We describe a streamlined one-pot strategy to construct highly functionalized protoberberine frameworks through a tandem Mannich/intramolecular acylation and copper-promoted decarboxylative olefination sequence. This operationally straightforward process delivers a wide array of derivatives in consistently high yields. Mechanistic investigations, including isolation of key intermediates, support a radical-driven decarboxylative olefination pathway that develops complex protoberberine-type alkaloids with exceptional efficiency. The utility of this synthetic platform is demonstrated through concise total syntheses of the neuroactive tetrahydroprotoberberine alkaloids (±)-isocorypalmine and (±)-stepholidine. Collectively, this chemistry establishes a concise and practical avenue for accessing valuable protoberberine scaffolds from readily available precursors.

Dopamine receptors (DRs) have been implicated in numerous disorders and diseases (e.g., Alzheimer's disease, Parkinson's disease, schizophrenia, and substance use disorders) and have served as attractive drug targets for these ailments. Despite their potential clinical utility, the development of selective DR ligands has been challenging due to difficulties in selectivity among the DR subtypes as well as other biogenic amine receptors and poor pharmacokinetic properties. The realization of their full potential necessitates continued advancements in DR ligands as investigative tools. This review aims to highlight the recent developments made in the chemical biology of DR ligands (e.g., bivalent ligands, photoactivatable ligands, photoswitchable probes, and fluorescent probes).

There is significant interest in identifying dopamine D3 receptor (D3R) antagonists and partial agonists with favorable drug-like properties as in vivo chemical tools and as experimental therapeutics for substance use disorders. We hypothesized that preserving the classical D3R antagonist pharmacophore while incorporating metabolically favorable structural features into the secondary pharmacophore ("tail") region would enable the discovery of selective D3R-targeted ligands with good ADME characteristics. Specifically, orthosteric binding site ("head") fragments known to confer D3R affinity were coupled with "tail" motifs inspired by non-catechol D1 receptor agonists, which have demonstrated enhanced pharmacokinetic stability. From this work, we have identified a new D3R ligand compound 8o. Compound 8o exhibits high affinity for D3R (2.7 nM) with >30-fold binding preference versus other dopamine receptors. Preliminary functional activity studies suggest that compound 8o is a D3R partial agonist. Docking studies indicate that 8o exhibits D3R interactions that are consistent with a bitopic mode of binding. Compound 8o also shows favorable in vitro ADME properties, that are predictive of good drug-like behavior, making it a promising lead molecule for future D3R in vivo probe and therapeutic optimization.

An intramolecular iridium-catalyzed hydroarylation process is described for the synthesis of the title scaffold from phenacyl enamide substrates. This method achieves high yields while eliminating the requirement for prefunctionalized substrates, as compared to Heck-type cyclization methodologies. Substrate scope and mechanistic studies indicate that the presence of electron-donating substituents in the incipient ring D moiety is essential for the cyclization, consistent with a pathway involving electrophilic aromatic substitution of iridium in ring D.

Aporphines are a class of isoquinoline alkaloids that are endowed with a range of biological activities. The 5-HT7R is an emerging biological target for prostate cancer therapeutics. In this manuscript, we report the synthesis and evaluation of aporphine enantiomers as 5-HT7R ligands, as well as their activity in inhibiting the proliferation of prostate cancer cells (specifically, PC3). The (S)-enantiomers displayed higher affinity at the 5-HT7R than the racemates and the (R)-enantiomer counterparts. The (S)-enantiomers were found to be antagonists at the 5-HT7R. Racemates as well as their respective enantiomers were selective for the 5-HT7R receptor over other serotonin and dopamine receptors evaluated. In the anticancer activity assays, the compounds showed more potent cytotoxic effects than the selective 5-HT7R antagonist control SB269970. However, no correlation was observed between the 5-HT7R affinity or 5-HT7R antagonist activity and anticancer potency, suggesting that other non-5-HT7R mechanisms play a role in the anticancer effects of the compounds. Compounds (R)-1 and (R)-4 were identified as the most potent anti-proliferative compounds and will be useful as lead molecules for prostate cancer therapeutic development in future studies.

