N-Benzyl-5-methoxytryptaminesas Potent Serotonin 5-HT2 Receptor Family Agonistsand Comparison with a Series of Phenethylamine Analogues
David E Nichols
M Flori Sassano
Adam L Halberstadt
Landon M Klein
Simon D Brandt
Simon P Elliott
Divisionof Chemical Biology andMedicinal Chemistry, Genetic Medicine Building, 2078, 120 Mason FarmRoad, University of North Carolina, Chapel Hill, NC 27599. Phone: (765) 404-0350. E-mail:denichol@email.unc.edu.
Received 2014 Nov 10; Collection date 2015 Jul 15.
This is an open access article published under an ACS AuthorChoiceLicense, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
Abstract
A seriesofN-benzylated-5-methoxytryptamine analogueswas prepared and investigated, with special emphasis on substituentsin the meta position of the benzyl group. A parallel series of severalN-benzylated analogues of 2,5-dimethoxy-4-iodophenethylamine(2C-I) also was included for comparison of the two major templates(i.e., tryptamine and phenethylamine). A broad affinity screen atserotonin receptors showed that most of the compounds had the highestaffinity at the 5-HT2 family receptors. Substitution at the para positionof the benzyl group resulted in reduced affinity, whereas substitutionin either the ortho or the meta position enhanced affinity. In general,introduction of a large lipophilic group improved affinity, whereasfunctional activity often followed the opposite trend. Tests of thecompounds for functional activity utilized intracellular Ca2+ mobilization. Function was measured at the human 5-HT2A, 5-HT2B, and 5-HT2C receptors, as well asat the rat 5-HT2A and 5-HT2C receptors. Therewas no general correlation between affinity and function. Severalof the tryptamine congeners were very potent functionally (EC50 values from 7.6 to 63 nM), but most were partial agonists.Tests in the mouse head twitch assay revealed that many of the compoundsinduced the head twitch and that there was a significant correlationbetween this behavior and functional potency at the rat 5-HT2A receptor.
Keywords: Serotonin, 5-HT2 receptors, 5-HT2A, agonist, phenethylamine, 5-methoxytryptamine, mouse head twitch
Introduction
Recently, an extremely potent hallucinogenicphenethylamine, 25I-NBOMe(N-(2-methoxybenzyl)-2,5-dimethoxy-4-iodophenethylamine;“smiles”)1 has been available on the illicitdrug market.1 For purposes of enforcement,it is presently considered by the Drug Enforcement Administration(DEA) to be an analogue of 2C-I (2), which is currentlya Schedule I controlled substance. The procedure to classify1 as a Schedule I substance has been initiated, and it hasbeen placed temporarily into Schedule I.2 Unfortunately, several deaths have been associated with the useof1,3−5 but it is not clear whether the deaths resulted fromthe ingestion of lethal amounts of pure solid drug, or whether thedrug has some inherent toxicity that is not normally associated withother hallucinogens.
There has been increasing global interestin1 andclosely related analogues. For example, the European Monitoring Centrefor Drugs and Drug Addiction (EMCDDA) has received a range of notificationsfrom EU Member States about analytically confirmed nonfatal and fatalintoxications associated with1. This was then followedby a risk assessment conducted by the Scientific Committee of theEMCDDA in order to assess health and social risks associated withthis particular analogue.6 In addition,the World Health Organization’s Expert Committee on Drug Dependencereviewed the status of a range of new substances for its 36th meetingin June 2014, which included1 and its 4-bromo and 4-chloroanalogues.7 In September 2014, the Councilof the European Union decided to subject1 to controlmeasures and criminal penalties throughout the European Union.8
Typically, simple N-alkylation dramaticallyattenuates or abolisheshallucinogenic activity in phenethylamines.9,10 TheN-benzyl moiety, however, confers exceptionally high potencyonto the molecule,11−15 and we have presented evidence that theN-benzylmay engage F339 in the human 5-HT2A receptor.14 We also examined variousN-arylmethylsubstituents and found that a variety of aryl groups were effectivein enhancing potency.16,17 In addition, the presence ofa polar substituent at the ortho position of the aryl ring (a possiblehydrogen bond acceptor) further enhances activity.18 Silva et al.18 also have reportedthat in an in vitro cylindrical rat tail artery strip1 had a pEC50 of 10.09 and anEmax of 30%.
Two decades ago, Glennon et al.19 reportedthat the affinities of theN-benzyl compound3a, as well as the 4-bromo- and 4-iodo-N-benzylcompounds,3b and3c, respectively, were2–3 times higher than that of the parent primary amine. Therehave been no further reports on these compounds, and in our own work,we had never examined 3- or 4-substituted benzyl substituents in thephenethylamine series.
In addition to the phenethylaminetype 5-HT2A agonists,certain simple tryptamines possess similar pharmacology, particularly4- or 5-oxygenated molecules. In the report by Glennon et al., placinganN-benzyl moiety on the amine of 5-methoxytryptaminehad essentially no effect on affinity. Interestingly,N-benzyl-5-methoxytryptamine previously had been reported to be anantagonist of serotonin-induced contraction in the rat stomach fundus,the isolated guinea pig uterus, and the isolated guinea pig taeniacecum.20 In addition, Leff et al.21 had shown thatN-benzyl-5-methoxytryptaminehad only weak partial agonist activity at 5-HT2 type receptorsin rabbit aorta and rat jugular vein.
Surprisingly, however,in the Glennon report,19 a 5-HT2A receptor affinity of 0.1 nM was reportedfor theN-4-bromobenzyl compound (compound33 in the Glennon report, numbered here as5f), with 1000-fold selectivity for 5-HT2A over 5-HT2C receptors. We found these data particularly intriguing.This degree of selectivity was overestimated, however, because affinityat the 5-HT2A receptor was measured by displacement ofan agonist ragioligand, whereas affinity at the 5-HT2C receptorwas measured by displacement of an antagonist radioligand. Nonetheless,no specific 5-HT2A-selective agonist has been available,although such a compound would be very valuable for serotonin neuroscienceresearch.
Although it was reported19 that 4-bromocompound5f had 0.1 nM affinity at the human 5-HT2A receptor, the 4-fluoro-, 4-chloro-, and 4-iodo-substitutedbenzyl congeners had reported affinities of 40, 105, and 120 nM, respectively,in that same report. We found this discontinuity in the structure–activityrelationship (SAR) puzzling, where the 4-bromo compound would be suchan outlier in the family of halogen-substituted benzyls. Further investigationby Jensen, however, revealed that the authentic 4-bromo compound5f actually had relatively low affinity for the 5-HT2A receptor, more consistent with the reported affinities of the otherhalogenated compounds.22 Although spectroscopicdata were not reported by Glennon et al.19 that might explain the basis for this discrepancy, their publicationindicated elemental analysis data to be consistent with the proposedstructure. If the elemental analysis data were correct, the mostlylikely explanation for the discordant biological data therefore seemedto be that5f might have been an isomer other than the4-substituted compound.
On the basis of the hypothesis that the original datawere associatedwith an isomer other than the 4-bromo compound, we subsequently discoveredthatN-3-bromobenzyl compound5e didhave higher affinity for the 5-HT2A receptor (Ki 1.48 nM), compared to that of the 4-bromocongener5f (Ki 11.2 nM). Further, the effect of an ortho-oxygenatedN-benzyl appeared not to be significant for affinity inthe tryptamine series, suggesting perhaps different binding orientationsof theN-benzyltryptamines versus theN-benzylphenethylamines within the receptor. That is, compound5a has been reported to have agonist potency (pEC50 7.08) in a rat tail artery assay not significantly different fromthe compound with an unsubstitutedN-benzyl moiety(pEC50 7.00), although theEmax was slightly higher for the 2′-methoxy compound.18 These findings prompted us to synthesize a smallseries of structurally related congeners to determine whether othersubstitutions might have even greater affinity and/or selectivityfor the 5-HT2A receptor.
Thus, in this article wedescribe the facile synthesis of compounds1,4a–4e, and5a–5l, preliminary screening at a variety of 5HTfamily receptors, and more detailed testing at human 5-HT2A, 5-HT2B, and 5-HT2C receptors, including affinitymeasurements using displacement of the agonist radioligand [125I]-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-HT2A receptor activation.23
Compound1 has been previouslyreported,24 and the NMR and electron ionizationmass spectraof4a and4b have been reported but withoutany biological data.25 We thus decidedto compare all of the series members at the same time to elucidatea consistent SAR.
Chemistry
All of the compounds weremost easily prepared using a modificationof the facile method first reported by Abdel-Magid et al.26 The free base of2 was stirredin 3 mL of MeOH for 30 min with the appropriate aldehyde, followedby reduction of the intermediate enamine with NaBH4. Followingappropriate workup, the bases were converted to their HCl or maleatesalts and crystallized in good to excellent yields.
Pharmacology
Affinities at a panel of 5-HT receptors were determined by theNIMH-sponsored PDSP program (http://pdsp.med.unc.edu/kidb.php). Affinities at both the human and rat 5-HT2A and 5-HT2C receptors also were determined, using both agonist and antagonistradioligands. As a measure of functional potency and efficacy, changesin intracellular Ca2+ levels were measured using a fluorometricimaging plate reader (FLIPRTETRA, Molecular Devices), atthe human 5-HT2A, 5-HT2B, and 5-HT2C receptors, and at the rat 5-HT2A and 5-HT2C receptors. Finally, as a measure of in vivo 5-HT2A receptoractivation, we assessed the ability of all compounds to induce themouse HTR.23 We hypothesized that functionalpotency at the rat 5-HT2A receptor might correlate bestwith the mouse head twitch behavioral data because ligand affinitiesat the rat 5-HT2A receptor correlate with the mouse 5-HT2A receptor but not with the human 5-HT2A receptor.27
Results
Further exploration of asmall library of 3-substitutedN-benzyl tryptaminesallowed us to develop a tentative SARfor this series, and it is clear that substituents on theN-benzyl 3-position do modulate affinity in the tryptamineseries. In the broad screening of 5-HT receptor types, all of thecompounds had the highest affinity at the 5-HT2 family of receptors(Tables1 and2).
