Synthesis and Characterization of 5-Hydroxy-2-(2-phenylethyl

Aug 13, 2015 - The involvement of the neurotransmitter serotonin (5-HT) in numerous physiological functions is often attributed to the diversity of re...
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Synthesis and Characterization of 5‑Hydroxy-2-(2phenylethyl)chromone (5-HPEC) and Its Analogues as Nonnitrogenous 5‑HT2B Ligands Dwight A. Williams,*,† Saheem A. Zaidi,‡ and Yan Zhang*,‡ †

Department of Pharmacology and Toxicology, Virginia Commonwealth University, 410 North 12th Street, P.O. Box 980613, Richmond, Virginia 23298-0613, United States ‡ Department of Medicinal Chemistry, Virginia Commonwealth University, BioTech One, Suite 205, 800 E. Leigh Street, P.O. Box 980540, Richmond, Virginia 23298-0540, United States S Supporting Information *

ABSTRACT: The involvement of the neurotransmitter serotonin (5-HT) in numerous physiological functions is often attributed to the diversity of receptors with which it interacts. Ligands targeting serotonin receptor 2B (5HT2B) have received renewed interest for their potential to help understand the role of 5-HT2B in migraines, drug abuse, neurodegenerative diseases, and irritable bowel syndrome. To date, most of the ligands targeting 5-HT2B have been nitrogen-containing compounds. The natural product 5-hydroxy-2-(2phenylethyl)chromone (5-HPEC, 5) has been shown previously to act as a non-nitrogenous antagonist for the 5-HT2B receptor (pKi = 5.6). This report describes further progress on the study of the structure−activity relationship of both naturally occurring and synthetic compounds bearing the 2-(2-phenylethyl)chromone scaffold at the 5-HT2B receptor. The inhibitory activity of the newly synthesized compounds (at 10 μM) was tested against each of the 5-HT2 receptors. Following this assay, the binding affinity and antagonism of the most promising compounds were then evaluated at 5-HT2B. Among all the analogues, 5-hydroxy-2(2-phenylpropyl)chromone (5-HPPC, 22h) emerged as a new lead compound, showing a 10-fold improvement in affinity (pKi = 6.6) over 5-HPEC with reasonable antagonist properties at 5-HT2B. Additionally, ligand docking studies have identified a putative binding pocket for 5-HPPC and have helped understand its improved affinity.

C

hromones (1) are heterocyclic compounds identified by their benzoannelated γ-pyrone core. They can be found throughout Nature and have been isolated from both terrestrial and marine sources. Naturally occurring chromones are often divided into subfamilies depending upon the position and type of substituent on the A ring.1,2 Examples of such subfamilies are the well-known flavones (2) and isoflavones (3) (Figure 1).1 Flavones and isoflavones have been of interest for many years, as they have shown a broad range of biological activities, which include but are not limited to antioxidant,3 anti-inflammatory,4 and antiviral effects.5 Recently, a relatively new class of naturally occurring chromones, the 2-(2-phenylethyl)chromones (4), have begun to receive growing attention. 2-(2-Phenylethyl)chromones were first described in 1978 and have a rare phenylethyl substituent at the C-2 position.6 Since their initial discovery, less than 100 naturally occurring 2-(2-phenylethyl)chromones have been isolated and characterized.7 Accordingly, there are very limited reports related to the biological activity of 2-(2-phenylethyl)chromones.7−11 Of interest is 5-hydroxy-2-(2phenylethyl)chromone (5-HPEC, 5), which was isolated from Imperata cylindrica P. Beauv. (Poaceae) by Yoon et al.9 The impetus for isolating and characterizing 5-HPEC (5) was its neuroprotective activity against glutamate excitotoxicity in primary cultures of rat cortical cells. Glutamate excitotoxicity describes the process of neuronal cell death that occurs when ionotropic glutamate receptors (iGluR) are overstimulated due © XXXX American Chemical Society and American Society of Pharmacognosy

Figure 1. Chromone core (1), flavone (2), isoflavone (3), 2-(2phenylethyl)chromone (4), and 5-hydroxy-2-(2-phenylethyl)chromone (5).

to disruptions in glutamate homeostasis that increase extracellular glutamate concentrations.12 Glutamate excitotoxReceived: February 4, 2015

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Figure 2. Structural scaffolds of known 5-HT2B antagonists.

differ structurally, a unifying feature of the aforementioned ligands is the presence of at least one basic nitrogen atom. A long-standing hypothesis has been that ligands targeting the 5HT receptors or transporter should contain a basic nitrogen atom in order to facilitate ligand binding and pharmacological action.29 Challenging this idea, Madras and associates have demonstrated that non-nitrogenous compounds maintained not only the pharmacological profile of their nitrogenous counterparts but also equipotent-binding affinities at the monoamine transporters.30−33 Thus, it is interesting that 5HPEC (5), which lacks a basic nitrogen atom, can target the 5HT2B receptor with moderate affinity and selectivity. This could suggest that 2-(2-phenylethyl)chromones such as 5 may represent a novel class of naturally occurring non-nitrogenous ligands for the 5-HT2B receptor. Given the interesting biological activity and binding profile of 5-HPEC (5), the goal of the current study was to explore the affinity, selectivity, and functional activity of other naturally occurring and non-natural 2-(2-phenylethyl)chromones at the 5-HT2B receptor. Such a systematic study would be useful for increasing the structural diversity of ligands targeting the 5HT2B receptor families and could also provide insight into how non-nitrogenous ligands interact with the 5-HT2B receptor to induce selectivity. Such compounds may also present unique pharmacological profiles. The structural features investigated included alkyl chain length and composition at C-2, the nature of the aromatic C ring, and substitution patterns of the B and C rings. Following their synthesis, each compound was screened for inhibition of radioligand binding at all three 5-HT2 receptors. Compounds showing significant inhibition were further evaluated for their binding affinity at the appropriate receptor. Compounds showing significant improvements in affinity over 5-HPEC (5) were then screened for their functional activity at the 5-HT2B. Finally, molecular modeling studies were performed to examine potential correlations between the structural modifications and the ligands’ 5-HT2B affinity.

