Conjugates of transthyretin ligands with opioid agonists and antagonists

Abstract

Provided herein are compounds of Formula (I): (I), or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, wherein T, L, LB, E, and D1 are as defined elsewhere herein. Also provided are methods of making compounds of Formula (I).

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 572,100, filed March 29, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] Aspects of the present invention generally relate to a delivery system for an active agent having a ligand (binding agent) selective for thyroxine carrier protein (TTR) in the serum of a subject; and a cleavable connector configured to operatively covalently link the ligand to an opioid antagonist or opioid agonist to increase the plasma circulation time and duration of action of the opioid antagonist or opioid agonist in serum. Background Technology

[0003] Opioids have significant adverse effects, highlighted by the large number of opioid use disorder (OUD) patients and the rising number of overdose deaths in the United States and elsewhere. An estimated 3 million Americans aged 12 and older have OUD, including 2.3 million prescription OUD patients. Approximately 66% of these deaths involve illicit synthetic opioids such as fentanyl (about 50 times more potent than heroin and 100 times more potent than morphine), a major driver of the current opioid epidemic. Many opioid overdose deaths are attributed to the mixing of fentanyl with other illicit drugs such as heroin, cocaine, and methamphetamine. The potential lethal dose of fentanyl is about two milligrams and is particularly dangerous for opioid-intolerant individuals who have never received opioids. Since 2019, overdose deaths among U.S. high school students have more than doubled, attributed to counterfeit drugs (e.g., Xanax, Percocet, Adderall) combined with lethal doses of fentanyl.

[0004] Although the half-lives of fentanyl (about 7 hours) and morphine (2 to 3 hours) are... t 1 / 2 While the duration of action (DOA) of fentanyl is relatively short, fentanyl overdose can last up to 24 hours, and morphine overdose can last up to 120 hours (depending on the usually unknown opioid dose). The μ-opioid receptor antagonist naloxone has a relatively short DOA (approximately 1 hour), which poses a major challenge to its efficacy against fentanyl overdose. Salvage of synthetic opioid overdose requires larger doses or repeated doses of naloxone compared to existing doses used by first responders (2 mg intramuscularly and 4 to 8 mg intranasally). Due to the high risk of recurrent respiratory depression, patients are typically given repeated small doses of naloxone as needed and monitored continuously for 6 hours before discharge.

[0005] Naloxone is not very effective in reversing respiratory depression caused by partial agonists such as buprenorphine and may require larger doses or repeated doses. Nalmefone's higher potency (5 times stronger than naloxone) and relatively longer DOA (up to 8 hours) make it a better alternative to naloxone. However, it is difficult to imagine how naloxone and nalmefone could be effectively deployed in mass casualty situations involving synthetic opioids, where the overdose could last up to 24 hours. Therefore, there is a strong, long-acting antidote (with a longer DOA than nalmefone) for use in conventional first aid or as a preventative measure in mass casualty events.

[0006] Furthermore, a large number of overdose patients discharged from hospitals after emergency treatment with antagonists die within the following month, with 20% of those deaths occurring within two days (most in private residences where patients are often alone and unable to administer the antidote themselves). Therefore, an effective opioid antidote with a prolonged DOA that can be administered to emergency overdose patients discharged from hospitals can provide the necessary extended protection against re-anesthesia. Summary of the Invention

[0007] Surprisingly, it has been found that the duration of action (DOA) of opioid antagonists or opioid agonists can be significantly prolonged by covalently linking the opioid antagonist or opioid agonist to a ligand selective for thyroxine transporter (TTR) via a cleavable linker. Therefore, this disclosure relates to a novel prodrug method for enhancing the DOA of opioid drugs without affecting their biological activity.

[0008] In one respect, this paper provides a conjugate of formula (I): Or its pharmaceutically acceptable salt, wherein: T is a selective ligand for the thyroxine transporter protein TTR; L stands for connector; L B Click products that do not exist or are formed through a click reaction between the first click handle and the second click handle; E is a cleavable ester; and D 1 It is an opioid antagonist or an opioid agonist.

[0009] In one embodiment, the cleavable ester moiety (E) of the formula (I) conjugate comprises an aromatic ring. In some embodiments, the aromatic ring may optionally be substituted with one or more substituents as set forth herein. In some embodiments, p-aminobenzoic acid (PABA) is used as the basic structural unit for forming an ester with an opioid antagonist or opioid agonist. In some embodiments, p-hydroxybenzoic acid (PHBA) is used as the basic structural unit for forming an ester with an opioid antagonist or opioid agonist. In some embodiments, substituted PABA is used as the basic structural unit for forming an ester with an opioid antagonist or opioid agonist. In some embodiments, the aromatic ester group (e.g., PABA or PHBA) of the formula (I) conjugate is hydrolyzed by carboxylesterase 2 (CES2) to a greater extent than by carboxylesterase 1 (CES1). In some embodiments, the aromatic ester group (e.g., PABA or PHBA) of the formula (I) conjugate is hydrolyzed by CES2, but only minimally by CES1. In some embodiments, the aromatic ester group (e.g., PABA) of the formula (I) conjugate is completely resistant to CES1.

[0010] Surprisingly, in some embodiments, TTR ligands have been found to influence the degree of hydrolysis of aromatic ester groups (e.g., PABA or PHBA groups) in the conjugate and the selectivity of carboxylesterases. For example, in some embodiments, the TTR ligand (binding agent) of the conjugate of formula (I) is capable of increasing the CES2 selectivity of the conjugate of formula (I). This CES2 selectivity provides a more controlled or prolonged release of opioid antagonists or opioid agonists from the conjugate. Furthermore, the hydrolysis rate can be effectively modulated (e.g., increased or decreased) by specific substitutions on the aromatic ester groups (e.g., PABA or PHBA groups) of the conjugate.

[0011] In some embodiments, the conjugate of formula (I) comprises the following ester moiety: in Indicates with L or L B The connection point.

[0012] In some embodiments, the conjugate of formula (I) comprises the following ester moiety: in Indicates with L or L B The connection point. R 1x It is H, F, Cl, NO2, CF3 or OMe, and R 1y It is H or Me. In some implementations, R 1x It is H or Me, and R 1yIt is H, F, Cl, Br, NO2, CF3, Me, or OMe. In some implementations, R 1x It is H and R 1y It's me.

[0013] In some embodiments, the conjugate of formula (I) comprises the following ester moiety: in Indicates with L or L B The connection point.

[0014] In some embodiments, the conjugate of formula (I) comprises the following ester moiety: in Indicates with L or L B The connection point.

[0015] In some embodiments, the conjugate of formula (I) comprises the following ester moiety: in Indicates with L or L B The connection point, R 1x It is H, F, Cl, NO2, CF3 or OMe, and R 1y It is H or Me. In some implementations, R 1x It is H or Me, and R 1y It is H, F, Cl, Br, NO2, CF3, Me, or OMe. In some implementations, R 1x It is H and R 1y It's me.

[0016] In some implementations, the ligand selective for thyroxine transporter TTR (T) is a compound of formula (E). (E) Or a pharmaceutically acceptable salt thereof, wherein variable R 1 R 2 R 3 R a R b X and a are defined herein. In some embodiments, the formula (I) conjugate comprises a ligand of formula (E) and an aromatic ester group (e.g., PABA or PHBA). In some embodiments, the formula (E) compound increases the CES2 selectivity of the aromatic ester group (e.g., PABA or PHBA) in vivo.

[0017] In one embodiment, this document provides a conjugate of formula (II): (II), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, 3, or 4 R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L stands for connector; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the following groups: hydrogen, halogen atoms (e.g., Cl, Br, or F), NO2, CF3, OMe, O(C 1-4 Alkyl), hydroxyl, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; and D 1 It is an opioid antagonist or an opioid agonist.

[0018] Any embodiment of the conjugate of formula (I) or formula (II) provided herein, or a pharmaceutically acceptable salt thereof, is applicable where applicable to any other formula detailed herein, as if each embodiment were specifically and individually listed. Therefore, it should be understood and described that each embodiment of the conjugate of formula (I) or formula (II) provided herein is applicable to the conjugates of formulas (III) to (XI) or (III') to (VIII') or a pharmaceutically acceptable salt thereof, as if each embodiment were specifically and individually listed. It should also be understood and described that all such embodiments are applicable to any of the pharmaceutical compositions, methods, kits, uses, or other aspects detailed herein.

[0019] In one aspect, this document provides a method for increasing the plasma circulation time and DOA of an opioid antagonist or opioid agonist, said method comprising administering to a subject (e.g., a human patient) a therapeutically effective amount of a conjugate of this disclosure (e.g., a conjugate of formula (I) or (II)). In some embodiments, administration of a conjugate of this disclosure (e.g., a conjugate of formula (I) or (II)) reduces the immunogenicity of the active agent relative to administration alone of the opioid antagonist or opioid agonist in its free (unconjugated) form. In other embodiments, the conjugate of this disclosure is administered orally.

[0020] In some embodiments of subcutaneous administration of the conjugates of this disclosure, the conjugates form a reservoir in the subcutaneous tissue. In some such embodiments, the solubility of the conjugate at physiological pH is less than that of the unconjugated drug at physiological pH. Therefore, the slow dissolution of the conjugate relative to the unconjugated drug slows the release of the drug into systemic circulation, thus prolonging the duration of action of the drug. Subsequent binding of the conjugate to the TTR in systemic circulation further prolongs the duration of action of the drug. As shown in Example 11, in some embodiments, the ability of the conjugate to form a reservoir is attributed to the zwitterionic nature of the conjugate at physiological pH. In some embodiments, despite the reduced solubility of the conjugate relative to the unconjugated drug, the conjugate is rapidly absorbed, resulting in rapid action. Therefore, the conjugates of this disclosure provide a drug with rapid onset of action and prolonged duration of action.

[0021] In some embodiments, the conjugates of this disclosure are administered parenterally to subjects in need. For example, the conjugates of this disclosure may be administered subcutaneously, intravenously, intramuscularly, or intrathecally.

[0022] Partly due to the balanced hydrophilicity of the TTR selective ligand, the conjugates of this disclosure (particularly the formula (II) conjugate) exhibit enhanced adsorption from tissues (particularly subcutaneous tissue). Following adsorption from tissues (e.g., subcutaneous tissue), the reversible binding of the TTR selective ligand of the conjugate significantly prolongs the duration of action of the active agent. As illustrated herein, cleavage of the linker between the TTR selective ligand and the active agent (opioid antagonist or opioid agonist) releases the active agent, thus allowing it to exert its pharmacological effects.

[0023] Furthermore, it has been found that the release of the active agent can be effectively controlled by appropriately selecting the cleavable moiety in the conjugate. For example, specific ester moieties, as described herein, undergo slow hydrolysis after administration, thereby releasing the active agent in a controlled manner for a prolonged period. Slower hydrolysis and high binding to TTR effectively prolong plasma circulation time and duration of action (DOA).

[0024] Therefore, the conjugates of this disclosure exhibit several beneficial properties of novel prodrug methods. The balanced hydrophilicity of the conjugates allows for rapid adsorption from tissues into the bloodstream, the binding of TTR prolongs plasma circulation time, and the cleavable moiety allows for modulated drug release. The desired release rate of a specific active drug from the conjugates of this disclosure will vary depending on the active agent.

[0025] In some implementations, the active agent (D) of the conjugate 1 This is an opioid antagonist. In some embodiments, the opioid antagonist is naloxone, nalmefene, naltrexone, methylnaltrexone, nalbuphrine, or nalirphine. In some embodiments, the plasma circulation and duration of action (DOA) of the opioid antagonist after administration of a conjugate of this disclosure (e.g., conjugate of formula (I) or (II)) is at least 12 hours. In some such embodiments, the DOA of the opioid antagonist after administration of a conjugate of this disclosure (e.g., conjugate of formula (I) or (II)) is at least 20 hours. In some such embodiments, the DOA of the opioid antagonist after administration of a conjugate of this disclosure is at least 96 hours. In some such embodiments, the DOA of the opioid antagonist after administration of a conjugate of this disclosure is at least 1 week.

[0026] The active agent (D) in the conjugate of formula (I) or formula (II) 1In some embodiments of opioid antagonists, administration of the conjugate reverses and protects against re-anesthesia (ventilation depression) due to an overdose of synthetic opioids. In some embodiments, the synthetic opioid is fentanyl. In some embodiments, the synthetic opioid is a fentanyl analogue, such as carfentanil, acetylfentanyl, butyrylfentanyl, or furanylfentanyl. In some embodiments, the synthetic opioid is hydromorphone. In some embodiments, the synthetic opioid is heroin. In some embodiments, the synthetic opioid is morphine. In some embodiments, the synthetic opioid is meperidine. In some embodiments, administration of the conjugate of formula (I) or formula (II) does not result in the deposition of opioid withdrawal symptoms.

[0027] In some embodiments, a conjugate containing an opioid antagonist is administered in combination with a free (unconjugated) opioid antagonist. In such an iteration, the free opioid antagonist effectively rescues opioid-induced respiratory depression (OIRD), while the opioid antagonist released from the conjugate increases the duration of action of the opioid antagonist and prevents re-anesthesia. In some embodiments, the free opioid antagonist is the same as the opioid antagonist in the conjugate. In some embodiments, the free opioid antagonist is different from the opioid antagonist in the conjugate. In some embodiments, the free opioid antagonist is naloxone. In some embodiments, the free opioid antagonist is nalmefene.

[0028] In some embodiments, the conjugates of this disclosure can be used to treat or mitigate opioid use disorder (OUD) in a subject of need (e.g., a human patient). In some embodiments, a method of treating OUD includes administering a therapeutically effective amount of a conjugate of this disclosure (e.g., a conjugate of formula (I) or (II)) containing an opioid antagonist as the active pharmaceutical ingredient (D). In some such embodiments, the opioid antagonist of the conjugate is naloxone, nalmefone, naltrexone, methylnaltrexone, nalbuphine, or nalprofen. In some embodiments, the opioid antagonist of the conjugate is naloxone. In some embodiments, the opioid antagonist of the conjugate is naltrexone. In some embodiments, the conjugate provides controlled and / or prolonged release of the opioid antagonist.

[0029] In other embodiments, a method of treating OUD includes administering a conjugate of this disclosure (e.g., a conjugate of formula (I) or formula (II)) comprising an opioid agonist as the active pharmaceutical ingredient (D). In some such embodiments, the opioid agonist of the conjugate is buprenorphine. In some such embodiments, the opioid agonist of the conjugate is aripiprazole. In some such embodiments, the opioid agonist of the conjugate is norclozapine. In some such embodiments, the opioid agonist of the conjugate is buspirone.

[0030] In some embodiments, the conjugates disclosed herein (e.g., conjugates of formula (I) or (II)) are applied to treat OUD (where D 1 (These are opioid antagonists or opioid agonists) that make withdrawal effects less severe compared to administration of opioid antagonists or opioid agonists in their unconjugated form. In some embodiments, the conjugate provides controlled and / or prolonged release of the opioid agonist.

[0031] In some embodiments, the conjugates of this disclosure (e.g., conjugates of formula (I) or (II)) may be administered in combination with one or more opioid antagonists or opioid agonists to treat OUD. In some embodiments, the conjugates of this disclosure may be administered in combination with buprenorphine (e.g., concurrently or sequentially). In some embodiments, the conjugates of this disclosure may be administered in combination with naloxone (e.g., concurrently or sequentially). In some embodiments, the conjugates of this disclosure may be administered in combination with naloxone (e.g., concurrently or sequentially). In some embodiments, the conjugates of this disclosure may be administered in combination with methylnaloxone (e.g., concurrently or sequentially). In some embodiments, the conjugates of this disclosure (wherein the opioid antagonist (D) in the conjugate is naloxone) may be administered in combination with buprenorphine (e.g., concurrently or sequentially) to treat OUD in patients of need. In some embodiments, the conjugate of this disclosure (wherein the opioid antagonist (D) in the conjugate is naltrexone or methylnaltrexone) can be administered in combination with buprenorphine (e.g., simultaneously or sequentially) to treat OUD in patients of need. In any of the foregoing embodiments, the conjugate can be administered subcutaneously.

[0032] The conjugates disclosed herein provide extended-release prodrugs for opioid antagonists or opioid agonists and provide opioid abuse control with minimal withdrawal effects. In some embodiments, the conjugates are administered to newborns of opioid-addicted mothers. In some embodiments, the conjugates are administered to children. In some embodiments, the conjugates are administered to children. In some embodiments, the conjugates are administered to elderly patients. In some embodiments, the conjugates are administered to patients with cardiovascular complications. In some embodiments, the conjugates are administered postoperatively to patients recovering from anesthesia / analgesia. In any of the foregoing embodiments, the conjugates may be administered subcutaneously.

[0033] In some embodiments, the conjugates disclosed herein can provide controlled and / or prolonged release of an opioid agonist. In some such embodiments, the slow release of the opioid agonist from the conjugate can be safely administered without causing euphoria or respiratory depression. In some embodiments, the opioid agonist is morphine, oxycodone, oxymorphone, codeine, hydrocodone, hydromorphone, methadone, thebaine, or fentanyl. In some embodiments, the conjugate is used to treat mild or acute pain.

[0034] In some embodiments, the conjugates of this disclosure are administered subcutaneously to human subjects at a dose of about 0.005 mg / kg to about 40 mg / kg. In some embodiments, the conjugates of this disclosure are administered subcutaneously to human subjects at a dose of about 0.01 mg / kg to about 40 mg / kg. In some embodiments, the conjugates of this disclosure are administered subcutaneously to human subjects at a dose of about 10 mg / kg to about 40 mg / kg. In some embodiments, the conjugates of this disclosure are administered subcutaneously to human subjects at a dose of about 10 mg / kg to about 20 mg / kg. Attached Figure Description

[0035] Figure 1 illustrates how the prodrug of this disclosure selectively binds to TTR in human plasma and releases naloxone in rat plasma. Figure 1a The chemical structures of naloxone, AG10-L, and the prodrug are described. AG10-L-E1-naloxone is an alkyl ester prodrug. AG10-L-E2-naloxone is a PABA aryl ester prodrug. C12-E2-naloxone is a control fatty acid-conjugated PABA aryl ester prodrug. Figure 1b The water solubility of the compound (incubated at 100 μM) in potassium phosphate buffer (pH 7.4) and the logarithmic properties of the compound in potassium phosphate buffer (pH 7.4) and n-octanol at physiological pH 7.4 were determined. D7.4 value. Figure 1c The binding affinity of the compounds to purified human TTR in buffer was evaluated using fluorescence polarization assays. The compounds were tested at concentrations ranging from 0.01 μM to 20 μM. The Cheng-Prusoff equation was used, and IC50 was employed. 50 Value calculation combined with constant ( K d Data represents the mean ± sd (). n = 3 independent repetitions). Figure 1d The binding selectivity of the compound (tested at 10 μM) for TTR (approximately 5 μM) in human serum was evaluated. The modification of TTR in human serum by the covalent FPE probe was monitored for 6 hours in the presence of the FPE probe alone (DMSO) or the probe and the TTR ligand. The ligand exhibiting lower FPE probe fluorescence showed higher binding selectivity for TTR. Figure 1e The percentage of occupancy was calculated using 3-hour readings relative to individual probes. Data are expressed as mean ± sd ( n = 4 independent repetitions). Figure 1f The plasma stability of the prodrugs AG10-L-E1-naloxone, AG10-L-E2-naloxone, and C12-E2-naloxone (as tested at 5 μM) in human plasma, and Figure 1g Plasma stability in rat plasma. Residual concentrations in each culture medium were determined by HPLC at specified time points. Bar graphs represent the mean ± SD of the percentage of residual compound. n = 3 independent repetitions).

[0036] Figure 2 shows the bioactivation of AG10-L-E2-naloxone via CES2 enzyme hydrolysis. Figure 2a The chemical structures of AG10-L-E2-naloxone and its hydrolysis product AG10-L-E2-COOH and naloxone. Figure 2b AG10-L-E2-naloxone and human TTR (pdb id: 4HIQ) 6 Docking studies between AG10-L-E2-naloxone and mouse CES2 enzyme (pdb id: 8AXC). The linker length of AG10-L-E2-naloxone (approximately 29 Å) is long enough to allow AG10-L-E2-naloxone to bind to both CES2 and TTR simultaneously. Figure 2c AG10-L-E2-naloxone was hydrolyzed by purified CES2 in buffer but not by CES1. Figure 2dMetabolic bioactivation of AG10-L-E2-naloxone in liver microsomes from humans (HLM), rats (RLM), and monkeys (MLM). HLM efficiently generates naloxone from AG10-L-E2-naloxone. The selective CES2 inhibitor loperamide significantly reduced naloxone release, while the CES1 inhibitor digitalis saponin had no effect. The concentration of naloxone released from AG10-L-E2-naloxone in HLM, MLM, and RLM was determined using a validated LC-MS / MS method. Figure 2e The stability of AG10-L-E2-naloxone in rat plasma (tested at 5 μM) in the absence and presence of the CES2 inhibitor loperamide and the CES1 inhibitor digitoxin. Figure 2f The stability of AG10-L-E2-naloxone (tested at 5 μM) in monkey plasma (plasma without CES1 or CES2) and rabbit plasma (plasma with high abundance of CES1 and CES2) was assessed. The residual concentration in each culture medium was determined by HPLC at specified time points. Figures 2c to 2f The bar chart represents the average ± sd of the remaining compound percentage. n = 3 independent repetitions).

[0037] Figure 3 shows the pharmacokinetic / pharmacodynamic evaluation of AG10-L-E2-naloxone in rats. Figure 3a Male Spriggdalen rats (each group) n = 3 Apply 15 µmol kg -1 The test compound (equivalent to 5 mg kg) -1 Naloxone and 15.8 mg / kg -1 A single subcutaneous (sc) injection of AG10-L-E2-naloxone was administered. Plasma concentrations of the test compound were measured at different time points and expressed as the mean ± sd of three biological replicates. Figure 3b brain concentration, and Figure 3c Cerebrospinal fluid (CSF) naloxone (administered in free form) and naloxone released from AG10-L-E2-naloxone. Male Spriggdalene rats (each group) n = 3 ) Administer 15 µmol kg -1 The test compound (equivalent to 5 mg kg) -1 Naloxone and 15.8 mg / kg -1 A single subcutaneous (sc) injection of AG10-L-E2-naloxone was administered. Naloxone concentrations in brain tissue and CSF were measured at 10 and 30 minutes post-administration. Bar graphs show the corresponding mean ± sd from three biological replicates. Figure 3d Male Spriggdale rats (per group) n = 9Morbidity data and antagonist rescue from morphine overdose in a biological repeat (sub-biological replication). Morphine (100 mg / kg at 0 hours) -1 And at 6 hours, 50 mg / kg -1 (subcutaneous dose) and antagonists (naloxone and AG10-L1-E2-naloxone; 15 µmol kg) -1 Dosage schedule (single subcutaneous dose at 5 minutes). Figure 3e Male Spriggdale rats (per group) n = 8 Mortality data from fentanyl overdose and antagonist rescue in a biological repeat (sub-biological replication). Fentanyl (3 mg / kg at 0 hours) -1 And at 6 hours, 3 mg kg -1 (subcutaneous dose) and antagonists (naloxone and AG10-L1-E2-naloxone; 15 µmol kg) -1 Dosage schedule (single subcutaneous dose at 5 minutes). Figures 3f to 3i Naloxone, AG10-L-E1-naloxone, AG10-L-E2-naloxone, and C12-E2-naloxone (15 µmol kg) -1 (A single subcutaneous dose at 1 minute) in male Spriggdalene rats (per group) n= 6 The reversal of fentanyl OIRD. Figure 3f The dosing time of fentanyl and its antagonist was plotted. Reversal of OIRD was expressed as minute ventilation relative to control (minute ventilation in rats before fentanyl injection was considered as baseline). Figure 3g Minute ventilation (mean ± SD) after the first dose of fentanyl and its antagonist, Figure 3 at 6 hours after the second dose of fentanyl, and Figure 3 at 24 hours after the third dose of fentanyl; per group n= 6 (Number of biological replicates). Animals were monitored in a plethysmography chamber 90 minutes after each fentanyl dose. Data were analyzed using GraphPad Prism V9.3.1.471 software (La Jolla, CA, USA).

[0038] Figure 4 shows the reversal of fentanyl OIRD by naloxone, nalmefenone, and AG10-L-E2-naloxone. Figure 4a Male Spriggdalen rats (each group) n = 3 Apply 60 µmol kg -1 The test compound (equivalent to 20 mg kg) -1 Naloxone and 63 mg kg -1A single subcutaneous (sc) injection of AG10-L-E2-naloxone was administered. Plasma concentrations of the test compound were measured at different time points and expressed as the mean ± sd of three biological replicates. Figure 4b fentanyl and antagonist (60 µmol kg) -1 The dosing time of fentanyl was determined. Reversal of OIRD was expressed as minute ventilation relative to control (minute ventilation in rats prior to fentanyl injection was considered baseline). Plots were drawn at... Figure 4c Minute ventilation over time after the first dose of fentanyl and its antagonist, the second dose of fentanyl 6 hours later (Figure 4d), and the third dose of fentanyl 24 hours later (Figure 4e) (mean ± sd; per group) n= 6 (Biological repetition). Animals were monitored in a plethysmography chamber 90 minutes after each fentanyl dose. Figure 4f Male Spriggdalen rats (n = 3 per group) were administered 60 µmol kg -1 The test compound (equivalent to 20 mg kg) -1 Naloxone and 63 mg kg -1 A single intravenous (iv) injection of AG10-L-E2-naloxone was administered. The concentration of the test compound in plasma was measured at different time points and expressed as the mean ± sd of three biological replicates. Figures 4g to 4i The reversal of fentanyl-induced OIRD by intravenous (iv) administration of naloxone and AG10-L-E2-naloxone. Plotted in... Figure 4g The first dose of fentanyl (3 mg kg) -1 (intravenous dose) and the antagonist 1 minute later (60 µmol kg) -1 Minute ventilation over time (mean ± sd) following a single intravenous dose; per group n= 6 (sub-biological repetition). Figure 4h The second dose of fentanyl (1.5 mg kg) -1 minute ventilation as a function of intravenous dose (intravenous dose) over time. Figure 4i The third dose of fentanyl (1.5 mg kg) -1 minute ventilation over time following an intravenous dose. Animals were monitored in a plethysmography chamber 90 minutes after each fentanyl dose. (See above in...) Figures 3f to 3i The volumetric data described in the text are analyzed.

[0039] Figure 5 shows the preclinical evaluation and pharmacokinetic study of AG10-L-E2-naloxone in monkeys. Figures 5a to 5dThe efficacy of a mixture of AG10-L-E2-naloxone and low-dose free naloxone against fentanyl-induced OIRD in rats was evaluated. AG10-L-E2-naloxone (60 µmol kg -1 Equivalent to 63 mg kg -1 ) and naloxone (3 µmol kg -1 Equivalent to 1 mg / kg -1 A single subcutaneous dose combination yielded excellent efficacy data, with complete reversal of OIRD after all three fentanyl doses. Minute ventilation (mean ± sd; per group) n= 6 (sub-biological repetition). Figures 5e to 5g To evaluate the potential of using AG10-L-E2-naloxone as a prophylactic agent against fentanyl in rats. Rats were first administered AG10-L-E2-naloxone (60 µmol kg). -1 ) or naloxone (15 µmol kg -1 and 60 µmol kg -1 Six hours later, the rats were given a single dose of fentanyl (1.5 mg kg). -1 (Subcutaneous dose) stimulation. Minute ventilation (mean ± sd; per group) n= 6 (Secondary biological repetition). As mentioned above... Figures 3f to 3i The volumetric data described in the text are analyzed. Figure 5h AG10-L-E2-naloxone (60 µmol / kg) -1 Effects of subcutaneous dose (60 µmol / kg) on ​​rat body weight over four weeks. Three groups of rats were used. The first group was treated with the medium. The second group was treated with AG10-L-E2-naloxone (60 µmol / kg). -1 The third group was treated with fentanyl, followed by AG10-L-E2-naloxone (60 µmol kg). -1 Treatment was administered, followed by fentanyl doses at 6 hours and 24 hours. Weight was expressed as mean ± SD (per group). n= 6 (sub-biological repetition). Figures 5i to 5k After 72 hours, the cytotoxicity of AG10-L-E2-naloxone and its metabolite AG10-L-E2-COOH (100 μM) to various cell lines was assessed using the MTT assay. Doxorubicin was used as a positive control (10 μM). Figure 5i , HeLa; cervical cancer cell lineage. Figure 5j Hep3B; human liver cancer cell line. Figure 5k MCF7; breast cancer cell line. Each bar shows mean ± sd ( n= 4 (Self-independent repetition). Figure 5lThree male cynomolgus macaques were administered AG10-L-E2-naloxone (30 µmol kg). -1 Equivalent to 31 mg kg -1 A single subcutaneous (sc) injection was administered. The concentrations of AG10-L-E2-naloxone and naloxone released from AG10-L-E2-naloxone in the plasma of each monkey were measured at different time points and expressed as the mean ± sd of three biological replicates.

[0040] Figure 6 This shows AG10-L-E2-naloxone ( Figure 6 a) AG10-L-E2-COOH ( Figure 6 b) and AG10-L-E2-ethyl ( Figure 6 c) Calculation of pKa values ​​and prediction of ionization in water (acidic pH of approximately 3.5 obtained by dissolving the HCl salts of these conjugates in water) and physiological pH (7.4).

[0041] Figure 7 This study illustrates the docking of AG10-L-E2-naloxone and known CES2-selective prodrug esters with CES2 and CES1. Mouse CES2 enzyme (pdb id: 8AXC) 1 Catalytic triplet residues S230, E347, and H459 are shown as black rods. Magnification highlights show the distance between the nucleophile S230 and the carbonyl ester of AG10-L-E2-naloxone is 2.9 Å. The distances between S230 and the carbonyl esters of the prodrugs irinotecan, procaine, and monoupivir are 3.3 Å, 3.1 Å, and 3.5 Å, respectively. The prodrugs are associated with CES1 (pdbid: 1MX9). 2 Docking studies showed that irinotecan, procaine, and monoupivir had longer distances, at 9.2 Å, 6.8 Å, and 4.8 Å, respectively. Docking studies also showed that AG10-L-E2-naloxone did not bind to CES1.

[0042] Figure 8 shows the chemical structure and stability of the newly synthesized aryl-based naloxone prodrug. Figure 8a The chemical structures of newly synthesized aryl-based naloxone prodrugs BA-naloxone, Boc-PABA-naloxone, PABA-naloxone, and known CES2 selective prodrugs are described. Figure 8b and Figure 8c To assess the stability of the prodrug in human plasma (tested at 100 μM), and Figures 8d-8iStability in rat plasma was investigated. The stability of the compound (tested at 100 μM) in rat plasma was studied in the absence and presence of the CES2 inhibitor loperamide and the CES1 inhibitor digitoxin. Known CES2-selective substrates procaine (local anesthetic), monoupivir (therapeutic for COVID-19), and irinotecan (anticancer drug) were included for comparison. The acyl moiety of the ester is shown in red. The residual concentration in each culture medium was determined by HPLC at specified time points. Bar graphs represent the mean ± SD of the residual compound percentage. n = 3 independent replicates). Two-way ANOVA (mixed model) was used, followed by a Durkheim post-hoc test to determine statistical differences.

[0043] Figure 9 This study evaluates the effect of TTR on the in vitro pharmacokinetics of AG10-L-E2-naloxone. Figure 9 a. Stability of AG10-L-E2-naloxone (tested at 5 μM) in rat plasma in the absence and presence of the selective TTR ligand AG10 (10 μM). Figure 9 b. Metabolic bioactivation of AG10-L-E2-naloxone (5 μM) in human liver microsomes (HLM) with and without human TTR (5 μM). Figure 9 c. The effect of rapid equilibrium dialysis on the binding of AG10-L-E2-naloxone (5 μM) to human TTR (5 μM) in buffer was evaluated using a molecular weight cutoff of 8 kDa (MWCO). Figure 9 a- Figure 9 The bar chart for c represents the average value of the remaining compound percentage ± sd ( n = 3 independent repetitions). For Figure 9 a, Figure 9 b. A two-way ANOVA (mixed model) was used, followed by the Holm-Šídák test to determine statistical differences, and for Figure 9 c. One-way ANOVA followed by a Durkheim post-hoc test was used to determine statistical differences. Figure 9 a, F(2,8) = 62.29, (*Compared to AG10-L-E2-naloxone, P <0.0001). For Figure 9 b, F(1,4) = 223.4, (*Compared to AG10-L-E2-naloxone, P <0.0001). For Figure 9 c, F(2,6) = 3216, (*Compared to AG10-L-E2-naloxone or AG10 without TTR, P<0.0001).

