WO2024211360A2

WO2024211360A2 - Specific reversal agents to treat acute and chronic toxicity of fentanyls

Info

Publication number: WO2024211360A2
Application number: PCT/US2024/022762
Authority: WO
Inventors: Yan Zhang
Original assignee: Virginia Commonwealth University
Current assignee: Virginia Commonwealth University
Priority date: 2023-04-04
Filing date: 2024-04-03
Publication date: 2024-10-10
Anticipated expiration: 2025-10-04
Also published as: WO2024211360A3

Abstract

Mu opioid receptor (MOR) antagonists that specifically and effectively reverse the acute and chronic toxicity of fentanyl and its analogs are provided. The antagonists were developed by modifying the structural skeleton of fentanyl and/or phenylfentanil. The antagonists compete with fentanyls at the MOR binding site and when bound, they reverse the toxicity of fentanyls more effectively and selectively or specifically than naloxone, naltrexone, and other epoxymorphinan-type opioids.

Description

SPECIFIC REVERSAL AGENTS TO TREAT ACUTE AND CHRONIC TOXICITY

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of United States provisional patent application 63/456,837 filed April 4, 2023. STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under grant number UG3DA054785 awarded by the National Institutes of Health. The United States government has certain rights in the invention. BACKGROUND OF THE INVENTION Technical Field The invention generally relates to agents to treat fentanyl toxicity and methods of using the agents. In particular, the invention provides agents having a structural skeleton similar to that of fentanyl and/or phenylfentanil and which recognize and antagonize the mu opioid receptor (MOR) selectively, thereby competing with fentanyl at the MOR without activating the MOR. Description of Related Art Opioid use disorders (OUDs) remain a pervasive public health issue in the United States and worldwide with exorbitant financial burden to our health care networks. There are currently over 10 million American adults that consume opioids within a week’s span and over four million of them consume said opioids regularly. Further, it is estimated that two percent of all adults in the U.S. meet the criterion for an opioid use disorder; this translates to over two million people in the U.S. battling an opioid use disorder, a number that is thought to exceed fifteen million worldwide. The terminal marker of the state of the overdose crisis is the number of deaths attributable to overdose itself. In the U.S., from 1999 to 2018, over 750,000 people lost their lives due to a drug overdose – the majority of these deaths involved an opioid. From 2014 to present, the opioid crisis has been driven by a dramatic surge of synthetic opioids, which primarily include fentanyl (Figure 8) and its analogs. In the years 2016 and 2017, the fentanyl-associated deaths were a staggering 19,413 and 28,466, respectively. Providing a slim ray of hope, there was a slight decrease in opioid- related fatalities in the period from 2017 – 2018. This was a trend that appeared to be primarily driven by a decrease in misuse of prescription opioids as the number of overdose deaths due to synthetic opioids continued to grow (notably carfentanil became less available during this period as well). However, this seems to have been a brief reprieve as provisional data from the CDC show the number of overdose deaths increasing once again from 2019 – 2020 with the most dramatic increase of 53.1% as a result of synthetic opioids. From 2020 – 2021, there was a 26% increase in the number of opioid-related overdose deaths with an estimated 76,000 in the U.S. alone. The number is expected to grow rapidly due to the increased availability and use of illegally made fentanyl and its analogs culminating in increased toxicity of the drug supply. Such a dire situation has driven the FDA to approve two drugs for opioid overdose, Narcan® (naloxone, NLX) and Opvee® (nalmefene) (Figure 8). However, despite efforts to increase the availability and awareness surrounding NLX and nalmefene, the overdose death toll induced by fentanyl and its agonist analogs is an ongoing national crisis. Further, both NLX and nalmefene have limitations. Administration of either overdose reversal agent to those with opioid agonists on board results in opioid withdrawal symptoms. In addition, the duration of action of these opioid agonists may exceed that of NLX due to its short half-life of approximately 1 h, which may result in renarcotization wherein an overdose patient revived from naloxone goes into an overdose state once again after the naloxone wears off due to remaining opioid agonist. What is more concerning is that NLX has a relatively low potency to reverse the respiratory depression caused by fentanyls, which has been perceived as the major cause for overdose deaths. Nalmefene has a half-life of 11 h, much longer than naloxone, but it is not yet clear whether nalmefene will prevent renarcotization. Also, nalmefene has high affinity towards the kappa opioid receptor, which may lead to some off-target effects. Further, fentanyl showed binding affinity in the single digit micromolar range at the ^1A and ^1B adrenoceptor subtypes as well as the dopamine D4.4 and D1 receptor subtypes. In contrast, morphine and other epoxymorphinan derivatives, including naloxone, naltrexone, and nalmefene, do not carry such an affinity to those receptors. Thus, it has been suggested that naloxone may be ineffective against the centrally mediated noradrenergic and cholinergic effects of fentanyls. Clinically these effects manifest as severe muscle rigidity and airway compromise (i.e., wooden chest syndrome) that is rapid and distinct from the respiratory depression seen with morphine-like epoxymorphinan derivatives. Altogether, it is reasonable to speculate that non-opioid mechanisms may underly the respiratory depression caused by fentanyl and its agonist analogs. Therefore, development of a novel reversal agent sufficient for fentanyl overdoses may rely on distinguishing these two pharmacological profiles, i.e., opioidergic and adrenergic, at a molecular level. SUMMARY OF THE INVENTION Other features and advantages of the present invention will be set forth in the description of invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. The invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof. It is imperative to develop efficacious countermeasures for the foreseeable ‘fourth wave’ of the opioid overdose crisis largely driven by fentanyl and its analogs. Accordingly, described herein are biologically (physiologically) active compounds which are mu opioid receptor (MOR) antagonists. In some aspects, the compounds bind specifically or selectively. In some aspects, the compounds specifically and effectively reverse the acute and chronic toxicity of fentanyl and its analogs. The antagonists were developed by modifying the structural skeleton of fentanyl and/or phenylfentanil. The antagonists compete with fentanyls at the MOR binding site. It is an object of this invention to provide a compound having the general formula

where R = substituted C1-C16 alkyl, unsubstituted C1-C16 alkyl, aryl, heteroaryl or substituted aryl; R1 = substituted C1-C16 alkyl, unsubstituted C1-C16 alkyl, aryl, heteroaryl or substituted aryl; n = 0, 1, 2 or 3; m = 0, 1, 2 or 3; o = 0, 1, 2, or 3; wherein spacers associated with n, m, or o, when present, may be or include one or more methylenes, or O, N, or S atoms, and wherein X = or

the compound is not fentanyl, phenylfentanil, mirfentanil or thiophentanil. In some aspects, the compound is and R1 = .

In other aspects, the compound is .

In additional aspects, R is substituted C1-C16 alkyl, unsubstituted C1-12 alkyl, aryl, heteroaryl or substituted aryl. And in further aspects, R1 is substituted C1-C16 alkyl, unsubstituted C1-16 alkyl, aryl, heteroaryl or substituted aryl. In some aspects, n = 0. In other aspects, n = 0, 1 or 2. In some aspects, the substituted C1-C16 alkyl is substituted with N, O or S. Also provided is a method of toxicity or overdose in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one of the compounds disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Antinociceptive effects of VZFN compounds using the warm water tail immersion study in mice (n=6) at 56 ± 0.1 °C. All compounds were administered subcutaneously at a single dose of 10 mg/kg. Saline was adopted as the negative control while morphine and fentanyl were adopted as positive controls. Tail withdrawal latency was monitored 20 min post-injection in this agonism study. Data is represented as mean values ± SD. Figure 2. Blockage of morphine-mediated antinociceptive effects by VZFN compounds using the warm water tail immersion study in mice (n=6) at 56 ± 0.1 °C. All VZFN compounds were administered subcutaneously at a single dose of 10 mg/kg. Saline and naloxone were adopted as the negative and positive controls respectively. Morphine (10 mg/kg) was administered 5 min post compound administration followed by measurement of tail withdrawal latency after a 20 min period in this antagonism study. Data is presented as mean values ± SD. *P < 0.05, **P < 0.01, and ***P < 0.0005, ****P < 0.0001, compared to vehicle (s.c.). Figure 3. Blockage of fentanyl-mediated antinociceptive effects by VZFN compounds using the warm water tail immersion study in mice (n=6) at 56 ± 0.1 °C. All VZFN compounds were administered subcutaneously at a single dose of 10 mg/kg. Saline was adopted as the negative control respectively. Fentanyl (0.1 mg/kg) was administered 5 min post compound administration followed by measurement of tail withdrawal latency after a 20 min period in this antagonism study. Data is presented as mean values ± SD. *P < 0.05, **P < 0.01, and ***P < 0.0005, ****P < 0.0001, compared to vehicle (s.c.). Figure 4. Potency determination of selected VZFN compounds in the warm water tail immersion studies. Compounds at varying doses were administered s.c. to groups of six Swiss Webster mice each 5 min before administration of 10 mg/kg morphine (s.c.). Tail withdrawal latencies were measured 20 min post morphine administration. Data is presented as mean values ± SD. Figure 5. Potency determination of selected VZFN compounds in the warm water tail immersion studies. Compounds at varying doses were administered s.c. to groups of six Swiss Webster mice each 5 min before of 0.1 mg/kg fentanyl (s.c.). Tail withdrawal latencies were measured 20 min post fentanyl administration. Data is presented as mean values ± SD. Figure 6. Effects of VZFN compounds on ventilation in mice. Error bars represent the standard error of minute volume (MVb) mean values within individual 5 min bins. Open symbols indicate significant differences compared to the MVb or saline (SAL) treated controls at individual timepoints (P ^ 0.05) via one-way ANOVA. Figure 7A-C. Dose dependent effects of (A)VZFN093, (B) VZFN094 and (C) VZFN202 on fentanyl induced respiratory depression in mice. Error bars represent the standard error of normalized mean values within individual 5 min bins. Open symbols indicate significant differences compared to the fentanyl (0.3 FEN + SAL)-treated controls at individual timepoints (P ^ 0.05) via one-way ANOVA. Figure 8. Chemical structures of fentanyl and its analogs, morphine, and the FDA approved opioid antagonists. Figure 9. Design strategy of target phenylfentanil analogs. Figure 10A and B. Calcium mobilization assay of Compound 3 as an agonist (A) or as an antagonist (B) in the presence of DAMGO or fentanyl. Naltrexone (NTX) was used as the control. (Data are presented as mean values ± S.E.M.; n = 3) Figure 11A-C. Warm-water tail immersion assay of compounds 3-19 in mice (n = 6) at 56 ± 0.1 °C. (A) Antinociceptive effects of compounds 3-19 (10 mg/kg). (B) Blockage of morphine-mediated antinociception by selected compounds (10 mg/kg) in the presence of morphine (10 mg/kg). (C) Blockage of fentanyl-mediated antinociception by selected compounds (10 mg/kg) in the presence of fentanyl (0.1 mg/kg). All compounds were administered subcutaneously. For tests of agonism (A), tail withdrawal latency was monitored 20 min post-injection. For tests of antagonism (B-C), morphine or fentanyl were administered five min post compound administration followed by measurement of tail withdrawal latency after a 20-min period Saline and morphine were adopted as negative and positive controls, respectively. Data are presented as mean values ± SEM. Figure 12. Warm-water tail immersion study of compound 3. (Compound 3 at varying doses was given s.c. to groups of six mice each 5 min before 10 mg/kg morphine or 0.1 mg/kg fentanyl was administrated (s.c.). Tail withdrawal latencies were measured 20 min post morphine or fentanyl administration. ns: no significant difference at p < 0.05. Figure 13. Effects of fentanyl paired with either vehicle or naloxone on ventilation in mice. Open symbols indicate significant differences compared to the MVb of the Fentanyl + Vehicle (saline)-treated control group at individual timepoints (p ^ 0.03). Error bars represent the standard error of normalized MVb mean values. Figure 14A and B. (A) Effects of compound 3 (10 mg/kg s.c.) and fentanyl on mouse respiration and (B) lack of effect of NLX to increase the respiration post administration of compound 3. Open symbols indicate significant differences compared to the MVb of the fentanyl control group at individual timepoints (p ^ 0.03). Error bars represent the standard error of normalized MVb mean values. Figure 15. Highest scored docking poses of fentanyl at the (A) active ^1A- AR and (B) inactive ^1A- AR and Compound 3 at the (C) active ^1A- AR and (D) inactive ^1A- AR. ^1A-AR^active and ^1A-AR^inactive receptors are shown in grey and cyan cartoon representations. Interacting residues in the orthosteric binding pocket are shown as orange sticks and in the secondary pocket are shown as blue sticks. Fentanyl and Compound 3 are shown as magenta and green sticks, respectively. Figure 16. Highest scored docking pose of fentanyl at the (A) active ^1B- AR (gray) and (B) inactive ^1B- AR (cyan) and Compound 3 at the (C) active ^1B- AR (gray) and (D) inactive ^1B- AR (cyan). Interacting residues occupying the orthosteric site are shown as orange sticks and those parts of the secondary pocket are shown as salmon sticks. Fentanyl and Compound 3 are shown as magenta and green sticks, respectively. DETAILED DESCRIPTION DEFINITIONS Fentanyl (also known as fentanil) is a potent synthetic narcotic analgesic with a rapid onset and short duration of action. It is a strong agonist at the µ-opioid receptors. It is manufactured under the trade names of SUBLIMAZE®, ACTIQ®, DUROGESIC®, DURAGESIC®, FENTORA®, ONSOLIS INSTANYL®, ABSTRAL® and others. Congeners refers to chemical substances related to each other by origin, structure, or function. Generally, the congeners discussed herein are structural and/or chemical analogs and/or derivatives of fentanyl. Structural and/or chemical analogs (analogues) as used herein are compounds in which one or more individual atoms have been replaced, either with a different atom, or with a different functional group. A derivative as used herein a compound that is derived from a similar compound by at least one chemical reaction. As used herein, a C1-C16 carbon group is, for example, a C1-C16 carbon group that is saturated (e.g., C1-C16 alkyl) or unsaturated (having one or more double or triple bonds between C atoms), substituted (e.g., C1-C16 heteroalkyl) or unsubstituted, or cyclic (which can also be substituted or non-substituted and saturated or unsaturated) or non-cyclic (straight- chain or branched). “C1-C16 alkyl” as used herein means a straight or branched (non-cyclic) hydrocarbon chain having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16 carbon atoms. In some aspects, the C1-C16 alkyl is unsubstituted, examples of which include but are not limited to: methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 2- methylbutyl, 3-methylbutyl, 1,2-dinnethylpropyl, 1 ,1-dimethylpropyl, 2,2-dimethylpropyl, 1- ethylpropyl, n-hexyl, 2-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1 ,1- dimethylbutyl, 1 ,2-dimethylbutyl, 2,2-dimethylbutyl, 1 ,3-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1,1 ,2-trimethylpropyl, 1 ,2,2-trimethylpropyl, 1-ethylbutyl, 1-ethyl-2- methylpropyl, n-heptyl, 2-heptyl, 3-heptyl, 2-ethylpentyl, 1-propylbutyl, n-octyl, 2-ethylhexyl, 2-propylheptyl, nonyl, decyl, tridecane, tetradecane, pentadecane and hexadecane. In other aspects, the branched or unbranched C1-C16-alkyl group is substituted (C1- C16 heteroalkyl) so that at least one atom of the group is not C, but is a heteroatom (or heteroatomic group), for example O, S, N, N(=O), C(=O) or S(=O), etc. In some aspects, the C1-C62-alkyl group is cyclic and can include or consist of a 3C- 16C mono- or polycyclic cycloalkyl group containing only C atoms. 3C-C62-cycloalkyl encompasses mono-, bi- or tricyclic hydrocarbyl groups having 3 to 16 carbon atoms. Preferably, these groups are (C5-C16)-cycloalkyl. The 3C-C16-cycloalkyl groups have preferably 3 to 8, more preferably 5 or 6, atoms in each ring. The 3C-C16-cycloalkyl groups have preferably 3 to 8, more preferably 5 or 6, ring atoms. Suitable C3-C16 cycloalkyl groups include but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, undecanyl, cyclododecyl, cyclopentadecyl, norbornyl, and adamantly, and including macrocycles, i.e., molecules and ions containing a ring of twelve or more atoms. Classical examples include crown ethers, calixarenes, porphyrins, and cyclodextrins. In other aspects, the 3C-C16-cycloalkyl group is a heterocyclic group in which one or more C atoms is substituted by a heteroatom (or heteroatomic group) such as O, S, N, N(=O), C(=O) or S(=O). Examples not limited to: -O-(C3-012)-cycloalkyl, - S-(C3-012)-cycloalkyl, -N-(C3-012)-cycloalkyl, -COOH-(C3-C12)-cycloalkyl, CONH-(C3- C12)-cycloalkyl, etc. Suitable C3-C16-heterocycloalkyl groups include but are not limited to tetrahydrothiophenyl, tetrahydrofuryl, tetrahydropyranyl and dioxanyl. In further aspects, the expression C3-C16-cycloalkyl or C3-C16-heterocycloalkyl encompasses nonaromatic, saturated or partly unsaturated cycloaliphatic groups having 3 to 16 carbon atoms. In C3-C16-heterocycloalkyl groups, one or more of the ring carbon atoms is/are replaced by a heteroatom or a heteroatomic group such as O, S, N, N(=O), C(=O) or S(=O). The term “aryl” means an aromatic group having up to 14 carbon atoms. Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl, naphthacenyl, and the like. “Substituted phenyl” and “substituted aryl” denote a phenyl group and aryl group, respectively, substituted with one, two, three, four or five (e.g. 1-2, 1-3 or 1-4 substituents) chosen from halo (F, Cl, Br, I), hydroxy, hydroxy, cyano, nitro, alkyl (e.g., C1-6 alkyl), alkoxy (e.g., C1-6 alkoxy), benzyloxy, carboxy, aryl, and so forth. As used herein, “heteroaryl” refers an aromatic heterocarbocycle having 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including both mono- and polycyclic ring systems. Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic. Representative heteroaryls are furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It is contemplated that the use of the term “heteroaryl” includes N-alkylated derivatives such as a 1-methylimidazol-5-yl substituent. The term “aryl” when used without the “substituted” modifier also refers to a monovalent group, having a aromatic carbon atom as the point of attachment, said carbon atom forming part of a six-membered aromatic ring structure wherein the ring atoms are all carbon, and wherein the monovalent group consists of no atoms other than carbon and hydrogen. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C₆H₄CH₂CH₃ (ethylphenyl), -C₆H₄CH₂CH₂CH₃ (propylphenyl), - C6H4CH(CH3)2, -C6H4CH(CH2)2, -C6H3(CH3)CH2CH3 (methylethylphenyl), -C6H4CH=CH2 C₆H₄C ^ CCH₃, naphthyl, and the

group aryl” refers to a monovalent group, having a aromatic carbon atom as the point of attachment, said carbon atom forming part of a six-membered aromatic ring structure wherein the ring atoms are all carbon, and wherein the monovalent group further has at least one atom independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. Non-limiting examples of substituted aryl groups include the groups: -C6H4F, -C6H4Cl, -C6H4Br, -C6H4I, -C6H4OH, -C6H4OCH3, - C₆H₄OCH₂CH₃, -C₆H₄OC(O)CH₃, -C₆H₄NH₂, -C₆H₄NHCH₃, -C₆H₄N(CH₃)₂, -C6H₄CH₂OH, -C6H4CH2OC(O)CH3, -C6H4CH2NH2, -C6H4CF3, -C6H4CN, -C6H4CHO, -C6H4CHO, - C₆H₄C(O)CH₃, -C₆H₄C(O)C₆H5, -C₆H₄4CO₂H, -C₆H₄CO₂CH₃, -C₆H₄CONH₂, -C₆H₄CONHCH₃, and -C₆H₄CON(CH₃) ₂. A “biologically active” or “physiologically active” compound is one that has an effect on living matter, usually within the body of a mammal, particularly one which is an antagonist of fentanyls at the MOR binding site. An “isomer” of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs. A “stereoisomer” or “optical isomer” is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs. “Enantiomers” are stereoisomers of a given compound that are mirror images of each other, like left and right hands. “Diastereomers” are stereoisomers of a given compound that are not enantiomers. “Pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use. Spacer or linker refers to a short chain of atoms (e.g., from about 1-10 atoms). Spacers may contain C atoms (e.g. a C1-C6 alkyl chain) or may be heteroatomic. The expression "heteroatomic" in relation to "heteroatomic linker" refers to a linker group comprising carbon and one or more heteroatoms. Exemplary heteroatoms include one or more atoms selected from the group consisting of O, N, P and S, N=O, C=O, OH, etc., and combinations thereof, typically interspersed within a short carbon chain. Specificity describes the extent to a drug produces only the desired therapeutic effect without causing any other physiological changes. A drug with high specificity exhibits a strong drug–receptor interaction, ensuring targeted action and minimal side effects. Selectivity describes a drug's ability to affect a particular cell population in preference to others. COMPOUNDS Provided herein are novel fentanyl antagonists having the general Formula I

where R = substituted C1-16 alkyl, unsubstituted C1-16 alkyl, aryl, heteroaryl or substituted aryl; R1 = substituted C1-16 alkyl, unsubstituted C1-16 alkyl, aryl, heteroaryl or substituted aryl; n = 0, 1, 2 or 3; m = 0, 1, 2 or 3; o = 0, 1, 2, or 3; wherein spacers associated with n, m, or o, when present, may be or include one or more methylenes, or O, N, or S atoms, and wherein X =

or with the caveat that the compound is not fentanyl, phenylfentanil, mirfentanil or thiophentanil. In some aspects, the compound is ,

, 5 A depiction of a generic synthesis scheme of compounds of these aspects is shown below:

In further aspects, the compound .

PHARMACEUTICAL COMPOSITIONS The compounds described herein are generally delivered (administered) as a pharmaceutical composition. Such pharmaceutical compositions generally comprise at least one of the disclosed compounds, i.e., one or more than one (a plurality) of different compounds (e.g., 2 or more such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) may be included in a single formulation. Accordingly, the present invention encompasses such formulations/compositions. The compositions generally include one or more substantially purified compounds as described herein, and a pharmacologically suitable (physiologically compatible, biologically compatible) carrier, which may be aqueous or oil-based. In some aspects, such compositions are prepared as liquid solutions or suspensions, or as solid forms such as tablets, pills, powders and the like. Solid forms suitable for solution in, or suspension in, liquids prior to administration are also contemplated (e.g., lyophilized forms of the compounds), as are emulsified preparations. In some aspects, the liquid formulations are aqueous or oil-based suspensions or solutions. In some aspects, the active ingredients are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients, e.g. pharmaceutically acceptable salts. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, preservatives, and the like. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like are added. The composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of compound in the formulations varies, but is generally from about 1-99%. Still other suitable formulations for use in the present invention are found, for example in Remington's Pharmaceutical Sciences, 22nd ed. (2012; eds. Allen, Adejarem Desselle and Felton). Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as Tween® 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. "Pharmaceutically acceptable salts" of the compounds refers to the relatively non- toxic, inorganic and organic acid addition salts and base addition salts of compounds of the present disclosure. In some aspects, these salts are prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulfamates, malonates, salicylates, propionates, methylene-bis-^-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates and laurylsulfonate salts, and the like. See, for example S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 66, 1- 19 (1977) which is incorporated herein by reference. Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. ammonia, ethylenediamine, N- methyl-glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, and dicyclohexylamine, and the like. In some aspects, the salt is an HCl (hydrochloride) salt. The pharmaceutical preparation is administered in vivo by any suitable route including but not limited to: inoculation or injection (e.g. intravenous, intraperitoneal, intramuscular, subcutaneous, intra-aural, intraarticular, and the like); or by absorption through epithelial or mucocutaneous linings (e.g., nasal, oral, gastrointestinal mucosa, and the like). Other suitable means include but are not limited to: inhalation (e.g. intranasally as a mist or spray), orally (e.g. as a pill, capsule, liquid, etc.), etc. In preferred embodiments, the mode of administration is topical or oral or by injection. In addition, the compositions may be administered in conjunction with other treatment modalities such as pain medications, buprenorphine (to treat respiratory depression), oxygen, adenaline, and the like. In emergency setting, administration is generally by inhalation or IM injection. METHODS Provided herein are methods of reversing the acute and chronic toxicity of fentanyl or analogs thereof. In some aspects, the reversal involves treating an overdose of fentanyl or analog (analogue) thereof and/or fentanyl poisoning/toxicity (or poisoning with a fentanyl analog) in a subject in need thereof. Such methods generally comprise administering to the subject a therapeutically effective amount of at least one novel compound disclosed herein. A therapeutically effective amount is typically an amount sufficient to eliminate or lessen at least one symptom of the toxicity or overdose. In some aspects, the methods comprise first (prior to the step of administering) a step of identifying a subject suffering from or suspected of suffering from toxicity and/or an overdose of or poisoning with fentanyl or a fentanyl analog. Such subjects generally exhibit one or more symptoms of overdose/toxicity such as: stupor, changes in pupillary size, cold and clammy skin, cyanosis, coma, and respiratory depression (which may lead to respiratory failure and death). The presence of triad of symptoms such as coma, pinpoint pupils, and respiratory depression are strongly suggestive of opioid poisoning. The methods of the present disclosure encompass administering a therapeutically effective amount of at least one compound described herein to eliminate or lessen at least one of such symptoms. In some aspects, such cases are caused by the recreational use of fentanyl and analogs thereof. In other aspects, the toxicity and/or overdose is caused inadvertently or accidentally when fentanyl and/or an analog thereof is administered as part of a pain management program. These may be emergency situations, e.g., situations in which a subject is “found” or identified by a friend, family member, etc., and an ambulance or other emergency service is contacted, or the subject is transported e.g., to an emergency room. However, the problem of toxicity or overdose may be identified by a medical professional caring for the subject, or even by the subject his-or herself. In these situations, the subject is thus frequently treated in an emergency setting and administration must be immediate and facile, such as, for example, by: 1) spraying the pharmaceutical into the nose (intranasal ; 2) auto injecting using a pre- filled, ready to use dose of the medication (e.g., by pressing the auto injector against a person’s upper leg; intramuscular or IM); or 3) injecting the naloxone via a needle and syringe (intramuscular or IM). All such means of administration are encompassed by the present methods. The dose of a compound that is administered may be any that is suitable for the particular patient. Generally, the dose ranges from about 1 to about 500 mg/kg of body weight, such as about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 mg/kg, or even more, including all single digit integers between these values. In emergency overdose situations, generally one dose of a pharmaceutical composition is administered as soon as possible after the subject is identified as having had an overdose. The amount administered may generally ranges from about 0.01 to about 500 mg/kg of body weight of the recipient, such as from about 0.05 to 400 mg.kg, or 0.1 to about 300 mg per kg, or about 0.5 to about 200 mg/lg, or about 1 to about 100 mg/kg, such as about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100 mg/kg, inclusive, including all decimal fractions within these ranges. However, administration of more than one dose is also encompassed, depending on the amount of fentanyl or analog thereof that was taken by the subject. In some aspects, methods of preventing the death of a suspect who has overdosed and/or who has been poisoned with fentanyl or an analog thereof are provided. Subjects who are treated with the methods described herein are generally mammals, such as humans. However, veterinary applications of this technology are also encompassed. For example, carfentanil or carfentanyl, sold under the brand name Wildnil®, is a very potent opioid analgesic which is used in veterinary medicine to anesthetize large animals such as elephants, rhinoceroses, horses, cattle, various animals living in zoos or preserves, etc. In this context, administration may be by tranquilizer dart. If an inadvertent overdose occurs, one or more of the present compounds may be administered to the animal, usually also by dart or another suitable means (e.g. IM, inhalation, etc.). Generally, the drugs that cause addiction and/or overdose and/or symptoms of withdrawal that can be treated or prevented as described herein are fentanyl or synthetic congeners of fentanyl. Examples include but are not limited to: fentanyl and its derivatives including but not limited to: Alfentanil; Sufentanil; Remifentanil; Carfentanil; acetylfentanyl; des-methylfentanyl; butyrfentanyl; ocfentanil; acrylfentanyl; para- fluorofentanyl; para-fluoroisobutyrfentanyl; cyclopropyl fentanyl; cis-3-methyl fentanyl; etc. The fentanyl overdose or addiction that may be prevented or treated as described herein may be due to the intake of fentanyl alone, or fentanyl that is mixed with or added to other drugs such as heroin, cocaine, methamphetamine, and MDMA (3,4-methylenedioxy methamphetamine, i.e. ecstasy), etc. In further aspects, the compounds disclosed herein are used to prevent or treat addiction to fentanyl and congeners thereof. The compounds can be used in treatment centers, for example, as safe substitutes or replacements for fentanyl and congeners thereof. Typically, in these scenarios, specific doses of the compounds are prescribed for and provided to an addict or recovering addict under trained medical supervision. Methods for Manufacturing, Formulating, and Packaging Dose Units and Kits Dose units of the present compounds can be made using manufacturing methods available in the art and can be of a variety of forms suitable for administration, and can include an enteric coating or other component(s) to facilitate protection from stomach acid, where desired. Dose units can be of any suitable size or shape. The dose unit can be of any shape suitable for enteral administration, e.g., ellipsoid, lenticular, circular, rectangular, cylindrical, and the like. Dose units provided as dry dose units can have a total weight of from about 1 microgram to about 1 gram and can be from about 5 micrograms to 1.5 grams, from about 50 micrograms to 1 gram, from about 100 micrograms to 1 gram, from 50 micrograms to 750 milligrams, and may be from about 1 microgram to 2 grams. Dose units can comprise components in any relative amounts. For example, dose units can be from about 0.1% to 99% by weight of active ingredients per total weight of dose unit. In some embodiments, dose units can be from 10% to 50%, from 20% to 40%, or about 30% by weight of active ingredients per total weight dose unit. Dose units can be provided in a variety of different forms and optionally provided in a manner suitable for storage, for example, a bottle (e.g., with a closure device, such as a cap), a blister pack (e.g., which can provide for enclosure of one or more dose units per blister), a vial, flexible packaging (e.g., sealed Mylar or plastic bags), an ampule (for single dose units in solution), a dropper, thin film, a tube and the like. Containers can include a cap (e.g., screw cap) that is removably connected to the container over an opening through which the dose units disposed within the container can be accessed. Containers can include a seal serve as a tamper-evident and/or tamper- resistant element, which seal is disrupted upon access to a dose unit disposed within the container. Such seal elements can be, for example, a frangible element that is broken or otherwise modified upon access to a dose unit disposed within the container. Examples of such frangible seal elements include a seal positioned over a container opening such that access to a dose unit within the container requires disruption of the seal (e.g., by peeling and/or piercing the seal). Examples of frangible seal elements include a frangible ring disposed around a container opening and in connection with a cap such that the ring is broken upon opening of the cap to access the dose units in the container. Dry and liquid dose units can be placed in a container (e.g., bottle or package, e.g., a flexible bag) of a size and configuration adapted to maintain stability of dose units over a period during which the dose units are dispensed into a prescription. For example, containers can be sized and configured to contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more single dry or liquid dose units. The containers can be sealed or resealable. The containers can be packaged in a carton or kit (e.g., for shipment from a manufacturer to a pharmacy or other dispensary). Such cartons can be boxes, tubes, or of other configuration, and may be made of any material (e.g., cardboard, plastic, and the like). The packaging system and/or containers disposed therein can have one or more affixed labels (e.g., to provide information such as lot number, dose unit type, manufacturer, and the like). The container can include a moisture barrier and/or light barrier, e.g., to facilitate maintenance of stability of the active ingredients in the dose units contained therein. Where the dose unit is a dry dose unit, the container can include a desiccant pack which is disposed within the container. The container can be adapted to contain a single dose unit or multiples of a dose unit. The container can include a dispensing control mechanism, such as a lock out mechanism that facilitates maintenance of dosing regimen. Dose units can be provided in a container in which they are disposed and may be provided as part of a packaging system or kit (optionally with instructions for use). For example, one or more dose units as described herein can be provided in separate containers, where dose units of different composition are provided in separate containers, and the separate containers disposed within package for dispensing. It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Representative illustrative methods and materials are herein described; methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual dates of public availability and may need to be independently confirmed. It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as support for the recitation in the claims of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitations, such as "wherein [a particular feature or element] is absent", or "except for [a feature or element]", or "wherein [a particular feature or element] is not present (included, etc.)...". As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention. EXAMPLES EXAMPLE 1.4,6-spiro fentanyl derivatives 4,6-spiro fentanyl derivatives were synthesized as described below in Scheme 1. Scheme 1. General Synthesis Scheme.

