (2.06 g, 6.31 mmol, 1.1 equiv) was added at -78°C. The reaction was stirred at room temperature for 16 hours. LC-MS showed the reaction was complete. The reaction mixture was quenched with saturated ammonium chloride (5 mL) and extracted with ethyl acetate. The organic layer was dried over Na₂SO₄, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography (PE/EA = 92:8) to give compound B27-03 (1.7 g) as a colorless liquid. Yield: 63.8%. [0637] Step 4: A mixture of compound 27-03 (1.16 g, 1.95 mmol, 1.0 equiv), (2,3- dimethylphenyl)(1-trityl-4-imidazolyl)methanone (1 g, 2.15 mmol, 1.1 equiv), and Pd(PPh₃)₂Cl₂ (68 mg, 0.0975 mmol, 0.05 equiv) in toluene (10 mL) was stirred at 95°C for 16 hours. LC-MS showed the reaction was complete. The reaction mixture was filtered, and the organic solution was concentrated under vacuum. The crude product was purified by silica gel column chromatography (PE/EA = 3:1) to give compound compound B27-04 (1.19 g) as a white solid. Yield: 88.7%. [0638] Step 5: A mixture of compound B27-04 (1.19 g, 1.0 equiv) and TFA (5 mL) in DCM (10 mL) was stirred at room temperature for 1 hour. LC-MS showed the reaction was complete. The reaction mixture was concentrated under vacuum, and the crude product was purified by silica gel column chromatography (MeOH/DCM = 13:87) to give compound B27-05 (550 mg) as a white solid. Yield: 56.8%. [0639] Step 6: A mixture of compound B27-05 (290 mg, 0.518 mmol, 1.0 equiv), TEA (157 mg, 1.554 mmol, 3.0 equiv), and (Boc)₂O (169 mg, 0.777 mmol, 1.5 equiv) in THF (3 mL) was stirred at room temperature for 16 hours. LC-MS showed the reaction was complete. The reaction mixture 133 Attorney Docket No.: 071741.11025/5WO1 was concentrated, and the crude product was purified by silica gel column chromatography (PE/EA = 3:1) to give compound B27-06 (243 mg) as a white solid. Yield: 85.9%. [0640] Step 7: A mixture of compound B27-06 (243 mg) and Pd(OH)₂ (243 mg) in THF (2.5 mL) and methanol (2.5 mL) was stirred at 45°C for 16 hours. LC-MS showed the reaction was complete. The reaction mixture was filtered, and the organic solution was concentrated under vacuum. The crude product was purified by preparative TLC (PE/EA = 5:1) to give compound B27-07 (130 mg) as a colorless liquid. Yield: 66.1%. [0641] Step 8: To a mixture of compound B27-07 (110 mg, 0.249 mmol, 1.0 equiv) in 2.5 mL of THF, NaH (11 mg, 0.274 mmol, 1.1 equiv) was added at 0°C. The reaction was stirred at room temperature for 0.5 hours under nitrogen, then BPO (6 mg, 0.0249 mmol, 0.1 equiv) was added. The mixture was stirred at room temperature for 5 days. LC-MS showed the reaction was incomplete. The reaction mixture was quenched with ice water (3 mL) and adjusted to pH 6 with 1 M HCl. The mixture was extracted with ethyl acetate, and the organic layer was dried over Na₂SO₄, filtered, and concentrated under vacuum. The residue was added to 2 mL of DCM and 1 mL of TFA, then stirred at room temperature for 0.4 hours. The mixture was concentrated, and the crude product was purified by preparative HPLC to give compound B27 (2 mg) as a white solid. Yield: 1.7%. [0642] Example 25. Synthesis of Compound B34 O O O O O O O P O H [06 3] Step : 50 m react on as conta n ng a mxture o -[( ,3-d met y p enyl)carbonyl]- 1-(triphenylmethyl)imidazole (500 mg, 1.12 mmol), 1,4-dioxaspiro[4.5]decane-8-carbaldehyde 134 Attorney Docket No.: 071741.11025/5WO1 (192.30 mg, 1.12 mmol), and Zn (738.99 mg, 11.29 mmol) in THF (10 mL) was cooled to 0°C. TiCl₄ (1.2 mL, 11.29 mmol) was added dropwise at 0°C. The reaction mixture was stirred at 0°C for 30 minutes and then at 70°C for 4 hours. LC-MS analysis confirmed the reaction was complete. The mixture was diluted with water and extracted with EA. The combined organic phases were washed with brine, dried over Na₂SO₄, and concentrated under vacuum. The crude product was purified by flash column chromatography (DCM/MeOH = 50:1 to 5:1) to afford B34-1 (160 mg) as a yellow solid. Yield: 48.19%. [0644] Step 2: A 50 mL reaction flask containing NaH (407.61 mg, 10.19 mmol) in DMF (5 mL) was cooled to 0°C under a nitrogen atmosphere. Ethyl 2-(diethoxyphosphoryl)propanoate (2.43 g, 10.19 mmol) in DMF (7 mL) was added dropwise. The mixture was stirred at 0°C for 1 hour. A solution of B34-1 (300 mg, 1.0191 mmol) in DMF (3 mL) was added dropwise at 0°C, and the reaction mixture was stirred at 90°C for 2 hours. LC-MS confirmed the reaction was complete. The mixture was quenched with saturated NH₄Cl solution (100 mL) and extracted with EA (3 × 50 mL). The combined organic phases were washed with brine (2 × 80 mL), dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by silica gel column chromatography (MeOH/DCM = 4%) to afford B34-2 (350 mg) as a yellow solid. Yield: 83.4%. [0645] Step 3: A sealed tube containing a solution of B34-2 (350 mg, 0.9247 mmol) in ethanol (10 mL), Pd/C (350 mg, 3.2889 mmol), and Pd(OH)₂/C (350 mg, 2.4923 mmol) was evacuated and backfilled with hydrogen three times, then charged with hydrogen (3 MPa). The reaction mixture was stirred at 50°C for 16 hours under a hydrogen atmosphere. LC-MS confirmed the reaction was complete. The mixture was filtered, and the filter cake was washed with ethanol (30 mL). The filtrate was concentrated to dryness to afford B34-3 (300 mg) as a colorless oil. Yield: 78.03%. [0646] Step 4: A 10 mL reaction flask containing a solution of B34-3 (200 mg, 0.5228 mmol) in THF (3 mL), H₂O (1.5 mL), and methanol (3 mL) was stirred with NaOH (41.82 mg, 1.4056 mmol) at 25°C for 12 hours. LC-MS confirmed the reaction was complete. The solvent was removed, and the mixture was diluted with water (100 mL) and adjusted to pH 4 with 1 M HCl. The mixture was extracted with EA (30 mL), and the organic phase was dried and concentrated. The crude product was purified by preparative HPLC (Gemini column, ACN-H₂O with 0.05% NH₄OH) to give B34 (40 mg) as a white solid. Yield: 20.7%. 135 Attorney Docket No.: 071741.11025/5WO1 [0647] Example 26. Synthesis of Compound B37 O N 3 N O HO OH O Trt C, 0 g, 140.7 mmol), ethane-1,2-diol (17.47 g, 281.4 mmol), and 4-methylbenzenesulfonic acid (2.42 g, 14 mmol) in toluene (200 mL) was heated at 110°C under a nitrogen atmosphere for 4 hours. After cooling to ambient temperature, the reaction mixture was quenched with NaHCO₃ (200 mL) and extracted with EtOAc (3 × 500 mL). The combined organic layers were washed with brine, dried with sodium sulfate, and concentrated under vacuum. The residue was purified by flash chromatography (0–25% EtOAc in hexanes) to afford B37-1 (3.2 g, 16.3 mmol) as a yellow oil. Yield: 12%. [0649] Step 2: A solution of B37-1 (3.7 g, 19.9 mmol) in THF (30 mL) was cooled to 0°C, and LAH (39.8 mL, 39.8 mmol) was added dropwise. The mixture was stirred at 25°C for 3 hours. The reaction was quenched with water (20 mL), 15% NaOH (20 mL), and water (60 mL). The organic phase was extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine, dried with sodium sulfate, and concentrated under vacuum to afford B37-2 (2.1 g, 12.6 mmol) as a yellow oil. Yield: 63.32%. [0650] Step 3: A mixture of B37-2 (1 g, 6.3 mmol) in DCM (10 mL) and Dess-Martin periodinane (3.21 g, 7.5 mmol) was stirred at 25°C for 2 hours. TLC showed the reaction was complete. The mixture was quenched with NaHCO₃ (30 mL) and extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine, dried with sodium sulfate, and concentrated under vacuum. The residue was purified by flash chromatography (0–30% EtOAc in hexanes) to afford B37-3 (0.6 g, 3.6 mmol) as a yellow oil. Yield: 57.14%. 136 Attorney Docket No.: 071741.11025/5WO1 [0651] Step 4: A mixture of 4-[(2,3-dimethylphenyl)carbonyl]-1-(triphenylmethyl)imidazole (1700.15 mg, 3.84 mmol), B37-3 (600 mg, 3.84 mmol), and Zn (1507.69 mg, 23.05 mmol) in THF (20 mL) was cooled to 0°C. TiCl₄ (4372.16 mg, 23.05 mmol) was added dropwise. The mixture was stirred for 1 hour at 0°C and then heated to 70°C for 3 hours. LC-MS showed the reaction was complete. The mixture was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine, dried with sodium sulfate, and concentrated under vacuum. The residue was purified by flash chromatography (0–5% MeOH in DCM) to afford B37-4 (200 mg, 0.57 mmol) as a yellow solid. Yield: 15%. [0652] Step 5: A mixture of TosMIC (208.91 mg, 1.07 mmol) and t-BuOK (350.20 mg, 3.12 mmol) in DMSO (3 mL) was stirred at 25°C for 30 minutes. LC-MS showed the reaction was complete. The mixture was quenched with water, and MeOH (66.09 mg, 0.8917 mmol) and B37-4 (250 mg, 0.8917 mmol) in DMSO (2 mL) were added dropwise. The reaction mixture was stirred at 25°C for 16 hours. LC-MS showed the reaction was complete. The mixture was quenched with water (30 mL) and extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine, dried with sodium sulfate, and concentrated under vacuum to afford B37-5 (200 mg, 0.5834 mmol) as a yellow solid. Yield: 65%. [0653] Step 6: A mixture of B37-5 (120 mg, 0.4118 mmol) in 5 N KOH (5 mL) was heated at 80°C for 16 hours. LC-MS showed the reaction was complete. The mixture was quenched with water (20 mL) and extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with water and brine, dried with sodium sulfate, and concentrated under vacuum to afford B37-6 (90 mg, 0.2755 mmol) as a white solid. Yield: 67%. [0654] Step 7: A mixture of B37-6 (90 mg, 0.29 mmol), Pd/C (92.59 mg, 0.87 mmol), and Pd(OH)₂/C (122.17 mg, 0.87 mmol) in methanol (3 mL) was heated at 50°C under a hydrogen atmosphere (3 MPa) for 16 hours in a high-pressure reactor. After cooling to ambient temperature, the mixture was filtered through Celite, and the filtrate was concentrated under vacuum. The residue was purified on a Biotage Isolera One (C18 column, eluting with 10–90% MeCN/H₂O containing 0.1% TFA) to provide B37 (33.01 mg, 0.1046 mmol) as a white solid. Yield: 36%. 