Potential modes of interaction of 9-aminomethyl-9,10-dihydroanthracene (AMDA) derivatives with the 5-HT2A receptor: A ligand structure-affinity relationship, receptor mutagenesis and receptor modeling investigation

6808

J. Med. Chem. 2008, 51, 6808–6828

Potential Modes of Interaction of 9-Aminomethyl-9,10-dihydroanthracene (AMDA) Derivatives

with the 5-HT_2AReceptor: A Ligand Structure-Afﬁnity Relationship, Receptor Mutagenesis and

Receptor Modeling Investigation

Scott P. Runyon,^†,‡Philip D. Mosier,^‡Bryan L. Roth,^§Richard A. Glennon,^‡and Richard B. Westkaemper*^,‡

Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth UniVersity, Richmond, Virginia 23298, Department of

Pharmacology, UniVersity of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

ReceiVed June 24, 2008

The effects of 3-position substitution of 9-aminomethyl-9,10-dihydroanthracene (AMDA) on 5-HT_2Areceptor

afﬁnity were determined and compared to a parallel series of DOB-like 1-(2,5-dimethoxyphenyl)-2-

aminopropanes substituted at the 4-position. The results were interpreted within the context of 5-HT_2Areceptor

models that suggest that members of the DOB-like series can bind to the receptor in two distinct modes that

correlate with the compounds’ functional activity. Automated ligand docking and molecular dynamics suggest

that all of the AMDA derivatives, the parent of which is a 5-HT_2Aantagonist, bind in a fashion analogous

to that for the sterically demanding antagonist DOB-like compounds. The failure of the F340^6.52L mutation

to adversely affect the afﬁnity of AMDA and the 3-bromo derivative is consistent with the proposed modes

of orientation. Evaluation of ligand-receptor complex models suggest that a valine/threonine exchange

between the 5-HT_2Aand D₂receptors may be the origin of selectivity for AMDA and two substituted

derivatives.

Introduction

class of high-afﬁnity 5-HT₂agents^5-9has been described, the

parent structure of which (1a, 9-aminomethyl-9,10-dihydroan-

thracene, AMDA) is a 5-HT₂selective⁶antagonist⁸that appears

to bind with the 5-HT_2Areceptor in a fashion distinct from

classical tricyclic agents.^6,9As an anthracene derivative, AMDA

is a fairly conformationally restrained molecule. Its 9-aminom-

ethyl group is preferentially oriented in a pseudoaxial¹⁰con-

formation, and its tricyclic ring system exhibits a fold angle of

about 147°.⁵Because AMDA (1a) shares a phenylethylamine

skeleton with phenylethylamine agonists such as DOB (2b),

potential binding mode relationships between AMDA and

phenylethylamines were evaluated by exploring the effects of

substitution at the 3-position of AMDA and the effects of the

structurally analogous 4-position substitution of DOB-like

phenylethylamine derivatives upon the binding afﬁnity. Possible

modes of binding of AMDA analogues and phenylethylamines

were identiﬁed using 5-HT_2Areceptor models constructed from

the crystal structure of bovine rhodopsin. These studies have

allowed us to formulate some useful generalizations about

binding modes of agonists versus antagonists as well as to

identify potential explanations for the observed receptor selec-

tivity in the AMDA series.

Serotonin has been implicated in a large number of processes

including the regulation of sleep, appetite, mood, aggression,

perception, memory, and anxiety.¹Thirteen distinct 5-HT G

protein-coupled receptors (GPCRs^a) have evolved that are

divided into six main families.²Not surprisingly, alterations of

5-HT receptor activity have been shown to occur in many

psychiatric diseases including anxiety, depression, eating dis-

orders, schizophrenia, personality disorders, and many drug-

induced psychotic states.²Additionally, a number of effective

psychopharmacologic agents for diseases as diverse as depres-

sion, schizophrenia, and anxiety have been developed that either

speciﬁcally alter brain levels of serotonin or bind to 5-HT

receptor subtypes.^1,3Over the past few years, all of the 5-HT

receptor subtypes have been cloned and sequenced.^3,4Among

the ﬁrst to be studied were the 5-HT_2Aand 5-HT_2Creceptors,

and a signiﬁcant body of reliable information has been ac-

cumulated regarding these 5-HT₂receptors. Nevertheless, it is

still not known with certainty how serotonergic agents (or, for

that matter, how the endogenous ligand 5-HT itself) interact at

5-HT receptors. Crucial to an understanding of how serotonergic

agents act, whether agonists, partial agonists, or antagonists, is

some understanding of this drug-receptor interaction. A novel

Results and Discussion

Chemistry. The structures of the target compounds 1a-h,

3a, 3b, 3d, 4a, 4b, and 4d are shown in Table 1. Compound 1a

was prepared using a literature procedure.⁸Compounds 2a-e

and 2g have also been previously reported in the literature, and

their syntheses are discussed elsewhere.^11-13Compounds 3a

and 4a were obtained from commercial sources. Compound 4a

was purchased as the free base and subsequently converted to

the HCl salt¹⁴using ethereal HCl. Compound 3c was prepared

as previously described.¹⁵

The synthesis of compounds 1b-h was not without difﬁculty.

The initial plan was to convert 3-substituted anthrones to the

desired aminomethanes in a straightforward fashion. This route

proved unsuccessful due to the rapid isomerization of anthrone

* To whom correspondence should be addressed. Phone: (804)828-6449.

Fax: (804)828-7625. E-mail: rbwestka@vcu.edu. Address: Department of

Medicinal Chemistry, P.O. Box 980540, School of Pharmacy, Virginia

Commonwealth University, Richmond, VA 23298-0540.

†

Current address: Organic and Medicinal Chemistry, Research Triangle

Institute.

‡

Department of Medicinal Chemistry, School of Pharmacy, Virginia

Commonwealth University.

§

Department of Pharmacology, University of North Carolina School of

Medicine.

^aAbbreviations: AMDA, 9-aminomethyl-9,10-dihydroanthracene; DOB,

2,5-dimethoxy-4-bromoamphetamine; DOI, 2,5-dimethoxy-4-iodoamphet-

amine; 5-HT, serotonin; LSD, lysergic acid diethylamide; GPCR, G protein-

coupled receptor; 5-HT_2A, serotonin receptor subtype 2A; D₂, dopamine

receptor subtype 2; SERT, serotonin transporter; NET, norepinephrine

transporter; TM, transmembrane; PDB, Protein Data Bank.

10.1021/jm800771x CCC: $40.75

2008 American Chemical Society

Published on Web 10/11/2008

Interaction of AMDA DeriVatiVes with the 5-HT_2AReceptor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6809

Table 1. Effects of Aromatic Substitution on 5-HT_2AReceptor Afﬁnity

R₁

R₂

compd

K_i, nM^a

compd

K_i, nM^b

compd

K_i, nM^a

compd

K_i, nM^a

-H

1a

1b

1c

1d

1e

1f

20

1.3

3.2

7.0

7.5

23

2a

2b

2c

2d

2e

5200

41

10

2.5

1200

3a

3b

3c

3d

16800

1770

60^c

4a

4b

4610

260

-Br

-H

-(CH₂)₃Ph

-C₆H₁₃

-OCH₃

-O(CH₂)₄CH₃

-OH

-C₆H₁₃

-H

78

4d

200

-H

1g

1h

107

43

2g

>50000

-OCH₃

^a[³H]Ketanserin labeled cloned 5-HT_2Asites. Values represent the mean of computer-derived K_iestimates (using LIGAND) of quadruplicate determinations.

Standard errors typically range between 15-25% of the K_ivalue. ^b[³H]Ketanserin-labeled 5-HT_2Asites.^{13 c}[³H]Ketanserin-labeled 5-HT_2Asites.¹⁵

Scheme 1^a

^aReagents and conditions: (a) (1) 2-amino-2-methyl-1-propanol, CH₂Cl₂; (2) SOCl₂, toluene. (b) sec-Butyllithium, THF -78 °C. (c) 5% HCl 10 h. (d)

10% Pd/Charcoal, HClO₄(cat), 2-PrOH. (e) BH₃-THF. (f) PCC, CH₂Cl₂. (g) Trimethylsilyl cyanide, CH₂Cl₂. (h) LiAlH₄, THF. (i) Eaton’s reagent, PPA, or

methane sulfonic acid. (j) BBr₃, CH₂Cl₂.

to 9-anthrol under acidic and basic conditions. Under most

nucleophilic conditions, anthrone was converted to the 9-alkyl

anthracenes by dehydration of the intermediate 9-alkyl-9-

hydroxy anthracene. Treatment of anthrone with the Tebbe

reagent¹⁶did, however, provide 9-methylene-9,10-dihydroan-

thracene in modest yields. It was expected that 9-methylene-

9,10-dihydroanthracene could then be oxidized to the 9-ami-

nomethyl-9,10-dihydroanthracene with BH₃and Chloramine T.

Unfortunately, we were unable to generate large enough

quantities of the 9-methylene-9,10-dihydroanthracene for this

route to prove practical. Our focus changed following a review

of the synthetic strategy employed by the Nichols group.¹⁷The

synthetic utility of an oxazoline as an ortho lithiating agent

provided the key intermediates essential to the synthesis of

compounds 1d-h (Scheme 1). 4-Substituted phenyloxazolines

were used to provide compounds 1e and 1f. In these cases, it

was reasoned that strongly electron donating groups (methoxy

and pentyloxy) in the para position would facilitate the

Friedel-Crafts cyclodehydration reaction ((i) Scheme 1). Per-

forming the cyclodehydration reaction of the aminoalcohol

intermediates 20 with weakly electron donating substituents (R₂

) hexyl, phenylpropyl) could lead to a mixture of two

regioisomers. Separation of the resulting 1- and 3-substituted

isomers would prove difﬁcult because the free bases of

compounds 1a-h are remarkably unstable. Exposure to air and/

or aqueous conditions causes a rapid (30 min-1 h) degradation

to unknown highly colored compounds. Thus, 3-substituted

benzaldehydes were used to generate compounds 1e, 1f, and

1g.

The general synthetic approach¹⁷(Scheme 1) began with ortho

lithiation of 16 using sec-butyl lithium. The appropriate ben-

zaldehydes were then added to the lithium anion at 0 °C. The

6810 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21

Runyon et al.

Scheme 2^a

Scheme 3^a

^aReagents and conditions: (a) NBS, CCl₄; (b) AlCl₃, benzene; (c) KOH,

ethylene glycol; (d) BH₃-THF; (e) PCC, CH₂Cl₂; (f) 1; allylbenzene, 9-BBN

2; PdCl₂(dppf), NaOH, THF.

THF.²¹The synthesis of 1c from 14 was identical to that

previously described for 1d,e,f,h (Scheme 1) by conversion of

the aldehyde to the 2-amino-1-hydroxy-[2-benzyl-4-(3-n-phe-

nylpropyl)phenyl]ethanol followed by cyclodehydration using

Eaton’s reagent.

^aReagents and conditions: (a) THF, 10 °C; (b) conc HCl, MeOH; (c)

PPA, room temp; (d) SnCl₂, EtOH.

4-Bromophenylethylamine (3b) was prepared by BH₃-THF

reduction of 4-bromophenyl acetonitrile. 4-n-Hexylbenzoyl

cyanide 26 was prepared as per the method of Olah et al.²²

using SnCl₄and TMSCN. The benzoyl cyanide was reduced to

the target 4-n-hexylphenylethylamine (3d) using catalytic

hydrogenation (10% Pd/C in acetic acid).²³Friedel-Crafts

alkylation of 2-bromo-2-(4-bromophenyl)acetonitrile with ben-

zene²⁴provided the diphenyl acetonitrile 27, which was reduced

with BH₃-THF complex to provide the target 4b. Compound

4d was prepared by the reaction of 4-n-hexylbenzoyl chloride

with benzene and AlCl₃to provide 28 (4-n-hexylphenyl)(phe-

nyl)methanone. Trimethylsilyl cyanide was then employed in

the preparation of the cyanohydrin 29 followed by reduction of

the hydroxy group with NaBH₄in TFA²⁵to provide 30 (2-(4-

n-hexylphenyl)-2-phenylacetonitrile). Reduction of the nitrile

was then carried out using Raney nickel under a hydrogen

atmosphere to provide 4d in moderate yield.

Biological Evaluation/5-HT_2AReceptor Afﬁnities. Radio-

ligand binding data (5-HT_2Areceptor afﬁnities) were obtained

for each of the target compounds (Table 1). The 5-HT_2Areceptor

can accommodate a wide range of substituents associated with

the 3-position of AMDA (1a-g; Table 1). Afﬁnities varied only

about 80-fold (1b, K_i) 1.3 nM; 1g, K_i) 107 nM) within the

series. With the exception of the 3-hydroxy compound (1g, K_i

) 107 nM), monosubstitution of AMDA (1a, K_i) 20 nM) either

does not change (1f, K_i) 23 nM) or increases afﬁnity to a

maximum of 15-fold (1b, K_i) 1.3) regardless of steric bulk or

electronic character of the substituent. The effects of 4-position

substitution on the afﬁnities of 1-(2,5-dimethoxy)-2-aminopro-

panes (DOX; 2a-e) are qualitatively similar in that each of

these, with the exception of the hydroxy substituent (2g, K_i>

50000 nM), retains or enhances afﬁnity. However, in the DOX

series, the range of afﬁnity enhancement is much greater (2d,

K_i) 2.5 nM; 2a, K_i) 5200 nM) than for the AMDA series

with a maximum range of about 2000-fold, excluding the

4-hydroxy compound (2g) that shows no measurable afﬁnity.

Consistent with these observations, the lipophilic character of

the 4-position substituent of DOX has been shown to modulate

afﬁnity over a broad range.¹³These results suggest that the

AMDA and DOX series may interact differently with the

5-HT_2Areceptor. The principal structural feature distinguishing

crude reaction mixtures were subjected to acidic hydrolysis

giving rise to the lactones 17d,e,f,h in moderate overall yield.

The lactones were hydrogenated in 2-PrOH with a catalytic

amount of HClO₄to give the acids 18d,e,f,h in good yields.

The acids were then reduced with BH₃-THF and reoxidized with

PCC to give the aldehydes 19d,e,f,h in excellent yields. The

aldehydes were converted to the appropriately substituted

2-amino-1-hydroxy-(2-benzylphenyl)ethanols 20d,e,f,h using

TMSCN with a catalytic amount of ZnI₂followed by LiAlH₄

reduction in THF. The target compounds 1d-h were then

prepared through a cyclodehydration reaction using either PPA,

Eaton’s reagent, or methanesulfonic acid. Compound 1g was

prepared from the hydrobromide salt of compound 1e with BBr₃

in CHCl₃.¹⁷

An alternate route was chosen for the synthesis of 1b due to

the presence of a bromine capable of undergoing lithium

insertion (Scheme 1). The Grignard 5 reacted in a 1,4 manner

with 4-bromophenyl nitrostyrene (Scheme 2) to provide 6 by

the method of Ashwood et al.¹⁸Deprotection of the alcohol 6

with HCl in methanol followed by the PPA mediated cyclode-

hydration provided 8 in very low yield. The poor yield of the

cyclodehydration reaction can be attributed to the deactivating

nature of the bromo substituent. However, this route provided

8 in a regiochemically unambiguous manner. SnCl₂reduction

of the nitro group in 8 was chosen to eliminate any potential

halogen loss.¹⁹

Compound 14, the precursor of 1c, was prepared as shown

in Scheme 3. Compound 10 was obtained by benzylic bromi-

nation (NBS in CCl₄) of 4-bromo-2-methylbenzonitrile, followed

by Friedel-Crafts alkylation with benzene. Conversion of the

cyano group 10 to the aldehyde was carried out in a stepwise

manner using KOH in ethylene glycol,²⁰followed by reduction

of the acid with BH₃-THF and reoxidation to the aldehyde 13

with PCC in CH₂Cl₂. This method was found to be superior to

DIBAL reduction of the nitrile due to difﬁculties encountered

in the separation of the aldehyde from the starting materials

even with the use of aldehyde conjugation reagents such as

sodium hydrogen sulﬁte. A modiﬁed Suzuki coupling reaction

was employed to introduce the phenylpropyl substituent using

allylbenzene/9-BBN followed by PdCl₂(dppf) and NaOH in

Interaction of AMDA DeriVatiVes with the 5-HT_2AReceptor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6811

AMDA from other phenylethylamines is the presence of a

second, fused aromatic group. Introduction of a second nonfused

phenyl group to phenylethylamine, (i.e., 2,2-diphenylethylamine)

slightly increases afﬁnity (3a, K_i) 16800 nM; 4a, K_i) 4610

nM). Introduction of 4-substituents can enhance the afﬁnity of

phenylethylamine by about 280-fold (3a, K_i) 16800 nM; 3c,

K_i) 60 nM; Table 1) and 2,2-diphenylethylamine by about

23-fold (4a, K_i) 4610 nM; 4d, K_i) 200 nM). These increases

in afﬁnity (particularly with respect to the phenylethylamines)

are greater than the increases seen in the AMDA series (1a,

1b; 15-fold), suggesting that there are differences in the modes

of receptor interaction. Thus, it appears that DOB-like com-

pounds, AMDA derivatives, and ring-opened AMDA derivatives

(i.e., 3 and 4) behave differently with respect to their binding

at the 5-HT_2Areceptor. This is perhaps not surprising given the

fact that DOB is an agonist,¹⁵whereas AMDA is an antagonist.⁸

At the very least, even if the two series bind in a comparable

fashion, they must interact preferentially with functionally and

conformationally distinct forms of the receptor. An alternative

possibility is that the binding sites of agonists and antagonists

only share a common ammonium ion binding site with the

remaining bulk of each type of agent occupying completely

different domains within the receptor.

Receptor Complex Models. There are numerous examples

of similar compounds binding quite differently to a common

receptor as well as ligands with multiple binding modes at a

single receptor.^9,26Analysis of early 5-HT_2Areceptor models

led us to consider two general areas of steric accessibility, as

depicted in Figure 1: site 1 (TM3 ﬂanked by TM4, TM5, and

TM6) and site 2 (TM3 ﬂanked by TM1, TM2, TM6, and TM7).

