Discovery of Novel Oleamide Analogues as Brain-Penetrant Positive Allosteric Serotonin 5-HT_2C Receptor and Dual 5-HT_2C/5-HT_2A Receptor Modulators

Abstract

The serotonin 5-HT_2A receptor (5-HT_2AR) and 5-HT_2CR localize to the brain and share overlapping signal transduction facets that contribute to their roles in cognition, mood, learning, and memory. Achieving selective targeting of these receptors is challenged by the similarity in their 5-HT orthosteric binding pockets. A fragment-based discovery approach was employed to design and synthesize novel oleamide analogues as selective 5-HT_2CR or dual 5-HT_2CR/5-HT_2AR positive allosteric modulators (PAMs). Compound 13 (JPC0323) exhibited on-target properties, acceptable plasma exposure and brain penetration, as well as negligible displacement to orthosteric sites of ~50 GPCRs and transporters. Furthermore, compound 13 suppressed novelty-induced locomotor activity in a 5-HT_2CR-dependent manner, suggesting 5-HT_2CR PAM, but not 5-HT_2AR, activity at the level of the whole organism at the employed doses of 13. We discovered new selective 5-HT_2CR PAMs and first-in-class 5-HT_2CR/5-HT_2AR dual PAMs that broaden the pharmacological toolbox to explore the biology of these vital receptors.

Graphical Abstract

INTRODUCTION

Fourteen serotonin (5-HT) receptors are identified with 1 ionotropic receptor (5-HT₃R) and 13 class A G protein-coupled receptors (GPCRs) designated as 5-HT_1–7R based on structural and pharmacological criteria.^1,2 Serotonin 5-HT₂ receptors (5-HT₂Rs) are a pharmacologically important 5-HT receptor family that includes the three subtypes 5-HT_2AR, 5-HT_2BR, and 5-HT_2CR, which share approximately 80% sequence homology in the transmembrane (TM) ligand-binding regions. Among them, 5-HT_2CR and 5-HT_2AR have generated immense interest from pharmacologists and medicinal chemists in recent decades.^2–6 5-HT_2AR and 5-HT_2CR are broadly distributed in the mammalian central nervous system (CNS) and mediate various brain functions including cognition, mood, learning, and memory.^2,7–9

The three 5-HT₂R subtypes (5-HT_2AR, 5-HT_2BR, and 5-HT_2CR) display similar molecular structures with a highly conserved endogenous agonist (5-HT) binding site and intersecting signal transduction pathways and pharmacology.¹⁰ Traditional agonists target the orthosteric ligand binding site within the seven-transmembrane bundle (7TM) of the receptor that is highly conserved, challenging the goal to achieve individual subtype selectivity. Allosteric modulators targeting a spatially and topographically distinct site provide a novel pharmacological paradigm for GPCR drug discovery.^11–15 The design of allosteric modulators that selectively target 5-HT₂R subtypes is a viable and practical approach for avoiding ligand binding to other 5-HT receptors as well as the serotonin reuptake transporter and is especially critical for avoiding 5-HT_2BR stimulation, which is known to be associated with cardiac valvulopathy and pulmonary hypertension adverse effects.¹⁶

The goal to develop 5-HT₂R allosteric modulators is a recent endeavor with a focus on 5-HT_2CR positive allosteric modulators (PAMs);^17–21 as depicted in Figure 1, the complex natural product derivative compound 1 (PNU-69176E) was the first reported 5-HT_2CR PAM and was characterized by a stereo-dependent functional activity profile (Figure 1).^22,23 We demonstrated two medicinal chemistry design iterations toward the optimization of 1 (PNU-69176E). Given the poor drug-likeness of 1, we focused on the α-d-galactopyrano-side fragment, termed the polar head (PH), and the undecyl substituent at the 4-position of the piperidine, which is termed the lipophilic tail (LT). These efforts provided efficacious 5-HT_2CR PAMs 2 (CYD-1–79) and 3 (CTW0415).^18,20 These 5-HT_2CR PAMs displayed improved pharmacokinetic (PK) profiles and demonstrated in vivo activity in preclinical animal models. Recent discovery efforts by others produced the N-benzyl-indole 4 (VA012) and piperazine-linked phenyl cyclopropyl methanone 5 as 5-HT_2cR PAMs.^17,19 Among them, compound 5 also exhibited 5-HT_2BR negative allosteric modulation (NAM) activity.¹⁹

Figure 1.

Chemical structures of representative small molecule 5-HT₂R allosteric modulators 1–5. Compounds 1–4 are 5-HT_2CR PAMs. Compound 5 is a 5-HT_2CR PAM and 5-HT_2BR NAM.

Compound 6 (oleamide, (Z)-9-octadecenamide; Figure 2), an endogenous fatty acid amide, was identified in the cerebrospinal fluid of sleep-deprived cats as well as human plasma.^24,25 Compound 6 is implicated in several biological and behavioral phenomena such as sleep induction, feeding regulation, and hypothermia;^26–29 6 was noted to act nonselectively as an agonist or allosteric modulator at 5-HT_1AR, 5-HT_2AR, and 5-HT_2CR and an inhibitor at 5-HT₇R as well as at other receptor systems.^{26,27,29–34} The activity of 6 in these systems is structurally sensitive, and only modest chemical modifications have been attempted thus far.^31,33,35

Figure 2.

Design strategy for novel 5-HT_2CR and/or dual 5-HT_2CR/5-HT_2AR PAMs. (A) The minimized three-dimensional structures of compound 2 (CYD-1–79, blue dashes) and 7 (red dashes) are presented in an overlay. Energy minimization was carried out using the Schrödinger Drug Discovery Suite (Schrödinger Release 2015–4: LigPrep, version 3.6, Schrödinger, LLC, New York, NY, 2015). (B) The overlay poses of compounds 2 (CYD-1–79) (red) and 7 (orange) docked to the 5-HT_2CR X-ray crystal structure (PDB ID: 6BQG) are illustrated as a ribbon representation, and compounds are shown in stick representation. (C) The current structural modification strategy is presented with new probes designed by hybridization of compounds 6 (oleamide) and 2 (CYD-1–79).

Compound 6 shares the common feature of a long LT and a terminal PH with the 5-HT_2CR PAM 2 (CYD-1–79).¹⁸ Although the LT of 6 is longer than that of PAM 2 (18- vs 15-carbon tail, respectively), an energy minimization overlay of 2 and 7 (the analogue of 6 modified by coupling 6 with a privileged 1,2-diol PH fragment) shows that, because of the cis conformation of the double bond, the tail lengths are approximately the same in maximal length (17.98 vs 17.78 Å; Figure 2A). Molecular docking of 2 and 7 to the 5-HT_2CR (PDB ID: 6BQG) X-ray crystal structure demonstrates a similar binding pose and directionality of both the LT and PH, as expected (Figure 2B).^18,20 We previously demonstrated that the proper size and volume of the LT and hydroxyl-rich PH are important to the activity of 5-HT_2cR PAMs,^18,20 which together contribute to the formation of hydrophobic and hydrogen bond interactions with the receptor extracellular loop 2 (ECL2) and TM helices. Given the similarity in the structural scaffolds of compounds 2 and 7, along with the molecular docking predictions, we reasoned that these two molecules may potentially act at the same allosteric site on the 5-HT_2CR and potentiate 5-HT signaling as a PAM. Additionally, we rationalized that the substitution of 4-alkylpiperidine-2-carboxamide within the oleamide scaffold to remove the multiple chiral centers of 5-HT_2CR PAMs compounds 2 and 3 would reduce the chemical complexity of the PAMs and improve the drug-likeness. Herein, we describe our recent progress in the design, synthesis, and pharmacological evaluation of a novel series of structurally optimized analogues by chemical hybridization of compounds 2 and 6, providing PAMs of 5-HT₂R subtypes. This series of 5-HT_2CR PAMs and dual 5-HT_2CR/5-HT_2AR PAMs exhibited in vitro functional activity and are valuable chemical probes for exploring the mechanisms underlying 5-HT_2CR and 5-HT_2AR allosteric modulation (Figure 2C).

RESULTS AND DISCUSSION

Chemistry and Cellular Signaling Analyses.

The synthesis of new oleamide analogues 7–30 with simplified PHs is depicted in Scheme 1. An effective and convenient one-step coupling of oleic acid, a long-chain unsaturated omega-9 fatty acid, with various aminol alcohol analogues, amino acid residues, or monoamine-like fragments was conducted via adapting the common condensation reagent N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) or 1-ethyl-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDCI) in combination with 1-hydroxybenzotriazole and the organic base N,N-diisopropylethylamine (DIPEA). Compounds 16–19 underwent saponification according to standard protocols to yield the corresponding carboxylic acid compounds 20–23. Compounds 7–30 were accomplished in 42–94% yields and then subjected to in vitro functional assessment.

Scheme 1.

Synthesis of Oleamide Analogues 7–30

Agonist binding to 5-HT_2CR and 5-HT_2AR recruits phospholipase Cβ (PLCβ) via G_αq/11 proteins and in turn elicits the efflux of intracellular calcium (Ca_i²⁺).³⁶ We employed a fluorescence-based assay to measure 5-HT-evoked Ca_i²⁺ levels as a measure of receptor activity in Chinese hamster ovary (CHO) cells stably transfected with the unedited (INI) isoform of human (h) h5-HT_2CR (h5-HT_2CR-CHO cells), h5-HT_2AR (h5-HT_2AR-CHO cells), or h5-HT_2BR (h5-HT_2BR-CHO cells).^{18 20} The capacity of 5-HT to promote Ca_i²⁺ release (E_max) was established and set as the 100% response.

Previous studies demonstrated that the 5-HT_2CR PAM 2 at the concentration of 1 nM evokes ~23% upward regulation of 5-HT-evoked Ca_i²⁺ release.¹⁸ Thus, compounds were screened at 1 nM in the presence of an increasing concentration of 5-HT to assess the enhancement of 5-HT-induced Ca_i²⁺ release (5-HT E_max). At 1 nM, none of the tested compounds exhibited intrinsic agonist activity to induce Ca_i²⁺ release in h5-HT_2CR-CHO, h5-HT_2AR-CHO, or h5-HT_2BR-CHO cells when administered 15 min prior to the addition of 5-HT (for details, see Figure S1, sections A–Y; Figure S2, sections A–J; and Figure S3, sections A–F).

Privileged PH fragments are instrumental in forming crucial interactions with the ECL2 and certain TM helices of the receptor, as noted previously.^18,20 The new series of oleamide analogues 7–30 with varied PHs were first screened in vitro in h5-HT_2CR-CHO cells (Table 1). Oleamide (6) exhibited moderate enhancement (~11%) of 5-HT-evoked Ca_i²⁺ release under the test conditions, whereas 7, which possesses the privileged 1,2-diol PH as 2 and 3, did not significantly increase 5-HT_2CR-evoked Ca_i²⁺ (Table 1; Figure S1). Exclusion of the middle hydroxyl group of 7 afforded the less-bulky oleyl propanolamide 8 that potentiated 5-HT-evoked Ca_i²⁺ release (Table 1; Figure 3A). Compound 9 with ethanolamide PH maintained 5-HT_2CR PAM activity (Table 1; Figure 4A).

Table 1.