137 Attorney Docket No.: 071741.11025/5WO1 [0655] Example 27. Synthesis of Compound B40-A and B40-B Pd(OAc)2,dppp O Py,Tf2O,DCM TEA,CO,DMSO O Pd(PPh3)4,dioxane O DMP,DCM OH rt,2h OTf MeOH,78°C,7h 85°C,16h rt,1H O O ep2 s O step4 O step1 st tep3 Br Br B HO O OH HCl ers1 OH Cl rs2 pyridine (11.87 g, 0.15 mol, 3.0 eq) in DCM (100 mL) was cooled to 0°C. Trifluoromethanesulfonic anhydride (23.97 g, 0.085 mol, 1.7 eq) was added at 0°C, and the mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. LC-MS analysis confirmed the reaction was complete. The reaction mixture was poured into 100 mL of ice water and extracted with DCM. The organic phase was washed with brine, dried over Na₂SO₄, and concentrated under vacuum. The residue was purified by silica gel column chromatography to afford compound B40-01 (15.53 g) as a colorless liquid. Yield: 93.6%. [0657] Step 2: A mixture of B40-01 (8 g, 0.024 mol, 1.0 eq), TEA (4.62 g, 0.0461 mol, 1.9 eq), Pd(OAc)₂ (1.1 g, 0.00482 mmol, 0.2 eq), and dppp (1.88 g, 0.0058 mmol, 0.24 eq) in a 1:1 mixture of DMSO and MeOH (260 mL) was stirred at 78°C under a 5 MPa CO atmosphere for 7 hours. LC-MS analysis confirmed the reaction was complete. The reaction mixture was poured into 1.2 L of ice water and extracted with DCM. The organic phase was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by silica gel column chromatography to afford compound B40-02. [0658] Step 3: A mixture of B40-02 (3.357 g, 0.0139 mol, 1.0 eq), (tributylstannyl)methanol (6.68 g, 0.0208 mol, 1.5 eq), and Pd(PPh₃)₄ (1.2 g, 0.104 mmol, 0.075 eq) in dioxane (40 mL) was stirred at 85°C for 16 hours under a nitrogen atmosphere. LC-MS analysis showed the reaction was 138 Attorney Docket No.: 071741.11025/5WO1 incomplete. The mixture was concentrated under vacuum, and the residue was purified by silica gel column chromatography to afford compound B40-03 (674 mg) as a white solid. Yield: 25%. [0659] Step 4: A mixture of B40-03 (610 mg, 3.14 mmol, 1.0 eq) and Dess-Martin periodinane (DMP) (2 g, 4.71 mmol, 1.5 eq) in DCM (6 mL) was stirred at room temperature for 1 hour under a nitrogen atmosphere. LC-MS analysis confirmed the reaction was complete. The reaction mixture was poured into 6 mL of saturated sodium bicarbonate solution, filtered, and extracted with DCM. The organic phase was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by silica gel column chromatography to afford compound B40-04 (496 mg) as a white solid. Yield: 82.3%. [0660] Step 5: A mixture of B40-04 (496 mg, 2.58 mmol, 1.0 eq), (2,3-dimethylphenyl)(1-trityl- 4-imidazolyl)methanone (1.14 g, 2.58 mmol, 1.0 eq), and Zn (1.26 g, 19.35 mmol, 7.5 eq) in THF (10 mL) was cooled to 0°C, and TiCl₄ (1.81 g, 9.55 mmol, 3.7 eq) was added dropwise. The mixture was stirred at 80°C for 1 hour under a nitrogen atmosphere. LC-MS analysis confirmed the reaction was complete. The mixture was poured into 50 mL of ice water, filtered, and extracted with ethyl acetate (EA). The organic phase was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by silica gel column chromatography to afford compound B40-05 (210 mg) as a white solid. Yield: 13.5%. [0661] Step 6: A mixture of B40-05 (210 mg, 1 eq) and Pd(OH)₂ (210 mg, 1.0 wt) in a 1:1 mixture of MeOH and THF (6 mL) was stirred at 45°C under a hydrogen atmosphere overnight. LC-MS analysis confirmed the reaction was complete. The mixture was filtered and concentrated under vacuum to afford compound B40-06 (82.5 mg) as a white solid. Yield: 65.3%. [0662] Step 7: A total of 285 mg of B40-06 was separated on a column with dimensions 30 × 250 mm packed with CHIRALPAK® IB-10 (10 µm particle size). A mixture of 70% mobile phase A (CO₂) and 30% mobile phase B (0.2% ammonia in MeOH/EtOH) was used. The operational conditions were: 35°C, flow rate 3 g/min, detection at 214 nm and 254 nm. The first eluting enantiomer, B40-06-P1 (125 mg), was isolated with an enantiomeric excess of 100%. Yield: 87.7%. The second eluting enantiomer, B40-06-P2 (139 mg), was isolated with an enantiomeric excess of 100%. Yield: 97.5%. [0663] Step 8: A mixture of B40-06-P1 (125 mg, 0.345 mmol, 1.0 eq) and NaOH (55 mg, 1.38 mmol, 4.0 eq) in a 2:2:1 mixture of MeOH, THF, and H₂O (2 mL) was stirred at 45°C for 16 hours under a nitrogen atmosphere. LC-MS analysis confirmed the reaction was complete. The mixture 139 Attorney Docket No.: 071741.11025/5WO1 was concentrated under vacuum, adjusted to pH 1 with 1 M HCl, and concentrated again under vacuum. The residue was purified by preparative HPLC to afford compound B40-A (86.1 mg) as a white solid. Yield: 65%. Overall yield: 0.6%. [0664] Step 8A: A mixture of B41-06-P2 (109mg, 0.345mmol, 1.0eq), NaOH (55mg, 1.38mmol, 4.0eq) in MeOH/THF/H2O=2/2/1 (2mL) was stirred at 45°C for 16h under N2. LCMS showed the reaction was completed. The reaction was concentrated under vacuum. The crude was adjusted to pH=1 with 1M HCl. The mixture was concentrated under vacuum and the residue was purified by prep-Hplc to give the compound B41-B (80.8mg) as a white solid. Yield: 70% Overall yield: 0.7% [0665] Example 28. Synthesis of Compound B43 Br Br 1.1eq benzyl mercaptan, O 1eqAG28915-02,2.4eqAG28 2eq DIPEA, Br N 1 Z Ti l4 THF o915-01, C1h+3h 1.2eqZnCN,2eqTEA, 01e Pd2(dba)3 , 1, [0666] Step 1: To a 250 mL reaction flask was added 60 mL THF, Zn (6.4 g, 98.74 mmol, 19 eq), cooled to 0°C, slowly added TiCl₄ (9.5 g, 49.89 mmol, 9.6 eq) while controlling the temperature at 0-10°C. The mixture was refluxed for 1 hour, cooled to room temperature, and then a solution of 4- 140 Attorney Docket No.: 071741.11025/5WO1 bromo-3-methyl-benzaldehyde (2.5 g, 12.56 mmol, 2.4 eq) and (2,3-dimethylphenyl)[1-(trityl)-1H- imidazol-4-yl]methanone (2.3 g, 5.23 mmol, 1 eq) in 40 mL THF was added slowly. The reaction was refluxed for 3 hours. LC-MS showed the reaction was completed. The mixture was filtered and concentrated under vacuum. The residue was purified by silica gel column chromatography (DCM/MeOH = 94/6) to afford B43-03 (1.9 g). Yield: 100%. [0667] Step 2: To a 100 mL reaction flask was added 36 mL DMF, B43-03 (1.85 g, 5.03 mmol, 1.0 eq), triphenylmethyl chloride (1.68 g, 6.04 mmol, 1.2 eq), and TEA (1.02 g, 10.07 mmol, 2 eq). The reaction mixture was stirred at 25°C for 15 hours. LC-MS showed the reaction was completed. The mixture was poured into water and extracted with EA. The residue was purified by silica gel column chromatography (PE/EA = 93/7) to afford B43-04 (2.1 g). Yield: 68.4%. [0668] Step 3: To a 50 mL reaction flask was added 20 mL toluene, B43-04 (1 g, 1.64 mmol, 1.0 eq), DIPEA (424 mg, 3.28 mmol, 2 eq), benzyl mercaptan (224 mg, 1.80 mmol, 1.1 eq), Pd₂(dba)₃ (150 mg, 0.164 mmol, 0.1 eq), and xantphos (190 mg, 0.328 mmol, 0.2 eq). The reaction mixture was stirred at 110°C for 15 hours. LC-MS showed the reaction was completed. The mixture was poured into water and extracted with EA. The residue was purified by silica gel column chromatography (PE/EA = 89/11) to afford B43-05 (720 mg). Yield: 67.2%. [0669] Step 4: To a 25 mL reaction flask was added 6 mL acetic acid and 2 mL water, B43-05 (400 mg, 0.612 mmol, 1 eq). After the mixture was cooled to 0°C, NCS (409 mg, 3.06 mmol, 5 eq) was added. The reaction mixture was stirred at 20°C for 2 hours. LC-MS showed the reaction was completed. The mixture was poured into water and extracted with EA. The organic phase was dried over Na₂SO₄ and concentrated under vacuum to afford B43-06 (520 mg). Yield: 100%. [0670] Step 5: To a 25 mL three-necked flask was added 5 mL DCM, B43-06 (theoretical yield: 385 mg, 0.612 mmol, 1 eq), TEA (620 mg, 6.12 mmol, 10 eq), and methyl 3-aminopropionate hydrochloride (427 mg, 3.06 mmol, 5 eq). The reaction mixture was stirred at 25°C for 2 hours. LC-MS showed the reaction was completed. The mixture was poured into water and extracted with DCM. The residue was purified by silica gel column chromatography to afford B43-07 (130 mg). Yield: 30.5%. [0671] Step 6: A mixture of B43-07 (130 mg), Pd(OH)₂/C (50 mg), CH₃OH (7 mL), and THF (7 mL) was stirred at 40°C for 4 days under a hydrogen atmosphere. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to afford B43-08 (50 mg). Yield: 58.7%. 141 Attorney Docket No.: 071741.11025/5WO1 [0672] Step 7: A mixture of B43-08 (50 mg, 0.109 mmol, 1 eq), NaOH (9 mg, 0.219 mmol, 2 eq), CH₃OH (4 mL), THF (4 mL), and H₂O (2 mL) was stirred at 40°C for 18 hours. LC-MS showed the reaction was completed. The mixture was concentrated under vacuum. Then 10 mL DCM and 5 mL TFA were added, and the solution was stirred for 10 minutes before evaporating the solvent. The residue was purified by preparative HPLC to give B43 (25 mg) as a white solid. Yield: 41.0%. Overall yield: 3.37%. [0673] Example 29. Synthesis of Compound B50 O O ydride h eOH , , , , g, 63 mmol, 3 eq), and MeI (6 g, 42 mmol, 2 eq) in DMF (50 mL) was stirred at 30°C overnight under N₂. LC-MS showed the reaction was completed. The reaction mixture was diluted with water (100 mL) and extracted with EA (100 mL × 2). The organic phase was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-01 (5 g). Yield: 95.24%. 142 Attorney Docket No.: 071741.11025/5WO1 [0675] Step 2: A mixture of B50-01 (5 g, 20 mmol, 1 eq), 2,4-Dimethoxyaniline (3.7 g, 22 mmol, 1.1 eq), and K₂CO₃ (4 g, 30 mmol, 1.5 eq) in DMF (50 mL) was stirred at 110°C overnight under N₂. LC-MS showed the reaction was completed. The reaction mixture was diluted with water (50 mL) and extracted with EA (50 mL × 2). The organic phase was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-02 (6 g). Yield: 70%. [0676] Step 3: A mixture of B50-02 (5 g, 13 mmol, 1 eq) and TFA (40 mL) in DCM (50 mL) was stirred at 30°C for 2 hours. LC-MS showed the reaction was completed. The reaction mixture was concentrated under vacuum. The mixture was diluted with saturated sodium carbonate solution and extracted with DCM (50 mL × 1). The organic phase was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-03 (3.6 g). Yield: 100%. [0677] Step 4: A mixture of B50-03 (3.6 g, 15 mmol, 1 eq) and acetic anhydride (2.2 g, 21 mmol, 1.5 eq) in toluene (40 mL) was stirred at 90°C for 16 hours under N₂. LC-MS showed the reaction was completed. The reaction mixture was diluted with saturated sodium bicarbonate solution (20 mL) and extracted with EA (20 mL × 2). The organic phase was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-04 (3.7 g). Yield: 84.7%. [0678] Step 5: A mixture of B50-04 (3.7 g, 12.7 mmol, 1 eq), (tributylstannyl)methanol (6 g, 19 mmol, 1.5 eq), and Pd(PPh₃)₄ (733 mg, 0.635 mmol, 0.05 eq) in 1,4-dioxane (40 mL) was refluxed at 90°C overnight under N₂. LC-MS showed the reaction was incomplete. The mixture was filtered and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-05 (3.8 g). Yield: 77.6%. [0679] Step 6: To a solution of B50-05 (3.8 g, 15.8 mmol, 1 eq) in DCM (25 mL), Dess-Martin periodinane (8 g, 19 mmol, 1.2 eq) was added at 0°C. The reaction mixture was stirred for 2 hours at room temperature. LC-MS showed the reaction was completed. The mixture was filtered and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-06 (2.4 g). Yield: 63.6%. [0680] Step 7: To a solution of B50-06 (2.2 g, 9.2 mmol, 2.4 eq), Zn (1.7 g, 26.6 mmol, 7 eq), and (2,3-Dimethylphenyl)(1-trityl-4-imidazolyl)methanone (1.7 g, 3.8 mmol, 1 eq) in THF (170 mL), titanium tetrachloride (2.5 g, 13.3 mmol, 3.5 eq) was added at 0°C. The reaction mixture was 143 Attorney Docket No.: 071741.11025/5WO1 stirred for 3 hours at 80°C. LC-MS showed the reaction was completed. The