Development of novel Mu opioid receptor antagonists based on a 6,4-spiro bicyclic fentanyl-derived scaffold.

Introduction

Opioid analgesics remain essential for pain management but are associated with severe adverse effects, including respiratory depression and high abuse potential.1–3 These effects are primarily mediated by the Mu opioid receptor (MOR), making it a central target for the development of safer opioid therapeutics.1,4 Ligands with reduced intrinsic efficacy at the MOR have attracted considerable interest because partial agonists and antagonists can modulate opioid signaling while potentially avoiding severe adverse effects associated with full agonists.5–8 However, controlling MOR intrinsic efficacy through rational ligand design remains a significant challenge, particularly for scaffolds derived from highly potent opioids such as fentanyl and its analogues.

The fentanyl scaffold provides a versatile platform for exploring structure–activity relationships due to its well-defined pharmacophore and high receptor affinity. Structural modifications of fentanyl have demonstrated that both core-ring topology and substituent variation substantially influence MOR signaling. As illustrated in Fig.

1, the exploration began with peripheral modifications, where benzylfentanyl exhibits reduced MOR affinity,9 whereas furanylfentanyl displays altered MOR signaling, including partial agonism in calcium mobilization assays and absent β-arrestin2 recruitment in BERT studies.10 Earlier work on mirfentanil further showed that heteroaryl amide modification could retain analgesic activity while producing less respiratory impairment than fentanyl in rats.11 These variations further extended to phenylfentanyl, which was initially noted for a ceiling effect on heart-rate depression at higher doses,12 but has more recently been characterized as a neutral MOR antagonist in calcium mobilization and BRET assays.8

Following these substituent variations, the focus has been shifted toward direct modification on the piperidine core. In our previous work, expansion of the piperidine ring to an 8-membered scaffold (e.g., VZFN342) produced ligands with antagonist-dominant properties while maintaining MOR binding and, notably, the ability to reverse fentanyl-induced respiratory depression.13 Most recently, the replacement of the piperidine ring with a 2-azaspiro[3.3]heptane core further demonstrated the structural tolerance of the fentanyl pharmacophore to core-ring re-engineering.14 Taken together, these findings demonstrate that core-ring geometry of fentanyl analogues is also a determinant of MOR signaling and suggest that conformational control of the core scaffold may provide a viable strategy to finetune receptor efficacy.

We therefore hypothesized that introducing a 6,4-spiro bicyclic core may restrict accessible ligand geometries while preserving key pharmacophoric features for MOR recognition, thereby providing a means to modulate MOR pharmacology. Structurally, the six-membered cyclohexyl ring maintains the upper portion of the parent scaffold bearing the N-phenyl amide motif, while allowing diversification of the acyl substituent with the heteroaryl groups investigated in this study. The four-membered N-containing ring introduces the spiro junction and geometric restriction.

Importantly, the basic amine is retained within this ring because it is a critical pharmacophoric feature for MOR recognition, including the characteristic ionic interaction with Asp1473.32. This nitrogen atom also provides a site for N-alkyl substitution and subsequent SAR exploration. In addition to core-ring modification, systematic variations were introduced at two key positions: (i) replacement of the amide substituent with heteroaryl-derived acyl groups, and (ii) diversification of the N-alkyl substituent with bulkier hydrophobic groups to explore interactions within the MOR orthosteric binding pocket.

The designed analogues were synthesized and evaluated through in vitro and in vivo pharmacological assays, complemented by ADME profiling, blood–brain barrier (BBB) penetration studies, and in vivo respiratory assessment. Conformational analysis and molecular docking were further employed to provide structural insight into the effects of the 6,4-spiro scaffold on receptor engagement.

Results and discussion

Molecular design and syntheses

As shown in Fig. 2, the molecular design was centered on introducing a 6,4-spiro-bicyclic core scaffold based on the parent fentanyl framework. This design preserves the overall spatial arrangement of key features of the previously reported 8-membered scaffold (VZFN342) while reducing accessible conformational space through the 6,4-spiro architecture.13 This design was intended to evaluate how conformational restriction on the fentanyl-derived core ring would modulate MOR signaling while preserving receptor engagement.

To explore the structure–activity relationship, substitutions were introduced at the R1 and R2 positions. At the R1 position, the original 3-furanyl moiety was replaced with furan-, thiophene-, and pyrrole-derived substituents, introduced at either the 2- or 3-position, to investigate the effect of heteroaryl orientation on receptor interactions. At the R2 position, following previously reported strategies, the N-alkyl substituent was varied to include allyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, benzyl, and phenylethyl groups.

These modifications were adopted to explore hydrophobic interactions within the MOR orthosteric binding pocket while maintaining the overall geometry and topology of the parent scaffold VZFN342.

As depicted in Scheme 1, the synthesis of the target compounds began with 1-Boc-4-piperidone (a). Reductive amination with aniline in the presence of AcOH and NaBH (OAc)3 afforded the secondary amine intermediate b.15 Subsequent acylation with the corresponding acyl chlorides provided intermediates c1–c6.8 The acyl chlorides were either commercially available (furanyl and thiophenyl derivatives) or prepared in situ for the pyrrolyl analogues.

Removal of the Boc protecting group using TFA generated the free amine intermediates d1–d6, which were used directly in the next step without purification. The resulting amines were then subjected to N-alkylation with various alkyl halides in the presence of K2CO3 in acetonitrile to furnish the desired products 1–42.16,17 Reactions proceeded at room temperature for allyl, cyclopropylmethyl, and benzyl derivatives, whereas reflux conditions were required for cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl derivatives. The final products were isolated as free bases in 12–84% yield, followed by conversion to their corresponding hydrochloride salts to improve aqueous solubility.

All compounds were purified and confirmed with ≥95% purity by HPLC prior to biological evaluations (Table 1).

Biological studies

In vivo single dose agonism study

Although in vitro assays are commonly used as an initial screening step in opioid drug discovery, an in vivo single-dose agonism assay was employed in this study following a previously reported protocol.8 This approach was adopted due to the limited availability of radioligands for MOR binding assays at the time of screening, and because in vivo evaluation allows simultaneous assessment of pharmacological activity together with pharmacokinetic factors influencing functional outcomes. 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. The antinociceptive activity of the synthesized compounds was evaluated using the warm-water tail immersion assay.

As shown in Fig. 3, none of the newly synthesized analogues produced measurable antinociceptive effects at a dose of 10 mg kg−1 (s.c.). These results indicate that the compounds do not exhibit detectable opioid agonist activity under the tested conditions, thereby supporting their further evaluation in opioid antagonism studies.

In vivo single-dose blockade of morphine-induced antinociception

To further evaluate the opioid antagonist activity of the synthesized compounds, an in vivo single-dose blockade assay against morphine-induced antinociception was performed following a previously reported protocol.8 The antagonistic activity was assessed using the warm-water tail immersion assay, in which the ability of the test compounds to attenuate the antinociceptive effect of morphine (10 mg kg−1, s.c.) was measured. As shown in Fig. 4, compounds that significantly attenuated morphine-induced antinociception were considered to exhibit antagonist-like activity.

Based on this criterion, compounds 2, 3, 5, 6, 12, 15, 16, 26, 27, 28, 29, 36, 37, 39, and 40 demonstrated significant attenuation of morphine-induced antinociception and were selected for further pharmacological evaluation.

In vivo single-dose blockade of fentanyl-induced antinociception

To further characterize the opioid pharmacological profile of the synthesized analogues, compounds that exhibited antagonist-like activity in the single-dose morphine blockade assay were subsequently evaluated in a fentanyl challenge experiment using the warm-water tail immersion assay.8 Among the 15 compounds that demonstrated antagonist-like activity in the morphine assay, only compounds 3, 4, and 6 exhibited statistically significant attenuation of fentanyl-induced (0.1 mg kg−1, s.c.) antinociception and were therefore selected for subsequent pharmacological characterization (Fig. 5).