Effects of Oleamide Analogues 7–30 (1 nM) on 5-HT-Induced Ca_i²⁺ Release in h5-HT_2CR-CHO Cells

Compound	PH	Emax RFU (%5-HT)a	Compound	PH	Emax RFU (%5-HT)a
5-HT		100.0%	5-HT		100.0%
6	H	111.4 ± 4.3b	19		125.4 ± 5.2b
7		108.0 ± 6.7
8		126.3 ± 8.5b	20		111.9 ± 4.4
9		124.0 ± 6.8b	21		117.8 ± 6.9
10		100.1 ± 8.8	22		106.6 ± 9.0
11		143.9 ± 14.1b	23		117.4 ± 12.4
12		126.0 ± 6.9b	24		111.9 ± 11.8
13		113.2 ± 3.7b	25		122.8 ± 6.7b
14		111.1 ± 5.4	26		116.0 ± 18.1
15		106.2 ± 2.1b	27		120.6 ± 15.5
16		109.5 ± 1.6b	28		121.4 ± 5.2b
17		126.8 ± 11.4	29		118.5 ± 6.4
18		127.8 ± 15.3	30		92.9 ± 1.3b

^aAddition of the synthetic compound (1 nM) occurred 15 min prior to assessment of Ca_i²⁺ release evoked by increasing concentrations of 5-HT (vehicle, 10⁻¹¹ to 10⁻⁵ M) in h5-HT_2CR-CHO cells. Data are presented as maximal 5-HT-induced Ca_i²⁺ release (E_max).

^bp < 0.05. Comparisons between means for E_max were conducted with an unpaired t test with Welch’s correction (GraphPad Prism). All statistical analyses were conducted with an experiment-wise error rate of α = 0.05.

Figure 3.

Concentration–response curves for compounds 8, 12, 15, 16, and 25 for 5-HT-induced Ca_i²⁺ release in (A) h5-HT_2CR-CHO cells or (B) h5-HT_2AR-CHO cells. Representative curves demonstrate the concentration–response curve for 5-HT in the absence (black circles) and in the presence (closed triangles) of the test compounds, vehicle (open circle), and test compound assessed alone (open triangle). The maximum 5-HT-induced Ca_i²⁺ release in the absence of the test compounds was set as 100%, and the E_max values of the test compounds are listed in Tables 1 and 2.

Figure 4.

Concentration–response curve of compounds 9, 11, 13, 19, and 28 for 5-HT-induced Ca_i²⁺ release in live (A) h5-HT_2CR-CHO cells or (B) h5-HT_2AR-CHO cells. Representative curves demonstrate test compounds against concentration–response curves for 5-HT in the absence (black circles) and in the presence (closed triangles), vehicle (open circle), and vehicle in the presence of test compound (open triangle). The maximum 5-HT-induced Ca_i²⁺ release in the absence of the test compounds was set as 100%, and the E_max values of the test compounds are listed in Tables 1 and 2.

Because compound 2 exhibited a stereo conformation preference for 5-HT_2CR PAM activity, compound 10, furnished by introducing an S configurational 1,2-diol to the amide, was then explored and identified as inactive (Table 1), suggesting that the privileged 1,2-diol PH of 2 and 3 is not favorable for the oleamide derivatives to produce 5-HT_2CR PAM activity. Compound 11, which has a 1,3-diol moiety vs a 1,2-diol PH, promoted a ~44% increase in 5-HT_2CR-evoked Ca_i²⁺ (Table 1; Figure 4A). Compound 12, which incorporates a phenyl into the diol PH of 11, maintained 5-HT_2CR PAM activity (Table 1; Figure 3A), as does compound 13 (Table 1; Figure 4A) with one more hydroxymethyl group to 11. Compound 14, which includes the hydroxyl group with ether, did not evoke PAM activity, most likely due to the loss of the terminal H-bond donor and the bulkier volume of the PH (Table 1). Meanwhile, lengthening the PH of 9 by etherification with another ethanol fragment was less favorable (15, Figure 3A). These findings illustrate that the hydroxyl containing moiety is important for 5-HT_2CR allosteric potentiation, and a proper size of the PH is required for 5-HT_2CR PAM activity.

We then synthesized the polar head with other chemical elements, including chiral amino acids (16–23), terminal phenol (24 and 26), catechol (25 and 27), and morpholino (28) or amino (29–30) substituted alkylamines (Table 1). As the introduction of a hydroxyl-containing moiety is beneficial for 5-HT_2CR PAM efficacy, hydroxyl or terminal phenol containing chiral amino acids were applied by condensation of the methyl ester of threonine, serine, and tyrosine, affording compounds 16–18. Among them, compound 16 (Table 1; Figure 3A) demonstrated 5-HT_2CR PAM activity. Interestingly, compound 19 (Table 1; Figure 4A), incorporating the 5-HT indole-like tryptophan methyl ester in the terminal position of the PH, resulted in a 5-HT-evoked E_max of 125.4% (Figure 4A). Saponification of the methyl esters provided free acids 20–23 potentially capable of additional H-bonding and salt interactions with the target receptor. However, these carbonyl acid derivatives 20–23 at 1 nM did not potentiate 5-HT efficacy (Table 1). Furthermore, both 4-aminoalkyl phenolic and catechol moieties were explored as potential PHs (24–27). Terminal phenol compound 25 with a 4-aminomethyl catechol PH promoted 5-HT_2CR PAM activity (Table 1; Figure 3A), whereas other terminal phenol compounds with less phenolic hydroxyl or more carbon spaced chain did not display 5-HT_2CR PAM efficacy (the 4-aminoalkyl phenolic 24, 26, and two-carbon spaced catechol 27; Table 1). Intriguingly, when aliphatic amines were employed as PHs, the two-carbon spaced morpholino compound (28) displayed 5-HT_2CR PAM activity (Table 1; Figure 4A), whereas the n-butylamine compound (29) with a bulky amine terminus did not induce a 5-HT_2CR PAM effect (Table 1). To our surprise, when 4-(aminomethyl)piperidine was incorporated at the end of oleamide, a comparable ~10% decrease in 5-HT_2CR-mediated Ca_i²⁺ release was observed, which may signal the potential for 30 to function as a negative allosteric modulator (NAM) (Table 1).

Oleamide (6) itself has a complex pharmacological profile,^{26,27,29–34} and a subset of the 10 oleamide-like compounds characterized as 5-HT_2CR PAMs (Table 1) was further evaluated in the in vitro Ca_i²⁺ efflux assay using h5-HT_2AR-CHO cells (Table 2). After screening this subset of 10 analogues at 1 nM in the h5-HT_2AR-CHO cells, we identified compounds with two pharmacological profiles. Compounds 8, 12, 15, 16, and 25 were identified as 5-HT_2CR PAMs (Table 1; Figure 3A) and exhibited no efficacy as allosteric modulators of the 5-HT_2AR (Table 2; Figure 3B). However, compounds 9, 11, 13, 19, and 28 were identified as 5-HT_2CR PAMs (Table 1; Figure 4A) and also exhibited efficacy as 5-HT_2AR PAMs (Table 2; Figure 4B). Thus, we differentiated 5-HT_2CR PAMs (8, 12, 15, 16, and 25) and dual 5-HT_2CR/5-HT_2AR PAMs (9, 11, 13, 19, and 28) within this current series of oleamide-like compounds.

Table 2.

Effects of Identified Oleamide-like 5-HT_2CR PAMs (1 nM) on 5-HT-Induced Ca_i²⁺ Release in h5-HT_2AR-CHO Cells

Compound	PH	Emax RFU (%5-HT)a
5-HT		100.0%
8		99.2 ± 2.6
9		121.0 ± 3.7b
11		119.0 ± 3.2b
12		98.8 ± 3.8
13		132.4 ± 6.1b
15		99.6 ± 0.6
16		106.0 ± 5.3
19		109.2 ± 3.5b
25		102.5 ± 2.7
28		105.7 ± 2.0b

^aAddition of the synthetic compound (1 nM) occurred 15 min prior to assessment of Ca_i²⁺ release evoked by increasing concentrations of 5-HT (vehicle, 10⁻¹¹ to 10⁻⁵ M) in h5-HT_2AR-CHO cells. Data are presented as maximal 5-HT-induced Ca_i²⁺ release (E_max).

^bp < 0.05. Comparisons between means for E_max were conducted with an unpaired t test with Welch’s correction (GraphPad Prism). All statistical analyses were conducted with an experiment-wise error rate of α = 0.05.

Compound 9, which possesses an ethanol PH, displayed 5-HT_2AR PAM activity (Table 2; Figure 4B). Compounds 11 and 13, introducing a second and a third methanol to the PH of 9, respectively, maintained 5-HT_2AR allosteric effects (Table 2; Figure 4B). This result suggests that the incorporation of the two-carbon spaced hydroxyl moiety is favorable for producing 5-HT_2AR PAM activity. Compound 19 bearing a methyl tryptophan as PH and compound 28 with a two-carbon linked morpholino PH also act as 5-HT_2AR PAMs (Table 2; Figure 4B). Lengthening the linker length of 9 by one more carbon (8), etherification with another ethanol fragment (15), or retaining the 1,3-diol moiety of 11 with the addition of a phenyl (12) did not result in 5-HT_2AR PAM activity (Table 2; Figure 4A). Compounds 16 and 25 with longer PHs as methyl threonine and 4-aminomethyl catechol did not result in 5-HT_2AR PAM activity (Table 2; Figure 3B). These findings indicate that the structure of 5-HT_2AR PAMs is more sensitive to the length and volume change of PH than 5-HT_2CR PAMs (Tables 1 and 2). Representative 5-HT_2CR PAMs 8, 12, and 25 and dual 5-HT_2CR/5-HT_2AR PAMs 9, 11, and 13 were next assessed in the in vitro functional assay in h5-HT_2BR-CHO cells (Table 3; Figure S3). None of the compounds altered 5-HT_2BR-evoked Ca_i²⁺ alone or in the presence of 5-HT (Table 3).

Table 3.

Effects of Representative 5-HT_2CR PAMs and Dual 5-HT_2CR/5-HT_2AR PAMs (1 nM) on 5-HT-Induced Ca_i²⁺ Release in h5-HT_2BR-CHO Cells^b

Compound	PH	Emax RFU (%5-HT)a
8		95.6 ± 4.6
9		106.1 ± 7.5
11		97.2 ± 9.2
12		92.4 ± 3.2
13		98.4 ± 4.6
25		100.6 ± 4.4

^aAddition of the synthetic compound (1 nM) occurred 15 min prior to assessment of Ca_i²⁺ release evoked by increasing concentrations of 5-HT (vehicle, 10⁻¹¹ to 10⁻⁵ M) in h5-HT_2BR-CHO cells. Data are presented as maximal 5-HT-induced Ca_i²⁺ release (E_max).

^bp < 0.05. Comparisons between means for E_max were conducted with an unpaired t test with Welch’s correction (GraphPad Prism). All statistical analyses were conducted with an experiment-wise error rate of α = 0.05.

Molecular Docking to 5-HT_2CR and 5-HT_2AR.

The molecular docking of the receptor–ligand interaction provides useful information for structure-based drug design. To obtain comprehensive docking poses of these PAMs at the 5-HT_2CR and 5-HT_2AR allosteric sites, we employed the Schrödinger Drug Discovery Suite for modeling the 5-HT_2CR and 5-HT_2AR and selected PAM molecules by utilizing recently reported X-ray crystal structures (see SI for details on docking protocol).^37,38 Docking studies for representative molecules 8, 9, 11, 12, 13, and 25 to the 5-HT_2CR followed the same protocol we reported previously.^18,20 Briefly, we preprocessed and optimized the 5-HT_2CR X-ray crystal structure (PDB: 6BQG), except for using the newly released OPLS4 force field in Maestro 12.7, to provide the docking model for 5-HT_2CR PAMs herein.^37,39 The docking model for 5-HT_2AR PAMs was obtained by utilizing the 5-HT_2AR X-ray crystal structure (PDB: 6WGT), which is in complex with d-lysergic acid diethylamide (LSD).³⁸ The structure was preprocessed and optimized with the Schrödinger protein preparation wizard, including filling loops with missing residues, replacing stabilizing mutations, and removing stabilization domains not part of the receptor. Subsequently, we ran the Schrödinger Induced Fit Docking (IFD) protocol to replace LSD with 5-HT in the orthosteric binding site to provide a suitable canvas upon which to dock our molecules. Selected compounds 9, 11, and 13 were then docked into the 5-HT_2AR–5-HT complex model using the Schrödinger Glide software platform, employing extra-precision (XP), standard-precision (SP), and IFD docking protocols to obtain reliable and reproducible poses. The representative pose for each molecule was selected from a top-scoring, enriched cluster, along with the biological and chemical rationality evaluation (e.g., interactions at the binding site and physicochemical properties).