reaction mixture was diluted with water (300 mL) and extracted with EA (100 mL × 2). The organic phase was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-07 (920 mg). Yield: 51.6%. [0681] Step 8: A mixture of B50-07 (800 mg, 1.96 mmol, 1 eq), Pd(OH)₂/C (700 mg), TFA (200 mg), and water (10 drops) in MeOH (16 mL) was stirred at 50°C for 16 hours under H₂. LC-MS showed the reaction was completed. The mixture was filtered and concentrated under vacuum to afford B50-08 (848 mg) as a crude product. It was used for the next step without further purification. Yield: 100%. [0682] Step 9: A mixture of B50-08 (848 mg, 2.07 mmol, 1 eq) and SOCl₂ (400 mg, 4.1 mmol, 2 eq) in MeOH (16 mL) was stirred at 80°C for 16 hours under N₂. LC-MS showed the reaction was completed. The reaction mixture was concentrated under vacuum. The mixture was diluted with saturated sodium bicarbonate solution and extracted with EA (20 mL × 2). The organic phase was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-09 (330 mg). Yield: 41.3%. [0683] Step 10: A mixture of B50-09 (200 mg, 0.54 mmol, 1 eq) in 4 M HCl (6 mL) was stirred at 45°C for 0.5 hours. NaNO₂ (45 mg, 0.65 mmol, 1.2 eq) in water (0.5 mL) was added at 0°C. The reaction mixture was stirred for 1 hour at 0°C. CuCl (270 mg, 2.7 mmol, 5 eq) in water (0.5 mL) was added at 0°C. The reaction mixture was stirred for 2 hours at 30°C. LC-MS showed the reaction was completed. The mixture was diluted with saturated sodium bicarbonate solution and extracted with EA (20 mL × 2). The organic phase was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by column chromatography on silica gel to afford B50-10 (90 mg). Yield: 40.7%. [0684] Step 11: A mixture of B50-10 (85 mg, 0.22 mmol, 1 eq), LiOH (28 mg, 0.66 mmol, 3 eq), and water (1 mL) in MeOH (1 mL) was stirred at 30°C for 3 hours under N₂. LC-MS showed the reaction was completed. The mixture was concentrated under vacuum as a crude product. DCM:TFA = 2:1 (3 mL) was added, and the mixture was concentrated under vacuum. The residue was purified by preparative HPLC to afford B50 (30 mg). Yield: 36.7%. 144 Attorney Docket No.: 071741.11025/5WO1 [0685] Example 30. Synthesis of Compound B53 O O O KSC(S)OEt, acetone DLP, Vinyl pivalate, Dichloroethane DLP, Dichloroethane, CSA B 0-10o C 16h S O 80o Br opiv TFA - bromophenyl)propan-1-one (10 g, 0.0343 mol, 1 eq) was cooled to 0°C. Potassium O-ethyl xanthate (6.05 g, 0.03773 mol, 1.1 eq) was added. The reaction mixture was stirred until the starting material disappeared. Acetone was evaporated, and the resulting mixture was partitioned between water and DCM. The organic phase was dried with brine and Na₂SO₄. The crude product was purified by flash chromatography on silica gel to obtain 9 g of B53-01. Yield: 78.95%. [0687] Step 2: A 100 mL three-necked flask containing 45 mL dichloroethane, B53-01 (9 g, 0.0271 mol, 1 eq), and vinyl pivalate (6.95 g, 0.0542 mol, 2 eq) was refluxed under nitrogen. Lauroyl peroxide (DLP) (540 mg, 1.36 mmol, 0.05 eq) was then added to the refluxing solution followed by additional portions (0.03 eq every 1-1.5 hours). When TLC monitoring showed that the starting material was consumed (after seven additions of DLP), the solution was cooled to room 145 Attorney Docket No.: 071741.11025/5WO1 temperature. The reaction mixture was purified by a fast silica gel column to give 14 g (crude) of B53-02. Yield: 100%. [0688] Step 3: A 500 mL three-necked flask containing 140 mL dichloroethane, B53-02 (14 g, 0.0271 mol, 1 eq), and camphorsulfonic acid (629 mg, 0.00271 mol, 0.1 eq) was refluxed under nitrogen. Lauroyl peroxide (DLP) was then added to the refluxing solution followed by additional portions (2.16 g, 5.42 mmol, 0.2 eq, every 1-1.5 hours). When TLC monitoring showed that the starting material was consumed (after 1 eq of DLP), the solution was cooled to room temperature. The organic phase was evaporated. The crude product was purified by flash chromatography on silica gel to obtain 1.1 g of B53-03. Yield: 12.02%. [0689] Step 4: A 100 mL three-necked flask containing 19 mL toluene, B53-03 (1.1 g, 4.66 mmol, 1 eq), and p-TsOH·H₂O (3.1 g, 16.3 mmol, 2.9 eq) was refluxed for 4 hours. When the starting material was totally consumed, the mixture was allowed to cool to room temperature, neutralized with saturated Na₂CO₃, extracted with DCM, dried, and evaporated under reduced pressure. The residue was purified by a fast silica gel column to give 1.1 g of B53-04. Yield: 83.33%. [0690] Step 5: A 100 mL three-necked flask containing 11 mL DCM, B53-04 (1.1 g, 4.66 mmol, 1 eq), pyridine (1.11 g, 13.98 mmol, 3 eq), and trifluoromethanesulfonic anhydride (Tf₂O) (2.23 g, 7.92 mmol, 1.7 eq) was stirred for 1.5 hours at room temperature. TLC showed the reaction was complete. The organic phase was washed with water, and the aqueous phase was extracted with DCM. The organic phases were combined, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by a fast silica gel column to give 1.33 g of B53-05. Yield: 77.78%. [0691] Step 6: A 200 mL autoclave containing 22 mL MeOH, 22 mL DMSO, B53-05 (1.3 g, 3.53 mmol, 1 eq), TEA (679.2 mg, 6.71 mmol, 1.9 eq), dppp (274.75 mg, 0.847 mmol, 0.24 eq), and Pd(OAc)₂ (159.92 mg, 0.706 mmol, 0.2 eq) was stirred for 6 hours at 78°C under CO (5 MPa). GC-MS showed the reaction was complete. The mixture was diluted with water and extracted with EA. The organic phase was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by a fast silica gel column to give 690 mg of B53-06. Yield: 70.34%. [0692] Step 7: A 25 mL three-necked flask containing 7 mL 1,4-dioxane, B53-06 (683 mg, 2.46 mmol, 1 eq), tri-n-butylstannylmethanol (1.183 g, 3.69 mmol, 1.5 eq), and Pd(PPh₃)₄ (142.14 mg, 0.123 mmol, 0.05 eq) was stirred for 16 hours at 80°C under nitrogen. LC-MS showed the reaction 146 Attorney Docket No.: 071741.11025/5WO1 was complete. The mixture was diluted with water (20 mL) and extracted with EA (40 mL × 2). The organic phase was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by a fast silica gel column to give 377 mg of B53-07. Yield: 66.61%. [0693] Step 8: A 25 mL three-necked flask containing 7.5 mL DCM, B53-07 (370 mg, 1.61 mmol, 1 eq), and MnO₂ (1.4 g, 16.1 mmol, 10 eq) was stirred for 16 hours at 45°C. The mixture was filtered and evaporated to dryness. The residue was purified by a fast silica gel column to give 287 mg of B53-08. Yield: 78.20%. [0694] Step 9: A 50 mL three-necked flask containing 14 mL THF and powdered Zn (763 mg, 11.67 mmol, 19 eq) was cooled to 0°C under nitrogen. TiCl₄ (1.12 g, 5.89 mmol, 9.6 eq) was added dropwise, and the mixture was stirred for 1 hour at 70°C. The mixture was cooled to 0°C. A solution of (2,3-dimethylphenyl)(1-trityl-4-imidazolyl)methanone (280 mg, 0.614 mmol, 1 eq) and B53-08 (280 mg, 1.23 mmol, 2 eq) in 14 mL THF was added. The reaction mixture was stirred for 3 hours at 70°C. LC-MS showed the reaction was complete. The mixture was diluted with water (25 mL) and extracted with EA (30 mL × 3). The organic phase was dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by a fast silica gel column to give 200 mg of B53-09. Yield: 25.48%. [0695] Step 10: A 10 mL single-necked flask containing 1 mL MeOH, 4 drops of water, 4 drops of TFA, B53-09 (100 mg, 0.252 mmol, 1 eq), and 10% Pd/C (100 mg) was stirred for 6 hours at 50°C under a hydrogen atmosphere. LC-MS showed the reaction was complete. The mixture was evaporated to dryness to give 154 mg of B53-10. Yield: 100%. [0696] Step 11: A 10 mL single-necked flask containing 5 mL of a solution (MeOH:THF:H₂O = 2:2:1), NaOH (77.4 mg, 1.9 mmol, 5 eq), and B53-10 (154 mg, 0.387 mmol, 1 eq) was stirred in a microwave at 120°C for 30 minutes. LC-MS showed the reaction was complete. A mixture of 1 mL MeCN, 1 mL TFA, and the residue was stirred at 15°C for 5 minutes under nitrogen. The mixture was concentrated under vacuum. The residue was purified by preparative HPLC to afford B53 (13.6 mg). Yield: 9.19%. Overall yield: 0.053%.LCMS: [M-C2HF3O2+1]+=385.2 [0697] Other compounds were synthesized similarly as the above compounds. The charaterization data of compounds are lsited in the Table 2 below. 147 Attorney Docket No.: 071741.11025/5WO1 [0698] Table 2 # 1H-NMR and MS LC-MS:[M-TFA+1]+ =336.2,1H NMR (400 MHz, DMSO) δ 14.33 (s, 2H), 11.79 (s, 1H), 9.00 (d, J = 0.9 Hz, 1H), 1 7.67 (d J = 7.8 Hz 1H) 7.60 (s 1H) 7.46 (t J = 7.7 Hz 1H) 7.35 (d J = 7.7 Hz 1H) 7.17 – 7.06 (m 2H) 6.94 (s = , ), = 3 z, .4 1 3 3 8 , J , z, 56 z, d, 148 Attorney Docket No.: 071741.11025/5WO1 [M+1]+ =279.21H NMR (400 MHz, DMSO) δ 11.21 (d, J = 18.6 Hz, 1H), 7.96 – 7.74 (m, 1H), 7.66 (d, J = 7.8 Hz, 1H), 6.40 (d, J = 25.1 Hz, 2H), 6.08 – 5.85 (m, 2H), 5.53 (dt, J = 19.2, 9.6 Hz, 1H), 4.41 – 4.24 (m, 1H), 4.10 (dd, J = 13 27.9, 21.9 Hz, 1H), 3.64 (s, 2H), 3.51 (s, 12H), 3.43 – 3.35 (m, 4H), 3.16 (dd, J = 19.1, 5.7 Hz, 5H), 2.99 (d, J = 6.1 (s, J , , , – ), ), , J 5, ), d, , 8, – , , , J = 2 8 8, 8, 149 Attorney Docket No.: 071741.11025/5WO1 LCMS: [M-C2HF3O2+1]+=324.2,1H NMR (400 MHz, DMSO) δ 14.36 (s, 5H), 8.97 (s, 3H), 7.77 (s, 3H), 7.64 (s, 27 3H), 7.60 (t, J = 6.7 Hz, 3H), 7.24 (t, J = 7.7 Hz, 3H), 7.15 (d, J = 7.3 Hz, 3H), 7.13 – 6.98 (m, 9H), 6.71 (d, J = 7.5 Hz, 3H), 6.01 (s, 3H), 2.27 (s, 9H), 2.12 (s, 9H) J J d, 3 , , , 3 , ), , , J 5 , 4 5 150 Attorney Docket No.: 071741.11025/5WO1 44- [M+1]+=356.2. ,1H NMR (400 MHz, DMSO) δ 11.86 (s, 1H), 9.64 (s, 1H), 7.58 (d, J = 1.0 Hz, 1H), 7.23 (t, J = 7.8 A Hz, 1H), 7.08 – 6.96 (m, 4H), 6.87 (d, J = 7.7 Hz, 1H), 6.79 (s, 1H), 6.43 (s, 1H), 5.55 (s, 1H), 2.93 (s, 3H), 2.23 (s, 3H), 2.12 (s, 3H). 50 50 m, z, s, , , m, , 151 Attorney Docket No.: 071741.11025/5WO1 [M+1] +=355.15,1H NMR (400 MHz, DMSO) δ 14.31 (s, 1H), 9.27 (s, 1H), 9.08 (s, 1H), 8.08 (d, J = 4.0 Hz, 1H), 59 7.68 (d, J = 8.0 Hz, 1H), 7.60 (t, J = 7.2 Hz, 1H), 7.34 (s, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.16 – 7.06 (m, 2H), 7.02 (s, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.80 – 6.74 (m, 2H), 6.71 (d, J = 7.2 Hz, 1H), 5.78 (s, 1H), 2.27 (s, 3H), 2.16 (s, 3H). , 61 – , 3 , , J , 3 , , ), 1 6 9, 152 Attorney Docket No.: 071741.11025/5WO1 [M-C2HF3O2+1]+=395.1,1H NMR (400 MHz, DMSO) δ 14.33 (s, 2H), 10.08 (s, 1H), 9.07 (d, J = 1.0 Hz, 1H), 7.34 74 (t, J = 7.9 Hz, 1H), 7.19 – 7.11 (m, 2H), 7.09 (t, J = 7.6 Hz, 1H), 7.00 (d, J = 14.5 Hz, 2H), 6.93 (d, J = 7.8 Hz, 1H), 6.66 (d, J = 7.3 Hz, 1H), 5.82 (s, 1H), 3.45 (t, J = 6.8 Hz, 2H), 2.89 (t, J = 6.8 Hz, 2H), 2.26 (s, 3H), 2.11 (d, J = 14.5 z, 0 8 J ), = s, 3 s, ), z, 3 s, 56 s, 1 153 Attorney Docket No.: 071741.11025/5WO1 [M-C2HF3O2+1]+=312.2 ,1H NMR (400 MHz, DMSO) δ 14.27 (s, 2H), 8.97 (d, J = 0.8 Hz, 1H), 7.65 (s, 1H), 7.28 92 (d, J = 7.6 Hz, 1H), 7.15 (s, 1H), 7.12 – 6.99 (m, 2H), 6.62 (s, 1H), 4.12 (d, J = 10.6 Hz, 1H), 2.25 (d, J = 4.1 Hz, 6H), 2.14 (dd, J = 22.9, 11.7 Hz, 1H), 2.03 (t, J = 10.0 Hz, 1H), 1.80 – 1.65 (m, 2H), 1.45 (dd, J = 31.5, 12.5 Hz, 2H), 1.31 5 .4 0 , (t, 5, m, z, , 6 .4 , d, s, J 76 = z, 0 65 6 5 7 s, z, , 154 Attorney Docket No.: 071741.11025/5WO1 [M-C2HF3O2+1]+=397.2,1H NMR (400 MHz, DMSO) δ 14.36 (s, 1H), 10.37 (s, 1H), 9.07 (s, 1H), 8.68 (d, J = 5.5 103 Hz, 2H), 7.64 (d, J = 5.4 Hz, 2H), 7.55 (d, J = 8.1 Hz, 1H), 7.39 (s, 1H), 7.31 (t, J = 7.9 Hz, 1H), 7.10 (dt, J = 15.1, 7.4 Hz, 2H), 6.97 – 6.86 (m, 2H), 6.66 (d, J = 7.4 Hz, 1H), 5.80 (s, 1H), 3.85 (s, 2H), 2.26 (s, 3H), 2.12 (s, 3H). 