Design and Synthesis of New Acyl Urea Analogs as Potential σ1R Ligands

In search of synthetically accessible open-ring analogs of PD144418 or 5-(1-propyl-1,2,5,6-tetrahydropyridin-3-yl)-3-(p-tolyl)isoxazole, a highly potent sigma-1 receptor (σ1R) ligand, we herein report the design and synthesis of sixteen arylated acyl urea derivatives. Design aspects included modeling the target compounds for drug-likeness, docking at σ1R crystal structure 5HK1, and contrasting the lower energy molecular conformers with that of the receptor-embedded PD144418—a molecule we opined that our compounds could mimic pharmacologically. Synthesis of our acyl urea target compounds was achieved in two facile steps which involved first generating the N-(phenoxycarbonyl) benzamide intermediate and then coupling it with the appropriate amines weakly to strongly nucleophilic amines. Two potential leads (compounds 10 and 12, with respective in vitro σ1R binding affinities of 2.18 and 9.54 μM) emerged from this series. These leads will undergo further structure optimization with the ultimate goal of developing novel σ1R ligands for testing in neurodegeneration models of Alzheimer’s disease (AD).


Introduction
Inspired by the highly potent σ1R ligand [PD144418 or 5-(1-propyl-1,2,5,6-tetrahydropyridin-3-yl)-3-(p-tolyl)isoxazole] which requires multi-step synthesis, we opted to deconstruct its isoxazole ring and convert it into a more readily accessible acyl urea scaffold. This structural change facilitates quick synthetic access to a broad range of open-ring acyl-urea analogs, with patentable space, as potential σ1R ligands-evidenced by compound SW-43 [1]. The acyl urea scaffold also allows access to analogs with additional rotatable bonds, conformations, and aqueous solubility due to H-donor/acceptor capabilities [2]. Interestingly, small molecules containing urea pharmacophores have adaptable clinical applications, therefore medicinal chemists are also exploring the utility of this functionality in potential anti-neurodegenerative agents [2][3][4]. In fact, literature not only indicates that σ1R is a promising druggable target in neurodegenerative pathologies like AD, but it also suggests additional roles in other disease pharmacologies (addiction, pain, cancer proliferation, HIV, COVID-19, etc.) [1,[5][6][7][8]. Now, the unsatisfactory level of the urea's blood-brain barrier (BBB) penetration due to H-bonding/ionization/polarity/molecular flexibility [2] suggests making structural elaborations to endow derivatives with "drug-like" pharmacokinetics (absorption, distribution, metabolism, elimination, toxicity or ADMET). Drug-like pharmacokinetics are determined by the molecule's hydrophobicity, flexibility, size, and electronic nature [9][10][11][12], and computational predictions can help identify such in early drug design. To that end, our sixteen compounds were computationally profiled for pharmacokinetics and docked at σ1R prior to their synthesis. Structural design aspects of our arylated acyl-ureas are illustrated in Figure 1.
computationally profiled for pharmacokinetics and docked at σ1R prior to their synthesis. Structural design aspects of our arylated acyl-ureas are illustrated in Figure 1. Online Molinspiration [13] and Osiris property explorer [14] were used to calculate/predict drug-like molecular properties for the target compounds. These studies involved retrieving the σ1R crystal structure (5HK1) with embedded ligand (PD144418), preparation (e.g., H2O and ligand removal), and docking the target molecules via the PyRx and AutoDock Vina suite [15,16]. All sixteen compounds comparably docked to the 5HK1 ligand binding site, suggesting that acyl ureas could also bind to σ1R. Amino acid (AA) interactions in the receptor binding site were also revealed. Synthetically, a variety of approaches for obtaining acyl ureas were reviewed [17][18][19][20][21][22][23][24][25][26][27][28][29] and the most feasible conditions were attempted with limited success-our goal was to obtain our compounds using the shortest and most cost-effective approach. In our hands, even the reported convenient Stokes' method [23] which required obtaining the same key N-(phenoxycabonyl)benzamide intermediate from diphenyl carbonate, proved futile. Instead, 1 H NMR analysis of reaction mixtures revealed phenol, methyl benzoate, and remaining starting materials. We therefore slightly modified Stokes' conditions [23] by preparing the N-(phenoxycabonyl)benzamide intermediate from phenol and benzoyl isocynate. Coupling the intermediate with amines or amides or thioamides via simple nucleophilic displacement afforded our series of N-acyl ureas plus N-acyl thioureas (Table 1) in good yields (>64%). To confirm our pharmacological assumptions regarding these molecules behaving like PD144418, the compounds were sent to the National Institute of Mental Health's Psychoactive Drug Screening Program and evaluated in radioligand displacement assays [30] to determine their in vitro σ1R affinities or Ki values. Table 1. Synthesized acyl ureas plus starting amines/amide/thioamides. a Characterized by 1 H, 13 C NMR, HRMS. b Yields of pure isolated products. ** Known compounds.

