Dual-Target Mu Opioid-Dopamine D₃ Receptor (MOR-D₃R) Ligands Based on Etonitazene: New Leads for Transforming “Nitazenes” into Novel Analgesics

Abstract

The development of opioid analgesics with low abuse liability remains a challenge. As dopamine D₃ receptor (D₃R) antagonists/partial agonists can reduce opioid self-administration and reinstatement in animals while augmenting antinociceptive effects, a dual-target ligand strategy was investigated. Here, the high affinity mu opioid receptor (MOR) agonist etonitazene was modified by incorporating various D₃R pharmacophores with varying linking chains. N-substituted etonitazene analogs 31 (MOR K_i = 33.4 nM; D₃R K_i = 90.6 nM), 35 (MOR K_i = 29.7 nM; D₃R K_i = 53.8 nM), and 43 (MOR K_i = 89.6 nM; D₃R K_i = 45.4 nM) achieved balanced binding affinities and were chosen as lead molecules. In vitro bioluminescent resonance energy transfer (BRET) assays confirmed that all three compounds functioned as MOR agonists and D₃R antagonists/partial agonists. Notably, compound 31 demonstrated metabolic stability and maximal antinociceptive effects (ED₅₀=29.1 mg/kg s.c.) with reduced motor stimulation in C57BL/6J mice. Although compound 31 produced respiratory depression at the analgesic dose of 30 mg/kg, this was comparable to morphine which has lower intrinsic efficacy and a safer profile compared to etonitazene. Overall, our findings support the feasibility of developing MOR-D₃R dual-target ligands, based on etonitazene, as efficacious analgesics that may have reduced addictive liability.

Graphical Abstract

Introduction

Analgesics are widely used to mitigate physical pain, and opioid agonists are particularly effective for treating moderate to severe pain^1–4. The primary molecular target of clinically prescribed opioids is the mu opioid receptor (MOR), which is abundantly expressed in central nervous system (CNS) regions involved in pain modulation, reward processing, and respiratory control. In the periphery, MOR modulates gastrointestinal (GI) tract motility^{1, 5–8}. While activation of MOR produces pain relief, it is also associated with euphoria, the development of tolerance⁹, and respiratory depression^10–13, all of which contribute to the high risk of opioid use disorder (OUD) and fatal overdose^14–18.

According to the Substance Abuse and Mental Health Services Administration (SAMHSA), approximately 125 million opioid prescriptions were dispensed in the United States (US) during 2023, with notable variation across states. Alarmingly, nearly 7% (8.6 million) of Americans aged 12 and older reported misusing prescription opioids in the past year. The US Centers for Disease Control and Prevention (CDC) reported that an average of 224 lives were lost each day during 2022 due to opioid overdose¹⁹.

Although recent data from the CDC show a decrease in opioid-related deaths, developing safer opioid analgesics with a reduced risk of addiction remains a national priority. As it has proven difficult to identify MOR agonists that are effective analgesics without problematic side effects, we have focused attention on designing and synthesizing bivalent and dual-target ligands that incorporate both MOR and dopamine D₃ receptor (D₃R) pharmacophores. The D₃R is a member of the dopamine D₂-like receptor subfamily of G protein-coupled receptors (GPCRs) that is activated by the endogenous neurotransmitter dopamine and predominantly expressed in mesolimbic^20–24 regions of the brain. The mesolimbic circuitry is critically involved in the regulation of reward, motivation, reinforcement, and drug-related behaviors^25–29. In 2019, D₃R antagonists and partial agonists were among the highest-priority pharmacotherapies identified by the National Institute on Drug Abuse (NIDA) for the prevention and treatment of OUD and opioid overdose³⁰. Indeed, significant progress has been made in the development of highly selective D₃R ligands as potential medications for substance use disorders (SUD) in general, and OUD in particular, with the highly selective D₃R antagonist R-VK4–116 moving toward clinical trials³¹.

Here, we hypothesized that modification of a potent MOR agonist to incorporate the pharmacophore of a D₃R antagonist/partial agonist might yield a single molecular entity providing effective antinociception with reduced addiction potential^32–34. In our earlier series^{32, 33}, we synthesized dual-target compounds incorporating several D₃R primary pharmacophores (e.g., 2,3-dichlorophenylpiperazine) into various MOR agonist scaffolds (Figure 1) ranging from full to partial agonists. These scaffolds included: loperamide (1), a peripherally restricted MOR agonist prescribed for the treatment of diarrhea, which has been investigated for managing opioid-induced hyperalgesia and neuropathic pain^35–41; methadone (2), a MOR agonist used for moderate to severe pain and as a medication for the treatment of OUD; and oliceridine (TRV130, 3)^{42, 43}, a centrally active partial MOR agonist used for acute pain. Our structure-activity relationship (SAR) studies revealed that the dual-target MOR agonists based on loperamide or TRV130 (5–7 in Figure 1 and Table 1) achieved moderate to high binding affinities for both MOR and D₃R.

Figure 1:

Selected examples of MOR agonists and previous lead dual target ligands based on Loperamide and TRV130

Table 1.

Binding affinities for the Etonitazene based MOR-D₃R ligands and previous lead molecules^a

Parent Structure	Cpds	R	vs. [3H]-DAMGO	vs. [3H]-NMSP	vs. [3H]-NMSP	CNS-MPO Scorec
			MOR	D2R	D3R
			Ki ± S.E.M. (nM)	Ki ± S.E.M. (nM)	Ki ± S.E.M. (nM)
	5b		0.832 ± 0.121 (n=3)	74.7 ± 7.50 (n=3)	171 ± 49.2 (n=5)	2.3
	6b		85.2 ± 32.7 (n=7)	5520 ± 1960 (n=5)	361 ± 131 (n=5)	3.2
	7b		23.8 ± 4.91 (n=3)	43.9 ± 9.04 (n=3)	39.2 ± 11.3 (n=5)	2.1
	4		0.149 ± 0.0169 (n=3)	NA	NA	3.9
	14		181± 75.4 (n=5)	24100 ± 927 (n=3)	2990 ± 572 (n=3)	2.7
	15		64.9 ± 12.6 (n=4)	34300 ± 2290 (n=3)	15700 ± 2120 (n=3)	2.5
	16		45.3 ± 12.5 (n=4)	NA	24100 ± 7700 (n=3)	2.6
	21		12.1 ± 1.68 (n=4)	91800 ± 13900 (n=3)	11000 ± 260 (n=3)	3.0
	23		26.8 ± 6.41 (n=4)	26500 ± 869 (n=3)	3930 ± 373 (n=3)	3.0
	31		33.4 ± 13.9 (n=3)	150 ± 14 (n=3)	90.6 ± 18.6 (n=3)	2.3
	35		29.7 ± 0.678 (n=3)	8.70 ± 0.398 (n=3)	53.8 ± 2.19 (n=3)	2.8
	39		46.1 ± 6.6 (n=5)	328.7 ± 47.3 (n=3)	206.2 ± 12.0 (n=3)	2.6
	43		89.6 ± 6.50 (n=3)	16.5 ± 6.94 (n=3)	45.4 ± 6.30 (n=3)	2.4
	55		69.2 ± 8.51 (n=3)	122 ± 48.0 (n=4)	100 ± 39.9 (n=4)	3.0
	59		41.8 ± 6.32 (n=3)	318 ± 149 (n=4)	350 ± 172 (n=4)	3.0
	63		80.6 ± 10.7 (n=4)	809 ± 328 (n=4)	668 ± 240 (n=4)	3.0
	67		38.0 ± 1.46 (n=3)	3320 ± 1090 (n=3)	2010 ± 523 (n=3)	3.0
	71		46.9 ± 6.84 (n=3)	233 ± 21.4 (n=3)	183 ± 30.3 (n=3)	2.4

^aAll the affinity values are expressed as K_i ± standard error of the mean (S.E.M.), derived from IC₅₀ values using the Cheng−Prusoff⁶⁹ equation and calculated as the mean of at least three independent experiments (n = number of independent experiments), each performed in triplicate.

^bData previously reported^{32, 33}.

^cThe CNS-MPO score for each compound is calculated using Chemicalize and Chemdraw. Detailed values are reported in the Supporting Information (Table S1). NA = Not active (>100 μM).

In the current investigation, a new series of dual-target ligands was designed using the potent MOR agonist etonitazene (4, Figure 1) as the primary MOR pharmacophore. Etonitazene, a 2-benzylbenzimidazole derivative first developed by Chemische Industrie Basel (CIBA) in the late 1950s,^{44, 45} was selected for its exceptionally high potency and selectivity for MOR over other opioid receptors.⁴⁶ In mice, etonitazene exhibits antinociceptive effects approximately 1,000 times more potent than morphine^47–49 and has a CNS multiparameter optimization (CNS-MPO) score of 3.9 indicating robust BBB permeability.^50,51 Our rationale for choosing such a potent opioid analgesic was based on previous work,^{32, 33, 52} where appending a D₃R pharmacophore typically reduced MOR binding affinity, potency and sometimes decreased efficacy. In the case of etonitazene, both reduced MOR potency and efficacy would be desirable. We hypothesized that a decreased affinity and/or potency could still have analgesic effects. Moreover, in the case of decreased efficacy, partial MOR agonists, such as TRV130, have proven effective as analgesics with a better side effect profile.^53–55 Recently, a MOR “superagonist”, N-desethyl-fluornitrazene (DFNZ), a fluoro-analog of etonitazene with limited brain permeability, was reported to have surprisingly potent analgesic activity with minimal side effects compared to classical opioid agonists.⁵⁶ In this study, we anticipated that coupling etonitazene with a D₃R antagonist/partial agonist might also prove advantageous. All newly synthesized dual-target compounds were initially evaluated using radioligand binding assays to determine affinities for the target receptors, MOR, D₃R, and D₂R. A subset of compounds (31, 35, 43) was evaluated for in vitro functional efficacies in bioluminescence resonance energy transfer (BRET) assays. In addition, the metabolic stability of 31, 35, and 43 was evaluated in rat and mouse liver microsomes. To investigate opioid-like effects in vivo, the antinociceptive, locomotor, and thermic effects of the compounds were determined in C57BL/6J mice. Finally, to assess potential respiratory side effects, we measured respiratory depression in mice (C57BL/6J) using whole-body plethysmography, comparing the results to those of the classical opioid agonist morphine.

Results and Discussion

Chemistry

We initiated our SAR study to modify the etonitazene core (4, Figure 1) by appending classic D₃R primary pharmacophores such as 1-(2,3-dichlorophenyl)piperazine, 1-(2-fluoro-3-methoxyphenyl)piperazine, and 1-(6-(trifluoromethyl)pyridin-2-yl)piperazine⁵⁷. In our earlier loperamide series³², compound 5 (Figure 1), featuring a 1-(2,3-dichlorophenyl)piperazine and a 2-C linking chain, demonstrated high binding affinity for MOR (K_i = 0.832 nM) with moderate affinity at D₂R and D₃R (K_i = 74.7 nM and 171 nM, respectively).

Building upon these findings, we initiated the synthesis of a key intermediate, compound 12, based on the etonitazene scaffold. This intermediate was prepared by modifying previously reported procedures^{44, 58, 59}, starting from commercially available 2-(4-ethoxyphenyl)acetic acid (8) and 2-((2-amino-4-nitrophenyl)amino)ethan-1-ol (10). The intermediate 12 was then designed to undergo either nucleophilic substitution (Scheme 1) or reductive amination (Scheme 2) with suitable amines to yield the desired target compounds.

Scheme 1.

a) Thionyl Chloride (SOCl₂), Dichloromethane (DCM), rt, 12 h, >99% yield; b) NaHCO₃, CHCl₃: H₂O (3:1), from 0 °C to RT, 84% yield; c) aqueous HCl (6M), 100 °C, 3 h, 90% yield; d) CBr₄, PPh₃, DCM, RT, 12 h, 68% yield; e) Appropriate aryl piperazine, K₂CO₃, acetonitrile (ACN), reflux, 12 h, 29–32% yield.

Scheme 2.

a) DMP, DCM, from 0 °C to RT, 2 h; b) CH₃CHO, NaBH₄, MeOH, from 0 °C to RT, 2 h, 21–26% yield; c) Compound 17, STAB, cat. AcOH, DCE, RT, 12 h, 13–38% yield over two steps.

To access the first series of dual target MOR–D₃R ligands using an etonitazene-based MOR pharmacophore, the hydroxyl group of 12 was converted to a bromide 13 via the Appel reaction. This transformation enabled subsequent N-alkylation in the presence of base to afford compounds, 14, 15, and 16 by using 1-(2,3-dichlorophenyl)piperazine, 1-(6-(trifluoromethyl)pyridin-2-yl)piperazine or 1-(2-fluoro-3-methoxyphenyl)piperazine, respectively (Scheme 1).

The next dual-target series was based on an etonitazene–tranylcypromine hybrid. Tranylcypromine and its analogs are reported to exhibit significant dopaminergic activity, with a binding preference for D₃R over D₂R^60–62. In our previous series³³, we investigated various substituted trans-cyclopropylamine analogs. Among these, the most promising results were obtained with the rel-trans-2-(4-chlorophenyl)cyclopropan-1-amine, compound 6 (Figure 1).

Based on these findings, synthesis of compound 21 was initiated using rel-trans-2-(4-chlorophenyl)cyclopropan-1-amine 18. Notably, in the context of etonitazene-based dual-target ligands, we observed an increase in the CNS-MPO score when switching from a secondary to a tertiary amine, consistent with decreased basicity and reduced number of hydrogen bond donors (HBDs) (detailed values are reported in S.I., Table S1). To begin the synthesis, intermediate 12 was oxidized using Dess–Martin periodinane (DMP) to give 17 and in the parallel synthetic route, commercially available compound 18 was converted to 20 by performing reductive amination in the presence of acetaldehyde and sodium borohydride (NaBH₄). Subsequently, compound 20 underwent further reductive amination with compound 17 in the presence of sodium triacetoxyborohydride (STAB) and catalytic acetic acid to yield the desired product 21. Following a similar approach, we synthesized 23 using compound 17 and 22, which was obtained by successfully alkylating compound 19 with acetaldehyde (Scheme 2).

In the next step, we evaluated the impact of the linker chain on both binding affinity and CNS-MPO score. The CNS-MPO score is a theoretical value used to predict drug-like properties and particularly blood brain barrier (BBB) permeability^{55, 57}. In this series, we aimed to achieve a CNS-MPO score of >3. As previously noted, tertiary amines yielded better CNS-MPO scores than secondary amines (Table S1). Therefore, tertiary amines were employed in our design. It was reported that longer linker chains – typically consisting of four to five methylene units – enhanced binding affinities for the D₃R³². In our previous series³³, one of the lead compounds, 7 (Figure 1), demonstrated promising dual-target activity, exhibiting binding affinities of K_i = 23.8 nM for MOR and K_i = 39.2 nM for D₃R, with a four-methylene linker. Encouraged by this result, the synthesis of a new series of analogs incorporating extended carbon chains was initiated, aiming to retain the tertiary amine functionality and further examine the role of linker length and basicity in optimizing binding affinities and CNS-MPO scores. As outlined in Scheme 3, canonical D₃R pharmacophores, including 1-(2,3-dichlorophenyl)piperazine (24) and 1-(2-methoxyphenyl)piperazine (25) were first functionalized via N-alkylation with 2-(4-bromobutyl)isoindoline-1,3-dione (26) by following the reported procedures^63–66. Subsequent deprotection with hydrazine yielded the free amine intermediates 29 and 33, respectively. Following the strategy previously described, these primary amines were converted to their corresponding secondary amines, 30 and 34, which were then subjected to reductive amination with the aldehyde 17, affording the final target compounds 31 and 35, respectively.