Chemistry. All nonaqueous reactions were carried out under a pre-dried nitrogen gas atmosphere. All other solvents and purchased from Sigma-Aldrich, Alfa Aesar, Bepharm Scinetific and Fisher Scientific and were used as received without further purification. Melting points were measured on an MPA100 OptiMelt automated melting point apparatus without correction. The IR spectra were recorded on a Thermo Scientific Nicolet iS10 FT-IR spectrometer. Analytical thin-layer chromatography (TLC) analyses were carried out on Analtech Uniplate F254 plates, and flash column chromatography (FCC) was performed over silica gel (230−400 mesh, Merck). The ¹H (400 MHz) and ¹³C (100MHz) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ultrashield 400 Plus spectrometer, and chemical shifts were expressed in parts per million. The high-resolution mass spectra were obtained on an Applied BioSystems 3200 Q trap with a turbo V source for TurbolonSpray. Analytical reversed-phase high-performance liquid chromatography (HPLC) was performed on a Varian ProStar 210 system using an Agilent Microsorb-MV 100−5 C18 column (250 × 4.6 mm). All analyses were conducted at ambient temperature with a flow rate of 0.4 mL/min. The mobile phase is acetonitrile/water (90:10) with 0.1% trifluoroacetic acid (TFA). The UV detector was set up at 210 nm. Compound purities were calculated as the percentage peak area of the analyzed compound, and retention times (Rt) were presented in minutes. The purity of all newly synthesized compounds was identified as ^95%. General Procedure for the synthesis of fentanyl derivatives. In a solution of I in anhydrous DCM was added acetic acid and followed by aniline at 0 °C under nitrogen. stirred the reaction mixture for 5 minutes and slowly added sodium triacetoxyhydroborate. stirred the reaction mixture at rt for additional 16h and quenched with adding methanol. Washed the organic layer with water, sat. NaHCO3 and brine, dried over MgSO₄ and purified by silica gel column chromatography, by using 10:1 to 4:1 hexanes: ethyl acetate as a mobile phase to get pure compound II. Compound II was dissolved in anhydrous DCM and added triethylamine at 0 °C, followed by slow addition of acyl chloride. Stirred the reaction mixture overnight and washed the organic layer with water and brine, dried over MgSO4 and purified by silica gel column chromatography, by using 4:1 to 1:1 hexanes: ethyl acetate as a mobile phase to get pure compound III. After purification, dissolved the compound III in DCM and slowly added trifluoroacetic acid at 0 °C. Stirred the reaction mixture overnight and dried it on rotary evaporator and used for the next step without further purification. The product from the previous step in anhydrous acetonitrile and added K₂CO_3, followed by alkyl bromide. Reflux the reaction mixture and progress monitored by TLC plate. After completion of the reaction, washed the organic layer with water and brine, dried dried over MgSO4 and purified by silica gel column chromatography, by using 99:1:1 to 95:5:1 dichloromethane: methanol: ammonium hydroxide as a mobile phase to get pure compounds which immediately transfers to its salt form by treating with HCl/MeOH to get final VZFN compounds. N-(7-phenethyl-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-3-carboxamide hydrochloride VZFN030:

was prepared following the general procedure as a pale solid in 20% yield. ^{^}^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^!^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^{^^}"^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^ ^^ ^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^ #^^ ^^$%^&'^^^(^^{^^}^^^^^^^^^ ^ ^^^^^^^^^^^^^^^^^^^^ ^ ^^^^^^)^^^(%*(^^^^^^^^ ^^+^,^-^,.^&/^^^^^^^^^^^^^+^,^-^,^^ N-(7-benzyl-7-azaspiro[3.5]nonan-2-yl)-N-phenylfuran-3-carboxamide hydrochlorideVZFN031: The title compound was prepared following the general procedure as a pale solid in 25% yield. ^^^^^^^^^^^^^^^^^^^^^^^^_^^^^^^^^^^^^^^^^^^^ ^ ^^ ^^^ ^^^^^^^^^^^^^ ^^^^ ^^^^^^^^^^^^^^^^ ^0^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^!^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^{^^}"^^^^^^^^^^^^^^^^^^^^^_^^^^^^^^^^^^^ ^^^^ ^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^#^^^^$%^&'^^^(^^{^^}^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^)^^^(%*(^^ ^^^^^^^^^+^,^-^,.^&/^^^^^^^^^^^^^+^,^-^,^^ N-(7-phenethyl-7-azaspiro[3.5]nonan-2-yl)-N-phenylfuran-3-carboxamide hydrochloride VZFN032:

was prepared following the general procedure as a pale solid in 25% yield. 1H NMR (400 MHz, DMSO d-6): 9.43 (s, 1H), 7.53-7.49 (m, 3H), 7.48 (t, J=1.86 Hz, 1H), 7.36-7.33 (m, 2H), 7.28-7.24 (m, 5H), 6.86 (s, 1H), 6.02 (dd, J= 1.91, 0.93 Hz, 1H), 4.99 (p, 1H), 3.48-3.45 (m, 1H), 3.27-3.21 (m, 3H), 2.99-2.95 (m, 3H), 2.77-2.74 (m, 1H), 2.33-2.30 (m, 1H), 2.12- 2.09 (m, 1H), 2.06-2.02 , 1.78-1.72 (m, 2H), 1.65-1.60 (m, 2H), 1.36- 1.33 (m, 1H).¹³C NMR (100 MHz, DMSO-d6): 162.4, 145.4, 143.3, 139.5, 137.5, 131.1, 130.0, 129.3, 129.1, 127.3, 122.8, 111.2, 56.6, 49.5, 49.1, 46.7, 38.4, 36.1, 35.7, 32.4, 30.7, 29.9. IR (diamond, cm^-1): 2931, 2563, 1622, 749, 699. HRMS m/z: calc. 415.2380 [M+H]⁺; obs., 415.2367 [M+H]⁺. N-(7-benzyl-7-azaspiro[3.5]nonan-2-yl)-N-phenylfuran-2-carboxamide hydrochloride VZFN033:

was prepared following the general procedure as a pale solid in 44% yield. 1H NMR (400 MHz, DMSO d-₆): 10.68 (s, 1H), 7.65-7.65 (m, 1H), 7.60-7.58 (m, 2H), 7.52- 7.48 (m,3H), 7.45-7.43 (m,3H), 7.28-7.25 (m, 2H), 6.33 (dd, J=3.53, 1.71 Hz, 1H), 5.54 (d, J=3.63 Hz, 1H), 4.94 (p, 1H), 4.22-4.19 (m, 2H), 3.19-3.16 (m, 1H), 3.03-3.00 (m, 1H), 2.92- 2.90 (m, 1H), 2.74-2.68 (m, 1H), 2.28-2.25 (m, 1H), 2.10-2.04 (m, 1H), 1.99-1.96 (m, 1H), 1.91-1.84 (m, 1H), 1.78-1.69 (m, 2H), 1.61-1.56 (m, 1H), 1.29-1.25 (m, 1H).¹³C NMR (100 MHz, DMSO-d6): 158.5, 147.3, 145.5, 139.4, 131.8, 130.8, 130.4, 129.9, 129.8, 129.2, 116.1, 111.6, 59.0, 49.2, 48.6, 47.1, 38.4, 36.3, 36.0, 35.3, 32.0, 30.8. IR (diamond, cm^-1): 2979, 2499, 1627, 786, 695. HRMS m/z: calc.401.2224 [M+H]⁺; obs., 401.2205 [M+H]⁺. N-(7-phenethyl-7-azaspiro[3.5]nonan-2-yl)-N-phenylfuran-2-carboxamide hydrochloride VZFN034: procedure as a pale solid in 39% yield. ¹H NMR (400 MHz, DMSO d-6): 10.00 (s, 1H), 7.64 (dd, J= 1.79, 0.74 Hz, 1H), 7.52-7.49 (m, 3H), 7.36-7.32 (m, 2H), 7.29- 7.24 (m, 5H), 6.34 J= 3.55, 1.78 Hz, 1H), 5.57 (d,

J=3.42 Hz, 1H), 4.96 (p, 1H), 3.46-3.43 (m, 1H), 3.23-3.19 (m, 2H), 3.02-2.93 (m, 3H), 2.78- 2.70 (m, 1H), 2.33-2.27 (m, 1H), 2.14-2.08 (m, 1H), 2.04-2.01 (m, 1H), 1.82-1.60 (m, 5H), 1.35-1.32 (m, 1H).¹³C NMR (100 MHz, DMSO-d6): 158.5, 147.3, 145.5, 139.4, 137.6, 130.8, 130.0, 129.2, 129.1, 129.1, 127.2, 116.1, 111.6, 56.6, 49.4, 49.1, 49.0, 47.0, 38.3, 36.0, 35.6, 32.3, 30.8, 29.9. IR (diamond, cm^-1): 2929, 2555, 1620, 752, 699. HRMS m/z: calc.415.2380 [M+H]⁺; obs., 415.2366 [M+H]⁺. N-(7-benzyl-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-2-carboxamide hydrochloride VZFN035:

was prepared following the general procedure as a pale solid in 57% yield. ¹H NMR (400 MHz, DMSO d-6): 10.42 (s, 1H), 7.62-7.60 (dd, J= 4.95, 1.12 Hz, 1H), 7.58- 7.56 (m, 2H), 7.53-7.51 (m, 3H), 7.45-7.43 (m, 3H), 7.32-

2H), 6.86-6.83 (dd, J= 5.08, 3.96 Hz, 1H), 6.48-6.47 (dd, J=3.85, 1.03 Hz, 1H), 4.96 (p, 1H), 4.23-4.20 (m, 2H), 3.21-3.18 (m, 1H), 3.05-3.02 (m, 1H), 2.93-2.90 (m, 1H), 2.72-2.69 (m, 1H), 2.28-2.27 (m, 1H), 2.12- 2.06 (m, 1H), 2.00-1.97 (m, 1H), 1.89-1.81 (m, 1H), 1.76-1.74 (m, 1H), 1.72-1.71 (m, 1H), 1.63-1.58 (m, 1H), 1.31-1.28 (m, 1H).¹³C NMR (100 MHz, DMSO-d6): 161.6, 139.4, 139.1, 132.0, 131.8, 131.3, 130.4, 130.1, 129.9, 129.5, 129.2, 127.4, 59.0, 49.2, 48.6, 47.6, 38.4, 36.0, 35.4, 32.1, 30.8. IR (diamond, cm^-1): 2929, 2472, 1613, 739, 696. HRMS m/z: calc.417.1995 [M+H]⁺; obs., 417.1978 [M+H]⁺. N-(7-phenethyl-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-2-carboxamide hydrochloride VZFN036:

The title compound was prepared following the general procedure as a pale solid in 43% yield. 1H NMR (400 MHz, DMSO d-6): 10.16 (s, 1H), 7.62-7.61 (dd, J= 5.02, 1.15 Hz, 1H), 7.54- 7.52 (m, 3H), 7.36-7.31 (m, 4H), 7.28-7.24 (m, 3H), 6.86-6.84 (dd, J= 5.24, 3.95 Hz, 1H), 6.49-6.48 (dd, J=3.91, 1.07 Hz, 1H), 4.98 (p, 1H), 3.47-3.44(m, 1H), 3.34-3.29 (m, 1H), 3.22- 3.18 (m, 2H), 3.03-2.99 (m, 2H), 2.96-2.93 (m, 1H), 2.76-2.73 (m, 1H), 2.31-2.28 (m, 1H), 2.15-2.09 (m, 1H), 2.04-2.00 (m, 1H), 1.87-1.84 (m, 1H), 1.80-1.74 (m, 2H), 1.66-1.61 (m, 1H), 1.35-1.32 (m, 1H).¹³C NMR (100 MHz, DMSO-d₆): 161.7, 139.5, 139.2, 137.6, 132.0, 131.8, 131.3, 130.1, 129.5, 129.1, 129.1, 127.4, 127.3, 56.6, 49.5, 49.0, 47.6, 38.4, 36.1, 35.7, 32.4, 30.8, 29.9. IR (diamond, cm^-1): 2961, 2530, 1626, 778, 753. HRMS m/z: calc.431.2152 [M+H]⁺; obs., 431.2139 [M+H]⁺. N-(7-benzyl-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-2-carboxamide hydrochloride VZFN037: The title compound was prepared following the general procedure as a pale solid in 43% yield. 1H NMR (400 MHz, DMSO d-₆): 11.41 (s, 1H), 10.15 (s, 1H), 7.56-7.50 (m, 5H), 7.46-7.44 (m, 3H), 7.26-7.24 (m, 2H), 6.77-6.75 (m, 1H), 5.76-5.74 (m, 1H), 5.02 (p, 1H), 4.62-4.60 (m, 1H), 4.23-4.21 (m, 2H), 3.22-3.19 (m, 1H), 3.06-3.03 (m, 1H), 2.96-2.88 (m, 1H), 2.75-2.67 (m, 1H), 2.29-2.23 (m, 1H), 2.10-2.04 (m, 1H), 2.02-1.98 (m, 1H), 1.86-1.79 (m, 1H), 1.75- 1.70 (m, 1H), 1.62-1.57 (m, 1H), 1.30-1.27 (m, 1H).¹³C NMR (100 MHz, DMSO-d₆): 161.0, 140.0, 131.8, 131.3, 130.4, 129.9, 129.2, 129.1, 125.5, 121.9, 113.2, 109.0, 59.1, 49.3, 48.7, 46.6, 38.5, 36.0, 35.4, 32.2, 30.7. IR (diamond, cm^-1): 2938, 2569, 1605, 752, 697. HRMS m/z: calc.400.2389 [M+H]⁺; obs., 400.2364 [M+H]⁺. N-(7-phenethyl-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-2-carboxamide hydrochloride VZFN038:

was prepared following the general procedure as a pale solid in 30% yield. 1H NMR (400 MHz, DMSO d-6): 11.39 (s, 1H), 9.85 (s, 1H), 7.55-7.52 (m, 3H), 7.36-7.32 (m, 2H), 7.27-7.24 (m, 5H), 6.77-6.75 (m, 1H), 5.77-5.75 (m, 1H), 5.04 (p, 1H), 4.63-4.61 (m, 1H), 3.47-3.44 (m, 1H), 3.23-3.19 (m, 3H), 3.01-2.97 (m, 3H), 2.79-2.71 (m, 1H), 2.31-2.25 (m, 1H), 2.14-2.08 (m, 1H), 2.05-2.01 (m, , 1.78-1.62 (m, 4H), 1.34-1.30 (m, 1H). ¹³C NMR (100 MHz, DMSO-d6): 161.0, 140.1, 137.6, 131.3, 129.9, 129.1, 129.1, 127.3, 125.5, 121.9, 113.2, 109.0, 56.6, 49.5, 49.1, 46.6, 38.5, 36.1, 35.7, 32.4, 30.7, 29.9. IR (diamond, cm- ¹): 2934, 2574, 1607, 747, 700. HRMS m/z: calc.414.2545 [M+H]⁺; obs., 414.2524 [M+H]⁺. N-(7-benzyl-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-3-carboxamide hydrochloride VZFN039:

was prepared following the general procedure as a pale solid in 33% yield. 1H NMR (400 MHz, DMSO d-₆): 10.92 (s, 1H), 10.37 (s, 1H), 7.58-7.56 (m, 2H), 7.49-7.44 (m, 6H), 7.21-7.18 (m, 2H), 6.49-6.48 (m, 1H), 6.12-6.11 (m, 1H), 5.64-5.62 (m, 1H), 4.99 (p, 1H), 4.22-4.20 (m, 2H), 3.21-3.18 (m, 1H), 3.04-3.02 (m, 1H), 2.95-2.87 (m, 1H), 2.71-2.68 (m, 1H), 2.27-2.21 (m, 1H), 2.08-2.02 (m, 1H), 1.99-1.96 (m, 1H), 1.88-1.80 (m, 1H), 1.73- 1.67 (m, 2H), 1.59-1.54 (m, 1H), 1.27-1.24 (m, 1H).¹³C NMR (100 MHz, DMSO-d₆): 164.7, 140.8, 131.8, 131.4, 130.4, 129.9, 129.7, 129.2, 128.6, 122.5, 119.4, 117.8, 110.2, 59.0, 49.3, 48.7, 46.5, 38.6, 36.2, 35.4, 32.2, 30.7. IR (diamond, cm^-1): 2930, 2565, 1580, 750, 699. HRMS m/z: calc.400.2389 [M+H]⁺; obs., 400.2389 [M+H]⁺. ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^ !"#^$%$&

procedure as a pale solid in 28% yield. ^{^}^^^^^^^^^^^^^^^^^^^^^^^_^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ^ ^^^^^^^^^^^^^ ^^^^^!^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^{^^}"^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^ ^^^^^^^^^^^^^^ ^^^^^^^^ ^^^^^^^^^^^ ^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^ #^^^^$%^&'^^^(^^{^^}^^^ ^^^ ^^^ ^^^^^^^^^^^ ^^^^^^^^^^^^^^)^^^(%*(^^^^^^^ ^ ^+^,^-^,.^&/^^^^^^^^^ ^^^+^,^-^,^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^ !"#^$%^&

The title compound was prepared following the general procedure as a pale solid in 23% yield. ^{^}^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^ ^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^12^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^12 ^^^^^ ^^^^^^^^^^^^^^^^^^^^!^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^ ^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^{^^}"^^^^^^^^^^^^^^^^^^^^^_^^^^ ^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^ ^^^^^^^^^^^ ^^^^ ^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^#^^^^$%^&'^^^(^^{^^}^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^)^^^(%*(^^^^^^^^^^^+^,^-^,.^&/^^^^^^^^^^^ ^+^,^-^,^^ N-(7-(cyclohexylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-3-carboxamide hydrochloride (VZFN080)

The title compound was prepared following the general procedure as a white solid in 34% yield.¹H NMR (400 MHz, DMSO) 39.65 (s, 1H), 7.40 (dddd, J = 11.7, 7.0, 4.7, 2.3 Hz, 3H), 7.31 (dd, J = 5.0, 3.0 Hz, 1H), 7.25 (dd, J = 3.0, 1.3 Hz, 1H), 7.22 – 7.17 (m, 2H), 6.81 (dd, J = 5.0, 1.3 Hz, 1H), 4.94 (t, J = 8.6 Hz, 1H), 3.18 (d, J = 12.4 Hz, 1H), 2.94 – 2.78 (m, 3H), 2.66 (dt, J = 13.8, 10.4 Hz, 1H), 2.30 (ddd, J = 12.0, 8.3, 4.5 Hz, 1H), 2.10 (ddd, J = 12.3, 8.1, 4.4 Hz, 1H), 1.97 – 1.90 (m, 2H), 1.87 – 1.56 (m, 10H), 1.28 – 1.13 (m, 4H), 0.92 (q, J = 12.3 Hz, 2H).¹³C NMR (101 MHz, CDCl₃) 3164.6, 139.8, 136.9, 130.1, 129.8, 129.5, 128.6, 128.4, 124.3, 53.4, 50.6, 50.4, 47.5, 37.9, 36.5, 35.0, 33.2, 31.9, 31.7, 30.7, 25.6, 25.5. IR (diamond, cm^-1): 3407, 2924, 2552, 1618, 739. Mass m/z: calculated for C26H34N2OS: 422.2392; found [M + H]⁺: 423.2471. N-(7-(cyclohexylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-2-carboxamide hydrochloride (VZFN081)

The title compound was prepared following the general procedure as a white solid in 33% yield. ¹H NMR (400 MHz, CDCl3) 311.78 (s, 1H), 7.39 (d, J = 5.3 Hz, 3H), 7.07 (d, J = 6.2 Hz, 2H), 7.04 – 6.99 (m, 2H), 6.88 (dd, J = 4.9, 1.4 Hz, 1H), 5.08 (s, 1H), 3.40 (d, J = 61.6 Hz, 2H), 2.73 (s, 3H), 2.46 (d, J = 39.8 Hz, 4H), 2.22 (s, 1H), 1.95 – 1.64 (m, 10H), 1.32 – 1.04 (m, 5H).¹³C NMR (101 MHz, CDCl3) 3162.42, 139.04, 138.49, 132.12, 130.97, 130.78, 129.78, 129.20, 126.75, 63.33, 50.60, 50.27, 47.59, 37.76, 36.36, 35.01, 33.14, 31.85, 31.72, 30.71, 25.62, 25.50. IR (diamond, cm^-1): 3407, 2923, 2494, 1616, 736. Mass m/z: calculated for C₂₆H₃₄N₂OS: 422.2392; found [M + H]⁺: 423.2458. N-(7-(cyclohexylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenylfuran-2-carboxamide hydrochloride (VZFN082)

was prepared following the general procedure as a white solid in 30% yield.¹H NMR (400 MHz, DMSO) 39.65 (s, 1H), 7.65 (d, J = 1.7 Hz, 1H), 7.57 – 7.45 (m, 3H), 7.32 – 7.20 (m, 2H), 6.34 (dd, J = 3.6, 1.7 Hz, 1H), 5.56 (d, J = 3.5 Hz, 1H), 4.95 (t, J = 8.7 Hz, 1H), 3.33 (d, J = 11.8 Hz, 1H), 3.19 (d, J = 8.9 Hz, 1H), 2.93 – 2.77 (m, 3H), 2.66 (dt, J = 13.1, 10.5 Hz, 1H), 2.29 (q, J = 12.0, 8.1, 4.4 Hz, 1H), 2.11 - 2.05 (m, 1H), 1.93 (q, J = 4.0 Hz, 2H), 1.87 – 1.57 (m, 9H), 1.30 – 1.07 (m, 4H), 0.94 (t, J = 11.9 Hz, 2H). ¹³C NMR (101 MHz, DMSO) 3 158.53, 147.29, 145.49, 139.42, 130.83, 129.94, 129.16, 116.07, 111.63, 61.96, 50.04, 49.61, 36.16, 35.26, 32.54, 31.94, 31.13, 30.76, 25.93, 25.44. IR (diamond, cm^-1): 3110, 2923, 2739, 1635, 751. Mass m/z: calculated for C₂₆H₃₄N₂O₂: 406.2620; found [M + H]⁺: 407.2677. N-(7-(cyclohexylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-2-carboxamide hydrochloride (VZFN083)

was prepared following the general procedure as a white solid in 36% yield.¹H NMR (400 MHz, CDCl3) 311.42 (s, 1H), 7.52 – 7.40 (m, 3H), 7.33 (s, 1H), 7.13 (dd, J = 6.3, 3.1 Hz, 2H), 6.16 (d, J = 3.6 Hz, 1H), 5.53 (dd, J = 12.3, 3.5 Hz, 1H), 5.11 (t, J = 8.8 Hz, 1H), 3.76 – 3.24 (m, 3H), 2.93 (d, J = 6.7 Hz, 1H), 2.81 – 2.67 (m, 1H), 2.60 – 2.31 (m, 4H), 2.26 – 2.13 (m, 1H), 2.01 – 1.58 (m, 10H), 1.43 – 1.25 (m, 3H).¹³C NMR (101 MHz, DMSO) 3 158.52, 147.27, 145.51, 139.40, 130.84, 129.95, 129.17, 116.08, 111.64, 49.78, 49.31, 47.05, 38.34, 36.09, 35.36, 34.87, 32.03, 31.43, 30.74, 25.07.IR (diamond, cm^-1):3407, 2929, 2682, 1636, 751. Mass m/z: calculated for C₂₅H₃₂N₂O₂: 392.2464; found [M + H]⁺: 393.2519. N-(7-(cyclohexylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenylfuran-3-carboxamide hydrochloride (VZFN084)