The presence of two distinct binding sites for GPCRs has been

noted in the literature.²⁷Previously, consideration of ligand SAR

and receptor mutagenesis data prompted us to provisionally

consider site 1 the “agonist site” and site 2 the “antagonist

site.”^28-30Similar suggestions have also been made for the

5-HT_1Areceptor.³¹Subsequently, the method used here to select

“agonist-biased” and “antagonist-biased” receptor models from

a population of conformationally distinct receptor models

(described in detail in the Experimental Section) has identiﬁed

site 1 as an agonist binding site and site 2 as an antagonist

binding site. The selected models identiﬁed in this way are thus

referred to in this work as the agonist and antagonist receptor

models, respectively. The computational methodology used in

this work was designed to mimic the current model of

protein-ligand binding in which the ligand selects a particular

receptor conformation from an ensemble of metastable states.³²

Site 1 and site 2 overlap, and the shared region between these

sites includes residues that are a part of helices TM3 (D155^3.32

and S159^3.36) and TM6 (W336^6.48and F339^6.51).³³

Figure 1. (A) Schematic representation of sterically accessible binding

sites within the 5-HT_2Areceptor provisionally considered to be the

agonist site (site 1) and the antagonist site (site 2).^28,29(B) Connolly

channel depicting site 1 and site 2 within the 5-HT_2Areceptor model.

The GOLD-generated docking mode for AMDA is also shown (CPK

space-ﬁlling model) to highlight the complementarity between the shape

of site 2 and the fold angle of the AMDA ring system. D155^3.32is

shown for reference (ball-and-stick model).

binding region: Salom et al.³⁴have obtained a crystal structure,

at 4.15 Å resolution, of the deprotonated form of metarhodopsin

II (Meta II), the fully activated state of rhodopsin. This structure

is strikingly similar to the structure of ground-state rhodopsin

in the transmembrane and extracellular loop regions, where

ligand binding sites are located. The authors thus conclude that

“rhodopsin is a good template for homology models of other

GPCRs used in docking calculations of both agonists and

antagonists because ground-state and photoactivated rhodopsin

are structurally similar”.³⁴Other studies have proposed that a

cluster of residues on TM6 in site 1 form a molecular “toggle

switch” that is responsible for the activation of rhodopsin-like

GPCRs.³⁵This work utilizes the ground-state conformation of

bovine rhodopsin (A chain of 1U19; 2.2 Å resolution) as a

homology modeling template. However, the conformations of

the side chains (and the backbone to a lesser degree) are allowed

to vary from that of the template rhodopsin structure.

Viewed from the perspective of the ligand, in the most general

terms it is usually observed that structures of antagonists differ

from the endogenous neurotransmitters and other agonists in

that they either lack key functional groups or present molecular

features in areas of space not occupied/utilized by any portion

of the agonist (i.e., an “accessory site”), or both.³⁶For example,

while 5-methoxytryptamine is a serotonin agonist, tryptamine

is a partial agonist (see the review by Glennon, Westkaemper,

Whenever receptor homology models are generated whose

purpose is to model the interaction of an agonist with the

receptor, the accuracy of these models is called into question

(more so than for antagonist-interaction models) since, until

very recently, only inactive or ground-state rhodopsin crystal

structures were available as homology modeling templates. More

precisely, the additional requirement of an agonist to activate

or trigger the receptor is thought to involve large-scale move-

ments of at least part of the secondary structure of the receptor,

making the activated receptor’s conformation signiﬁcantly

different from the inactive state’s conformation. Currently, such

large-scale changes in conformation are not routinely incorpo-

rated into homology models. Recent crystallographic evidence,

however, suggests that the conformation of the activated form

of bovine rhodopsin does not signiﬁcantly change in the ligand

6812 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21

Runyon et al.

and Bartyzel³⁷); also, it has been shown that 2-phenyltryptamines

are high-afﬁnity 5-HT_2Areceptor antagonists.³⁸Similarly, LSD

is an agonist or partial agonist whereas 2-bromo LSD is an

antagonist.³⁹In the DOX series, compounds with small sub-

stituents at the 4-position are agonists and those with bulky

substituents such as phenylpropyl are antagonists.^13,15In the

latter case, the 2,5-dimethoxy groups of 1-(2,5-dimethoxy-4-

(3-phenylpropyl)phenyl)-2-aminopropane (2c), functional groups

characteristically required for agonist activity, are no longer

required for binding and, in fact, the desmethoxy parent 3c has

comparableafﬁnitytothe2,5-dimethoxysubstitutedderivative.^13,15

A similar observation can be made for 2d and 3d. It has been

hypothesized that phenylalkylamines with small 4-position

substituents (e.g., 2a, 2b, 2e) bind differently from those with

bulky 4-position substituents (e.g., 2c, 2d). Models of complexes

of the 5-HT_2Areceptor and DOB support the notion that there

may be limited bulk tolerance at the 4-position for some modes

of binding. Bound within site 1, substituents at the 4-position

of DOX project into the interfacial region between TM5 and

TM6 (see Figure 2A for an example). Preliminary modeling

studies have indicated that, whereas 4-methyl and 4-ethyl

substituents appear to be tolerated in the DOB-like series,

successively adding methylene units to the 4-position of 1-(2,5-

dimethoxy-4-ethylphenyl)-2-aminopropane bound to the receptor

actually causes a displacement of the of the aromatic ring (2.3

Å) from the initial site on minimization. The bound ligand 2d

is rapidly displaced from its initial site during dynamics

simulations (100 ps, 300 K, range constraint NH-OD155,

1.3-2.6 Å, helix backbone constrained) whereas DOB (2b) is

not. Another possible binding mode would place large 4-position

substituents in site 2 (Figure 1). A 5-HT_2Areceptor model with

the phenylethylamine 3d bound in site 2 did not show

displacement of the aromatic ring, and the ligand remained in

the binding site on dynamics simulation.

noethane compounds (Table 2) are either unchanged or increased

by F340L mutation (4a, K_i) 4,140 nM; 4b, K_i) 3.5 nM)

relative to the wild type (4a, K_i) 4,610 nM; 4b, K_i) 260

nM). Again, these results support the notion that, with respect

to F340, the diphenyl compounds (4a,b) behave differently from

phenylethylamines and most likely bind in a mode distinct from

that of the analogous tricyclic compounds 1a and 1b. Changes

in the side chain conformation of F340 have been previously

invoked to explain afﬁnity enhancement for some classes of

compounds with the F340L mutant.³⁰

Model Construction. In the following subsections, compu-

tationally derived 5-HT_2AGPCR models are described that

separately model the binding characteristics of selected agonists

and antagonists. “Agonist-biased” and “antagonist-biased”

receptor models were generated in the following way: Using

the MODELER software package, a population of 100 5-HT_2A

conformationally distinct receptor models derived from bovine

rhodopsin was generated. The automated docking program

GOLD was then used to separately dock both stereoisomers of

a high-afﬁnity agonist (DOB, 2b) and an antagonist (ketanserin)

into each of the 100 5-HT_2Areceptor models (Chart 1). On the

basis of the quality of the docked receptor-ligand complexes

and information from site-directed mutagenesis, one of the 100

models was selected to be the “agonist” 5-HT_2Areceptor model

and another was selected as the “antagonist” 5-HT_2Amodel

(Supporting Information Figure 1). Both stereoisomers of each

of the compounds listed in Table 1 were then docked into both

the agonist and antagonist receptor models. Docking scores and

information from mutagenesis data were then used to select the

most appropriate receptor (agonist or antagonist) for each ligand.

Agonist Receptor Complex Models. Both isomers of DOB

were found to favorably interact with the selected agonist

receptor model and in a nearly identical fashion (Supporting

Information Figure 2). The most signiﬁcant difference in the

binding modes of the stereoisomers is in the position of the

protonated amine; however, both isomers are able to form a

salt bridge with D155^3.32. The proposed binding pocket for

R(-)-DOB (R(-)-2b) is shown in Figure 2A, and residues that

can potentially interact with it are reported (Supporting Informa-

tion Table 1). The aromatic ring is associated most closely with

W336^6.48, F339^6.51, and F340^6.52. The 4-bromo substituent is

oriented toward the interfacial region between TM5 and TM6

and the 2-methoxy group of DOB accepts a hydrogen bond from

N343^6.55. The 5-methoxy group is near S159^3.36, T160^3.37, and

S242^5.46and can potentially form hydrogen bonds with these

residues to further stabilize the receptor-ligand complex.

Additionally, a lipophilic interaction occurs between the methyl

of the 5-methoxy group and W336^6.48. Other nearby aromatic

residues that can potentially interact with the aromatic ring of

DOB include F243^5.47and F340^6.52. F339^6.51is in a position to

further stabilize the ammonium-D155^3.32-S159^3.36complex

via a π-cation interaction.

The effects of N-alkylation and N-benzylation appear to

support the notion that DOB and AMDA interact with the

receptors differently. In the case of both 5-methoxytryptamine

and DOB, successive N-methylation decreased afﬁnity but

N-benzyl DOB and N-benzyl-5-methoxytryptamine have slightly

higher afﬁnities (2- to 6-fold) than their parents.⁴⁰In the AMDA

series, successive N-methylation also decreased afﬁnity but,

unlike the DOB and 5-methoxytryptamine series, N-benzylation

decreased afﬁnity (36-fold).⁶

Information from mutagenesis experiments further suggests

that AMDA and phenylalkylamines (i.e., 3) or DOX analogues

(i.e., 2) with small 4-position substituents (e.g., DOB, DOI) bind

differently, at least with respect to F340^6.52. In the current

models, the side chain of F340^6.52is at the interface between

TM5 and TM6, π-stacked with the side chain of F243^5.47(Figure

2A). Any effect that an F340 mutation might have on ligand

afﬁnity could either be due to changes in a direct, ligand-receptor

van der Waals interaction or an indirect effect caused by a

change in the shape of the helical bundle. The mutation F340L

has been shown to decrease afﬁnity of agonists but generally

has no effect on the binding of classical antagonists.⁴¹AMDA

(1a) and the bromo analogue 1b both bind to the mutant receptor

equally well (3-fold decrease and no change in afﬁnity,

respectively) compared to their afﬁnity at the wild type receptor

(Table 2). The same mutation has little effect on ketanserin

afﬁnity but essentially abolishes DOI binding (an approximately

14000-fold decrease).⁴¹This is entirely consistent with AMDA

and AMDA derivatives binding in a completely different fashion

from DOI, at least with respect to the F340^6.52position in the

receptor structure. The afﬁnities of the two 2,2-diphenylami-

Recently, it was reported⁴²that R(-)-DOB and S(+)-DOB

both have high afﬁnity for the 5-HT_2Areceptor but with the

R(-)-isomer showing a somewhat lower K_ithan the S(+)-isomer

(R(-)-DOB, K_i) 0.29 nM; S(+)-DOB, K_i) 1.9 nM).

Employing modeling techniques, the authors showed that the

R and S isomers of DOX phenylethylamines can bind in a very

similar fashion but that the orientation of S239^5.43, F240^5.44

,

F243^5.47, F244^5.48, and F340^6.52differed depending on which

isomer was docked into the receptor. Although our modeling

technique places DOB in the same location as the previous

authors, the model described here features a π-stacked interac-

tion between F340^6.52and F243^5.47. Disruption of this associa-

Interaction of AMDA DeriVatiVes with the 5-HT_2AReceptor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6813

Figure 2. The proposed binding mode of selected compounds docked and energy-minimized in the 5-HT_2Areceptor models. Carbon atoms of the ligand

are colored green. Residues whose heavy atoms fall within 4 Å of the bound ligand heavy atoms are displayed. A light-blue transparent trace indicates the

position of the receptor backbone. Hydrogen bonding interactions are indicated with a thin black line, and H-bond donor-acceptor distances (in Å) are

indicated in blue. The antagonist models are displayed from a common point of view in which TMs 6, 7 and 1 are closest to the viewer. (A) R(-)-DOB

(R(-)-2b); agonist model. (B) Ketanserin; antagonist model. (C) AMDA (1a); antagonist model. (D) (S)-3-hydroxy-AMDA (1g); antagonist model. (E)

(S)-3-phenylpropylAMDA (1c); antagonist model. (F) (R)-2c; antagonist model.

tion (either through a point mutation or via interactions with a

ligand) may alter the location and orientation of the helices

within the helix bundle. As mentioned by Parrish, et al.,⁴²the

have been shown to have highly coupled evolution as part of a

physically connected network that links distant functional sites

in the tertiary structure of GPCRs.⁴³The close association of

the small-substituent DOX ligands with F340^6.52would also

cognate residues in bovine rhodopsin, F212^5.47and A269^6.52

,

6814 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21

Runyon et al.

Table 2. Effects of the 5-HT_2AReceptor F340^6.52L Mutation on Ligand

Afﬁnity

AMDA (1a) was successfully docked into site 1 in the agonist

model with the basic amine H-bonded to both D155^3.32and

S159^3.36. One of the aromatic rings is oriented toward the cluster

K_I(nM)^a

of hydrophobic residues on TM6, interacting with I163^3.40

,

compd

1a

1b

wild type

F340L

F243^5.47, W336^6.48, and F340^6.52; the second aromatic ring of

AMDA interacts with V156^3.33, I206^4.56, and L229^xl2.52. Im-

portantly, it is shown here (Table 2) that mutation of F340^6.52

to leucine has little effect on the binding of antagonists like

AMDA (1a). In contrast, the F340^6.52L mutation had dramatic

effects on the binding of DOI, an agonist very similar to DOB

(2b). This would indicate that 1a and 2b bind differently with

20

1.3

57

1.8

4a

4b

DOI

ketanserin

4610

260

0.92

0.4

4140

3.5

13700

0.23

^aK_ivalues at the wild type receptors are from Table 1. Standard errors

typically range between 15% and 25% of the K_ivalue. [³H]Ketanserin

labeled cloned 5-HT_2Asites.

respect to F340^6.52

.

AMDA analogues with small substituents at the 3-position

(1b,e,g) are oriented in site 1 in a manner analogous to that of

AMDA. However, for 1e and 1g, there are no nearby H-bond

donors or acceptors to effectively interact with the polar

functionality at the 3-position (W336^6.48is close, but with poor

H-bond geometry). For the larger, more ﬂexible hydrophobic

analogues (1c,d,f), the 3-position substituent is either directed

toward the opening of the receptor cavity (for S-isomers) or

folds back onto the dihydroanthracene core (for R-isomers), a

characteristic that is statistically unlikely based upon an analysis

of crystal structures of ligand-receptor complexes.^57,58In the

phenylisopropylamine series, DOB analogues with small sub-

stituents at the 4-position (2a,e,g) dock into the receptor in a

similar fashion as DOB, with the aromatic ring associated with

W336^6.48, F339^6.51, and F340^6.52and the methoxy groups

interacting with S159^3.36, T160^3.40, S242^5.46, and N343^6.55. The

position of the aromatic ring for these compounds is close to

the position of the aromatic ring in AMDA most closely

associated with the aromatic cluster on TM6. As with the

AMDA analogues substituted with small polar substituents at

the 3-position, there is no H-bonding partner for the small polar

substituents at the 4-position of the DOB analogues. This is

consistent with the low observed binding afﬁnities for these

compounds. For DOB analogues 2c and 2d, the large 4-position

substituents are folded back onto the ligand’s aromatic ring (both

isomers), analogous to the AMDA analogues (R-isomer) with

large 3-position substituents. To accommodate the bulk of the

large substituent, the aromatic ring is displaced toward TM4

and the H-bonds with the methoxy groups are diminished or

eliminated. The phenylethylamine analogues 3a-d dock with

conformations that are similar to their corresponding DOB

analogues, and the diphenylmethylamine analogues 4a, 4b, and

4d dock in the receptor like their corresponding AMDA

analogues 1a, 1b, and 1d.

Chart 1. Structures of AMDA (1a), DOB (2b), and Ketanserin

explain why the F340L mutation nearly abolished⁴⁴the afﬁnity

of DOI for the 5-HT_2Areceptor. Both enantiomers of DOB and

DOI have a high afﬁnity for the 5-HT_2Areceptor⁴²and were

docked into the agonist receptor model in a nearly identical

manner, the only difference being the position of the R-methyl

group (Supporting Information Figure 2). In our agonist receptor

model, DOB occupies the same binding pocket that has been

predicted for the endogenous ligand 5-HT.^45-47

The proposed binding mode for DOB features the ligand

accepting a hydrogen bond from N343^6.55. An analysis of the

primary sequences of the known human 5-HT receptor subtypes

reveals that only 5-HT_2A, 5-HT_2B, 5-HT_2C, 5-HT₄, and 5-HT₆

receptors feature asparagine at 6.55, although other subtypes

have side chains capable of donating a hydrogen bond at this

position (6.55 ) Ser in 5-HT_1B, 5-HT_1D, and 5-HT₇). The

Psychoactive Drug Screening Program (PDSP) K_iDatabase

(http://pdsp.med.unc.edu/pdsp.php) contains entries for DOB as

the test ligand for 5-HT₁and 5-HT₂receptor subtypes. In all

cases, the afﬁnity of DOB for the 5-HT₂subtypes was much

greater (0.6 to 152 nM) than that for the 5-HT₁subtypes

(556-6327 nM). This is consistent with the hypothesis that N^6.55

contributes signiﬁcantly to the observed high afﬁnity of DOB

at the 5-HT₂subtypes. The 6.55 position has also been shown

to be important for the binding of other agonists and antagonists

at serotonergic and other closely related aminergic GPCR

subtypes.^48-53

Antagonist Receptor Complex Models. The energy-

minimized ketanserin-receptor model is depicted in Figure 2B

(nearby residues are listed in Supporting Information Table 1).