We undertook the rational design and synthesis of a novel series of ligands intended to function as selective dual dopamine D1 receptor (D1R) partial agonists and D3 receptor (D3R) antagonists. The molecular architecture of these compounds was derived by integrating key pharmacophoric features from established D1R partial agonists and D3R antagonists. Specifically, the 6-(2-methylphenyl)-1,5-dimethylpyrimidine-2,4(1H,3H)-dione scaffold was employed as the core "tail" region associated with D1R partial agonism, while various substituted phenyl piperazine moieties were introduced as "head" groups to confer D3R antagonistic activity. A pyridine ring was utilized as a central linker across the series. Contrary to the intended dopaminergic profile, these compounds exhibited markedly higher binding affinities for α2-adrenergic receptors (α2-ARs) relative to their activity at dopamine receptor subtypes. Several analogues demonstrated potent α2C-AR binding affinities in the low nanomolar range (Ki = 7-30 nM), with moderate selectivity (up to 17-fold) over other α2-AR subtypes. Notably, compounds bearing ortho-substituted aryl groups within the "head" domain generally displayed enhanced α2C-AR binding compared to their para-substituted counterparts. Molecular docking studies conducted at both α2A-AR and α2C-AR suggested that multiple receptor-ligand interactions contribute to the observed binding profiles. In particular, an anion-pi interaction between Asp131 of α2C-AR and the phenyl ring of the phenyl piperazine "head" moiety was identified as a possible determinant of the increased α2C-AR affinity observed in ortho-substituted analogues. Given the therapeutic potential of selective α2C-AR targeting in treating various disorders, coupled with the limited availability of clinically approved selective α2C-AR ligands, the discovery of this new scaffold offers new prospects for drug discovery targeting α2C-ARs.

Due to their evolutionary bias as ligands for biologically relevant drug targets, natural products offer a unique opportunity as lead compounds in drug discovery. Given the involvement of dopamine receptors in various physiological and behavioral functions, they are linked to numerous diseases and disorders such as Parkinson's disease, schizophrenia, and substance use disorders. Consequently, ligands targeting dopamine receptors hold considerable therapeutic and investigative promise. As this perspective will highlight, dopamine receptor targeting natural products play a pivotal role as scaffolds with unique and beneficial pharmacological properties, allowing for natural product-inspired drug design and lead optimization. As such, dopamine receptor targeting natural products still have untapped potential to aid in the treatment of disorders and diseases related to central nervous system (CNS) and peripheral nervous system (PNS) dysfunction.

To illuminate the tolerance of fluoroalkoxylated groups at the C-3 and C-9 positions of tetrahydroprotoberberines (THPBs) on D1R activity, C-3 and C-9 fluoroalkoxylated analogues of (S)-12-bromostepholidine were prepared and evaluated. All compounds examined were D1R antagonists as measured by a cAMP assay. Our structure-activity studies herein indicate that the C-3 position tolerates a 1,1-difluoroethoxy substituent for D1R antagonist activity. Compound 13a was the most potent cAMP-based D1R antagonist identified and was also found to antagonize 8-arrestin translocation in a TANGO assay. Affinity assessments at other dopamine receptors revealed that 13a is selective for D1R and unlike other naturally-occurring THPBs such as (S)-stepholidine, lacks D2R affinity. In preliminary biopharmaceutical assays, excellent BBB permeation was observed for 13a. Further pharmacological studies are warranted on (S)-stepholidine congeners to harvest their potential as a source of novel, druggable D1R-targeted agents.