Each of the agonists and oleamide-like PAM compounds modeled was prepared with the Maestro LigPrep module, ensuring biologically accurate ionization states and conformations with the OPLS4 force field. As the global overview of the docking poses shows in Figure 5A,B, the six oleamide-like compounds—8, 9, 11, 12, 13, 25—occupy a similar docking cavity for both 5-HT_2CR and 5-HT_2AR, distinct from the 5-HT binding pocket and differentiated by a somewhat more restricted, smaller volume in the 5-HT_2AR. The six compounds localize in a spatially distinct allosteric binding pocket near the top of the 7TM bundle formed by helices I–III, V–VII, and ECL2 and directly above the orthosteric binding site. Consistent with our previous docking studies,²⁰ the six molecules exhibited the flat stretch pose over the binding cavity with the PH responsible for key hydrogen bonding interactions to ECL2 and TM helix residues and the long LT oriented within a spatially constrained hydrophobic pocket, generating beneficial hydrophobic interactions (Figure 5A,B). The calculated distance between the 5-HT indole N-atom and the nearest carbon in LT of oleamide-derived compounds in both 5-HT₂Rs was carefully measured. For the 5-HT_2CR, the average distance between the six compounds and 5-HT was calculated to be 8.59 Å, and for the 5-HT_2AR, this average distance for the three compounds modeled was 8.13 Å. This measurement indicates an occupancy deeper into the TM bundle for the new 5-HT_2CR PAMs compared to the previous PAM 3 into the hydrophobic pocket (measured at 13.5 Å between 5-HT and 3), along with a tighter packed profile.²⁰ Moreover, as shown in Figure 5C,D, the dual 5-HT_2CR/5-HT_2AR PAMs reach deeper into the more compact binding pocket of 5-HT_2AR, displaying a slightly closer distance to the orthosteric ligand 5-HT (8.13 Å) compared to the allosteric site in 5-HT_2CR. This may be the reason that 5-HT_2AR PAMs are more sensitive to the length and volume than 5-HT_2CR PAMs. This feature may be related to distinctions in the sequence of the ECL2 between the two receptors; the 5-HT_2CR structure has a VAL208 in the same spatial position that the 5-HT_2AR contains the more voluminous LEU229 (Figure 5C,D).

Figure 5.

Extracellular overview of the crystal structure of 5-HT_2CR (PDB ID: 6BQG) or 5-HT_2AR (PDB ID: 6WGT)-PAMs complex with serotonin (teal) binding in the orthosteric site. (A) Global overview of 5-HT_2CR with representative 5-HT_2CR PAMs 8 (magenta), 12 (orange), and 25 (red) and dual 5-HT_2CR/5-HT_2AR PAMs 9 (blue), 11 (purple), and 13 (green). (B) Global overview of 5-HT_2AR with representative dual 5-HT_2CR/5-HT_2AR PAMs 9 (blue), 11 (purple), and 13 (green). (C) Docking poses of dual 5-HT_2CR/5-HT_2AR PAMs 9 (blue), 11 (purple), and 13 (green) in 5-HT_2CR. (D) Docking poses of dual 5-HT_2CR/5-HT_2AR PAMs 9 (blue), 11 (purple), and 13 (green) in 5-HT_2AR. The 5-HT_2CR is gray colored, and the 5-HT_2AR is gold colored. PAM compounds are shown in stick representation. Hydrogen bonds are in yellow dotted lines.

Figure 6 illustrates the detailed docking pose of dual 5-HT_2CR/5-HT_2AR PAMs 9 and 13. The amide carbonyl and terminal hydroxyl groups of 9 accept two hydrogen binding interactions with residues ASN331 (TMVI) and LEU209 (ECL2) in the 5-HT_2CR–5-HT complex, respectively (Figure 6A). Identical interactions with ASN343 (TMVI) and LEU228 (ECL2) are observed for the 5-HT_2AR–5-HT model (Figure 6B). The LT moiety of our oleamide-like compounds had been difficult to precisely dock into our previous allosteric model for 5-HT_2CR due to its lack of functional groups and overall flexibility.^18,20 However, with new structures and model improvements, the hydrophobic cavities created by the active-state receptor provide useful constraints for modeling the LT orientation. This is evident in the comparison between pose differences for compound 13 at the two receptors (Figure 6C,D). The binding pocket tunnel is created in 5-HT_2AR by LEU229 compared to the less imposing VAL208 in 5-HT_2CR in the corresponding ECL2 location. This spatial difference is likely to be advantageous for designing new 5-HT_2AR- or 5-HT_2CR-selective allosteric modulators. An additional difference includes a flipped orientation of ASN331 (5-HT_2CR) and ASN343 (5-HT_2AR) in the TMVI helix between the two 5-HT₂Rs (Figure 6A,B). The docking study for compound 13 also demonstrates the relative openness of the 5-HT_2CR allosteric pocket in comparison (Figure 6E,F). The binding poses are essentially flipped primarily because of crowding in 5-HT_2AR by LEU229 (5-HT_2CR has VAL208) as denoted by the bar (5-HT_2CR) and arrow (5-HT_2AR). Importantly, in the docking poses for both the 5-HT_2AR and 5-HT_2CR, compound 13 interacts via hydrogen bonds with residues in the ECL2 and additional residues in the TM helices. Specifically, the amide and terminal hydroxyl groups form interactions with residues LEU209 (ECL2), GLU347 (TMVII), and SER334 (TMVI) in 5-HT_2CR and residues LEU229 (ECL2), ASN363 (TMVII), and SER131 (TMII) in 5-HT_2AR. This work suggests that the stabilization of the ECL2 of both 5-HT₂Rs by bridging interactions between key LEU209 (5-HT_2CR) and LEU229 (5-HT_2AR) residues toward adjacent TM helix residues results in a conformational state by which positive allosteric modulation is achieved. Further ECL2 stabilization by filling the resultant hydrophobic allosteric pocket with a complimentary LT moiety likely plays a role in the observed PAM activity. In addition, we compared the 5-HT_2BR structure (PDB ID: 4IB4) with the 5-HT_2CR and 5-HT_2AR X-ray crystal structures by carrying out a superimposition and pairwise sequence alignment. Structural analysis of these receptors shows that the 5-HT_2CR/5-HT_2AR PAM 9 and 13 binding site in the corresponding area of 5-HT_2BR presents structural distinctions. Whereas the key residues of ECL2 remain almost the same among three structures (ECL2: 2A_LEU229/2B_LEU209/2C_LEU209), the key residues on TMVI, TMVII, and TMII that are forming H-bonds with the amide and terminal hydroxy groups of ligands are substantially different from those on 5-HT_2BR (TMVI: 2A_ALA346/2B_LEU347/2C_SER334; TMVII: 2A_GLY359/2B_GLN359/2C_GLU347; TMVII: 2A_ASN363/2B_GLU363/2C_ASN351; TMII: 2A_SER131/2B_ALA111/2C_SER110). Ligand-mediated stabilization does not appear to be effectively achieved between ECL2 and the adjacent TM helix for 5-HT_2BR due to structural differences, which might contribute to the selectivity of compound 13.

Figure 6.

Detailed interaction diagram of dual 5-HT_2CR/5-HT_2AR PAMs 9 and 13 to the 5-HT_2CR (PDB ID: 6BQG) or 5-HT_2AR (PDB ID: 6WGT) X-ray crystal structure. Detailed interaction diagram of 9 (blue) to the (A) 5-HT_2CR X-ray crystal structure or (B) 5-HT_2AR X-ray crystal structure. Top view of 13 (green) to the (C) 5-HT_2CR X-ray crystal structure or (D) 5-HT_2AR X-ray crystal structure. Side view of 13 (green) to the (E) 5-HT_2CR X-ray crystal structure or (F) 5-HT_2AR X-ray crystal structure. The 5-HT_2CR is gray colored, and the 5-HT_2AR is gold colored. PAM compounds are shown in stick representation. Hydrogen bonds are noted in yellow dotted lines.

In Vitro Radioligand Binding Displacement Study.

Considering the dual in vitro activity at the 5-HT_2CR and 5-HT_2AR, compounds 11 and 13 were selected as representative tool compounds out of the active analogues for further pharmacological evaluation. To explore the off-target profile of the two compounds, we utilized the National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program (PDSP) to assess a broad-panel of GPCRs and monoamine transporters (Table 4; Table S1).⁴⁰ Generally, compounds 11 and 13 exhibited no off-target effects for most of the receptors and monoamine transporters evaluated, except that 11 exhibited a K_i of 0.46 μM at 5-HT_2CR (Table S1). Compound 13 did not displace 5-HT_2AR or 5-HT_2CR binding, suggesting an important feature for selectivity of a 5-HT_2A/2CR PAM. Both compounds 11 and 13 did not displace binding to the human ether-a-go-go-related gene (hERG) potassium channel, suggesting a low risk of cardiac adverse events.⁴¹ Compound 13 featured micromolar displacement at the H₃ receptor (K_i = 5.3 μM) and σ₂ receptor (K_i = 3.6 μM) (Table S1). Nonetheless, the off-target profile of compound 13 is considered promising, and compound 13 was selected as a representative dual PAM for further evaluation (for the full panel results, see Table S1).

Table 4.

Displacement of Radioligand Binding by Compound 13 in a Broad Panel of Receptors and Transporters

receptor/transporter	radioligand	% inhibitiona	Ki (μM)b
5-HT1A	[3H]-8-OH-DPAT	13.99	NT
5-HT1B	[3H]-GR125743	−5.9	NT
5-HT1D	[3H]-GR125743	−6.35	NT
5-HT1E	[3H]-5-HT	6.48	NT
5-HT2A	[3H]-ketanserin	2.03	NT
5-HT2B	[3H]-LSD	33.25	NT
5-HT2C	[3H]-mesulergine	44.64	NT
5-HT3	[3H]-LY278584	5.63	NT
5-HT5A	[3h]-LSD	−7.85	NT
5-HT6	[3H]-LSD	−8.61	NT
5-HT7	[3H]-LSD	−8.95	NT
D1	[3H]-SCH23390	22.64	NT
D2	[3H]-N-methylspiperone	2.44	NT
D3	[3H]-N-methylspiperone	−6.88	NT
D4	[3H]-N-methylspiperone	19.02	NT
D5	[3H]-SCH23390	56.32	>10Avg
DAT	[3H]-WIN35428	4.01	NT
SERT	[3H]-citalopram	−20.8	NT
NET	[3H]-nisoxetine	−0.8	NT
α 2A	[3H]-rauwolscine	28.38	NT
α 2B	[3H]-rauwolscine	14.82	NT
α 2C	[3H]-rauwolscine	20.26	NT
HERG	[3H]-dofetilide	1.6	NT

^aThe binding replacement test was conducted at 10 μM of compound 13. A binding inhibition result >50% was considered reliable replacement of target receptor radioligand by the test compound.

^bThe K_i value was calculated via a nonlinear regression analysis of radioligand competition isotherms for ligand binding inhibition >50%. NT = not tested, Avg = average K_i from repeated experiments.