Table 1. Affinities of New Compounds for theHuman 5-HT2A and 5-HT2C Receptors Using BothAgonist and Antagonist Radioligandsa.
| h5-HT2A pKi ±SEM (Ki nM) | h5-HT2C pKi ± SEM (Ki nM) | |||
|---|---|---|---|---|
| cmpd | [3H]ketanserin | [125I]DOI | [3H]mesulergine | [125I]DOI |
| 1 | 9.28 ± 0.11 (0.52) | 9.80 ± 0.15 (0.16) | 9.16 ± 0.09 (0.69) | 9.30 ± 0.16 (0.50) |
| 4a | 8.81 ± 0.17 (1.5) | 9.57 ± 0.09 (0.27) | 8.38 ± 0.01 (4.17) | 9.90 ± 0.07 (0.13) |
| 4b | 7.93 ± 0.13 (11.7) | 9.15 ± 0.16 (0.70) | 7.85 ± 0.02 (14.1) | 8.44 ± 0.14 (3.63) |
| 4c | 8.63 ± 0.18 (2.34) | 9.42 ± 0.09 (0.38) | 8.06 ± 0.07 (8.71) | 8.99 ± 0.18 (1.02) |
| 4d | 8.40 ± 0.04 (3.98) | 9.24 ± 0.12 (0.57) | 8.12 ± 0.02 (7.59) | 8.79 ± 0.08 (1.62) |
| 4e | 7.28 ± 0.14 (52.5) | 8.49 ± 0.09 (3.24) | 7.34 ± 0.02 (45.7) | 8.48 ± 0.25 (3.31) |
| 5a | 7.78 ± 0.05 (16.6) | 8.82 ± 0.19 (1.51) | 7.49 ± 0.14 (32.4) | 8.47 ± 0.10 (3.39) |
| 5b | 8.11 ± 0.10 (7.76) | 8.98 ± 0.14 (1.05) | 7.42 ± 0.12 (38.0) | 8.23 ± 0.09 (5.89) |
| 5c | 7.16 ± 0.16 (69.2) | 7.98 ± 0.04 (10.5) | 6.90 ± 0.03 (126) | 7.85 ± 0.13 (14.1) |
| 5d | 7.60 ± 0.12 (25.1) | 8.63 ± 0.19 (2.34) | 7.00 ± 0.01 (100) | 7.85 ± 0.10 (14.1) |
| 5e | 8.17 ± 0.11 (6.76) | 8.83 ± 0.10 (1.48) | 7.58 ± 0.05 (26.3) | 8.25 ± 0.11 (5.62) |
| 5f | 6.37 ± 0.12 (427) | 7.95 ± 0.22 (11.2) | 6.60 ± 0.15 (251) | 7.54 ± 0.19 (28.8) |
| 5g | 7.67 ± 0.04 (21.4) | 8.58 ± 0.17 (2.63) | 7.32 ± 0.09 (47.9) | 8.06 ± 0.14 (8.71) |
| 5h | 8.28 ± 0.08 (5.25) | 8.98 ± 0.10 (1.05) | 7.55 ± 0.06 (28.2) | 8.37 ± 0.05 (4.27) |
| 5i | 8.46 ± 0.09 (3.47) | 9.21 ± 0.16 (0.62) | 8.19 ± 0.09 (6.46) | 8.98 ± 0.08 (1.05) |
| 5j | 8.32 ± 0.17 (4.79) | 8.93 ± 0.11 (1.17) | 7.65 ± 0.03 (22.4) | 8.47 ± 0.08 (3.39) |
| 5k | 7.55 ± 0.05 (28.2) | 8.53 ± 0.19 (2.95) | 6.99 ± 0.06 (102) | 7.83 ± 0.26 (14.8) |
| 5l | 8.05 ± 0.15 (8.91) | 8.51 ± 0.17 (3.09) | 7.88 ± 0.23 (13.2) | 8.68 ± 0.30 (2.09) |
pKi ± SEM (affinities in nM);n = 3–5 separate displacement curves.
Table 2. PDSP Screening Affinitiesfor AllCompounds at Other Human Serotonin Receptor Typesa.
| cmpd | 5-HT2B | 5-HT1A | 5-HT1B | 5-HT1D | 5-ht1e | 5-HT3 | 5-ht5a | 5-HT6 | 5-HT7 |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 8.86 ± 0.03 (1.4) | 5.99 ± 0.05 (1033) | 5.23 ± 0.06 (5886) | 6.27 ± 0.05 (533) | >10,000 | >10,000 | 5.55 ± 0.07 (2795) | 7.5 ± 0.06 (32) | 5.81 ± 0.06 (1542) |
| 4a | 8.34 ± 0.03 (4.6) | 6.03 ± 0.05 (925) | 5.49 ± 0.05 (3232) | 6.36 ± 0.05 (439) | 5.77 ± 0.05 (1707) | >10,000 | 7.24 ± 0.06 (57) | 7.17 ± 0.06 (67) | 6.23 ± 0.06 (583) |
| 4b | 7.78 ± 0.03 (17) | 5.97 ± 0.05 (1064) | 5.8 ± 0.05 (1592) | 6.49 ± 0.05 (325) | 5.89 ± 0.05 (1285) | >10,000 | 5.99 ± 0.06 (1020) | 7.12 ± 0.03 (75) | 5.8 ± 0.06 (1575) |
| 4c | 7.7 ± 0.04 (20) | 5.94 ± 0.06 (1155) | >10,000 | 6.37 ± 0.05 (423) | >10,000 | >10,000 | 5.64 ± 0.09 (2290) | 6.59 ± 0.06 (257) | 5.59 ± 0.05 (2547) |
| 4d | 7.89 ± 0.04 (13) | 6.17 ± 0.06 (670) | 5.80 ± 0.05 (1568) | 6.79 ± 0.05 (162) | 6.10 ± 0.04 (792) | >10,000 | 6 ± 0.08 (1009) | 6.76 ± 0.06 (175) | 6.45 ± 0.05 (355) |
| 4e | 7.17 ± 0.04 (68) | 6.19 ± 0.06 (649) | 5.22 ± 0.05 (5093) | 6.51 ± 0.05 (311) | >10,000 | 5.61 ± 0.05 (2460) | 5.73 ± 0.06 (1848) | 6.46 ± 0.05 (350) | 6.19 ± 0.05 (641) |
| 5a | 8.04 ± 0.03 (9) | 6.64 ± 0.05 (231) | >10,000 | 5.89 ± 0.05 (1292) | >10,000 | >10,000 | >10,000 | 7.06 ± 0.03 (87) | 5.75 ± 0.06 (1770) |
| 5b | 8.6 ± 0.03 (2.5) | 6.48 ± 0.05 (335) | >10,000 | 6.48 ± 0.06 (334) | >10,000 | >10,000 | 5.9 ± 0.06 (1261) | 7.6 ± 0.03 (25) | 6.39 ± 0.05 (406) |
| 5c | 7.49 ± 0.03 (33) | 7.12 ± 0.06 (76) | 5.97 ± 0.04 (1060) | 6.79 ± 0.06 (161) | >10,000 | >10,000 | 5.62 ± 0.09 (2388) | 6.45 ± 0.03 (353) | 7.44 ± 0.05 (37) |
| 5d | 7.62 ± 0.03 (24) | 6.54 ± 0.05 (286) | >10,000 | 6.11 ± 0.05 (782) | >10,000 | 5.21 ± 0.07 (6169) | >10,000 | 6.69 ± 0.05 (203) | 5.96 ± 0.05 (1086) |
| 5e | 8.45 ± 0.03 (3.6) | 6.81 ± 0.05 (155) | 5.19 ± 0.06 (6433) | 6.42 ± 0.05 (381) | >10,000 | >10,000 | 6.21 ± 0.06 (612) | 7.34 ± 0.03 (45) | 6.93 ± 0.06 (116) |
| 5f | 6.83 ± 0.03 (150) | 7.11 ± 0.05 (78) | 5.35 ± 0.05 (4374) | 6.57 ± 0.05 (271) | >10,000 | >10,000 | 5.99 ± 0.08 (1034) | 6.25 ± 0.03 (566) | 6.45 ± 0.06 (358) |
| 5g | 7.66 ± 0.03 (22) | 6.53 ± 0.04 (295) | 5.57 ± 0.06 (2674) | 6.50 ± 0.06 (319) | >10,000 | >10,000 | 5.61 ± 0.05 (2450) | 7.23 ± 0.03 (59) | 6.62 ± 0.05 (242) |
| 5h | 8.16 ± 0.02 (6.6) | 6.71 ± 0.05 (195) | 5.36 ± 0.06 (4392) | 6.55 ± 0.06 (282) | >10,000 | >10,000 | 5.64 ± 0.06 (2310) | 7.30 ± 0.03 (50) | 6.55 ± 0.05 (281) |
| 5i | 9.12 ± 0.03 (0.76) | 6.91 ± 0.05 (122) | 5.53 ± 0.05 2963) | 6.70 ± 0.06 (199) | >10,000 | >10,000 | 5.81 ± 0.05 (1536) | 7.58 ± 0.03 (27) | 7.66 ± 0.05 (22) |
| 5j | 8.71 ± 0.03 (1.9) | 6.57 ± 0.04 (271) | 5.37 ± 0.07 (4241) | 6.55 ± 0.06 (283) | 5.41 ± 0.05 (3876) | >10,000 | 5.41 ± 0.06 (3852) | 7.21 ± 0.03 (62) | 6.67 ± 0.05 (212) |
| 5k | 7.56 ± 0.02 (28) | 6.62 ± 0.05 (240) | >10,000 | 6.56 ± 0.06 (278) | >10,000 | >10,000 | 5.51 ± 0.06 (3091) | 7.06 ± 0.03 (87) | 6.58 ± 0.05 (262) |
| 5l | 8.39 ± 0.04 (4.1) | 6.90 ± 0.05 (127) | >10,000 | 6.18 ± 0.05 (659) | >10,000 | >10,000 | 6.08 ± 0.08 (841) | 8.01 ± 0.06 (9.7) | 6.87 ± 0.05 (136) |
pKi ± SEM, (affinity in nM).
At the 5-HT2A and 5-HT2C receptors, the highestaffinity was observed in the competition displacements with [125I]-DOI. Except for5c and5f,all of the tryptamine compounds had low nanomolar or subnanomolaraffinity for the human 5-HT2A receptor. The known phenethylamine1 had by far the highest affinity at 5-HT2A/2C receptors, with subnanomolar affinity at both subtypes.We have previously reported an affinity for1 at thehuman 5-HT2A receptor of 0.04 nM.14 Of the tryptamines, only the 3-iodobenzyl compound5i, had subnanomolar affinity at the 5-HT2A receptor, althoughall of the tryptamines had high affinity at this receptor. It shouldbe noted that N-methylation of5e completely abolishedaffinity at the 5-HT2A receptor (Ki > 10 μM; data not shown), indicatingthat tertiary amines are not tolerated in theN-benzyltryptamines.