The title compound was prepared following the general procedure as a white solid in 29% yield.¹H NMR (400 MHz, CDCl3) 311.69 (s, 1H), 7.47 (s, 3H), 7.32 (s, 1H), 7.14 (s, 2H), 6.16 (d, J = 3.3 Hz, 1H), 5.55 (d, J = 3.4 Hz, 1H), 5.12 (s, 1H), 3.57 – 3.28 (m, 2H), 2.74 (s, 2H), 2.45 (d, J = 42.4 Hz, 3H), 2.18 (d, J = 8.8 Hz, 1H), 1.96 – 1.62 (m, 10H), 1.10 (s, 3H), 0.87 (d, J = 9.9 Hz, 4H). ¹³C NMR (101 MHz, DMSO) 3158.52, 147.27, 145.50, 139.41, 130.83, 129.94, 129.16, 116.07, 111.64, 61.95, 50.03, 49.60, 47.06, 38.31, 36.15, 35.26, 32.53, 31.94, 31.12, 30.76, 25.93, 25.44. IR (diamond, cm^-1): 3407, 2923, 2739, 1637, 751. Mass m/z: calculated for C26H34N2O2: 406.2620; found [M + H]⁺: 407.2691. N-(7-(cyclobutylmethyl)-7-azaspiro[3.5]nonan-3-yl)-N-phenylfuran-3-carboxamide hydrochloride (VZFN085)

was prepared following the general procedure as a white solid in 32% yield.¹H NMR (400 MHz, DMSO) 310.16 (s, 1H), 7.61 – 7.41 (m, 4H), 7.34 – 7.21 (m, 2H), 6.86 (t, J = 1.1 Hz, 1H), 6.01 (d, J = 1.9 Hz, 1H), 4.97 (t, J = 8.7 Hz, 1H), 3.21 (d, J = 12.6 Hz, 1H), 3.11 – 2.97 (m, 3H), 2.84 (dt, J = 13.1, 10.0 Hz, 1H), 2.73 (p, J = 7.2 Hz, 1H), 2.62 (dt, J = 13.1, 10.3 Hz, 1H), 2.28 (ddd, J = 12.0, 8.1, 4.4 Hz, 1H), 2.07 (ddt, J = 14.0, 10.8, 5.1 Hz, 3H), 1.98 – 1.70 (m, 8H), 1.58 (t, J = 10.4 Hz, 1H), 1.25 (dd, J = 14.3, 2.8 Hz, 1H).¹³C NMR (101 MHz, DMSO) 3162.41, 145.41, 143.28, 139.48, 131.09, 130.02, 129.26, 122.82,

yield.¹H NMR (400 MHz, CDCl₃) 312.02 (s, 1H), 7.47 (s, 3H), 7.33 (d, J = 1.6 Hz, 1H), 7.13 (s, 2H), 6.16 (dd, J = 3.6, 1.6 Hz, 1H), 5.51 (d, J = 3.5 Hz, 1H), 5.12 (s, 1H), 3.37 (s, 1H), 3.21 (s, 1H), 3.00 (d, J = 48.9 Hz, 2H), 2.63 (s, 1H), 2.40 (s, 3H), 2.19 (s, 3H), 1.99 (s, 2H), 1.83 (s, 5H), 1.60 (s, 2H), 1.31 (d, J = 19.5 Hz, 1H), 0.87 (d, J = 7.0 Hz, 1H).¹³C NMR (101 MHz, DMSO) 3 158.52, 147.28, 145.49, 139.40, 130.83, 129.94, 129.16, 116.07, 111.63, 60.71, 49.33, 48.81, 47.05, 38.42, 35.97, 35.51, 32.16, 30.67, 30.55, 27.39, 27.30, 18.56. IR (diamond, cm^-1): 3471, 2958, 2472, 1638, 751. Mass m/z: calculated for C24H30N2O2: 378.2307; found [M + H]⁺: 379.2371 N-(7-(cyclobutylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenylfuran-2-carboxamide hydrochloride (VZFN087)

general procedure as a white solid in 33% yield.¹H NMR (400 MHz, DMSO) 39.72 (s, 1H), 7.55 – 7.43 (m, 4H), 7.25 (dd, J = 6.9, 2.5 Hz, 2H), 6.85 (s, 1H), 6.01 (d, J = 1.8 Hz, 1H), 4.97 (p, J = 8.7 Hz, 1H), 3.23 – 3.15 (m, 1H), 3.02 – 2.81 (m, 3H), 2.74 – 2.60 (m, 1H), 2.28 (ddt, J = 11.5, 7.9, 3.7 Hz, 1H), 2.19 (q, J = 7.7 Hz, 1H), 2.09 (ddd, J = 12.1, 8.0, 4.3 Hz, 1H), 1.98 – 1.86 (m, 2H), 1.76 (ddd, J = 30.6, 11.5, 6.6 Hz, 4H), 1.65 – 1.41 (m, 6H), 1.22 (dq, J = 15.1, 10.5 Hz, 3H).¹³C NMR (101 MHz, DMSO) 3 162.43, 145.40, 143.27, 139.51, 131.09, 130.02, 129.25, 122.83, 111.16, 60.93, 49.82, 49.37, 46.80, 38.46, 36.17, 35.39, 34.88, 32.09, 31.41, 30.71, 25.07, 25.05. IR (diamond, cm^-1): 3416, 2931, 2503, 1628, 731. Mass m/z: calculated for C25H32N2O2: 392.2464; found [M + H]⁺: 393.2539. N-(7-(cyclopentylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-3- carboxamide hydrochloride (VZFN088)

was prepared following the general procedure as a white solid in 30% yield.¹H NMR (400 MHz, DMSO) 39.73 (s, 1H), 7.45 – 7.33 (m, 3H), 7.30 (dd, J = 5.0, 3.0 Hz, 1H), 7.24 (dd, J = 3.0, 1.3 Hz, 1H), 7.22 – 7.14 (m, 2H), 6.81 (dd, J = 5.0, 1.3 Hz, 1H), 4.93 (p, J = 8.7 Hz, 1H), 3.20 (d, J = 12.6 Hz, 1H), 3.01 – 2.83 (m, 3H), 2.75 – 2.61 (m, 1H), 2.31 (ddd, J = 12.1, 8.2, 4.4 Hz, 1H), 2.21 (p, J = 7.7 Hz, 1H), 2.10 (ddd, J = 12.2, 8.1, 4.3 Hz, 1H), 1.93 (td, J = 14.2, 3.3 Hz, 2H), 1.78 (ddt, J = 20.9, 11.5, 7.3 Hz, 4H), 1.66 – 1.43 (m, 5H), 1.31 – 1.14 (m, 4H). ¹³C NMR (101 MHz, DMSO) 3164.50, 140.16, 137.91, 130.77, 129.69, 129.53, 128.52, 128.43, 125.74, 60.94, 55.39, 49.83, 49.37, 47.38, 38.63, 36.35, 35.40, 34.89, 32.08, 31.42, 30.67, 25.07, 25.06. IR (diamond, cm^-1): 3334, 2951, 2504, 1628, 728. Mass m/z: calculated for C25H32N2OS: 408.2235; found [M + H]⁺: 409.2319. N-(7-(cyclobutylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-3-carboxamide hydrochloride (VZFN089)

was prepared following the general procedure as a white solid in 30% yield.¹H NMR (400 MHz, DMSO) 310.28 (s, 1H), 7.44 – 7.34 (m, 3H), 7.30 (dd, J = 5.1, 2.9 Hz, 1H), 7.24 (dd, J = 3.0, 1.3 Hz, 1H), 7.21 – 7.16 (m, 2H), 6.81 (dd, J = 5.1, 1.3 Hz, 1H), 4.92 (p, J = 8.8 Hz, 1H), 3.21 (d, J = 12.7 Hz, 1H), 3.10 – 3.02 (m, 1H), 3.00 (t, J = 5.7 Hz, 2H), 2.91 – 2.79 (m, 1H), 2.78 – 2.70 (m, 1H), 2.62 (dtd, J = 12.4, 9.4, 4.7 Hz, 1H), 2.30 (ddd, J = 12.1, 8.2, 4.5 Hz, 1H), 2.13 – 2.01 (m, 3H), 1.96 – 1.69 (m, 8H), 1.60 (t, J = 10.4 Hz, 1H), 1.26 (dd, J = 14.1, 3.0 Hz, 1H).¹³C NMR (101 MHz, DMSO) 3140.16, 137.91, 130.77, 129.69, 129.52, 128.52, 128.43, 125.73, 60.71, 49.32, 48.82, 47.37, 38.72, 36.21, 35.51, 32.17, 30.62, 30.55, 27.40, 27.33, 18.56. IR (diamond, cm^-1): 3408, 2932, 2532, 1621, 739. Mass m/z: calculated for C24H30N2OS: 394.2079; found [M + H]⁺: 395.2159. N-(7-(cyclopentylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-2- carboxamide hydrochloride (VZFN090)

general procedure as a white solid in 43% yield.¹H NMR (400 MHz, DMSO) 39.69 (s, 1H), 7.60 (d, J = 5.0 Hz, 1H), 7.52 (dd, J = 5.0, 1.8 Hz, 3H), 7.30 (dd, J = 6.6, 2.9 Hz, 2H), 6.88 – 6.81 (m, 1H), 6.48 (d, J = 3.8 Hz, 1H), 4.97 (p, J = 8.7 Hz, 1H), 3.37 – 3.30 (m, 1H), 3.22 – 3.13 (m, 1H), 2.85 (d, J = 31.1 Hz, 3H), 2.66 (q, J = 11.9 Hz, 1H), 2.29 (ddd, J = 12.1, 8.2, 4.4 Hz, 1H), 2.09 (ddd, J = 12.1, 8.2, 4.4 Hz, 1H), 1.93 (t, J = 5.1 Hz, 2H), 1.75 (qd, J = 7.6, 3.8 Hz, 4H), 1.63 (dt, J = 20.3, 9.6 Hz, 4H), 1.31 – 1.14 (m, 3H), 0.92 (q, J = 11.2 Hz, 2H).¹³C NMR (101 MHz, DMSO) 3161.67, 139.49, 139.17, 131.97, 131.77, 131.29, 130.12, 129.49, 127.42, 65.37, 61.97, 50.05, 49.62, 47.62, 38.43, 36.23, 35.26, 32.55, 31.98, 31.15, 30.75, 25.93, 25.45. IR (diamond, cm^-1): 3231, 2928, 2475, 1608, 735. Mass m/z: calculated for C25H32N2OS: 408.2235; found [M + H]⁺: 409.2293. N-(7-(cyclobutylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-2-carboxamide hydrochloride (VZFN091)

was prepared following the general procedure as a white solid in 38% yield. ¹H NMR (400 MHz, DMSO) 310.10 (s, 1H), 7.60 (dd, J = 5.1, 1.2 Hz, 1H), 7.51 (dd, J = 4.9, 1.9 Hz, 3H), 7.34 – 7.24 (m, 2H), 6.84 (dd, J = 5.1, 3.8 Hz, 1H), 6.47 (dd, J = 3.8, 1.2 1H), 4.95 (p, J = 8.8 Hz, 1H), 3.21 (d, J = 12.5 Hz, 1H), 3.06 (d, J = 12.5 Hz, 1H), 3.00 (t, J = 6.1 Hz, 2H), 2.83 (dt, J = 12.7, 9.9 Hz, 1H), 2.73 (p, J = 7.2 Hz, 1H), 2.63 (dt, J = 13.6, 11.3 Hz, 1H), 2.28 (ddd, J = 12.1, 8.2, 1H), 2.13 – 2.00 (m, 3H), 1.94 (d, J = 14.2 Hz, 1H), 1.90 – 1.68 (m, 7H), 1.59 (t, J = 10.4 Hz, 1H), 1.27 (d, J = 14.1 Hz, 1H).¹³C NMR (101 MHz, DMSO) 3 161.66, 139.48, 139.16, 131.97, 131.77, 131.28, 130.13, 129.49, 127.42, 60.72, 49.35, 48.84, 47.60, 38.53, 36.05, 35.52, 32.21, 30.65, 30.55, 27.38, 27.30, 18.56. IR (diamond, cm^-1): 3032, 2959, 2453, 1609, 732. Mass m/z: calculated for C₂₄H₃₀N₂OS: 394.2079; found [M + H]⁺: 395.2154 N-(7-(cyclohexylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-3- carboxamide hydrochloride (VZFN092)

was prepared following the general procedure as a white solid in 24% yield. ¹H NMR (400 MHz, DMSO) 310.93 (s, 1H), 9.67 (s, 1H), 7.56 – 7.38 (m, 3H), 7.19 (dd, J = 7.6, 1.8 Hz, 2H), 6.48 (q, J = 2.4 Hz, 1H), 6.11 (dt, J = 3.5, 1.8 Hz, 1H), 5.63 (q, J = 2.4 Hz, 1H), 5.06 – 4.88 (m, 1H), 3.32 (d, J = 13.8 Hz, 1H), 3.16 (d, J = 7.5 Hz, 1H), 2.93 – 2.75 (m, 3H), 2.73 – 2.57 (m, 1H), 2.25 (ddd, J = 12.0, 8.3, 4.5 Hz, 1H), 2.05 (ddd, J = 12.2, 8.2, 4.4 Hz, 1H), 1.92 (q, J = 3.7 Hz, 2H), 1.84 – 1.54 (m, 9H), 1.25 – 1.09 (m, 4H), 0.92 (q, J = 11.7 Hz, 2H). ¹³C NMR (101 MHz, DMSO) 3164.71, 140.79, 131.38, 129.72, 128.62, 122.47, 119.40, 117.80, 110.18, 61.98, 50.10, 49.69, 46.53, 38.62, 36.37, 35.32, 32.56, 32.07, 31.15, 30.64, 25.93, 25.45. IR (diamond, cm^-1): 3193, 2926, 2575, 1574, 731. Mass m/z: calculated for C₂₆H₃₅N₃O: 405.2780; found [M + H]⁺: 406.2833. N-(7-(cyclopentylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-3- carboxamide hydrochloride (VZFN093) general procedure as a white solid in 21% yield. ¹H NMR (400 MHz, DMSO) 310.92 (s, 1H), 9.82 (s, 1H), 7.48 (q, J = 6.4 Hz, 3H), 7.25 – 7.16 (m, 2H), 6.48 (q, J = 2.4 Hz, 1H), 6.11 (q, J = 2.1 Hz, 1H), 5.63 (q, J = 2.4 Hz, 1H), 4.99 (q, J = 8.7 Hz, 1H), 3.19 (d, J = 12.3 Hz, 1H), 3.01 – 2.81 (m, 4H), 2.73 – 2.52 (m, 2H), 2.24 (ddd, J = 20.8, 10.3, 5.7 Hz, 3H), 2.05 (td, J = 7.8, 4.0 Hz, 1H), 1.92 (q, J = 4.0 Hz, 2H), 1.80 (dq, J = 14.5, 5.3 Hz, 4H), 1.28 – 1.17 (m, 4H). ¹³C NMR (101 MHz, DMSO) 3 164.72, 140.79, 131.38, 129.73, 128.62, 122.48, 119.40, 117.80, 110.18, 60.93, 49.86, 49.41, 46.53, 38.64, 36.31, 35.43, 34.89, 32.16, 31.44, 30.61, 25.07, 25.05. IR (diamond, cm^-1): 3357, 2935, 2567, 1580, 732. Mass m/z: calculated for C₂₅H₃₃N₃O: 391.2624; found [M + H]⁺: 392.2697 N-(7-(cyclobutylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-3- carboxamide hydrochloride (VZFN094)

was prepared following the general procedure as a white solid in 16% yield. ¹H NMR (400 MHz, DMSO) 310.92 (s, 1H), 10.13 (s, 1H), 7.53 – 7.40 (m, 3H), 7.23 – 7.14 (m, 2H), 6.48 (q, J = 2.4 Hz, 1H), 6.11 (dt, J = 3.3, 1.8 Hz, 1H), 5.63 (q, J = 2.3 Hz, 1H), 4.99 (t, J = 8.7 Hz, 1H), 3.21 (d, J = 12.5 Hz, 1H), 3.11 – 2.97 (m, 3H), 2.83 (dt, J = 13.2, 9.8 Hz, 1H), 2.74 (q, J = 7.3 Hz, 1H), 2.67 – 2.57 (m, 1H), 2.25 (ddd, J = 12.1, 8.2, 4.4 Hz, 1H), 2.05 (tq, J = 7.9, 4.3 Hz, 3H), 1.97 – 1.65 (m, 9H), 1.55 (t, J = 10.4 Hz, 1H), 1.28 – 1.17 (m, 1H). ¹³C NMR (101 MHz, DMSO) 140.79, 131.38, 129.73, 128.62, 122.47, 119.40, 117.80, 110.18, 60.72, 49.40, 48.89, 46.51, 38.73, 36.18, 35.57, 32.29, 30.55, 27.39, 27.30, 18.56. IR (diamond, cm^-1): 3184, 2932, 2544, 1581, 731. Mass m/z: calculated for C24H31N3O: 377.2467; found [M + H]⁺: 378.2529. N-(7-(cyclohexylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-2-

carboxamide

The title compound was prepared following the general procedure as a white solid in 25% yield.¹H NMR (400 MHz, DMSO) 311.45 – 11.32 (m, 1H), 9.58 (s, 1H), 7.58 – 7.42 (m, 3H), 7.35 – 7.15 (m, 2H), 6.83 – 6.61 (m, 1H), 5.80 – 5.69 (m, 1H), 5.02 (p, J = 8.8 Hz, 1H), 4.67 – 4.57 (m, 1H), 3.21 – 3.14 (m, 1H), 2.92 – 2.76 (m, 3H), 2.66 (tdd, J = 12.7, 9.7, 4.0 Hz, 1H), 2.26 (ddd, J = 12.1, 8.2, 4.4 Hz, 1H), 2.08 (ddd, J = 12.2, 8.1, 4.3 Hz, 1H), 1.93 (q, J = 4.1 Hz, 2H), 1.85 – 1.55 (m, 10H), 1.30 – 1.20 (m, 3H), 1.19 – 1.11 (m, 1H), 0.94 (t, J = 11.6 Hz, 2H).¹³C NMR (101 MHz, DMSO) 3160.99, 140.05, 131.33, 129.93, 129.07, 125.51, 121.89, 113.22, 109.05, 61.99, 50.10, 49.67, 46.60, 38.49, 36.21, 35.32, 32.55, 32.07, 31.11, 30.67, 25.92, 25.44. IR (diamond, cm^-1): 3242, 2926, 2478, 1582, 729. Mass m/z: calculated for C26H35N3O: 405.2780; found [M + H]⁺: 406.2843. N-(7-(cyclopentylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-2- carboxamide hydrochloride (VZFN096) The title compound was prepared following the general procedure as a white solid in 21% yield. ¹H NMR (400 MHz, DMSO) 311.46 – 11.35 (m, 1H), 9.67 (s, 1H), 7.52 (qd, J = 4.4, 1.9 Hz, 3H), 7.30 – 7.20 (m, 2H), 6.76 (td, J = 2.7, 1.4 Hz, 1H), 5.03 (p, J = 8.8 Hz, 1H), 4.66 – 4.57 (m, 1H), 3.20 (d, J = 12.6 Hz, 1H), 3.02 – 2.83 (m, 3H), 2.67 (dt, J = 13.8, 10.4 Hz, 1H), 2.32 – 2.16 (m, 2H), 2.08 (ddd, J = 12.2, 8.1, 4.3 Hz, 1H), 2.02 – 1.87 (m, 2H), 1.87 – 1.70 (m, 5H), 1.59 (ddd, J = 10.9, 8.3, 2.9 Hz, 3H), 1.50 (ddt, J = 7.5, 4.2, 2.6 Hz, 2H), 1.31 – 1.16 (m, 3H). ¹³C NMR (101 MHz, DMSO) 3 160.99, 140.05, 131.33, 129.93, 129.07, 125.52, 121.88, 113.22, 109.04, 60.94, 55.38, 49.86, 49.40, 46.59, 38.51, 36.16, 35.43, 34.89, 32.17, 31.41, 30.65, 25.07, 25.05. IR (diamond, cm^-1): 3245, 2949, 2479, 1583, 730. Mass m/z: calculated for C25H33N3O: 391.2624; found [M + H]⁺: 392.2686. N-(7-(cyclobutylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-2- carboxamide hydrochloride (VZFN097)

The title compound was prepared following the general procedure as a white solid in 19% yield. ¹H NMR (400 MHz, DMSO) 311.43 – 11.30 (m, 1H), 9.79 (s, 1H), 7.52 (dd, J = 5.2, 1.9 Hz, 3H), 7.29 – 7.20 (m, 2H), 6.75 (td, J = 2.8, 1.4 Hz, 1H), 5.75 (q, J = 2.6 Hz, 1H), 5.02 (p, J = 8.8 Hz, 1H), 4.69 – 4.56 (m, 1H), 3.09 – 2.99 (m, 3H), 2.84 (dt, J = 13.0, 9.6 Hz, 1H), 2.75 – 2.59 (m, 2H), 2.26 (ddd, J = 12.1, 8.2, 4.4 Hz, 1H), 2.06 (qd, J = 9.6, 3.3 Hz, 3H), 2.01 – 1.93 (m, 1H), 1.91 – 1.64 (m, 8H), = 10.4 Hz, 1H), 1.10 (t, J = 7.0 Hz, 1H). ¹³C NMR (101 MHz, DMSO) 3160.98, 140.03, 131.32, 129.93, 129.07, 125.51, 121.89, 113.22, 109.05, 65.37, 60.75, 49.06, 48.94, 46.56, 38.57, 36.05, 35.60, 32.32, 30.57, 30.56, 27.25, 18.55, 15.63. IR (diamond, cm^-1): 3245, 2934, 2467, 1580, 738. Mass m/z: calculated for C₂₄H₃₁N₃O: 377.2467; found [M + H]⁺: 378.2546. N-(7-(cyclopropylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-3- carboxamide hydrochloride (VZFN130)

was prepared following the general procedure as a white solid in 40% yield.¹H NMR (400MHz, DMSO) 39.89 (s, 1H), 7.61 (d, J = 5.0 Hz, 1H), 7.53 (dd, J = 5.1, 1.8 Hz, 3H), 7.31 (dd, J= 6.5, 2.9 Hz, 2H), 6.85 (t, J = 4.5 Hz, 1H), 6.48 (d, J = 3.8 Hz, 1H), 4.98 (p, J = 8.8 Hz, 1H),3.50 (s, 1H), 3.45 – 3.36 (m, 1H), 3.28 (d, J = 12.6 Hz, 1H), 2.93 – 2.85 (m, 2H), 2.76 – 2.64 (m,1H), 2.30 (m, J = 12.1, 8.2, 4.5 Hz, 1H), 2.11 (m, J = 12.2, 8.1, 4.3 Hz, 1H), 2.06 – 1.96 (m, 1H),1.85 (m, J = 14.4, 7.2 Hz, 1H), 1.81 – 1.68 (m, 2H), 1.62 (t, J = 10.4 Hz, 1H), 1.38 – 1.27 (m,1H), 1.14 – 1.02 (m, 1H), 0.69 – 0.54 (m, 2H), 0.36 (t, J = 5.0 Hz, 2H). ¹³C NMR (100 MHz,DMSO) 3161.65, 139.47, 139.16, 132.00, 131.79, 131.30, 130.14, 129.51, 127.44, 60.10, 49.13, 48.61, 47.59, 38.52, 36.05, 35.58, 32.26, 30.81, 5.75, 4.60, 4.54. IR (diamond, cm^-1): 2929, 2502, 1615, 731. Mass m/z: calculated for C22H28N2OS: 380.1922; found [M + H]⁺: 381.1988. N-(7-allyl-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-3-carboxamide hydrochloride (VZFN131) was general procedure as a white solid in 35% yield.¹H NMR (400 MHz, DMSO) 310.46 (s, 1H), 7.60 (dd, J = 5.0, 1.2 Hz, 1H), 7.52 (dd, J = 5.0, 1.9 Hz, 3H), 7.35 – 7.26 (m, 2H), 6.89 – 6.79 (m, 1H), 6.48 (dd, J = 3.8, 1.2 Hz, 1H), 5.96 (m, J = 13.8, 9.7, 7.0 Hz, 1H), 5.53 – 5.37 (m, 2H), 4.97 (p, J = 8.8 Hz, 1H), 3.62 (d, J = 6.3 Hz, 2H), 3.27 (d, J = 9.4 Hz, 1H), 3.13 (d, J = 12.3 Hz, 1H), 2.85 (m, J = 12.6, 9.2, 3.2 Hz, 1H), 2.70 – 2.58 (m, 1H), 2.29 (m, J = 12.1, 8.2, 4.5 Hz, 1H), 2.10 (m, J = 12.2, 8.1, 4.4 Hz, 1H), 1.99 (m, J = 14.7, 3.1 Hz, 1H), 1.85 (dd, J = 13.2, 4.0 Hz, 1H), 1.75 (m, J = 13.8, 9.3 Hz, 2H), 1.61 (t, J = 10.4 Hz, 1H), 1.31 (m, J = 14.5, 2.9 Hz, 1H).¹³C NMR (101 MHz, DMSO) 3161.67, 139.47, 139.16, 131.97, 131.78, 131.28, 130.13, 129.50, 128.13, 127.42, 124.94, 57.96, 48.99, 48.47, 47.60, 38.47, 36.03, 35.54, 32.23, 30.74. (diamond, cm^-1): 2929, 2502, 1615, 724. Mass m/z: calculated for C₂₂H₂₆N₂OS: 366.1766; found [M + H]⁺: 367.1823. N-(7-(cyclopropylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-2- carboxamide hydrochloride (VZFN132)

was prepared following the general procedure as a white solid in 38% yield.1H NMR (400 MHz, CDCl3) 311.79 (s, 1H), 7.55 – 7.43 (m, 3H), 7.29 (dd, J = 5.0, 1.1 Hz, 1H), 7.22 – 7.11 (m, 2H), 6.77 (dd, J = 5.0, 3.8 Hz, 1H), 6.65 (dd, J = 3.9, 1.2 Hz, 1H), 5.12 (q, J = 8.3 Hz, 1H), 3.61 (d, J = 11.1 Hz, 1H), 3.48 (d, J = 7.4 Hz, 1H), 3.12 (s, 2H), 2.85 (s, 1H), 2.61 (d, J = 65.6 Hz, 2H), 2.37 (d, J = 32.9 Hz, 2H), 2.19 (s, 1H), 1.99 (d, J = 14.2 Hz, 1H), 1.85 (t, J = 10.6 Hz, 1H), 1.77 (t, J Hz, 1H), 1.35 (s, 1H), 1.28 (s, 1H), 0.76 (d, J = 7.5 Hz, 2H), 0.40 (d, J = 4.4 Hz, 2H).¹³C NMR (101 MHz, DMSO) 3161.63, 139.43, 139.16, 132.02, 131.78, 131.31, 130.14, 129.52, 127.45, 48.95, 48.43, 47.57, 45.73, 38.56, 35.98, 35.49, 30.86, 8.87, 4.64, 4.57. (diamond, cm^-1): 2929, 2502, 1615, 734. Mass m/z: calculated for C₂₂H₂₈N₂OS: 380.1922; found [M + H]⁺: 381.1996. N-(7-allyl-7-azaspiro[3.5]nonan-2-yl)-N-phenylthiophene-2-carboxamide hydrochloride (VZFN133)

was prepared following the general procedure as a white solid in 32% yield.¹H NMR (400 MHz, DMSO) 310.52 (s, 1H), 7.60 (dd, J = 5.1, 1.2 Hz, 1H), 7.51 (m, J = 6.3, 2.8 Hz, 3H), 7.34 – 7.26 (m, 2H), 6.84 (dd, J = 5.1, 3.8 Hz, 1H), 6.48 (dd, J = 3.8, 1.2 Hz, 1H), 6.04 – 5.89 (m, 1H), 5.46 (m, J = 13.9, 1.6 Hz, 2H), 4.97 (p, J = 8.8 Hz, 1H), 3.70 – 3.57 (m, 2H), 3.27 (d, J = 11.9 Hz, 1H), 3.18 – 3.08 (m, 1H), 2.85 (m, J = 12.7, 9.6, 3.3 Hz, 1H), 2.64 (m, J = 12.7, 9.3, 2.9 Hz, 1H), 2.29 (m, J = 12.2, 8.2, 4.5 Hz, 1H), 2.10 (m, J = 12.2, 8.1, 4.5 Hz, 1H), 1.99 (dd, J = 14.4, 2.9 Hz, 1H), 1.91 – 1.80 (m, 1H), 1.74 (m, J = 13.8, 11.4, 5.7 Hz, 2H), 1.61 (t, J = 10.4 Hz, 1H), 1.30 (dt, J = 14.2, 2.8 Hz, 1H). ¹³C NMR (100 MHz, DMSO) 3161.67, 139.47, 139.16, 131.97, 131.78, 131.29, 130.13, 129.50, 128.11, 127.43, 124.97, 57.96, 49.01, 48.49, 47.59, 38.46, 36.03, 35.55, 32.25, 30.74. (diamond, cm^-1): 2934, 2468, 1615, 738. Mass m/z: calculated for C22H26N2OS: 366.1766; found [M + H]⁺: 367.1855. N-(7-(cyclopropylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenylfuran-3-carboxamide hydrochloride (VZFN134) The title compound was prepared following the general procedure as a white solid in 36% yield.¹H NMR (400 MHz, DMSO) 310.23 (s, 1H), 7.59 – 7.44 (m, 4H), 7.33 – 7.20 (m, 2H), 6.86 (s, 1H), 6.02 (d, J = 1.8 Hz, 1H), 5.00 (q, J = 8.8 Hz, 1H), 3.44 – 3.35 (m, 1H), 3.25 (s, 1H), 2.88 (dq, J = 9.7, 5.1 Hz, 2H), 2.68 (tdd, J = 12.8, 9.2, 3.0 Hz, 1H), 2.29 (ddd, J = 12.0, 8.2, 4.4 Hz, 1H), 2.10 (ddd, J = 12.2, 8.2, 4.4 Hz, 1H), 2.04 – 1.94 (m, 1H), 1.89 (dd, J = 13.3, 4.0 Hz, 1H), 1.78 (td, J = 12.7, 3.2 Hz, 2H), 1.59 (t, J = 10.4 Hz, 1H), 1.29 (dd, J = 14.2, 3.0 Hz, 1H), 1.08 (ddq, J = 12.4, 8.0, 4.4 Hz, 1H), 0.66 – 0.52 (m, 2H), 0.36 (t, J = 4.9 Hz, 2H). 1³C NMR (101 MHz, DMSO) 3 162.42, 145.41, 143.27, 139.50, 131.10, 130.02, 129.26, 122.83, 111.17, 60.04, 49.05, 48.54, 46.78, 36.02, 35.55, 32.21, 30.83, 5.73, 4.61, 4.55. (diamond, cm^-1): 2928, 2499, 1617, 733. Mass m/z: calculated for C23H28N2O2: 364.2151; found [M + H]⁺: 365.2215. N-(7-allyl-7-azaspiro[3.5]nonan-2-yl)-N-phenylfuran-3-carboxamide hydrochloride (VZFN135)