Scheme 3.

a) 2-(4-bromobutyl)isoindoline-1,3-dione (26) or 2-(2-bromoethyl)isoindoline-1,3-dione (27), K₂CO₃, acetonitrile (ACN), reflux, 12 h, 78–91% yield; b) NH₂NH₂, EtOH, reflux, 12 h, 59–99% yield; c) CH₃CHO, NaBH₄, MeOH, from 0 °C to RT, 2 h, 31–53% yield; d) 17, STAB, cat. AcOH, DCE, RT, 12 h, 18–30% yield over two steps.

To further evaluate the influence of linker length on CNS-MPO score and receptor binding affinity, analogs featuring a two-carbon linker were also investigated. Using a similar synthetic strategy, compound 24 was alkylated with 2-(2-bromoethyl)isoindoline-1,3-dione (27) to obtain 36, followed by hydrazine-mediated deprotection to yield the primary amine intermediate 37. This intermediate was subsequently converted to the corresponding secondary amine (38), which then underwent reductive amination with the aldehyde 17 to afford the final target compound (39). In a parallel synthesis, compound 43 was obtained through reductive amination of 17 with the secondary amine intermediate 42 (Scheme 3).

In our next series, we explored analogs containing 2- and 4-methylene linkers tethered to a pyrrolidine ring instead of a piperazine ring as a D₃R pharmacophore. This design was inspired by the structure of eticlopride, a well-characterized D₂R/D₃R antagonist/inverse agonist⁶⁷. Based on our previous work that involved modifying the pyrrolidine ring of eticlopride, we aimed to assess how variations in linker length might influence receptor binding affinity and functional activity⁶⁸. To construct the 3-substituted pyrrolidine core 48, commercially available tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate (44) was converted to 46 by performing Suzuki coupling with 2-methoxy phenyl bromide (45). Then compound 46 underwent hydrogenation in the presence of 10% Pd/C (47) followed by Boc deprotection with TFA to give the desired product 48 (Scheme 4).

Scheme 4.

a) Pd(PPh₃)₄, Na₂CO₃, toluene/H₂O (10:1), 100 °C, 12 h, 90% yield; b) 10% Pd/C, H₂ (35 atm), EtOAc, 3h, 99% yield; c) TFA, DCM, RT, 2 h, 99% yield.

Using a similar synthetic strategy, pyrrolidines 48–51 were separately alkylated with either 2-(4-bromobutyl)isoindoline-1,3-dione (26) or 2-(2-bromoethyl)isoindoline-1,3-dione (27) to afford 52, 56, 60, 64, and 68. In parallel, the phthalimide groups of these intermediates were removed with hydrazine followed by alkylation to give secondary amines 54, 58, 62, 66, and 70 which undergo reductive amination separately with compound 17 to obtain the desired products 55, 59, 63, 67 and 71 respectively. (Scheme 5).

Scheme 5.

a) 2-(4-bromobutyl)isoindoline-1,3-dione (26) or 2-(2-bromoethyl)isoindoline-1,3-dione (27), K₂CO₃, acetonitrile (ACN), reflux, 12 h, 42–79% yield; b) NH₂NH₂, EtOH, reflux, 12 h, 67–89% yield; c) CH₃CHO, NaBH₄, MeOH, from 0 °C to RT, 2 h, 41% yield; d) 17, STAB, cat. AcOH, DCE, RT, 12 h, 11–40% yield over two steps.

Radioligand competition binding assays and CNS-MPO analysis

The binding affinities of all newly synthesized compounds at hMOR, hD₃R, and hD₂R were determined in human embryonic kidney 293 (HEK293) cell lines stably expressing a single receptor subtype. Binding assays were performed using [³H]DAMGO for hMOR and [³H]N-methylspiperone for hD_2LR and hD₃R. Affinity values are reported as K_i ± S.E.M. Binding affinities and CNS-MPO scores for all new compounds, as well as reference compounds etonitazene and previously identified leads, are included for comparison (Table 1).

Compounds 14, 15, and 16 were synthesized based on the favorable binding affinity profile of our earlier lead compound, 5 (Table 1). In our new series, we obtained compounds with moderate MOR binding affinities, but a significant loss in D₃R affinity was observed compared to compound 5. Among these, 14 demonstrated the best overall profile, with K_i = 181 nM for MOR and ~3 μM for D₃R. While 15 and 16 showed higher MOR affinities (K_i = 64.9 nM and 45.3 nM, respectively), but both lacked measurable binding at D₂R and D₃R (>10 μM).

Inspired by our previously developed TRV-trans-aryl cyclopropyl dual-target ligand, 6 (Table 1), the tertiary amines inserted into etonitazene provided compounds 21 and 23 with promising CNS-MPO scores (CNS-MPO = 3). Nevertheless, although 21 and 23 exhibited high MOR binding affinities (K_i = 12.1 and 26.8 nM, respectively), their D₃R binding affinities were markedly reduced compared to previous leads.

While MOR binding affinities remained promising, the D₃R affinities were very low, prompting us to consider modifications to the linker length. In our previous dual-target design strategy, compounds incorporating a four-carbon (4C) linker demonstrated better overall MOR and D₃R binding profiles (e.g., compound 7 in Table 1). Accordingly, compounds 31 and 35 were synthesized to incorporate a 4C linker with classical D₃R pharmacophores such as 1-(2,3-dichlorophenyl)piperazine and 1-(2-methoxyphenyl)piperazine respectively; both compounds exhibited moderate affinities at MOR (K_i = 33.4 nM and 29.7 nM, respectively) and D₃R (K_i = 90.6 nM and 53.8 nM, respectively), albeit with relatively low CNS-MPO scores. Notably, 35 displayed higher D₂R affinity (K_i = 8.70 nM) than D₃R.

We also explored shorter methylene linkers to obtain compounds 39 and 43, maintaining the same D₃R pharmacophore. Compound 43 showed reduced MOR binding (K_i = 89.6 nM) compared to its 4C analog 35, while D₃R binding remained similar (K_i = 45.4 nM). In contrast, compound 39 retained reasonable MOR affinity (K_i = 46.1 nM) but exhibited significantly weaker D₃R binding (K_i = 206 nM) than 31.

Next, the effect of replacing the piperazine with a pyrrolidine ring was investigated, while still maintaining the 4C linker. The pyrrolidine substitution improved the CNS-MPO scores across all analogs, 55, 59, 63 and 67. The most promising compound from this series, 55, bearing a 2-methoxyphenyl pyrrolidine moiety, showed moderate binding affinities at both MOR and D₃R (K_i = 69.2 and 100 nM, respectively). However, replacing the 2-methoxyphenyl ring with a new D₃R pharmacophore, such as thiophene (59), furan (63), or pyrrole (67), progressively reduced D₃R binding, while MOR affinity remained largely unchanged. Of note, compound 71 with a shorter methylene linker had a low CNS-MPO score, despite similar binding affinities for both MOR and D₃R. Based on these results, compounds 31, 35 and 43 were chosen for further evaluation.

Off Target Binding Data

The three lead compounds, 31, 35 and 43 were also screened for competitive inhibition of the Kappa (KOR) and Delta opioid receptors (DOR). A fixed supramaximal concentration of 10 μM for each compound was used to test for displacement of [³H]U69,593 (1 nM for KOR) or [³H]Naltrindole (1 nM for DOR) (Table S2). Inhibition of >50% at a 10 μM concentration for the three analogues tested at DOR was not observed. Hence these compounds were deemed inactive at DOR. For KOR, the compounds displaced [³H]U69,593 from a range of ~72–98%, which prompted further analysis to determine the affinities (K_i) of these compounds for KOR. The KOR affinities were determined from two independent competitive binding assays measuring concentrations ranging from 0.01 nM to 10 μM. All the compounds showed low μM affinity (K_i=2.0–5.6 μM) for KOR, with compound 31 showing the highest MOR selectivity (MOR/KOR=169; Table S3).

Additionally, these analogues were also evaluated for off-target binding affinities (Supporting Information (S.I.), Table S4). Compound 31 showed very low binding affinities for both dopamine D1 and D4 receptors (D₁R and D₄R), respectively as well as serotonin receptor subtypes including 5-HT_1A, 5-HT_2A and 5-HT_2C. Like 31, compound 35 had very low binding affinities for D₁R, 5-HT_2A and 5-HT_2C but had moderate binding affinities for D₄R and 5-HT_1A. Both compounds 31 and 35 showed high binding affinity for the 5-HT_2B receptor. Although activation of this serotonin receptor subtype has been associated with cardiotoxic effects, all tested compounds behaved as 5-HT_2B antagonists in the functional assays (Table S4). Compound 31 is a low potency full agonist at the 5-HT_1A, whereas 35 is a moderate potency partial agonist at the 5-HT_1A (Table S5).

BRET Functional Studies at MOR and D₃R

Bioluminescence resonance energy transfer (BRET) experiments were conducted to evaluate functional activities of the three lead compounds 31, 35, and 43. Potencies and efficacies at MOR (in Nb33 recruitment (a measure of MOR activation), G_i G protein activation, and arrestin-3 (β-arrestin2) recruitment assays) were determined and compared to the standard MOR agonists DAMGO, morphine, and TRV130. Potencies and efficacies at D₃R (G_o activation) were determined and compared to dopamine, the D2-like agonist quinpirole, and the D2-like antagonist haloperidol. Results are shown in Figure 2 and in tabular form in Table 2. All compounds showed MOR agonist activity but with varying efficacies. The order of MOR efficacy (as shown in low amplification assays of Nb33 and Arr3 recruitment) is from high to low 31 > 35 > 43. MOR potencies across assays were similar for all three lead compounds and typically <10-fold lower than controls, DAMGO, morphine, and TRV130.

Figure 2.

Functional profiles of selected MOR-D₃R ligands. Each panel shows a different signaling readout: A. Nb33 recruitment at MOR, B. Arrestin-3 recruitment at MOR (in the presence of overexpressed GRK2), C. MOR-mediated Gα_i2 protein activation, D. D₃R-mediated Gα_oA protein activation and E. Antagonism at D₃R using Gα_oA G protein activation assay in the presence of 3 nM of quinpirole. Data represent mean ± S.E.M. of 3 independent experiments performed in duplicate.

Table 2.

BRET functional studies for Etonitazene-based MOR-D ₃ R ligands ^a

Compound	MOR Nb33 recruitment		MOR Gi2 activation		MOR arr3 recruitment (GRK2)		D3R GoA activation
	pEC50 ± S.E.M. [EC50, nM]	Emax ± S.E.M. (%)	pEC50 ± S.E.M. [EC50, nM]	Emax ± S.E.M. (%)	pEC50 ± S.E.M. [EC50, nM]	Emax ± S.E.M. (%)	pEC50 ± S.E.M. [EC50, nM]	Emax ± S.E.M. (%)	pIC50 ± S.E.M. [EC50, nM]
DAMGO	7.02 ± 0.06 [95.6]	99.8 ± 2.5	8.86 ± 0.05 [1.4]	100	7.67 ± 0.03 [21]	100 ± 1.1	-	-	-
Morphine	6.35 ± 0.09 [442]	64.9 ± 2.6	7.89 ± 0.07 [12.9]	106.9 ± 2.7	6.77 ± 0.03 [172]	78.7 ± 1.1	-	-	-
TRV130 (3)	6.99 ± 0.08 [102.2]	33.9 ± 1.1	8.38 ± 0.09 [4.15]	101.5 ± 2.6	7.45 ± 0.09 [35]	55.1 ± 1.6	-	-	-
Quinpirole	-	-	-	-	-	-	8.37 ± 0.05 [4.2]	100 ± 1.4	-
Dopamine	-	-	-	-	-	-	9.03 ± 0.17 [0.94]	87.8 ± 4.4	-
Haloperidol	-	-	-	-	-	-	-	-	7.99 ± 0.22 [10.1]
31	5.91 ± 0.04 [1240]	116.7 ± 2.5	7.49 ± 0.06 [33]	108.1 ± 2.1	6.29 ± 0.03 [513]	109.4 ± 1.6	6.24 ± 0.18 [573]	42.6 ± 4.4	-
35	6.25 ± 0.04 [560]	74.4 ± 1.5	7.60 ± 0.07 [25]	104.5 ± 2.5	6.46 ± 0.05 [350]	89.6 ± 2.1	-	-	5.14 ± 0.45 [7299]
43	6.02 ± 0.06 [962]	48.5 ± 1.7	7.27 ± 0.07 [54]	101 ± 2.7	6.01 ± 0.04 [971]	68.5 ± 1.5	-	-	6.43 ± 0.25 [374.5]

^aAll values are the mean of three (n=3) independent experiments

At D₃R, 31 displayed partial agonist activity with an efficacy ~40% that of quinpirole. The other two compounds, 35 and 43, were D₃R antagonists inhibiting quinpirole (3 nM), with 43 displaying higher potency than 35.

Mouse and Rat Liver Microsome Metabolic Stability

Phase I metabolic stability studies were conducted in both rat and mouse liver microsomes for 31, 35, and 43 over a 60-min incubation period with and without NADPH as a cofactor. As shown in Figure 3 (+NADPH), 31 demonstrated the highest metabolic stability with ~45% of the parent compound remaining at 60 min in both species. In contrast, both 35 and 43 were rapidly metabolized with only 0–5% of the parent compound remaining at 30 min in both mouse and rat liver microsomes. All compounds were >90% stable in incubations lacking NADPH, indicating a CYP-(cytochrome P450)-dependent metabolism. Although 35 and 43 were metabolically less stable than 31, we tested all three compounds in a series of behavioral models in mice to determine their effects on locomotion, analgesia and body temperature as compared to the classic opioid agonists, fentanyl and morphine.

Figure 3. Metabolic stability of compounds 31, 35, and 43 in mouse and rat liver microsomes.

Compounds were incubated for 60 min in the presence of NADPH, and % parent remaining was measured over time.

Evaluation of acute antinociceptive, locomotor, and hypothermic effects in mice

Given the in vitro profiles of 31, 35, and 43 at MOR and D₃R, we hypothesized that the compounds may have reduced or modified opioid-like effects in vivo due to hybrid target activity. Therefore, the drugs were administered subcutaneously (s.c.) to mice at various doses (1, 10, 30, and 100 mg/kg) to examine acute opioid-like effects on antinociception, locomotor activity, and body temperature. Figure 4 depicts the dose-related effects of the compounds across these measures relative to previously published values for fentanyl and morphine using the same experimental setup⁴⁷. The potencies and maximum observed effects across measures are shown in S.I. (Table S7). Time-course plots for the effects measured are shown in Figure 5, with tabulated mean data for each shown in S.I. (Table S8).

Figure 4.

Dose-response curves for 31, 35 and 43 for opioid-like effects in mice relative to published values under the same experimental conditions for fentanyl and morphine. Drugs were administered s.c. Data represent mean ± S.E.M. of n = 4 – 5 mice/dose for effects of the drugs on antinociception in the hot plate assay (A), distance traveled (B), and body temperature change (C). Fentanyl and morphine data are replotted from reference 47. Dose-related statistical differences for each analogue are reported in Table S6.

Figure 5.

Time-course plots for dose-related opioid-like effects of 31 (A – C), 35 (D – F) and 43 (G – I) after s.c. administration in mice. Data represent mean ± S.E.M. of n = 4 –5 mice/dose for effects of the drugs on distance traveled (A, D, G), antinociception in the hot plate assay (B, E, H), and body temperature change (C, F, I).