icity has been proposed as a common end point for neuronal cell loss in glaucoma, retinal ischemia, Alzheimer’s disease, stroke, and HIV-associated neurocognitive disorder. Despite years of intense research developing iGluR antagonists to treat excitotoxicity, this approach has had little success, as prolonged antagonism of iGluRs is also neurotoxic. Therefore, alternative strategies are being sought to mediate the effects of glutamate excitotoxicity. As stated, 5-HPEC (5) showed neuroprotective activity against glutamate excitotoxicity; however, the potential mechanisms of action leading to the protective activity of 5HPEC are not fully known. Through a series of biological screening experiments, it was possible to show that 5-HPEC (5) can act as a selective serotonin receptor 2B (5-HT2B) antagonist with reasonable affinity (pKi = 5.6).13 The biological activity of chromones has largely been attributed to their antioxidative properties, and only recently have they been considered as potential ligands for G-protein coupled receptors like 5-HT2B.14−16 The 5-HT2B receptor is one of three subtypes found in the serotonin receptor 2 (5-HT2) family of receptors, with the others being 5-HT2A and 5-HT2C.17 Studies have shown that 5-HT2B is involved in the development of migraine,18 modulating the 5-HT transport system,19 and the rewarding and reinforcing effects of the abused drug 3,4methylenedioxymethamphetamine (MDMA).20,21 The endogenous ligand serotonin (5-HT) and selective ligands targeting the 5-HT2A receptor have been shown to be protective against glutamate neurotoxicity, but the potential role of 5-HT2B in protection against glutamate excitotoxicity has yet to be fully determined.22−25 This could be in part due to the notable challenge in developing ligands specific for subtypes within the 5-HT2 family.26 5-HT2B shares approximately 46−50% sequence identity with 5-HT2A and 5-HT2C. The homology within the transmembrane domain of the 5-HT2 receptors (which contains the 5-HT binding pocket) is higher than 70%.17 Thus, the design of selective ligands targeting 5-HT2B over 5-HT2A/2C presents a considerable obstacle. Among the available 5-HT2B selective ligands, commonly used structural scaffolds are arylureas (6), arylpiperazines (7), indolonapthyridines (8), pyrimidines (9), and tetrahydro-β-carbolines (10) (Figure 2).27,28 Although they

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RESULTS AND DISCUSSION Chemistry. The synthesis of the 5-HPEC analogues in this study was based on a previous route established for the B

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synthesis of 2-(2-phenylethyl)chromones.34 The first step was to synthesize the esters needed to explore changes in C-2 substitution (Scheme 1). Several approaches were employed for

(benzyloxy)acetate (19) was synthesized by reaction of benzyl alcohol with sodium hydride at 0 °C followed by the addition of ethyl 2-bromoacetate. In the previous report on the synthesis of 5-HPEC (5), it was determined that monoprotection of the starting dihydroxyacetophenone (20a) is required to give high yields of the target chromone via a direct Claisen condensation approach.34 Accordingly, the same strategy was employed in the synthesis of the proposed analogues (Scheme 2). The appropriate acetophenone (20) was protected with MOM as previously described to give 21.36 The protected acetophenone was then refluxed with sodium hydride to generate the needed enolate. Following enolate formation the prepared ester (12, 15, 17, or 19) was added and the mixture was refluxed further for 4 h. Upon workup, the crude condensation product was dissolved in MeOH with catalytic hydrochloric acid and refluxed for 45 min to give the desired analogues in good to excellent yields after purification (5, 22a−n, Table 1). Biological Testing. The goal of this study was to investigate the affinity, selectivity, and functional activity of naturally occurring and synthetic compounds bearing the 2-(2phenylethyl)chromone scaffold at the 5-HT2B receptor. Screening of these analogues was done in three phases.13 The initial screening measured the inhibition of radioligand binding at each of the 5-HT2 receptors by the newly synthesized analogues at 10 μM. Those compounds showing greater that 50% inhibition at this concentration were further tested for their affinity at the designated receptor. Analogues demonstrating significant improvement in 5-HT2B binding affinity over 5HPEC (5) were then screened for their functional activity. The results of the initial screening are shown in Table 1. 5HPEC (5) showed only significant inhibitory activity (62.6%) for the 5-HT2B receptor. Replacement of C-7′ with an oxygen atom (22a) maintained significant inhibitory activity at 5-HT2B (50.0%) as well the apparent selectivity over 5-HT2A/2C. Several naturally occurring 2-(2-phenylethyl)chromones contain hydroxy group substituents on the C ring. Substitution at C-3′ with a hydroxy group (22c) increased inhibitory activity at 5HT2B to 76.3% and maintained selectivity to some degree. Hydroxy group substitution at C-4′ (22d) maintained inhibitory activity at 5-HT2B (70.9%); however, selectivity to 5-HT2B was reduced as the inhibitory activity at 5-HT2C was increased to 56.6%. This observation suggested that the C-4′ position is important for 5-HT2B/2C selectivity over 5-HT2A. Replacement of the phenyl ring with a thiophene (22e) or furan (22f) ring system maintained significant inhibitory activity and selectivity for 5-HT2B. In addition to the above structural changes, the length of the C-2 alkyl chain was also explored. Decreasing the C-2 alkyl chain length (22g) decreased inhibitory activity at the 5-HT2B receptor. Conversely, when the alkyl spacer was extended to

Scheme 1. Synthesis of Esters for Claisen Condensationa

a Reagents and conditions: (a) H2SO4 (cat.), EtOH, reflux, 24 h. (b) CH 2 Cl 2 , DIPEA, MOMCl, rt, 3 h. (c) 1. DBU, triethyl phosphonoacetate, rt, 4 h. 2. H2, Pd/C (10 mol %), 60 atm, 2 h. (d) 1. 1,1′-Carbonyldimidazole, THF, rt, 30 min. 2. EtOH, rt, 16 h. (e) 1. NaH, THF, 0 °C 1 h. 2. Ethyl 2-bromoacetate, 0 °C, 16 h.

ester synthesis depending upon the available starting materials. The synthesis of ethyl 2-phenylacetate (12a), ethyl 3phenylpropanoate (12b), ethyl 4-phenylbutanoate (12c), ethyl 5-phenylpentanoate (12d), and ethyl 3-(thiophen-2yl)propanoate (12e) was accomplished via Fischer esterification.35 Refluxing the appropriate carboxylic acid in ethanol with catalytic sulfuric acid produced the desired ester in quantitative yield, which after workup was used without further purification. The esters adopted to examine effects of substitution on the C ring were synthesized in three steps. First, the hydroxy group of 13 was protected with methoxylmethyl (MOM) by reaction with methyoxymethyl chloride (MOMCl) in dichloromethane (CH2Cl2).36 The protected aldehyde (14) was then reacted in a modified Horner−Wadsworth−Emmons reaction with triethyl phosphonoacetate to yield the α,β-unsaturated ester that was immediately reduced to afford the substituted alkanes 15a−c.37 Ethyl 2-phenoxyacetate (17) was synthesized by activation of phenoxyacetic acid (16) with 1,1′-carbonyldiimidazole (CDI) followed by the addition of absolute ethanol.38 Finally, ethyl 2-