Compds.
Amine Acyl Urea a % Yield b MP (°C) 1 ** [25,26]  Online Molinspiration [13] and Osiris property explorer [14] were used to calculate/predict drug-like molecular properties for the target compounds. These studies involved retrieving the σ1R crystal structure (5HK1) with embedded ligand (PD144418), preparation (e.g., H 2 O and ligand removal), and docking the target molecules via the PyRx and AutoDock Vina suite [15,16]. All sixteen compounds comparably docked to the 5HK1 ligand binding site, suggesting that acyl ureas could also bind to σ1R. Amino acid (AA) interactions in the receptor binding site were also revealed. Synthetically, a variety of approaches for obtaining acyl ureas were reviewed [17][18][19][20][21][22][23][24][25][26][27][28][29] and the most feasible conditions were attempted with limited success-our goal was to obtain our compounds using the shortest and most cost-effective approach. In our hands, even the reported convenient Stokes' method [23] which required obtaining the same key N-(phenoxycabonyl)benzamide intermediate from diphenyl carbonate, proved futile. Instead, 1 H NMR analysis of reaction mixtures revealed phenol, methyl benzoate, and remaining starting materials. We therefore slightly modified Stokes' conditions [23] by preparing the N-(phenoxycabonyl)benzamide intermediate from phenol and benzoyl isocynate. Coupling the intermediate with amines or amides or thioamides via simple nucleophilic displacement afforded our series of N-acyl ureas plus N-acyl thioureas (Table 1) in good yields (>64%). To confirm our pharmacological assumptions regarding these molecules behaving like PD144418, the compounds were sent to the National Institute of Mental Health's Psychoactive Drug Screening Program and evaluated in radioligand displacement assays [30] to determine their in vitro σ1R affinities or K i values. computationally profiled for pharmacokinetics and docked at σ1R prior to their synthesis. Structural design aspects of our arylated acyl-ureas are illustrated in Figure 1. Online Molinspiration [13] and Osiris property explorer [14] were used to calculate/predict drug-like molecular properties for the target compounds. These studies involved retrieving the σ1R crystal structure (5HK1) with embedded ligand (PD144418), preparation (e.g., H2O and ligand removal), and docking the target molecules via the PyRx and AutoDock Vina suite [15,16]. All sixteen compounds comparably docked to the 5HK1 ligand binding site, suggesting that acyl ureas could also bind to σ1R. Amino acid (AA) interactions in the receptor binding site were also revealed. Synthetically, a variety of approaches for obtaining acyl ureas were reviewed [17][18][19][20][21][22][23][24][25][26][27][28][29] and the most feasible conditions were attempted with limited success-our goal was to obtain our compounds using the shortest and most cost-effective approach. In our hands, even the reported convenient Stokes' method [23] which required obtaining the same key N-(phenoxycabonyl)benzamide intermediate from diphenyl carbonate, proved futile. Instead, 1 H NMR analysis of reaction mixtures revealed phenol, methyl benzoate, and remaining starting materials. We therefore slightly modified Stokes' conditions [23] by preparing the N-(phenoxycabonyl)benzamide intermediate from phenol and benzoyl isocynate. Coupling the intermediate with amines or amides or thioamides via simple nucleophilic displacement afforded our series of N-acyl ureas plus N-acyl thioureas (Table 1) in good yields (>64%). To confirm our pharmacological assumptions regarding these molecules behaving like PD144418, the compounds were sent to the National Institute of Mental Health's Psychoactive Drug Screening Program and evaluated in radioligand displacement assays [30] to determine their in vitro σ1R affinities or Ki values. computationally profiled for pharmacokinetics and docked at σ1R prior to their synthesis. Structural design aspects of our arylated acyl-ureas are illustrated in Figure 1. Online Molinspiration [13] and Osiris property explorer [14] were used to calculate/predict drug-like molecular properties for the target compounds. These studies involved retrieving the σ1R crystal structure (5HK1) with embedded ligand (PD144418), preparation (e.g., H2O and ligand removal), and docking the target molecules via the PyRx and AutoDock Vina suite [15,16]. All sixteen compounds comparably docked to the 5HK1 ligand binding site, suggesting that acyl ureas could also bind to σ1R. Amino acid (AA) interactions in the receptor binding site were also revealed. Synthetically, a