Effects on hot plate latencies revealed that all three compounds produced antinociceptive effects but to differing degrees (Figure 4A). For example, compound 31 produced significantly greater hot plate latencies at 30 and 100 mg/kg relative to vehicle controls, but compounds 35 and 43 did not (Table S6). The relative potencies of the drugs to produce antinociceptive effects were similar (ED₅₀ = 21 – 29 mg/kg), but maximal observed latencies (%MPE) were different (Table S7). Compounds 35 and 43 only produced partial effects on hot plate latencies (E_max = 59 and 31% MPE, respectively), while compound 31 produced near maximal antinociception (E_max = 95% MPE) similar to reported values for fentanyl and morphine (E_max = 100 and 85% MPE, respectively)⁴⁷. Notably, the potencies of the test drugs were ~1/3 less potent compared to morphine, with 31 being the only new compound to match efficacy of the reference compounds. Compound 31 also had the quickest onset of action for antinociceptive effects, with peak effects occurring at the 15 min timepoint versus the 45 or 60 min timepoint for the other compounds (Figure 5B, 5E and 5H, Table S8). The compounds all produced dose-related effects on locomotion, and significantly increased motor activity relative to vehicle controls at 30 and/or 100 mg/kg (Figure 4B, Table S6). The relative potencies for inducing hyperlocomotion were similar, between 8 – 17 mg/kg, but the maximal distance traveled differed between compounds (Table S7). Compound 31 was more efficacious for this endpoint (distance traveled = 17,631 cm) relative to 35 and 43 (distance traveled = 11,347 and 9,217 cm respectively). Nevertheless, the maximal locomotor effects of 31 were reduced relative to fentanyl and morphine (distance traveled = 35,454 and 23,531 cm, respectively). The onset of peak locomotor effects was quickest with compounds 31 and 35 (15 – 30 min) compared to compound 43 (30 – 45 min; Figure 5A, 5D and 5G, Table S8).

Acute effects of the drugs on body temperature revealed dose-related hypothermic effects, similar to the effects of fentanyl and morphine (Figure 4C). However, only compounds 31 and 35 produced statistically significant differences in body temperature relative to vehicle controls (Table S6). The potencies of the drugs for hypothermic effects varied (ED₅₀ = 11.5 – 32.7 mg/kg), with a rank order of 31 ≥ 35 ≥ 43 (Table S7). Maximal observed temperature reductions varied, with 31 and 35 displaying similar degrees of hypothermia compared to morphine (4 – 6 °C reductions), and lower values observed for compound 43 and fentanyl (2 – 3 °C reductions). The time-courses for hypothermic effects of 31, 35, and 43 were delayed relative to other measures of opioid-like effects. Peak hypothermic effects were not reached until 30 – 60 min post drug administration (Figure 5C, 5F and 5I, Table S8) and may reflect D₂R effects or active metabolites.

Overall, the novel etonitazene MOR-D₃R dual-target ligands displayed potencies for opioid-like effects, comparable to available data for morphine, as opposed to fentanyl and etonitazene. Compound 31 produced maximal antinociceptive effects in the hot plate assay, whereas compounds 35 and 43 were partially active up to 100 mg/kg. All three compounds displayed reduced hyperlocomotion relative to morphine, fentanyl, and etonitazene, which suggests they may differ from traditional opioids in terms of abuse potential.

Naloxone reversibility of acute opioid-like effects in mice

We next examined the ability of naloxone (0.3 mg/kg) to reverse opioid-like effects induced by compounds 31 and 35, given these compounds were the most active of the three new drugs tested. Mice were treated with compound 31 or 35 (100 mg/kg) followed by naloxone (0.3 mg/kg 15 min later) to test the reversibility of acute opioid-like effects, and similar experiments were conducted with a high-dose of fentanyl (0.3 mg/kg). Prior to naloxone administration, all three treatments produced statistically greater locomotion, hot plate latencies, and hypothermia relative to vehicle controls (Figure S1, Table S9). Naloxone administration reversed the antinociceptive (Figure 6A – C) and locomotor effects (Figure 6D – F) of the drugs, but hypothermic effects were only partially attenuated (Figure S2A – C). Naloxone reversed the hypothermic effects of 31 and 35 but effects were partially to fully reduced at the 60 min time point (Figure S2A, Table S9 – 10). In contrast, hypothermic effects of fentanyl were attenuated quicker and reversed by the 30 – 45 min time points (Figure S2B, Table S9 – 10). Overall, these data suggest that acute opioid-like effects of compounds 31 and 35 can be reversed, or at least reduced, by blockade of MOR with naloxone.

Figure 6.

Time-course plots (A, B, D, E) and comparisons of effects of NLX (0.3 mg/kg) on opioid-like effects of 31 and 35 in mice (30 – 60 min timepoints, C, F) relative to fentanyl (0.3 mg/kg). All drugs were administered s.c. Data represent mean ± S.E.M. of n = 5 – 8 mice/dose for effects of the drugs on antinociception in the hot plate assay (A, B, C) and distance traveled (D, E, F). Descriptive statistics and statistical comparisons can be found in Table S10 & Table S11. Asterisks (*) represent statistical differences vs. the veh, veh control group, while the pound symbol (#) represents differences between treatments with vs. without naloxone (NLX, all p <0.05).

Evaluation of Respiratory Depression

Based on the behavioral and metabolic stability data, compound 31 was tested for its effects on respiration in mice. We first examined the effects of morphine (as a control), administered at the same doses (3 and 10 mg/kg, i.p.) that produced analgesia (as shown in Figure 4A), on respiration with whole-body plethysmography. We chose to administer morphine or 31 intraperitoneally in the respiratory depression experiments because both analgesic and respiration depression experiments were conducted using established assay-specific protocols in two collaborating laboratories. Subcutaneous administration is commonly used in nociceptive assays because it provides reliable systemic absorption and a sustained analgesic time course during behavioral testing, whereas intraperitoneal administration is widely employed in acute respiratory pharmacology studies because it produces rapid systemic exposure suitable for assessing opioid-induced respiratory depression. We acknowledge that differences in route of administration may influence pharmacokinetic parameters, including absorption rate and time-to-peak effect. However, the purpose of these experiments was to assess morphine responsiveness within each assay rather than to make direct quantitative comparisons of morphine potency or efficacy across behavioral and respiratory endpoints.

Systemic administration of morphine produced a dose-dependent reduction in respiration, as indicated by decreased respiratory frequency (breaths per min, BPM; Figure 7A), prolonged inspiratory duration (milliseconds per inspiration; Figure 7B), increased tidal volume (milliliters per breath; Figure 7C), and decreased minute ventilation (milliliters per min; Figure 7D). Two-way ANOVA for repeated measures across time and morphine dose revealed significant main effects of morphine treatment and time, as well as a treatment × time interaction (see F and p values in S. I., Table S12). Post hoc group comparisons showed significant differences between the vehicle and 10 mg/kg morphine treatment groups during the 30–90 minute period (Figure 7, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 7. Effects of morphine on respiration in mice.

Systemic administration of morphine (3, 10 mg/kg, i.p.) produced a dose-dependent depression on respiration as assessed by decreased respiratory frequency (breath per min, BPM) (A), prolonged inspiratory duration (ms) (B), increased tidal volume (mL per breath) (C), and decreased minute ventilation (mL/min) (D).

Next, we used the same methods to examine the effects of compound 31 on mouse respiration. Systemic administration of 31 at doses of 30 and 100 mg/kg, which produced significant analgesic effects (as shown in Figures 4A and 5B), also significantly inhibited respiration in a dose-dependent manner, as assessed by decreased respiratory frequency (BPM; Figure 8A), prolonged inspiratory duration (milliseconds (ms); Figure 8B), increased tidal volume (milliliters (mL); Figure 8C), and decreased minute ventilation (milliliters per min; Figure 8D). Two-way repeated measures ANOVA across time and compound 31 dose revealed significant main effects of treatment and time, as well as a treatment × time interaction (see F and p values in S.I. Table S12). Post hoc group comparisons showed significant differences between the vehicle and both 30 and 100 mg/kg 31 treatment groups across all measures (Figure 8, *p < 0.05, **p < 0.01, ***p < 0.001), with effects persisting for more than 3 h at the higher dose.

Figure 8. Effects of compound 31 on respiration in mice.

Systemic administration of 31 (30, 100 mg/kg, i.p.) produced a dose-dependent depression on respiration, as assessed by decreased respiratory frequency (breath per min, BPM) (A), prolonged inspiratory duration (ms) (B), increased tidal volume (mL per breath) (C), and decreased minute ventilation (mL/min) (D).

Notably, both morphine and 31 produced respiratory depression but with distinct profiles. Morphine induced a transient reduction in respiration (~90 min) at 10 mg/kg, whereas 31 elicited a more potent and longer-lasting effect on both the amplitude and duration of respiratory depression at a dose of 100 mg/kg. Nevertheless, it should be noted that at 30 mg/kg, compound 31 showed antinociception, albeit not maximal, but equivalent to that level of antinociception demonstrated with the 10 mg/kg dose of morphine (Figure 4A). Hence, although 31 produced respiratory depression at 30 mg/kg (Figure 8) these levels are comparable to those of morphine at the 10 mg/kg dose and considerably lower than those at the maximally effective antinociceptive dose of 100 mg/kg.

Summary

Etonitazene is an ultrapotent MOR agonist that has selectivity for MOR over other opioid receptors and is ~1,000 times more potent in vivo than classic opioids such as morphine⁵⁸. Herein we report the incorporation of different D₃R primary pharmacophores into the etonitazene scaffold by replacing the N,N-diethyl moiety. Based on our previous work, we hypothesized that by incorporating the D₃R primary pharmacophore into etonitazene, the resulting molecules would retain moderate MOR affinity and possibly reduced efficacy compared to the parent molecule, resulting in a compound that retained antinociceptive effects but with a lower side effect profile. In this report, we expanded etonitazene SAR and discovered compounds 31, 35, and 43, which presented balanced binding affinities for both MOR and D₃R in the nanomolar range. Metabolic stability studies in rat and mouse liver microsomes indicated that compound 31 was more stable (~45% left) than 35 and 43 (0–5% left) after 60 min. All compounds were >90% stable in incubations lacking NADPH, indicating CYP-dependent metabolism.

The antinociceptive, locomotor, and hypothermic effects of the lead compounds 31, 35, and 43 were evaluated and compared to previously reported values for fentanyl and morphine using the same acute opioid-like effect paradigm in mice. We observed that compound 31 produced significantly greater hot plate latencies both at 30 and 100 mg/kg doses relative to vehicle controls and produced near maximal antinociception (E_max = 95% MPE) similar to reported values for fentanyl and morphine (E_max = 100 and 85% MPE, respectively). In contrast, compounds 35 and 43 only produced partial effects on hot plate latencies (E_max = 59 and 31% MPE, respectively) at the highest doses tested (100 mg/kg). Notably, the test drugs were less potent than morphine, with 31 being the only new compound to induce full maximal antinociceptive effects. Compound 31 also showed the fastest onset of action for antinociception compared to compounds 35 and 43. For acute effects on locomotor activity, compound 31 was similarly more efficacious (distance traveled = 17,631 cm) compared to 35 and 43 (distance traveled = 11,347 and 9,217 cm respectively). Importantly, the maximal locomotor effects of 31 were lower than fentanyl and morphine (distance traveled = 35,454 and 23,531 cm, respectively), perhaps suggesting lower abuse potential. Despite some evidence showing correlations between hyperlocomotor activity of morphine and conditioned place preference effects, future studies will have to unravel whether hyperlocomotor activity of opioids can be used as a definitive predictive measure for abuse liability by comparing hyperlocomotor potencies with potencies for drug self-administration or other more well-established abuse liability measures.

The naloxone reversibility of the acute effects of compounds 31 and 35 were also evaluated. As expected, naloxone reversed the antinociceptive and locomotor effects of fentanyl and 31. These data suggest that acute opioid-like effects of compound 31 were reversed by blockade of MOR with naloxone. Finally, one of the most challenging side effects of MOR agonists is respiratory depression, which is the primary culprit for overdose lethality. While there is no data to our knowledge that suggests D₃R antagonists or partial agonists would have any effect on mitigating respiratory depression elicited by this class of drugs, we wanted to evaluate this potentially lethal side effect. Indeed, compound 31 produced respiratory depression in mice, but its effects were distinct compared to the those reported for other nitazenes¹³. In this study, morphine induced a transient reduction in respiration (~90 min) at 10 mg/kg, whereas 31 elicited a more potent and longer-lasting effect on both the amplitude and duration of respiratory depression at a 10 times higher dose of 100 mg/kg. Nevertheless, at the dose (30 mg/kg) that produced the same antinociception as morphine (10 mg/kg), the measures of respiratory depression were significantly lower and at essentially the same levels as morphine. Hence, at the very least, this etonitazene dual target ligand showed similar levels of respiratory depression and antinociception as morphine, which is an improvement as compared to the nitazene class, in general.

In summary, a series of novel etonitazene MOR-D₃R dual-target ligands were designed, synthesized, and evaluated for binding affinities at MOR, D₃R, and D₂R and functional efficacies at MOR and D₃R. Compounds 31, 35, and 43 were selected for further evaluation based on their balanced MOR and D₃R binding profiles and functional efficacies. Compound 31 was a D₃R partial agonist, whereas 35 and 43 were D₃R antagonists. Compound 43 had a desirable partial agonist profile at MOR but was metabolized rapidly, as was 35. Neither of these analogues were efficacious as analgesics in the hot plate assay. Compound 31 was a full agonist at MOR. It produced maximal antinociceptive effects in the hot plate assay with reduced hyperlocomotion and similar respiratory depression measures to morphine. Additional investigation into MOR-D₃R dual target ligand SAR with a goal of discovering partial agonists at MOR, and potentially higher affinity at D₃R than MOR, are underway. It is anticipated that a MOR partial agonist may produce lower respiratory depression while maintaining maximal antinociception and that a high affinity D₃R partial agonist or antagonist may reduce addictive liability. These findings will open a new window to developing safer pain medications in the future.

EXPERIMENTAL METHODS

Chemistry

General Information.