Scheme 2. Synthesis of 2-(2-Phenylethyl)chromones via Claisen Condensation and Cyclizationa

a

Reagents and conditions: (a) CH2Cl2, DIPEA, MOMCl, rt, 1 h. (b) 1. NaH, THF, reflux 1 h. 2. Ester, THF, reflux, 4 h. (c) MeOH, HCl (12 drops), reflux, 45 min. C

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Table 1. Percent Inhibition of Radioligand Binding at 5-HT2 Receptors by Compounds 5 and 22a−n at 10 μM (Inhibition ≥50% Is Deemed Significant)

a

Denotes compound previously isolated from natural sources.

was investigated by examining the activity of other 2-(2phenylethyl)chromone types. Although naturally occurring, no biological investigations of 7-hydroxy-2-(2-phenylethyl)chromone (22k, 7-HPEC) have been reported. In the testing performed, none of the 7-HPEC analogues (22k−n) showed significant inhibitory activity at 5-HT2B. This suggested that the C-5 hydroxy group may be critical for activity at the 5-HT2B receptor. Moreover, the selectivity for these 7-HPEC analogues was reduced. Interestingly, compound 22n, carrying a hydroxy group at the C-4′ position, showed significant inhibitory activity (53.1%) for the 5-HT2C receptor. Compound 22d, which also has a hydroxy group at C-4′, showed higher inhibitory activity

give 5-hydroxy-2-(3-phenylpropyl)chromone (5-HPPC, 22h), inhibitory activity at 5-HT2B was increased and selectivity was maintained. 2-[(Benzyloxy)methyl]-5-hydroxychromone (22i), in which C-8′ is replaced with an oxygen atom, also showed significant inhibitory activity (77.7%) at the 5-HT2B receptor, although selectivity over the 5-HT2C receptor appeared to be diminished. Further extension of the alkyl chain (22j) decreased inhibitory activity. In the initial isolation of 5-HPEC (5) it was noted that removal of the C-5 hydroxy group dramatically reduced neuroprotective activity.9 Therefore, the necessity of the C-5 hydroxy substituent for inhibitory activity at 5-HT2 receptors D

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HT2C = 5.9). Substitution of the phenyl ring with a thiophene (22e) or furan (22f) gave a 2-fold improvement in affinity (pKi = 5.9 and 5.7, respectively) over 5-HPEC (5). The most dramatic improvement in affinity was observed with 5-HPPC (22h). 5-HPPC showed a 10-fold (pKi = 6.6) increase in 5-HT2B affinity, suggesting that a propyl chain at C2 may be optimal. The replacement of C-8′ to give the benzyloxy derivative 22i maintained the relative chain length; however, a 6-fold decrease in 5-HT2B affinity compared to that of 5-HPPC (22h) was observed. This, in conjunction with the inhibition data, suggested that nonpolar moieties at C-2 would be preferred. Encouraged by the improvement in affinity with 5-HPPC (22h), the functional activity of 5-HPPC was investigated. Presently, only 5-HT2B receptor antagonists are clinically beneficial, as agonists of this receptor have been shown to increase significantly the risk of developing valvular heart disease.39,40 Our group determined previously that 5-HPEC (5) behaves functionally as a 5-HT2B antagonist.13 Given this increase in affinity, 5-HPPC (22h) was tested to determine if antagonist activity at 5-HT2B was maintained. As conducted earlier, a calcium mobilization assay in Flp-In HEK cells was performed to investigate the functional activity of 5-HPPC (22h) at 10 μM. At this concentration, 5-HPPC (22h) showed negligible efficacy (0.23% of the maximal response of 5-HT) when compared to the endogenous agonist 5-HT. Conversely, when challenged with an EC50 dose (1.6 nM) of 5-HT, 5HPPC (22h) demonstrated improved antagonist activity (20.5% inhibition) compared to 5-HPEC (5) (6.9% inhibition). Taken together, these results suggest that 5-HPPC maintains antagonist activity at the 5-HT2B receptor. Ligand Docking. To gain further insight into plausible reasons for the changes seen in the inhibitory activity and affinity upon structural modification, ligand docking experiments on the 5-HT2B receptor crystal structure (PDB ID-4IB4) were undertaken. A generic algorithm docking program (GOLDv51) was used to explore docking poses for each of the synthesized analogues. The generated binding modes were then scored using the Hydropathic INTeraction (HINT) scoring function.41 The highest scored poses for 5-HPEC (5) and 5-HPPC (22h) inside the 5-HT2B receptor are shown in Figure 3, with the associated HINT scores shown in Table 3. The chromone ring in each compound occupied a hydrophobic pocket formed by Val136, Phe217, Phe340, Phe341, and Val336. Within this pocket, the C-5 hydroxy group is able to participate in hydrogen-bonding interactions with Asp135 and Ser139. Compounds 22k−n would be unable to participate in the described hydrogen-bonding interaction due to the lack of a hydroxy group at C-5, and this could explain their decreased 5HT2B inhibitory activities. In addition to the importance of the C-5 hydroxy group observed from the inhibitory activity data, the affinity data showed that the length and composition of the C-2 alkyl chain may also be important. As shown in this model, the C-2 alkyl chain units of 5-HPEC (5) and 5-HPPC (22h) are directed toward the cytoplasmic surface of the receptor. The phenyl ring substituents of 5HPEC (5) and 5-HPPC (22h) can be positioned toward one of the two possible hydrophobic pockets: one may be formed between helices 5, 6, and 7 (Met218, Leu347, Leu348, and Leu362) and the other formed between extracellular loop 2 and helix 3 (Trp131, Leu132, Val208, and Leu209) (Figure S15, Supporting Information). Overlapping of 5-HPPC (22h) and 5-HPEC (5) showed that the extended nonpolar phenyl ring of

toward the 5-HT2C receptor as well. Such results lend further support to the hypothesis that the C-4′ hydroxy group may be favorable for 5-HT2C receptor selectivity. Following the inhibition studies, analogues showing greater than 50% inhibitory activity at 5-HT2B were examined for their binding affinity toward this receptor. These data are shown in Table 2. While having inhibitory activity at 5-HT2B in the Table 2. pKi Values for Analogues Showing ≥50% Inhibitory Activity in the Primary Assay

primary assay at 10 μM, 5-hydroxy-2-(phenoxymethyl)chromone (22a) showed insignificant binding affinity at this receptor (pKi < 5). Hydroxy group substitution at C-3′ (22c) provided a 2-fold improvement in affinity over 5-HPEC (5) (pKi = 5.9). The C-4′ hydroxy group (22d) also improved 5HT2B affinity (pKi = 6.0); however, as was observed in the inhibition assay, selectivity over 5-HT2C was lost (pKi at 5E

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activity at 5-HT2B. To understand the basis for this improved affinity, ligand docking studies were conducted. These results were consistent with the observed biological data and revealed a primarily hydrophobic binding pocket with significant hydrogen-bonding contribution by the Asp135 and Ser139 residues with the C-5 hydroxy group. Extension of the C-2 alkyl chain allowed 5-HPPC (22h) to take greater advantage of this hydrophobic pocket. Accordingly, these data support an initial hypothesis that the natural product 5-HPEC (5) can serve as a novel scaffold to design and develop novel non-nitrogenous 5HT2B antagonists. These new ligands are anticipated to help shed new light on understanding the correlation of the neuroprotective properties of 2-(2-phenylethyl)chromones and their antagonism at the 5-HT2B receptor.