variety of approaches for obtaining acyl ureas were reviewed [17][18][19][20][21][22][23][24][25][26][27][28][29] and the most feasible conditions were attempted with limited success-our goal was to obtain our compounds using the shortest and most cost-effective approach. In our hands, even the reported convenient Stokes' method [23] which required obtaining the same key N-(phenoxycabonyl)benzamide intermediate from diphenyl carbonate, proved futile. Instead, 1 H NMR analysis of reaction mixtures revealed phenol, methyl benzoate, and remaining starting materials. We therefore slightly modified Stokes' conditions [23] by preparing the N-(phenoxycabonyl)benzamide intermediate from phenol and benzoyl isocynate. Coupling the intermediate with amines or amides or thioamides via simple nucleophilic displacement afforded our series of N-acyl ureas plus N-acyl thioureas (Table 1) in good yields (>64%). To confirm our pharmacological assumptions regarding these molecules behaving like PD144418, the compounds were sent to the National Institute of Mental Health's Psychoactive Drug Screening Program and evaluated in radioligand displacement assays [30] to determine their in vitro σ1R affinities or Ki values. computationally profiled for pharmacokinetics and docked at σ1R prior to their synthesis. Structural design aspects of our arylated acyl-ureas are illustrated in Figure 1. Online Molinspiration [13] and Osiris property explorer [14] were used to calculate/predict drug-like molecular properties for the target compounds. These studies involved retrieving the σ1R crystal structure (5HK1) with embedded ligand (PD144418), preparation (e.g., H2O and ligand removal), and docking the target molecules via the PyRx and AutoDock Vina suite [15,16]. All sixteen compounds comparably docked to the 5HK1 ligand binding site, suggesting that acyl ureas could also bind to σ1R. Amino acid (AA) interactions in the receptor binding site were also revealed. Synthetically, a variety of approaches for obtaining acyl ureas were reviewed [17][18][19][20][21][22][23][24][25][26][27][28][29] and the most feasible conditions were attempted with limited success-our goal was to obtain our compounds using the shortest and most cost-effective approach. In our hands, even the reported convenient Stokes' method [23] which required obtaining the same key N-(phenoxycabonyl)benzamide intermediate from diphenyl carbonate, proved futile. Instead, 1 H NMR analysis of reaction mixtures revealed phenol, methyl benzoate, and remaining starting materials. We therefore slightly modified Stokes' conditions [23] by preparing the N-(phenoxycabonyl)benzamide intermediate from phenol and benzoyl isocynate. Coupling the intermediate with amines or amides or thioamides via simple nucleophilic displacement afforded our series of N-acyl ureas plus N-acyl thioureas (Table 1) in good yields (>64%). To confirm our pharmacological assumptions regarding these molecules behaving like PD144418, the compounds were sent to the National Institute of Mental Health's Psychoactive Drug Screening Program and evaluated in radioligand displacement assays [30] to determine their in vitro σ1R affinities or Ki values. computationally profiled for pharmacokinetics and docked at σ1R prior to their synthesis. Structural design aspects of our arylated acyl-ureas are illustrated in Figure 1. Online Molinspiration [13] and Osiris property explorer [14] were used to calculate/predict drug-like molecular properties for the target compounds. These studies involved retrieving the σ1R crystal structure (5HK1) with embedded ligand (PD144418), preparation (e.g., H2O and ligand removal), and docking the target molecules via the PyRx and AutoDock Vina suite [15,16]. All sixteen compounds comparably docked to the 5HK1 ligand binding site, suggesting that acyl ureas could also bind to σ1R. Amino acid (AA) interactions in the receptor binding site were also revealed. Synthetically, a variety of approaches for obtaining acyl ureas were reviewed [17][18][19][20][21][22][23][24][25][26][27][28][29] and the most feasible conditions were attempted with limited success-our goal was to obtain our compounds using the shortest and most cost-effective approach. In our hands, even the reported convenient Stokes' method [23] which required obtaining the same key N-(phenoxycabonyl)benzamide intermediate from diphenyl carbonate, proved futile. Instead, 1 H NMR analysis of reaction mixtures revealed phenol, methyl benzoate, and remaining starting materials. We therefore slightly modified Stokes' conditions [23] by