In addition to the hydrogen bond formed between the am-

monium ion in the ketanserin piperidine ring and the conserved

D155^3.32, three other hydrogen bonds are evident: S131^2.61bonds

with the p-ﬂuorobenzoyl carbonyl oxygen, S159^3.36bonds with

the N1 quinazolinedione nitrogen atom, and S373^7.46bonds with

the carbonyl oxygen at position 2 of the quinazolinedione ring

system (Figure 2B). Hydrophobic residues surrounding the

The ligand-accessible and highly conserved residues W336^6.48

and F340^6.52of the “aromatic cluster”⁵⁴in site 1 have been

proposed to be part of a rotameric “toggle switch”^35,55in which

the ꢀ₁torsion angles of these residues determine, in conjunction

with the proline kink in TM6, the proximity of the intracellular

ends of TM3 and TM6 (i.e., the “ionic lock”⁵⁶). The ꢀ₁

conformations that correspond to the “activated” receptor are

trans for both 6.48 and 6.52, which is consistent with the putative

agonist model presented here. Taken together, this information

along with the examples given above provide additional

evidence that the agonist model described here is accurate.

remainder of the ligand include W151^3.28, I152^3.29, V156^3.33

L229^xl2.52, W336^6.48, V366^7.39, W367^7.40, and Y370^7.43

,

.

To provide an indication of the correctness of the docked

solution, relevant mutagenesis binding data were collected from

the literature; these are listed in Table 3. As [³H]ketanserin is

often used as the radioligand in competitive binding assays

involving the 5-HT_2Areceptor and its mutants, K_dvalues were

frequently available. Many of the mutations listed in Table 3

Interaction of AMDA DeriVatiVes with the 5-HT_2AReceptor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6815

indirect destabilization of the binding site. The S159^3.36A and

S159^3.36C mutations were found to have almost no effect on

the afﬁnity of ketanserin. While this may seem to contradict

our proposed model because S159^3.36participates in a hydrogen

bond, the actual situation is probably more subtle. For example,

it has been shown⁵⁹that the entire quinazolinedione ring system

may be replaced with a phenylethyl fragment lacking H-bonding

capability without signiﬁcant loss of afﬁnity (less that 2-fold

decrease in K_i). If ketanserin binds as proposed, then this would

suggest that the hydrogen-bonding capability of S159^3.36is not

required and thus consistent with the mutagenesis data.

W336^6.48A was found to have one of the largest effects on

ketanserin binding (a 900-fold decrease). This is consistent with

the model because there is a substantial amount of hydrophobic

surface contact area between the quinazolinedione ring system

and the indole ring of W336^6.48. Similarly, mutation of F339^6.51

to alanine or leucine results in moderate decrease (8- to 25-

fold) in ketanserin’s binding afﬁnity due to the loss of

hydrophobic bulk in the region. The F339^6.51Y mutation

introduces a phenolic group into an area occupied by the fused

phenyl ring of the quinazolinedione moiety, resulting in a

moderate decrease in binding afﬁnity. Mutation of F340^6.52to

alanine or leucine has no signiﬁcant effect. This is also consistent

with the model because this residue is at a distant location in

site 1 and is not expected to interact with ketanserin. The effect

of the F340^6.52Y mutation is substantial, with a 70-fold decrease

in binding afﬁnity. Mutation to tyrosine at this position would

introduce a hydroxyl group into the lipid bilayer. This could

possibly facilitate the disruption of the F243^5.47-F340^6.52

interaction and the binding cavity as a result because the tyrosine

OH group would presumably prefer to be located in the more

polar interior of the receptor. The W367^7.40L mutation abolished

ketanserin binding and nearly abolished the binding of small

agonists like 5-HT and DOM that would be expected to bind

completely within site 1.⁴⁴As well as providing a site of

interaction for ketanserin, this would seem to indicate that

W367^7.40forms part of an extended site 1, as mentioned earlier.

Alternatively, W367^7.40may also interact with W76^1.34. Such

an interaction may serve to stabilize the helical bundle, at least

for the serotonin receptor subtypes, in which tryptophan is

uniformly conserved at the 7.40 position, and a hydrophobic

residue (tryptophan for the 5-HT₂subtypes) appears at the 1.34

position in all but the 5-HT_1Dreceptor (serine for 5-HT_1D).

Finally, the Y370^7.43A mutation decreases ketanserin’s afﬁnity

by nearly 20-fold. This seems reasonable, considering the

relatively close proximity of Y370^7.43to the piperidine ring of

the ligand. In summary, these results are consistent with our

proposed binding mode for the antagonist ketanserin.

Table 3. Effect of Various Mutations on the Binding Afﬁnity of

Ketanserin for 5-HT_2AMutants

mutation

W76^1.34

effect

ref

comments^a

A

10-fold V in afﬁnity

44 interacts with W367^7.40

D120^2.50

N

10-fold V in afﬁnity

105 widely conserved across

GPCRs

F125^2.55

L

S

no effect

2-fold V in afﬁnity

no effect

104 not in binding pocket

44 not in binding pocket

106 not in binding pocket

104 not in binding pocket

107 not in binding pocket

107 Inaccessible when e2 loop

is in cavity.

M132^2.62

L

no effect

T134^2.64

A

D155^3.32

A

E

N

Q

no detectable binding 108 ammonium binding site

75-fold V in afﬁnity

105 ammonium binding site

no detectable binding 108 ammonium binding site

S159^3.36

A

C

no effect

45 one turn below D155^3.32

105 conserved D/ERY motif

D172^3.49

N

W200^4.50

A

no effect

44 widely conserved; not in

binding site

S239^5.43

F240^5.44

S242^5.46

F243^5.47

F244^5.48

A

<2-fold V in afﬁnity

2-fold V in afﬁnity

∼2-fold V in afﬁnity

4.5-fold V in afﬁnity

2-fold V in afﬁnity

47 in site 1

47 not in binding pocket

109 in site 1

47 interacts with F340^6.52

47 not in binding pocket.

W336^6.48

A

900-fold V in afﬁnity

10-fold V in afﬁnity

25-fold V in afﬁnity

8-fold V in afﬁnity

25-fold V in afﬁnity

20-fold V in afﬁnity

7-fold V in afﬁnity

2-fold V in afﬁnity

no effect

2-fold V in afﬁnity

no effect

2-fold V in afﬁnity

70-fold V in afﬁnity

44 “toggle switch”; site 1/site 2.

104 one turn above W336^6.48

44 one turn above W336^6.48

106 one turn above W336^6.48

107 one turn above W336^6.48

104 one turn above W336^6.48

104 interacts with F243^5.47

44 interacts with F243^5.47

107 interacts with F243^5.47

106 interacts with F243^5.47

104 interacts with F243^5.47

F339^6.51

F340^6.52

A

L

Y

A

L

Y

F365^7.38

L

A

4-fold V in afﬁnity

no detectable binding

18-fold V in afﬁnity

3.5-fold V in afﬁnity

44 not in binding site

44 interacts with W76^1.34

44 in site 2

44 in the turn between TM7

and helix 8

W367^7.40

Y370^7.43

F383A

^aComments refer to the antagonist model described here.

involve residues that are not located in the binding crevice of

the receptor. Others involve residues that are conserved across

all GPCRs and are probably required to maintain the structural

integrity and/or basic functioning of the receptor. Several of

the mutations involve the conserved aspartate D155^3.32, and the

result of mutating this residue to something other than aspartate

is a near or complete loss of afﬁnity for ketanserin. Presumably,

this mutation would also disrupt the binding of many other small

basic amine-containing compounds, agonists and antagonists

alike. Other mutations are more relevant to the binding of

ketanserin itself. Mutation of serines S239^5.43and S242^5.46on

TM5 to alanine has no signiﬁcant effect upon the binding of

ketanserin. This is consistent with the proposed model, as

ketanserin does not approach TM5. The F243^5.47and F340^6.52

mutations each minimally decrease the binding of ketanserin.

While neither F243^5.47nor F340^6.52are within van der Waals

interaction distance with ketanserin, the small decreases in

afﬁnity at these mutated positions could be accounted for by

Our approach in selecting a receptor for antagonists involved

choosing the most highly ranked (as measured by the Chem-

Score ﬁtness function) receptor-ligand complex that exhibited

reasonable conformations for both ligand and receptor; in this

particular case, the ketanserin test ligand adopted a twist-boat

conformation. Twist-boat forms of cyclohexane are known to

have energies that are about 5.5 kcal/mol higher than the

corresponding “chair” forms;⁶⁰those for piperidine would be

expected to exhibit a similar increase in energy. At ﬁrst glance,

then, this particular docked solution for ketanserin may seem

unreasonable. However, it has been noted that in many cases,

the conformation of the bound ligand is one that may not even

be close to a local energy minimum.^57,58,61,62

Very recently, Dezi, et al.⁶³have generated a 5-HT_2Amodel

suited to the binding of butyrophenone antipsychotics using

methodology that is quite similar to that described here. In their

6816 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21

Runyon et al.

study, they present a ketanserin binding mode that is essentially

“backwards” when compared to the ketanserin model proposed

here (i.e., the p-ﬂuorobenzoyl group is oriented toward TM5

instead of toward TM2). However, the authors go on to mention

that there are likely to be multiple binding modes that contribute

to the observed afﬁnity for slender, roughly symmetric ligands

(wherein a centrally positioned cation is ﬂanked by two sets of

roughly equivalent hydrogen bonding groups) such as ketanserin

and the butyrophenones. Indeed, in our own experience, the

GOLD-derived solutions for ketanserin usually dock in either

of these two major orientations. Thus, it is possible that there

exist alternate valid docked solutions for ketanserin.

same as for the parent AMDA. For the small polar groups (1e,g),

H-bonding takes place with either T81^1.39and S131^2.61(S-

isomers) or with S159^3.36and S373^7.46(R-isomers). The top-

ranked GOLD-docked solution for (S)-1g features a hydrogen

bond to both T81^1.39and S131^2.61(Figure 2D). The more

elongated shape of site 2 relative to site 1 allows for larger

3-position substituents (1c,d,f,h) to dock in a more fully

extended conformation. For S-isomers, the substituent is directed

toward and interacts with Y370^7.43. To accommodate the large

substituent, the tricyclic core is shifted away from the e2 loop

and toward site 1. This is possible due to the basic amine’s

ready accessibility to both side chain oxygen atoms on D155^3.32

.

The docked and minimized (S)-3-phenlypropyl-AMDA (S-1c)

interaction model is depicted in Figure 2E (nearby residues are

listed in Supporting Information Table 1). The tricyclic core of

compound 1c interacts with residues on TM3 (D155^3.32and

S159^3.36) and on TM6 (M335^6.47, W336^6.48, and F339^6.51). The

bulky 3-position substituent is located in the cavity bounded

by TM1, TM2, and TM7 (site 2), with the aromatic portion

anchored at Y370^7.43. For R-isomers with large 3-position

Qualitatively, ketanserin and 3-phenylpropyl-AMDA (1c) are

docked in much the same way (Figure 2E), with the p-

ﬂuorobenzoyl group occupying nearly the same region of space

as the phenyl group of the phenylpropyl substituent of 1c, and

the quinazolinedione ring system is in roughly the same area

as the tricyclic ring system of 1c (the rings of 1c are oriented

toward TM6; the ketanserin quinazolinedione rings are oriented

toward TM5). Signiﬁcantly, all four isomers of 1c (most likely

acting as antagonists) received very high scores when docked

into the ketanserin-selected antagonist model (Supporting

Information Figure 3), indicating that both ketanserin and 1c

can recognize and engage the same receptor conformation.

substituents, the R-group is either directed toward S159^3.36

,

W336^6.48, and M365^6.47(1d,f) or the AMDA core is inverted

in an “upside-down” fashion, with the R-group interacting with

Y370^7.43as described for the S-isomers (1c).

The bis-substituted compound 1h (Table 1) was initially

evaluated in an attempt to bridge and interact simultaneously

with both sites 1 and 2. The 6-methoxy group was expected to

interact with a hydrogen bond donating residue of site 1, with

the n-hexyl group anchored in site 2. While monosubstitution

with either an n-hexyl (K_i) 7.0 nM, 1d) or a methoxy group

(K_i) 7.5 nM, 1e) enhances afﬁnity relative to AMDA (K_i)

20 nM, 1a) to a small extent, the bis-substituted compound (K_i

) 43 nM, 1h) has a lower afﬁnity than either the unsubstituted

compound (1a) or the monosubstituted derivatives (1c,d). At

the very least, the bifunctional nature of 1h does not greatly

enhance afﬁnity. The docked solution of (R)-1h (not shown) in

the antagonist model reveals that the ligand primarily occupies

site 2 and that the n-hexyl group is situated in a similar fashion

to the phenylpropyl group of (S)-1c. The methoxy group of (R)-

1h is directed toward TM6 and interacts with M335^6.47, but

there are no H-bond donors nearby with which the methoxy

oxygen can interact. For (S)-1h, the positions of the substituents

are reversed: the methoxy group H-bonds with T81^1.39and/or

S131^2.61and the n-hexyl group is associated with W336^6.48and

The docked and minimized AMDA-5-HT_2Aantagonist model

is depicted in Figure 2C (nearby residues are listed in Supporting

Information Table 1). When compared to 5-HT and traditional

phenylethylamine-derived agonists, AMDA lacks both agonist-

like functional groups (e.g., the 5-OH group of 5-HT or the

2,5-dimethoxy substituents of DOB) and presents an added

feature, the “second” aromatic ring. The shape of the binding

pocket in the antagonist receptor model exquisitely compliments

the general shape of the AMDA molecule and that of its tricyclic

core in particular (Figure 1B). The AMDA molecule is situated

in the receptor in a distinctly different location than DOB,

although AMDA and DOB do interact with common residues

on TM3 (D155^3.32) and TM6 (Y339^6.51). Effectively situated

between TM3 and TM7, AMDA binds in the receptor such that

one aromatic ring orients toward TM6 and the other is oriented

toward TM1. The ammonium group of AMDA interacts with

D155^3.32and also can interact (as suggested by molecular

dynamics experiments) with the backbone carbonyl oxygen atom

of C227^xl2.50, one of the cysteines of the disulﬁde bridge

anchoring the e2 loop to the extracellular end of TM3. The

aromatic ring that is oriented toward TM1 is sandwiched

between W151^3.28and Y370^7.43; the ring oriented toward TM6

forms π-π interactions with D155^3.32. The docked AMDA

M335^6.47

.

DOB-like isopropylamines with small substitutions at the

4-position of the phenyl ring (2a,b,e,g) are docked into the

antagonist model such that the aromatic ring is situated between

W151^3.28and Y370^7.43. Those that are nonpolar (2a,b) orient

either the 2-methoxy group toward S226^xl2.49or the 5-methoxy

group toward S77^1.35(both isomers). Those that are polar (2e,g)

orient the 4-substituent to interact with either T81^1.39or S131^2.61

(both isomers). DOB-like isopropylamines with large substit-

uents at the 4-position place the phenyl ring between D155^3.32

and V366^7.39(both isomers) as depicted in Figure 2F for

compound (R)-2c. Such compounds are stabilized in the binding

site via hydrogen bonds with D155^3.32and S159^3.36(ligand

ammonium group) as well as W151^3.28and S226^xl2.49(5-

methoxy group), alough the latter H-bonds are far from ideal.

An intramolecular hydrogen bond is also possible between the

2-methoxy group oxygen atom and the ammonium group. The

large 4-position substituent is directed toward the same pocket

in site 2 as large 3-position substituents of the AMDA analogues

and is stabilized in an analogous manner. Supporting Information

solution also forms close hydrophobic contacts with V366^7.39

,

which could have implications for the selectivity of AMDA for

5-HT_2Aover the dopaminergic D₂receptor (vide infra).

Site 2 is lined most notably with several polar residues

(T81^1.39, S131^2.61, S159^3.36, S373^7.46) and hydrophobic residues

(M128^2.58, W151^3.28, V366^7.39, Y370^7.43). The distribution of

polar and hydrophobic residues is such that an amphiphilic

cavity is created between the relatively polar faces of TM1 and

TM2 and the lipophilic face of TM7. It is possible that the

amphiphilic nature of the site is the characteristic that allows

both relatively polar (e.g., 1e,g), nonpolar (e.g., 1c,d), and mixed

(e.g., 1f) groups to bind with reasonably high afﬁnity almost

without discrimination, as described in the following paragraphs.

The 3-position substituents of AMDA are directed either

toward TM1 and TM2 (for S-isomers) or toward TM3 and TM6

(for R-isomers). The tricyclic core of the AMDA analogues with

small substituents (1b,e,g) adopts a position in site 2 that is the

Interaction of AMDA DeriVatiVes with the 5-HT_2AReceptor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6817

Table 4. Receptor and Transporter Selectivity for Compounds 1a, 1b,

and 1d

less pronounced for 1d (60- and 120-fold). Because selectivity

against the D₂receptor is not strongly inﬂuenced by the nature

of the 3-position substituent, the observed selectivity is probably

attributable to the AMDA nucleus itself. Examination of the

docked AMDA structure (Figure 2C) along with an alignment

of the 5-HT_2Aand D₂receptor sequences (Figure 3) allows the

identiﬁcation of residues that are likely responsible for the

selectivity of AMDA for the 5-HT_2A/Creceptors. Residues in

the antagonist model that possess a heavy atom within a 4.5 Å

radius of any heavy atom in AMDA are highlighted in yellow

boxes in Figure 3. Within this set of residues, one variant

position in TM1 (S77^1.35), one position in TM2 (S131^2.61), three

in TM3 (W151^3.28, I152^3.29and S159^3.36), one in the e2 loop

(S226^xl2.49), and one in TM7 (V366^7.39) that face the central

cavity of the 5-HT_2Areceptor model can be identiﬁed. It is

possible that each of these residues contribute to the selectivity

of ligands for 5-HT_2Aover D₂. The cognate residue of S77^1.35

in the 5-HT_2Areceptor is Y37^1.35in the D₂receptor. The

additional steric bulk of the tyrosine side chain (compared to

serine) would be sufﬁcient to displace AMDA from its preferred

binding site. The cognate residue of W151^3.28in the D₂receptor

is F110^3.28. Although tryptophan and phenylalanine are both

aromatic, the close association of the side chain at this position

with the aromatic ring system of AMDA (3.6 Å) would suggest

that even small differences in side chain topology could take

on greater signiﬁcance for binding. S226^xl2.49, whose equivalent

residue in D₂is E181^xl2.49, is adjacent to the disulﬁde bond

anchoring the e2 loop to the top of TM3. In this position, the

side chain of a glutamic acid residue could extend into AMDA’s

proposed binding site, placing its polar carboxyl group in

approximately the same location as one of the aromatic rings

of AMDA. Perhaps the most inﬂuential residue in determining

5-HT_2A/C/D₂selectivity is V366^7.39, which is equivalent to

position to T412^7.39of the D₂receptor, and whose side chain

heavy atoms are within 4 Å of three heavy atoms (3.54, 3.85,

and 3.93 Å) of the site 2 aromatic ring system of AMDA in the

antagonist model. As mentioned earlier, in adrenergic receptors,

the presence of an alanine or threonine instead of the asparagine

residue at this position has been shown to be responsible for

subtype selectivity within the serotonin receptor family, par-

ticularly with respect to the ability to bind ꢁ-adrenergic

antagonists such as propranolol.^72,73It is possible that placement

of a polar threonine near the AMDA aromatic ring may be

unfavorable enough to account for the lower afﬁnity of AMDA

and AMDA derivatives with the D₂receptor. This hypothesis

could be tested by evaluation of the V366T mutant of the

5-HT_2Areceptor.