[0044] Figure 10 Evaluation of blood-brain barrier penetration of AG10-L-E2-naloxone and AG10-L-E2-COOH. Figure 10 a, the brain concentration to plasma concentration ratio of AG10-L-E2-naloxone, AG10-L-E2-COOH, naloxone derived from AG10-L-E2-naloxone, and naloxone administered in free form, and Figure 10 b, CSF to plasma concentration ratio. Male Spriggdalen rats (each group) n = 3) Give 15 µmol kg -1 The test compound (equivalent to 5 mg kg) -1 Naloxone and 15.8 mg / kg -1 A single subcutaneous injection of AG10-L-E2-naloxone. Bar graphs show the corresponding mean ± sd of three biological replicates. Statistical differences were determined using two-way ANOVA followed by the Holm-Sidak test (* compared to naloxone). P <0.0001). For the brain-to-plasma ratio experiment, F(1,4) = 59.34. P <0.0001, and for the CSF to plasma ratio experiment, F(1,4) = 35.87. P <0.0001.

[0045] Figure 11 The pharmacodynamic evaluation of repeated doses of AG10-L-E2-naloxone in rats is shown. Male Spriggdalene rats (each group) n Mortality data from fentanyl overdose in 8 biological replicates and multiple low-dose naloxone rescues. Fentanyl dosing time (3 mg / kg at 0 hours). -1 and 3 mg kg at 6 hours -1 Subcutaneous dose). Group 1 (red line): Administered medium. Group 2 (grey line): 5 minutes after the first fentanyl dose, administer a single subcutaneous dose of 3 µmol / kg. -1 Naloxone (equivalent to 1 mg kg) -1 Then, five smaller doses of naloxone (0.6 µmol kg per dose) were administered at 1.5, 2.5, 3.5, 4.5, and 5.5 hours. -1 Naloxone; equivalent to 0.2 mg / kg per dose. -1 Group 3 (blue line): Five minutes after the first fentanyl dose, administer a single subcutaneous dose of 3 µmol / kg. -1 Naloxone (equivalent to 1 mg / kg) -1Then, ten smaller doses of naloxone (0.6 µmol kg per dose) were administered at 1.5, 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, and 10.5 hours. -1 Naloxone; equivalent to 0.2 mg / kg per dose. -1 Statistical differences were determined using a log-rank (Mantel-Cox) test followed by Bonferroni correction for multiple comparisons, with Bonferroni correction α = 0.016. *Compared to the medium, P <0.0001, and compared to the medium, P <0.0026.

[0046] Figure 12 A comparison of the pharmacokinetic characteristics of naloxone administered subcutaneously at low and high doses over a period of up to 6 hours is shown. Naloxone pharmacokinetic characteristics (data from...) Figure 3a and Figure 5a ) shows with 5 mg kg -1 The difference in dosage is likely due to temporary saturation of the clearance pathway, with higher naloxone doses (20 mg / kg) being more likely to be attributed to this. -1 (Subcutaneous) showed slower clearance of 0.5 to 2 hours. After 2 hours, both doses exhibited similar clearance rates and linear kinetics, indicating limited tissue redistribution at higher doses.

[0047] Figure 13 This study demonstrates the reversal of intravenous (iv) fentanyl OIRD in male Spriggdalene rats by multiple low doses of naloxone or AG10-L-E2-naloxone. Figure 13 a. Dosage schedule of fentanyl and its antagonist. At the first fentanyl dose (3 mg / kg) -1 Administer naloxone (0.6 µmol kg / intravenous dose) over 1 minute. -1 Equivalent to 0.2 mg kg -1 ), followed by five small intravenous doses at 2, 3, 4, 5, and 6 hours, and then a second dose of fentanyl (1.5 mg kg) at 6 hours. -1 Intravenous dose) (per group) n= 6 At the first fentanyl dose (3 mg kg) -1 Administer AG10-L-E2-naloxone (15 µmol kg / intravenous dose) over 1 minute. -1 Equivalent to 5 mg / kg -1 A second dose of fentanyl (1.5 mg / kg) was administered 4 hours later.-1 (Intravenous dose). Reversal of OIRD is represented by minute ventilation relative to baseline (minute ventilation in rats prior to fentanyl injection is considered baseline). Plotted in Figure 13 b. First dose of fentanyl and antagonist, Figure 13 Minute ventilation over time after the second fentanyl dose 6 hours later (mean ± sd; per group) n= 6 (Number of biological replicates). Animals were monitored in a plethysmography chamber 90 minutes after each fentanyl dose. Data were analyzed using GraphPad Prism V9.3.1.471 software (La Jolla, CA, USA). Prior analysis revealed that, to achieve statistical power of 0.8 at α = 0.05, the number of animals in each group must be at least 3, considering our experimental SD value. Power analysis was performed using G*Power software (version 3.0.10). Statistical differences were determined using two-way ANOVA with repeated measures followed by a Durkheimer post-hoc test. Significant differences were determined at (#) P < 0.05.

[0048] Figures 14a-14e The results of rat plasma stability assays for AG10-L-E2-naloxone, AG10-L-PHBA-E-naloxone, PEG-linked-E2-naloxone, AG10-short-L-E2-naloxone and AG10-L-m-amino-E-naloxone are shown respectively.

[0049] Figure 15 The results of human plasma stability assays for AG10-L-E2-naloxone, AG10-L-PHBA-E-naloxone, PEG-linker-E2-naloxone, AG10-short-L-E2-naloxone, and AG10-L-m-amino-E-naloxone are shown. Detailed Implementation

[0050] Current opioid antagonists are insufficient in the new era of synthetic opioids such as fentanyl, primarily due to their short duration of action (DOA) and limited efficacy against reanesthesia. Here, we develop a novel prodrug approach to enhance the DOA of opioid antagonists or opioid agonists without compromising their biological activity. Our approach involves conjugating an opioid antagonist or opioid agonist to a selective TTR ligand via an ester moiety. The use of a selective TTR ligand, as described herein, utilizes endogenous TTR, representing a major advantage of this novel prodrug approach. TTR is a 55 kDa homotetrameric protein primarily secreted from the liver into the bloodstream with a circulating half-life of approximately 2 days. The primary function of TTR in humans (plasma concentration of approximately 5 μM) is to transport holo-retinol-binding proteins (RBPs that bind to retinol or vitamin A) in the blood. Using orthogonal sites to holo-retinol, TTR acts as a backup carrier for thyroxine (<1% thyroxine binding).

[0051] Our method possesses several unique features that make it an ideal choice for prodrug methods for antidotes. First, the prodrug can bind to the TTR, thereby extending its DOA to 2 days or longer. Second, the prodrug has balanced hydrophilicity, which is crucial for rapid absorption from subcutaneous tissue to provide sufficient concentrations of the antidrug for effective rescue. Third, the balanced hydrophilicity, small size, and ease of synthesis of our conjugate (we do not use polymers) will make it ideal for subcutaneous (sc) administration, offering the advantage of ease of administration while simultaneously providing a prolonged prodrug duration compared to intravenous routes. Fourth, the hydrophilicity of the prodrug and subcutaneous administration will help avoid the dramatic increase of free opioid antagonists (or opioid agonists) in the brain, thereby reducing life-threatening withdrawal symptoms. Fifth, the modular approach will allow us to produce prodrugs with adjustable hydrolysis rates, which can be used for rescue, protection (prevention), or both.

[0052] definition "Individual" or "subject" refers to a mammal and includes both human and non-human mammals. Examples of individuals include, but are not limited to, mice, rats, hamsters, guinea pigs, pigs, rabbits, cats, dogs, goats, sheep, cattle, monkeys, and humans. In some implementations, an individual or subject refers to a human.

[0053] As used in this article, the "about" parameter or value includes and describes the parameter or value itself. For example, "about X" includes and describes X itself.

[0054] "Treatment" or "treating" is a method used to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired outcomes may include one or more of the following: reducing one or more symptoms caused by a disease or condition; reducing the severity of a disease or condition; slowing or preventing the development of one or more symptoms associated with a disease or condition (e.g., stabilizing the disease or condition, preventing or delaying the worsening or progression of the disease or condition); and alleviating the disease, for example by causing the disappearance of clinical symptoms (e.g., improving the disease state, enhancing the effect of another medication, delaying the progression of the disease, improving quality of life, and / or prolonging survival).

[0055] As used herein, “delay” the development of a disease or disorder means postponing, hindering, slowing, delaying, stabilizing, and / or postponing the development of the disease or disorder. Depending on the medical history and / or the individual being treated, such delay can vary in length. As will be apparent to those skilled in the art, sufficient or significant delay can effectively encompass prevention, as the individual does not develop the disease or disorder.

[0056] As used herein, the terms "therapeutic effective amount" or "effective amount" mean an amount sufficient to achieve a therapeutic effect when a compound of this disclosure or a pharmaceutically pharmaceutically acceptable salt thereof is administered to an individual. As understood in the art, an effective amount can be one or more doses, for example, a single dose or multiple doses may be required to achieve the desired therapeutic endpoint. An effective amount may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to have been administered in an effective amount if a desired or beneficial result is achieved or realized in combination with one or more other agents.

[0057] As used herein, “unit dosage form” refers to a physically discrete unit suitable as a unit dose, each unit containing a predetermined amount of active ingredient or compound in a pharmaceutically acceptable carrier.

[0058] As used herein, “pharmaceutically acceptable” means a substance that is not biologically or otherwise undesirable, for example, a substance that can be incorporated into a pharmaceutical composition administered to an individual without causing significant undesirable biological effects.

[0059] As used herein, the term "half-life" can be used to refer to the loss of half of its activity (e.g., its pharmacological, physiological, or radiological activity). The term "half-life" can be used interchangeably with the terms "biological half-life" or "terminal half-life." In some embodiments, the half-life of a substance is the time it takes for a substance (e.g., a metabolite, drug, signaling molecule, radionuclide, or other substance) to lose half of its pharmacological, physiological, or radiological activity. In some embodiments, the term "half-life" can be used to refer to bodily purification, which eliminates a substance from the body through renal and hepatic function plus excretion. In some embodiments, the term "half-life" can be used to refer to the time it takes for the plasma concentration of a substance to halve to its steady-state level (plasma half-life).

[0060] As used herein, the terms “joint,” “link,” and “linking group” refer to a chemical linking portion that connects two groups and has a main chain length of 50 atoms or less. In some embodiments, a joint or link can be a covalent bond connecting two groups, or a chemical portion of a chain having a length between 1 and 50 atoms, between 2 and 20 atoms, between 3 and 30 atoms, between 4 and 40 atoms, between 10 and 50 atoms, between 10 and 30 atoms, between 12 and 26 atoms, between 14 and 30 atoms, or increasing in length by an amount of 1 atom. For example, a joint or a portion thereof may have a length of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45, or 50 carbon atoms, wherein the joint may be a straight chain, branched chain, cyclic chain, or a single atom. In some embodiments, the length of the joint may be 10 angstroms, 11 angstroms, 12 angstroms, 13 angstroms, 14 angstroms, 15 angstroms, 16 angstroms, 17 angstroms, 18 angstroms, 19 angstroms, 20 angstroms, 22 angstroms, 23 angstroms, 24 angstroms, 25 angstroms, 26 angstroms, 27 angstroms, 28 angstroms, 29 angstroms, 30 angstroms, 32 angstroms, 34 angstroms, 36 angstroms, 38 angstroms, 40 angstroms, 45 angstroms, 50 angstroms, or any amount in increments of 1 angstrom. In some embodiments, one, two, three, four, or five or more carbon atoms in the linker backbone may optionally be substituted with sulfur, nitrogen, or oxygen heteroatoms. The bonds between backbone atoms may be saturated or unsaturated, typically with no more than one, two, or three unsaturated bonds present in the linker backbone. The linker may include one or more substituents, such as alkyl, aryl, or alkenyl groups. The linker may include, but is not limited to, oligomers (ethylene glycol); ethers; thioethers; tertiary amines; and alkyl groups that may be straight-chain or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linker backbone may include cyclic groups, such as aryl, heterocyclic, or cycloalkyl groups, wherein the backbone includes two or more atoms of the cyclic group, such as two, three, or four atoms. The linker may be cleavable or non-cleavable.

[0061] The linker can use any suitable functional group (carboxylic acid, amine, alcohol, carbamate, ester, amide, ether, thioether, maleimide, etc.) and the linker chemical can be conjugated with the TTR ligand and Y. For example, the conjugation chemistry described by GT Hermanson (“Bioconjugate Techniques”, Academic Press, 2nd edition, 2008) is readily applicable to the preparation of the subject heterobifunctional compounds and is hereby incorporated herein by reference in its entirety.

[0062] As used herein, the term "alkyl" refers to an unbranched or branched saturated monovalent hydrocarbon chain. Alkyl groups as used herein have 1–20 carbon atoms (i.e., C60). 1-20 Alkyl groups), 1-16 carbons (i.e., C16) 1-16 Alkyl groups), 1-12 carbons (i.e., C46) 1-12 Alkyl groups), 1-10 carbons (i.e., C46) 1-10 Alkyl groups), 1-8 carbons (i.e., C464-C5 ... 1-8 Alkyl groups), 1-6 carbons (i.e., C46) 1-6 Alkyl groups), 1-4 carbons (i.e., C46) 1-4 Alkyl groups or 1-3 carbons (i.e., C46) 1-3 Alkyl groups. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl. When an alkyl residue having a specific number of carbon atoms is named by its chemical name or molecular formula, it may include all positional isomers having that number of carbon atoms; for example, "butyl" covers n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl. Certain commonly used alternative names may be used, which will be understood by those skilled in the art. For example, a divalent group, such as a divalent "alkyl", may be referred to as an "alkylene".

[0063] As used herein, the term "alkenyl" refers to a branched or unbranched monovalent hydrocarbon chain containing at least one carbon-carbon double bond. The alkenyl groups used herein have 2–20 carbons (i.e., C60 ... 2-20 alkenyl), 2-16 carbons (i.e., C10), 2-16 alkenyl), 2-12 carbons (i.e., C10), 2-12 alkenyl), 2-10 carbons (i.e., C10), 2-10 alkenyl), 2-8 carbons (i.e., C10), 2-8 alkenyl), 2-6 carbons (i.e., C10), 2-6 alkenyl), 2-4 carbons (i.e., C10), 2-4 Alkenyl) or 2-3 carbons (i.e., C) 2-3 Alkenyl groups (including, but not limited to, vinyl, propenyl, propenyl, 1,2-butadienyl, and 1,3-butadienyl). When naming an alkenyl residue with a specific number of carbon atoms using a chemical name or molecular formula, all positional isomers having that number of carbon atoms may be included; for example, "propenyl" encompasses both propenyl and propenyl. Certain commonly used alternative names may be used, which will be understood by those skilled in the art. For example, a divalent group, such as a divalent "alkenyl," may be called an "alkenylene."

[0064] As used herein, the term "alkynyl" refers to a branched or unbranched monovalent hydrocarbon chain containing at least one carbon-carbon triple bond. The alkynyl groups used herein have 2–20 carbons (i.e., C60 ... 2-20 alkynyl group), 2-16 carbons (i.e., C16-C26) 2-16 alkynyl group), 2-12 carbons (i.e., C12-C22) 2-12 alkynyl group), 2-10 carbons (i.e., C10-C20) 2-10 alkynyl group), 2-8 carbons (i.e., C10-C20) 2-8 alkynyl group), 2-6 carbons (i.e., C10-C20) 2-6 alkynyl group), 2-4 carbons (i.e., C10-C20) 2-4 (alkynyl group) or 2-3 carbons (i.e., C10) 2-3 (Alynyl group). Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl-1-alkynyl, propynyl-2-alkynyl, butynyl-1-alkynyl, butynyl-2-alkynyl, and butynyl-3-alkynyl. When an alkynyl residue having a specific number of carbon atoms is named by its chemical name or molecular formula, it may include all positional isomers having that number of carbon atoms; for example, "propynyl" includes both propynyl-1-alkynyl and propynyl-2-alkynyl. Certain commonly used alternative names may be used, which will be understood by those skilled in the art. For example, a divalent group, such as a divalent "alkynyl," may be called an "alkynylene".

[0065] As used herein, the term "alkoxy" refers to the -O-alkyl moiety. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexyloxy, and 1,2-dimethylbutoxy.

[0066] As used herein, the term "aryl" refers to a fully unsaturated carbocyclic moiety. The term "aryl" encompasses both monocyclic and polycyclic fused-ring moieties. As used herein, aryl encompasses a moiety containing, for example, 6 to 20 ring carbon atoms (i.e., C24 carbon atoms). 6-20 aryl), 6 to 16 ring carbon atoms (i.e., C 6-16 aryl), 6 to 12 ring carbon atoms (i.e., C 6-12 aryl group or 6 to 10 ring carbon atoms (i.e., C46, ​​C56, C6 ... 6-10 The ring moiety of an aryl group. Examples of aryl moiety include, but are not limited to, phenyl, naphthyl, fluorenyl, and anthracene.

[0067] As used herein, the term "cycloalkyl" refers to a saturated or partially unsaturated carbocyclic moiety. The term "cycloalkyl" encompasses both monocyclic and polycyclic moietyes, wherein the polycyclic moiety may be fused, branched, or spirocyclic. Cycloalkyl includes cycloalkenyl groups, wherein the cyclic moiety contains at least one cyclic double bond. Cycloalkyl includes any polycyclic carbocyclic moiety containing at least one non-aromatic ring, regardless of its connection point to the rest of the molecule. As used herein, cycloalkyl includes, for example, a moiety containing 3 to 20 cyclic carbon atoms (i.e., C64 ... 3-20cycloalkyl groups), 3 to 16 cyclic carbon atoms (i.e., C164 ...64646646466646666666666666666666666666666 3-16 cycloalkyl groups), 3 to 12 cyclic carbon atoms (i.e., C12+ ... 3-12 cycloalkyl groups), 3 to 10 cyclic carbon atoms (i.e., C14 and C24). 3-10 cycloalkyl groups), 3 to 8 cyclic carbon atoms (i.e., C1646-C ... 3-8 cycloalkyl groups), 3 to 6 cyclic carbon atoms (i.e., C164-C ... 3-6 cycloalkyl groups or 3 to 5 cyclic carbon atoms (i.e., C1646-C ... 3-5 The cycloalkyl group is a ring. Monocyclic cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic groups include, for example, bicyclic [2.2.1]heptyl, bicyclic [2.2.2]octyl, adamantyl, norbornyl, decahydronaphthyl, 7,7-dimethyl-bicyclic [2.2.1]heptyl, etc. In addition, cycloalkyl groups also include spirocycloalkyl ring groups, such as spiro[2.5]octyl, spiro[4.5]decyl, or spiro[5.5]undecyl.

[0068] As used herein, the term "halogen" refers to an atom occupying Group VIIA of the periodic table and includes fluorine (fluorinated), chlorine (chloroinated), bromine (brominated), and iodine (iodinated). Furthermore, terms such as "halogenated alkyl" are intended to include both monohalogenated and polyhalogenated alkyl groups. For example, the term "C" 1-4 "Halogenated alkyl" is intended to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, difluoromethyl, etc.

[0069] As used herein, the term "heteroaryl" refers to an aromatic (fully unsaturated) ring moiety containing one or more cyclic heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. The term "heteroaryl" includes both monocyclic and polycyclic fused-ring moieties. As used herein, heteroaryls include, for example, 5 to 20 ring atoms (i.e., 5 to 20-membered heteroaryls), 5 to 16 ring atoms (i.e., 5 to 16-membered heteroaryls), 5 to 12 ring atoms (i.e., 5 to 12-membered heteroaryls), 5 to 10 ring atoms (i.e., 5 to 10-membered heteroaryls), 5 to 8 ring atoms (i.e., 5 to 8-membered heteroaryls), or 5 to 6 ring atoms (i.e., 5 to 6-membered heteroaryls). Any monocyclic or polycyclic aromatic ring moiety containing one or more cyclic heteroatoms is considered a heteroaryl, regardless of its connection point to the rest of the molecule (i.e., the heteroaryl moiety can be connected to the rest of the molecule via any cyclic carbon or any cyclic heteroatom of the heteroaryl moiety). Examples of heteroaryl groups include, but are not limited to, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, and pyridazinyl.

[0070] As used herein, the term "heterocyclic group" refers to a saturated or partially unsaturated cyclic moiety encompassing one or more cyclic heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. The term "heterocyclic group" includes monocyclic and polycyclic moieties, wherein polycyclic moieties can be fused, bridged, or spirocyclic. Any non-aromatic monocyclic or polycyclic moieties containing at least one cyclic heteroatom are considered heterocyclic groups, regardless of their connection points to the rest of the molecule (i.e., the heterocyclic group moieties can be connected to the rest of the molecule via any cyclic carbon or any cyclic heteroatom of the heterocyclic group moieties). Furthermore, the term "heterocyclic group" is intended to encompass any polycyclic moieties containing at least one cyclic heteroatom, wherein the polycyclic moieties contain at least one non-aromatic ring, regardless of their connection points to the rest of the molecule. As used herein, heterocyclic groups comprise, for example, 3 to 20 ring atoms (i.e., 3-20 membered heterocyclic groups), 3 to 16 ring atoms (i.e., 3-16 membered heterocyclic groups), 3 to 12 ring atoms (i.e., 3-12 membered heterocyclic groups), 3 to 10 ring atoms (i.e., 3-10 membered heterocyclic groups), 3 to 8 ring atoms (i.e., 3-8 membered heterocyclic groups), 3 to 6 ring atoms (i.e., 3-6 membered heterocyclic groups), 3 to 5 ring atoms (i.e., 3-5 membered heterocyclic groups), 5 to 8 ring atoms (i.e., 5-8 membered heterocyclic groups), or 5 to 6 ring atoms (i.e., 5-6 membered heterocyclic groups). Examples of heterocyclic groups include, for example, aza-butyl, piperidinyl, piperazine, pyrrolyl, pyrazolyl, thiazolyl, tetrahydrofuranyl, or tetrahydropyranyl. Examples of spiroheterocyclic rings include, but are not limited to, bicyclic and tricyclic systems, such as oxabicyclo[2.2.2]octyl, 2-oxa-7-azaspiro[3.5]nonyl, 2-oxa-6-azaspiro[3.4]octyl, and 6-oxa-1-azaspiro[3.3]heptyl. Examples of fused heterocyclic rings include, but are not limited to, 5,6,7,8-tetrahydropyridino[4,3-d]pyrimidinyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidinyl, 5,7-dihydrofurano[3,4-d]pyrimidinyl, and 5,7-dihydrothieno[3,4-d]pyrimidinyl, wherein the heterocyclic group can be bonded via any ring of the fused system.

[0071] As used in this article, the term "oxo group" refers to the =O part.

[0072] As used herein, the terms “optional” or “optionally” mean that the event or situation described below may or may not occur, and the description includes instances where the event or situation occurs and instances where it does not occur. Thus, the term “optionally substituted” means that any one or more (e.g., 1, 2, 1 to 5, 1 to 3, 1 to 2, etc.) hydrogen atoms on a specified atom or part or group may or may not be substituted by atoms or parts or groups other than hydrogen. By way of example only and not limitation, the phrase “methyl group optionally substituted with one or more chlorine atoms” covers the -CH3, -CH2Cl, -CHCl2, and -CCl3 moieties.

[0073] It should be understood that aspects and implementation schemes described herein as “comprising” include “consisting of implementation schemes” and “substantially consisting of implementation schemes”.

[0074] As used herein, the term "pharmaceutically acceptable salt" for a given compound means a salt that retains the biological efficacy and properties of the given compound and is not biologically or otherwise undesirable. "Pharmaceutically acceptable salt" includes, for example, salts formed with inorganic acids and salts formed with organic acids. Furthermore, if the compound described herein is obtained as an acid addition salt, the free base can be obtained by alkalizing a solution of the acid salt. Conversely, if the product is a free base, the addition salt, particularly a pharmaceutically acceptable addition salt, can be produced by dissolving the free base in a suitable organic solvent and treating the solution with acid, following conventional procedures for preparing acid addition salts from basic compounds. See, for example, Handbook of Pharmaceutical Salts Properties, Selection, and Use [Reference to John Wiley & Sons (2008), International Union of Pure and Applied Chemistry, which is incorporated herein by reference in its entirety.] Those skilled in the art will recognize the various synthetic methods that can be used to prepare non-toxic, pharmaceutically acceptable addition salts. Pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Salts derived from inorganic acids include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Salts derived from organic acids include, for example, acetic acid, propionic acid, gluconic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, trifluoroacetic acid, etc. Similarly, pharmaceutically acceptable base addition salts can be prepared from inorganic or organic bases. Salts derived from inorganic bases include (by way of example only) sodium, potassium, lithium, aluminum, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines. Specific examples of suitable amines include (by way of example only) isopropylamine, trimethylamine, diethylamine, tri(isopropyl)amine, tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol, piperazine, piperidine, morpholine, N-ethylpiperidine, etc.

[0075] Some of the compounds described herein can exist as tautomers. These tautomers exist in equilibrium with each other. For example, amide-containing compounds can exist in equilibrium with imine tautomers. Regardless of which tautomer is exhibited and regardless of the equilibrium nature between the tautomers, those skilled in the art will understand that the compounds disclosed herein comprise both amide and imine tautomers. Thus, for example, amide-containing compounds are understood to include their imine tautomers. Similarly, imine-containing compounds are understood to include their amide tautomers.

[0076] The compounds disclosed herein, or pharmaceutically acceptable salts thereof, may include an asymmetric center and thus may produce enantiomers, diastereomers, and other stereoisomers, which can be defined according to absolute stereochemistry as ( R )-or( S (or (D)- or (L)- for amino acids). This disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms and mixtures thereof in any ratio. Optically active (+) and (-), ( R )-and( S (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as chromatography and / or fractional crystallization. Conventional techniques for preparing / separating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemic mixtures (or racemic mixtures of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC) and chiral supercritical fluid chromatography (SFC). When the compounds described herein contain olefinic double bonds or other geometrically asymmetric centers, this disclosure is intended to include both E-geometric isomers and Z-geometric isomers, unless otherwise specified. Similarly, cis and trans are used in their conventional sense to describe relative spatial relationships.

[0077] Conjugate In one respect, this paper provides a conjugate of formula (I): Or its pharmaceutically acceptable salt, wherein: T is a selective ligand for the thyroxine transporter protein TTR; L stands for connector; L B Click products that do not exist or are formed through a click reaction between the first click handle and the second click handle; E is a cleavable ester; and D 1 It refers to the drug component (opioid antagonists or opioid agonists).

[0078] In some embodiments, the connector (L) has a size of about 10 Å to about 40 Å. In some embodiments, the connector (L) has a size of about 10 Å to about 40 Å. In some embodiments, the connector (L) has a size of about 15 Å to about 30 Å. In some embodiments, the connector (L) has a size of about 20 Å to about 30 Å. In some embodiments, the connector (L) has a size of about 20 Å. In some embodiments, the connector (L) has a size of about 25 Å. In some embodiments, the connector (L) has a size of about 30 Å.

[0079] In some implementations, the connector (L) and the click product (L) B Both have a size of approximately 10 Å to approximately 50 Å. In some embodiments, the connector (L) and the click product (L) have a size of approximately 10 Å to approximately 50 Å. B Both have a size of approximately 15 Å to approximately 45 Å. In some embodiments, the connector (L) and the click product (L) have a size of approximately 15 Å to approximately 45 Å. B Together, they have a size of approximately 25 Å to approximately 40 Å. In some embodiments, the connector (L) and the click product (L) B Together, they have a size of approximately 20 Å. In some embodiments, the connector (L) and the click product (L) B Together, they have a size of approximately 25 Å. In some embodiments, the connector (L) and the click product (L) B Together, they have a size of approximately 30 Å. In some embodiments, the connector (L) and the click product (L) B Together, they have a size of approximately 35 Å. In some embodiments, the connector (L) and the click product (L) B They together have a size of approximately 40 Å.

[0080] In some implementations, L B The following are the click products: (i) Copper-catalyzed reactions between azides and alkynes; (ii) The reaction between azides and dibenzocyclooctene (DBCO); (iii) Reverse electron-demanding Diels-Alder cycloaddition (IEDDA) between the trans-cyclooctene (TCO) moiety and the tetrazine ring, or (iv) Staudinger reaction between azides and phosphine.

[0081] In some implementations, the product L is used to form the click product L. BThe first and second clickers can be any suitable click chemical pair (e.g., azide-DBCO, azide-BCN, Tz-TCO, meTz-TCO, etc.). In some embodiments, the click product can be formed using a copper-catalyzed click reaction. One such copper-catalyzed click reaction is the Hughesgen 1,3-dipolar cycloaddition (CuAAC) between an azide and an alkyne (see, for example, Torn). e et al. J. Org. Chem. 2002, 67 (9), 3057-3064 and Rostovtsev et al., Angew. Chem. Int. Ed. (2002, 41(14), 2596-2599). In some embodiments, the click product is a triazole moiety. In some embodiments, the click product can be formed using a bisorthogonal chemistry method.

[0082] In some implementations, click product L B It can be formed by any suitable electron-demanding Diels-Alder reaction. In some embodiments, the click product can be formed by an electron-demanding Diels-Alder reaction between the trans-cyclooctene (TCO) moiety on the first or second click shank and the tetrazine (Tz) ring on the first or second click shank (see, for example, Selvaraj et al.). Curr. Opin. Chem. Biol. 2013, 17 (5), 753-760 and Rossin et al., Bioconjugate Chem (2013, 24(7), 1210-1217). In some embodiments, the first click handle comprises a tetrazine ring and the second click handle comprises a TCO moiety. In some embodiments, the tetrazine ring is unsubstituted. In some such embodiments, the tetrazine ring is methyltetrazine. In some embodiments, the tetrazine ring is a 6-methyl-substituted tetrazine. In some embodiments, the click product is a dihydropyridazine moiety.

[0083] In some implementations, click product L B It is a triazine moiety. In some embodiments, the conjugate of formula (I) comprises one of the following moieties: , where X 2 It is -CH2-, -NR x -, -S-, or -O-, where R x As defined elsewhere herein. In some embodiments, the conjugate comprises a portion In some implementations, the conjugate comprises a portion .

[0084] In some implementations, L is C 4-20 alkylene, wherein the C 4-20Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally selected from -O-, -NR. x Substitution of - and -S- groups, where L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 Alkyl group, wherein R x It is H or C that is optionally substituted with a halogen group. 1-6 Alkyl group. In some embodiments, L is C. 10-20 alkylene, wherein the C 10-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, -N(CH3)-, and -S-. In some embodiments, L is C 10-15 alkylene, wherein the C 10-15 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, and -N(CH3)-. In some embodiments, L is C 5-10 alkylene, wherein the C 5-10 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O- and -NH-.

[0085] In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion .

[0086] In some implementation schemes, LL B Includes part In some implementation schemes, LL B Includes part In some implementation schemes, LL B Includes part In some implementation schemes, LL B Includes part In some implementation schemes, LL B Includes part In some implementation schemes, LL B Includes part In some implementation schemes, LL B Includes part .

[0087] In some implementation schemes, LL B Includes part or , where R 9 Is it H or C? 1-4 Alkyl, R 10 Is it H or C? 1-4 Alkyl group, and Q is -CH2-, -O-, -S-, -NH-, or -N(C 1-4 alkyl)-.

[0088] In some embodiments, the formula (I) conjugate contains a portion , , , , , , , or ,in: Ring A is C 6-10 Aryl or 5 to 10-membered heteroaryl, among which The C 6-10 aryl or 5- to 10-membered heteroaryl groups are optionally surrounded by one or more R groups. 7 Replace, where each R 7 Independently selected from the following groups: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NO2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl and Indicator and connector (L or LL) B ( ) connection points.

[0089] In some embodiments, ring A is an optionally substituted 5- to 10-membered heteroaryl group. Examples of heteroaryl rings include, but are not limited to: azacyclic butyl, pyrazolyl, pyrroleyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyridazinyl, indolizyl, isoindolyl, indolyl, dihydroindolyl, indolyl, purine, quinazinyl, isoquinolinyl, quinolinyl, phthalazinyl, naphthylpyridinyl, quinoxalinyl, quinazolinyl, terpineyl, pteridinyl, carbazole, carbolinyl, phenanthridine, acridineyl, phenanthridine The following groups are included: pyrrolinyl, isothiazolyl, phenazinyl, isoxazolyl, phenoxazinyl, phenthiazinyl, imidazoalkyl, imidazolinyl, piperidinyl, piperazinyl, indolinyl, phthalimide, 1,2,3,4-tetrahydroisoquinolinyl, 4,5,6,7-tetrahydrobenzo[b]thiophenyl, thiazoyl, thiazoalkyl, thiophenyl, benzo[b]thiophenyl, morpholinyl, thiomorpholinyl, 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolinyl, and tetrahydrofuranyl. In some embodiments, ring A is selected from the group consisting of: phenyl, pyridinyl, pyrimidinyl, oxazolyl, pyrazolyl, and thiazoyl, wherein the phenyl, pyridinyl, pyrimidinyl, oxazolyl, pyrazolyl, or thiazoyl is optionally surrounded by one or more C... 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, halogen, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 3-6 Cycloalkyl or -NO2-substituted, wherein the C 1-6 Alkyl or C 1-6 The alkoxy group is optionally substituted with one or more halogen groups. In some embodiments, ring A is selected from the group consisting of phenyl, pyridyl, and pyrimidinyl, wherein the phenyl, pyridyl, and pyrimidinyl groups are optionally substituted with one or more C groups. 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, halogen, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 3-6 Cycloalkyl or nitro-substituted, wherein the C 1-6 Alkyl or C1-6 The alkoxy group may optionally be substituted with one or more halogen groups.