All chemicals and solvents were purchased from chemical suppliers unless otherwise stated and used without further purification. All melting points were determined (when obtainable) on an OptiMelt automated melting point system and are uncorrected. Reactions were not yield optimized. All nuclear magnetic resonance (NMR) spectra (¹H and ¹³C) were acquired in deuterated solvents (CDCl₃ or CD₃OD) on JOEL JNM-ECZL 400S spectrometer and a Varian Mercury Plus 400 spectrometer. Chemical shifts are reported in parts per million (ppm) and were adjusted using the residual solvents (CDCl₃: 7.26 ppm for ¹H NMR, 77.2 ppm for ¹³C NMR; CD₃OD: 3.31 ppm for ¹H NMR, 49.0 ppm for ¹³C NMR) as an internal reference. Coupling constants are reported in hertz (Hz) and peak multiplicities as either a singlet (s), doublet (d), triplet (t), quartet (q), or multiplet (m).. Chemical shifts, multiplicities and coupling constants (J) have been reported and calculated using JEOL Delta v6.0 or MNova 9.0 software. Gas chromatography-mass spectrometry (GC/MS) data were acquired (where obtainable) using an Agilent Technologies (SantaClara, CA) 7890B GC equipped with an HP-5MS column (cross-linked 5% PH ME siloxane, 30 m× 0.25 mm i.d. × 0.25 μm film thickness) and a 5977B mass-selective ion detector in the electron-impact mode. Ultrapure grade helium was used as the carrier gas at a flow rate of 1.2 mL/min. The injection port and transfer line temperatures were 250 and 280 °C, respectively, and the oven temperature gradient used was as follows: the initial temperature (70 °C) was held for 1 min and then increased to 300 °C at 20 °C/min and maintained at 300 °C for 4 min, total run time 16.5 min. Column chromatography was performed using a Teledyne Isco CombiFlash RF flash chromatography system, or a Teledyne Isco EZ-Prep chromatography system. When % DMA is reported as eluting system, it stands for % of methanol in DCM, in presence of 1–10% NH₄OH. High-performance liquid chromatography (HPLC) was conducted on an Agilent Technologies 1260 Infinity system coupled to a diode-array detector (DAD). A Phenonmenex Gemini C18 100 Å LC column (50 mm × 4.6 mm, 3 μm particle size) was used as the stationary phase. For basic conditions, the mobile phase consisted of H₂O with 0.1% diethylamine (solvent A) and ACN with 0.1% diethylamine (solvent B). For acidic conditions, solvents contained 0.1% TFA instead of diethylamine. All samples were prepared at a concentration of ca. 1 mg/mL in ACN, and 20 μL of each solution was injected onto the column, which was maintained at 40 °C. Using a flow rate of 1.0 mL/min, the solvent gradient was as follows: 10% B held for 10 min, 10–40% B ramped over 10 min, 40% B held for 10 min, 40–80% B over 10 min, 80% B held for 20 min. Compound purity was determined based on peak integration (area under the curve) of the absorbance signals at 254 and 230 nm. Analytical HPLC columns were purchased from Daicel corporation, Agilent or Phenomenex. Microanalyses were performed by Robertson Microlit Laboratories (Ledgewood, NJ) and agree with ± 0.4% of calculated values (Table S1). In Electrospray Ionization Mass Spectrometry (ESI-MS) neat solutions of samples were dissolved and diluted in acetonitrile (ACN). Samples were analyzed by direct injection (10 μL) using a Vanquish UHPLC system (ThermoFisher, Waltham, MA) with tandem Orbitrap Exploris 120 mass spectrometer (ThermoFisher). The flow rate was 200 μL/min with an isocratic mobile phase of 80% ACN for the four-minute run. Analysis was performed using a heated electron spray ionization (HESI) source in positive ion mode. In MS mode, the mass resolution was set at 120,000 while in MS/MS mode the mass resolution was set at 15,000. Unless otherwise stated, all the test compounds were evaluated to be >95% pure based on combustion analysis, NMR, HPLC-DAD-HRMS-MS/MS. The detailed analytical results are reported in the characterization of each final compound and in the S.I.

General Procedure A for N-alkylation:

The appropriate amine (1 eq) was dissolved in MeOH (10 mL) under an Argon atmosphere, and acetaldehyde (5–10 eq) was added dropwise by using a pre-cooled syringe at 0 °C. The progress of the reaction was monitored by GC/MS. After 45 min, NaBH₄ (2 eq) was added to the mixture at 0 °C, and the reaction was allowed to warm to RT while stirring for an additional 45 min. After the reaction was complete as assessed by GC/MS, the mixture was quenched with H₂O, and the organic solvents were evaporated under reduced pressure. The remaining H₂O phase was extracted with DCM/i-PrOH (3:1, 3×15 mL). The organic phase was dried over Na₂SO₄ or MgSO₄, filtered, and evaporated under reduced pressure to obtain the crude product, which was either purified by column chromatography or used immediately in the next step.

2-(4-Ethoxyphenyl)-N-(2-((2-hydroxyethyl)amino)-5-nitrophenyl)acetamide (11):

NaHCO₃ (3.6 g, 43 mmol) was added to a stirred solution of 10 (0.94 g, 4.8 mmol) in CHCl₃: H₂O (2:1, 25 mL). 2-(4-ethoxyphenyl)acetyl chloride, 9 (0.86 g, 4.3 mmol) (prepared by treating 8 (0.93 g, 5.2 mmol) with SOCl₂ (4 mL, 54 mmol) in DCM (10 mL)) was dissolved in CHCl₃ (20 mL) and added to the reaction mixture dropwise at 0 °C for 30 min. The reaction mixture was allowed to warm to room temperature (RT) and stirred for 3 h. After confirming the reaction was completed by TLC, the aq phase was extracted with EtOAc (3×20 mL). The combined organic phase was dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude material was dissolved in acetone: DCM: MeOH (10:2.5:0.5). Hexanes were added to obtain the product by trituration. The product was collected by filtration, washed with hexanes (2×10 mL), and dried under vacuum at 40 °C to obtain 11 (1.3 g, 84% yield) as a light yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.02 (dd, J = 2.6, 9.2 Hz, 1H), 7.96 (s, 1H), 7.27 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 9.2 Hz, 1H), 4.00 (q, J = 7.0 Hz, 2H), 3.70 (t, J = 5.6 Hz, 2H), 3.66 (s, 2H), 3.36 (t, J = 5.6 Hz, 2H), 1.37 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CD₃OD) δ 174.5, 159.6, 150.8, 137.8, 131.3, 128.4, 125.6, 124.5, 122.9, 115.8, 110.5, 64.5, 61.1, 46.2, 43.2, 15.2.

2-(2-(4-Ethoxybenzyl)-5-nitro-1H-benzo[d]imidazol-1-yl)ethan-1-ol (12):

HCl (1.6 g, 7.3 mL, 6M, 44 mmol) was added to 11 (450 mg, 1.25 mmol). The mixture was stirred at 100 °C for 2.5 h. After completion, the reaction mixture was cooled in an ice bath and then conc. NH₄OH was slowly added to pH 9–10. A pale-yellow solid formed and was collected by filtration, washed with hexanes (2×10 mL), and dried at RT under vacuum. The compound was purified by flash chromatography eluting with 50% EtOAc/ hexanes to obtain 12 (385 mg, 90% yield) as a light yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.49 (d, J = 2.1 Hz, 1H), 8.18 (dd, J = 2.2, 9.0 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.15 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 4.36 (s, 2H), 4.31 (t, J = 5.2 Hz, 2H), 3.97 (q, J = 7.0 Hz, 2H), 3.69 (t, J = 5.2 Hz, 2H), 1.33 (t, J = 7.0 Hz, 3H).¹³C NMR (101 MHz, CD₃OD) δ 160.4, 159.7, 145.0, 142.4, 141.2, 130.9 (2C), 128.7, 119.2, 116.0 (2C), 115.5, 112.1, 64.5, 61.4, 47.9, 34.0, 15.2.

1-(2-Bromoethyl)-2-(4-ethoxybenzyl)-5-nitro-1H-benzo[d]imidazole (13):

Triphenylphosphine (PPh₃; 403 mg, 1.54 mmol) was added to a stirred solution of 12 (350 mg, 1.03 mmol) in DCM (15 mL), followed by portion-wise addition of CBr₄ (510 mg, 1.54 mmol) at 0 °C. The reaction mixture allowed to warm to RT and stirred for 12 h. The volatiles were evaporated, and the residue was purified by flash chromatography eluting with 40% EtOAc/hexanes to obtain 13 (280 mg, 68% yield) as a light yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.68 (s, 1H), 8.22 (d, J = 10.0 Hz, 1H), 7.37 (d, J = 8.7 Hz, 1H), 7.17 (d, J = 8.3 Hz, 2H), 6.86 (d, J = 7.8 Hz, 2H), 4.45 (t, J = 6.7 Hz, 2H), 4.33 (s, 2H), 4.01 (q, J = 7.1 Hz, 2H), 3.25 (t, J = 7.3 Hz, 2H), 1.40 (t, J = 6.9 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 158.6, 157.4, 144.0, 142.2, 139.5, 129.7 (2C), 126.9, 118.8, 116.6, 115.4, 109.3, 96.8, 63.8, 45.8, 34.2, 27.8, 15.0.

1-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)-2-(4-ethoxybenzyl)-5-nitro-1H-benzo[d]imidazole (14):

K₂CO₃ (308 mg, 2.23 mmol) was added to a stirred solution of 13 (180 mg, 0.45 mmol) and 1-(2,3-dichlorophenyl)piperazine (257 mg, 1.11 mmol) in ACN (10 mL), and the mixture was stirred at reflux for 12 h. The mixture was filtered, the solvent was evaporated, and the desired product isolated by flash chromatography eluting with 5% MeOH containing 10% NH₄OH/DCM to obtain 14 (80 mg, 32% yield) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.64 (d, J = 2.0, 1H), 8.18 (dd, J = 2.2, 8.9 Hz, 1H), 7.36 (d, J = 8.9 Hz, 1H), 7.17–7.11 (m, 4H), 6.91 (dd, J = 2.6, 6.9 Hz, 1H), 6.85–6.82 (m, 2H), 4.34 (s, 2H), 4.16 (t, J = 6.7 Hz, 2H), 3.98 (q, J = 7.0 Hz, 2H), 2.99 (brs, 4H), 2.57–2.50 (m, 6H), 1.37 (t, J = 7.0 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 158.3, 157.5, 150.8, 143.6, 141.9, 139.7, 134.0, 129.5, 127.48, 127.45, 127.1, 124.8, 118.6, 118.2, 116.2, 115.0, 109.3, 63.5, 56.8, 53.7, 51.2, 42.5, 33.9, 14.8. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 mm; gradient 10%-80% ACN in H₂O; 60 min run; injection 20 μL (1 mg/mL); temperature 40 °C; ^tR 37.730 min, purity >95%. HRMS-MS/MS [C₂₈H₂₉Cl₂N₅O₃ + H]⁺ calculated 554.17202, found 554.17103. The free base was converted into the corresponding HCl salt, which was obtained as a white solid (m.p. 145–147°C). Elemental analysis (C₂₈H₂₉Cl₂N₅O₃ • HCl•1.25 H₂O) calculated C 54.82, H 5.34, N 11.42, found C 54.99, H 5.12, N 11.34.

2-(4-Ethoxybenzyl)-5-nitro-1-(2-(4-(6-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)ethyl)-1H-benzo[d]imidazole (15):

The compound was prepared following the same procedure described for 14, starting from 13 (102 mg, 0.252 mmol), 1-(6-(trifluoromethyl)pyridin-2-yl)piperazine (146 mg, 0.631 mmol) and K₂CO₃ (174 mg, 1.26 mmol). The crude product was purified by flash chromatography eluting with 5% MeOH containing 10% NH₄OH/ DCM to obtain 15 (40 mg, 29% yield) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.67 (d, J = 1.7 Hz, 1H), 8.20 (dd, J = 8.9, 1.9 Hz, 1H), 7.58 (t, J =7.9 Hz, 1H), 7.38 (d, J = 8.9 Hz, 1H) 7.15 (d, J = 8.5 Hz, 2H), 6.96 (d, J = 7.4 Hz, 1H), 6.86 (d, J = 8.5 Hz, 2H), 6.75 (d, J = 8.6 Hz, 1H), 4.36 (s, 2H), 4.19 (t, J = 6.5 Hz, 2H), 3.99(q, J = 6.9 Hz,, 2H), 3.56 (m, 4H), 2.49 (m, 6H), 1.39 (t, J = 6.9 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 159.6, 159.3, 158.5, 144.6, 143.0, 140.6, 139.3, 130.4 (3C), 128.0, 119.2, 117.2 (2C), 116.0 (2C), 110.4, 110.2 64.5, 57.8, 54.2, 45.7, 43.6, 34.9, 15.7. ¹⁹F NMR (376 MHz, CDCl₃) δ −101.1. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 μm; gradient 10%-80% ACN in H₂O; 60 min run; injection 20 μL (1 mg/mL); temperature 40°C; ^tR 36.510 min, purity >99%. HRMS-MS/MS (C₂₈H₂₉F₃N₆O₃ + H]⁺ calculated 555.23260, found 555.23160.

2-(4-Ethoxybenzyl)-1-(2-(4-(2-fluoro-3-methoxyphenyl)piperazin-1-yl)ethyl)-5-nitro-1H-benzo[d]imidazole (16):

The compound was prepared following the same procedure described for 14, starting from 13 (106 mg, 0.262 mmol), 1-(2-fluoro-3-methoxyphenyl)piperazine hydrochloride (162 mg, 0.656 mmol) and K₂CO₃ (362 mg, 2.62 mmol). The desired product was isolated by flash chromatography eluting with 5% MeOH containing 10% NH₄OH/ DCM to obtain 16 (40 mg, 29% yield) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.68–8.64 (m, 1H), 8.22–8.17 (m, 1H), 7.40–7.34 (m, 1H), 7.17–7.12 (m, 2H), 6.99–6.95 (m, 1H), 6.87–6.82(m, 2H), 6.66–6.53 (m, 2H), 4.36–4.33 (m, 2H), 4.19–4.15 (m, 2H), 4.01–3.96 (m, 2H), 3.88–3.84 (m, 3H), 3.05 (brs, 4H), 2.59–2.49 (m, 6H), 1.42–1.35 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.4, 158.6, 149.7, 148.0, 145.5, 144.7, 143.1, 141.8, 140.8, 130.6, 128.2, 124.7, 124.6, 119.3, 117.3, 116.2, 112.2, 110.4, 108.4, 64.6, 58.0, 57.5, 54.8, 51.6, 43.6, 35.0, 15.9. ¹⁹F NMR (376 MHz, CDCl₃) δ −146.4, −146.4, −146.4. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 μm; gradient 10%-80% ACN in H₂O; 60 min run; injection 20 μL (1 mg/mL); temperature 40°C; ^tR 34.175 min, purity >99%. HRMS-MS/MS [C₂₉H₃₂FN₅O₄ + H]⁺ calculated 534.25111, found 534.24893.

2-(2-(4-Ethoxybenzyl)-5-nitro-1H-benzo[d]imidazol-1-yl) acetaldehyde (17):

In an open vessel, 12 (427 mg, 1.25 mmol) was dissolved in DCM (50 mL) and placed in an ice bath. DMP (1.06 g, 2.50 mmol) was added to the solution portion wise at 0 °C. The reaction mixture was allowed to warm to RT and stirred for 2 h. NaHCO₃ was added to pH 9, and the reaction was extracted with DCM (3×20mL). The organic phase was dried over Na₂SO₄, filtered, and evaporated under reduced pressure to give the crude product, which was used in next step without purification.

Rel-trans-2-(4-chlorophenyl)-N-ethylcyclopropan-1-amine (20):

The compound was prepared following the general procedure A starting from 18 (300 mg, 1.470 mmol), acetaldehyde (0.50 mL, 7.35 mmol), and NaBH₄ (111 mg, 2.94 mmol). The desired product was isolated by flash chromatography eluting with 50% EtOAc containing 5% TEA/hexanes to obtain 20 (76 mg, 26% yield) as a viscous liquid. ¹H-NMR (400 MHz, CDCl₃) δ 7.21 (d, J = 8.5 Hz, 2H), 6.75 (d, J = 8.4 Hz, 2H), 2.78 (q, J = 7.2 Hz, 2H), 2.33–2.29 (m, 1H), 1.89–1.85 (m, 1H), 1.14–1.07 (m, 4H), 0.97–0.93 (m, 1H). GC/MS (EI) ^tR 7.487 min, 195.10 m/z.