3 10 9 7 6 9 8 ), 4 7 6 s, 6 68 155 Attorney Docket No.: 071741.11025/5WO1 [M-C2HF3O2+1]+=452.2,1H NMR (400 MHz, DMSO) δ 14.32 (s, 2H), 9.97 (s, 1H), 9.07 (d, J = 1.0 Hz, 1H), 7.57 117 (d, J = 8.2 Hz, 1H), 7.38 (s, 1H), 7.29 (t, J = 7.9 Hz, 1H), 7.16 – 7.05 (m, 2H), 6.97 (s, 1H), 6.86 (d, J = 7.7 Hz, 1H), 6.67 (d, J = 7.3 Hz, 1H), 5.79 (s, 1H), 3.15 (dd, J = 18.4, 7.9 Hz, 2H), 3.00 (d, J = 12.5 Hz, 2H), 2.28 (d, J = 8.9 Hz, = ). 6 s, 36 – z, z, 55 , t, , , J z, 3 ), 156 Attorney Docket No.: 071741.11025/5WO1 LC-MS= [M-C2HF3O2+1]+= 371.1,1H NMR (400 MHz, DMSO) δ 14.31 (s, 2H), 9.67 (s, 1H), 9.07 (d, J = 0.8 Hz, 127 1H), 7.28 (dt, J = 13.3, 6.3 Hz, 2H), 7.17 – 7.05 (m, 3H), 6.96 (d, J = 8.0 Hz, 2H), 6.79 (d, J = 7.7 Hz, 1H), 6.66 (d, J = 7.3 Hz, 1H), 5.76 (s, 1H), 2.43 (d, J = 4.9 Hz, 3H), 2.26 (s, 3H), 2.13 (s, 3H). d, 1 = , .8 ), z, 82 J 8 6 6 7 7 8 , , ), 157 Attorney Docket No.: 071741.11025/5WO1 [M-C2HF3O2+1]+=356.2,1H NMR (400 MHz, DMSO) δ 14.37 (s, 2H), 9.77 (s, 1H), 9.09 (s, 1H), 7.33 (t, J = 7.9 Hz, 140 1H), 7.28 (d, J = 4.5 Hz, 2H), 7.24 – 7.17 (m, 1H), 7.14 (d, J = 7.6 Hz, 1H), 7.01 (s, 2H), 6.89 (t, J = 7.4 Hz, 2H), 5.82 (s, 1H), 2.96 (s, 3H), 2.71 – 2.55 (m, 2H), 1.07 (t, J = 7.5 Hz, 3H). z, 07 = . = = , , 67 .4 8 9 m, m, ), 82 ), 0 = ), 58 , (s, 1 45 158 Attorney Docket No.: 071741.11025/5WO1 [M-C2HF3O2+1]+=376.2,1H NMR (400 MHz, DMSO) δ 14.33 (s, 2H), 11.57 (s, 1H), 9.05 (s, 1H), 7.71 (s, 1H), 7.54 154 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 7.09 (d, J = 7.3 Hz, 1H), -B 6.86 (s, 1H), 4.36 (d, J = 10.9 Hz, 1H), 4.09 (t, J = 11.3 Hz, 1H), 3.66 (s, 3H), 2.95 – 2.84 (m, 1H), 2.82 – 2.75 (m, J z, ), J 56 4 9 z, m, , 7 9 t, , z, 0 ), J , z, = 159 Attorney Docket No.: 071741.11025/5WO1 LC-MS: [M-C2HF3O2+1]+=426.2.,1H NMR (400 MHz, DMSO) δ 14.19 (s, 2H), 9.76 (d, J = 7.4 Hz, 1H), 9.00 (s, 166 1H), 7.31 (t, J = 7.9 Hz, 1H), 7.14 (d, J = 7.5 Hz, 2H), 7.08 (t, J = 7.5 Hz, 1H), 7.00 (s, 1H), 6.91 (d, J = 11.9 Hz, 2H), 6.66 (d, J = 7.7 Hz, 1H), 5.79 (s, 1H), 4.17 – 4.08 (m, 1H), 3.57 (ddd, J = 22.3, 15.0, 7.7 Hz, 2H), 3.21 (d, J = 5.9 Hz, = 2 ), J (t, p, J , , J , = , J = 160 Attorney Docket No.: 071741.11025/5WO1 LC-MS: [M-C2HF3O2+1]+=404.2,1H NMR (400 MHz, DMSO) δ 14.24 (s, 2H), 9.82 (s, 1H), 8.95 (s, 1H), 7.59 (s, 1H), 7.43 (d, J = 8.3 Hz, 2H), 7.08 (t, J = 7.4 Hz, 5H), 4.67 (t, J = 7.8 Hz, 1H), 4.19 – 4.10 (m, 1H), 3.76 (dd, J = 14.3, 176 7.3 Hz, 1H), 3.59 (dd, J = 14.6, 7.5 Hz, 1H), 3.30 (dd, J = 13.7, 8.5 Hz, 1H), 3.09 (dd, J = 13.8, 7.2 Hz, 1H), 2.47 (d, J z, ), , 3 2 – 0 , J 5 , J J 7 J 161 Attorney Docket No.: 071741.11025/5WO1 LC-MS: [M-C2HF3O2+1]+=352.2,1H NMR (400 MHz, DMSO) δ 14.24 (s, 2H), 9.62 (s, 1H), 8.96 (s, 1H), 7.65 (dd, 193 J = 21.2, 12.9 Hz, 2H), 7.06 (dd, J = 12.2, 8.4 Hz, 4H), 6.92 (d, J = 8.2 Hz, 1H), 4.71 (t, J = 7.7 Hz, 1H), 3.33 (dd, J = 13.6, 8.7 Hz, 1H), 3.12 (dd, J = 13.7, 7.2 Hz, 1H), 2.22 (s, 3H), 2.14 (s, 3H), 2.04 (s, 3H). = 9 2 5 d, , 20 = 6 9 .9, 4 d, 0 , J z, , 53 5 , s, 162 Attorney Docket No.: 071741.11025/5WO1 [M+1] +=420.2,1H NMR (400 MHz, MeOD) δ 7.55 (s, 1H), 7.51 – 7.49 (m, 2H), 7.14 – 7.12 (m, 1H), 7.04 – 6.99 (m, 212 3H), 6.96 – 6.94 (m, 1H), 6.76 (s, 1H), 4.59 (t, J = 8.0 Hz, 1H), 4.02 – 3.98 (m, 2H), 3.52 – 3.46 (m, 2H), 3.44 – 3.39 (m, 1H), 3.15 – 3.10 (m, 1H), 2.98 – 2.87 (m, 1H), 2.21 (s, 3H), 2.14 – 2.05 (m, 2H), 2.04 (s, 3H), 1.70 – 1.67 (m, = d, J = 16 = z, z, 4 , (t, ), t, 0 , J 7 9 163 Attorney Docket No.: 071741.11025/5WO1 [M-C2HF3O2-1]-=376.21H NMR (400 MHz, DMSO) δ 14.20 (s, 2H), 8.95 (s, 1H), 8.74 (t, J = 5.9 Hz, 1H), 7.72 (d, 229 J = 8.2 Hz, 2H), 7.61 (s, 1H), 7.26 (d, J = 8.2 Hz, 2H), 7.15 – 7.01 (m, 3H), 4.75 (t, J = 7.9 Hz, 1H), 3.42 (dd, J = 13.7, 8.4 Hz, 1H), 3.21 (dd, J = 13.7, 7.4 Hz, 1H), 2.21 (s, 3H), 2.13 (s, 3H). 5 ), t, , 0 7 J dt, s, = (t, z, 7, J 91 , 164 Attorney Docket No.: 071741.11025/5WO1 246 LC-MS: [M-C2HF3O2+1]+=333.2,1H NMR (400 MHz, DMSO) δ 14.25 (s, 2H), 8.96 (s, 1H), 7.57 (s, 1H), 7.10 – 6.99 (m, 3H), 6.61 (s, 1H), 6.48 (d, J = 21.4 Hz, 2H), 4.62 (t, J = 7.5 Hz, 1H), 3.29 (s, 4H), 3.21 – 3.14 (m, 1H), 2.97 (dd, J = 13.9, 6.7 Hz, 1H), 2.23 (s, 3H), 2.18 (s, 3H). ). 2 ), - , J , , J z, 1 z, 33 , 1, J , 165 Attorney Docket No.: 071741.11025/5WO1 [M+1] +=383.9,1H NMR (400 MHz, DMSO) δ 11.69 (s, 1H), 7.65 (s, 1H), 7.54 (d, J = 6.8 Hz, 1H), 7.45 – 7.24 (m, 511 4H), 7.07 (t, J = 7.6 Hz, 1H), 7.00 (d, J = 7.2 Hz, 1H), 5.17 (s, 1H), 3.73 – 3.61 (m, 5H), 3.10 (t, J = 7.2 Hz, 2H), 2.85 (qd, J = 14.0, 7.2 Hz, 2H), 2.21 (s, 3H), 2.16 (s, 3H). 8 ), , – , = , .9, 9, t, J , z, ), = 4 , 166 Attorney Docket No.: 071741.11025/5WO1 LCMS: [M-C2HF3O2+1]+=360.2,1H NMR (400 MHz, DMSO) δ 14.24 (s, 2H), 11.36 (s, 1H), 8.95 (s, 1H), 7.75 – B21 7.56 (m, 3H), 7.28 (d, J = 8.1 Hz, 2H), 7.08 (dt, J = 9.6, 6.2 Hz, 3H), 6.47 (s, 1H), 4.75 (t, J = 7.8 Hz, 1H), 3.43 (d, J = 5.4 Hz, 1H), 3.20 (dd, J = 13.7, 7.5 Hz, 1H), 2.21 (s, 3H), 2.12 (s, 3H). , – ), , 2 2 – ), d, – J ), 10 ), 6 6 , 7 , , 167 Attorney Docket No.: 071741.11025/5WO1 B39- LCMS: [M-HCL+1]+=335.2.,1H NMR (400 MHz, DMSO) δ 12.12 (d, J = 292.4 Hz, 2H), 7.62 (d, J = 7.9 Hz, 1H), A 7.49 (s, 1H), 7.20 (d, J = 7.1 Hz, 1H), 6.99 (ddd, J = 25.4, 12.8, 5.2 Hz, 4H), 6.75 (s, 1H), 4.54 (t, J = 7.6 Hz, 1H), 3.37 (dd, J = 12.3, 6.9 Hz, 1H), 3.07 (dd, J = 13.7, 7.2 Hz, 1H), 2.41 (s, 3H), 2.19 (s, 3H), 2.15 (s, 3H). , .0, 0, .4 2 .7 J ), ), , , , 8, z, 56 8 6 s, 168 Attorney Docket No.: 071741.11025/5WO1 LCMS: [M-C2HF3O2+1]+= 369.1/371.1,1H NMR (400 MHz, DMSO) δ 14.17 (s, 3H), 8.96 (s, 1H), 7.61 (s, 1H), B51 7.19 – 6.96 (m, 5H), 4.75 (dd, J = 18.2, 10.3 Hz, 1H), 3.35 – 3.29 (m, 1H), 3.13 (dd, J = 13.9, 7.1 Hz, 1H), 2.22 (d, J = 9.7 Hz, 6H), 2.15 (s, 3H). , J 52 [0699] Example 1. α2AAR FLIPR assay [0700] This experimental protocol involved cell seeding and a FLIPR assay using the α2AAR (α2A-adrenergic receptor) cell line hosted in HEK293 cells. The growth media used is DMEM (11965-092, Gibco) supplemented with 10% FBS (FSP500, Excell), 300μg/mL G418 (10131-027, Gibco), and 2μg/mL Blasticidin S HCl (BS) (A11139-03, Gibco). On Day 1, the cell seeding process started with the removal of the culture medium, followed by rinsing the cells with DPBS (21-031-CVC, Corning). Cells were then treated with 0.05% EDTA-Trypsin (25300-062, Gibco), incubated at 37°C for 1-2 minutes, and monitored under an inverted microscope. The cells were detached, resuspended in growth media, and centrifuged at room temperature at 1000 rpm for 5 minutes. After discarding the supernatant, the cell pellet was resuspended in growth media to a concentration of 10×105 cells per mL. This suspension was added to 384-well plates (19-Jul-38, Greiner) at 20 µL per well and incubated overnight at 37℃ in 5% CO2. [0701] On Day 2, the FLIPR assay began with the preparation of the assay buffer comprising 20 mM HEPES (15630-106, Invitrogen), 1× HBSS (14025-076, Invitrogen), and 0.5% BSA (B2064, Sigma). A 250 mM Probenecid solution was prepared in this buffer. The Fluo-4 DirectTM Loading Buffer was made by dissolving Fluo-4 DirectTM crystals (F10471, Invitrogen) in the FLIPR Assay Buffer and adding Probenecid. The buffer was then vortexed and allowed to stand for over 5 minutes, shielded from light. For the FLIPR procedure, testing compounds for agonist activite were serially diluted and transferred to a 384-well compound plate (25-Jan-39, Greiner). The cell plate was then treated with 2× Fluo-4 DirectTM loading buffer and incubated for 50 minutes at 37 ºC in a 5% CO2 atmosphere, followed by 10 minutes at room temperature. Subsequently, the FLIPR assay buffer was added to the compound plate, which is then centrifuged. 169 Attorney Docket No.: 071741.11025/5WO1 [0702] The cell plate was analyzed in the FLIPR Tetra+ System for fluorescence signals. For the agonist test, reference compounds were added to the cell plates, and fluorescence was measured. The “Max-Min” calculation began from Read 1 to the maximum allowed. The data were analyzed using Prism software to calculate activation percentage for agonists and inhibition percentage for antagonists. The results were then fitted using specific models to determine EC50 for agonists. [0703] The experimental protocol utilized various reagents and apparatus, including Penicillin/Streptomycin (100x) (SV30010, Hyclone), Poly-L-lysine hydrobromide (P1399, Sigma), and different types of 384-well plates such as the 384-Well PP 2.0 Microplate (PP-0200, LABCYTE) and 384 well Low Dead Volume Microplate (LP-0200, LABCYTE). The use of specific reference compounds like UK14304 was also integral to the assay. [0704] Example 2. α2AAR binding assay [0705] The α2AR Binding Assay was conducted using a stable HEK293 cell line, specifically constructed by WuXi AppTec for targeting α2AAR. This assay primarily focused on the binding activity of the radioligand [3H]-RX 821002 (PerkinElmer, NET1153250UC) to α2AAR, with the membrane concentration set at 0.5 μg/well and the radioligand concentration at 0.5 nM. Essential equipment for this assay includes Unifilter-96 GF/C filter plates (Perkin Elmer, 6005174), 96 well conical polypropylene plates (Agilent, 5042-1385), TopSeal-A sealing film (Perkin Elmer, 6050185), a MicroBeta2 reader (CNLL0153, Perkin Elemer, 1310887), and a cell harvester (UNIFILTER-96, Perkin Elemer, 1951369), all procured from Perkin Elmer. Both the assay and wash buffers consist of 50 mM Tris-HCl at a pH of 7.4 (Tris base, Sigma, T1503-1KG). [0706] The procedure initiated with the preparation of test compounds and a reference compound, yohimbine (Sigma, Y3125), through an 8-point 4-fold serial dilution, transferring 1 μL of each to the assay plate. The assay involved adding 100 μL of membrane stocks (0.5μg/well) and 100 μL 0.5nM of [3H]-RX 821002 to each well. After sealing, the plates were agitated at room temperature for one hour. Subsequently, the Unifilter-96 GF/C filter plates were pre-soaked with 0.3% PEI (Sigma, P3143) for at least half an hour. The reaction mixtures were then filtered and washed four