[0090] In some embodiments, ring A is phenyl or a substituted phenyl group. In some such embodiments, ring A is substituted with a methyl, methoxy, halogen (e.g., Cl, Br, F, or I), nitro, or CF3 group.

[0091] In some embodiments, the formula (I) conjugate contains a portion , , , , , or .

[0092] In some embodiments, the formula (I) conjugate contains a portion , or .

[0093] In some embodiments, the formula (I) conjugate contains a portion , , , , , , , or ,in: Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 Cycloalkyl, p is 0, 1, 2 or 3, and Indicator and connector (L or LL) B ( ) connection points.

[0094] In some embodiments, the formula (I) conjugate contains a portion , , or , in: Ring A is C6-10 Aryl or 5 to 10-membered heteroaryl, among which The C 6-10 aryl or 5- to 10-membered heteroaryl groups are optionally surrounded by one or more R groups. 7 Replace, where each R 7 Independently selected from the following groups: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; and Indicates the connection point with the connector (L).

[0095] In some embodiments, the formula (I) conjugate contains a portion , , or , Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 Cycloalkyl, p is 0, 1, 2 or 3, and Indicates the connection point with the connector (L).

[0096] In some embodiments, the formula (I) conjugate contains a portion , In some embodiments, the formula (I) conjugate contains a portion Each methylene group may optionally be replaced by a halogen atom (e.g., Cl, Br, or F), a hydroxyl group, -OCF3, -NH2, or -NHC. 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy or C 3-6 Cycloalkyl substitution.

[0097] In some embodiments, the formula (I) conjugate contains a portion , or , Each methylene group may optionally be replaced by a halogen atom (e.g., Cl, Br, or F), a hydroxyl group, -OCF3, -NH2, or -NHC. 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy or C 3-6 Cycloalkyl substitution.

[0098] And among them Indicator and connector (L or LL) B The connection point of ) where Indicates the connection point with the connector (L).

[0099] In some embodiments, the formula (I) conjugate contains a portion , or ,and Indicates the connection point with the connector (L).

[0100] In some implementation schemes, D 1 Choose from the following groups: , , , , and In some implementations, D is... or .

[0101] In some embodiments, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (A): (A), Or its pharmaceutically acceptable salt, wherein: Xa X b and X c Independently selected from C(R) 8a (R) 8b ), O, NR 8b Or S; where R 8a and R 8b Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino and cyano; The A ring is a 4- to 12-membered ring, and in some embodiments, the 4- to 12-membered ring is an aromatic or heteroaromatic ring; Each Y is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted amino, amino, substituted amino, acylamino, substituted acylamino, sulfonamide, sulfonyl fluoride, thioester and cyano; c is an integer in the range of 0 to 5; and The B ring is a heterocycle selected from the following (h1-h30): Where R 11 -R 16 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino, and cyano; R 17 Selected from hydroxyl, alkyl, amino, and alkylamino; and R 11 -R 16 At least one of them is with X c The linking group.

[0102] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (B): (B), Or its pharmaceutically acceptable salt, wherein: n is an integer in the range of 0 to 8; R 18 R 19 and R 20 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino, sulfonamide, sulfonyl fluoride, thioester and cyano; X a It is C(R) 21 (R) 22 ), O, NR 22 Or S; where R 21 and R 22 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino and cyano; A is a 5- to 12-membered ring, and in some embodiments, the 5- to 12-membered ring is an aromatic or heteroaromatic ring; Each Y is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino, sulfonamide, sulfonyl fluoride, thioester, and cyano; and c is a number from 0 to 5.

[0103] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (C): (C), Or its pharmaceutically acceptable salt, wherein: n is an integer in the range of 1 to 4; R 23 It is a short-chain alkyl group having 1 to 4 carbon atoms; R 24 It is hydrogen; R 25 It is a short-chain alkyl group having 1 to 4 carbon atoms; X a It is C(R) 21 (R) 22 ), O, NR 22Or S; where R 21 and R 22 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino and cyano; Each Y is independently selected from hydrogen, halogen, acyl, substituted acyl, carboxyl, heterocyclic group, alkoxycarbonyl, sulfonamide, sulfonyl fluoride, thioester, and substituted alkoxycarbonyl; and c is 2.

[0104] In some embodiments, the ligand of compound (C) is R. 23 It is methyl and R 24 Xa is a methyl group; Xa is O; and Y is a fluorine or carboxyl group.

[0105] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (D): (D), Or its pharmaceutically acceptable salt, wherein: n is 1 to 8; R 23 R 24 and R 25 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino and cyano; X a It is C(R) 21 (R) 22 ), O, NR 22 Or S; where R 21 and R 22 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino and cyano; R a It is CHO, COOH, COOCH3, COOR 6 CONR 7R 8 Tetrazolyl, CONHOH, B(OH)2, CONHSO2Ar, CONHCH(R) 9 COOH, CF3, hydrogen, halogen, alkyl, substituted alkyl, acyl, substituted acyl, carboxyl, heterocyclic group, sulfonamide, sulfonyl fluoride, thioester, alkoxycarbonyl or substituted alkoxycarbonyl; R b It is CHO, COOH, COOCH3, COOR2 6 CONR2 7 R2 8 Tetrazolyl, CONHOH, B(OH)2, CONHSO2Ar, CONHCH(R2) 9 COOH, CF3, hydrogen, halogen, alkyl, substituted alkyl, acyl, substituted acyl, carboxyl, heterocyclic group, sulfonamide, sulfonyl fluoride, thioester, alkoxycarbonyl or substituted alkoxycarbonyl; R2 6 It is an alkyl, haloalkyl, cycloalkyl, or heterocyclic group; R 27 and R 28 Each is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, or heteroaryl; and, R 29 It is a side chain of a naturally occurring α-aminocarboxylic acid.

[0106] In some embodiments, the ligand of compound (D) is R. b The structure is selected from bromine, chlorine and fluorine.

[0107] In some implementations, the ligand of compound (D) has the structure of compound (E): (E) Or its pharmaceutically acceptable salt.

[0108] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (F): (F) Or its pharmaceutically acceptable salt.

[0109] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (G): (G) Or its pharmaceutically acceptable salt, wherein R a It is OH, CHO, COOH, CONH2, CONH(OH), COOR30 CONHR 30 ; R 30 It is a straight-chain or branched alkyl group with 1-3 carbon atoms.

[0110] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (H): (H) Or its pharmaceutically acceptable salt.

[0111] In some embodiments, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (I): Or its pharmaceutically acceptable salt.

[0112] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (K): Or its pharmaceutically acceptable salt.

[0113] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (L): Or its pharmaceutically acceptable salt.

[0114] In some embodiments, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (M): Or its pharmaceutically acceptable salt.

[0115] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (N): Or its pharmaceutically acceptable salt.

[0116] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (O): Or its pharmaceutically acceptable salt.

[0117] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (P): Or its pharmaceutically acceptable salt.

[0118] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (Q): Or its pharmaceutically acceptable salt.

[0119] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (R): Or its pharmaceutically acceptable salt.

[0120] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (S): Or its pharmaceutically acceptable salt.

[0121] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (T): Or its pharmaceutically acceptable salt.

[0122] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (U): Or its pharmaceutically acceptable salt.

[0123] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (V): Or its pharmaceutically acceptable salt.

[0124] In some embodiments, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (W): Or its pharmaceutically acceptable salt.

[0125] In some implementations, T (a ligand selective for thyroxine transporter TTR) has the structure of compound (Y): (Y) Or its pharmaceutically acceptable salt.

[0126] In some embodiments of compound (Y), the linker (L) is attached to a ligand at the meta-C16 position of the carboxyl carbon at C14, such as conjugates of formula (Ia): (Ia) In some embodiments of compound (Y), the linker (L) is attached to a ligand at the C15 position adjacent to the carboxyl carbon at C14, such as conjugates of formula (Ib): (Ib) In some implementations, T (a ligand selective for thyroxine transporter TTR) has one of the following structures: In some implementations, T (a ligand selective for thyroxine transporter TTR) has one of the following structures: In some embodiments of the conjugate of formula (Ia) or formula (Ib), L is C 4-20 alkylene, wherein the C 4-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally selected from -O-, -NR. x Substitution of - and -S- groups, where L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 Alkyl group. In some embodiments, L is C. 10-20 alkylene, wherein the C 10-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, -N(CH3)-, and -S-. In some embodiments, L is C 10-15 alkylene, wherein the C 10-15 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, and -N(CH3)-. In some embodiments, L is C 5-10 alkylene, wherein the C 5-10 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O- and -NH-.

[0127] In some embodiments of formula (Ia) or (Ib), L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion .

[0128] In some implementation schemes, LL B Includes part In some implementation schemes, LL B Includes part In some implementation schemes, LL B Includes part In some implementation schemes, LL B Includes part In some implementation schemes, LL B Includes part In some implementation schemes, LL B Includes part .

[0129] In some embodiments of formula (Ia) or formula (Ib), LL B Includes part or , where R 9 Is it H or C? 1-4 Alkyl, R 10 Is it H or C? 1-4 Alkyl group, and Q is -CH2-, -O-, -S-, -NH-, or -N(C 1-4 alkyl)-.

[0130] In one embodiment, this document provides a conjugate of formula (II'): (II'), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, 3 or 4; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L stands for connector; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the following groups: hydrogen, halogen atoms (e.g., Cl, Br, or F), NO2, CF3, OMe, O(C 1-4 Alkyl), hydroxyl, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; and D 1 It's the pharmaceutical part.

[0131] In one embodiment, this document provides a conjugate of formula (II): (II), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, 3 or 4; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L stands for connector; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the following groups: hydrogen, halogen atoms (e.g., Cl, Br, or F), NO2, CF3, OMe, O(C 1-4 Alkyl), hydroxyl, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; and D 1 It refers to the drug component (opioid antagonists or opioid agonists).

[0132] In some embodiments, the conjugate of formula (II) has formula (IIa): (IIa) Or its pharmaceutically acceptable salt.

[0133] In some embodiments, the conjugate of formula (II) has formula (IIb): (IIb) Or its pharmaceutically acceptable salt.

[0134] In some embodiments, the formula (II) conjugate has formula (IIc): (IIc) Or its pharmaceutically acceptable salt.

[0135] In some embodiments, the formula (II) conjugate has formula (IId): (IId) Or its pharmaceutically acceptable salt.

[0136] In some embodiments, the formula (II) conjugate has formula (IIe): (IIe) Or its pharmaceutically acceptable salt.

[0137] In some embodiments, the conjugate of formula (II) has formula (IIf): (IIf) Or its pharmaceutically acceptable salt.

[0138] In some embodiments, the formula (II) conjugate has the formula (IIg): (IIg) Or its pharmaceutically acceptable salt.

[0139] In some embodiments, the formula (II) conjugate has formula (IIh): (IIh) Or its pharmaceutically acceptable salt.

[0140] In some embodiments, the conjugate of formula (II) has formula (IIi): (IIi) Or its pharmaceutically acceptable salt.

[0141] In some implementations, the formula (II') conjugate has the formula (IIj): (IIj) Or its pharmaceutically acceptable salt.

[0142] In some implementations, the formula (II') conjugate has the formula (IIk): (IIk) Or its pharmaceutically acceptable salt.

[0143] In some embodiments of conjugates of formula (II) or (II') or any variant thereof (such as formulas (IIa)-(IIi)), R 2 Is it H or C? 1-2 Alkyl group. In some embodiments, R 2 It is H or -CH3. In some implementations, R 2 It is H.

[0144] In some embodiments of conjugates of formula (II) or (II') or any variant thereof (such as formulas (IIa)-(IIk)), R 3 Is it H or C? 1-2 Alkyl group. In some embodiments, R 3 It is H or -CH3. In some implementations, R 3 It is H.

[0145] In some embodiments of conjugates of formula (II) or (II') or any variant thereof (such as formulas (IIa)-(IIk)), R 4 Is it H or C? 1-2 Alkyl group. In some embodiments, R 4 It is H or -CH3. In some implementations, R 4 It is -CH3.

[0146] In some embodiments of conjugates of formula (II) or (II') or any variant thereof (such as formulas (IIa)-(IIk)), R 5 Is it H or C? 1-2 Alkyl group. In some embodiments, R 5 It is H or -CH3. In some implementations, R 5 It is -CH3.

[0147] In some embodiments of conjugates of formula (II) or (II') or any variant thereof (such as formulas (IIa)-(IIh), (IIj) or (IIk)), n is 2 or 3. In some embodiments, n is 3.

[0148] In some embodiments of conjugates of formula (II) or (II') or any variant thereof (such as formulas (IIa)-(IIh) or (IIj)), m is 0, 1, or 2. In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1.

[0149] In some embodiments of conjugates of formula (II) or (II') or any variant thereof (such as formulas (IIa)-(IIh) or (IIj)), p is 0, 1, or 2. In some embodiments, p is 0 or 1. In some embodiments, p is 0. In some embodiments, p is 1.

[0150] In some embodiments of conjugates of formula (II) or (II') or any variant thereof (such as formulas (IIa)-(IIk)), L is C 4-20 alkylene, wherein the C 4-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally selected from -O-, -NR. x Substitution of - and -S- groups, where L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 Alkyl group. In some embodiments, L is C. 10-20 alkylene, wherein the C 10-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, -N(CH3)-, and -S-. In some embodiments, L is C 10-15 alkylene, wherein the C 10-15 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, and -N(CH3)-. In some embodiments, L is C 5-10 alkylene, wherein the C 5-10 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O- and -NH-.

[0151] In some embodiments of conjugates of formula (II) or (II') or any variant thereof (such as formulas (IIa)-(IIk)), L contains a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion .

[0152] In some embodiments of conjugates of formula (II) or (II') or any variant thereof (such as formulas (IIa)-(IIk)), D 1 Choose from the following groups: , , , , and In some implementations, D 1 yes or .

[0153] In one embodiment, this document provides a conjugate of formula (III'): (III') Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, 3 or 4; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; L B It is a click product formed by the click reaction between the first click handle and the second click handle; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the following groups: hydrogen, halogen atoms (e.g., Cl, Br, or F), NO2, CF3, OMe, O(C 1-4 Alkyl), hydroxyl, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; and D 1 It's the pharmaceutical part.

[0154] In one embodiment, this document provides a conjugate of formula (III): (III) Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, 3 or 4; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; L B It is a click product formed by the click reaction between the first click handle and the second click handle; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the following groups: hydrogen, halogen atoms (e.g., Cl, Br, or F), NO2, CF3, OMe, O(C 1-4 Alkyl), hydroxyl, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; and D 1 It refers to the drug component (opioid agonists or opioid antagonists).

[0155] In some embodiments, the formula (III) conjugate has formula (IIIa): (IIIa) Or its pharmaceutically acceptable salt.

[0156] In some embodiments, the formula (III) conjugate has formula (IIIb): (IIIb) Or its pharmaceutically acceptable salt.

[0157] In some embodiments, the formula (III) conjugate has formula (IIIc): (IIIc) Or its pharmaceutically acceptable salt.

[0158] In some embodiments, the formula (III) conjugate has formula (IIId): (IIId) Or its pharmaceutically acceptable salt.

[0159] In some embodiments, the formula (III) conjugate has formula (IIIe): (IIIe) Or its pharmaceutically acceptable salt.

[0160] In some embodiments, the formula (III) conjugate has formula (IIIf): (IIIf) Or its pharmaceutically acceptable salt.

[0161] In some embodiments, the formula (III) conjugate has the formula (IIIg): (IIIg) Or its pharmaceutically acceptable salt.

[0162] In some embodiments, the formula (III) conjugate has formula (IIIh): (IIIh) Or its pharmaceutically acceptable salt.

[0163] In some embodiments, the formula (III) conjugate has formula (IIIi): (IIIi) Or its pharmaceutically acceptable salt.

[0164] In some implementations, the (III') conjugate has the formula (IIIj): (IIIj) Or its pharmaceutically acceptable salt.

[0165] In some implementations, the (III') conjugate has the formula (IIIk): (IIIk) Or its pharmaceutically acceptable salt.

[0166] In some embodiments of conjugates of formula (III) or (III') or any variant thereof (such as formulas (IIIa)-(IIIk)), R 2 Is it H or C? 1-2 Alkyl group. In some embodiments, R 2 It is H or -CH3. In some implementations, R 2 It is H.

[0167] In some embodiments of conjugates of formula (III) or (III') or any variant thereof (such as formulas (IIIa)-(IIIk)), R 3 Is it H or C? 1-2 Alkyl group. In some embodiments, R 3 It is H or -CH3. In some implementations, R 3 It is H.

[0168] In some embodiments of conjugates of formula (III) or (III') or any variant thereof (such as formulas (IIIa)-(IIIk)), R 4 Is it H or C? 1-2 Alkyl group. In some embodiments, R 4 It is H or -CH3. In some implementations, R 4 It is -CH3.

[0169] In some embodiments of conjugates of formula (III) or (III') or any variant thereof (such as formulas (IIIa)-(IIIk)), R 5 Is it H or C? 1-2 Alkyl group. In some embodiments, R 5 It is H or -CH3. In some implementations, R 5 It is -CH3.

[0170] In some embodiments of conjugates of formula (III) or (III') or any variant thereof (such as formulas (IIIa)-(IIIk)), n is 2 or 3. In some embodiments, n is 3.

[0171] In some embodiments of conjugates of formula (III) or (III') or any variant thereof (such as formulas (IIIa)-(IIIj)), m is 0, 1, or 2. In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1.

[0172] In some embodiments of conjugates of formula (III) or (III') or any variant thereof (such as formulas (IIIa), (IIIb) or (IIId)-(IIIj)), p is 0, 1, or 2. In some embodiments, p is 0 or 1. In some embodiments, p is 0. In some embodiments, p is 1.

[0173] In some embodiments of conjugates of formula (III) or (III') or any variant thereof (such as formulas (IIIa)-(IIIk)), L A It is C 4-20 alkylene, wherein the C 4-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally selected from -O-, -NR. x Substitution of - and -S- groups, where L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 Alkyl, and wherein R x It is H or C that is optionally substituted with a halogen group. 1-6 Alkyl group. In some embodiments, L A It is C 10-20 alkylene, wherein the C 10-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, -N(CH3)-, and -S-. In some embodiments, L A It is C 10-15 alkylene, wherein the C 10-15 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, and -N(CH3)-. In some embodiments, L A It is C 5-10 alkylene, wherein the C 5-10 Alkylene is optionally surrounded by one or more L 1aSubstitution, and one or more alkylene units are optionally replaced by groups selected from -O- and -NH-.

[0174] In some embodiments of conjugates of formula (III) or (III') or any variant thereof (such as formulas (IIIa)-(IIIk)), L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion .

[0175] In some embodiments of conjugates of formula (III) or (III') or any variant thereof (such as formulas (IIIa)-(IIIk)), the click product L B It is a triazine moiety. In some embodiments, the conjugate of any of formula (III) or (III'), or formulas (IIIa)-(IIIk), comprises one of the following moieties: , where X 2 It is -CH2-, -NR x -, -S-, or -O-, where R x As defined elsewhere herein. In some embodiments, the conjugate comprises a portion In some implementations, the conjugate comprises a portion .

[0176] In some embodiments of conjugates of formula (III) or (III') or any variant thereof (such as formulas (IIIa)-(IIIk)), L A -L B Includes part In some implementations, L A -L B Includes part In some implementations, L A -L B Includes part In some implementations, L A -L B Includes part In some implementations, L A -L B Includes part In some implementations, L A -L B Includes part .

[0177] In some embodiments of conjugates of formula (III) or (III') or any variant thereof (such as formulas (IIIa)-(IIIk)), L A -L B Includes part or , where R 9 Is it H or C? 1-4 Alkyl, R 10 Is it H or C? 1-4 Alkyl group, and Q is -CH2-, -O-, -S-, -NH-, or -N(C 1-4 alkyl)-.

[0178] In some embodiments of conjugates of formula (III) or (III') or any variant thereof (such as formulas (IIIa)-(IIIk)), D 1 Choose from the following groups: , , , , and In some implementations, D 1 yes or .

[0179] In one embodiment, this document provides a (IV') conjugate: (IV') Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C?1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, 3 or 4; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; L B It is a click product formed by the click reaction between the first click handle and the second click handle; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the following groups: hydrogen, halogen atoms (e.g., Cl, Br, or F), NO2, CF3, OMe, O(C 1-4 Alkyl), hydroxyl, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C1-6 Alkyl, C 1-6 Alkoxy and C 3-6 Cycloalkyl; p is 0, 1, 2 or 3; and D 1 It's the pharmaceutical part.

[0180] In one embodiment, this document provides a formula (IV) conjugate: (IV) Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, 3 or 4; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; L B It is a click product formed by the click reaction between the first click handle and the second click handle; Y 1 Is it a key or C? 1-4 Alkylene; Y2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the following groups: hydrogen, halogen atoms (e.g., Cl, Br, or F), NO2, CF3, OMe, O(C 1-4 Alkyl), hydroxyl, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 Cycloalkyl; p is 0, 1, 2 or 3; and D 1 It refers to the drug component (opioid agonists or opioid antagonists).

[0181] In some implementations, the (IV) conjugate has the formula (IVa): (IVa) Or its pharmaceutically acceptable salt.

[0182] In some implementations, the (IV) conjugate has the formula (IVb): (IVb) Or its pharmaceutically acceptable salt.

[0183] In some implementations, the (IV) conjugate has the formula (IVc): (IVc) Or its pharmaceutically acceptable salt.

[0184] In some implementations, the (IV) conjugate has the formula (IVd): Or its pharmaceutically acceptable salt.

[0185] In some implementations, the (IV) conjugate has the formula (IVe): (IVe) Or its pharmaceutically acceptable salt.

[0186] In some implementations, the (IV) conjugate has the formula (IVf): (IVf) Or its pharmaceutically acceptable salt.

[0187] In some implementations, the (IV) conjugate has the formula (IVg): (IVg) Or its pharmaceutically acceptable salt.

[0188] In some implementations, the (IV) conjugate has the formula (IVh): (IVh) Or its pharmaceutically acceptable salt.

[0189] In some implementations, the (IV) conjugate has the formula (IVi): (IVi) Or its pharmaceutically acceptable salt.

[0190] In some implementations, the (IV') conjugate has the formula (IVj): (IVj) Or its pharmaceutically acceptable salt.

[0191] In some implementations, the (IV') conjugate has the formula (IVk): (IVk) Or its pharmaceutically acceptable salt.

[0192] In some embodiments of conjugates of formula (IV) or (IV') or any variant thereof (such as formulas (IVa)-(IVk)), R 2 Is it H or C? 1-2 Alkyl group. In some embodiments, R 2 It is H or -CH3. In some implementations, R 2 It's H.

[0193] In some embodiments of conjugates of formula (IV) or (IV') or any variant thereof (such as formulas (IVa)-(IVk)), R 3 Is it H or C? 1-2 Alkyl group. In some embodiments, R 3 It is H or -CH3. In some implementations, R 3 It's H.

[0194] In some embodiments of conjugates of formula (IV) or (IV') or any variant thereof (such as formulas (IVa)-(IVk)), R4 Is it H or C? 1-2 Alkyl group. In some embodiments, R 4 It is H or -CH3. In some implementations, R 4 It is -CH3.

[0195] In some embodiments of conjugates of formula (IV) or (IV') or any variant thereof (such as formulas (IVa)-(IVk)), R 5 Is it H or C? 1-2 Alkyl group. In some embodiments, R 5 It is H or -CH3. In some implementations, R 5 It is -CH3.

[0196] In some embodiments of conjugates of formula (IV) or (IV') or any variant thereof (such as formulas (IVa)-(IVk)), n is 2 or 3. In some embodiments, n is 3.

[0197] In some embodiments of conjugates of formula (IV) or (IV') or any variant thereof (such as formulas (IVa)-(IVj)), m is 0, 1, or 2. In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1.

[0198] In some embodiments of conjugates of formula (IV) or (IV') or any variant thereof (such as formulas (IVa)-(IVj)), p is 0, 1, or 2. In some embodiments, p is 0 or 1. In some embodiments, p is 0. In some embodiments, p is 1.

[0199] In some embodiments of conjugates of formula (IV) or (IV') or any variant thereof (such as formulas (IVa)-(IVk)), L A It is C 4-20 alkylene, wherein the C 4-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally selected from -O-, -NR. x Substitution of - and -S- groups, where L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 Alkyl group, wherein R x It is H or C that is optionally substituted with a halogen group. 1-6 Alkyl group. In some embodiments, L A It is C 10-20 alkylene, wherein the C 10-20 Alkylene is optionally surrounded by one or more L 1aSubstitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, -N(CH3)-, and -S-. In some embodiments, L A It is C 10-15 alkylene, wherein the C 10-15 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, and -N(CH3)-. In some embodiments, L A It is C 5-10 alkylene, wherein the C 5-10 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O- and -NH-.

[0200] In some embodiments of conjugates of formula (IV) or (IV') or any variant thereof (such as formulas (IVa)-(IVk)), L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part .

[0201] In some embodiments of conjugates of formula (IV) or (IV') or any variant thereof (such as formulas (IVa)-(IVk)), D 1Choose from the following groups: , , , , and In some implementations, D 1 yes or .

[0202] In some implementations, this document provides a conjugate of formula (V'): (V'), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; Y 1 Is it a key or C? 1-4 Alkylene; Y2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 Cycloalkyl; p is 0, 1, 2 or 3; and D 1 It's the pharmaceutical part.

[0203] In some implementations, this document provides a conjugate of formula (V): (V), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 Cycloalkyl; p is 0, 1, 2 or 3; and D 1 It refers to the drug component (opioid agonists or opioid antagonists).

[0204] In some implementations, the formula (V) conjugate has the formula (Va): (Va) Or its pharmaceutically acceptable salt.

[0205] In some implementations, the (V) conjugate has the formula (Vb): (Vb) Or its pharmaceutically acceptable salt.

[0206] In some implementations, the formula (V) conjugate has the formula (Vc): (Vc) Or its pharmaceutically acceptable salt.

[0207] In some implementations, the formula (V) conjugate has the formula (Vd): (Vd) Or its pharmaceutically acceptable salt.

[0208] In some implementations, the (V) conjugate has the formula (Ve): (Ve) Or its pharmaceutically acceptable salt.

[0209] In some implementations, the (V) conjugate has the formula (Vf): (Vf) Or its pharmaceutically acceptable salt.

[0210] In some implementations, the (V) conjugate has the formula (Vg): (Vg) Or its pharmaceutically acceptable salt.

[0211] In some implementations, the (V') conjugate has the formula (Vh): (Vh) Or its pharmaceutically acceptable salt.

[0212] In some implementations, the formula (V') conjugate has the formula (Vi): (Vi) Or its pharmaceutically acceptable salt.

[0213] In some embodiments of conjugates of formula (V) or (V') or any variant thereof (such as formulas (Va)-(Vi)), R 2 Is it H or C? 1-2 Alkyl group. In some embodiments, R 2 It is H or -CH3. In some implementations, R 2 It's H.

[0214] In some embodiments of conjugates of formula (V) or (V') or any variant thereof (such as formulas (Va)-(Vi)), R 3 Is it H or C? 1-2 Alkyl group. In some embodiments, R 3 It is H or -CH3. In some implementations, R 3 It's H.

[0215] In some embodiments of conjugates of formula (V) or (V') or any variant thereof (such as formulas (Va)-(Vi)), R 4 Is it H or C? 1-2 Alkyl group. In some embodiments, R4 It is H or -CH3. In some implementations, R 4 It is -CH3.

[0216] In some embodiments of conjugates of formula (V) or (V') or any variant thereof (such as formulas (Va)-(Vi)), R 5 Is it H or C? 1-2 Alkyl group. In some embodiments, R 5 It is H or -CH3. In some implementations, R 5 It is -CH3.

[0217] In some embodiments of conjugates of formula (V) or (V') or any variant thereof (such as formula (Va)-(Vi)), n is 2 or 3. In some embodiments, n is 3.

[0218] In some embodiments of conjugates of formula (V) or (V') or any variant thereof (such as formulas (Va)-(Vh)), m is 0, 1, or 2. In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1.

[0219] In some embodiments of conjugates of formula (V) or (V') or any variant thereof (such as formulas (Va)-(Vh)), p is 0, 1, or 2. In some embodiments, p is 0 or 1. In some embodiments, p is 0. In some embodiments, p is 1.

[0220] In some embodiments of conjugates of formula (V) or (V') or any variant thereof (such as formulas (Va)-(Vi)), L A It is C 4-20 alkylene, wherein the C 4-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally selected from -O-, -NR. x Substitution of - and -S- groups, where L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 Alkyl group, wherein R x It is H or C that is optionally substituted with a halogen group. 1-6 Alkyl group. In some embodiments, L A It is C 10-20 alkylene, wherein the C 10-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, -N(CH3)-, and -S-. In some embodiments, L A It is C10-15 alkylene, wherein the C 10-15 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, and -N(CH3)-. In some embodiments, L A It is C 5-10 alkylene, wherein the C 5-10 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O- and -NH-.

[0221] In some embodiments of conjugates of formula (V) or (V') or any variant thereof (such as formulas (Va)-(Vi)), L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part .

[0222] In some embodiments of conjugates of formula (V) or (V') or any variant thereof (such as formulas (Va)-(Vi)), D 1 Choose from the following groups: , , , , and In some implementations, D 1 yes or .

[0223] In some implementations, this document provides a conjugate of formula (VI'): (VI') Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; and D 1 It's the pharmaceutical part.

[0224] In some implementations, this document provides a conjugate of formula (VI): (VI) Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; and D 1 It refers to the drug component (opioid agonists or opioid antagonists).

[0225] In some implementations, the formula (VI) conjugate has formula (VIa): (VIa) Or its pharmaceutically acceptable salt.

[0226] In some implementations, the formula (VI) conjugate has the formula (VIb): (VIb) Or its pharmaceutically acceptable salt.

[0227] In some implementations, the formula (VI) conjugate has the formula (VIc): (VIc) Or its pharmaceutically acceptable salt.

[0228] In some implementations, the formula (VI) conjugate has the formula (VId): (VId) Or its pharmaceutically acceptable salt.

[0229] In some implementations, the formula (VI) conjugate has the formula (VIe): (VIe) Or its pharmaceutically acceptable salt.

[0230] In some implementations, the formula (VI) conjugate has the formula (VIf): (VIf) Or its pharmaceutically acceptable salt.

[0231] In some implementations, the (VI') conjugate has the formula (VIg): (VIg) Or its pharmaceutically acceptable salt.

[0232] In some implementations, the formula (VI') conjugate has the formula (VIh): (VIh) Or its pharmaceutically acceptable salt.

[0233] In some embodiments of conjugates of formula (VI) or (VI') or any variant thereof (such as formulas (VIa)-(VIh)), R 2 Is it H or C? 1-2 Alkyl group. In some embodiments, R 2 It is H or -CH3. In some implementations, R 2 It's H.

[0234] In some embodiments of conjugates of formula (VI) or (VI') or any variant thereof (such as formulas (VIa)-(VIh)), R 3 Is it H or C? 1-2 Alkyl group. In some embodiments, R 3 It is H or -CH3. In some implementations, R 3 It's H.

[0235] In some embodiments of conjugates of formula (VI) or (VI') or any variant thereof (such as formulas (VIa)-(VIh)), R 4 Is it H or C? 1-2 Alkyl group. In some embodiments, R 4 It is H or -CH3. In some implementations, R 4 It is -CH3.

[0236] In some embodiments of conjugates of formula (VI) or (VI') or any variant thereof (such as formulas (VIa)-(VIh)), R 5 Is it H or C? 1-2Alkyl group. In some embodiments, R 5 It is H or -CH3. In some implementations, R 5 It is -CH3.

[0237] In some embodiments of conjugates of formula (VI) or (VI') or any variant thereof (such as formulas (VIa)-(VIh)), n is 2 or 3. In some embodiments, n is 3.

[0238] In some embodiments of conjugates of formula (VI) or (VI') or any variant thereof (such as formulas (VIa)-(VIg)), m is 0, 1, or 2. In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1.

[0239] In some embodiments of conjugates of formula (VI) or (VI') or any variant thereof (such as formulas (VIa)-(VIg)), p is 0, 1, or 2. In some embodiments, p is 0 or 1. In some embodiments, p is 0. In some embodiments, p is 1.

[0240] In some embodiments of conjugates of formula (VI) or (VI') or any variant thereof (such as formulas (VIa)-(VIh)), L A It is C 4-20 alkylene, wherein the C 4-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally selected from -O-, -NR. x Substitution of - and -S- groups, where L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 Alkyl group, wherein R x It is H or C that is optionally substituted with a halogen group. 1-6 Alkyl group. In some embodiments, L A It is C 10-20 alkylene, wherein the C 10-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, -N(CH3)-, and -S-. In some embodiments, L A It is C 10-15 alkylene, wherein the C 10-15 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, and -N(CH3)-. In some embodiments, L A It is C5-10 alkylene, wherein the C 5-10 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O- and -NH-.

