Novel Dual-Target Mu Opioid (MOR) and Dopamine D₃ Receptors (D₃R) Ligands as Potential Non-Addictive Pharmacotherapeutics for Pain Management

Abstract

The need for safer pain-management therapies with decreased abuse liability inspired a novel drug design that retains μ-opioid receptor (MOR)-mediated analgesia, while minimizing addictive liability. We recently demonstrated that targeting dopamine D₃ receptors (D₃R) with highly selective antagonists/partial agonists can reduce opioid self-administration and reinstatement to drug seeking in rodent models without diminishing antinociceptive effects. The identification of D₃R as a target for the treatment of opioid use disorders, prompted the idea of generating a class of ligands presenting bitopic or bivalent structures, allowing the dual-target binding of MOR and D₃R. Structure-activity relationship studies using computationally aided drug-design, and in vitro binding assays led to the identification of potent dual-target leads (23, 28, and 40), based on different structural templates and scaffolds, with moderate (sub-micromolar) to high (low nanomolar/sub-nanomolar) binding affinities. BRET-based functional studies revealed MOR agonist-D₃R antagonist/partial agonist efficacies that suggest potential for maintaining analgesia with reduced opioid-abuse liability.

Graphical Abstract

INTRODUCTION

Over the last decade, the United States has faced a devastating opioid epidemic with an estimate of over 130 people dying from opioid overdose every day.¹ The misuse and often consequent addiction to opioids (e.g., prescription pain relievers, and synthetic opioids in general) is a serious national health, social, and economic emergency. Coupled with the novel coronavirus pandemic, the mortality rate involving opioid overdose is rising, and the need for new therapeutic strategies is more urgent than ever^{2, 3}. According to recent reports⁴, >20% of patients being treated for chronic pain will misuse their opioid prescriptions⁵, and 8–12% develop opioid use disorders (OUD)⁵. Ultimately, an estimated 5% of patients is reported to transition from misuse of prescription opioids to heroin^6–8, or other synthetic opioids.

The National Institutes of Health (NIH), launched the Helping to End Addiction Long-Term^SM (HEAL) initiative to promote and support innovative research addressing this national health emergency. Moreover, the National Institute on Drug Abuse (NIDA) recently proposed a list of the “ten most wanted” medication development strategies to tackle the opioid epidemic/crisis⁹. Among them, dopamine D₃ receptor (D₃R) antagonists and partial agonists are a “new” proposed class of ligands as therapeutics to attenuate opioid self-administration.

In the past, D₃R selective antagonists, such as GSK598809, have been investigated as potential treatments for psychostimulant use disorder (e.g., cocaine)¹⁰, however, the potentiation of hypertensive effects observed in dogs produced by cocaine in the presence of this selective D₃R antagonist prevented further development¹¹ and suggested this may be a class effect. We recently demonstrated that our novel D₃R antagonists and partial agonists look promising for the treatment of OUD.^12–14 Highly selective antagonists, such as VK4–116 (1) and VK4–40 (2) (Figure 1), attenuate oxycodone self-administration and reinstatement to drug seeking, without compromising oxycodone’s antinociceptive effects, in rodents. Importantly, these D₃R antagonists/partial agonists do not potentiate the cardiovascular effects induced by cocaine or oxycodone in rats.¹⁵ In combination, these studies support the development of D₃R antagonists/partial agonists to reduce the risk of opioid misuse and the consequent development of opioid use disorders.

Figure 1.

Drug design based on structural modification of canonical synthons inspired by agonists, antagonists, and partial agonists selectively targeting MOR and D₃R. Orange boxes denote bivalent templates, green boxes denote bitopic templates. Variables A, B, and C correspond to the aromatic substitutions seen in compounds 1, 2, 7, 10 or 11. General Ar group corresponds to the aromatic substituents in compounds 7, 9, 10, and 11. General R¹ and R² groups represent H, Me or O. Variables Y and Z represent C or N, variable X is H, OMe, or O.

Until now, the main target for pain-management therapies and drug development has been the opioid system, and in particular the μ-opioid receptors (MOR), belonging to the G-protein coupled receptors (GPCR) family. However, due to the abuse liability and development of tolerance associated with the most common MOR agonists used in pain therapy, including chronic pain, for which opioid agonists are largely ineffective in the long-term^16–18, research efforts have been directed toward identifying specific physiological responses associated with MOR agonists’ cellular activation pathways. Functionally biased agonists have been posited to reduce the side effect profile of classic opioid analgesics and augment their utility not only as analgesics, but also in the treatment of OUD^19–22.

Design, synthesis, and pharmacological characterization of signaling-pathway biased agonists targeting the μ-opioid receptors (MOR) allowed the identification of highly selective G-protein biased agonists, with limited activation of β-arrestin pathways, highlighting different physiological effects mediated by each independent pathway^{19, 23}. It has been suggested that the MOR G-protein pathway seems to be the predominant mediator of the analgesic effects of MOR agonists, meanwhile it has been posited that the simultaneous hindering of β-arrestin recruitment might reduce the respiratory depression and other side effects, such as constipation, associated with opioid-like drugs^24–28. However, this has been a topic of intensive investigation, and recently it has been reported that classical MOR agonists, such as morphine and fentanyl, induce dose-dependent respiratory depression and constipation in β-arrestin2 knock-out mice, similarly to what is observed in wild-type mice^29–31. Subsequent pharmacological evaluation has also shown that MOR G-protein biased agonists still have abuse potential^{29, 32–34}. It is still unclear whether the optimal outcome and pharmacotherapeutic potential of MOR agonists can be gained through selective activation of a particular downstream signaling pathway (functional selectivity) or whether an optimal level partial agonism at multiple pathways may instead provide a route for the development of safer opioids^{31, 35}. Independent of the signaling pathways and cellular mechanisms associated with respiratory depression and constipation, abuse liability remains as a serious concern that must be addressed with different drug design approaches.

The recognition of D₃R antagonism/partial agonism as an alternative and non-opioid approach for treatment of OUD, modulating the abuse potential of common prescription opioids (e.g., oxycodone)^{12, 13, 15}, combined with the well-established antinociceptive properties of MOR agonists, prompted the idea of generating a novel class of dual-target ligands directed to both MOR and D₃R (Figure 1). In theory, compounds that are both MOR agonists/partial agonists and D₃R antagonist/partial agonists would have analgesic activity without concomitant abuse liability. Our approach aimed at maintaining the analgesic effects of the classic MOR agonists, while reducing the rewarding properties and subsequent abuse liability as a result of D₃R antagonism. This drug design may lead to the development of safer dual-target drugs, bridging the most promising pharmacological effects of two classes of molecules/targets previously developed independently. Of note, our drug design was to develop new small molecules endowed with differing ranges of affinities for both targets, independently modulating their physiology/pharmacology rather than towards simultaneous binding of MOR and D₃R when in close proximity or in a heteromeric conformation. Nevertheless, if MOR-D₃R heteromers are demonstrated to exist and are physiologically relevant, these molecules may be interesting tools to probe their pharmacology.

Furthermore, several well-known MOR agonists, such as loperamide (3) (peripherally limited potent MOR agonist, FDA approved for anti-diarrhea treatment), and diphenoxylate (4), share similar structural motifs with the highly potent non-selective D₂R/D₃R antagonist haloperidol (5) (Figure 1)³⁶. Substituted phenyl-piperazine and/or phenyl-piperidine synthons, common to both classes of ligands, can exploit the structural similarities between MOR and D₃R proteins, thus achieving dual-target binding. Of note, a similar approach was taken previously toward more effective peripherally limited analgesics, based on loperamide, although implementing a bivalent or bitopic drug design or binding to D₃R was not described or presumably intended³⁷.

Moreover, methadone (6), (Figure 1), also showed low micromolar D₂R and D₃R affinity in our binding assays (Table 1), supporting the hypothesis that some of its structural fragments, could be used to target not only the MOR orthosteric binding site (OBS), but the D₂R and D₃R as well. Unlike 6, the synthetic opioid fentanyl, with its completely different structural features, showed an interesting moderate affinity for the dopamine D₄R subtype, but total lack of recognition for either D₂R or D₃R (Table 1). This template was not considered in our drug design for this reason and also because, recently, bivalent ligands using fentanyl have been reported to lack antinociceptive activity^{38, 39}.

Table 1.

Radioligand competition binding affinity data, for all the MOR diphenyl PP analogs based on 3, 4, and 6 at hMOR and hD₂-like receptor subtypes. All the affinity values are expressed as K_i ± SEM, derived from IC₅₀ values using the Cheng-Prusoff equation⁶³, and calculated as the mean of at least three independent experiments (n = number of independent experiments), each performed in triplicate.

ND = Not Determined.

^aNo inhibition of specific radioligand binding was observed at the highest tested concentration in one to three independent experiments, each performed in triplicate.

^bK_i value obtained from reference⁶¹.

Due to the limited availability of reported structure-activity relationships (SAR) for dual-target MOR-D₃R ligands^{38, 40–42}, we used a fragment-based drug design approach, supported by molecular docking, computer-aided drug design (CADD), and extensive in-vitro pharmacology to guide SAR, hit optimization and lead identification.

Herein, we refer to bivalent dual-target analogs when a MOR agonist primary pharmacophore (PP) is tethered with a D₃R antagonist PP; both can bind their respective OBS, eliciting their corresponding opioid agonist and dopaminergic antagonist effects. Consistent with our definition of bitopic ligands⁴³, we classify these new analogs as bitopic when they incorporate a MOR agonist PP, targeting its corresponding OBS that also has structural features suitable for D₃R OBS recognition, tethered to a D₃R secondary pharmacophore (SP), identifying the D₃R secondary binding pocket (SBP)⁴⁴. Of note, this D₃R SP may also elicit binding interactions within the MOR SBP. All new compounds represent carefully designed linkers, as tethering fragments, with specific SAR focusing on the linkers’ regiochemistry, stereochemistry and substituents⁴³.

All newly synthesized analogs were tested for their on-target and off-target affinities at MOR, D₂R, D₃R, and D₄R, in a combination of agonist and antagonist radioligand competition binding assays. Compounds selected as hits, for their promising dual-target and sub-micromolar affinities, were further evaluated in functional bioluminescence resonance energy transfer (BRET) studies, to assess their agonist and/or antagonist potencies for the target of interest, and possible functional selectivity (biased agonism) for specific signaling pathways.

RESULTS AND DISCUSSION

Chemistry

This novel class of compounds can be subdivided, as depicted in Figure 1 (A–C), in three general templates: A) the N,N-dimethyl-2,2-diphenylacetamide, 2,2-diphenylacetonitrile, and 1,1-diphenylbutan-2-one MOR PPs, derived from 3, 4, and 6 respectively (Figure 1), were tethered with suitably substituted PPs, inspired by selective and non-selective D₃R antagonists (e.g., 1, 2, PG648 (7), eticlopride (8) and SB269,652 (9); Figure 1), via short ethyl linker chain; B) the same MOR OBS-binding agonist PPs were linked with D₃R OBS antagonist PPs via a longer and more complex butyl linker, substituted with a 3-hydroxyl group, or the piperazine or pyrrolidine basic function in several regiochemical combinations; C) the MOR agonist PPs were replaced with the more rigid, stereochemically complex and hindered ethyl-2-(-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ylidene)acetate and 5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ol PP (10 and 11; Figure 1), with structural features reminiscent of previously published D₃R ligands^{45, 46} that we have found to have low micromolar affinity for the MOR as well. This new MOR PP presented key functional groups for extending linkers of multiple lengths, substitutions, rigidity and chirality, and tethering D₃R PPs (for bivalent dual-target compounds) or D₃R SPs (e.g., 2-indoleamide for bitopic dual-target compounds). When possible and appropriate, a complete resolution of the chiral centers at PP, SP or linkers was performed, and the stereochemical properties of the new analogs have been taken into consideration when generating detailed SAR.

The first series of compounds with the 2,2-diphenylbutanenitrile as the MOR PP, inspired by 4, were prepared as depicted in Scheme 1. Starting from the commercially available 4-bromo-2,2-diphenylbutanenitrile, simple N-alkylation under basic conditions yielded the first group of bivalent MOR-D₃R hybrids, where the D₃R PP was represented by 2-phenylethan-1-amine (12), 1-(2,3-dichlorophenyl)piperazine (13), 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine (14), and 4-amino-1-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-2-ol (15). This provided initial SAR deduction, by increasing the structural complexity of the D₃R PP, and simultaneously modifying the linker length and substitution. To investigate the optimal regiochemistry for introducing the linker and D₃R PP, compound 18 was prepared, where the butyl-4-(2,3-dichlorophenyl)piperazine synthon was introduced to replace the nitrile, while maintaining the MOR 4-(dimethylamino)-2,2-diphenylbutanamide moiety, as seen in both 3 and 6 (Figure 1). In detail, 4-bromo-2,2-diphenylbutanenitrile was first N-alkylated with dimethylamine hydrochloride (Me₂NH·HCl) under basic conditions, followed by hydrolysis of the nitrile in the presence of 48% HBr (aq solution) to yield the HBr salt of amino acid 17. Subsequent amidation mediated by EDC, HOBt, and 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine⁴⁷ yielded the desired product 18. Despite the importance of the α-methyl group of 6 being well reported in the literature⁴⁸, favoring the optimal binding pose within the MOR OBS, we decided to study SAR of structurally simplified analogs. Thus, via simple Grignard addition to the nitrile 16, we prepared the desmethyl-methadone 19.

Scheme 1.

a) appropriate primary or secondary amine, K₂CO₃, acetonitrile (ACN), 130 ° C, overnight, 8 – 53%; b) Me₂NH·HCl, K₂CO₃, ACN, 130 ° C, overnight, 79%; c) HBr 48% in H₂O, 100 ° C, overnight; d) N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole hydrate (HOBt), 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine⁴⁷, N,N-diisopropylethylamine (Hünig’s base, DIPEA), dichloromethane (DCM), 25 ° C, overnight, 6%; e) ethyl magnesium bromide (EtMgBr) 3M in diethyl ether (Et₂O), toluene, 0 -> 110 ° C, 3 hrs, 59%.

In Scheme 2, in order to investigate the structural requirement of the D₃R pharmacophore, and in particular the effect of replacing the basic butyl-4-(2,3-dichlorophenyl)piperazine, with the corresponding amide analog, HCTU mediated amide coupling of 5-((tert-butoxycarbonyl)amino)pentanoic acid, followed by removal of the Boc-protecting group, and consequent mono N-alkylation with 4-bromo-2,2-diphenylbutanenitrile afforded the desired product, 21. The longer 5 carbon atom linker, instead of the canonical butyl chain, was chosen because of the increased rigidity of the cyclic amide function and need for extending the tethered PP to an optimal distance.

Scheme 2.

a) 1-(2,3-dichlorophenyl)piperazine, 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), DCM, 25 ° C, 3 hrs, 59%; b) trifluoroacetic acid (TFA), DCM, 25 ° C, 24 hrs; c) 4-bromo-2,2-diphenylbutanenitrile, K₂CO₃, ACN, 82 ° C, overnight, 3% (over two steps).

The switch from 2,2-diphenylbutanenitrile (4-like analogs) to N,N-dimethyl-2,2-diphenylbutanamide (3-like analogs) as the MOR PP was achieved as described in Scheme 3. Starting from the commercially available N-(3,3-diphenyldihydrofuran-2(3H)-ylidene)-N-methylmethanaminium bromide, simple ring opening with the appropriate primary or secondary amines afforded compounds 22, presenting the 6-like dimethylamino function, 23, where the 2,3-dichlorophenyl piperazine D₃R scaffold was introduced, as well as 24, whose 1,2,3,4-tetrahydroisoquinoline-7-carbonitrile is another common D₂R-D₃R antagonist PP fragment, present in well-characterized ligands, such as 9^49–52.

Scheme 3.

a) appropriate secondary amine, K₂CO₃, DIPEA (for 23 and 24), ACN, tert-butyl methyl ether (TBME), reflux, 24 hrs, 16 – 91%; b) 2N NaOH in H₂O, 25 ° C, 5 min, 100%; c) Dess-Martin periodinane (DMP), DCM, 0 to 25 °C, 1 hr, 60%; d) appropriate primary or secondary amine, catalytic acetic acid (cat. AcOH), sodium triacetoxyborohydride (STAB), 1,2-dichloroethane (DCE), 25 ° C, 2.5 hrs, 12 – 100%.

To investigate the effect of the linker length and substitution on the bivalent hybrid analogs, N-(3,3-diphenyldihydrofuran-2(3H)-ylidene)-N-methylmethanaminium bromide was simply washed with 2N NaOH in H₂O, and extracted with DCM, to obtain the alcohol intermediate 25, which was then oxidized to the aldehyde using Dess-Martin periodinane and then mono-N-alkylated via reductive amination conditions with the appropriate primary and secondary amines. This small library of compounds (27, 28, 29, 30, 31, and 32) covered a large structural variety of canonical D₃R antagonist PPs, as well as butyl and hydroxyl substituted linkers, known for increasing D₃R subtype selectivity⁵³.

Compound 34, containing the 1,2,3,4-tetrahydroisoquinoline-7-carbonitrile pharmacophore was synthesized according to Scheme 4, via preparation of intermediate 33, and reductive amination with 26.

Scheme 4.

a) N-(4-bromobutyl)phthalimide, K₂CO₃, cat. KI, ACN, 82 ° C, overnight; b) hydrazine (NH₂NH₂), ethanol (EtOH), 80 ° C, 3 hrs, 94% (over 2 steps); c) N,N-dimethyl-4-oxo-2,2-diphenylbutanamide (26), cat. AcOH, STAB, DCE, 25 ° C, 2.5 hrs, 46%.