K_i, nM ((SEM)

a

b

c

compd 5-HT_2A

5-HT_2C

43 (5)

D₂

SERT^d

NET^e

1a^f

1b

1d

20

1.3

7.0

>10000

1200 (160) 4490 (1080)

845 (270)

>10000

3.3 (0.4) >10000

62 (6)

6280 (820) 490 (9)

^aRadioligand [³H]ketanserin. ^bRadioligand [³H]mesulergine. ^cRadioligand

[³H]spiperone. ^dRadioligand [³H]paroxitine. ^eRadioligand [³H]nisoxitine.

^fData from ref 6.

Figure 4 shows the similarity in binding modes of 1c and 2c in

the antagonist receptor binding site. The lack of H-bonding with

the methoxy groups is consistent with the observation that they

are not needed when the 4-position is phenylpropyl.¹⁵The

phenylethylamine derivatives 3a-d dock into site 2 in the same

way as do the isopropylamine analogues 2a-d, and the

diphenylmethylamine derivatives (4a,b,d) dock similarly to their

respective AMDA derivatives.

There is very little mutagenesis data available to lend insight

into the nature of the interaction between the AMDA class of

compounds and the 5-HT_2Areceptor speciﬁcally. However,

residues that contribute to the amphiphilic nature of site 2 and

have been implicated to be important for the binding of ligands

into the antagonist model have also been implicated to be

important for ligands binding in other closely related aminergic

GPCRs. These include the residues at positions S131^{2.61 64,65}

,

W151^{3.28 65-68}Y370^{7.43 44,69-71}and V366^{7.39 68,72-78}

,

.

Once

again, when taken together, these examples provide evidence

that site 2 is accessible in the 5-HT_2Areceptor and that

the antagonist model described here is accurate.

The results of the docking experiments may be summarized

as follows: Each of the tested compounds containing a phenyl-

propyl, n-hexyl, or n-pentyloxy group exhibited signiﬁcant

binding afﬁnity (K_ie 200 nM), and it was these compounds

that tended to show the greatest preference for site 2 (antagonist

model) over site 1 (agonist model) based on the ChemScore

ﬁtness function used by the GOLD docking program (Supporting

Information Table 2). Compound 2c has been shown to have

antagonist character,¹⁵which is consistent with its preference

for the antagonist model. The relatively high afﬁnity of AMDA

derivatives with small polar substituents (1e,g) is likely due to

the stabilization of both isomers in the amphiphilic site 2. The

high-afﬁnity compounds with small nonpolar R-groups (1a,b,

2b) tended to favor the agonist model to a greater degree. This

makes sense for the agonist 2b, where there is more extensive

and effective hydrogen bonding with the methoxy groups, which

have been shown to be necessary for high afﬁnity in these

compounds.^13,15In addition, the F340^6.52L mutation has been

shown to abolish the afﬁnity of DOI, a compound closely related

to 2b (DOB). The preference of the antagonist 1a for site 1

may be an anomaly, however, because the AMDA core is in

close proximity to F340^6.52, yet the F340^6.52L mutation has little

effect on its binding afﬁnity. Similarly, 4b may actually bind

in site 1 rather that in site 2, as predicted from the ﬁtness

function. Further mutagenesis testing and functional assays will

be necessary, however, in order to unequivocally resolve the

binding modes of these compounds.

Conclusion

Previous investigations have provided evidence that phenyl-

alkylamines can be agonists or antagonists depending on the

nature of the 4-position substituent.^13,15It has been speculated

that the difference in functional behavior is a reﬂection of the

possibility that agonist and antagonist phenylalkylamines bind

in a different fashion with the 5-HT_2Areceptor. A comparison

of the effects of a parallel series of aromatic substituents based

on the tricyclic 5-HT_2Aantagonist AMDA suggests that the

AMDA series may bind in a fashion similar to that of antagonist

phenylalkylamines with bulky aromatic substituents. Differential

effects of the F340L mutation observed for the AMDA series

and phenylethylamine agonists supports this hypothesis. Auto-

mated docking studies with ligands docked into 5-HT_2Amodels

are consistent with the hypothesis that agonists bind such that

the aromatic rings are oriented toward the ﬁfth and sixth

Receptor Selectivity. As shown in Table 4, AMDA and two

of its high afﬁnity analogues are quite selective for 5-HT₂

receptors. 5-HT_2Aafﬁnity is between 900- and 7000-fold higher

than D₂receptor afﬁnity. There is little selectivity for 5-HT_2A

versus 5-HT_2Creceptors (2- to 9-fold). Selectivity for the 5-HT_2A

receptor over the serotonin and norepinephrine transporters is

pronounced for 1a and 1b (between 500- and 3000-fold) and

6818 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21

Runyon et al.

Figure 3. Alignment of the bovine rhodopsin, 5-HT_2A, and D₂receptor sequences. Sequence positions highlighted in red indicate highly conserved

amino acids among the class A GPCR family that serve as reference positions in the general Ballesteros-Weinstein¹¹⁰numbering system. Traditional

numbering is also given for the 5-HT_2A(top) and D₂(bottom) sequences. Bovine rhodopsin residues highlighted in green indicate positions that are

within 12.0 Å of bound retinal; these were mutated to alanine prior to the 3-D model building phase. Bovine rhodopsin residues highlighted in

purple indicate positions in the third intracellular loop that were mutated to glycine in the 5-HT_2Asequence and in subsequent 5-HT_2Amodels.

Residues highlighted in yellow boxes in the 5-HT_2Asequence represent those that are closest to AMDA (within 4.5 Å heavy atom to heavy atom)

in the antagonist model. Note: the D₃, M₁, V_1a, ꢁ₂, and δ-opioid GPCR sequences have been omitted for brevity and clarity (see text for details).

The ﬁgure was created using ALSCRIPT.¹¹¹

resonance (¹H NMR and ¹³C NMR) spectra were obtained with a

transmembrane helices (site 1), a region of limited bulk

Varian Gemini 300 spectrometer using tetramethylsilane as an

internal standard. Infrared spectra were recorded on a Nicolet Avatar

360 ESP FT-infrared spectrometer. Elemental analysis was per-

formed by Atlantic Microlab, Inc., and determined values are within

0.4% of theory (Supporting Information Table 3). Thin-layer

chromatography (TLC) was performed using silica gel-coated

GHLF plates (250 µm, 2.5 cm × 10 cm, Analtech, Inc., Newark,

DE). Anhydrous solvents were purchased and stored under nitrogen

over molecular sieves. Medium pressure column chromatography

was carried out using Silica gel 60, 0.040-0.063 mm, (230-400

mesh), Lancaster Synthesis.

tolerance, whereas antagonists place the substituted aromatic

ring near the seventh and toward the ﬁrst, second, and seventh

transmembrane helices (site 2) in a region of greater bulk

tolerance. AMDA and two substituted derivatives were found

have a high degree of selectivity against the D₂receptor and

the serotonin (SERT) and norepinephrine (NET) transporters

but were nonselective with respect to the 5-HT_2Creceptor.

Analysis of the putative binding modes of AMDA and related

derivatives indicate that a valine/threonine exchange between

5-HT_2A/Cand D₂receptors contributes signiﬁcantly to the

observed selectivity of these compounds.

3-Bromo-9-aminomethyl-9,10-dihydroanthracene

hydrochloride (1b). Tin(II) chloride dihydrate (1.06 g, 4.70 mmol)

was added to a well stirred solution of 3-bromo-9-nitromethyl-9,10-

dihydroanthracene (8, 0.300 g, 0.940 mmol) in absolute EtOH (3

mL). The suspension was heated at 70 °C on an oil bath (5 h). The

Experimental Methods

Synthesis.MeltingpointsweredeterminedusingaThomas-Hoover

melting point apparatus and are uncorrected. Proton magnetic

Interaction of AMDA DeriVatiVes with the 5-HT_2AReceptor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6819

resulting bright-yellow solution was allowed to cool to room

temperature and the solvent removed under reduced pressure to

provide a yellow oil. The oil was dissolved in EtOAc (10 mL), sat

NaHCO₃solution (5 mL) was added, the suspension was ﬁltered,

and the ﬁlter cake was washed with EtOAc (5 × 30 mL). The ﬁltrate

was collected, H₂O (20 mL) was added, and the mixture was

extracted with EtOAc. The EtOAc portion was washed with H₂O

(2 × 20 mL) and brine (20 mL), dried (MgSO₄), and concentrated

under reduced pressure to provide a viscous yellow oil. The resulting

yellow oil was puriﬁed using medium pressure column chroma-

tography (CH₂Cl₂/MeOH, 9:1) to provide an opaque semisolid. The

semisolid was dissolved in EtOAc (10 mL) and ethereal HCl was

added until no further precipitate formed. The resulting suspension

was ﬁltered and washed with EtOAc to provide a white solid that

was recrystallized from MeOH/CHCl₃to provide 1b (0.10 g, 33%)

129.78, 129.98, 130.83, 136.79, 137.56, 138.51, 140.08. Anal.

(C₁₆H₁₇NO·C₄H₄O₄) C, H, N.

3-n-Pentyloxy-9-aminomethyl-9,10-dihydroanthracene fumarate

(1f). 2-Amino-1-{2-[3-(n-pentyloxy)benzyl]phenyl}-1-ethanol ox-

alate (20f, 0.500 g, 1.16 mmol) was added to methanesulfonic acid

(20 mL) under N₂. The suspension was allowed to stir at room

temperature (2 h), and water (50 mL) was added. The suspension

was made basic to pH 13 with 10% NaOH and extracted with

EtOAc (3 × 50 mL). The combined extracts were washed with

water and brine, dried (MgSO₄), and concentrated to provide a

brown oil. The oil was puriﬁed using medium pressure column

chromatography (CH₂Cl₂/MeOH, 9:1) to provide a yellow oil (0.190

g, 55%). The fumarate salt was prepared by adding fumaric acid

(0.750 g, 0.650 mmol) to the amine in 2-PrOH (30 mL). The

suspension was heated until the solid dissolved, cooled, and then

ﬁltered. The yellow solid was recrystallized from EtOAc/2-PrOH

to provide 1f (0.210 g, 44%) as pale-yellow crystals; mp 178-180

°C. ¹H NMR (CD₃OD) δ 0.70-0.75 (t, J ) 7 Hz, 3H, CH₃),

1.16-1.26 (m, 4H, CH₂), 1.50-1.57 (m, 2H, CH₂), 2.84-2.87 (d,

J ) 8 Hz, 2H, CH₂-NH₂), 3.10-3.17 (m, 2H, CH₂-O), 3.64-3.70

(d, J ) 19 Hz, 1H, Ar-CH₂-Ar), 3.91-3.97 (d, J ) 19 Hz, 1H,

Ar-CH₂-Ar), 4.11-4.16 (t, J ) 8 Hz, 1H, Ar-CH-Ar), 6.49 (s,

2H, fumarate), 6.59-6.62 (d, J ) 8 Hz, 1H, Ar-H), 6.72 (s, 1H,

Ar-H), 7.04-7.23 (brm, 5H, Ar-H). ¹³C NMR (CD₃OD) δ 14.95,

19.38, 24.08, 29.96, 30.68, 36.63, 45.51, 48.71, 48.99, 50.7, 53.28,

69.62, 114.59, 115.91, 128.5, 129.98, 130.86, 136.85, 137.8, 138.75,

140.30, 160.58, 171.87. Anal. (C₂₀H₂₅NO·C₄H₄O₄) C, H, N.

3-Hydroxy-9-aminomethyl-9,10-dihydroanthracene hydrobro-

mide (1g). The hydrobromide salt of 1e was prepared by adding

ethereal HBr to 3-methoxy-9-aminomethyl-9,10-dihydroanthracene

in anhydrous Et₂O until no further precipitate formed. The

suspension was ﬁltered, and the ﬁlter cake was washed with

anhydrous Et₂O. The white powder was recrystallized from EtOAc/

2-PrOH to provide 3-methoxy-9-aminomethyl-9,10-dihydroan-

thracene hydrobromide as a white powder; mp 258-261 °C dec.

A 1.0 M solution of BBr₃in CH₂Cl₂(1.0 mL, 0.960 mmol) was

added under N₂in a dropwise manner to 3-methoxy-9-aminomethyl-

9,10-dihydroanthracene hydrobromide (0.100 g, 0.320 mmol) in

CHCl₃at -78 °C (dry ice/acetone). The suspension was allowed

to warm to room temperature over 2 h and was then allowed to

stir for 5 h. The suspension was cooled to -78 °C, anhydrous

MeOH (7 mL) was added, and the suspension was allowed to warm

to room temperature. The suspension was concentrated under

reduced pressure to provide a white solid that was recrystallized

from 2-PrOH/CHCl₃to provide 1g (0.430 g, 43%) as a white

powder; mp 278-281 °C dec. ¹H NMR (DMSO-d₆) δ 2.88-2.90

(d, J ) 6 Hz), 2H, CH₂-NH₂), 3.77-3.84 (d, J ) 19 Hz, 1H,

Ar-CH₂-Ar), 4.03-4.10 (d, J ) 19 Hz, 1H, Ar-CH₂-Ar),

4.10-4.16 (t, J ) 6 Hz, 1H, Ar-CH-Ar), 6.64-6.67 (d, J ) 8 Hz,

1H, Ar-H), 6.77 (s. 1H, Ar-H), 7.17-7.39 (brm, 7H, Ar-H).

Anal. (C₁₅H₁₅NO·HBr·0.25 H₂O) C, H, N.

1

as white crystals; mp 282-284 °C dec. H NMR (DMSO-d₆) δ

2.92-2.95 (d, J ) 8 Hz, 2H, CH₂-NH₃), 3.92-3.98 (d, J ) 19

Hz, 1H, Ar-CH₂-Ar), 4.12-4.19 (d, J ) 19 Hz, 1H, Ar-CH₂-Ar),

4.28-4.34 (t, J ) 8 Hz, 1H, Ar-CH). Anal. (C₁₅H₁₄N·HCl·0.25

H₂O) C, H, N.

3-(3-Phenylpropyl)-9-aminomethyl-9,10-dihydroanthracene fu-

marate (1c). Compound 1c was prepared from 15 in a manner

analogous to 1e. The fumarate salt was recrystallized from acetone/

CHCl₃to provide 1c (0.042 g, 13%) as pale-yellow crystals; mp

1

172-175 °C. H NMR (DMSO-d₆) δ 1.83-1.93 (m, 2H, CH₂),

2.56-2.63 (m, 4H, Ar-CH₂), 2.82-2.85 (d, J ) 8 Hz, 2H,

CH₂-NH₂), 3.83-3.89 (d, J ) 18 Hz, 1H, Ar-CH₂-Ar), 4.06-4.12

(d, J ) 18 Hz, 1H, Ar-CH₂-Ar), 4.12-4.17 (t, J ) 8 Hz, 1H,

Ar-CH-Ar), 6.48 (s, 2H, fumarate), 7.05-7.43 (brm, 12H, Ar-H).

Anal. (C₂₄H₂₅N·C₄H₄O₄·0.25 acetone) C, H, N.

3-n-Hexyl-9-aminomethyl-9,10-dihydroanthracene fumarate (1d).

2-Amino-1-(2-benzyl-4-n-hexylphenyl)-1-ethanol oxalate (20d, 0.350

g, 1.13 mmol) was added to PPA (10.0 g), and the viscous mixture

was stirred at room temperature by hand for 30 min. Water (100

mL) was slowly added, and the mixture was made basic to pH 12

with sat NaHCO₃. The aqueous solution was extracted with EtOAc

(3 × 70 mL), and the organic extracts were combined, dried

(MgSO₄), and concentrated under reduced pressure to provide a

brown oil. The oil was puriﬁed using medium pressure column

chromatography (CH₂Cl₂/MeOH, 9:1) to provide (0.100 g, 30%)

as a pale-orange oil. The oil was dissolved in anhydrous acetone

(20 mL), and anhydrous fumaric acid (0.030 g, 0.340 mmol) was

added. The mixture was heated until the solid dissolved and the

mixture was then cooled and ﬁltered to provide 1d (0.070 g, 15%)

as a white powder; mp 185-186 °C. ¹H NMR (DMSO-d₆) δ

0.85-0.86 (s, 2H, CH₃), 1.24 (brs, 6H, CH₂), 1.55-1.62 (m, 2H,

CH₂), 2.52-2.57 (t, J ) 8 Hz, 2H, CH₃), 2.83-2.85 (d, J ) 8 Hz,

2H, CH₂-NH₂), 3.82-3.88 (d, J ) 18.5 Hz, 1H, Ar-CH₂-Ar),

4.06-4.12 (d, J ) 18.5 Hz, 1H, Ar-CH₂-Ar), 4.12-4.19 (t, J )

7 Hz, 1H, Ar-CH-Ar), 6.45 (s, 2H, fumarate), 7.05-7.38 (brm,

7H, Ar-H). Anal. (C₂₁H₂₇N·C₄H₄O₄·0.25 H₂O) C, H, N.