Rel-trans-2-(4-chlorophenyl)-N-(2-(2-(4-ethoxybenzyl)-5-nitro-1H-benzo[d]imidazol-1-yl)ethyl)-N-ethylcyclopropan-1-amine (21):

Cat. AcOH (3–4 drops) and a solution of 20 (76 mg, 0.39 mmol) in DCE (6 mL) were added to a stirred solution of 17 (130 mg, 0.39 mmol) in DCE (4 mL) at RT. The mixture was stirred for 30 min, then STAB (99 mg, 0.47 mmol) was added portion-wise. The reaction was stirred overnight at RT. After consumption of all the starting material as judged by TLC, the reaction was basified to pH 9 with NaHCO₃, and the aq phase was extracted with DCM (3×10 mL). The combined organic phases were dried over Na₂SO₄, filtered, and evaporated under reduced pressure to give the crude product, which was purified by flash chromatography eluting with 50% EtOAc/hexanes to obtain 21 (76 mg, 38% yield) as a gummy solid. ¹H-NMR (400 MHz, CD₃OD) δ 8.52 (s, 1H), 8.18–8.12 (m, 1H), 7.52 (d, 1H), 7.26–7.08 (m, 4H), 6.89–6.81 (m, 4H), 4.33–4.26 (m, 2H), 4.22 (s, 2H), 4.02–3.95 (m, 4H), 2.83–2.62 (m, 3H), 1.95–1.92 (m, 1H),, 1.38–1.34 (m, 3H), 1.05 (t, J = 7.1, Hz, 3H), 0.92–0.88 (m, 1H), 0.77–0.72 (m, 1H). ¹³C-NMR (101 MHz, CD₃OD) δ 158.6, 158.4, 143.7, 140.9, 139.4, 129.6, 129.4, 129.4, 128.0, 126.8, 124.7, 117.7, 114.7, 114.6, 110.5, 63.2, 63.1, 53.2, 42.9, 34.1, 32.7, 24.8, 16.8, 13.8, 10.7. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 μm; gradient 10%-80% ACN in H₂O; 60 min run; injection 20 μL (1 mg/mL); temperature 40°C; ^tR 39.104 min, purity >95%. HRMS-MS/MS [C₂₉H₃₁ClN₄O₃+H]⁺calculated 519.21575, found 519.21486.

Rel-trans-N-ethyl-2-(2-methoxyphenyl)cyclopropan-1-amine (22):

The compound was prepared following general procedure A, starting with 19 (250 mg, 1.25 mmol), acetaldehyde (552 mg, 700 μL, 12.5 mmol), and NaBH₄ (95 mg, 2.50 mmol). The crude compound was purified by column chromatography using 50% EtOAc containing 5% TEA/hexanes to obtain 22 (50 mg, 21% yield) as a viscous liquid. ¹H-NMR (400 MHz, CDCl₃) δ 7.12 (t, J = 6.9 Hz, 1H), 6.86–6.79 (m, 3H), 3.84 (s, 3H), 2.87–2.74 (m, 2H), 2.27–2.23 (m, 1H), 2.18–2.13 (m, 1H), 1.12(t, J = 7.1 Hz, 3H), 1.01–0.94 (m, 2H). GC/MS (EI) ^tR 7.495 min, 191.10 m/z.

Rel-trans-N-(2-(2-(4-ethoxybenzyl)-5-nitro-1H-benzo[d]imidazol-1-yl)ethyl)-N-ethyl-2-(2-methoxyphenyl)cyclopropan-1-amine (23):

The compound was prepared following the same procedure described for 21, starting from 17 (99 mg, 0.29 mmol), cat. AcOH acid (3–5 drops), 22 (56 mg, 0.29 mmol), and STAB (74 mg, 0.35 mmol). The crude product was purified by flash chromatography eluting with 50% EtOAc/hexanes to obtain 23 (20 mg, 13% yield) as a gummy solid. ¹H-NMR (400 MHz, CDCl₃) δ 8.64 (d, J = 1.98 Hz, 1H), 8.11 (dd, J = 8.9, 2.0 Hz, 1H), 7.24 (d, J = 7.3 Hz, 1H), 7.15(t, J= 8.0 Hz, 1H), 7.01 (d, J= 8.5 Hz, 2H), 6.90–6.83 (m, 2H), 6.74 (dd, J = 14.9, 8.0 Hz, 3H), 4.30– 4.11 (m, 4H), 3.95 (q, J = 6.9 Hz, 2H), 3.73 (s, 3H), 2.72–2.65 (m, 4H), 2.19–2.14 (m, 1H), 1.90–1.86 (m, 1H), 1.37 (t, J =6.9 Hz, 3H), 1.01–0.95 (m, 4H), 0.89–0.84 (m, 1H). ¹³C-NMR (101 MHz, CDCl₃) δ 158.2, 157.8, 157.6, 143.5, 142.0, 139.8, 129.6, 129.5, 127.2, 126.9, 125.1, 120.6, 118.3, 116.3, 115.0, 110.3, 109.4, 63.6, 55.4, 53.2, 49.2, 46.5, 43.4, 33.8, 19.6, 15.8, 14.9, 11.6. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 μm; gradient 10%-80% ACN in H₂O; 60 min run; injection 20 μL (1 mg/mL); temperature 40°C; ^tR 38.400 min, purity >99%. HRMS-MS/MS [C₃₀H₃₄N₄O₄ + H]⁺calculated 515.26528, found 515.26470.

2-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)isoindoline-1,3-dione (28):

By following the previous reports⁶⁴, 26 (2.3 g, 8.1 mmol) and K₂CO₃ (5.0 g, 37 mmol) were added to a stirred solution of 1-(2,3-dichlorophenyl)piperazine (1.7 g, 7.5 mmol) in ACN (50 mL). The reaction mixture was stirred in a sealed vessel at 90 °C overnight, cooled to RT, and filtered rinsing with ACN. The solvent was evaporated under reduced pressure, and the crude product was purified by flash chromatography eluting with 50% EtOAc/hexanes to obtain 28(2.89 g, 89% yield) as a white solid. ¹H-NMR (400 MHz,CD₃OD) δ 7.85–7.80 (m, 2H), 7.79–7.76 (m, 2H), 7.22–7.16 (m, 2H), 7.05 (dd, J = 7.2, 2.3 Hz, 1H), 3.70 (t, J = 6.9 Hz, 2H), 3.03 (brs, 4H), 2.64 (bs, 4H), 2.48–2.44 (m, 2H), 1.74 (p, J = 7.0 Hz, 2H), 1.61–1.53 (m, 2H). The ¹H NMR is consistent with the reported literature^{64, 65}.

4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butan-1-amine (29):

By following the reported procedure⁶⁵, 28 (2.89 g, 6.68 mmol) was dissolved in EtOH (15 mL) and hydrazine (2.0 mL, 66.8 mmol) was added to the solution. The reaction mixture was stirred at reflux for 12 h. The solvent was evaporated under vacuum. The residue suspended in 20% aq. K₂CO₃ and extracted with DCM/i-PrOH (3:1). The organic phase was dried over Na₂SO₄, filtered, and evaporated under vacuum to obtain 29 (2.0 g, 99% yield) as a viscous liquid. The crude material obtained was used in the next step without further purification. ¹H-NMR (400 MHz, CD₃OD) δ 7.23–7.17 (m, 2H), 7.06 (dd, J = 7.3, 2.2 Hz, 1H), 3.05 (brs, 4H), 2.68–2.55 (m, 6H), 2.45–2.41 (m, 2H), 1.61–1.48 (m, 4H).

4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-N-ethylbutan-1-amine (30):

The compound was prepared following the general procedure A starting from 29 (2.0 g, 6.6 mmol), acetaldehyde (1.8mL, 33 mmol), and NaBH₄ (0.50 g, 13 mmol). The desired product was isolated by flash chromatography eluting with 10% MeOH containing 10% NH₄OH/DCM, to obtain 30 (820 mg, 38% yield) as a gummy solid. ¹H-NMR (400 MHz, CD₃OD) δ 7.23–7.17 (m, 2H), 7.07 (dd, J = 7.3, 2.2 Hz, 1H), 3.05 (brs, 4H), 2.68–2.58 (m, 8H), 2.45–2.41 (m, 2H), 1.58–1.52 (m, 4H), 1.11 (t, J = 7.2 Hz, 3H).

4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-N-(2-(2-(4-ethoxybenzyl)-5-nitro-1H-benzo[d]imidazol-1-yl)ethyl)-N-ethylbutan-1-amine (31):

The compound was prepared following the same procedure described for 21, starting from 17 (300 mg, 884 μmol), cat. AcOH acid (3–5 drops), 30 (292 mg, 884 μmol), and STAB (225 mg, 1.06 mmol). The desired product was isolated by flash chromatography eluting with 5% MeOH containing 10% NH₄OH/ DCM to obtain 31 (118 mg, 20% yield) as a pale-yellow solid. ¹H-NMR (400 MHz, CD₃OD) δ 8.53 (s, 1H), 8.23 (d, J = 8.6, 1H), 7.67 (d, J = 8.6 Hz, 1H), 7.23–7.18 (m, 4H), 7.08–7.07 (m, 1H), 6.89 (d, J = 8.6 Hz, 2H), 4.39 (s, 2H), 4.29 (t, J =8.0 Hz, 2H), 3.99 (q, J = 6.9 Hz, 2H), 3.05 (brs, 4H), 2.67–2.55 (m, 8H), 2.34–2.24 (m, 4H), 1.35 (t, J =6.9 Hz, 3H), 1.34–1.29(m, 2H), 1.13–1.09 (m, 2H), 0.95 (t, J = 7.1 Hz, 3H). ¹³C-NMR (101 MHz, CD₃OD) δ 158.8, 158.4, 150.9, 143.7, 140.9, 139.7, 133.6, 129.5, 127.8, 127.2, 127.1, 124.7, 118.8, 117.9, 114.7, 114.3, 110.7, 63.2, 57.9, 53.2, 52.8, 52.5, 50.3, 42.9, 32.8, 24.7, 23.4, 20.7, 13.8, 10.7. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 μm; gradient 10%-80% ACN in H₂O; 60 min run; injection 20 μL (1 mg/mL); temperature 40°C; ^tR 21.527 min, purity >99%. HRMS-MS/MS [C₃₄H₄₂Cl₂N₆O₃+H]⁺calculated 653.27682, found 653.27616. The free base was converted into the corresponding HCl salt, which was obtained as a white solid (m.p. 229–231 °C). Elemental analysis (C₃₄H₄₂Cl₂N₆O₃• 3HCl• H₂O) calculated C 52.29, H 6.06, N 10.76, found C 52.55, H 5.92, N 10.37.

2-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butyl)isoindoline-1,3-dione (32):

The compound was prepared following the same procedure described for 28, starting from 25 (2.0 g, 10 mmol), 26 (3.2 g, 11 mmol), and K₂CO₃ (7.2 g, 52 mmol). The desired product was isolated by flash chromatography eluting with 50% EtOAc/hexanes to obtain 32 (3.2 g, 78% yield) as an off-white solid. ¹H-NMR (400 MHz, CD₃OD) δ 7.85–7.81 (m, 2H), 7.80–7.76 (m, 2H), 7.00–6.84 (m, 4H), 3.82 (s, 3H), 3.69 (t, J = 6.9 Hz, 2H), 3.02 (brs, 4H), 2.63 (brs, 4H), 2.47–2.43 (m, 2H), 1.70 (p, J = 7.1, 6.99 Hz, 2H), 1.60–1.53 (m, 2H). The ¹H NMR is consistent with the reported literature⁶³.

4-(4-(2-Methoxyphenyl)piperazin-1-yl)butan-1-amine (33):

The compound was prepared following the same procedure described for 29 starting from 32 (3.2 g, 8.1 mmol) and hydrazine (2.6 mL, 81 mmol). The crude material obtained was used in the next step without further purification (1.3 g, 59% yield). ¹H-NMR (400 MHz, CDCl₃) δ 6.97–6.84 (m, 4H), 3.84 (s, 3H), 3.09 (brs, 4H), 2.70–2.64 (m, 6H), 2.40 (brs, 2H), 1.55–1.47 (m, 4H). The ¹H NMR is consistent with the reported literature⁷⁰.

N-Ethyl-4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-amine (34):

The compound was prepared following the general procedure A starting from 33 (2.13 g, 8.09 mmol), acetaldehyde (2.5 mL, 40 mmol), and NaBH₄ (612 mg, 16.2 mmol). The desired product was isolated by flash chromatography on silica gel eluting with 10% MeOH containing 10% NH₄OH/DCM to obtain 34 (725 mg, 31% yield) as a gummy solid. ¹H-NMR (400 MHz, CDCl₃) δ 6.96–6.83 (m, 4H), 3.83 (s, 3H), 3.07 (brs, 4H), 2.62 (brs, 8H), 2.39 (brs, 2H), 1.52 (brs, 4H), 1.09 (brs, 3H). ¹³C-NMR (101 MHz, CDCl₃) δ 152.3, 141.4, 122.9, 121.0, 118.3, 111.2, 58.7, 55.4, 53.6, 50.8, 49.9, 44.3, 28.3, 24.9, 15.5.

N-(2-(2-(4-Ethoxybenzyl)-5-nitro-1H-benzo[d]imidazol-1-yl)ethyl)-N-ethyl-4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-amine (35):

The compound was prepared following the same procedure described for 21 starting from 17 (300 mg, 884 μmol), cat. AcOH acid (3–5 drops), 34 (283 mg, 972 μmol), and STAB (225 mg, 1.06 mmol). The desired product was isolated by flash chromatography eluting with 10% MeOH containing 10% NH₄OH/DCM to obtain 35 (135 mg, 25% yield) as a pale-yellow solid. ¹H-NMR (400 MHz, CDCl₃) δ 8.51 (s, 1H), 8.23–8.16 (m, 1H), 7.39–7.30 (m, 1H), 7.19–7.10 (m, 2H), 7.04–6.78 (m, 6H), 4.34 (s, 2H), 4.12–3.94 (m, 4H), 3.87 (s, 3H), 3.17–2.96 (m, 4H), 2.68–2.28 (m, 12 h), 1.47–1.34 (m, 5H), 1.32–1.19 (m, 2H), 0.95–0.86 (m, 3H). ¹³C-NMR (101 MHz, CD₃OD) δ 162.7, 162.4, 156.5, 147.6, 144.9, 144.6, 143.7, 133.4, 131.2, 127.4, 124.8, 122.0, 121.8, 118.7 (2C), 118.2, 115.4, 114.7, 67.2, 62.0, 58.5, 57.1, 56.8, 56.4, 53.9, 47.0, 36.7, 28.8, 27.5, 17.7, 14.7. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 μm; gradient 10%-80% ACN in H₂O + 0.1% DEA; 60 min run; injection 20 μL (1 mg/mL); temperature 40°C; ^tR 37.106 min, purity >99%. HRMS-MS/MS [C₃₅H₄₆N₆O₄+H]⁺calculated 615.36533, found 615.36407. The free base was converted into the corresponding HCl salt, obtained as a white solid (m.p.151–153 °C). Elemental analysis (C₃₅H₄₆N₆O₄• 3HCl• H₂O) calculated C 56.64, H 6.93, N 11.32, found C 56.77, H 6.70, N 10.97.

2-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)isoindoline-1,3-dione (36):

The compound was prepared following the same procedure described for 28 starting with 24 (1.0 g, 4.3 mmol), 27 (1.2 g, 4.3 mmol), and K₂CO₃ (3.0 g, 22 mmol). The desired product was isolated by flash chromatography eluting with 50% EtOAc/hexanes to obtain 36 (1.6 g, 91% yield) as a pale-yellow solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.85–7.83 (m, 2H), 7.72–7.70 (m, 2H), 7.13–7.08 (m, 2H), 6.91 (dd, J = 7.1, 2.5 Hz, 1H), 3.85 (t, J = 6.5 Hz, 2H), 2.99 (brs, 4H), 2.70 (t, J = 6.3 Hz, 6H).

2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethan-1-amine (37):

The compound was prepared following the same procedure described for 29 starting from 36 (1.71 g, 4.23 mmol) and hydrazine (1.33 mL, 42.3 mmol). The crude material obtained was used in the next step without further purification (1.14 g, 98% yield).

2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-N-ethylethan-1-amine (38):

The compound was prepared following the same procedure described for 20 starting from 37 (480 mg, 1.75 mmol), acetaldehyde (0.50 mL, 8.75 mmol), and NaBH₄ (132 mg, 3.50 mmol). The desired product was purified by flash chromatography eluting with 8% MeOH containing 10% NH₄OH/DCM to obtain 38 (282 mg, 53% yield) as a gummy solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.16–7.11 (m, 2H), 6.97–6.89 (m, 1H), 3.05 (brs, 4H), 2.77–2.53 (m, 10H), 1.12 (t, J =7.3 Hz, 3H).