Systematically discovering protein-ligand interactions across the entire human and pathogen genomes is critical in chemical genomics, protein function prediction, drug discovery, and many other areas. However, more than 90% of gene families remain "dark"-i.e., their small-molecule ligands are undiscovered due to experimental limitations or human/historical biases. Existing computational approaches typically fail when the dark protein differs from those with known ligands. To address this challenge, we have developed a deep learning framework, called PortalCG, which consists of four novel components: (i) a 3-dimensional ligand binding site enhanced sequence pre-training strategy to encode the evolutionary links between ligand-binding sites across gene families; (ii) an end-to-end pretraining-fine-tuning strategy to reduce the impact of inaccuracy of predicted structures on function predictions by recognizing the sequence-structure-function paradigm; (iii) a new out-of-cluster meta-learning algorithm that extracts and accumulates information learned from predicting ligands of distinct gene families (meta-data) and applies the meta-data to a dark gene family; and (iv) a stress model selection step, using different gene families in the test data from those in the training and development data sets to facilitate model deployment in a real-world scenario. In extensive and rigorous benchmark experiments, PortalCG considerably outperformed state-of-the-art techniques of machine learning and protein-ligand docking when applied to dark gene families, and demonstrated its generalization power for target identifications and compound screenings under out-of-distribution (OOD) scenarios. Furthermore, in an external validation for the multi-target compound screening, the performance of PortalCG surpassed the rational design from medicinal chemists. Our results also suggest that a differentiable sequence-structure-function deep learning framework, where protein structural information serves as an intermediate layer, could be superior to conventional methodology where predicted protein structures were used for the compound screening. We applied PortalCG to two case studies to exemplify its potential in drug discovery: designing selective dual-antagonists of dopamine receptors for the treatment of opioid use disorder (OUD), and illuminating the understudied human genome for target diseases that do not yet have effective and safe therapeutics. Our results suggested that PortalCG is a viable solution to the OOD problem in exploring understudied regions of protein functional space.

We evaluated C-3 alkoxylated and C-3/C-9 dialkoxylated (-)-stepholidine analogues to probe the tolerance at the C-3 and C-9 positions of the tetrahydroprotoberberine (THPB) template toward affinity for dopamine receptors. A C-9 ethoxyl substituent appears optimal for D1R affinity since high D1R affinities were observed for compounds that contain an ethyl group at C-9, with larger C-9 substituents tending to decrease D1R affinity. A number of novel ligands were identified, such as compounds 12a and 12b, with nanomolar affinities for D1R and no affinity for either D2R or D3R, with compound 12a being identified as a D1R antagonist for both G-protein- and β-arrestin-based signaling. Compound 23b was identified as the most potent and selective D3R ligand containing a THPB template to date and functions as an antagonist for both G-protein- and β-arrestin-based signaling. Molecular docking and molecular dynamics studies validated the D1R and D3R affinity and selectivity of 12a, 12b, and 23b.

The 1-phenylbenzazepine template has yielded a number of D1R-like ligands, which, though useful as pharmacological tools, have significant drawbacks in terms of selectivity versus D5R as well as pharmacokinetic behavior. A number of 1-phenylbenzazepines contain a 6-chloro functional group, but extensive SAR studies around the 6-chloro-1-phenylbenzazepine framework have not been reported in the literature. To further understand the tolerance of the 6-chloro-1-phenylbenzazepine template for various substituent groups towards affinity and selectivity at D1R, we synthesized two series of analogs with structural variations at the C-7, C-8, N-3, C-3′ and C-4′ positions. The series 2 analogs differed from series 1 analogs in possessing a nitrogenated functionality at C-8 and lacked a C-4′ substituent, but were otherwise similar. Analogs were assessed for affinity at D1R, D2R and D5R. For both series, we found that the analogs lacked affinity for D2R and showed modest D1R versus D5R selectivity. For series 1 analogs, an N-3 methyl substituent group was better tolerated than N-H or an N-3 allyl substituent. The C-8 position appears to be tolerant of amino and methanesulfonamide substituents for high D1R affinity, but C-8 amides displayed low to moderate D1R affinities. A C-3′ methyl substituent appeared to be critical for the D1R affinity of some analogs, but the C-4′ substituents tried (hydroxy and methoxy; series 1) did not result in any significant boost in D1R affinity. Compound 15a was the most potent and selective D1R ligand identified from these studies (Ki at D1R = 30 nM; 6-fold selectivity versus D5R). Further functional activity assessments indicate that 15a functions as a D1R antagonist towards cAMP-mediated signaling. The predicted drug-like properties of 15a are encouraging for further pharmacological assessments on the compound.