The rank order of affinity of all compounds at the [125I]-DOI-labeled h5-HT2C receptor generally paralleled thatmeasured at the 5-HT2A receptor, although the affinitiestended to be somewhat lower. Again, among the tryptamines studied5i had the highest affinity at this receptor, as well as atthe 5-HT2B receptor. Affinities measured at the [125I]-DOI site tended to be on the order of 5–10 times higherthan that at the antagonist labeled sites at both receptors.
Functional potencies at the rat and human 5-HT2A and5-HT2C receptors and the human 5-HT2B receptorare shown in Table3. Compound1 was a nearly full agonist at both receptor types, with a 4.2 nMEC50 at the human 5-HT2A receptor and 11 nMEC50 at the rat 5-HT2A receptor. The most potentcompound was5a, with an EC50 of 1.9 nM and85% efficacy at the h5-HT2A. Notably, this compound hastheN-2-methoxybenzyl substituent, the same as themost potent phenethylamine1, suggesting that it maybe optimal for activation of the 5-HT2A receptor when placedat the 2-position of theN-benzyl moiety. Efficaciesof the tryptamines at the rat and human 5-HT2A receptorsand human 5-HT2C receptor varied from about 40% to 80%,with a few compounds that were full agonists (e.g.,5a and5c), whereas at the rat 5-HT2C receptorall of the compounds were full agonists.
Table 3. FunctionalData for New Compoundsin Rat and Human 5-HT2A and 5-HT2C and Human5-HT2B Receptorsa.
| r5-HT2A | h5-HT2A | h5-HT2B | r5-HT2C | h5HT2C | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| cmpd | pEC50 (EC50 nM) | Emax % | pEC50 (EC50 nM) | Emax % | pEC50 (EC50 nM) | Emax % | pEC50 (EC50 nM) | Emax % | pEC50 (EC50 nM) | Emax % |
| 5-HT | 8.3 ± 0.04 (5.4) | 100 ± 1.5 | 8.7 ± 0.05 (2.0) | 100 ± 1.6 | 9.31 ± 0.04 (0.49) | 99.9 ± 1.1 | 9.70 ± 0.03 (0.20) | 99.6 ± 0.73 | 9.52 ± 0.08 (0.30) | 98.5 ± 2.4 |
| 1 | 8.0 ± 0.04 (11) | 79.4 ± 1.1 | 8.4 ± 0.05 (4.2) | 86.4 ± 1.4 | 7.81 ± 0.09 (15) | 65 ± 2 | 7.02 ± 0.05 (95) | 104 ± 2 | 7.38 ± 0.12 (41.7) | 92 ± 0 |
| 4a | 7.6 ± 0.04 (27) | 51.7 ± 0.9 | 7.6 ± 0.03 (28) | 71.6 ± 0.9 | 7.4 ± 0.3 (38) | NAb | 6.88 ± 0.06 (133) | 91 ± 3 | 7.47 ± 0.36 (33.8) | 41 ± 6 |
| 4b | 7.3 ± 0.04 (50) | 53.8 ± 0.9 | 7.2 ± 0.03 (60) | 74.1 ± 0.8 | 7.1 ± 0.1 (87) | 38 ± 2 | 6.88 ± 0.05 (132) | 97 ± 2 | 7.36 ± 0.31 (43.2) | 50 ± 7 |
| 4c | 7.4 ± 0.06 (36) | 65.6 ± 1.6 | 7.4 ± 0.04 (42) | 88.0 ± 1.5 | 6.82 ± 0.07 (134) | 83 ± 3 | 6.98 ± 0.02 (105) | 104 ± 1 | 7.24 ± 0.13 (57.6) | 87 ± 5 |
| 4d | 7.8 ± 0.03 (14) | 68.5 ± 0.9 | 7.8 ± 0.04 (17) | 87.5 ± 1.3 | 7.05 ± 0.05 (85) | 90 ± 2 | 7.44 ± 0.05 (36) | 101 ± 2 | 7.28 ± 0.17 (57.6) | 74 ± 5 |
| 4e | 6.8 ± 0.03 (150) | 67.3 ± 0.9 | 6.8 ± 0.03 (170) | 88.0 ± 1.4 | 6.21 ± 0.04 (610) | 90 ± 2 | 6.54 ± 0.04 (290) | 105 ± 2 | 6.66 ± 0.14 (200) | 77 ± 5 |
| 5a | 7.7 ± 0.03 (21) | 80.9 ± 1.1 | 8.7 ± 0.05 (1.9) | 85.2 ± 1.4 | 8.2 ± 0.1 (6.7) | 52 ± 2 | 7.79 ± 0.04 (16) | 102 ± 2 | 7.24 ± 0.12 (57.1) | 119 ± 6 |
| 5b | 7.5 ± 0.04 (34) | 52.2 ± 0.9 | 8.2 ± 0.04 (6.2) | 70.0 ± 1.0 | 6.0 ± 0.4 (949) | NAb | 6.78 ± 0.05 (168) | 102 ± 2 | 6.75 ± 0.15 (178) | 65 ± 5 |
| 5c | 6.7 ± 0.03 (190) | 75.0 ± 1.3 | 7.4 ± 0.04 (42) | 84.1 ± 1.3 | 7.64 ± 0.04 (23) | 81 ± 1 | 7.73 ± 0.04 (19) | 102 ± 2 | 7.12 ± 0.11 (75.1) | 112 ± 5 |
| 5d | 6.3 ± 0.04 (450) | 49.7 ± 1.2 | 7.5 ± 0.05 (30) | 74.7 ± 1.5 | 6.8 ± 0.3 (168) | NAb | 6.05 ± 0.05 (898) | 104 ± 3 | 6.36 ± 0.09 (439) | 94 ± 5 |
| 5e | 6.9 ± 0.03 (130) | 65.5 ± 0.8 | 7.9 ± 0.04 (13) | 73.8 ± 1.1 | 7.5 ± 0.2 (29) | 20 ± 2 | 6.38 ± 0.04 (422) | 112 ± 3 | 6.49 ± 0.23 (321) | 64 ± 8 |
| 5f | 5.8 ± 0.04 (1500) | 77.6 ± 2.4 | 6.4 ± 0.02 (430) | 90.3 ± 1.2 | 6.54 ± 0.05 (290) | 90 ± 2 | 6.69 ± 0.03 (204) | 108 ± 2 | 6.28 ± 0.14 (529) | 83 ± 7 |
| 5g | 7.1 ± 0.04 (80) | 69.1 ± 1.3 | 8.0 ± 0.1 (10) | 89.3 ± 1.1 | 7.42 ± 0.08 (38) | 37 ± 1 | 7.34 ± 0.07 (46) | 100 ± 3 | 6.72 ± 0.13 (192) | 83 ± 5 |
| 5h | 7.1 ± 0.03 (83) | 70.1 ± 1.0 | 7.9 ± 0.04 (14) | 81.2 ± 1.3 | 7.3 ± 0.2 (50) | NAb | 6.54 ± 0.04 (286) | 105 ± 2 | 6.50 ± 0.13 (316) | 85 ± 6 |
| 5i | 6.9 ± 0.04 (120) | 73.4 ± 1.4 | 7.8 ± 0.04 (16) | 79.0 ± 1.1 | 7.4 ± 0.2 (43) | 31 ± 2 | 6.51 ± 0.05 (313) | 110 ± 3 | 6.35 ± 0.09 (445) | 94 ± 5 |
| 5j | 7.6 ± 0.04 (26) | 56.2 ± 0.9 | 8.2 ± 0.04 (6.5) | 73.3 ± 1.0 | NAb | 6.72 ± 0.04 (192) | 104 ± 2 | 6.54 ± 0.10 (289) | 75 ± 4 | |
| 5k | 6.1 ± 0.03 (770) | 69.6 ± 1.4 | 7.1 ± 0.04 (87) | 75.5 ± 1.2 | 6.97 ± 0.07 (107) | 51 ± 2 | 6.79 ± 0.03 (162) | 104 ± 2 | 6.29 ± 0.11 (512) | 75 ± 5 |
| 5l | 6.9 ± 0.05 (120) | 32.0 ± 0.7 | 7.5 ± 0.04 (32) | 46.9 ± 0.8 | NAb | 6.69 ± 0.05 (205) | 101 ± 2 | 6.55 ± 0.11 (283) | 60 ± 4 | |
Values are pEC50 ±SEM, with (EC50) values in nM andEmax given in percentage of the maximum response to 5-HT.
NA, not active;Emax ≤ 15%.
It is noteworthy that the functional potencies in the rat and human5-HT2A receptors are essentially identical for phenethylaminecompounds1, and4a–4e, yet the potencies for tryptamine compounds5a–5l are 4–10-fold higher at the human 5-HT2A receptor than at the rat 5-HT2A receptor. This findingmay reflect the single amino acid difference in the orthosteric bindingsite of these two receptors at position 5.46. In the rat or mouse5-HT2A receptor, residue 5.46 is an alanine, whereas inthe human receptor it is a serine. We have previously shown that mutationof this residue in the human receptor from serine to alanine has littleeffect on affinity or function for phenethylamine 5-HT2A agonists but does have a significant effect for tryptamines.28 One might infer, therefore, from these potencydifferences that the indole NH in the present series also engagesthis serine in the human receptor but not the alanine in the rat receptor,consistent with mutagenesis studies reported by others.29,30
Figure1 shows an illustrative dose–responsecurve for compound5h in the mouse HTR. HTR data forall compounds are given in Table4. Althoughsome of the compounds failed to induce the HTR at doses up to 30 mg/kg,most of the “inactive” compounds displayed relativelylow potency at 5-HT2A (see Figure2), so it is possible that they would induce the HTR if tested athigher doses. Importantly, for the subset of compounds that inducedthe HTR, behavioral potency was significantly correlated with functionalpotency at the r5-HT2A receptor (r = 0.69,p < 0.03; Figure2), but there was no correlationwith functional EC50 values at the r5-HT2C receptor(r = 0.17,p > 0.1). Despite the overall correlationbetween mouse HTR and r5-HT2A potency, the relationshipwas not always orderly for individual compounds. Compound1 was by far the most potent compound in that assay, with an ED50 of 0.078 mg/kg (data taken from Halberstadt and Geyer31). It is not clear why1 shouldbe so much more potent than any other compound because, for example,4d is inactive but appears nearly comparable functionally,with an EC50 of 14 nM and efficacy of 69%, compared withan EC50 of 11 nM for1 with an efficacy of79%. The next most potent compounds in the mouse HTR are4c and5j, with identical ED50s of 2.31 mg/kg,about 300-fold less potent than1. Although they havesimilar functional EC50 values (36 and 26 nM), nothingin the functional or binding data can explain their lower potencycompared to that of1. Further, compounds5a,5b, and5g have virtually identical ED50 values in the mouse HTR, yet their functional EC50s at the rat 5-HT2A receptor are 21, 34, and 80 nM, respectively.