preparing the N-(phenoxycabonyl)benzamide intermediate from phenol and benzoyl isocynate. Coupling the intermediate with amines or amides or thioamides via simple nucleophilic displacement afforded our series of N-acyl ureas plus N-acyl thioureas (Table 1) in good yields (>64%). To confirm our pharmacological assumptions regarding these molecules behaving like PD144418, the compounds were sent to the National Institute of Mental Health's Psychoactive Drug Screening Program and evaluated in radioligand displacement assays [30] to determine their in vitro σ1R affinities or Ki values. computationally profiled for pharmacokinetics and docked at σ1R prior to their synthesis. Structural design aspects of our arylated acyl-ureas are illustrated in Figure 1. Online Molinspiration [13] and Osiris property explorer [14] were used to calculate/predict drug-like molecular properties for the target compounds. These studies involved retrieving the σ1R crystal structure (5HK1) with embedded ligand (PD144418), preparation (e.g., H2O and ligand removal), and docking the target molecules via the PyRx and AutoDock Vina suite [15,16]. All sixteen compounds comparably docked to the 5HK1 ligand binding site, suggesting that acyl ureas could also bind to σ1R. Amino acid (AA) interactions in the receptor binding site were also revealed. Synthetically, a variety of approaches for obtaining acyl ureas were reviewed [17][18][19][20][21][22][23][24][25][26][27][28][29] and the most feasible conditions were attempted with limited success-our goal was to obtain our compounds using the shortest and most cost-effective approach. In our hands, even the reported convenient Stokes' method [23] which required obtaining the same key N-(phenoxycabonyl)benzamide intermediate from diphenyl carbonate, proved futile. Instead, 1 H NMR analysis of reaction mixtures revealed phenol, methyl benzoate, and remaining starting materials. We therefore slightly modified Stokes' conditions [23] by preparing the N-(phenoxycabonyl)benzamide intermediate from phenol and benzoyl isocynate. Coupling the intermediate with amines or amides or thioamides via simple nucleophilic displacement afforded our series of N-acyl ureas plus N-acyl thioureas (Table 1) in good yields (>64%). To confirm our pharmacological assumptions regarding these molecules behaving like PD144418, the compounds were sent to the National Institute of Mental Health's Psychoactive Drug Screening Program and evaluated in radioligand displacement assays [30] to determine their in vitro σ1R affinities or Ki values. computationally profiled for pharmacokinetics and docked at σ1R prior to their synthesis. Structural design aspects of our arylated acyl-ureas are illustrated in Figure 1. Online Molinspiration [13] and Osiris property explorer [14] were used to calculate/predict drug-like molecular properties for the target compounds. These studies involved retrieving the σ1R crystal structure (5HK1) with embedded ligand (PD144418), preparation (e.g., H2O and ligand removal), and docking the target molecules via the PyRx and AutoDock Vina suite [15,16]. All sixteen compounds comparably docked to the 5HK1 ligand binding site, suggesting that acyl ureas could also bind to σ1R. Amino acid (AA) interactions in the receptor binding site were also revealed. Synthetically, a variety of approaches for obtaining acyl ureas were reviewed [17][18][19][20][21][22][23][24][25][26][27][28][29] and the most feasible conditions were attempted with limited success-our goal was to obtain our compounds using the shortest and most cost-effective approach. In our hands, even the reported convenient Stokes' method [23] which required obtaining the same key N-(phenoxycabonyl)benzamide intermediate from diphenyl carbonate, proved futile. Instead, 1 H NMR analysis of reaction mixtures revealed phenol, methyl benzoate, and remaining starting materials. We therefore slightly modified Stokes' conditions [23] by preparing the N-(phenoxycabonyl)benzamide intermediate from phenol and benzoyl isocynate. Coupling the intermediate with amines or amides or thioamides via simple nucleophilic displacement afforded our series of N-acyl ureas plus N-acyl thioureas (Table 1) in good yields (>64%). To confirm our pharmacological assumptions regarding these molecules behaving like PD144418, the compounds were sent to the National Institute of Mental Health's Psychoactive Drug Screening Program and evaluated in radioligand displacement assays [30] to determine their in vitro σ1R affinities or Ki values.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation.