In order to expand the library towards different D₃R PP, the canonical piperazine and 1,2,3,4-tetrahydroisoquinoline, inspired by selective D₃R antagonists and partial agonists (1, 2, 7, and 9; Figure 1), were replaced in Scheme 5 with the highly decorated phenyl-N-(pyrrolidin-2-ylmethyl)benzamide derived from the eticlopride pharmacophore (8, Figure 1). Alkylation of (S)-nor-eticlopride (obtained from the NIDA Drug Supply Program) with 4-bromo-2,2-diphenylbutanenitrile yielded compound 35, introducing the 2,2-diphenyl-nitrile MOR PP, tethered by an ethyl linker. Meanwhile, formation of intermediate 36, allowed the synthesis of analog 37, tethered via the longer butyl-amino linker.

Scheme 5.

a) N-(4-bromobutyl)phthalimide, K₂CO₃, ACN, 82 ° C, overnight; b) NH₂NH₂, EtOH, 80 ° C, 3 hrs, 36% (over 2 steps); c) K₂CO₃, ACN, 130 ° C, overnight, 10 – 23%.

Analogous to the approach described above for the phenylpiperazine series, we investigated the replacement of the diphenyl nitrile MOR PP, with the 3-like N,N-dimethylamide functional group for the 8-based bivalent analogs. In Scheme 6, the substituted benzoic acid intermediate was prepared following previous literature procedures^{14, 54}. The benzoic acid was coupled with 2-(4-(2-(aminomethyl)pyrrolidin-1-yl)butyl)isoindoline-1,3-dione, which was freshly prepared in situ via selective Boc- deprotection of 38, prior to the HCTU mediated amide coupling. The phthalimide group in intermediate 39 was removed and the resulting primary amine was mono-N-alkylated via reductive amination to yield 40 as the racemic mixture.

Scheme 6.

a) N-(4-bromobutyl)phthalimide, K₂CO₃, ACN, 82 ° C, overnight, 85%; b) TFA, DCM, 25 ° C, 3 hrs; c) HCTU, DCM, 25 ° C, 48 hrs, 26%; d) NH₂NH₂, EtOH, 80 ° C, 3 hrs; e) cat. AcOH, STAB, DCE, 25 ° C, 12 hrs, 21% (over two steps).

In this case, we decided to synthesize the racemic mixture, starting from the commercially available tert-butyl (pyrrolidin-2-ylmethyl)carbamate, because although 8 favors the (S) absolute configuration at the pyrrolidine ring, we didn’t want to exclude the possibility of different stereochemical requirements for this bivalent analog, as we have seen in another series of bivalent/bitopic D₃R ligands⁴⁶.

SAR for an alternative MOR PP, with a 3,3-diphenyl substituted pyrrolidine, were investigated through the synthesis of analogs in Scheme 7. The common starting material 4-bromo-2,2-diphenylbutanenitrile was reacted with lithium aluminum hydride (LAH), to give the primary amine, and consequent ring-closure in a one-pot step. Intermediate 41 was either methylated via treatment with methyl chloroformate followed in situ LAH reduction to afford 42, or reacted with propionyl chloride to yield the cyclic amide synthon 43. This rather simple PP allowed us to evaluate the effect of structural rigidity in 6- and 3-like MOR PP, and the effect of replacing the protonatable cyclic amine to a cyclic amide. To prepare the bivalent analog 46, 41 was initially acylated with 2-chloroacetyl chloride. Subsequent alkylation of racemic tert-butyl (pyrrolidin-2-ylmethyl)carbamate yielded 45. Finally, Boc- deprotection and amide coupling afforded the desired product.

Scheme 7.

a) LAH, THF, 0 to 25 °C, 15 hrs, 40%; b) 2-chloroacetyl chloride, DIPEA, THF, 0 to 25 °C, 1 hr; c) cat. KI, K₂CO₃, ACN, 82 °C, 3 hrs, 74% (over two steps); d) TFA, DCM, 25 °C, 2 hrs; e) HCTU, DIPEA, DCM, 25 °C, 3 hrs, 15% (over two steps); f) methyl chloroformate, DIPEA, DCM, 25 °C, 1 hr; g) LAH, THF, 0 to 25 °C, 56% (over two steps); h) propionyl chloride, DIPEA, DCM, 40 °C, overnight, 29%.

As consistently observed in previous work⁴³, when generating bitopic or bivalent ligand SAR, it is essential to study structural requirements not only for the PP and/or SP, but regiochemistry and stereochemistry of the linkers, which can play a crucial role in their biological activity.

In Scheme 8, we approached a modification of the regiochemistry for the pyrrolidine D₃R PP scaffold. The final compound 48 presents the MOR diphenyl-N,N-dimethyl amide PP tethered to the D₃R PP, via a butyl ether linker fused in postion-4 of the pyrrolidine nucleus, in a rel-trans stereochemistry configuration with respect to the 8 amide PP appended in position-2. This was easily introduced as shown in Scheme 8, starting from 47 via reductive amination and Boc-deprotection to ultimately yield 48.

Scheme 8.

A) cat. AcOH, STAB, DCE, 25 °C, 4 hrs; b) TFA, DCM, 25 °C, overnight, 8.5% (over two steps).

Extensive in silico docking studies and optimization (see the section below) were directed towards improving the dual-target affinity of these new MOR-D₃R analogs and guided us to the synthesis of 51 (Scheme 9). In particular, the hydroxy substitution of the MOR diphenyl PP was based on the in silico observation that the meta-hydroxy groups will engage a water-mediated hydrogen bond network observed in several known opioid ligands while being well-tolerated by the D₃R SP binding site.

Scheme 9.

a) titanium tetrachloride (TiCl₄), triethylamine (TEA), DCM, 0 to 25 ° C, overnight, 58%; b) 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine, cat. AcOH, STAB, DCE, 25 °C, 4 hrs, 88%; c) H₂ (50 psi), Pd/C (20% wet support), EtOH, 25 ° C, 12 hrs; d) HBr 33% in AcOH; 118 ° C, 48 hrs, 13 – 31%; e) 1-(2-chloro-3-ethylphenyl)piperazine¹⁴, cat. AcOH, STAB, DCE, 25 ° C, 4 hrs, 88%; f) H₂ (30 psi), Pd/C (20% wt.), EtOAc:EtOH (2:1), 25 ° C, 3 hrs, 75%.

Homologation of the commercially available bis(3-methoxyphenyl)methanone in presence of TiCl₄ and triethylamine⁵⁵, allowed the formation of the α,β-unsaturated aldehyde 49. Reductive amination in presence of 2,3-dichlorophenyl piperazine yielded 50, which was hydrogenated in the presence of Pd/C. The crude mixture was then subsequently O-demethylated with 33% HBr in AcOH (Scheme 9).

High resolution mass spectroscopy (HRMS) and NMR analyses revealed the loss of both chlorine atoms in final product 51. Retrospective analyses of the previous synthetic steps and intermediates showed that the loss of both chlorine atoms occurred during hydrogenation while reducing the styrenyl olefin. Unfortunately, dehalogenation and concomitant hydrogenation appears to occur faster than the reduction of the desired olefin. Despite replacing Pd/C with PtO₂, solvent (from EtOH to EtOAc), and H₂ pressure (from 50 psi to 15–20 psi), the same reaction outcome was observed. Nevertheless, we proceeded in testing 51, as a proof of concept to validate the biological activity of the newly proposed bis-phenol PP to target MOR.

The 1-(2-chloro-3-ethylphenyl)piperazine scaffold proved to be resistant to hydrogenation conditions¹⁴, and was introduced as the D₃R PP tethered to the MOR bis-phenol scaffold (Scheme 9). Intermediate 52 was readily prepared by reductive amination, followed by milder hydrogenation conditions (30 psi H₂, using a mixture of EtOAc:EtOH 3:1 as solvent, for 3 h of total reaction time) to obtain the saturated analog 53. Partial and total O-demethylation was again achieved in the presence of 33% HBr in AcOH, and respective products 54 (obtained and tested as the racemic mixture) and 55 were isolated and tested in vitro.

To further expand SAR on the MOR PP, in Scheme 10 we synthesized a new library of bitopic analogs presenting different phenylmorphan nuclei as PPs to target MOR, and the well-known 2-indoleamide SP to target D₃R SBP. We were motivated to use this combination of pharmacophores because phenylmorphans are a well-characterized scaffold for obtaining highly potent and selective MOR ligands⁵⁶, and they also structurally resemble phenyl/pyridine morpholino moieties that we extensively studied as a D₃R OBS ligands⁴⁵. Moreover, we recently demonstrated how the pyridine morpholine scaffold designed as a D₃R PP, can also be structurally tweaked via the bitopic approach and linker tethering to target other families of GPCRs, including MOR⁴⁶. This suggested that simple structural modification of the phenylmorphan scaffold might also exploit affinity for both targets, and ultimately be suitable for the generation of bitopic and bivalent hybrids for both MOR and D₃R.

Scheme 10.

a) N-(4-oxobutyl)-1H-indole-2-carboxamide⁵⁷, cat. AcOH, STAB, DCE, 25 ° C, 1.5 hrs, 26 – 88%; b) trans-N-(2-(2-formylcyclopropyl)ethyl)-1H-indole-2-carboxamide⁴⁵, cat. AcOH, STAB, DCE, 25 ° C, 1.5 hrs, 65%; c) preparative enantiontioselective high-performance liquid chromatography (PREP-HPLC), ChiralPak AD-H column.

In Scheme 10, nor-56 was N-alkylated via reductive amination with N-(4-oxobutyl)-1H-indole-2-carboxamide⁵⁷, and trans-N-(2-(2-formylcyclopropyl)ethyl)-1H-indole-2-carboxamide⁴⁵, to obtain 57 and 58, respectively. We have previously published⁴⁵ the importance of the rigid cyclopropyl linker to achieve unique pharmacological profiles, and its stereochemistry is essential in modulating favorable poses for D₃R target recognition, binding affinity, selectivity and functional efficacy. Thus, both trans enantiomers of 58 were resolved via preparative chiral HPLC (58a and 58b), and pharmacologically evaluated. Due to the lack of significant differences between their biological profiles we didn’t proceed any further in assigning the absolute configuration of each trans-cyclopropyl enantiomers for these analogs.

In silico docking predicted that replacing the ethyl acrylate on the phenylmorphan ring with a less sterically hindered group (e.g., ketone or hydroxyl group), would be tolerated within the MOR OBS and could increase the affinity of the new hybrids for D₃R. Moreover, since the bitopic analogs 57 and 58 showed high affinity for MOR, but moderate to low binding at D₂-like receptors (Table 2), we aimed to study and invert the stereochemistry of the phenylmorphan ring, while simultaneously evaluating all the possible stereochemical combinations with the hydroxyl group. We synthesized both diastereoisomers 61 and 62, starting from the fully resolved 59 and 60, to study not only the effect of the hydroxyl substitution, but of its stereochemistry too, in the dual-target binding profile.

Table 2.

Radioligand competition binding affinity data, for all the MOR substituted phenylmorphan PP analogs, at hMOR and hD₂-like receptor subtypes. All the affinity values are expressed as K_i ± SEM, derived from IC₅₀ values using the Cheng-Prusoff equation⁶³, and calculated as the mean of at least three independent experiments (n = number of independent experiments), each performed in triplicate.

ND = Not Determined.

^aNo inhibition of specific radioligand binding was observed at the highest tested concentration in one to three independent experiments, each performed in triplicate.

Lastly, as shown in Scheme 11, we prepared the bivalent analog of this phenylmorphan series, 65, presenting both the MOR PP and the canonical D₃R PP 2,3-dichlorophenyl piperazine for D₃R OBS, instead of the 2-indoleamide for SBP binding. The alcohol intermediate 63 was prepared starting from 1-(2,3-dichlorophenyl)piperazine hydrochloride and 2-bromoethan-1-ol, under basic conditions. Tosylation of the hydroxy group and subsequent nucleophilic substitution with 56 yielded the desired product 65.

Scheme 11.

A) 2-bromoethan-1-ol, K₂CO₃, cat. KI, ACN, 82 ° C, overnight, 60%; b) p-toluenesulfonyl chloride (p-TsCl), DIPEA, DCM, 25 °C, overnight, 36%; c) NaHCO₃, ACN, 82 ° C, 6 hrs, 43%.

Molecular Docking and Computer Aided Drug Design (CADD)

We employed structure-based molecular modeling methods to guide the rational design of our dual-target compounds. All atom docking studies on inactive state D₃R (PDB: 3PBL)⁴⁴, and active state MOR (PDB: 5C1M)⁵⁸ were performed, followed by local optimization of receptor-ligand interactions via energy-based Monte Carlo minimization protocols⁵⁹ using ICM-Pro (Molsoft LLC). Figure 2 shows optimized binding modes of 5 and 3 inside D₃R and MOR, respectively, indicating salt bridge interactions between basic nitrogen and conserved D^3.32 of the receptors. The fluorophenyl butanone moiety of 5 occupies the OBS between TM3, TM5, and TM6 in the D₃R, lined by hydrophobic residues such as V111^3.33, V189^5.39, F345^6.51, F346^6.52, and H349^6.55. In MOR, diphenyl butanamide moiety of 3 also occupies the OBS pocket similarly to canonical MOR ligands such as morphine and methadone, interacting with residues Y150^3.33, M153^3.35, V238^5.42, W295^6.48, I298^6.51, H299^6.52, V302^6.55, W320^7.35, I324^7.39, and Y328^7.43. Though the chlorophenyl and hydroxy substituents on the piperidine rings are common for both 5 and 3, these moieties occupy distinct SBPs and interact with different residues in their cognate receptors. Attempts at crossdocking these ligands, i.e. dock 5 to MOR and 3 to D₃R were unsuccessful, corroborating selectivity of the PPs to their corresponding OBS pockets.

Figure 2.

Binding mode of 5 (orange sticks) inside A) D₃R (cyan; PDB: 3PBL), and B) binding mode of 3 (green sticks) MOR (white; PDB: 5C1M).

Figure 3 shows an overlay of MOR and D₃R docking modes of 23, a compound with the N,N-dimethyl-diphenylbutanamide PP of MOR tethered to a 2,3-dichlorophenyl-piperazine PP of canonical D₃R antagonists/partial agonists. In docking to MOR (Figure 3b), we observed a perfect fit of the MOR PP in the corresponding OBS, while the D₃R PP of 23 was accommodated in the secondary site of MOR. In D₃R, the lack of flexibility of the N-linked 2,3-dichlorophenyl of 23 prevented this D₃R PP from reaching its OBS site (Figure 3a). However, the 2,3-dichlorophenyl moiety still comfortably fit the hydrophobic pocket lined by V111^3.33, W342^6.48, F345^6.51, F346^6.52, and H349^6.55. The MOR PP moiety of 23, N,N-dimethyl-diphenylbutanamide, is placed in a hydrophobic region of D₃R surrounded by V86^2.61, L89^2.64, C103^3.25, F106^3.28, C181^ECL2, Y365^7.35, and T369^7.39, and makes additional interactions resulting in comparable docking scores for MOR and D₃R, supporting the synthesis of this compound.

Figure 3.

Binding modes of 23 (green) and 5 (orange) inside A) D₃R (cyan; PDB: 3PBL), and B) Docking mode of 23 (green) and 13 (orange) inside MOR (white; PDB: 5C1M).

In contrast, despite D₃R SAR that supported introduction of the butyl linker between the tethered N,N-dimethyl-diphenylbutanamide or 2,2-diphenylbutanenitrile MOR PP and the 2,3-dichlorophenyl-piperazine, as seen in compounds such as 14 and 28, this design had no effect on the placement of the compounds in the OBS of D₃R, while the corresponding SP motif moves further towards the extracellular region, away from the hydrophobic residues of TM2, TM3 and ECL2 (Figure 4).

Figure 4.

Binding mode of 14 (orange sticks) and 28 (green sticks) inside A) D₃R (cyan; PDB: 3PBL), and B) MOR (white; PDB: 5C1M).

The meta-hydroxy compounds were designed to avail the water mediated hydrogen bonding network close to H299^6.52 and Y150^3.33 in MOR, such as 51, 54 and 55, indeed indicate the presence of those predicted hydrogen bonds, while the 2-chloro-3-ethyl-substituted phenyl-piperazine D₃R PP motif is tolerated in the voluminous and solvent accessible MOR pocket (Figure S1). Further, the highly decorated 8-based D₃R PP of compounds 40 and 48 are placed expectedly in the OBS of D₃R and like the substituted phenyl analogs, the N,N-dimethyl-diphenylbutanamide motif occupies hydrophobic sub-pockets between TM2, TM3, ECL2 and TM7 (Figure 5).

Figure 5.

Binding mode of 40 (orange sticks) inside A) D₃R (cyan; PDB: 3PBL), and B) MOR (white; PDB: 5C1M).

The conformationally restricted ethyl-2-(-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ylidene)acetate MOR PP motif of 10 expectedly docks in the MOR hydrophobic pocket formed between TM3-TM5-TM6-TM7 and 3-hydroxyphenyl forms water mediated hydrogen bonding interactions with H299^6.52 and Y150^3.33. The ethyl phenyl tail of 10 shares the same hydrophobic sub-pocket between TM2-TM3 engaged by fentanyl-like compounds. The docking mode of 65 overlaps completely with the binding mode of 10, except the extended 2,3-dichlorophenyl piperazine of 65 moves slightly towards the extracellular region and is placed in the sub-pocket between TM2-TM3-ECL2. The 2,3-dichlorophenyl piperazine of 65 is docked in the OBS of D₃R, while the ethyl-2-(-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ylidene)acetate moiety is placed in the hydrophobic SBP region (Figure 6).