times with cold wash buffer using a Perkin Elmer Cell harvester. Post-filtration, the plates were dried at 50 ℃ for one hour. The next step involved sealing the bottom of the filter plate wells with Perkin Elmer Unifilter-96 backing seal tape and adding 50 μL of MicroScint-O cocktail (PerkinElmer, 6013611) to each well. The top of the plates was then sealed with TopSeal-A sealing 170 Attorney Docket No.: 071741.11025/5WO1 film. The trapped 3H was quantified using a Perkin Elmer MicroBeta2 Reader. The inhibition rate was calculated using the formula: %Inhibition= (1-(Assay well Average_LC)/ (Average_HC- Average_LC)) × 100%. Finally, the data were analyzed with Prism 5.0 software, employing the “log (inhibitor) vs. response -- Variable slope” model for data fitting. This comprehensive process ensured precise assessment of the binding affinity of compounds to the α2AAR. [0707] The result of the α2AAR FLIPR assay and binding assay result are listed in Table 3 and Table 4 below. [0708] Table 3. α2A AR agonist activity (EC50) and affinity (Ki) α2A α2A Compound racemate/ agonist α2A Compound racemate/ α2A affinity (Ki, agonist affinity (Ki, 171 Attorney Docket No.: 071741.11025/5WO1 18 -A D 154 -B E 18 -B B B 155 -A B B 172 Attorney Docket No.: 071741.11025/5WO1 50 - C C 182 - C 51 - C B 183 - D 173 Attorney Docket No.: 071741.11025/5WO1 85 - C B 219 - A A 86 - B A 220 - A A 174 Attorney Docket No.: 071741.11025/5WO1 120 - A A 504 - C 121 - A B 505 - C A C: 50nM-250nM D: 250nM-1000nM E: >1000nM [0709] Table 4. α2A AR agonist activity (EC50) and affinity (Ki) Compound α2A AR α2A AR Compound α2A AR α2A AR N nit ffinit (Ki N nit ffinit (Ki 175 Attorney Docket No.: 071741.11025/5WO1 B27 A B42-B C B28 A A B43 A A B: 50nM-250nM C: 250nM-1000nM D: >1000nM [0710] Example 3. MDR1-MDCK Permeability Assay [0711] MDR1-MDCK II cells (obtained from Piet Borst at the Netherlands Cancer Institute) were seeded onto Polycarbonate membranes (PC) in 96-well insert systems at 3.33 x 105 cells/ mL until to 4-7 days for confluent cell monolayer formation. Selected α2AR agonist from Table 3 and Table 4 were diluted with the transport buffer (HBSS with 10.0 mM Hepes, pH7.4) from DMSO stock solution to a concentration of 2µM (DMSO<1%) and applied to the apical or basolateral side of the cell monolayer. Digoxin was used as a positive control for the P-glycoprotein (P-gp) substrate, while clonidine, dexmedetomidine, faldomidine and brimonidine were used as negative control. Permeation of the test compounds from A to B direction and/or B to A direction was determined in duplicate. Digoxin was tested at 10.0 μM from A to B direction and B to A direction in duplicate. The plate was incubated for 2.5 hours in CO2 incubator at 37.0±1.0°C, with 5.0% CO2 at saturated humidity without shaking. In addition, the efflux ratio of each compound was also determined. Test and reference compounds were quantified by LC/MS/MS analysis based on the peak area ratio of analyte/IS. [0712] After transport assay, lucifer yellow rejection assay was applied to determine the cell monolayer integrity. Buffers were removed from both apical and basolateral chambers, followed by the addition of 75 µL of 100 µM lucifer yellow in transport buffer and 250 µL transport buffer in apical and basolateral chambers, respectively. The plate was incubated for 30 minutes at 37.0°C 176 Attorney Docket No.: 071741.11025/5WO1 with 5.0% CO2 and 95.0% relative humidity without shaking. After 30 minutes incubation, 20 µL of lucifer yellow samples were taken from the apical sides, followed by the addition of 60 µL of transport Buffer. And then 80 µL of lucifer yellow samples were taken from the basolateral sides. The relative fluorescence unit (RFU) of lucifer yellow was measured at 425/528 nm (excitation/emission) with an Envision plate reader. [0713] The apparent permeability coefficient Papp (cm/s) was calculated using the equation: Papp = (dCr/dt) × Vr / (A x C0) wherein dCr/dt is the cumulative concentration of compound in the receiver chamber as a function of time (µM/s); Vr is the solution volume in the receiver chamber (0.075 mL on the apical side, 0.25 mL on the basolateral side); A is the surface area for the transport, i.e.0.143 cm2 for the area of the monolayer; and C0 is the initial concentration in the donor chamber (µM). [0714] The efflux ratio was calculated using the equation: Efflux Ratio = Papp (BA) / Papp (AB) [0715] The results of the MDR1-MDCK permeability assay are listed in Table 5 below. [0716] Table 5 Efflux ratio of P-gp from MDCK-MDR1 assay compound racemate / Efflux compound racemate / Efflux name enantiomer ratio name enantiomer ratio Digoxin 13.74 109 94.75 Clonidine 0.71 111 172.99 Dexmedetomidine 0.64 112 58.40 Fadolmidine 0.80 118 25.26 Brimonidine 0.83 121 -B 30.89 1 -B 51.02 124 -B 10.26 5 -B 33.20 136 -B 7.69 18 -B 27.47 138 7.85 44 -B 41.00 141 2.61 45 -B 14.0 2.86 46 -B 34.01 157 -B 10.85 47 -B 18.17 158 23.66 55 26.39 163 9.08 56 11.51 165 14.79 64 186 3.38 67 28.97 187 2.60 72 32.93 190 4.91 73 96.13 200 27.73 74 93.45 205 61.80 80 112.12 208 8.88 81 62.48 213 4.92 177 Attorney Docket No.: 071741.11025/5WO1 95 102.22 214 56.11 98 82.58 215 4.77 99 140.52 216 3.21 101 103.83 217 17.52 103 149.19 219 26.89 105 65.78 401 22.02 108 72.22 [0717] Example 4. in vivo drug distribution [0718] The binding affinity of various compounds to plasma proteins was evaluated, including clonidine HCl, dexmedetomidine HCl, 1-B HCl, and 44-B HCl, with warfarin serving as a control. The experiment utilized a HT-Dialysis plate (HTD 96 b) and a dialysis membrane with a molecular weight cutoff of 12-14 kDa. The plasma was derived from male C57BL/6J mice, treated with EDTA-K2 as an anticoagulant. The experimental procedure commenced with plasma thawing under cold tap water, followed by centrifugation at 3220 ×g for 5 minutes to eliminate clots, and pH adjustment to 7.4 ± 0.1. [0719] Dialysis membranes were initially hydrated in ultra-pure water for about one hour and then treated in a 20:80 ethanol-water mixture for 20 minutes. These prepared membranes could be used immediately or stored at 2-8°C for up to a month. Membranes were rinsed in ultra-pure water before use. [0720] Test and control compounds were prepared at a 400 μM concentration by diluting stock solutions with DMSO. Working solutions were further diluted to create 2 μM loading matrix solutions, which were thoroughly mixed. In the assay, 50 μL aliquots of these solutions were dispensed in triplicate into a Sample Collection Plate, balanced with blank PBS to a final volume of 100 μL per well. A stop solution containing acetonitrile, tolbutamide, and labetalol was added, and samples were mixed and cooled at 2 to 8°C. [0721] During the dialysis, 100 μL aliquots from the loading matrix were placed in the dialysis well's donor side, matched with an equal volume of PBS on the receiver side, and incubated at 37°C for 4 hours. Post-dialysis, samples from both sides were collected, balanced to 100 μL with corresponding blank fluids, treated with stop solution, vortexed, and centrifuged to prepare for LC- MS/MS analysis. [0722] Data analysis involved calculating the percentages of Unbound, Bound, and Recovery of the compounds post-dialysis. %Unbound was calculated as the ratio of the compound's peak area 178 Attorney Docket No.: 071741.11025/5WO1 on the receiver side to its internal standard, reflecting the fraction that crossed the membrane. %Bound was the complement of %Unbound, representing the fraction retained on the donor side. %Recovery was determined from the peak area ratios on both sides of the membrane, assessing the dialysis efficiency in retaining the compound. These metrics provided insights into the compound's free, bound, and recoverable quantities, elucidating its behavior in the dialysis system. The plasma protein binding ratio result is shown in Table 6. [0723] Table 6. The plasma protein binding ratio Sample Name %Unbound SD %Bound %Recovery SD (n = 3) (n = 3) Clonidine HCl 70.8 4.8 29.2 93.2 3.7 Dexmedetomidine HCl 6.8 0.2 93.2 88.9 1.8 1-B HCl 5.1 0.3 94.5 88.8 3.5 44-B HCl 6.4 0.8 93.6 86.7 7.0 Warfarin 2.3 0.1 97.7 82.7 2.2 [0724] The binding affinity of various compounds to brain proteins was evaluated including clonidine HCl, dexmedetomidine HCl, 1-B HCl, and 44-B HCl, with propranolol serving as a control. The initial preparation of the dialysis membrane involved thawing brain homogenate in a water bath at room temperature and subsequently heating it at 37°C for 10 minutes. The dialysis setup utilized was from HT Dialysis LLC, featuring a HT-Dialysis plate (Model HTD 96 b) and a dialysis membrane with a molecular weight cutoff of 12-14 kDa. [0725] The membrane underwent a comprehensive pretreatment which included hydration in ultra-pure water at room temperature for approximately one hour. This was followed by separation and immersion in a 20:80 ethanol:water solution for about 20 minutes. After this treatment, the membranes were either used immediately or stored at 2-8°C for up to one month, with a final rinse in ultra-pure water prior to experimental use. [0726] For compound preparation, test and control substances were first dissolved to create 400μM working solutions by mixing 4μL of stock solution with 96μL of DMSO. These working solutions were then further diluted to 2μM in a blank matrix by combining 3μL of the prepared solution with 597μL of matrix, ensuring thorough mixing. [0727] During the assay, 50μL aliquots of the 2μM compound-matrix mixture were dispensed in triplicate into a Sample Collection Plate. Each aliquot was paired with an equal volume of blank 179 Attorney Docket No.: 071741.11025/5WO1 PBS to standardize the total volume to 100μL per well at a 1:1 matrix to PBS ratio. A stop solution comprising 500μL of acetonitrile with tolbutamide and labetalol at 250nM each was added to stabilize the samples at T0. The samples were then shaken at 800 rpm for 10 minutes and stored at 2-8°C. [0728] The dialysis procedure included assembling the dialysis device according to the manufacturer's specifications, loading the matrix aliquots into the donor side of the dialysis wells, and conducting the dialysis under a humidified atmosphere with 5% CO2 at 37°C for 4 hours. [0729] Post-dialysis, 50 μL samples were collected from both the receiver and donor sides into new 96-well plates. Volumes were adjusted to 100 μL by adding an equivalent amount of the opposite blank matrix or PBS. The samples were prepared for LC-MS/MS analysis after thorough vertexing and centrifugation. Blank control samples were prepared and processed similarly to mirror the test conditions. [0730] Data analysis involved calculating the percentages of undiluted unbound and bound fractions, and recovery of the compounds. The %Undiluted Unbound was determined using the formula: %Undiluted Unbound = 100 × 1/D / ((1 / (F/T) - 1) + 1/D), where D is the dilution factor (10). %Undiluted Bound was derived as 100 - %Undiluted Unbound. %Recovery was calculated using: %Recovery = 100 × (F + T) / T0, with F and T representing the peak area ratios of the compound to the internal standard on the receiver and donor sides respectively, after 4 hours of incubation. The brain protein binding result is shown in Table 7. [0731] Table 7: The brain protein binding result Sample Name %Undiluted SD %Undiluted %Recovery SD Unbound Bound (n = 3) (n = 3) (n = 3) Clonidine HCl 23.0 5.4 77.0 105.2 1.3 Dexmedetomidine HCl 6.3 0.9 93.7 93.8 2.7 1-B HCl 5.1 0.3 94.9 88.8 3.5 44-B HCl 2.4 0.3 97.6 88.6 3.7 Propranolol 2.6 0.2 97.4 108.6 1.8 [0732] Male C57BL/6J mouse was used in the in vivo distribution assay. The sample of brain, spinal cord and serum were collected for the drug distribution calculation. 