[0241] In some embodiments of conjugates of formula (VI) or (VI') or any variant thereof (such as formulas (VIa)-(VIh)), L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part .

[0242] In some embodiments of conjugates of formula (VI) or (VI') or any variant thereof (such as formulas (VIa)-(VIh)), D 1 Choose from the following groups: , , , , and In some implementations, D 1 yes or .

[0243] In some implementations, this document provides a conjugate of formula (VII'): (VII'), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; L B It is a click product formed by the click reaction between the first click handle and the second click handle; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; r is 1, 2, 3, 4, 5, 6, or 7; s is 1, 2, 3, 4, 5, 6, or 7; and D 1 It's the pharmaceutical part.

[0244] In some implementations, this document provides a conjugate of formula (VII): (VII), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; L B It is a click product formed by the click reaction between the first click handle and the second click handle; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; r is 1, 2, 3, 4, 5, 6, or 7; s is 1, 2, 3, 4, 5, 6, or 7; and D 1 It refers to the drug component (opioid agonists or opioid antagonists).

[0245] In some embodiments, the conjugate of formula (VII) has formula (VIIa): (VIIa) Or its pharmaceutically acceptable salt.

[0246] In some embodiments, the conjugate of formula (VII) has formula (VIIb): (VIIb) Or its pharmaceutically acceptable salt.

[0247] In some embodiments, the conjugate of formula (VII) has formula (VIIc): (VIIc) Or its pharmaceutically acceptable salt.

[0248] In some implementations, the conjugate of formula (VII) has formula (VIId): (VIId) Or its pharmaceutically acceptable salt.

[0249] In some embodiments of conjugates of formula (VII) or (VII') or any variant thereof (such as formulas (VIIa)-(VIId)), R 2 Is it H or C? 1-2 Alkyl group. In some embodiments, R 2 It is H or -CH3. In some implementations, R 2 It's H.

[0250] In some embodiments of conjugates of formula (VII) or (VII') or any variant thereof (such as formulas (VIIa)-(VIId)), R 3 Is it H or C? 1-2 Alkyl group. In some embodiments, R 3 It is H or -CH3. In some implementations, R 3 It's H.

[0251] In some embodiments of conjugates of formula (VII) or (VII') or any variant thereof (such as formulas (VIIa)-(VIId)), R 4 Is it H or C? 1-2 Alkyl group. In some embodiments, R 4 It is H or -CH3. In some implementations, R4 It is -CH3.

[0252] In some embodiments of conjugates of formula (VII) or (VII') or any variant thereof (such as formulas (VIIa)-(VIId)), R 5 Is it H or C? 1-2 Alkyl group. In some embodiments, R 5 It is H or -CH3. In some implementations, R 5 It is -CH3.

[0253] In some embodiments of conjugates of formula (VII) or (VII') or any variant thereof (such as formulas (VIIa)-(VIId)), n is 2 or 3. In some embodiments, n is 3.

[0254] In some embodiments of conjugates of formula (VII) or (VII') or any variant thereof (such as formulas (VIIa)-(VIId)), m is 0, 1, or 2. In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1.

[0255] In some embodiments of conjugates of formula (VII) or (VII') or any variant thereof (such as formulas (VIIa)-(VIId)), p is 0, 1, or 2. In some embodiments, p is 0 or 1. In some embodiments, p is 0. In some embodiments, p is 1.

[0256] In some embodiments of conjugates of formula (VII) or (VII') or any variant thereof (such as formulas (VIIa)-(VIId)), the click product L B It is a triazine moiety. In some embodiments, the conjugate of any of formula (VII) or (VII'), or formulas (VIIa)-(VIId) comprises one of the following moieties: , where X 2 It is -CH2-, -NR x -, -S-, or -O-, where R x As defined elsewhere herein. In some embodiments, the conjugate comprises a portion In some implementations, the conjugate comprises a portion .

[0257] In some embodiments of conjugates of formula (VII) or (VII') or any variant thereof (such as formulas (VIIa)-(VIId)), L A -L B Includes part or , where R9 Is it H or C? 1-4 Alkyl, R 10 Is it H or C? 1-4 Alkyl group, and Q is -CH2-, -O-, -S-, -NH-, or -N(C 1-4 alkyl)-.

[0258] In some embodiments of conjugates of formula (VII) or (VII') or any variant thereof (such as formulas (VIIa)-(VIId)), D 1 Choose from the following groups: , , , , and In some implementations, D 1 yes or .

[0259] In some implementations, this document provides a conjugate of formula (VIII'): (VIII') Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; L B It is a click product formed by the click reaction between the first click handle and the second click handle; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; r is 1, 2, 3, 4, 5, 6, or 7; s is 1, 2, 3, 4, 5, 6, or 7; t is 1, 2, 3, or 4; u is 1, 2, 3, or 4; and D 1 It's the pharmaceutical part.

[0260] In some implementations, this document provides a conjugate of formula (VIII): (VIII) Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; L B It is a click product formed by the click reaction between the first click handle and the second click handle; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; r is 1, 2, 3, 4, 5, 6, or 7; s is 1, 2, 3, 4, 5, 6, or 7; t is 1, 2, 3, or 4; u is 1, 2, 3, or 4; and D 1 It refers to the drug component (opioid agonists or opioid antagonists).

[0261] In some embodiments, the (VIII) conjugate has the formula (VIIIa): (VIIIa) Or its pharmaceutically acceptable salt.

[0262] In some embodiments, the (VIII) conjugate has the formula (VIIIb): (VIIIb) Or its pharmaceutically acceptable salt.

[0263] In some embodiments, the (VIII) conjugate has the formula (VIIIc): (VIIIc) Or its pharmaceutically acceptable salt.

[0264] In some embodiments, the (VIII) conjugate has the formula (VIIId): (VIIId) Or its pharmaceutically acceptable salt.

[0265] In some embodiments, the (VIII) conjugate has the formula (VIIIe): (VIIIe) Or its pharmaceutically acceptable salt.

[0266] In some implementations, the (VIII') conjugate has the formula (VIIIf): (VIIIf) Or its pharmaceutically acceptable salt.

[0267] In some embodiments of conjugates of formula (VIII) or (VIII') or any variant thereof (such as formulas (VIIIa)-(VIIIf)), R 2 Is it H or C? 1-2 Alkyl group. In some embodiments, R 2 It is H or -CH3. In some implementations, R 2 It's H.

[0268] In some embodiments of conjugates of formula (VIII) or (VIII') or any variant thereof (such as formulas (VIIIa)-(VIIIf)), R 3 Is it H or C? 1-2 Alkyl group. In some embodiments, R 3 It is H or -CH3. In some implementations, R 3 It's H.

[0269] In some embodiments of conjugates of formula (VIII) or (VIII') or any variant thereof (such as formulas (VIIIa)-(VIIIf)), R 4 Is it H or C? 1-2 Alkyl group. In some embodiments, R 4 It is H or -CH3. In some implementations, R 4 It is -CH3.

[0270] In some embodiments of conjugates of formula (VIII) or (VIII') or any variant thereof (such as formulas (VIIIa)-(VIIIf)), R 5 Is it H or C? 1-2 Alkyl group. In some embodiments, R 5 It is H or -CH3. In some implementations, R 5 It is -CH3.

[0271] In some embodiments of conjugates of formula (VIII) or (VIII') or any variant thereof (such as formulas (VIIIa)-(VIIIf)), n is 2 or 3. In some embodiments, n is 3.

[0272] In some embodiments of conjugates of formula (VIII) or (VIII') or any variant thereof (such as formulas (VIIIa)-(VIIIf)), m is 0, 1, or 2. In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1.

[0273] In some embodiments of conjugates of formula (VIII) or (VIII') or any variant thereof (such as formulas (VIIIa) or (VIIIc)-(VIIIf)), p is 0, 1, or 2. In some embodiments, p is 0 or 1. In some embodiments, p is 0. In some embodiments, p is 1.

[0274] In some embodiments of conjugates of formula (VIII) or (VIII') or any variant thereof (such as formulas (VIIIa)-(VIIIb)), the click product L B It is a triazine moiety. In some embodiments, the conjugate of any of formula (VIII) or (VIII'), or formulas (VIIIa)-(VIIIb) comprises one of the following moieties: , where X 2 It is -CH2-, -NR x -, -S-, or -O-, where R x As defined elsewhere herein. In some embodiments, the conjugate comprises a portion In some implementations, the conjugate comprises a portion .

[0275] In some embodiments of conjugates of formula (VIII) or (VIII') or any variant thereof (such as formulas (VIIIa)-(VIIIb)), L A -L B Includes part or , where R 9 Is it H or C? 1-4 Alkyl, R 10 Is it H or C? 1-4 Alkyl group, and Q is -CH2-, -O-, -S-, -NH-, or -N(C 1-4 alkyl)-.

[0276] In some embodiments of conjugates of formula (VIII) or (VIII') or any variant thereof (such as formulas (VIIIa)-(VIIIf)), D 1 Choose from the following groups: , , , , and In some implementations, D 1 yes or .

[0277] In some implementations, this document provides a conjugate of formula (IX): (IX), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L stands for connector; Z 1 It is C 1-6 Alkyl, C 6-10 Aryl or 5 to 10-membered heteroaryl, among which The C 1-6 Alkyl groups are optionally surrounded by one or more Z 1a Replace, and The C 6-10 Aryl or 5- to 10-membered heteroaryl groups are optionally surrounded by one or more Z groups. 1b replace; Each Z 1a or Z 1b Independently halogenated, C 1-6alkoxy or C-group optionally substituted with a halogen group 1-6 Alkyl; and D 1 It refers to the drug component (opioid agonists or opioid antagonists).

[0278] In some embodiments of the formula (IX) conjugate, R 2 Is it H or C? 1-2 Alkyl group. In some embodiments, R 2 It is H or -CH3. In some implementations, R 2 It's H.

[0279] In some embodiments of the formula (IX) conjugate, R 3 Is it H or C? 1-2 Alkyl group. In some embodiments, R 3 It is H or -CH3. In some implementations, R 3 It's H.

[0280] In some embodiments of the formula (IX) conjugate, R 4 Is it H or C? 1-2 Alkyl group. In some embodiments, R 4 It is H or -CH3. In some implementations, R 4 It is -CH3.

[0281] In some embodiments of the formula (IX) conjugate, R 5 Is it H or C? 1-2 Alkyl group. In some embodiments, R 5 It is H or -CH3. In some implementations, R 5 It is -CH3.

[0282] In some embodiments of the formula (IX) conjugate, n is 2 or 3. In some embodiments, n is 3.

[0283] In some embodiments of the conjugate of formula (IX), m is 0, 1, or 2. In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1.

[0284] In some embodiments of the formula (IX) conjugate, L is C 4-20 alkylene, wherein the C 4-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally selected from -O-, -NR. x Substitution of - and -S- groups, where L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6Alkyl group. In some embodiments, L is C. 10-20 alkylene, wherein the C 10-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, -N(CH3)-, and -S-. In some embodiments, L is C 10-15 alkylene, wherein the C 10-15 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, and -N(CH3)-. In some embodiments, L is C 5-10 alkylene, wherein the C 5-10 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O- and -NH-.

[0285] In some embodiments of the formula (IX) conjugate, L contains a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion In some implementations, L includes a portion .

[0286] In some embodiments of conjugates of formula (IX) or any variant thereof, D 1 Choose from the following groups: , , , , and In some implementations, D 1 yes or .

[0287] In some implementations, this document provides a conjugate of formula (X): (X), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; L B It is a click product formed by the click reaction between the first click handle and the second click handle; Z 1 It is C 1-6 Alkyl, C 6-10 Aryl or 5 to 10-membered heteroaryl, among which The C 1-6 Alkyl groups are optionally surrounded by one or more Z 1a Replace, and The C 6-10 Aryl or 5- to 10-membered heteroaryl groups are optionally surrounded by one or more Z groups.1b replace; Each Z 1a or Z 1b Independently halogenated, C 1-6 alkoxy or C-group optionally substituted with a halogen group 1-6 Alkyl; and D 1 It refers to the drug component (opioid antagonists or opioid agonists).

[0288] In some embodiments of the (X) conjugate, R 2 Is it H or C? 1-2 Alkyl group. In some embodiments, R 2 It is H or -CH3. In some implementations, R 2 It's H.

[0289] In some embodiments of the (X) conjugate, R 3 Is it H or C? 1-2 Alkyl group. In some embodiments, R 3 It is H or -CH3. In some implementations, R 3 It's H.

[0290] In some embodiments of the (X) conjugate, R 4 Is it H or C? 1-2 Alkyl group. In some embodiments, R 4 It is H or -CH3. In some implementations, R 4 It is -CH3.

[0291] In some embodiments of the (X) conjugate, R 5 Is it H or C? 1-2 Alkyl group. In some embodiments, R 5 It is H or -CH3. In some implementations, R 5 It is -CH3.

[0292] In some embodiments of the (X) conjugate, n is 2 or 3. In some embodiments, n is 3.

[0293] In some embodiments of the (X) conjugate, m is 0, 1, or 2. In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1.

[0294] In some embodiments of the (X) conjugate, L A It is C 4-20 alkylene, wherein the C 4-20 Alkylene is optionally surrounded by one or more L 1aSubstitution, and one or more alkylene units are optionally selected from -O-, -NR. x Substitution of - and -S- groups, where L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 Alkyl group, wherein R x It is H or C that is optionally substituted with a halogen group. 1-6 Alkyl group. In some embodiments, L A It is C 10-20 alkylene, wherein the C 10-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, -N(CH3)-, and -S-. In some embodiments, L A It is C 10-15 alkylene, wherein the C 10-15 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, and -N(CH3)-. In some embodiments, L A It is C 5-10 alkylene, wherein the C 5-10 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O- and -NH-.

[0295] In some embodiments of the (X) conjugate, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part .

[0296] In some embodiments of the formula (X) conjugate, the click product L B It is a triazine moiety. In some embodiments, the (X) conjugate comprises one of the following moieties: , where X 2 It is -CH2-, -NR x -, -S-, or -O-, where R x As defined elsewhere herein. In some embodiments, the conjugate comprises a portion In some implementations, the conjugate comprises a portion .

[0297] In some embodiments of the (X) conjugate, L A -L B Includes part or , where R 9 Is it H or C? 1-4 Alkyl, R 10 Is it H or C? 1-4 Alkyl group, and Q is -CH2-, -O-, -S-, -NH-, or -N(C 1-4 alkyl)-.

[0298] In some embodiments of the (X) conjugate, D 1 Choose from the following groups: , , , , and In some implementations, D 1 yes or .

[0299] In some implementations, this document provides a conjugate of formula (XI): (XI), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; Z 1 It is C 1-6 Alkyl, C 6-10 Aryl or 5 to 10-membered heteroaryl, among which The C 1-6 Alkyl groups are optionally surrounded by one or more Z 1a Replace, and The C 6-10 Aryl or 5- to 10-membered heteroaryl groups are optionally surrounded by one or more Z groups. 1b replace; Each Z 1a or Z 1b Independently halogenated, C 1-6 alkoxy or C-group optionally substituted with a halogen group 1-6 Alkyl; and D 1 It refers to the drug component (opioid antagonists or opioid agonists).

[0300] In some embodiments of the conjugate of formula (XI), R 2 Is it H or C? 1-2Alkyl group. In some embodiments, R 2 It is H or -CH3. In some implementations, R 2 It's H.

[0301] In some embodiments of the conjugate of formula (XI), R 3 Is it H or C? 1-2 Alkyl group. In some embodiments, R 3 It is H or -CH3. In some implementations, R 3 It's H.

[0302] In some embodiments of the conjugate of formula (XI), R 4 Is it H or C? 1-2 Alkyl group. In some embodiments, R 4 It is H or -CH3. In some implementations, R 4 It is -CH3.

[0303] In some embodiments of the conjugate of formula (XI), R 5 Is it H or C? 1-2 Alkyl group. In some embodiments, R 5 It is H or -CH3. In some implementations, R 5 It is -CH3.

[0304] In some embodiments of the conjugate of formula (XI), n is 2 or 3. In some embodiments, n is 3.

[0305] In some embodiments of the conjugate of formula (XI), m is 0, 1, or 2. In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1.

[0306] In some embodiments of the conjugate of formula (XI), L A It is C 4-20 alkylene, wherein the C 4-20 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally selected from -O-, -NR. x Substitution of - and -S- groups, where L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 Alkyl group, wherein R x It is H or C that is optionally substituted with a halogen group. 1-6 Alkyl group. In some embodiments, L A It is C 10-20 alkylene, wherein the C 10-20 Alkylene is optionally surrounded by one or more L 1aSubstitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, -N(CH3)-, and -S-. In some embodiments, L A It is C 10-15 alkylene, wherein the C 10-15 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O-, -NH-, and -N(CH3)-. In some embodiments, L A It is C 5-10 alkylene, wherein the C 5-10 Alkylene is optionally surrounded by one or more L 1a Substitution, and one or more alkylene units are optionally replaced by groups selected from -O- and -NH-.

[0307] In some embodiments of the conjugate of formula (XI), L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part In some implementations, L A Includes part .

[0308] In some embodiments of the conjugate of formula (XI), D 1 Choose from the following groups: , , , , and In some implementations, D 1 yes or .

[0309] In some embodiments, this document provides a conjugate having the structure shown below: , Where D 1 As defined elsewhere in this document.

[0310] In some embodiments, the conjugate is selected from the conjugates in Table A or their salts.

[0311] Table A.

[0312] In some embodiments, this document provides a conjugate having the structure shown below: .

[0313] In some embodiments, this document provides a conjugate having the structure shown below: .

[0314] In some embodiments, this document provides a conjugate having the structure shown below: .

[0315] In some embodiments, this document provides a conjugate having the structure shown below: .

[0316] In some embodiments, this document provides a conjugate having the structure shown below: .

[0317] In some embodiments, this document provides a conjugate having the structure shown below: .

[0318] In some embodiments, this document provides a conjugate having the structure shown below: .

[0319] Pharmaceutical Composition This document provides pharmaceutical compositions comprising one or more conjugates of this disclosure (e.g., conjugates of formulas (I) to (XI) or (II') to (VIII') or any variations or embodiments thereof as described elsewhere herein) or a pharmaceutically acceptable salt of any of the foregoing. In some embodiments, this document provides a pharmaceutical composition comprising (i) a compound of any of formulas (I) to (XI) or (II') to (VIII') or any variations or embodiments thereof as described elsewhere herein, or a pharmaceutically acceptable salt of any of the foregoing, and (ii) one or more pharmaceutically acceptable excipients.

[0320] Suitable pharmaceutically acceptable excipients may include, for example, fillers, diluents, sterile aqueous solutions and various organic solvents, permeation enhancers, solubilizers and adjuvants. The term excipient may cover a wide range of substances, including but not limited to any substance used as a binder, disintegrant, coating, tableting / encapsulation aid, cream or lotion, lubricant, solution for parenteral administration, material for chewable tablets, sweetener or flavoring agent, suspending / gelling agent or wet granulation agent. Examples of suitable excipients are well known to those skilled in the art. See, for example, Handbook of Pharmaceutical Excipients ,Pharmaceutical Press (2017), which is incorporated herein by reference in its entirety. Such compositions are prepared in a manner well known in the pharmaceutical field. See, for example, Remington's Pharmaceutical Sciences , Academic Press, 23rd edition (2020), which is incorporated herein by reference in its entirety.

[0321] Treatment In one aspect, this document provides a method for increasing the plasma circulation time of an active agent, the method comprising covalently linking the active agent to a plasma protein conjugate to form a conjugate of the present disclosure (e.g., a conjugate of formula (I)-(XI) or (II')-(VIII') or any variant thereof).

[0322] In one aspect, this document provides a method for increasing the in vivo half-life of an active agent, the method comprising covalently linking the active agent to a plasma protein binding agent to form a conjugate of the present disclosure (e.g., a conjugate of formula (I)-(XI) or (II')-(VIII') or any variant thereof).

[0323] In one aspect, this document provides a method for increasing the duration of action (DOA) of an active agent, the method comprising covalently linking the active agent to a plasma protein binder to form a conjugate of this disclosure.

[0324] In one aspect, this document provides a method for reducing the immunogenicity of an active agent in a subject, the method comprising obtaining a delivery system having a ligand (i) highly selective for an endogenous plasma protein of the subject, the plasma protein having a molecular weight in the range of about 30 kDa to about 80 kDa; (ii) having a high binding affinity (Kd) of at least 10⁻⁶ M for the plasma protein; and (iii) having a molecular weight in the range of about 200 Da to about 2000 Da; and a linker having a length in the range of about 10 angstroms to about 50 angstroms or 10 atoms to 50 atoms. The method further comprises covalently linking the delivery system to the active agent to produce a conjugated active agent; and administering the conjugated active agent to the subject; wherein the plasma protein masks the active agent to prevent the generation of antibodies in the subject after administration.

[0325] In one aspect, this document provides methods for treating diseases, symptoms, or ailments, the methods comprising administering to an individual in need a conjugate of the present disclosure (e.g., a conjugate of any of formulas (I)-(XI) or (II')-(VIII') or any variant or embodiment thereof, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof), or a pharmaceutical composition comprising a conjugate of the present disclosure, any variant or embodiment thereof, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0326] In some implementation methods, a therapeutically effective amount of the compound is administered.

[0327] In some embodiments, when administered to a subject using the delivery system provided herein, the half-life of the active agent (opioid antagonist or opioid agonist) can range from about 30 minutes to about 90 days. In some embodiments, when administered to a subject using the delivery system provided herein, the half-life of the active agent (opioid antagonist or opioid agonist) can range from about 30 minutes to about 30 days. For example, in some embodiments, when administered to a subject using a delivery system taught herein with a ligand highly selective for thyroxine transporter, the half-life of the active agent can range from about 30 minutes to about 2 days. Similarly, in some embodiments, when administered to a subject using a delivery system taught herein with a ligand highly selective for human serum albumin, the half-life of the active agent can range from about 30 minutes to about 2 days. Therefore, in some implementations, when the delivery system taught herein is administered to a subject, the half-life of the active agent may be about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 18 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 10 days, about 12 days, about 16 days, about 20 days, about 30 days, or any amount in increments of 1 hour. Those skilled in the art will understand that the half-life of the activity of an active agent administered to a subject using the delivery system taught herein can be within the following ranges: about 30 minutes to about 2 days, about 1 hour to about 36 hours, about 2 hours to about 20 hours, about 2 hours to about 10 hours, about 3 hours to about 30 hours, about 3 hours to about 15 hours, about 4 hours to about 40 hours, about 4 hours to about 20 hours, about 5 hours to about 50 hours, about 5 hours to about 25 hours, or any range or amount thereof in increments of 1 hour.

[0328] In one aspect, this document provides a method for reducing the risk of recurrent respiratory depression, the method comprising administering the conjugate of this disclosure to an individual in need prior to exposure to a drug.

[0329] In one aspect, this document provides a preventative method comprising administering the disclosed conjugate to an individual in need prior to exposure to the drug.

[0330] In some embodiments, the drug is an opioid. In some embodiments, the drug is a weaponized opioid. In some embodiments, the drug is selected from the group consisting of fentanyl, carfentanil, or remifentanil.

[0331] In some embodiments, the conjugates of this disclosure are administered parenterally to subjects in need. For example, the conjugates of this disclosure may be administered subcutaneously, intravenously, intramuscularly, or intrathecally.

[0332] Partly due to the hydrophilicity of the TTR selective ligand, the conjugates of this disclosure (particularly those of formula (II)-(XI) or (II')-(VIII')) exhibit enhanced tissue (particularly subcutaneous) adsorption. Following tissue (e.g., subcutaneous) adsorption, the reversible binding of the TTR selective ligand of the conjugate significantly prolongs the duration of action of the active agent. As illustrated herein, cleavage of the linker between the TTR selective ligand and the active agent (opioid antagonist or opioid agonist) releases the active agent, thus allowing it to exert its pharmacological effects.

[0333] Furthermore, it has been found that the release of active agents (opioid antagonists or opioid agonists) can be effectively controlled by appropriately selecting the cleavable moiety in the conjugate. For example, specific ester moieties (e.g., PABA or PHBA) as described herein undergo slow hydrolysis after administration, thereby releasing the active agent in a controlled manner for a prolonged period. Slower hydrolysis and high binding to TTR effectively prolong the plasma circulation time of the active agent.

[0334] Therefore, the conjugates of this disclosure exhibit several beneficial properties of novel prodrug methods. The balanced hydrophilicity of the conjugates allows for rapid adsorption from tissues into the bloodstream, the binding of TTR prolongs the half-life, and the cleavable moiety allows for modulated drug release. The desired release rate of a specific active drug from the conjugates of this disclosure will vary depending on the active agent.

[0335] In some embodiments, the active agent (D) of the conjugate is an opioid antagonist. In some embodiments, the opioid antagonist is naloxone, nalmefen, naltrexone, methylnaltrexone, nalbuphine, or nalprofen.

[0336] In some embodiments, the DOA of the opioid antagonist or opioid agonist is at least 12 hours after administration of the conjugates of this disclosure. In some embodiments, the DOA of the opioid antagonist or opioid agonist is at least 20 hours after administration of the conjugates of this disclosure. In some embodiments, the DOA of the opioid antagonist or opioid agonist is at least 24 hours after administration of the conjugates of this disclosure. In some embodiments, the DOA of the opioid antagonist or opioid agonist is at least 30 hours after administration of the conjugates of this disclosure. In some such embodiments, the DOA of the opioid antagonist or opioid agonist is at least 40 hours after administration of the conjugates of this disclosure. In some such embodiments, the DOA of the opioid antagonist or opioid agonist is at least 48 hours after administration of the conjugates of this disclosure. In some such embodiments, the DOA of the opioid antagonist or opioid agonist is at least 72 hours after administration of the conjugates of this disclosure. In some such embodiments, the DOA of the opioid antagonist or opioid agonist after administration of the conjugate of this disclosure is at least 96 hours. In some such embodiments, the DOA of the opioid antagonist or opioid agonist after administration of the conjugate of this disclosure is at least 1 week. In some such embodiments, the DOA of the opioid antagonist or opioid agonist after administration of the conjugate of this disclosure is at least 2 weeks. In some such embodiments, the DOA of the opioid antagonist or opioid agonist after administration of the conjugate of this disclosure is at least 3 weeks. In some such embodiments, the DOA of the opioid antagonist or opioid agonist after administration of the conjugate of this disclosure is at least 1 month. In some such embodiments, the DOA of the opioid antagonist or opioid agonist after administration of the conjugate of this disclosure is from about 12 hours to about 48 hours. In some such embodiments, the DOA of the opioid antagonist or opioid agonist after administration of the conjugate of this disclosure is about 24 hours to about 48 hours. In some embodiments, the DOA of the opioid antagonist or opioid agonist after administration of the conjugate of this disclosure is about 48 hours to about 96 hours. In some embodiments, the DOA of the opioid antagonist or opioid agonist after administration of the conjugate of this disclosure is about 24 hours to about 1 week.

[0337] In some embodiments, the plasma circulation of the opioid antagonist or opioid agonist is at least 12 hours after administration of the conjugates of this disclosure. In some embodiments, the plasma circulation of the opioid antagonist or opioid agonist is at least 20 hours after administration of the conjugates of this disclosure. In some embodiments, the plasma circulation of the opioid antagonist or opioid agonist is at least 24 hours after administration of the conjugates of this disclosure. In some embodiments, the plasma circulation of the opioid antagonist or opioid agonist is at least 30 hours after administration of the conjugates of this disclosure. In some such embodiments, the plasma circulation of the opioid antagonist or opioid agonist is at least 40 hours after administration of the conjugates of this disclosure. In some such embodiments, the plasma circulation of the opioid antagonist or opioid agonist is at least 48 hours after administration of the conjugates of this disclosure. In some such embodiments, the plasma circulation of the opioid antagonist or opioid agonist is at least 72 hours after administration of the conjugates of this disclosure. In some such embodiments, the plasma circulation of the opioid antagonist or opioid agonist is at least 96 hours after administration of the conjugate of this disclosure. In some such embodiments, the plasma circulation of the opioid antagonist or opioid agonist is at least 1 week after administration of the conjugate of this disclosure. In some such embodiments, the plasma circulation of the opioid antagonist or opioid agonist is at least 2 weeks after administration of the conjugate of this disclosure. In some such embodiments, the plasma circulation of the opioid antagonist or opioid agonist is at least 3 weeks after administration of the conjugate of this disclosure. In some such embodiments, the plasma circulation of the opioid antagonist or opioid agonist is at least 1 month after administration of the conjugate of this disclosure. In some such embodiments, the plasma circulation of the opioid antagonist or opioid agonist is from about 12 hours to about 48 hours after administration of the conjugate of this disclosure. In some such embodiments, plasma circulation of the opioid antagonist or opioid agonist is from about 24 hours to about 48 hours after administration of the conjugate of this disclosure. In some embodiments, plasma circulation of the opioid antagonist or opioid agonist is from about 48 hours to about 96 hours after administration of the conjugate of this disclosure. In some embodiments, plasma circulation of the opioid antagonist or opioid agonist is from about 24 hours to about 1 week after administration of the conjugate of this disclosure.

[0338] In some embodiments where the active agent (D) of the conjugate of formula (I) or formula (II) is an opioid antagonist, administration of the conjugate reverses and protects against re-anesthesia (ventilation depression) due to overdose of synthetic opioids. In some embodiments, the synthetic opioid is fentanyl. In some embodiments, the synthetic opioid is a fentanyl analogue, such as carfentanil, acetylfentanyl, butyrylfentanyl, or furazolidone. In some embodiments, the synthetic opioid is heroin. In some embodiments, the synthetic opioid is morphine. In some embodiments, the synthetic opioid is meperidine. In some embodiments, administration of the conjugate of this disclosure does not result in the deposition of opioid withdrawal symptoms.

[0339] In some implementations, the disorder is a drug overdose. In some implementations, the disorder is an opioid overdose. In some implementations, the disorder is a fentanyl overdose, heroin overdose, morphine overdose, or carfentanil overdose.

[0340] In some embodiments, the conjugate is administered in combination with a free (unconjugated) opioid antagonist. In such an iteration, the free opioid antagonist effectively rescues opioid-induced respiratory depression (OIRD), while the opioid antagonist released from the conjugate increases the duration of action of the opioid antagonist and prevents re-anesthesia. In some embodiments, the free opioid antagonist is the same as the opioid antagonist in the conjugate. In some embodiments, the free opioid antagonist is different from the opioid antagonist in the conjugate. In some embodiments, the free opioid antagonist is naloxone. In some embodiments, the free opioid antagonist is nalmefene.

[0341] In some embodiments, the conjugates of this disclosure can be used to treat or mitigate opioid use disorder (OUD) in a subject of need (e.g., a human patient). In some embodiments, a method of treating OUD includes administering a therapeutically effective amount of a conjugate of this disclosure (e.g., a conjugate of formula (I) or (II)) containing an opioid antagonist as the active pharmaceutical ingredient (D). In some such embodiments, the opioid antagonist of the conjugate is naloxone, nalmefone, naltrexone, methylnaltrexone, nalbuphine, or nalprofen. In some embodiments, the opioid antagonist of the conjugate is naloxone. In some embodiments, the opioid antagonist of the conjugate is naltrexone. In some embodiments, the conjugate provides controlled and / or prolonged release of the opioid antagonist.

[0342] In other embodiments, a method of treating OUD includes administering a conjugate of the present disclosure comprising an opioid agonist as the active pharmaceutical ingredient (D). In some such embodiments, the opioid agonist of the conjugate is buprenorphine. In some such embodiments, the opioid agonist of the conjugate is aripiprazole. In some such embodiments, the opioid agonist of the conjugate is norclozapine. In some such embodiments, the opioid agonist of the conjugate is buspirone. In some embodiments, administering a conjugate of the present disclosure to treat OUD (where D is an opioid antagonist or an opioid agonist) results in a less severe withdrawal effect compared to administration of an opioid antagonist or opioid agonist in a non-conjugated pharmaceutical form. In some embodiments, the conjugate provides controlled and / or prolonged release of the opioid agonist.

[0343] In some embodiments, the conjugates of this disclosure may be administered in combination with one or more opioid antagonists or opioid agonists to treat OUD. In some embodiments, the conjugates of this disclosure may be administered in combination with buprenorphine (e.g., concurrently or sequentially). In some embodiments, the conjugates of this disclosure may be administered in combination with naloxone (e.g., concurrently or sequentially). In some embodiments, the conjugates of this disclosure may be administered in combination with naloxone (e.g., concurrently or sequentially). In some embodiments, the conjugates of this disclosure may be administered in combination with methylnaloxone (e.g., concurrently or sequentially). In some embodiments, the conjugates of this disclosure (wherein the opioid antagonist (D) in the conjugate is naloxone) may be administered in combination with buprenorphine (e.g., concurrently or sequentially) to treat OUD in patients of need. In some embodiments, the conjugates of this disclosure (wherein the opioid antagonist (D) in the conjugate is naltrexone or methylnaltrexone) may be administered in combination with buprenorphine (e.g., concurrently or sequentially) to treat OUD in patients of need. In any of the foregoing embodiments, the conjugate may be administered subcutaneously.