In vivo dose–response antagonism study

To further confirm the antagonist activity observed in the single-dose screening assays, compounds 3, 4, and 6 were evaluated in dose–response studies against morphine- and fentanyl-induced antinociception.8 Dose ranges for the dose–response studies were selected based on the %MPE observed in the single dose antagonism at 10 mg kg−1, with doses adjusted upward or downward from 10 mg kg−1 accordingly to achieve a reliable estimation of the AD50 values. As shown in Fig. 6, all three compounds exhibited dose-dependent antagonism of both morphine- and fentanyl-induced antinociceptive effects.

Among them, compound 3 displayed the highest potency, with AD50 values of 2.04 mg kg−1 and 5.72 mg kg−1 against morphine and fentanyl, respectively (Table 2). Notably, the potency of compound 3 was comparable to that of the parent 8-membered compound VZFN342, indicating that reorganization into the 6,4-spiro scaffold preserved in vivo antagonist activity.

In vitro MOR binding and functional characterization

To further investigate the receptor pharmacology of the most potent analogue, compound 3, in vitro opioid receptor binding and functional assays were performed (Table 3).8,18,19 Compound 3 exhibited high affinity for the MOR (Ki = 25.0 ± 2.6 nM) and maintained selectivity over the KOR and DOR. Compared with the parent 8-membered scaffold VZFN342, compound 3 exhibited a threefold improvement in MOR binding affinity, indicating that reorganization into the 6,4-spiro scaffold enhanced receptor recognition. Compared with phenylfentanyl, compound 3 retained MOR binding while displaying reduced intrinsic efficacy in the GTPγS functional assay.

Specifically, compound 3 produced minimal receptor activation (Emax = 3.05% of DAMGO), consistent with a low efficacy MOR ligand. These results indicate that the 6,4-spiro scaffold preserves MOR engagement while substantially limiting receptor activation. The minimal intrinsic efficacy observed for compound 3 is consistent with its antagonist-like behavior in the in vivo antinociception assays, where low efficacy ligands can competitively attenuate the effects of full agonists.

Conformational analysis

To evaluate the reduction in conformational flexibility associated with the 6,4-spiro bicyclic scaffold, conformational analyses were performed for compound 3 and its parent 8-membered ring analogue VZFN342. Conformational searches were carried out in Schrödinger software package. Conformers were generated and energy-minimized using the OPLS force field with implicit water solvation.

For each conformer, the minimized potential energy was recorded, and the root-mean-square deviation (RMSD) was calculated relative to the lowest-energy conformer. The resulting conformational ensembles were visualized as relative potential energy (ΔE) versus RMSD plots (Fig. 7).

The flexible 8-membered scaffold (VZFN342) generated 508 unique minimized conformations with RMSD values extending to approximately 3.5 Å (Fig. 7A), consistent with the substantial torsional flexibility of the ring system. In contrast, the 6,4-spiro scaffold of compound 3 yielded only 299 unique minimized conformations with RMSD values extending to approximately 4.2 Å (Fig.

7B). Although the RMSD distribution of the 6,4-spiro scaffold appeared slightly broader, the reduced number of accessible conformations indicates that this framework populates fewer discrete conformational states, reflecting greater geometric preorganization of the ligand. The broader RMSD range observed for the 6,4-spiro scaffold therefore reflects conformational differences from a small number of conformational subfamilies, rather than increased flexibility.

Collectively, these results suggest that reorganization into the 6,4-spiro scaffold reduces overall conformational freedom of the fentanyl-derived scaffold, favoring a more restricted set of accessible geometries that may facilitate productive engagement with the MOR binding pocket.

Molecular docking

To further examine how the 6,4-spiro scaffold may interact with the MOR binding site, molecular docking studies were performed. Compound 3 was docked into the inactive MOR crystal structure (PDB: 4DKL).20 Compound 3 adopted a binding orientation within the orthosteric pocket broadly consistent with that of VZFN342 (Fig. 8A), with the tertiary amine forming a conserved salt bridge with Asp147, a key interaction observed for classical opioid ligands.

Hydrophobic contacts were observed with Val143, Ile146, Trp293, Ile296, and Tyr326, recapitulating core interactions characteristic of opioid ligands within the MOR binding pocket. Additional contacts with Trp133, Asn127, Val236, and Ile322 suggest a subtly altered binding geometry relative to the parent scaffold, potentially reflecting the structural reorganization into the 6,4-spiro framework (Fig. 8).

Metabolic stability and permeability studies

To further characterize the drug-like properties of compound 3, its metabolic stability and permeability were evaluated. Metabolic stability was evaluated in human liver S9 fractions following a standard protocol.21 As shown in Table 4, compound 3 exhibited excellent metabolic stability in human liver S9 (t1/2 = 728 min, CLint = 1.1 μL min−1 mg−1), demonstrating substantially slower intrinsic clearance than the reference compounds clozapine,22 diclofenac,23 estrone,24 and terfenadine.25 In contrast, more rapid clearance was observed in mouse liver S9, consistent with known species-dependent metabolic differences. Permeability was evaluated using Caco-2 assays (Table 5).

Compound 3 displayed low efflux ratios (∼2) for both P-gp26 and BCRP,27 which were markedly reduced in the presence of selective inhibitors, indicating potential transporter-mediated efflux. Collectively, these results indicate that the 6,4-spiro scaffold maintains a favorable in vitro ADME profile, particularly with respect to its human metabolic stability, supporting its further exploration as a fentanyl-derived MOR ligand. Given these encouraging in vitro ADME properties, the central nervous system (CNS) exposure of compound 3 was next evaluated in vivo through a blood–brain barrier (BBB) penetration study.

Time-dependent BBB penetration study

An in vivo time-dependent BBB penetration study was performed to assess the CNS exposure of compound 3.28,29 Brain and plasma concentrations were measured at multiple time points following subcutaneous (s.c.) administration (10 mg kg−1) in mice. As shown in Table 6, compound 3 and naltrexone (NTX) were administered at the same dose for comparison. Brain and plasma concentrations were determined at the indicated time points, and brain-to-plasma ratios were calculated to assess relative CNS exposure.

Across all time points examined, compound 3 exhibited substantially lower brain exposure relative to plasma compared with naltrexone, with brain-to-plasma ratios ranging from 0.05–0.11, whereas NTX displayed ratios greater than 1. These results indicate that compound 3 shows limited penetration across the BBB, suggesting reduced CNS exposure relative to the clinically used MOR antagonist NTX.

In vivo respiratory depression study

Respiratory depression is a leading factor contributing to fatalities in opioid overdose. Whole-body plethysmography (WBP) is commonly used to evaluate opioid-induced respiratory depression.1,2,8 In this study, respiratory parameters were monitored following administration of compound 3. The primary outcome measure was minute volume (Fig.

9A), calculated as the product of respiratory rate (Fig. 9B) and tidal volume (Fig. 9C).

Administration of compound 3 alone did not produce measurable respiratory depression at the tested dose, indicating minimal intrinsic respiratory liability. This observation may be related to the limited CNS exposure of compound 3, although other factors including MOR potency and overall pharmacological profile, may also contribute. In fentanyl-challenge experiments, compound 3 did not produce a measurable reversal of fentanyl-induced respiratory depression under the conditions tested (Fig.

S1).

