Compoundhas been previously reported, (24) and the NMR and electron ionization mass spectra ofandhave been reported but without any biological data. (25) We thus decided to compare all of the series members at the same time to elucidate a consistent SAR.

Thus, in this article we describe the facile synthesis of compounds, and, preliminary screening at a variety of 5HT family receptors, and more detailed testing at human 5-HT, 5-HT, and 5-HTreceptors, including affinity measurements using displacement of the agonist radioligand [I]-DOI and functional effects in elevating intracellular calcium. We also present behavioral data for the mouse head twitch response (HTR) as a measure of in vivo 5-HTreceptor activation. (23)

On the basis of the hypothesis that the original data were associated with an isomer other than the 4-bromo compound, we subsequently discovered that-3-bromobenzyl compounddid have higher affinity for the 5-HTreceptor (1.48 nM), compared to that of the 4-bromo congener11.2 nM). Further, the effect of an ortho-oxygenated-benzyl appeared not to be significant for affinity in the tryptamine series, suggesting perhaps different binding orientations of the-benzyltryptamines versus the-benzylphenethylamines within the receptor. That is, compoundhas been reported to have agonist potency (pEC7.08) in a rat tail artery assay not significantly different from the compound with an unsubstituted-benzyl moiety (pEC7.00), although thewas slightly higher for the 2′-methoxy compound. (18) These findings prompted us to synthesize a small series of structurally related congeners to determine whether other substitutions might have even greater affinity and/or selectivity for the 5-HTreceptor.

Although it was reported (19) that 4-bromo compoundhad 0.1 nM affinity at the human 5-HTreceptor, the 4-fluoro-, 4-chloro-, and 4-iodo-substituted benzyl congeners had reported affinities of 40, 105, and 120 nM, respectively, in that same report. We found this discontinuity in the structure–activity relationship (SAR) puzzling, where the 4-bromo compound would be such an outlier in the family of halogen-substituted benzyls. Further investigation by Jensen, however, revealed that the authentic 4-bromo compoundactually had relatively low affinity for the 5-HTreceptor, more consistent with the reported affinities of the other halogenated compounds. (22) Although spectroscopic data were not reported by Glennon et al. (19) that might explain the basis for this discrepancy, their publication indicated elemental analysis data to be consistent with the proposed structure. If the elemental analysis data were correct, the mostly likely explanation for the discordant biological data therefore seemed to be thatmight have been an isomer other than the 4-substituted compound.

Surprisingly, however, in the Glennon report, (19) a 5-HTreceptor affinity of 0.1 nM was reported for the-4-bromobenzyl compound (compoundin the Glennon report, numbered here as), with 1000-fold selectivity for 5-HTover 5-HTreceptors. We found these data particularly intriguing. This degree of selectivity was overestimated, however, because affinity at the 5-HTreceptor was measured by displacement of an agonist ragioligand, whereas affinity at the 5-HTreceptor was measured by displacement of an antagonist radioligand. Nonetheless, no specific 5-HT-selective agonist has been available, although such a compound would be very valuable for serotonin neuroscience research.

In addition to the phenethylamine type 5-HTagonists, certain simple tryptamines possess similar pharmacology, particularly 4- or 5-oxygenated molecules. In the report by Glennon et al., placing an-benzyl moiety on the amine of 5-methoxytryptamine had essentially no effect on affinity. Interestingly,-benzyl-5-methoxytryptamine previously had been reported to be an antagonist of serotonin-induced contraction in the rat stomach fundus, the isolated guinea pig uterus, and the isolated guinea pig taenia cecum. (20) In addition, Leff et al. (21) had shown that-benzyl-5-methoxytryptamine had only weak partial agonist activity at 5-HTtype receptors in rabbit aorta and rat jugular vein.

Two decades ago, Glennon et al. (19) reported that the affinities of the-benzyl compound, as well as the 4-bromo- and 4-iodo--benzyl compounds,and, respectively, were 2–3 times higher than that of the parent primary amine. There have been no further reports on these compounds, and in our own work, we had never examined 3- or 4-substituted benzyl substituents in the phenethylamine series.