The 1-phenylbenzazepine scaffold has yielded several D1R targeting ligands, but some gaps remain in our understanding of the structure-activity relationships in this scaffold. In particular, there is a paucity of studies that have investigated the effects of substituents at the C2' position of 1-phenylbenzazepines on their affinity and selectivity towards D1R. In this study, a set of methyl- and fluoro- C2'-substituted 1-phenylbenzazepines, with ring A catechol or 8-hydroxy-7-methoxy moieties in tandem with N-methyl or N-allyl substituent groups, was synthesized and evaluated for affinity at a subset of dopamine receptors - D1R, D2R and D5R. These studies indicate that an N-methyl group is generally preferred over N-unsubstituted or N-allyl groups for strong D1R affinity. In addition, it was revealed that compounds with a ring A 8-hydroxy-7-methoxy motif displayed stronger D1R affinity than analogous compounds with a ring A catechol moiety. Furthermore, the presence of a C2' substituent does not significantly impact D1R selectivity over D5R. However, for all analogs assessed, D1R selectivity over D2R was maintained. D1R vs D5R selectivity was generally poor or modest (less than 10-fold) among members of the series. A new high affinity selective D1R ligand - 10b (K-i = 5.7 nM), was identified in this study; further pharmacological characterization indicates that 10b is an antagonist at D1R (IC50 = 10.7 nM). Docking studies on 10b indicate that a number of interactions with hydrophobic residues (Trp321, Val317, Phe313, Phe289, Phe288, Phe285, Phe203, Tyr194, Leu190, Ser188, His 164, Ile104, Val100 and Trp99) in addition to the typical N-Asp103 salt bridge are important for its D1R affinity.

Aporphine alkaloids have shown affinity for serotonin receptors (5-HTRs), and there has been a recent upsurge of interest in aporphines as 5-HT2CR ligands. 1,2,9,10-Tetraoxygenated aporphine alkaloids in particular have demonstrated good affinity for 5-HTRs. In continued efforts to understand the impacts of structural modification of the 1,2,9,10-tetraoxygenated aporphine template on affinity, selectivity, and activity at 5-HT2R subtypes, we used (+)-boldine (8) as a semisynthetic feedstock in the preparation of C-2-alkoxylated (+)-predicentrine analogues. Compound 10n, which contains a benzyloxy group at C-2, has been identified as a novel 5-HT2CR ligand with strong affinity (4 nM) and moderate selectivity versus 5-HT2BR and 5-HT2AR (12-fold and 6-fold, respectively). Compound 10n functions as an antagonist at 5-HT2A and 5-HT2C receptors. Computational experiments indicate that several hydrophobic interactions as well as strong H-bond and salt bridge interactions between the protonated amine moiety in 10n and Asp134 are responsible for the potent 5-HT2CR affinity of this compound. Furthermore, compound 10n displays favorable predicted drug-like characteristics, which is encouraging toward future optimization.