Figure 1.
Representativedose–response plot in the mouse head twitchassay for compound5h. *p < 0.05versus vehicle (Tukey’s test).
Table 4. Activity of New Compounds in Producingthe Mouse Head Twitch.
| ED50 mg/kg (95% CI) | test duration(min) | N | dose range | active doses (mg/kg) | maxcounts | maximallyeffective dose(mg/kg) | magnitudeof peak effect × vehicle | |
|---|---|---|---|---|---|---|---|---|
| 1 | 0.078 (0.055–0.111) | 30 | 5 | 0.03–1.0 | 0.1, 0.3, 1 | 102.6 | 1 | 16.0 |
| 4a | 4.34 (1.41–13.32) | 10 | 10 | 0.3–30 | 3, 10, 30 | 11.4 | 30 | 5.7 |
| 4b | inactive | 5 | 0.3–30 | |||||
| 4c | 2.31 (1.41–3.77) | 20 | 5 | 0.3–30 | 3, 10, 30 | 23.2 | 10 | 3.0 |
| 4d | inactive | 5–7 | 0.3–10 | |||||
| 4e | inactive | 6 | 1–30 | |||||
| 5a | 3.15 (1.94–5.12) | 20 | 10 | 0.3–30 | 10, 30 | 25.4 | 10 | 3.9 |
| 5b | 3.28 (1.53–7.04) | 10 | 5–6 | 1–30 | 10 | 9.2 | 10 | 3.7 |
| 5c | inactive | 5 | 30 | |||||
| 5d | inactive | 5 | 0.3–30 | |||||
| 5e | 5.18 (2.35–11.38) | 10 | 5–6 | 1–30 | 10, 30 | 14.2 | 30 | 4.4 |
| 5f | inactive | 5 | 0.3–30 | |||||
| 5g | 3.33 (2.25–4.93) | 10 | 6 | 1–30 | 10, 30 | 14.5 | 10 | 7.3 |
| 5h | 4.43 (2.03–9.69) | 10 | 5–6 | 1–30 | 10, 30 | 10.6 | 30 | 8.0 |
| 5i | 7.77 (3.40–17.53) | 10 | 6 | 1–30 | 10, 30 | 20.2 | 30 | 3.4 |
| 5j | 2.31 (0.82–6.51) | 10 | 5 | 0.3–30 | 10 | 14.6 | 10 | 3.5 |
| 5k | inactive | 5 | 30 | |||||
| 5l | inactive | 4–5 | 30 |
Figure 2.
Plotsof active and inactive compounds as a function of potencyand efficacy at the rat 5-HT2A receptor (panel A) and thehuman 5-HT2A receptor (panel B).
With the exception of5k and5l, whichhad relatively low functional potencies at the r5-HT2A (EC50 values of 770 and 120 nM, respectively), all of the meta-substitutedN-benzyl derivatives of 5-methoxytryptamine induced theHTR. That included the 3-methyl (5j; ED50 =2.31 mg/kg), 3-methoxy (5b; ED50 = 3.28 mg/kg),3-fluoro (5g; ED50 = 3.33 mg/kg), 3-chloro(5h; ED50 = 4.43 mg/kg), 3-bromo (5e; ED50 = 5.18 mg/kg), and 3-iodo (5i; ED50 = 7.77 mg/kg) compounds.
The HTR produced by compounds5b and5j showed a biphasic bell-shaped dose–responsefunction (theresponse peaked at 10 mg/kg and 30 mg/kg was inactive). Other 5-HT2A agonists, including DOI, DOM, 2C-T-7, and 5-MeO–DIPT,have been shown to produce similar nonmonotonic responses.32−ref34 Fantegrossi et al.ref34 have argued thatthe descending arm of the biphasic HTR dose–response is a consequenceof 5-HT2C activation, which attenuates the response to5-HT2A activation. Recently, however, it was reported thatN-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenethylamine(25CN-NBOH), a 5-HT2A agonist with 100-fold selectivityover 5-HT2C, also induces the HTR with a biphasic dose–response.ref35 The fact that the descending arm of the responseto 25CN-NBOH was not affected by a 5-HT2C antagonistref35 demonstrates that the inhibition of the HTRat high doses does not necessarily result from competing activityat 5-HT2C. One potential alternative explanation for thebiphasic HTR is that high levels of 5-HT2A activation mayproduce competing behaviors that interfere with expression of headshaking. Along those lines, it has been reported that high doses ofquipazine, 5-MeO-DMT, and (+)-LSD produce stereotypic behaviors thatpreclude head shakes and wet dog shakes in rats.ref36,ref37
Discussion
Unfortunately, despite the report by Glennonet al.,19 compound5e wasnot selective forthe h5-HT2A receptor versus the h5-HT2C receptor.Using affinity at the [125I]-DOI-labeled receptors, theselectivity of5e was slightly less than 4-fold. Evenusing affinity at the [125I]-DOI-labeled h5-HT2A receptor and the [3H]-mesulergine-labeled h5-HT2C receptor, “selectivity” was only about 18-fold. Themost selective compound in the entire series, with respect to affinity,was5d, but with only 6-fold selectivity.
Withrespect to selectivity in function at the h5-HT2A vs h5-HT2C, the most selective tryptamine was5j, with44-fold selectivity and less than a 3-fold difference in affinityat the agonist-labeled receptors. Indeed, we were disappointed thatnone of the compounds had high selectivity for the h5-HT2A receptor.
Overall, with the exception of compound1, none ofthe compounds was particularly potent in producing the HTR. This lowpotency is somewhat surprising, given that many known hallucinogenswith high affinity for the 5-HT2A 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 ≤1mg/kg.32,33,34−36 However, certain tryptamine hallucinogens, including 5-MeO-N,N-dimethyltryptamine (5-MeO-DMT) and α-methyltryptamine,are active within the same dose range (3–30 mg/kg) as theN-benzyltryptamines tested herein.36−38 It is unlikelythat the lowin vivo potencies of the compounds studiedhere are related to the use of an automated HTR detection system becausewe have confirmed that the results obtained using this system areconsistent with published data based on visual scoring.23 For example, the potency of LSD measured usingthe automated system (ED50 = 0.13 μmol/kg)23 is almost exactly the same as the potency assessedusing direct observation (ED50 = 0.14 μmol/kg).37 One possible explanation for the low potenciesmight be rapid first pass metabolism ofN-benzyl-analoguesin general39 combined with a slow releasefrom subcutaneous tissue due to the highly hydrophobic nature of thecompounds.
Substitution on theN-benzyl ringhas differenteffects, depending on whether the phenethylamines or the tryptaminesare being studied. For example,ortho-bromo-substitutedtryptamine congener5d failed to induce the HTR when tested at dosesup to 30 mg/kg (∼60 μmol/kg), yetN-3-bromobenzyl5e is active. By contrast,N-2-bromobenzylphenethylamine4c is active, whereasN-3-bromobenzyl4d is inactive in the HTR assay.
None of the phenethylamines or tryptamines with 4-substitutedN-benzyl groups,4b,4e,5c, or5f, was active in the HTR. All of thesecompounds were partial agonists with relatively low potency in ther5-HT2A functional assay. Although5e, witha 3-substitutedN-benzyl, has an EC50 andEmax virtually identical to4e,it is active in the HTR assay. It is possible that differences inpharmacokinetics 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-HT2A receptor and potency in the HTR assay, as shown in Figure3.
Figure 3.
Regression analysis of pED50 for the mousehead twitchresponse on the pEC50 for function for active compoundsat the rat 5-HT2A receptor;n = 10.
Taken together, these data showthat forN-benzylphenethylaminesthe highest in vivo potency in mice is associated with an ortho-substituenton the benzyl group, whereas theN-benzyltryptaminesare more active in vivo when a meta-substituent is present. Hence,there are SAR differences between theN-benzyltryptaminesand theN-benzylphenethylamines for the inductionof the HTR, which likely reflect different binding orientations inthe 5-HT2A receptor. Obviously, the indole system is largerthan a simple phenyl ring, something that would clearly affect thebinding modes for the two different series at the orthosteric site.For example, the distance from the indole C(3) atom to the 5-oxygenatom is 4.94 Å, whereas the corresponding distance from the 5-methoxyoxygen to C(1) of the aryl ring is only 3.70 Å. Even the distanceof 4.85 Å from C(1) of the aryl ring to the 4-iodo atom of thephenethylamines is less than the 4.94 Å distance measured fromC(3) of the indole to the 5-methoxy.
One exception is that forboth theN-benzyltryptaminesandN-benzylphenethylamines, oxygenated substituentsare tolerated at the ortho- and meta-positions of the benzyl moiety.For example,1,4a,5a, and5b are all active in the HTR assay, whereas4d and5d are inactive over a range of doses. This observationagain would be consistent with some structural feature in the 5-HT2A receptor that could engage a polar oxygen atom at the ortho-positionof theN-benzyl moiety. There has been speculation,based on virtual docking studies with phenethylamines and tryptamines,that an oxygen atom in the ortho-position of theN-benzyl moiety may interact with a hydrogen bond donor (possiblythe OH of Tyr 370(7.43) in the h5-HT2A receptor.14,18 It is conceivable that an oxygen atom at the meta-position inN-benzyltryptamines also could form a hydrogen bond withTyr 370, possibly involving a water molecule.