Typically, simple N-alkylation dramatically attenuates or abolishes hallucinogenic activity in phenethylamines. (9, 10) The-benzyl moiety, however, confers exceptionally high potency onto the molecule, (11-15) and we have presented evidence that the-benzyl may engage F339 in the human 5-HTreceptor. (14) We also examined various-arylmethyl substituents and found that a variety of aryl groups were effective in enhancing potency. (16, 17) In addition, the presence of a polar substituent at the ortho position of the aryl ring (a possible hydrogen bond acceptor) further enhances activity. (18) Silva et al. (18) also have reported that in an in vitro cylindrical rat tail artery striphad a pECof 10.09 and anof 30%.

There has been increasing global interest inand closely related analogues. For example, the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) has received a range of notifications from EU Member States about analytically confirmed nonfatal and fatal intoxications associated with. This was then followed by a risk assessment conducted by the Scientific Committee of the EMCDDA in order to assess health and social risks associated with this particular analogue. (6) In addition, the World Health Organization’s Expert Committee on Drug Dependence reviewed the status of a range of new substances for its 36th meeting in June 2014, which includedand its 4-bromo and 4-chloro analogues. (7) In September 2014, the Council of the European Union decided to subjectto control measures and criminal penalties throughout the European Union. (8)

Recently, an extremely potent hallucinogenic phenethylamine, 25I-NBOMe (-(2-methoxybenzyl)-2,5-dimethoxy-4-iodophenethylamine; “smiles”)has been available on the illicit drug market. (1) For purposes of enforcement, it is presently considered by the Drug Enforcement Administration (DEA) to be an analogue of 2C-I (), which is currently a Schedule I controlled substance. The procedure to classifyas a Schedule I substance has been initiated, and it has been placed temporarily into Schedule I. (2) Unfortunately, several deaths have been associated with the use of (3-5) but it is not clear whether the deaths resulted from the ingestion of lethal amounts of pure solid drug, or whether the drug has some inherent toxicity that is not normally associated with other hallucinogens.

All of the compounds were most easily prepared using a modification of the facile method first reported by Abdel-Magid et al. (26) The free base ofwas stirred in 3 mL of MeOH for 30 min with the appropriate aldehyde, followed by reduction of the intermediate enamine with NaBH. Following appropriate workup, the bases were converted to their HCl or maleate salts and crystallized in good to excellent yields.

Affinities at a panel of 5-HT receptors were determined by the NIMH-sponsored PDSP program ( http://pdsp.med.unc.edu/kidb.php ). Affinities at both the human and rat 5-HTand 5-HTreceptors also were determined, using both agonist and antagonist radioligands. As a measure of functional potency and efficacy, changes in intracellular Calevels were measured using a fluorometric imaging plate reader (FLIPR, Molecular Devices), at the human 5-HT, 5-HT, and 5-HTreceptors, and at the rat 5-HTand 5-HTreceptors. Finally, as a measure of in vivo 5-HTreceptor activation, we assessed the ability of all compounds to induce the mouse HTR. (23) We hypothesized that functional potency at the rat 5-HTreceptor might correlate best with the mouse head twitch behavioral data because ligand affinities at the rat 5-HTreceptor correlate with the mouse 5-HTreceptor but not with the human 5-HTreceptor. (27)

The HTR produced by compoundsandshowed a biphasic bell-shaped dose–response function (the response peaked at 10 mg/kg and 30 mg/kg was inactive). Other 5-HTagonists, including DOI, DOM, 2C-T-7, and 5-MeO–DIPT, have been shown to produce similar nonmonotonic responses. (32-34) Fantegrossi et al. (34) have argued that the descending arm of the biphasic HTR dose–response is a consequence of 5-HTactivation, which attenuates the response to 5-HTactivation. Recently, however, it was reported that-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenethylamine (25CN-NBOH), a 5-HTagonist with 100-fold selectivity over 5-HT, also induces the HTR with a biphasic dose–response. (35) The fact that the descending arm of the response to 25CN-NBOH was not affected by a 5-HTantagonist (35) demonstrates that the inhibition of the HTR at high doses does not necessarily result from competing activity at 5-HT. One potential alternative explanation for the biphasic HTR is that high levels of 5-HTactivation may produce competing behaviors that interfere with expression of head shaking. Along those lines, it has been reported that high doses of quipazine, 5-MeO-DMT, and (+)-LSD produce stereotypic behaviors that preclude head shakes and wet dog shakes in rats. (36, 37)

With the exception of 5k and 5l , which had relatively low functional potencies at the r5-HT 2A (EC 50 values of 770 and 120 nM, respectively), all of the meta-substituted N -benzyl derivatives of 5-methoxytryptamine induced the HTR. That included the 3-methyl ( 5j ; ED 50 = 2.31 mg/kg), 3-methoxy ( 5b ; ED 50 = 3.28 mg/kg), 3-fluoro ( 5g ; ED 50 = 3.33 mg/kg), 3-chloro ( 5h ; ED 50 = 4.43 mg/kg), 3-bromo ( 5e ; ED 50 = 5.18 mg/kg), and 3-iodo ( 5i ; ED 50 = 7.77 mg/kg) compounds.