The title compound was prepared following the general procedure as a white solid in 30% yield.¹H NMR (400 MHz, DMSO) 310.92 (s, 1H), 7.38 (m, J = 14.0, 7.0 Hz, 3H), 7.30 (dd, J = 5.1, 2.9 Hz, 1H), 7.24 (d, J = 2.8 Hz, 1H), 7.18 (d, J = 7.2 Hz, 2H), 6.80 (d, J = 4.9 Hz, 1H), 5.98 (m, J = 16.8, 6.9 Hz, 1H), 5.54 – 5.33 (m, 2H), 4.93 (p, J = 8.7 Hz, 1H), 3.62 (q, J = 7.1 Hz, 2H), 3.26 (d, J = 12.3 Hz, 1H), 3.11 (d, J = 12.3 Hz, 1H), 2.84 (tt, J = 12.2, 7.0 Hz, 1H), 2.63 (q, J = 11.0 Hz, 1H), 2.29 (m, J 8.2, 4.3 Hz, 1H), 2.10 (m, J = 12.0, 8.2, 4.3 Hz, 1H), 2.01 – 1.85 (m, 2H), 1.77 (m, J = 20.6, 12.1, 6.2 Hz, 2H), 1.60 (t, J = 10.3 Hz, 1H), 1.28 (d, J = 13.9 Hz, 1H). ¹³C NMR (100 MHz, DMSO) 3164.51, 140.14, 137.90, 130.77, 129.70, 129.52, 128.53, 128.43, 128.20, 125.74, 124.85, 57.96, 48.94, 48.43, 47.37, 38.66, 36.20, 35.51, 32.18, 30.72. (diamond, cm^-1): 2929, 2464, 1634, 734. Mass m/z: calculated for C22H26N2O2: 350.1994; found [M + H]⁺: 351.2059. N-(7-allyl-7-azaspiro[3.5]nonan-2-yl)-N-phenylfuran-2-carboxamide hydrochloride (VZFN136)

was prepared following the general procedure as a white solid in 33% yield. ¹H NMR (400 MHz, DMSO) 39.68 (s, 1H), 7.65 (d, J = 1.7 Hz, 1H), 7.59 – 7.43 (m, 3H), 7.32 – 7.22 (m, 2H), 6.34 (dd, J = 3.6, 1.7 Hz, 1H), 5.56 (d, J = 3.5 Hz, 1H), 4.96 (p, J = 8.7 Hz, 1H), 3.41 (d, J = 9.9 Hz, 1H), 3.26 (s, 1H), 2.90 (d, J = 11.3 Hz, 3H), 2.69 (q, J = 11.8 Hz, 1H), 2.29 (ddd, J = 12.1, 8.2, 4.5 Hz, 1H), 2.10 (m, J = 12.2, 8.1, 4.4 Hz, 1H), 2.01 (d, J = 14.4 Hz, 1H), 1.82 (d, J = 9.7 Hz, 1H), 1.74 (dd, J = 11.5, 9.3 Hz, 2H), 1.60 (d, J = 10.4 Hz, 1H), 1.31 (d, J = 14.0 Hz, 1H), 1.13 – 0.99 (m, 1H), 0.61 (d, J = 7.7 Hz, 2H), 0.35 (t, J = 4.9 Hz, 2H). ¹³C NMR (100 MHz, DMSO) 3158.53, 147.28, 145.50, 139.42, 130.83, 129.95, 129.16, 116.08, 111.64, 49.16, 48.64, 47.07, 36.01, 32.25, 30.82, 5.76, 4.59, 4.53. (diamond, cm^-1): 2930, 2499, 1635, 734. Mass m/z: calculated for C23H28N2O2: 364.2151; found [M + H]⁺: 365.2214. N-(7-(cyclobutylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-2- carboxamide hydrochloride (VZFN137) The title compound was prepared following the general procedure as a white solid in 27% yield.¹H NMR (400 MHz, DMSO) 310.82 (s, 1H), 7.64 (d, J = 1.7 Hz, 1H), 7.55 – 7.44 (m, 3H), 7.31 – 7.19 (m, 2H), 6.33 (dd, J = 3.6, 1.8 Hz, 1H), 6.07 – 5.90 (m, 1H), 5.56 (d, J = 3.5 Hz, 1H), 5.51 – 5.38 (m, 2H), 4.95 (t, J = 8.7 Hz, 1H), 3.62 (d, J = 6.4 Hz, 2H), 3.26 (d, J = 12.7 Hz, 1H), 3.12 (d, J = 12.5 Hz, 1H), 2.90 – 2.77 (m, 1H), 2.72 – 2.57 (m, 1H), 2.28 (ddd, J = 12.1, 8.2, 4.4 Hz, 1H), 2.08 (ddd, J = 12.2, 8.2, 4.4 Hz, 1H), 1.99 (dd, J = 14.3, 2.9 Hz, 1H), 1.95 – 1.81 (m, 1H), 1.76 (tt, J = 11.4, 6.6 Hz, 2H), 1.60 (t, J = 10.4 Hz, 1H), 1.29 (dt, J = 14.1, 2.6 Hz, 1H).¹³C NMR (100 MHz, DMSO) 3158.54, 147.30, 145.47, 139.41, 130.82, 129.93, 129.15, 128.18, 124.85, 116.06, 111.62, 57.95, 48.94, 48.41, 47.07, 38.37, 35.97, 35.50, 32.17, 30.78. (diamond, cm^-1): 2929, 2464, 1663, 752 Mass m/z: calculated for C22H26N2O2: 350.1994; found [M + H]⁺: 351.2067. N-(7-(cyclopropylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-3- carboxamide hydrochloride (VZFN138)

was prepared following the general procedure as a white solid in 19% yield. 1H NMR (400 MHz, DMSO) 310.55 (s, 1H), 10.46 (s, 1H), 7.65 (d, J = 1.7 Hz, 1H), 7.55 – 7.45 (m, 3H), 7.27 (dd, J = 7.3, 2.2 Hz, 2H), 6.34 (dd, J = 3.6, 1.7 Hz, 1H), 5.56 (d, J = 3.5 Hz, 1H), 4.95 (q, J = 8.7 Hz, 1H), 3.44 – 3.36 (m, 1H), 3.26 (d, J = 13.1 Hz, 1H), 3.08 – 3.04 (m, 3H), 2.87 (dd, J = 13.0, 7.8 Hz, 2H), 2.73 – 2.63 (m, 1H), 2.29 (m, J = 12.1, 8.2, 4.5 Hz, 1H), 2.09 (m, J = 12.2, 8.1, 4.4 Hz, – 1.92 (m, 1H), 1.81 – 1.73 (m, 1H), 1.60 (t, J = 10.4 Hz, 1H), 1.28 (d, J = 13.2 Hz, 1H), 1.08 (m, J = 7.4, 3.3 Hz, 1H), 0.64 – 0.56 (m, 2H), 0.36 (t, J = 5.0 Hz, 2H).¹³C NMR (100 MHz, DMSO) 3158.52, 147.28, 145.49, 139.40, 130.84, 129.94, 129.16, 116.07, 111.63, 59.99, 48.97, 48.44, 47.06, 45.78, 35.96, 35.50, 32.13, 30.88, 8.88, 5.71, 4.63. (diamond, cm^-1): 2930, 2497, 1636, 754. Mass m/z: calculated for C23H29N3O: 363.2311; found [M + H]⁺: 364.2379. N-(7-allyl-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-3-carboxamide hydrochloride (VZFN139)

was prepared following the general procedure as a white solid in 15% yield. ¹H NMR (400 MHz, DMSO) 310.95 (s, 1H), 10.73 (s, 1H), 6.48 (q, J = 2.4 Hz, 1H), 6.11 (q, J = 2.1 Hz, 1H), 5.98 (m, J = 17.3, 12.0, 4.9 Hz, 1H), 5.63 (q, J = 2.4 Hz, 1H), 5.47 – 5.41 (m, 2H), 5.01 (q, J = 8.8 Hz, 1H), 3.26 (d, J = 12.4 Hz, 2H), 3.11 (d, J = 12.5 Hz, 1H), 2.85 (m, J = 9.0, 4.6 Hz, 1H), 2.62 (m, J = 9.6, 6.1 Hz, 1H), 2.27 – 2.22 (m, 1H), 2.05 (m, J = 7.9, 4.1 Hz, 1H), 1.98 – 1.85 (m, 2H), 1.72 (m, J = 10.9, 6.7 Hz, 2H), 1.56 (t, J = 10.3 Hz, 1H), 1.31 – 1.14 (m, 2H).¹³C NMR (100 MHz, DMSO) 3164.74, 140.75, 131.37, 129.73, 128.14, 124.92, 122.49, 119.37, 110.18, 57.97, 49.02, 48.50, 46.52, 38.66, 36.15, 35.57, 32.30, 30.64. (diamond, cm^-1): 3192, 2930, 1644, 732. Mass m/z: calculated for C₂₂H₂₇N₃O: 349.2154; found [M + H]⁺: 350.2210. N-(7-(cyclopropylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-2- carboxamide hydrochloride (VZFN140) The title compound was prepared following the general procedure as a white solid in 35% yield. 1H NMR (400 MHz, DMSO) 311.41 (s, 1H), 10.16 (s, 1H), 7.53 (dd, J = 5.1, 1.8 Hz, 3H), 7.31 – 7.20 (m, 2H), 6.76 (d, J = 3.1 Hz, 1H), 5.76 (q, J = 2.6 Hz, 1H), 5.04 (p, J = 8.8 Hz, 1H), 4.61 (p, J = 1.6 Hz, 1H), 3.45 – 3.37 (m, 2H), 3.27 (d, J = 12.4 Hz, 1H), 2.87 (m, J = 10.1, 5.4 Hz, 2H), 2.68 (tdd, J = 12.7, 9.3, 2.9 Hz, 1H), 2.27 (m, J = 12.1, 8.2, 4.4 Hz, 1H), 2.10 (m, J = 12.1, 8.2, 4.4 Hz, 1H), 2.00 (dd, J = 14.1, 2.9 Hz, 1H), 1.88 (dt, J = 13.8, 6.9 Hz, 1H), 1.77 – 1.72 (m, 1H), 1.59 (t, J = 10.4 Hz, 1H), 1.28 (dd, J = 14.1, 2.8 Hz, 1H), 1.14 – 1.02 (m, 2H), 0.65 – 0.54 (m, 2H), 0.40 – 0.30 (m, 2H).¹³C NMR (101 MHz, DMSO) 3160.98, 140.03, 131.34, 129.94, 129.08, 125.51, 121.89, 113.22, 109.05, 65.38, 60.07, 49.10, 48.58, 46.57, 36.01, 35.59, 32.30, 30.77, 5.74, 4.61, 4.55. (diamond, cm^-1): 3242, 2546, 1604, 738. Mass m/z: calculated for C23H29N3O: 363.2311; found [M + H]⁺: 364.2367. N-(7-(cyclobutylmethyl)-7-azaspiro[3.5]nonan-2-yl)-N-phenyl-1H-pyrrole-2- carboxamide hydrochloride (VZFN141)

The title compound was prepared following the general procedure as a white solid in 30% yield.¹H NMR (400 MHz, DMSO) 311.38 (s, 1H), 9.73 (s, 1H), 7.52 (m, J = 4.3, 1.8 Hz, 3H), 7.31 – 7.18 (m, 2H), 6.76 (td, J = 2.7, 1.4 Hz, 1H), 5.98 – 5.86 (m, 1H), 5.75 (dt, J = 3.8, 2.4 Hz, 1H), 5.57 – 5.37 (m, 2H), 5.03 (p, J = 8.8 Hz, 1H), 4.62 (m, J = 3.9, 2.5, 1.4 Hz, 1H), 3.66 (d, J = 6.9 Hz, 2H), 3.29 (s, 1H), 3.16 (d, J = 15.9 Hz, 1H), 2.87 (d, J = 11.5 Hz, 1H), 2.72 – 2.60 (m, 1H), 2.27 (m, J = 12.0, 8.2, 4.4 , 2.09 (m, J = 12.1, 8.1, 4.3 Hz, 1H), 2.02 (d, J = 14.5 Hz, 1H), 1.74 (dd, J = 11.5, 9.3 Hz, 2H), 1.69 – 1.55 (m, 2H), 1.31 (d, J = 14.0 Hz, 1H).¹³C NMR (101 MHz, DMSO) 3160.99, 140.04, 131.32, 129.93, 129.08, 127.98, 125.50, 125.20, 121.89, 113.23, 109.05, 48.66, 46.56, 38.49, 36.05, 30.64. (diamond, cm^-1): 3258, 2934, 2401, 1584, 733. Mass m/z: calculated for C₂₂H₂₇N₃O: 349.2154; found [M + H]⁺: 350.2225. EXAMPLE 2.6,4-spiro fentanyl derivatives 6,4-spiro fentanyl derivatives were synthesized as described below. Scheme 2. General synthesis scheme.

Chemistry. All nonaqueous reactions were carried out under a pre-dried nitrogen gas atmosphere. All other solvents and purchased from Sigma-Aldrich, Alfa Aesar, Bepharm Scinetific and Fisher Scientific and were used as received without further purification. Melting points were measured on an MPA100 OptiMelt automated melting point apparatus without correction. The IR spectra were recorded on a Thermo Scientific Nicolet iS10 FT-IR spectrometer. Analytical thin-layer chromatography (TLC) analyses were carried out on Analtech Uniplate F254 plates, and flash column chromatography (FCC) was performed over silica gel (230−400 mesh, Merck). The ¹H (400 MHz) and ¹³C (100MHz) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ultrashield 400 Plus spectrometer, and chemical shifts were expressed in parts per million. The high-resolution mass spectra were obtained on an Applied BioSystems 3200 Q trap with a turbo V source for TurbolonSpray. Analytical reversed-phase high-performance liquid chromatography (HPLC) was performed on a Varian ProStar 210 system using an Agilent Microsorb-MV 100−5 C18 column (250 × 4.6 mm). All analyses were conducted at ambient temperature with a flow rate of 0.4 mL/min. The mobile phase is acetonitrile/water (90:10) with 0.1% trifluoroacetic acid (TFA). The UV detector was set up at 210 nm. Compound purities were calculated as the percentage peak area of the analyzed compound, and retention times (Rt) were presented in minutes. The purity of all newly synthesized compounds was identified as ^95%. General Procedure for the synthesis of fentanyl derivatives. In a solution of 1 in anhydrous DCM was added acetic acid and followed by aniline at 0 °C under nitrogen. stirred the reaction mixture for 5 minutes and slowly added sodium triacetoxyhydroborate. stirred the reaction mixture at rt for additional 16h and quenched with adding methanol. Washed the organic layer with water, sat. NaHCO3 and brine, dried over MgSO₄ and purified by silica gel column chromatography, by using 10:1 to 4:1 hexanes: ethyl acetate as a mobile phase to get pure compound 2. Compound 2 was dissolved in anhydrous DCM and added triethylamine at 0 °C, followed by slow addition of acyl chloride. Stirred the reaction mixture overnight and washed the organic layer with water and brine, dried over MgSO4 and purified by silica gel column chromatography, by using 4:1 to 1:1 hexanes: ethyl acetate as a mobile phase to get pure compound 3. After purification, dissolved the compound 3 in DCM and slowly added trifluoroacetic acid at 0 °C. Stirred the reaction mixture overnight and dried it on rotary evaporator and used for the next step without further purification. The product 4 from the previous in anhydrous acetonitrile and added K2CO3, followed by alkyl bromide. Reflux the reaction mixture and progress monitored by TLC plate. After completion of the reaction, washed the organic layer with water and brine, dried dried over MgSO4 and purified by silica gel column chromatography, by using 99:1:1 to 95:5:1 dichloromethane: methanol: ammonium hydroxide as a mobile phase to get pure compounds 5, which immediately transfers to its salt form by treating with HCl/MeOH to get final VZFN compounds.

[3.5]nonan-7-yl)-N-phenylfuran-2-carboxamide hydrochloride (VZFN200) The title compound was prepared following the general procedure as a white powder in 31.9% yield.¹H NMR (400 MHz, DMSO-d6) 311.25 (s, 1H), 7.62 (d, J = 1.7 Hz, 1H), 7.53 – 7.43 (m, 3H), 7.31 – 7.20 (m, 2H), 6.30 (dd, J = 3.6, 1.7 Hz, 1H), 5.85 – 5.71 (m, 1H), 5.52 – 5.36 (m, 3H), 4.46 (m, J = 12.1, 3.6 Hz, 1H), 3.70 (dt, J = 12.2, 4.1 Hz, 3H), 3.62 (dd, J = 10.2, 6.5 Hz, 2H), 3.56 (dd, J = 10.2, 6.4 Hz, 1H), 2.29 (m, J = 13.7, 3.2 Hz, 1H), 2.08 – 1.97 (m, 1H), 1.81 (m, J = 28.2, 12.5, 3.0 Hz, 2H), 1.58 (td, J = 13.3, 3.6 Hz, 2H), 1.07 (m, J = 57.6, 12.8, 3.5 Hz, 2H).¹³C NMR (100 MHz, DMSO-d₆) 3158.24, 147.34, 145.32, 138.97, 131.01, 129.71, 129.19, 127.97, 124.08, 115.86, 111.56, 62.11, 60.98, 56.61, 55.37, 53.86, 34.87, 34.67, 33.01, 27.13, 27.06. Mass m/z: calculated for C22H26N2O2: 350.1994; found [M + H]⁺: 351.2081. HPLC data: purity 99.51%, retention time 2.663 min. 7-yl)-N-phenylfuran-2-carboxamide The title compound was prepared following the general procedure as a white powder in 16.3% yield.¹H NMR (400 MHz, DMSO-d₆) 310.93 (s, 1H), 7.62 (d, J = 1.6 Hz, 1H), 7.52 – 7.43 (m, 3H), 7.25 (dd, J = 6.7, 2.9 Hz, 2H), 6.30 (dd, J = 3.6, 1.7 Hz, 1H), 5.40 (d, J = 3.5 Hz, 1H), 4.46 (m, J = 12.0, 8.5, 3.6 Hz, 1H), 3.67 (m, J = 17.0, 13.4, 8.2 Hz, 4H), 2.94 (t, J = 6.6 Hz, 2H), 2.41 – 2.31 (m, 1H), 2.06 – 1.95 (m, 1H), 1.81 (m, J = 32.6, 12.8, 3.1 Hz, 2H), 1.58 (td, J = 13.4, 3.6 Hz, 2H), 1.26 – 1.08 (m, 1H), 0.98 (m, J = 20.4, 7.8, 4.1 Hz, 2H), 0.49 (dd, J = 8.0, 1.9 Hz, 2H), 0.33 (d, J = 4.7 Hz, 2H).¹³C NMR (100 MHz, DMSO-d6) 3 158.25, 147.33, 145.32, 138.98, 131.01, 129.73, 129.20, 115.86, 111.56, 62.40, 61.26, 58.82, 53.93, 34.99, 34.96, 32.89, 27.17, 27.08, 6.12, 3.57, 3.45. Mass m/z: calculated for C₂₃H₂₈N₂O₂: 364.2151; found [M + H]⁺: 365.2209. HPLC data: purity 99.83%, retention time 2.700 min.

-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-2-carboxamide hydrochloride (VZFN202) The title compound was prepared following the general procedure as a white powder in 18.6% yield.¹H NMR (400 MHz, DMSO-d₆) 310.66 (s, 1H), 7.65 – 7.57 (m, 1H), 7.46 (dd, J = 5.0, 1.9 Hz, 3H), 7.30 – 7.19 (m, 2H)

(dd, J = 3.6, 1.7 Hz, 1H), 5.40 (d, J = 3.5 Hz, 1H), 4.44 (tt, J = 12.1, 3.6 Hz, 1H), 3.75 – 3.54 (m, 5H), 3.10 (td, J = 6.6, 2.3 Hz, 2H), 2.33 (dd, J = 13.6, 3.0 Hz, 1H), 2.05 – 1.91 1.89 – 1.70 (m, 6H), 1.56 (m, J = 9.8, 7.2, 3.8 Hz, 2H), 1.06 (m, J = 57.0, 12.8, 3.5 Hz, 2H).¹³C NMR (100 MHz, DMSO-d6) 3158.25, 147.32, 145.32, 138.97, 131.01, 129.73, 129.21, 115.86, 111.56, 63.09, 61.94, 59.69, 53.91, 35.05, 34.99, 32.84, 30.97, 27.15, 27.05, 26.30, 26.23, 18.70. Mass m/z: calculated for C₂₄H₃₀N₂O₂: 378.2307; found [M + H]⁺: 379.2386. HPLC data: purity 99.74%, retention time 2.770 min.

-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-2-carboxamide hydrochloride (VZFN203) The title compound was prepared following the general procedure as a white powder in 27.5% yield.¹H NMR (400 MHz, DMSO-d₆) 310.29 (s, 1H), 7.62 (dd, J = 1.7, 0.8 Hz, 1H), 7.47 (dd, J = 5.0, 1.9 Hz, 3H), 7.29 – 7.21 (m, 2H), 6.30 (dd, J = 3.5, 1.7 Hz, 1H), 5.40 (d, J = 3.5 Hz, 1H), 4.51 – 4.40 (m, 1H), 3.85 – 3.59 (m, 4H), 3.06 (t, J = 6.7 Hz, 2H), 2.32 (dd, J = 13.3, 3.1 Hz, 1H), 2.00 (td, J = 15.8, 14.1, 5.5 Hz, 2H), 1.90 – 1.66 (m, 4H), 1.64 – 1.40 (m, 6H), 1.22 – 1.10 (m, 3H), 1.00 (qd, J = 12.8, 3.5 Hz, 1H).¹³C NMR (100 MHz, DMSO- d6) 3158.25, 147.33, 145.32, 138.98, 131.02, 129.73, 129.20, 115.86, 111.56, 63.59, 62.36, 60.07, 53.90, 35.95, 35.15, 34.62, 32.98, 30.48, 27.15, 27.07, 24.94, 24.89. Mass m/z: calculated for C25H32N2O2: 392.2464; found [M + H]⁺: 393.2522. HPLC data: purity 98.98%, retention time 2.847 min.

7-yl)-N-phenylfuran-2-carboxamide hydrochloride (VZFN204) The title compound was prepared following the general procedure as a white powder in 28.7% yield.¹H NMR (400 MHz, DMSO-d6) 310.32 (s, 1H), 7.62 (dd, J = 1.8, 0.8 Hz, 1H), 7.47 (dd, J = 5.0, 1.9 Hz, 3H), 7.29 – 7.21 (m, 2H), 6.30 (dd, J = 3.5, 1.7 Hz, 1H), 5.40 (d, J = 3.5 Hz, 1H), 4.51 – 4.40 (m, 1H), 3.80 (dt, J = 16.2, 8.8 Hz, 2H), 3.63 (m, J = 15.7, 10.1, 5.9 Hz, 2H), 2.94 (t, J = 6.5 Hz, 2H), 2.33 (dd, J = 13.3, 3.0 Hz, 1H), 2.03 (dd, J = 13.1, 3.0 Hz, 1H), 1.81 (dd, J = 26.4, 12.5 Hz, 2H), 1.72 – 1.48 (m, 8H), 1.23 – 1.06 (m, 4H), 1.06 – 0.97 (m, 1H), 0.94 – 0.81 (m, 2H).¹³C NMR (100 MHz, DMSO-d6) 3158.25, 147.33, 145.31, 138.97, 131.02, 129.72, 129.20, 115.85, 111.56, 63.89, 62.60, 61.49, 53.89, 35.21, 34.60, 33.82, 32.97, 30.30, 30.24, 27.13, 27.07, 25.94, 25.44, 25.38. Mass m/z: calculated for C₂₆H₃₄N₂O₂: 406.2620; found [M + H]⁺: 407.2673. HPLC data: purity 99.49%, retention time 2.945 min.

[3.5]nonan-7-yl)-N-phenylfuran-2-carboxamide hydrochloride (VZFN205) The title compound was prepared following the general procedure as a white powder in 32.9% yield.¹H NMR (400 MHz, DMSO-d₆) 310.98 (s, 1H), 7.61 (dd, J = 1.8, 0.8 Hz, 1H), 7.55 – 7.49 (m, 2H), 7.49 – 7.38 (m, 6H), 7.29 – 7.17 (m, 2H), 6.30 (dd, J = 3.5, 1.7 Hz, 1H), 5.45 – 5.36 (m, 1H), 4.51 – 4.38 (m, – 4.21 (m, 2H), 3.76 (dd, J = 10.3, 6.4 Hz, 1H), 3.72 – 3.64 (m, 2H), 3.58 (t, J = 8.1 Hz, 1H), 2.32 – 2.23 (m, 1H), 2.06 (dd, J = 12.7, 3.2 Hz, 1H), 1.80 (t, J = 15.3 Hz, 2H), 1.65 – 1.51 (m, 2H), 1.16 (dd, J = 12.7, 3.4 Hz, 1H), 1.06 – 0.93 (m, 1H).¹³C NMR (100 MHz, DMSO-d6) 3158.24, 147.33, 145.31, 138.98, 131.13, 130.98, 130.59, 129.70, 129.67, 129.23, 129.17, 115.85, 111.55, 62.59, 61.33, 57.94, 53.94, 34.99, 34.56, 32.94, 27.14, 27.03. Mass m/z: calculated for C26H28N2O2: 400.2151, found [M + H]⁺: 401.2205. HPLC data: purity 99.61%, retention time 2.775 min.

azaspiro[3.5]nonan-7-yl)-N-phenylfuran-2-carboxamide hydrochloride (VZFN206) The title compound was prepared following the general procedure as a white powder in 52.4% yield.¹H NMR (400 MHz, DMSO-d6) 310.90 (s, 1H), 7.62 (dd, J = 1.8, 0.8 Hz, 1H), 7.47 (dd, J = 5.1, 1.9 Hz, 3H), 7.37 – 7.21 (m, 7H), 6.30 (dd, J = 3.5, 1.7 Hz, 1H), 5.41 (d, J = 3.5 Hz, 1H), 4.46 (tt, J = 12.0, 3.5 Hz, 1H), 3.70 (m, J = 10.6, 10.1, 5.8 Hz, 4H), 3.31 (dd, J = 8.8, 6.1 Hz, 2H), 2.79 (t, J = 8.1 Hz, 2H), 2.27 (dd, J = 13.4, 3.1 Hz, 1H), 2.04 (dt, J = 13.3, 3.1 Hz, 1H), 1.92 – 1.73 (m, 2H), 1.59 (m, J = 13.0, 8.2, 3.5 Hz, 2H), 1.07 (m, J = 64.9, 12.8, 3.5 Hz, 2H).¹³C NMR (100 MHz, DMSO-d6) 3158.25, 147.33, 145.33, 138.97, 137.20, 131.02, 129.73, 129.22, 129.20, 129.04, 127.27, 115.88, 111.57, 63.05, 61.83, 56.00, 53.84, 49.06, 34.89, 34.78, 33.01, 30.46, 27.15, 27.06. Mass m/z: calculated for C₂₇H₃₀N₂O₂: 414.2307, found [M + H]⁺: 415.2382. HPLC data: purity 99.43%, retention time 2.840 min. 2-carboxamide hydrochloride The title compound was prepared following the general procedure as a white powder in 20.5% yield.¹H NMR (400 MHz, DMSO-d₆) 310.88 (s, 1H), 7.58 (dd, J = 5.1, 1.2 Hz, 1H), 7.51 – 7.44 (m, 3H), 7.32 – 7.25 (m, 2H), 6.82 (dd, J = 5.1, 3.8 Hz, 1H), 6.36 (dd, J = 3.9, 1.3 Hz, 1H), 5.77 (m, J = 16.9, 10.3, 6.6 Hz, 1H), 5.52 – 5.36 (m, 2H), 4.48 (m, J = 12.2, 8.7, 3.7 Hz, 1H), 3.77 – 3.68 (m, 3H), 3.65 (d, J = 7.4 Hz, 2H), 3.59 (d, J = 5.7 Hz, 1H), 2.24 (d, J = 13.4 Hz, 1H), 2.08 – 1.98 (m, 1H), 1.83 (dd, J = 28.2, 12.7 Hz, 2H), 1.66 – 1.52 (m, 2H), 1.17 – 1.08 (m, 1H), 1.00 (q, J = 12.5 Hz, 1H).¹³C NMR (100 MHz, DMSO-d6) 3 161.33, 139.36, 138.99, 131.90, 131.65, 131.44, 129.92, 129.54, 127.93, 127.37, 124.14, 62.31, 61.21, 56.73, 54.48, 46.03, 34.88, 34.68, 33.12, 27.11, 8.99. Mass m/z: calculated for C₂₂H₂₆N₂OS: 366.1766, found [M + H]⁺: 367.1826. HPLC data: purity 99.87%, retention time 2.772 min.

-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-2- carboxamide hydrochloride (VZFN208) The title compound was prepared following the general procedure as a white powder in 20.6% yield.¹H NMR (400 MHz, DMSO-d₆) 310.10 (s, 1H), 7.58 (dd, J = 5.1, 1.2 Hz, 1H), 7.49 (dd, J = 5.5, 1.7 Hz, 3H), 7.34 – 7.24 (m, 2H), 6.82 (dd, J = 5.0, 3.8 Hz, 1H), 6.36 (dd, J = 3.8, 1.2 Hz, 1H), 4.52 – 4.43 (m, 1H), 3.74 (dd, J = 13.0, 6.3 Hz, 4H), 2.97 (t, J = 6.6 Hz, 2H), 2.23 (d, J = 13.3 Hz, 1H), 2.05 – , 1.89 (d, J = 12.9 Hz, 1H), 1.80 (d, J = 12.8 Hz, 1H), 1.68 – 1.53 (m, 2H), 1.25 – 1.14 (m, 1H), 1.01 (dd, J = 12.9, 3.3 Hz, 1H), 0.92 (tt, J = 8.2, 4.8 Hz, 1H), 0.59 – 0.46 (m, 2H), 0.32 (dq, J = 4.7, 2.5, 2.0 Hz, 2H).¹³C NMR (100 MHz, DMSO-d6) 3161.36, 139.35, 139.03, 131.90, 131.66, 131.43, 129.94, 129.56, 127.37, 62.83, 61.74, 59.18, 54.59, 34.99, 34.94, 33.13, 27.18, 27.07, 6.21, 3.58, 3.44. Mass m/z: calculated for C23H28N2OS: 380.1922, found [M + H]⁺: 381.1992. HPLC data: purity 97.53%, retention time 2.817 min.