We calculated the central nervous system (CNS) multi-parameter optimization (MPO) value for compound 13.⁴² The CNS MPO was adopted to increase the odds of prospectively designing CNS-targeted molecules that achieve CNS exposure.^42,43 The calculated score for compound 13 is 3.3 out of a collective score range from 0 to 6. Of note, for this series of molecules, the predictive nature is inherently limited by a lower number of structurally comparable molecules in the training data set. The literature suggests that a higher MPO value is desirable for the CNS drugs (Table S2).

Toxicity Profile and Pharmacokinetics (PK) Profile of Compound 13.

In silico toxicity predictions were conducted to evaluate compound 13 in various toxicity end points such as acute toxicity, hepatotoxicity, cytotoxicity, carcinogenicity, mutagenicity, immunotoxicity, adverse outcome (ProTox-II) pathways, and toxicity targets (Table 5; Table S3). The results suggest that compound 13 exhibits a profile consistent with a low expectation of adverse drug reactions or toxic effects and was ranked in a nontoxic class VI (LD₅₀ > 5000 mg/kg). A study of CYP450 inhibition was then carried out in human liver microsomes to assess the inhibitory potential of compound 13 (10 μM) against CYP450 isoforms (Table 5). Compound 13 displayed ≤20% inhibition of CYP3A4, CYP1A2, CYP2C8, CYP2C19, CYP2D6, and CYP2C9 and ~50% inhibition of CYP2B6.

Table 5.

Toxicity Profile and Human Liver Microsome P450 Inhibition Profile for 13^a

In silico toxicity profile
predicted LD50					10,000 mg/kg
toxicity classb					6 (low)
prediction accuracy					70.97%
targetc		prediction (probability)			targetc		prediction (probability)
hepatotoxicity		inactive (0.84)			AR-LBD		inactive (0.98)
carcinogenicity		inactive (0.59)			aromatase		inactive (0.99)
immunotoxicity		inactive (0.96)			PPAR-gamma		inactive (0.98)
mutagenicity		inactive (0.89)			Nrf2/ARE		inactive (0.94)
cytotoxicity		inactive (0.82)			HSE		inactive (0.94)
aryl hydrocarbon receptor		inactive (0.98)			MMP		inactive (0.95)
estrogen receptor α		inactive (0.89)			phosphoprotein p53		inactive (0.96)
ER-LBD		inactive (0.97)			ATAD5		inactive (0.99)
androgen receptor		inactive (0.98)
Human liver microsome P450 inhibition profile
P450 isoforms	CYP3A4d	CYP3A4e	CYP1A2	CYP2B6	CYP2C8	CYP2C9	CYP2C19	CYP2D6
inhibitory %	18.72	11.11	3.4	53.31	11.87	21.2	18.34	10.13

^aFor detailed in silico toxicity prediction profile (ProTox-II), see SI Table S3 (more information at http://tox.charite.de/protox II/).⁴⁴ Cytochrome P450 enzymatic inhibition assays for compound 13 performed at 10 μM and represented as percent inhibition.

^bToxicity class ranks from 1 to 6: 1 = high, 6 = low.

^cER-LBD = estrogen receptor ligand binding domain; AR-LBD = androgen receptor ligand binding domain; PPAR-gamma = peroxisome proliferator activated receptor gamma; Nrf2/ARE = nuclear factor (erythroid-derived 2)-like 2/antioxidant responsive element; HSE = heat shock factor response element; MMP = mitochondrial membrane potential; ATAD5 = ATPase family AAA domain-containing protein 5.

^dCYP3A4 (midazolam).

^eCYP3A4 (testosterone).

A membrane permeability evaluation of compound 13 was carried out in hMDRI-MDCKII cells to investigate potential CNS permeability and drug efflux (Table 6). Compound 13 demonstrated a moderate permeability and a low efflux ratio of 0.6 and 0.4, respectively, in the absence or presence of a P-glycoprotein (P-gp) inhibitor, indicating that 13 is not a predicted substrate for P-gp. Compound 13 displayed a kinetic solubility in the PBS buffer (pH = 7.2) of 48.55 μg/mL. The rate of disappearance of 13 following incubation with rat or human liver microsomes was monitored to determine the in vitro intrinsic clearance; 13 showed a higher clearance rate than prior 5-HT_2CR PAMs.^18,20 Nonetheless, in vivo PK evaluation in male Sprague–Dawley rats after a single dose of 10 mg/kg by intraperitoneal (ip) or 20 mg/kg by oral (po) administration was performed to assess the drug-like properties and in vivo probe potential of 13. As summarized in Table 7, 10 mg/kg of 13 administered ip (T_1/2 = 2.41 ± 1.73 h) or 20 mg/kg of 13 administered po (T_1/2 = 2.14 ± 0.18 h) resulted in a similar plasma exposure (AUC_0–inf; ip: 1885 ± 232 ng·h·mL⁻¹; po: 615 ± 94 ng·h·mL⁻¹) to 5-HT_2CR PAM 2 (AUC_0–inf; intravenous: 939 ± 108 ng·h·mL⁻¹; po: 737 ± 56 ng·h·mL⁻¹), although it was slightly inferior to 5-HT_2CR PAM 3.^18,20 Compound 13 exhibited brain/plasma (B/P) ratios of 0.589 (15 min) and 2.05 (1 h) after intraperitoneal administration, significantly higher than 0.3, which was reported as the cutoff to classify CNS drugs. These data suggest that 13 has a comparable plasma exposure to that of PAM 2 and good brain permeability as a suitable in vivo pharmacological tool compound for further in vivo animal studies.

Table 6.

In Vitro PK Data for Compound 13

MDCK-MDR1 permeability	without P-gp inhibitor: PappA → B = 5.86 × 10−6 cm/s; PappB → A = 3.23 × 10−6 cm/s; efflux ratio: 0.6
	with P-gp inhibitor:PappA → B = 10.64 × 10−6 cm/s; PappB → A = 4.77 × 10−6 cm/s; efflux ratio: 0.4
kinetic solubility (PBS pH = 7.2)	48.55 μg/mL
liver microsomal clearance	CLr = 174.66 μL/min/mg
	CLh = 148.85 μL/min/mg

Table 7.

In Vivo PK and Brain Penetrability for Compound 13^a

In vivo PK profile
dose (mg/kg)	T1/2 (h)	Tmax (h)	Cmax (ng/mL)	AUC0–inf (ng·h·mL−1)
10, ip	2.41 ± 1.73	0.5	878 ± 236	1885 ± 232
20, po	2.14 ± 0.18	0.667 ± 0.289	181 ± 36	615 ± 94
Brain penetration analysis
dose (mg/kg)	time (h)	brain conc (ng/g)	plasma conc (ng/mL)	brain/plasma ratio
10, ip	0.25	378 ± 58.8	642 ± 133	0.589
10, ip	1.0	1120 ± 65	547 ± 18.3	2.05

^aT_1/2, half-life; T_max, time of maximum concentration; C_max, maximum concentration; AUC_0–inf, area under the plasma concentration–time curve; time, hours after dose for brain collection; brain conc, averaged concentration of 13 in tissue sample. Experiments were studied in biological triplicates, and data values are shown as the mean ± SEM (± standard error of the mean) from male Sprague–Dawley rats. Vehicle, 10% dimethyl sulfoxide (DMSO)/90% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD).

Effects of Compound 13 on Spontaneous Locomotor Activity.

Locomotor activity in an unfamiliar environment is frequently measured to assess novelty-evoked exploratory behavior that predicts vulnerability to maladaptive psychiatric-like states (e.g., anxiety, initiation of problematic drug use) in rodent models.^45–49 Ligands that activate the 5-HT_2CR or augment its functional efficacy are well-described to dose-dependently suppress novelty-induced motor activity in rodents.^17,18,50,51 Preferential 5-HT_2AR agonists are noted to increase or decrease novelty-induced motor activity in rodents depending on the dose administered.^50,52 We surmised that 13, a proposed dual 5-HT_2CR/5-HT_2AR PAM in vitro, would impact novelty-induced motor activity in vivo through enhancement of endogenous 5-HT actions at 5-HT_2CR and/or 5-HT_2AR. Thus, in the present experiment, we tested the hypothesis that 13 would dose-dependently alter novelty-induced motor activity in male Sprague–Dawley rats. We assessed the effects of 13 (3, 10, or 30 mg/kg ip) on total ambulations over a 90 min session (mean ± S.E.M) (Figure 7A).^18,53,54 A main effect of treatment (F_3,44 = 7.826, p = 0.0003) was observed for total ambulations; a priori comparisons revealed that 30 mg/kg of 13 significantly reduced total ambulations vs vehicle (p < 0.0001).

Figure 7.

Impact of compound 13 on novelty-induced locomotor activity in Sprague–Dawley rats. (A) Male rats (n = 12/treatment) naïve to the activity monitors were injected with vehicle (ip) or compound 13 (3, 10, or 30 mg/kg, ip) 30 min prior to the start of the 90 min locomotor assessment. The mean total ambulations (±SEM) for the 90 min session are plotted with open circles representing individual rats (*p < 0.05 vs Veh). (B) In a separate cohort (n = 11–12/treatment), Veh, the 5-HT_2CR antagonist SB242084 (0.5 mg/kg, ip), or the 5-HT_2AR antagonist M100907 (0.1 mg/kg, ip) plus Veh or 13 (30 mg/kg) was administered 30 min prior to the 90 min locomotor assessment. Average activity (mean ± SEM) is shown for each treatment group and compared to animals receiving Veh + Cmpd 13 (*p < 0.05). The average basal activity (Veh + Veh; dashed line) is shown for reference. Please see Experimental Section for Veh descriptions.

A separate cohort of rats was then employed to assess the impact of the selective 5-HT_2AR (M100907, 0.1 mg/kg, ip) or the 5-HT_2CR antagonist (SB242084, 0.5 mg/kg, ip) on the suppressive motor effects of 13 to illuminate 5-HT_2CR- and 5-HT_2AR-specific contributions. The employed doses of both 5-HT₂R antagonists were selected based on their lack of effect on novelty-evoked motor activity in naïve male rats (data not shown).^55,56 Total ambulations in a novel environment for the 90 min session are presented (mean ± SEM) (Figure 7B). Veh + Cmpd 13 (30 mg/kg ip) reduced activity (~34%) relative to Veh + Veh baseline (dashed line) as seen in Figure 7A (~31%). A main effect of treatment (F_2,32 = 8.971, p = 0.0008) was observed for ambulations across the entire 90 min session (Figure 7B). SB242084 (0.5 mg/kg) pretreatment reversed 13-evoked suppression of motor activity (p = 0.0039 vs Veh + Cmpd 13), whereas M100907 (0.1 mg/kg) treatment was ineffective (p = 0.7664 vs Veh + Cmpd 13). This behavioral profile has been described for previously identified 5-HT_2CR PAMs, suggesting that 13 is a preferential 5-HT_2CR PAM in vivo.¹⁸

CONCLUSIONS

We report the molecular design, chemical synthesis, and pharmacological evaluation of a novel series of oleamide-based analogues as novel selective 5-HT₂R PAMs in vitro. Several compounds (8, 12, 15, 16, and 25) were identified as selective 5-HT_2CR PAMs, whereas 9, 11, 13, 19, and 28 were characterized as dual 5-HT_2CR/5-HT_2AR PAMs. Further in vitro evaluation of representative 5-HT_2CR PAMs and dual 5-HT_2CR/5-HT_2AR PAMs suggested acceptable 5-HT subtype selectivity with no efficacy at 5-HT_2BR. Molecular docking studies identified a spatially distinct allosteric binding site for 5-HT_2CR and 5-HT_2AR differentiated by a narrower pocket with a reduced volume for 5-HT_2AR vs 5-HT_2CR, providing a clue for the discovery of subtype selective allosteric 5-HT_2CR and 5-HT_2AR modulators. Compound 13 (JPC0323) showed negligible displacement of the orthosteric binding sites of roughly 50 GPCRs and transporters, including 5-HT_2AR and 5-HT_2CR. Therefore, 13 is a first-in-class dual 5-HT_2CR/5-HT_2AR PAM selected from an extended medicinal chemistry endeavor. Collectively, 13 exhibited a suitable CNS MPO value, acceptable plasma exposure at 10 mg/kg (ip), and decent brain penetrability and behavioral efficacy in a spontaneous locomotor activity assay. These cumulative findings suggest that 13 maintains favorable drug-like properties for utility as an in vivo probe of allosteric modulation of the 5-HT_2CR/5-HT_2AR.