Figure 6.

Binding mode of 65 (orange sticks) and 10 (green sticks) inside A) D₃R (cyan; PDB: 3PBL), and B) MOR (white; PDB: 5C1M)

Binding Studies and Structure-Activity Relationships (SAR)

All the newly synthesized compounds were tested for their binding affinities at hMOR (in competition with [³H]-DAMGO), hD₂R, hD₃R, and hD₄R (in competition with [³H]-N-methylspiperone ([³H]-NMSP) for all the hD₂-like subtypes). Moreover, a subset of selected hits were further studied at hD₂R and hD₃R using the agonist [³H]-(R)-(+)-7-OH-DPAT as the competing radioisotope. We have previously observed and reported that differences in affinity due to the radioligand being an agonist or antagonist can predict functional efficacy profiles for the tested compounds^{45, 60}.

In Table 1 the binding data are reported for the first series of MOR-D₃R hybrid analogs, based on, 3-, 4-, and 6-like PPs. Reference compounds, including fentanyl, 3, 4, 5, and 6 are reported in the table for useful comparisons. Among the reference compounds, it was interesting to observe how 3, a well-known potent MOR agonist, despite presenting an SP identical to the D₂-like antagonist 5 (Figure 1), binds with low micromolar affinity to all the D₂-like subtypes, as predicted in the CADD studies. Fentanyl, 4, and 6, all have low nanomolar affinities for MOR, as expected and consistent with the literature^{61, 62}. Fentanyl, however, presents moderate affinity for D₄R (K_i = 554 nM), while being inactive at both D₂R and D₃R (K_is >10,000 nM), meanwhile 6 is endowed with a preferential low micromolar affinity for D₃R, being completely inactive at D₂R and >10-fold selective over D₄R. These data highlight how subtle structural changes in well-characterized MOR agonists, can induce different binding profiles and subtype selectivity for the D₂-like dopamine receptors, and that binding affinities can be directed toward dual-target profiles with well-designed structural modifications.

The docking studies showed that the MOR PP motifs (N,N-dimethyl-diphenylbutanamide and 2,2-diphenylbutanenitrile) occupy primarily hydrophobic pockets in both D₃R and MOR. In case of MOR, this pocket formed between TM3, TM5, TM6, and TM7, is an accumulation of three sub-pockets lined by: a) V238^5.42, M153^3.33, H299^6.52, I298^6.51, and V302^6.55 b) M153^3.33, W295^6.48, and Y328^7.43 c) W320^7.35, I324^7.39, I298^6.51, and V302^6.55. In D₃R, these MOR PP motifs occupy a hydrophobic SBP between TM2, TM3, and ECL2 (Figure 3 and 4). In general, replacing the nitrile group with the 3-like N,N-dimethylamide synthon significantly increased the affinity profiles of all the analogs. In particular, 23 presents one of the highest MOR affinities among all the new analogs (MOR K_i = 0.832 nM), and despite the shorter linker, which is generally less favorable for D₂-like receptors affinity, we still obtained a potent D₂-like ligand (D₂R K_i = 74.7 nM, D₃R K_i = 171 nM, and D₄R K_i = 102 nM). A similar nanomolar binding profile across D₂R and D₃R was also confirmed when 23 was tested in the presence of [³H]-(R)-(+)-7-OH-DPAT. The analogous nitrile compound, 13, showed reduced MOR binding (~320-fold; K_i = 266 nM) and reduced D₃R binding (~13-fold; K_i = 2,240 nM).

The docking studies of butyl linked compounds show that, in D₃R, the N,N-dimethyl-diphenylbutanamide and 2,2-diphenylbutanenitrile MOR PP motifs move further towards the extracellular region, away from the hydrophobic residues of TM2, TM3, and ECL2 (Figure 4). Perhaps, because of this conformational change, N,N-dimethylamide to cyano substitutions on the extended linker molecules such as 14 (D₂R K_i = 149 nM, D₃R K_i = 132 nM) are better tolerated at D₃R than the shorter linker compounds such as 13 (D₂R K_i = 2630 nM, D₃R K_i = 2240 nM). As in D₃R, the extended linker compounds are reasonably well tolerated inside MOR. However, in contrast to its binding profile at D₃R, even the extended linker molecule with nitrile substitutions 14 shows reduced MOR binding (K_i = 490 nM). This was also observed with the nitrile analogues, 35 and 37. Compound 28, an analogous compound with substituted N,N-dimethylamide in the MOR PP motif, shows significant improvement in both D₃R affinity (K_i = 39.2 nM) and a MOR binding (K_i = 23.8 nM). This was the first hit analog in this series to show a low nanomolar dual-target affinity for both the MOR and the D₂-like receptors. In contrast, compound 18 showed similarly high affinity for the D₂-like receptors, but MOR affinity was diminished (K_i = 1470 nM). Whereas compounds 15, 19, 21, 24 and 27 showed the opposite profile, having higher affinities for MOR than D₂R or D₃R. Compounds 16 and 22 were poorly active at all receptors tested, reflecting an inability to bind the OBS of either MOR or the D₂-like receptors.

Introduction of the hydroxy substituent in the butylamine linker (compounds 29 and 32), as well as replacement of the 2,3-dichlorophenyl piperazine, with the 2-chloro-3-ethyl-phenylpiperazine (compounds 31 and 32) either maintained or slightly decreased the overall affinity for all the D₂-like receptor subtypes, when compared to 28 and shown in Figure S2. The introduction of 1,2,3,4-tetrahydroisoquinoline-7-carbonitrile as D₃R PP (34), decreased affinity for the D₂-like receptors into the micromolar range. None of the diphenyl-pyrrolidine analogues (compounds 42, 43 and 46) were active. However, the only bivalent compound 46 did have moderate affinity for D₃R (K_i = 288 nM)

Removal of either nitrile or amide functions from the diphenyl MOR PP, and introduction of the meta-hydroxy substituents to the bis-phenyl system, 51, was tolerated as suggested from the docking predictions (Figure S1). Despite the presence of a simple unsubstituted phenylpiperazine pharmacophore, 51 still maintained moderate affinities at both MOR (K_i = 213 nM) and D₃R (K_i = 249 nM).

When the bis-meta-phenol MOR PP was used in combination with the 2-chloro,3-ethyl-phenylpiperazine D₃R PP, tethered via the shorter (two methylene units) linker (55), moderate affinity for all the targets of interest was maintained, but this analogue was not significantly different from the longer linker analog 51. The presence of the meta-hydroxy substituents also retained D₂-like affinity for 55, similar to that observed for 23 and 30 (containing the 3-like MOR PP). In a continuation of a trend noted previously, we observed a significant loss of affinity at MOR, with 55 presenting a binding K_i >300-fold less than 30 and 23. These results suggest that while the bis-phenol MOR PP is well-tolerated as an alternative to the more canonical 3-like di-phenyl-N,N-dimethylamide, the binding profile is still dependent on the linker length, rigidity and overall substitutions on the D₃R PP as well.

In agreement with the CADD, the meta-substitutions in bis-phenyl containing compounds are important for MOR recognition. The methoxy analogs result in partial loss of MOR affinity with a general binding profile of 53 (di-methoxy) < 54 (mono-methoxy) ~ 55 (di-hydroxy). This, however, doesn’t apply for D₂-like binding, where all three analogs show moderate affinities for all the subtypes independent of meta-methoxy or meta-hydroxy substitutions on the bis-phenyl rings, interestingly with higher affinities at D₄R.

Shifting from a phenylpiperazine-based D₃R PP, to a highly decorated 8-based D₃R PP, to develop SAR around the pyrrolidine scaffold, 40, containing a racemic pyrrolidin-2-ylmethyl-amide linker, and 48, presenting a butyl ether linker chain in position 4 of the trans-pyrrolidine nucleus were synthesized. Compound 48 showed the highest D₂R/D₃R affinity among all the new analogs (D₂R K_i = 9.41 nM; D₃R K_i = 2.21 nM), however the regio- and stereochemistry of the substituted pyrrolidine ring was detrimental for MOR binding, with a K_i of 559 nM. On the other hand, 40 emerged as our third lead, alongside 23 and 28, with its almost identical affinities for both MOR (K_i = 106 nM) and D₃R (K_i = 135 nM), ~4- and ~25-fold selectivity over D₂R and D₄R respectively. This profile distinguished 40 as one of the most promising dual-target MOR-D₃R compounds in the series.

The binding data for the phenylmorphan analogs are reported in Table 2. Compounds 10, 11, and the nor-analogs 56, 59 and 60 were tested as reference compounds, since most of them were key MOR PP building blocks for our bivalent and bitopic drug design. Compound 10 showed the highest MOR affinity as an OBS PP, with K_i = 0.633 nM, similar to the affinities of 3 and the hybrid bivalent analogue, 23. Interestingly, 10 also showed moderately low micromolar and sub-micromolar affinities for D₂R and D₃R, supporting the hypothesis that the phenylmorphan ring structurally resembles more flexible phenyl-morpholino moieties, canonical scaffolds for D₃R ligands⁴⁵.

Docking studies suggested that the steric clash between the ethyl acrylate group and the backbone of the D₃R receptor would limit and potentially challenge the development of bitopic ligands 57 and 58 targeting D₃R, despite being tolerated in the MOR OBS. Indeed, bitopic analogs 57, 58, and the resolved enantiomers 58a and 58b, all showed high affinities at MOR (K_is ranging from 13.5 nM to 57 nM), but micromolar K_is for all the D₂-like subtypes, independent of linker rigidity or stereochemistry. The shorter bivalent analog 65, with a simple ethyl linker chain (n=2 was predicted by docking studies to be the optimal spacer to resolve steric clashes of the MOR PP motif in D₃R; Figure 6). Indeed, tethering the MOR phenylmorphan PP and the D₃R 2,3-dichlorophenyl piperazine PP presented one of the most interesting dual-target candidates with equivalent affinities at MOR (K_i = 92.7 nM) and D₃R (K_i = 139 nM).

Replacement of the sterically hindered ethyl acrylate group with a smaller hydroxy substituent allowed validation of the docking observation that a small substituent would significantly improve D₃R binding. Overall, 61 and 62 adopt the same docking as 65 inside MOR but lose hydrophobic interactions of the ethyl acrylate moiety with M153^3.36 and Y328^7.43 (Figure S3). The hydrogen-bonding interactions between the 9-OH and Y328^7.43 provide limited compensation for 62. However, for 61, this hydrogen bond is accompanied by negative interactions due to proximity of the carboxylate oxygen of D149^3.32 (3.2 Å compared to 4.5 Å of S-chiral 62). Further, 61 and 62 adopt an interesting conformation inside D₃R with the indole carboxamide ring situated in the OBS. In the absence of positively charged nitrogen, the nitrogen of the amide forms a hydrogen bond with the D110^3.32 residue. Interestingly, the 9-OH is placed very close to the backbone of TM7 for 62 and TM2 for 61. Therefore, the substitution of this position with ethyl acrylate would cause steric clashes with D₃R, as indicated by the loss of affinity of 57. Bitopic analog 62, with all the resolved stereochemical configurations around the phenylmorphan moiety inverted with respect to 57, showed a D₃R K_i of 79.6 nM, ~172-fold improved affinity with respect to the ethyl acrylate analog 57, maintaining a moderate affinity (K_i = 464 nM) for MOR.

Across all the tested compounds, no significant differences were observed in the D₂-like affinities determined using [³H]-NMSP and [³H]-(R)-(+)-7-OH-DPAT binding assays were observed, unlike our previous observations for efficacious agonist ligands^{43, 45}, consistent with our hypothesis that all the new analogs are likely antagonists or low efficacy partial agonists at D₃R.

Based on their binding profiles, a select group of hits were tested in functional assays, to determine their agonist and antagonist potencies for the multiple GPCR-related signaling pathways, as well as to validate and confirm the MOR agonism and D₃R antagonism/partial agonism profile we were seeking with these new hybrid molecules.

BRET Functional Studies at MOR and D₃R

With the binding studies and SAR established, we then characterized the action of selected ligands to signal through both MOR and D₃R; these results are shown in Figure 7 and Table 3. The action of these ligands was assessed through arrestin-3 (or β-arrestin-2) recruitment at MOR and G protein activation (GPA) at MOR (Gα_i2) and D₃R (Gα_oA) assays. In addition, the ability of the ligands to induce the active state of the MOR was determined by measuring recruitment of a conformationally selective nanobody that recognizes and binds to the active conformation of MOR, nanobody 33 (Nb33)⁶⁴. Seven of the newly synthesized MOR-D₃R hybrids (14, 23, 28, 40, 57, 58a and 58b) were tested together with 10. The efficacious agonists DAMGO (D-Ala2, N-MePhe4, Gly5-ol-enkephalin), quinpirole and dopamine were used as reference agonists to normalize data at the MOR and D₃R, respectively. We included the MOR partial agonist morphine to illustrate the relative coupling efficiency and amplification of the different assays (Figure 7A,B and D). Both known antagonists, naloxone (MOR) and 5 (D₃R), inhibited agonist-stimulated GPA in a concentration-dependent manner (Figure 7C and F).

Figure 7. Functional profiles of selected MOR-D₃R hybrids.

Each panel shows a different signaling readout: A. Nb33 recruitment at MOR, B. MOR-mediated Gα_i2 protein activation, C. Antagonism at MOR using GPA in the presence of 100 nM of DAMGO, D. Arrestin-3 recruitment at MOR in the presence of overexpressed GRK2, E. D₃R-mediated Gα_oA protein activation and F. Antagonism at D₃R using GPA in the presence of 3 nM of quinpirole. In order to ensure that test ligands achieved maximal receptor occupancy possible before agonist addition, all ligands were added to the cells 3h prior to activating D₃R with quinpirole and measuring BRET signals with the exception of 5. Dotted lines represent fits where the bottom asymptote was constrained to be 0%.

Table 3.

Potencies and efficacies at MOR and D₃R. All data represent the mean of at least three independent experiments (n = number of independent experiments), each performed in duplicate. Potency values are expressed as pEC₅₀ ± SEM with the corresponding EC₅₀ in nM in brackets. Efficacy values are calculated as a percentage of a reference ligand (DAMGO or quinpirole for MOR and D₃R respectively) and expressed as E_max ± SEM (%).

Compound	MOR Nb33 recruitment		MOR Gi2 activation			MOR arr3 recruitment (+ GRK2)		D3R GoA activation
	pEC50 ± SEM [nM]	Emax ± SEM (%)	pEC50 ± SEM [nM]	Emax ± SEM (%)	pIC50 ± SEM [nM]	pEC50 ± SEM [nM]	Emax ± SEM (%)	pEC50 ± SEM [nM]	Emax ± SEM (%)	pIC50 ± SEM [nM]
DAMGO	7.02 ± 0.04 [95.9] (n=3)	100	8.83 ± 0.03 [1.50] (n=9)	100	-	7.97 ± 0.03 [11.1] (n=3)	100	-	-	-
Morphine	6.38 ± 0.05 [416] (n=3)	73.4 ± 1.7	8.06 ± 0.03 [8.66] (n=6)	97.4 ± 1.1	-	7.10 ± 0.04 [79.8] (n=3)	92.1 ± 1.4	-	-	-
Naloxone	-	-	-	-	6.52 ± 0.1 [305] (n=3)	-	-	-	-	-
Quinpirole	-	-	-	-	-	-	-	8.62 ± 0.05 [2.39] (n=7)	100	-
Dopamine	-	-	-	-	-	-	-	9.05 ± 0.05 [0.896] (n=3)	88.0 ± 1.3	-
Haloperidol (5)	-	-	-	-	-	-	-	-	-	8.46 ± 0.07 [3.49] (n=3)
10	7.51 ± 0.10 [31.3] (n=3)	33.3 ± 1.2	8.28 ± 0.05 [5.29] (n=9)	91.9 ± 1.3	-	7.70 ± 0.07 [20.0] (n=3)	69.3 ± 1.7	NA	NA	5.70 ± 0.06 [1997] (n=3)
14	NA	NA	4.98 ± 0.29 [10530] (n=6)	114.9 ± 39.2	-	NA	NA	6.78 ± 0.15 [165] (n=4)	63.8 ± 4.2	-
23	6.58 ± 0.06 [266] (n=3)	95.5 ± 2.7	7.91 ± 0.06 [12.3] (n=3)	100.8 ± 2.1	-	7.08 ± 0.03 [83.3] (n=3)	102.7 ± 1.3	6.13 ± 0.16 [747] (n=3)	20.2 ± 2.1	-
28	NA	NA	5.96 ± 0.09 [1107] (n=3)	44.5 ± 2.4	-	NA	NA	7.58 ± 0.16 [26.4] (n=3)	55.0 ± 3.2	-
40	5.67 ± 0.20 [2136] (n=3)	16.6 ± 2.1	6.68 ± 0.08 [211] (n=3)	84.4 ± 2.9	-	6.32 ± 0.11 [484] (n=3)	25.3 ± 1.3	NA	NA	5.82 ± 0.15 [1515] (n=3)
57	NA	NA	NA	NA	5.45 ± 0.05 [3522] (n=5)	NA	NA	NA	NA	>5 (n=3)
58a	NA	NA	NA	NA	5.68 ± 0.05 [2102] (n=6)	NA	NA	5.83 ± 0.08 [1498] (n=4)	110.6 ± 6.0	-
58b	NA	NA	NA	NA	5.24 ± 0.06 [5770] (n=6)	NA	NA	45.6 % of DA at 10μM (n=4)	ND	-

NA = Not Active, the compound presents no agonist activity at the highest tested concentration.