Conclusion

In this study, we designed and evaluated a 6,4-spiro bicyclic scaffold derived from the fentanyl framework to investigate how reorganization of the flexible core into a spirocyclic architecture influences Mu opioid receptor (MOR) pharmacology. The most potent analogue, compound 3, exhibited a threefold improvement in MOR binding affinity related to the parent scaffold VZFN342, demonstrated antagonist-like activity in vivo against morphine- and fentanyl-induced antinociception while exhibiting very low intrinsic efficacy in MOR functional assays. Conformational analysis indicated that the 6,4-spiro scaffold samples fewer accessible conformational states relative to the parent 8-membered scaffold, suggesting greater geometric preorganization of the ligand.

Molecular docking further showed that compound 3 maintains key interactions within the MOR orthosteric binding pocket while adopting a binding pose compatible with the inactive receptor conformation.

In vitro ADME studies revealed excellent metabolic stability in human liver S9 fractions and minimal transporter-mediated efflux in Caco-2 assays, while in vivo BBB studies indicated limited CNS exposure compared to naltrexone. Consistent with these properties, compound 3 did not produce measurable respiratory depression at the tested dose in whole-body plethysmography studies.

Together, these findings demonstrate that reorganization of the fentanyl derived scaffold into a 6,4-spiro framework may preserve MOR engagement while limiting intrinsic receptor activation and respiratory liability. The 6,4-spiro framework thus represents a promising structural platform to develop MOR ligands with tunable efficacy profiles.

Materials and methods

Chemistry

General experimental procedures

All nonaqueous reactions were performed under a nitrogen atmosphere in oven-dried glassware. Unless otherwise noted, all reagents and solvents were purchased from Sigma-Aldrich, Alfa Aesar, Bepharm Scientific or Fisher Scientific and used as received without further purification. Analytical thin-layer chromatography (TLC) was carried out on Analtech Uniplate F254 plates, and flash column chromatography (FCC) was performed using silica gel (230–400 mesh, Merck).

The 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Bruker Ultrashield 400 Plus spectrometer, and chemical shifts (δ) are reported in parts per million (ppm) relative to residual solvent signals. High-resolution mass spectra (HRMS) were obtained on an Applied BioSystems 3200 QTRAP mass spectrometer equipped with a turbo V ion source operating in TurbolonSpray mode. Analytical reversed-phase high-performance liquid chromatography (HPLC) was performed on a Waters Arc HPLC system equipped with an XBridge™ C18 column (3.5 μm, 4.6 × 50 mm).

Analyses were conducted at ambient temperature with a flow rate of 0.2 mL min−1. The mobile phase is acetonitrile/water (70 : 30) containing 0.1% trifluoroacetic acid (TFA). UV detection was performed at 210 nm.

Compound purities were determined by peak area integration, and retention times (Rt) were reported in minutes. All newly synthesized compounds were determined to have ≥95% purity prior to biological evaluation.

General procedure for the synthesis of 6,4-spiro bicyclic analogues

The synthesis procedures for compounds 1–42 are described as follows. To a solution of a in anhydrous dichloromethane (DCM), acetic acid was added, followed by aniline at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred for 10 minutes, after which sodium triacetoxyborohydride (NaBH(OAc)3) was added portion wise.

The mixture was stirred at room temperature for 16 h, then quenched with water. The layers were separated, and the organic phase was washed sequentially with saturated sodium bicarbonate (NaHCO3) and brine, dried over anhydrous sodium sulfate (Na2SO4), and purified by flush column chromatography (FCC) on silica gel using hexanes/ethyl acetate (10 : 1, v/v) to afford intermediate b. Intermediate b was dissolved in anhydrous DCM and cooled to 0 °C.

Triethylamine (TEA) was added, followed by slow addition of an appropriate acyl chloride. The reaction mixture was stirred at room temperature for 12 h. The reaction was quenched with water, washed with sat.

NaHCO3, then brine, dried over Na2SO4 and purified by FCC using hexanes/ethyl acetate (5 : 1, v/v) to afford intermediate c1–c6. Intermediate c was dissolved in DCM, and trifluoroacetic acid (TFA) was added dropwise at 0 °C. The reaction mixture was stirred overnight and concentrated under reduced pressure to yield intermediates d1–d6, which were used in the next step without further purification.

Intermediate d1–d6 was dissolved in anhydrous acetonitrile, followed by addition of potassium carbonate (K2CO3) and an appropriate alkyl chloride/bromide. The reaction mixture was stirred at room temperature or reflux, and progress was monitored by TLC. Upon completion, the mixture was cooled to room temperature, concentrated under reduced pressure, redissolved in DCM, and purified by FCC using DCM/MeOH/NH3·H2O (20 : 1 : 0.1, v/v/v) as the eluent to afford the target compounds as free bases.

Tert-butyl-7-(phenylamino)-2-azaspiro[3.5]nonane-2-carboxylate (b)

Yield 90%, light yellow powder. 1H NMR (400 MHz, chloroform-d) δ 7.14 (dd, J = 8.6, 7.2 Hz, 2H), 6.70–6.63 (m, 1H), 6.59–6.54 (m, 2H), 3.62 (s, 2H), 3.59 (s, 2H), 3.31–3.18 (m, 1H), 1.95 (ddd, J = 32.7, 13.5, 3.6 Hz, 4H), 1.56 (td, J = 13.0, 3.6 Hz, 2H), 1.44 (s, 9H), 1.17 (d, J = 12.2 Hz, 2H). 13C NMR (101 MHz, chloroform-d) δ 156.54, 146.98, 129.32, 118.51, 117.34, 115.09, 113.32, 79.28, 50.61, 34.59, 34.21, 31.92, 29.48, 28.43, 22.68, 14.11.

HRMS (ESI): m/z [M + H]+ calculated for C19H29N2O2: 317.2229; found: 339.2032.

Tert-butyl-7-(N-phenylfuran-2-carboxamido)-2-azaspiro[3.5]nonane-2-carboxylate (c1)

Yield 92%, light yellow powder. 1H NMR (400 MHz, chloroform-d) δ 7.48–7.37 (m, 3H), 7.34–7.29 (m, 1H), 7.14 (dd, J = 7.8, 1.8 Hz, 2H), 6.13 (dd, J = 3.6, 1.7 Hz, 1H), 5.42 (d, J = 3.6 Hz, 1H), 4.77–4.63 (m, 1H), 3.54 (s, 2H), 3.42 (s, 2H), 2.00–1.83 (m, 4H), 1.72–1.57 (m, 2H), 1.41 (s, 9H), 1.27–1.10 (m, 2H). 13C NMR (101 MHz, chloroform-d) δ 158.84, 156.47, 147.23, 144.27, 138.80, 130.60, 129.23, 128.78, 115.93, 110.86, 54.04, 35.03, 34.23, 28.40, 27.42.

HRMS (ESI): m/z [M + Na]+ calculated for C24H30N2NaO2: 433.2103; found: 433.2083.

Tert-butyl-7-(N-phenylfuran-3-carboxamido)-2-azaspiro[3.5]nonane-2-carboxylate (c2)

Yield 95%, light yellow powder. 1H NMR (400 MHz, chloroform-d) δ 7.48–7.38 (m, 3H), 7.13 (ddd, J = 7.3, 3.4, 1.6 Hz, 3H), 6.49 (s, 1H), 6.15 (dd, J = 1.9, 0.8 Hz, 1H), 4.69 (ddd, J = 12.2, 8.6, 3.2 Hz, 1H), 3.53 (s, 2H), 3.42 (s, 2H), 1.90 (tdd, J = 15.9, 15.1, 8.0, 2.7 Hz, 4H), 1.63 (td, J = 13.3, 3.3 Hz, 2H), 1.41 (s, 9H), 1.16 (qd, J = 13.0, 12.1, 2.7 Hz, 2H). 13C NMR (101 MHz, chloroform-d) δ 162.74, 156.47, 145.17, 141.80, 130.84, 129.39, 128.99, 122.50, 111.34, 79.27, 35.08, 34.25, 28.41, 27.52.