Compound 13 suppressed spontaneous ambulations in a 5-HT_2CR-, but not 5-HT_2AR-, dependent manner, suggesting that 13 demonstrates a preferential 5-HT_2CR PAM profile in vivo. However, these observations do not definitively demonstrate a lack of efficacy of 13 to engage 5-HT_2AR signaling. Both 5-HT_2CR agonists and PAMs reliably blunt horizontal activity in rodents, whereas the impact of 5-HT_2AR activation on motor activity is ligand- and dose-dependent.^{17,18,50−52} Further, 5-HT_2CR agonists block the 5-HT_2AR-mediated head twitch response in rodents,⁵⁷ whereas selective 5-HT_2CR antagonists can unmask 5-HT_2AR activity produced by nonselective 5-HT_2CR agonists.⁵⁸ These data as well as cellular studies suggest that functional cross talk between the 5-HT_2AR and 5-HT_2CR is biologically meaningful.^6,59–61 Stimulation of the 5-HT_2AR is necessary for the powerful effects of psychedelics to alter perception and cognition.^62,63 Our in vivo results support the involvement of the 5-HT_2CR, but not the 5-HT_2AR, in the behavioral effects of compound 13, and future studies are required to characterize the effects of 13 in 5-HT_2AR-dependent preclinical models. PAM actions at 5-HT_2AR would distinguish from full 5-HT_2AR agonists that are hallucinogenic and may provide an alternative, potentially safer therapeutic approach in this light.^62,63 5-HT_2CR agonists and 5-HT_2AR agonists have been proposed for the treatment of anxiety, obsessive–compulsive, and substance use disorders.^62–65 As a dual 5-HT_2CR/5-HT_2AR PAM, compound 13 would be expected to amplify signaling at the 5-HT_2CR and 5-HT_2AR, with a reduced likelihood of agonist-induced adverse side effects, such as 5-HT_2AR agonist-evoked hallucinations.

Our phased success in the chemical modification of oleamide enriches the 5-HT₂R allosteric modulatory library with a novel series of structurally diversified scaffolds. This series will facilitate pharmacological investigations to examine 5-HT_2CR and 5-HT_2AR allosteric modulation to promote the development of novel therapeutics for chronic diseases with impaired serotonergic function. Further chemical optimization studies focusing on modifying the long lipophilic tail based on these identified chemical leads will improve the overall physicochemical and pharmacokinetic profiles and provide optimized drug-like compounds for further in vitro cellular and in vivo analysis.

EXPERIMENTAL SECTION

Chemistry.

General.

All commercially available reaction reagents and solvents were reagent grade and used directly. Preparative column chromatography was carried out using silica gel 60, particle size 0.063–0.200 mm (70–230 mesh, flash). Analytical TLC was performed employing silica gel 60 F254 plates (Merck, Darmstadt). NMR spectra were recorded on a Bruker-600 (¹H, 600 MHz; ¹³C, 150 MHz) spectrometer or Bruker-300 (¹H, 300 MHz; ¹³C, 75 MHz). ¹H and ¹³C NMR spectra were recorded with tetramethylsilane (TMS) as an internal reference. Chemical shifts were presented in ppm, and J values were expressed in Hz. Melting points were obtained on a Thermo Scientific electrothermal digital melting point apparatus. High-resolution mass spectra (HRMS) were acquired with a Thermo Fisher LTQ Orbitrap Elite mass spectrometer. Parameters were as follows: the nano ESI spray voltage was 1.8 kV, capillary temperature was 275 °C, resolution was 60,000, and ionization was achieved by positive mode. Purity of final compounds was carried out on a Shimadzu HPLC system (model CBM-20A LC-20 AD SPD-20A UV/vis) with analytical conditions as follows: Waters μBondapak C18 (300 × 3.9 mm), flow rate 0.5 mL/min, UV detection at 254 and 210 nm, and linear gradient from 30% acetonitrile in water (0.1% TFA) to 100% acetonitrile (0.1% TFA) in 20 min followed by 30 min of the last-named solvent. All newly synthesized compounds were characterized with ¹H NMR, ¹³C NMR, HRMS, and HPLC analysis. All biologically evaluated compounds are >95% pure.

General Procedure for the Synthesis of Oleamide Analogues 7–30.

To a solution of oleic acid (1.0 equiv) in dichloromethane (2 mL) was added HBTU (1.3 equiv) or EDCI (1.5 equiv) in combination with 1-hydroxybenzotriazole (1.5 equiv) and stirred under room temperature; then alcohol analogues, amino acid analogues, and monoamine-like fragments (1.1 equiv) along with DIPEA (2.5 equiv) were added to the solution. The reaction mixture was stirred for another 8 h, and a TLC plate was used to detect the reaction with the potassium permanganate chromogenic agent. After the completion of reaction, saturated ammonium chloride aqueous solution (10 mL) was titrated to the solution to quench the reaction, and then the mixture system was extracted with ethyl acetate (20 mL × 3), and washed with water and brine, dried over anhydrous Na₂SO₄, and filtered. The organic solvent was concentrated under reduced pressure and purified on a silica gel column (DCM/MeOH = 99:1), which afforded the desired products 7–30.

N-(2,3-Dihydroxypropyl)oleamide (7).

Compound 7 (57 mg, 80%) was prepared from oleic acid (0.20 mmol) following the general synthetic procedure for 7–30 as a white wax-like material. ¹H NMR (300 MHz, CDCl₃) δ 6.13 (s, 1H), 5.36 (h, J = 4.0 Hz, 2H), 3.77 (q, J = 5.2 Hz, 1H), 3.57 (t, J = 4.1 Hz, 2H), 3.42 (q, J = 5.8 Hz, 2H), 2.23 (t, J = 7.6 Hz, 2H), 2.02 (q, J = 6.2 Hz, 4H), 1.64 (t, J = 7.4 Hz, 2H), 1.30 (d, J = 10.6 Hz, 20H), 0.89 (t, J = 6.4 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 175.3, 130.0, 129.7, 71.2, 63.6, 42.2, 36.6, 31.9, 29.8, 29.7, 29.5, 29.3, 29.2, 29.1, 27.22, 27.16, 25.7, 22.7, 14.1. HRMS (ESI) calcd for C₂₁H₄₁NO₃ [M + H]⁺ 356.3159; found 356.3158.

N-(3-Hydroxypropyl)oleamide (8).

Compound 8 (25 mg, 52%) was prepared from oleic acid (0.14 mmol) following the general synthetic procedure for 7–30 as a white solid; mp 63.0–63.5 °C. ¹H NMR (300 MHz, CDCl3) δ 5.91 (s, 1H), 5.43–5.30 (m, 2H), 3.64 (t, J = 5.6 Hz, 2H), 3.46–3.37 (m, 2H), 2.20 (t, J = 7.6 Hz, 2H), 2.02 (q, J = 6.2 Hz, 4H), 1.66 (dt, J = 14.6, 6.9 Hz, 4H), 1.42–1.18 (m, 20H), 0.95–0.82 (m, 3H). ¹³C NMR (75 MHz, CDCl₃/MeOD) δ 175.1, 129.9, 129.6, 59.1, 38.5, 36.5, 36.1, 36.0, 31.9, 31.8, 29.7, 29.6, 29.4, 29.23, 29.19, 29.1, 27.13, 27.10, 25.8, 22.6, 14.0. HRMS (ESI) calcd for C₂₁H₄₁NO₂ [M + H]⁺ 340.3210; found 340.3352.

N-(2-Hydroxyethyl)oleamide (9).

Compound 9 (52 mg, 71%) was prepared from oleic acid (0.27 mmol) following the general synthetic procedure for 7–30 as a white solid; mp 63.0–63.5 °C. ¹H NMR (300 MHz, CDCl₃) δ 6.34 (s, 1H), 5.41–5.26 (m, 2H), 3.70 (t, J = 4.9 Hz, 2H), 3.56 (s, 1H), 3.40 (dd, J = 10.2, 5.4 Hz, 2H), 2.24–2.15 (m, 2H), 2.06–1.95 (m, 4H), 1.73–1.51 (m, 2H), 1.29 (d, J = 10.0 Hz, 20H), 0.88 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.6, 130.0, 129.7, 62.1, 42.4, 36.6, 31.9, 29.8, 29.7, 29.5, 29.31, 29.28, 29.2, 27.21, 27.17, 25.7, 22.7, 14.1. HRMS (ESI) calcd for C₂₀H₄₀NO₂ [M + H]⁺ 326.3054; found 326.3570.

(S)-N-(2,3-Dihydroxypropyl)oleamide (10).

Compound 10 (32 mg, 60%) was prepared from oleic acid (0.15 mmol) following the general synthetic procedure for 7–30 as a white wax-like material. ¹H NMR (300 MHz, CDCl₃) δ 6.44 (t, J = 6.1 Hz, 1H), 5.41–5.29 (m, 2H), 3.98 (bs, 1H), 3.85 (s, 1H), 3.76 (t, J = 5.2 Hz, 1H), 3.55 (bs, 2H), 3.40 (tq, J = 14.1, 8.2, 6.8 Hz, 2H), 2.22 (t, J = 7.6 Hz, 2H), 2.02 (q, J = 6.4 Hz, 4H), 1.77–1.54 (m, 2H), 1.40–1.19 (m, 20H), 0.93–0.84 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 175.4, 130.0, 129.7, 71.1, 63.6, 42.1, 36.6, 31.9, 29.8, 29.7, 29.5, 29.32, 29.29, 29.2, 27.23, 27.18, 25.7, 22.7, 14.1. HRMS (ESI) calcd for C₂₁H₄₁NO₃ [M + H]⁺ 356.3159; found 356.3154.

N-(1,3-Dihydroxypropan-2-yl)oleamide (11).

Compound 11 (58 mg, 60%) was prepared from oleic acid (0.27 mmol) following the general synthetic procedure for 7–30 as a whitish wax. ¹H NMR (300 MHz, CDCl₃) δ 6.69 (d, J = 7.9 Hz, 1H), 5.46–5.16 (m, 2H), 3.97–3.77 (m, 1H), 3.65 (ddd, J = 28.2, 11.3, 4.8 Hz, 4H), 2.86 (s, 2H), 2.26–2.09 (m, 2H), 2.07–1.85 (m, 4H), 1.71–1.50 (m, 2H), 1.26 (d, J = 9.6 Hz, 20H), 0.86 (t, J = 6.6 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 175.0, 130.0, 129.7, 61.7, 52.4, 52.3, 36.6, 36.5, 31.8, 29.71, 29.69, 29.5, 29.3, 29.2, 29.1, 27.2, 27.1, 25.7, 22.6, 14.0. HRMS (ESI) calcd for C₂₁H₄₁NO₃ [M + H]⁺ 356.3159; found 356.3150.

N-((2S)-1,3-Dihydroxy-1-phenylpropan-2-yl)oleamide (12).