The three substituted phenylmorphan MOR PP analogs (57, 58a and 58b), despite showing improved affinities for MOR compared to 56, did not activate MOR in any of the three pathways tested (Figure 7A,B and D). Thus, we tested the ability of these bitopic compounds to inhibit the action of 100 nM of DAMGO in the GPA i2 assay. As shown in Figure 7C, all three ligands were able to inhibit DAMGO-mediated GPA to the same extent as naloxone albeit with lower potencies. When tested at the D₃R, 57 failed to demonstrate any D₃R activity (up to 10 μM, Figure 7E–F), which is likely due to the low affinity of this compound for this receptor (Table 2). Compounds 58a and 58b showed agonist activity at D₃R but with low (micromolar) potencies that reflect their affinity for the D₃R (Figure 7E, Table 3). Thus, these two phenylmorphan hybrids display MOR antagonism and D₃R agonism, rather than the MOR agonist/D₃R antagonist or partial agonist profile we targeted.

In contrast all four MOR diphenyl PP analogs tested (14, 23, 28 and 40), showed agonist activity at MOR, with 23 being the most potent and efficacious compound. Compound 14 showed low potency MOR agonism that could only be detected at the highest concentration used of 10 μM in the most amplified and sensitive GPA i2 assay. 40 displayed higher potency and efficacy in assays of MOR activation than 28, with 28 displaying no detectable agonism in the less amplified Nb33 and arrestin-3 recruitment assays, but an E_max of 50% that of DAMGO in the GPA i2 assay. Compound 40 gave a robust response (E_max = 84.4% of DAMGO) in the GPA i2 assay but much weaker responses in the arrestin and Nb33 assays, indicating it is a less efficacious partial agonist than morphine. All four bivalent compounds share a similar MOR diphenyl PP based on 3 and 4, the N,N-dimethyl-diphenylbutanamide PP being more favorable than the diphenylbutanenitrile PP for MOR agonism. The major structural differences are present in the D₃R PP as well as the type and length of the linker between the two pharmacophores: a shorter linker being more favorable for MOR agonism. In general, across the series of compounds and morphine we observe higher maximal effects (E_max) and potencies in the GPA i2 assay as compared to that in the arrestin-3 recruitment and Nb33 assays. Such behavior is consistent with the action of partial agonists at signaling endpoints with different levels of amplification and coupling efficiency of the pathway. In agreement with this, when these data were analyzed using the Black and Leff operational model of agonism and assessed for biased agonism using DAMGO as the reference ligand, none of the compounds displayed significant bias between these two pathways relative to the action of DAMGO (Supplementary Table S2).

When these four MOR diphenyl PP ligands were tested for their ability to activate the D₃R (Figure 7E–F), 14 and 28 showed similar efficacies (64% and 55% of dopamine, respectively), with 28 being the most potent compound in agreement with their relative affinities (Table 1). Although 14 and 40 display similar affinities for D₃R, 40 acted as an antagonist with micromolar potency (IC₅₀ = 1.5 μM), whereas 14 acted as a robust partial agonist (E_max = 63.8% of quinpirole). Lastly, 23, which was the most potent MOR agonist and shares the same D₃R PP structure as 28 but with a shorter linker, displayed weak partial agonism (E_max = 20% of quinpirole) at the D₃R and sub-micromolar potency consistent with its binding affinity.

CONCLUSIONS

The data obtained highlight a series of hit to lead candidates as MOR-D₃R dual-target ligands. We have synthesized multiple combinations of bivalent or bitopic ligands based on carefully designed structural modifications and in silico guided SAR around the MOR PP, D₃R PP and SP, as well as linkers, with a particular focus on regio- and stereochemistry. Importantly, we have identified compounds with a range of sub-nanomolar to sub-micromolar binding affinities for each receptor of interest and thus provide a new approach to modulate the pharmacological profiles of highly selective MOR agonists through concomitant dual-target D₃R antagonism.

The functional studies revealed three lead analogs, 23, 28 and 40, which are partial agonists at MOR and partial agonists or antagonists at D₃R. We and others have suggested that low intrinsic efficacy could explain the improved therapeutic window observed on the most recent MOR agonists, such as PZM21 and TRV-130³¹ and our three lead compounds fit this desired functional profile with the added feature of D₃R low efficacy partial agonism or antagonism, which may prove beneficial in avoiding the addictive liability of opioid receptor targeted drugs. Further, evaluation and thus a better understanding of the desired kinetic profiles at both targets for optimal pharmacological effect will be crucial in the development of future generations of dual-target MOR-D₃R ligands. Indeed, current drug design is focused on improving drug-like characteristics as well as blood brain barrier penetrability as the current lead compounds have central nervous system multiparameter optimization (MPO)^{65, 66} scores of ~2 and are predicted to be peripherally limited. This indeed was what Komoto and colleagues³⁷ found with their loperamide analogues, which is perhaps unsurprising. Nevertheless, with proof-of-concept in hand, we have laid the groundwork for an alternative pharmacological approach, using bivalent drug design to engage both MOR and D₃R in the pursuit of a novel class of opioid analgesics with lower abuse potential.

EXPERIMENTAL METHODS

Chemistry

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. The ¹H and ¹³C NMR spectra were recorded on a Varian Mercury Plus 400 instrument. Proton chemical shifts are reported as parts per million (δ ppm) relative to tetramethylsilane (0.00 ppm) as an internal standard, or to deuterated solvents. Coupling constants are measured in Hz. Chemical shifts for ¹³C NMR spectra are reported as parts per million (δ ppm) relative to deuterated CHCl₃ or deuterated MeOH (CDCl₃ 77.5 ppm, CD₃OD 49.3 ppm). Chemical shifts, multiplicities and coupling constants (J) have been reported and calculated using Vnmrj Agilent-NMR 400Mr or MNova 9.0 software. Gas chromatography-mass spectrometry (GC/MS) data were acquired (where obtainable) using an Agilent Technologies (Santa Clara, 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 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. Preparative thin layer chromatography was performed on Analtech silica gel plates (1000 μm). When %DMA is reported as eluting system, it stands for % of methanol in DCM, in presence of 0.5%−1% NH₄OH. Preparative chiral HPLC was performed using a Teledyne Isco EZ-Prep chromatography system with DAD (Diode Array Detector) and ELS detectors. HPLC analysis was performed using an Agilent Technologies 1260 Infinity system coupled with DAD (Diode Array Detector). For each analytical HPLC run multiple DAD λ absorbance signals were measured in the range of 210–280 nm. Separation of the analyte, purity and enantiomeric/diastereomeric excess determinations were achieved at 40 °C using the methods reported in each detailed reaction description. Preparative and analytical HPLC columns were purchased from Daicel corporation or Phenomenex. HPLC methods and conditions are reported in the descriptions of the chemical reactions where they were applied. Microanalyses were performed by Atlantic Microlab, Inc. (Norcross, GA) and agree with ± 0.4% of calculated values. HRMS (mass error within 5 ppm) and MS/MS fragmentation analysis were performed on a LTQ-Orbitrap Velos (Thermo-Scientific, San Jose, CA) coupled with an ESI source in positive ion mode to confirm the assigned structures and regiochemistry. Unless otherwise stated, all the test compounds were evaluated to be >95% pure on the basis of combustion analysis, NMR, GC-MS, and HPLC-DAD. The detailed analytical results are reported in the characterization of each final compound.

4-(Phenethylamino)-2,2-diphenylbutanenitrile (12).

A suspension of 4-bromo-2,2-diphenylbutanenitrile (500 mg, 1.67 mmol), 2-phenylethan-1-amine (605 mg, 5 mmol), and K₂CO₃ (230 mg, from 1.67 mmol up to 10 equivalents) in ACN (50 mL), was heated at 130 ° C in a sealed vessel overnight. The mixture was filtered, the solvent removed under vacuum, and the residue purified by flash chromatography eluting with 15% DMA. The desired product was obtained as a colorless oil (300 mg, 53% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.36 – 7.18 (m, 15H), 3.74 (t, J = 7.1 Hz, 2H), 3.07 (t, J = 6.2 Hz, 2H), 2.95 (t, J = 7.2 Hz, 2H), 2.60 (t, J = 6.2 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 171.34, 142.74, 139.55, 128.86, 128.57, 128.46, 128.38, 128.36, 128.12, 126.98, 126.18, 61.47, 53.40, 46.66, 46.18, 37.35, 33.47. The free base was converted into the corresponding oxalate salt. HRMS (C₂₄H₂₄N₂ + H⁺) calculated 341.20123, found 341.20048 (error −0.7 ppm). CHN (C₂₄H₂₄N₂ + 1.5 H₂C₂O₄ + H₂O) calculated C 65.71, H 5.92, N 5.68; found C 65.76, H 5.77, N 5.57. mp: Salt too hygroscopic to determine melting point.

4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-2,2-diphenylbutanenitrile (13).

The reaction was performed following the same procedure described for 12, starting from 1-(2,3-dichlorophenyl)piperazine (250 mg, 0.9 mmol) and 4-bromo-2,2-diphenylbutanenitrile (350 mg, 1.17 mmol). The desired product was purified by flash chromatography eluting with hexanes/ethyl acetate (hex/EtOAc 5:5), and obtained as a colorless oil (30 mg, 7.5% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.45 – 7.23 (m, 10H), 7.19 – 7.08 (m, 2H), 6.94 (dd, J = 6.8, 2.8 Hz, 1H), 3.04 (br s, 4H), 2.65 (dd, J = 10.5, 4.3 Hz, 6H), 2.57 – 2.49 (m, 2H). The free base was converted into the corresponding oxalate salt. CHN (C₂₆H₂₅N₃Cl₂ + H₂C₂O₄ + 0.25 H₂O) calculated C 61.71, H 5.09, N 7.71; found C 61.67, H 5.09, N 7.85. mp: 202–207 ° C.

4-((4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)amino)-2,2-diphenylbutanenitrile (14).

The reaction was performed following the same procedure described for 12, starting from 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine (200 mg, 0.7 mmol) and 4-bromo-2,2-diphenylbutanenitrile (105 mg, 0.35 mmol). The desired product was purified by flash chromatography eluting with 15% DMA, and obtained as a colorless oil (58 mg, 31% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.37 – 7.29 (m, 10H), 7.29 – 7.18 (m, 2H), 6.95 (dd, J = 6.4, 3.2 Hz, 1H), 4.84 (br s, 1H), 3.52 (t, J = 7.2 Hz, 2H), 3.26 (t, J = 6.2 Hz, 2H), 3.05 (t, J = 4.8 Hz, 4H), 2.71 (t, J = 6.2 Hz, 2H), 2.59 (br s, 4H), 2.44 (dd, J = 8.2, 6.7 Hz, 2H), 1.70 – 1.53 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 171.46, 151.32, 142.37, 133.99, 128.51, 128.45, 127.48, 127.39, 127.15, 124.48, 118.56, 61.65, 58.24, 53.24, 51.31, 46.34, 44.72, 37.36, 25.14, 24.26. The free base was converted into the corresponding oxalate salt. HRMS (C₃₀H₃₄N₄Cl₂ + H⁺) calculated 521.22333, found 521.22363 (error 0.3 ppm). CHN (C₃₀H₃₄N₄Cl₂ + 3 H₂C₂O₄ + 0.5 H₂O) calculated C 54.01, H 5.16, N 7.00; found C 53.97, H 5.33, N 7.15. mp: Salt decomposes above 80 ° C.

4-((4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)amino)-2,2-diphenylbutanenitrile (15).

The reaction was performed following the same procedure described for 12, starting from 4-amino-1-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-2-ol⁴⁷ (223 mg, 0.7 mmol) and 4-bromo-2,2-diphenylbutanenitrile (105 mg, 0.35 mmol). The desired product was purified by flash chromatography eluting with 15% DMA, and obtained as a colorless oil (53 mg, 28% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.48 – 7.32 (m, 6H), 7.24 – 7.10 (m, 6H), 6.95 (dd, J = 7.2, 2.3 Hz, 1H), 4.22 – 4.06 (m, 3H), 3.75 – 3.67 (m, 2H), 3.12 (br s, 4H), 2.96 (tt, J = 8.1, 4.2 Hz, 4H), 2.76 (d, J = 10.7 Hz, 2H), 2.67 (dd, J = 12.5, 3.2 Hz, 1H), 2.61 – 2.50 (m, 1H), 2.01 (s, 1H), 1.82 (ddd, J = 16.4, 13.9, 7.1 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 169.96, 150.78, 138.44, 138.40, 134.10, 129.50, 129.48, 128.91, 128.89, 127.88, 127.78, 127.58, 127.43, 124.83, 118.64, 64.45, 63.72, 62.31, 53.36, 50.83, 50.62, 45.63, 37.82, 31.51. HRMS (C₃₀H₃₄ON₄Cl₂ + H⁺) calculated 537.21824, found 537.21935 (error 1 ppm). HPLC analysis method A: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 30% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 4.944 min, purity >94.3% (absorbance at 254 nm). HPLC analysis method B: Chiralpak OZ-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 30% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 9.072 and 10.862 min, purity >95%, er 43:57 (absorbance at 254 nm).

4-(Dimethylamino)-2,2-diphenylbutanenitrile (16).

The reaction was performed following the same procedure described for 12, starting from dimethylamine hydrochloride (2 g, 25 mmol) and 4-bromo-2,2-diphenylbutanenitrile (5 g, 16.7 mmol). The desired product was purified by flash chromatography eluting with 5% DMA, and obtained as a colorless oil (3.5 g, 79% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.66 – 6.75 (m, 10H), 2.68 – 2.50 (m, 2H), 2.50 – 2.33 (m, 2H), 2.25 (s, 6H). GC/MS (EI), Rt 10.499 min; 264.1 (M⁺), purity >95%. The free base was converted into the corresponding oxalate salt. CHN (C₁₈H₂₀N₂ + H₂C₂O₄) calculated C 67.78, H 6.26, N 7.90; found C 67.66, H 6.43, N 7.96. mp: 163–169 ° C.

3-Carboxy-N,N-dimethyl-3,3-diphenylpropan-1-aminium bromide (17).

Compound 16 (2 g, 7.6 mmol) was dissolved in 48% HBr aq solution (50 mL) and stirred under reflux overnight. The solution was concentrated to half-volume, decanted and the residue washed multiple times with Et₂O. The dried crude material was used in the next step without further purification.

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)-4-(dimethylamino)-2,2-diphenylbutanamide (18).

A solution of 17 (460 mg, 1.26 mmol), EDC hydrochloride (240 mg, 1.26 mmol), HOBt (170 mg, 1.26 mmol) and DIPEA (2.2 mL; 12.6 mmol) in DCM (20 mL) was stirred at RT for 1 h, followed by dropwise addition of 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine (380 mg, 1.26 mmol) in DCM (20 mL). The mixture was stirred at RT overnight, the solvent evaporated under vacuum and the residue purified by flash chromatography eluting with 10% DMA. The desired product was obtained as a yellow oil (40 mg, 6% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.36 – 7.20 (m, 10H), 7.18 – 7.08 (m, 2H), 6.94 (dd, J = 6.6, 3.0 Hz, 1H), 6.74 (br s, 1H), 3.31 – 3.14 (m, 2H), 3.03 (br s, 4H), 2.62 – 2.53 (m, 6H), 2.40 – 2.27 (m, 2H), 2.19 (m, 8H), 1.53 – 1.36 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 173.99, 151.26, 143.81, 133.99, 128.74, 128.25, 127.47, 127.40, 126.81, 124.51, 118.55, 60.22, 58.03, 55.98, 53.23, 51.28, 45.53, 45.42, 44.87, 39.71, 36.80, 29.40, 27.36, 24.09. The free base was converted into the corresponding oxalate salt. HRMS (C₃₂H₄₀ON₄Cl₂ + H⁺) calculated 567.26519, found 567.26524 (error 1.3 ppm). CHN (C₃₂H₄₀ON₄Cl₂ + 2 H₂C₂O₄ + 3 H₂O) calculated C 53.93, H 6.29, N 6.99; found C 53.89, H 5.90, N 7.31. mp: Salt too hygroscopic to determine melting point.

6-(Dimethylamino)-4,4-diphenylhexan-3-one (19).

Ethyl magnesium bromide (3 M solution in diethyl ether, 10 mmol) was added dropwise to a solution of 16 (2 g, 7.5 mmol) in toluene (30 mL) at 0 °C. The mixture was slowly warmed to RT and then stirred at reflux for 3 h. The reaction was quenched with 10 mL of 2N HCl (aq solution) at 0 °C and stirred at reflux for for 30 min. The suspension was basified with 2N NaOH at 0 °C, the toluene removed under vacuum and the aq phase extracted with DCM/2-PrOH (3:1). The organic layers were combined, dried over Na₂SO₄, filtered and dried under vacuum to afford the crude material, which was purified by flash chromatography eluting with 10% DMA. The desired product was obtained as a colorless oil (1.3 g, 59%). ¹H NMR (400 MHz, CDCl₃) δ 7.51 – 7.06 (m, 10H), 2.52 (m, 2H), 2.30 (m, 2H), 2.15 (d, J = 0.9 Hz, 6H), 1.95 (m, 2H), 0.88 (td, J = 7.3, 0.9 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 211.02, 141.48, 129.14, 128.26, 126.94, 65.04, 55.67, 45.52, 35.41, 32.47, 9.05. The free base was converted into the corresponding oxalate salt. HRMS (C₂₀H₂₅NO + H⁺) calculated 296.20089, found 296.20150 (error 0.4 ppm). CHN (C₂₀H₂₅NO + H₂C₂O₄) calculated C 68.55, H 7.06, N 3.63; found C 68.51, H 7.15, N 3.62. mp: 161–165 ° C.

tert-Butyl (5-(4-(2,3-dichlorophenyl)piperazin-1-yl)-5-oxopentyl)carbamate (20).