Figure 1 shows an illustrative dose–response curve for compoundin the mouse HTR. HTR data for all compounds are given in Table 4 . Although some of the compounds failed to induce the HTR at doses up to 30 mg/kg, most of the “inactive” compounds displayed relatively low potency at 5-HT(see Figure 2 ), so it is possible that they would induce the HTR if tested at higher doses. Importantly, for the subset of compounds that induced the HTR, behavioral potency was significantly correlated with functional potency at the r5-HTreceptor (r = 0.69,< 0.03; Figure 2 ), but there was no correlation with functional ECvalues at the r5-HTreceptor (r = 0.17,> 0.1). Despite the overall correlation between mouse HTR and r5-HTpotency, the relationship was not always orderly for individual compounds. Compoundwas by far the most potent compound in that assay, with an EDof 0.078 mg/kg (data taken from Halberstadt and Geyer (31) ). It is not clear whyshould be so much more potent than any other compound because, for example,is inactive but appears nearly comparable functionally, with an ECof 14 nM and efficacy of 69%, compared with an ECof 11 nM forwith an efficacy of 79%. The next most potent compounds in the mouse HTR areand, with identical EDs of 2.31 mg/kg, about 300-fold less potent than. Although they have similar functional ECvalues (36 and 26 nM), nothing in the functional or binding data can explain their lower potency compared to that of. Further, compounds, andhave virtually identical EDvalues in the mouse HTR, yet their functional ECs at the rat 5-HTreceptor are 21, 34, and 80 nM, respectively.

It is noteworthy that the functional potencies in the rat and human 5-HTreceptors are essentially identical for phenethylamine compounds, and, yet the potencies for tryptamine compoundsare 4–10-fold higher at the human 5-HTreceptor than at the rat 5-HTreceptor. This finding may reflect the single amino acid difference in the orthosteric binding site of these two receptors at position 5.46. In the rat or mouse 5-HTreceptor, residue 5.46 is an alanine, whereas in the human receptor it is a serine. We have previously shown that mutation of this residue in the human receptor from serine to alanine has little effect on affinity or function for phenethylamine 5-HTagonists but does have a significant effect for tryptamines. (28) One might infer, therefore, from these potency differences that the indole NH in the present series also engages this serine in the human receptor but not the alanine in the rat receptor, consistent with mutagenesis studies reported by others. (29, 30)

Functional potencies at the rat and human 5-HTand 5-HTreceptors and the human 5-HTreceptor are shown in Table 3 . Compoundwas a nearly full agonist at both receptor types, with a 4.2 nM ECat the human 5-HTreceptor and 11 nM ECat the rat 5-HTreceptor. The most potent compound was, with an ECof 1.9 nM and 85% efficacy at the h5-HT. Notably, this compound has the-2-methoxybenzyl substituent, the same as the most potent phenethylamine, suggesting that it may be optimal for activation of the 5-HTreceptor when placed at the 2-position of the-benzyl moiety. Efficacies of the tryptamines at the rat and human 5-HTreceptors and human 5-HTreceptor varied from about 40% to 80%, with a few compounds that were full agonists (e.g.,and), whereas at the rat 5-HTreceptor all of the compounds were full agonists.

The rank order of affinity of all compounds at the [ 125 I]-DOI-labeled h5-HT 2C receptor generally paralleled that measured at the 5-HT 2A receptor, although the affinities tended to be somewhat lower. Again, among the tryptamines studied 5i had the highest affinity at this receptor, as well as at the 5-HT 2B receptor. Affinities measured at the [ 125 I]-DOI site tended to be on the order of 5–10 times higher than that at the antagonist labeled sites at both receptors.