180 Attorney Docket No.: 071741.11025/5WO1 [0733] Before commencing the study, the mice were acclimated to the test facility for at least 3 days. During this period, their general health was assessed by veterinary staff or other authorized personnel. The mice were housed in groups of up to four per cage in polysulfone cages, using either certified aspen shaving bedding or corncob bedding. This bedding was regularly tested for environmental contaminants by the manufacturer. The facility's environment was carefully controlled to maintain a temperature range of 20-26℃, relative humidity between 40 to 70%, and a 12-hour light/12-hour dark cycle, although this cycle can be interrupted for study-related activities. Temperature and humidity were continuously monitored by the Vawasala ViewLinc Monitoring system. [0734] For dosing, an appropriate amount of the compounds was accurately weighed and mixed with a suitable volume of vehicle to achieve a clear solution. This process may require vertexing or sonication in a water bath. The animals were dosed within four hours of formulation preparation. Samples from each formulation were then collected for dose validation using either LC/UV or LC- MS/MS analysis. [0735] Oral gavage was employed for dosing following the facility's SOPs, with the dose volume based on the animal's body weight measured on the morning of the dosing day. Compounds such as 5 mg/kg Clonidine HCl, 5 mg/kg dexmedetomidine HCl, 5 mg/kg and 80 mg/kg compound 1-B, and 5 mg/kg and 80 mg/kg compound 44-B were administered in a 20% HP-β-CD solution in water, with sample collections scheduled at 0.5, 1, 2, and 8 hours post-dosing. [0736] Blood collections were performed from the saphenous vein or another suitable site, with approximately 0.1 mL collected per time point into pre-chilled commercial EDTA-K2 tubes. The samples were kept on wet ice until centrifugation at 4°C and 3,200 g for 10 minutes. The plasma was then transferred into pre-labeled 96-well plates or polypropylene tubes, quick-frozen over dry ice, and stored at -60℃ or lower until LC-MS/MS analysis. [0737] The brain and spinal cord tissues were harvested immediately, washed with cold saline, dried, and weighed. These samples were homogenized in a cold 15 mM PBS (pH 7.4):MeOH=2:1 solution at a 1:9 tissue-to-buffer ratio. The homogenates were split into two aliquots: one for immediate LC-MS/MS analysis and one stored at -70±10 °C as a backup. This comprehensive method ensures the detailed and standardized collection and analysis of pharmacokinetic data in a controlled and scientifically rigorous manner. 181 Attorney Docket No.: 071741.11025/5WO1 [0738] We calculated AUC ratio from the equation AUC ratio = Tissue AUC0-last / plasma AUC0- last. LogBB=log10(brain AUC0-last / plasma AUC0-last), LogSB= log10(brain AUC0-last / spinal cord AUC0-last), Kp= brain AUC0-last / plasma AUC0-last and Kp,uu,brain = AUCb,u/AUCp,u= AUCbrain/AUCplasma × (fu,brain/fu,plasma). The in vivo drug distribution result is shown in Table 8. [0739] Table 8: the in vivo drug distribution Brain Spinal cord Kp, uu, Compounds dosage logBB logSB /plasma, Kp /plasma brain Clonidine HCl 5 mg/kg 2.27 1.78 0.356 0.252 0.739 Dexmedetomidine HCl 5 mg/kg 1.26 1.14 0.101 0.133 1.17 5 mg/kg below detection limit* 1-B HCl 80 mg/kg 0.0598 0.0153 -1.22 -0.732 0.032 5 mg/kg below detection limit* 44-B HCl 80 mg/kg 0.0334 0.0511 -1.39 -1.29 0.013 * drug in brain and spinal cord is below the detection limit (DL). DL=10ng/mL. The distribution parameters are not available [0740] Example 5. Efficacy study on spared nerve injury in mice [0741] 50 male C57BL/6 mice weighing between 20-30 g were subjected to spared nerve injury (SNI) surgery, of which 6 mice were as sham surgery and the others were of SNI surgery. A few days after SNI surgery, all animals were subjected to mechanical allodynia test to obtain baseline paw withdrawal threshold (PWT). The qualified mice baseline PWT < 0.6 g were randomly assigned to different groups (Vehicle group and test articles groups) based on baseline PWT and 6 sham mice as Sham group for evaluating efficacy of the test compounds, 8 mice in each group. [0742] The animals were acclimated to the environment for 3-7 days after arriving at the animal facility. Three days before 1st mechanical allodynia test, the animals were habituated to the test environment for 15 minutes per day. [0743] Aseptic techniques were employed by all surgeons, and all surgical instruments, including scissors, sharp forceps, scalpels, sterile cotton pads, needles, and metal clips, were sterilized prior to surgery. The animals were anesthetized with Zoletil 50 (50 mg/kg, 2.5 mL/kg, i.p.) and Xylazine Hydrochloride (8 mg/kg, 2.5 mL/kg, i.p.), with a toe pinch used to ensure full anesthesia before incision, and ophthalmic ointment applied to the rodents' eyes to prevent drying of the corneas. The 182 Attorney Docket No.: 071741.11025/5WO1 fur on the posterior thigh was closely shaved, and the surgical area's skin was swabbed with three rounds of alternating Betadine and 70% ethanol, then allowed to dry. An incision was made on the lateral surface of the thigh, cutting through the biceps femoris muscle to expose the sciatic nerve and its terminal branches: the sural, common peroneal, and tibial nerves, with the common peroneal and tibial nerves being cut, leaving the sural nerve intact. The wound was closed in layers, with the skin sutured. Surgical instruments were cleaned and sterilized using a glass bead sterilizer post-operation. The animals recovered from anesthesia on a warm pad, were injected with 1 mL sterile saline subcutaneously to prevent dehydration, and returned to their home cage once fully awake and mobile. [0744] On day 11, the animals were individually placed in plastic enclosures with mesh bottoms, allowing full paw access. For three consecutive days, mice were acclimated for 15 minutes each day. Mechanical allodynia baseline measurements were performed on day 14. Animals not exhibiting allodynia (PWT>0.6 g) were excluded, leaving 24 qualified animals (PWT<0.6 g) who were then randomly divided into three groups based on their baseline PWT, in addition to 6 sham mice forming a Sham group, totaling four groups with 6-8 mice each. [0745] The administration route for the therapeutic intervention for compounds 1-B with a dosage from 1 mg/mL to 20 mg/mL, and 10-B, 44-B, 45-B, 46-B, 47-B, 121, 136, 118, 156 and 175 was oral (p.o.) with a dosage of 1 mg/mL, while the ones for 1 mg/kg morphine via s.c. and 3mg/kg pregabalin via p.o. as positive control, which were prepared in a 20% HP-β-CD solution. 1-B, 10-B, 44-B 45-B, 46-B and 47-B are the active enantiomers of 1, 10, 44, 45, 46, 47, respectively, while 121, 136, 118, and 156 are racemate. The solution was vortexed to ensure thorough mixing until homogeneous. The dosage administered to the mice was 10 ml/kg. [0746] Mechanical allodynia tests were conducted on the left hind paw of mice, which were individually placed in plastic enclosures with mesh bottoms for full paw access and acclimated for 15 minutes prior to testing. Following acclimation, the mid-plantar hind paw was probed using a series of eight Von Frey filaments with logarithmically incremental stiffness: 0.02 g (2.36), 0.04 g (2.44), 0.07 g (2.83), 0.16 g (3.22), 0.4 g (3.61), 0.6 g (3.84), 1 g (4.08), and 1.4 g (4.17). The filaments were applied perpendicularly to the paw's plantar surface with enough force to slightly buckle against it, maintaining contact for 6-8 seconds. Tests were spaced by 5-second intervals to ensure clear resolution of any response to the prior stimulus, with a sharp withdrawal or flinching upon filament removal indicating a positive response. Ambulatory reactions were deemed 183 ambiguous, prompting a repeat of the stimulus. Testing began with the 0.16 g (3.22) fdament, adjusting the force of subsequent fdaments up or down depending on the mouse's response, following the Dixon up-down method. The maximum force used was the 1.4 g (4.17) filament, with the criteria for a positive response being a distinct withdrawal of the paw or flinching immediately after the filament's removal.
[0747] Data were presented in Prism 8.0 (Graph Pad Software, Inc.) by one-way ANOVA or two-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison or by t test followed by two-tailed comparison test. The results are demonstrated by FIGs. 1A-1H.
[0748] A series of experiments were conducted to determine the combination index for a peripheral alpha-2 agonist in conjunction with various analgesics. The study included doses of 1 mg/kg 1-B p.o. and 1 mg/kg 44-B p.o. Additionally, analgesia drugs, including 1 mg/kg morphine s.c., 3 mg/kg pregabalin p.o., 3 mg/kg suzetrigine p.o., 7 mg/kg duloxetine p.o., 7 mg/kg amitriptyline p.o., and lOmg/kg ketorolac i.p. were administered, as well as their respective combination therapies. Furthermore, the analgesia ratio was calculated using the following equation:
[0749] The synergistic effects of these combinations compared to single-drug treatments were evaluated. Combination indexes (CI) were calculated based on Bliss independence model (CI = EA+EBEAB EA^- represents the analgesic ratio of compound 1-B or compound 44-B. EB indicates the analgesic ratio of the other analgesia drugs, such as morphine, pregabalin, suzetrigine, duloxetine, amitriptyline, and ketorolac. EAB denotes the analgesic ratio resulting from a combination treatment involving compound 1-B or compound 44-B and one of the following drugs: morphine, pregabalin, suzetrigine, duloxetine, amitriptyline, and ketorolac.