A solution of 5-((tert-butoxycarbonyl)amino)pentanoic acid (470 mg, 2.16 mmol), 1-(2,3-dichlorophenyl)piperazine (500 mg, 2.16 mmol) and HCTU (895 mg, 2.16 mmol) in DCM (25 mL) was stirred at RT for 3 h. The solvent was removed under vacuum and the residue purified by flash chromatography eluting with hex/EtOAc (40/60). The desired product was obtained as a yellow oil (550 mg, 59% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.23 – 7.11 (m, 2H), 6.92 (dd, J = 7.7, 1.8 Hz, 1H), 4.63 (br s, 1H), 3.79 (t, J = 4.9 Hz, 2H), 3.68 – 3.60 (m, 2H), 3.15 (q, J = 6.6 Hz, 2H), 3.00 (dq, J = 10.7, 5.2 Hz, 4H), 2.39 (t, J = 7.4 Hz, 2H), 1.76 – 1.62 (m, 2H), 1.60 – 1.51 (m, 2H), 1.43 (s, 9H).

4-((5-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-5-oxopentyl)amino)-2,2-diphenylbutanenitrile (21).

Trifluoroacetic acid (1 mL, 12.8 mmol) was added to a solution of 20 (550 mg, 1.28 mmol) in DCM (10 mL). The mixture was stirred at RT for 24 h, basified with 2N NaOH and extracted with DCM. The organic layers were combined, dried over Na₂SO₄, filtered and dried under vacuum to afford the crude primary amine intermediate, which was dissolved in ACN (20 mL), followed by addition of 4-bromo-2,2-diphenylbutanenitrile (384 mg, 1.28 mmol) and K₂CO₃ (10 equivalents). The reaction mixture was stirred at reflux overnight, filtered and the solvent removed under vacuum. The residue was purified by flash chromatography eluting with 15% DMA, and the desired product obtained as a yellow oil (20 mg, 3% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.37 – 7.10 (m, 12H), 6.90 (dd, J = 7.8, 1.8 Hz, 1H), 3.78 (d, J = 5.8 Hz, 2H), 3.63 (t, J = 4.9 Hz, 2H), 3.59 – 3.51 (m, 2H), 3.28 (t, J = 6.2 Hz, 2H), 2.97 (dt, J = 9.3, 5.0 Hz, 4H), 2.72 (t, J = 6.2 Hz, 2H), 2.50 – 2.41 (m, 2H), 1.71 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 171.48, 171.42, 150.64, 142.20, 134.16, 128.51, 128.47, 128.43, 127.74, 127.50, 127.25, 125.13, 118.75, 61.72, 51.69, 51.20, 46.41, 45.80, 44.41, 41.73, 37.36, 32.69, 26.68, 22.47. HRMS (C₃₁H₃₄ON₄Cl₂ + H⁺) calculated 549.21824, found 549.21825 (error 0.0 ppm). HPLC analysis method: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 30% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 27.693 min, purity >95% (absorbance at 254 nm).

4-(Dimethylamino)-N,N-dimethyl-2,2-diphenylbutanamide (22).

Dimethylamine hydrochloride (1.18 g, 14.5 mmol) was added to a suspension of N-(3,3-diphenyldihydrofuran-2(3H)-ylidene)-N-methylmethanaminium bromide (500 mg, 1.45 mmol) and K₂CO₃ (2.0 g, 14.5 mmol) in TBME/ACN (25 mL/10 mL). The reaction mixture was heated in a sealed vessel for 24 h, the solvent removed under vacuum, and the residue purified by flash chromatography eluting with EtOAc/MeOH (95/5). The desired product was obtained as a colorless oil (70 mg, 16% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.43 – 7.24 (m, 10H), 2.97 (br s, 3H), 2.57 – 2.46 (m, 2H), 2.30 (m + br s, 2H + 9H). GC/MS (EI), Rt 11.256 min; 310.1 (M⁺), purity >95%. The free base was converted into the corresponding oxalate salt. HRMS (C₂₀H₂₆ON₂ + H⁺) found 311.21098 (error −2.4 ppm). CHN (C₂₀H₂₆ON₂ + 1.5 H₂C₂O₄ + 0.75 H₂O) calculated C 60.19, H 6.70, N 6.10; found C 60.09, H 6.36, N 6.13. mp: 174–178 ° C.

4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-N,N-dimethyl-2,2-diphenylbutanamide (23).

1-(2,3-Dichlorophenyl)piperazine hydrochloride (400 mg, 1 mmol) was added to a suspension of N-(3,3-diphenyldihydrofuran-2(3H)-ylidene)-N-methylmethanaminium bromide (500 mg, 1 mmol), K₂CO₃ (1 g, 7 mmol) and DIPEA (1 mL, 7 mmol) in ACN (20 mL). The reaction mixture was stirred at reflux overnight, the solvent removed under vacuum and the residue purified by flash chromatography eluting with 5% DMA. The desired product was obtained as a colorless oil (640 mg, 91% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.46 – 7.34 (m, 10H), 7.34 – 7.20 (m, 2H), 7.15 – 7.03 (m, 1H), 2.97 (br s, 6H), 2.56 – 2.43 (m, 8H), 2.19 – 2.10 (m, 2H), 1.69 (s, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 173.41, 151.42, 140.76, 133.89, 128.35, 128.10, 127.44, 127.30, 126.70, 124.29, 118.54, 59.70, 55.71, 53.16, 51.33, 42.24. The free base was converted into the corresponding oxalate salt. HRMS (C₂₈H₃₁ON₃Cl₂ + H⁺) calculated 496.19169, found 496.19052 (error −2.3 ppm). CHN (C₂₈H₃₁ON₃Cl₂ + 1.5 H₂C₂O₄) calculated C 58.96, H 5.43, N 6.65; found C 58.69, H 5.33, N 6.65. mp: 199–205 ° C.

4-(7-Cyano-3,4-dihydroisoquinolin-2(1H)-yl)-N,N-dimethyl-2,2-diphenylbutanamide (24).

The reaction was performed following the same procedure described for 23, starting from 1,2,3,4-tetrahydroisoquinoline-7-carbonitrile (100 mg, 0.63 mmol). The crude material was purified by flash chromatography eluting with 5% DMA and the desired product was obtained as a colorless oil (130 mg, 40% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.46 – 7.18 (m, 12H), 7.10 (d, J = 7.9 Hz, 1H), 3.49 (br s, 2H), 2.98 (br s, 3H), 2.82 (t, J = 5.9 Hz, 2H), 2.63 (t, J = 5.9 Hz, 2H), 2.55 – 2.46 (m, 2H), 2.40 – 2.26 (br s, 3H), 2.26 – 2.17 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 173.44, 140.62, 140.46, 136.71, 130.29, 129.39, 129.33, 128.42, 128.39, 128.07, 126.80, 119.18, 109.08, 59.74, 55.45, 55.42, 50.18, 42.84, 29.55. The free base was converted into the corresponding oxalate salt. HRMS (C₂₈H₂₉ON₃ + H⁺) calculated 424.23834, found 424.23765 (error −1.6 ppm). CHN (C₂₈H₂₉ON₃ + 1.5 H₂C₂O₄ + 0.5 H₂O) calculated C 65.60, H 5.86, N 7.40; found C 65.76, H 5.87, N 7.29. mp: 178–181 ° C.

4-Hydroxy-N,N-dimethyl-2,2-diphenylbutanamide (25).

N-(3,3-Diphenyldihydrofuran-2(3H)-ylidene)-N-methylmethanaminium bromide (700 mg, 2 mmol) was suspended in 2N NaOH (15 mL aq solution) and then stirred at RT for 5 min. The mixture was extracted with DCM, the organic layers were combined, dried over Na₂SO₄, filtered and evaporated under vacuum to afford the pure desired product in quantitative yield, as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.48 – 7.25 (m, 10H), 3.17 (q, J = 5.0 Hz, 2H), 3.04 – 2.95 (m, 3H), 2.57 – 2.49 (m, 2H), 2.35 – 2.27 (m, 3H). GC/MS (EI), Rt 11.847 min; 283.1 (M⁺).

N,N-Dimethyl-4-oxo-2,2-diphenylbutanamide (26).

DMP was added portion-wise to a solution of 25 (200 mg, 0.71 mmol) in DCM (10 mL) at 0 °C. The reaction mixture was slowly warmed to RT and stirred for 1 h. The suspension was washed with 10% NaHCO₃ (aq solution), the organic layer dried over Na₂SO₄, filtered and evaporated under vacuum. The residue was purified by flash chromatography eluting with Hex/EtOAc (50/50) to afford the desired product as a white solid (120 mg, 60% yield). ¹H NMR (400 MHz, CDCl₃) δ 9.17 (t, J = 2.2 Hz, 1H), 7.46 – 7.25 (m, 10H), 3.11 – 2.99 (m, 6H), 2.33 (s, 2H). GC/MS (EI), Rt 11.744 min; 281.1 (M⁺).

N,N-Dimethyl-4-(phenethylamino)-2,2-diphenylbutanamide (27).

A solution of 26 (90 mg, 0.32 mmol), 2-phenylethan-1-amine (77 mg, 0.64 mmol) and cat. AcOH in DCE (5 mL) was stirred at RT for 30 min. STAB (97 mg, 0.48 mmol) was added portion-wise and the mixture stirred for additional 2 h. The solvent was removed under vacuum and the residue purified by flash chromatography eluting with 5% DMA. The desired product was obtained as a colorless oil (quantitative yield). ¹H NMR (400 MHz, CDCl₃) δ 7.39 – 7.10 (m, 15H), 2.96 (m, 3H), 2.75 (br s, 7H), 2.48 – 2.28 (m + br s, 4H + 1H); ¹³C NMR (101 MHz, CDCl₃) δ 176.89, 173.88, 140.40, 139.18, 128.82, 128.73, 128.54, 128.45, 128.04, 126.94, 126.22, 126.17, 60.51, 50.11, 46.28, 44.40, 43.46, 40.00, 35.13, 23.25. The free base was converted into the corresponding oxalate salt. HRMS (C₂₆H₃₀ON₂ + H⁺) calculated 387.24309, found 387.24226 (error −2.1 ppm). CHN (C₂₆H₃₀ON₂ + 1.5 H₂C₂O₄ + 0.1 NH₄OH) calculated C 66.33, H 6.43, N 5.60; found C 66.10, H 6.61, N 5.91. mp: 112–117 ° C.

4-((4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)amino)-N,N-dimethyl-2,2-diphenylbutanamide (28).

The reaction was performed following the same procedure described for 27, starting from 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine (272 mg, 0.9 mmol). The desired product was purified by flash chromatography eluting with 25% DMA and obtained as a colorless oil (300 mg, 65% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.41 – 7.33 (m, 7H), 7.28 – 7.26 (m, 3H), 7.15 – 7.13 (m, 2H), 7.00 – 6.90 (m, 1H), 3.05 (s, 4H), 2.98 (s, 3H), 2.62 (br s, J = 6.0 Hz, 3H), 2.51 (t, J = 6.8 Hz, 6H), 2.45 – 2.37 (m, 4H), 2.29 (br s, 3H), 1.62 – 1.49 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 178.09, 173.97, 151.17, 139.95, 133.94, 128.69, 128.65, 127.97, 127.48, 127.38, 127.15, 124.55, 118.73, 60.48, 57.93, 53.14, 51.06, 50.67, 47.97, 45.70, 43.25, 25.94, 24.19, 23.98. The free base was converted into the corresponding oxalate salt. HRMS (C₃₂H₄₀ON₄Cl₂ + H⁺) calculated 567.26519, found 567.26458 (error −1.1 ppm). CHN (C₃₂H₄₀ON₄Cl₂ + 2.5 H₂C₂O₄ + H₂O) calculated C 54.82, H 5.84, N 6.91; found C 54.89, H 5.68, N 6.83. mp: initial decomposition ~119 ° C, complete melting 150–160 ° C.

4-((4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)amino)-N,N-dimethyl-2,2-diphenylbutanamide (29).

The reaction was performed following the same procedure described for 27, starting from 4-amino-1-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-2-ol (130 mg, 0.41 mmol). The desired product was purified by flash chromatography eluting with 25% DMA and obtained as a colorless oil (80 mg, 34% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.37 (m, 8H), 7.31 – 7.22 (m, 2H), 7.16 – 7.07 (m, 2H), 6.93 (dd, J = 6.5, 3.1 Hz, 1H), 3.82 (m, 1H), 3.04 (br s, 6H), 2.97 (br s, 1H), 2.83 – 2.67 (m, 4H), 2.59 (dt, J = 11.8, 6.1 Hz, 4H), 2.50 – 2.30 (m, 4H), 2.30 – 2.17 (m, 4H), 1.56 – 1.32 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 173.53, 151.32, 140.75, 140.71, 133.98, 128.38, 128.33, 128.12, 128.09, 128.04, 127.48, 127.36, 126.71, 126.68, 124.45, 118.56, 71.43, 68.18, 64.64, 62.95, 59.92, 53.04, 52.17, 51.35, 47.60, 47.30, 45.64, 43.84, 34.82, 34.04. HPLC analysis method A: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 11.906 and 12.953 min, purity >99%, er 38:62 (absorbance at 254 nm). HPLC analysis method B: Chiralcel OD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 12.011 min, purity >99% (absorbance at 254 nm). HPLC analysis method C: Chiralcel OZ-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 12.172 min, purity >95% (absorbance at 254 nm). The free base was converted into the corresponding oxalate salt. HRMS (C₃₂H₄₀O₂N₄Cl₂ + H⁺) calculated 583.26011, found 583.26204 (error 2.3 ppm). CHN (C₃₂H₄₀O₂N₄Cl₂ + 2 H₂C₂O₄ + 1.5 H₂O) calculated C 54.69, H 5.99, N 7.09; found C 54.75, H 5.71, N 7.16. mp: Salt decomposes above 116 ° C.

4-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)-N,N-dimethyl-2,2-diphenylbutanamide (30).

The reaction was performed following the same procedure described for 27, starting from 1-(2-chloro-3-ethylphenyl)piperazine¹⁴ (120 mg, 0.53 mmol). The desired product was purified by flash chromatography eluting with 15% DMA and obtained as a colorless oil (130 mg, 50% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.44 – 7.34 (m, 8H), 7.29 (m, 2H), 7.13 (t, J = 7.8 Hz, 1H), 6.91 (ddd, J = 19.7, 7.8, 1.5 Hz, 2H), 3.07 (br s, 6H), 2.99 (br s, 2H), 2.74 (br s + q, 4H + 2H), 2.61 – 2.52 (m, 2H), 2.38 (m, 4H), 1.20 (t, J = 7.5 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 175.46, 173.36, 148.98, 143.16, 140.12, 128.63, 128.54, 128.49, 128.06, 126.98, 126.87, 124.19, 118.14, 59.68, 55.36, 52.67, 50.17, 40.52, 27.39, 22.19, 14.03. The free base was converted into the corresponding oxalate salt. HRMS (C₃₀H₃₆ON₃Cl + H⁺) found 490.26239 (error 0.9 ppm). CHN (C₃₀H₃₆ON₃Cl + 1.5 H₂C₂O₄) calculated C 63.40, H 6.29, N 6.72; found C 63.35, H 6.46, N 6.67. mp: 183–186 ° C.

4-((4-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)butyl)amino)-N,N-dimethyl-2,2-diphenylbutanamide (31).

The reaction was performed following the same procedure described for 27, starting from 4-(4-(2-chloro-3-ethylphenyl)piperazin-1-yl)butan-1-amine (390 mg, 1.33 mmol). The desired product was purified by flash chromatography eluting with 15% DMA and obtained as a colorless oil (90 mg, 12% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.43 – 7.30 (m, 8H), 7.30 – 7.21 (m, 2H), 7.13 (t, J = 7.8 Hz, 1H), 6.92 (ddd, J = 8.1, 6.6, 1.6 Hz, 2H), 3.03 (br s, 6H), 2.97 (br s, 2H), 2.76 (q, J = 7.5 Hz, 2H), 2.48 – 2.38 (m, 4H), 2.38 – 2.30 (m, 6H), 2.30 – 2.22 (m, 4H), 1.51 – 1.40 (m, 2H), 1.40 – 1.31 (m, 2H), 1.21 (t, J = 7.5 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 173.52, 149.69, 143.14, 140.85, 128.66, 128.32, 128.07, 126.83, 126.64, 123.85, 117.99, 59.90, 58.56, 53.42, 51.55, 49.76, 47.50, 45.75, 28.20, 27.46, 24.72, 23.03, 14.09. The free base was converted into the corresponding oxalate salt. HRMS (C₃₄H₄₅ON₄Cl + H⁺) calculated 561.33547, found 561.33350 (error −4.4 ppm). CHN (C₃₄H₄₅ON₄Cl + 2 H₂C₂O₄ + 1.75 H₂O) calculated C 59.06, H 6.85, N 7.25, found C 59.09, H 6.65, N 7.17.

4-((4-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)-3-hydroxybutyl)amino)-N,N-dimethyl-2,2-diphenylbutanamide (32).