-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-2-carboxamide hydrochloride (VZFN209) The title compound was prepared following the general procedure as a white powder in 52.4% yield.¹H NMR (400 MHz, DMSO-d6) 310.57 (s, 1H), 7.58 (dd, J = 5.0, 1.2 Hz, 1H), 7.51 – 7.43 (m, 3H), 7.32 – 7.24 (m, 2H), 6.81 (dd, J = 5.1, 3.8 Hz, 1H), 6.35 (dd, J = 3.9, 1.2 Hz, 1H), 4.53 – 4.39 (m, 1H), 3.65 (m, J = 33.9, 23.4, 10.6, 7.5 Hz, 4H), 3.15 – 3.05 (m, 2H), 2.31 (dd, J = 13.4, 3.0 Hz, 1H), 2.05 – 1.91 (m, 3H), 1.91 – 1.81 (m, 2H), 1.81 – 1.70 (m, 4H), 1.56 (td, J = 13.7, 7.2 Hz, 2H), 1.21 – 1.06 (m, 1H), 0.99 (qd, J = 12.9, 3.5 Hz, 1H). 1³C NMR (100 MHz, DMSO-d₆) 3161.31, 139.40, 139.02, 131.89, 131.62, 131.45, 129.91, 129.54, 127.36, 63.14, 62.01, 59.72, 54.54, 35.08, 34.99, 32.90, 31.15, 30.97, 27.17, 27.06, 26.28, 26.22, 18.69. Mass m/z: calculated for C24H30N2OS: 394.2079, found [M + H]⁺: 395.2132. HPLC data: purity 99.17%, retention time 2.879 min. 7-yl)-N-phenylthiophene-2- carboxamide hydrochloride (VZFN210) The title compound was prepared following the general procedure as a white powder in 49.5% yield.¹H NMR (400 MHz, DMSO-d6) 310.44 (s, 1H), 7.58 (dd, J = 5.0, 1.2 Hz, 1H), 7.51 – 7.44 (m, 3H), 7.32 – 7.25 (m, 2H), 6.81 (dd, J = 5.1, 3.8 Hz, 1H), 6.35 (dd, J = 3.8, 1.2 Hz, 1H), 4.47 (tt, J = 12.0, 3.5 Hz, 1H), 3.85 – 3.71 (m, 2H), 3.64 (m, J = 17.4, 10.2, 6.4 Hz, 2H), 3.05 (t, J = 6.7 Hz, 2H), 2.34 (dd, J = 13.5, 3.0 Hz, 1H), 2.06 – 1.95 (m, 2H), 1.83 (m, J = 28.6, 12.5, 3.0 Hz, 2H), 1.70 (dq, J = 10.2, 5.6, 3.6 Hz, 2H), 1.57 (m, J = 14.0, 10.3, 5.4 Hz, 4H), 1.46 (dp, J = 11.3, 3.4 Hz, 2H), 1.21 – 1.09 (m, 3H), 1.00 (qd, J = 12.7, 3.4 Hz, 1H).¹³C NMR (100 MHz, DMSO-d₆) 3161.31, 139.40, 139.02, 131.89, 131.62, 131.46, 129.91, 129.54, 127.36, 63.52, 62.30, 60.03, 54.52, 35.95, 35.18, 34.63, 32.96, 30.51, 27.17, 27.09, 24.94, 24.89. Mass m/z: calculated for C₂₅H₃₂N₂OS: 408.2235, found [M + H]⁺: 409.2297. HPLC data: purity 98.49%, retention time 2.993 min.

-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-2-carboxamide hydrochloride (VZFN211) The title compound was prepared following the general procedure as a white powder in 55.0% yield.¹H NMR (400 MHz, DMSO-d₆) 310.38 (s, 1H), 7.58 (dd, J = 5.0, 1.2 Hz, 1H), 7.51 – 7.44 (m, 3H), 7.32 – 7.24 (m, 2H), 6.81 (dd, J = 5.1, 3.8 Hz, 1H), 6.35 (dd, J = 3.8, 1.2 Hz, 1H), 4.47 (tt, J = 12.1, 3.5 Hz, (m, J = 14.9, 10.9, 7.6 Hz, 2H), 3.63 (m, J = 14.9, 10.0, 6.2 Hz, 2H), 2.94 (t, J = 6.5 Hz, 2H), 2.40 – 2.30 (m, 1H), 2.08 – 1.98 (m, 1H), 1.82 (m, J = 26.3, 12.4, 3.0 Hz, 2H), 1.72 – 1.49 (m, 8H), 1.19 – 1.10 (m, 3H), 1.00 (dd, J = 12.8, 3.3 Hz, 1H), 0.88 (td, J = 11.6, 5.1 Hz, 2H).¹³C NMR (100 MHz, DMSO-d6) 3161.31, 139.40, 139.01, 131.88, 131.61, 131.46, 129.91, 129.54, 127.36, 63.90, 62.62, 61.49, 54.51, 35.23, 34.61, 33.82, 33.00, 30.30, 30.24, 27.15, 27.08, 25.94, 25.44, 25.38. Mass m/z: calculated for C₂₆H₃₄N₂OS: 422.2392, found [M + H]⁺: 423.2455. HPLC data: purity 100%, retention time 3.118 min.

[3.5]nonan-7-yl)-N-phenylthiophene-2-carboxamide hydrochloride (VZFN212) The title compound was prepared following the general procedure as a white powder in 41.8% yield.¹H NMR (400 MHz, DMSO-d₆) 310.90 (s, 1H), 7.57 (dd, J = 5.1, 1.2 Hz, 1H), 7.53 – 7.49 (m, 2H), 7.48 – 7.43 (m, 3H), 7.41 (m, J = 4.6, 3.2, 2.0 Hz, 3H), 7.29 – 7.24 (m, 2H), 6.80 (dd, J = 5.0, 3.8 Hz, 1H), 6.34 (dd, J = 3.9, 1.2 Hz, 1H), 4.45 (m, J = 12.1, 8.6, 3.5 Hz, 1H), 4.36 – 4.22 (m, 2H), 3.76 (dd, J = 10.3, 6.5 Hz, 1H), 3.68 (p, J = 5.4 Hz, 2H), 3.59 (dd, J = 9.8, 6.9 Hz, 1H), 2.26 (dd, J = 13.5, 3.1 Hz, 1H), 2.06 (dd, J = 13.4, 3.2 Hz, 1H), 1.89 – 1.74 (m, 2H), 1.65 – 1.50 (m, 2H), 1.20 – 0.94 (m, 2H).¹³C NMR (100 MHz, DMSO- d₆) 3161.31, 139.40, 139.03, 131.88, 131.61, 131.42, 131.14, 130.57, 129.90, 129.69, 129.51, 129.25, 127.35, 62.68, 61.43, 58.00, 54.57, 35.02, 34.57, 33.00, 27.16, 27.04. Mass m/z: calculated for C₂₆H₂₈N₂OS: 416.1922, found [M + H]⁺: 417.2008. HPLC data: purity 98.78%, retention time 2.905 min. phenylthiophene-2-carboxamide hydrochloride The title compound was prepared following the general procedure as a white powder in 80.9% yield.¹H NMR (400 MHz, DMSO-d₆) 311.07 (s, 1H), 7.58 (dd, J = 5.0, 1.2 Hz, 1H), 7.52 – 7.44 (m, 3H), 7.35 – 7.20 (m, 7H), 6.81 (dd, J = 5.0, 3.8 Hz, 1H), 6.36 (dd, J = 3.9, 1.2 Hz, 1H), 4.48 (tt, J = 12.0, 3.5 Hz, 1H), 3.76 – 3.61 (m, 4H), 3.30 (tt, J = 12.9, 6.1 Hz, 2H), 2.79 (t, J = 8.1 Hz, 2H), 2.35 – 2.25 (m, 1H), 2.04 (m, J = 13.3, 9.6, 4.5 Hz, 1H), 1.84 (m, J = 33.1, 12.6, 3.4 Hz, 2H), 1.58 (tt, J = 13.4, 4.4 Hz, 2H), 1.21 – 1.10 (m, 1H), 0.98 (qd, J = 12.8, 3.5 Hz, 1H).¹³C NMR (100 MHz, DMSO-d6) 3161.31, 139.39, 138.99, 137.27, 131.90, 131.63, 131.48, 129.92, 129.55, 129.19, 129.03, 127.36, 127.24, 62.94, 61.70, 55.93, 54.44, 34.91, 34.81, 32.96, 30.44, 27.17, 27.09. Mass m/z: calculated for C₂₇H₃₀N₂OS: 430.2079, found [M + H]⁺: 431.2105. HPLC data: purity 98.54%, retention time 2.988 min.

[3.5]nonan-7-yl)-N-phenylfuran-3-carboxamide hydrochloride (VZFN214) The title compound was prepared following the general procedure as a white powder in 31.1% yield.¹H NMR (400 MHz, DMSO-d₆) 310.98 (s, 1H), 7.54 – 7.41 (m, 4H), 7.24 (dd, J = 6.7, 2.9 Hz, 2H), 6.71 (s, 1H), 5.98 (d, J = 1.9 Hz, 1H), 5.77 (m, J = 17.0, 10.4, 6.6 Hz, 1H), 5.52 – 5.35 (m, 2H), 4.54 – 4.41 (m, – 3.67 (m, 3H), 3.64 (t, J = 5.3 Hz, 2H), 3.57 (dd, J = 10.2, 6.5 Hz, 1H), 2.25 (dd, J = 13.6, 3.0 Hz, 1H), 2.02 (dd, J = 13.3, 3.0 Hz, 1H), 1.90 – 1.73 (m, 2H), 1.58 (m, J = 16.8, 11.8, 3.8 Hz, 2H), 1.21 – 1.07 (m, 1H), 1.06 – 0.92 (m, 1H).¹³C NMR (100 MHz, DMSO-d6) 3162.01, 145.08, 143.07, 139.18, 131.27, 129.73, 129.23, 122.97, 111.31, 64.33, 62.70, 54.24, 35.42, 35.16, 27.61. Mass m/z: calculated for C22H26N2O2: 350.1994, found [M + H]⁺: 351.2080. HPLC data: purity 98.74%, retention time 2.683 min.

-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-3-carboxamide hydrochloride (VZFN215) The title compound was prepared following the general procedure as a white powder in 43.5% yield.¹H NMR (400 MHz, DMSO-d6) 310.53 (s, 1H), 7.52 – 7.42 (m, 4H), 7.28 – 7.20 (m, 2H), 6.71 (s, 1H), 5.98 (d, J = 1.9 Hz, 1H), 4.54 – 4.41 (m, 1H), 3.70 (m, J = 20.7, 16.6, 10.2 Hz, 4H), 2.95 (t, J = 6.6 Hz, 2H), 2.29 (dd, J = 13.3, 3.0 Hz, 1H), 2.01 (dd, J = 13.2, 3.1 Hz, 1H), 1.82 (dd, J = 34.0, 13.1 Hz, 2H), 1.58 (m, J = 17.2, 11.6, 6.4 Hz, 2H), 1.17 (td, J = 12.8, 3.5 Hz, 1H), 1.06 – 0.87 (m, 2H), 0.55 – 0.43 (m, 2H), 0.32 (dq, J = 4.6, 2.5, 2.0 Hz, 2H).¹³C NMR (100 MHz, DMSO-d₆) 3162.10, 145.16, 143.14, 139.10, 131.29, 129.82, 129.32, 122.91, 111.29, 62.61, 61.49, 58.98, 53.75, 35.02, 34.95, 33.03, 27.26, 27.15, 6.16, 3.58, 3.44. Mass m/z: calculated for C23H28N2O2: 364.2151, found [M + H]⁺: 365.2239. HPLC data: purity 100%, retention time 2.723 min. 7-yl)-N-phenylfuran-3-carboxamide The title compound was prepared following the general procedure as a white powder in 12.4% yield.¹H NMR (400 MHz, DMSO-d₆) 310.01 (s, 1H), 7.46 (m, J = 9.3, 4.8, 2.2 Hz, 4H), 7.24 (dd, J = 6.8, 2.8 Hz, 2H), 6.71 (s, 1H), 6.01 – 5.93 (m, 1H), 4.54 – 4.40 (m, 1H), 3.77 – 3.65 (m, 3H), 3.64 – 3.57 (m, 2H), 3.12 (q, J = 6.9, 5.5 Hz, 2H), 2.25 – 2.17 (m, 1H), 1.99 (dd, J = 16.8, 10.8 Hz, 3H), 1.88 – 1.81 (m, 2H), 1.76 (dd, J = 8.1, 5.5 Hz, 4H), 1.57 (t, J = 13.4 Hz, 2H), 1.13 (dd, J = 12.6, 3.3 Hz, 1H), 1.02 – 0.94 (m, 1H).¹³C NMR (100 MHz, DMSO-d6) 3145.16, 143.14, 131.28, 129.82, 129.32, 111.28, 62.25, 59.88, 34.98, 30.95, 27.12, 26.20, 18.66. Mass m/z: calculated for C₂₄H₃₀N₂O₂: 378.2307, found [M + H]⁺: 379.2379. HPLC data: purity 100%, retention time 2.795 min.

-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-3-carboxamide hydrochloride (VZFN217) The title compound was prepared following the general procedure as a white powder in 23.8% yield.¹H NMR (400 MHz, DMSO-d6) 310.34 (s, 1H), 7.53 – 7.41 (m, 4H), 7.24 (dd, J = 6.7, 2.9 Hz, 2H), 6.71 (s, 1H), 5.98 (d, J = 1.9 Hz, 1H), 4.52 – 4.41 (m, 1H), 3.78 (dt, J = 18.0, 8.4 Hz, 2H), 3.64 (m, J = 17.4, 10.1, 6.3 Hz, 2H), 3.06 (t, J = 6.7 Hz, 2H), 2.33 (dd, J = 13.4, 3.1 Hz, 1H), 2.07 – 1.94 (m, 2H), 1.84 (dd, J = 10.8, 5.3 Hz, 1H), 1.80 – 1.74 (m, 1H), 1.73 – 1.65 (m, 2H), 1.56 (qt, J = 6.7, 4.0 Hz, 4H), 1.50 – 1.40 (m, 2H), 1.21 – 1.10 (m, 3H),

hydrochloride (VZFN218) The title compound was prepared following the general procedure as a white powder in 32.8% yield.¹H NMR (400 MHz, DMSO-d6) 310.30 (s, 1H), 7.54 – 7.40 (m, 4H), 7.29 – 7.18 (m, 2H), 6.71 (s, 1H), 6.01 – 5.95 (m, 1H), 4.52 – 4.40 (m, 1H), 3.79 (d, J = 12.9 Hz, 2H), 3.70 – 3.56 (m, 2H), 2.94 (t, J = 6.3 Hz, 2H), 2.33 (d, J = 12.9 Hz, 1H), 2.03 (d, J = 13.1 Hz, 1H), 1.80 (dd, J = 26.1, 12.7 Hz, 2H), 1.72 – 1.47 (m, 8H), 1.23 – 1.06 (m, 5H), 1.01 (t, J = 13.3 Hz, 1H), 0.87 (d, J = 13.1 Hz, 2H).¹³C NMR (100 MHz, DMSO-d6) 3 162.08, 145.15, 143.14, 139.08, 131.30, 129.81, 129.31, 122.91, 111.29, 63.95, 62.66, 61.51, 53.69, 35.23, 34.61, 33.82, 33.03, 30.28, 27.21, 25.93, 25.42. Mass m/z: calculated for C26H34N2O2: 406.2620, found [M + H]⁺: 407.2633. HPLC data: purity 100%, retention time 2.978 min.

3-carboxamide hydrochloride The title compound was prepared following the general procedure as a white powder in 54.3% yield.¹H NMR (400 MHz, DMSO-d6) 310.75 (s, 1H), 7.54 – 7.48 (m, 2H), 7.48 – 7.38 (m, 7H), 7.27 – 7.18 (m, 2H), 6.70 (s, 1H), 6.00 – 5.94 (m, 1H), 4.45 (m, J = 12.1, 8.6, 3.5 Hz, 1H), 4.29 (m, J = 13.0, 6.1 Hz, 2H), 3.77 (dd, J = 10.3, 6.4 Hz, 1H), 3.73 – 3.65 (m, 2H), 3.60 (t, J = 8.0 Hz, 1H), 2.24 (dd, J = 13.4, 3.0 Hz, 1H), 2.12 – 2.01 (m, 1H), 1.80 (t, J = 15.6 Hz, 2H), 1.58 (dt, J = 14.0, 11.6 Hz, 2H), 1.17 – 1.10 (m, 1H), 1.00 (m, J = 12.7, 3.5 Hz, 1H).¹³C NMR (100 MHz, DMSO-d₆) 3162.07, 145.15, 143.13, 139.10, 131.26, 130.52, 129.79, 129.66, 129.28, 129.25, 122.91, 111.28, 62.81, 61.55, 53.77, 34.58, 27.17. Mass m/z: calculated for C₂₆H₂₈N₂O₂: 400.2151, found [M + H]⁺: 401.2106. HPLC data: purity 99.26%, retention

2.807 min.

azaspiro[3.5]nonan-7-yl)-N-phenylfuran-3-carboxamide hydrochloride (VZFN220) The title compound was prepared following the general procedure as a white powder in 43.5% yield.¹H NMR (400 MHz, DMSO-d6) 311.06 (s, 1H), 7.52 – 7.42 (m, 4H), 7.36 – 7.29 (m, 2H), 7.28 – 7.20 (m, 5H), 6.72 (s, 1H), 6.02 – 5.95 (m, 1H), 4.54 – 4.42 (m, 1H), 3.68 (dt, J = 13.5, 6.7 Hz, 4H), 2.79 (t, J 2H), 2.29 (dd, J = 13.3, 3.0 Hz, 1H), 2.03 (dd, J = 13.0, 3.1 Hz, 1H), 1.92 – 1.73 (m, 2H), 1.58 (m, J = 13.3, 4.4 Hz, 2H), 1.21 – 1.10 (m, 1H), 0.97 (m, J = 12.9, 3.5 Hz, 1H).¹³C NMR (100 MHz, DMSO-d₆) 3162.09, 145.16, 143.14, 139.06, 137.26, 131.30, 129.81, 129.32, 129.19, 129.03, 127.24, 122.90, 111.29, 62.95, 61.71, 55.94, 53.62, 34.92, 34.81, 32.96, 30.43, 27.24, 27.15. Mass m/z: calculated for C27H30N2O2: 414.2307, found [M + H]⁺: 415.2241. HPLC data: purity 100%, retention time 2873 min.

^^^^^^^^^^^^^ ^!"#^^^^^ The title compound was prepared following the general procedure as a white powder in 84.2% yield.¹H NMR (400 MHz, DMSO d-6): 10.94 (s, 1H), 7.38-7.22 (m, 9H), 7.18-7.16 (m, 3H), 6.78-6.77 (m, 1H), 4.48-4.42 (m, 1H), 3.71-3.68 (m, 4H), 2.80-2.76(m, 3H), 2.29- 2.26 (m, 1H), 2.04-2.01 (m, 1H), 1.89-1.78 (m, 2H), 1.61-1.58(m, 2H), 1.55-1.54 (m, 1H), 1.01-1.01 (m, 2H).¹³C NMR (100 MHz, DMSO-d₆): 164.50, 140.15, 137.88, 137.60, 130.78, 129.70, 129.58, 129.13, 129.11, 128.54, 128.43, 127.26, 125.76, 56.57, 49.48, 49.01, 47.33, 38.62, 36.24, 35.65, 32.32, 30.72, 29.89. Mass m/z: calculated for C27H30N2OS: 430.2079; found [M+H]⁺:431.2154. HPLC data: purity 100.00%, retention time 2.967 min.

^^^^^^^^^^^^^ The title compound was prepared following the general procedure as a white powder in 68.8% yield.¹H NMR (400 MHz, DMSO d-6): 10.31 (s, 1H), 7.37-7.33 (m, 3H), 7.28-7.26 (m, 1H), 7.18-7.15 (m, 3H), 6.78-6.77 (d, 1H), 4.46-4.40 (m, 1H), 3.81-3.79 (m, 2H), 3.67-3.63 (m, 2H), 2.96-2.93 (m, 2H), 2.32-2.31 (m, 1H), 2.01-2.00 (m, 1H), 1.84-1.70 (m, 2H), 1.66-1.51 (m, 9H), 1.17-1.04 (m, 4H), 0.90-0.85 (m, 2H).¹³C NMR (100 MHz, DMSO-d6): 164.27, 139.78, 138.04, 130.99, 129.46, 129.13, 128.55, 128.47, 125.55, 63.94, 62.67, 61.51, 54.29, 35.29, 34.63, 33.82, 33.08, 30.28, 30.22, 27,30, 27.25, 25.93, 25.44, 25.37. Mass m/z: calculated for C26H34N2OS: 422.2392; found [M + H]⁺: 423.2485. HPLC data: purity 98.80%, retention time 3.065 min.

^^^^^^^^^^^^^ ^!"#^^^^^ The title compound was prepared following the general procedure as a white powder in 51.6% yield.¹H NMR (400 MHz, DMSO d-₆): 10.61 (s, 1H), 7.37-7.33 (m, 3H), 7.28-7.26 (m, 1H), 7.17-7.15 (d, 3H), 6.78-6.76 (d, 1H), 4.46-4.40 (m, 1H), 3.72-3.58 (m, 4H), 3.39- 3.38 (m, 1H), 3.12-3.08 (m, 2H), 1.95-1.81 (m,1H), 1.81-1.78 (m, 3H), 1.77-1.74 (m, 6H), 1.56-1.54 (m, 2H), 1.21-0.97(m, 2H).¹³C NMR (100 MHz, DMSO-d6): 164.27, 139.80, 138.04, 130.98, 129.45, 129.14, 128.54, 125.55, 63.14, 62.00, 59.71, 54.31, 49.06, 35.15, 35.02, 32.95, 30.98, 27.32, 27.23, 26.29, 26.23, 18.69. Mass m/z: calculated for C₂₄H₃₀N₂OS: 394.2079; found [M + H]⁺: 395.2158. HPLC data: purity 99.35%, retention time 2.862 min.

^^^^^^^^^^^ ^^^^^^^^^^^^^ ^!"#^^^%^ The title compound was prepared following the general procedure as a white powder in 72.2% yield.¹H NMR (400 MHz, DMSO d-6): 10.02 (s, 1H), 7.36-7.33 (m, 3H), 7.28-7.26 (m, 1H), 7.18-7.15(m, 3H), 6.78-6.77 (m, 1H), 4.46-4.40 (m, 1H), 3.80-3.78 (m, 2H), 3.70-3.65 (m, 2H), 3.08-3.05(m, 2H), 2.28-2.24 (m, 1H), 2.00-1.98 (m, 2H), 1.88-1.85 (m, 1H), 1.80-1.77 (m, 1H), 1.72-1.69(m, 2H), 1.61-1.46 (m, 6H), 1.15-1.09(m, 4H).¹³C NMR (100 MHz, DMSO-d6): 162.43, 145.43, 143.29, 139.48, 137.52, 131.09, 130.03, 129.28, 129.12, 127.28, 122.81, 111.16, 56.57, 49.54, 49.06, 46.74, 38.43, 36.07, 35.68, 32.38, 30.74, 29.93. Mass m/z: calculated for C₂₅H₃₂N₂OS: 408.2235; found [M + H]⁺: 409.2295. HPLC data: purity 97.11%, retention time 2.940 min.

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^

powder in 42% yield.¹H NMR (400 MHz, DMSO d-₆): 10.62 (s,1H), 7.50-7.48 (m, 2H), 7.42-7.41 (m, 3H), 7.34-7.31 (m, 3H), 7.28-7.26 (m, 1H), 7.16-7.14 (m, 3H), 6.77-6.75 (m, 1H), 4.42- 4.31 (m, 1H), 4.30-4.27 (m, 2H), 3.78-3.63 (m, 2H), 3.61-3.60 (m, 1H), 2.23-2.20 (m, 1H), 2.09-2.05 (m, 1H), 1.85-1.81 (m, 2H), 1.77-1.57 (m, 2H), 1.56-1.54 (m, 1H), 1.18-1.02 (m, 1H).¹³C NMR (100 MHz, DMSO-d₆): 161.64, 139.43, 139.14, 132.00, 131.79, 131.28, 130.38, 130.13, 129.87, 129.50, 129.19, 127.44, 59.00, 49.22, 48.63, 47.57, 38.44, 36.02, 35.36, 32.05, 30.81. Mass m/z: calculated for C₂₆H₂₈N₂OS: 416.1922; found [M + H]⁺: 417.1978. HPLC data: purity 100.00%, retention time 2.858 min.

^^^^^^^^^^^^^ ^!"#^^^(^ The title compound was prepared following the general procedure as a white powder in 33% yield.¹H NMR (400 MHz, DMSO d-₆): 11.08 (s, 1H), 7.37-7.33 (m, 3H), 7.28-7.26 (m, 1H), 7.22-7.15 (m, 3H), 6.78-6.77 (m, 1H), 5.82-5.72(m, 1H), 5.49-5.44 (m, 1H), 5.40-5.37 (m, 1H), 4.47-4.41(m, 1H), 3.70-3.57(m, 6H), 2.29-2.25 (m, 1H), 2.04-2.01(m, 1H), 1.88- 1.77(m, 2H), 1.61-1.55 (m, 2H), 1.22-0.98 (m, 2H).¹³C NMR (100 MHz, DMSO-d6): 160.98, 140.02, 131.77, 131.32, 130.36, 129.92, 129.22, 129.07, 125.50, 121.89, 113.22, 109.04, 59.07, 49.31, 48.73, 46.57, 38.51, 36.02, 35.44, 32.17, 30.74. Mass m/z: calculated for C₂₂H₂₆N₂OS: 366.1766; found [M + H]⁺: 367.1839. HPLC data: purity 97.04%, retention time 2.725 min. The title compound was prepared following the general procedure as a white powder in 42% yield.¹H NMR (400 MHz, DMSO d-₆): 10.46 (s, 1H), 7.38-7.33(m, 3H), 7.28-7.26(m, 1H), 7.18-7.16 (m, 3H), 6.78-6.77 (m, 1H), 4.47-4.41 (m, 1H), 3.74-3.70 (m, 4H), 2.97- 2.93 (m, 2H), 2.30-2.26 (m, 1H), 2.03-1.99 (m, 1H), 1.89-1.77 (m, 2H), 1.60-1.57 (m, 2H), 1.25-1.14 (m, 1H), 1.11-1.07 (m, 1H), 0.97-0.93 (m, 1H), 0.51-0.49 (m, 2H), 0.33-0.31 (m, 2H).¹³C NMR (100 MHz, DMSO-d₆): 160.99, 140.06, 137.59, 131.33, 129.93, 129.12, 120.20, 127.26, 125.52, 121.89, 113.22, 109.05, 56.58, 49.53, 49.06, 38.52, 36.09, 35.69, 32.44, 30.70, 29.91. Mass m/z: calculated for C₂₃H₂₈N₂OS: 380.1922; found [M + H]⁺: 381.1988. HPLC data: purity 100.00%, retention time 2.768 min.

-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-2- carboxamide hydrochloride (VZFN228) The title compound was prepared following the general procedure as a pale solid in 12% yield.¹H-NMR (400 MHz, 3 ppm, DMSO-d₆) 11.31 (bs, 1H), 10.42 (bs, 1H), 7.47-7.41 (m, 3H), 7.24-7.22 (m, 2H), 6.74-6.73 (m, 1H), 5.73-5.71 (m, 1H), 4.53-4.48 (m, 1H), 4.44 (s, 1H), 3.84-3.75 (m, 2H), 3.66-3.58 (m, 2H), 2.94 (t, J= 6.5 Hz, 2H), 2.37-2.33 (m, 1H), 2.04- 2.00 (m, 1H), 1.83-1.70 (m, 2H), 1.66-1.51 (m, 7H), 1.20-1.06 (m, 6H), 0.99-0.85 (m, 2H). ¹³C-NMR (100 MHz, 3 ppm, DMSO-d6).160.58, 139.53, 131.50, 129.70, 129.11, 125.52, 121.69, 113.00, 109.00, 63.91, 62.60, 35.31, 34.63, 33.81, 33.05, 30.29, 30.24, 27.32, 27.24, 25.93, 25.43, 25.37. Mass spectrum (m/z): calculated for C26H35N3O: 405.2780; found (M+H)⁺: 406.28. HPLC data: purity: 99.16 %, Retention time: 2.997 min. -2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-2-

carboxamide hydrochloride (VZFN229) The title compound was prepared following the general procedure as a pale solid in 25% yield.¹H-NMR (400 MHz, 3 ppm, DMSO-d6) 11.31 (bs, 1H), 10.31 (bs, 1H), 7.48-7.45 (m, 3H), 7.24-7.21 (m, 2H), 6.74-6.72 (m, 1H), 5.72-5.70 (m, 1H), 4.53-4.47 (m, 1H), 4.43 (s, 1H), 3.70-3.58 (m, 4H), 3.10-3.09 (m, 2H), 2.27-2.24 (m, 1H), 2.01-1.92 (m, 3H), 1.86-1.72 (m, 7H), 1.59-1.53 (m, 2H), 1.12-0.95 (m, 2H).¹³C-NMR (100 MHz, 3 ppm, DMSO-d₆) 160.58, 139.57, 131.49, 129.70, 129.10, 125.52, 121.69, 112.99, 108.99, 63.31, 62.13, 59.85, 53.37, 35.19, 35.02, 33.06, 30.99, 27.30, 26.22, 18.68. Mass spectrum (m/z): calculated for C24H31N3O: 377.2467; found (M+H)⁺: 378.2533. HPLC data: purity: 95.47 %, Retention time: 2.788 min.

2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-2-carboxamide hydrochloride (VZFN230) The title compound was prepared general procedure as a pale solid in 19% yield.¹H-NMR (400 MHz, 3 ppm, DMSO-d6) 11.31 (bs, 1H), 10.20 (bs, 1H), 7.49-7.46 (m, 3H), 7.34-7.30 (m, 2H), 7.25-7.22 (m, 5H), 6.74-6.73 (m, 1H), 5.73-5.71 (m, 1H), 4.55-4.49 (m, 1H), 4.44 (s, 1H), 3.75-3.69 (m, 4H), 2.76-2.74 (m, 2H), 2.17-2.14 (m, 1H), 2.04-2.01 (m, 1H), 1.86-1.75 (m, 3H), 1.62-1.58 (m, 2H), 1.17-0.93 (m, 3H).¹³C-NMR (100 MHz, 3 ppm, DMSO-d6) 160.57, 139.57, 131.51, 129.70, 129.18, 129.11, 128.99, 127.16, 125.53, 121.69, 113.00, 109.00, 63.39, 62.07, 53.36, 34.85, 27.35. Mass spectrum (m/z): calculated for C27H31N3O: 413.2467; found (M+H)⁺: 414.2551. HPLC data: purity: 99.26 %, Retention time: 2.872 min.