[0750] For interpretation, a CI value within the range of 0.95 to 1.1 was considered additive, reflecting expected outcomes under independent drug action while accounting for experimental variability. CI values < 0.95 were considered synergistic, indicating that the observed combination effect exceeded the predicted additive effect. In contrast, CI values > 1.05 were considered antagonistic, suggesting that the combination was less effective than expected based on individual drug activities. Attorney Docket No.: 071741.11025/5WO1 [0751] The results were recorded in FIGs.4A-4J, and summarized in Table 9. The results show that all CIs are either below or close to 1, which indicates that compound 1-B exhibits synergistic or additive effects when combined with morphine, pregabalin and suzetrigine, respectively, while compound 44-B demonstrate synergy or additivity when combined with morphine, pregabalin, suzetrigine, duloxetine, amitriptyline, and ketorolac, respectively. [0752] Table 9: the combination indexes calculation EA: EB: EAB: A drug B drug Time CI 3 8 5 9 4 9 2 2 8 5 [0753] Example 6. Efficacy on bone cancer pain model in mice [0754] Animals were acclimatized to the environment for 3-7 days upon arrival at the facility. Male C3H/He mice were anesthetized with a combination of Zoletil 50 (50 mg/kg) and Xylazine 185 Attorney Docket No.: 071741.11025/5WO1 Hydrochloride (8 mg/kg) administered via intraperitoneal injection, and positioned supinely. The right hind limb was shaved and sterilized. A minimal incision was made on the right hind leg to sever the patellar ligaments and expose the condyles of the distal femur. The proximal femur was perforated using a 0.3 mL syringe needle. A 10 µL suspension containing 2×104 NCTC-2472 cells (suspended in a pellet formed from 2 mL of cell stock by centrifugation at 1000 rpm for 4 minutes, washed twice with 2 mL PBS, and resuspended in PBS at a concentration of 2×106 cells/mL) was slowly injected into the intramedullary cavity of the femur. Control group animals received a 10 µL PBS injection (day 0). Animals were subsequently acclimatized to the testing environment for an additional three days before baseline PWT measurements were initiated. [0755] On day 14, baseline measurements for mechanical allodynia were conducted. Animals not exhibiting allodynia (PWT > 0.6 g) were excluded. The remaining qualified animals were then randomly assigned into four groups based on their baseline PWT values. The animals received a single injection of test compounds, including pregabalin 3mg/kg p.o., morphine 1mg/kg s.c., 44-B 1mg/kg p.o., as well as a group for 1-B 20mg/kg p.o. and 44-B 20mg/kg p.o. at a dose of 10 mL/kg based on body weight, and mechanical allodynia tests were performed at various time points post- administration as dictated by different experimental requirements with sham group and vehicle group. Each mouse was placed in a separate plastic enclosure with a mesh floor to freely access the paws and allowed to acclimate for 15 minutes prior to testing. [0756] Mechanical allodynia tests were conducted and analyzed as described in the SNI model in Example 5. The results are demonstrated by FIGs.2A-2D. [0757] The synergistic effect was evaluated by comparing the combination treatment with single- agent treatments, following the protocol described in Example 5. [0758] The results were recorded in FIGs.4K-4O and FIGs.3B-3C and summarized in Table 9 and Table 10. The results show combination indices (CIs) are below 1.1, indicating that compound 44-B exhibits synergistic or additive effects when administered in combination with morphine, pregabalin, duloxetine, and amitriptyline, respectively. 186 Attorney Docket No.: 071741.11025/5WO1 [0759] Table 10: Combination Indexes Calculation EAB: EA: EB: A dr B dr Tim A+B CI 6 1 6 8 7 [ ] xampe . va uat on o t e e cacy n post-surgery pa n mo e n m ce [0761] Upon arrival at the facility, the animals were adaptively fed for 3 to 7 days. Additionally, for three days preceding the surgical procedures, all animals were placed in the test environment and acclimated daily for at least 15 minutes. [0762] Aseptic techniques were rigorously applied by all surgeons. All surgical tools—including scissors, sharp forceps, scalpels, sterile cotton pads, needles, and metal clips—were sterilized prior to use. Animals were anesthetized using Zoletil 50 (50 mg/kg, 2.5 mL/kg, intraperitoneal) and Xylazine Hydrochloride (8 mg/kg, 2.5 mL/kg, intraperitoneal). A toe pinch confirmed deep anesthesia before any incisions were made. Ophthalmic ointment was applied to the animals’ eyes to prevent corneal drying. The plantar aspect of the left hind paw was cleansed with three rounds of alternating Betadine and 70% ethanol applications, allowing the surface to air-dry. A 0.5-mm longitudinal incision was then made through the skin and fascia from 2 mm proximal to the heel towards the toes. The plantar muscle was longitudinally incised while preserving the origin and insertion points. Hemostasis was achieved with gentle pressure, and the skin was closed with two mattress sutures. Post-surgery, all surgical instruments were cleaned and re-sterilized using a glass bead sterilizer. Animals were allowed to recover from anesthesia on a heated recovery pad and 187 Attorney Docket No.: 071741.11025/5WO1 were hydrated with 1 mL of sterile saline orally to prevent dehydration. Once fully awake and mobile, the animals were returned to their home cages. [0763] On the first day post-surgery, all animals, including those in the Naive group, were assessed for mechanical allodynia using a Touch-Test Sensory Evaluator. Surgical animals not displaying allodynia (PWT > 0.6 g) were excluded, leaving only 24 qualified surgical animals who were randomly assigned into three groups based on their baseline PWT, forming a total of four groups including the Naive group. [0764] Animals were administered test compounds: morphine at 3 mg/mL s.c., 1-B HCl at 10 mg/mL p.o. and 44-B at 10 mg/mL p.o., all at a dosage of 10 mL/kg based on body weight. Animals in the Naive group were also assessed but received no treatment. [0765] Mechanical allodynia tests were conducted and analyzed as described in SNI model in Example 5. The results are demonstrated by FIGs.3A-3C. [0766] Example 8. in vivo Efficacy Study in the Treatment of Subcutaneous Colorectal Cancer Syngeneic Model MC38 in Female C57BL6/J Mice [0767] The objective of this study is to evaluate the in vivo efficacy study of test srticles in the Treatment of Subcutaneous Colorectal Cancer Syngeneic Model MC38 in Female C57BL6/J mice. The mice are Mus musculus C57BL6/J, female, supplied by Beijing HFK Bioscience Co. LTD, with an average age of 6-8 weeks. The cage is polysulfone IVC cage, with a temperature 20-26°C and humidity 40 – 70%. The light cycle is 12 hours light and 12 hours dark. The mice is feed by a diet of standard rodent chow, irradiated, ad libitum. The water is autoclaved filtered RO (reverse osmosis) softened, filtered water, ad libitum. [0768] Clonidine and compound 1-B HCl were used as control and test articles, respectively, and the study is designed according to the following table. Due to the poor state of mice caused by high dose, the dose of clonidine in G2 group and 1-B HCl in G4 group was adjusted from 5mg/mL to 2mg/mL and 10mg/kg to 5mg/kg, respectively, starting from Day 4. The detailed design and formulation is in Table 11. 188 Attorney Docket No.: 071741.11025/5WO1 [0769] Table 11. Study design and formulation Group No. of Treatment Dose Dosing Dosing ROA Dosing Frequency & mice Level Solution Volume Duration (mg/kg) (mg/mL) (μL/g) 1 6 Vehicle -- -- 10 p.o. QD×Day0~Day17 2 6 Clonidine 5 0.5 10 p.o. QD×Day0~Day3 2* 0.2 10 p.o. QD×Day4~Day17 3 6 1-B HCl 5 0.5 10 p.o. BID×Day0~Day17 4 6 1-B HCl 10 1 10 p.o. BID×Day0~Day3 5* 0.5 10 p.o. QD×Day4~Day17 *dosage adjusted on day 4 [0770] The MC38 cancer cells were maintained in vitro with DMEM medium supplemented with 10% fetal bovine serum and 50μg/mL Hygromycin B at 37ºC in an atmosphere of 5% CO2 in air. The cells in exponential growth phase were harvested and quantitated by cell counter before tumor inoculation. Each mouse was inoculated subcutaneously at the right rear flank region with MC38 tumor cells (1x 106) in 0.1 mL of PBS mixed with PBS for tumor development. The randomization started when the mean tumor size reached approximately 121.36 mm3.30 mice were enrolled in the study. All animals were randomly allocated to 5 study groups, 6 mice in each group. Randomization was performed based on “Matched distribution” method. The date of randomization was denoted as day 0. [0771] The treatment was initiated on the same day of randomization (day 0) per study design. After tumor cells inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss (Body weights were measured twice per week after randomization), eye/hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail. Tumor volumes were measured twice per week after randomization in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: “V = (L × W × W)/2, where V was tumor volume, L was tumor length (the longest tumor dimension) and W was tumor width (the longest tumor dimension perpendicular to L). Dosing as well as tumor and body weight measurements 189 Attorney Docket No.: 071741.11025/5WO1 were conducted in a Laminar Flow Cabinet. The body weights and tumor volumes were measured by using StudyDirectorTM software (version 3.1.399.19). [0772] The body weights of all animals were monitored throughout the study and animals were euthanized if they lost over 20% of their body weight relative to the weight on the day of randomization. Meanwhile, the individual mouse was euthanized if its tumor volume exceeds 3000 mm3. To deter cannibalization, any animal exhibiting an ulcerated or necrotic tumor were separated immediately and singly housed and monitored daily before the animal was euthanized or until tumor regression was completed. The mouse was euthanized rapidly if a) tumor ulcerates, and the ulceration diameter was greater than 5 mm, or pus or necrosis observed, and b) tumor burden, including metastasis, compromises animal’s normal physiologic performances, e.g., orientation, access to food or water, etc. [0773] The body weight between randomization grouping is shown in FIG.5A. The tumor growth of each treatment group and control group is shown in FIG.5B. In day17, the mice were sacrificed and the tumors were removed and measured. The data are shown in FIG.5C. The tumor growth inhibition (TGI) and T/C were calculated based on the tumor size data of day 17, which is the last dosing day of the treatment. The Tumor volume was shown as the mean ± SEM, while the T/C% = tumor volume of treatment group / tumor volume of control group × 100%. The TGI% = (1-T/C) × 100%. The pharmacodynamic analysis result is shown in Table 12. [0774] Table 12: Pharmacodynamic analysis of each group in Subcutaneous Colorectal Cancer Syngeneic Model MC38 D17 Group Tumor volume (mm3) M) TGI (% P Value (mean ± SE ) T/C (%) (Compared with control group) G1 2478.07±439.10 -- -- -- G2 766.52±147.17 72.62% 27.38 <0.05 G3 1009.16±258.31 62.32% 37.68 <0.05 G4 996.54±270.05 62.86% 37.14 <0.05 [0775] Another in vivo efficacy study of clonidine, 44-B and their combination therapy with Anti-mPD-1 in the treatment of subcutaneous colorectal cancer syngeneic model MC38 in female C57BL6/J mice was carried out with same protocol but different treatment design.10mg/kg Anti- mPD-1 i.p. BIWx5 does, 2mg/kg clonidine p.o. QDx15days, and 2mg/mL 44-B HCl p.o. 190 Attorney Docket No.: 071741.11025/5WO1 QD×15days were administrated as the single therapy, while the 10mg/kg Anti-mPD-1 i.p. BIWx5 does and 2mg/kg clonidine p.o. QD×15days, as well as the combination of 10mg/kg Anti-mPD-1 i.p. BIWx5 does and 5mg/kg 44-B p.o. QDx15days were administrated as the combination therapy. The experiment design is in Table 13. [0776] Table 13. Study design and formulation Group No. of Treatment Dose Level Dosing Solution Dosing Volume ROA Dosing Frequency & mice (mg/kg) (mg/ml) (μL/g) Duration 1 6 Vehicle -- -- * 2 6 Anti-mPD-1 10 1 3 6 Clonidine 2 0.2 p. . y 4 6 44-B HCl 2 0.2 10 p.o. QD*15days 5 6 44-B HCl 5 0.5 10 p.o. QD*15days 6 6 Clonidine 2 0.2 10 p.o. QD*15days Anti-mPD-1 10 1 10 i.p. BIW×5 doses 7 6 44-B HCl 2 0.2 10 p.o. QD*15days Anti-mPD-1 10 1 10 i.p. BIW×5 doses 8 6 44-B HCl 5 0.5 10 p.o. QD*15days Anti-mPD-1 10 1 10 i.p. BIW×5 doses [0777] The body weight between randomization grouping is shown in FIG.6A, indicating both group 3 and group 6 have body weight loss. The tumor growth of each treatment group and control group is shown in FIG.6B. In day17, the mice were sacrificed and the tumors were removed and measured. The data is shown in FIG.6C. [0778] The tumor growth inhibition (TGI) and T/C were calculated based on the tumor size data of day 14, which is the last dosing day of the treatment. The Tumor volume was shown as the mean ± SEM, while the T/C% = tumor volume of treatment group / tumor volume of control group × 100%. The TGI% = (1/T/C) × 100%. Combination indexes (CI) were calculated based on Bliss independence model (^^^^^^^^ = ^^^^^^^^+^^^^^^^^×(1−^^^^^^^^) ^^^^^^^^^^^^ )). EA represents the TGI of compound 44-B. EB indicates the TGI of An notes the analgesic ratio resulting from a combined treatment involving compound 44-B and Anti-mPD-1. The data is shown in Table 14. The combination group of 10mg/kg Anti-mPD-1 i.p. BIW×5 does and 5mg/kg 44-B p.o. QD×15days showed a synergy effect. 