Discovering chemical-protein interactions for millions of chemicals across the entire human and pathogen genomes is instrumental for chemical genomics, protein function prediction, drug discovery, and other applications. However, more than 90% of gene families remain dark, i.e., their small molecular ligands are undiscovered due to experimental limitations and human biases. Existing computational approaches typically fail when the unlabeled dark protein of interest differs from those with known ligands or structures. To address this challenge, we developed a deep learning framework PortalCG. PortalCG consists of four novel components: (i) a 3-dimensional ligand binding site enhanced sequence pre-training strategy to represent the whole universe of protein sequences in recognition of evolutionary linkage of ligand binding sites across gene families, (ii) an end-to-end pretraining-fine-tuning strategy to simulate the folding process of protein-ligand interactions and reduce the impact of inaccuracy of predicted structures on function predictions under a sequence-structure-function paradigm, (iii) a new out-of-cluster meta-learning algorithm that extracts and accumulates information learned from predicting ligands of distinct gene families (meta-data) and applies the meta-data to a dark gene family, and (iv) stress model selection that uses different gene families in the test data from those in the training and development data sets to facilitate model deployment in a real-world scenario. In extensive and rigorous benchmark experiments, PortalCG considerably outperformed state-of-the-art techniques of machine learning and protein-ligand docking when applied to dark gene families, and demonstrated its generalization power for off-target predictions and compound screenings under out-of-distribution (OOD) scenarios. Furthermore, in an external validation for the multi-target compound screening, the performance of PortalCG surpassed the human design. Our results also suggested that a differentiable sequence-structure-function deep learning framework where protein structure information serve as an intermediate layer could be superior to conventional methodology where the use of predicted protein structures for predicting protein functions from sequences. We applied PortalCG to two case studies to exemplify its potential in drug discovery: designing selective dual-antagonists of Dopamine receptors for the treatment of Opioid Use Disorder, and illuminating the undruggable human genome for targeting diseases that do not have effective and safe therapeutics. Our results suggested that PortalCG is a viable solution to the OOD problem in exploring the understudied protein functional space. Author Summary Many complex diseases such as Alzheimer’s disease, mental disorders, and substance use disorders do not have effective and safe therapeutics due to the polygenic nature of diseases and the lack of thoroughly validate drug targets and their ligands. Identifying small molecule ligands for all proteins encoded in the human genome will provide new opportunity for drug discovery of currently untreatable diseases. However, the small molecule ligand of more than 90% gene families is completely unknown. Existing protein-ligand docking and machine learning methods often fail when the protein of interest is dissimilar to those with known functions or structures. We develop a new deep learning framework PortalCG for efficiently and accurately predicting ligands of understudied proteins which are out of reach of existing methods. Our method achieves unprecedented accuracy over state-of-the-arts by incorporating ligand binding site information and sequence-to-structure-to-function paradigm into a novel deep meta-learning algorithms. In a case study, the performance of PortalCG surpassed the human design. The proposed computational framework will shed new light into how chemicals modulate biological system as demonstrated by applications to drug repurposing and designing polypharmacology. It will open a new door to developing effective and safe therapeutics for currently incurable diseases. PortalCG can be extended to other scientific inquiries such as predicting protein-protein interactions and protein-nucleic acid recognition.

The effect of rigidification of the n-butyl linker region of tetrahydroisoquinoline-containing D3R ligands via inclusion of an o-xylenyl motif was examined in this study. Generally, rigidification with an o-xylenyl linker group reduces D3R affinity and negatively impacts selectivity versus D2R for compounds possessing a 6-methoxy-1,2,3,4,-tetrahydroisoquinolin-7-ol primary pharmacophore group. However, D3R affinity appears to be regulated by the primary pharmacophore group and high affinity D3R ligands with 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline primary pharmacophore groups were identified. The results of this study also indicate that D3R selectivity versus the σ2R is dictated by the benzamide secondary pharmacophore group, this being facilitated with 4-substituted benzamides. Compounds 5s and 5t were identified as high affinity (Ki < 4 nM) D3R ligands. Docking studies revealed that the added phenyl ring moiety interacts with the Cys181 in D3R which partially accounts for the strong D3R affinity of the ligands.

A series of 1-phenylbenzazepines containing bromine or chlorine substituents at the ortho position of the appended phenyl ring (2'-monosubstituted or 2',6'- disubstituted patterns) were synthesized and evaluated for affinity towards dopamine D1R, D2R and D5R. As is typical of the 1-phenylbenzazepine scaffold, the compounds displayed selectivity towards D1R and D5R; analogs generally lacked affinity for D2R. Interestingly, 2',6'-dichloro substituted analogs showed modest D5R versus D1R selectivity whereas this selectivity was reversed in compounds with a 2'-halo substitution pattern. Compound 10a was identified as a D1R antagonist (K-1 = 14 nM; IC50 = 9.4 nM).