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Figure 3. Plausible ligand binding modes for 5-HPEC (5) and 5HPPC (22h) inside the 5-HT2B receptor. Cartoon representation of the 5-HT2B receptor in cyan. Ligands are shown as ball-and-stick representations: 5-HPEC (green), 5-HPPC (orange). Amino acid residues involved in interactions are shown in stick representation (cyan).

Table 3. Optimal Docking Scores of 5-HPEC (5) and 5HPPC (22h) in the 5-HT2B Receptor Crystal Structure compound

HINT score

5-HPEC (5) 5-HPPC (22h)

507 777

EXPERIMENTAL SECTION

General Experimental Procedures. Chemical reagents were purchased from Sigma-Aldrich, Alfa Aesar, Combi-blocks, or AK Scientific and used without further purification. TLC analyses were carried out on Analtech Uniplate F254 plates. Chromatographic purification was accomplished on silica gel columns (230−400 mesh, Bodman). IR spectra were recorded on a Nicolet Avatar 360 FT-IR instrument with ATR attachment. 1H (400 MHz) and 13C (100 MHz) NMR spectra were acquired at 30 °C on a Bruker Ultrashield 400 Plus spectrometer. HRMS analysis was performed on a Quattro II triple quadrupole mass spectrometer, a Waters Micromass QTOF-II instrument (ESI source), or an Applied Bio Systems 3200 Q trap with a turbo V source for TurbolonSpray. Fischer Esterification. Sulfuric acid (4.0 mL, 122 mmol) was slowly added to a solution of the carboxylic acid (122 mmol) in ethanol (150 mL) previously cooled to 0 °C on an ice bath. After warming to room temperature, the mixture was heated to reflux and allowed to stir for 16 h. On cooling to room temperature, the mixture was concentrated by rotary evaporation at room temperature. The residue was then dissolved in EtOAc. The organic layer was washed three times with half the volume of saturated NaHCO3 solution. The organic layer was then washed with brine and dried over MgSO4. The organic layer was then evaporated to dryness to afford the desired ester (in >90% yield), which was used in the Claisen condensation without further purification. Ethyl 2-phenylacetate (12a): colorless oil; 1H NMR (CDCl3, 400 MHz) δ 7.33−7.23 (5H, m), 4.14 (2H, q, J = 7.14 Hz), 3.59 (2H, s), 1.24 (3H, t, J = 7.14 Hz); 13C NMR (CDCl3, 100 MHz) δ 171.6, 134.2, 129.3, 128.5, 127.1, 60.8, 41.5, 14.2. Ethyl 3-phenylpropanoate (12b): colorless oil; 1H NMR (CDCl3, 400 MHz) δ 7.29−7.18 (5H, m), 4.12 (2H, q, J = 7.14 Hz), 2.94 (2H, t, J = 7.60 Hz), 2.61 (2H, t, J = 7.56 Hz), 1.22 (3H, t, J = 7.12 Hz); 13C NMR (CDCl3, 100 MHz) δ 172.9, 140.6, 128.5, 128.3, 126.2, 60.4, 35.9, 31.0, 14.2. Ethyl 4-phenylbutanoate (12c): colorless oil; 1H NMR (CDCl3, 400 MHz) δ 7.29−7.17 (5H, m), 4.12 (2H, q, J = 7.13 Hz), 2.65 (2H, t, J = 7.60 Hz), 2.32 (2H, t, J = 7.5 Hz), 1.95 (2H, p, J = 7.54 Hz), 1.25 (3H, t, J = 7.08 Hz); 13C NMR (CDCl3, 100 MHz) δ 173.5, 141.5, 128.5, 128.4, 125.9, 60.3, 35.2, 33.7, 26.6, 14.3. Ethyl 5-phenylpentanoate (12d): colorless oil; 1H NMR (CDCl3, 400 MHz) δ 7.29−7.15 (5H, m), 4.11 (2H, q, J = 7.13 Hz), 2.62 (2H, t, J = 7.10 Hz), 2.31 (2H, t, J = 7.16 Hz), 1.66 (4H, m), 1.24 (3H, t, J = 7.14 Hz); 13C NMR (CDCl3, 100 MHz) δ 173.7, 142.2, 128.4, 128.3, 125.8, 60.2, 35.6, 34.2, 30.9, 24.6, 14.3. Ethyl 3-(thiophen-2-yl)propanoate (12e): pale yellow oil; 1H NMR (CDCl3, 400 MHz) δ 7.11 (1H, d, J = 5.2 Hz), 6.91 (1H, dd, J = 5.2, 3.4 Hz), 6.81 (1H, d, J = 3.4 Hz), 4.14 (2H, q, J = 7.13 Hz), 3.16 (2H, t, J = 7.62 Hz), 2.67 (2H, t, J = 7.62 Hz), 1.25 (3H, t, J = 7.16 Hz); 13C NMR (CDCl3, 100 MHz) δ 172.5, 143.1, 126.8, 124.6, 123.5, 60.6, 36.2, 25.2, 14.2. General Procedure for Protection with MOM.36 To a solution of phenol (4.0 g, 32.8 mmol) and DIPEA (17.1 mL, 98.2 mmol) in CH2Cl2 (45 mL) was added MOMCl (4.1 g, 50.5 mmol) at room