Unfortunately,a 5-HT2A selective agonist did not emergefrom this small library of compounds. There are now only two selective5-HT2A agonists reported,40,41 but they havenot been available for extensive study. Thus, research on 5-HT2A receptor function has been forced to employ either a mixed5-HT2A/2C agonist such as DOI in combinationwith a specific 5-HT2C antagonist, or to administer antagonistsalone, the latter paradigm really being appropriate to study receptorfunction only when there are high levels of endogenous receptor activationor constitutive activity of the receptors. Genetic knockout mice havenot revealed particular behavioral phenotypes and have served primarilyto demonstrate that a particular drug depends on the presence of 5-HT2A or 5-HT2C receptors for its effect. Hence, thepsychopharmacology of a “pure” 5-HT2A agonistremains completely unknown. Furthermore, the tremendous present interestin the role of the 5-HT2A receptor in normal brain functionmakes it imperative that scientists in the field gain access to a5-HT2A specific agonist so that research into the rolesof the 5-HT2A receptor can be more fully elucidated.
Methods
Chemistry
General Methods
Reagents were purchased from Sigma-AldrichCo. (St. Louis, MO) or Alfa Aesar (Ward Hill, MA) and used as delivered,unless otherwise specified. Thin layer chromatography was carriedout using J. T. Baker flexible sheets (silica gel IB2-F) with fluorescentindicator, visualizing with UV light at 254 nm or iodine stain. Meltingpoints were determined using a Mel-Temp apparatus and are uncorrected.NMR experiments were carried out using a Bruker Advance 300 MHz instrument,and the chemical shift (δ) values are in parts per million (ppm)relative to tetramethylsilane at 0.00 ppm. The solvent was CD3OD. NMR samples were dissolved in MeOD. Ph = aromatic protons/carbonsof benzyl group; In = aromatic protons/carbons of the indole nucleus;Ar = either phenyl or indole resonances, or phenyl in the case ofcompounds1–4f. Coupling constants (J) are presented in Hertz. Abbreviations used in the reporting ofNMR spectra include: br = broad, s = singlet, d = doublet, t = triplet,q = quartet, and quint = quintuplet.
Mass spectra were performedby high resolution LC-QTOF-MS on protonated molecules [M + H]+. UHPLC-Q-TOF-MS conditions for UHPLC separation employeda mobile phase consisting of 100% MeCN that included 1% formic acid(organic phase) and an aqueous solution of 1% formic acid (aqueousphase). The column was maintained at 40 °C with a 0.6 mL/minflow rate and 5.5 min acquisition time. The elution was a 5–70%MeCN gradient ramp over 3.5 min, then up to 95% MeCN in 1 min andheld for 0.5 min before returning to 5% MeCN in 0.5 min. Q-TOF-MSdata were acquired in positive mode scanning from 100 to 1000m/z with and without auto MS/MS fragmentation.Ionization was achieved with an Agilent JetStream electrospray sourceand infused internal reference masses. Agilent 6540 Q-TOF-MS parameters:gas temperature, 325 °C; drying gas, 10 L/min; and sheath gastemperature, 400 °C. Internal reference masses of 121.05087 and922.00979m/z were used.
Forcompounds1 and4a–4e, 0.5 mmol of the free base of 4-iodo-2,5-dimethoxyphenethylamine10,42 was stirred for 30 min at room temperature with 0.55 mmol of theappropriate aldehyde in 3 mL of methanol. The reaction was then placedon an ice bath, and 48 mg (1.25 mmol) of NaBH4 was addedin three portions over 15 min. The ice bath was removed and the reactionallowed to stir for an additional 15 min. The reaction was then transferredto a separatory funnel with 50 mL of EtOAc. The organic phase waswashed three times with saturated NaCl, then dried overnight overNa2SO4. The drying agent was removed by suctionfiltration, and the filtrate was concentrated under reduced pressure.EtOH (1 mL) was added to the amber residue, and the HCl salt was preparedby acidification with 0.5 mL of 1 N HCl/EtOH. Dilution with EtOAcor diethyl ether then led to crystallization of the HCl salts, generallyin good yields. In most cases, the supernatant was simply decantedfrom the crystalline product, followed by resuspension of the crystalsin Et2O and decantation, then air drying to afford theproducts as white to off-white fine needles. No attempt was made tooptimize the yields, but in one case the supernatant was reduced todryness and the residue crystallized from EtOH/Et2O toafford an additional 6% of product. This small additional recoverywas not deemed sufficient to warrant the extra effort. Thus, all reportedyields are those obtained after the first crystallization.
Thesynthesis of tryptamines5a–5l followedessentially the same procedure, except that maleate saltswere prepared. As an example, 1.0 mmol of 5-methoxytryptamine freebase (Aldrich) was stirred for 30 min with 1.10 mmol of the appropriatealdehyde in 5 mL of methanol. The reaction was then placed on an icebath, and 96 mg (2.5 mmol) of NaBH4 was added in threeportions over 15 min. The ice bath was removed and the reaction allowedto stir for an additional 15 min. The reaction was then transferredto a separatory funnel with 50 mL of EtOAc and was washed three timeswith saturated NaCl. The organic phase was dried overnight over Na2SO4, then filtered and concentrated under reducedpressure. Maleic acid (116 mg, 1 mmol) and 1.0 mL of acetone werethen added to the residual amber oil, and the solution swirled untilall of the maleic acid had dissolved. The reaction was then dilutedwith 10 mL of EtOAc, and Et2O was added nearly to the cloudpoint. In most cases, crystallization occurred rapidly and spontaneously,and the product solution was stored overnight in a cold room. Crystallineproducts were collected by suction filtration, washed on the filterwith EtOAc, and then air-dried to afford white to off-white fine needles.
N-(2-Methoxybenzyl)-2-(4-iodo-2,5-dimethoxyphenyl)ethan-1-amineHydrochloride (1)
Obtained as needles followingcrystallization from acetone/EtOAc/Et2O; yield 86%; mp168–170 °C, Lit24 mp 162–166°C, 166.131H NMR (300MHz, CD3OD) δ ppm 7.46 (1H, td,J = 8.2, 1.7 Hz, Ar–H), 7.37 (1H, dd,J =7.6, 1.6 Hz, Ar–H), 7.35 (1H, s, Ar–H), 7.09 (1H, d,J = 8.3 Hz, Ar–H), 7.02 (1H, td,J = 7.5, 1.0 Hz, Ar–H), 6.86 (1H, s, Ar–H), 4.24 (2H,s, NB-CH2), 3.88 (3H, s, OCH3), 3.81 (3H, s,OCH3), 3.78 (3H, s, OCH3), 3.20–3.25(2H, m, α-CH2), 3.03–2.98 (2H, m, β-CH2).13C NMR (CD3OD): δ ppm 159.37(Ar–Cq), 154.44 (Ar–Cq), 153.60 (Ar–Cq), 132.81(Ar–CH), 132.73 (Ar–CH), 126.99 (Ar–Cq), 123.19(Ar–CH), 122.13 (Ar–CH), 120.29 (Ar–Cq), 114.98(Ar–CH), 112.16 (Ar–CH), 85.04 (Ar–Cq-iodine),57.59 (OCH3), 56.71 (OCH3), 56.24 (OCH3), 48.1 (NB-CH2), 48.0 (α-CH2), 28.49(β-CH2). HRMS calculated for C18H23INO3 [M + H]+, 428.07171; observed[M + H]+, 428.07239. The EI mass spectrum also has beenreported by Casale and Hays.25
N-(3-Methoxybenzyl)-2-(4-iodo-2,5-dimethoxyphenyl)ethan-1-amineHydrochloride (4a)
Obtained as needles followingcrystallization from acetone/EtOAc/Et2O; yield 85%; mp171–2 °C.1H NMR (300 MHz, CD3OD)δ ppm 7.38 (1H, t,J = 7.7 Hz, Ar–H),7.34 (1H, s, Ar–H), 6.98–7.10 (3H, m, Ar–H),6.86 (1H, s, Ar–H), 4.19 (2H, s, NB-CH2), 3.83 (3H,s, OCH3), 3.81 (3H, s, OCH3), 3.79 (3H, s, OCH3), 3.22–3.27 (2H, m, α-CH2), 2.99–3.04(2H, m, β-CH2).13C NMR (CD3OD): δ ppm 161.77 (Ar–Cq), 154.43 (Ar–Cq), 153.63(Ar–Cq), 133.82 (Ar–Cq), 131.48 (Ar–CH), 127.01(Ar–Cq), 123.14 (Ar–CH), 122.92 (Ar–CH), 116.53(Ar–CH), 116.13 (Ar–CH), 114.95 (Ar–CH), 85.00(Ar–Cq-iodine), 57.59 (OCH3), 56.68 (OCH3), 55.93 (OCH3), 52.23 (NB-CH2), 48.1 (α-CH2), 28.65 (β-CH2). HRMS calculated for C18H23INO3 [M + H]+, 428.07171;observed [M + H]+, 428.07319. The EI mass spectrum hasalso been reported by Casale and Hays.25
N-(4-Methoxybenzyl)-2-(4-iodo-2,5-dimethoxyphenyl)ethan-1-amineHydrochloride (4b)
This particular compoundwas extremely difficult to crystallize, providing unfilterable gelsupon attempts to crystallize it from EtOH, EtOH/Et2O, orMeOH/Et2O. It was finally obtained by dissolving in a minimumamount of boiling acetonitrile and allowing the solution to cool.Upon cooling, the solution also took on a gel-like appearance, butunlike other attempts, this material could be collected by vacuumfiltration through a sintered glass filter funnel. The voluminouswhite solid was washed on the filter with a small amount of cold acetonitrile,then left on the funnel with suction until dry; yield 72%; mp 180–182°C.1H NMR (300 MHz, CD3OD): δ ppm7.41 (2H, d,J = 8.7 Hz, 2 x Ar–H), 7.34 (1H,s, Ar–H), 7.00 (2H, d,J = 8.5 Hz, 2 x Ar–H),6.85 (1H, s, Ar–H), 4.15 (2H, s, NB-CH2), 3.82 (3H,s, OCH3), 3.81 (3H, s OCH3), 3.79 (3H, s OCH3), 3.18–3.23 (2H, m, α-CH2), 2.96–3.01(2H, m, β-CH2).13C NMR (CD3OD): δ ppm 162.27 (Ar–Cq), 154.42 (Ar–Cq), 153.64(Ar–Cq), 132.59 (2 x Ar–CH), 127.05 (Ar–Cq),124.22 (Ar–Cq), 123.16 (Ar–CH), 115.64 (2 x Ar–CH),114.94 (Ar–CH), 84.98 (Ar–Cq-iodine), 57.59 (OCH3), 56.68 (OCH3), 55.90 (OCH3), 51.90(NB-CH2), 47.8 (α-CH2), 28.67 (β-CH2). HRMS calculated for C18H23INO3 [M + H]+, 428.07171; observed [M + H]+, 428.07320. The EI mass spectrum has also been reported by Casaleand Hays.25
N-(2-Bromobenzyl)-2-(4-iodo-2,5-dimethoxyphenyl)ethan-1-amineHydrochloride (4c)
Obtained as needles followingcrystallization from acetone/EtOAc/Et2O; yield 79%; mp170–1 °C.1H NMR (300 MHz, CD3OD):δ ppm 7.74 (1H, dd,J = 7.9, 1.3 Hz, Ar–H),7.61 (1H, dd,J = 7.7, 1.7 Hz, Ar–H), 7.49(1H, td,J = 7.5, 1.3 Hz), 7.39 (1H, td,J = 7.9, 1.9 Hz), 7.35 (1H, s, Ar–H), 6.89 (1H, s,Ar–H), 4.42 (2H, s, NB-CH2), 3.82 (3H, s, OCH3), 3.81 (3H, s, OCH3), 3.31–3.36 (2H, m,α-CH2), 3.03–3.08 (2H, m, β-CH2).13C NMR (CD3OD): δ ppm 154.46 (Ar–Cq),153.62 (Ar–Cq), 134.74 (Ar–CH), 133.03 (Ar–CH),132.81(Ar–CH), 132.32 (Ar–Cq), 129.70 (Ar–CH),126.87 (Ar–Cq), 125.94 (Ar–Cq), 123.19 (Ar–CH),114.98 (Ar–CH), 85.08 (Ar–Cq-iodine), 57.60 (OCH3), 56.73 (OCH3), 51.99 (NB-CH2), 48.7(α-CH2), 28.62 (β-CH2). HRMS calculatedfor C17H20BrINO2 [M + H]+, 475.97166; observed [M + H]+, 475.97212.