Chemistry
Literature is replete with synthetic options for obtaining acyl ureas. For this project, we reviewed and tried several established approaches [17][18][19][20][21][22][23][24][25][26][27][28][29]. However, our attempts at synthesizing the target compounds were besieged by complex reaction mixtures and poor reaction yields after tedious purifications. For instance, classical conditions (e.g., Scheme 1) which involve initially reacting benzamides and reactive oxalyl chloride followed by the addition of corresponding amines, lead us to dark reaction mixtures with multiple product spots by thin layer chromatography (TLC). Perhaps the acidic conditions were too reactive or unstable and therefore thwarted desired product formation. We eventually settled on the reported convenient Stokes' method [23] and slightly modified it by first generating the N-(phenoxycarbonyl)benzamide intermediate from benzoyl isocyanate and phenol (rather than from diphenylcarbonate). Obtaining this key intermediate, in this manner, facilitated the synthesis of our target acyl urea or N-benzoyl urea compounds in Table 1 conveniently and cost-effectively, in two steps (Scheme 2) and in good yields (64-94%). Essentially, our compounds were synthesized by directly coupling amines or amides, or thioamides with a readily scalable N-(phenoxycarbonyl)benzamide intermediate, under reflux, via simple nucleophilic displacements. This synthetic approach tolerated both weakly and strongly nucleophilic amines, under mild reaction conditions, and is amenable to both parallel and high throughput access to diverse arylated acyl ureas for different applications. All compounds were purified (via filtration and/or combi-Flash) and structurally confirmed by spectroscopic methods [ 1 H and 13 C-NMR, high-resolution MS (ES)]. 1 H NMRs of this family of compounds showed the presence of an exchangeable signal characterizing the -NH proton at the region δ 9 to 15 ppm DMSO-d6. 13 C NMRs ascertained the presence of two to three C=O groups.