2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-N-(2-(2-(4-ethoxybenzyl)-5-nitro-1H-benzo[d]imidazol-1-yl)ethyl)-N-ethylethan-1-amine (39):

The compound was prepared following the same procedure described for 21 starting from 17 (195 mg, 576 μmol), cat. AcOH acid (3–5 drops), 38 (150 mg, 496 μmol), and STAB (158 mg, 744 μmol). The desired product was isolated by flash chromatography eluting with 10% MeOH containing 10% NH₄OH/DCM to obtain 39 (57 mg, 18% yield) as a pale yellow solid. ¹H-NMR (400 MHz, CDCl₃) δ 8.65 (d, J = 1.6 Hz, 1H), 8.18 (dd, J = 8.8, 1.6 Hz, 1H), 7.39 (d, J = 8.9 Hz, 1H), 7.15–7.10 (m, 4H), 6.90–6.82 (m, 3H), 4.34 (s, 2H), 4.10 (t, J = 6.4 Hz, 2H), 3.98 (q, J = 6.9 Hz, 2H), 2.98 (brs, 4H), 2.64–2.47 (m, 10H), 2.29 (t, J = 7.0 Hz, 2H), 1.37 (t, J = 7.0 Hz, 3H), 0.91 (t, J = 7.0 Hz, 3H). ¹³C-NMR (101 MHz, CDCl₃) δ 158.3, 157.8, 151.1, 143.6, 142.1, 139.9, 134.1 (2C), 129.6, 127.6, 127.3, 124.8, 118.6, 118.2, 116.3, 115.1, 109.5, 63.6, 56.7, 53.7, 53.3, 51.3, 51.2, 48.8, 43.9, 33.9, 14.9, 11.9. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 μm; gradient 10%-80% ACN in H₂O + 0.1% DEA; 60 min run; injection 20 μL (1 mg/mL); temperature 40°C; ^tR 40.227 min, purity >99%. HRMS-MS/MS [C₃₂H₃₈Cl₂N₆O₃+H]⁺ calculated 625.24552, found 625.24390. The free base was converted into the corresponding HCl salt, which was obtained as a yellow solid (m.p.143–145°C). Elemental analysis (C₃₂H₃₈Cl₂N₆O₃• 3HCl• 1.75 H₂O) calculated C 50.14, H 5.85, N 10.96, found C 50.36, H 5.55, N 10.35.

2-(2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl)isoindoline-1,3-dione (40):

The compound was prepared following the same procedure described for 28 starting from 25 (2.0 g, 10 mmol), 27 (2.9 g, 11 mmol), and K₂CO₃ (7.2 g, 52 mmol). The desired product was isolated by flash chromatography eluting with 50% EtOAc/hexanes to obtain 40 (3.0 g, 81% yield) as an off-white solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.87–7.80 (m, 2H), 7.74–7.66 (m, 2H), 7.03–6.76 (m, 4H), 4.03–3.72 (m, 5H), 3.02 (brs, 4H), 2.84–2.49 (m, 6H). The ¹H NMR is consistent with the reported literature⁷⁰.

2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethan-1-amine (41):

The compound was prepared following the same procedure described for 29 starting from 40 (3.07 g, 8.40 mmol) and hydrazine (2.5 mL, 84.0 mmol). The crude material obtained was used in the next step without further purification (1.93 g, 98% yield). ¹H-NMR (400 MHz, CDCl₃) δ 6.97–6.84 (m, 4H), 3.84 (s, 3H), 3.08 (brs, 4H), 2.92–2.75 (m, 2H), 2.65 (brs, 4H), 2.51–2.38 (m, 2H). The ¹H NMR is consistent with the reported literature⁷⁰.

N-Ethyl-2-(4-(2-methoxyphenyl)piperazin-1-yl)ethan-1-amine (42):

The compound was prepared following the general procedure A starting from 41 (1.45 g, 6.2 mmol), acetaldehyde (1.8mL, 31 mmol), and NaBH₄ (466 mg, 12.3 mmol). The desired product was isolated by flash chromatography eluting with 20% MeOH containing 1% NH₄OH/DCM to obtain 42 (800 mg, 49% yield) as a gummy solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.00–6.84 (m, 4H), 3.84 (s, 3H), 3.08 (brs, 4H), 2.75 (t, J = 6.2 Hz, 2H), 2.70–2.65 (m, 6H), 2.56 (t, J = 6.2 Hz, 2H), 1.12 (t, J = 7.1 Hz, 3H).

2-(2-(4-Ethoxybenzyl)-5-nitro-1H-benzo[d]imidazol-1-yl)-N-ethyl-N-(2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl)ethan-1-amine (43):

The compound was prepared following the same procedure described for 21 starting from 17 (515 mg, 1.52 mmol), cat. AcOH acid (3–5 drops), 42 (400 mg, 1.52 mmol), and STAB (386 mg, 1.82 mmol). The desired product was isolated by flash chromatography eluting with 5% MeOH containing 10% NH₄OH/DCM followed by a second purification using EtOAc containing 5% MeOH to obtain 43 (267 mg, 30% yield) as a light yellow solid. ¹H-NMR (400 MHz, CD₃OD) δ 8.66 (m, 1H), 8.19 (m, 1H), 7.40 (m, 1H), 7.15 (m, 2H), 7.00 (m, 1H), 6.91–6.78 (m, 5H), 4.35 (s, 2H), 4.11 (m, 2H), 3.99 (m, 2H), 3.86 (s, 3H), 3.03 (brs, 4H), 2.64–2.50 (m, 10H), 2.30 (m, 2H), 1.39 (m, 3H), 0.91 (m, 3H). ¹³C-NMR (101 MHz, CD₃OD) δ 158.3, 157.9, 152.4, 143.6, 142.1, 141.3, 139.9, 129.7, 127.4, 123.2, 121.1, 118.3, 116.3, 115.1, 111.3, 109.6, 63.6, 56.9, 55.5, 54.0, 53.6, 53.3, 51.5, 50.7, 48.9, 44.1, 34.0, 15.0, 12.0. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 μm; gradient 10%-80% ACN in H₂O + 0.1% TFA; 60 min run; injection 20 μL (1 mg/mL); temperature 40°C; ^tR 19.477 min, purity >99%. HRMS-MS/MS [C₃₃H₄₂N₆O₄ +H]⁺ calculated 587.33403, found 587.33286. The free base was converted into the corresponding HCl salt, which was obtained as a light yellow solid (m.p. 166–168°C). Elemental analysis (C₃₃H₄₂N₆O₄ • 3HCl) calculated C 56.94, H 6.52, N 12.07, found C 57.24, H 6.69, N 11.68.

tert-Butyl 3-(2-methoxyphenyl)-2,5-dihydro-1H-pyrrole-1-carboxylate (46):

An oven-dried, resealable Schlenk flask was evacuated, backfilled with Argon and charged with tetrakis(triphenylphosphine)palladium(0) (92.7 mg, 80.2 μmol). 1,4-Dioxane (10 mL) was added and degassed for 5 min, followed by the addition of 44 (355 mg, 1.20 mmol), K₃PO₄ (511 mg, 2.41 mmol), and H₂O (1.0 mL), maintaining an Argon atmosphere. Finally, 1-bromo-2-methoxybenzene (45, 0.1 mL, 802 μmol) was added dropwise. The flask was sealed with a Teflon screwcap, and the reaction was heated to 60–70°C, while stirring, until the aryl halide was completely consumed, as determined via GC/MS analysis. The reaction mixture was cooled to RT, then filtered through Celite and washed with EtOAc. The solvent was evaporated to afford the crude compound, which was purified by column chromatography eluting with 10% EtOAc/hexanes to obtain 46 (200 mg, 90% yield) as an off-white solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.25 – 7.14 (m, 2H), 6.93 – 6.87 (m, 2H), 6.37 (dt, J = 1.86, 32.19 Hz, 1H), 4.50 (d, J = 30.51 Hz, 2H), 4.28 (d, J = 27.73 Hz, 2H), 3.84 (d, J = 5.19 Hz, 3H), 1.50 (d, J = 4.56 Hz, 9H). GC/MS (EI) ^tR 10.775 min, 275.20 m/z.

tert-Butyl 3-(2-methoxyphenyl)pyrrolidine-1-carboxylate (47):

In a hydrogenation bottle, 46 (300 mg, 1.09 mmol) was dissolved in EtOAc (10 mL), followed by addition of 10% Pd/C (34.8 mg, 327 μmol). The reaction was shaken in a Parr apparatus for 3 h under H₂ (35 psi). After the reaction completion was confirmed by GC/MS, the mixture was filtered through Celite and washed with EtOAc. The solvent was evaporated to afford the crude product, which was used in the next step without purification (300 mg, 99% yield).

3-(2-Methoxyphenyl)pyrrolidine (48):

TFA (1.05 mL, 13.7 mmol) was added to a solution of 47 (292 mg, 1.05 mmol) in DCM (10 mL) dropwise. After the reaction was completed as determined by TLC, the excess TFA was removed under reduced pressure, the residue was basified with 2N aq NaOH to pH 9–10, and extracted with DCM:i-PrOH (3:1). The organic phase was dried over Na₂SO₄, filtered, and evaporated under vacuum to get the crude product, which was used in the next step without further purification (180 mg, 99% yield).

2-(4-(1-(2-Methoxyphenyl)pyrrolidin-3-yl)butyl)isoindoline-1,3-dione (52):

The compound was prepared following the same procedure described for 28 starting from 48 (300 mg, 1.03 mmol), 26 (320 mg, 1.13 mmol), and K₂CO₃ (1.42 g, 10.3 mmol). The desired product was isolated by flash chromatography eluting with EtOAc to obtain 52 (269 mg, 69% yield) as an off-white solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.84–7.81 (m, 2H), 7.72–7.70 (m, 2H), 7.26–7.17 (m, 2H), 6.93–6.86 (m, 2H), 3.84 (s, 3H), 3.80–3.65 (m, 5H), 3.10 (brs, 4H), 2.50–2.10 (m, 2H), 1.77 (brs, 4H).

4-(3-(2-Methoxyphenyl)pyrrolidin-1-yl)butan-1-amine (53):

The compound was prepared following the same procedure described for 29 starting from 52 (250 mg, 0.66 mmol) and hydrazine (0.20 mL, 6.61 mmol). The crude material obtained was used in the next step without further purification (110 mg, 67% yield).

N-Ethyl-4-(3-(2-methoxyphenyl)pyrrolidin-1-yl)butan-1-amine (54):

The compound was prepared following the general procedure A starting from 53 (110 mg, 443 μmol), acetaldehyde (0.1 mL, 2.21 mmol), and NaBH₄ (33.5 mg, 886 μmol). The desired product was isolated by flash chromatography eluting with 20% MeOH containing 10% NH₄OH/DCM to obtain 54 (50 mg, 41% yield) as a gummy solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.26 (d, J = 5.8 Hz, 1H), 7.16 (t, J = 7.8 Hz, 1H), 6.90 (t, J = 7.5 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 3.79 (s, 3H), 3.71–3.65 (m, 1H), 3.03 (t, J = 8.0 Hz, 1H), 2.86–2.81 (m, 1H), 2.70–2.45 (m, 8H), 2.28–2.19 (m, 1H), 1.91–1.82 (m, 1H), 1.61–1.56 (m, 4H), 1.13 (t, J = 7.2 Hz, 3H). GC/MS RT 10.824 min, 276.20 m/z.

N-(2-(2-(4-Ethoxybenzyl)-5-nitro-1H-benzo[d]imidazol-1-yl)ethyl)-N-ethyl-4-(3-(2-methoxyphenyl)pyrrolidin-1-yl)butan-1-amine (55):

The compound was prepared following the same procedure described for 21, starting from 17 (61 mg, 0.18 mmol), cat. AcOH (3–5 drops), 54 (50 mg, 0.18 mmol), and STAB (46 mg, 0.22 mmol). The desired product was isolated by flash chromatography eluting with 8% MeOH containing 10% NH₄OH/DCM to obtain 55 (40 mg, 37% yield) as a light yellow solid. ¹H-NMR (400 MHz, CDCl₃) δ 8.64 (d, J = 2.1 Hz, 1H), 8.17 (dd, J = 8.9, 2.1 Hz, 1H), 7.33–7.27 (m, 2H), 7.17–7.11 (m, 3H), 6.91–6.81 (m, 4H), 4.32 (s, 2H), 4.06 (t, J = 6.7 Hz, 2H), 3.97 (q, J = 7.0 Hz, 2H), 3.72 (s, 3H), 3.70 (p, J = 7.9 Hz, 1H), 2.96 (t, J = 8.4 Hz, 1H), 2.76 (q, J = 7.5 Hz, 1H), 2.66–2.30 (m, 10H), 2.23 (dq, J = 6.5, 7.6, 13.3 Hz, 1H), 1.84 (dq, J = 7.7, 13.6 Hz, 1H), 1.45–1.24 (m, 7H), 0.88 (t, J = 7.1 Hz, 3H). ¹³C-NMR (101 MHz, CDCl₃) δ 158.3, 157.8, 157.2, 143.5, 142.0, 139.8, 129.6 (2C), 127.4, 127.3, 127.1, 120.6, 118.3, 116.3, 115.1, 110.5, 109.4, 63.6, 60.7, 56.5, 55.4, 54.7, 54.1, 52.8, 48.2, 43.9, 36.5, 33.9, 31.6, 26.8, 25.3, 14.9, 12.0. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 μm; gradient 10%-80% ACN in H₂O + 0.1% DEA; 60 min run; injection 20 μL (1 mg/mL); temperature 40°C; ^tR 37.758 min, purity >99%. HRMS-MS/MS [C₃₅H₄₅N₅O₄+H]⁺ calculated 600.35443, found 600.35352.

2-(4-(3-(Thiophen-2-yl) pyrrolidin-1-yl)butyl)isoindoline-1,3-dione (56):

The compound was prepared following the same procedure described for 28 starting with commercially available 49 (109 mg, 575 μmol), 26 (178 mg, 632 μmol), and K₂CO₃ (794 mg, 5.75 mmol). The desired product was isolated by flash chromatography eluting with 50% EtOAc/hexanes to obtain 56 (124 mg, 61% yield) as an off-white solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.84–7.80 (m, 2H), 7.72–7.68 (m, 2H), 7.10 (d, J = 5.0 Hz, 1H), 6.89 (t, J = 4.0 Hz, 1H), 6.80 (d, J = 3.1 Hz, 1H), 3.71 (t, J = 7.2 Hz, 2H), 3.60 (dt, J = 7.4, 15.1 Hz, 1H), 3.09–3.04 (m, 1H), 2.83–2.77 (m, 1H), 2.59–2.43 (m, 4H), 2.37–2.28 (m, 1H), 1.92–1.84 (m, 1H), 1.74 (p, J = 7.3 Hz, 2H), 1.58–1.51 (m, 2H).

4-(3-(Thiophen-2-yl)pyrrolidin-1-yl)butan-1-amine (57):

The compound was prepared following the same procedure described for 29 starting from 56 (124 mg, 350 μmol) and hydrazine (112 mg, 3.50 mmol). The crude material obtained was used in the next step without further purification (70 mg, 89% yield).

N-Ethyl-4-(3-(thiophen-2-yl)pyrrolidin-1-yl)butan-1-amine (58):

The compound was prepared following the general procedure A starting from 57 (70.0 mg, 312 μmol), acetaldehyde (0.10 mL, 1.56 mmol), and NaBH₄ (23.0 mg, 624 μmol). The crude material was used in the next step without purification.