-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-2- carboxamide hydrochloride (VZFN231) The title compound was prepared following the general procedure as a pale solid in 24% yield.¹H-NMR (400 MHz, 3 ppm, DMSO-d6) 11.31 (bs, 1H), 10.04 (bs, 1H), 7.48-7.44 (m, 3H), 7.25-7.21 (m, 2H), 6.74-6.72 (m, 1H), 5.72-5.70 (m, 1H), 4.54-4.48 (m, 1H), 4.43 (s, 1H), 3.82-3.73 (m, 2H), 3.69-3.60 (m, 2H), 3.07-3.04 (t, J= 6.72 Hz, 2H), 2.27-2.24 (m, 1H), 2.03-1.91 (m, 2H), 1.84-1.81 (m, 1H), 1.77-1.64 (m,

1.60-1.42 (m, 6H), 1.17-1.07 (m, 3H), 1.03-0.92 (m, 1H).¹³C-NMR (100 MHz, 3 ppm, DMSO-d6) 160.58, 139.56, 131.50, 129.70, 129.10, 125.52, 121.69, 112.99, 109.00, 63.72, 62.45, 60.12, 53.33, 35.95, 35.25, 34.66, 33.13, 30.45, 27.34, 27.24, 24.93, 24.89. Mass spectrum (m/z): calculated for C₂₅H₃₃N₃O: 391.2624; found (M+H)⁺: 392.2677. HPLC data: purity: 98.06 %, Retention time: 2.872 min. 7-yl)-N-phenyl-1H-pyrrole-2- The title compound was prepared following the general procedure as a pale solid in 31% yield.¹H-NMR (400 MHz, 3 ppm, DMSO-d₆) 11.32 (bs, 1H), 10.77 (bs, 1H), 7.51-7.44 (m, 3H), 7.25-7.22 (m, 2H), 6.74-6.72 (m, 1H), 5.73-5.70 (m, 1H), 4.55-4.48 (m, 1H), 4.44 (s, 1H), 3.75-3.60 (m, 4H), 2.93 (t, J= 6.51 Hz, 2H), 2.34-2.31 (m, 1H), 2.01-1.98 (m, 1H), 1.85-1.73 (m, 2H), 1.61-1.54 (m, 2H), 1.19-1.11 (m, 1H), 1.05-0.88 (m, 2H), 0.51-0.46 (m, 2H) 0.35-0.28 (m, 2H).¹³C-NMR (100 MHz, 3 ppm, DMSO-d₆) 160.58, 139.58, 131.50, 129.70, 129.10, 125.53, 121.68, 112.99, 108.99, 65.36, 62.46, 61.29, 58.82, 53.39, 35.10, 34.99, 32.99, 27.36, 27.25, 15.63. Mass spectrum (m/z): calculated for C₂₃H₂₉N₃O: 363.2311; found (M+H)⁺: 364.2398. HPLC data: purity: 99.93 %, retention time: 2.713 min.

[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-2-carboxamide hydrochloride (VZFN233) The title compound was prepared following the general procedure as a pale solid in 17% yield.¹H-NMR (400 MHz, 3 ppm, DMSO-d₆) 11.31 (bs, 1H), 10.62 (bs, 1H), 7.48-7.40 (m, 8H), 7.23-7.20 (m, 2H), 6.73-6.72 (m, 1H), 5.73-5.70 (m, 1H), 4.53-4.47 (m, 1H), 4.42 (s, 1H), 4.33-4.23 (m, 2H), 3.79-3.59 (m, 4H), 2.22-2.20 (m, 1H), 2.04-2.03 (m, 1H), 1.82-1.74 (m, 2H), 1.63-1.54 (m, 2H), 1.15-1.07 (m, 1H), 1.02-0.94 (m, 1H).¹³C-NMR (100 MHz, 3 ppm, DMSO-d6) 160.57, 139.57, 131.47, 131.13, 130.54, 129.68, 129.26, 129.08, 125.52, 121.68, 112.98, 108.99, 62.81, 61.55, 35.10, 34.59, 33.12, 27.33, 27.19. Mass spectrum (m/z): calculated for C26H29N3O: 399.2311; found (M+H)⁺: 400.2396. HPLC data: purity: 99.07 %, retention time: 2.793 min. [3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-2-carboxamide

hydrochloride (VZFN234) The title compound was prepared following the general procedure as a pale solid in 22% yield.¹H-NMR (400 MHz, 3 ppm, DMSO-d6) 11.32 (bs, 2H), 7.48-7.44 (m, 3H), 7.24-7.22 (m, 2H), 6.74-6.73 (m, 1H), 5.81-5.73 (m, 1H), 5.72-5.71 (m, 1H), 5.48-5.37 (m, 2H), 4.54- 4.48 (m, 1H), 4.44 (s, 1H), 3.68-3.56 (m, 6H), 2.32-2.29 (m, 1H), 2.03-2.01 (m, 1H), 1.79 (s, 2H), 1.61-1.54 (m, 2H), 1.18-1.00 (m, 2H).¹³C-NMR (100 MHz, 3 ppm, DMSO-d₆) 160.57, 139.55, 131.49, 129.69, 129.09, 127.96, 125.52, 124.09, 121.69, 113.00, 108.99, 56.67, 53.31, 49.06, 34.97, 34.71, 33.17, 31.14, 27.31, 27.23. Mass spectrum (m/z): calculated for C22H27N3O: 349.2154; found (M+H)⁺: 350.2225. HPLC data: purity: 96.70 %, retention time: 2.670 min.

-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-3- carboxamide hydrochloride (VZFN235) The title compound was prepared following the general procedure as a pale solid in 32% yield.¹H NMR (400 MHz, DMSO-d₆) 2H), 7.43 (s, 3H), 7.18 (s, 2H), 6.44 (s, 1H), 5.98 (s, 1H), 5.58 (s, 1H), 4.48 (t, J = 12.3 Hz, 1H), 3.72 – 3.58 (m, 3H), 3.16 (s, 1H), 3.03 (t, J = 6.8 Hz, 2H), 2.42 (d, J = 14.1 Hz, 1H), ), 2.09 – 1.99 (m, 2H), ), 1.80 – 1.70 (m, 5H), ), 1.55 – 1.45 (m, 7H), ), 1.14 – 1.10 (m, 2H).¹³C NMR (100 MHz, DMSO-d6) 3164.7, 140.7, 131.3, 129.7, 128.6, 122.4, 119.3, 117.7, 110.1, 60.9, 49.8, 49.4, 46.5, 38.6, 36.3, 35.4, 34.8, 32.1, 31.4, 30.6, 25.0, 25.0. Mass m/z: calculated for C25H33N3O: 391.2624; found [M + H]⁺: 392.2687. HPLC data: purity 95.84%, retention time 2.745 min.

-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-3- carboxamide hydrochloride (VZFN236) The title compound was prepared following the general procedure as a pale solid in 30% yield.¹H NMR (400 MHz, DMSO-d6) 310.85 (s, 1H), 10.26 (bs, 1H), 7.44 – 7.43 (m, 3H), 7.19 – 7.17 (m, 2H), 6.46 (q, J = 4.8, 3.2 Hz, 1H), 6.60 – 6.99 (m, 1H), 5.58 (q, J = 4.8, 2.2 Hz, 1H), 4.51 – 4.45 (m, 1H), 3.84 – 3.75 (m, 2H), 3.66 – 3.58 (m, 2H), 2.94 (t, J = 6.6 Hz, 2H), 2.31 (d, J = 15.2 Hz, 1H), 2.03 (dd, J = 15.2, 3.0 Hz, 1H), 1.81 – 1.50 (m, 10H), 1.17 – 1.07 (m, 4H), 0.92 – 0.85 (m, 2H).¹³C NMR (100 MHz, DMSO-d₆) 3164.7, 140.7, 131.3, 129.7, 128.6, 122.4, 119.3, 117.7, 110.1, 61.9, 50.0, 49.6, 49.0, 46.5, 45.8, 38.6, 36.3, 35.3, 32.5, 32.0, 31.1, 30.6, 25.9, 25.4. Mass m/z: calculated for C₂₆H₃₅N₃O: 405.2780; found [M + H]⁺: 406.2833. HPLC data: purity 100%, retention time 2.755 min.

7-yl)-N-phenyl-1H-pyrrole-3- carboxamide hydrochloride The title compound was prepared following the general procedure as a pale solid in 24% yield.¹H NMR (400 MHz, DMSO-d6) 310.85 (bs, 1H), 10.52 (bs, 1H), 7.43 (t, J = 3.5 Hz, 3H), 7.18 – 7.16 (m, 2H), 6.44 (q, J = 4.7, 3.1 Hz, 1H), 5.99 (bs, 1H), 5.58 (q, J = 4.2, 2.0 Hz, 1H), 4.50 – 4.44 (m, 1H), 3.73 – 3.54 (m, 4H), 3.10 – 3.04 (m, 2H), 2.28 (d, J = 14.5 Hz, 1H), 2.00 – 1.93 (m, 3H), 1.88 – 1.70 (m, 7H), 1.57 – 1.50 (m, 2H), 1.13 – 0.91 (m, 2H.¹³C NMR (100 MHz, DMSO-d6) 3164.1, 140.4, 131.53, 129.4, 128.6, 122.3, 119.5, 117.6, 110.2, 63.2, 62.0, 59.7, 53.3, 35.2, 35.0, 33.0, 30.9, 27.4, 27.3, 26.2, 18.6. Mass m/z: calculated for C₂₄H₃₁N₃O: 377.2467; found [M + H]⁺: 378.2531. HPLC data: purity 99.75%, retention time 2.617 min.

-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-3- carboxamide hydrochloride (VZFN238) The title compound was prepared following the general procedure as a pale solid in 18% yield.¹H NMR (400 MHz, DMSO-d6) 310.85 (s, 1H), 10.52 (bs, 1H), 7.43 (s, 3H), 7.18 (s, 2H), 6.45 (s, 1H), 5.99 (s, 1H), 5.58 (s, 1H), 4.49 (t, J = 12.8 Hz, 1H), 3.70– 3.63 (m, 5H), 2.94 (s, 2H), 2.26 (d, J = 13.6 Hz, 1H), 1.98 (d, J = 13.6 Hz, 1H), 1.83 – 1.72 (m, 2H), 1.56 (bs, 2H), 0.99 – 0.93 (m, 2H), 0.49 (d, J = 8.7 Hz, 2H), 0.31 (bs, 2H).¹³C NMR (100 MHz, DMSO-d6) 3164.2, 140.4, 131.5, 129.5, 128.6, 122.3, 119.5, 117.6, 110.28, 62.6, 61.49, 58.9, 53.3, 35.2, 35.0, 33.1, 27.4, 6.1, 3.5, m/z: calculated for C₂₃H₂₉N₃O: 363.2311; found [M + H]⁺: 364.2397. HPLC data: purity 96.34%, retention time 2.573 min. azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-3-carboxamide

(VZFN239) The title compound was prepared following the general procedure as a pale solid in 38% yield.¹H NMR (400 MHz, DMSO-d6) 310.85 (bs, 2H), 7.43 (t, J = 3.2 Hz, 3H), 7.33 – 7.29 (m, 2H), 7.25 – 7.23 (m, 3H), 7.19 – 7.17 (m, 2H), 6.45 (q, J = 4.8, 2.5 Hz, 1H), 6.00 (bs, 1H), 5.58 (q, J = 4.0, 1.6 Hz, 1H), 4.52 – 4.46 (m, 1H), 3.74– 3.36 (m, 4H), 3.41 – 3.26 (m, 2H), 2.27 (t, J = 9.3 Hz, 2H), 2.23 (d, J = 16.5Hz, 1H), 2.03– 1.99 (m, 1H), 1.83 – 1.72 (m, 2H), 1.53 – 1.53 (m, 2H), 1.19 – 1.07 (m, 1H), 1.00 – 0.90 (m, 2H). 1³C NMR (100 MHz, DMSO-d₆) 3164.1, 140.3, 137.2, 131.5, 129.5, 129.1, 129.0, 128.6, 127.2, 122.3, 119.5, 117.6, 110.2, 63.0, 61.8, 55.9, 53.2, 35.1, 34.8, 33.1, 30.4, 27.4, 27.3. Mass m/z: calculated for C₂₇H₃₁N₃O: 413.2367; found [M + H]⁺: 414.2559. HPLC data: purity 95.50%, retention time 2.673 min.

N-(2-benzyl-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-3-carboxamide hydrochloride (VZFN240) The title compound was prepared following the general procedure as a pale solid in 38.3% yield.¹H NMR (400 MHz, DMSO-d₆) 310.94 (s, 1H), 10.84 (s, 1H), 7.55 – 7.47 (m, 2H), 7.41 (m, J = 3.8, 1.9 Hz, 6H), 7.21 – 7.13 (m, 2H), 6.45 (q, J = 2.4 Hz, 1H), 5.99 (dt, J = 3.3, 1.8 Hz, 1H), 5.57 (td, J = 2.7, 1.6 Hz, 1H), 4.53 – 4.42 (m, 1H), 4.35 – 4.21 (m, 2H), 3.74 (dd, J = 10.3, 6.5 Hz, 1H), 3.67 (dd, J = 9.8, 6.6 Hz, 2H), 3.56 (t, J = 8.3 Hz, 1H), 2.26 (dd, J = 13.2, 3.1 Hz, 1H), 2.04 (dd, J = 12.6, 2.8 Hz, 1H), 1.75 (t, J = 15.4 Hz, 2H), 1.63 – 1.48 (m, 2H), 0.96 (td, J = 12.8, 3.3 Hz, 1H).¹³C NMR (100 MHz, DMSO-d6) 3164.19, 140.42, 131.52, 131.15, 130.58, 129.68, 129.49, 129.24, 128.63, 122.34, 119.51, 117.66, 110.29, 65.37, 62.70, 61.44, 57.98, 53.34, 35.22, 34.64, 33.16, 27.44, 27.33, 15.63. Mass m/z: calculated for C26H29N3O: 399.2311, found [M + H]⁺: 400.2364. HPLC data: purity 99.79%, retention time 2.616 min.

[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-3-carboxamide hydrochloride (VZFN241) The title compound was prepared following the general procedure as a pale solid in 20.7% yield.¹H NMR (400 MHz, DMSO-d₆) 310.84 (s, 1H), 10.66 (s, 1H), 7.47 – 7.38 (m, 3H), 7.22 – 7.14 (m, 2H), 6.45 (q, J = 2.4 Hz, 1H), 6.00 (dt, J = 3.3, 1.8 Hz, 1H), 5.75 (m, J = 17.0, 10.3, 6.6 Hz, 1H), 5.58 (td, J = 2.7, 1.5 Hz, 1H), 5.50 – 5.36 (m, 2H), 4.56 – 4.43 (m, 1H), 3.68 (dt, J = 25.3, 4.7 Hz, 5H), 3.57 (t, J = 8.2 Hz, 1H), 2.23 – 2.13 (m, 1H), 2.01 (dd, J = 13.3, 3.0 Hz, 1H), 1.77 (dd, J = 28.5, 12.7 Hz, 2H), 1.64 – 1.51 (m, 2H), 1.19 – 1.05 (m, 1H), 1.05 – 0.92 (m, 1H).¹³C NMR (100 MHz, DMSO-d₆) 3164.19, 140.41, 131.55, 129.50, 128.66, 127.95, 124.14, 122.36, 119.51, 117.67, 110.29, 62.47, 61.35, 56.77, 53.25, 35.09, 34.76, 33.36, 27.35. Mass m/z: calculated for C₂₂H₂₇N₃O: 349.2154, found [M + H]⁺: 350.2214. HPLC data: purity 99.39%, retention time 2.532 min. EXAMPLE 3. Chemical synthesis data fused ring system fentanyl derivatives 5,5-fused ring system fentanyl derivatives were synthesized according to the general synthesis scheme shown below: Scheme 3. General Synthesis Scheme for 5,5-fused ring system fentanyl derivatives

In a solution of I in anhydrous DCM was added acetic acid and followed by aniline at 0 °C under nitrogen. stirred the reaction mixture for 5 minutes and slowly added sodium triacetoxyhydroborate. stirred the reaction mixture at rt for additional 16h and quenched with adding methanol. Washed the organic layer with water, sat. NaHCO3 and brine, dried over MgSO4 and purified by silica gel column chromatography, by using 10:1 to 4:1 hexanes: ethyl acetate as a mobile phase to get pure compound II (Yield = 91%) . Compound II was dissolved in anhydrous DCM and added triethylamine at 0 °C, followed by slow addition of acyl chloride. Stirred the reaction mixture overnight and washed the organic layer with water and brine, dried over MgSO₄ and purified by silica gel column chromatography, by using 4:1 to 1:1 hexanes: ethyl acetate as a mobile phase to get pure compound III (Yield 60-86%). After purification, dissolved the compound III in DCM and slowly added trifluoroacetic acid at 0 °C. Stirred the reaction mixture overnight and dried it on rotary evaporator and used for the next step without further purification. The product from the previous step dissolved in anhydrous acetonitrile and added K2CO3, followed by alkyl bromide. Reflux the reaction mixture and progress monitored by TLC plate. After completion of the reaction, washed layer with water and brine, dried dried over MgSO4 and purified by silica gel column chromatography, by using 99:1:1 to 95:5:1 dichloromethane: methanol: ammonium hydroxide as a mobile phase to get pure compounds which immediately transfers to its salt form by treating with HCl/MeOH to get final VZFN (Yield = 65-90%) compounds. EXAMPLE 4. Chemical synthesis data for 6,6-spiro ring system fentanyl derivatives 6,6-spiro ring system fentanyl derivatives were synthesized according to the general synthesis scheme shown below in Scheme 4. Scheme 4. General Synthesis Scheme for 5,5-fused ring system fentanyl derivatives

In a solution of 1 in anhydrous DCM was added acetic acid and followed by aniline at 0 °C under nitrogen. stirred the reaction mixture for 5 minutes and slowly added sodium triacetoxyhydroborate. stirred the reaction mixture at rt for additional 16h and quenched with adding methanol. Washed the organic layer with water, sat. NaHCO3 and brine, dried over MgSO₄ and purified by silica gel column chromatography, by using 10:1 to 4:1 hexanes: ethyl acetate as a mobile phase to get pure compound 2. Compound 2 was dissolved in anhydrous DCM and added triethylamine at 0 °C, followed by slow addition of acyl chloride. Stirred the reaction mixture overnight and washed the organic layer with water and dried over MgSO₄ and purified by silica gel column chromatography, by using 4:1 to 1:1 hexanes: ethyl acetate as a mobile phase to get pure compound 3. After purification, dissolved the compound 3 in DCM and slowly added trifluoroacetic acid at 0 °C. Stirred the reaction mixture overnight and dried it on rotary evaporator and used for the next step without further purification. The product 4 from the previous step dissolved in anhydrous acetonitrile and added K₂CO_3, followed by alkyl bromide. Reflux the reaction mixture and progress monitored by TLC plate. After completion of the reaction, washed the organic layer with water and brine, dried dried over MgSO₄ and purified by silica gel column chromatography, by using 99:1:1 to 95:5:1 dichloromethane: methanol: ammonium hydroxide as a mobile phase to get pure compounds 5, which immediately transfers to its salt form by treating with HCl/MeOH to get final VZFN compounds. EXAMPLE 5. Chemical synthesis data for 7-member ring system fentanyl analogues 7-member ring system fentanyl derivatives were synthesized according to the general synthesis scheme shown below: Scheme 5. General Synthesis Scheme for 7-member ring system fentanyl derivatives

aniline was added and followed by acetic acid dropwise. The reaction mixture was stirred for 5 minutes and slowly added sodium triacetoxyhydroborate in a portion. The resulting brown reaction mixture was stirred at ambient temperature for additional 16h and quenched with adding methanol, and then diluted with DCM and all contents were transferred to a separatory funnel. The mixture was partitioned by DCM. The organic layer was washed with water, sat. NaHCO3 and brine, dried over MgSO4 and purified by silica gel column chromatography, by using 10:1 to 4:1 hexanes: ethyl acetate as a mobile phase to get pure compound B. Compound B was dissolved in a pre-dried anhydrous DCM and added triethylamine, followed by slow addition of acyl chloride on ice bath. The reaction mixture was allowed to come to RT with stirring then left for stirring overnight under nitrogen gas. The progress was checked by TLC and filtered through celite and washed the organic layer with water and brine, dried over MgSO4 and purified by silica gel column chromatography, by using 4:1 to 1:1 hexanes: ethyl acetate as a mobile phase to get pure compound C. After purification, compound C was dissolved in DCM and slowly added trifluoroacetic acid at 0 °C. Stirred the reaction mixture overnight and dried it on rotary evaporator and used for the next step without further purification. To a solution of compound C, the previous step dissolved in anhydrous acetonitrile and was added K2CO3, followed by alkyl bromide at ambient temperature. The suspension was vigorously stirred and refluxed overnight and progress monitored by TLC plate. After completion of the reaction, washed the organic layer with water and brine, dried over MgSO₄ and purified by silica gel column chromatography, by using 99:1:1 to 95:5:1 dichloromethane: methanol: ammonium hydroxide as a mobile phase to get pure compounds D which immediately transfers to its salt form E by treating with HCl/MeOH to get final VZFN compounds. EXAMPLE 6. Chemical synthesis data for 8-member ring system fentanyl analogues 8-member ring system fentanyl derivatives were synthesized according to the general synthesis scheme shown below: Scheme 6. General Synthesis Scheme for 8-member ring system fentanyl derivatives

bath, aniline was added and followed by acetic acid dropwise. The reaction mixture was stirred for 5 minutes and slowly added sodium triacetoxyhydroborate in a portion. The resulting brown reaction mixture was stirred at ambient temperature for additional 16h and quenched with adding methanol, and then diluted with DCM and all contents were transferred to a separatory funnel. The mixture was partitioned by DCM. The organic layer was washed with water, sat. NaHCO₃ and brine, dried over MgSO₄ and purified by silica gel column chromatography, by using 10:1 to 4:1 hexanes: ethyl acetate as a mobile phase to get pure compound B. Compound B was dissolved in a pre-dried anhydrous DCM and added triethylamine, followed by slow addition of acyl chloride on ice bath. The reaction mixture was allowed to come to RT with stirring then left for stirring overnight under nitrogen gas. The progress was checked by TLC and filtered through celite and washed the organic layer with water and brine, dried over MgSO4 and purified by silica gel column chromatography, by using 4:1 to 1:1 hexanes: ethyl acetate as a mobile phase to get pure compound C. After purification, compound C was dissolved in DCM and slowly added trifluoroacetic acid at 0 °C. Stirred the reaction mixture overnight and dried it on rotary evaporator and used for the next step without further purification. To a solution of compound C, from the previous step dissolved in anhydrous acetonitrile and was added K2CO3, followed by alkyl bromide at ambient temperature. The suspension was vigorously stirred and refluxed overnight and progress monitored by TLC plate. After completion of the reaction, washed the organic layer with water and brine, dried over MgSO₄ and purified by silica gel column chromatography, by using 99:1:1 to 95:5:1 dichloromethane: methanol: ammonium hydroxide as a mobile phase to get pure compounds D which immediately transfers to its salt form E by treating with HCl/MeOH to get final VZFN compounds. EXAMPLE 7. VZFN compounds in vivo studies Methods Warm water tail immersion. Antinociceptive effect of compounds was determined using the warm-water tail immersion assay. 6–8-week 25-35 g male Swiss Webster mice were housed in cages (5 maximal per cage) in animal care quarters and maintained at 22 ± 2 °C on a 12 h light-dark cycle. Food (standard chow) and water were available ad libitum. The mice were brought to the lab (22 ± 2 °C, 12 h light-dark cycle) and allowed 18 h to recover from the transport. The tail-flick test was performed using a water bath with the temperature maintained at 56 ± 0.1°C. The baseline latency (control) was determined before administration of the compounds to the mice, and only mice with a baseline latency of 2 to 4 s were used. In the agonism study, the tail immersion was done 20 min (time that morphine effect starts to peak) post injection of the test compounds subcutaneously (s.c.). To prevent tissue damage, a 10 s maximum cutoff time was imposed. Antinociceptive response was calculated as the percentage of maximum possible effect (%MPE), where %MPE = [(test – control latency)/ (10 – control latency)] × 100. When being studied for their antagonist effects to morphine or fentanyl, the test compounds were given (s.c.) 5 min before morphine or fentanyl. The tail immersion test was then conducted 20 min after giving morphine or fentanyl (s.c.). %MPE was calculated for each mouse. AD50 values were calculated using the least-squares linear regression analysis followed by calculation of 95% confidence interval by the Bliss method. Whole body plethysmography.6–8-week 25-35 g male Swiss Webster mice were housed in cages (5 maximal per cage) in animal care quarters and were maintained at 22 ± 2 °C on a reversed 12-hour dark-light cycle. All experiments were conducted in the dark (active) phase. Ventilatory parameters were captured for freely moving mice in individual test chambers using whole-body plethysmography (FinePointe WBP Chamber with Halcyon Technology, Data Sciences International, St. Paul, MN, USA). The test chambers (0.5 L volume with adjustable 0.5 L/min room air bias flow) were housed in a room illuminated by custom- built, 660 nM-emitting T8-style ceiling-mounted light tubes each with 120, 0.2-watt Epistar 2835 SMD LEDs (Benwei Electronics Co., Ltd., Shenzhen, China), a wavelength with limited visibility to mice, to enable maintenance of the dark cycle during testing. The test chambers were continuously supplied 5% CO₂, 21% O₂, and balance N₂ (AirGas, Radnor, PA, USA) to minimize variability of baseline ventilatory activity, and to increase the sensitivity and capacity of the assay to detect meaningful differences in ventilation as determined in preliminary tests as well as reported and used by others. Subjects were tested no earlier than at least 1 h after the start of the dark phase to further enhance the capacity to detect perturbations to ventilatory parameters. Subjects (n= 8) were first acclimatized to the chambers for 20 min (Baseline) after which saline or fentanyl was administered, and respiration was recorded for 20 min (Agonist phase). Following this, VZFN compounds or saline was administered s.c. and respiration was recorded for 35 min (Reversal phase). Respiratory rate, tidal volume, and minute volume as a function of dose and time were recorded using software (FinePointe Software Research Suite; Data Sciences International, St. Paul, MN, USA). Statistical Analysis. One-way ANOVA followed by the post-hoc Dunnett test were performed to assess the significance using GraphPad Prism software (GraphPad Software, San Diego, CA). Results The results are shown in Figure 1-7. As can be seen from Figure 1, none of the compounds showed antinociceptive effects. As can be seen from Figure 2, 27 compounds potently antagonized the antinociception of 10 mg/kg morphine. As can be seen from Figure 3, 8 compounds potently antagonized the antinociception of 0.1 mg/kg fentanyl. Out of these, 5 compounds were selected for further studies. Figures 4 and 5 show potency determinations of selected VZFN compounds in the warm water tail immersion studies. As can be seen from Figure 6, the tested compounds did not induce depression in respiration with doses up to 60 mg/kg. As can be seen from Figure 7, VZFN093 and VZFN094 did not exhibit a significant increase in minute volume (MVb) at doses up to 32 mg/kg and at 32 mg/kg while they showed a significant increase in tidal volume (TVb) at both 20 min and 10 min post administration. This indicated these compounds have the potential to increase the air volume per respiration cycle. VZFN202 did not show a significant increase in the respiratory parameters observed including MVb, TVb and respiratory rate up to a dose of 30 mg/kg while the trend is positive. EXAMPLE 8. Molecular Pharmacology Profiling of Phenylfentanil and its Analogs to Understand the Putative Involvement of an Adrenergic Mechanism in Fentanyl Induced Respiratory Depression While there are approved therapeutics to treat opioid overdoses, the need for treatments to reverse overdoses due to ultrapotent fentanyls remains unmet. This may be due in part to an adrenergic mechanism of fentanyls in addition to their stereotypical mu opioid receptor (MOR) effects. Herein, we report our efforts to further understanding of the functions these distinct mechanisms impart. Employing the known MOR neutral antagonist phenylfentanil as a lead, seventeen analogs were designed based on the concept of isosteric replacement. To probe mechanisms of analogs were pharmacologically evaluated in vitro and in vivo, while in silico modeling studies were also conducted on phenylfentanil. While it did not indicate MOR involvement in vivo, phenylfentanil yielded respiratory minute volumes similar to those caused by fentanyl. Taken together with molecular modeling studies, these results indicated that respiratory effects of fentanyls may also correlate to inhibition of both ^1A- and ^1B-adrenergic receptors. It is well recognized that a minor but unique structural change on the epoxymorphinan skeleton at the 17-N position, e.g., from methyl to cyclopropylmethyl, may switch an opioid ligand from an MOR agonist to an antagonist, i.e., morphine vs. naloxone or naltrexone (Figure 8). These structural similarities make naloxone and nalmefene effective antidotes to reverse overdoses caused by morphine and heroin due to the resultant similarities in their binding modes to their target protein, the MOR. In stark contrast, among thousands of fentanyl analogs, such a structurally matching pair is missing. For example, mirfentanil (Figure 8) seemed to be a promising reversal agent for fentanyl at lower doses, but its agonistic properties at higher doses on top of its potential off-target effects disqualified its candidacy for such a role. Structurally, the fentanyl scaffold can be classified into four modifiable moieties, including the N-alkyl chain, piperidine ring, acyl group and aniline ring (Figure 9). Since the original disclosure of fentanyl by Paul Janssen in 1960, a broad array of fentanyl analogs have been developed by modifying these moieties, primarily seeking analgesics with superior pharmacokinetic properties, onset time, and effective dosage. During the continuous search for fentanyl-based MOR agonists as potent analgesics, a few fentanyl analogs have also been serendipitously characterized as low to intermediate efficacy MOR modulators. As depicted in Table 1, these representative compounds possess sub-nanomolar to nanomolar MOR binding affinity while manifesting varying degrees of efficacy at the MOR. Structural examination shows that these fentanyl analogs all share the fentanyl scaffold whilst carrying varied acyl substituents (Figures 8 and 9). This observation seemingly suggests the key role of the structural nature of the acyl substitution in binding and recognition at the MOR, and more importantly, it indicates that replacing the propionyl group of fentanyl with other acyl groups may be a feasible approach to obtain structurally diverse MOR ligands with low efficacy or even neutral antagonism that might be capable of counteracting the effects of fentanyl on the MOR. Meanwhile, it would be intriguing to investigate if such a structure activity study could provide insights into fentanyl’s pharmacology at the adrenergic receptors, another putative target for fentanyl induced respiratory depression. Additionally, this study could aid in differentiating the structural contributors towards the pharmacological effects of fentanyl on the opioid and adrenergic receptors. Table 1. Pharmacological profile comparison of phenylfentanil and its analogs along with clinically approved epoxymorphinans. Compounds MOR binding MOR [³⁵S]GTP4S binding Ki (nM) EC50 (nM) % Emax of DAMGO Morphine 6.55 ± 0.74 34.4 ± 5.1 89 ± 17 Naloxone (NLX) 1.1 ± 0.1 8.64 ± 0.33 11.7 ± 3.57 Naltrexone (NTX) 0.33 ± 0.02 0.16 ± 0.04 5.4 ± 0.8 Nalmefene 0.3± 0.15 -- -- Fentanyl 0.083 ± 0.016 22.6 ± 4.7 87.5 ± 8.2% Thiophene Fentanyl 0.333 ± 0.087 31.6 ± 8.1 36.5 ± 2.4% Valeryl Fentanyl 2.16 ± 0.84 58.5 ± 7.9 29.7 ± 4.5% Phenylfentanil 3.55 ± 0.99 29.4 ± 2.6 8.8 ± 2.2 Therefore, we decided to adopt phenylfentanil, a neutral antagonist at the MOR, as the starting point and applied the concept of isosteric replacement to replace the phenyl ring on the acyl group with its isosteric counterparts: furan, thiophene and pyrrole (Figure 9). In addition, compounds possessing an acetamido (n = 1) or N-propanamido (n = 2) side chains were incorporated to probe the distance and flexibility requirement of the introduced heteroaromatic ring for binding affinity and efficacy. Moreover, side chain substitution position was also evaluated to study the possible role of orientation of the heteroaromatic ring. Herein we report the synthesis of a series of phenylfentanil analogs containing various heteroaromatic rings on the acyl group, their binding affinity and efficacy profiles at the MOR and the other two canonical opioid receptors; that is the kappa opioid receptor (KOR) and delta opioid receptor (DOR), as well as their potential to reverse the analgesic effects of and respiratory depression induced by fentanyl. Further, we also report the binding profile of fentanyl and compound 3 (phenylfentanil) as well as in silico modeling studies at the alpha-adrenergic receptors to facilitate our understanding on the putative adrenergic mechanism of fentanyl and phenylfentanil. Results and Discussion Chemical Synthesis. To date, several synthetic routes have been devised to access biologically and structurally diversified fentanyl analogs since the initial disclosure of fentanyl. We looked for synthetically tractable and operationally straightforward transformations amenable to rapid SAR exploration. As such, an optimized synthetic route reported by Mayer and coworkers was adopted in our case (Scheme 7). Briefly, commercially available 4-piperidone hydrochloride was treated with 2-(bromoethyl)- benzene under basic conditions to yield the alkylated piperidone 1. A reductive amination reaction was then carried out through reacting intermediate 1 with aniline mediated by sodium triacetoxyborohydride in the presence of acetic acid to furnish the 4-anilino-N- phenethylpiperidine precursor, 2, in good yield. Next, 4-anilino-N-phenethylpiperidine 2 was acylated using different acyl chlorides either purchased from vendors or prepared in- house following previously published procedures to produce fentanyl analog free bases, which were then converted to hydrochloride form for better aqueous solubility.