Compound 12 (61 mg, 94%) was prepared from oleic acid (0.15 mmol) following the general synthetic procedure for 7–30 as an off-white wax-like material. ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ 7.41–7.14 (m, 5H), 6.68–6.77 (d, J = 8.4 Hz, 1H), 5.39–5.25 (m, 2H), 4.97 (d, J = 3.5 Hz, 1H), 4.08–3.99 (m, 1H), 3.89 (d, J = 2.5 Hz, 2H), 3.63 (qd, J = 11.1, 5.8 Hz, 2H), 2.08 (t, J = 7.3 Hz, 2H), 1.99 (q, J = 7.2 Hz, 4H), 1.44 (p, J = 7.4 Hz, 2H), 1.26 (d, J = 9.4 Hz, 20H), 0.92–0.78 (m, 3H). ¹³C NMR (75 MHz, CDCl₃/CD₃OD) δ 175.0, 141.5, 129.9, 129.7, 128.1, 127.4, 125.7, 71.8, 62.2, 56.4, 56.3, 36.5, 36.4, 31.8, 29.69, 29.66, 29.4, 29.24, 29.19, 29.1, 29.0, 27.1, 25.7, 22.6, 14.0. HRMS (ESI) calcd for C₂₇H₄₅NO₃ [M + H]⁺ 432.3472; found 432.3465.

N-(1,3-Dihydroxy-2-(hydroxymethyl)propan-2-yl)oleamide (13).

Compound 13 (73 mg, 94%) was prepared from oleic acid (0.20 mmol) following the general synthetic procedure for 7–30 as a white wax. ¹H NMR (300 MHz, CDCl₃) δ 6.51 (s, 1H), 5.36 (s, 2H), 3.60 (s, 6H), 2.24 (t, J = 7.6 Hz, 2H), 2.02 (d, J = 6.1 Hz, 4H), 1.62 (s, 2H), 1.30 (d, J = 11.4 Hz, 20H), 0.90 (d, J = 6.0 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 175.3, 130.0, 129.7, 61.8, 61.6, 37.0, 31.9, 29.7, 29.5, 29.3, 29.2, 29.1, 27.2, 25.8, 22.7, 14.1. HRMS (ESI) calcd for C₂₂H₄₄NO₄ [M + H]⁺ 386.3265; found 386.3262.

N-(2,2-Diethoxyethyl)oleamide (14).

Compound 14 (56 mg, 70%) was prepared from oleic acid (0.20 mmol) following the general synthetic procedure for 7–30 as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 5.78–5.65 (m, 1H), 5.41–5.25 (m, 2H), 4.50 (t, J = 5.2 Hz, 1H), 3.70 (dq, J = 9.6, 7.1 Hz, 2H), 3.53 (dq, J = 15.9, 6.8 Hz, 2H), 3.38 (t, J = 5.5 Hz, 2H), 2.17 (t, J = 7.6 Hz, 2H), 2.00 (q, J = 6.2 Hz, 4H), 1.62 (t, J = 7.4 Hz, 2H), 1.42–1.14 (m, 26H), 0.88 (t, J = 6.5 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 173.2, 130.0, 129.7, 100.8, 62.8, 41.9, 36.7, 31.9, 29.73, 29.68, 29.5, 29.3, 29.2, 29.1, 27.2, 27.1, 25.7, 22.6, 15.3, 14.1. HRMS (ESI) calcd for C₂₄H₄₇NO₃Na [M + Na]⁺ 420.3448; found 420.3446.

N-(2-(2-Hydroxyethoxy)ethyl)oleamide (15).

Compound 15 (62 mg, 83%) was prepared from oleic acid (0.20 mmol) following the general synthetic procedure for 7–30 as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 6.21–6.03 (m, 1H), 5.33 (q, J = 6.3 Hz, 2H), 3.82–3.69 (m, 2H), 3.57 (q, J = 4.4 Hz, 4H), 3.46 (q, J = 5.4 Hz, 2H), 2.59 (s, 1H), 2.18 (t, J = 7.6 Hz, 2H), 2.01 (q, J = 6.4 Hz, 4H), 1.63 (t, J = 7.4 Hz, 2H), 1.29 (d, J = 9.6 Hz, 20H), 0.88 (t, J = 6.3 Hz, 3H).¹³C NMR (75 MHz, CDCl₃) δ 173.5, 130.0, 129.7, 72.2, 70.0, 61.7, 39.2, 36.7, 31.9, 29.7, 29.5, 29.3, 29.1, 27.1, 25.7, 22.6, 14.1. HRMS (ESI) calcd for C₂₂H₄₄NO₃ [M + H]⁺ 370.3316; found 370.3314.

Methyl Oleoyl-l-allothreoninate (16).

Compound 16 (113 mg, 68%) was prepared from oleic acid (0.42 mmol) following the general synthetic procedure for 7–30 as a white solid; mp 63.0–63.5 °C. ¹H NMR (300 MHz, CDCl₃) δ 6.67–6.32 (m, 1H), 5.47–5.10 (m, 2H), 4.69–4.48 (m, 1H), 4.33 (s, 1H), 3.74 (s, 3H), 3.26 (s, 1H), 2.27 (t, J = 7.6 Hz, 2H), 2.11–1.89 (m, 4H), 1.79–1.51 (m, 2H), 1.28 (d, J = 11.6 Hz, 20H), 1.19 (d, J = 6.4 Hz, 3H), 0.87 (t, J = 6.6 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.1, 171.7, 130.0, 129.7, 67.8, 57.3, 52.4, 38.6, 36.5, 31.9, 29.74, 29.71, 29.5, 29.29, 29.27, 29.24, 29.15, 27.19, 27.16, 25.7, 22.6, 20.0, 14.1. HRMS (ESI) calcd for C₂₃H₄₃NO₄ [M + H]⁺ 398.3265; found 398.3453.

Methyl Oleoyl-l-serinate (17).

Compound 17 (100 mg, 62%) was prepared from oleic acid (0.42 mmol) following the general synthetic procedure for 7–30 as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 6.62 (s, 1H), 5.44–5.18 (m, 2H), 4.77–4.54 (m, 1H), 4.08–3.82 (m, 2H), 3.78 (s, 3H), 3.36 (s, 1H), 2.33–2.15 (m, 2H), 2.12–1.91 (m, 4H), 1.78–1.51 (m, 2H), 1.28 (d, J = 10.1 Hz, 20H), 0.88 (t, J = 6.6 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 173.9, 171.1, 130.0, 129.7, 63.2, 54.6, 52.7, 36.4, 31.9, 29.8, 29.7, 29.5, 29.30, 29.26, 29.2, 29.1, 27.21, 27.17, 25.6, 22.7, 14.1. HRMS (ESI) calcd for C₂₂H₄₁NO₄ [M + H]⁺ 384.3108; found 384.3457.

Methyl Oleoyl-l-tyrosinate (18).

Compound 18 (143 mg, 74%) was prepared from oleic acid (0.42 mmol) following the general synthetic procedure for 7–30 as a white solid; mp 71.5–72.3 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.16 (s, 1H), 6.95 (d, J = 8.5 Hz, 2H), 6.75 (d, J = 8.5 Hz, 2H), 6.05 (d, J = 8.0 Hz, 1H), 5.47–5.23 (m, 2H), 4.90 (dt, J = 8.0, 6.0 Hz, 1H), 3.75 (s, 3H), 3.04 (ddd, J = 30.8, 14.0, 5.9 Hz, 2H), 2.29–2.11 (m, 2H), 2.12–1.90 (m, 4H), 1.71–1.48 (m, 2H), 1.28 (s, 20H), 0.89 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 173.5, 172.5, 155.7, 130.2, 130.0, 129.8, 126.9, 115.6, 53.2, 52.4, 37.3, 36.6, 31.9, 29.8, 29.7, 29.5, 29.3, 29.2, 29.1, 27.23, 27.19, 25.6, 22.7, 14.1. HRMS (ESI) calcd for C₂₈H₄₅NO₄ [M + H]⁺ 460.3421; found 460.3436.

Methyl Oleoyl-l-tryptophanate (19).

Compound 19 (152 mg, 75%) was prepared from oleic acid (0.42 mmol) following the general synthetic procedure for 7–30 as a yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 8.43 (s, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.17 (dtd, J = 14.8, 7.1, 1.1 Hz, 2H), 6.98 (d, J = 2.4 Hz, 1H), 6.03 (d, J = 7.8 Hz, 1H), 5.49–5.24 (m, 2H), 5.00 (dt, J = 7.9, 5.4 Hz, 1H), 3.71 (s, 3H), 3.34 (dd, J = 5.3, 1.4 Hz, 2H), 2.22–2.11 (m, 2H), 2.03 (dd, J = 7.8, 4.7 Hz, 4H), 1.69–1.50 (m, 2H), 1.30 (s, 20H), 0.91 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 172.9, 172.6, 136.2, 130.0, 129.8, 127.7, 122.7, 122.2, 119.6, 118.5, 111.3, 110.0, 52.9, 52.3, 36.6, 31.9, 29.8, 29.7, 29.5, 29.34, 29.26, 29.2, 29.1, 27.7, 27.3, 27.2, 25.5, 22.7, 14.1. HRMS (ESI) calcd for C₃₀H₄₆N₂O₃ [M + H]⁺ 483.3581; found 483.3417.

Oleoyl-l-allothreonine (20).

Solid LiOH monohydrate (16.8 mg, 0.4 mmol) was added to a solution of 16 (40 mg, 0.10 mmol) in THF/H₂O 3:1 (2 mL) at rt. The reaction mixture was stirred for 48 h and determined complete by TLC. The reaction mixture was neutralized with HCl and extracted with EtOAc (3 × 10 mL). The combined organic extracts were washed with brine (5 mL) and concentrated under reduced pressure to afford 20 (25 mg, 66%) as a colorless gel. ¹H NMR (600 MHz, CDCl₃) δ 6.99–6.71 (bs, 2H), 5.53–5.11 (m, 2H), 4.52 (d, J = 6.9 Hz, 1H), 4.42 (s, 1H), 2.40–2.25 (m, 2H), 2.10–1.89 (m, 4H), 1.65 (s, 2H), 1.30 (d, J = 18.5 Hz, 20H), 1.22 (d, J = 5.2 Hz, 3H), 0.90 (t, J = 6.9 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃) δ 175.4, 174.3, 130.0, 129.6, 67.5, 57.7, 36.4, 31.9, 29.8, 29.6, 29.35, 29.32, 29.2, 27.24, 27.21, 25.8, 22.7, 19.4, 14.1. HRMS (ESI) calcd for C₂₂H₄₁NO₄ [M + H]⁺ 384.3108; found 384.3166.

Oleoyl-l-serine (21).

Compound 21 (20 mg, 54%) was prepared from 17 by a procedure similar to that used to prepare compound 20, as a white wax-like material. ¹H NMR (300 MHz, CDCl₃) δ 5.31 (td, J = 4.7, 2.1 Hz, 2H), 4.52 (t, J = 3.8 Hz, 1H), 3.95 (dd, J = 11.6, 3.9 Hz, 1H), 3.80 (dd, J = 11.5, 3.7 Hz, 1H), 3.61 (s, 3H), 2.25 (dt, J = 10.4, 7.5 Hz, 2H), 1.98 (q, J = 6.3 Hz, 4H), 1.71–1.50 (m, 2H), 1.38–1.18 (m, 20H), 0.94–0.78 (m, 3H). ¹³C NMR (75 MHz, CDCl₃/MeOD) δ 174.4, 172.7, 130.0, 129.7, 62.6, 36.3, 31.9, 29.71, 29.68, 29.5, 29.3, 29.24, 29.20, 29.1, 27.2, 25.5, 22.6, 14.0. HRMS (ESI) calcd for C₂₁H₃₉NO₄ [M + H]⁺ 370.2952; found 370.2995.