N-(2-(2-(4-Ethoxybenzyl)-5-nitro-1H-benzo[d]imidazol-1-yl)ethyl)-N-ethyl-4-(3-(thiophen-2-yl)pyrrolidin-1-yl)butan-1-amine (59):

The compound was prepared following the same procedure described for 21 starting from 17 (94 mg, 0.28 mmol), cat. AcOH acid (3–5 drops), 58 (70 mg, 0.28 mmol), and STAB (71 mg, 0.33 mmol). The desired product was isolated by flash chromatography eluting with 8% MeOH containing 10% NH₄OH/DCM to obtain 59 (24 mg, 15.0% yield) as a gum. ¹H-NMR (400 MHz, CD₃OD) δ 8.46 (d, J = 2.1 Hz, 1H), 8.16 (dd, J = 8.9, 2.3 Hz, 1H), 7.60 (d, J = 8.9 Hz, 1H), 7.16–7.13 (m, 3H), 6.88–6.83 (m, 4H), 4.34 (s, 2H), 4.23 (t, J = 6.3 Hz, 2H), 3.95 (q, J = 6.9 Hz, 2H), 3.60 (q, J = 8.2 Hz, 1H), 3.07–3.03 (m, 1H), 2.80–2.74 (m, 1H), 2.59–2.24 (m, 11H), 1.91–1.83 (m, 1H), 1.33–1.23 (m, 5H), 1.15–1.08 (m, 2H), 0.89 (t, J = 7.1 Hz, 3H). ¹³C-NMR (101 MHz, CD₃OD) δ 158.7, 158.4, 147.4, 143.6, 140.9, 139.6, 129.4 (2C), 127.2, 126.4, 123.3, 122.9, 117.9, 114.7, 114.3, 110.6, 63.2, 61.8, 55.9, 53.5, 53.3, 52.4, 43.0, 38.3, 33.2, 32.8, 25.6, 24.8, 13.8, 10.8. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 μm; gradient 10%-80% ACN in H₂O + 0.1% DEA; 60 min run; injection 20 μL (1 mg/mL); temperature 40°C; ^tR 39.284 min, purity >99%. HRMS-MS/MS [C₃₅H₄₅N₅O₄ + H]⁺ calculated 576.30029, found 576.29984.

2-(4-(3-(Furan-2-yl)pyrrolidin-1-yl)butyl)isoindoline-1,3-dione (60):

The compound was prepared following the same procedure described for 28 starting with commercially available 50 (100 mg, 729 μmol), 26 (226 mg, 802 μmol), and K₂CO₃ (504 mg, 3.64 mmol). The desired product was isolated by flash chromatography eluting with 50% EtOAc/hexanes to obtain 60 (195 mg, 79% yield) as an off-white solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.83–7.81 (m, 2H), 7.70–7.68 (m, 2H), 7.28 (m, 1H), 6.25 (dd, J = 3.1, 1.9 Hz, 1H), 6.00 (d, J = 3.2 Hz, 1H), 3.70 (t, J = 7.1 Hz, 2H), 3.40 (dt, J = 17.3, 7.5 Hz, 1H), 3.00 (t, J = 8.5 Hz, 1H), 2.80 (q, J = 8.2 Hz, 1H), 2.58–2.42 (m, 4H), 2.24–2.15 (m, 1H), 1.96–1.88 (m, 1H), 1.76–1.68 (m, 2H), 1.59–1.51 (m, 2H).

4-(3-(Furan-2-yl)pyrrolidin-1-yl)butan-1-amine (61):

The compound was prepared following the same procedure described for 29 starting from 60 (195 mg, 576 μmol) and hydrazine (0.20 mL, 5.76 mmol). The crude material obtained was used in the next step without further purification (99 mg, 83% yield). ¹H-NMR (400 MHz, CDCl₃) δ 7.29 (m, 1H), 6.26 (d, J = 3.2 Hz, 1H), 6.01 (d, J = 3.2 Hz, 1H), 3.42 (p, J = 7.7 Hz, 1H), 3.02 (t, J = 8.5 Hz, 1H), 2.83–2.78 (m, 1H), 2.70 (t, J = 6.7 Hz, 2H), 2.53–2.39 (m, 4H), 2.26–2.16 (m, 1H), 1.97–1.89 (m, 1H), 1.58–1.43 (m, 6H).

N-Ethyl-4-(3-(furan-2-yl)pyrrolidin-1-yl)butan-1-amine (62):

The compound was prepared following the general procedure A starting from 61 (400 mg, 1.92 mmol), acetaldehyde (0.50 mL, 9.6 mmol), and NaBH₄ (145 mg, 3.84 mmol). The crude product was used in the next step without purification.

N-(2-(2-(4-Ethoxybenzyl)-5-nitro-1H-benzo[d]imidazol-1-yl)ethyl)-N-ethyl-4-(3-(furan-2-yl)pyrrolidin-1-yl)butan-1-amine (63):

The compound was prepared following the same procedure described for 21 starting from 17 (287 mg, 846 μmol), cat. AcOH acid (3–5 drops), 62 (200 mg, 846 μmol), and STAB (215 mg, 1.02 mmol). The desired product was isolated by flash chromatography on silica gel eluting with 7% MeOH containing 10% NH₄OH/DCM to obtain 63 (95 mg, 20% yield) as a pale-yellow solid. ¹H-NMR (400 MHz, CD₃OD) δ 8.44 (d, J = 2.1 Hz, 1H), 8.14 (dd, J = 8.9, 2.1 Hz, 1H), 7.58 (d, J = 8.9 Hz, 1H), 7.32 (d, J = 4.0 Hz, 1H), 7.14 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 6.25 (dd, J = 3.1, 1.9 Hz, 1H), 6.04 (d, J = 3.2 Hz, 1H), 4.33 (s, 2H), 4.22 (t, J = 6.4 Hz, 2H), 3.95 (q, J = 7.0 Hz, 2H), 3.38–3.32 (m, 1H), 2.92–2.88 (m, 1H), 2.72–2.66 (m, 1H), 2.53–2.38 (m, 6H), 2.31–2.10 (m, 5H), 1.91–1.87 (m, 1H), 1.31 (t, J =7.0 Hz, 3H), 1.21 (m, 2H), 1.10 (p, J = 7.2 Hz, 2H), 0.88 (t, J = 7.1 Hz, 3H). ¹³C-NMR (101 MHz, CD₃OD) δ 158.7, 158.4, 157.0, 143.6, 141.2, 140.9, 139.6, 129.4 (2C), 127.2, 117.9, 114.7, 114.3, 110.6, 109.8, 104.0, 63.2, 58.6, 56.0, 53.5, 53.3, 52.4, 43.0, 36.2, 32.8, 29.4, 25.8, 24.9, 13.8, 10.8. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 μm; gradient 10%-80% ACN in H₂O + 0.1% DEA; 60 min run; injection 20 μL (1 mg/mL); temperature 40°C; ^tR 38.105 min, purity >99%. HRMS-MS/MS [C₃₂H₄₁N₅O₄+H]⁺ calculated 560.32245, found 560.32313.

2-(4-(3-(1H-Pyrrol-1-yl)pyrrolidin-1-yl)butyl)isoindoline-1,3-dione (64):

The compound was prepared following the same procedure described for 28 starting with commercially available 51 (541 mg, 3.13 mmol), 26 (1.06 g, 3.76 mmol), and K₂CO₃ (3.03 g, 21.9 mmol). The desired product was isolated by flash chromatography eluting with 50% EtOAc/hexanes to obtain 64 (634 mg, 60% yield) as an off-white solid. ¹H-NMR (400 MHz,CDCl₃) δ 7.85–7.81 (m, 2H), 7.72–7.67 (m, 2H), 6.77 (t, J = 2.1 Hz, 2H), 6.11 (t, J = 2.1 Hz, 2H), 4.59–4.52 (m, 1H), 3.70 (t, J = 7.2 Hz, 2H), 2.86–2.72 (m, 3H), 2.55–2.34 (m, 4H), 2.01–1.95 (m, 1H), 1.74 (p, J = 7.5, 7.4 Hz, 2H), 1.54 (p, J = 7.4, 7.3 Hz, 2H).

4-(3-(1H-Pyrrol-1-yl)pyrrolidin-1-yl)butan-1-amine (65):

The compound was prepared following the same procedure described for 29 starting from 64 (634 mg, 1.88 mmol) and hydrazine (0.30 mL, 9.39 mmol). The crude material obtained was used in the next step without further purification (347 mg, 89% yield).

4-(3-(1H-Pyrrol-1-yl)pyrrolidin-1-yl)-N-ethylbutan-1-amine (66):

The compound was prepared following the general procedure A starting from 65 (324 mg, 1.56 mmol), acetaldehyde (0.50mL, 8.0 mmol), and NaBH₄ (118 mg, 3.13 mmol). The crude product was used in the next step without purification.

4-(3-(1H-Pyrrol-1-yl)pyrrolidin-1-yl)-N-(2-(2-(4-ethoxybenzyl)-5-nitro-1H-benzo[d]imidazol-1-yl)ethyl)-N-ethylbutan-1-amine (67):

The compound was prepared following the same procedure described for 21 starting 17 (130 mg, 383 μmol), cat. AcOH acid (3–5 drops), 66 (90.0 mg, 383 μmol), and STAB (97.0 mg, 460 μmol). The desired product was isolated by flash chromatography eluting with 7% MeOH containing 10% NH₄OH/DCM to obtain 67 (24 mg, 11% yield) as a gummy solid. ¹H-NMR (400 MHz,CD₃OD) δ 8.48 (d, J = 2.3 Hz, 1H), 8.17 (dd, J = 8.9, 2.1 Hz, 1H), 7.61 (d, J = 8.9 Hz, 1H), 7.14 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 6.75 (t, J = 2.1 Hz, 2H), 5.98 (t, J = 2.1 Hz, 2H), 4.61–4.57 (m, 1H), 4.35 (s, 2H), 4.24 (t, J = 6.4 Hz, 2H), 3.96 (q, J = 6.9 Hz, 2H), 2.81–2.69 (m, 2H), 2.61 (dd, J = 10.1, 5.4 Hz, 1H), 2.55–2.44 (m, 5H), 2.38–2.27 (m, 4H), 2.24–2.17 (m, 1H), 1.98–1.93 (m, 1H), 1.32 (t, J = 7.0 Hz, 3H), 1.26–1.19 (m, 2H), 1.16–1.08 (m, 2H), 0.90 (t, J = 7.1 Hz, 3H). ¹³C-NMR (101 MHz, CD₃OD) δ 158.7, 158.4, 143.6, 140.9, 139.6, 129.4 (2C), 127.2, 118.6, 117.9, 114.7, 114.3, 110.6, 107.6, 63.2, 60.7, 57.4, 55.7, 53.3, 53.2, 52.4, 43.0, 32.7, 32.5, 25.7, 24.8, 20.7, 13.8, 10.7. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 μm; gradient 10%-80% ACN in H₂O + 0.1% DEA; 60 min run; injection 20 μL (1 mg/mL); temperature 40°C; ^tR 41.905 min, purity >99%. HRMS-MS/MS [C₃₂H₄₂N₆O₃+H]⁺ calculated 559.33912, found 559.33854.

2-(2-(3-(2-Methoxyphenyl)pyrrolidin-1-yl)ethyl)isoindoline-1,3-dione (68):

The compound was prepared following the same procedure described for 28 starting with 48 (800 mg, 3.74 mmol), 27 (1.14 g, 4.49 mmol), and K₂CO₃ (5.2 g, 37.4 mmol). The desired product was isolated by flash chromatography eluting with 95% EtOAc/ hexanes to obtain 68 (550 mg, 42% yield) as a pale yellow solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.87–7.82 (m, 2H), 7.74–7.69 (m, 2H), 7.22 (d, J = 7.8 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 6.80 (d, J = 7.2 Hz, 2H), 3.85 (t, J = 6.8 Hz, 2H), 3.83 (s, 3H), 3.67 (p, J = 7.4, 7.3 Hz, 1H), 3.01 (t, J = 8.4 Hz, 1H), 2.87–2.72 (m, 4H), 2.59 (t, J = 8.1 Hz, 1H), 2.26–2.16 (m, 1H), 1.81 (td, J = 12.9, 7.5 Hz, 1H).

2-(3-(2-Methoxyphenyl)pyrrolidin-1-yl)ethan-1-amine (69):

The compound was prepared following the same procedure described for 29 starting from 68 (550 mg, 1.57 mmol) and hydrazine (503 mg, 493 μL, 15.7 mmol). The crude material obtained was used in the next step without further purification (293 mg, 85% yield).

N-Ethyl-2-(3-(2-methoxyphenyl)pyrrolidin-1-yl)ethan-1-amine (70):

The compound was prepared following the general procedure A starting from 69 (293 mg, 1.33 mmol), acetaldehyde (0.50 mL, 8.68 mmol), and NaBH₄ (101 mg, 2.66 mmol). The crude material was used in the next step without purification.

2-(2-(4-Ethoxybenzyl)-5-nitro-1H-benzo[d]imidazol-1-yl)-N-ethyl-N-(2-(3-(2-methoxyphenyl)pyrrolidin-1-yl)ethyl)ethan-1-amine (71):

The compound was prepared following the same procedure described for 21 starting from 17 (195 mg, 576 μmol), cat. AcOH acid (3–5 drops), 70 (143 mg, 576 μmol), and STAB (146 mg, 691 μmol). The desired product was isolated by flash chromatography eluting with 5% MeOH containing 10% NH₄OH/DCM to obtain 71 (130 mg, 40% yield) as a gummy solid. ¹H-NMR (400 MHz,CD₃OD) δ 8.46 (d, J = 2.1 Hz, 1H), 8.17 (dd, J = 8.9, 2.1 Hz, 1H), 7.65 (d, J = 8.9 Hz, 1H), 7.17–7.11 (m, 4H), 6.86 (dd, J = 16.2, 8.6 Hz, 4H), 4.36 (s, 2H), 4.26 (t, J =6.2 Hz, 2H), 3.94 (q, J = 7.0 Hz, 2H), 3.77 (s, 3H), 3.59–3.50 (m, 1H), 2.83 (t, J =8.7 Hz, 1H), 2.70 (q, J = 7.7Hz, 1H), 2.60 (t, J =6.2 Hz, 2H), 2.5–2.40 (m, 5H), 2.26–2.05 (m, 4H), 1.81–1.73 (m, 1H), 1.32–1.18 (m, 3H), 1.03–0.83 (m, 3H). ¹³C-NMR (101 MHz, CD₃OD) δ 158.7, 158.4, 157.3, 143.6, 140.9, 139.6, 139.0, 130.5, 129.5 (2C), 127.4, 127.3, 127.1, 126.9, 120.4, 117.9, 114.7, 114.3, 110.7, 110.3, 63.2, 59.8, 54.5, 54.3, 53.9, 52.7, 51.6, 43.1, 36.9, 32.7, 30.2, 13.8, 10.8. Analytical HPLC: Phenomenex Gemini C18 4.6 × 50 mm, 3 μm; gradient 10%-80% ACN in H₂O + 0.1% TFA; 60 min run; injection 20 μL (1 mg/mL); temperature 40°C; ^tR 22.634 min, purity >99%. HRMS-MS/MS [C₃₃H₄₁N₅O₄+H]⁺calculated 572.32313, found 572.32253. The free base was converted into the corresponding HCl salt, which was obtained as a pale yellow solid (m.p. 181–183°C). Elemental analysis (C₃₃H₄₁N₅O₄•3HCl• 0.25 H₂O) calculated 57.81, H 6.54, N 10.22, found C 57.83, H 6.59, N 10.02.