3-Methoxy-9-aminomethyl-9,10-dihydroanthracene fumarate

(1e). 2-Amino-1-[2-(3-methoxybenzyl)phenyl]-1-ethanol oxalate

(20e, 0.130 g, 0.370 mmol) was added to a well-stirred solution of

Eaton’s reagent (20 mL) under N₂. The mixture was allowed to

stir (1 h), and water (50 mL) was added. The suspension was made

basic to pH 13 with 10% NaOH and extracted with EtOAc (3 ×

35 mL). The extracts were combined, dried (MgSO₄), and concen-

trated under reduced pressure to provide a pale-yellow oil (0.080

g, 0.330 mmol). The oil was dissolved in anhydrous 2-PrOH, and

fumaric acid (0.0420 g, 0.360 mmol) was added. The suspension

was heated until all of the solid dissolved, allowed to cool to room

temperature, and concentrated under reduced pressure. The solid

residue was then recrystallized (EtOAc/2-PrOH) to provide 1e

(0.060 g, 45%); mp 195-197 °C. ¹H NMR (CD₃OD) δ 2.79-2.82

(d/d, J ) 8 Hz, J ) 8 Hz, 2H, CH₂-NH₂), 3.58 (s, 3H, OCH₃),

3.65-3.71 (d, J ) 19 Hz, 1H, Ar-CH₂-Ar), 3.88-3.94 (d, J ) 19

Hz, 1H, Ar-CH₂-Ar), 3.98-4.2 (t, J ) 8 Hz, 1H, Ar-CH-Ar), 6.47

(s, 2H, Fumarate), 6.62-6.64 (d, J ) 8 Hz, 1H, Ar-H), 6.72 (s,

1H, Ar-H), 7.04-7.12 (brm, 5H, Ar-H). ¹³C NMR (CD₃OD) δ

36.49, 45.51, 46.08, 56.30, 114.05, 115.26, 128.46, 129.0, 129.16,

3-n-Hexyl-6-methoxy-9-aminomethyl-9,10-dihydroanthracene fu-

marate (1h). Compound 1h was prepared from 20h in a manner

analogous to that of 1f. The resulting yellow oil was puriﬁed using

medium pressure column chromatography (CH₂Cl₂/MeOH, 9:1) to

provide 1h (0.0580 g, 20%) as a white powder; mp 169-171 °C.

¹H NMR (DMSO-d₆) δ 0.85-0.86 (t, J ) 6 Hz, 3H, CH₃), 1.27

(m, 6H, CH₂), 1.53-1.58 (m, 2H, CH₂), 2.79-2.84 (d, J ) 7 Hz,

2H, CH₂-NH₂), 3.74 (s, 3H, OCH₃), 3.78-3.84 (d, J ) 19 Hz,

1H, Ar-CH₂-Ar), 4.04-4.08 (d, J ) 19 Hz, 1H, Ar-CH₂-Ar),

4.08-4.12 (t, J ) 7 Hz, 1H, Ar-CH-Ar), 6.44 (s, 2H, fumarate),

6.79-6.82 (d, J ) 8 Hz, 1H, Ar-H), 6.93 (s, 1H, Ar-H),

7.04-7.06 (d, J ) 8 Hz, 1H, Ar-H), 7.14 (s, 1H, Ar-H),

7.25-7.30 (m, 2H, Ar-H) Anal. (C₂₂H₃₀NO·C₄H₄O₄) C, H, N.

2-(4-Bromophenyl)-1-aminoethane hydrochloride (3b). A 1.0 M

solution of borane-THF complex (30.6 mL, 30.6 mmol) was added

to 4-bromophenyl acetonitrile (2.00 g, 10.2 mmol) in anhydrous

THF (10 mL). The solution was heated at reﬂux for 8 h. The

solution was allowed to cool to room temperature, 6 M HCl (10

mL) was cautiously added, and the solution was heated at reﬂux

for 30 min. The solution was allowed to cool to room temperature,

6820 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21

Runyon et al.

10% NaOH (45 mL) was added, and the suspension was extracted

with CH₂Cl₂(3 × 50 mL). The organic extracts were combined,

dried (MgSO₄), and concentrated under reduced pressure to provide

(1.50 g) of a colorless oil. The oil was dissolved in anhydrous Et₂O

(50 mL), and ethereal HCl was added until no more precipitate

formed. The suspension was ﬁltered and washed with anhydrous

Et₂O to provide a white solid that was recrystallized from 2-PrOH

to provide 3b (1.20 g, 58%) as white needles; mp 239-241 °C

manner to a well-stirred solution of 2-bromobenzyl alcohol (3.00

g, 16.0 mmol) in N,N-diisopropylethylamine (10 mL) at 0 °C under

N₂. The yellow solution was allowed to stir at 0 °C (2 h), allowed

to warm to room temperature, and stirred (5 h). The solvent was

removed under reduced pressure to provide a yellow oil. The oil

was dissolved in CHCl₃(30 mL) and washed with water (3 × 75

mL). The organic extract was dried (MgSO₄) and concentrated

under reduced pressure to provide a pale-yellow oil that was puriﬁed

using medium pressure column chromatography (petroleum ether/

acetone, 8:2) to provide 5 (2.79 g, 75%) as a colorless oil. ¹H NMR

(CDCl₃) δ 3.49 (s, 3H, CH₃), 4.65 (s, 2H, CH₂), 4.75 (s, 2H, CH₂),

7.16-7.57 (brm, 4H, Ar-H). Compound 5 was used without further

characterization in the preparation of 6.

1-[1-(4-Bromophenyl)-2-nitroethyl]-2-[(methoxymethoxy)meth-

yl]benzene (6). A crystal of I₂was added to clean, dry magnesium

turnings (0.160 g, 6.58 mmol) in anhydrous THF (10 mL).

1-Bromo-2-[(methoxymethoxy)methyl]benzene (5, 1.58 g, 6.58

mmol) was slowly added (maintaining a gentle reﬂux) to the THF/

Mg suspension, and the mixture was heated at reﬂux (≈ 45 min or

until most of the Mg was dissolved). The suspension was allowed

to cool to room temperature, the solvent was decanted from the

unreacted Mg turnings and slowly added in a dropwise manner

(not allowing the temperature to rise above 10 °C) to an ice-cold

well-stirred solution of trans-2-(4-bromophenyl)-1-nitroethene (1.50

g, 6.58 mmol) in anhydrous THF (15 mL). After the addition was

complete, the reaction was allowed to warm to room temperature

(1 h) and stirring was continued (10 h). HCl (1.0 M, 15 mL) was

added, and the suspension was concentrated under reduced pressure.

Water (25 mL) was added, and the yellow suspension was extracted

with EtOAc (3 × 20 mL) and washed with sat NaHCO₃(40 mL)

and brine (40 mL). The organic extracts were combined, dried

(MgSO₄), and concentrated under reduced pressure to provide a

viscous yellow oil. The resulting oil was puriﬁed by medium

pressure column chromatography (petroleum ether/acetone, 8:2) to

provide 6 (1.00 g, 40%) as a viscous yellow oil. ¹H NMR (CDCl₃):

δ 3.45 (s, 3H, OCH₃), 4.56-4.98 (brm, 6H, CH₂), 5.29-5.35 (t, J

) 8 Hz, 1H, Ar-CH-Ar), 7.1-7.46 (brm, 9H, Ar-H). ¹³C NMR

(CDCl₃) δ 44.07, 56.25, 68.06, 79.35, 96.32, 127.68, 128.39, 129.46,

130.05, 131.55, 132.66. IR (ﬁlm) 1556, 1376 cm^-1. Compound 6

was used in the preparation of 7.

1

dec (lit⁴⁷mp 240-243 °C). H NMR (CD₃OD) δ 2.71-2.76 (m,

2H, CH₂), 2.93-2.98 (m, 2H, CH₂), 7.00-7.03 (d, J ) 8 Hz, 2H,

Ar-H), 7.27-7.30 (d, J ) 8 Hz, 2H, Ar-H). ¹³C NMR (CD₃OD)

δ 34.43, 42.19, 132.37, 133.59.

2-(4-n-Hexylphenyl)-1-aminoethane hydrochloride (3d). Pd on

charcoal (10%, 0.50 g) and concentrated sulfuric acid (0.300 g)

were added to 26 (0.300 g, 1.40 mmol) in glacial acetic acid (5

mL). The mixture was hydrogenated (10 h) at 50 psi. The mixture

was ﬁltered through a celite pad, and the ﬁlter cake was washed

with glacial acetic acid (15 mL). The ﬁltrate was concentrated under

reduced pressure and made basic to pH 10 with sat NaHCO₃. The

suspension was extracted with EtOAc (3 × 40 mL), and the organic

extracts were combined, dried (MgSO₄), and concentrated under

reduced pressure to provide (0.210 g, 1.00 mmol) of a pale-yellow

oil. The oil was dissolved in anhydrous Et₂O (25 mL), and a 1.0

M solution of HCl in Et₂O (1 mL) was added. The solution was

cooled and ﬁltered to provide a white powder. The powder was

recrystallized from 2-PrOH/Et₂O to provide 3d (0.180 g, 53%) as

white plates; mp 176-178 °C. (lit³¹175-177 °C). ¹H NMR

(DMSO-d₆) δ 0.85-0.87 (t, J ) 8 Hz, 3H, CH₃), 1.12-1.14 (s,

6H, CH₂) 1.61-1.63 (m, 2H, CH₂), 2.49-2.54 (t, J ) 7.7 Hz, 2H,

Ar-CH₂), 2.83-2.87 (m, 2H, CH₂), 2.95-2.99 (m, 2H, CH₂), 7.13

(s, 4H, Ar-H). ¹³C NMR (DMSO-d₆) δ 14.33, 22.44, 28.71, 31.34,

31.48, 32.91, 35.13, 40.27, 128.84, 134.37, 141.02.

2-(4-Bromophenyl)-2-phenyl-1-aminoethane hydrochloride (4b).

A 1.0 M solution of borane-THF complex (7.28 mL, 7.28 mmol)

was added at room temperature to a well stirred solution of 2-(4-

bromophenyl)-2-phenylacetonitrile (27 0.500 g, 1.82 mmol) in

anhydrous THF (5 mL). The solution was then heated at reﬂux (5

h) and allowed to cool. A 6.0 M solution of HCl (7 mL) was

cautiously added, and the suspension was heated at reﬂux (30 min).

The suspension was allowed to cool, made basic with 10% NaOH

(≈35 mL), and extracted with Et₂O (3 × 35 mL). The organic

extracts were combined, dried (MgSO₄), and concentrated under

reduced pressure to provide an oily solid. The solid was dissolved

in anhydrous Et₂O (40 mL), and ethereal HCl was added. The white

suspension was ﬁltered and washed with anhydrous Et₂O (10 mL).

The white solid was recrystallized from 2-PrOH to provide 4b

(0.290 g, 51%) as white crystals; mp 216-218 °C. ¹H NMR

(DMSO-d₆) δ 3.44-3.58 (m, 2H, CH₂-NH₂), 4.37-4.42 (t, J ) 8

Hz, 1H, CH), 7.23-7.53 (brm, 9H, Ar-H). Anal. (C₁₄H₁₄BrN·HCl)

C, H, N.

{2-[1-(4-Bromophenyl)-2-nitroethyl]phenyl}methanol (7). Con-

centrated HCl (4 drops) was added to 1-[1-(4-bromophenyl)-2-

nitroethyl]-2-[(methoxymethoxy)methyl]benzene (6, 1.00 g, 2.63

mmol) in MeOH (15 mL). The reaction mixture was heated in an

oil bath at 65 °C (5 h). The solution was allowed to cool to room

temperature and concentrated under reduced pressure to provide a

viscous yellow oil. The oil was dissolved in EtOAc (20 mL), the

suspension was made basic with sat NaHCO₃(≈ 50 mL), and the

mixture was extracted with EtOAc (3 × 20 mL). The organic

extracts were combined, dried (MgSO₄), and concentrated under

reduced pressure to provide a viscous yellow oil that was puriﬁed

using medium pressure column chromatography (petroleum ether/

acetone, 8:2) to provide 7 (0.800 g, 90%) as a viscous yellow oil.

¹H NMR (CDCl₃) δ 4.64-4.92 (m, 2H, Ar-CH₂-OH), 4.93-5.06

(m, 2H, CH₂-NO₂), 5.3-5.36 (t, J ) 8 Hz, 1H, Ar-CH-Ar),

2-(4-n-Hexylphenyl)-2-phenyl-1-aminoethane hydrochloride (4d).

A methanolic solution containing Raney nickel (≈ 0.500 g) was

added to 2-(4-n-hexylphenyl)-2-phenylacetonitrile (30, 0.400 g, 1.44

mmol) in anhydrous MeOH (20 mL) and sat NH₃/MeOH solution

(5 mL). The suspension was hydrogenated at 40 psi for 10 h. The

suspension was ﬁltered through a celite pad, and the ﬁlter cake

was washed with anhydrous MeOH (25 mL). The ﬁltrate was

concentrated under reduced pressure to provide a colorless oil. The

oil was dissolved in anhydrous Et₂O (35 mL), and ethereal HCl

was added until no further precipitate formed. The white suspension

was ﬁltered and washed with anhydrous Et₂O (10 mL). The white

solid was recrystallized from 2-PrOH/Et₂O to provide 4d (0.200

g, 44%) as white crystals; mp 174-175 °C. ¹H NMR (CD₃OD) δ

0.65-0.69 (t, J ) 8 Hz, 2H, CH₃), 1.09 (s, 6H, CH₂), 1.32-1.39

(m, 2H, CH₂), 2.34-2.39 (t, J ) 7.7 Hz, 2H, CH₂), 3.38-3.41 (d,

J ) 7.8 Hz, 2H, CH₂), 4.02-4.07 (t, J ) 8.25 Hz, 1H, CH),

6.95-7.17 (brm, 9H, Ar-H). ¹³C NMR (DMSO-d₆) δ 14.51, 20.41,

30.48, 33.17, 33.21, 37.02, 45.08, 50.87, 129.33, 129.42, 130.68,

130.73. Anal. (C₂₀H₂₇N·HCl): C, H, N.

7.1-7.4 (brm, 8H, Ar-H). IR (ﬁlm) 3384, 1550, 1375 cm^-1

.

3-Bromo-9-nitromethyl-9,10-dihydroanthracene (8). PPA (5 mL)

was added to {2-[1-(4-bromophenyl)-2-nitroethyl]phenyl}methanol

(7, 1.02 g, 3.04 mmol), and the viscous mixture was stirred by

hand and heated in an oil bath at 65 °C (30 min). After the reaction

was complete, ice (40.0 g) was added to the white/brown semisolid

and the solution was made basic to pH 12 with sat NaHCO₃

solution. The suspension was extracted with EtOAc (3 × 25 mL)

and the organic extracts were combined, dried (MgSO₄), and

concentrated under reduced pressure to provide a brown semisolid

(0.540 g). The crude solid was then recrystallized from methanol

to provide 8 (0.480 g, 49%) as pale-yellow crystals; mp 113-114

°C. ¹H NMR (CDCl₃): δ 3.80-3.95 (d, J ) 19 Hz, 1H, CH₂),

4.07-4.13 (d, J ) 19 Hz, 1H, Ar-CH₂-Ar), 4.37-4.49 (m, 2H,

CH₂-NO₂) 4.76-4.82 (t, J ) 9 Hz, 1H, CH), 7.15-7.39 (brm,

7H, Ar-H). ¹³C NMR (CDCl₃) δ 35.22, 45.5, 80.15, 122.27,

1-Bromo-2-[(methoxymethoxy)methyl]benzene (5). Chlorom-

ethylmethyl ether (1.61 g, 20.0 mmol) was added in a dropwise

Interaction of AMDA DeriVatiVes with the 5-HT_2AReceptor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6821

127.85, 128.56, 128.66, 128.85, 130.24, 130.61, 131.75, 133.91,

38.62, 126.50, 126.80, 127.70, 128.97, 129.13, 129.29, 130.23,

132.49, 132.98, 141.13, 142.36, 143.60, 149.73, 192.55.

134.28, 136.05, 139.17. IR (KBr) 1537, 1382, cm^-1

.

4-Bromo-2-(bromomethyl)benzonitrile (9). NBS (3.49 g, 19.6

mmol) was added to 4-bromo-2-methylbenzonitrile (3.50 g, 17.9

mmol) in CCl₄(25 mL) under N₂. The reaction was slowly warmed

with an IR lamp and heated at reﬂux (6 h). The suspension was

cooled and ﬁltered and the ﬁltrate was concentrated under reduced

pressure to provide an oily solid. The oily solid was puriﬁed using

medium pressure column chromatography (petroleum ether/acetone

9:1) to provide a white solid that was recrystallized (toluene/

petroleum ether) to provide 9 (3.45 g, 70%) as white needles; mp

88-91 °C. ¹H NMR (CDCl₃) δ 4.55 (s, 2H, CH₂-Br), 7.54-7.58

(m, 2H, Ar-H), 7.73 (s, 1H, Ar-H). ¹³C NMR (CDCl₃) δ 28.78,

128.72, 132.90, 133.81, 134.26, 134.78, 142. 67. The product was

used in the preparation of 10.

2-Benzyl-4-bromobenzonitrile (10). 4-Bromo-2-(bromomethyl-

)benzonitrile (9, 3.25 g, 11.82 mmol) was added under N₂to AlCl₃

(3.14 g, 23.6 mmol) in anhydrous benzene (30 mL) at 0 °C. The

reaction mixture was allowed to warm to room temperature and

heated at reﬂux (45 min). The reaction was allowed to cool, poured

onto ice (50.0 g), and made acidic to pH 2 with 5% HCl. The

suspension was extracted with CH₂Cl₂(3 × 75 mL) and the

combined extracts were washed with water and brine, dried

(MgSO₄), and concentrated under reduced pressure to provide a

white solid. The white solid was recrystallized from toluene/

petroleum ether to provide 10 (3.10 g, 96%) as white crystals; mp

73-75 °C. ¹H NMR (CDCl₃) δ 4.13 (s, 2H, Ar-CH₂-Ar),

7.07-7.50 (brm, 8H, Ar-H). ¹³C NMR (CDCl₃) δ 40.56, 127.63,

129.49, 129.55, 130.88, 133.82, 134.55. Compound 10 was used

without further characterization in the preparation of 11.