The reaction was performed following the same procedure described for 27, starting from 4-amino-1-(4-(2-chloro-3-ethylphenyl)piperazin-1-yl)butan-2-ol (166 mg, 0.53 mmol). The desired product was purified by flash chromatography eluting with 25% DMA and obtained as a colorless oil (110 mg, 36% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.43 – 7.32 (m, 7H), 7.32 – 7.22 (m, 3H), 7.13 (t, J = 7.8 Hz, 1H), 6.92 (ddd, J = 11.9, 7.8, 1.6 Hz, 2H), 3.82 (m, 1H), 3.03 (s, 6H), 2.98 (s, 2H), 2.82 – 2.69 (m, 5H), 2.61 (ddd, J = 11.7, 8.1, 5.3 Hz, 3H), 2.47 – 2.19 (m, 8H), 1.58 – 1.34 (m, 2H), 1.22 (t, J = 7.5 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 173.55, 149.65, 143.17, 140.71, 128.67, 128.39, 128.05, 126.82, 126.72, 123.88, 117.98, 67.91, 64.64, 59.97, 53.73, 51.60, 47.48, 47.22, 45.49, 34.03, 27.45, 14.07. HPLC analysis method A: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 8.242 and 9.055 min, purity >99%, er 37:63 (absorbance at 254 nm). HPLC analysis method B: Chiralcel OD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 8.516 and 9.788 min, purity >99%, er 57:43 (absorbance at 254 nm). HPLC analysis method C: Chiralcel OZ-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 8.886 and 9.524 min, purity >99%. er 50:50 (absorbance at 254 nm). The free base was converted into the corresponding oxalate salt. HRMS (C₃₄H₄₅O₂N₄Cl + H⁺) found 577.33059 (error 0.4 ppm). CHN (C₃₄H₄₅O₂N₄Cl + 2 H₂C₂O₄ + H₂O) calculated C 58.87, H 6.63, N 7.23, found C 59.06, H 6.46, N 7.14. mp: 126–130 ° C.

2-(4-Aminobutyl)-1,2,3,4-tetrahydroisoquinoline-7-carbonitrile (33).

A suspension of 1,2,3,4-tetrahydroisoquinoline-7-carbonitrile (300 mg, 1.9 mmol), N-(4-bromobutyl)phthalimide (535 mg, 1.9 mmol), cat. KI (3.15 mg, 19 μmol) and K₂CO₃ (2.6 g, 19 mmol) in ACN (20 mL) was stirred under reflux overnight. The reaction mixture was cooled down to RT, filtered, and the solvent removed under vacuum and the residue dissolved in EtOH (10 mL), followed by addition of hydrazine (0.175 mL). The solution was stirred under reflux for 3 h, EtOH was evaporated, the residue diluted with 20% K₂CO₃ aq solution and extracted with DCM. The organic layers were combined, dried over Na₂SO₄, filtered and evaporated under vacuum. The crude material was used in the next step without further purification (300 mg, 94% yield).

4-((4-(7-Cyano-3,4-dihydroisoquinolin-2(1H)-yl)butyl)amino)-N,N-dimethyl-2,2-diphenylbutanamide (34).

The reaction was performed following the same procedure described for 27, starting from 33 (300 mg, 1.31 mmol) and 26 (368 mg, 1.31 mmol). The desired product was purified by flash chromatography eluting with 10% DMA and obtained as a colorless oil (300 mg, 46% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.42 – 7.21 (m, 12H), 7.16 (d, J = 7.9 Hz, 1H), 3.57 (s, 2H), 3.47 (d, J = 0.7 Hz, 6H), 2.99 – 2.87 (m, 4H), 2.68 (t, J = 5.9 Hz, 2H), 2.49 – 2.35 (m, 7H), 1.50 (q, J = 7.7 Hz, 2H), 1.41 (q, J = 7.3 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 173.55, 140.74, 140.36, 136.37, 130.37, 129.50, 129.46, 128.35, 128.04, 126.69, 119.13, 109.29, 59.89, 58.00, 55.45, 50.77, 50.10, 49.57, 47.45, 45.64, 29.41, 27.93, 24.82. The free base was converted into the corresponding oxalate salt. HRMS (C₃₂H₃₈ON₄ + H⁺) found 495.31212 (error 0.5 ppm). CHN (C₃₄H₄₅O₂N₄Cl + 2 H₂C₂O₄ + 1.5 H₂O) calculated C 61.61, H 6.46, N 7.98, found C 61.59, H 6.36, N 7.98. mp: Salt decomposes above 134 ° C.

(S)-3-Chloro-N-((1-(3-cyano-3,3-diphenylpropyl)pyrrolidin-2-yl)methyl)-5-ethyl-6-hydroxy-2-methoxybenzamide (35).

The reaction was performed following the same procedure described for 12, starting from (S)-nor-eticlopride (250 mg, 0.8 mmol) and 4-bromo-2,2-diphenylbutanenitrile (240 mg, 0.8 mmol). The desired product was purified by flash chromatography eluting with hex/EtOAc (60/40) and obtained as a colorless oil (98 mg, 23% yield). ¹H NMR (400 MHz, CDCl₃) δ 13.78 (s, 1H), 8.74 (s, 1H), 7.44 – 7.19 (m, 11H), 3.86 (s, 3H), 3.62 (ddd, J = 14.0, 6.9, 2.6 Hz, 1H), 3.29 – 3.12 (m, 2H), 2.89 – 2.78 (m, 1H), 2.72 – 2.55 (m, 5H), 2.49 – 2.37 (m, 1H), 2.28 (q, J = 8.8 Hz, 1H), 1.90 (dq, J = 12.3, 8.1 Hz, 1H), 1.75 (t, J = 7.8 Hz, 2H), 1.67 – 1.51 (m, 1H), 1.32 – 1.15 (m, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 169.44, 160.15, 152.40, 140.02, 139.45, 132.90, 130.91, 128.95, 128.01, 127.97, 126.64, 122.00, 116.06, 108.18, 62.15, 61.44, 54.04, 50.33, 50.08, 40.43, 38.52, 29.68, 28.12, 22.71, 22.55, 13.43. CHN (C₃₁H₃₄N₃O₃Cl + 0.4 hexanes) calculated C 70.81, H 7.05, N 7.42; found C 70.49, H 7.15, N 7.08. HPLC analysis method A: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: gradient from 10% to 40% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 7.799 min, purity >99%, ee >99% (absorbance at 254 nm). HPLC analysis method B: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 10% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 9.043 min, purity >99%, ee >99% (absorbance at 254 nm). HRMS (C₃₁H₃₄N₃O₃Cl + H⁺) calculated 532.23615, found 532.23691 (error 0.4 ppm).

(S)-N-((1-(4-Aminobutyl)pyrrolidin-2-yl)methyl)-3-chloro-5-ethyl-6-hydroxy-2-methoxybenzamide (36).

The reaction was performed following the same procedure described for 33, starting from (S)-3-chloro-5-ethyl-6-hydroxy-2-methoxy-N-(pyrrolidin-2-ylmethyl)benzamide ((S)-nor-eticlopride 250 mg, 0.8 mmol). The crude material was used in the next step without further purification (110 mg, 36% yield).

(S)-3-Chloro-N-((1-(4-((3-cyano-3,3-diphenylpropyl)amino)butyl)pyrrolidin-2-yl)methyl)-5-ethyl-6-hydroxy-2-methoxybenzamide (37).

The reaction was performed following the same procedure described for 12, starting from 36 (110 mg, 0.29 mmol) and 4-bromo-2,2-diphenylbutanenitrile (78 mg, 0.26 mmol). The desired product was purified by flash chromatography eluting with 10% DMA and obtained as a colorless oil (15 mg, 10% yield). ¹H NMR (400 MHz, CDCl₃ + CD₃OD) δ 7.39 – 7.23 (m, 6H), 7.18 – 7.06 (m, 5H), 3.83 (s, 3H), 3.82 – 3.50 (m, 6H), 3.36 – 3.26 (m, 6H), 2.82 (p, J = 6.9 Hz, 2H), 2.53 (q, J = 7.5 Hz, 2H), 1.74 (m, 5H), 1.11 (ddd, J = 8.2, 7.1, 0.8 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 174.69, 163.12, 156.56, 143.10, 142.99, 137.03, 134.45, 132.70, 132.67, 132.08, 132.06, 131.89, 131.85, 120.19, 112.47, 67.26, 66.39, 64.97, 57.87, 54.60, 49.82, 43.72, 42.16, 31.41, 27.77, 27.47, 26.07, 25.77, 16.57. HPLC analysis method A: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: gradient from 10% to 40% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 22.362 min, purity >95%, ee >99% (absorbance at 254 nm). HPLC analysis method B: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 30% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 14.389 min, purity >99%, ee >99% (absorbance at 254 nm). HRMS (C₃₅H₄₃N₄O₃Cl + 2H)²⁺ found 302.15901; (C₃₅H₄₃N₄O₃Cl + H⁺) calculated 603.30965, found 603.31020 (error 0.4 ppm).

tert-Butyl ((1-(4-(1,3-dioxoisoindolin-2-yl)butyl)pyrrolidin-2-yl)methyl)carbamate (38).

A suspension of tert-butyl (pyrrolidin-2-ylmethyl)carbamate (500 mg, 2.5 mmol), N-(4-bromobutyl)phthalimide (775 mg, 2.75 mmol), cat. KI (4.15 mg, 25 μmol) and K₂CO₃ (3.45 g, 25 mmol) in ACN (20 mL) was stirred under reflux overnight. The reaction mixture was cooled down to RT, filtered, and the solvent removed under vacuum, the desired product, presenting both N-Boc and N-phthalimide protecting groups, was purified by flash chromatography eluting with 10% DMA and obtained as a yellow oil (940 mg, 85% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.71 (dd, J = 5.5, 3.1 Hz, 2H), 5.00 – 4.95 (m, 1H), 3.77 – 3.64 (m, 2H), 3.27 (d, J = 10.9 Hz, 1H), 3.15 – 2.99 (m, 2H), 2.73 (dt, J = 11.9, 8.1 Hz, 1H), 2.47 (br s, 1H), 2.23 – 2.02 (m, 2H), 1.89 – 1.43 (m, 8H), 1.43 (s, 9H).

3-Chloro-N-((1-(4-(1,3-dioxoisoindolin-2-yl)butyl)pyrrolidin-2-yl)methyl)-5-ethyl-6-hydroxy-2-methoxybenzamide (39).

A solution of 38 (940 mg, 2.34 mmol) and TFA (2.5 mL) in DCM (15 mL) was stirred at RT for 3 h. The reaction was quenched with NaHCO₃ (sat. aq Solution) and extracted with DCM/2-PrOH (3:1). The organic layers were combined, dried over Na₂SO₄, filtered and evaporated under vacuum. The crude material was dissolved in DCM (15 mL), followed by dropwise addition of a solution of 3-chloro-5-ethyl-6-hydroxy-2-methoxybenzoic acid⁵⁴ (647 mg, 2.81 mmol) and HCTU (1.16 g, 2.81 mmol) in DCM (15 mL). The reaction mixture was stirred at RT for 48 h, the solvent was removed under vacuum and the crude material purified by flash chromatography eluting with 5% DMA. The desired product was obtained as a brown oil (310 mg, 26% yield). ¹H NMR (400 MHz, CDCl₃) δ 13.12 (s, 1H), 9.11 (s, 1H), 7.80 (dd, J = 5.4, 3.0 Hz, 2H), 7.69 (dd, J = 5.5, 3.0 Hz, 2H), 7.26 – 7.15 (m, 1H), 3.96 – 3.78 (m, 5H), 3.72 (m, J = 14.0 Hz, 4H), 3.57 (br s, 1H), 3.48 (br s, 1H), 3.26 (br s, 2H), 2.66 – 2.51 (m, 3H), 2.22 (dt, J = 15.0, 7.6 Hz, 1H), 1.99 (tt, J = 13.4, 6.9 Hz, 2H), 1.80 – 1.60 (m, 3H), 1.18 (dt, J = 10.7, 7.5 Hz, 3H).

3-Chloro-N-((1-(4-((4-(dimethylamino)-4-oxo-3,3-diphenylbutyl)amino)butyl)pyrrolidin-2-yl)methyl)-5-ethyl-6-hydroxy-2-methoxybenzamide (40).

Hydrazine (0.2 mL, 50–60% wt. in H₂O) was added to a solution of 39 (310 mg, 0.6 mmol) in EtOH (20 mL) and the solution was stirred at reflux for 3 h. The solvent was removed under vacuum, the residue diluted with 20% K₂CO₃ aq solution and extracted with DCM. The organic layers were combined, dried over Na₂SO₄, filtered and evaporated under vacuum. The obtained crude material was dissolved in DCE (10 mL) and added to a solution of 26 (169 mg, 0.6 mmol) and catalytic AcOH in DCE (10 mL). The mixture was stirred for 10 min at RT and STAB (190 mg, 0.9 mmol) was added portionwise. The reaction was stirred for additional 12 h, the solvent was removed under vacuum and the residue purified by flash chromatography eluting with 10% DMA. The desired product was obtained as a colorless oil (80.5 mg, 21% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.79 (br s, 1H), 7.40 – 7.22 (m, 11H), 3.82 (s, 3H), 3.72 (dt, J = 12.1, 4.0 Hz, 2H), 3.28 – 3.19 (m, 1H), 3.14 (dt, J = 9.5, 4.5 Hz, 1H), 2.95 (br s, 3H), 2.74 – 2.52 (m, 3H), 2.47 – 2.35 (m, 4H), 2.25 (d, J = 14.1 Hz, 4H), 2.12 (q, J = 8.6 Hz, 3H), 1.93 – 1.80 (m, 1H), 1.72 (d, J = 7.8 Hz, 2H), 1.69 – 1.52 (m, 1H), 1.40 (dt, J = 16.4, 8.3 Hz, 4H), 1.20 (dt, J = 27.3, 7.3 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 173.62, 169.49, 160.11, 152.51, 140.71, 140.65, 132.82, 130.67, 128.37, 128.17, 128.16, 128.03, 126.72, 116.01, 108.17, 62.27, 61.41, 60.04, 54.01, 53.80, 49.38, 47.15, 45.23, 40.32, 28.10, 27.73, 26.57, 22.58, 22.51, 13.41. HPLC analysis method A: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 30% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 9.538 and 10.664 min, purity >99%, er 46:54 (absorbance at 254 nm). HPLC analysis method B: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 15% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 22.250 and 25.814 min, purity >99%, er 50:50 (absorbance at 254 nm). HRMS (C₃₇H₄₉N₄O₄Cl + 2H)²⁺ found 325.18021, (C₃₇H₄₉N₄O₄Cl + H⁺) calculated 649.35151, found 649.35221 (error 1.0 ppm).

3,3-Diphenylpyrrolidine (41).

A suspension of LAH (0.56 g, 14.8 mmol) in THF (50 mL) was cooled to 0 °C and a solution of 4-bromo-2,2-diphenylbutanenitrile (1.5 g, 5 mmol) in THF (20 mL) was added dropwise. The mixture was stirred at RT for 15 h, quenched with MeOH (5 mL) and sat. aq NaHCO₃ solution (5 mL), filtered over celite and concentrated under vacuum. The residue was suspended in DCM and washed with sat. Na₂CO₃ solution. The organic layer was dried over Na₂SO₄, filtered and evaporated under vacuum. The crude material was purified by flash chromatography eluting with 10% DMA. The desired product was obtained as a yellow oil (0.450 g, 40% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.34 – 7.22 (m, 8H), 7.22 – 7.13 (m, 2H), 3.52 (s, 2H), 3.13 (t, J = 7.2 Hz, 2H), 2.80 – 2.70 (br s, 1H), 2.52 (t, J = 7.2 Hz, 2H). GC/MS (EI), Rt 9.987 min; 223.2 (M⁺).

1-Methyl-3,3-diphenylpyrrolidine (42).

Methyl chloroformate (85 mg, 69 μL, 0. 9 mmol) was added dropwise to a solution of 41 (100 mg, 0.45 mmol) in THF (10 mL), followed by excess of DIPEA (5 eq.). The mixture was stirred at RT for 1 h, the solvent evaporated under vacuum and the residue dissolved in THF (10 mL). This solution was added dropwise to a suspension of LAH (17 mg, 0.45 mmol) in THF (10 mL), at 0 °C. The mixture was warmed to RT, quenched with MeOH/2N aq NaOH (1:1 ratio, 2 mL), filtered over celite, and the solvents were evaporated under vacuum. The crude material was purified by flash chromatography eluting with 10% DMA to afford the desired product as a colorless oil (60 mg, 56% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.26 (d, J = 4.6 Hz, 8H), 7.20 – 7.11 (m, 2H), 3.22 (s, 2H), 2.82 (td, J = 7.0, 0.6 Hz, 2H), 2.60 (dd, J = 7.7, 6.7 Hz, 2H), 2.42 (s, 3H). GC/MS (EI), Rt 9.650 min; 237.2 (M⁺), purity >99%.

1-(3,3-Diphenylpyrrolidin-1-yl)propan-1-one (43).

A solution of 41 (70 mg, 0.31 mmol), propionyl chloride (58 mg, 0.63 mmol) and DIPEA (5 eq) in DCM (10 mL) was stirred under reflux overnight. The solvent was removed under vacuum and the residue purified by flash chromatography eluting with hex/EtOAc (70/30). The desired product was obtained as a colorless oil (25 mg, 29% yield). ¹H NMR (400 MHz, CDCl₃) rotamer conformations observed (60:40; rotamer A:rotamer B) δ 7.34 – 7.14 (m, 10H), 4.16 (s, 2H, rotamer A), 4.04 (s, 2H, rotamer B), 3.53 (t, J = 6.9 Hz, 2H, rotamer B), 3.42 (t, J = 6.7 Hz, 2H, rotamer A), 2.63 (t, J = 6.7 Hz, 2H, rotamer A), 2.52 (t, J = 6.9 Hz, 2H, rotamer B), 2.41 (q, J = 7.5 Hz, 2H, rotamer B), 2.26 (q, J = 7.5 Hz, 2H, rotamer A), 1.21 (t, J = 7.5 Hz, 3H, rotamer B), 1.16 (t, J = 7.5 Hz, 3H, rotamer A). GC/MS (EI), Rt 12.154 min; 279.1 (M⁺), purity >99%.