5-HPPC (22h) reached further into the first hydrophobic pocket. This difference was perceived as a favorable interaction and was reflected by an increased HINT score, and it could provide an explanation for the increased affinity of 5-HPPC (22h) over 5-HPEC (5). Taken together, these docking studies were consistent with the observed biological data and may be applied to guide future molecular design to further improve the affinity of this scaffold for the 5-HT2B receptor. To summarize, previous work from this laboratory has demonstrated that the naturally occurring 2-(2-phenylethyl)chromone 5-HPEC (5) has the potential to serve as a scaffold for the development of non-nitrogenous ligands at the 5-HT2B receptor. The focus of the present study was to examine structural features of the 2-(2-phenylethyl)chromone scaffold required to improve affinity and maintain selectivity toward 5HT2B. A series of naturally occurring and synthetic 2-(2phenylethyl)chromones bearing the 5-HPEC scaffold was synthesized and screened for this purpose. Key observations from these studies include the following: the C-5 hydroxy group is required for affinity at 5-HT2B; substitution at C-3′ is tolerated, while substitution at C-4′ decreases selectivity over 5HT2C; replacing the phenyl C ring with smaller aromatic moieties is well tolerated; and hydrocarbon alkyl chains of three carbons at C-2 may be preferred. Among all the analogues synthesized so far, 5-HPPC (22h) has emerged as a new lead compound, showing a 10-fold improvement in 5-HT2B affinity over 5-HPEC (5). 5-HPPC (22h) also maintained antagonist F

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= 7.13 Hz), 4.08 (2H, s), 1.28 (3H, t, J = 7.16 Hz); 13C NMR (CDCl3, 100 MHz) δ 170.3, 137.2, 128.5, 128.1, 128.0, 73.3, 67.3, 60.8, 14.2. General Procedure for the Preparation of Hydroxychromones (22a−n). To a slurry of sodium hydride (4.0 mmol) at reflux in THF was added dropwise over 10 min the acetophenone 21 (1.0 mmol), which was dissolved in 10 mL of THF. The solution was then allowed to reflux for 1 h past the addition of the last drop. While still at reflux, the appropriate ester (12, 15, 17, or 19) (1.5 mmol) was then added dropwise over 15 min, and the resulting solution stirred at reflux for 4 h. The reaction mixture was then poured over 50 mL of saturated NH4Cl while still hot and extracted three times with EtOAc. The combined organic layers were dried over Na2SO4 and concentrated to yield the crude Claisen product. Without purification, the Claisen product was then dissolved in 20 mL of MeOH. While vigorously stirring, 15 drops of concentrated HCl were added, and the solution was placed in an oil bath that was preheated to 90 °C and was refluxed for 45 min. On cooling to room temperature, saturated Na2CO3 was added until neutral. Next, 50 mL of EtOAc was added. The organic layer was washed with H2O (25 mL) and brine (25 mL), dried over Na2SO4, and concentrated to give the crude hydroxychromone products (22), which were purified by the appropriate measure to give the desired product in fair to excellent yield (20−90%). 5-Hydroxy-2-(2-phenylethyl)chromone (5): purified by column chromatography by gradient elution from 1:1 CH2Cl2−hexanes to 100% CH2Cl2; IR (diamond) νmax 2934, 1653, 1616, 1480, 1409, 1258, 845, 802 cm−1; 1H NMR (CDCl3, 400 MHz) δ 12.51 (1H, s, OH-5), 7.44 (1H, t, J = 8.32 Hz), 7.18−7.32 (5H, m), 6.85 (1H, dd, J = 8.4, 0.80 Hz), 6.76 (1H, dd, J = 8.4, 0.80 Hz), 6.06 (1H, s), 3.05 (2H, t, J = 7.0 Hz), 2.92 (2H, t, J = 6.8 Hz); 13C NMR (CDCl3, 100 MHz) δ 183.5, 169.8, 160.8, 156.7, 139.4, 135.1, 128.7, 128.2, 126.6, 111.2, 110.6, 108.8, 106.6, 36.0, 32.8; HRMS m/z 265.0909 (calcd for C17H14O3, 265.0870). 5-Hydroxy-2-(phenoxymethyl)chromone (22a): purified by column chromatography eluting with 3:1 CH2Cl2−hexanes and precipitation from acetone; IR (diamond) νmax 3082, 2903, 1655, 1628, 1497, 1431, 1384, 1240, 863, 803 cm−1; 1H NMR (DMSO-d6, 400 MHz) δ 12.47 (1H, s), 7.67 (1H, t, J = 8.36 Hz), 7.34 (2H, dt, J = 2.04, 8.72 Hz), 7.11−6.99 (4H, m), 6.84 (1H, dd J = 7.6, 8.24 Hz), 6.54 (1H, s), 5.17 (2H, s); 13C NMR (DMSO-d6, 100 MHz) δ 182.8, 166.1, 159.9, 157.3, 156.0, 136.1, 129.6, 121.6, 114.8, 111.1, 110.1, 108.1, 107.3, 65.3; HRMS m/z 267.0664 (calcd for C16H12O4, 267.0663). 5-Hydroxy-2-(2′-hydroxy-2-phenylethyl)chromone (22b): purified by stirring the crude solid in minimal CH2Cl2 and collecting the undissolved solid; IR (diamond) νmax 3153, 2944, 1650, 1618, 1583, 1487, 1259, 845, 801 cm−1; 1H NMR (DMSO-d6, 400 MHz) δ 12.63 (1H, br s, OH-5), 9.48 (1H, br s, OH-2′), 7.62 (1H, t, J = 8.32 Hz), 7.08−6.99 (3H, m), 6.78 (2H, t, J = 8.4 Hz), 6.69 (1H, t, J = 7.4 Hz), 6.23 (1H, s), 2.95 (4H, s); 13C NMR (DMSO-d6, 100 MHz) δ 182.9, 171.4, 159.9, 156.2, 155.2, 135.6, 129.8, 127.4, 125.8, 118.8, 114.9, 110.7, 109.8, 108.1, 107.0, 33.5, 27.0; HRMS m/z 281.0854 (calcd for C17H14O4, 281.0819). 5-Hydroxy-2-(3′-hydroxy-2-phenylethyl)chromone (22c): purified by stirring the crude solid in minimal CH2Cl2 and collecting the undissolved solid; IR (diamond) νmax 3220, 2947, 1654, 1614, 1578, 1565, 1245, 843, 801 cm−1; 1H NMR (CDCl3, 400 MHz) δ 12.67 (1H, s, OH-5), 7.61 (1H, t, J = 8.32 Hz), 7.09 (1H, t, J = 7.4 Hz), 6.98 (1H, dt, J = 2.4, 8.4 Hz), 6.69 (3H, m), 6.65 (1H, dt, J = 2.4, 0.84 Hz), 6.18 (1H, s), 3.05 (4H, m); 13C NMR (CDCl3 100 MHz) δ 183.6, 170.1, 160.7, 156.7 156.1, 141.2, 135.3 129.9, 120.4, 115.2, 113.7, 111.3, 110.5, 108.7, 106.9, 53.8, 32.6; HRMS m/z 283.0985 (calcd for C17H14O4, 283.0971). 5-Hydroxy-2-(4′-hydroxy-2-phenylethyl)chromone (22d): purified by stirring the crude solid in minimal CH2Cl2 and collecting the undissolved solid; IR (diamond) νmax 3381, 2947, 1650, 1616, 1579, 1569, 1250, 845, 800 cm−1; 1H NMR (DMSO-d6, 400 MHz) δ 12.62 (1H, s, OH-5), 7.64 (1H, t, J = 8.32 Hz), 7.03 (3H, d, J = 8.44 Hz), 6.78 (1H, d, J = 8.2 Hz), 6.65 (2H, d, J = 8.44 Hz), 6.27 (1H, s), 2.93 (4H, m); 13C NMR (DMSO-d6, 100 MHz) δ 182.8, 171.1, 159.5,