2-Benzyl-4-bromobenzoic acid (11). 2-Benzyl-4-bromobenzoni-

trile (10, 0.90 g, 3.31 mmol) was added to KOH (1.15 g, 24.2 mmol)

in ethylene glycol (7 mL) and water (0.5 mL). The solution was

heated at reﬂux (3 h), allowed to cool to room temperature, and

made acidic to pH 2 with 5% HCl. The suspension was extracted

with CHCl₃(3 × 50 mL), and the extracts were washed with water

and brine, dried (MgSO₄), and concentrated under reduced pressure

to provide a white solid. The solid was recrystallized from formic

acid/acetic acid to provide 11 (0.950 g, 100%) as white crystals;

mp 131-134 °C. ¹H NMR (DMSO-d₆) δ 4.36 (s, 2H, Ar-CH2-Ar),

6.67 (s, 1H, Ar-H), 7.01-7.04 (d, J ) 8 Hz, 1H, Ar-H),

7.16-7.78 (brm, 6H, Ar-H).

2-Amino-1-[2-benzyl-4-(3-n-phenylpropyl)phenyl]-1-ethanol ox-

alate (15). Compound 15 was prepared from 14 in a manner

analogous to that of 20d. The oxalate salt was recrystallized from

2-PrOH to provide 15 (0.630 g, 64%) as a white powder; mp

1

168-170 °C. H NMR (DMSO-d₆) δ 1.78-1.88 (m, 2H, CH₂),

2.45-2.57 (m, 4H, CH₂-Ar), 2.70-2.79 (m, 2H, CH₂-NH₂),

3.92-3.97 (d, J ) 16 Hz, 1H, Ar-CH₂-Ar), 4.02-4.07 (d, J ) 16

Hz, 1H, Ar-CH₂-Ar), 5.05-5.08 (d, J ) 9 Hz, CH-OH), 6.96 (s,

1H, Ar-H), 7.15-7.53 (brm, 12H, Ar-H). Anal.

(C₂₄H₂₇NO·C₂H₂O₄) C, H, N.

2-(4-n-Hexylphenyl)-4,4-dimethyl-2-oxazoline (16d). A solution

of 4-n-hexylbenzoyl chloride (3.00 g, 13.3 mmol) in CH₂Cl₂(6

mL) was added under N₂to 2-amino-2-methyl-1-propanol (2.49 g,

27.9 mmol) in CH₂Cl₂(6 mL) at such a rate as to maintain a

temperature of 0 °C. The suspension was allowed to stir for 4 h

and was ﬁltered. The ﬁlter cake was washed with CH₂Cl₂(2 × 20

mL), and the ﬁltrate was washed with 3N HCl (100 mL) and water

(100 mL). The solvent was removed under reduced pressure to

provide (3.60 g, 97%) a yellow oil. Thionyl chloride (1.83 g, 15.31

mmol) was added slowly to the crude 4-n-hexyl-N-(2-hydroxy-1,1-

dimethylethyl) benzamide (3.60 g, 13.0 mmol) in anhydrous toluene

(25 mL). The solution was stirred at room temperature (10 h),

poured into water (30 mL), made basic to pH 10 with sat NaHCO₃,

and extracted with EtOAc (3 × 40 mL). The extracts were

combined, dried (MgSO₄), and concentrated under reduced pressure

to provide a pale-orange oil. The oil was puriﬁed by Kuhgelrohr

bulb to bulb distillation (bp 141 °C at 0.10 mmHg) to provide 16d

(3.33 g, 98%) as a colorless oil. ¹H NMR (CDCl₃) δ 0.85-0.89 (t,

J ) 7 Hz, 3H, CH₃), 1.28 (s, 6H, CH₂), 1.37 (s, 6H, CH₃),

1.53-1.62 (m, 2H, CH₂), 2.6-2.65 (t, J ) 7 Hz, 2H, Ar-CH₂),

4.09 (s, 2H, CH₂-O), 7.19-7.22 (d, J ) 8 Hz, 2H, Ar-H), 7.83

(d, J ) 8 Hz, 2H, Ar-H). ¹³C NMR (CDCl₃) δ 14.64, 23.14, 28.99,

29.43, 31.76, 32.23, 36.48, 79.59, 128.74, 128.92, 147.09. IR (ﬁlm)

1655 cm^-1

.

5-n-Hexyl-3-phenyl-1,3-dihydro-1-isobenzofuranone (17d). A

well-stirred solution of 2-(4-n-hexylphenyl)-4,4-dimethyl-2-oxazo-

line (16d, 1.00 g, 3.85 mmol) in anhydrous THF (5 mL) was cooled

to -78 °C (dry ice/acetone) under N₂. A 2.5 M solution of n-butyl

lithium in cyclohexane (1.77 mL, 4.43 mmol) was added slowly

to the solution so as to maintain the temperature at -78 °C. The

solution was allowed to warm to room temperature and stirred (1

h). The red solution was then cooled to 0 °C, and a solution of

benzaldehyde (0.410 g, 3.85 mmol) in anhydrous THF (2 mL) was

added over 30 min. The reaction mixture was allowed to stir for

5 h at room temperature, and H₂O (20 mL) was added. The mixture

was extracted with EtOAc (3 × 30 mL). The combined fractions

were dried (MgSO₄) and concentrated under reduced pressure to

provide a yellow oil. HCl (5%, 25 mL) was added to the oil, and

the suspension was heated at reﬂux (10 h). The suspension was

allowed to cool and then extracted with EtOAc (3 × 40 mL). The

combined extracts were dried (MgSO₄) and concentrated under

reduced pressure to provide an orange oil that was puriﬁed by

medium pressure column chromatography (petroleum ether/acetone,

9:1) to provide 17d (0.910 g, 80%) as a colorless oil that solidiﬁed

on standing. The solid was recrystallized from EtOAc/petroleum

ether; mp 69-70 °C. ¹H NMR (CDCl₃) δ 0.85 (s, 3H, CH₃), 1.28

(s, 6H, CH₂), 1.53-1.62 (m, 2H, CH₂), 2.6-2.65 (s, 2H, Ar-CH₂),

6.35 (s, 1H, Ar-CH-Ar), 7.26-7.86 (brm, 8H, Ar-H). IR (KBr)

2-Benzyl-4-bromobenzyl alcohol (12). Compound 12 was pre-

pared from 11 in a manner analogous to that of 21. The resulting

oil (1.01 g, 96%) was used in the next step without further

puriﬁcation.

2-Benzyl-4-bromobenzaldehyde (13). Compound 13 was pre-

pared from 12 in a manner analogous to that of 19d. The resulting

oil was puriﬁed using medium pressure column chromatography

(petroleum ether/acetone, 9:1) to provide 13 (0.750 g, 75%) as a

colorless oil. ¹H NMR (CDCl₃) δ 4.27 (s, 2H, Ar-CH₂-Ar),

7.02-7.29 (brm, 7H, Ar-H), 7.69-7.74 (d, J ) 6 Hz, 1H, Ar-H),

10.11 (s, 1H, COH).

2-Benzyl-4-(3-phenylpropyl)benzaldehyde (14). A 0.5 M solution

of 9-BBN in THF (8 mL, 4.00 mmol) was added under N₂in a

dropwise manner to a solution of allylbenzene (0.470 g, 4.01 mmol)

in anhydrous THF (2.5 mL) at 0 °C. The mixture was then allowed

to stir for 12 h at room temperature. 2-Benzyl-4-bromobenzaldehyde

(13, 1.00 g, 3.34 mmol) in THF (12 mL), PdCl₂(dppf) (0.080 g,

0.100 mmol), and NaOH (3 M, 3.34 mL) were then added to the

ﬂask containing the 9-phenylpropyl-9-BBN. The mixture was heated

at reﬂux (12 h), allowed to cool to room temperature, and water

(20 mL) was added. The suspension was extracted with EtOAc (3

× 50 mL), and the combined extracts were washed with water and

brine, dried (MgSO₄), and concentrated under reduced pressure.

The resulting brown oil was puriﬁed using medium pressure column

chromatography (petroleum ether/acetone, 9:1) to provide 14 (0.700

1743 cm^-1

.

3-(3-Methoxyphenyl)-1,3-dihydro-1-isobenzofuranone (17e). Com-

pound 17e was prepared from 3-methoxybenzaldehyde and 2-phen-

yl-4,4-dimethyl-2-oxazoline in a manner analogous to 17d. The

resulting yellow solid was recrystallized from EtOH/MeOH to

provide 17e (5.60 g, 81%) as yellow needles; mp 112-114 °C. ¹H

NMR (CDCl₃) δ 3.77 (s, 3H, OCH₃), 6.37 (s, 1H, Ar-CH-Ar),

6.78-6.91 (m, 3H, Ar-H), 7.27-7.96 (brm, 5H, Ar-H). ¹³C NMR

(CDCl₃) δ 54.89, 82.08, 111.98, 114.20, 118.65, 122.41, 125.02,

125.20, 128.94, 129.63, 133.90, 137.50, 149.16, 159.57, 170.06.

1

g, 66%) as a colorless oil. H NMR (CDCl₃) δ 1.90-2.00m (m,

2H, CH₂), 2.60-2.68 (m, 4H, Ar-CH₂), 4.40 (s, 2H, Ar-CH₂-Ar),

7.06-7.30 (brm, 12H, Ar-H), 7.76-7.79 (d, J ) 8 Hz, 1H, Ar-H),

10.17 (s, 1H, COH). ¹³C NMR (CDCl₃) δ 33.00, 35.90, 36.03,

6822 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21

Runyon et al.

3-[3-(n-Pentyloxy)phenyl]-1,3-dihydro-1-isobenzofuranone (17f).

Compound 17f was prepared from 21 (3-n-pentyloxybenzaldehyde)

and 2-phenyl-4,4-dimethyl-2-oxazoline in a manner analogous to

17d. The resulting yellow solid was recrystallized from toluene/

petroleum ether to provide 17f (4.78 g, 55%) as yellow needles;

6.73-6.78 (m, 3H, Ar-H) 7.05-7.21 (brm, 3H, Ar-H), 7.99-8.0

(d, J ) 8 Hz, 1H, Ar-H). ¹³C NMR (CDCl₃) δ 14.64, 23.14, 29.77,

31.50, 32.21, 36.43, 40.22, 55.63, 111.83, 115.32, 122.01, 126.23,

127.06, 129.75, 132.55, 132.60, 143.16, 143.84, 149.23, 160.00,

173.44.

1

mp 94-96 °C. H NMR (CDCl₃) δ 0.89-0.94 (t, J ) 7 Hz, 3H,

2-Benzyl-4-n-hexylbenzaldehyde (19d). A solution of 2-benzyl-

4-n-hexylbenzyl alcohol (21, 0.400 g, 1.42 mmol) in anhydrous

CH₂Cl₂(5 mL) was added over 30 min at room temperature to a

well-stirred suspension of PCC (0.460 g, 2.13 mmol) and celite

(1.0 g in 50 mL CH₂Cl₂). The solution was allowed to stir at room

temperature for 2 h, anhydrous Et₂O (25 mL) was added, and the

dark-brown suspension was ﬁltered through a Florisil column. The

solution was concentrated under reduced pressure to provide 19d

(0.320 g, 80%) as a colorless oil. The product was used without

CH₃), 1.3-1.46 (m, 4H, CH₂), 1.70-1.79 (m, 2H, CH₂), 3.88-3.93

(t, J ) 7 Hz, 3H, CH₂-O), 6.36 (s, 1H, Ar-CH-Ar), 6.77 (s, 1H,

Ar-H), 6.85-6.90 (m, 2H, Ar-H) 7.25-7.67 (brm, 4H, Ar-H),

7.94-7.96 (d, J ) 8 Hz, 1H, Ar-H). ¹³C NMR (CDCl₃) δ 14.56,

23.0, 28.71, 29.45, 68.63, 83.15, 113.55, 115.65, 119.46, 123.42,

126.04, 126.19, 129.91, 130.57, 134.88, 138.42, 150.23, 160.16.

5-n-Hexyl-3-(3-methoxyphenyl)-1,3-dihydro-1-isobenzofura-

none (17h). Compound 17h was prepared from 16d (2-(4-n-

hexylphenyl)-4,4-dimethyl-2-oxazoline) and 3-methoxybenzalde-

hyde in a manner analogous to that of 17d. The resulting orange

oil was puriﬁed using medium pressure column chromatography

(petroleum ether/acetone, 9:1) to provide 17h (4.82 g, 77%) as a

yellow oil. ¹H NMR (CDCl₃) δ 0.83-0.87 (t, J ) 6 Hz, 3H, CH₃),

1.28 (m, 6H, CH₂), 1.57-1.60 (m, 2H, CH₂), 2.64-2.69 (t, J ) 8

Hz, 2H, CH₂-Ar), 3.77 (s, 3H, OCH₃), 6.31 (s, 1H, Ar-CH-Ar),

6.79-6.90 (m, 3H, Ar-H), 7.12 (s, 1H, Ar-H), 7.27-7.36 (m,

2H, Ar-H), 7.82-7.85 (d, J ) 8 Hz, 1H, Ar-H). ¹³C NMR

(CDCl₃) δ 14.59, 23.08, 29.44, 31.74, 32.12, 36.93, 55.88, 82.91,

112.99, 115.14, 119.73, 122.99, 123.68, 126.00, 130.52, 130.58,

1

further puriﬁcation. H NMR (CDCl₃) δ 0.85-0.87 (t, J ) 7 Hz,

2H, CH₃), 1.28 (brs, 6H, CH₂), 1.55-1.60 (m, 2H, CH₂), 2.60-2.65

(t, J ) 8 Hz, 2H, CH₃), 4.14 (s, 2H, Ar-CH₂-Ar), 7.07-7.29 (brm,

7H, Ar-H), 7.75-7.78 (d, J ) 8 Hz, 1H, Ar-H), 10.17 (s, 1H,

COH). ¹³C NMR (CDCl₃) δ 14.65, 23.15, 29.50, 31.52, 32.21,

36.66, 38.65, 126.76, 127.68, 129.08, 129.28, 132.47, 132.93,

140.85, 143.48, 150.55, 192.59. IR (ﬁlm) 1693 cm^-1

.

2-(3-Methoxybenzyl)benzaldehyde (19e). Compound 19e was

prepared from 23 in a manner analogous to that of 19d. The

resulting oil was puriﬁed using medium pressure column chroma-

tography (petroleum ether/acetone, 8:2) to provide 19e (0.490 g,

98%) as a pale-yellow oil. ¹H NMR (CDCl₃) δ 3.75 (s, 3H, OCH₃),

4.42 (s, 2H, Ar-CH₂-Ar), 6.68 (s, 1H, Ar-H), 6.72-6.75 (d, J )

8.2 Hz, 1H, Ar-H), 7.17-7.53 (brm, 5H, Ar-H), 7.84-7.85 (d,

J ) 3 Hz, 1H, Ar-H), 10.24 (s, 1H, COH). ¹³C NMR (CDCl₃)

38.59, 55.69, 111.96, 115.34, 121.77, 127.60, 130.10, 132.21,

138.78, 150.69, 151.24, 160.62. IR (ﬁlm) 1772 cm^-1

.

2-Benzyl-4-n-hexylbenzoic acid (18d). Five drops of HClO₄(70%

in water), 10% Pd/C (0.100 g), and 5-n-hexyl-3-phenyl-1,3-dihydro-

1-isobenzofuranone 17d (0.400 g, 1.36 mmol) was hydrogenated

in 2-PrOH (11 mL) at 55 psi for 12 h. The suspension was ﬁltered

through a celite pad and concentrated under reduced pressure. The

resulting oil was dissolved in CHCl₃(50 mL) and extracted with

10% NaOH (40 mL). The aqueous layer was made acidic to pH 3

with 5% HCl and extracted with CH₃Cl (3 × 50 mL). The organic

extracts were combined, dried (MgSO₄), and concentrated under

reduced pressure to provide a white solid. The solid was recrystal-

lized from formic acid/acetic acid to provide 18d (0.300 g, 74%)

132.56, 134.50, 192.95. IR (ﬁlm) 1699 cm^-1

.

2-[3-(n-Pentyloxy)benzyl]benzaldehyde (19f). Compound 19f was

prepared from 24 in a manner analogous to that of 19d. The

resulting oil was puriﬁed using medium pressure column chroma-

tography (petroleum ether/acetone, 8:2) to provide 19f (3.56 g,

100%) as a pale-yellow oil. ¹H NMR (CDCl₃) δ 0.85-0.93 (t, J )

7 Hz, 3H, CH₃), 1.32-1.42 (m, 4H, CH₂), 1.69-1.76 (m, 2H, CH₂),

3.86-3.90 (t, J ) 7 Hz, 2H, CH₂-O), 4.40 (s, 2H, Ar-CH₂-Ar),

6.68-6.73 (m, 3H, Ar-H), 7.14-7.54 (brm, 4H, Ar-H), 7.83-7.87

(d, J ) 8 Hz, 1H, Ar-H), 10.24 (s, 1H, COH).

1

as white needles; mp 67-68 °C. H NMR (CDCl₃) δ 0.84-0.89

(t, J ) 7 Hz, 2H, CH₃), 1.27 (brs, 6H, CH₂), 1.53-1.58 (m, 2H,

CH₂), 2.56-2.61 (t, J ) 6 Hz, 2H, CH₃), 4.43 (s, 2H, Ar-CH₂-Ar),

7.03-7.28 (brm, 7H, Ar-H), 7.98-8.01 (d, J ) 8 Hz,1H, Ar-H).

¹³C NMR (CDCl₃) δ 14.65, 23.15, 29.47, 31.51, 32.20, 36.43,

40.23, 126.42, 127.04, 128.84, 129.52, 132.56, 132.65, 149.22,

4-n-Hexyl-2-(3-methoxybenzyl)benzaldehyde (19h). Compound

19h was prepared from 25 in manner analogous to that of 19d.