N-(3-Bromobenzyl)-2-(4-iodo-2,5-dimethoxyphenyl)ethan-1-amineHydrochloride (4d)
Obtained as needles followingcrystallization from acetone/EtOAc/Et2O; yield 89%; mp199–201 °C.1H NMR (300 MHz, CD3OD): δ ppm 7.69–7.74 (1H, m, Ar–H), 7.60–7.66(1H, m, Ar–H), 7.45–7.51 (1H, m, Ar–H), 7.41(1H, d,J = 7.7 Hz, Ar–H), 7.35 (1H, s, Ar–H),6.86 (1H, s, Ar–H), 4.22 (2H, s, NB-CH2), 3.81 (3H,s, OCH3), 3.79 (3H, s, OCH3), 3.22–3.27(2H, m, α-CH2), 2.98–3.03 (2H, m, β-CH2).13C NMR (CD3OD): δ ppm 154.44(Ar–Cq), 153.63 (Ar–Cq), 134.97 (Ar–Cq), 134.03(Ar–CH), 133.87, (Ar–CH), 132.12 (Ar–CH), 129.89(Ar–CH), 126.93 (Ar–Cq), 124.00 (Ar–Cq), 123.18(Ar–CH), 114.95 (Ar–CH), 85.06 (Ar–Cq-iodine),57.59 (OCH3), 56.70 (OCH3), 51.51 (NB-CH2), 48.3 (α-CH2), 28.68 (β-CH2). HRMS calculated for C17H20BrINO2 [M + H]+, 475.97166; observed [M + H]+, 475.97281.
N-(4-Bromobenzyl)-2-(4-iodo-2,5-dimethoxyphenyl)ethan-1-amineHydrochloride (4e)
Obtained as needles followingcrystallization from acetone/EtOAc/Et2O; yield 81%; mp196–7 °C.1H NMR (300 MHz, CD3OD)δ ppm 7.64 (2H, d,J = 8.7 Hz, 2 x Ar–H),7.42 (2H, d,J = 8.5 Hz, 2 x Ar–H), 7.34 (1H,s, Ar–H), 6.86 (1H, s, Ar–H), 4.21 (2H, s, NB-CH2), 3.81 (3H, s, OCH3), 3.79 (3 H, s, OCH3), 3.22–3.27 (2H, m, α-CH2), 2.98–3.03(2H, m, β-CH2).13C NMR (CD3OD): δ ppm 154.43 (Ar–Cq), 153.62 (Ar–Cq), 133.50(2 x Ar–CH), 133.00 (2 x Ar–CH), 131.71 (Ar–Cq),126.92 (Ar–Cq), 124.92 (Ar–Cq), 123.16 (Ar–CH),114.94 (Ar–CH), 85.02 (Ar–Cq-iodine), 57.60 (OCH3), 56.68 (OCH3), 51.58 (NB-CH2), 48.2(α-CH2), 28.66 (β-CH2). HRMS calculatedfor C17H20BrINO2 Calculated [M +H]+, 475.97166; observed [M + H]+, 475.97268.
N-(2-Methoxybenzyl)-2-(5-methoxy-1H-indol-3-yl)ethan-1-amine Hydrochloride (5a)
Obtained as needles following crystallization from EtOH/EtOAc; yield91%; mp 232–4 °C.1H NMR (300 MHz, CD3OD): δ ppm 7.42 (1H, td,J = 7.9, 1.7 Hz,Ph-H), 7.33 (1H, dd,J = 7.4, 1.6 Hz, Ph-H), 7.28(1H, dd,J = 8.9, 0.6 Hz, In–H), 7.16 (1H,s, In–H), 6.97–7.03 (2H, m, Ph-H), 6.95 (1H, d,J = 2.4 Hz, In–H), 6.81 (1H, dd,J = 8.8, 2.4 Hz, In–H), 4.23 (2H, s, NB-CH2), 3.78(3H, s, OCH3), 3.67 (3H, s, OCH3), 3.28–3.33(2H, m, α-CH2, overlapping with solvent), 3.12–3.17(2H, m, β-CH2).13C NMR (CD3OD): δ ppm 159.25 (Ph–Cq), 155.42 (In-Cq), 133.67 (Ar–Cq),132.77 (Ph–CH), 132.68 (Ph–CH), 128.34 (Ar–Cq),125.35 (In-CH), 122.12 (In-CH), 120.13 (Ar–Cq), 113.41 (Ph–CH),113.21 (In-CH), 112.06 (Ph–CH), 109.51 (Ar–Cq), 101.00(In-CH), 56.35 (OCH3), 55.93 (OCH3), 48.90 (α-CH2), 48.3 (NB-CH2), 23.21 (β-CH2). HRMS calculated for C19H23N2O2 [M + H]+, 311.17540; observed [M + H]+, 311.17548.
N-(3-Methoxybenzyl)-2-(5-methoxy-1H-indol-3-yl)ethan-1-amine Maleate (5b)
Obtainedas needles following crystallization from acetone/EtOAc/Et2O; yield 84%; mp 124–5 °C.1H NMR (300 MHz,CD3OD): δ ppm 7.33–7.39 (1H, m, Ph-H), 7.26(1H, dd,J = 8.9, 0.6 Hz, In–H), 7.13 (1H,s, In–H), 6.99–7.01 (4H, m, overlapping 3 x Ph-H, 1x In–H), 6.80 (1H, dd,J = 8.8, 2.4 Hz, In–H),6.24 (2H, s, maleate), 4.18 (2H, s, NB-CH2), 3.81 (3H,s, OCH3), 3.80 (3H, s, OCH3), 3.28–3.35(2H, α-CH2, overlapping with solvent), 3.11–3.16(2H, m, β-CH2).13C NMR (CD3OD): δ ppm 170.89 (maleate), 161.79 (Ph–Cq), 155.39(In-Cq), 136.79 (maleate), 133.89 (Ar–Cq), 133.60 (Ar–Cq),131.50 (Ph–CH), 128.44 (Ar–Cq), 125.00 (In-CH), 122.87(Ph–CH), 116.40 (Ph–CH), 116.15 (Ph–CH), 113.35(In-CH), 113.07 (In-CH), 109.84 (Ar–Cq), 101.04 (In-CH), 56.39(OCH3), 55.88 (OCH3), 52.16 (NB-CH2), 49.0 (α-CH2), 23.36 (β-CH2).HRMS calculated for C19H23N2O2 [M + H]+, 311.17540; observed [M + H]+, 311.17572
N-(4-Methoxybenzyl)-2-(5-methoxy-1H-indol-3-yl)ethan-1-amine Maleate (5c)
Obtainedas needles following crystallization from acetone/EtOAc/Et2O; yield 82%; mp 172–3 °C.1H NMR (300 MHz,CD3OD): δ ppm 7.36 (2H, d,J = 8.0Hz, 2 x Ph-H), 7.26 (1 H, dd,J = 8.8, 0.5 Hz, In–H),7.12 (1H, s, In–H), 6.99 (1H, d,J = 2.5 Hz,In–H), 6.97 (2H, d,J = 6.6 Hz, 2 x Ph-H),6.80 (1H, dd,J = 8.9, 2.4 Hz, In–H), 6.24(2H, s, maleate), 4.15 (2H, s, NB-CH2), 3.81 (3H, s, OCH3), 3.80 (3H, s, OCH3), 3.27–3.32 (2H, m,α-CH2, overlapping with solvent), 3.09–3.14(2H, m, β-CH2).13C NMR (CD3OD): δ ppm 170.90 (maleate), 162.24 (Ph–Cq), 155.37(In-Cq), 136.78 (maleate), 133.60 (Ar–Cq), 132.52 (2 x Ph–CH),128.45 (Ar–Cq), 124.97 (In-CH), 124.27 (Ar–Cq), 115.64(2 x Ph–CH), 113.34 (In-CH), 113.06 (In-CH), 109.89 (Ar–Cq),101.06 (In-CH), 56.39 (OCH3), 55.89 (OCH3),51.78 (NB-CH2), 48.5 (α-CH2), 23.38 (β-CH2). HRMS calculated for C19H23N2O2 [M + H]+, 311.17540; observed [M + H]+, 311.17632
N-(2-Bromobenzyl)-2-(5-methoxy-1H-indol-3-yl)ethan-1-amineMaleate (5d)
Obtained as needles following crystallizationfrom acetone/EtOAc/Et2O; yield 72%; mp 93–5 °C.1H NMR (300 MHz, CD3OD): δ ppm 7.70 (1H, dd,J = 7.9, 1.3 Hz, Ph-H), 7.54 (1H, dd,J = 7.7, 1.9 Hz, Ph-H), 7.45 (1H, td,J = 7.5, 1.4Hz, Ph-H), 7.36 (1H, td,J = 7.8, 1.8 Hz, Ph-H),7.26 (1H, dd,J = 8.9, 0.6 Hz, In–H), 7.16(1H, s, In–H), 7.02 (1H, d,J = 2.3 Hz, In–H),6.80 (1H, dd,J = 8.9, 2.4 Hz, In–H), 6.24(2H, s, maleate), 4.41 (2H, s, NB-CH2), 3.82 (3H, s, OCH3), 3.40–3.45 (2H, m, α-CH2), 3.16–3.21(2H, m, β-CH2).13C NMR (CD3OD): δ ppm 170.89 (maleate), 155.41 (In-Cq), 136.75 (maleate),134.71 (Ph–CH), 133.64 (Ar–Cq), 133.04 (Ph–CH),132.76 (Ph–CH), 132.40 (Ar–Cq), 129.65 (Ph–CH),128.44(Ar–Cq), 125.94 (Ar–Cq), 125.12 (In-CH), 113.38 (In-CH),113.10 (In-CH), 109.67 (Ar–Cq), 101.06 (In-CH), 56.40 (OCH3), 51.90 (NB-CH2), 49.3 (α-CH2), 23.32 (β-CH2). HRMS calculated for C18H20BrN2O [M + H]+, 359.07535; observed[M + H]+, 359.07581.