temperature. This solution was stirred at room temperature for 1−2 h. After the allotted time, water was added to the mixture and the aqueous layer was extracted with CH2Cl2. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by chromatography over silica gel eluted with 1:1 CH2Cl2−hexanes. For 2-methoxymethoxybenzaldehyde (13a), 3-methoxymethoxybenzaldehyde (13b), 4-methoxymethoxybenzaldehyde (13a), 2hydroxy-4-methoxymethoxyacetophenone (20a), and 2-hydroxy-6methoxymethoxyacetophenone (20b), all spectroscopic data and yields were consistent with those previously reported.36 Ethyl 3-(2-(methoxymethoxy)phenyl)propanoate (15a). To a mixture of 13 (6.6 mmol) and triethyl phosphonoacetate (6.0 mmol) was added DBU (8.2 mmol). The resulting mixture was stirred for 4 h at room temperature. The reaction was quenched with H2O (25 mL), and the aqueous layer was extracted with EtOAc (20 mL, 3×). The organic layer was washed with brine, dried over Na2SO4, and concentrated to give a crude reddish-brown oil. This crude oil was filtered over a silica gel pad with 1:1 CH2Cl2−hexanes to remove the baseline residue. The filtrate was then concentrated to give a lightcolored oil. This oil was then redissolved in ethanol (30 mL), and Pd/ C (10% wt) was added. The mixture was then hydrogenated at 60 psi for 2 h. The reaction was then filtered over Celite, and the Celite layer was washed with ethanol. After the filtrate was concentrated down, the resulting oil was purified on silica gel with 4:1 hexanes−EtOAC to give the desired product as a colorless oil: 1H NMR (CDCl3, 400 MHz) δ 7.16 (2H, m), 7.05 (1H), 6.62 (1H), 5.29 (2H, s), 4.1 (2H, q, J = 7.16 Hz), 3.48 (3H, s), 2.96 (2H, t, J = 7.56 Hz), 2.61 (2H, t, J = 7.56 Hz), 1.23 (3H, t, J = 7.12 Hz); 13C NMR (CDCl3, 100 MHz) δ 173.3, 155.1, 130.0, 129.4, 127.6, 121.6, 113.7, 94.2, 60.3, 56.0, 34.4, 26.1, 14.2. Ethyl 3-(3-(methoxymethoxy)phenyl)propanoate (15b): prepared in the same manner as 15a; colorless oil; 1H NMR (CDCl3, 400 MHz) δ 7.17 (1H, m), 6.87 (3H, m), 5.15 (2H, s), 4.13 (2H, q, J = 7.12 Hz), 3.47 (3H, s), 2.92 (2H, t, J = 7.60 Hz), 2.61 (2H, t, J = 7.52 Hz), 1.23 (3H, t, J = 7.12 Hz); 13C NMR (CDCl3, 100 MHz) δ 172.8, 157.4, 142.2, 129.4, 121.8, 116.3, 114.0, 94.5, 60.4, 55.9, 35.8, 30.9, 14.2. Ethyl 3-(4-(methoxymethoxy)phenyl)propanoate (15c): prepared in the same manner as 15a; colorless oil; 1H NMR (CDCl3, 400 MHz) δ 7.12 (2H, d, J = 8.5 Hz), 6.96 (2H, d, J = 8.6 Hz), 5.14 (2H, s), 4.13 (2H, q, J = 7.16 Hz), 3.46 (3H, s), 2.89 (2H, t, J = 7.6 Hz), 2.58 (2H, t, J = 7.52 Hz), 1.23 (3H, t, J = 7.12 Hz); 13C NMR (CDCl3, 100 MHz) δ 172.9, 155.6, 134.0, 129.2, 116.3, 94.5, 60.3, 55.9, 36.1, 30.1, 14.2. Ethyl 2-phenoxyacetate (17). To a slurry of 1,1′-carbonyldimidazole in anhydrous THF was added a solution of phenoxyacetic acid 16 (anhydrous). Upon addition, rapid effervescence was observed. This solution was allowed to stir for 30 min past the end of effervescence. At this time, absolute ethanol was added, and the mixture was allowed to stir at room temperature for 16 h. The mixture was then concentrated, and the residue was taken up in dichloromethane. The organic layer was washed three times with half the volume of saturated NaHCO3, then washed with brine, dried over MgSO4, and concentrated to give the desired product as a colorless oil: 1 H NMR (CDCl3, 400 MHz) δ 7.28 (2H, dd, J = 3.0, 1.8 Hz), 6.97 (1H, t, J = 3.1 Hz), 6.89 (2H, d, J = 2.0 Hz), 4.61 (2H, s), 4.26 (2H, q, J = 7.15 Hz), 1.29 (3H, t, J = 7.13 Hz); 13C NMR (CDCl3, 100 MHz) δ 168.9, 157.8, 129.6, 121.7, 114.7, 65.4, 61.3, 14.2. Ethyl 2-(benzyloxy)acetate (19). To a slurry of sodium hydride (60% dispersion, 17.8 mmol) cooled to −10 °C was added benzyl alcohol (13.7 mmol) dropwise over 5 min. This solution was allowed to stir at this temperature for 1 h. Following this, ethyl 2-bromoacetate (13.7 mmol) was added. The solution was then allowed to come to room temperature and stirred at room temperature for 20 h. The reaction mixture was then concentrated. The residue was taken up in EtOAc. The organic layer was washed with NaHCO3 and brine. After drying over Na2SO4, the organic layer was concentrated to give a crude pale yellow oil. The oil was purified over silica gel by eluting with 1:1 CH2Cl2−hexanes to give the pure product as a colorless oil: 1H NMR (CDCl3, 400 MHz) δ 7.36−7.33 (5H, m), 4.63 (2H, s), 4.22 (2H, t, J G