Oleoyl-l-tyrosine (22).

Compound 22 (40 mg, 90%) was prepared from 18 by a procedure similar to that used to prepare compound 20, as a white solid; mp 170.0–170.5 °C. ¹H NMR (600 MHz, CDCl₃) δ 6.90 (d, J = 8.0 Hz, 2H), 6.62 (d, J = 8.0 Hz, 2H), 5.41–5.11 (m, 2H), 4.38 (s, 1H), 3.94–3.59 (bs, 1H), 3.02–2.91 (m, 1H), 2.90–2.74 (m, 1H), 2.04–1.93 (m, 6H), 1.44 (d, J = 6.2 Hz, 2H), 1.36–1.10 (m, 20H), 0.85 (t, J = 7.0 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃/MeOD) δ 177.9, 174.3, 155.3, 130.2, 129.9, 129.7, 128.3, 115.3, 56.1, 37.0, 36.4, 31.9, 29.7, 29.5, 29.30, 29.27, 29.2, 27.2, 25.7, 22.6, 14.0. HRMS (ESI) calcd for C₂₇H₄₃NO₄ [M + H]⁺ 446.3265; found 446.3216.

Oleoyl-l-tryptophan (23).

Compound 23 (22 mg, 42%) was prepared from 19 by a procedure similar to that used to prepare compound 20, as a white wax-like material. ¹H NMR (300 MHz, CDCl₃) δ 8.42 (s, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.19 (t, J = 7.2 Hz, 1H), 7.11 (t, J = 7.2 Hz, 1H), 6.97 (s, 1H), 6.18 (d, J = 7.6 Hz, 1H), 5.46–5.26 (m, 2H), 4.92 (dd, J = 12.4, 5.4 Hz, 1H), 3.48–3.09 (m, 2H), 2.12–1.93 (m, 6H), 1.57–1.41 (m, 2H), 1.29 (s, 15H), 1.20 (s, 5H), 0.90 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 175.5, 174.3, 136.1, 130.0, 129.8, 127.8, 123.3, 122.1, 119.7, 118.4, 111.5, 109.5, 53.6, 36.4, 31.9, 29.8, 29.7, 29.6, 29.4, 29.3, 29.2, 29.1, 27.3, 27.2, 27.0, 25.4, 22.7, 14.1. HRMS (ESI) calcd for C₂₉H₄₄N₂O₃ [M + H]⁺ 469.3425; found 469.3500.

N-(4-Hydroxybenzyl)oleamide (24).

Compound 24 (40 mg, 69%) was prepared from oleic acid (0.15 mmol) following the general synthetic procedure for 7–30 as a white solid; mp 71.5–72.3 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.85–7.67 (m, 1H), 7.14 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H), 6.03 (t, J = 5.5 Hz, 1H), 5.61–5.25 (m, 2H), 4.39 (d, J = 5.6 Hz, 2H), 2.37–2.18 (m, 2H), 2.06 (dd, J = 7.9, 4.2 Hz, 4H), 1.79–1.61 (m, 2H), 1.34 (s, 20H), 0.95 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 173.8, 156.2, 130.0, 129.7, 129.20, 129.17, 115.8, 43.4, 36.8, 31.9, 29.8, 29.7, 29.5, 29.32, 29.25, 29.2, 29.1, 27.23, 27.18, 25.8, 22.7, 14.1. HRMS (ESI) calcd for C₂₅H₄₁NO₂ [M + H]⁺ 388.3210; found 388.3480.

N-(3,4-Dihydroxybenzyl)oleamide (25).

Compound 25 (30 mg, 50%) was prepared from oleic acid (0.15 mmol) following the general synthetic procedure for 7–30 as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 6.93–6.79 (m, 2H), 6.67 (dd, J = 8.1, 1.9 Hz, 1H), 6.17 (t, J = 5.7 Hz, 1H), 5.59–5.24 (m, 2H), 4.34 (d, J = 5.8 Hz, 2H), 2.34–2.21 (m, 2H), 2.15–1.95 (m, 4H), 1.81–1.55 (m, 2H), 1.34 (s, 20H), 0.95 (t, J = 6.7 Hz, 3H).¹³C NMR (75 MHz, CDCl₃) δ 174.4, 144.6, 144.3, 130.0, 129.8, 129.7, 119.7, 115.1, 114.9, 43.6, 36.8, 31.9, 29.8, 29.7, 29.5, 29.3, 29.22, 29.19, 29.1, 27.23, 27.17, 25.8, 22.7, 14.1. HRMS (ESI) calcd for C₂₅H₄₁NO₃ [M + H]⁺ 404.3159; found 404.3338.

N-(4-Hydroxyphenethyl)oleamide (26).

Compound 26 (24 mg, 42%) was prepared from oleic acid (0.14 mmol) following the general synthetic procedure for 7–30 as a white wax-like material. ¹H NMR (300 MHz, CDCl₃) δ 7.72 (s, 1H), 7.04 (d, J = 8.5 Hz, 2H), 6.94–6.74 (m, 2H), 5.83–5.68 (m, 1H), 5.49–5.25 (m, 2H), 3.52 (q, J = 6.9 Hz, 2H), 2.77 (t, J = 7.0 Hz, 2H), 2.25–2.14 (m, 2H), 2.13–1.97 (m, 4H), 1.77–1.53 (m, 2H), 1.32 (s, 20H), 0.93 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.0, 155.4, 130.0, 129.7, 129.71, 129.66, 115.7, 41.0, 36.8, 34.8, 31.9, 29.8, 29.7, 29.5, 29.3, 29.2, 29.1, 27.23, 27.18, 25.8, 22.7, 14.1. HRMS (ESI) calcd for C₂₆H₄₃NO₂ [M + H]⁺ 402.3367; found 402.3293.

N-(3,4-Dihydroxyphenethyl)oleamide (27).

Compound 27 (45 mg, 68%) was prepared from oleic acid (0.14 mmol) following the general synthetic procedure for 7–30 as a white solid; mp 69.1–71.0 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.94 (s, 1H), 6.85 (d, J = 8.0 Hz, 1H), 6.79 (d, J = 2.0 Hz, 1H), 6.59 (dd, J = 8.0, 2.0 Hz, 1H), 5.86 (t, J = 5.1 Hz, 1H), 5.49–5.25 (m, 2H), 3.51 (dd, J = 13.1, 6.9 Hz, 2H), 2.72 (t, J = 7.1 Hz, 2H), 2.29–2.14 (m, 2H), 2.14–1.97 (m, 4H), 1.75–1.52 (m, 2H), 1.32 (s, 20H), 0.93 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.6, 144.5, 143.3, 130.4, 130.0, 129.7, 120.4, 115.5, 115.3, 41.0, 36.8, 34.9, 31.9, 29.8, 29.7, 29.5, 29.33, 29.31, 29.21, 29.18, 29.1, 27.23, 27.18, 25.8, 22.7, 14.1. HRMS (ESI) calcd for C₂₆H₄₃NO₃ [M + H]⁺ 418.3316; found 418.3624.

N-(2-Morpholinoethyl)oleamide (28).

Compound 28 (71 mg, 67%) was prepared from oleic acid (0.27 mmol) following the general synthetic procedure for 7–30 as an off-white wax-like material. ¹H NMR (300 MHz, CDCl₃) δ 5.99 (s, 1H), 5.44–55.26 (m, 2H), 3.78–3.64 (m, 4H), 3.37 (q, J = 6.0 Hz, 2H), 2.57–2.41 (m, 6H), 2.19 (t, J = 6.0 Hz, 2H), 2.08–1.94 (m, 4H), 1.73–1.54 (m, 2H), 1.30 (d, J = 11.9 Hz, 20H), 0.89 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 173.2, 130.0, 129.7, 66.9, 57.1, 53.3, 36.8, 35.5, 31.9, 29.8, 29.7, 29.5, 29.32, 29.29, 29.2, 27.22, 27.17, 25.8, 22.7, 14.1. HRMS (ESI) calcd for C₂₄H₄₆N₂O₂ [M + H]⁺ 395.3632; found 395.3628.

tert-Butyl (4-Oleamidobutyl)carbamate (29).

Compound 29 (73 mg, 81%) was prepared from oleic acid (0.20 mmol) following the general synthetic procedure for 7–30 as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 5.78 (s, 1H), 5.33 (d, J = 5.3 Hz, 2H), 4.66 (s, 1H), 3.26 (q, J = 6.2 Hz, 2H), 3.13 (d, J = 6.4 Hz, 2H), 2.16 (t, J = 7.6 Hz, 2H), 2.08–1.94 (m, 4H), 1.62 (t, J = 7.5 Hz, 2H), 1.51 (d, J = 4.5 Hz, 4H), 1.44 (s, 9H), 1.28 (d, J = 9.2 Hz, 20H), 0.93–0.83 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 173.2, 156.1, 130.0, 129.7, 40.1, 39.0, 36.8, 31.9, 29.74, 29.70, 29.5, 29.29, 29.25, 29.1, 28.4, 27.6, 27.20, 27.16, 26.7, 25.8, 22.7, 14.1. HRMS (ESI) calcd for C₂₇H₅₃N₂O₃ [M + H]⁺ 453.4051; found 453.4059.

tert-Butyl 4-(Oleamidomethyl)piperidine-1-carboxylate (30).

Compound 30 (81 mg, 85%) was prepared from oleic acid (0.20 mmol) following the general synthetic procedure for 7–30 as a colorless oil. ¹H NMR (300 MHz, CDCl3) δ 5.59 (t, J = 6.1 Hz, 1H), 5.34 (td, J = 7.5, 4.7 Hz, 2H), 4.20–4.02 (m, 2H), 3.15 (s, 2H), 2.68 (t, J = 12.8 Hz, 2H), 2.18 (t, J = 7.6 Hz, 2H), 2.02 (q, J = 6.7 Hz, 4H), 1.65 (tt, J = 8.3, 4.3 Hz, 5H), 1.46 (s, 9H), 1.29 (d, J = 9.9 Hz, 20H), 1.19–1.09 (m, 2H), 0.89 (t, J = 6.5 Hz, 3H). ¹³C NMR (75 MHz, CDCl3) δ 173.3, 154.8, 130.0, 129.7, 79.4, 44.8, 43.6, 36.9, 36.4, 31.9, 29.8, 29.7, 29.5, 29.3, 29.24, 29.17, 29.13, 29.10, 28.4, 27.21, 27.16, 25.8, 22.7, 14.1. HRMS (ESI) calcd for C₂₉H₅₅N₂O₃ [M + H]⁺ 479.4207; found 479.4216.

In Vitro Pharmacology.

Intracellular Calcium (Ca_i²⁺) Release Assay in h5-HT₂R-CHO Cells.

The assays were conducted with Chinese hamster ovary (CHO) cells stably transfected with the human unedited (INI) h5-HT_2CR (h5-HT_2CR-CHO cells), human h5-HT_2AR (h5-HT_2AR-CHO cells), or h5-HT5-HT_2BR5-HT_2BR-CHO cells, which were generous gifts from Drs. Kelly A. Berg and William P. Clarke (University of Texas Health Science Center, San Antonio). The h5-HT_2BR (5-HT_2BR1/5-HT_2BR; h5-HT_2BR-CHO) cells were purchased from GenScript, Piscataway, NJ. The cellular growth environment was as follows: 37 °C, 5% CO₂, and 85% relative humidity. The h5-HT_2CR-CHO and h5-HT_2AR-CHO cells were cultured in a GlutaMax-MEM medium (Invitrogen, Carlsbad, CA) containing 5% fetal bovine serum (Atlanta Biologicals, Atlanta, GA) and 100 μg/mL hygromycin (Mediatech, Manassas, VA). The h5-HT_2BR-CHO cells were cultured in Ham’s F12 media supplemented with 10% FBS and 200 μg/mL Zeocin (Thermo Fisher Scientific, Carlsbad, CA). All cells were passaged when they reached 80% confluency.