HRMS (ESI): m/z [M + Na]+ calculated for C24H30N2NaO2: 433.2103; found: 433.2103.

Tert-butyl-7-(N-phenylthiophene-2-carboxamido)-2-azaspiro[3.5]nonane-2-carboxylate (c3)

Yield 99%, light yellow powder. 1H NMR (400 MHz, chloroform-d) δ 7.46–7.39 (m, 3H), 7.28–7.24 (m, 1H), 7.17 (dt, J = 6.5, 1.7 Hz, 2H), 6.74 (dd, J = 5.0, 3.8 Hz, 1H), 6.56 (dd, J = 3.9, 1.2 Hz, 1H), 4.69 (tt, J = 12.3, 3.4 Hz, 1H), 3.53 (s, 2H), 3.42 (s, 2H), 1.99–1.84 (m, 4H), 1.63 (td, J = 14.3, 13.6, 3.8 Hz, 2H), 1.41 (s, 9H), 1.17 (td, J = 12.4, 12.0, 3.3 Hz, 2H). 13C NMR (100 MHz, chloroform-d) δ 162.02, 156.45, 139.02, 131.89, 131.06, 130.72, 129.41, 129.05, 126.61, 79.23, 35.08, 34.25, 28.40, 27.47.

HRMS (ESI): m/z [M + Na]+ calculated for C24H30N2NaO3S: 449.1875; found: 449.1861.

Tert-butyl-7-(N-phenylthiophene-3-carboxamido)-2-azaspiro[3.5]nonane-2-carboxylate (c4)

Yield 85%, light yellow powder. 1H NMR (400 MHz, chloroform-d) δ 7.39–7.31 (m, 3H), 7.11–7.04 (m, 2H), 6.98 (dd, J = 5.0, 3.0 Hz, 1H), 6.95 (dd, J = 3.0, 1.4 Hz, 1H), 6.88 (dd, J = 5.0, 1.4 Hz, 1H), 4.67 (t, J = 12.3 Hz, 1H), 3.54 (s, 2H), 3.44 (s, 2H), 1.97–1.85 (m, 4H), 1.69–1.60 (m, 2H), 1.41 (s, 9H), 1.29–1.16 (m, 2H). HRMS (ESI): m/z [M + H]+ calculated for C24H31N2O3S: 427.2055; found: 427.2036; m/z [M + Na]+ calculated for C24H30N2NaO3S: 449.1875; found: 449.1850.

Tert-butyl-7-(N-phenyl-1H-pyrrole-2-carboxamido)-2-azaspiro[3.5]nonane-2-carboxylate (c5)

Yield 78%, off white powder. 1H NMR (400 MHz, DMSO-d6) δ 11.32 (bs, 1H), 7.49–7.47 (m, 3H), 7.23–7.21 (m, 2H), 6.74–6.72 (m, 1H), 5.73–5.71 (m, 1H), 4.55–4.49 (m, 1H), 4.44 (s, 1H), 3.43 (s, 2H), 3.32–3.30 (m, 2H), 1.88–1.84 (m, 2H), 1.78–1.75 (m, 2H), 1.59–1.53 (m, 2H), 1.34 (s, 9H), 1.18–1.04 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 160.62, 156.15, 139.66, 131.47, 129.66, 129.05, 125.60, 121.61, 112.94, 108.97, 78.79, 34.98, 34.33, 28.53, 27.60.

HRMS (ESI): m/z [M + H]+ calculated for C24H32N3O3: 410.2444; found: 410.2444.

Tert-butyl-7-(N-phenyl-1H-pyrrole-3-carboxamido)-2-azaspiro[3.5]nonane-2-carboxylate (c6)

Yield 39%, steaky brown solid. 1H NMR (400 MHz, chloroform-d) δ 8.32 (s, 1H), 7.39 (dd, J = 5.2, 2.1 Hz, 3H), 7.20–7.08 (m, 2H), 6.44 (q, J = 2.5 Hz, 1H), 6.32 (dt, J = 3.4, 1.8 Hz, 1H), 5.74 (d, J = 2.1 Hz, 1H), 4.80–4.65 (m, 1H), 3.52 (s, 2H), 3.42 (s, 2H), 1.97–1.82 (m, 4H), 1.68–1.57 (m, 2H), 1.40 (s, 9H), 1.16 (qd, J = 13.1, 12.4, 3.7 Hz, 2H). 13C NMR (100 MHz, chloroform-d) δ 164.81, 156.50, 140.09, 131.07, 129.05, 128.30, 122.76, 120.01, 117.16, 110.61, 53.81, 35.20, 34.33, 28.41, 27.65.

N-phenyl-N-(2-azaspiro[3.5]nonan-7-yl)furan-2-carboxamide (d1)

Quantitative, steaky brown solid. 1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 2H), 7.62 (d, J = 1.6 Hz, 1H), 7.52–7.41 (m, 3H), 7.29–7.20 (m, 2H), 6.30 (dd, J = 3.6, 1.6 Hz, 1H), 5.39 (d, J = 3.4 Hz, 1H), 4.45 (td, J = 10.7, 9.5, 6.4 Hz, 1H), 3.56 (s, 2H), 3.49 (s, 2H), 2.10 (d, J = 13.0 Hz, 2H), 1.80 (d, J = 12.3 Hz, 2H), 1.63–1.50 (m, 2H), 1.07 (q, J = 12.6 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 158.25, 158.03, 147.33, 145.31, 138.98, 130.99, 129.72, 129.20, 115.85, 111.55, 55.12, 54.19, 53.96, 53.80, 37.22, 34.06, 27.04.

HRMS (ESI): m/z [M + H]+ calculated for C19H23N2O2: 311.1760; found: 311.1767.

N-phenyl-N-(2-azaspiro[3.5]nonan-7-yl)furan-3-carboxamide (d2)

Quantitative, steaky brown solid. 1H NMR (400 MHz, chloroform-d) δ 9.26 (s, 2H), 7.44 (q, J = 6.5 Hz, 3H), 7.18–7.05 (m, 3H), 6.62–6.50 (m, 1H), 6.11 (dd, J = 1.9, 0.8 Hz, 1H), 4.69–4.54 (m, 1H), 3.77–3.68 (m, 2H), 3.62 (d, J = 6.8 Hz, 2H), 2.24 (d, J = 13.2 Hz, 2H), 1.91 (d, J = 12.6 Hz, 2H), 1.64 (t, J = 13.2 Hz, 2H), 1.15 (q, J = 12.4 Hz, 2H). HRMS (ESI): m/z [M + H]+ calculated for C19H23N2O2: 311.1760; found: 311.1755.

N-phenyl-N-(2-azaspiro[3.5]nonan-7-yl)thiophene-2-carboxamide (d3)

Quantitative, steaky brown solid. 1H NMR (400 MHz, chloroform-d) δ 7.44 (s, 3H), 7.27 (d, J = 5.0 Hz, 1H), 7.15 (s, 2H), 6.74 (d, J = 4.4 Hz, 1H), 6.57 (d, J = 3.7 Hz, 1H), 4.66 (s, 1H), 3.73 (s, 2H), 2.86 (s, 2H), 2.26 (s, 2H), 1.93 (s, 2H), 1.64 (s, 2H), 1.15 (s, 2H). HRMS (ESI): m/z [M + H]+ calculated for C19H23N2OS: 327.1531; found: 327.1521.