The resulting brown oil was puriﬁed using medium pressure column

chromatography (petroleum ether/acetone, 9:1) to provide 19h (1.98

173.23. IR (KBr) 2924, 1687 cm^-1

.

1

g, 92%) as a pale-yellow oil. H NMR (CDCl₃) δ 0.85-0.89 (t, J

2-(3-Methoxybenzyl)benzoic acid (18e). Compound 18e was

prepared from 17e in a manner analogous to 18d. The resulting

white solid was recrystallized from formic acid/acetic acid to

) 6 Hz, 3H, CH₃), 1.26-1.29 (m, 6H, CH₂), 1.58-1.63 (m, 2H,

CH₂), 2.59-2.65 (t, J ) 8 Hz, 2H, CH₂-Ar), 3.75 (s, 3H, OCH₃),

4.39 (s, 2H, Ar-CH₂-Ar), 6.68-6.74 (m, 3H, Ar-H), 7.07-7.23

(m, 3H, Ar-H), 7.75-7.78 (d, J ) 8 Hz, 1H, Ar-H), 10.17 (s,

1H, COH). ¹³C NMR (CDCl₃) δ 14.62, 23.12, 29.48, 31.50, 32.20,

36.64, 38.62, 55.66, 111.89, 115.25, 121.70, 127.69, 130.03, 132.44,

1

provide 18e (1.81 g, 89%) as white needles; mp 95-96 °C. H

NMR (CDCl₃) δ 3.77 (s, 3H, OCH₃), 4.46 (s, 2H, Ar-CH₂-Ar),

6.75-6.77 (d, J ) 7 Hz, 1H, Ar-H), 6.8 (s, 1H, Ar-H), 7.18-7.5

(brm, 5H, Ar-H), 8.08-8.10 (d, J ) 7.8 Hz, 1H, Ar-H). ¹³C

NMR (CDCl₃) 40.19, 55.67, 111.92, 115.48, 122.13, 126.97,

129.87, 132.29, 133.62, 142.89, 143.89, 173.79.

132.87, 142.68, 143.27, 150.41, 160.0, 192.54. IR (ﬁlm) 1700 cm^-1

.

2-Amino-1-(2-benzyl-4-n-hexylphenyl)-1-ethanol oxalate (20d).

Trimethylsilyl cyanide (0.270 g, 2.78 mmol) was added to a

suspension of 2-benzyl-4-n-hexylbenzaldehyde (19d, 0.650 g, 2.32

mmol) and ZnI₂(catalytic amount) in anhydrous CH₂Cl₂(3 mL).

The solution was allowed to stir at room temperature (3 h) and

heated at reﬂux (1 h). The solution was allowed to cool to room

temperature and concentrated under reduced pressure to give a pale-

yellow oil. The oil in anhydrous THF (5 mL) was added under N₂

to a suspension of LiAlH₄(0.260 g, 6.85 mmol) in anhydrous THF

(10 mL) at 0 °C. The solution was warmed to room temperature

and heated at reﬂux (5 h). The solution was allowed to cool to

room temperature, and water, (0.25 mL), 10% NaOH (0.25 mL),

and celite (1.5 g) were added. The suspension was ﬁltered through

a sintered glass ﬁlter, and the ﬁlter cake was washed with CH₂Cl₂

(75 mL). The ﬁltrate was dried (MgSO₄) and concentrated under

reduced pressure to provide (0.460 g) a colorless oil. The oil was

dissolved in anhydrous acetone (20 mL), and oxalic acid (0.130 g,

1.38 mmol) was added. The solution was heated until the solid

dissolved, and the solution was cooled and ﬁltered to provide 20d

2-[3-(n-Pentyloxy)benzyl]benzoic acid (18f). Compound 18f was

prepared from 17f in a manner analogous to that of 18d. The

resulting white solid was recrystallized from formic acid/acetic acid

1

to provide 18f (4.18 g, 96%) as white needles; mp 89-91 °C. H

NMR (DMSO-d₆) δ 0.85-0.89 (t, J ) 6 Hz, 3H, CH₃), 1.27-1.33

(m, 4H, CH₂), 1.61-1.68 (m, 2H, CH₂), 3.84-3.88 (t, J ) 6 Hz,

2H, CH₂-O), 4.30 (s, 2H, Ar-CH₂-Ar), 6.69 (m, 2H, Ar-H), 6.71

(s, 1H, Ar-H), 7.11-7.48 (brm, 4H, Ar-H), 7.79-7.82 (d, J ) 8

Hz, 1H, Ar-H). ¹³C NMR (DMSO-d₆) 14.26, 22.26, 28.09, 28.76,

38.74, 67.53, 111.83, 115.44, 121.18, 126.65, 129.59, 130.57,

131.06, 131.70, 132.07, 141.80, 143.03, 159.03, 169.22.

4-n-Hexyl-2-(3-methoxybenzyl)benzoic acid (18h). Compound

18h was prepared from 17h in a manner analogous to that of 18d.

The resulting yellow semisolid was recrystallized from formic acid/

acetic acid to provide 18h (2.21 g, 84%) as a white powder; mp

58-60 °C. ¹H NMR (CDCl₃) δ 0.86-0.91 (t, J ) 6 Hz, 3H, CH₃),

1.29 (m, 6H, CH₂), 1.55-1.62 (m, 2H, CH₂), 2.58-2.63 (t, J ) 8

Hz, 2H, CH₂-Ar), 3.76 (s, 3H, OCH₃), 4.43, (s, 2H, Ar-CH₂-Ar),

Interaction of AMDA DeriVatiVes with the 5-HT_2AReceptor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6823

(0.420 g, 45%) as white crystals; mp 164-166 °C. ¹H NMR

(DMSO-d₆) δ 0.85-0.87 (s, 2H, CH₃), 1.28 (brs, 6H, CH₂),

1.55-1.60 (brs, 2H, CH₂), 2.48-2.53 (t, J ) 6 Hz, 2H, CH₃),

2.69-2.78 (m, 2H, CH₂-NH₂), 3.91-3.96 (d, J ) 16 Hz, 1H, Ar-

CH₂-Ar), 4.00-4.06 (d, J ) 16 Hz, 1H, Ar-CH₂-Ar), 5.03-5.06

(d, J ) 8.5 Hz, 1H, CH-OH), 6.97-7.44 (brm, 8H, Ar-H). ¹³C

NMR (DMSO-d₆) δ 14.67, 23.20, 29.60, 31.93, 32.27, 36.14, 38.79,

46.38, 67.06, 126.55, 126.91, 127.67, 129.01, 129.46, 131.11,

137.95, 136.09, 141.01, 143.45.

2-Amino-1-[2-(3-methoxybenzyl)phenyl]-1-ethanol oxalate (20e).

Compound 20e was prepared from 19e in a manner analogous to

20d. The oxalate salt was prepared and recrystallized from 2-PrOH

to provide 20e (0.130 g, 33%) as a white powder; mp 159-161

°C. ¹H NMR (CD₃OD) δ 2.54-2.62 (m, 2H, CH₂-NH₂), 3.51 (s,

3H, OCH₃), 3.86 (s, 2H, Ar-CH₂-Ar), 4.93-4.98 (d, J ) 9 Hz,

1H, Ar-CH-OH), 6.47-6.54 (m, 2H, Ar-H), 6.94-7.10 (brm, 5H,

Ar-H), 7.38-7.40 (m, 1H, Ar-H). ¹³C NMR (CD₃OD) δ 39.65,

47.19, 56.11, 67.91, 113.02, 116.15, 122.61, 127.99, 128.75, 129.77,

131.13, 132.50, 139.18, 141.31, 144.10, 166.95.

2-Amino-1-{2-[3-(n-pentyloxy)benzyl]phenyl}-1-ethanol oxalate

(20f). Compound 20f was prepared from 19f in a manner analogous

to that of 20d. The oxalate salt was prepared and recrystallized

from 2-PrOH to provide 20f (2.68 g, 51%) as a white powder; mp

140-142 °C. ¹H NMR (DMSO-d₆) δ 0.85-0.90 (t, J ) 7 Hz, 3H,

CH₃), 1.13-1.18 (m, 2H, CH₂), 1.28-1.34 (m, 2H, CH₂), 2.7-2.84

(m, 2H, CH₂-NH₂), 3.83-3.92 (t, J ) 7 Hz, 2H, CH₂-O),

3.93-3.98 (d, J ) 16 Hz, 1H, Ar-CH₂-Ar), 4.02-4.07 (s, J ) 16

Hz, 1H, Ar-CH₂-Ar), 5.2-5.23 (d, J ) 8 Hz, 1H, CH-OH),

6.66-6.78 (m, 3H, Ar-H), 7.14-7.35 (brm, 4H, Ar-H), 7.53-7.67

(d, J ) 8 Hz, 1H, Ar-H).

reduced pressure to provide 22 (0.450 g, 79%) as a colorless oil.

The product was used without further puriﬁcation. ¹H NMR

(CDCl₃) δ 0.85-0.89 (t, J ) 6 Hz, 2H, CH₃), 1.28 (brs, 6H, CH₂),

1.53-1.60 (m, 2H, CH₂), 2.54-2.59 (t, J ) 8 Hz, 2H, CH₃), 4.06

(s, 2H, CH₂), 4.58 (s, 2H, CH₂), 7.0-7.30 (brm, 8H, Ar-H). ¹³C

NMR (CDCl₃) δ 14.69, 23.20, 29.56, 32.03, 32.29, 36.19, 39.16,

63.72, 126.66, 127.37, 129.08, 129.17, 129.28, 131.48, 136.71,

138.52, 141.12, 143.42. IR (ﬁlm) 3334 cm^-1

.

2-(3-Methoxybenzyl)benzyl alcohol (23). Compound 23 was

prepared from 18e in a manner analogous to that of 22. The resulting

oil was puriﬁed using medium pressure column chromatography

(petroleum ether/acetone, 8:2) to provide 23 (0.540 g, 75%) as a

colorless oil. ¹H NMR (CDCl₃) δ 3.76 (s, 3H, OCH₃), 4.07 (s, 2H,

Ar-CH₂-OH), 4.65 (s, 2H, Ar-CH₂-Ar), 6.7-6.76 (brm, 2H,

Ar-H), 7.18-7.41 (brm, 6H, Ar-H). ¹³C NMR (CDCl₃) 39.06,

55.70, 63.79, 111.87, 115.21, 121.66, 127.44, 128.57, 128.96,

130.08, 131.15, 138.94, 139.41, 142.77, 159.88. IR (ﬁlm) 3340

cm^-1

.

2-[3-(n-Pentyloxy)benzyl]benzyl alcohol (24). Compound 24 was

prepared from 18f in a manner analogous to that of 22. The resulting

oil was puriﬁed using medium pressure column chromatography

(petroleum ether/acetone, 8:2) to provide 24 (3.58 g, 100%) as a

1

colorless oil. H NMR (CDCl₃) δ 0.89-0.93 (t, J ) 7 Hz, 3H,

CH₃), 1.33-1.43 (m, 4H, CH₂), 1.56 (brs, 1H, OH), 1.70-1.76

(m, 2H, CH₂), 3.86-3.90 (t, J ) 7 Hz, 2H, CH₂-O), 4.04 (s, 2H,

Ar-CH₂-Ar), 4.64 (s, 2H, CH₂-OH), 6.68-6.71 (m, 2H, Ar-H),

6.74 (s, 1H, Ar-H), 7.14-7.28 (brm, 4H, Ar-H), 7.39-7.40 (m,

1H, Ar-H). ¹³C NMR (CDCl₃) δ 14.59, 23.05, 28.77, 29.55, 39.11,

63.81, 68.42, 112.46, 115.76, 121.43, 127.41, 128.55, 128.95,

130.02, 131.17, 139.14, 139.24, 142.68, 159.51.

4-n-Hexyl-2-(3-methoxybenzyl)benzyl alcohol (25). Compound

25 was prepared from 18h in a manner analogous to that of 22.

The resulting oil was puriﬁed using medium pressure column

chromatography (petroleum ether/acetone, 8:2) to provide 25 (2.21

2-Amino-1-[4-n-hexyl-2-(3-methoxybenzyl)phenyl]-1-ethanol fu-

marate (20h). Compound 20h was prepared from 19h in a manner

in a manner analogous to that of 20d. The fumarate salt was

recrystallized from EtOAc/2-PrOH to provide 20h (0.510 g, 46%)

as a white powder; mp 162-164 °C. ¹H NMR (DMSO-d₆) δ

0.81-0.85 (t, J ) 6 Hz, 3H, CH₃), 1.24 (m, 6H, CH₂), 1.49-1.53

(m, 2H, CH₂), 2.64-2.83 (m, 2H, CH₂-NH₂), 3.69 (s, 3H, OCH₃),

3.86-3.91 (d, J ) 16 Hz, 1H, Ar-CH₂-Ar), 3.98-4.03 (d, J ) 16

Hz, 1H, Ar-CH₂-Ar), 5.05-5.08 (d, J ) 10 Hz, 1H, CH-OH),

6.46 (s, 2H, fumarate), 6.72-6.74 (m, 3H, Ar-H), 6.96 (s, 1H,

Ar-H), 7.06-7.19 (brm, 2H, Ar-H), 7.41-7.43 (d, J ) 8 Hz,

1H, Ar-H). ¹³C NMR (DMSO-d₆) δ 14.31, 22.43, 28.60, 31.25,

31.46, 35.10, 37.46, 45.97, 55.22, 66.21, 111.59, 114.80, 121.23,

126.54, 126.87, 129.66, 130.54, 135.68, 137.21, 138.04, 141.82,

142.62, 159.66, 168.84.

1

g, 92%) as a colorless oil. H NMR (CDCl₃) δ 0.85-0.89 (t, J )

6 Hz, 3H, CH₃), 1.26-1.29 (m, 6H, CH₂), 1.53-1.60 (m, 2H, CH₂),

2.54-2.59 (t, J ) 8 Hz, 2H, CH₂-Ar), 3.76 (s, 3H, OCH₃), 4.03

(s, 2H, Ar-CH₂-Ar), 4.52 (s, 2H, CH₂-OH), 6.68-6.74 (m, 3H,

Ar-H), 7.00-7.30 (brm, 4H, Ar-H). ¹³C NMR (CDCl₃) δ 14.62,

23.18, 29.54, 32.01, 32.28, 36.17, 39.16, 55.66, 63.75, 111.82,

115.10, 121.10, 127.41, 129.30, 130.02, 131.47, 136.70, 138.79,

143.03, 143.44. IR (ﬁlm) 3365 cm^-1

.

4-n-Hexyl-benzoylcyanide (26). Trimethysilyl cyanide (0.62 mL,

4.67 mmol) was slowly added under N₂to a well stirred solution

of 4-n-hexylbenzoyl chloride (1.0 g, 4.45 mmol) in anhydrous

CH₂Cl₂(10 mL). Tin(IV) chloride (0.10 mL, 0.850 mmol) was

added over 30 min to the solution at room temperature and the

mixture was allowed to stir for 2.5 h, during which time the solution

gradually turned from yellow to a dark brown. Ice-cold water (30

mL) was added, and the mixture was extracted with CH₂Cl₂(3 ×

30 mL). The organic extracts were combined, dried (MgSO₄), and

concentrated under reduced pressure to provide a brown oil. The

oil was puriﬁed using medium pressure column chromatography

(petroleum ether/acetone, 9.5:0.5) to provide 26 (0.820 g, 85%) as

3-n-Pentyloxybenzaldehyde (21). KOH (3.25 g, 49.1 mmol) was

added to 3-hydroxybenzaldehyde (5.00 g, 40.9 mmol) in absolute

EtOH (125 mL). The mixture was allowed to stir for 30 min at

room temperature, and 1-bromopentane (6.80 g, 45.0 mmol) was

added. The reaction mixture was heated at reﬂux (12 h), the

suspension was allowed to cool to room temperature, water (75

mL) was added, and the solution was extracted with Et₂O (3 × 75

mL). The combined extracts were washed with water and brine,

dried (MgSO₄), and concentrated under reduced pressure to provide

a pale-orange oil. The oil was puriﬁed using medium pressure

column chromatography (petroleum ether/acetone, 9:1) to provide

21 (5.82 g, 74%) as an orange oil. ¹H NMR (CDCl₃) δ 0.91-0.96

(t, J ) 7 Hz, 3H, CH₃), 1.31-1.48 (m, 4H, CH₂), 1.17-1.85 (m,

2H, CH₂), 3.98-4.03 (t, J ) 6 Hz, 2H, CH₂-O), 7.15-7.17 (m,

1

a pale-yellow oil. H NMR (CDCl₃) δ 0.86-0.88 (t, J ) 8 Hz,

3H, CH₃), 1.13 (s, 6H, CH₂), 1.60-1.65 (m, 2H, CH₂), 2.7-2.75

(t, J ) 7.8 Hz, 2H, Ar-CH₂), 7.38-7.41 (d, J ) 8 Hz, 2H, Ar-H),

8.04-8.07 (d, J ) 8 Hz, 2H, Ar-H). ¹³C NMR (CDCl₃) δ 14.59,

23.08, 29.43, 31.37, 32.14, 36.92, 130.17, 131.25, 154.24. IR (ﬁlm)

2221, 1687 cm^-1

.

1H, Ar-H), 7.37-7.44 (brm, 3H, Ar-H), 9.96 (s, 1H, COH). ¹³

C

2-(4-Bromophenyl)-2-phenylacetonitrile (27). Bromine (0.82 g,

5.1 mmol) was slowly added over 1 h to 4-bromophenylacetonitrile

(1.00 g, 5.10 mmol) at 110 °C (oil bath). The temperature was

maintained between 105 and 110 °C for 30 min until the evolution

of HBr had ceased. The solution was allowed to cool to room

temperature, and a steady stream of nitrogen was passed over the

solution (30 min). The resulting yellow oil was dissolved in

anhydrous benzene (1.20 g, 15.0 mmol), and AlCl₃(0.680 g, 5.10

mmol) was added. The solution was heated at reﬂux (3 h), cooled

to room temperature, and poured onto ice (25 g). The solution was

made acidic to pH 2 with 5% HCl and extracted with Et₂O (3 ×

NMR (CDCl₃) δ 14.56, 22.98, 28.71, 29.38, 68.86, 113.35, 122.50.