DOI: 10.1021/acs.jnatprod.5b00118 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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7-Hydroxy-2-(2′-hydroxy-2-phenylethyl)chromone (22l): purified by stirring the crude solid in acetone for 24 h and then filtering the precipitate; IR (diamond) νmax 3176, 2945, 1626, 1546, 1443, 1227, 842, 818 cm−1; 1H NMR (DMSO-d6, 400 MHz) δ 10.69 (1H, s), 9.38 (1H, s), 7.94 (1H, d, J = 8.72 Hz), 7.06 (1H, dd, J = 7.44, 1.44 Hz), 6.99 (1H, dt, J = 1.64, 7.9 Hz), 6.86 (1H, dd, J = 8.7, 2.2 Hz), 6.79− 6.78 (2H, m), 6.68 (1H, t, J = 7.4 Hz), 2.90 (4H, m); 13C NMR (DMSO-d6, 100 MHz) δ 176.4, 168.6, 162.3, 157.7, 154.9, 129.8, 127.3, 126.5, 125.9, 118.9, 115.8, 114.8, 114.7, 108.9, 102.1, 33.3, 27.0; HRMS m/z 283.0946 (calcd for C17H14O4, 283.0971). 7-Hydroxy-2-(3′-hydroxy-2-phenylethyl)chromone (22m): purified by stirring the crude solid in CH2Cl2 and collecting the precipitate; IR (diamond) νmax 3145, 2926, 1626, 1590, 1558, 1402, 1250, 1225, 856, 834 cm−1; 1H NMR (acetone-d6, 400 MHz) δ 9.89 (1H, br s), 8.56 (1H, br s), 7.91 (1H, d, J = 8.68 Hz), 7.09 (1H, t, J = 7.8 Hz), 6.93 (1H, dd, J = 8.72, 2.28 Hz), 6.87 (1H, d, J = 2.24 Hz), 6.68 (2H, m), 6.66 (1H, m), 6.00 (1H, s), 3.00 (4H, m); 13C NMR (acetone-d6, 100 MHz) δ 177.2, 168.7, 163.1, 159.1, 158.4, 142.8, 130.3, 127.7, 120.4, 117.9, 116.2, 115.2, 114.1, 110.4, 103.3, 36.2, 33.4; HRMS m/z 283.0957 (calcd for C17H14O4, 283.0971). 7-Hydroxy-2-(4′-hydroxy-2-phenylethyl)chromone (22n): purified by column chromatography eluting with 7:3 hexanes−EtOAc; IR (diamond) νmax 3181, 2942, 1622, 1596, 1547, 1256, 848, 808 cm−1; 1 H NMR (acetone-d6, 400 MHz) δ 9.49 (1H, s), 8.11 (1H, s), 7.92 (1H, d, J = 8.68 Hz), 7.11 (2H, d, J = 8.48 Hz), 6.94 (1H, dd, J = 8.7, 2.24 Hz), 6.89 (1H, d, J = 2.24 Hz), 6.77 (2H, d, J = 8.48 Hz), 5.99 (1H, m), 3.01 (2H, t, J = 8.36 Hz), 2.89 (2H, t, J = 8.4 Hz); 13C NMR (acetone-d6, 100 MHz) δ 177.1, 168.8, 159.1, 131.9, 130.2, 127.7, 117.9, 116.0, 115.1, 110.4, 103.2, 36.7, 32.7; HRMS m/z 305.0787 (calcd for C17H14O4Na, 305.0790). Biological Testing. Percentage inhibition studies, Ki determinations, receptor binding profiles, and agonist and/or antagonist functional data were conducted by the National Institute of Mental Health’s Psychoactive Drug Screening Program (PDSP), contract number HHSN-271-2008-00025-C (NIMH PDSP). This facility is directed by Dr. Bryan L. Roth at the University of North Carolina at Chapel Hill, NC, and Project Officer Dr. Jamie Driscol at NIMH, Bethesda, MD. For experimental details please refer to the PDSP Web site http://pdsp.med.unc.edu/ and click on “Binding Assay” or “Functional Assay” on the menu bar. Molecular Modeling Studies. The chemical structures of ligands 5-HPEC (5) and 5-HPPC (22h) were sketched within SYBYL-X2.0, and their Gasteiger−Hückel charges were assigned before energy minimization (10 000 iterations) under the TAFF. The generic algorithm docking program GOLD42 was used to perform the docking studies with standard default settings unless otherwise specified. The binding site was defined to include all atoms within 10 Å of the αcarbon atom of Asp135 for the 5-HT2B crystal structure (PDB ID 4IB4).43 The obtained GOLD-docked solutions were merged into the receptor and the combined receptor−ligand structures were energyminimized using the parameters described above in order to remove clashes and minimize strain energy, thus optimizing the interactions between ligand and receptor within the binding pocket. These models were then subjected to hydropathic analysis with the HINT41 program, and best-scored HINT solutions were obtained. The pictures shown were generated using the PyMOL Molecular Graphics System, version 1.5.0.4, Schrödinger, LLC.