N-phenyl-N-(2-azaspiro[3.5]nonan-7-yl)thiophene-3-carboxamide (d4)

Quantitative, steaky brown solid. 1H NMR (400 MHz, chloroform-d) δ 8.70 (s, 2H), 7.36 (dd, J = 5.2, 2.0 Hz, 3H), 7.06 (dq, J = 4.4, 2.1 Hz, 3H), 7.01 (dd, J = 5.1, 3.0 Hz, 1H), 6.82 (dd, J = 5.1, 1.3 Hz, 1H), 4.62 (t, J = 12.0 Hz, 1H), 3.73 (d, J = 31.2 Hz, 4H), 2.16 (d, J = 13.3 Hz, 2H), 1.97 (d, J = 12.7 Hz, 2H), 1.68 (t, J = 13.1 Hz, 2H), 1.23 (q, J = 12.3 Hz, 2H). 13C NMR (101 MHz, chloroform-d) δ 137.40, 130.48, 129.29, 129.14, 128.76, 128.32, 124.05, 35.14, 34.27, 28.41, 27.61.

HRMS (ESI): m/z [M + H]+ calculated for C19H23N2OS: 327.1531; found: 327.1517.

N-phenyl-N-(2-azaspiro[3.5]nonan-7-yl)-1H-pyrrole-2-carboxamide (d5)

Quantitative, steaky brown solid. White powder. 92%.

1H NMR (400 MHz, DMSO-d6) δ 11.31 (bs, 1H), 8.54 (bs, 2H), 7.48–7.47 (m, 2H), 7.30–7.21 (m, 3H), 6.73–6.73 (m, 1H), 5.72–5.70 (m, 1H), 4.54–4.48 (m, 1H), 4.42 (s, 1H), 3.60–3.57 (m, 2H), 3.53–3.50 (m, 2H), 2.04–2.00 (m, 2H), 1.80–1.78 (m, 2H), 1.61–1.54 (m, 2H), 1.10–1.04 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 160.59, 139.58, 131.49, 130.40, 129.71, 129.19, 129.12, 128.17, 127.57, 125.51, 124.27, 121.71, 119.30, 112.99, 110.97, 65.36, 55.69, 54.79, 53.41, 37.28, 34.21, 34.13, 27.23, 15.62. HRMS (ESI): m/z [M + H]+ calculated for C19H32N3O3: 310.1919; found: 310.1916.

N-phenyl-N-(2-azaspiro[3.5]nonan-7-yl)-1H-pyrrole-3-carboxamide (d6)

Quantitative, steaky brown solid. 1H NMR (400 MHz, DMSO-d6) δ 10.83 (s, 1H), 8.53 (s, 2H), 7.51–7.38 (m, 3H), 7.24–7.14 (m, 2H), 6.50–6.43 (m, 1H), 6.04–5.95 (m, 1H), 5.62–5.54 (m, 1H), 4.49 (ddt, J = 12.2, 7.1, 3.6 Hz, 1H), 3.59 (t, J = 6.3 Hz, 4H), 2.02 (d, J = 13.1 Hz, 2H), 1.78 (d, J = 12.8 Hz, 2H), 1.57 (td, J = 13.1, 3.6 Hz, 2H), 1.04 (dd, J = 14.6, 11.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 164.21, 140.44, 131.54, 129.52, 128.67, 122.36, 119.51, 117.68, 110.29, 55.76, 54.85, 53.38, 37.33, 34.32, 27.35.

The resulting free bases were converted to their hydrochloride salts (HCl salt) by treatment with HCl in methanol, affording the final compounds 1–42.

N-(2-allyl-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-2-carboxamide hydrochloride (1)

White powder, 32%. 1H NMR (400 MHz, DMSO-d6) δ 11.25 (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) δ 158.24, 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 C22H27N2O2: 351.2073; found [M + H]+: 351.2081. HPLC data: purity 99.51%, retention time 2.663 min.

N-(2-(cyclopropylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-2-carboxamide hydrochloride (2)

White powder, 16%. 1H NMR (400 MHz, DMSO-d6) δ 10.93 (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) δ 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 C23H29N2O2: 365.2229; found [M + H]+: 365.2209. HPLC data: purity 99.83%, retention time 2.700 min.

N-(2-(cyclobutylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-2-carboxamide hydrochloride (3)

White powder, 19%. 1H NMR (400 MHz, DMSO-d6) δ 10.66 (s, 1H), 7.65–7.57 (m, 1H), 7.46 (dd, J = 5.0, 1.9 Hz, 3H), 7.30–7.19 (m, 2H), 6.30 (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 (m, 3H), 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) δ 158.25, 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 C24H31N2O2: 379.2386; found [M + H]+: 379.2386. HPLC data: purity 99.74%, retention time 2.770 min.

N-(2-(cyclopentylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-2-carboxamide hydrochloride (4)

White powder, 28%. 1H NMR (400 MHz, DMSO-d6) δ 10.29 (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) δ 158.25, 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 C25H33N2O2: 393.2542; found [M + H]+: 393.2522. HPLC data: purity 98.98%, retention time 2.847 min.

N-(2-(cyclohexylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-2-carboxamide hydrochloride (5)

White powder, 29%. 1H NMR (400 MHz, DMSO-d6) δ 10.32 (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) δ 158.25, 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 C26H35N2O2: 407.2699; found [M + H]+: 407.2673. HPLC data: purity 99.49%, retention time 2.945 min.

N-(2-benzyl-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-2-carboxamide hydrochloride (6)

White powder, 33%. 1H NMR (400 MHz, DMSO-d6) δ 10.98 (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, 1H), 4.36–4.21 (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) δ 158.24, 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 C26H29N2O2: 401.2229, found [M + H]+: 401.2205. HPLC data: purity 99.61%, retention time 2.775 min.

N-(2-phenethyl-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-2-carboxamide hydrochloride (7)

White powder, 52%. 1H NMR (400 MHz, DMSO-d6) δ 10.90 (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) δ 158.25, 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 C27H31N2O2: 415.2386, found [M + H]+: 415.2382. HPLC data: purity 99.43%, retention time 2.840 min.

N-(2-allyl-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-3-carboxamide hydrochloride (8)

White powder, 31%. 1H NMR (400 MHz, DMSO-d6) δ 10.98 (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, 1H), 3.74–3.67 (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) δ 162.01, 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 C22H27N2O2: 351.2081, found [M + H]+: 351.2080. HPLC data: purity 98.74%, retention time 2.683 min.

N-(2-(cyclopropylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-3-carboxamide hydrochloride (9)

White powder, 44%. 1H NMR (400 MHz, DMSO-d6) δ 10.53 (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) δ 162.10, 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 C23H29N2O2: 365.2229, found [M + H]+: 365.2239. HPLC data: purity 100.00%, retention time 2.723 min.

N-(2-(cyclobutylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-3-carboxamide hydrochloride (10)

White powder, 12%. 1H NMR (400 MHz, DMSO-d6) δ 10.01 (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) δ 145.16, 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 C24H31N2O2: 379.2342, found [M + H]+: 379.2379. HPLC data: purity 100.00%, retention time 2.795 min.

N-(2-(cyclopentylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-3-carboxamide hydrochloride (11)

White powder, 24%. 1H NMR (400 MHz, DMSO-d6) δ 10.34 (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), 0.99 (qd, J = 12.7, 3.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 162.09, 145.15, 143.13, 139.08, 131.29, 129.81, 129.31, 122.90, 111.29, 63.56, 62.33, 60.05, 53.70, 35.95, 35.18, 34.63, 32.97, 30.49, 27.23, 27.15, 24.93, 24.89.