Scheme 7. Synthesis of phenylfentanil analogs bearing heterocyclic rings. Reagents and conditions: (a) (2-bromoethyl)benzene, K2CO3, CH3CN, 80 ^oC; (b) Aniline, AcOH, Na(OAc)₃BH, DCM, r.t.; (c) acyl chlorides commercially available or prepared in- house, TEA, toluene, reflux; (d) HCl/MeOH, Et2O Overall, 17 target compounds were synthesized while due to the synthetic feasibility of the acetamido and N-propionamido derivatives of pyrrole their corresponding final products could not be prepared. All target compounds were subjected to full characterization via analytical approaches including ¹H NMR, ¹³C NMR, MS and HPLC before advancing to pharmacological assessments. In Vitro Pharmacological Studies. We first characterized the target fentanyl analogs with the radioligand competition binding assay and the MOR [³⁵S]-GTP^S functional assay following previously described procedures. The purpose of these two experiments was to determine the binding affinity at the three opioid receptors and to assess their functional potencies and efficacies at the MOR, respectively. As outlined in Table 1, the parent compound 3 (phenylfentanil) possessed nanomolar binding affinity at the MOR and micromolar binding affinity at the KOR and DOR, values which were similar to those reported in prior studies. With the same linker (n=0), the isosteric replacement of the phenyl group with its counterparts furan, thiophene and pyrrole, resulted in improvement of binding affinity at all three receptors while maintaining selectivity over KOR and DOR (see compounds 3, 6, 9, 12, 15 and 19). For instance, compound 6 featuring a 2’-furoyl moiety exhibited >20-fold enhancement in binding affinity at all three receptors and maintained an almost identical selectivity profile as compared to parent compound 3. In addition to increased binding affinity, compound 15 also yielded increased selectivity over the KOR and DOR by ~5 fold and ~3 fold, respectively, when compared to parent compound 3. For those target compounds with identical heteroaromatic groups at the amide portion, the alteration from carboxamido to acetamido and n-propanamido side chains generally resulted in reduced binding affinity at the MOR while maintaining the binding affinity at the KOR and DOR (see compounds 6, 7 and 8; compounds 9, 10 and 11; compounds 12, 13 and 14). This observation suggested the necessity of omitting heteroaromatic rings equipped with linear and flexible linkers on the acyl chain of fentanyl in order to achieve favorable MOR binding selectivity. Interestingly, a similar trend was not observed with phenyl substituted analogs (see compounds 3, 4, and 5). In this case, the binding affinity and selectivity appeared to be independent of the amide chain length. Additionally, the attachment points of the linker, 2’ or 3’ of the heteroaromatic ring, appeared to have only marginal effects on binding affinity and selectivity. As seen in Table 2, after exchanging the phenyl group in compound 3 with furan, thiophene and pyrrole, the resulting derivatives showed drastically decreased potency and largely enhanced the activation at the MOR, indicating the critical role of hydrophobicity rather than aromaticity at this position. Meanwhile, increase in the amide linker length also appeared to decrease potency significantly while having no effect on the efficacy. Table 2. The binding affinity, binding selectivity, and MOR [³⁵S]-GTP^S functional assay results of synthesized phenylfentanil analogs.

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^^ ^ a These compounds have been previously reported in literature. b At least four replicates were collected for all data points. On the other hand, there seems to be no obvious effect of substitution position on the heterocyclic rings on compound efficacy. Although all other analogs were observed with lower potency than the parent compound 3 (EC₅₀ = 5.63 nM), they exhibited much higher efficacy than compound 3, ranging between 21 and 65%, again demonstrating the critical role of phenyl ring in achieving near to neutral antagonism in the MOR. Overall, among the 17 compounds tested, compound 3 was determined to be a low efficacy agonist or neutral MOR antagonist with high MOR binding affinity and selectivity over the KOR and DOR. Though [³⁵S]-GTP^S binding provides a direct assessment of GPCR-mediated G- protein activation- the initial phase in MOR signaling- cytosolic Ca²⁺ concentration can be assessed as a downstream secondary messenger. Hence, besides the membrane based [³⁵S]- GTP^S functional assay, a calcium mobilization assay was also employed to verify the antagonism of compound 3 at the MOR. As presented in Figure 10A, compound 3, at concentrations as high as 100 µM, showed no significant agonism at the MOR in the calcium mobilization assay (EC50 of DAMGO and fentanyl in the same assay was 68.4 ± 11.1 nM and 133 ± 19.2 nM, respectively). This result corresponds with a previously reported functional assessment of compound 3, i.e., no significant G-protein recruitment or β-arrestin recruitment was detected. The ability of compound 3 to block MOR agonists was then evaluated in the calcium assay in the presence of a fixed concentration (EC₈₀) of DAMGO and fentanyl, respectively (Figure 10B). As shown, compound 3 inhibited the Ca²⁺ flux induced by DAMGO and fentanyl in a concentration dependent manner and demonstrated its antagonistic effects with IC50 values of 4.1 ± 0.6 µM and 20.4 ± 1.3 µM, respectively. This result further confirmed the antagonist characteristic of compound 3 though its antagonistic potency seemed moderate. While most compounds were observed as partial agonists in vitro, all of them were subjected to the warm-water tail immersion assays to assess their agonist potential or their potential to block the MOR-activating function caused by morphine or fentanyl in mice following previously described protocol. In this assay, a higher % MPE (maximum possible effect) value represents a stronger antinociceptive effect. As depicted in Figure 11A, among the tested fentanyl derivatives, compounds 4, 5, 6, 9, 12, 15 and 19 (10 mg/kg) exhibited noticeable antinociception with high % MPE values, which appeared to be in line with their moderate to high efficacies seen in the [³⁵S]-GTP^S functional assay (Table 2). Further, compounds 3, 7, 8, 10, 11, 13, 14, 16, 17 and 18 (10 mg/kg) did not manifest obvious antinociception and were subsequently tested as antagonists against morphine and fentanyl. As shown in Figure 11B, 8 out of the 10 compounds partially blocked the antinociceptive effect of morphine at a dose of 10 mg/kg. Additionally, when tested against 0.1 mg/kg fentanyl, only compound 17 weakly blocked its antinociceptive effect (Figure 11C). Unexpectedly, compound 3, previously determined as an antagonist in vitro, only exhibited moderate antagonism of morphine’s effects and did not counteract the antinociceptive effects of fentanyl. These seemingly contradictory outcomes could potentially stem from ADME (absorption, distribution, metabolism, and excretion) concerns, leading to the possibilities that compound 3 might either lack CNS permeability or undergo rapid metabolism in vivo. These aspects necessitate further investigation. Due to the fact that compound 3 showed no significant agonism at the MOR and displayed blocking effects to both fentanyl and DAMGO in the calcium flux assays while at a dose of 10 mg/kg it did not suppress both morphine and fentanyl’s antinociceptive activity (Figure 11C), it was further tested at higher doses in the warm-water tail immersion assay. However, at doses up to 60 mg/kg, compound 3 was still not able to block morphine or fentanyl’s antinociceptive effects significantly (Figure 12). This was in agreement with its low potency in the calcium flux assay (Figure 10B) while as mentioned above, its potentially suboptimal pharmacokinetic properties might also play a role. In Vivo Whole-Body Plethysmography Study. A leading factor contributing to fatalities in opioid overdoses is respiratory depression. The fentanyl scaffold exhibits a greater inclination to induce respiratory depression compared to other mu-opioid receptor (MOR) agonists such as morphine or heroin. Fentanyl and its agonist analogs predominantly induce respiratory depression by diminishing the reaction to elevated pCO₂ and reduced pO₂ levels. Consequently, this diminishes the urge to breathe. This subdued respiratory drive leads to a decrease in breathing rate and instances of apnea (temporary cessation of breathing), which, in severe instances, can lead to fatality. Although it did not show any MOR activation potential in the warm-water tail immersion study, compound 3 was studied for its potential liability to cause respiratory depression in mice. This was done in order to further understand the role of the MOR in regard to the stereotypical side effects associated with fentanyl and fentanyl analogs via use of an analog that does not activate the MOR in vivo. Whole-body plethysmography (WBP) has routinely been used for evaluating opioid induced respiratory depression (OIRD) by many research groups in both mice and rats. WBP can provide extensive respiration parameters including tidal volume, minute volume and respiration rate. In WBP tests, the main outcome measures, presented as a percentage relative to the control group, include: 1) Respiratory rate (BPM), which is defined as the number of breaths per minute; 2) Tidal volume (TVb), which is defined as the lung volume that represents the typical amount of air displaced between inhalation and exhalation; and 3) Minute volume (MVb), defined as the volume of air inspired or expired within a minute. Here we report minute volume as a representative as it is the product of respiratory rate and tidal volume. The assay was previously validated using 0.15 mg/kg fentanyl. Respiration in freely moving Swiss Webster mice was measured using whole body plethysmography chambers supplied with an air mixture containing 5% CO2. A 10-min baseline respiration period was recorded prior to any compound administration (data not shown). After the acclimatization period, 0.15 mg/kg fentanyl was administered subcutaneously and respiration was recorded for 5 minutes. Following this, vehicle, or 3 mg/kg NLX was administered subcutaneously, and respiration was recorded for a period of 30 minutes. As shown in Figure 13, 3 mg/kg NLX treatment post fentanyl administration resulted in a significant increase in minute volume within 10 minutes. However, this increase lasted for less than 15 minutes. Following this, compound 3 was studied for its potential to result in respiratory depression. As seen in Figure 14A, while compound 3 (10 mg/kg s.c.) had a slower onset, there was no significant difference between the minute volumes after 10 minutes post- administration of fentanyl or compound 3. This observation suggested that compound 3, not an MOR agonist, could possess the ability to induce respiratory depression in mice. Intriguingly, unlike as seen in fentanyl, up to 3 mg/kg NLX did not significantly increase the post-compound 3 administration minute volume (Figure 14B). Thus, considering the pharmacological characteristics of compound 3, which displayed no MOR-activation in vitro or in vivo (as shown in Table 2 and Figures 10 and 11), while resulting in respiratory minute volumes similar to that of fentanyl, indicated the involvement of an additional non- MOR mediated mechanism of compound 3 and possibly the fentanyl scaffold in general. Adrenergic binding and function of compound 3 Studies in animal models have shown that fentanyl not only binds to the mu opioid receptors but also alpha-1 adrenergic receptors within the locus coeruleus releasing norepinephrine. It is also known that the alpha-1 adrenergic receptor antagonist prazosin prevents fentanyl induced wooden chest syndrome in animals. Additionally, fentanyl and some of its known derivatives induce rapid and profound muscle rigidity. This effect is thought to occur through cerulospinal fibers that are either innervated by or under the control of postsynaptic ^1A adrenergic receptors (^1A-Adr). Table 3. Binding affinity and functionality of compound 3 and comparator compounds on the ^1A- and ^1B-adrenergic receptors. Binding affinity Antagonism function Compound Ki (nM) IC50 (nM) ^1A-Adr ^1B- ^1A-Adr ^{^1B-Adr} Fentanyl 1100 3660 3690

Compound 110 4750 > 5000 Prazosin 0.13 0.027 4.02 8.7 Thus, to further understand the supposed involvement of the adrenergic system in the pharmacological profile of compound 3, its binding affinity and function at the ^1A- and ^1B-Adr were determined. As shown in Table 3, compound 3 (phenylfentanil) bound to both receptors with higher affinity than fentanyl while showing reasonable inhibitory potency on the ^1A-Adr, supporting the involvement of the adrenergic system in its respiratory depression outcomes. Molecular docking studies of fentanyl and compound 3 at the +1A- and +1B-Adr. The binding and functional studies concerning ^1A-Adr and ^1B-Adr strongly suggest that both fentanyl and compound 3 interact with ^1-adrenergic receptors. To propose their plausible binding modes and offer a rationale for their antagonistic effects, molecular modeling investigations were carried out. Due to the unavailability of x-ray or cryo-EM structures of the active ^1B-Adr, active ^1A-Adr and inactive ^1A-Adr, a database search was performed using the basic local alignment search tool (BLAST) in order to identify a suitable template for homology modeling operation. From these results, it was revealed that the cryo-EM structure of agonist bound ^2B-Adr (PDB ID 6K41) and the x-ray crystal structure of inverse-agonist bound ^1B-Adr (PDB ID 7B6W) had the highest sequence identity (39 and 63%, respectively) and homology (57 and 78%, respectively) with the ^1A-Adr. These were thus chosen as the templates for construction of the active and inactive conformations of the ^1A-Adr, respectively. Similarly, the cryo-EM structure of agonist bound ^2B-Adr (PDB ID 6K41) showed high sequence identity and homology (39%, 59%) with ^1B-Adr and was chosen as a template for constructing the homology model of the active conformation of ^1B-Adr. Multiple sequence alignment revealed that the transmembrane residues were highly conserved between the two template proteins and ^1A-Adr and ^1B-Adr (data not shown). Homology models were constructed for all three receptors using SwissModel and model quality was assessed via MolProbity and Protein Structure Analysis (ProSA). As seen from the Ramachandran plots (not shown), for ^1A-Adr^active 90% and 98% of the amino acid residues were in the favored and for ^1A-Adr^inactive 91% and 99% of the residues were in the favored and allowed regions and, for ^1B-Adr^active 92% and 97% of the residues were in the favored and allowed regions, respectively. The z-scores of these models were -2.25, -2.15 and -3.83 for ^1A-Adr^active, ^1A-Adr^inactive and, ^1B-Adr^active, respectively (not shown) which were well within similar experimentally determined structures. Following this, fentanyl, and compound 3 were docked in the active and inactive conformations of the ^1A- and ^1B- Adr. Protein structures were prepared for docking in Sybylx2.1 and GOLD 2020, a genetic algorithm docking program was used to dock the ligands. The binding site was defined to include all atoms within 10Å of co-crystallized inverse agonist in the inactive ^1B-Adr. This binding pocket was retained for docking of the compounds to the active conformation of ^1B-Adr as well as for both ^1A-Adr. A distance constraint between the 10-N of the compounds and the carboxylate group of D106 in ^1A- Adr and D125 in ^1B-Adr was applied. The molecules were docked into the proteins with a total of 100 iterations. CHEMPLP score was used to obtain plausible docking poses and the docking poses were then rescored with HINT (Hydropathic INTeractions) scoring. Clustering of the docking poses of fentanyl and compound 3 in the active and inactive conformations of ^1B-Adr and ^1A-Adr resulted in a single high scoring cluster family. Overall, overlap of the structures of the two receptors revealed that the orientation of the ECL2 was significantly different in both receptor subtypes. In the ^1A-Adr^active, the ECL2 appeared to be oriented outwards, resulting in a larger binding pocket compared to that seen in the ^1B-Adr^active. This in turn oriented the two glutamate residues away from the binding pocket eliminating the large negative hydrophobic-polar interaction that was seen in case of binding of fentanyl and compound 3 to the ^1B-Adr^active. This allowed for stronger binding interactions of both molecules to the ^1A-Adr compared to the ^1B-Adr as reflected by their binding affinities. It was observed that overall, the docking scores of fentanyl and Compound 3 binding to ^1A-Adr were higher than those for ^1B-Adr binding (Table 4) indicating that the molecules would show higher binding for the ^1A-Adr over ^1B-Adr as seen by their affinity (Table 3). Additionally, across both receptor conformations, the docking scores for the inactive conformations were higher compared to their respective active conformations (Table 4) suggesting their preference to behave as antagonists at the two ^-Adr subtypes. The ^1A-and ^1B-Adr binding pockets have been known to comprise of a conserved orthosteric binding pocket defined Adr bound to epinephrine and, a secondary pocket defined based on the co-crystal structure of the inverse agonist (+)- cyclazosin. The ^1A-Adr orthosteric binding pocket was largely hydrophobic comprising of residues from TM3, TM6 and TM7 including A103^TM3, V107^TM3, W285^TM6, F288^TM6, F289^TM6 and F312^TM7. This orthosteric pocket was conserved between the two receptors with the ^1B-Adr orthosteric pocket comprising of corresponding residues from TM3, TM6 and TM7 including A122^TM3, V126^TM3, W307^TM6, F310^TM6, F311^TM6, F334^TM7(data not shown). Additionally, the residues E87^TM2 and E180^ECL2 in ^1A-Adr were also conserved and corresponded to E106^TM2 and E199^ECL2 in ^1B-Adr (data not shown). Table 4. ChemPLP and HINT scores are of the optimal binding modes of fentanyl and Compound 3 at the two ^-adrenergic receptors. Compd. Fentanyl Compound 3 Adr ^1A^active ^1A^inactive ^1B^active ^1B^inactive ^1A^active ^1A^inactive ^1B^active ^1B^inactive

In ^1A-Adr two secondary pockets were observed based on the conformation of the receptor (addressed below). This resulted in a difference in the binding of the compounds and may possibly provide an explanation for their functional activity. On the other hand, in ^1B-Adr the secondary pocket formed between TM1, TM2 and TM7 and contained residues E106^TM2 and K331^TM7 which are critical for receptor activation. E106^TM2 and K331^TM7 form intramolecular H-bonding in the active conformation of ^1B-Adr and it is known that abolishing the positive charge on K331^TM7 results in constitutively active receptor. Binding of fentanyl and compound 3 to the ^1A-Adr: The piperidine ring of both fentanyl and compound 3 bound in a chair conformation occupying the orthosteric binding pocket while the phenethyl-piperidine nitrogen atom formed electrostatic interactions with D106^TM3 (Figure 15). However, the binding mode of the phenethyl group of both molecules to the ^1A-Adr^active and ^1A-Adr^inactive were significantly different. In the ^1A-Adr^inactive the phenethyl group occupied the known secondary pocket formed between the ECL2 and TM5 comprising of hydrophobic residues I175^ECL2, C176^ECL2, Q177^ECL2, I178^ECL2, (Figure 15). In the ^1A-Adr^active, due to the ECL2 orientation, this group bound deeper in the receptor and occupied a new secondary pocket formed by TM3, TM5 and TM6 and comprised of residues T111^TM3, I114^TM3, S192^TM5 W285^TM6 and F289^TM6. In addition, strong pi-stacking interactions were observed between F86^TM2 and the N-phenyl propionamido / N-phenyl benzamido group of fentanyl/ compound 3 in the ^1A-Adr^inactive as compared to ^1A-Adr^active. Binding of fentanyl and compound 3 to the ^1B-Adr: Similar to ^1A-Adr binding, the phenethyl-piperidine ring in fentanyl and compound 3 occupied the conserved orthosteric pocket and the phenethyl-piperidine nitrogen atom formed electrostatic interactions with D125^TM3 (Figure 16). It was observed that the binding mode of the phenethyl group of Compound 3 in the orthosteric pocket was significantly different than that seen in fentanyl’s binding to ^1B-Adr^active (Figure 16). In the ^1B- Adr^active, the binding of both molecules to the orthosteric pocket resulted in a large negative hydrophobic-polar interaction between the phenyl group of both molecules and the carboxyl side chain of E199^ECL2 (3.5 Å) (Figure 16). In the ^1B-Adr^inactive, this negative interaction was replaced by positive pi-stacking interactions with Y203^TM5 (Figure 16). Interestingly, this strong pi-stacking interaction corresponded to that

in the ^1A-Adr^inactive between the phenylethyl group of both molecules and Y184^TM5 (Figures 15 and 16). Thus, we hypothesized that this TM5 tyrosine as well as the TM2 phenylalanine may be essential for the antagonistic function of fentanyl and compound 3 at the ^1-Adrs. The N-phenyl propionamido / N- phenyl benzamido group of fentanyl / compound 3 occupied the secondary pocket of ^1B-Adr. In case of the ^1B-Adr^active, this resulted in steric hindrance disrupting the E106^TM2-K331^TM7 intramolecular H-bonding (Figure 16). In the ^1B- Adr^inactive, the E106^TM2-K331^TM7 interaction is absent resulting in a larger secondary pocket enabling the N-phenyl

N-phenyl benzamido groups to comfortably occupy the secondary binding pocket as reflected by their high docking scores (Figure 16). Through extensive molecular modeling endeavorsand the recent elucidation of the cryo-EM structure of fentanyl bound to the MOR, a distinct fentanyl binding mode has come to light. This has offered a rational framework to comprehend the structure-activity relationships (SAR) of various fentanyl derivatives from literature. The MOR^active-fentanyl complex structure (PDB ID: 8EF5) revealed fentanyl's unique binding pose compared to morphine and DAMGO, occupying a secondary pocket adjacent to the TM2/3 region. This divergence in binding partially fentanyl's heightened potency relative to morphine. Moreover, docking studies involving fentanyl's agonist derivatives mirror this binding pose at the MOR. Given their structural resemblance, it can be assumed that these fentanyl agonist derivatives likely trigger MOR activation through a mechanism similar to that of fentanyl. However, our molecular modeling studies suggest notable differences in the binding modes of fentanyl at the MOR^active and ^1A-Adr^inactive. The phenethyl group of fentanyl binds to the secondary pocket in both receptors, yet in the MOR^active, it aligns with TM2 and TM3, while in the ^1A-Adr^inactive, it corresponds to TM5 and ECL2. Consequently, the orientation of fentanyl skeleton appears to be inverted between these receptors. While further structural biology studies are warranted to verify such a hypoothesis, these results may potentially explain why fentanyl as an MOR agonist and compound 3 as an MOR antagonist potentially both manifest as antagonists at ^1A- and ^1B-Adr, highlighting the divergent functions of these compounds across different receptors. Conclusions It is imperative to develop efficacious countermeasures for the foreseeable ‘fourth wave’ of the opioid overdose crisis largely driven by fentanyl and its analogs. To this end, we applied phenylfentanil (compound 3), a previously identified MOR neutral antagonist, as the starting point and employed the concept of isosteric replacement to swap the phenyl ring on the acyl group with its isosteric counterparts: furan, thiophene and pyrrole. As such, seventeen fentanyl analogs, including seven reported ones, were synthesized, and pharmacologically evaluated. Their structure-activity relationships reflected well with literature reports while phenylfentanil acted as a neutral antagonist on the MOR. More intriguingly phenylfentanil did not reverse the in vivo antinociceptive effects of morphine or fentanyl effectively while it yielded respiratory minute volumes not significantly different from those caused by fentanyl 10 minutes post-administration but were not significantly increased by naloxone. This indicated a potential secondary non-opioidergic mechanism of action. Further studies on the adrenergic system through binding and molecular modeling studies suggested that phenylfentanil may act as an inhibitor on both ^1A- and ^1B- adrenergic receptors to potentially induce said respiratory effects. To summarize, development of fentanyl counteracting agents by applying fentanyl skeleton is a reasonable route to pursue but is challenging due to the putative involvement of adrenergic systems. Experimental Section. Chemistry. All nonaqueous reactions out under a pre-dried nitrogen gas atmosphere. All solvents and reagents were purchased from either Sigma-Aldrich or Alfa Aesar and were used as received without further purification. Melting points were measured on a MPA100 OptiMelt automated melting point apparatus without correction. IR spectra were recorded on a Thermo Scientific Nicolet iS10 FT-IR Spectrometer. Analytical thin- layer chromatography (TLC) analyses were carried out on Analtech Uniplate F254 plates and flash column chromatography (FCC) was performed over silica gel (230−400 mesh, Merck).¹H (400 MHz) and ¹³C (100 MHz) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ultrashield 400 Plus spectrometer, and chemical shifts were expressed in ppm. Mass spectra were obtained on an Applied BioSystems 3200 Q trap with a turbo V source for TurbolonSpray. Analytical reversed-phase high performance liquid chromatography (HPLC) was performed on a Waters Arc HPLC system using XBridge^TM C183.5 µm (4.6 x 50 mm) column. All analyses were conducted at ambient temperature with a flow rate of 0.2 mL/min. Mobile phase is acetonitrile (70%)/water with 0.1% trifluoroacetic acid (TFA; 30%). The UV detector was set up at 210 nm. Compound purities were calculated as the percentage peak area of the analyzed compound, and retention times (Rt) were presented in minutes. The purity of all newly synthesized compounds was identified as ^ 95%. Synthesis of N-phenylethylpiperidin-4-one (1) To a solution of 4-piperidone monohydrate hydrochloride (1g, 7.41 mmol) in acetonitrile (15 mL) was added K2CO3 (2.6 g, 18.25 mmol) and (2-bromoethyl)benzene (1.21 mL, 8.89 mmol) at room temperature. The suspension was vigorously stirred and refluxed overnight at 80^oC. After cooling downs, the mixture was diluted with DCM (200 mL) and water (100 mL), and then transferred to a separatory funnel and extracted, the organic layer was washed with saturated NaHCO3 (3 × 50 mL), brine (3 × 50 mL) and dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash column chromatography (Hexane / EtOAc, 2 / 1) to afford compound 1 (1.1 g, 73%) as a yellow solid. Synthesis of N-[1-(2-phenylethyl)-4-piperidinyl]aniline (2) To a solution of compound 1 (600 mg, 2.95 mmol), AcOH (169 CL, 2.95 mmol), aniline (270 CL, 2.95 mmol) in DCM (10 mL) was added sodium triacetoxyborohydride (939 mg, 4.43 mmol) in portions at 0^oC, the resulting brown mixture was then stirred at r.t. overnight. MeOH (5 mL) was then added and diluted with DCM (100 mL). The organic phase was washed with saturated NaHCO₃ (3 × 50 (3 × 50 mL) and dried over Na₂SO₄ and concentrated in vacuo. The residue was then purified by flash column chromatography (Hexane / EtOAc, 1 / 10) to afford compound 2 (675 mg, 82%) as a yellow solid. General synthesis of fentanyl analogs in free base form The mixture of carboxylic acid (0.5 equiv) and thionyl chloride (2 mL) was refluxed for 3 h. After cooling , all solvent was removed under vacuum, followed by the addition of a solution of compound 2 (0.25 equiv.) and TEA (1 equiv.) in toluene (3 mL). The resulting mixture was then refluxed for 2 days. After cooling , all solvent was removed and the residue was purified by flash column chromatography (DCM / MeOH, 20 / 1) to give title compounds with yields of 28-44%. General procedure for synthesis of final salt To a solution of free base (1 equiv.) in MeOH (1 mL) was added a solution of HCl/MeOH (4 equiv.) dropwise at 0^oC. The clear solution was stirred for another 15 min at the same temperature following which white solid precipitated out as the addition of ethyl ether (10 mL). The suspension was allowed to stir at r.t. for another 3 h and then filtered to give the target salt. Synthesis of N-(1-phenethylpiperidin-4-yl)-N-phenylbenzamide (3) The title compound was prepared following the general procedure as an off-white solid in 54% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d6) ^ 9.77 (s, 1H), 7.34–7.16 (m, 15H), 4.82 (t, J = 12.0 Hz, 1H), 3.62-3.59 (m, 2H), 3.25 – 3.15 (m, 4H), 3.00 – 2.95 (m, 2H), 2.15-2.12 (m, 2H), 1.82-1.73 (m, 2H).¹³C NMR (400 MHz, DMSO-d6) ^ 169.9, 138.7, 137.0, 136.8, 130.8 (2 C), 128.9, 128.8 (2 C), 128.6 (4 C), 127.8, 127.7 (2 C), 127.5 (2 C), 126.8, 56.4, 51.0 (2 C), 50.5, 29.4, 27.2 (2 C). HRMS calcd. for C26H29N2O [M + H]⁺: 385.22744. Found: 385.2258. Synthesis of N-(1-phenethylpiperidin-4-yl)-N,2-diphenylacetamide (4) The title compound was prepared following the general procedure as white powder in 74% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d6) ^ 9.86 (s, 1H), 7.53 – 7.48 (m, 3H), 7.34 – 7.31 (m, 2H), 7.26 – 7.19 (m, 8H), 7.01 – 6.99 (m, 2H), 4.76-4.70 (m, 1H), 3.55- 3.53 (m, 2H), 3.21- 3.08 (m, 4H), 2.97-2.93 (m, 2H), 1.99-1.96 (m, 2H), 1.66-1.56 (m, 2H). ¹³C NMR (400 MHz, DMSO-d6) ^ 169.6, 137.9, 137.0, 135.6, 130.5 (2 C), 129.5 (2 C), 129.0 (2 C), 128.7, 128.6 (4 C), 128.0 (2 C), 126.8, 126.3, 56.4, 50.9 (2 C), 49.4, 40.9, 29.4, 27.2 (2 C). HRMS calcd. for C27H31N2O [M + H]⁺ 399.2431. Found: 399.2422. Synthesis of N-(1-phenethylpiperidin-4- diphenylpropanamide (5) The title compound was prepared following the general procedure as a white sold in 90% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d₆) ^ 9.84 (s, 1H), 7.48–7.43 (m, 3H), 7.34–7.31 (m, 2H), 7.26–7.20 (m, 5H), 7.16-7.11 (m, 3H), 7.02-7.01 (m, 2H) 4.77–4.71 (m, 1H), 3.55-3.52 (m, 2H), 3.21–3.08 (m, 4H), 2.97-2.93 (m, 2H), 2.75 (t, J = 8.0 Hz, 2H), 2.12 (t, J = 8.0 Hz, 2H), 1.95-1.92 (m, 2H), 1.62–1.53 (m, 2H).¹³C NMR (100 MHz, DMSO-d₆) ^ 170.7, 141.0, 137.9, 137.0, 130.3 (2 C), 129.4 (2 C), 128.6 (4 C), 128.2 (2 C), 128.1 (2 C), 126.8, 125.9 (2 C), 56.4, 50.9 (2 C), 49.1, 36.3, 30.9, 29.4, 27.2 (2 C). HRMS calcd. for C₂₈H₃₃N₂O [M + H]⁺: 413.2587. Found: 413.2606. Synthesis of N-(1-phenethylpiperidin-4-yl)-N-phenylfuran-2-carboxamide (6) The title compound was prepared following the general procedure as a white solid in 70% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d6) ^ 10.00 (s, 1H), 7.66 (dd, J = 1.6, 0.6 Hz, 1H), 7.53–7.51 (m, 3H), 7.33–7.31 (m, 4H), 7.26–7.24 (m, 3H), 6.33 (dd, J = 3.5, 1.7 Hz, 1H), 5.44 (d, J = 3.0 Hz, 1H), 4.85–4.81 (m, 1H), 3.58 (d, J = 12.6 Hz, 2H), 3.22– 3.19 (m, 4H), 3.00–2.96 (m, 2H), 2.07 (d, J = 12.4 Hz, 2H), 1.73 (qd, J = 13.2, 3.2 Hz, 2H). 1³C NMR (400 MHz, DMSO-d₆) ^ 158.0, 146.5, 145.2, 137.9, 137.0, 130.8 (2 C), 129.5 (2