[0344] The conjugates disclosed herein provide extended-release prodrugs for opioid antagonists or opioid agonists and provide opioid abuse control with minimal withdrawal effects. In some embodiments, the conjugates are administered to newborns of opioid-addicted mothers. In some embodiments, the conjugates are administered to children. In some embodiments, the conjugates are administered to children. In some embodiments, the conjugates are administered to elderly patients. In some embodiments, the conjugates are administered to patients with cardiovascular complications. In some embodiments, the conjugates are administered postoperatively to patients recovering from anesthesia / analgesia. In any of the foregoing embodiments, the conjugates may be administered subcutaneously.

[0345] In some embodiments, the conjugates disclosed herein can provide controlled and / or prolonged release of an opioid agonist. In some such embodiments, the slow release of the opioid agonist from the conjugate can be safely administered without causing euphoria or respiratory depression. In some embodiments, the opioid agonist is morphine, oxycodone, oxymorphone, codeine, hydrocodone, hydromorphone, methadone, thebaine, or fentanyl. In some embodiments, the conjugate is used to treat mild or acute pain.

[0346] In some embodiments, the conjugates of this disclosure are administered subcutaneously to human subjects at a dose of about 0.005 mg / kg to about 40 mg / kg. In some embodiments, the conjugates of this disclosure are administered subcutaneously to human subjects at a dose of about 0.01 mg / kg to about 40 mg / kg. In some embodiments, the conjugates of this disclosure are administered subcutaneously to human subjects at a dose of about 0.005 mg / kg to about 10 mg / kg. In some embodiments, the conjugates of this disclosure are administered subcutaneously to human subjects at a dose of about 0.1 mg / kg to about 10 mg / kg. In some embodiments, the conjugates of this disclosure are administered subcutaneously to human subjects at a dose of about 0.5 mg / kg to about 10 mg / kg. In some embodiments, the conjugates of this disclosure are administered subcutaneously to human subjects at a dose of about 5 mg / kg to about 40 mg / kg. In some embodiments, the conjugates of this disclosure are administered subcutaneously to human subjects at a dose of about 10 mg / kg to about 30 mg / kg. In some embodiments, the conjugates of this disclosure are administered subcutaneously to human subjects at a dose of about 15 mg / kg to about 25 mg / kg.

[0347] medicine box This disclosure further provides a pharmaceutical kit for carrying out the methods described herein. The kit may contain a conjugate of this disclosure as described herein, or a pharmaceutically acceptable salt thereof, and suitable packaging. The kit may contain one or more containers containing any of the compounds described herein. In one aspect, the kit includes a compound of this disclosure or a pharmaceutically acceptable salt thereof, and a label and / or instructions for using said compound to treat the disease or condition described herein. The kit may contain a unit dosage form of the compound.

[0348] This document provides a kit comprising (i) a conjugate of the present disclosure, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula (I) or any variant or embodiment thereof, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and (ii) instructions for therapeutic use.

[0349] In some implementation schemes, an individual is a person.

[0350] The present invention also provides articles comprising, in a suitable container, a conjugate thereof, any variation or embodiment thereof, a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof.

[0351] In some implementations, the container may be a vial, a wide-mouth bottle, an ampoule, a preloaded syringe, or an intravenous infusion bag.

[0352] Preparation method This document provides methods for preparing the conjugates of this disclosure, any variations or embodiments thereof, or pharmaceutically acceptable salts of any of the foregoing. The synthetic methods described below, as well as those detailed in the examples, are merely illustrative of some methods by which the compounds of this disclosure, or embodiments or aspects thereof, can be synthesized. Various modifications to these synthetic reaction schemes will be apparent to those skilled in the art. Although certain exemplary embodiments are depicted and described herein, the compounds of this disclosure, or any variations or embodiments thereof, can be prepared using suitable starting materials according to the methods generally described herein and / or methods available to those skilled in the art.

[0353] When the following general procedure shows a specified stereoisomer, or an unspecified stereoisomer, or a mixture of stereoisomers, it should be understood that a similar chemical transformation can be performed on other specific stereoisomers, or unspecified stereoisomers, or mixtures thereof.

[0354] When a particular reagent or solvent is specified for a reaction in a general procedure, a skilled technician will recognize that other reagents or solvents may be substituted as needed.

[0355] Example The synthetic reaction schemes detailed in the examples are merely illustrative of some methods by which the disclosed compounds or embodiments or aspects thereof can be synthesized. As will be apparent to those skilled in the art, various modifications can be made to these synthetic reaction schemes.

[0356] If necessary, conventional techniques, including but not limited to filtration, distillation, crystallization, and chromatography, can be used to separate and purify the starting materials and intermediates of the synthetic reaction. Conventional methods (including physical constants and spectroscopic data) can be used to characterize these materials.

[0357] Although certain exemplary embodiments are depicted and described herein, the compounds of this disclosure or any variations or embodiments thereof may be prepared using appropriate starting materials in accordance with the methods generally described herein and / or methods available to those skilled in the art.

[0358] All publications, patent applications, patents and other references mentioned in this article are explicitly incorporated in their entirety by reference, as if each reference were incorporated individually by reference.

[0359] Example 1: Synthesis and Characterization of the Conjugates of this Disclosure Materials and methods Materials and reagents. Naloxone hydrochloride (#N285000) was purchased from Toronto Research Chemicals, Toronto, Ontario. Nalmefen hydrochloride (#408550) was purchased from Tocris Bioscience™. Naloxone-D5 100 µg / mL (#N-063) was purchased from Cerilliant. Morphine sulfate (CII) (#M1167) and fentanyl citrate (CII) (#F1147) were purchased from Spectrum Chemical Manufacturing Corporation. Digoxin saponins (#D0540) and loperamide hydrochloride (#L0154) were purchased from Fisher Scientific. Dodecyl azide (#A1887) was purchased from Synthonix. Prealbumin from human plasma (human TTR) (#P1742) and prealbumin from human serum (#H4522) were purchased from Sigma-Aldrich. Innovative-grade US-derived rat plasma (#IGRTSDPLANAH), rabbit plasma (#IGRBPLANAH), cynomolgus monkey plasma (#IGMNCYPLANAH), and mixed human plasma (#IPLAWBNAH) were purchased from Innovative Research. XTreme200 mixed human liver microsomes (#1910096), male IGS SD rat liver microsomes (#2210274), male cynomolgus monkey liver microsomes (#2210248), and the NADPH RapidStart regeneration system for prolonging metabolism (#K5100-5) were purchased from Xenotech. Human carboxylesterase 1 (CES1, #E0287-1VL) and carboxylesterase 2 (CES2, #C4749) were purchased from Sigma-Aldrich. RPMI-1640 medium and minimum limit Eagle's medium (HyClone, Utah, USA), fetal bovine serum (Gemini), penicillin / streptomycin (100 units / mL and 100 µg / mL (Gibco, NY, USA) and L-glutamine (Glutamax-100X, #35-050-061, Gibco, NY, USA) were purchased from the companies mentioned in parentheses. CellBIND® 96-well clear plates (#CLS3340-50EA) were purchased from Corning®. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide) of the test compounds in the cell lines was determined using the CellTiter 96 non-radioactive cell proliferation assay kit (#G4000, Promega, WI, USA).

[0360] Chemical synthesis and HPLC purity analysis General chemical procedure. Unless otherwise stated, all reactions were carried out under an argon or nitrogen atmosphere, using dry solvents under anhydrous conditions. Solvents used were ACS grade from Fisher. Unless otherwise indicated, yields refer to chromatographic and spectroscopic results. 1 H NMR and 13 Homogeneous material (C10 NMR). Reagents were purchased from Aldrich and Fisher and used directly without further purification. UV light was used, with the iodine chamber as the visualization agent, through a fluorescent indicator F10 ... 254 Thin-layer chromatography (TLC) was performed on an EMD Millipore® silica gel 60 plastic TLC plate (#1.05735.0001) to monitor the reaction. Normal-phase rapid column chromatography was performed using a Combi Flash® Rf+Lumen instrument (Teledyne ISCO) with a high-efficiency silica rapid column (RediSep® Rf+ Gold), and preparative thin-layer chromatography (PTLC) separations were performed on an Analtech® 2 mm (60F-254) (#P02015) spectrometer. Recordings were performed on a JEOL JNM-ECA 600 spectrometer. 1 H NMR and 13 C10 NMR spectroscopy was performed, calibrated using residual undeuterated solvent as an internal reference. High-resolution mass spectrometry (HRMS) was performed at the UCBerkley mass spectrometry facility using electrospray ionization from the solution. Coupling constants (J) are expressed in Hertz.

[0361] A preparative HPLC method was used to purify the synthesized compounds. Purification was performed using Waters Chromesope V2.1 software on a Waters 150Q LC system with a PDA and a FlexInject system connected to a photodiode array detector operating in the UV range of 190–800 nm. After injecting 5 mL of each sample, HPLC analysis was performed at ambient temperature on a SunFire® Prep C18 OBD column (19 x 250 mm, 5 μm) to obtain chromatograms at a UV absorbance of 310 nm. The mobile phase consisted of solvent A and solvent B, and was injected at 20 mL / min. -1 The flow rate was determined by solvent A, which consisted of methanol-water (5:95, v / v) containing 0.1% trifluoroacetic acid, and solvent B, which consisted of methanol-water (95:5, v / v) containing 0.1% trifluoroacetic acid. The HPLC program was an isocratic gradient of 0% solvent B for the first 4 minutes, followed by a linear increase from 0% to 100% solvent B over 4–20 minutes, then isocratic elution with 100% solvent B up to 24 minutes, followed by a linear decrease to 0% B at 26 minutes, and then a hold at 0% solvent B for 4 minutes.

[0362] An analytical HPLC method was used to evaluate the purity of synthesized compounds. Detailed HPLC information (trace, retention time, and purity %) for key compounds is included below. Purity analysis (all compounds >95%) of key compounds was performed using a C18 reversed-phase HPLC column on an Arc Premier Waters modular HPLC system connected to a Waters 2998 photodiode array detector operating in the UV range of 190–800 nm, and quantification was performed using Empower 3 software. After injecting 20 μL of each sample, the solution was precipitated at ambient temperature at 1 mL / min. -1 Elution was performed by HPLC on a Waters™ XBridge C18 column (4.6 x 250 mm, 5 μm) to obtain chromatograms at 310 nm UV absorbance. The mobile phase consisted of solvent A (5:95, v / v) of methanol-water containing 0.1% ammonium hydroxide and solvent B (95:5, v / v) of methanol-water containing 0.1% ammonium hydroxide. The HPLC program for the C18 column was a gradient method: linear increase from 0% to 100% solvent B over 0–20 min, followed by isocratic elution with 100% solvent B up to 25 min, decreasing to 0% B at 27 min, and holding at 0% B for 3 min.

[0363] General Procedure A. Preparation of Naloxone Free Base.

[0364] At ambient temperature, a tertiary amine base, N,N-diisopropylethylamine (1.25 equivalents), is added to a solution of naloxone hydrochloride (1 equivalent) in dichloromethane. After stirring for a short time (e.g., 5-10 minutes), the solvent is removed under reduced pressure to obtain the corresponding free base of naloxone.

[0365] General Procedure B. Ester Synthesis Under an inert atmosphere (e.g., N2 or argon), the selected drug (D) is... 1 The selected carboxylic acid (1.2 equivalents) and a catalytic amount of acylation catalyst (e.g., 4-dimethylaminopyridine, 0.2 equivalents) dissolved in dichloromethane are added. The reaction mixture is stirred briefly to allow pre-activation, after which a coupling agent, such as N,N'-dicyclohexylcarbodiimide (approximately 1 equivalent), is added. The reaction mixture is stirred at ambient temperature for an extended period (e.g., 12–24 hours) to ensure complete conversion. The mixture is then filtered and the solvent is removed under reduced pressure. The crude product is purified using chromatographic techniques (e.g., rapid silica gel column chromatography using an appropriate gradient of ethyl acetate and hexane) to obtain the target ester. The structure and purity of the product are confirmed using analytical methods such as nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS).

[0366] General procedure C. Copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC).

[0367] Click (CuAAC) coupling was performed by coupling naloxone-alkyne / drug-alkyne (1.2 equivalents), azide-connector-AG10 / azide-connector (1 equivalent), CuSO4·5H2O (0.5 equivalents), and sodium ascorbate (1.2 equivalents) in a mixture of tetrahydrofuran / water (2:1). The reaction mixture was stirred overnight at room temperature. Tetrahydrofuran and water were removed under reduced pressure. The residue was purified by silica gel chromatography or dissolved in methanol and purified by preparative reversed-phase HPLC to obtain the desired product.

[0368] General procedure D. Removal of the Boc protecting group The Boc-protected conjugate and precursor were treated with a mixture of dichloromethane and trifluoroacetic acid (3:1). The reaction mixture was stirred at room temperature under an inert atmosphere for 2 hours. Then, ammonium hydroxide (28%–36%) dissolved in water was slowly added dropwise along with methanol. This solution was then concentrated under reduced pressure and purified by preparative reversed-phase HPLC to obtain the desired product.

[0369] Synthesis of AG10-L-E2-naloxone Scheme 1 shows a schematic diagram of the synthesis of AG10-L-E2-naloxone.

[0370] Scheme 1. Synthesis of AG10-L-E2-naloxone.

[0371] Ethyl 4-((tert-butoxycarbonyl)(prop-2-yn-1-yl)amino)benzoate (1). At room temperature under a nitrogen atmosphere, triethylamine (1.2 g, 1.64 mL, density 0.73 g / mL) was added to a solution of ethyl 4-(prop-2-yn-1-yl)benzoate (2 g, 9.84 mmol, 1 equivalent), di-tert-butyl dicarbonate (2.6 g, 11.9 mmol, 1.2 equivalent), and 4-dimethylaminopyridine (0.24 g, 1.96 mmol, 0.2 equivalent) in anhydrous tetrahydrofuran (12 mL). -1 (11.86 mmol, 1.2 equivalents). The reaction mixture was stirred overnight at 80 °C. The reaction mixture was cooled to room temperature, and then formic acid (906 mg, 0.742 mL, density 1.22 g / mL) in 3 mL of ethyl acetate was added at room temperature. -119.68 mmol (2 equivalents) was added dropwise to a stirred reaction vessel. The solvent and excess formic acid were then dried under reduced pressure. The remaining residue was dissolved in ethyl acetate (30 mL), filtered, and the solvent was removed under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 0%-100% ethyl acetate / hexane) to give compound 1 (2.76 g, 92.4% separation yield) as a clear yellow viscous liquid. 1 H NMR (600 MHz, CDCl3) δ 7.98 (d, 2H, J = 9), 7.38 (d, 2H, J = 9), 4.39 (s, 2H), 4.33 (q, 2H, J =7.2), 2.24 (s, 1H), 1.44 (s, 9H), 1.35 (t, 3H, J = 7.2); 13 C-NMR (150 MHz, CDCl3)δ 166.12, 153.39, 146.16, 130.16, 127.75, 125.23, 100.00, 81.83, 79.64,72.22, 61.02, 39.49, 28.29, 14.40. HRMS (DART) m / z: C 17 H 21 NO4[M+Na] + The calculated value is 326.1363; the measured value is 326.1364.

[0372] (4aS,7aR,12bS)-4-((tert-butoxycarbonyl)(prop-2-yn-1-yl)amino)benzoic acid 3-allyl-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methylbridged benzofurano[3,2-e]isoquinoline-9-yl ester (2). Sodium hydroxide (729 mg, 18.2 mol, 2 equivalents) in water (3.75 mL) was added to a stirred solution of 1 (2.76 g, 9.1 mmol, 1 equivalent) in methanol (7.5 mL) at room temperature. This reaction mixture was stirred overnight at 50 °C, followed by drying of the water and methanol solvent under reduced pressure. The remaining residue was treated with a solution containing formic acid (2.44 g, 2 mL, density 1.22 g / mL). -1The acetic acid was redissolved in ethyl acetate (30 mL) at 53 mmol, filtered, and the solvent was removed under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 0%–100% ethyl acetate / hexane) to give a white powder of the acid hydrolysis product of 1 (2.46 g), which was used directly in the next step. N,N-diisopropylethylamine (0.88 g, 1.2 mL, density 0.742 g / mL) was added to a solution of naloxone hydrochloride (1.32 g, 3.63 mmol, 1 equivalent) in CH2Cl2 (30 mL) at room temperature. -1 4.5 g (1.25 mmol, equivalent) and stirred for five minutes. Then, CH2Cl2 was removed under reduced pressure, leaving a dry residue. At room temperature under a nitrogen atmosphere, a solution of the acid hydrolysis product of 1 (1.2 g, 4.36 mmol, 1.2 mmol) and 4-dimethylaminopyridine (89 mg, 0.72 mmol, 0.2 mmol) dissolved in 30 mL of CH2Cl2 was added to this residue. The reaction mixture was stirred for five minutes, and then, under a nitrogen atmosphere, N-N'-dicyclohexylcarbodiimide (0.75 g, 3.63 mL, 3.63 mmol, 1 mmol) was added to a 1 M solution of anhydrous CH2Cl2. The reaction mixture was stirred at room temperature for 48 hours, then filtered, and excess CH2Cl2 was removed under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 0%-100% ethyl acetate / hexane) to give compound 2 (1.5 g, 70% separation yield) as a white powder. 1 H NMR (600 MHz, CDCl3) δ 8.16 (d, 2H, J = 7.8), 7.44 (d, 2H, J =8.4), 6.93 (d, 1H, J = 8.4), 6.71 (d, 1H, J = 8.4), 5.83-5.75 (m, 1H), 5.28-5.15(m, 2H), 4.68 (s,1H), 4.41 (s,2H), 3.20-3.12 (m, 3H), 3.04-2.96 (m, 2H),2.66-2.57 (m, 2H), 2.43-2.36 (m, 1H), 2.31-2.24 (m, 2H), 2.19-2.11 (m, 1H), 1.88-1.82 (m, 1H), 1.67-1.59 (m, 2H), 1.46 (s, 9H); 13C-NMR (150 MHz, CDCl3) δ207.82, 163.73, 153.47, 148.11, 146.93, 135.19, 132.86, 131.27, 130.52,130.34, 126.30, 125.43, 123.20, 119.63, 118.44, 100.00, 90.78, 82.08, 79.62,72.59, 70.24, 62.06, 57.70, 50.80, 43.38, 39.51, 36.25, 31.34, 30.74, 28.42, 23.13; HRMS (DART) m / z: C 34 H 36 N₂O₇[M+H] + The calculated value is 585.2595; the measured value is 585.2593.

[0373] 3-((6-((2-(2-(2-(4-(((4-((((4aS,7aR,12bS)-3-allyl-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methylbridged benzofurano[3,2-e]isoquinoline-9-yl)oxy)carbonyl)phenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)amino)hexyl)oxy)-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid (AG10-L-E2-naloxone). Click (CuAAC) coupling was performed by coupling compound 2 (561 mg, 0.96 mmol, 1.2 equivalents), compound 3 (515 mg, 0.8 mmol, 1 equivalent; synthesized as previously reported), CuSO4·5H2O (100 mg, 0.4 mmol, 0.5 equivalents), and sodium ascorbate (190 mg, 0.96 mmol, 1.2 equivalents) in a) 9 mL of a mixture of tetrahydrofuran / water (2:1). The reaction mixture was stirred overnight at room temperature. Tetrahydrofuran and water were removed under reduced pressure. The residue was dissolved in water and extracted with CH2Cl2. The organic extract was concentrated under reduced pressure and purified by rapid column chromatography (silica gel, 0%–100% CH2Cl2 / methanol) to give a di-Boc protected AG10-L-E2-naloxone intermediate, which was directly treated with a mixture of 4 mL of CH2Cl2 / trifluoroacetic acid (3:1). The reaction mixture was stirred at room temperature under an inert atmosphere for 2 hours. Then, ammonium hydroxide dissolved in water (28%-36%) was slowly added dropwise with 3 mL of methanol until no hydrogen formation was observed. This solution was then concentrated under reduced pressure and purified by preparative reversed-phase HPLC to obtain AG10-L-E2-naloxone as a viscous solid (633 mg, 64% separation yield). 1H NMR (600MHz, CD3OD) δ 7.84-7.79 (m, 3H), 7.03-6.99 (m, 2H), 6.95-6.87 (m, 1H), 6.84-6.76 (m, 1H), 6.65-6.60 (m, 2H), 6.59-6.54 (m, 1H), 5.90-5.77 (m, 1H), 5.64-5.49 (m, 2H), 4.80 (s, 1H), 4.48-4.42 (m, 1H), 4.40-4.37 (m, 1H), 3.94-3.80(m, 6H), 3.79-3.72 (m, 2H), 3.56-3.34 (m, 6H), 3.23 (s, 1H), 3.1-2.96 (m,4H), 2.92-2.82 (m, 3H), 2.75-2.59 (m, 2H), 2.59-2.5 (m, 3H), 2.16 (s, 6H),1.94-1.27 (m, 16H); 13 C-NMR (150 MHz, CD3OD) δ 207.09, 167.44, 163.90, 160.28,160.22, 153.67, 148.33, 145.07, 133.37, 133.27, 132.33, 128.84, 128.72,128.17, 125.69, 124.44, 123.78, 120.62, 114.76, 111.96, 107.85, 107.80,106.17, 89.73, 89.69, 70.17, 70.07, 69.88, 69.25, 68.15, 67.12, 66.13, 61.90,55.64, 52.98, 49.85, 48.99, 47.38, 46.69, 46.10, 38.21, 35.16, 30.86, 29.64,28.85, 27.72, 26.15, 25.76, 25.49, 23.38, 18.86, 10.62; HRMS (DART) m / z: C 56 H 70 N8O 11 [M+H] + The calculated value is 1031.5237; the measured value is 1031.5241.

[0374] Synthesis of AG10-L-E2-COOH Scheme 2 shows a schematic diagram of the synthesis of AG10-L-E2-COOH.

[0375] Scheme 2. Synthesis of AG10-L-E2-COOH.

[0376] 3-((6-((2-(2-(2-(4-((((4-carboxyphenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)amino)hexyl)oxy)-5-(3-(3,5-dimethyl-1H-pyrazole-4-yl)propoxy)benzoic acid (AG10-L-E2-COOH). Sodium hydroxide (77.6 mg, 1.94 mmol, 2 equivalents) in water (1 mL) was added to a solution of AG10-L-E2-naloxone (1000 mg, 0.97 mmol, 1 equivalent) in methanol (4 mL). The solution was stirred overnight at 50 °C. The solvent was then removed under reduced pressure. The remaining residue was purified by reversed-phase HPLC to give AG10-L-E2-COOH (420 mg, 60% separation yield); HRMS (DART). m / z: C 37 H 51 N7O8[M+H] + The calculated value is 722.3872; the measured value is 722.3869.

[0377] Synthesis of C12-L-E2-naloxone Scheme 3 shows a schematic diagram of the synthesis of AG10-L-E2-COOH.

[0378] Scheme 3. Synthesis of C12-E2-naloxone.

[0379] (4aS,7aR,12bS)-4-(((1-dodecyl-1H-1,2,3-triazol-4-yl)methyl)amino)benzoic acid 3-allyl-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methylbridged benzofurano[3,2-e]isoquinoline-9-yl ester (C12-E2-naloxone). Click (CuAAC) coupling was performed by reacting compound 2 (500 mg, 0.86 mmol, 1 equivalent), dodecyl azide (182 mg, 0.85 mmol, 1 equivalent), CuSO4·5H2O (106.7 mg, 0.425 mmol, 0.5 equivalent), and sodium ascorbate (169.4 mg, 0.855 mmol, 1 equivalent) in a mixture of 3 mL THF / H2O (4:1). The reaction mixture was stirred overnight at room temperature. The solution was concentrated under reduced pressure. The residue was extracted with ethyl acetate. The organic extract was concentrated under reduced pressure to give an intermediate, which was used directly in the next step. Then, a mixture (1 mL) containing TFA and CH2Cl2 (1:4 ratio) was added to the solution of the intermediate, and the reaction mixture was stirred at room temperature for 2 hours. The solution was concentrated under reduced pressure and purified by preparative HPLC to give compound C12-E2-naloxone (255 mg, 43% yield after two steps). 1 H NMR (600 MHz, CD3OD) δ 7.87-7.78 (m, 3H), 6.94 (t, 1H, J= 8.4), 6.85-6.78 (m, 1H), 6.61 (s, 2H), 5.91-5.79 (m, 1H), 5.64-5.52 (m,2H), 4.82 (s, 1H), 4.58-4.34 (m, 1H), 4.27 (t, 2H, J = 7.2), 3.92-3.66 (m,2H), 3.57-3.32 (m, 2H), 3.17-2.50 (m, 3H), 2.21-1.46 (m, 9H), 1.26-1.06 (m, 18H), 0.79 (t, 3H, J = 7.2); 13C-NMR (150 MHz, CD3OD) δ 206.56, 164.97, 148.36,133.63, 133.04, 131.95, 128.21, 127.65, 126.50, 125.52, 125.30, 124.27,123.05, 120.21, 119.64, 111.58, 97.09, 93.49, 89.43, 69.73, 62.55, 55.47,50.14, 49.02, 46.31, 33.42, 31.73, 29.89, 29.39, 29.19, 28.69, 27.59, 26.97,26.08, 25.41, 24.73, 22.93, 22.40, 13.12; HRMS (DART) m / z: C 41 H 53 N5O5[M+H] + The calculated value is 696.4119; the measured value is 696.4121.

[0380] Synthesis of AG10-L-E1-naloxone Scheme 4 shows a schematic diagram of the synthesis of AG10-L-E1-naloxone.

[0381] Scheme 4. Synthesis of AG10-L-E1-naloxone.

[0382] 3-((6-((2-(2-(2-(4-(3-(((4aS,7aR,12bS)-3-allyl-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methylbridged benzofurano[3,2-e]isoquinoline-9-yl)oxy)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)amino)hexyl)oxy)-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid (AG10-L-E1-naloxone). N,N-diisopropylethylamine (0.24 mL, 1.374 mmol, 1 equivalent) was added to a solution of naloxone hydrochloride (500 mg, 1.374 mmol, 1 equivalent) in CH₂Cl₂ (4 mL) at room temperature and stirred for 10 min. The solution was concentrated under reduced pressure to give naloxone, which was used directly in the next step. 4-pentyneic acid (134.8 mg, 1.374 mmol, 1 equivalent) and DMAP (33.6 mg, 0.275 mmol, 0.2 equivalent) were added to naloxone containing CH₂Cl₂ (3 mL) under an argon atmosphere. Then, DCC (0.46 mL, 2.748 mmol, 2 equivalent) was added to the solution under an argon atmosphere and the reaction mixture was stirred overnight at room temperature. The reaction mixture was purified by rapid column chromatography (silica gel, 0%–100% ethyl acetate / hexane) and the intermediate was used directly in the next step. Click (CuAAC) coupling was performed by coupling the residue with deBoc compound 3 (300 mg, 0.549 mmol, 1 equivalent; previously reported), CuSO4·5H2O (68.5 mg, 0.2745 mmol, 0.5 equivalent), and sodium ascorbate (108.8 mg, 0.549 mmol, 1 equivalent) in a mixture of 3 mL THF / H2O (4:1). The reaction mixture was stirred overnight at room temperature. The solution was concentrated under reduced pressure and purified by preparative HPLC to give compound AG10-L-E1-naloxone (250 mg, 47% yield after two steps). 1H NMR (600 MHz, DMSO-d6) δ 7.95 (s, 1H), 7.05-6.95 (m, 3H), 6.84 (d, 1H, J = 8.4), 6.72-6.68(m, 1H), 5.95-5.87 (m, 1H), 5.62-5.54 (m, 2H), 5.11 (s, 1H), 4.52-4.46 (m,2H), 4.03-3.91 (m, 5H), 3.86-3.76 (m, 3H), 3.65 (d, 1H, J = 6), 3.64-3.61 (m,2H), 3.59-3.49 (m, 5H), 3.24-3.16 (m, 1H), 3.12-3.04 (m, 3H), 3.04-2.80 (m,7H), 2.70-2.56 (m, 1H), 2.5 (s, 1H), 2.29-2.08 (m, 8H), 2.06-1.91 (m, 1H),1.87-1.80 (m, 2H), 1.75-1.66 (m, 2H), 1.65-1.57 (m, 2H), 1.55-1.15 (m, 7H); 13 C-NMR (150 MHz, DMSO-d6) δ 206.93, 170.46, 167.45, 160.28, 158.82, 158.60,147.92, 145.46, 133.38, 132.69, 129.15, 128.96, 128.14, 125.67, 124.18,123.07, 120.70, 107.81, 106.19, 89.91, 70.13, 70.07, 69.91, 69.28, 68.15,67.10, 66.14, 61.85, 55.63, 55.44, 49.81, 49.03, 47.38, 46.65, 46.06, 35.16,33.41, 30.88, 29.49, 28.86, 27.79, 26.15, 25.81, 25.49, 23.38, 21.18, 18.73,10.42. HRMS (DART) m / z: C 51 H 67 N7O 11 [M+H] + The calculated value is 954.4971; the measured value is 954.4971.

[0383] Synthesis of AG10-short-L-E2-naloxone According to the general procedure B, use and Then, according to the general procedure, C is used. Then, AG10-short-L-E2-naloxone was prepared according to the general procedure D.

[0384] Synthesis of AG10-L-PHBA-E-naloxone According to the general procedure B, use and Then, according to the general procedure, C is used. Then, AG10-L-PHBA-E-naloxone was prepared according to the general procedure D.

[0385] Synthesis of AG10-L-m-amino-L-naloxone According to the general procedure B, use and Then, according to the general procedure, C is used. Preparation of AG10-L-m-amino-L-naloxone.

[0386] Synthesis of PEG-connector-E2-naloxone According to the general procedure B, use and Then, according to the general procedure, C is used. Then, PEG-E2-naloxone was prepared according to the general procedure D.

[0387] Analytical HPLC method. The test compounds were analyzed using a Waters™ XBridge C18 column (4.6 x 250 mm, 5 μm) on an Arc PremierWaters modular HPLC system connected to a Waters 2998 photodiode array detector operating in the UV range of 190–800 nm. HPLC analysis was performed using a gradient method with linear increases in solvent B from 0% to 100% over 25 minutes. The mobile phase consisted of solvent A (5:95, v / v) of methanol-water containing 0.1% ammonium hydroxide and solvent B (95:5, v / v) of methanol-water containing 0.1% ammonium hydroxide, at a flow rate of 1.0 mL / min. -1 .

[0388] Solubility of the compound in potassium phosphate buffer (pH 7.4). A stock solution (10 mM) of the test compound was prepared in DMSO and stored at -20 °C. The compound (200 µM) was incubated in 50 mM potassium phosphate buffer (pH 7.4) at 37 °C for 72 h. The sample was vortexed, centrifuged at 12,000 rpm for 5 min, and the supernatant was filtered. Aliquots (100 µL) of the sample were then taken and quenched by adding 200 µL of 0.1% formic acid in methanol. The mixture was centrifuged again at 12,000 rpm for 5 min. The clarified supernatant was then analyzed by analytical HPLC as described. Calibration curves of the prodrug were generated by dissolving the DMSO solution of the test compound in 0.1% formic acid in methanol (concentrations of 200, 100, 50, 25, 12.5, and 6.25 µM) and then analyzing by HPLC. The concentration of the soluble test compound in potassium phosphate buffer was quantified using the calibration curves. Data is expressed as mean ± sd ( n = 3 independent replicates) and show representative HPLC traces.

[0389] Using shake bottle D 7.4 Determination. Aqueous potassium phosphate buffer (10 mM, pH 7.4) solutions were prepared and then saturated with n-octanol. Similarly, n-octanol was saturated with aqueous potassium phosphate buffer at pH 7.4. Both solutions were vigorously vortexed and then allowed to stand to ensure complete separation of the two phases. Shake-flask experiments were performed at room temperature. Once each phase was prepared, 1 mL of 1-octanol and 1 mL of aqueous potassium phosphate buffer were added to a glass vial. 20 µL of the test compound stock solution (10 mM stock solution in DMSO; final concentration 100 µM in the pre-saturated mixture) was added and the vial was shaken at room temperature for one hour. After one hour, the layers were separated and each aliquot (100 µL) of the n-octanol and aqueous buffer layers was treated by adding 200 µL of 0.1% formic acid in methanol. The mixture was centrifuged again at 12,000 rpm for 5 min. The clarified supernatant was then analyzed by analytical HPLC as described. The concentration of the test compound was quantified using a calibration curve. Data represent mean ± SD ( n = 3 independent repetitions).

[0390] Calculate Log using the following equation. D 7.4 .