The Ca_i²⁺ release assay was performed according to our previous publications, with minor modifications.^18,20,23 Specifically, cells (150 μL; passages 9–15) were plated in serum-replete medium at a density of 14,000–16,000 (FlexStation 3; Molecular Devices) or 30,000 cells/well (FLIPR^TETRA; Molecular Devices) in black-wall 96-well culture plates with optically clear flat bottoms. After ~24 h, the medium was replaced with serum-free (SF) GlutaMax-MEM medium (h5-HT_2CR-CHO and h5-HT_2AR-CHO cells) or serum free HAM’s F12 (h5-HT_2BR-CHO cells) supplemented with 20 nM to 100 μM putrescine (Sigma-Aldrich, St. Louis, MO), 20 nM to 100 μM progesterone (Sigma-Aldrich), and 1:100 ITS (1000 mg/L human recombinant insulin, 550 mg/L human recombinant transferrin, 0.67 mg/L selenious acid; Corning Inc., Corning, NY) (SF+ medium). After an incubation for another 3 h, SF+ medium for the h5-HT_2CR-CHO and h5-HT_2AR-CHO cells was replaced with 40 μL of Hank’s balanced saline solution (HBSS; without CaCl₂ or MgCl₂, pH 7.4) plus 40 μL of Calcium 6 dye solution (FLIPR No-wash kit, Molecular Devices, Sunnyvale CA) supplemented with 2.5 mM of water-soluble probenecid (Sigma-Aldrich), and then the plate was incubated with dye solution in the dark for 2 h at 37 °C followed by 15 min at room temperature. For h5-HT_2BR-CHO cells, Calcium 6 dye was incubated in the presence of serum-free Ham’s F12 medium supplemented with progesterone, putrescine, and ITS as described above. The drug was diluted at 5× concentration in 1× HBSS, and controls contained the same final concentration of diluent. The delivery of the compound (20 μL/well) was 15 min prior to the addition of 5-HT (10 pM to 100 μM; 25 μL/well), and a baseline was established for each well before the addition of the compound and 5-HT. The fluorescence readings were then adopted to evaluate the allosteric modulation of 5-HT-induced Ca_i²⁺ release. FlexStation 3 (Molecular Device) or FLIPR^TETRA (80–130 gain, 80% intensity, 0.3 s exposure) was used to measure fluorescence. For FlexStation 3, a 17 s baseline was established before the compound was added, and fluorescence was recorded every 1.7 s thereafter for 240 s. The maximum peak height of each well was determined by SoftMax software (Pro 5.4.5). For FLIPR^TETRA, a 10 s baseline was established before adding the compound, and then the fluorescence was recorded every 1 s for 120 s after the compound or 360 s after 5-HT. The maximum peak height of each well was determined by ScreenWorks 4.0 software. After the final reading, the cells were fixed in 2% paraformaldehyde (Sigma) overnight.

A four-parameter nonlinear regression analysis (GraphPad Prism 7 or 9) was used to determine the 5-HT-induced Ca_i²⁺ maximum release (E_max) in the presence of the test compound and calculated from four to six biological replicates, with each biological replicate performed in technical triplicates. The E_max of the test compound plus 5-HT was normalized to the E_max of 5-HT alone. Subsequently, Welch’s unpaired t test (GraphPad prism) was used for post hoc comparison of the E_max means. All statistical analyses were performed with an experimental error rate of α = 0.05. All treatment assignments were blinded to investigators who performed in vitro assays and end point statistical analyses.

In Vivo PK and Brain Penetration Analyses.

Male Sprague–Dawley rats (n = 3/treatment group; Beijing Vital River Laboratory, Animal Technology Co., Ltd., Beijing, China) weighing 200–250 g at the beginning of the experiment were housed three per cage in a pathogen-free, temperature-controlled (20–26 °C), and humidity-controlled (40–70%) environment with a 12 h light–dark cycle and ad libitum access to food and filtered water. Rats were randomly assigned to treatment groups. Vehicle [10% dimethyl sulfoxide (DMSO) and 90% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD); Cyclodextrin Technologies Development, Inc., High Springs, FL, USA] or compound 13 dissolved in vehicle was administered to rats ip at 10 mg/kg or po at 20 mg/kg. Blood samples (300 μL) were collected from the jugular vein before dosing and at 0.08, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h postdosing for ip administration and 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h postdosing for po administration. The blood samples were placed in heparinized tubes and centrifuged at 6000 rpm for 5 min at 4 °C. Brain samples were collected at 0.25 and 1 h postdosing. All samples were stored at −20 °C. The concentration of 13 in each sample was analyzed by Sundia MediTech Co., Ltd. The study and the related standard operating procedures were reviewed and approved by the Bioduro-Sundia Institutional Animal Care and Use Committee. The Bioduro-Sundia animal facility is approved with yearly inspection by the Shanghai Laboratory Animal Management Committee. The PK parameters of compound 13 were calculated according to a noncompartmental model using WinNonlin 8.1 (Pharsight Corporation, ver 5.3, Mountain View, CA, USA). The peak concentration (C_max) and time of peak concentration (T_max) were directly obtained from the plasma concentration–time profile. The elimination rate constant (λ) was obtained by the least-squares fitted terminal log-linear portion of the slope of the plasma concentration–time profile. The elimination half-life (T_1/2) was evaluated according to 0.693/λ. The area under the plasma concentration–time curve from 0 to time t (AUC_0–t) was evaluated using the linear trapezoidal rule and further extrapolated to infinity (AUC_0–inf) following the equation AUC_0–inf = AUC_0–t + Clast/λ. The PK parameters and brain concentrations are presented as mean ± SEM.

Effects of Compound 13 on Spontaneous Locomotor Activity.

Animals.

A total of 96 male Sprague–Dawley rats (Envigo RMS, LLC; Indianapolis, IN) weighing 175–199 g at the start of experiments were used. Rats were housed two per cage and allowed to acclimate for 7 days in a colony room at a constant temperature (21–23 °C) and humidity (45–50%) on a 12 h light–dark cycle (lights on 0600–1800 h). Food and water were available ad libitum, and rats were handled for 7 days prior to the start of behavioral testing. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (2011) and with the approval of the UTMB Institutional Animal Care and Use Committee. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, when available.

Drugs.

SB242084 (6-chloro-5-methyl-1[[2-(2-methylpyrid-3-yloxy)pyrid-5-yl]carbamoyl]indoline dihydrochloride; Cayman Chemical, Ann Arbor, MI) was dissolved in 0.9% NaCl containing 10 mM citric acid (Sigma Chemical Co.) and 8% HP-β-CD (Trappsol, Cyclodextrin Technologies Development Inc., High Springs, FL) with the final pH of the solution adjusted to 5.6. M100907 ((R)-(2,3-dimethoxyphenyl)-[1-[2-(4-fluorphenyl)ethyl]-piperidin-4-yl]methanol) (synthesized by Kenner Rice, National Institute on Drug Abuse, Bethesda, MD) was dissolved in 1% Tween-80 in 0.9% NaCl. Compound 13 was dissolved in 4% Tween 80 and 1% DMSO in 0.9% NaCl. To facilitate dissolution, prepared doses of compound 13 were briefly sonicated and maintained at ~37 °C until injection. SB242084 and M100907 were administered ip at a volume of 1 mL/kg. Because of solubility limits, compound 13 was injected at a volume of 2 mL/kg to achieve the desired dose.

Locomotor Activity Assessments.

Locomotor activity was monitored and quantified under low light conditions using a modified open field activity system (San Diego Instruments, San Diego, CA) as previously described.¹⁸ Clear plexiglass chambers (40 × 40 × 40 cm³) were surrounded by a 4 × 4 photobeam matrix positioned 4 cm from the chamber floor. Total ambulations were quantified as the sum of consecutive photobeam breaks that occurred within the inner central 16 × 16 cm² perimeter and in the surrounding outer peripheral 16 × 16 cm² perimeter of the activity monitor.

1. A between-subjects design was implemented to evaluate the dose–effect relationship of compound 13 on spontaneous motor activity. On test day, rats (n = 48) were administered vehicle (4% Tween 80 and 1% DMSO in 0.9% NaCl, 2 mL/kg, ip) or compound 13 (3, 10, or 30 mg/kg, 2 mL/kg, ip) 30 min prior to placement in activity monitors; ambulations and vertical activity were assessed for 90 min.

2. In a separate cohort, a between-subjects design was employed to ascertain 5-HT₂R subtypes mediating locomotor suppression induced by compound 13. Rats (n = 48) underwent a 7 day drug washout period before the start of experiment ii. On test day, rats were administered the 5-HT_2AR-selective antagonist M100907⁶⁶ (0.1 mg/kg, 1 mL/kg, ip), the 5-HT_2CR-selective antagonist SB242084⁶⁷ (0.5 mg/kg, 1 mL/kg, ip), or saline (1 mL/kg, ip) immediately followed by administration of vehicle (4% Tween 80 and 1% DMSO in 0.9% NaCl, 2 mL/kg, ip) or compound 13 (30 mg/kg, 2 mL/kg, ip) 30 min prior to placement in activity monitors; ambulations and vertical activity were assessed for 90 min. One animal was removed from the analysis because of a photobeam failure in its activity monitor.

Statistical Analyses.

Locomotor activity data are presented as mean ambulations (± SEM) totaled over the 90 min session for both the compound 13 dose–response curve and combination treatments with a 5-HT_2AR or 5-HT_2CR antagonist. The main effect of treatment on total ambulations was analyzed with a one-way analysis of variance (ANOVA); predetermined comparisons between treatment groups were made with Dunnett’s procedure. All statistical analyses were conducted with an experiment-wise error rate of α = 0.05.

ABBREVIATIONS

5-HT

serotonin

5-HT_2AR

5-HT_2A receptor

5-HT_2BR

5-HT_2B receptor

5-HT_2CR

5-HT_2C receptor

5-HT₂Rs

serotonin 5-HT₂ receptors

PAMs

positive allosteric modulators

CNS

central nervous system

GPCRs

G protein-coupled receptors

LSD

d-lysergic acid diethylamide

LT

lipophilic tail

PH

polar head

ECL2

extracellular loop 2

7TM

seven-transmembrane bundle

SAR

structure–activity relationship

HBTU

N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate

EDCI

1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

DIPEA

N,N-diisopropylethylamine

TFA

trifluoroacetic acid

PTLC

preparative thin layer chromatographic

PLCβ

phospholipase Cβ

Ca_i²⁺

intracellular calcium

CHO

Chinese hamster ovary

E _max

maximum 5-HT-induced Ca_i²⁺ release

NAM

negative allosteric modulator

NIMH

National Institute of Mental Health

PDSP

psychoactive drug screening program

MPO

multiparameter optimization

P-gp

P-glycoprotein

PK

pharmacokinetics

IFD

Induced Fit Docking

XP

extra-precision

SP

standard-precision

ECL

extracellular loop

TM

transmembrane helix

hERG

human ether-a-go-go-related gene

T _1/2

half-life

T _max

time of maximum concentration

C _max

maximum concentration

ip

intraperitoneal

AUC_0–inf

area under the concentration–time curves from time zero to infinity

B/P

brain/plasma

DMSO

dimethyl sulfoxide

HP-β-CD

2-hydroxypropyl-β-cyclodextrin