Heterocycles that bear the novel 5,6,14,14a-tetrahydro-8H-benzo[6,7][1,4] thiazepino[3,4-a]isoquinoline and the 5,6,14,14a-tetrahydro-8H-13l2-benzo [6,7][1,4]diazepino[3,4-a]isoquinoline frameworks were synthesized in a facile manner. These tetrahydroprotoberberine (THPB)-inspired scaffolds demonstrate selective affinity for the σ1R in contrast to the naturally occurring THPB congeners that show D1R and σ2R selectivity.

The purpose of this study was to investigate the effects of (-)-stepholidine (SPD), a compound with dopamine D1 partial agonist and D2/D3 antagonist properties, on the development and expression of cocaine conditioned place preference (CPP). Subjects (N = 65; male Long Evans rats) were tested using a CPP procedure consisting of 3 phases: (1) a 15-min pre-exposure session where animals could explore each compartment freely, (2) eight 30-min conditioning sessions where animals were restricted to one side or the other with cocaine (10 mg/kg) or saline, respectively, on alternating days and (3) a 15-minute preference test session where animals could explore each compartment freely. To test the effects of SPD on expression of cocaine CPP, rats were administered vehicle (distilled water with 20 % DMSO), 10, 15 or 20 mg/kg SPD (intraperitoneally) 30 min prior to the test session. We found that 20 mg/kg of SPD significantly blocked the expression of cocaine CPP. To test the effects of SPD on the development of CPP, 0 (vehicle), 10, 15 or 20mg/kg SPD were administered 30 min prior to each cocaine conditioning session and vehicle before each saline conditioning session; no treatment was given prior to the test session. A preference test showed that each SPD group maintained a CPP similar to the vehicle group. These data indicate that SPD can block the expression of a cocaine CPP but has no effect on its development, suggesting that it inhibits the effects of cocaine cues on cocaine incentive motivated behavior. These results suggest that SPD may be a potential treatment for cue-driven aspects of cocaine use disorder.

The novel 1,3,4,11b-tetrahydro-1H-fluoreno[9,1-cd]azepine framework, a structurally rigidified variant of the 1-phenylbenzazepine template, was synthesized via direct arylation as a key reaction. Evaluation of the binding affinities of the rigidified compounds across a battery of serotonin, dopamine, and adrenergic receptors indicates that this scaffold unexpectedly has minimal affinity for D-1 and other dopamine receptors and is selective for the 5-HT6 receptor. The affinity of these systems at the 5-HT6 receptor is significantly influenced by electronic and hydrophobic interactions as well as the enhanced rigidity of the ligands. Molecular docking studies indicate that the reduced D-1 receptor affinity of the rigidified compounds may be due in part to weaker H-bonding interactions between the oxygenated moieties on the compounds and specific receptor residues. Key receptor-ligand H-bonding interactions, salt bridges, and pi-pi interactions appear to be responsible for the 5-HT6 receptor affinity of the compounds. Compounds 10 (6,7-dimethoxy-2,3,4,11b-tetrahydro-1H-fluoreno[9,1-cd]azepine) and 12 (6,7-dimethoxy-2-methyl-2,3,4,11b-tetrahydro-1H-fluoreno[9,1-cd]azepine) have been identified as structurally novel, high affinity (K-i = 5 nM), selective 5-HT6 receptor ligands.

New aporphines containing C10 nitrogen substituents (viz. nitro, aniline or amide moieties), were synthesized and evaluated for affinity at human serotonin 5-HT1A and 5-HT2A receptors and at human dopamine D1, D2 and D5 receptors. Two series of analogs were investigated: series A which contain a sole C10 nitrogen substituent on the tetracyclic aporphine core and series B which are 1,2,10-trisubstituted aporphines. Remarkably, compounds from both series lacked affinity for the D5 receptor, thus attaining D1 versus D5 selectivity. Compound 20c was the most potent D1 ligand identified. Docking studies at D1 and D5 receptors indicate that the binding mode of 20c at the D1 receptor allows for stronger hydrophobic contacts, (primarily with Phe residues) as compared to the D5 receptor, accounting for its D1 versus D5 selectivity. Considering the lack of affinity for the D5 receptor (and low affinity at other receptors tested), compound 20c represents an interesting starting point for further structural diversification of aporphines as sub-type selective D1 receptor tools.