At the 5-HTand 5-HTreceptors, the highest affinity was observed in the competition displacements with [I]-DOI. Except forand, all of the tryptamine compounds had low nanomolar or subnanomolar affinity for the human 5-HTreceptor. The known phenethylaminehad by far the highest affinity at 5-HTreceptors, with subnanomolar affinity at both subtypes. We have previously reported an affinity forat the human 5-HTreceptor of 0.04 nM. (14) Of the tryptamines, only the 3-iodobenzyl compound, had subnanomolar affinity at the 5-HTreceptor, although all of the tryptamines had high affinity at this receptor. It should be noted that N-methylation ofcompletely abolished affinity at the 5-HTreceptor (> 10 μM; data not shown), indicating that tertiary amines are not tolerated in the-benzyltryptamines.

Further exploration of a small library of 3-substituted-benzyl tryptamines allowed us to develop a tentative SAR for this series, and it is clear that substituents on the-benzyl 3-position do modulate affinity in the tryptamine series. In the broad screening of 5-HT receptor types, all of the compounds had the highest affinity at the 5-HT2 family of receptors (Tables 1 and 2 ).

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5e was not selective for the h5-HT 2A receptor versus the h5-HT 2C receptor. Using affinity at the [125I]-DOI-labeled receptors, the selectivity of 5e was slightly less than 4-fold. Even using affinity at the [125I]-DOI-labeled h5-HT 2A receptor and the [3H]-mesulergine-labeled h5-HT 2C receptor, “selectivity” was only about 18-fold. The most selective compound in the entire series, with respect to affinity, was 5d, but with only 6-fold selectivity. Unfortunately, despite the report by Glennon et al., (19) compoundwas not selective for the h5-HTreceptor versus the h5-HTreceptor. Using affinity at the [I]-DOI-labeled receptors, the selectivity ofwas slightly less than 4-fold. Even using affinity at the [I]-DOI-labeled h5-HTreceptor and the [H]-mesulergine-labeled h5-HTreceptor, “selectivity” was only about 18-fold. The most selective compound in the entire series, with respect to affinity, was, but with only 6-fold selectivity.

With respect to selectivity in function at the h5-HT 2A vs h5-HT 2C , the most selective tryptamine was 5j, with 44-fold selectivity and less than a 3-fold difference in affinity at the agonist-labeled receptors. Indeed, we were disappointed that none of the compounds had high selectivity for the h5-HT 2A receptor.

1, none of the compounds was particularly potent in producing the HTR. This low potency is somewhat surprising, given that many known hallucinogens with high affinity for the 5-HT 2A receptor, such as 2,5-dimethoxy-4-iodoamphetamine (DOI), R-(−)-2,5-dimethoxy-4-methylamphetamine (R-DOM), R-(−)-2,5-dimethoxy-4-bromoamphetamine (R-DOB), 2,5-dimethoxy-4-propylthiophenethylamine (2C-T-7), psilocin, and 5-MeO-N,N-diisopropyltryptamine (5-MeO–DIPT) produce the head twitch in mice at doses of ≤1 mg/kg.N,N-dimethyltryptamine (5-MeO-DMT) and α-methyltryptamine, are active within the same dose range (3–30 mg/kg) as the N-benzyltryptamines tested herein.in vivo potencies of the compounds studied here are related to the use of an automated HTR detection system because we have confirmed that the results obtained using this system are consistent with published data based on visual scoring. 50 = 0.13 μmol/kg) 50 = 0.14 μmol/kg).N-benzyl-analogues in general Overall, with the exception of compound, none of the compounds was particularly potent in producing the HTR. This low potency is somewhat surprising, given that many known hallucinogens with high affinity for the 5-HTreceptor, such as 2,5-dimethoxy-4-iodoamphetamine (DOI),-(−)-2,5-dimethoxy-4-methylamphetamine (-DOM),-(−)-2,5-dimethoxy-4-bromoamphetamine (-DOB), 2,5-dimethoxy-4-propylthiophenethylamine (2C-T-7), psilocin, and 5-MeO--diisopropyltryptamine (5-MeO–DIPT) produce the head twitch in mice at doses of ≤1 mg/kg. (32, 33, 38-40) However, certain tryptamine hallucinogens, including 5-MeO--dimethyltryptamine (5-MeO-DMT) and α-methyltryptamine, are active within the same dose range (3–30 mg/kg) as the-benzyltryptamines tested herein. (40-42) It is unlikely that the lowpotencies of the compounds studied here are related to the use of an automated HTR detection system because we have confirmed that the results obtained using this system are consistent with published data based on visual scoring. (23) For example, the potency of LSD measured using the automated system (ED= 0.13 μmol/kg) (23) is almost exactly the same as the potency assessed using direct observation (ED= 0.14 μmol/kg). (41) One possible explanation for the low potencies might be rapid first pass metabolism of-benzyl-analogues in general (43) combined with a slow release from subcutaneous tissue due to the highly hydrophobic nature of the compounds.