Mass m/z: calculated for C25H33N2O2: 393.2542, found [M + H]+: 393.2555. HPLC data: purity 100.00%, retention time 2.873 min.

N-(2-(cyclohexylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-3-carboxamide hydrochloride (12)

White powder, 33%. 1H NMR (400 MHz, DMSO-d6) δ 10.30 (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) δ 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 C26H35N2O2: 407.2699, found [M + H]+: 407.2633. HPLC data: purity 100.00%, retention time 2.978 min.

N-(2-benzyl-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-3-carboxamide hydrochloride (13)

White powder, 54%. 1H NMR (400 MHz, DMSO-d6) δ 10.75 (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) δ 162.07, 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 C26H29N2O2: 401.2229, found [M + H]+: 401.2106. HPLC data: purity 99.26%, retention time 2.807 min.

N-(2-phenethyl-2-azaspiro[3.5]nonan-7-yl)-N-phenylfuran-3-carboxamide hydrochloride (14)

White powder, 44%. 1H NMR (400 MHz, DMSO-d6) δ 11.06 (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 = 8.1 Hz, 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) δ 162.09, 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 C27H31N2O2: 415.2386, found [M + H]+: 415.2241. HPLC data: purity 100.00%, retention time 2.873 min.

N-(2-allyl-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-2-carboxamide hydrochloride (15)

White powder, 21%. 1H NMR (400 MHz, DMSO-d6) δ 10.88 (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) δ 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 C22H27N2OS: 367.1844, found [M + H]+: 367.1826. HPLC data: purity 99.87%, retention time 2.772 min.

N-(2-(cyclopropylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-2-carboxamide hydrochloride (16)

White powder, 21%. 1H NMR (400 MHz, DMSO-d6) δ 10.10 (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), 2.05–1.96 (m, 1H), 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) δ 161.36, 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 C23H29N2OS: 381.2001, found [M + H]+: 381.1992. HPLC data: purity 97.53%, retention time 2.817 min.

N-(2-(cyclobutylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-2-carboxamide hydrochloride (17)

White powder, 52%. 1H NMR (400 MHz, DMSO-d6) δ 10.57 (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) δ 161.31, 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 C24H31N2OS: 395.2157, found [M + H]+: 395.2132. HPLC data: purity 99.17%, retention time 2.879 min.

N-(2-(cyclopentylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-2-carboxamide hydrochloride (18)

White powder, 50%. 1H NMR (400 MHz, DMSO-d6) δ 10.44 (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) δ 161.31, 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 C25H33N2OS: 409.2314, found [M + H]+: 409.2297. HPLC data: purity 98.49%, retention time 2.993 min.

N-(2-(cyclohexylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-2-carboxamide hydrochloride (19)

White powder, 55%. 1H NMR (400 MHz, DMSO-d6) δ 10.38 (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, 1H), 3.80 (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) δ 161.31, 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 C26H35N2OS: 423.2470, found [M + H]+: 423.2455. HPLC data: purity 100.00%, retention time 3.118 min.

N-(2-benzyl-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-2-carboxamide hydrochloride (20)

White powder, 42%. 1H NMR (400 MHz, DMSO-d6) δ 10.90 (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) δ 161.31, 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 C26H29N2OS: 417.2001, found [M + H]+: 417.2008. HPLC data: purity 98.78%, retention time 2.905 min.

N-(2-phenethyl-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-2-carboxamide hydrochloride (21)

White powder, 81%. 1H NMR (400 MHz, DMSO-d6) δ 11.07 (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) δ 161.31, 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 C27H31N2OS: 431.2157, found [M + H]+: 431.2105. HPLC data: purity 98.54%, retention time 2.988 min.

N-(2-allyl-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-3-carboxamide hydrochloride (22)

White powder, 33%. 1H NMR (400 MHz, DMSO-d6): 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 C22H27N2OS: 367.1844; found [M + H]+: 367.1839. HPLC data: purity 97.04%, retention time 2.725 min.

N-(2-(cyclopropylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-3-carboxamide hydrochloride (23)

Yellow solid, 42%. 1H NMR (400 MHz, DMSO-d6): 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 C23H29N2OS: 381.2001; found [M + H]+: 381.1988. HPLC data: purity 100.00%, retention time 2.768 min.

N-(2-(cyclobutylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-3-carboxamide hydrochloride (24)

White powder, 52%. 1H NMR (400 MHz, DMSO-d6): 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, 128.47, 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 C24H31N2OS: 395.2157; found [M + H]+: 395.2158. HPLC data: purity 99.35%, retention time 2.862 min.

N-(2-(cyclopentylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-3-carboxamide hydrochloride (25)

White powder, 72%. 1H NMR (400 MHz, DMSO-d6): 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 C25H33N2OS: 409.2314; found [M + H]+: 409.2295. HPLC data: purity 97.11%, retention time 2.940 min.

N-(2-(cyclohexylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-3-carboxamide hydrochloride (26)

White powder, 69%. 1H NMR (400 MHz, DMSO-d6): 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 C26H35N2OS: 423.2470; found [M + H]+: 423.2485. HPLC data: purity 98.80%, retention time 3.065 min.

N-(2-benzyl-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-3-carboxamide hydrochloride (27)

White powder, 42%. 1H NMR (400 MHz, DMSO-d6): 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 C26H29N2OS: 417.2001; found [M + H]+: 417.1978. HPLC data: purity 100.00%, retention time 2.858 min.

N-(2-phenethyl-2-azaspiro[3.5]nonan-7-yl)-N-phenylthiophene-3-carboxamide hydrochloride (28)

White powder, 84%. 1H NMR (400 MHz, DMSO-d6): 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 C27H31N2OS: 431.2157; found [M + H]+: 431.2154. HPLC data: purity 100.00%, retention time 2.967 min.

N-(2-allyl-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-2-carboxamide hydrochloride (29)

White powder. 22%. 1H NMR (400 MHz, δ 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, δ 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 C22H28N3O: 350.2232; found [M + H]+: 350.2225. HPLC data: purity: 96.70%, retention time: 2.670 min.

N-(2-(cyclopropylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-2-carboxamide hydrochloride (30)

White powder. 31%. 1H NMR (400 MHz, δ 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, δ 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 C23H30N3O: 364.2389; found [M + H]+: 364.2398. HPLC data: purity: 99.93%, retention time: 2.713 min.

N-(2-(cyclobutylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-2-carboxamide hydrochloride (31)

White powder. 25%. 1H NMR (400 MHz, δ 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, δ 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 C24H32N3O: 378.2545; found [M + H]+: 378.2533. HPLC data: purity: 95.47%, retention time: 2.788 min.

N-(2-(cyclopentylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-2-carboxamide hydrochloride (32)

White powder. 24%. 1H NMR (400 MHz, δ 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, 3H), 1.60–1.42 (m, 6H), 1.17–1.07 (m, 3H), 1.03–0.92 (m, 1H).

13C NMR (100 MHz, δ 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 C25H34N3O: 392.2702; found [M + H]+: 392.2677. HPLC data: purity: 98.06%, retention time: 2.872 min.

N-(2-(cyclohexylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-2-carboxamide hydrochloride (33)

White powder. 12%. 1H NMR (400 MHz, δ 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, δ 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, 61.49, 53.33, 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 C26H36N3O: 406.2858; found [M + H]+: 406.2860.

HPLC data: purity: 99.16%, retention time: 2.997 min.

N-(2-benzyl-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-2-carboxamide hydrochloride (34)

White powder. Yield 17%. 1H NMR (400 MHz, δ 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, δ 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, 58.07, 53.39, 35.10, 34.59, 33.12, 27.33, 27.19. Mass spectrum (m/z): calculated for C26H30N3O: 400.2389; found [M + H]+: 400.2396. HPLC data: purity: 99.07%, retention time: 2.793 min.