Drug-Likeness Predictions
At a minimum, drug-likeness implies ligands exhibiting Lipinski's rule of five (Ro5), topological polar surface area (TPSA), and the number of rotatable bond (NRB) limit considerations [9][10][11][12]31]. Table 2 itemizes partial molecular descriptors and drug-likeness properties obtained via Molinspiration /Osiris [13,14] predictions on our target compounds (entries 1-16). Ro5 upper limits are listed in the bottom row. We eventually settled on the reported convenient Stokes' method [23] and slightly modified it by first generating the N-(phenoxycarbonyl)benzamide intermediate from benzoyl isocyanate and phenol (rather than from diphenylcarbonate). Obtaining this key intermediate, in this manner, facilitated the synthesis of our target acyl urea or N-benzoyl urea compounds in Table 1 conveniently and cost-effectively, in two steps (Scheme 2) and in good yields (64-94%). Essentially, our compounds were synthesized by directly coupling amines or amides, or thioamides with a readily scalable N-(phenoxycarbonyl)benzamide intermediate, under reflux, via simple nucleophilic displacements. We eventually settled on the reported convenient Stokes' method [23] and slightly modified it by first generating the N-(phenoxycarbonyl)benzamide intermediate from benzoyl isocyanate and phenol (rather than from diphenylcarbonate). Obtaining this key intermediate, in this manner, facilitated the synthesis of our target acyl urea or N-benzoyl urea compounds in Table 1 conveniently and cost-effectively, in two steps (Scheme 2) and in good yields (64-94%). Essentially, our compounds were synthesized by directly coupling amines or amides, or thioamides with a readily scalable N-(phenoxycarbonyl)benzamide intermediate, under reflux, via simple nucleophilic displacements. This synthetic approach tolerated both weakly and strongly nucleophilic amines, under mild reaction conditions, and is amenable to both parallel and high throughput access to diverse arylated acyl ureas for different applications. All compounds were purified (via filtration and/or combi-Flash) and structurally confirmed by spectroscopic methods [ 1 H and 13 C-NMR, high-resolution MS (ES)]. 1 H NMRs of this family of compounds showed the presence of an exchangeable signal characterizing the -NH proton at the region δ 9 to 15 ppm DMSO-d6. 13 C NMRs ascertained the presence of two to three C=O groups.

Drug-Likeness Predictions
At a minimum, drug-likeness implies ligands exhibiting Lipinski's rule of five (Ro5), topological polar surface area (TPSA), and the number of rotatable bond (NRB) limit considerations [9][10][11][12]31]. Table 2 itemizes partial molecular descriptors and drug-likeness properties obtained via Molinspiration /Osiris [13,14] predictions on our target compounds (entries 1-16). Ro5 upper limits are listed in the bottom row. This synthetic approach tolerated both weakly and strongly nucleophilic amines, under mild reaction conditions, and is amenable to both parallel and high throughput access to diverse arylated acyl ureas for different applications. All compounds were purified (via filtration and/or combi-Flash) and structurally confirmed by spectroscopic methods [ 1 H and 13 C-NMR, high-resolution MS (ES)]. 1 H NMRs of this family of compounds showed the presence of an exchangeable signal characterizing the -NH proton at the region δ 9 to 15 ppm DMSO-d 6 . 13 C NMRs ascertained the presence of two to three C=O groups.
All target compounds were of low molecular weight (MW < 500), their range was 231.21-396.44. Low molecular weight drug molecules are readily absorbed, diffuse, and are easier to transport versus heavier molecules. Lipophilicity (logP) and TPSA values are predictors of drug oral bioavailability. Calculated log p values of 1.53-3.53 were in the acceptable range of drug molecules capable of penetrating biological membranes including the blood-brain barrier (BBB). The numbers of H-bond acceptors (O and N atoms) and H-bond donors (OH and NH) in the target compounds were within the Ro5 guidelines. TPSA values for all molecules were within the acceptable range. TPSA values are used to characterize potential drug absorption (including intestinal absorption), bioavailability, Caco-2 permeability, and BBB penetration. These values are calculated from the surface areas occupied by oxygen, nitrogen, and the hydrogen atoms attached to them. Thus, the TPSA is closely related to the hydrogen bonding potential of a compound. Interestingly, all compounds exhibited 52.65-119.7 Å TPSA values, indicating potential good oral bioavailabilities. Good bioavailability is more likely for compounds with ≤10 rotatable bonds and TPSA of ≤140 Å. As the NRBs increase, the molecule becomes more flexible and more adapt- able for efficient interactions in particular binding pockets. Interestingly all compounds have 1-4 rotatable bonds and are flexible. Positive drug-likeness scores suggest that the designed molecules retain predominant structural features of known/common commercial drugs. All but two of the sixteen target molecules (compounds 2 and 9) exhibited positive drug-likeness scores.