N-(3-Bromobenzyl)-2-(5-methoxy-1H-indol-3-yl)ethan-1-amine Maleate (5e)
Obtainedas needles following crystallization from acetone/EtOAc/Et2O; yield 86%; mp 137–8 °C.1H NMR (300 MHz,CD3OD): δ ppm 7.67–7.68 (1H, m, Ph-H), 7.61(1H, dt,J = 7.7, 1.6 Hz, Ph-H), 7.34–7.45(2H, m, Ph-H), 7.27 (1H, d,J = 8.7 Hz, In–H),7.14 (1H, s, In–H), 7.01 (1H, d,J = 2.3 Hz,In–H), 6.80 (1H, dd,J = 8.9, 2.4 Hz, In–H),6.24 (2H, s, maleate), 4.21 (2H, s, NB-CH2), 3.82 (3H,s, OCH3), 3.31–3.36 (2H, m, α-CH2, overlapping with solvent), 3.11–3.16 (2H, m, β-CH2).13C NMR (CD3OD): δ ppm 170.92(maleate), 155.40 (In-Cq), 136.78 (maleate), 135.10 (Ar–Cq),134.00 (Ph–CH), 133.82 (Ph–CH), 133.61 (Ar–Cq),132.10 (Ph–CH), 129.81 (Ph–CH), 128.45 (Ar–Cq),125.01 (In-CH), 124.03 (Ar–Cq), 113.37 (In-CH), 113.08 (In-CH),109.82 (Ar–Cq), 101.06 (In-CH), 56.42 (OCH3), 51.52(NB-CH2), 49.1 (α-CH2), 23.40 (β-CH2). HRMS calculated for C18H20BrN2O [M + H]+, 359.07535; observed [M + H]+, 359.07547
N-(4-Bromobenzyl)-2-(5-methoxy-1H-indol-3-yl)ethan-1-amine Maleate (5f)
Obtainedas needles following crystallization from acetone/EtOAc/Et2O; yield 75%; mp 181–3 °C.1H NMR (CD3OD): δ ppm 7.60 (2H, d,J = 8.5 Hz,2 x Ph-H), 7.37 (2H, d,J = 8.5 Hz, 2 x Ph-H), 7.26(1H, d,J = 8.9 Hz, In–H), 7.13 (1H, s, In–H),6.99 (1H, d,J = 2.3 Hz, In–H), 6.80 (1H,dd,J = 8.9, 2.4 Hz, In–H), 6.24 (2H, s, maleate),4.20 (2H, s, NB-CH2), 3.81 (3H, s, OCH3), 3.31–3.36(2H, m, α-CH2, overlapping with solvent), 3.11–3.16(2H, m, β-CH2).13C NMR (CD3OD): δ ppm 170.89 (maleate), 155.38 (In-Cq), 136.7 5 (maleate),133.61 (Ar–Cq), 133.51 (2 x Ph–CH), 132.92 (2 x Ph–CH),131.77 (Ar–Cq), 128.43 (Ar–Cq), 125.02 (In-CH), 124.90(Ar–Cq), 113.36 (In-CH), 113.06 (In-CH), 109.76 (Ar–Cq),101.06 (In-CH), 56.41 (OCH3), 51.50 (NB-CH2),48.90 (α-CH2), 23.40 (β-CH2). HRMScalculated for C18H20BrN2O [M + H]+, 359.07535; observed [M + H]+, 359.07597.
N-(3-Fluorobenzyl)-2-(5-methoxy-1H-indol-3-yl)ethan-1-amineMaleate (5g)
Obtainedas needles following crystallization from acetone/EtOAc/Et2O; yield 78%; mp 150–2 °C.1H NMR (300 MHzCD3OD): δ ppm 7.44–7.51 (1H, m, Ph-H), 7.16–7.29(4H, m, overlapping 3 x Ph-H, 1 x In–H), 7.14 (1H, s, In–H),7.01 (1H, d,J = 2.4 Hz, In–H), 6.80 (1H,dd,J = 8.9, 2.4 Hz, In–H), 6.24 (2H, s, maleate),4.24 (2H, s, NB-CH2), 3.81 (3H, s, OCH3), 3.31–3.37(2H, m, α-CH2, overlapping with solvent), 3.12–3.17(2H, m, β-CH2).13C NMR (CD3OD): δ ppm 170.89 (maleate), 164.38 (Ph–Cq-3′,d,J = 246.2 Hz), 155.40 (In-Cq), 136.74 (maleate),135.08 (Ph–Cq-1′, d,J = 7.5 Hz), 133.61(In-Cq), 132.31 (Ph–C-5′, d,J = 8.3Hz), 128.45 (In-Cq), 126.88 (Ph–C-6′, d,J = 3.0 Hz), 125.00 (In-CH), 117.79 (Ph–C-2′, d,J = 22.5 Hz), 117.60 (Ph–C-4′, d,J = 21.8 Hz), 113.37 (In-CH), 113.08 (In-CH), 109.79 (In-Cq),101.06, (In-CH), 56.41 (OCH3), 51.58 (NB-CH2,J = 1.5 Hz), 49.1 (α-CH2), 23.38(β-CH2). HRMS calculated for C18H20FN2O [M + H]+, 299.15542; observed[M + H]+, 299.15602.
N-(3-Chlorobenzyl)-2-(5-methoxy-1H-indol-3-yl)ethan-1-amine Maleate (5h)
Obtainedas needles following crystallization from acetone/EtOAc/Et2O; yield 79%; mp 116–8 °C.1H NMR (300 MHz,CD3OD): δ ppm 7.52 (1H, br, s, Ph-H), 7.34–7.49(3H, m, Ph-H), 7.26 (1H, d,J = 8.9 Hz, In–H),7.14 (1H, s, In–H), 7.01 (1H, d,J = 2.4 Hz,In–H), 6.80 (1H, dd,J = 8.9, 2.4 Hz, In–H),6.24 (2H, s, maleate), 4.22 (2H, s, NB-CH2), 3.82 (3H,s, OCH3), 3.31–3.37 (2H, m, α-CH2, overlapping with solvent), 3.12–3.17 (2H, m, β-CH2).13C NMR (CD3OD): δ ppm 155.41(In-Cq), 136.76 (maleate), 136.10 (Ar–Cq), 134.82 (Ar–Cq),133.60 (Ar–Cq), 131.89 (Ph–CH), 131.04 (Ph–CH),130.84 (Ph–CH), 129.38 (Ph–CH), 128.45 (Ar–Cq),125.01 (In-CH), 113.36 (In-CH), 113.08 (In-CH), 109.78 (In-Cq), 101.04(In-CH), 56.40 (OCH3), 51.54 (NB-CH2), 49.1(α-CH2), 23.39 (β-CH2). HRMS calculatedfor C18H20ClN2O [M + H]+, 315.12587; observed [M + H]+, 315.12666
N-(3-Iodobenzyl)-2-(5-methoxy-1H-indol-3-yl)ethan-1-amineMaleate (5i)
Obtainedas needles following crystallization from acetone/EtOAc/Et2O; yield 84%; mp 131–2 °C.1H NMR (300 MHz,CD3OD): δ ppm 7.87 (1H, brs, Ph-H), 7.81 (1H, d,J = 7.9 Hz, Ph-H), 7.45 (1H, d,J = 7.7Hz, Ph-H), 7.27 (1H, d,J = 8.3 Hz, In–H),7.21 (1H, t,J = 7.8 Hz, Ph-H), 7.13 (1H, s, In–H),7.01 (1H, d,J = 2.3 Hz, In–H), 6.80 (1H,dd,J = 8.9, 2.3 Hz, In–H), 6.24 (2H, s, maleate),4.18 (2H, s, NB-CH2), 3.82 (3H, s, OCH3), 3.31–3.36(2H, m, α-CH2, overlapping with solvent), 3.11–3.16(2H, m, β-CH2).13C NMR (CD3OD): δ ppm 155.39 (In-Cq), 139.98 (Ph–CH), 139.88 (Ph–CH),136.76 (maleate), 134.98 (Ar–Cq), 133.58 (Ar–Cq), 132.03(Ph–CH), 130.31 (Ph–CH), 128.46 (Ar–Cq), 124.99(In-CH), 113.36 (In-CH), 113.08 (In-CH), 109.80 (Ar–Cq), 101.03(In-CH), 95.42 (Ar–Cq-iodine), 56.42 (OCH3), 51.41(NB-CH2), 49.1 (α-CH2), 23.37 (β-CH2). HRMS calculated for C18H20IN2O [M + H]+, 407.06148; observed [M + H]+, 407.06188.