2-Chloro-1-(3,3-diphenylpyrrolidin-1-yl)ethan-1-one (44).

2-Chloroacetyl chloride (126 mg, 1.12 mmol) was added dropwise to a solution of 41 (250 mg, 1.12 mmol) in THF (10 mL) at 0 °C, followed by dropwise addition of DIPEA (0.3 mL, 1.68 mmol). The mixture was allowed to warm to RT and stirred for 1 h. The solvent was removed under vacuum and the residue used in the following step without further purification. GC/MS (EI), Rt 12.740 min; 299.1 (M⁺).

tert-Butyl ((1-(2-(3,3-diphenylpyrrolidin-1-yl)-2-oxoethyl)pyrrolidin-2-yl)methyl)carbamate (45).

A mixture of 44 (290 mg, 0.97 mmol), tert-butyl (pyrrolidin-2-ylmethyl)carbamate (194 mg, 0.97 mmol), KI (161 mg, 0.97 mmol) and K₂CO₃ (1.34 g, 9.67 mmol) in ACN (25 ml) was stirred under reflux for 3 h. The mixture was filtered, the solvent evaporated under vacuum and the residue purified by flash chromatography eluting with 10% DMA. The desired product was obtained as a yellow oil (330 mg, 74% yield). ¹H NMR (400 MHz, CDCl₃) rotamer conformations observed δ 7.34 – 7.15 (m, 10H), 5.30 (m, 1H), 3.69 – 3.38 (m, 4H), 3.29 – 2.93 (m, 3H), 2.71 – 2.17 (m, 4H), 1.90 (tt, J = 19.2, 8.6 Hz, 2H), 1.73 (m, J = 17.3, 8.5 Hz, 4H), 1.50 – 1.38 (m, 9H).

3-Chloro-N-((1-(2-(3,3-diphenylpyrrolidin-1-yl)-2-oxoethyl)pyrrolidin-2-yl)methyl)-5-ethyl-6-hydroxy-2-methoxybenzamide (46).

TFA (0.3 mL) was added to a solution of 45 (330 mg, 0.71 mmol) in DCM (10 mL). The mixture was stirred at RT for 2 h, basified with NH₄OH (28% aq solution) and extracted with DCM. The organic layers were combined, dried over Na₂SO₄, filtered and evaporated under vacuum to afford the crude material, which was filtered over a silica pad, eluting and washing with 25% DMA to isolate the desired primary amine intermediate. The amine was dissolved in DCM (10 mL) and added dropwise to a solution of 3-chloro-5-ethyl-6-hydroxy-2-methoxybenzoic acid (70 mg, 0.3 mmol), HCTU (0.2 g, 0.33 mmol) and DIPEA (1.5 eq) in DCM (10 mL). The mixture was stirred at RT for 3 h, the solvent was removed under vacuum and the residue purified by flash chromatography eluting with 5% DMA. The desired product was obtained as a colorless oil (26 mg, 15% yield). ¹H NMR (400 MHz, CDCl₃) rotamer conformations observed δ 13.68 (s, 1H), 8.82 (s, 1H), 7.31 – 7.12 (m, 11H), 4.23 (t, J = 11.0 Hz, 1H), 4.02 (dd, J = 26.1, 11.5 Hz, 1H), 3.85 (ds, J = 17.4 Hz, 3H), 3.79 – 3.68 (m, 1H), 3.62 – 3.21 (m, 6H), 2.96 (br s, 1H), 2.68 – 2.52 (m, 4H), 2.14 – 1.97 (m, 1H), 1.92 – 1.66 (m, 4H), 1.28 – 1.14 (m, 3H); ¹³C NMR (101 MHz, CDCl₃) rotamer conformations observed δ 169.75, 160.07, 160.04, 152.47, 152.46, 144.94, 144.88, 144.79, 133.07, 130.79, 130.77, 128.57, 128.53, 128.51, 126.67, 126.62, 126.59, 126.58, 126.52, 116.16, 116.11, 107.98, 61.57, 61.49, 56.60, 56.14, 56.12, 55.10, 54.87, 54.68, 54.45, 53.40, 52.22, 44.57, 44.39, 40.88, 40.46, 37.52, 35.57, 29.68, 28.38, 28.26, 23.05, 22.86, 22.51, 13.39. HPLC analysis method A: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 10% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 20.539 and 23.859 min, purity >99%, er 51:49 (absorbance at 254 nm). HRMS (C₃₃H₃₈N₃O₄Cl + H⁺) calculated 576.26236, found 576.26297 (error 1.0 ppm).

3-Chloro-N-(((2S,4R)-4-(4-((4-(dimethylamino)-4-oxo-3,3-diphenylbutyl)amino)butoxy)pyrrolidin-2-yl)methyl)-5-ethyl-6-hydroxy-2-methoxybenzamide (48).

A solution of 47 (400 mg, 0.8 mmol), 26 (225 mg, 0.8 mmol) and cat. AcOH (0.05 eq) in DCE (20 mL) was stirred at RT for 1 h, followed by portion-wise addition of STAB (339 mg, 1.6 mmol). The mixture was stirred at RT for 3 h, basified with 10% NH₄OH in MeOH, the solvent evaporated under vacuum and the residue purified by flash chromatography eluting with 10% DMA. The obtained intermediate was dissolved in DCM (20 mL) and TFA (10 mL), and the solution was stirred at RT overnight. The excess of TFA was removed under vacuum, the residue resuspended in aq NH₄OH (pH 9) and extracted with DCM/2-PrOH (3:1). The organic layers were combined, dried over Na₂SO₄, filtered and evaporated to afford the crude material, which was purified by flash chromatography eluting with 25% DMA. The desired product was obtained as a colorless oil (45 mg, 8.5% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.92 (s, 1H), 7.43 – 7.21 (m, 11H), 3.97 (t, J = 4.9 Hz, 1H), 3.89 (s, 3H), 3.57 (dq, J = 9.6, 4.7 Hz, 3H), 3.36 (dt, J = 5.8, 2.8 Hz, 3H), 3.22 (tt, J = 8.9, 4.5 Hz, 2H), 3.02 – 2.90 (m, 5H), 2.69 – 2.55 (m, 4H), 2.45 (dd, J = 21.5, 5.7 Hz, 4H), 2.30 (br s, 2H), 2.00 (dd, J = 13.7, 6.9 Hz, 1H), 1.70 – 1.50 (m, 5H), 1.31 – 1.14 (m, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 174.57, 169.21, 160.06, 152.48, 139.97, 132.89, 130.72, 128.78, 127.96, 127.93, 127.26, 116.16, 108.17, 80.67, 68.17, 61.57, 56.10, 51.78, 43.34, 36.07, 27.16, 22.50, 21.93, 13.41. HPLC analysis method: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 18.518 min, purity >95%, ee >95% (absorbance at 210 nm). The free base was converted into the corresponding oxalate salt. HRMS (C₃₇H₄₉N₄O₅Cl + 2H)²⁺ calculated 333.17685, found 333.17656 (error 0.9 ppm), (C₃₇H₄₉N₄O₅Cl + H)⁺ calculated 665.34642, found 665.34552 (error 1.4 ppm). CHN (C₃₇H₄₉N₄O₅Cl + 2 H₂C₂O₄ + 1.5 H₂O) calculated C 56.45, H 6.47, N 6.42; found C 56.25, H 6.24, N 6.40. mp: Salt decomposes above 90 ° C.

3,3-Bis(3-methoxyphenyl)acrylaldehyde (49).

A solution of bis(3-methoxyphenyl)methanone (1 g, 4.13 mmol) and TiCl₄ (1.0 M solution in DCM, 16.5 mL, 16.5 mmol) in DCM was cooled to 0 °C. TEA (2.3 mL, 16.5 mmol) was added dropwise, the mixture was warmed to RT and stirred overnight⁵⁵. The reaction was quenched with sat. NH₄Cl aq solution and stirred 30 min. The mixture was extracted with DCM, the organic layers were combined, dried over Na₂SO₄, filtered and concentrated under vacuum. The crude material was purified by flash chromatography eluting with hex/EtOAc (95/5). The desired product was obtained as a colorless oil (645 mg, 58% yield). ¹H NMR (400 MHz, CDCl₃) δ 9.60 – 9.53 (m, 1H), 7.42 – 7.25 (m, 2H), 7.07 – 6.77 (m, 6H), 6.64 – 6.56 (m, 1H), 3.89 – 3.74 (m, 6H). GC/MS (EI), Rt 11.647 min; 268.1 (M⁺).

N-(3,3-Bis(3-methoxyphenyl)allyl)-4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine (50).

A solution of 49 (302 mg, 1.12 mmol), 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine (340 mg, 1.12 mmol) and cat. AcOH (0.1 eq) in DCE (20 mL) was stirred for 1 h at RT, followed by portion-wise addition of STAB (715 mg, 3.37 mmol). The reaction was stirred for 3 h, the solvent evaporated under vacuum and the residue purified by flash chromatography eluting with 5% DMA. The desired product was obtained as a colorless oil (550 mg, 88% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.26 (m, 1H), 7.16 (q, J = 7.6 Hz, 2H), 6.90 – 6.79 (m, 3H), 6.79 – 6.67 (m, 4H), 6.67 – 6.60 (m, 1H), 6.20 – 6.11 (m, 1H), 3.74 (d, J = 5.4 Hz, 6H), 3.38 – 3.33 (m, 4H), 3.08 (br s, 4H), 2.74 (m, 2H), 2.58 (br s, 2H), 2.42 (m, 2H), 1.47 (m, 2H), 1.28 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 175.73, 159.49, 159.43, 150.75, 142.64, 140.20, 134.01, 129.39, 129.11, 127.48, 127.47, 124.83, 122.01, 119.88, 118.71, 115.22, 113.32, 112.98, 112.85, 57.48, 55.20, 55.16, 53.39, 52.57, 52.48, 51.42, 50.27, 30.90, 23.33, 23.10, 21.27. HPLC analysis method: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 6.538 min, purity >95% (absorbance at 254 nm). HRMS (C₃₁H₃₇N₃O₂Cl₂ + H)⁺ calculated 554.23356, found 554.23390.

3,3’-(3-((4-(4-Phenylpiperazin-1-yl)butyl)amino)propane-1,1-diyl)diphenol (51).

A suspension of 50 (250 mg, 0.45 mmol) and Pd/C (20% wt. wet, 0.05 eq) in EtOH (10 mL), was shaken in a Parr apparatus under 50 psi pressure of hydrogen gas for 12 h. The mixture was filtered through a pad of celite, the solvent evaporated under vacuum and the residue purified trough a pad of silica eluting with 5% DMA. This intermediate was dissolved in 33% HBr (solution in AcOH, 2 mL) and stirred under reflux for 48 h. The solvent was removed under vacuum and the residue basified with 10% v/v NH₄OH solution in methanol. The crude material was purified by flash chromatography eluting with 25% DMA. The desired product was obtained as a yellow oil (6.8 mg, 29% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.28 – 7.19 (m, 1H), 7.09 (t, J = 7.8 Hz, 2H), 6.90 – 6.80 (m, 2H), 6.71 – 6.60 (m, 4H), 6.55 (s, 2H), 5.44 (br s, 2H), 3.81 (t, J = 7.9 Hz, 1H), 3.09 (t, J = 5.0 Hz, 4H), 2.61 (s, 2H), 2.49 (dt, J = 11.8, 5.4 Hz, 6H), 2.25 (p, J = 7.5 Hz, 4H), 1.30 – 1.16 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 172.49, 157.37, 150.94, 145.43, 129.75, 129.07, 119.90, 119.43, 116.11, 114.54, 114.47, 65.83, 58.11, 52.97, 34.26, 26.57, 25.34, 24.12, 22.59, 15.25. HPLC analysis method: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes +0.1% DEA; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 38.741 min, purity >95% (absorbance at 254 nm). HRMS (C₂₉H₃₇N₃O₂ + H)⁺ found 460.29628.

1-(3,3-Bis(3-methoxyphenyl)allyl)-4-(2-chloro-3-ethylphenyl)piperazine (52).

The reaction was performed following the same procure described for 50, starting from 1-(2-chloro-3-ethylphenyl)piperazine¹⁴ (180 mg, 0.78 mmol). The desired product was obtained as a colorless oil (330 mg, 88% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.24 – 7.11 (m, 3H), 7.00 – 6.66 (m, 8H), 6.31 (t, J = 7.0 Hz, 1H), 3.79 (d, J = 14.0 Hz, 6H), 3.34 (d, J = 6.9 Hz, 2H), 3.14 (br s, 4H), 3.00–2.76 (br s, 4H), 2.76 (q, J = 7.5 Hz, 2H), 1.21 (t, J = 7.5 Hz, 3H).

1-(3,3-Bis(3-methoxyphenyl)propyl)-4-(2-chloro-3-ethylphenyl)piperazine (53).

A suspension of 52 (330 mg, 0.69 mmol) and Pd/C (20% wt. wet, 0.05 eq) in EtOAc:EtOH (10:5 mL), was shaken in a Parr apparatus under 30 psi pressure of H₂ for 3 h. The mixture was filtered through a pad of celite, the solvent evaporated under vacuum and the desired product obtain as a colorless oil (250 mg, 75% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.18 (dt, J = 20.9, 7.8 Hz, 3H), 6.94 (ddd, J = 11.1, 7.8, 1.5 Hz, 2H), 6.90 – 6.67 (m, 6H), 3.96 – 3.86 (m, 1H), 3.82 – 3.67 (br s, 6H), 3.10 (br s, 4H), 2.76 (br s + q, J = 7.5 Hz, 6H), 2.50 (dd, J = 9.9, 5.8 Hz, 2H), 2.06 (m, 2H), 1.29 – 1.15 (t, J = 7.5 Hz, 3H). HRMS (C₂₉H₃₅ClN₂O₂ + H)⁺ calculated 479.24598, found 479.24505 (error 1.9 ppm). The free base (50 mg) was converted into the corresponding oxalate salt. mp: Salt decomposes above 100 ° C.

3-(3-(4-(2-chloro-3-ethylphenyl)piperazin-1-yl)-1-(3-methoxyphenyl)propyl)phenol (54) and 3,3’-(3-(4-(2-chloro-3-ethylphenyl)piperazin-1-yl)propane-1,1-diyl)diphenol (55)

A solution of 53 (200 mg, 0.42 mmol) in 33% HBr (solution in AcOH, 5 mL) and DCM (10 mL) was stirred under reflux for 24 h. The solvent was removed under vacuum, the residue basified with 28–30% v/v aq NH₄OH and extracted with DCM:2-PrOH (3:1). The crude material was purified by flash chromatography eluting with 10% DMA. 54 eluted first as a yellow oil (60 mg, 31% yield): ¹H NMR (400 MHz, CDCl₃) δ 7.20 – 7.07 (m, 3H), 6.94 (dd, J = 7.6, 1.5 Hz, 1H), 6.91 – 6.66 (m, 4H), 6.64 (d, J = 1.0 Hz, 1H), 6.64 – 6.47 (m, 2H), 3.83 (t, J = 7.6 Hz, 1H), 3.73 (s, 3H), 3.01 (br s, 4H), 2.76 (br s + q, J = 7.5 Hz, 6H), 2.49 – 2.36 (m, 2H), 2.35 – 2.15 (m, 2H), 1.36 – 1.17 (t, J = 7.5 Hz, 3H). HRMS (C₂₈H₃₃ClN₂O₂ + H)⁺ calculated 465.23033, found 465.22945 (error 1.9 ppm). 55 eluted second as a yellow oil (25 mg, 13% yield): ¹H NMR (400 MHz, CDCl₃) δ 7.09 (td, J = 7.8, 2.4 Hz, 3H), 6.93 (dd, J = 7.6, 1.5 Hz, 1H), 6.85 – 6.69 (m, 3H), 6.63 (ddd, J = 8.1, 2.5, 0.9 Hz, 2H), 6.55 (t, J = 1.9 Hz, 2H), 3.75 (q, J = 7.9 Hz, 1H), 2.97 (br s, 4H), 2.75 – 2.60 (br s + q, J = 7.6 Hz, 6H), 2.43 (br s, 2H), 2.26 – 2.15 (m, 2H), 1.30 – 1.16 (t, J = 7.5 Hz, 3H). HRMS (C₂₇H₃₁ClN₂O₂ + H)⁺ calculated 451.21468, found 451.21429 (error 0.9 ppm).

trans-Ethyl (Z)-2-((1S,5S)-2-(4-(1H-indole-2-carboxamido)butyl)-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ylidene)acetate (57).