Molecular Docking
Docking was conducted to evaluate protein-ligand interactions and predict binding affinities of the target compounds versus the comparator potent σ1R ligand PD144418. Our target molecules were docked using the PyRx and AutoDock Vina platforms [15,16]. Analysis of the docking scores and prediction of binding modes between the ligand and σ1R was conducted using BIOVIA Discovery Studio 4.5 visualizer [32]. Figure 2 contrasts the before (A) and after (B) preparation visualizations of PD14418 in σ1R or 5HK1 binding pocket. All target compounds were of low molecular weight (MW < 500), their range was 231. 21-396.44. Low molecular weight drug molecules are readily absorbed, diffuse, and are easier to transport versus heavier molecules. Lipophilicity (logP) and TPSA values are predictors of drug oral bioavailability. Calculated log p values of 1.53-3.53 were in the acceptable range of drug molecules capable of penetrating biological membranes including the blood-brain barrier (BBB). The numbers of H-bond acceptors (O and N atoms) and H-bond donors (OH and NH) in the target compounds were within the Ro5 guidelines. TPSA values for all molecules were within the acceptable range. TPSA values are used to characterize potential drug absorption (including intestinal absorption), bioavailability, Caco-2 permeability, and BBB penetration. These values are calculated from the surface areas occupied by oxygen, nitrogen, and the hydrogen atoms attached to them. Thus, the TPSA is closely related to the hydrogen bonding potential of a compound. Interestingly, all compounds exhibited 52.65-119.7 Å TPSA values, indicating potential good oral bioavailabilities. Good bioavailability is more likely for compounds with ≤10 rotatable bonds and TPSA of ≤140 Å. As the NRBs increase, the molecule becomes more flexible and more adaptable for efficient interactions in particular binding pockets. Interestingly all compounds have 1-4 rotatable bonds and are flexible. Positive drug-likeness scores suggest that the designed molecules retain predominant structural features of known/common commercial drugs. All but two of the sixteen target molecules (compounds 2 and 9) exhibited positive drug-likeness scores.

Molecular Docking
Docking was conducted to evaluate protein-ligand interactions and predict binding affinities of the target compounds versus the comparator potent σ1R ligand PD144418. Our target molecules were docked using the PyRx and AutoDock Vina platforms [15,16]. Analysis of the docking scores and prediction of binding modes between the ligand and σ1R was conducted using BIOVIA Discovery Studio 4.5 visualizer [32]. Figure 2 contrasts the before (A) and after (B) preparation visualizations of PD14418 in σ1R or 5HK1 binding pocket.
(A) (B) Docking yielded the most stable conformer free-binding energy scores (i.e., binding affinities in kcal/mol) and H-bonding interactions within the active site of σ1R (Table 3). Docking yielded the most stable conformer free-binding energy scores (i.e., binding affinities in kcal/mol) and H-bonding interactions within the active site of σ1R (Table 3).
Additionally, docking revealed relevant amino acids for bonding interactions in the active site of σ1R (5HK1). Example interactions are illustrated in Figure 3.  Additionally, docking revealed relevant amino acids for bonding interactions in the active site of σ1R (5HK1). Example interactions are illustrated in Figure 3. The binding affinity values of the most stable/best-docked 5HK1 bound conformers ranged from −11.40 to −8.6 kcal/mol. The binding energies of all compounds were significant and comparable to the reference ligand PD14418 (−10.2 kcal/mol) and were commonly due to conventional H-bonding interactions between GLU 172 and the amide bond hydrogen atom. Representative compound 11 exhibited the strongest (three conventional hydrogen bonds via -OH, carbonyl, and -NH) polar interactions with ASP126, THR 181, and TYR 120 residues. In addition to the H-bond interactions illustrated in Figure 3, the 5HK1-compound 11 complexes were also stabilized by π-sigma (Tyr 120) and π-anion (Glu 172, and Asp 126) interactions which also contributed to the calculated lowest root mean square deviation (RMSD) value and provided the best-fit result. On the other hand, the comparator (PD14418) formed: π-sigma with His 194; π-alkyl with Val 84, Leu 105, Phe 133, Val 162, and Ala 185; π-anion with Asp 126; π-donor with Phe 107, Ser 117, Tyr 120, Ile 124, Trp 164, Glu 172 and Thr 181residues. The binding affinity values of the most stable/best-docked 5HK1 bound conformers ranged from −11.40 to −8.6 kcal/mol. The binding energies of all compounds were significant and comparable to the reference ligand PD14418 (−10.2 kcal/mol) and were commonly due to conventional H-bonding interactions between GLU 172 and the amide bond hydrogen atom. Representative compound 11 exhibited the strongest (three conventional hydrogen bonds via -OH, carbonyl, and -NH) polar interactions with ASP126, THR 181, and TYR 120 residues. In addition to the H-bond interactions illustrated in Figure 3, the 5HK1-compound 11 complexes were also stabilized by π-sigma (Tyr 120) and π-anion (Glu 172, and Asp 126) interactions which also contributed to the calculated lowest root mean square deviation (RMSD) value and provided the best-fit result. On the other hand, the comparator (PD14418) formed: π-sigma with His 194; π-alkyl with Val 84, Leu 105, Phe 133, Val 162, and Ala 185; π-anion with Asp 126; π-donor with Phe 107, Ser 117, Tyr 120, Ile 124, Trp 164, Glu 172 and Thr 181residues.