[0391] Example 2: Evaluation of the binding affinity of the conjugate The binding affinity of the compound to TTR in buffer was evaluated. Fluorescence polarization (FP) assays were used to determine the ability of AG10-L, AG10-L-E2-COOH, AG10-L-E2-naloxone, and AG10-L-E1-naloxone to displace the FP probe from TTR in buffer. Serial dilutions of the compound (0.010 µM to 20 µM) were added to 384-well plates containing the FP probe (50 nM) and TTR (300 nM) in assay buffer (PBS pH 7.4, 0.01% Triton X100, 1% DMSO in a final volume of 25 μL). The samples were equilibrated by stirring on a plate shaker for 20 min at room temperature. Fluorescence polarization (excitation λ 485 nm, emission λ 525 nm, cutoff λ 515 nm) measurements were performed using a SpectraMax M5 microplate reader (Molecular Devices). Fluorescence data were collected using SoftMax® Pro software v5.4.1 (Molecular Devices, Inc.). IC50 values ​​were obtained by fitting the data to the following equation [y = (AD) / (1 +(x / C)^B) + D], where A = maximum FP signal, B = slope, and C = apparent binding constant (…). K app ), and D = minimum FP signal. Using the Proussov equation, from IC 50 Value calculation combined with constant ( K d Value. Data represents the average ± sd ( n = 3 independent repetitions).

[0392] The binding affinity and selectivity of the test compounds for TTR in human serum were evaluated. The binding affinity and selectivity of AG10-L, AG10-L-E2-COOH, AG10-L-E2-naloxone, and AG10-L-E1-naloxone to TTR in human serum were determined by their ability to compete with fluorescent probe exclusion (FPE) probes for binding to TTR. AG10 and tafamidis were used as controls. An aliquot of human serum (98 μL) was mixed with 1 μL of the test compound (1.0 mM stock solution in DMSO; final concentration in serum: 10 μM) and 1 μL of the FPE probe (0.36 mM stock solution in DMSO; final concentration in serum: 3.6 μM). Fluorescence changes (λ) were monitored every 15 minutes at 25 °C using a SpectraMax M5 microplate reader. ex = 328 nm and λ em= 384 nm), lasting for 6 hours. Data represent mean ± sd ( n = 4 independent repetitions).

[0393] Results: Fluorescence polarization (FP) was used to determine (…). K d = 394.3 nM and 258.8 nM respectively; Figure 1c The binding affinity of the prodrugs AG10-L-E1-naloxone and AG10-L-E2-naloxone was evaluated. This binding affinity is related to the binding affinity of AG10-L and AG10-L-E2-COOH (…). K d The binding affinities (262.9 nM and 213 nM, respectively) were similar. We then evaluated the selectivity of the prodrugs for binding to TTR relative to >4,000 other human serum proteins using a well-developed TTR serum fluorescent probe exclusion (FPE) selectivity assay. Our data showed that all tested compounds maintained excellent binding selectivity for TTR in human serum (approximately 50% to 70% TTR occupancy). Figure 3d , Figure 3e The performance of AG10-L-E2-COOH (48.1% TTR occupancy) is similar to that of the TTR stabilizer chlorpromazine (an approved drug for TTR amyloidosis; 49.2% TTR occupancy).

[0394] Example 3: Stability Analysis Stability analysis of the test compound in human and rat plasma. The test compound (5 μM) was incubated in human or rat plasma at 37 °C. 100 μL plasma samples were analyzed at 0 h, 0.25 h, 2 h, 4 h, and 24 h. All samples were processed by adding 200 μL of quenching buffer (0.1% formic acid in methanol) followed by centrifugation at 15,000 rpm for 5 min. The supernatant was incubated at -20 °C for 5 min and centrifuged again at 15,000 rpm for 5 min. The supernatant was analyzed by analytical HPLC as described. Data are expressed as mean ± SD. n = 3 independent repetitions).

[0395] Results: We first evaluated the stability of the prodrug in human plasma. AG10-L-E1-naloxone was unstable (0% remained after 1 minute of serum incubation). Figure 1fThis aligns with the instability of alkyl-based ester prodrugs. In contrast, PABA-based esters (AG10-L-E2-naloxone and C12-E2-naloxone) are completely protected from hydrolysis in human plasma. While aryl-based esters are more stable than their alkyl counterparts, we are still surprised by the complete stability of these prodrugs in human plasma. Our modeling studies show that the linker we used is long enough to extend the PABA ester in the TTR T4 binding bag, which in principle would allow plasma esterases to target our prodrug, even if it is bound to the TTR. Figure 2b To investigate these observations, we evaluated the stability of the prodrug in rat serum. As expected, the alkyl ester prodrug (AG10-L-E1-naloxone) was hydrolyzed most rapidly (0% remaining after 1 minute of serum incubation). Figure 1g The PABA ester AG10-L-E2-naloxone exhibited higher stability (60% remaining after 15 minutes and 3.8% after 2 hours). The hydrolysis rate of C12-E2-naloxone (34.2% remaining after 15 minutes and 0% after 2 hours) was slightly faster than that of AG10-L1-E2-naloxone. The difference in prodrug hydrolysis between human and rat plasma points to carboxylesterases 1 and 2 (CES1 and CES2), which are present in rat plasma but not in human plasma.

[0396] Docking with TTR and CES2. Computer simulations of mouse Ces2c docking with AG10-L-E2-naloxone were performed using Dock6. Crystal structures of TTR (pdb id: 4HIQ) and Ces2c (pdb id: 8AXC) were obtained from the RCSB.org protein database. The geometry of AG10-L-E2-naloxone was optimized at the B3LYP / 6-311G* level using Gaussian09. For docking with TTR and Ces2c, AG10-L-E2-naloxone was split into two: AG10-connector and naloxone-connector. Geometry optimization of both ligands with shorter connectors was performed at the same B3LYP / 6-311G* level. Molden was used to analyze ligand geometry and prepare ligand files for docking. The UCSF Chimer program was used for receptor docking preparation and analysis of docking results. AG10-L A and L B -E2-naloxone was docked to TTR and Ces2c, respectively. TTR--- AG10-L A And Ces2c--- L B A docking complex of -E2-naloxone was stacked on an optimized structure of AG10-L-E2-naloxone to observe the possibility of forming a ternary complex of TTR-AG10-L-E2-naloxone-Ces2c.

[0397] Example 4: Stability of the conjugate to carboxylesterase hydrolysis Materials and methods Metabolic studies of AG10-L-E2-naloxone in recombinant human carboxylesterase. CES1 (30 µg / mL) was prepared from 50 mM potassium phosphate buffer. -1 Or 100 µg mL -1 ) or CES2 (30 µg mL -1 The reaction mixture was incubated at 30°C for 5 minutes. The reaction was initiated by adding 5 µM AG10-L-E2-naloxone (or DMSO as a negative control). After incubation, 100 μL of sample from the reaction mixture was quenched at 0 h and 1 h by adding 100 μL of quenching buffer (0.1% formic acid in methanol) to the sample to terminate the reaction at each specified time point. The sample was then centrifuged at 12,000 rpm for 5 minutes at 4°C. The supernatant (150 μL) was further diluted by adding 75 μL of 0.1% formic acid aqueous solution. The sample was stored at -20°C until analyzed by HPLC as described above. Data represent mean ± SD. n = 3 (Self-independent repetition).

[0398] Microsomal stability assay. 2 µL of AG10-L-E2-naloxone stock solution (1 mM in DMSO) was added to a microcentrifuge tube containing 3.2 µL acetonitrile and 194.8 µL potassium phosphate buffer (50 mM, pH 7.4, containing 3 mM MgCl2). The tube was pre-incubated at 37°C for 10 min, followed by the addition of liver microsomes. Human, monkey, or rat liver microsomes and NADPH regeneration solution (NADP, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase; concentration 2 mg / mL) were then added. -1 And respectively 2 mM, 10 mM and 2 units mL -1Add to potassium phosphate buffer (water is used instead of NADPH in the negative control). Preheat the reaction mixture at 37°C for 10 minutes. NADPH is generated using the RapidSTART NADPH regeneration system (Xenotech) by adding 0.7 mL of high-purity water to the frozen component in the RapidStart vial, vortexing, and then adding activated RapidStart solution to the incubation. Add 200 µL of this NADPH regeneration solution to each pre-incubation assay tube containing 200 µL of AG10-L-E2-naloxone in phosphate buffer. The final reaction concentrations of AG10-L-E2-naloxone, microsomes, NADP, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase are 5 µM, 1 mg / mL, and 1 mg / mL, respectively. -1 1 mM, 5 mM and 1 unit mL -1 Incubate the vials at 37°C. At 0 and 1 hour, remove 100 µL aliquots and add 100 µL of quenching buffer (containing 15 ng / mL) to each aliquot. -1 The microsomal reaction of naloxone-D5 was terminated with 0.1% formic acid in methanol as an internal standard. The sample was centrifuged at 12,000 rpm for 5 min and the supernatant was further diluted (150 µL) with 75 µL of 0.1% formic acid aqueous solution. The sample was stored at -20 °C until analysis using a validated LC-MS / MS method with a triple quadrupole mass spectrometer (QQQ). Data are expressed as mean ± SD. n = 3 (Self-independent repetition).

[0399] Microsomal stability analysis was performed using loperamide or digitalis saponins. The microsomal assays described above were also performed in HLM in the presence of loperamide or digitalis saponins (final concentration 100 µM). Loperamide or digitalis saponins were added to the microsomal buffer before being added to the test compound.

[0400] Stability analysis of AG10-L-E2-naloxone in rat plasma in the presence of CES inhibitors. Rat plasma was pre-incubated for 5 min at room temperature with either the CES2 inhibitor loperamide (100 µM) or the CES1 inhibitor digitoxin (100 µM) or DMSO (negative control). Then, AG10-L-E2-naloxone (5 μM) was added to the incubation mixture. Samples were measured at 0 h, 0.5 h, and 1 h. All samples were processed by adding 200 μL of quenching buffer (0.1% formic acid in methanol) followed by centrifugation at 15,000 rpm for 5 min. The supernatant was incubated at -20 °C for 5 min and centrifuged again at 15,000 rpm for 5 min. The supernatant was analyzed by analytical HPLC as described. Data are expressed as mean ± SD. n = 3 independent repetitions).

[0401] Stability analysis of AG10-L-E2-naloxone in monkey and rabbit plasma. AG10-L-E2-naloxone (5 μM) was incubated in monkey or rabbit plasma at 37 °C. 100 μL of sample was analyzed at 0 h and 24 h. All samples were processed by adding 200 μL of quenching buffer (0.1% formic acid in methanol) followed by centrifugation at 15,000 rpm for 5 min. The supernatant was incubated at -20 °C for 5 min and centrifuged again at 15,000 rpm for 5 min. The supernatant was analyzed by analytical HPLC as described. Data are expressed as mean ± SD ( n = 3 independent repetitions).

[0402] result Carboxylesterases (CESs) play a crucial role in the hydrolytic inactivation of many clinically used ester drugs and the bioactivation of prodrugs. CES1 and CES2 are the two most prevalent forms of CES in humans. Human CES1 plays a major role in the bioactivation of many prodrugs, while CES2 plays a minor role. Significant species differences exist in the tissue distribution and catalytic activity of CESs, making human translation challenging. CES1 is highly expressed in the liver, while CES2 is mainly found in the small intestine, but is also present in the colon, kidneys, and liver (at protein levels 10-15 times lower than CES1).

[0403] We investigated the potential role of CES1 / CES2 in the bioactivation of AG10-L-E2-naloxone. C12-E2-naloxone is insoluble in buffer solution. Figure 1b This prevented us from evaluating its CES selectivity. Our in vitro data on purified human CES clearly demonstrate that AG10-L-E2-naloxone is a substrate with specificity for CES2. Figure 2c The CES2 specificity of AG10-L-E2-naloxone was entirely unexpected, as only a few prodrugs (such as procaine and irinotecan) were selectively bioactivated by CES2 (which is also bioactivated by CES1 and other plasma proteins). For example, procaine undergoes rapid hydrolysis by butyrylcholinesterase in human plasma in addition to hepatic CES2. In contrast, the bioactivation of irinotecan by CES2 was extremely slow (less than 2% of the dose was bioactivated by CES2). To further investigate CES2 specificity and evaluate potential human translation, we evaluated the metabolic bioactivation of AG10-L-E2-naloxone in liver microsomes from humans, rats, and monkeys. Figure 2d Human liver microsomes (HLM) efficiently produce naloxone from AG10-L-E2-naloxone. The selective CES2 inhibitor loperamide significantly reduced naloxone release, while the CES1 inhibitor digitalis saponin had no effect. The amount of naloxone released from AG10-L-E2-naloxone in rat liver microsomes (RLM) was lower than that formed in HLM, primarily due to higher metabolic activity (via CYP dealkylation) of naloxone released from RLM compared to HLM. Since CES enzyme expression is similar in monkeys and humans, we evaluated the bioactivation of AG10-L-E2-naloxone in monkey liver microsomes (MLM). The amount of naloxone released from MLM was similar to that from HLM, suggesting that monkeys can be a suitable animal model for evaluating AG10-L-E2-naloxone.

[0404] The specificity of CES2 for the bioactivation of AG10-L-E2-naloxone was further confirmed by measuring the plasma stability of CES1 and CES2 inhibitors in rats. Figure 2e We also confirmed our results by evaluating the stability of AG10-L-E2-naloxone in monkey plasma (plasma without CES1 or CES2; 100% stability after 24 hours) and rabbit plasma (plasma with high abundance of CES1 and CES2; 0% residual after 24 hours).

[0405] Although the crystal structure of human CES1 is available, the first crystal structure of mouse CES2 has recently been reported as a potential ortholog of human CES2. The overall structure of mouse CES2 is similar to that of human CES1, differing mainly in the proximity of the active site. We conducted docking studies of AG10-L-E2-naloxone with both human CES1 and mouse CES2. AG10-L-E2-naloxone did not bind to CES1. In contrast, AG10-L-E2-naloxone showed a highly compatible active site with CES2, and the nucleophilic serine (S230) is adjacent to the carbonyl group of the ester group in AG10-L-E2-naloxone. Figure 2bThis data represents the first docking experiment between a specific CES2 substrate and a CES2 enzyme. Insights from the binding mode and selectivity of CES2 to AG10-L-E2-naloxone may help in the design of additional CES2-specific prodrugs. The lower concentration of hepatic CES2 compared to CES1 provides unique CES2 bioactivation for AG10-L-E2-naloxone. This may be advantageous for controlling the slow and continuous release of naloxone from AG10-L-E2-naloxone (discussed below in the monkey study in Example 10).

[0406] Example 5: Subcutaneous administration of AG10-L-E2-naloxone to rats to prolong and control the release of naloxone. There was no dramatic increase in plasma and brain concentration. Materials and methods Evaluation of the subcutaneous and intravenous pharmacokinetic characteristics of AG10-L-E2-naloxone and naloxone in rats. Subcutaneous (sc) and intravenous (iv) pharmacokinetic (PK) studies were conducted using male Spriggdalen rats (225–250 g; 7–8 weeks old) cannulated from the Charles River. For the intravenous PK studies, each animal received a single intravenous bolus dose of either naloxone or AG10-L-E2-naloxone (60 µmol kg) in 500 µL of dosing solution (10% DMSO, 20% PEG, 70% H2O). -1 Equivalent to 20 mg kg -1 Naloxone was administered, followed by an injection of 200 µL of sterile saline to flush the jugular vein cannula (n = 3 rats per group). At predetermined time points (0.033, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post-administration), 200 µL of blood samples were collected from each rat via the jugular vein cannula into heparinized tubes, and the volume was replaced with sterile saline. For subcutaneous pk studies, 15 µmol / kg naloxone was administered to the nape of the neck. -1 or 60 µmol kg -1 AG10-L-E2-naloxone 15 µmol kg -1 or 60 µmol kg -1500 µL of the drug solution (per 250 g rat) was administered. In each of the four groups (n = 3 rats per group), blood samples were collected from each rat via jugular cannulation into heparinized tubes at predetermined time points (0.033, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, and 96 hours post-administration), and the same volume was replaced with sterile saline. Plasma samples were prepared directly after blood collection by centrifugation at 1,500 g for 10 min at 4 °C. Aliquots of 50 µL plasma samples were then precipitated with 2 volumes of extraction buffer (containing 15 ng / mL naloxone-D5 in 0.1% formic acid in methanol). The samples were vortexed for 30 seconds and then held at -20 °C for 5 min. The samples were then centrifuged at 15,000 rpm for 5 minutes; the supernatant was collected and kept at -20°C for 5 minutes, and then centrifuged again at 15,000 rpm for 5 minutes. On the same day of the study, the samples were analyzed by triple quadrupole mass spectrometry (QQQ) using LC-MS / MS to quantify the concentrations of naloxone and AG10-L-E2-naloxone in all groups.

[0407] Sample preparation and LC-MS / MS analysis of the experimental compounds. Sample preparation. Stock solutions of naloxone (20 mM) and AG10-L-E2-naloxone (10 mM) were prepared in DMSO solution. Working solutions were prepared by serial dilution of the DMSO solutions mentioned above. Calibration curve standards were prepared by incorporating aliquots (1 µL) of each working solution into 49 µL of blank rat plasma. Quality control (QC) samples prepared at low, medium, and high concentrations were extracted simultaneously with animal samples. Extraction was performed using 2 volumes of extraction buffer (containing 15 ng / mL). -1 Naloxone-D5 was precipitated in 50 µL aliquots of plasma using 0.1% formic acid in methanol. The corresponding double blank samples were extracted with extraction buffer (i.e., 0.1% formic acid in methanol) without an internal standard. Samples were processed in the same manner described above. To quantify naloxone, 20 µL of sample was injected for LC-MS / MS analysis. Analytes in plasma samples were quantified using a triple quadrupole mass spectrometer (QQQ). The mobile phase consisted of solvent A (0.1% formic acid aqueous solution containing 0.5 mM ammonium formate) and solvent B (0.1% formic acid in methanol), with a flow rate of 0.5 mL / min. -1LC-MS / MS analysis was performed on a Waters™ XBridge C18 (4.6 x 150 mm, 5 µm). The turbine spray ionization source was set to positive ionization mode. The test compounds were identified and quantified using fragmented patterns and peak areas, respectively. LC-MS / MS data were collected from Agilent Masshunter software. Naloxone-D5 was used as an internal standard. Gas flow rate, sprayer, sheath gas temperature, sheath gas flow rate, capillary voltage, nozzle voltage, and gas temperature were set to 7 l / min, 30 psi, 250 °C, 12 l / min, 3500 V, 500 V, and 300 °C, respectively. LC-MS / MS analysis was performed using the following gradient method: linearly increasing from 0% to 100% solvent B over 0–3 min, holding at 100% solvent B for 5.5 min, then decreasing from 100% to 0% solvent B over 0.5 min, and finally holding at 0% solvent B for 1 min. The retention times for naloxone and naloxone-D5 were 4.62 and 4.62 minutes, respectively. The total run time was 7 minutes. The mean ± sd concentration of naloxone was determined based on a calibration curve of naloxone and internal standard in plasma generated by an LC-MS / MS analyst. Samples were diluted when their concentrations exceeded the calibration curve range. The table below lists the detailed mass spectrometer conditions for multiple reaction monitoring (MRM) for each compound. The limit of quantitation (LLOQ) for naloxone was 1 nM. The linear range was calculated based on regression analysis between the analyte / internal standard peak area ratio and the analyte concentration. In each case, the accuracy was between 80% and 120%.

[0408] Evaluation of AG10-L-E2-naloxone and naloxone brain uptake in rats. This study was conducted using male Spriggdalen rats with jugular vein cannulation. Animals were randomly assigned to four treatment groups (each group... n = 3 (Only one animal). Each animal received a single subcutaneous dose of 15 µmol / kg. -1 Naloxone or AG10-L-E2-naloxone was administered in 500 µL of dosing solution (10% DMSO, 20% PEG400, and 70% sterile deionized water). 90 mg / kg was injected intraperitoneally 10 and 30 minutes after administration. -1 Ketamine and 9 mg kg -1 Thiaprazine was used to anesthetize rats. CSF samples were collected from the cerebellomedullary cistern using a 22-gauge needle. Blood was collected by phlebotomy from the aorta using a 20-gauge needle, followed by decapitation and brain collection. Sample preparation and LC-MS / MS analysis of experimental compoundsSample Preparation. Immediately freeze the brain in liquid nitrogen. Process and analyze the plasma samples in the same manner as plasma samples described in pharmacokinetic studies. Store CSF and brain samples at -80°C for further analysis. Prepare stock solutions of naloxone (20 mM) and AG10-E2-naloxone (10 mM) in DMSO solution. Prepare working solutions by serially diluting the DMSO solution mentioned above. Prepare calibration curve standards by incorporating aliquots (1 µL) of each working solution into 49 µL of artificial CSF (125 mM NaCl, 2.5 mM KCl, 1.26 mM CaCl2, and 1.18 mM MgCl2) or blank rat brain homogenate. Extract quality control (QC) samples prepared at low, medium, and high concentrations simultaneously with animal samples. Prepare brain homogenates by homogenizing brain tissue with 2 volumes (w:v) of homogenization solution (PBS buffer). Use 2 volumes of extraction buffer (containing 0.1% formic acid and 15 ng / mL) -1 Naloxone-D5 was precipitated in 50 µL aliquots of brain homogenate or CSF sample using methanol-water (95:5, v / v). The corresponding double blank samples were extracted with extraction buffer containing 0.1% formic acid (i.e., methanol-water (95:5, v / v) without internal standard). The CSF and brain homogenate samples were vortexed for 30 seconds and then held at -20°C for 5 minutes. The samples were then centrifuged at 15,000 rpm for 5 minutes; the supernatant was collected and held at -20°C for 5 minutes and centrifuged again at 15,000 rpm for 5 minutes. The samples were stored at -80°C for further analysis. LC-MS / MS analysis was also performed as described in the pharmacokinetic studies of these molecules. The LLOQ for naloxone and AG10-L-E2-naloxone in brain and CSF matrices was 1 nM. (Brain (ng g) -1 ) relative to plasma concentration (ng / mL) -1 The ratio of CSF (ng / mL) is expressed as a percentage of the brain to blood plasma ratio. -1 ) relative to plasma concentration (ng / mL) -1 The ratio is expressed as a percentage of the CSF to plasma ratio.

[0409] Rats receiving LD 50 Survival rates following doses of fentanyl or morphine. Male Spriggdalene rats (225–250 g; 7–8 weeks old) were obtained from Charles River Laboratories. Rats were randomly assigned to three groups (carrier, naloxone, and AG10-L-E2-naloxone). Rats initially received a subcutaneous dose of 100 mg / kg fentanyl or morphine. -1 Morphine (per group) n = 9Five minutes after the morphine dose, rats were administered a single subcutaneous dose of the carrier (10% DMSO, 20% PEG400, and 70% H2O) and naloxone (15 µmol kg). -1 Equivalent to 5 mg kg -1 ) or AG10-L-E2-naloxone (15 µmol kg -1 Equivalent to 15.8 mg / kg -1 Naloxone and AG10-L-E2-naloxone were dissolved in the same mediator administration solution. Morphine was dissolved in water. The injection volumes of morphine and the antagonist were 200 and 500 µL, respectively. The rats were then returned to their cages under standard conditions. The number of dead rats and their approximate time of death were recorded. Six hours after the first morphine dose, all surviving rats received a second subcutaneous dose of 50 mg / kg. -1 Morphine. Return the rats to their cages and observe to record any deaths. Record the total number of dead rats and calculate the survival rate in each group.

[0410] In the fentanyl mortality study, rats were first given a subcutaneous dose of 3 mg / kg. -1 Fentanyl (per group) n = 8 Five minutes after the fentanyl dose, rats were administered a single subcutaneous dose of the carrier (10% DMSO, 20% PEG400, and 70% H2O) and naloxone (15 µmol kg). -1 Equivalent to 5 mg kg -1 ) or AG10-L-E2-naloxone (15 µmol kg -1 Equivalent to 15.8 mg kg -1 Naloxone and AG10-L-E2-naloxone were dissolved in the same mediator administration solution. Fentanyl was dissolved in water. The injection volumes of fentanyl and the antagonist were 200 µL and 500 µL, respectively. The rats were then returned to their cages under standard conditions. The number of dead rats and their approximate time of death were recorded. Six hours after the first morphine dose, all surviving rats received a second subcutaneous dose of 3 mg / kg. -1 Fentanyl. Return the rats to their cages and observe to record any deaths. Record the total number of dead rats and calculate the survival rate for each group.

[0411] Reversal and prevention of fentanyl-induced respiratory depression in rats. The efficacy of antagonists in reversing fentanyl-induced respiratory depression in rats was evaluated using a whole-body plethysmography device from DSI Buxco (St. Paul, MN; equipped with FinePointe software). Spriggdalen rats (225–250 g; 7–8 weeks old) (each group) n = 6Rats were acclimatized for 30 minutes in a silencing, enclosed plethysmography chamber under continuous fresh airflow (2.2 l / min). Rats were awake and allowed free movement in the test chamber during the monitoring period. For baseline measurements, rat respiration was recorded for 20 minutes prior to injection. Rats were then removed from the test chamber and sequentially administered fentanyl (3 mg / kg). -1 A single subcutaneous or intravenous dose), or a single subcutaneous or intravenous dose of the antagonist one minute later (15 µmol kg). -1 or 60 µmol kg -1 Rats were treated with fentanyl. They were returned to the testing chamber, and respiratory parameters were measured for 90 minutes. Six hours and 24 hours after antagonist administration, rats were given two additional subcutaneous or intravenous doses of fentanyl (1.5 mg kg each). -1 Rats were challenged. Respiratory parameters were measured for 90 minutes after each fentanyl injection. Rats' ventilation was measured by recording changes in ventricular pressure, which were then processed and digitized. For each rat, the software calculated the mean tidal volume (Vol. 1). T (ml / breath), respiratory rate (f: breaths / minute), and minute ventilation (V) E = V T * f: ml / min). Since minute ventilation is V T The product of f is therefore reported because it is considered a scientifically relevant and quantifiable net physiological mean. The baseline minute ventilation data for each rat are averaged and designated as 100% of the baseline value. The minute ventilation readings for each rat after administration are then standardized and reported at 5-minute intervals based on the following equation: Minute ventilation (% relative to control baseline) = Minute ventilation at a specific time / Baseline minute ventilation * 100.

[0412] To administer an antagonist prophylactically to counteract fentanyl-induced respiratory depression: rats (each group) n = 6 Rats were allowed to acclimatize to the plethysmography chamber for 30 minutes, and baseline measurements were recorded as described above. Rats received a single subcutaneous dose of the mediator and naloxone (15 µmol / kg). -1 and 60 µmol kg -1 ) or AG10-L-E2-naloxone (60 µmol kg -1 ), and respiratory parameters were measured for 90 minutes. Six hours later, a single subcutaneous dose of fentanyl (1.5 mg / kg) was administered. -1 Rats were challenged and returned to the plethysmography chamber. Respiratory parameters were measured for 90 minutes, and the rats were treated and data reported as described above. Treatment doses of morphine and fentanyl were determined in preliminary studies and also based on published literature. The dose of morphine used (100 mg / kg) -1The reported LD50 of morphine in rats is 64–140 mg / kg. -1 For fentanyl, we use the reported value of 3 mg / kg. -1 These dosages were confirmed in preliminary experiments in which we started with lower doses of morphine and fentanyl and no fatalities were observed. Overdose studies of morphine and fentanyl were approved by the University of the Pacific (IACUC).

[0413] Preliminary evaluation of AG10-L-E2-naloxone toxicity in rats. The in vivo toxicity of AG10-L-E2-naloxone in rats was evaluated. Adult male Charles River rats weighing 225–250 g were used in the study. Three groups of rats were used. The first group was treated with 500 µL of a medium (10% DMSO, 20% PEG, and 70% H2O). The second group was treated with AG10-L-E2-naloxone (60 µmol / kg) dissolved in 500 µL of the medium. -1 The third group was treated with fentanyl (3 mg / kg). -1 Treatment with a subcutaneous dose, followed by AG10-L-E2-naloxone (60 µmol kg) -1 Treatment was initiated, followed by two doses of fentanyl (1.5 mg kg) at 6 hours and 24 hours. -1 Treatment was administered subcutaneously each time. Animal weight was monitored weekly for four weeks. Weight is expressed as mean ± sd (per group). n= 6 (sub-biological repetition).

[0414] result AG10-L1-E2-Naloxone for the prevention of death in rats following two doses of morphine or fentanyl We evaluated the pharmacokinetic (PK) characteristics of naloxone and AG10-L-E2-naloxone in rats. Figure 3a Two compounds (15 µmol kg) were used. -1 Equivalent to 5 mg kg -1 The naloxone dose was administered to rats as a single subcutaneous dose, and blood samples were collected from the jugular vein cannula at predetermined time points (ranging from 5 minutes to 72 hours). Figure 3a Consistent with our hypothesis, the PK characteristics of AG10-L-E2-naloxone are significantly different from those of naloxone. Both compounds are rapidly absorbed (naloxone was detected in plasma 2 minutes after administration of free naloxone or AG10-L-E2-naloxone) and T... max It takes about 15 minutes. The C of naloxone administered in free form... max(1360 nM) significantly higher than the C of naloxone released from AG10-L-E2-naloxone. max (16.9 ± 2.1 nM). However, although no measurable amount of free naloxone was observed 4 hours after administration, AG10-L-E2-naloxone and its released naloxone were still present even after 72 hours. Figure 3a The pharmacokinetic characteristics of naloxone showed a typical spike curve (ranging from approximately 1360 nM at 15 minutes). max At 4 hours, the C content was 34 nM. min Surprisingly, the release of naloxone from AG10-L-E2-naloxone follows a flatter curve (ranging from approximately 17 nM C at 15 minutes). max C at 2.4 nM at 72 hours min We believe this will be superior to the early concentration spikes observed with naloxone (which may cause withdrawal symptoms). Importantly, human data show that naloxone has a lower C-level concentration after subcutaneous administration. max (3.5 nM for a 2 mg dose and 17 nM for a 5 mg dose, with dose checks performed on IM or SC doses) is approximately 10 nM. To the best of our knowledge, this is the first PK-characteristic example demonstrating a smooth release of therapeutic molecules without the presence of typical peaks and troughs or burst effects observed after multiple dosings. This data strongly supports and validates our novel approach, in which the hydrophilicity of our prodrug controls the release of these molecules from subcutaneous tissue. This combination of slow release and TTR binding results in a gradual and stable plasma concentration of naloxone released in rats.

[0415] Then, we evaluated the blood-brain barrier (BBB) ​​permeability of AG10-L-E2-naloxone (5 mg / kg; subcutaneous) in rats. Figure 3b The concentrations of naloxone released from AG10-L1-E2-naloxone in the brain (7.8 nM and 30.1 nM after 10 and 30 minutes, respectively) were approximately 30 to 60 times lower than the concentrations of naloxone administered in free form (236.3 nM and 1721.7 nM after 10 and 30 minutes, respectively). Similar data were observed for the two compounds in cerebrospinal fluid (CSF). Figure 3cThis clearly shows that AG10-L1-E2-naloxone releases a significant amount of naloxone across the BBB. No detectable concentrations of the parent AG10-L1-E2-naloxone were found in the brain or CSF. These data support our hypothesis that the hydrophilicity and selective binding to the TTR limits the ability of the AG10-conjugate to cross the BBB. The brain-to-plasma ratio of naloxone released from AG10-L1-E2-naloxone (250.3% after 30 minutes) and the CSF-to-plasma ratio (173.3% after 30 minutes) were remarkably similar to the percentage of naloxone administered in free form. This is significantly higher than 2% (the BBB penetration cutoff; molecules with plasma concentrations <2% in the brain or CSF have been determined not to cross the BBB).

[0416] We evaluated the potential of our prodrug as an antidote for rescuing opioid overdose deaths. Two opioids, morphine and fentanyl, were evaluated. Although the half-lives of fentanyl (approximately 7 hours) and morphine (2 to 3 hours) are... t 1 / 2 The duration of overdose is relatively short, but the duration of overdose can last up to 24 hours for fentanyl and up to 120 hours for morphine (depending on the usually unknown opioid dose). Treatment doses for morphine and fentanyl were determined in preliminary studies and also based on published literature. The dosage of morphine (100 mg / kg) -1 The reported LD50 of morphine in rats 50 64–140 mg kg -1 For fentanyl, we use the reported value of 3 mg / kg. -1 Three groups of rats (each group) n= 9 ) Administer morphine (100 mg / kg) -1 (Single subcutaneous dose). 5 minutes later: administer the mediator to group 1; administer naloxone (15 µmol kg) to group 2. -1 Equivalent to 5 mg kg -1 A single subcutaneous dose was administered, and a molar equivalent dose of AG10-L-E2-naloxone (15 µmol kg) was given to group 3. -1 Equivalent to 15.8 mg kg -1 (Single subcutaneous dose). 15 µmol kg of the selected naloxone and AG10-L-E2-naloxone. -1 The dose is higher than the dose used in the literature (1 mg kg). -1 Equivalent to 3 µmol kg -1This ensures that respiratory depression at morphine and fentanyl doses is almost completely reversed by the slower-released naloxone from our prodrug. All mediators in the control group died after the first morphine dose, while the naloxone and AG10-L-E2-naloxone groups remained alive. Figure 3d Six hours later, administer the second dose of morphine (50 mg / kg). -1 A single subcutaneous dose was administered to animals treated with naloxone and AG10-L-E2-naloxone to simulate re-anesthesia. All animals in the naloxone group died within 2 hours following the second morphine dose. Notably, all animals in the AG10-L1-E2-naloxone group survived and remained alive for several weeks after administration.