N-(2-phenethyl-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-2-carboxamide hydrochloride (35)

White powder. 19%. 1H NMR (400 MHz, δ 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, δ 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 C27H32N3O: 414.2545; found [M + H]+: 414.2551. HPLC data: purity: 99.26%, retention time: 2.872 min.

N-(2-allyl-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-3-carboxamide hydrochloride (36)

White powder, 21%. 1H NMR (400 MHz, DMSO-d6) δ 10.84 (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) δ 164.19, 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 C22H28N3O: 350.2232, found [M + H]+: 350.2214. HPLC data: purity 99.39%, retention time 2.532 min.

N-(2-(cyclopropylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-3-carboxamide hydrochloride (37)

White powder, 47%. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (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) δ 164.2, 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, 3.4.

Mass m/z: calculated for C23H30N3O: 364.2389; found [M + H]+: 364.2397. HPLC data: purity 96.34%, retention time 2.573 min.

N-(2-(cyclobutylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-3-carboxamide hydrochloride (38)

White powder, 69%. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (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) δ 164.1, 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 C24H32N3O: 378.2545; found [M + H]+: 378.2531. HPLC data: purity 99.75%, retention time 2.617 min.

N-(2-(cyclopentylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-3-carboxamide hydrochloride (39)

White powder, 82%. 1H NMR (400 MHz, DMSO-d6) δ 10.90 (bs, 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) δ 164.7, 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 C25H34N3O: 392.2702; found [M + H]+: 392.2687. HPLC data: purity 95.84%, retention time 2.745 min.

N-(2-(cyclohexylmethyl)-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-3-carboxamide hydrochloride (40)

White powder, 81%. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (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) δ 164.7, 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 C26H36N3O: 406.2858; found [M + H]+: 406.2833. HPLC data: purity 100.00%, retention time 2.755 min.

N-(2-benzyl-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-3-carboxamide hydrochloride (41)

White powder, 38%. 1H NMR (400 MHz, DMSO-d6) δ 10.94 (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) δ 164.19, 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 C26H30N3O: 400.2389, found [M + H]+: 400.2364. HPLC data: purity 99.79%, retention time 2.616 min.

N-(2-phenethyl-2-azaspiro[3.5]nonan-7-yl)-N-phenyl-1H-pyrrole-3-carboxamide (42)

White powder, 71%. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (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 = 16.5 Hz, 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) δ 164.1, 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 C27H32N3O: 414.2545; found [M + H]+: 414.2559. HPLC data: purity 95.50%, retention time 2.673 min.

Biological evaluations

Drugs and reagent

Morphine (morphine sulfate pentahydrate salt) was purchase from Mallinckrodt (St. Louis, MO, USA) or provided by the national Institute of Drug Abuse (NIDA). All drugs and tested compounds were dissolved in pyrogen-free isotonic saline (Baxter Healthcare, Deerfield, IL, USA) or sterile-filtered distilled/deionized water.

All other reagents were purchased from either Sigma-Aldrich or Thermo Fisher Scientific and used as received. Several in vitro metabolism studies described in this manuscript involved human subject samples. These studies were conducted through Contract Research Organizations (CROs).

The CROs provided documentation related to these samples, including an Ethics Policy Statement, Current Next of Kin Authorization, and Donor Consent Certification. According to these documents, the sample collection was approved by the respective local ethics committee (ID10851), and all participants gave informed consent.

In vitro competitive radioligand binding assay and functional assay

Competition binding and functional assays were performed using opioid receptors expressed in Chinese hamster ovary (CHO) cells, as previously described.8 The radioligands [3H]Naloxone, [3H]NTI, and [3H]norBNI (or [3H]DPN) were applied to label the μ, δ, and κ opioid receptors, respectively. For the binding assay, 20 μg of membrane protein was incubated with 1.4 nM of the corresponding radioligand in the presence of varying 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. Following incubation, bound radioligand was separated from free ligand by rapid filtration through GF/B glass fiber filters, followed by three washes with ice-cold wash buffer (50 mM Tris–HCl, pH 7.2) using a Brandel cell harvester.

Radioactivity was quantified using a liquid scintillation counter. Specific binding was determined as the difference between total binding and binding in the presence of 5 μM naltrexone. The IC50 values were determined and converted to Ki values using the Cheng–Prusoff equation.

Functional assays were assessed using a [35S]GPTγS binding assay in the same membrane penetrations used for the receptor binding studies. Membrane proteins (10 μg) were incubated with varying concentrations of testing compounds, GDP (20 μM), and 0.1 nM [35S]GPTγS in assay buffer for 1.5 h at 30 °C. Nonspecific binding was determined in the presence of 20 μM unlabeled GPTγS.

The full agonists DAMGO (3 μM), U50, 488H (5 μM), and SNC80 (5 μM) were included as reference controls to define maximal stimulation at the μ, κ, and δ opioid receptors, respectively.

Animals

Male Swiss-Webster mice (6–8 weeks, 25–35 g) were housed in cages (five maximal per cage) in temperature-controlled animal care quarters maintained at 22 ± 2 °C on a 12 h light–dark cycle, with food (standard chow) and water were available ad libitum. All experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee (IACUC, Animal Welfare Assurance Number D16-00180) at Virginia Commonwealth University Medical Center and were conducted in accordance with the guidelines of the International Association for the Study of Pain (IASP).

Male Swiss-Webster mice (6–8 weeks, 25–35 g) were housed in cages (five maximal per cage) in temperature-controlled animal care quarters maintained at 22 ± 2 °C on a 12 h light–dark cycle, with food (standard chow) and water were available ad libitum. All experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee (IACUC, Animal Welfare Assurance Number D16-00180) at Virginia Commonwealth University Medical Center and were conducted in accordance with the guidelines of the International Association for the Study of Pain (IASP).

Warm water immersion assay

Prior to experimentation, mice were brought to the laboratory (22 ± 2 °C, 12 h light–dark cycle) and allowed 18 h to acclimate. Antinociception was evaluated using the warm-water tail immersion assay. The tail-flick test was performed using a water bath with the temperature maintained at 56 ± 0.1 °C.

Each mouse was gently restrained in a cloth with only the tail exposed. Baseline latency was measured before subcutaneous (s.c.) administration of test compounds. The distal one-third of the tail was immersed perpendicularly into the water bath, and the latency for the mouse to withdraw its tail was recorded as the baseline latency.

Only mice displaying baseline latencies between 2 to 4 s were used.

Test latency was obtained 20 min later after agonist administration. A 10 s maximum cutoff latency was applied to prevent tissue damage. Antinociception was expressed as the percentage of maximal possible effect (% MPE) calculated using the following equation:Each compound was tested in six mice per dose.

For antagonist studies, the test compound was administered (s.c.) 5 min prior to morphine or fentanyl administration.

Measurement of respiration

Male Swiss-Webster mice (6–8 weeks, 25–35 g) were housed in cages (five maximal per cage) in temperature-controlled animal care quarters maintained at 22 ± 2 °C on a reversed 12 h light–dark cycle. All experiments were conducted in the dark (active) phase. Respiration was measured using WBP 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 drug administration. Respiratory rate and depth were recorded continuously and averaged over 1 to 5 min periods.

Tidal volume was calculated from the raw inspiration data and expiration data. Minute volume was then calculated as rate × tidal volume. The test compound was first administered (s.c.), and respiration was recorded for 5 min.

A second injection was then administered, and respiratory parameters were recorded for an additional 30 min.