Substitution on the N-benzyl ring has different effects, depending on whether the phenethylamines or the tryptamines are being studied. For example, ortho-bromo-substituted tryptamine congener 5d failed to induce the HTR when tested at doses up to 30 mg/kg (∼60 μmol/kg), yet N-3-bromobenzyl 5e is active. By contrast, N-2-bromobenzyl phenethylamine 4c is active, whereas N-3-bromobenzyl 4d is inactive in the HTR assay.

N-benzyl groups, 4b, 4e, 5c, or 5f, was active in the HTR. All of these compounds were partial agonists with relatively low potency in the r5-HT 2A functional assay. Although 5e, with a 3-substituted N-benzyl, has an EC 50 and E max virtually identical to 4e, it is active in the HTR assay. It is possible that differences in pharmacokinetics or metabolic lability could explain these data. Nevertheless, if only the compounds active in the mouse HTR assay are compared, one finds a significant correlation between potency in the rat 5-HT 2A receptor and potency in the HTR assay, as shown in Figure None of the phenethylamines or tryptamines with 4-substituted-benzyl groups,, or, was active in the HTR. All of these compounds were partial agonists with relatively low potency in the r5-HTfunctional assay. Although, with a 3-substituted-benzyl, has an ECandvirtually identical to, it is active in the HTR assay. It is possible that differences in pharmacokinetics or metabolic lability could explain these data. Nevertheless, if only the compounds active in the mouse HTR assay are compared, one finds a significant correlation between potency in the rat 5-HTreceptor and potency in the HTR assay, as shown in Figure 3

Figure 3 Figure 3. Regression analysis of pED 50 for the mouse head twitch response on the pEC 50 for function for active compounds at the rat 5-HT 2A receptor; n = 10.

Taken together, these data show that for N-benzylphenethylamines the highest in vivo potency in mice is associated with an ortho-substituent on the benzyl group, whereas the N-benzyltryptamines are more active in vivo when a meta-substituent is present. Hence, there are SAR differences between the N-benzyltryptamines and the N-benzylphenethylamines for the induction of the HTR, which likely reflect different binding orientations in the 5-HT 2A receptor. Obviously, the indole system is larger than a simple phenyl ring, something that would clearly affect the binding modes for the two different series at the orthosteric site. For example, the distance from the indole C(3) atom to the 5-oxygen atom is 4.94 Å, whereas the corresponding distance from the 5-methoxy oxygen to C(1) of the aryl ring is only 3.70 Å. Even the distance of 4.85 Å from C(1) of the aryl ring to the 4-iodo atom of the phenethylamines is less than the 4.94 Å distance measured from C(3) of the indole to the 5-methoxy.

N-benzyltryptamines and N-benzylphenethylamines, oxygenated substituents are tolerated at the ortho- and meta-positions of the benzyl moiety. For example, 1, 4a, 5a, and 5b are all active in the HTR assay, whereas 4d and 5d are inactive over a range of doses. This observation again would be consistent with some structural feature in the 5-HT 2A receptor that could engage a polar oxygen atom at the ortho-position of the N-benzyl moiety. There has been speculation, based on virtual docking studies with phenethylamines and tryptamines, that an oxygen atom in the ortho-position of the N-benzyl moiety may interact with a hydrogen bond donor (possibly the OH of Tyr 370(7.43) in the h5-HT 2A receptor.N-benzyltryptamines also could form a hydrogen bond with Tyr 370, possibly involving a water molecule. One exception is that for both the-benzyltryptamines and-benzylphenethylamines, oxygenated substituents are tolerated at the ortho- and meta-positions of the benzyl moiety. For example,, andare all active in the HTR assay, whereasandare inactive over a range of doses. This observation again would be consistent with some structural feature in the 5-HTreceptor that could engage a polar oxygen atom at the ortho-position of the-benzyl moiety. There has been speculation, based on virtual docking studies with phenethylamines and tryptamines, that an oxygen atom in the ortho-position of the-benzyl moiety may interact with a hydrogen bond donor (possibly the OH of Tyr 370in the h5-HTreceptor. (14, 18) It is conceivable that an oxygen atom at the meta-position in-benzyltryptamines also could form a hydrogen bond with Tyr 370, possibly involving a water molecule.