[0417] We also used two doses of fentanyl (3 mg kg each) -1 Subcutaneous, at times = 0 hours and 6 hours; each group n = 8 ) to stimulate rats to conduct similar experiments ( Figure 3e Following the first dose of fentanyl, six rats in the mediator group died, and the remaining rats died after the second dose. In the naloxone group, all eight rats died after the second dose of fentanyl. Importantly, after the second dose of fentanyl, only one of the eight rats in the AG10-L-E2-naloxone treatment group died. Figure 3e The data clearly demonstrate that AG10-L1-E2-naloxone is more effective than naloxone in reducing mortality in rats given two high doses of fentanyl. Given the urgent need to develop an effective antidote for fentanyl, the following studies will focus on evaluating our prodrug against fentanyl overdose.

[0418] Reversal of opioid-induced respiratory depression (OIRD) in rats Systemic side effects of opioid toxicity include life-threatening intraepithelial neoplasia (OIRD), which can have potentially fatal consequences for patients. OIRD is also a major obstacle to the safe administration of morphine for acute pain following injury or trauma in emergency situations. OIRD can be properly managed when monitored by trained medical personnel and when appropriate equipment is available. However, it is difficult to achieve in emergency situations where these options are unavailable (e.g., battlefield environments or weaponized synthetic opioids). Although long-term administration can lead to tolerance to respiratory depression, it can cause death in opioid-naïve patients, meaning it is a side effect of greatest concern to clinicians in the postoperative setting. We tested our prodrug (15 µmol kg) using a non-invasive whole-body plethysmography method. -1 It is equivalent to 5 mg kg -1 Naloxone; single subcutaneous dose, n = 6 Reversing high-dose fentanyl (LDL) 50 3 mg kg-1 The ability of OIRD (single subcutaneous dose) to ( Figures 3f-3i ).

[0419] After the first fentanyl dose ( Figure 3g No reversal of OIRD was observed after subcutaneous administration of the mediator or lipotropic naloxone prodrug containing PABA ester C12-E2-naloxone (although hydrolyzed in rat serum). Figure 1g This was predictable because lipogenesis slowed the release of the conjugate from subcutaneous tissue. We also had difficulty formulating C12-E2-naloxone into the dosing medium, requiring doubling the volume of the dosing medium. Two out of six rats in both the medium-treated and C12-E2-naloxone-treated groups died after the first fentanyl dose, so we did not continue testing the C12-E2-naloxone prodrug. As expected, naloxone and the alkyl ester AG10-L-E1-naloxone provided rapid reversal of OIRD after 10 minutes (confirmed by a rapid recovery of minute ventilation). Figure 3g For AG10-L-E2-naloxone, the minute ventilation in rats gradually recovered after administration, but did not reach levels close to those of normal rats (the minute ventilation at 90 minutes was 52.5% ± 4.5% of the control).

[0420] To test the ability of the prodrug to prevent fentanyl-induced ventilatory depression (i.e., re-anesthesia) after initial reversal, a second dose of fentanyl (1.5 mg / kg) was administered 6 hours after naloxone or the prodrug. -1 A single subcutaneous dose) stimulated rats ( Figure 3h As expected, naloxone did not effectively reverse OIRD, and four out of six rats in the naloxone group died after the second fentanyl dose. AG10-L-E1-naloxone was also ineffective (as confirmed by the stability of rat serum showing rapid hydrolysis of the alkyl ester). Figure 1g AG10-L-E2-naloxone was the only prodrug to show significant OIRD reversal 6 hours after the second dose of fentanyl. Figure 3h 24 hours after administering naloxone or the prodrug, administer the third dose of fentanyl (1.5 mg / kg). -1 A single subcutaneous dose was administered to stimulate rats. This again demonstrated that AG10-L-E2-naloxone was more effective than naloxone and AG10-L-E1-naloxone (one of six rats in the AG10-L-E1-naloxone group died after the third fentanyl dose) in preventing death and reversing OIRD. Figure 3iThe slow onset of action of AG10-L-E2-naloxone suggests that the initial concentration of naloxone released from the prodrug needs to be increased to achieve effective rescue from the first fentanyl dose. However, this slow-release characteristic may be helpful for certain therapeutic applications, such as the treatment of recovery from analgesia and in newborns (discussed below).

[0421] Example 6: AG10-L1-E2-Naloxone is more effective than naloxone and nalmefone in preventing fentanyl re-anesthesia. Our data show that low-dose AG10-L-E2-naloxone effectively reduced fentanyl levels after two doses (3 mg kg each). -1 The mortality rate afterward. However, the plethysmography results ( Figure 3g This indicates a need to increase the initial concentration of naloxone released. Therefore, we evaluated a higher dose of AG10-L-E2-naloxone (4-fold higher; 60 µmol kg). -1 Equivalent to 63mg / kg -1 Pharmacokinetic characteristics of ) Figure 4a For this extremely high dose, the C of naloxone administered in free form... max (6498 ± 2.15 nM) was significantly higher than the C of naloxone released from AG10-L-E2-naloxone. max (approximately 30 nM) Figure 4a In contrast to naloxone, and as observed for lower doses, the release of naloxone from AG10-L-E2-naloxone follows a similarly flatter curve (ranging from approximately 30 nM C at 30 minutes). max C at 15 nM at 96 hours min Then, we tested a higher dose of AG10-L-E2-naloxone (60 µmol kg). -1 Equivalent to 63 mg kg -1 The efficacy of ) Figures 4b-4e We also included extremely high doses of two FDA-approved antidotes, naloxone (60 µmol kg). -1 Equivalent to 20 mg kg -1 ) and nalmefene (60µmol kg -1 Equivalent to 21 mg kg -1 As a control, nalmefene effectively reversed OIRD within 2 minutes of administration after the first fentanyl dose (consistent with reported rapid absorption; T...). max (Approximately 20 minutes) Figure 4c However, in rats given nalmefone, significant hyperventilation and some spasms or seizures were observed, indicating the deposition of opioid withdrawal symptoms. Three rats in the fentanyl control group died. Higher doses of AG10-L-E2-naloxone (60 µmol kg) were also observed.-1 The efficacy of the (reversible within 20 minutes) dose was superior to the lower dose (15 µmol kg). -1 (Reversed within 50 minutes) Figure 4c Following stimulation of rats with a second dose of fentanyl, nalmefene and AG10-L-E2-naloxone were equally effective in reversing OIRD, indicating the presence of effective levels of naloxone release 6 hours after administration of AG10-L-E2-naloxone. Figure 4d Naloxone control rats did not effectively reverse OIRD after administration of the second fentanyl dose (i.e., 6 hours after naloxone administration; 2 rats in the naloxone control group died). We then challenged the rats with a third dose of fentanyl 24 hours after administration of the antagonist (to simulate re-anesthesia). Figure 4e It is noteworthy that while nalmefene did not effectively reverse fentanyl-induced OIRD, AG10-L-E2-naloxone effectively counteracted OIRD. This data demonstrates that we have developed what we believe to be the most potent μ-opioid antagonist prodrug (effective as a reversal agent and potentially an ideal prophylactic agent).

[0422] Example 7: Compared to lower doses, higher doses of naloxone did not provide additional efficacy. Surprisingly, there was a lack of correlation between the pharmacokinetic and pharmacodynamic characteristics of naloxone. Pharmacokinetic data showed a substantial plasma concentration of free naloxone (25 nM) at 6 hours, similar to the concentration of naloxone released from AG10-L-E2-naloxone (21 nM). Figure 4a However, rats treated with naloxone performed poorly in plethysmography after the second fentanyl dose, and two rats died despite sufficient naloxone in circulation. Figure 4a and Figure 4d This data suggests that the initial extremely high levels of naloxone (6498 nM at 15 minutes) may actually be harmful and make animals more susceptible to the toxic effects of a second dose of fentanyl compared to the low sustained levels of naloxone from AG10-L-E2-naloxone (concentration = 20 nM at 15 minutes).

[0423] To investigate this, we conducted another experiment in which fentanyl and its antagonist (naloxone and AG10-L-E2-naloxone) were administered intravenously at a concentration of 60 µmol kg. -1 Equivalent to 20 mg kg -1 (single intravenous dose) Figure 4fPharmacokinetic data showed that naloxone (concentration at 15 min = 20597 nM) and both naloxone and AG10-L-E2-naloxone (concentration at 15 min = 1599 nM) had extremely high concentrations at earlier time points after administration. Although OIRD was reversed after the first fentanyl dose in both treatment groups, rats treated with naloxone exhibited significant hyperventilation, and the animals experienced severe convulsions / seizures. No hyperventilation or seizures were observed in animals treated with AG10-L-E2-naloxone. Pharmacokinetic data showed that both free naloxone and naloxone released from AG10-L-E2-naloxone had significantly higher plasma concentrations at 6-form (750 nM and 60 nM, respectively) and 24-form (395 nM and 44 nM, respectively). Figure 4f However, although AG10-L-E2-naloxone showed mild activity after the second fentanyl dose, both treatment groups demonstrated effective protection against OIRD after the second (6 hours) and third (24 hours) doses of fentanyl. Figure 4g-Figure 4i This data suggests that exposing rats to very high doses of naloxone following fentanyl administration can actually cause some initial harm or alterations in μ / other receptors, thus reducing the animals' protection from naloxone at later time points. There are reports of pulmonary edema following high / rapid reversal. However, further investigation is needed.

[0424] Example 8: AG10-L1-E2-naloxone is a potential preventative and antiviral agent against fentanyl-induced death and OIRD. treatment To compensate for the lower efficacy of AG10-L-E2-naloxone in the first 30 minutes after administration, we tested the feasibility of combining the prodrug with a small amount of free naloxone. AG10-L-E2-naloxone (60 µmol kg) -1 Equivalent to 63 mg kg -1 ) and naloxone (3 µmol kg -1 Equivalent to 1 mg kg -1 A single subcutaneous dose combination yielded excellent efficacy data, with OIRD completely reversed after all three fentanyl doses. Figures 5a-5d Importantly, no rats experienced severe hyperventilation or convulsions / seizures, indicating that AG10-L-E2-naloxone poses a low risk of producing opioid withdrawal symptoms.

[0425] Finally, we explored the potential of using AG10-L-E2-naloxone as a prophylactic agent against fentanyl. Figures 5e-5g First, rats (each treatment group) were given... n = 6 ) Administer AG10-L-E2-naloxone (60 µmol kg) -1) or naloxone (15µmol kg) -1 and 60 µmol kg -1 Compared with animals treated with the medium, there was no change in minute ventilation. Six hours later, the rats were given a single dose of fentanyl (1.5 mg kg). -1 (Subcutaneous dose) stimulation. AG10-L-E2-naloxone is the only antidote that significantly protects rats from OIRD, and all animals ( n = 6 Survival. In contrast, neither of the two naloxone doses was effective in preventing OIRD or death (4 animals died in the vector group; 15 µmol kg). -1 Three animals died at a dose of naloxone; and 60 µmol kg -1 Two animals died at the dose of naloxone. This data supports the observation that higher doses of naloxone have lower efficacy despite the presence of considerable circulating concentrations (Figure 4), and highlights the potential challenges of using higher doses of μ-opioid antagonists to combat fentanyl overdose.

[0426] Example 9: Preclinical evaluation of AG10-L1-E2-naloxone – rat study We evaluated the effects of AG10-L-E2-naloxone on three groups of rats (each group) n = 6 The effect of weight. The first group was treated with the medium. The second group was treated with AG10-L-E2-naloxone (60 µmol kg). -1 The third group was treated with fentanyl, followed by AG10-L-E2-naloxone (60 µmol kg). -1 Rats were treated with fentanyl at 6 and 24 hours (same regimen as above). Rats were observed for four weeks, and no signs of toxicity or death were observed. All animals achieved their expected body weight throughout the study period, and no significant difference was observed between the AG10-L-E1-naloxone treatment group and the carcass treatment group. Figure 5h ).

[0427] We also tested high concentrations of AG10-L-E2-naloxone (100 µM, which was higher than the C40 concentration in rats). max The effects of 0.8 µM (100-fold higher) and its metabolite AG10-L-E2-COOH on the viability and proliferation of three cell lines were investigated. Cell viability (MTT) was measured using the CellTiter 96 non-radioactive cell proliferation assay. HeLa and MCF-7 cells were humidified at 37°C under a 5% CO2 and 95% air atmosphere supplemented with 10% fetal bovine serum and penicillin / streptomycin (100 units / mL, respectively). -1 and 100 μg / mL -1Hep3B cells were cultured in RPMI-1640 medium containing 1% L-glutamine and 1% FBS. Hep3B cells were cultured in minimal Eagle's medium containing 10% FBS. Cells were allowed to grow to approximately 75% confluence and seeded into 96-well plates at approximately 5000 cells / well for HeLa and MCF-7 cells and approximately 8000 cells / well for Hep3B cells. Cells were then treated with naloxone, AG10-L-E2-naloxone, and AG10-L-E2-COOH (tested at 100 µM) or doxorubicin (10 µM) as a positive control. Control cells were also treated with an appropriate concentration of DMSO. After incubation at 37°C and 5% CO2 for 72 hours, cell viability was determined according to the standard CellTiter 96 non-radioactive cell proliferation assay protocol.

[0428] Both compounds showed no cytotoxic effects on any of the cell lines tested. Figures 5i-5k We also confirmed that AG10-L-E2-COOH is stable in aqueous solution for at least two months at room temperature.

[0429] Example 10: Preclinical evaluation of AG10-L1-E2-naloxone – Monkey study Materials and methods Three male cynomolgus monkeys were administered AG10-L-E2-naloxone (30 µmol kg). -1 Equivalent to 31 mg kg -1 A single subcutaneous (sc) injection was administered. The concentrations of AG10-L-E2-naloxone and naloxone released from AG10-L-E2-naloxone in the plasma of each monkey were measured at different time points and expressed as the mean ± sd of three biological replicates.

[0430] result To evaluate the potential of translating our data into humans, we evaluated AG10-L-E2-naloxone in the non-human primate cynomolgus monkey (Cynodon dactylus). n = 3 Pharmacokinetic characteristics in ) Figure 5l Monkeys represent a possible model because their plasma lacks the CES enzyme (). Figure 2f Furthermore, the naloxone released from AG10-L-E2-naloxone in monkey liver microsomes is similar to that in humans. Figure 2d Monkeys were also used as a preclinical model to test the efficacy of the antidote against fentanyl and carfentanil. A single subcutaneous dose (30 µmol / kg) was administered. -1 Equivalent to 31 mg kg -1AG10-L-E2-naloxone was administered at half the dose in rats, and plasma concentrations of maternal AG10-L-E2-naloxone and released naloxone were measured over 72 hours. The enhanced pharmacokinetic characteristics of maternal AG10-L-E2-naloxone were similar to those in rats (compared to a typical 2 mg IM dose of naloxone; Cmax and disappeared within 4 hours). AG10-L-E2-naloxone was rapidly absorbed (plasma concentration at 5 minutes = 309 nM). Cmax was also rapidly reached within 15 minutes of administration. max (2392 nM). Importantly, the concentrations of AG10-L-E2-naloxone from form 4 to form 72 (the last test time point) were within a narrow range (303 nM to 141 nM). The pharmacokinetic profile of naloxone released from AG10-L-E2-naloxone was significant, ranging from 3 nM at 15 minutes post-dose to 4.5 nM at 72 hours. C60 at 8 hours max The concentration is 12.5 nM. Data regarding the lower concentrations of naloxone relative to its parent prodrug are consistent with in vitro data, highlighting the stability of the prodrug in monkey plasma but allowing for the release of naloxone via CES2 from the liver and potentially other organs with CES2 activity. This slow and prolonged release of stable concentrations of naloxone is thought to provide excellent efficacy characteristics in rats. We predict that AG10-L-E2-naloxone and the released naloxone will have a prolonged release time in monkeys, supported by pharmacokinetic studies in rats (at least 96 hours). Figure 4a ).

[0431] Example 11: Development and clinical application of a naloxone prodrug reservoir for prolonged protection against opioid overdose. Pre-test introduction In 2023, the United States reported more than 80,000 deaths from opioid overdoses, most of which were caused by synthetic opioids such as fentanyl. 1 Among high school students, an average of 22 teenagers died each week in 2022 from counterfeit drugs laced with fentanyl. 2 A lethal dose of approximately two milligrams of fentanyl poses a considerable risk, especially to individuals who have not previously used opioids. Although fentanyl has a relatively short half-life (approximately 7 hours), an overdose can last up to 24 hours. 3 Naloxone, used to reverse excess μ-opioid receptor antagonists, has a short duration of action (DOA of approximately 1 hour), requiring repeated dosing and continuous monitoring. 4 This highlights the need for long-acting opioid antagonists. Emerging approaches include pseudo-irreversible antagonists, covalent naloxone nanoparticles, and antibody-based strategies. 4-7However, these methods fail to address the key challenge: rapidly reversing opioid-induced respiratory depression and subsequently providing protection against re-anesthesia. Developing prolonged-release opioid antagonists is crucial, and one promising approach involves using prodrugs that slowly release the active drug over time. 8-11 However, this prodrug approach has so far achieved limited success with naloxone.

[0432] Our strategy involves administering naloxone prodrugs containing a hydrophilic derivative of a small molecule AG10 (Alhamadsheh-Graef molecule 10 or acoramidis). 12 It reversibly binds to the plasma protein thyroxine transporter (TTR). Our team discovered that acomide is the most potent and selective TTR ligand and has recently received FDA approval for the treatment of thyroxine transporter amyloid cardiomyopathy (ATTR-CM). 13 This embodiment introduces a naloxone prodrug delivery system utilizing the ionizable properties of our hydrophilic AG10 derivative. Following subcutaneous administration of a fully soluble prodrug solution, the prodrug forms a zwitterion at physiological pH, thereby reducing its solubility and creating a reservoir that prolongs the release and duration of action of naloxone. The prodrug is specifically bioactivated by carboxylesterase 2 (CES2) enzymes present in the human liver, allowing for controlled and prolonged naloxone release. Furthermore, its balanced hydrophilicity and ability to bind TTRs to plasma reduce passive diffusion across the blood-brain barrier (BBB), potentially lowering the risk of deposited opioid withdrawal symptoms and central nervous system (CNS) toxicity.

[0433] In this embodiment, we demonstrated the potential of our approach by developing a naloxone prodrug that exhibits a prolonged DOA compared to naloxone for fentanyl and morphine overdose in rats. The consistent pharmacokinetic characteristics of naloxone released from our prodrug in male rats and cynomolgus monkeys, along with its superior efficacy (in rats) and promising safety profile and chemical stability, highlight the potential for converting this prodrug to humans. This simple and feasible naloxone prodrug system could significantly advance the treatment of opioid overdose and potentially other opioid-related conditions.

[0434] result The concept of prodrug delivery systems The degree of ionization and solubility of ionizable drugs depend on the pH of the drug solution. The rate of drug absorption by subcutaneous (sc) tissue is also affected by the physiological pH of the subcutaneous interstitial fluid (approximately pH 7.4). One example is insulin glargine (Lantus). ®This is a long-acting insulin analog (DOA of approximately 24 hours after subcutaneous injection). The incorporation of two arginine residues shifts its isoelectric point from pH 5.4 to 6.7, making it soluble in weakly acidic solutions (pH 4). 14 Upon injection into the subcutaneous tissue, the acidic solution is neutralized by the interstitial fluid, forming a reservoir of microprecipitates that slowly dissolve to release insulin glargine over time. The slow absorption rate of insulin glargine acts as a rate-limiting step, influencing its pharmacokinetic profile by controlling the gradual decrease in plasma insulin levels. This behavior confirms flip-flop kinetics, where the final stage of concentration decline depends more on release and absorption at the injection site than on systemic clearance. 15 However, compared to conventional insulin, the low levels of soluble free glargine insulin and the slow dissolution of its reservoir result in delayed absorption and a slower onset of action. This delayed absorption is a drawback of long-acting lipid or polymer reservoirs. 16 This is especially true for applications requiring rapid onset of action, such as opioid antidotes. It is noteworthy that non-polymeric drugs (similar to insulin glargine) that form a reservoir after injection are extremely rare.

[0435] We recently developed a hydrophilic derivative of AG10 that enhances DOA and restricts BBB penetration of non-cleavable covalently linked opioid agonists and antagonists in rats. 17 Surprisingly, these highly hydrophilic covalent conjugates were rapidly absorbed after subcutaneous administration, appearing in rat plasma within 2 minutes, despite their expected limited membrane permeability. We hypothesize that these conjugates form zwitterions at physiological pH, thereby reducing their hydrophilicity / solubility in interstitial fluid and forming a reservoir similar to insulin glargine. This prompted us to investigate the potential zwitterionic properties of AG10 derivatives to generate prodrug delivery systems for prolonged subcutaneous drug administration, focusing on the opioid antagonist naloxone.

[0436] Design of naloxone prodrug Our prodrug approach involves conjugating a linker-modified hydrophilic derivative of AG10 to naloxone using esters with varying stabilization. Modeling studies have shown that the AG10 linker can extend to a TTR, allowing naloxone to be conjugated via different ester moieties. Esters commonly found in prodrugs can be activated by enzymatic or chemical hydrolysis, with esterases present throughout the body, including in the blood, liver, kidneys, adipose tissue, and brain. 18 .

[0437] We first explored the esterification of naloxone (AG10-L-E1-naloxone) with aliphatic acid derivatives. Figure 1aHowever, we found that the hydrolysis rate was very fast (discussed below). Recognizing that the metabolic fate of esters depends on their stereo and electronic properties, we turned to the use of aromatic para-aminobenzoic acid (PABA) to form an ester (AG10-L-E2-naloxone) with naloxone. Figure 1a PABA was chosen because of its steric (aromatic) and electronic (electron-donating) stability to the ester moiety. PABA is naturally present in humans, making it largely non-toxic (LD50 in dogs and rats). 50 >2 g kg -1 ) 19 We also synthesized the acyl hydrolysis product of AG10-L-E2-naloxone (i.e., AG10-L-E2-COOH). Figure 1a As a control for fatty acid conjugation (existing methods for extending the half-life of peptides via albumin binding), we synthesized a lipotropic naloxone prodrug containing the PABA ester C12-E2-naloxone. All compounds were synthesized as HCl salts (utilizing the presence of multiple basic amines in the conjugates).

[0438] To test our zwitterionic hypothesis, we evaluated the water solubility of the test compounds in water and phosphate buffer (pH 7.4, simulating the pH of subcutaneous tissue). Dissolving the HCl salt of the prodrug in water produced an acidic pH of approximately 3.5. All compounds except C12-E2-naloxone (5 µM) were soluble in water at 200 µM. Figure 1b Naloxone's solubility in phosphate buffer at pH 7.4 is slightly lower (170 µM) than in water. However, compared to water (200 µM), the solubility of AG10-L-E2-naloxone at pH 7.4 is significantly reduced (44 µM), supporting our hypothesis that the zwitterionic form of the AG10-connector reduces solubility at physiological pH. Figure 6 a). AG10-L-E2-COOH is completely soluble in water and buffer solution (200 µM), indicating no zwitterion formation. Figure 6 b). Conversely, zwitterions are formed ( Figure 6 c) AG10-L-E2-ethyl showed lower solubility at pH 7.4 (97 µM) than at pH 3.5 (200 µM). D 7.4 The value further supports the hypothesis, showing the hydrophilicity (log) of the zwitterionic AG10-L-E2-ethyl. D 7.4 = 1.2) and AG10-L-E2-COOH (log D 7.4 = -1.6) significantly lower than naloxone (log D 7.4= 1.2) and lipolyzed C12-E2-naloxone (log D 7.4 = 2.0) compared to AG10-L-E2-naloxone (log D 7.4 = 1.5) exhibits a balanced hydrophilicity / lipophilicity, thereby enhancing its subcutaneous absorption. Achieving this balance is important because it enables the formation of reservoirs while maintaining the important free soluble fraction (44 µM, Figure 1b This is crucial for effective and rapid prodrug absorption.

[0439] The prodrug selectively binds to TTR in buffer and human serum. Measurement using fluorescence polarization (FP) binding 20 ( K d = 394.3 nM and 258.8 nM respectively; Figure 1c The binding affinity of the prodrugs AG10-L-E1-naloxone and AG10-L-E2-naloxone to TTR was evaluated. Their affinity for AG10-L-E2-COOH ( K d =213 nM) equivalent. Selectivity was determined using TTR serum fluorescent probe exclusion (FPE). 21 These compounds were evaluated for selectivity for TTR against more than 4,000 other human serum proteins. All tested compounds exhibited strong binding selectivity for TTR in human serum (approximately 50% to 70% TTR occupancy). Figure 1d , Figure 1e AG10-L-E2-naloxone showed a TTR occupancy of 48.1%, similar to the approved TTR stabilizer chlorpromazine (49.2%).

[0440] AG10-L-E2-naloxone and C12-E2-naloxone prodrugs are stable in human plasma, but release into rat plasma. Naloxone In vivo efficacy studies of the prodrug were conducted in rats. Initially, we assessed the hydrolysis of the prodrug ester in human and rat plasma, taking into account its similar TTR plasma concentrations (approximately 5 µM) and high sequence homology (approximately 80%). 22-24 The TTR thyroxine binding site of AG10 and the prodrug is conserved between rats and humans. 23 In human plasma, the alkyl-based AG10-L-E1-naloxone prodrug is unstable and completely degrades within 1 minute, while the PABA-based esters (AG10-L-E2-naloxone and C12-E2-naloxone) are completely stable after 24 hours. Figure 1f This unexpected stability is surprising because modeling studies show that PABA esters extend into a TTR-binding pocket, thereby allowing esterases to enter ( Figure 2bTo investigate these observations, we evaluated the stability of the prodrug in rat plasma. AG10-L-E1-naloxone degraded rapidly, while AG10-L-E2-naloxone showed moderate stability (60% remaining after 15 minutes and 3.8% remaining after 2 hours). C12-E2-naloxone was slightly less stable than AG10-L-E2-naloxone. This difference in hydrolysis rate points to carboxylesterases 1 and 2 (CES1 and CES2), which are present in rat plasma but not in human plasma. 25 .

[0441] AG10-L-E2-naloxone is specifically bioactivated by the CES2 enzyme. Carboxylesterases (CES) CES1 and CES2 are crucial for the hydrolytic inactivation of many ester drugs. 25 Human CES1 is primarily responsible for the bioactivation of several prodrugs, while CES2 plays a secondary role. 26，27 CES1 is highly expressed in the liver, while CES2 is mainly found in the small intestine, kidneys, and liver. The expression level of CES2 in human liver is approximately 9 times lower than that of CES1. 27 We investigated the roles of CES1 and CES2 in the bioactivation of AG10-L-E2-naloxone. Due to their insolubility in buffer ( Figure 1b We were unable to evaluate the selectivity of CES for C12-E2-naloxone. In vitro studies of recombinant human CES showed that AG10-L-E2-naloxone is a specific substrate for CES2. Figure 2c This is surprising because very few prodrugs (such as procaine and irinotecan) are selectively hydrolyzed by CES2. Besides hepatic CES2, procaine is rapidly hydrolyzed by butyrylcholinesterase in human plasma, while irinotecan is slowly hydrolyzed by both CES1 and CES2, with a bioactivated dose of less than 5%. 27，28 To further investigate CES2 specificity and its potential for human transformation, we evaluated the metabolic bioactivation of AG10-L-E2-naloxone in liver microsomes from humans, rats, and cynomolgus monkeys. Figure 2d Human liver microsomes (HLM) efficiently produce naloxone from AG10-L-E2-naloxone, and the CES2 inhibitor loperamide significantly reduces naloxone release, while the CES1 inhibitor digitoxin has no protective effect. Rat liver microsomes (RLM) produce less naloxone due to higher metabolism and CYP3A4 dealkylation. Given similar CES2 expression in monkeys and humans... 25 Monkey liver microsomes (MLM) showed naloxone release comparable to that of HLM, indicating that monkeys are a suitable model for evaluating the efficacy of AG10-L-E2-naloxone. Specific CES2 bioactivation of AG10-L-E2-naloxone was confirmed using rat plasma containing CES1 and CES2 inhibitors. Figure 2e Additionally, AG10-L-E2-naloxone showed 100% stability in cynomolgus monkey plasma (without CES1 and CES2) after 24 hours. 25 and 0% stability in rabbit plasma (high CES1 and CES2 levels) Figure 2f ).

[0442] The liver concentration of CES2 was 9 times lower than that of CES1. 27 This provides a unique bioactivation for AG10-L-E2-naloxone. Furthermore, CES2 is absent from subcutaneous adipose tissue, while CES1 is present. 29 This reduces the potential explosive release of naloxone from the prodrug reservoir. Although the crystal structure of human CES1 is available... 30 However, a potential ortholog of human CES2 was recently reported: the first crystal structure of mouse CES2. 31 The mouse CES2 structure is similar to the human CES1, the difference being the entry of the active site. Docking studies showed that AG10-L-E2-naloxone does not bind to CES1, but it is extremely well-matched to the active site of CES2, and the nucleophilic serine (S230) is adjacent (2.9 Å) to the carbonyl carbon of the ester group in AG10-L-E2-naloxone. Figure 2b and Figure 7 This data is supported by docking studies of three known CES2 selective prodrugs, irinotecan, procaine, and monoprevir, with both CES1 and CES2. Figure 7 Three prodrugs showed excellent binding to CES2, with good distances between the nucleophilic S230 and the carbonyl carbon of the prodrug (3.3 Å, 3.1 Å, and 3.5 Å for irinotecan, procaine, and monoupivir, respectively). While AG10-L-E2-naloxone showed no binding to CES1, the three other prodrugs showed moderate to weak binding, with distances between the nucleophilic S230 and the carbonyl carbon of the prodrug (9.2 Å, 6.8 Å, and 4.8 Å for irinotecan, procaine, and monoupivir, respectively). Therefore, our study represents the first docking experiment with CES2-specific substrates, providing insights for designing additional CES2-specific prodrugs.

[0443] The specific hydrolysis of AG10-L-E2-naloxone by CES2 is driven by conjugation with the AG10-linker. We investigated whether the specific hydrolysis of AG10-L-E2-naloxone by CES2 is driven by a combination of PABA esters and the AG10-linker. We synthesized three aryl-based naloxone prodrugs without the AG10-linker. Naloxone benzoate (BA-naloxone) was hydrolyzed most rapidly in human plasma, while Boc-PABA-naloxone and PABA-naloxone were more stable (Figure 8). This highlights the stabilizing effect of the electron-donating amino group in PABA on the ester moiety. All three compounds were efficiently hydrolyzed in human plasma, indicating their substrate potential for esterases other than CES1 or CES2. CES2 selectivity was demonstrated in rat plasma stability studies using CES2 and CES1 inhibitors. Our data suggest that the specific hydrolysis of AG10-L-E2-naloxone by CES2 is driven by the conjugation of the PABA ester with the AG10-linker. We also tested known CES2 substrates procaine, monopivir, and irinotecan, and found that they were not specific for CES2 (Figure 8). To our knowledge, AG10-L-E2-naloxone is the first prodrug to be specifically bioactivated by CES2.

[0444] The specific hydrolysis of AG10-L-E2-naloxone by CES2 challenges our current understanding of the structural features required for CES2 substrates. 27 It has been proposed that compounds with larger acyl moieties are hydrolyzed by CES1, while CES2 preferentially hydrolyzes compounds with smaller acyl moieties, such as procaine and monoprevir. Our data indicate that compounds with much larger acyl moieties, such as AG10-L-E2-COOH, can also be hydrolyzed by CES2. These insights could help in designing novel prodrugs with higher specificity for CES2 hydrolysis.

[0445] Evaluation of the effect of TTR on the in vitro pharmacokinetics of AG10-L-E2-naloxone Although AG10-L-E2-naloxone is stable in human plasma, the presence of the CES2 enzyme in rat plasma hinders the precise evaluation of the role of TTR in the pharmacokinetics of AG10-L-E2-naloxone. Therefore, we performed in vitro experiments to investigate the role of TTR in our system. Figure 9 We evaluated the stability of AG10-L-E2-naloxone (tested at 5 μM) in rat plasma in the absence and presence of the selective TTR ligand AG10 (10 μM). Figure 9 a). We observed a small but significant reduction in the amount of residual AG10-L-E2-naloxone in the presence of AG10, suggesting that TTR provides some protection against CES2 in rat plasma. This result was expected because we designed the linker (approximately 29 Å) in AG10-L-E2-naloxone to be long enough to allow binding to both CES2 and TTR simultaneously (see [link to article]). Figure 2bThe protective effect of TTR was further supported by evaluating the metabolic stability of AG10-L-E2-naloxone in human liver microsomes in the presence and absence of TTR. Figure 9 b).