N-(3-Methylbenzyl)-2-(5-methoxy-1H-indol-3-yl)ethan-1-amine Maleate (5j)
Obtainedas needles following crystallization from acetone/EtOAc/Et2O; yield 78%; mp 125–7 °C.1H NMR (300 MHz,CD3OD): δ ppm 7.22–7.35 (5H, m, overlapping4 x Ph-H and 1 x In–H), 7.13 (1H, s, In–H), 6.99 (1H,d,J = 2.3 Hz, In–H), 6.80 (1H, dd,J = 8.9, 2.4 Hz, In–H), 6.24 (2H, s, maleate), 4.17(2H, s, NB-CH2), 3.82 (3H, s, OCH3), 3.29–3.35(2H, m, α-CH2, overlapping with solvent), 3.10–3.15(2H, m, β-CH2), 2.36 (3H, s, CH3).13C NMR (CD3OD): δ ppm 170.90 (maleate), 155.38(In-Cq), 140.47 (Ar–Cq), 136.80 (maleate), 133.61 (Ar–Cq),132.46 (Ar–Cq), 131.49 (Ph–CH), 131.40 (Ph–CH),130.27 (Ph–CH), 128.46 (Ar–Cq), 127.93 (Ph–CH),125.00 (In-CH), 113.35 (In-CH), 113.06 (In-CH), 109.87 (Ar–Cq),101.07 (In-CH), 56.40 (OCH3), 52.24 (NB-CH2),48.9 (α-CH2), 23.37 (β-CH2), 21.36(CH3). HRMS calculated for C19H23N2O [M + H]+, 295.18049; observed [M + H]+, 295.18090.
N-(3-Methylthiobenzyl)-2-(5-methoxy-1H-indol-3-yl)ethan-1-amine Maleate (5j)
Obtained as needles following crystallization from acetone/EtOAc/Et2O; yield 80%; mp 151–2 °C.1H NMR (300MHz, CD3OD): δ ppm 7.31–7.39 (3H, m, Ph-H),7.26 (1H, d,J = 8.9 Hz, In–H), 7.19 (1H,dt,J = 7.0, 1.9 Hz, Ph-H), 7.13 (1H, s, In–H),7.00 (1H, d,J = 2.4 Hz, In–H), 6.80 (1H,dd,J = 8.9, 2.4 Hz, In–H), 6.24 (2H, s, maleate),4.19 (2H, s, NB-CH2), 3.81 (3H, s, OCH3), 3.30–3.35(2H, m, α-CH2, overlapping with solvent), 3.11–3.16(2H, m, β-CH2), 2.48 (3H, s, CH3).13C NMR (CD3OD): δ ppm 170.91 (maleate), 155.40(Ar–Cq), 141.95 (Ar–Cq), 136.78 (maleate), 133.60 (Ar–Cq),133.34 (Ar–Cq), 130.74 (Ph–CH), 128.46 (Ar–Cq),128.40 (Ph–CH), 128.30 (Ph–CH), 127.20 (Ph–CH),125.00 (In-CH), 113.36 (In-CH), 113.08 (In-CH), 109.84 (Ar–Cq),101.04 (In-CH), 56.41 (OCH3), 52.05 (NB-CH2),49.1 (α-CH2), 23.38 (β-CH2), 15.37(CH3). HRMS calculated for C19H23N2OS [M + H]+, 327.15256; observed [M + H]+, 327.15362.
N-(3-Trifluoromethylbenzyl)-2-(5-methoxy-1H-indol-3-yl)ethan-1-amine Maleate (5k)
Obtained as needles following crystallization from acetone/EtOAc/Et2O; yield 62%; mp 161–2 °C.1H NMR (300MHz, CD3OD): δ ppm 7.84 (1H, brs, Ph-H), 7.62–7.78(3H, m, Ph-H), 7.26 (1H, d,J = 8.8 Hz, In–H),7.14 (1H, s, In–H), 7.02 (1H, d,J = 2.1 Hz,In–H), 6.80 (1H, dd,J = 8.9, 2.3 Hz, In–H),6.24 (2H, s, maleate), 4.32 (2H, s, NB-CH2), 3.81 (3H,s, OCH3), 3.35–3.40 (2H, m, α-CH2), 3.13–3.18 (2H, m, β-CH2).13C NMR (CD3OD): δ ppm 170.91 (maleate), 155.41 (In-Cq),136.74 (maleate), 134.85 (Ph–CH), 134.04 (Ph–Cq), 133.60(In-Cq), 132.56 (Ph-Cq, d,J = 32.3 Hz), 131.24 (Ph–CH),128.46 (In-Cq), 127.84 (Ph–CH, q,J = 4.0Hz), 127.46 (Ph–CH, q,J = 3.8 Hz), 125.4(CF3, q,J = 272 Hz), 125.01 (In-CH),113.36 (In-CH), 113.06 (In-CH), 109.79 (In-Cq), 101.06 (In-CH), 56.39(OCH3), 51.63 (NB-CH2), 49.20 (α-CH2), 23.41 (β-CH2). HRMS calculated for C19H20F3N2O [M + H]+, 349.15222; observed [M + H]+, 349.15259
Pharmacology
ReceptorAffinity
Receptor affinity values for a panelof human serotonin receptors were obtained for all compounds throughthe NIMH-sponsored PDSP program (www.pdsp.med.unc.edu).Affinity data from screening are reported in Table1. Following the initial screen, more detailed values wereobtained for affinity at the human 5-HT2A and 5-HT2C receptors using both an antagonist radioligand ([3H]ketanserin for 5-HT2A) and ([3H]mesulerginefor 5-HT2C) and an agonist radioligand ([3H]-DOI)for both receptors. Those data are reported in Table2.
Receptor Efficacy and Potency in the Ca2+ MobilizationAssay
Changes in intracellular Ca2+ levels weremeasured using a Fluorometric Imaging plate reader (FLIPRTETRA, Molecular Devices), essentially as described in the PDSP (NIMHPsychoactive Drug Screening Program) Assay Protocol Book (www.pdsp.med.unc.edu). PO1C cells stably transfected with r5-HT2C or r5-HT2A receptors, and HEK 293 cells stably transfected with h5-HT2A, h5-HT2B, or h5-HT2C receptors wereplated (20,000 cells/well) into poly-l-lysine coated 394-wellclear-bottom black-walled microplates (Greiner Bio-one) with 50 μLof media (DMEM media supplemented with 500 μg/mL Geneticin sulfate(G-418), 10% dialyzed fetal bovine serum, and 50 U of penicillin/50μg of streptomycin) and incubated overnight (37 °C, 5%CO2). The following day, media were replaced with 20 μLof FLIPR Calcium 4 Assay Kit (Molecular Devices) diluted in assaybuffer (HBSS, 2.5 mM probenecid, and 20 mM HEPES, pH 7.4–7.8)and incubated for 45 min at 37 °C and 15 min at room temperature.Compounds were initially dissolved in DMSO. The 16-point curves wereprepared as 3× serial dilutions for each compound with finalconcentrations ranging from 10 μM to 0.003 nM. Basal fluorescencewas measured for 10 s, then 10 μL of test or control compoundswas added followed by continued fluorescence measurement for an additional120 s. Raw data were normalized to baseline fluorescence (0%) and5HT at 10 μM (100%), expressed as percent activation, and plottedas a function of molar concentration of test compound using Prism5.0 (GraphPad Software). These data are reported in Table3.
Mouse Head Twitch Response
Animals
Male C57BL/6J mice (6–8 weeks old) wereobtained from Jackson Laboratories (Bar Harbor, ME, USA) and housedin a vivarium at the University of California, San Diego, an AAALAC-approvedanimal facility that meets Federal and State requirements for thecare and treatment of laboratory animals. Mice were housed up to fourper cage in a climate-controlled room with a reversed light-cycle(lights on at 1900 h, off at 0700 h). Food and water were providedad libitum, except during behavioral testing. Testing was performedbetween 1000 and 1830 h. Experiments were conducted in accord withNIH guidelines and were approved by the UCSD animal care committee.
Procedures
The HTR was assessed using a head-mountedmagnet and a magnetometer detection coil. Mice were anesthetized (100mg/kg ketamine, 3 mg/kg acepromazine, and 20 mg/kg xylazine, IP),and a neodymium magnet (4.57 × 4.57 × 2.03 mm, 375 mg) wasattached to the skull using dental cement. The magnet was positionedso that the N–S axis was parallel to the dorsoventral planeof the head. Mice were allowed to recover for 2 weeks after surgery.HTR experiments were conducted in a well-lit room. Test compoundswere dissolved in water containing 5% Tween-80 and administered SC(5 or 10 mL/kg). Mice were injected with drug or vehicle and placedin a glass cylinder surrounded by a magnetometer coil. Head movementswere recorded and analyzed for HTR as described previously.23,31 Coil voltage was low-pass filtered (5–10 kHz), amplified,and digitized (40 kHz sampling rate) using a Powerlab/8SP with LabChartv 7.3.2 (ADInstruments, Colorado Springs, CO, USA). The data werefiltered off-line (40–200 Hz band-pass), and HTRs were identifiedby manually searching for sinusoidal wavelets possessing at leasttwo bipolar peaks, spectrum in the 40–160 Hz range, amplitudeexceeding the background noise level, and duration <0.15 s, withstable coil voltage during the period immediately before and aftereach response.
Analysis
HTR counts were analyzedusing one-way analysesof variance (ANOVAs). Post-hoc comparisons were made using Tukey’sstudentized range method. Significance was demonstrated by surpassingan α-level of 0.05. ED50 values and 95% confidencelimits were calculated using nonlinear regression. These data arereported in Table4.
Author Contributions
D.N.directedthe project, synthesized all of the compounds, supervised the integrationof the various studies, and was responsible for the writing and finalediting of the manuscript, F.S. carried out the calcium mobilizationfunctional assays, A.H. supervised the mouse head twitch assays, L.M.K.assisted with the mouse assays, S.D.B. and S.P.E. carried out theanalytical chemistry assays, and W.F. made key suggestions for theproject and edited the manuscript.
This work wassupported by the NIMH Psychoactive Drug Screening Program, NIMH K01MH100644, NIDA R01 DA002925, and the Brain and Behavior Research Foundation.
The authors declarenocompeting financial interest.
Funding Statement
National Institutes of Health, United States
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