191 Attorney Docket No.: 071741.11025/5WO1 [0779] Table 14: The tumor size on the day 14 Dose Tumor Volume P Group Treatment Level (me T/C TGI CI (mg/kg) an ± SEM) Value 1 vehicle - 2139.68±321.06 - - - - 2 Anti-mPD-1 10 1211.81±107.44 56.63% 43.37% 0.0850 - 3 Clonidine 2 970.01±177.02 45.33% 54. 4 44-B HCl 2 1779.37±263.14 83.16% 16. . - 5 44-B HCl 5 1628.27±260.90 76.10% 23.90% 0.725 - 6 Clonidine+ 2+10 802.38±191.23 37.50% 62.50% 0.00261 1.19 Anti-mPD-1 44-B HCl + 7 Anti-mPD-1 2+10 1331.06±214.34 62.21% 37.79% 0.190 1.40 8 44-B HCl + Anti-mPD-1 5+10 774.11±156.24 36.18% 63.82 0.00200 0.89 [0780] Example 9. Spontaneous Locomotor Activity in Mice [0781] The study evaluated the effects of clonidine, brimonidine tartrate, compound 1-B HCl, compound 44-B HCl on spontaneous locomotor activity in male C57BL/6 mice. Initially, mice were acclimatized to the testing environment for 8 hours the day before the experiment, followed by at least 2 hours of habituation on the day of the test. The mice were then grouped randomly based on their body weight into six per group, ensuring a balanced distribution for the administration of the drug, which was dissolved in 20% HP-β-CD in water. On the test day, in one test, clonidine at a dose of 1mg/kg and compound 1-B HCl at concentrations of 1mg/kg, 10mg/kg, and 20mg/kg were freshly prepared and administered orally at a volume of 10mL/kg. In another test, clonidine at a dose of 1mg/kg, brimonidine tartrate at a dose of 1mg/kg and compound 44-B HCl at a dose of 1mg/kg were freshly prepared and administered orally at a volume of 10mL/kg. [0782] The locomotor activity was monitored by placing the mice in the center of a test box, with a video tracking system measuring the distance traveled every 5 minutes for 60 minutes. The testing began at T=0 minutes, immediately after administering the vehicle or compounds, and concluded at T=60 minutes. For data analysis, Prism 8.3.0 software was utilized, employing Two- way ANOVA followed by Bonferroni's multiple comparison test to analyze distance variations across different time points and One-way ANOVA followed by Dunnett’s multiple comparisons test for assessing the total distance covered by the groups. A significance level of p<0.05 was 192 Attorney Docket No.: 071741.11025/5WO1 established for determining significant differences. As demonstrated in FIG.7, no significant sedation was observed after the treatment of compound 1-B HCl at 1mg/kg, 10mg/kg and 20mg/kg in the first assay and compound 44-B HCl at 1mg/kg in the second assay, while 1mg/kg of clonidine in the first assay and 1mg/kg brimonidine tartrate in the second assay leads to a significant sedation. The total distance between 0 to 60 minutes of the two tests is shown in FIG. 7A and FIG.7B. [0783] Example 10. The effects on motor function in mice [0784] Upon arrival at the facility, the animals were acclimated for one week. The day before the rotarod training commenced, mice were randomly assigned to groups based on their body weight to ensure homogeneity across the groups in terms of weight before any treatment was administered. Rotarod training occurred two days prior to the testing phase. On the first training day, the mice underwent three trials on the rotarod at a speed of 6 rpm, each lasting 120 seconds, with 30-minute intervals between trials. If a mouse fell off before completing 120 seconds, it was immediately placed back on the rotarod to complete the training duration. The following day, the training consisted of a single trial at the same speed of 6 rpm but extended to 300 seconds. Mice that fell before the 300-second mark were similarly returned to the rotarod to ensure they reached the full training time. [0785] On the test day, treatments were administered orally to the mice at a dosage volume of 10 mL/kg based on their body weight. The treatments included a vehicle, clonidine (1 mg/kg), and 44- B HCl at three dosages (1 mg/kg, 10 mg/kg, and 20 mg/kg). The time of compound administration was designated as time zero. [0786] The rotarod test was conducted at 30, 60, and 120 minutes post-administration, with each session lasting 300 seconds at a speed of 6 rpm. The primary measure was the latency time until a mouse fell from the rotarod, which served as an indicator of the compounds' effects on motor function. [0787] Data were recorded in Microsoft Excel and subsequently analyzed using GraphPad Prism. Statistical significance was assessed with a threshold P-value of less than 0.05, indicating meaningful differences between treatment groups. [0788] The detailed analysis method is described as follows. Initially, the data are assessed for normal distribution and homogeneity of variance. If the data adhere to both normal distribution and 193 Attorney Docket No.: 071741.11025/5WO1 homogeneity of variance, a T-test is applied for comparisons involving two data sets, and a one- way ANOVA is utilized for analyses involving multiple data sets. In cases where the data exhibit normal distribution but heterogeneity of variance, Welch’s T-test is used for two data sets, and a nonparametric test is employed for multiple data sets. If the data do not fit a normal distribution, the Mann-Whitney test is applied for two data sets, and the Kruskal-Wallis test is used for multiple data sets. The results are displayed in FIGs.8A to 8D. 194

Claims

Attorney Docket No.: 071741.11025/5WO1 CLAIMS 1. A method for treating or preventing pain in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a first analgesic agent, and a therapeutically effective amount of a second analgesic agent, wherein the first analgesic agent is an a peripherally selective alpha-2 adrenergic receptor (α2AR) agonist, and the second analgesic agent is chosen from opioids, antidepressant medications, antiepileptic medications, local analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen (paracetamol), corticosteroids, NMDA receptor antagonists, cannabinoids, adenosine agonists, transient receptor potential (TRP) channel blockers, and voltage-gated sodium channel inhibitors. 2. The method of claim 1, wherein the peripherally selective α2AR agonist has a Kp,uu,brain lower than 0.05, 0.02, or 0.01. 3. The method of claim 1, wherein the pain is chosen from neuropathic pain, nociceptive pain, nociplastic pain, and mixed pain. 4. The method of claim 1, wherein the opioid is chosen from heroin, codeine, morphine, thebaine, diacetyl morphine, hydromorphone, hydrocodone, oxymorphone, fentanyl and methadone. 5. The method of claim 1, wherein the antidepressant medication is a selective serotonin and norepinephrine reuptake inhibitor (SNRI), a tricyclic antidepressant(TCAs), or a monoamine oxidases inhibitors (MAOIs). 6. The method of claim 1, wherein the antidepressant medication is chosen from amitriptyline, nortriptyline, desipramine, clomipramine, imipramine, duloxetine, venlafaxine, desvenlafaxine, bupropion, and milnacipran. 7. The method of claim 1, wherein the antiepileptic medication is chosen from gabapentin, pregabalin, mirogabalin, phenibut, baclofen, 4-fluorophenibut, 4-methylpregabalin, atagabalin (PD-200,390), imagabalin, PD-217,014, tolibut, oxcarbazepine, and carbamazepine. 195 Attorney Docket No.: 071741.11025/5WO1 8. The method of claim 1, wherein the nonsteroidal anti-inflammatory drug (NSAID) is chosen from aspirin, diflunisal, ibuprofen, naproxen, indomethacin, diclofenac, meclofenamate, mefenamic acid, meloxicam, piroxicam, nabumetone, celecoxib, and etoricoxib. 9. The method of claim 1, wherein the NMDA antagonists is chosen from ketamine, methadone, memantine, amantadine, and dextromethorphan. 10. The method of claim 1, wherein the voltage-gated sodium channel inhibitor is the inhibitor of NaV1.3, NaV1.7, NaV1.8 or NaV1.9. 11. The method of claim 10, wherein the voltage-gated sodium channel inhibitor is selective NaV1.8 inhibitor. 12. The method of claim 11, wherein the selective NaV1.8 inhibitor is chosen from suzetrigine (VX-548), ANP-230, VX-150, JMK-000623, HBW-004285, DSP-2230, VX-993, VX-937, LTG-001, kindolor, and FZ-008. 13. The method of claim 1, wherein the peripherally selective α2AR agonist comprises an α2AR activation moiety covalently linked to a peripheral distribution moiety. 14. The method of any one of claim 11, wherein the α2AR activation moiety is an α2AR agonist. 15. The method of any one of claims 11-14, wherein the peripheral distribution moiety comprises a substrate element for an active efflux transporter. 16. The method of claim 15, wherein the active efflux transporter is P-glycoprotein (P-gp). 17. The method of claim 16 , wherein the substrate element for P-glycoprotein (P-gp) is chosen from: 196 Attorney Docket No.: 071741.11025/5WO1 O H O N O H H O N N O O O NH N P H S O F3 S 18. The method of claim 15, wherein the active efflux transporter is breast cancer resistance protein (BCRP) transporter. 19. The method of claim 15, wherein the active efflux transporter is multidrug resistance protein 2 (MRP2) transporter. 20. The method of claim 11, wherein the α2AR activation moiety has formu B , wherein, A is one chosen from: S ; each R1 is independently chosen from hydrogen, halogen, haloalkyl, hydroxyl, hydroxyalkyl, alkoxy, alkyl, and -COOH; 197 Attorney Docket No.: 071741.11025/5WO1 B is chosen from: H N N H X S N N , wherein X is S, O, or NH. RT 21. The method of claim 11, wherein the peripheral distribution moiety has formula , wherein, R2 r 8 ; C6-12 aryl, or C1-12 heteroaryl, wherein the C3-12 cycloalkyl or C2-12 heterocyclyl is optionally fused with an aryl; r is 1 or 2; n2 is 0, 1, or 2; each R2 is independently chosen from hydrogen, halogen, hydroxyl, and alkoxy; R3 is chosen from CN, hydroxy, alkoxy, -C(O)-C0-12 alkylene-CN, -C0-12 alkylene-C2-12 heterocyclyl, -SO2-alkyl, -C(O)-NR4R4’, -SO2-NR4R4’, -C0-12 alkylene-R3’, -O-C0-12 alkylene- COOH, -C0-12 alkylene-N(R4)-C(O)-R5, -C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-O-C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-P(=O)(R4)(R4’), O O O R6 O CH3 ; wherein one -CH2- group in the -C0-12 alkylene- r , the -C0-12 alkylene-COOH is optionally substituted with one or more substitutes chosen from amino and alkylamino, and the C2-12 heterocyclyl and C1-12 heteroaryl are each optionally substituted with one or more R4a; R3’ is chosen from -C(O)-NR4R4’, -SO2-NR4R4’, -C0-12 alkylene-COOH, -C0-12 alkylene- N(R4)-C(O)-R5, -C0-12 alkylene-N(R4)-SO2-R5, C0-12 alkylene-C1-12 heteroaryl; each R4a is independently chosen from hydroxy, alkyl, oxo, ketone, and -C2-12 heterocyclyl; each of R4 and R4’ is independently hydrogen, alkyl, alkoxy, -SO2- N(R6a)t, -C0-12 alkylene- COOH, -C0-12 alkylene-N(R6a)t, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 198 Attorney Docket No.: 071741.11025/5WO1 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C0-12 alkylene-OR6a, or hydroxyalkyl, wherein the hydroxyalkyl is optionally substituted with alkoxy; wherein each of the alkyl, C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a; or R4 and R4’, together with the nitrogen atom that they are attached to, form a heterocycle comprising one or more heteroatoms chosen from O, N, and S; alternatively, when one R2 is adjacent to R3, the R2 and R3, together with the atoms that they are attached to, form a ring optionally substituted with one or more R4a;; R5 is amino, alkylamino, C1-12 haloalkyl, -C0-12 alkylene-OR6a, -C0-12 alkylene-N(R6a)t, -C0- 12 alkylene-SR6a, -C0-12 alkylene-CN, -C0-12 alkylene-C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C1-12 heteroaryl, -C2-12 alkenyl, or alkyl optionally substituted with cyano, amido, trialkylammonium, or thiolate; wherein each of the C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl is optionally substituted with one or more R4a; each R6a is independently chosen from hydrogen, C1-12 alkyl, C1-12 alkoxy, -C0-12 alkylene- C3-12 cycloalkyl, -C0-12 alkylene-C2-12 heterocyclyl, -C0-12 alkylene-C6-12 aryl, and -C0-12 alkylene- C1-12 heteroaryl; wherein each of the alkyl, C3-12 cycloalkyl, C2-12 heterocyclyl, and C1-12 heteroaryl, is optionally substituted with one or more R4a; R6 is alkoxy, amino, sulfonamide, carbamide, or alkyl optionally substituted with cyano; R7 is hydrogen, alkyl, -C0-12 alkylene-COOH, optionally substituted C3-12 cycloalkyl, C2-12 aryl, C1-12 heteroaryl, -C0-12 alkylene-N(R4)-SO2-R5, -C0-12 alkylene-P(=O)(R4) (R4’), -C0-12 alkylene-N(R4)-C(=S)-R5, -C(=S)-R5, or alkyl optionally substituted with cyano; R8 is alkoxy, amino, alkylamino, amide, sulfonamide, or carbamide; n3 is 0, 1, 2, 3, or 4; n4 is 1, 2, 3, 4, 5, or 6; t is 2 or 3; m is 0, 1, 2, 3, 4, or 5; and n is 0, 1, 2, 3, or 4. 199
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