111.3, 56.5, 51.0 (2 C), 50.1, 29.5, 27.0 (2 C). HRMS calcd. for C₂₄H₂₇N₂O₂ [M + H]⁺: 375.2067. Found: 375.2086. Synthesis of 2-(furan-2-yl)-N-(1-phenethylpiperidin-4-yl)-N-phenylacetamide (7) The title compound was prepared following the general procedure as a white sold in 85% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d6) ^ 9.40 (s, 1H), 7.54–7.49 (m, 4H), 7.35–7.29 (m, 4H),

(m, 3H), 6.34 (s, 1H), 6.06 (d, J = 2.7 Hz, 1H), 4.73 (m, 1H), 3.58–3.55 (m, 2H), 3.30 (s, 2H), 3.24–3.10 (m, 4H), 2.96–2.91 (m, 2H), 2.03–1.99 (m, 2H), 1.64–1.53 (m, 2H).¹³C NMR (400 MHz, DMSO-d₆) ^ 167.4, 159.1, 149.0, 141.9 (2 C), 137.7, 130.4 (2 C), 129.6 (2 C), 128.8, 128.6 (2 C), 110.5, 107.6, 56.4, 54.9 (2 C), 51.0, 34.2 (2 C), 29.5, 27.2.HRMS calcd for C25H29N2O2 [M + H]⁺: 389.2224. Found: 389.2225. Synthesis of 3-(furan-2-yl)-N-(1-phenethylpiperidin-4-yl)-N-phenylpropanamide (8) The title compound was prepared following the general procedure as a light yellow oil in 58% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d₆) ^ 9.81 (s, 1H), 7.50–7.44 (m, 4H), 7.34–7.31 (m, 3H), 7.26–7.20 (m, 4H), 6.29 (s, 1H), 5.95 (d, J = 2.8 Hz, 1H), 4.76– 4.71 (m, 1H), 3.56–3.53 (m, 2H), 3.22–3.08 (m, 4H), 3.01–2.93 (m, 2H), 2.78 (t, J = 7.5 Hz, 2H), 2.14 (t, J = 7.5 Hz, 2H), 2.00–1.93 (m, 2H), 1.64–1.53 (m, 2H).¹³C NMR (400 MHz, DMSO-d₆) ^ 170.7, 154.9, 141.8, (2 C), 130.0 (2 C), 129.9, 129.4 (2 C), 129.1 (2 C), 127.3, 110.8, 105.6, 56.9,

, 38.5, 30.0 (2 C), 27.7, 23.7.HRMS calcd for C₂₆H₃₁N₂O₂ [M + H]⁺: 403.2380. Found: 403.2361. Synthesis of N-(1-phenethylpiperidin-4-yl)-N-phenylfuran-3-carboxamide (9) The title compound was prepared following the general procedure as a white sold in 88% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d6) ^ 9.84 (s, 1H), 7.54–7.52 (m, 3H), 7.49 (s, 1H), 7.36–7.31 (m, 4H), 7.27–7.24 (m, 3H), 6.79 (s, 1H), 6.01 (s, 1H), 4.89– 4.83 (m, 1H), 3.61–3.58 (m, 2H), 3.25–3.19 (m, 4H), 3.00–2.95 (m, 2H), 2.09–2.06 (m, 2H), 1.76–1.67 (m, 2H).¹³C NMR (400 MHz, DMSO-d₆) ^ 162.4, 145.5, 143.3, 138.4, 137.4, 131.5 (2 C), 130.0 (2 C), 129.6, 129.1 (4 C), 127.3, 122.6, 111.2, 56.9, 51.5 (2 C), 49.1, 30.0, 27.6 (2 C). HRMS calcd for C24H27N2O2 [M + H]⁺: 375.2067. Found: 375.2058. Synthesis of 2-(furan-3-yl)-N-(1-phenethylpiperidin-4-yl)-N-phenylacetamide (10) The title compound was prepared following the general procedure as a pale solid in 80% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d₆) ^ 9.72 (s, 1H), 7.53–7.49 (m, 4H), 7.34–7.23 (m, 8H),

(s, 1H), 4.73 (t, J = 12.0 Hz, 1H), 3.55 (d, J = 12.0 Hz, 2H), 3.22–3.11 (m, 4H), 3.04 (s, 2H), 2.97–2.93 (m, 2H), 2.00–1.97 (m, 2H), 1.64–1.55 (m, 2H). ¹³C NMR (400 MHz, DMSO-d6) ^ 169.2, 142.7, 140.1 (2 C), 137.9, 136.9, 130.4 (2 C), 129.6 (2 C), 128.6 (4 C), 126.8, 118.6, 111.8, 56.4, 51.0 (2 C), 49.2, 30.8, 29.5, 27.2 (2 C).HRMS calcd for C25H29N2O2 [M + H]⁺: 389.2224. Found: 389.2222. Synthesis of 3-(furan-3-yl)-N-(1-phenethylpiperidin-4-yl)-N-phenylpropanamide (11) The title compound was prepared following the general procedure as a white solid in 94% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d₆) ^ 9.58 (s, 1H), 7.21–7.14 (m, 4H), 7.06–6.91 (m, 8H), 5.90 (s, 1H), 4.48-4.42 (m, 1H), 3.27–3.24 (m, 2H), 2.93–2.79 (m, 4H), 2.69-2.65 (m, 2H), 2.26 (t, J = 8.0 Hz, 2H), 1.77 (t, J = 8.0 Hz, 2H), 1.68–1.65 (m, 2H), 1.36-1.27 (m, 2H).¹³C NMR (400 MHz, DMSO-d6) ^ 170.7, 142.9, 138.9, 138.0, 137.0, 130.3 (2 C), 129.5 (2 C), 128.6 (4 C), 126.8, 123.8 (2 C), 111.1, 56.4, 51.0 (2 C), 49.1, 35.1, 29.4, 27.2 (2 C), 20.0.HRMS calcd for C26H31N2O2 [M + H]⁺: 403.2380. Found: 403.2361. Synthesis of N-(1-phenethylpiperidin-4-yl)-N-phenylthiophene-2-carboxamide (12) The title compound was prepared following the general procedure as a white sold in 97% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d6) ^ 10.04 (s, 1H), 7.63 (d, J = 5.0 Hz, 1H), 7.54–7.52 (m, 3H), 7.37–7.34 (m, 2H), 7.33–7.31 (m, 2H), 7.26–7.24 (m, 3H), 6.84 (dd, J = 4.1, 3.4 Hz, 1H), 6.43 (d, J = 3.4 Hz, 1H), 4.86 (m, 1H), 3.60–3.57 (m, 2H), 3.22–

was as a yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d₆) ^ 9.85 (s, 1H), 7.55–7.50 (m, 3H), 7.35–7.31 (m, 3H), 7.28–7.23 (m, 5H), 6.89 (dd, J = 5.0, 3.0 Hz, 1H), 6.67 (d, J = 3.0 Hz, 1H), 4.75-4.69 (m, 1H), 3.56–3.53 (m, 2H), 3.46 (s, 2H), 3.22–3.08 (m, 4H), 2.98–2.93 (m, 2H), 2.00–1.97 (m, 2H), 1.67–1.58 (m, 2H).¹³C NMR (400 MHz, DMSO-d6) ^ 168.6, 137.7, 137.0, 136.6, 130.4 (2 C), 129.6 (2 C), 128.9, 128.6 (3 C), 126.8, 126.3 (2 C), 126.2, 125.1, 56.4, 51.0 (2 C), 49.5, 35.4, 29.5, 27.2 (2 C).HRMS calcd for C25H29N2OS [M + H]⁺: 405.1995. Found: 405.1976. Synthesis of N-(1-phenethylpiperidin-4-yl)-N-phenyl-3-(thiophen-2-yl)propanamide (14) The title compound was prepared following the general procedure as a white solid in 97% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d₆) ^ 9.83 (s, 1H), 7.51–7.43 (m, 3H), 7.35–7.31 (m, 2H), 7.26–7.23 (m, 4H), 7.18–7.16 (m, 2H), 6.88 (dd, J = 4.9, 3.6 Hz, 1H), 6.71 (d, J = 3.6 Hz, 1H), 4.77-4.71 (m, 1H), 3.56–3.53 (m, 2H), 3.22–3.09 (m, 4H), 2.99–2.93 (m, 4H), 2.17 (t, J = 7.3 Hz, 2H), 1.97–1.93 (m, 2H), 1.65–1.54 (m, 2H).¹³C NMR (400 MHz, DMSO-d₆) ^ 170.3, 143.5, 137.9, 137.0, 130.3 (2 C), 129.5 , 128.6 (4 C), 126.8 (3 C), 124.6, 123.7 (2 C), 56.4, 51.0 (2 C), 49.1, 36.5, 29.4, 27.2 (2 C), 24.9. HRMS calcd for C₂₆H₃₁N₂OS [M + H]⁺: 419.2152. Found: 419.2143. Synthesis of N-(1-phenethylpiperidin-4-yl)-N-phenylthiophene-3-carboxamide (15) The title compound was prepared following the general procedure as a white sold in 91% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d6) ^ 9.97 (s, 1H), 7.42–7.39 (m, 3H), 7.36–7.30 (m, 3H), 7.27–7.24 (m, 6H), 6.82 (d, J = 4.9 Hz, 1H), 4.86-4,80 (m, 1H), 3.61–3.58 (m, 2H), 3.25–3.20 (m, 4H), 3.01–2.97 (m, 2H), 2.11–2.08 (m, 2H), 1.82–1.72 (m, 2H).¹³C NMR (400 MHz, DMSO-d6) ^ 164.1, 138.7, 137.1, 137.0, 130.8 (2 C), 129.2, 129.2 (2 C), 128.7 (2 C), 128.6 (2 C), 128.4, 128.0 (2 C), 126.8, 125.3, 56.5, 51.1, 48.6 (2 C), 29.5, 27.2 (2C). HRMS calcd for C24H27N2OS [M + H]⁺: 391.1839. Found: 391.1837. Synthesis of N-(1-phenethylpiperidin-4-yl)-N-phenyl-2-(thiophen-3-yl)acetamide (16) The title compound was prepared following the general procedure as a white solid in 82% yield. Hydrochloride salt: ¹H NMR d₆) ^ 9.88 (s, 1H), 7.52–7.50 (m, 3H), 7.42–7.40 (m, 1H), 7.35–7.31 (m, 2H), 7.25–7.24 (m, 5H), 7.04 (m, 1H), 6.85 (d, J = 4.8 Hz, 1H), 4.74 (m, 1H), 3.57–3.54 (m, 2H), 3.25 (s, 2H), 3.15–3.12 (m, 4H), 2.98–2.94 (m, 2H), 2.00–1.97 (m, 2H), 1.66–1.57 (m, 2H).¹³C NMR (400 MHz, DMSO-d6) ^ 169.3, 137.9, 137.0, 135.2, 130.4 (2 C), 129.5 (2 C), 128.7 (2 C), 128.6 (4 C), 126.8, 125.5, 122.3, 56.4, 51.0 (2 C), 49.3, 35.7, 29.5, 27.2 (2 C). HRMS calcd for C25H29N2OS [M + H]⁺: 405.1995. Found: 405.1976. Synthesis of N-(1-phenethylpiperidin-4-yl)-N-phenyl-3-(thiophen-3-yl)propanamide (17) The title compound was prepared following the general procedure as a white solid in 49% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d6) ^ 9.64 (s, 1H), 7.50–7.44 (m, 3H), 7.39 (m, 1H), 7.35–7.31 (m, 2H), 7.26–7.23 (m, 3H), 7.17–7.15 (m, 2H), 6.99 (s, 1H), 6.80 (d, J = 4.8 Hz, 1H), 4.75 (m, 1H), 3.56–3.53 (m, 2H), 3.23–3.09 (m, 4H), 2.99–2.92 (m, 2H), 2.76 (t, J = 7.5 Hz, 2H), 2.12 (t, J = 7.5 Hz, 2H), 1.99–1.93 (m, 2H), 1.61–1.52 (m, 2H).¹³C NMR (400 MHz, DMSO-d₆) ^ 170.7, 141.3, 138.0, 136.9, 130.3 (2 C), 129.5 (2 C), 129.4, 128.9, 128.6 (2 C), 128.2 (2 C), 126.8, 125.8, 120.6, 56.4, 51.1 (2 C), 48.9, 35.5, 29.5, 27.2 (2 C), 25.3. HRMS calcd for C₂₆H₃₁N₂OS [M + H]⁺: 419.2152. Found: 419.2143. Synthesis of N-(1-phenethylpiperidin-4-yl)-N-phenyl-1H-pyrrole-2-carboxamide hydrochloride (18) The title compound was prepared following the general procedure as a white solid in 76% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d₆) ^ 11.44 (s, 1H), 9.76 (s, 1H), 7.54–7.53 (m, 3H), 7.35–7.30 (m, 4H), 7.27–7.24 (m, 3H), 6.77 (s, 1H), 5.75 (dd, J = 6.4, 2.6 Hz, 1H), 4.93–4.86 (m, 1H), 4.50 (s, 1H), 3.57 (d, J = 11.3 Hz, 2H), 3.22–3.17 (m, 4H), 2.98 (dd, J = 10.4, 6.5 Hz, 2H), 2.06 (d, J = 13.0 Hz, 2H), 1.75–1.67 (m, 2H).¹³C NMR (400 MHz, DMSO-d₆) ^ 160.4, 138.5, 134.0, 131.3 (2 C), 129.4 (2 C), 129.0, 128.7 (4 C), 126.8, 124.7, 121.6, 112.9, 108.7, 56.5, 51.1 (2 C), 49.6, 29.5, 27.2 (2 C).HRMS calcd for C24H28N3O [M + H]⁺: 374.2227. Found: 374.2235. Synthesis of N-(1-phenethylpiperidin-4-yl)-N-phenyl-1H-pyrrole-3-carboxamide (19) The title compound was prepared following the general procedure as a light yellow solid in 94% yield. Hydrochloride salt: ¹H NMR (400 MHz, DMSO-d₆) ^ 10.95 (s, 1H), 9.84 (s, 1H), 7.50–7.48 (m, 3H), 7.35–7.31 (m, 2H), 7.26–7.24 (m, 5H), 6.49 (d, J = 2.0 Hz, 1H), 6.05 (s, 1H), 5.62 (d, J = 2.0 Hz, 1H), 4.87 (m, 1H), 3.56–3.56 (m, 2H), 3.21–3.17 (m, 4H), 2.99– 2.95 (m, 2H), 2.05–2.02 (m, 2H), 1.74–1.66 (m, 2H).¹³C NMR (400 MHz, DMSO-d6) ^ 164.0, 139.2, 137.0, 131.3 (2 C), 129.2 (2 C), 128.6 (2 C), 128.5, 126.8, 122.1, 118.6, 117.3, 109.8, 56.4, 51.1 (2 C), 49.6, 29.5, 27.3 (2 C). HRMS calcd for C24H28N3O [M + H]⁺: 374.2221. Found: 374.2217. Biological Evaluation. Drugs. Morphine (morphine sulfate pentahydrate salt) was purchased from Mallinckrodt (St. Louis, MO) or provided by the National Institute of Drug Abuse (NIDA). All drugs and test compounds were dissolved in pyrogen-free isotonic saline (Baxter Healthcare, Deerfield, IL) or sterile-filtered distilled/deionized water. All other reagents and radioligands were purchased from either Sigma-Aldrich or Thermo Fisher. Animals. Male Swiss-Webster mice (23-35 g, 7−8 weeks, Harlan Laboratories, Indianapolis, IN) were housed five to a cage in animal care quarters maintained at 22 °C on a 12-hour light/dark cycle with food and water available ad libitum. Protocols and procedures were approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University Medical Center and complied with the recommendations of the International Association for the Study of Pain. In Vitro Competitive Radioligand Binding Assay and Functional Assay. Competition binding was performed using the monoclonal opioid receptor expressed in Chinese hamster ovary (CHO) as previously described.¹⁸ [³H]Naloxone, [³H]NTI, and [³H]norBNI (or [³H]DPN) were applied to label the ^, ^, and ^ opioid receptors, respectively. In this assay, 20 µg of membrane protein was incubated with 1.4 nM the corresponding radioligand in the presence of different concentrations of test compounds in TME buffer (50 mM Tris, 3 mM MgCl2, and 0.2 mM EGTA, pH 7.7) for 1.5 h at 30 °C. After incubation, the bound radioactive ligand was separated from free radioligand by filtration through GF/B glass fiber filters and rinsed three times with ice-cold wash buffer (50 mM Tris-HCl, pH 7.2) using a Brandel harvester. The results were determined by utilizing a scintillation counter. Specific binding was determined as the difference in binding obtained in the absence and presence of 5 µM naltrexone. The IC50 values were determined and converted to Ki values using the Cheng–Prusoff equation. Functional assays were conducted in the same cell membranes used for the receptor binding assays. Membrane proteins (10 ^g) were incubated with varying concentrations of drugs, GDP (20 ^M), and 0.1 nM ³⁵S-GTP[^S] in assay buffer for 1.5 h at 30 °C. Nonspecific binding was determined with 20 ^M unlabeled GTP[^S]. DAMGO (3 ^M), U50,488H (5 ^M), and SNC80 (5 ^M)

included in the assay for a maximally effective concentration of a full agonist for the ^, ^, and ^ opioid receptors, respectively. Calcium mobilization assay. mMOR-CHO cells were cultured with DMEM/F-12 supplemented with 10% FBS at 37 °C and 5% CO₂. The cells were transfected with Gqi5 cDNA using Lipofectamine 2000 medium OPTI according to the manufacturer’s recommended procedure. Then the cells were incubated for 4 h before being plated to a clear bottom, black 96-well assay plate at 15,000 cells/well in cell growth media. Cells were ready for calcium mobilization assay after 16-20 h incubation.50 CL of loading buffer was added to each well in the assay plate, followed by 1 hour incubation. The positive control, and varying concentrations of the testing compound were added to a source plate. (For antagonist measurement, 20 CL of the testing compound was then added to each well and incubated for another 15 min.) Before the measurement, the loading buffer was decanted and 80 CL/well of the washing buffer was added to the 96 well plate. Subsequently, the assay plates were read on a FlexStation3 microplate reader at 494/516 ex/em. The changes in fluorescence were monitored and peak height values were obtained using SoftMaxPro software (Molecular Devices). Nonlinear regression curves and IC50 values were generated using GraphPad Prism 8.0. All concentrations were tested in triplicate and all experiments were repeated at least three times. Warm Water Immersion Assay.6-8 week 25-35 g male Swiss Webster mice were housed in cages (5 maximal per cage) in animal care quarters and maintained at 22 ± 2 °C on a 12 h light-dark cycle. Food (standard chow) and water were available ad libitum. The mice were brought to the lab (22 ± 2 °C, 12 h light-dark cycle) and allowed 18 h to recover from the transport. The tail-flick test was performed using a water bath with the temperature maintained at 56 ± 0.1°C. Each mouse was gently wrapped in a cloth with only the tail exposed. Baseline latency was measured before s.c. injection of the compounds. The distal one-third of the tail was immersed perpendicularly in water, and the mouse rapidly flicked his tail from the bath at the first sign of discomfort. The duration of time the tail remained in the water bath was counted as the baseline latency. Untreated mice with baseline latency reaction times ranging from 2 to 4 seconds were used. Test latency was obtained 20 min later after the agonist injection. A 10-second maximum cutoff latency was used to prevent any tissue damage. Antinociception was quantified as the percentage of maximal possible effect (%MPE), which was calculated as %MPE= [(test latency − control latency)/(10 − control latency)] × 100. The %MPE value was calculated for each mouse using 6 mice per compound. If the compound was antagonizing effects against morphine or fentanyl, the compound was s.c. injected 5 minutes prior to the agonist administration. Measurement of respiration.6–8-week 25-35 g male Swiss Webster mice were housed in cages (5 maximal per cage) in animal care quarters and were maintained at 22 ± 2 °C on a reversed 12-hour dark-light cycle. All experiments were conducted in the dark (active) phase. Respiration was measured using whole body plethysmography chambers (EMKA Technologies, France) in freely moving mice. The chambers were supplied with an air mixture containing 5% CO2. A 10-min baseline respiration period was recorded prior to any administration. The rate and depth of respiration were recorded and averaged over 1- or 5- min periods. Tidal volume was calculated from the raw inspiration data and expiration data. Minute volume was then calculated as rate x tidal volume. The first compound was administered s.c. and respiration was recorded for 5 minutes. Then respiration was recorded for a period of 30 minutes after the second injection. Statistical Analysis. One-way ANOVA followed by the post-hoc Dunnett test were performed to assess significance using Prism 6.0 software (GraphPad Software, San Diego, CA). Homology model construction: Due to the unavailability of structures for the active ^1B- Adr, active ^1A-Adr and inactive ^1A-Adr, a database search was performed using the basic local alignment search tool (BLAST) in order to identify a suitable template for homology modeling operation. The x-ray crystal structure of inverse-agonist bound ^1B-Adr (PDB ID 7B6W) was used as the inactive conformation of ^1B-Adr for docking studies. In order to identify a suitable template for homology modeling operation, database search was performed using the basic local alignment search tool (BLAST). The cryo-EM structure of agonist bound ^2B-Adr (PDB ID 6K41) was used as the template for the homology model of the active ^1A- and ^1B-Adr while the inverse-agonist bound ^1B-Adr (PDB ID 7B6W) was used as the template for inactive ^1A-Adr homology model. The sequence alignment performed using the program Clustal Omega.⁷⁴ The homology models were constructed for all three receptors using SwissModel and the local geometry of the optimized structure was checked using MolProbity and Protein Structure Analysis (ProSA-Web). Molecular docking studies: Fentanyl and Compound 3 (phenylfentanil) were drawn using Sybylx2.1, assigned Gasteiger-Huckel charges and energy minimized with the Tripos Force Field. Fentanyl and compound 3 were docked in both the active and inactive conformations of the ^1A- and ^1B- Adr. Protein prepared for docking by adding hydrogen atoms, deleting water molecules and bound ligands inside the binding pocket. The conserved disulfide bridges between C99 and C176 in inactive ^1A-Adr and between C118 and C195 in inactive ^1B-Adr were built. Further, the mutated L334 residue in the crystal structure of ^1B-Adr was changed back to its original F334. GOLD 2020, a genetic algorithm docking program was used to dock the ligands and binding site was defined to include all atoms within 10Å of co-crystallized inverse agonist in the inactive ^1B-Adr. This binding pocket was retained for docking of the compounds to the active conformation of ^1B-Adr as well for both ^1A-Adr. A distance constraint between the 10-N of the compounds and the carboxylate group of D106 in ^1A-Adr and D125 in ^1B-Adr was applied. The molecules were docked into the proteins with a total of 100 iterations. To optimize the structural models for the ligand-protein complexes, docking was followed by energy minimization under Tripos Force Field in Sybylx2.1. CHEMPLP score, which has been optimized for modeling steric complementarity between ligand and protein along with distance and angle dependent hydrogen bonding, was used to obtain plausible docking poses. These docking poses were then rescored with HINT (Hydropathic INTeractions)⁷⁵, an empirical free energy scoring tool based on the experimental measurements of logP for 1-octanol and water, to estimate atomic level free energies associated with noncovalent interactions. Optimal docking poses for each ligand-protein complex were chosen based on highest ChemPLP and HINT scores. Figures were generated using PyMOL version 1.7.4. ABBREVIATIONS USED CHO, Chinese hamster ovary; CL, confidence level; CNS, central nervous system; DAMGO, [D-Ala2-MePhe4-Gly(ol)5]enkephalin; DOR, delta opioid receptor; GPCRs, G protein-coupled receptors; KOR, kappa opioid receptor; MOR, mu opioid receptor; %MPE, percentage maximum possible effect; NIDA, National Institute of Drug Abuse; OUD, opioid use disorder. While the invention has been described in terms of its several exemplary embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

Claims

CLAIMS We claim:

1. A compound having the general formula where

R = substituted C1-C16 alkyl, or unsubstituted C1-C16 alkyl, aryl, heteroaryl or substituted aryl; ^ R₁ = substituted C1-C16 alkyl, unsubstituted C1-C16 alkyl, aryl, heteroaryl or substituted aryl; n = 0, 1, 2 or 3; m = 0, 1, 2 or 3; o = 0, 1, 2, or 3; wherein spacers associated with n, m, or o, when present, may be or include one or more methylenes, or O, N, or S atoms, and wherein X = ;

R2 = or with the caveat that the compound is not fentanyl, phenylfentanil, mirfentanil or thiophentanil. 2. The compound of claim 1, wherein the substituted C1-C16 alkyl is substituted with one or more of N, O or S. 3. The compound of claim 1, wherein the compound is and R₁ =

.

4. The compound of claim 1, wherein the compound is . 5. The compound of claim 1, wherein R is aryl, heteroaryl or substituted aryl. 6. The compound of claim 1, wherein R1 is aryl, heteroaryl or substituted aryl. 7. The compound of claim 1, wherein n = 0. 8. The compound of claim 1, wherein n = 0, 1 or 2. 9. A method of treating fentanyl toxicity or overdose in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound of any of claims 1-8.