Methods for Radioligand Binding Assays

hD₂R and hD₃R.

Radioligand binding assays were conducted similarly as previously described^{33, 60}. HEK293 cells stably expressing human D_2LR or D₃R were grown in a 50:50 mix of DMEM and Ham’s F12 culture media, supplemented with 20 mM HEPES, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1X antibiotic/antimycotic, 10% heat-inactivated fetal bovine serum, and 200 μg/mL hygromycin (Life Technologies, Grand Island, NY) and kept in an incubator at 37°C and 5% CO₂. Upon reaching 80−90% confluence, cells were harvested using premixed Earle’s balanced salt solution with 5 mM EDTA (Life Technologies) and centrifuged at 3000 rpm for 10 min at 21°C. The supernatant was removed, and the pellet was resuspended in 10 mL hypotonic lysis buffer (5 mM MgCl₂, 5 mM Tris, pH 7.4 at 4°C) and centrifuged at 14 500 rpm (~25000g) for 30 min at 4°C. The pellet was then resuspended in binding buffer. Bradford protein assay (Bio-Rad, Hercules, CA) was used to determine the protein concentration. For [³H]-N-methylspiperone binding studies membranes were diluted to 500 μg/mL, in fresh EBSS binding buffer made from 8.7 g/L Earle’s Balanced Salts without phenol red (US Biological, Salem, MA), 2.2 g/L sodium bicarbonate, pH to 7.4, and stored in a −80 °C freezer for later use. On the test day, each test compound was diluted into half-log serial dilutions using the 30% dimethyl sulfoxide (DMSO) vehicle. When it was necessary to assist solubilization of the drugs at the highest tested final concentration of 100 μM or 10 μM, 0.1% or 0.01% AcOH (final concentration v/v) was added alongside the vehicle, respectively. Membranes were diluted in fresh binding buffer. Radioligand competition experiments were conducted in 96-well plates containing 300 μL fresh binding buffer, 50 μL of the diluted test compound, 100 μL of membranes (for [³H]-N-methylspiperone assays: 10−20 μg/well total protein for both hD_2LR and hD₃R), and 50 μL of radioligand diluted in binding buffer ([³H]-N-methylspiperone: 0.4 nM final concentration for all the hD₂-like receptor subtypes; Novandi Chemistry AB). Aliquots of radioligands solution were also quantified accurately in each experiment replicate, to determine how much radioactivity was added, taking in account the experimentally determined counter efficiency. Nonspecific binding was determined using 10 μM (+)-butaclamol (Sigma-Aldrich, St. Louis, MO), and total binding was determined with the 30% DMSO vehicle (3% final concentration). All compound dilutions were tested in triplicate, and the reaction incubated for 60 min at RT. The reaction was terminated by filtration through PerkinElmer Uni-Filter-96 GF/C, presoaked for the incubation time in 0.5% polyethylenimine, using a Brandel 96-Well Plates Harvester Manifold (Brandel Instruments, Gaithersburg, MD). The filters were washed thrice with 3 mL (3 times ~1 mL/well) of ice-cold binding buffer. PerkinElmer MicroScint 20 Scintillation Cocktail (65 μL) was added to each well, and filters were counted using a PerkinElmer MicroBeta Microplate Counter. IC₅₀ values for each compound were determined from dose−response curves, and K_i values were calculated using the Cheng−Prusoff equation. K_d values for the radioligands were determined via separate homologous competitive binding experiments. When a complete inhibition could not be achieved at the highest tested concentrations, K_i values have been extrapolated by constraining the bottom of the dose−response curves (=0% residual specific binding) in the nonlinear regression analysis. These analyses were performed using GraphPad Prism version 9 for Macintosh (GraphPad Software, San Diego, CA). All results were rounded to the third significant figure. K_i values were determined from at least three independent experiments and are reported as the mean ± standard error of the mean (S.E.M.).

hMOR.

Radioligand binding experiments were conducted, and the results analyzed, as described above, and similarly as previously reported³³. HEK293 cells stably expressing hMOR were grown in a DMEM medium, supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin (or antibiotic/antimycotic) and hygromycin B (50 μg/mL). Upon reaching confluence the cells were harvested and the membranes prepared as detailed before. The binding buffer was made of 50 mM Tris and 5 mM MgCl₂ at pH 7.4. The experiments were performed in presence of [³H]-DAMGO (final concentration 3 nM; Novandi Chemistry AB) and ~30 μg/well of membranes (final concentration). The reactions were incubated for 60 min at RT and terminated by rapid filtration through Perkin Elmer Uni-Filter-96 GF/C, presoaked for 60 min in 0.5% polyethylenimine. The non-specific binding was determined using 10 μM naloxone. The radioligand K_d was measured via radioligand saturation experiments.

BRET assays

BRET assays were performed as described previously^{32, 33, 71}. All reagents were purchased from Sigma Aldrich-Merck, unless otherwise stated. Briefly, human embryonic kidney 293 T (HEK 293T) cells, were cultured at 37 °C, 5% CO₂ in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). Cells were seeded in 10 cm Petri dishes (3 × 10⁶ cells per dish) and allowed to grow overnight in full media at 37 °C, 5% CO₂. Cells were transiently transfected the next day, in media supplemented with antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, Gibco) using a 1:6 total DNA to PEI (PolySciences Inc) ratio. BRET constructs were as follows: 4 μg of Nb33-Venus and 1 μg of mMOR-Rluc8 for Nb33 recruitment, 4 μg of arrestin-3-Venus, 2 μg of WT-GRK2 and 1 μg of mMOR-Rluc8 for arrestin-3 recruitment and 2 μg of WT-Gα (i2 or oA), 1 μg of Gβ1-Venus(156–239), 1 μg of Gγ2-Venus(1–155), 1 μg of masGRK3ct-Rluc8 and 1 μg of receptor (SNAP-mMOR or hD₃R) for GPA assays⁷². The following day, cells were plated in Greiner poly-D-lysine-coated, white bottom 96-well plates (SLS) in full media. On the day of the assay (48h post-transfection), cells were washed once with D-PBS (Lonza, SLS) and incubated in D-PBS for 30 min at 37 °C. The Rluc substrate coelenterazine h (NanoLight) was added to each well (final concentration of 5 μM) and ligands (final concentration from 10 μM to 0.01 nM in D-PBS) were added to the wells before reading the plate at 37 °C in a PHERAstar FSX microplate reader (Venus and Rluc emission signals at 535 and 475 nm, respectively, BMG Labtech) every minute for 10 min, with an additional 30 min read at the end of the assay. For the D₃R antagonist-mode of the GPA assay, a final concentration of 3 nM of quinpirole was added to the wells just after compound addition, to induce a 50% response that can then be inhibited by D₃R antagonists or weak partial agonists. The ratio between Venus fluorescence and Rluc luminescence was used to quantify the BRET signal in each well. Data were normalized to maximal and minimal response of DAMGO or quinpirole for MOR or D₃R, respectively. All data points represent the mean of three independent experiments performed in duplicate. Error bars represent the standard error of the mean (S.E.M.) and data points were fitted using the built-in log(agonist) vs. response (three parameters) model in Prism 9.0 (GraphPad software Inc., San Diego, CA). For the antagonist-mode assays, data points were fitted using the built-in log(antagonist) vs. response (three parameters) model.

Mouse and Rat Liver Microsome Metabolic Stability

Phase I metabolic stability was conducted as previously described with minor modifications^{73, 74}. Briefly, reactions were carried out with 100 mM potassium phosphate buffer, pH 7.4, in the presence of NADPH regenerating system (1.3 mM NADPH, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl₂, 0.4 U/mL glucose-6-phosphate dehydrogenase, 50 μM sodium citrate). Reactions in triplicate were initiated by addition of the liver microsomes to the incubation mixture (compound final concentration was 10 μM; 0.5 mg/mL microsomes). Compound disappearance over time was monitored via LC/MS.

Chromatographic analysis was performed on a Dionex ultra high-performance LC system coupled with Q Exactive Focus orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham MA). Separation was achieved using Agilent Eclipse Plus column (100 × 2.1mm i.d; maintained at 35°C) packed with a 1.8 μm C18 stationary phase. The mobile phase used was composed of 0.1% Formic Acid in Acetonitrile and 0.1% Formic Acid in H₂O with gradient elution, starting with 2.5% organic phase (from 0 to 0.25 min) linearly increasing to 99% (from 0.25 to 1.25 min), and re-equilibrating to 2.5% by 4 min. The total run time for each analyte was 5 min. Pumps were operated at a flow rate of 0.4 mL/min. The mass spectrometer controlled by Xcalibur software 4.0.27.13 (Thermo Scientific) was operated with a HESI ion source in positive ionization mode for all compounds. Compounds were identified in the full-scan mode (from m/z 75 to 1125) and percent remaining was calculated by comparing t = 0 sample with t = 30 and 60 min samples.

Behavioral models in mice

Locomotor, antinociceptive and hypothermic effects

Animals and housing

Male and female C57BL/6J mice (2 – 6 months of age, 48 total) were provided by the NIDA IRP breeding program (24 mice) or purchased from The Jackson Laboratory (Bar Harbor, ME, USA; 24 mice). All mice were group-housed prior to the study with ad libitum food and H₂O, and maintained on a 12-h light-dark cycle (lights on at 7 am) throughout the study in facilities at the NIDA IRP in Baltimore, MD, USA. The facility is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, and all procedures were approved by the NIDA IRP Animal Care and Use Committee (23-OSD-35).

Transponder implants and assessment of opioid-like effects in mice

At least one week before behavioral testing, subcutaneous temperature transponders (model TP500, Avidity Science LLC, Waterford, WI, USA) were implanted on the backs of mice under brief isoflurane anesthesia/immobilization, as previously described^{75, 76}. These transponders enable non-invasive body temperature measurement using a handheld reader. Mice were single-housed post-implants for the entirety of the behavioral testing period.

On test days, mice were transported in home cages to the testing room and acclimated for 1 h. The total session lasted 90 min, with the first 15 min allotted for acclimation to test arenas. Baseline measurements of weight, body temperature, hot plate latency, and locomotor activity were taken 15 min prior to drug administration. At 0 min, baseline body temperature and hot plate latency were remeasured before administering s.c. injections of test drugs. Mice were then monitored for 75 min, with distance traveled, body temperature, and hot plate latency recorded every 15 min. Mice were tested every 7 days for up to 3 weeks, with random dose/condition assignments. Experiments were conducted during the light phase, between 9 am and 5 pm local time.

Hot plate latency was measured as previously described^{47, 77}. Mice were placed on a 52°C hot plate (IITC Life Sciences, Woodland Hills, CA, USA), and reactions such as paw licking, shaking, flicking, repeated genitalia licking, or jumping led to their immediate removal, with a 45 sec cutoff. Locomotor activity was measured using a TruScan locomotor chamber (Coulbourn Instruments, PA, USA) and continuously monitored. Body temperature changes were tracked with a handheld reader (Bio Medic Data Systems model # DAS-8027-IUS) interfacing with the implanted transponder. For NLX reversal studies, the general procedure was identical to dose-response testing except that mice received s.c. injection of vehicle or NLX (0.3 mg/kg) 15 min after administration of 31, 35, and 43 (100 mg/kg), or fentanyl (0.3 mg/kg).

Body temperature data were transformed to change from baseline (temperature Δ) using the time zero temperature value. Mean temperature Δ across all post-injection timepoints was used to calculate mean effects and plot dose-response curves. Hot plate latencies were normalized to percent maximum possible effect (%MPE) at each timepoint, calculated using the formula: (experimental latency – baseline latency) / (maximum possible latency – baseline latency) × 100, with a 45 sec maximum. Mean %MPE across the first 60 min post-injection was used for dose-response curves. Locomotor activity was measured as distance traveled (cm) continuously and summed in 15 min bins.

Drug potencies (ED₅₀) in mouse studies were determined using the ascending limb of dose-response curves using four-parameter variable slope fits for locomotor activity. The full dose-response curves were used for fitting hot plate latency and temperature Δ data. Data for fentanyl (locomotor) and 31 (temperature) were best visualized with bell-shaped nonlinear regression fits. Mean effects on motor activity and temperature change in mouse studies were compared to vehicle controls using Welch’s ANOVA with Dunnett's T3 post hoc test (p < 0.05), while hot plate latencies were compared using Kruskal-Wallis with Dunn’s post hoc test (p < 0.05). All graphs and statistical analyses were performed using GraphPad Prism 10 (La Jolla, CA, USA).

Respiratory Depression

Two groups of mice (n=8 per group) were used to evaluate the effects of morphine and 31 on respiration, respectively. Respiratory activity was assessed in conscious, freely moving mice using whole-body plethysmography⁷⁸. Mice were placed individually into a sealed plethysmography chamber and allowed to acclimate until a stable breathing pattern was observed. Chamber pressure fluctuations generated by inspiratory and expiratory airflow were continuously recorded using the manufacturer’s transducer and acquisition software. Calibration was performed according to the instrument’s standard procedure to permit conversion of pressure signals into respiratory volumes when appropriate. All recordings were conducted under consistent environmental conditions, and animals were monitored visually to minimize the inclusion of movement-related artifacts.

After stable respiration in testing chamber was achieved, each mouse randomly received three doses of morphine or 31, including vehicle. On the test day, mice were habituated to recording chambers for 30 min. Then mice were briefly removed from test chambers and given a single i.p. injection of drug or vehicle. Mice were then immediately returned to the chambers for 3 h to obtain post-dose respiratory parameters. The inter-injection intervals were 2–5 days. Breath-by-breath respiratory parameters were derived automatically from the pressure waveform and verified through inspection of raw traces. Primary measures included respiratory rate or frequency (breaths/min), inspiratory duration (ms), tidal volume (mL/breath), and minute ventilation (tidal volume × respiratory rate, mL/min). Additional variables – such as expiratory time (ms), peak inspiratory flow (mL/sec), peak expiratory flow (mL/sec), end inspiratory pause (time between inspiration and expiration; ms), and end expiratory pause (time between respiratory cycles; ms), and enhanced pause (Penh) total expiratory time (ms) – were recorded and extracted when needed.

Stable epochs free of grooming or exploratory movements were selected for analysis, and values were averaged over defined time windows for each mouse. All respiratory measures were analyzed using standardized settings to ensure consistency across animals and experimental groups.

Highlights:

• Dual-target mu opioid-dopamine D₃ receptor ligands were designed as novel analgesics

• Compound 31 showed balanced MOR and D₃R binding affinities in the nM range

• Compound 31 produced maximal antinociceptive effects in the mouse hot plate assay

• Compound 31 had reduced hyperlocomotion, which may indicate lower abuse liability

ABBREVIATIONS

ACN

acetonitrile

AcOH

acetic acid

BRET

bioluminescence resonance energy transfer

βarr

β-arrestin

BBB

blood brain barrier

CNS-MPO

central nervous system multiparameter optimization

CYP

cytochrome P450

DA

dopamine

DCE

Dichloroethane

DCM

dichloromethane

DIPEA

N,N-diisopropylethylamine

DA

Dopamine

D2-like R

dopamine D2-like receptors

DIBAL-H

diisobutylaluminium hydride

DMA

dichloromethane, methanol, and ammonium hydroxide

DMP

Dess-Martin periodinane

ESI

electrospray ionization

GPA

G-protein activation

HEK293 cells

human embryonic kidney 293 cells

HPLC

high performance liquid chromatography

HRMS

high-resolution mass spectrometry

MOR

μ-opioid receptor

OIH

opioid induced hyperalgesia

OUD

opioid use disorders

STAB

sodium triacetoxyborohydride

TFA

trifluoroacetic acid