123.82, 130.53, 138.34, 159.98, 192.75.

2-Benzyl-4-n-hexylbenzyl alcohol (22). A 1.0 M solution of

borane-THF complex (8 mL, 8 mmol) was added under N₂to a

well stirred solution of 2-benzyl-4-n-hexylbenzoic acid (18d, 0.600

g, 2.00 mmol) at 0 °C. The solution was heated at reﬂux (5 h) and

allowed to cool. HCl 6.0 M (5 mL) was added cautiously, and the

mixture was again heated at reﬂux (30 min). The solution was

allowed to cool to room temperature, made basic with 15% NaOH

(≈ 30 mL), and extracted with EtOAc (3 × 45 mL). The organic

extracts were combined, dried (MgSO₄), and concentrated under

6824 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21

Runyon et al.

35 mL). The organic extracts were combined, washed with water

(50 mL), sat NaHCO₃(50 mL), and brine (50 mL), dried (MgSO₄),

and concentrated under reduced pressure to provide a pale-ellow

semisolid. The semisolid was recrystallized from absolute EtOH

to provide 27 (0.650 g, 46%) as pale-yellow crystals; mp 80-82

created that reproduced the alignment of Bissantz et al.⁷⁹An

unambiguous (i.e., highly conserved residues previously identiﬁed^80,81

are aligned and there are no insertions or deletions in the helical

regions) alignment of the 5-HT_2Asequence with the aforementioned

proﬁle was performed using the ClustalX⁸²program. Within

ClustalX, the slow-accurate alignment algorithm was used, the

BLOSUM matrix series⁸³was employed, and the gap opening

penalty was increased from 10.0 to 15.0 to help maintain the

continuity of the transmembrane helical segments. As in Bissantz

et al.,⁷⁹the alignment was carried out in two separate steps: the

ﬁrst alignment included all residues from the N-terminus to the i3

loop and the second alignment included all residues from the i3

loop to the C-terminus. Manual adjustment of the ClustalX

alignment was also required to properly align the disulﬁde-forming

cysteine residues in the e2 loop. The D₂sequence was subsequently

aligned with that of 5-HT_2Ato identify loci within the binding sites

of the two receptors where cognate amino acids differ and to identify

those that may be responsible for 5-HT_2A/D₂selectivity. The

resulting alignment is presented in Figure 3.

The alignment described above was used as the basis for

subsequent homology modeling of the 5-HT_2Areceptor. In light of

the growing evidence^35,79,84-91that the binding of agonists versus

antagonists may be more effectively modeled using at least two

distinct static receptor models rather than a single static model,

and our own observation that the agonist/antagonist properties of

the 1-(2,5-dimethoxyphenyl)-2-aminopropane derivatives may be

modulated by the nature of the substituent at the 4-position of the

phenyl ring,¹⁵separate agonist and antagonist 5-HT_2Amodels were

generated. The automodel routine in the MODELER 8v1 soft-

ware package^92,93was thus used to generate an initial population

of 100 5-HT_2Areceptor models. These models were constructed

based on homology to bovine rhodopsin (chain A of PDB entry )

1U19), whose coordinates were downloaded from the RCSB Protein

Data Bank (http://www.rcsb.org).

1

°C. (lit⁴⁸mp 79-81 °C). H NMR (CDCl₃) δ 5.10 (s, 1H, CH),

7.21-7.53 (brm, 9H, Ar-H). IR (KBr) 2246 cm^-1

.

(4-n-Hexylphenyl)(phenyl)methanone (28). AlCl₃(2.36 g, 17.8

mmol) was added slowly at 0 °C to 4-n-hexylbenzoyl chloride (2.00

g, 8.90 mmol) in anhydrous benzene (50 mL). The suspension was

warmed to room temperature and heated at reﬂux (2 h). The solution

was then cooled to room temperature and poured onto ice (30.0

g). The suspension was made acidic with 5% HCl (≈ 50 mL) and

extracted with EtOAc (3 × 45 mL). The organic extracts were

combined, washed with sat NaHCO₃(50 mL), water (50 mL) and

brine (50 mL), dried (MgSO₄), and concentrated under reduced

pressure to provide 28 (2.00 g, 87%) as a pale-yellow oil. The

1

product was of sufﬁcient purity to use in the next step. H NMR

(CDCl₃) δ 0.86-0.91 (t, J ) 7 Hz, 2H, CH₃), 1.25 (s, 6H, CH₂),

1.62-1.67 (m, 2H, CH₂), 2.65-2.71 (t, J ) 8 Hz, 2H, CH₂),

7.25-7.80 (brm, 9H, Ar-H). IR (ﬁlm) 1662 cm^-1

.

2-(4-n-Hexylphenyl)-2-hydroxy-2-phenylacetonitrile (29). Tri-

methylsilyl cyanide (0.440 g, 4.51 mmol) was added to 4-n-

hexylphenyl)(phenyl)methanone (28, 1.00 g, 3.76 mmol) and zinc

iodide (cat. amount) in CH₂Cl₂(5 mL). The suspension was heated

at reﬂux (2 h), cooled to room temperature, and concentrated under

reduced pressure to provide a pale-yellow oil. The oil was dissolved

in THF (5 mL), and 3 M HCl (3 mL) was added. The suspension

was heated at reﬂux (1 h), cooled, and water (100 mL) was added.

The mixture was extracted with EtOAc (3 × 20 mL), and the

organic extracts were combined, dried (MgSO₄), and concentrated

under reduced pressure to provide a pale-yellow oil that was puriﬁed

using medium pressure column chromatography (petroleum ether/

1

acetone, 8:2) to provide 29 (0.680 g, 62%) as a colorless oil. H

NMR (CDCl₃) δ 0.83-0.87 (t, J ) 8 Hz, 2H, CH₃), 1.28 (s, 6H,

CH₂), 1.53-1.58 (m, 2H, CH₂), 2.57-2.62 (t, J ) 8 Hz, 2H, CH₂),

3.27 (brs, 1H, OH), 7.18-7.56 (brm, 9H, Ar-H). IR (ﬁlm) 3403

To maximize the variation in the side chain conformations of

the MODELER-generated receptors, the residues falling within 12.0

Å of the retinal ligand in 1U19 were mutated to alanine. These

residues are highlighted with a green background in Figure 3. This

approach, which has been used to successfully model R₂-adrenergic

receptors,⁹⁴allows MODELER to more fully explore side chain

conformational space because the side chains may then be placed

onto the backbone without the added constraints imposed by the

existing rhodopsin sidechains. This also ameliorates the problem

of retinal leaving behind a “ghost site” in the newly created 5-HT_2A

receptors.⁹⁵

In addition to mutating the residues lining the 1U19 binding

cavity to alanine, two other important aspects relating to the transfer

of 3-D information from the 1U19 template to the 5-HT_2Amodels

(particularly in the less well-conserved regions) were speciﬁcally

addressed. First, instead of attempting to accurately model the i3

loop of 5-HT_2A(which is 40 residues longer than rhodopsin’s), the

backbone of the i3 loop of rhodopsin was transferred to the 5-HT_2A

models and the residues therein mutated to glycine. These residues

are highlighted with a purple background in Figure 3. This

effectively created a tether that would keep the helices in place

while having a minimal impact on the remaining portion of the

receptor during minimizations and/or dynamics simulations. Pogozhe-

va, Lomize, and Mosberg⁹⁶have used a similar technique to build

opioid receptors in which a contiguous primary amino acid sequence

for the receptor was necessary. Second, because the N- and

C-terminal domains of bovine rhodopsin and 5-HT_2Aare very

dissimilar in terms of both sequence homology and length, and

because these domains are not believed to be important for the

binding of small molecules,^97,98these features were not included

in the 5-HT_2Amodels.

cm^-1

.

2-(4-n-Hexylphenyl)-2-phenylacetonitrile (30). A NaBH₄pellet

(1.00 g, 26.4 mmol) was added to a well-stirred solution of 2-(4-

n-hexylphenyl)-2-hydroxy-2-phenylacetonitrile (29, 0.550 g, 1.88

mmol) in triﬂuoroacetic acid (15 mL). The viscous purple mixture

was stirred at room temperature for 2 h and concentrated under

reduced pressure. Water (40 mL) was added, and the suspension

was extracted with EtOAc (3 × 20 mL). The organic extracts were

combined, dried (MgSO₄), and concentrated under reduced pressure

to provide a pale-purple oil that was puriﬁed using medium pressure

column chromatography (petroleum ether/acetone, 8:2) to pro-

vide 30 (0.350 g, 67%) as a colorless oil. ¹H NMR (CDCl₃) δ

0.85-0.89 (t, J ) 8 Hz, 2H, CH₃), 1.28 (s, 6H, CH₂), 1.52-1.59

(m, 2H, CH₂), 2.55-2.60 (t, J ) 7.8 Hz, 2H, CH₂), 5.20 (s, 1H,

CH), 7.15-7.36 (brm, 9H, Ar-H). ¹³C NMR (CDCl₃) δ 14.64,

23.14, 29.51, 31.86, 32.24, 36.10, 42.85, 128.15, 128.28, 128.70,

129.74, 129.75. IR (ﬁlm): 2246 cm^-1

.

Molecular Modeling. Molecular modeling investigations were

conducted using the SYBYL molecular modeling package (version

7.1, 2005, Tripos Associates, St. Louis, MO) on MIPS R14K- and

R16K-based IRIX 6.5 Silicon Graphics Fuel and Tezro worksta-

tions. Molecular mechanics-based energy minimizations were

performed using the Tripos Force Field with Gasteiger-Hu¨ckel

charges, a distance-dependent dielectric constant ε ) 4, and a

nonbonded interaction cutoff ) 8 Å and were terminated at an

energy gradient of 0.05 kcal/(mol ·Å).

The primary sequences of the human dopamine D₃(P35462),

human muscarinic cholinergic M₁(P11229), human vasopressin

V_1a(P37288), human adrenergic ꢁ₂(P07550), human δ-opioid

(P41143), human 5-HT_2A(P28223), human dopamine D₂(P14416),

and bovine rhodopsin (P02699) receptors were retrieved from the

ExPASy Proteomics Server (http://www.expasy.org/) at the Swiss

Institute of Bioinformatics. An alignment proﬁle consisting of the

D₃, M₁, V_1a, ꢁ₂, δ-opioid, and bovine rhodopsin receptors was

Each of the initial 100 5-HT_2Amodels thus generated was energy-

minimized as described above with a maximum of 100000 iterations

and with no constraints. The process of then selecting an agonist

and an antagonist model from the receptor population was facilitated

by the use of the automated docking program GOLD (version

3.0.1).^99,100A high-potency agonist (DOB, 2b, K_i) 41 nM) and

Interaction of AMDA DeriVatiVes with the 5-HT_2AReceptor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6825

an antagonist (ketanserin, K_i) 0.4 nM) were selected as reference

ligands, and each was docked into all 100 receptors using the GOLD

program. For DOB, separate docking runs were performed for each

explicitly represented stereoisomer ((R)- and (S)- forms of 2b ) 2

isomers) because each isomer shows low nanomolar binding afﬁnity

at the 5-HT_2Areceptor. Standard default settings were used (no

speed-up), early termination was disabled, and 10 genetic algorithm

(GA) runs were performed for each ligand. The ChemScore¹⁰¹

ﬁtness function was used, and the binding site was deﬁned to include

all residues within a 15.0 Å radius of the D155^3.32C^γcarbon atom.

A docking constraint was also enforced that biased the docking

results toward solutions in which the ligand was hydrogen bonded

to the conserved D155^3.32side chain. When these initial docking

runs were complete, the lists of ChemScores for each isomer were

tabulated (each reference ligand isomer was docked into each of

the 100 MODELER-generated receptors) and sorted best to worst

(highest to lowest for ChemScore in GOLD). The top-scoring

complexes for each reference ligand are summarized in Supporting

Information Table 4.

SYBYL to assess the geometric integrity of various structural

elements (bond lengths, torsion angles, etc.) within each receptor.

Unusual and unfavorable geometries were interactively corrected

as necessary. There were two such regions in both the agonist and

antagonist receptors. The ﬁrst involved the region of TM7 proximal

to position 7.43, where the retinal chromophore is covalently bound

in rhodopsin. Backbone geometries in this region were visibly

nonoptimal (kinked), so the residues in the region V364^7.37to

A374^7.47were assigned ideal R helix coordinates using SYBYL

7.1. A second visibly distorted region at location L236^5.40to

V241^5.45was reﬁned in a similar way. The modiﬁed receptors were

then energy-minimized as described for the initial 5-HT_2Areceptors

prior to docking. The ﬁnal models are depicted together with the

A chain of rhodopsin (1U19) in Supporting Information Figure 1.

Ligand molecules were sketched manually using SYBYL 7.1

(Tripos, Inc., St. Louis, MO) and assigned three-dimensional

coordinates using the CONCORD (v.6.1.0) facility within SYBYL.

Because the synthesized and tested compounds are racemic mixtures

in most if not all cases, R/S isomers were explicitly represented

for each ligand where necessary. GOLD was used to dock the

ligands into the agonist and antagonist 5-HT_2Amodels in an

automated fashion under the same conditions as described above

for the receptor selection phase. Ligand preference for a particular

receptor was determined by the difference in the ChemScore ﬁtness

function values for the agonist and antagonist receptor solutions

(Supporting Information Table 2). However, there was one excep-

tion: AMDA (1a), which was predicted to have a slight preference

for the agonist receptor, was associated with the antagonist model

on the strength of the F340^6.52L mutation data, as described earlier.

Final docked ligand-receptor complexes were energy-minimized

as described above for the initial 5-HT_2Amodels. The minimized

complexes were further subjected to a short molecular dynamics

simulation (Tripos Force Field, Gasteiger-Hu¨ckel charges, distance-

dependent dielectric ) 4.0, ﬁxed aggregate ) all residues >8.0 Å

from the GOLD-docked solution, 100 ps simulation time, 300 K)

to provide further evidence of the veracity of the docked solutions.

Afﬁnity Determinations. Binding assays and data analysis were

performed as previously described using [³H]ketanserin as the

radioligand and stably transfected NIH3T3 cells expressing the

5-HT_2Areceptor (GF-62 cells).¹⁰³The F339L and F340L mutants

were prepared and assayed as previously described.¹⁰⁴

The top-scoring receptor-ligand complexes for each of the

reference compounds were then inspected visually to ensure that

the receptor formed a chemically intuitive complex with the ligand

and that the receptor-ligand complex could account for the

observed point mutation data. High-scoring complexes that did not

meet this requirement were discarded. Ligand-receptor complexes

were discarded for a variety of reasons, including: (1) the docked

solution for DOB did not accept a hydrogen bond from the receptor

at either the 2- or 5-position (rationale: methoxy groups are

necessary for high afﬁnity of the smaller 4-substituted phenylethy-

lamines [K_i) 1770 nM, 3b; K_i) 41 nM, 2b]), (2) the docked

solution for DOB did not interact with F340 (rationale: DOB most

likely binds in the receptor site in the same manner as does DOI

(Supporting Information Figure 2), which loses all afﬁnity for the

F340A mutant (Table 2)), (3) the docked solution for ketanserin

could not explain the effects of mutagenesis at F340, Y370, and/or

W76 (Table 3), and (4) the e2 loop of the receptor was incorrectly

modeled (rationale: MODELER occasionally placed the e2 loop

segment that joins the top of TM4 to the disulﬁde bond farther

down in the receptor site than the segment joining the disulﬁde

bond to the top of TM5sthe reverse of what is seen in the bovine

rhodopsin crystal structure). A complete listing of the 12 top-scoring

receptor-reference ligand complexes and comments on their

binding modes is presented in Supporting Information Table 4.

The ChemScores for the 100 top complexes covered a wide range

(R(-)-DOB, 1.41-33.52; S(+)-DOB, 3.06-30.71; ketanserin,

-31.84-35.21), a result of the receptor population containing

members that exhibited both very high and very low degrees of

complementarity to the docked ligands. The top-scoring receptors

for a given ligand tended to have similar high scores (Supporting

Information Table 4), but the corresponding binding modes of the

docked ligands were very different. It was thus necessary to use

additional information from receptor mutagenesis and ligand SAR

to eliminate from consideration those that were not consistent with

this data and to select the most appropriate receptor models (vide

supra). Of course, reconciliation of relevant experimentally derived

data with molecular models is a necessary part of any modeling

study. Seven of the top 12 receptor models are common to both

R(-)-DOB and S(+)-DOB. Interestingly, ﬁve of the top 12 receptor

models for ketanserin are also found in the top receptor lists of

either R(-)-DOB or S(+)-DOB. It should also be noted that for a

given receptor-ligand model, the set of docked solutions from the

10 GA runs tended to be qualitatively similar, differing only slightly

in the ligands’ relative position and orientation within the binding

site. For each of the reference ligands, then, the ﬁnal receptor model

chosen was the highest scoring chemically intuitive complex. For

DOB, the same receptor model was coincidentally chosen for each

of the two stereoisomers. In this selected agonist model, the DOB

isomers docked in a similar fashion (Supporting Information

Figure 2).

Acknowledgment. This work was supported by United

States Public Health Service grant MH57969 (R.B.W.),

R01MH61887, U19MH082441 (B.L.R.), and the NIMH Psy-

choactive Drug Screening Program (B.L.R.).

Supporting Information Available: Receptor-ligand inter-

atomic distances; GOLD-ChemScore ﬁtness function values; el-

emental microanalysis; initial docking results for DOB and

ketanserin, rhodopsin template and 5-HT2A receptor model back-

bone traces; docked solutions for DOB and DOI: agonist receptor

model; docked solutions for the four isomers of 1c: antagonist

receptor model; docked solutions for S-1c and R-2c: antagonist

receptor model. This material is available free of charge via the

Internet at http://pubs.acs.org.

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JM800771X

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Potential modes of interaction of 9-aminomethyl-9,10-dihydroanthracene (AMDA) derivatives with the 5-HT2A receptor: A ligand structure-affinity relationship, receptor mutagenesis and receptor modeling investigation

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