A solution of 56 (20 mg, 66 μmol), N-(4-oxobutyl)-1H-indole-2-carboxamide⁵⁷ (18 mg, 80 μmol) and catalytic AcOH (0.01 eq) in DCE (10 mL) was stirred at RT for 1 h, followed by portion-wise addition of STAB (30 mg, 66 μmol). The reaction was stirred for additional 30 min and basified with 10% NH₄OH solution in MeOH (10 mL), the solvent was evaporated under vacuum and the residue purified by flash chromatography eluting with 20% DMA. The desired product was obtained as a colorless oil (30 mg, 88% yield). ¹H NMR (400 MHz, CDCl₃) δ 9.30 (s, 1H), 7.61 (dd, J = 8.0, 1.0 Hz, 1H), 7.46 – 7.38 (m, 1H), 7.32 – 7.08 (m, 3H), 6.97 – 6.78 (m, 4H), 6.78 – 6.70 (m, 1H), 5.30 (s, 1H), 5.16 (d, J = 17.9 Hz, 2H), 4.13 – 3.92 (m, 2H), 3.63 – 3.44 (m, 2H), 3.20 (ddd, J = 13.2, 8.9, 4.8 Hz, 1H), 2.73 (ddt, J = 17.8, 12.3, 5.8 Hz, 3H), 2.46 (ddd, J = 14.4, 8.7, 6.0 Hz, 1H), 2.27 – 2.00 (m, 5H), 1.90 – 1.50 (m, 6H), 1.14 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 167.91, 166.96, 161.79, 155.61, 148.65, 136.14, 131.04, 129.25, 127.68, 124.33, 121.83, 120.55, 119.46, 115.00, 114.38, 113.60, 111.89, 102.22, 59.92, 55.45, 53.31, 48.76, 45.69, 40.19, 39.34, 38.73, 30.66, 27.22, 24.50, 20.48, 14.13. HPLC analysis method A: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 30.677 min, purity >99%, ee >99% (absorbance at 254 nm). HPLC analysis method B: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 30% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 13.495 min, purity >99%, ee >99% (absorbance at 254 nm). HRMS (C₃₁H₃₇O₄N₃ + H⁺) calculated 516.28568, found 516.28475 (error −1.8 ppm).

Trans-Ethyl (Z)-2-((2S,6R)-6-(((2-(2-(1H-indole-2-carboxamido)ethyl)cyclopropyl)methyl)(ethyl)amino)-2-(3-hydroxyphenyl)-2-methylcyclohexylidene)acetate (58).

The reaction was performed following the same procure described for 57, starting from 56 (20 mg, 66 μmol) and N-(2-(2-formylcyclopropyl)ethyl)-1H-indole-2-carboxamide⁴⁵ (17 mg, 66 μmol). The desired product was obtained as a colorless oil (23 mg, 65% yield). ¹H NMR (400 MHz, CDCl₃) mixture of diastereomers observed δ 9.33 (s, 1H, d.r. 60:40), 7.62 (dd, J = 8.1, 3.8 Hz, 1H), 7.47 – 7.39 (m, 1H), 7.34 – 7.25 (m, 1H), 7.23 – 7.08 (m, 2H), 6.93 (d, J = 10.1 Hz, 1H), 6.89 – 6.69 (m, 3H), 6.60 (s, 1H), 5.25 – 5.10 (m, 2H), 4.13 – 3.95 (m, 2H), 3.72 – 3.49 (m, 2H), 3.16 (d, J = 12.3 Hz, 1H), 2.96 – 2.74 (m, 2H), 2.46 (dd, J = 14.2, 6.9 Hz, 1H), 2.37 – 2.27 (m, 1H), 2.25 – 1.98 (m, 7H), 1.64 – 1.49 (m, 4H), 1.38 – 1.24 (m, 1H), 1.15 (dt, J = 8.2, 7.1 Hz, 3H), 0.89 (d, J = 62.8 Hz, 1H, d.r. 60:40), 0.69 (d, J = 52.6 Hz, 1H, d.r. 60:40), 0.32 (dt, J = 17.8, 5.9 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 166.85, 166.83, 161.95, 161.69, 156.18, 155.88, 136.29, 136.20, 130.95, 130.50, 129.23, 129.13, 127.69, 127.56, 124.62, 124.40, 121.90, 121.85, 120.74, 120.58, 115.31, 115.13, 113.95, 113.82, 112.01, 111.90, 102.62, 60.90, 60.56, 59.87, 59.81, 55.32, 54.77, 50.85, 48.21, 48.08, 45.64, 45.60, 40.24, 40.17, 40.09, 39.74, 33.47, 33.42, 30.86, 20.19, 18.92, 17.04, 16.55, 15.72, 14.17, 14.15, 10.91, 9.23. HPLC analysis method: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 22.572 and 27.023 min, purity >99%, dr 45:55 (absorbance at 254 nm). HRMS (C₃₃H₃₉O₄N₃ + H⁺) calculated 542.30133, found 542.29988 (error −3.6 ppm). The two diastereoisomers 58a and 58b were resolved and separated by preparative chiral HPLC (Chiralpak AD-H 21 mm × 250 mm × 5 μm): mobile phase: gradient from 10% to 40% 2-PrOH in hexanes; temperature: 25 °C; flow rate: 15–18 mL/min; injection volume: 3 mL (~15–20 mg/mL sample concentration); detection at λ 230 nm and 254 nm with the support of ELS detector. 58a eluted first; HPLC analysis method: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 28.905 min, purity >99%, de >99% (absorbance at 254 nm). 58b eluted second; HPLC analysis method: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 34.205 min, purity >99%, de >99% (absorbance at 254 nm).

N-(4-((1S,5S,9R)-9-Hydroxy-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-2-yl)butyl)-1H-indole-2-carboxamide (61).

The reaction was performed following the same procure described for 57, starting from (1S,5S,9R)-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ol (59; 40 mg, 0.17 mmol). The desired product was purified by flash chromatography eluting with 20% DMA and obtained as a white solid (20 mg, 26% yield). ¹H NMR (400 MHz, CD₃OD) δ 7.57 (ddt, J = 9.0, 8.0, 1.0 Hz, 2H), 7.41 (dt, J = 8.2, 0.8 Hz, 2H), 7.24 – 6.94 (m, 4H), 6.89 – 6.73 (m, 2H), 6.66 – 6.58 (m, 1H), 4.25 (m, 1H), 4.07 (s, 1H), 3.74 (d, J = 0.5 Hz, 2H), 3.46 (d, J = 6.1 Hz, 1H), 3.44 – 3.25 (m, 3H), 2.96 (d, J = 9.8 Hz, 1H), 2.70 (br s, 3H), 2.48 (td, J = 13.5, 7.8 Hz, 2H), 2.31 (d, J = 12.8 Hz, 1H), 2.23 – 2.08 (m, 1H), 1.93 – 1.61 (m, J = 4.2, 3.2 Hz, 6H), 1.28 (br s, 1H). HPLC analysis method: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 50% 2-PrOH in hexanes + 0.1% DEA; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 29.790, purity >99%, ee >99% (absorbance at 254 nm). HRMS (C₂₇H₃₄O₃N₃ + H⁺) calculated 448.25947, found 448.25979.

N-(4-((1S,5S,9S)-9-Hydroxy-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-2-yl)butyl)-1H-indole-2-carboxamide (62).

The reaction was performed following the same procure described for 57, starting from (1S,5S,9S)-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ol (60; 40 mg, 0.17 mmol). The desired product was purified by flash chromatography eluting with 20% DMA and obtained as a white solid (20 mg, 26% yield). ¹H NMR (400 MHz, CD₃OD) δ 7.62 – 7.53 (m, 2H), 7.42 (t, J = 8.3 Hz, 2H), 7.25 – 7.12 (m, 2H), 7.12 – 6.80 (m, 4H), 6.40 (m, 1H), 4.30 (m, 1H), 4.07 (s, 1H), 3.75 (s, 2H), 3.49 – 3.37 (m, 4H), 2.98 (br s, 1H), 2.20 – 1.93 (m, 7H), 1.73 – 1.66 (m, 5H), 1.28 (br s, 2H). HPLC analysis method: Chiralpak AD-H analytical column (4.5mm × 250mm – 5 μm particle size); mobile phase: isocratic 50% 2-PrOH in hexanes + 0.1% DEA; flow rate: 1 mL/min; injection volume: 20 μL; sample concentration: ~1 mg/mL; multiple DAD λ absorbance signals measured in the range of 210–280 nm, Rt 32.837, purity >99%, ee >99% (absorbance at 254 nm). HRMS (C₂₇H₃₄O₃N₃ + H⁺) calculated 448.25947, found 448.25982.

2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethan-1-ol (63).

A solution of 1-(2,3-dichlorophenyl)piperazine hydrochloride (500 mg, 1.87 mmol) and K₂CO₃ (1.29 g, 9.34 mmol) in ACN (30 mL) was stirred for 25 min under gentle heating, followed by dropwise addition of 2-bromoethan-1-ol (257 mg, 2.06 mmol) and cat. KI. The suspension was stirred under reflux overnight, filtered, and the solvent evaporated under vacuum to afford the crude material. The desired product was purified by flash chromatography eluting with 100% EtOAc and obtained as a yellow oil (310 mg, 60% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.21 – 7.10 (m, 2H), 6.96 (dd, J = 6.7, 2.8 Hz, 1H), 3.72 – 3.62 (m, 2H), 3.08 (br s, 4H), 2.71 (br s, 4H), 2.67 – 2.60 (m, 2H).

2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl 4-methylbenzenesulfonate (64)

p-toluenesulfonyl chloride (p-TsCl, 200 mg, 1.05 mmol) was added portion wise to a solution of 63 (240 mg, 0.87 mmol) and DIPEA (0.310 mL, 225 mg, 1.74 mmol) in DCM (15 mL). The mixture was stirred at RT overnight, the solvent evaporated under vacuum, and the crude residue purified by flash chromatography eluting with hex/EtOAc (1/1). The desired product was obtained as a white solid (135 mg, 36% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.81 (d, J = 7.9 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 7.18 – 7.08 (m, 2H), 6.91 (dd, J = 6.8, 2.9 Hz, 1H), 4.16 (t, J = 5.7 Hz, 2H), 3.06 – 2.99 (br s, 4H), 2.72 (t, J = 5.8 Hz, 2H), 2.61 (br s, 4H), 2.44 (s, 3H).

Ethyl (Z)-2-((1S,5S)-2-(2-(4-(2,3-dichlorophenyl)piperazin-1-yl)ethyl)-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ylidene)acetate (65).

A suspension of 56 (50 mg, 0.175 mmol), 64 (71.5 mg, 0.166 mmol) and NaHCO₃ (100 mg, 1.2 mmol) in ACN (7 mL) was heated and stirred in a sealed vessel for 6 h. The reaction mixture was filtered, the solvent evaporated under vacuum and the residue purified by flash chromatography eluting with 10% DMA. The desired product was obtained as a yellow oil (40 mg, 43% yield). ¹H NMR (400 MHz, CD₃OD) δ 7.33 – 7.14 (m, 3H), 7.14 – 7.05 (m, 1H), 6.92 (m, 2H), 6.65 (m, 1H), 5.48 (s, 1H), 4.08 (dq, J = 21.4, 7.1 Hz, 2H), 3.34 – 3.21 (m, 2H), 3.08 (br s, 4H), 2.97 – 2.70 (m, 10H), 2.56 – 2.42 (m, 1H), 2.22 – 1.98 (m, 4H), 1.74 (m, 1H), 1.65 (s, 1H), 1.19 (t, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 168.38, 167.92, 158.33, 152.58, 149.48, 134.91, 130.18, 129.03, 128.40, 125.82, 120.17, 119.71, 116.02, 115.67, 114.48, 111.42, 59.50, 57.16, 56.04, 54.80, 54.16, 52.08, 46.63, 41.34, 39.27, 31.73, 21.35, 14.54. The free base was converted into the corresponding oxalate salt. HRMS (C₃₀H₃₇N₃O₃Cl₂ + H⁺) calculated 558.22847, found 558.22925 (error 1.4 ppm). CHN (C₃₀H₃₇N₃O₃Cl₂ + 2.5 H₂C₂O₄ + 2 H₂O) calculated C 51.29, H 5.66, N 5.13; found C 51.07, H 5.28, N 5.21. mp: Salt decomposes above 110 ° C.

Radioligand Binding Studies

hD₂R, hD₃R, and hD₄R.

Radioligand binding assays were conducted similarly as previously described^{45, 67}. HEK293 cells stably expressing human D₂LR or D₃R or D_4.4 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. For [³H]-(R)-(+)-7- OH-DPAT binding studies, membranes were harvested fresh; the binding buffer was made from 50 mM Tris, 10 mM MgCl₂, 1 mM EDTA, pH 7.4. 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 concentration, 0.1% AcOH (final concentration v/v) was added alongside the vehicle. 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, 10–20, and 20–30 μg/well total protein for hD_2LR, hD₃R, and hD_4.4R, respectively; for [³H]-(R)-(+)-7- OH-DPAT assays: 40–80, and 20–40 μg/well total protein for hD_2LR, and hD₃R, respectively), and 50 μL of radioligand diluted in binding buffer ([³H]-N-methylspiperone: 0.4 nM final concentration for all the hD₂-like receptor subtypes; [³H]-(R)-(+)-7-OH-DPAT: 1.5 nM final concentration for hD₂L, and 0.5 nM final concentration for hD₃; Perkin Elmer). 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. All compound dilutions were tested in triplicate, and the reaction incubated for 60 min ([³H]-N-methylspiperone assays) or 90 min ([³H]-(R)-(+)-7-OH-DPAT assays) at RT. The reaction was terminated by filtration through PerkinElmer Uni-Filter-96 GF/B, 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 Å~ 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 equation⁶³. K_d values 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 8 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 (SEM).

hMOR.

Radioligand binding experiments were conducted, and the results analyzed, as described above, and similarly as previously reported⁶². 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; Perkin Elmer) 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/B, presoaked for 60 min in 0.5% polyethylenimine. The non-specific binding was determined using 10 μM C-TOP or cold DAMGO. The radioligand K_d was measured via radioligand saturation experiments.

Bioluminescence Resonance Energy Transfer (BRET) Studies

All reagents were purchased from Sigma Aldrich-Merck unless otherwise stated. BRET experiments were performed in transiently transfected human embryonic kidney 293 T (HEK 293T) cells, as described previously.^{31, 68} Briefly, cells were grown and maintained at 37 °C in 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 (2.5 × 10⁶ cells per dish) and allowed to grow overnight in media at 37 °C, 5 % CO₂. The following day, cells were transiently transfected 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, 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 GPA⁶⁹ and 4 μg of arrestin-3-Venus, 2 μg of WT-GRK2 and 1 μg of mMOR-Rluc8 for arrestin-3 recruitment. Cells were then allowed to grow overnight at 37 °C, 5 % CO₂. The next day, cells were plated in Greiner poly-D-lysine-coated 96-well plates (SLS) in media and allowed to grow overnight. 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. For the antagonist-mode assays that require pre-incubation, the cells were washed once with D-PBS and incubated with ligands in D-PBS (supplemented with 10 mM glucose) at 37 °C, 5 % CO₂ for 3h prior to starting the assay. The Rluc substrate coelenterazine h (NanoLight) was added to each well (final concentration of 5 μM) and cells were incubated for 5 minutes at 37 °C. After 5 minutes, ligands (final concentration from 10 μM to 1 nM in D-PBS) were added to the plate and cells were incubated for a further 10 minutes at 37 °C before reading the plate in a PHERAstar FSX microplate reader (Venus and Rluc emission signals at 535 and 475 nm respectively, BMG Labtech). The ratio of Venus:Rluc counts was used to quantify the BRET signal in each well. Data were normalized to the wells containing 10 μM DAMGO/quinpirole or no drug for maximal or minimal response, respectively and as indicated in the figure legends. All experiments were performed in duplicate and at least three times independently. All data points represent the mean and error bars represent the standard error of the mean (SEM) and were fitted using the built-in log(agonist) vs. response (three parameters) model in Prism 8.0 (GraphPad software Inc., San Diego, CA).For agonist-mode assays data was fitted to a three parameter concentration-response model where EC₅₀ is the concentration of the agonist needed to elicit half the maximal response of the particular agonist, defined as E_max. For the antagonist-mode assays, data points were fitted using a three-parameter concentration-response model where IC₅₀ is the concentration required to inhibit half the maximum response of the agonist used at a particular concentration. Values of pEC₅₀ or pIC₅₀ plus/minus error are given as the error has a gaussian distribution whereas the error associated with the antilog value does not. For some ligands for which the lower asymptote of the curve was not well defined within the concentration range (represented by a dotted line) the bottom was constrained to be equal to 0%.

Molecular Docking and Computer Aided Drug Design

The receptor structures corresponding to PDB accession code 6CM4, 3PBL, and 5C1M were extracted from RCSB for inactive state D₂R, inactive state D₃R, and active state MOR, respectively. All the objects except the receptor protein subunit, the crystallized ligand, and in case of active state MOR three crystallographic waters were deleted, and this was followed by the addition of hydrogens and optimization of the side chain residues. Ligands were sketched, assigned formal charges, and energy-optimized prior to docking. The ligand docking box for potential grid docking was defined as the whole extracellular half of the protein, and all-atom docking was performed using the energy minimized structures for all ligands with a thoroughness value of 10. The best-scored docking solutions were further optimized by several rounds of minimization and Monte Carlo sampling of the ligand conformation, including the surrounding side-chain residues (within 5 A° of the ligand) and the three crystallographic waters in the MOR orthosteric sites. All the above molecular modeling operations were performed in ICM-Pro v3.8–5 molecular modeling package.

ABBREVIATIONS

DA

dopamine

D_2-likeR

dopamine D_2-like receptors

OUD

opioid use disorders

CADD

computer aided drug design

BRET

bioluminescence resonance energy transfer

HEK 293 cells

human embryonic kidney 293 cells

HPLC

high performance liquid chromatography

HRMS

high resolution mass spectrometry

DMA

dichloromethane, methanol, ammonium hydroxide

EDC

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

HOBt

hydroxybenzotriazole

HCTU

O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

DIPEA

N,N-diisopropylethylamine

STAB

sodium triacetoxyborohydride

DMP

dess–martin periodinane

ACN

acetonitrile

LAH

lithium aluminium hydride

GPA

G-protein Activation