Computational (kcal/mol) versus in vitro (µM, from the National Institute of Mental Health Psychoactive Drug Screening Program or NIMH-PDSP [https://pdsp.unc.edu/ pdspweb/ (accessed 28 February 2023)] σ1R binding affinity data are illustrated in Table 4. Table 4. Modeled (kcal/mol) versus in vitro (µM) binding affinity values. * PD144418 is a selective and high affinity (K i = 0.08 nM) σ1R ligand [33]. It is worth noting that although we used modeling/docking to guide our synthetic target prioritization, we did not observe a good correlation between predicted versus the in vitro affinity data. This discrepancy is perhaps a result of quantitative limitations of the free and open-source software (FOSS) platforms (e.g., Open Babel) we utilized. For example, ligand protonation states can affect virtual binding affinities [34], ergo, multiple ionization states needed to be included in the prediction data. Moving forward, we endeavor to utilize more robust computational platforms (either through collaborations or by subscription) to optimize the binding affinities of the two leads before synthesizing any follow-up compounds for the SAR studies.

Materials and General Methods
Reagents and solvents were purchased from vendors [Fischer Scientific and Acros Organics (Waltham, MA, USA)/Sigma Aldrich (St. Louis, MO, USA] and used as received unless otherwise indicated. All reactions were performed using established procedures. Melting points were taken on the Mel-temp capillary apparatus and reported uncorrected. Teledyne Combiflash Rf flash chromatography fitted with Redisep Rf silica gel cartridges was used for compound purifications. Analytical TLC plates from EM Science (silica Gel 60 F 254 ) were used. 1 H and 13 C spectra (Supplementary Materials) were recorded using CDCl 3 or DMSO-d 6 as solvents on a Bruker 300 MHz spectrometer at ambient probe temperature unless otherwise indicated. 1 H and 13 C chemical shifts are reported versus SiMe 4 and were determined by reference to the residual 1 H and 13 C solvent peaks. Coupling constants (J) are reported in hertz (Hz). Characterization data were reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants, number of protons, and mass-to-charge (m/z) ratios. High-Resolution Mass Spectrometry (HRMS) was carried out using an Agilent 6230 electrospray ionization time-of-flight instrument. Where necessary, the purity/mass of the target compounds was determined by analytical reversed-phase high-performance liquid chromatography/mass spectrometer (HPLC/MS) tandem on a Dionex Ultimate 3000 HPLC system. HPLC was conducted using Nova-Pak C18 Column, 60Å, 4 µm, (4.6 mm × 150 mm) at ambient temperature, and a flow rate of 1.0 mL/min., CH 3 CN & H 2 O eluent (containing 0.1% acetic acid); gradient, 5% CH 3 CN to 100% CH 3 CN; 8 min.; UV detection at 254 nm. Mass was obtained in positive ion mode using heated electrospray ionization (ISQ-HESI) source. MS conditions: capillary voltage, 3.0000 V; drying gas flow, 0.2mL/min; vaporizer gas temperature, 350 • C; and gas pressure, 28.8 psig. MS data were acquired with Chromeleon 7.