156.2, 155.4, 135.7, 129.9, 129.2, 115.0, 110.7, 109.7, 108.4, 107.3, 35.5, 31.1; HRMS m/z 283.0998 (calcd for C17H14O4, 283.0971). 5-Hydroxy-2-(2-(thiophen-2-yl)ethyl)chromone (22e): purified by column chromatography eluting with 1:1 CH2Cl2−hexanes; IR (diamond) νmax 3103, 3066, 2913, 1645, 1613, 1474, 1439, 1379, 1357, 1239, 840, 807 cm−1; 1H NMR (acetone-d6, 400 MHz) δ 12.64 (1H, s), 7.61 (1H, t, J = 8.36 Hz), 7.25 (1H, dd, J = 2.16, 4.2 Hz), 6.99 (1H, dd, J = 0.72, 8.44 Hz), 6.93−6.91 (2H, m), 6.75 (1H, dd, J = 0.60, 8.28 Hz), 6.22 (1H, s), 3.35 (2H, t, J = 7.24 Hz), 3.09 (2H, t, J = 7.64 Hz); 13C NMR (acetone-d6, 100 MHz) δ 184.3, 170.9, 161.8, 157.7, 143.1, 136.3, 127.8, 126.0, 124.7 111.7, 111.1, 109.6, 107.8, 36.7, 27.4; HRMS m/z 271.0436 (calcd for C15H12O3S, 271.0434). 5-Hydroxy-2-(2-(furan-2-yl)ethyl)chromone (22f): Purified by column chromatography eluting with 4:1 hexanes−EtOAc; IR (diamond) νmax 3144, 3121, 2917, 1645, 1615, 1593, 1581, 1313, 1295, 1262, 838, 808 cm−1; 1H NMR (acetone-d6, 400 MHz) δ 12.65 (1H, s, OH-5), 7.61 (1H, t, J = 8.32 Hz), 7.43 (1H, d, J = 1.68 Hz), 6.96 (1H, d, J = 8.4 Hz), 6.74 (1H, d, J = 8.24 Hz), 6.31 (1H, dd, 1.92, 8.4 Hz), 6.23 (1H, s), 6.15 (1H, d, J = 3.2 Hz), 3.13 (2H, t, 7.0 Hz), 3.03 (2H, t, 7.0 Hz); 13C NMR (acetone-d6, 100 MHz) δ 182.3, 171.1, 161.7, 157.7, 154.5, 142.5, 136.3, 111.7, 111.2, 109.3, 107.6, 106.8, 33.4, 25.7; HRMS m/z 255.0675 (calcd for C15H12O4, 255.0663). 5-Hydroxy-2-benzylchromone (22g): purified by column chromatography eluting with 4:1 hexanes−EtOAc; IR (diamond) νmax 3098, 3055, 2788, 1667, 1618, 1482, 1434, 1372, 1263, 864, 820 cm−1; 1H NMR (acetone-d6, 400 MHz) δ 12.62 (1H, s), 7.57 (1H, t, J = 8.4 Hz), 7.43- 7.28 (5H, m), 6.89 (1H, dd, J = 0.68, 8.48 Hz), 6.72 (1H, dd, J = 0.52, 8.24 Hz), 6.16 (1H, s), 4.05 (1H, s); 13C NMR (acetone-d6, 100 MHz) δ 184.4, 171.2, 161.8, 157.7, 136.4, 136.2, 130.1, 129.7, 128.2, 111.7, 111.1, 109.6, 107.7, 40.8; HRMS m/z 251.0747 (calcd for C16H12O3, 251.0714). 5-Hydroxy-2-(3-phenylpropyl)chromone (22h): purified by column chromatography eluting with 4:1 hexanes−EtOAc; IR (diamond) νmax 3025, 2930, 1652, 1618, 1475, 1459, 1367, 1252, 845, 802 cm−1; 1 H NMR (CDCl3, 400 MHz) δ 12.54 (1H, s), 7.47 (1H, t, J = 8.32 Hz), 7.31−7.17 (5H, m), 6.83 (1H, dd, J = 0.64, 8.4 Hz), 6.77 (1H, d, J = 8.24 Hz), 6.09 (1H, s), 2.72 (2H, t, J = 7.4 Hz), 2.62 (2H, t, J = 7.32 Hz), 2.07 (2H, t, J = 7.72 Hz); 13C NMR (CDCl3, 100 MHz) δ 184.5, 170.6, 160.8, 156.7, 140.7, 135.0, 128.5, 128.4, 126.2 111.1, 110.6, 108.5, 106.8, 34.9, 33.6, 28.2; HRMS m/z 279.1040 (calcd for C18H16O3, 279.1023). 5-Hydroxy-2-((benzyloxy)methyl)chromone (22i): purified by column chromatography eluting with 7:3 hexanes−EtOAc; IR (diamond) νmax 3030, 2951, 2871, 1649, 1585, 1480, 1457, 1420, 1255, 1220, 853, 802 cm−1; 1H NMR (acetone-d6, 400 MHz) δ 12.60 (1H, s), 7.57 (1H, t, J = 8.32 Hz), 7.43−7.28 (5H, m), 6.90 (1H, dd, J = 0.52, 8.48 Hz), 6.73 (1H, d, J = 8.28 Hz), 6.34 (1H, s), 4.71 (2H, s), 4.52 (2H, s); 13C NMR (acetone-d6, 100 MHz) δ 184.2, 168.5, 161.8, 157.4, 138.6, 136.5, 129.3, 128.7, 111.9, 111.5, 109.5, 108.5, 107.9, 73.8, 68.6, 38.7; HRMS m/z 281.0827 (calcd for C17H14O4, 281.0819). 5-Hydroxy-2-(4-phenylbutyl)chromone (22j): purified by column chromatography eluting with 7:3 hexanes−EtOAc; IR (diamond) νmax 3061, 2933, 2852, 1650, 1621, 1478, 1459, 1382, 1366, 1258, 873, 803 cm−1; 1H NMR (acetone-d6, 400 MHz) δ 12.70 (1H, s), 7.59 (1H, t, J = 8.32 Hz), 7.28−7.14 (5H, m), 6.95 (1H, dd, J = 0.68, 8.4 Hz), 6.74 (1H, d, J = 8.28 Hz), 6.20 (1H, s), 2.77−2.67 (4H, m), 1.83−1.74 (4H, m); 13C NMR (acetone-d6, 100 MHz) δ 184.5, 172.6, 161.8, 157.7, 143.0, 136.2, 129.2, 129.1, 126.6, 111.6, 111.1, 109.0, 107.7, 36.0, 34.5, 31.5, 27.0; HRMS m/z 293.1196 (calcd for C19H18O3, 293.1183). 7-Hydroxy-2-(2-phenylethyl)chromone (22k): purified by column chromatography eluting with 19:1 CH2Cl2−MeOH; IR (diamond) νmax 3026, 1621, 1548, 1495, 1421, 1256, 852, 823 cm−1; 1H NMR (acetone-d6, 400 MHz) δ 9.45 (1H, s, OH-7), 7.96−7.90 (1H, m), 7.28−7.18 (5H, m), 6.94 (1H, d, J = 8.68 Hz), 6.87 (1H, s), 6.00 (1H, s), 3.08 (2H, t, J = 7.76 Hz), 2.95 (2H, t, J = 6.08 Hz); 13C NMR (acetone-d6, 100 MHz) δ 177.2, 168.7, 163.1, 159.1, 141.2, 129.3, 129.3, 127.7, 127.1, 117.8, 115.2, 110.3, 103.3, 36.2, 33.4; HRMS m/z 265.0890 (calcd for C17H14O3, 265.0870).

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00118. 1 H and 13C spectra for all previously unreported compounds and alternative docking poses of 5-HPEC (5) and 5-HPPC (22h) in the 5-HT2B crystal structure (PDF) H

DOI: 10.1021/acs.jnatprod.5b00118 J. Nat. Prod. XXXX, XXX, XXX−XXX

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (D. Williams). *Tel: +1 804 828 0021. Fax: +1 804 828 7625. E-mail: [email protected] (Y. Zhang). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Y. Yuan for helpful discussions in preparing the data for this article. This work was supported by a NIH/NIDA training grant (DA007027) (D.A.W.) and PHS Research Funds (DA024022) (Y.Z.). Ki determinations, receptor binding profiles, and agonist and/or antagonist functional data were generously provided by the National Institute of Mental Health’s Psychoactive Drug Screening Program, contract number HHSN-271-2008-00025-C (NIMH PDSP).

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DOI: 10.1021/acs.jnatprod.5b00118 J. Nat. Prod. XXXX, XXX, XXX−XXX