[0446] Notably, the primary effect of TTR on enhancing the pharmacokinetic profile of AG10-L-E2-naloxone will stem from its ability to reduce renal filtration, a crucial factor in reducing the duration of action of hydrophilic molecules. The instability of AG10-L-E2-naloxone to CES2 in rat plasma hinders the in vivo evaluation of the effect of TTR on PK. To simulate this effect in vitro, we used rapid equilibrium dialysis to model the effect of TTR on reducing renal filtration of AG10-L-E2-naloxone. Figure 9 c). Data showed that binding to human TTR significantly limited the ability of AG10-L-E2-naloxone to passively cross dialysis membranes. Our previous in vivo work on the non-cleavable linker supports this finding, demonstrating the effect of TTR on reducing renal filtration of these conjugates in rats. 32，33 Therefore, we believe that the effect of TTR on enhancing the pharmacokinetic characteristics of AG10-L-E2-naloxone will be confirmed in humans, as we observed in cynomolgus monkeys below.

[0447] Subcutaneous administration of AG10-L-E2-naloxone to rats to prolong and control the release of naloxone without blood loss Plasma and brain concentration increased dramatically To investigate the zwitterionic properties of the AG10-connector and the effect of TTR binding on the prodrug DOA, we evaluated the pharmacokinetic properties of naloxone and AG10-L-E2-naloxone in male rats. (15 µmol kg) -1 Equivalent to 5 mg kg -1 All naloxone doses were administered as a single subcutaneous dose, with blood samples collected via a jugular vein catheter at predetermined time points (2 minutes to 72 hours). Figure 3a (See Table 2). Consistent with our hypothesis, the pharmacokinetic profile of AG10-L-E2-naloxone differs significantly from that of naloxone. Both are rapidly absorbed (detectable in plasma 2 minutes after administration), T... max For approximately 15 minutes. The C of naloxone administered in free form. maxThe concentration of naloxone (1,390 ± 187 nM) was significantly higher than that of naloxone released from AG10-L-E2-naloxone (16.9 ± 1.5 nM). The cutoff time for all time points was 72 hours. Naloxone administered in free form was below the lower limit of quantification (LLOQ) by 1 nM at the 6-hour time point and thereafter. AG10-L-E2-naloxone and its released naloxone were still present even after 72 hours. Free naloxone showed a typical peak / peak curve (C1 of approximately 1,390 nM at 15 minutes). max At 4 hours, the C content was 31 nM. min ), while AG10-L-E2-naloxone follows a flatter, peakless curve (approximately 17 nM C at 15 minutes). max C at 2.4 nM at 72 hours min ) ( Figure 3a This reflects the flipping kinetics. This could potentially prevent early withdrawal symptoms. Importantly, human data show that for a 2 mg dose, naloxone has a C10 concentration after subcutaneous administration. max It is approximately 10 nM 34 Therefore, our findings provide the first example of pharmacokinetic characteristics exhibiting the smooth release of therapeutic small molecules without the burst effects or typical peaks and troughs observed in the absence of multiple dosings or polymeric mediators. 35，36 These results validate our approach, demonstrating that the balanced hydrophilicity / lipophilicity of our prodrug controls its release from subcutaneous tissue. This slow release, combined with TTR binding (reduced renal filtration) and CES2 bioactivation, results in controlled and stable plasma concentrations of naloxone released in rats.

[0448] We then evaluated the BBB osmosis (15 µmol kg) of AG10-L-E2-naloxone in rats. -1 Subcutaneous Figure 3b The brain concentrations of naloxone released from AG10-L-E2-naloxone (7.8 ± 1.7 nM at 10 min and 30.1 ± 2.1 nM at 30 min) were 30 to 60 times lower than those of naloxone administered in free form (236.3 ± 50 nM at 10 min and 1721.7 ± 280 nM at 30 min). Similar results were observed in cerebrospinal fluid (CSF). Figure 3c The brain-to-plasma ratio (250.3% ± 24.1% at 30 min) and CSF-to-plasma ratio (137.7% ± 29.7% at 30 min) of naloxone derived from AG10-L-E2-naloxone were similar to those of naloxone administered in free form. No detectable levels of AG10-L-E2-naloxone or its hydrolysis product AG10-L-E2-COOH were found in the brain or CSF. Figure 10Molecules with plasma concentrations <2% in the brain or CSF are not considered to cross the BBB. These findings support our hypothesis that hydrophilicity and selective TTR binding limit the ability of AG10-conjugates to cross the BBB.

[0449] AG10-L-E2-Naloxone for the prevention of death in rats following two doses of morphine or fentanyl We evaluated AG10-L-E2-naloxone as an antidote for opioid (morphine and fentanyl) overdose. Opioid dosage was based on preliminary studies and literature, with morphine dosage at 100 mg / kg. -1 (in the report LD) 50 64-140 mg kg -1 )Inside) 37 And fentanyl is 3 mg / kg -1 (Reported by LD) 50 ) 38 Three groups of male rats (each group) n = 9) Receive morphine (100 mg / kg) -1 (Subcutaneous). 5 minutes later: Group 1 received the medium; Group 2 received naloxone (15 µmol kg). -1 Equivalent to 5 mg kg -1 Subcutaneous); Group 3 received AG10-L-E2-naloxone (15 µmol kg) -1 (Subcutaneous). All controls given the mediator died after the first morphine dose, while the naloxone and AG10-L-E2-naloxone groups survived. Figure 3d Six hours later, a second dose of morphine (50 mg / kg) was administered to the naloxone and AG10-L-E2-naloxone groups. -1 (Subcutaneous) to simulate re-anesthesia. All rats treated with naloxone died within 2 hours. Notably, all rats treated with AG10-L-E2-naloxone survived.

[0450] When using fentanyl (3 mg kg each) -1 Two doses, subcutaneously, at 0 hours and 6 hours; each group n In a similar experiment (= 8), six rats treated with the medium died after the first dose, and the remaining two died after the second dose. Figure 3b In the naloxone group, all rats died after the second fentanyl dose. The higher survival rates of two rats in the mediator group after the second fentanyl dose, compared to the naloxone group, may indicate acute tolerance, which typically develops rapidly over hours or days and can occur with a single high dose or short, repeated doses. 39，40This rapid tolerance can be attributed to receptor desensitization or internalization, which temporarily reduces opioid receptor responsiveness. Acute tolerance differs from chronic tolerance, which develops over time with prolonged use and involves more persistent receptor downregulation (reduced receptor number) and changes in gene expression.

[0451] In the AG10-L-E2-naloxone group, only one rat died after the second fentanyl dose. These results confirm that AG10-L-E2-naloxone is superior to naloxone in reducing mortality in rats given very high doses of morphine and fentanyl. The efficacy data also correlated well with pharmacokinetic studies using the same dose of the antagonist. Figure 3a At 6 hours, no naloxone administered in free form was detected, but 9 nM naloxone from AG10-L-E2-naloxone was present. These results suggest that the prolonged release of naloxone from AG10-L-E2-naloxone provides protection against re-anesthesia and death in rats. To confirm that the short half-life of naloxone is the reason for its lack of efficacy in mortality studies, we conducted experiments to replicate a clinical setting (…). Figure 11 (Instead of a single dose of 5 mg / kg) -1 Subcutaneous dose, both groups of rats received 1 mg kg -1 The initial loading subcutaneous dose of naloxone. Group 1 then received five smaller doses of naloxone (0.2 mg / kg subcutaneously per hour). -1 A total of 1 mg kg was obtained after 6 hours. -1 Group 2 received ten smaller doses of naloxone (0.2 mg / kg subcutaneously per hour). -1 A total of 2 mg kg was obtained after 12 hours. -1 Data showed that continuous administration of smaller doses of naloxone effectively reduced mortality. While continuous subcutaneous administration of naloxone for up to 12 hours reduced mortality after a second fentanyl dose at 6 hours (only 2 out of 8 rats died), Figure 11 (blue line in the diagram), but stopping naloxone administration 30 minutes before the second fentanyl dose at 6 hours significantly increased mortality (6 out of 8 rats died). Figure 11 (The gray line in the image). This result highlights the challenges of requiring continuous naloxone administration in the clinical setting, as discussed in detail below. Importantly, Figure 3e The data presented illustrate the potential clinical utility and advantages of a single subcutaneous dose of AG10-L-E2-naloxone in providing protection against reanesthesia. Given the urgent need for an effective antidote, the following studies will focus on evaluating the effects of AG10-L-E2-naloxone on fentanyl overdose.

[0452] Reversal of opioid-induced respiratory depression (OIRD) in rats Systemic side effects of opioid toxicity include life-threatening opioid-induced respiratory depression (OIRD), which can be fatal, especially in patients who have never received opioids. 3，41，42 We tested our prodrug (15 µmol kg) using a non-invasive whole-body plethysmography method. -1 Single subcutaneous dose, n = 6) Reversing the effects of high doses of fentanyl (LD50) in untreated male rats 50 3 mg kg -1 The ability of a single subcutaneous dose to induce OIRD ( Figures 3f-3i The usage is more than 10 times higher than reported in the literature. 5-7 This high dose of fentanyl is used to mimic the OIRD of effective synthetic opioid analogues. After the first fentanyl dose ( Figure 3g Neither the mediator nor the lipid-modified naloxone prodrug C12-E2-naloxone reversed OIRD (although hydrolysis was observed in rat plasma). Figure 1g This was expected, as lipogenesis slows the release of the prodrug from subcutaneous tissue. We also had difficulty formulating C12-E2-naloxone, requiring doubling the volume of the dosing medium. Two of the six rats in each group died, and therefore we did not continue testing the C12-E2-naloxone prodrug. Naloxone and the alkyl ester AG10-L-E1-naloxone provided rapid reversal of OIRD within 5 minutes. For AG10-L-E2-naloxone, recovery began at 65 minutes, but minute ventilation did not reach normal levels (55.2% ± 16.6% of baseline at 90 minutes).

[0453] To test the prevention of fentanyl-induced respiratory depression (re-anesthesia) after initial reversal, rats were given a second dose of fentanyl (1.5 mg kg) 6 hours after naloxone or prodrug administration. -1 (subcutaneous) Figure 3h Naloxone failed to prevent OIRD, and four out of six rats died. AG10-L-E1-naloxone was also ineffective in preventing OIRD. AG10-L-E2-naloxone significantly prevented OIRD after a second fentanyl dose. The rats received a third fentanyl dose (1.5 mg / kg) 24 hours later. -1 (Subcutaneous). Compared with naloxone and AG10-L-E1-naloxone (one of the six rats in the AG10-L-E1-naloxone group died), AG10-L-E2-naloxone again demonstrated higher efficacy in preventing death and reversing OIRD. Figure 3iThe slow onset of AG10-L-E2-naloxone suggests that effective rescue from a first fentanyl dose may require enhanced initial naloxone release from the prodrug. However, this slow-release characteristic may be advantageous for certain therapeutic applications (discussed below).

[0454] AG10-L-E2-Naloxone is more effective than naloxone and nalmefone in preventing fentanyl re-anesthesia. Our data show that low-dose AG10-L-E2-naloxone effectively reduced two fentanyl doses (3 mg kg each). -1 The mortality rate after [the initial release of naloxone] was [high / low]. However, plethysmography results indicated a need to increase the initial concentration of naloxone released. Therefore, we evaluated higher doses of AG10-L-E2-naloxone (4-fold higher; 60 µmol kg / kg). -1 Equivalent to 63 mg kg -1 Pharmacokinetic characteristics of a single subcutaneous dose Figure 4a (See Table 3). The cutoff time for all time points was 96 hours. At the corresponding 72 and 96 hours, the plasma concentrations of naloxone administered in free form were below the limit of quantification (BLQ). The C-value of naloxone administered in free form... max The AG10-L-E2-naloxone dose (6,498 nM) was significantly higher than that released from AG10-L-E2-naloxone (36 nM). The pharmacokinetic profile of the higher AG10-L-E2-naloxone dose followed a flat release curve similar to that of the lower dose (approximately 30 nM at 30 minutes). max C at 15 nM at 96 hours min The pharmacokinetic characteristics of AG10-L-E2-naloxone strongly support inverted pharmacokinetics, as demonstrated by other subcutaneously administered drugs such as insulin glargine and buprenorphine. 15，43 .

[0455] Although at a low 5 mg kg -1 At subcutaneous doses, no naloxone administered in free form was detected at 6 hours, but we hypothesize that at higher doses (20 mg / kg)... -1 The low levels of naloxone observed at later time points following the subcutaneous dose may be due to residual naloxone deposited in the subcutaneous tissue after administration. This hypothesis is supported. Figure 1b Supported by data from [reference needed], which showed that naloxone has lower solubility (170 µM) at physiological pH compared to a weakly acidic dosing solution (200 µM). Following administration, at physiological pH, the small insoluble fraction of naloxone gradually dissolves and is absorbed at a slower rate than the initial major soluble fraction, resulting in a more stable curve at later time points.

[0456] Another possible explanation for this observation is the redistribution of naloxone from tissue reservoirs following the initial high plasma concentration. To investigate this, we compared 5 mg / kg... -1 and 20 mg kg -1 Pharmacokinetic parameters for subcutaneous doses (see Tables 2 and 3). Increasing the dose fourfold resulted in C... max and AUC inf These increased by approximately 4.5-fold and 6-fold, respectively. To eliminate the effect of later time points on AUC, we compared 5 mg / kg... -1 With 20 mg kg -1 AUC between subcutaneous doses 0-6小时 ( Figure 12 ). 20 mg kg -1 AUC of dose 0-6小时 Also more than 5 mg kg -1 A six-fold increase in dosage indicates that higher naloxone doses exhibit dose nonlinearity due to decreased clearance, likely caused by enzyme saturation. This is reflected in a 20 mg / kg dose. -1 Subcutaneous dose and 5 mg kg -1 A slower slope for subcutaneous doses compared to those from 0.5 hours to 2 hours indicates reduced clearance. Figure 12 Importantly, the clearance of both doses of naloxone followed linear kinetics after 2 hours, indicating that tissue redistribution had the least impact at later time points, and that the dissolution of precipitated naloxone was a more plausible explanation.

[0457] We then tested higher doses (60 µmol kg). -1 The efficacy of AG10-L-E2-naloxone is included, and it also includes high doses of the FDA-approved antidote naloxone and nalmefene (60 µmol kg). -1 (as a control) Figures 4b-4e Following the first fentanyl dose, nalmefone reversed OIRD within 2 minutes, but withdrawal symptoms caused hyperventilation and mild spasticity. 44，45 ( Figure 4c Three control rats died. Higher doses of AG10-L-E2-naloxone were more effective than lower doses. After the second fentanyl dose, nalmefenone and AG10-L-E2-naloxone were equally effective in reversing OIRD, indicating that naloxone released 6 hours after administration of AG10-L-E2-naloxone reached effective levels. Figure 4d Naloxone was ineffective after the second fentanyl dose, and two rats died. After a third fentanyl dose at 24 hours (to simulate reanesthesia), AG10-L-E2-naloxone was effective against OIRD, while nalmefone was ineffective, and one rat died. Figure 4eTo the best of our knowledge, these data suggest that AG10-L-E2-naloxone is the longest-acting μ-opioid antagonist prodrug reported, with no withdrawal symptoms observed, and has the potential to act as both a reversal agent and a preventative agent.

[0458] High doses of naloxone do not provide additional efficacy relative to lower doses. The lack of correlation between the pharmacokinetics and pharmacodynamics of high-dose naloxone was unexpected. Although naloxone achieved a considerable plasma concentration (25 nM) at 6 hours, similar to AG10-L-E2-naloxone (21 nM), rats treated with high doses of naloxone performed poorly in plethysmography experiments following a second fentanyl dose, with two rats dying. Figure 4a and Figure 4d These data suggest that high initial levels of naloxone (6,498 nM at 15 minutes) can be harmful, making animals more susceptible to the toxic effects of a second dose of fentanyl. For further investigation, we conducted efficacy studies in which we administered fentanyl intravenously and high doses of the antagonist (naloxone and AG10-L-E2-naloxone) (60 µmol kg). -1 (Single intravenous dose) Figure 4g-Figure 4i Prior to beginning our efficacy studies, we conducted pharmacokinetic studies on high intravenous doses of naloxone and AG10-L-E2-naloxone. Figure 4f 60 µmol kg -1 (Single intravenous dose) (Table 4). Data showed high early concentrations of both naloxone (20,597 nM at 15 minutes) and naloxone released from AG10-L-E2-naloxone (1,599 nM at 15 minutes). The cutoff time for all time points in the pharmacokinetic studies was 24 hours. For 20 mg / kg -1 Dosage, intravenous / subcutaneous C max The ratios were 8-fold for naloxone and 500-fold for AG10-L-E2-naloxone, indicating a significant reservoir effect of AG10-L-E2-naloxone following subcutaneous administration due to flipping kinetics caused by slower dissolution and gradual absorption (see Tables 3 and 4). The high intravenous / subcutaneous AUC ratio of AG10-L-E2-naloxone (3-fold for naloxone and 50-fold for AG10-L-E2-naloxone) further supports this. To explain the 20 mg / kg... -1 We calculated the steady-state volume of distribution (V0.05) based on the high naloxone concentration at the 24-hour cutoff point after intravenous administration. ss And it was found to be 12.4 Lkg. -1 Its smaller dose (1.6 mg kg) -1 V ss = 1.23 L kg-1 10 times higher 17 This reflects nonlinear pharmacokinetics, with more naloxone distributed to tissues at higher doses. The decreased clearance (CL = 1.3 L / hr / kg for high doses vs. 3.4 L / hr / kg for low doses) also indicates nonlinear pharmacokinetics, similar to the toxicokinetics observed in opioid overdose, where zero-order elimination dominates. In zero-order elimination, small increases in opioid doses can disproportionately raise plasma levels, leading to toxicity, as a constant amount of drug is eliminated over time. This results in long-term and severe opioid toxicity, where standard pharmacokinetic parameters often become irrelevant. 3 .

[0459] For efficacy studies ( Figure 4g-Figure 4i ; Antagonist at 60 µmol kg -1 Administered; a single intravenous dose), although both treatments reversed OIRD after the first fentanyl dose, rats treated with naloxone experienced marked hyperventilation and severe convulsions, unlike animals treated with AG10-L-E2-naloxone. Figure 4g Four rats in the vector group died. Both treatments showed high plasma naloxone concentrations at 6 hours (750 nM naloxone and 60 nM naloxone released from AG10-L-E2-naloxone) and at 24 hours (395 nM and 44 nM, respectively). However, neither treatment effectively protected against OIRD after the second (6-hour) and third (24-hour) doses of fentanyl. Figure 4h , Figure 4i These findings suggest that extremely high initial naloxone levels may harm animals or alter μ-opioid receptors, thereby reducing the effectiveness of naloxone over time. Rare reports of pulmonary edema induced by high doses of naloxone in humans have also been reported. 46，47 We believe that prodrugs such as AG10-L-E2-naloxone can help in further research on these effects.

[0460] AG10-L-E2-naloxone is a potential preventative and control agent against fentanyl-induced death and OIRD. To compensate for the lower efficacy of AG10-L-E2-naloxone in the first 30 minutes after administration, we combined the prodrug with a small amount of free naloxone. A single subcutaneous dose of AG10-L-E2-naloxone (60 µmol kg) -1 ) and naloxone (3 µmol kg -1 Equivalent to 1 mg kg -1 It provided excellent efficacy, completely reversing OIRD after all three fentanyl doses without causing severe hyperventilation or spasms, indicating a low risk of withdrawal symptoms. Figures 5a-5d ).

[0461] We also explored AG10-L-E2-naloxone as a prophylactic agent against fentanyl. Rats were administered AG10-L-E2-naloxone (60 µmol kg). -1 (subcutaneous) or naloxone (15 µmol kg) -1 and 60 µmol kg -1 (subcutaneous) Figures 5e-5g Minute ventilation remained unchanged compared to animals treated with vectors. Figure 5f Six hours later, fentanyl (1.5 mg / kg) was administered. -1 (Subcutaneous) stimulation of rats. AG10-L-E2-naloxone significantly protected rats from OIRD, and all animals survived. In contrast, both doses of naloxone were ineffective, with multiple deaths in each group. Figure 5g This data further supports our hypothesis that higher doses of naloxone adversely affect efficacy and, even in the presence of large amounts of naloxone, increase susceptibility to fentanyl toxicity. These results highlight the challenges of using higher doses of naloxone to combat fentanyl overdose and support the possibility of AG10-L-E2-naloxone as a more effective antidote.

[0462] Multiple small doses of naloxone provide better efficacy against fentanyl-induced OIRD. Long-term opioid antagonist treatment leads to an increase in µ-opioid receptor density (receptor upregulation) without altering receptor affinity, resulting in increased sensitivity to opioid agonists. 48 In one study, 7 days of naloxone infusion in mice showed dose-dependent upregulation of the receptors. While low subcutaneous doses (0.1–1 mg / kg / day) caused minimal changes, a high dose of 10 mg / kg / day resulted in an 80% increase in µ-opioid receptor density. This upregulation also enhanced the analgesic potency of morphine, indicating ...

Claims

1. A compound of formula (I): Or its pharmaceutically acceptable salt, wherein: T is a selective ligand for the thyroxine transporter protein TTR; L stands for connector; L B Click products that do not exist or are formed through a click reaction between the first click handle and the second click handle; E is a cleavable ester; and D 1 It is an opioid antagonist or an opioid agonist.

2. The conjugate as claimed in claim 1, wherein: L B The following are the click products: (i) Copper-catalyzed reactions between azides and alkynes; (ii) The reaction between azides and dibenzocyclooctene (DBCO); (iii) Reverse electron-demanding Diels-Alder cycloaddition (IEDDA) between the trans-cyclooctene (TCO) moiety and the tetrazine ring, or (iv) Staudinger reaction between azides and phosphine.

3. The conjugate of claim 1, comprising the following portion: , , or , Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 Cycloalkyl, p is 0, 1, 2 or 3, and Indicates the connection point with the connector (L).

4. The conjugate of claim 1, wherein T has the structure of compound (A): (A)， Or its pharmaceutically acceptable salt, wherein: X a X b and X c Independently selected from C(R) 8a (R) 8b ), O, NR 8b Or S; where R 8a and R 8b Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino and cyano; The A ring is a 4- to 12-membered ring, and in some embodiments, the 4- to 12-membered ring is an aromatic or heteroaromatic ring; Each Y is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted amino, amino, substituted amino, acylamino, substituted acylamino, sulfonamide, sulfonyl fluoride, thioester and cyano; c is an integer in the range of 0 to 5; and The B ring is a heterocycle selected from the following (h1-h30): Where R 11 -R 16 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino, and cyano; R 17 Selected from hydroxyl, alkyl, amino, and alkylamino; and R 11 -R 16 At least one of them is with X c The linking group.

5. The conjugate of claim 1, wherein T has the structure of compound (B): Or its pharmaceutically acceptable salt, wherein: n is an integer in the range of 0 to 8; R 18 R 19 and R 20 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino, sulfonamide, sulfonyl fluoride, thioester and cyano; X a It is C(R) 21 (R) 22 ), O, NR 22 Or S; where R 21 and R 22 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino and cyano; A is a 5- to 12-membered ring, and in some embodiments, the 5- to 12-membered ring is an aromatic or heteroaromatic ring; Each Y is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino, sulfonamide, sulfonyl fluoride, thioester, and cyano; and c is a number from 0 to 5.

6. The conjugate of claim 1, wherein T has the structure of compound (C): (C)， Or its pharmaceutically acceptable salt, wherein: n is an integer in the range of 1 to 4; R 23 It is a short-chain alkyl group having 1 to 4 carbon atoms; R 24 It is hydrogen; R 25 It is a short-chain alkyl group having 1 to 4 carbon atoms; X a It is C(R) 21 (R) 22 ), O, NR 22 Or S; where R 21 and R 22 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino and cyano; Each Y is independently selected from hydrogen, halogen, acyl, substituted acyl, carboxyl, heterocyclic group, alkoxycarbonyl, sulfonamide, sulfonyl fluoride, thioester, and substituted alkoxycarbonyl; and c is 2.

7. The conjugate of claim 1, wherein T has the structure of compound (D): (D)， Or its pharmaceutically acceptable salt, wherein: n is 1 to 8; R 23 R 24 and R 25 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino and cyano; X a It is C(R) 21 (R) 22 ), O, NR 22 Or S; where R 21 and R 22 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, heterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino and cyano; R a It is CHO, COOH, COOCH3, COOR 6 CONR 7 R 8 Tetrazolyl, CONHOH, B(OH)2, CONHSO2Ar, CONHCH(R) 9 COOH, CF3, hydrogen, halogen, alkyl, substituted alkyl, acyl, substituted acyl, carboxyl, heterocyclic group, sulfonamide, sulfonyl fluoride, thioester, alkoxycarbonyl or substituted alkoxycarbonyl; R b It is CHO, COOH, COOCH3, COOR2 6 CONR2 7 R2 8 Tetrazolyl, CONHOH, B(OH)2, CONHSO2Ar, CONHCH(R2) 9 COOH, CF3, hydrogen, halogen, alkyl, substituted alkyl, acyl, substituted acyl, carboxyl, heterocyclic group, sulfonamide, sulfonyl fluoride, thioester, alkoxycarbonyl or substituted alkoxycarbonyl; R2 6 It is an alkyl, haloalkyl, cycloalkyl, or heterocyclic group; R 27 and R 28 Each is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, or heteroaryl; and, R 29 It is a side chain of a naturally occurring α-aminocarboxylic acid.

8. The conjugate of claim 7, wherein T has the structure of compound (E): (E) Or its pharmaceutically acceptable salt.

9. The conjugate of claim 7, wherein T has the structure of compound (Y): (Y) Or its pharmaceutically acceptable salt.

10. A compound of formula (II'): (II’), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, 3, or 4 R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L stands for connector; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the following groups: hydrogen, halogen atoms (e.g., Cl, Br, or F), NO2, CF3, OMe, O(C 1-4 Alkyl), hydroxyl, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; and D 1 It is an opioid antagonist or an opioid agonist.

11. A compound of formula (III'): (III’) Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, 3 or 4; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; L B It is a click product formed by the click reaction between the first click handle and the second click handle; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the following groups: hydrogen, halogen atoms (e.g., Cl, Br, or F), NO2, CF3, OMe, O(C 1-4 Alkyl), hydroxyl, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; and D 1 It is an opioid antagonist or an opioid agonist.

12. A compound of formula (IV'): (IV’) Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, 3 or 4; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; L B It is a click product formed by the click reaction between the first click handle and the second click handle; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the following groups: hydrogen, halogen atoms (e.g., Cl, Br, or F), NO2, CF3, OMe, O(C 1-4 Alkyl), hydroxyl, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 Cycloalkyl; p is 0, 1, 2 or 3; and D 1 It is an opioid antagonist or an opioid agonist.

13. A compound of formula (V'): (V’)， Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 Cycloalkyl; p is 0, 1, 2 or 3; and D 1 It is an opioid antagonist or an opioid agonist.

14. A compound of formula (VI'): (WE') Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl p is 0, 1, 2, or 3; and D 1 It is an opioid antagonist or an opioid agonist.

15. A compound of formula (VII'): (VII’), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; L B It is a click product formed by the click reaction between the first click handle and the second click handle; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; r is 1, 2, 3, 4, 5, 6, or 7; s is 1, 2, 3, 4, 5, 6, or 7; and D 1 It is an opioid antagonist or an opioid agonist.

16. A compound of formula (VIII'): (VIII’) Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; Y 1 Is it a key or C? 1-4 Alkylene; Y 2 It is NR 6 , O or S; L B It is a click product formed by the click reaction between the first click handle and the second click handle; R 6 Is it H or C? 1-4 alkyl; Each R 7 Independently selected from the group consisting of: hydrogen, halogen atoms (e.g., Cl, Br, or F), hydroxyl group, -OCF3, -NH2, -NHC 1-4 Alkyl, -N(C) 1-4 Alkyl group 2, -CONH2, -CONHC 1-4 Alkyl, -CON(C) 1-4 Alkyl)2、-NHCOC 1-4 Alkyl, C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 cycloalkyl; p is 0, 1, 2, or 3; r is 1, 2, 3, 4, 5, 6, or 7; s is 1, 2, 3, 4, 5, 6, or 7; t is 1, 2, 3, or 4; u is 1, 2, 3, or 4; and D 1 It is an opioid antagonist or an opioid agonist.

17. A compound of formula (IX): (IX), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L stands for connector; Z 1 It is C 1-6 Alkyl, C 6-10 Aryl or 5 to 10-membered heteroaryl, among which The C 1-6 Alkyl groups are optionally surrounded by one or more Z 1a Replace, and The C 6-10 Aryl or 5- to 10-membered heteroaryl groups are optionally surrounded by one or more Z groups. 1b replace; Each Z 1a or Z 1b Independently halogenated, C 1-6 alkoxy or C-group optionally substituted with a halogen group 1-6 Alkyl; and D 1 It is an opioid antagonist or opioid agonist (opioid agonist or opioid antagonist).

18. A compound of formula (X): (X)， Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; L B It is a click product formed by the click reaction between the first click handle and the second click handle; Z 1 It is C 1-6 Alkyl, C 6-10 Aryl or 5 to 10-membered heteroaryl, among which The C 1-6 Alkyl groups are optionally surrounded by one or more Z 1a Replace, and The C 6-10 Aryl or 5- to 10-membered heteroaryl groups are optionally surrounded by one or more Z groups. 1b replace; Each Z 1a or Z 1b Independently halogenated, C 1-6 alkoxy or C-group optionally substituted with a halogen group 1-6 Alkyl; and D 1 It is an opioid antagonist or an opioid agonist.

19. A compound of formula (XI): (XI), Or its pharmaceutically acceptable salt, wherein: Each R 1 Independently a halogenated or C 1-4 alkyl; m is 0, 1, 2, or 3; R 2 Is it H or C? 1-4 alkyl; X 1 Is it -O- or -NR? x -; n is 0, 1, 2, or 3; R 3 Is it H or C? 1-4 alkyl; R 4 Is it H or C? 1-4 alkyl; R 5 Is it H or C? 1-4 alkyl; R x Is it H or C? 1-4 alkyl; L A It is C 3-20 Alkylene, wherein The C 3-20 Alkylene is optionally surrounded by one or more L 1a Replace, and One or more alkylene units are optionally selected from -O-, -NR y - and -S- group substitutions; Each L 1a It is a halogen group, C 1-6 Alkyl or halogenated C 1-6 alkyl; Each R y Is it H or C? 1-4 alkyl; Z 1 It is C 1-6 Alkyl, C 6-10 Aryl or 5 to 10-membered heteroaryl, among which The C 1-6 Alkyl groups are optionally surrounded by one or more Z 1a Replace, and The C 6-10 Aryl or 5- to 10-membered heteroaryl groups are optionally surrounded by one or more Z groups. 1b replace; Each Z 1a or Z 1b Independently halogenated, C 1-6 alkoxy or C-group optionally substituted with a halogen group 1-6 Alkyl; and D 1 It is an opioid antagonist or an opioid agonist.

20. The conjugate according to any one of claims 1-19, wherein D 1 It is naloxone, nalmefen, naltrexone, methylnaltrexone, nalbuphine, or nalprofen.

21. The conjugate according to any one of claims 1-19, wherein D 1 It is an opioid antagonist.

22. The conjugate of claim 21, wherein the opioid antagonist is naloxone or nalmefene.

23. A method for reversing reanesthesia (ventilation depression) caused by an overdose of synthetic opioids, the method comprising administering the conjugate of claim 21 or claim 22.

24. The method of claim 23, wherein the synthetic opioid is fentanyl or a fentanyl analogue.

25. The method of claim 23 or claim 24, further comprising administering a free (unconjugated) form of the opioid antagonist.

26. The method of claim 25, wherein the opioid antagonist administered in free (unconjugated) form reacts with D in the conjugate. 1 same.

27. The method of claim 25, wherein the opioid antagonist administered in free (unconjugated) form reacts with D in the conjugate. 1 different.

28. The method of any one of claims 25-27, wherein the opioid drug administered in free (unconjugated) form is naloxone.

29. The method of any one of claims 25-27, wherein the opioid drug administered in free (unconjugated) form is nalmefene.

30. A method for treating the effects of opioid use disorder (OUD) in a human subject, the method comprising administering the conjugate of claim 21 or claim 22.

31. The method of claim 30, further comprising administering a free (unconjugated) form of the opioid antagonist.

32. The method of claim 31, wherein the opioid antagonist administered in free (unconjugated) form reacts with D in the conjugate. 1 same.

33. The method of claim 31, wherein the opioid antagonist administered in free (unconjugated) form reacts with D in the conjugate. 1 different.

34. The method of any one of claims 31-33, wherein the opioid drug administered in free (unconjugated) form is naloxone.

35. The method of any one of claims 31-33, wherein the opioid drug administered in free (unconjugated) form is nalmefene.

36. The method of any one of claims 23-35, wherein the conjugate is administered at a dose of about 0.01 mg / kg to about 40 mg / kg.

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