ADMET

In vitro metabolism intrinsic clearance (microsomes, S9, cryopreserved hepatocytes, recombinant CYP, recombinant UGT)

Metabolic stability, expressed as percent of the parent compound remaining, was calculated by comparing the peak area of the compound at the time point relative to that at time-0. The half-life (T1/2) was estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming the first-order kinetics. The apparent intrinsic clearance (CLint, in μL min−1 pmol−1, μL min−1 mg−1 or μL min−1 per Mcell) was calculated according to the following formula:

Permeability

The apparent permeability coefficient (Papp) of the test compound was calculated as follows:where VR is the volume of the receiver chamber. CR,end is the concentration of the test compound in the receiver chamber at the end time point, Δt is the incubation time and A is the surface area of the cell monolayer. CD,mid is the calculated mid-point concentration of the test compound in the donor side, which is the mean value of the donor concentration at time 0 minute and the donor concentration at the end time point.

CR,mid is the mid-point concentration of the test compound in the receiver side, which is one half of the receiver concentration at the end time point. Concentrations of the test compound were expressed as peak areas of the test compound.

Recovery of the test compound from the permeability assay. The recovery of the test compound was calculated as follows:where VD and VR are the volumes of the donor and receiver chambers, respectively. CD,end is the concentration of the test compound in the donor sample at the end time point.

CR,end is the concentration of the test compound in the receiver sample at the end time point. CD0 is the concentration of the test compound in the donor sample at time zero. Concentrations of the test compound are expressed as peak areas of the test compound.

Fluorescein assessment for permeability assays

Fluorescein was used as the cell monolayer integrity marker. Fluorescein permeability assessment (in the A–B direction at pH 7.4 on both sides) was performed after the permeability assay for the test compound. The cell monolayer that had a fluorescein permeability of less than 1.5 × 10−6 cm s−1 for Caco-2 and MDR1-MDCKII cells and 2.5 × 10−6 cm s−1 for MDCKII cells was considered intact, and the permeability result of the test compound from intact cell monolayer is reported.

Efflux transporter substrate assessment

The efflux ratio (ER) was calculated as follows:where Papp(B–A) is the apparent permeability coefficient in the B to A direction, and Papp(A–B) is the apparent permeability coefficient in the A to B direction. A compound is considered as a substrate of an efflux transporter if the efflux ratio ≥ 2; and the transporter selective inhibitor inhibits the efflux ratio more than 50%.

In vivo BBB penetration studies

Following our previously reported protocol,28,29 Swiss Webster mice (three mice each time point) were given compound 3 (10 mg kg−1, s.c.). At 5, 10, 30 and 60 min time points post administration, the mice were decapitated, and whole brain and blood samples were collected. Brain samples were washed with saline to ensure removal of any blood on the isolated brains.

They were then immersed in 300 μL saline. Blood samples were centrifuged for 10 min at 15 000g at 4 °C following which plasma was collected. Brain and plasma samples were stored at −80 °C until further analysis.

UPLC–MS/MS analysis

The identification and quantification of compound 3 in mouse plasma and brain were performed using a modification of a previously described method with naloxone-d5 as the internal standard.34 Prior to extraction, brain tissues were homogenized with deionized water at a 1 : 3 (w/w) ratio using an Omni Bead Ruptor (Omni International Inc., Kennesaw, GA). Each analytical run included seven-point calibration curves (10–1000 ng mL−1 or ng g−1) for compound 5, quality control samples at 30, 300, and 750 ng mL−1 or ng g−1, as well as negative and blank controls, all prepared in plasma or brain homogenate. An internal standard (ISTD) containing 10 ng of naloxone-d5 in methanol was added to 100 μL of plasma or 400 μL of brain homogenate for each calibrator, control, and specimen.

After mixing, 100 μL of 5 M ammonium hydroxide and 2 mL of a 25 : 75 methylene chloride–diethyl ether mixture was added. Samples were vortexed for 2 minutes and centrifuged at 3000 rpm for 5 minutes. The organic layer was evaporated under nitrogen and reconstituted with 100 μL of mobile phase before LC–MS/MS analysis.

Chromatography was performed on a Sciex ExionLC 2.0+ system coupled to a Sciex 6500 QTRAP with an IonDrive Turbo V source (Sciex, Ontario, Canada), using a Zorbax Eclipse column (4.6 × 75 mm, 3.5 μm; Agilent, USA) and an isocratic mobile phase of 10 mM ammonium formate: methanol (50 : 50, v/v) at 0.6 mL min−1. Source conditions included a temperature of 600 °C, curtain gas at 30 mL min−1, ion spray voltage of 5000 V, and ion source gases 1 and 2 at 50 and 30 mL min−1, respectively. Data were acquired in positive-ion mode using multiple reaction monitoring (MRM) with the following transitions (m/z), and collision energy (eV) in parenthesis: compound 3, 379 > 282 (24) and 379 > 188 (32); naloxone-d5, 333 > 212 (45), 333 > 315 (25), and 333 > 273.

Total run time was 4 minutes. Quantification was performed using linear regression of analyte-to-ISTD peak area ratios from the calibration curves.

Statistical analysis

One-way ANOVA followed by the post hoc Dunnett test was performed to assess significance using Prism 6.0 software (GraphPad Software, San Diego, CA).

Molecular modeling

Conformational searches were performed for compounds 3 and VZFN342 using the conformational search workflow implemented in Schrödinger Maestro. Three-dimensional structures were prepared for each ligand, and conformer ensembles were generated and energy-minimized using the OPLS force field with implicit water solvation. The lowest-energy conformer was designated as the reference structure, and heavy-atom RMSD values were calculated for all minimized conformers relative to this reference.

Conformational distributions were visualized as plots of relative potential energy (ΔE) versus RMSD.30

Docking studies were conducted using the glide module in Schrödinger Maestro. The inactive-state MOR structure (PDB: 4DKL)20 was prepared using the Protein Preparation Wizard: bond orders were assigned, hydrogens were added, missing side chains were modeled, and protonation states were assigned at physiological pH using Epik/PROPKA. The hydrogen-bonding network was optimized, and the structure was energy-minimized under harmonic restraints using the OPLS force field to relieve steric clashes while preserving the experimental backbone geometry.31,32

A receptor grid was generated using the receptor grid generation panel, centered on the orthosteric site. To enforce the conserved ionic interaction typical of opioids at MOR, a glide constraint was applied to D1473.32 by requiring interaction between the ligand's cationic nitrogen and the carboxylate of the aspartic acid. This constraint ensured that docked poses maintained the canonical salt-bridge geometry during pose generation and scoring.13,33–36 Compound 3 was prepared using LigPrep, generating 3D geometries and relevant protonation/tautomeric states at the selected pH range.

Docking was performed with Glide using standard precision settings with ligand flexibility enabled and post-docking minimization turned on.37,38 For each ligand, top poses were retained for analysis, and the top-ranked 10 poses were inspected visually to compare binding orientations and interactions within the orthosteric pocket.

Author contributions

Y. Z. conceived and oversaw the project and finalized the manuscript; E. L. drafted the manuscript; E.

L., L. T. N., N.

U., B. S. K., and Z.

L. performed the chemical synthesis under the supervision of Y. Z.; P. P.

P. performed warm-water tail immersion studies under the supervision of W. L. D.; L.

T. N. performed HPLC analyses under supervision of Y. Z.; A.

R. performed the molecular modeling studies under supervision of Y. Z.; A. O. performed HRMS measurements under supervision of Y.

Z.; S. W. and D. E.

S. performed the receptor binding affinity experiments.

Conflicts of interest

The authors declare no competing financial interest.

Abbreviations

Supplementary Material