Dihydrobenzofuran analogues of hallucinogens. 3. Models of 4-substituted (2,5-dimethoxyphenyl)alkylamine derivatives with rigidified methoxy groups

Article abstract of DOI:10.1021/jm960199j

Tetrahydrobenzodifuran functionalities were employed as conformationally restricted bioisosteres of the aromatic methoxy groups in prototypical hallucinogenic phenylalkylamines 1 and 2. Thus, a series of 8-substituted 1- (2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b']difuran-4-yl)-2-aminoalkanes (7a-e) were prepared and evaluated for activity in the two-lever drug discrimination paradigm in rats trained to discriminate saline from LSD tartrate (0.08 mg/kg) and for the ability to displace [³H]ketanserin from rat cortical homogenate 5-HT(2A) receptors and [³H]-8-OH-DPAT from rat hippocampal homogenate 5-HT(1A) receptors. In addition, 1-(8-bromo-2,3,6,7- tetrahydrobenzo[1,2-b:4,5-b']difuran-4-yl)-2-aminopropane (7b), which was found to be extremely potent in the rat in vivo assays, was evaluated for its ability to compete with [¹²⁵I]DOI and [³H]ketanserin binding to cells expressing cloned human 5-HT(2A), 5-HT(2B), and 5-HT(2C) receptors. All of the dihydrofuranyl compounds having a hydrophobic substituent para to the alkylamine side chain had activities in both the in vitro and in vivo assays that equaled or surpassed the activity of the analogous conformationally flexible parent compounds. For example, 7b substituted for LSD in the drug discrimination assay with an ED₅₀ of 61 nmol/kg and had K(i) values in the nanomolar to subnanomolar range for the displacement of radioligand from rat and human 5-HT₂ receptors, making it one of the most potent hallucinogen- like phenylalkylamine derivatives reported to date. The results suggest that the dihydrofuran rings in these new analogues effectively model the active binding conformations of the methoxy groups of the parent compounds 1 and 2. In addition, the results provide information about the topography and relative orientation of residues involved in agonist binding in the serotonin 5-HT₂ receptors.

Full text of DOI:10.1021/jm960199j

J . Med. Chem. 1996, 39, 2953-2961
2953
Dih yd r oben zofu r a n An a logu es of Ha llu cin ogen s. 3.1 Mod els of 4-Su bstitu ted
(2,5-Dim eth oxyp h en yl)a lk yla m in e Der iva tives w ith Rigid ified Meth oxy Gr ou p s2
Aaron P. Monte,‡ Danuta Marona-Lewicka, Matthew A. Parker, David B. Wainscott,† David L. Nelson,† and
David E. Nichols*
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences,
Purdue University, West Lafayette, Indiana 47907, and Lilly Research Laboratories, Eli Lilly and Company,
Indianapolis, Indiana 46285
Received March 11, 1996X
Tetrahydrobenzodifuran functionalities were employed as conformationally restricted bioiso-
steres of the aromatic methoxy groups in prototypical hallucinogenic phenylalkylamines 1 and
2. Thus, a series of 8-substituted 1-(2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b′]difuran-4-yl)-2-
aminoalkanes (7a -e) were prepared and evaluated for activity in the two-lever drug
discrimination paradigm in rats trained to discriminate saline from LSD tartrate (0.08 mg/kg)
and for the ability to displace [3H]ketanserin from rat cortical homogenate 5-HT2A receptors
and [3H]-8-OH-DPAT from rat hippocampal homogenate 5-HT1A receptors. In addition, 1-(8-
bromo-2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b′]difuran-4-yl)-2-aminopropane (7b), which was found
to be extremely potent in the rat in vivo assays, was evaluated for its ability to compete with
[
125I]DOI and [3H]ketanserin binding to cells expressing cloned human 5-HT2A, 5-HT2B, and
5-HT2C receptors. All of the dihydrofuranyl compounds having a hydrophobic substituent para
to the alkylamine side chain had activities in both the in vitro and in vivo assays that equaled
or surpassed the activity of the analogous conformationally flexible parent compounds. For
example, 7b substituted for LSD in the drug discrimination assay with an ED50 of 61 nmol/kg
and had Ki values in the nanomolar to subnanomolar range for the displacement of radioligand
from rat and human 5-HT2 receptors, making it one of the most potent hallucinogen-like
phenylalkylamine derivatives reported to date. The results suggest that the dihydrofuran rings
in these new analogues effectively model the active binding conformations of the methoxy groups
of the parent compounds 1 and 2. In addition, the results provide information about the
topography and relative orientation of residues involved in agonist binding in the serotonin
5-HT2 receptors.
In tr od u ction
Representative phenylisopropylamines 1a -d and
phenethylamines 2a -c remain some of the most potent
and selective serotonin 5-HT2 agonists available, having
nanomolar affinity for both 5-HT2A and 5-HT2C receptor
subtypes.3-10 With the exception of the hallucinogenic
ergolines such as d-lysergic acid N,N-diethylamide
(LSD), these agents also represent some of the most
potent compounds in behavioral assays for hallucinogen-
like activity.3,10-12 Because we have been interested in
exploring the molecular mechanisms of action of hal-
lucinogens for many years,13,14 compounds of general
structure 1 and 2 seemed logical starting points for
further structure-activity relationship (SAR) studies
involving a rigid analogue approach to probe the topog-
raphy of the serotonin receptor agonist binding site(s).
Previous SAR investigations have established that
three main structural features of 1 and 2 are required
for optimal in vivo and in vitro hallucinogen-like activ-
ity.13,14 These are (1) the primary amine functionality
separated from the phenyl ring by two carbon atoms,
(2) the presence of the 2- and 5-aromatic methoxy
groups, and (3) a hydrophobic 4-substituent (alkyl, halo,
alkylthio, trifluoromethyl, etc.). The presence of the
methyl group R to the amine in compounds such as 1
does not enhance in vitro receptor affinity but does lend
increased in vivo potency and duration of action to these
molecules, possibly due to the inhibition of metabolism
by deamination. We have recently suggested that the
R-methyl may also increase intrinsic efficacy at the
5-HT2A receptor.10 Given these basic structural require-
ments, rigid analogues of 1 and 2 have previously been
designed to examine the pharmacological consequences
of locking the various functional groups of these mol-
ecules into restricted conformations. For example, the
aminopropyl side chain of 1a has been incorporated into
aminotetralin and aminoindan rings to restrict mobility
of the alkylamine side chain, but these modifications
generally lead to inactive compounds.15
It has been proposed that serine residues are key
recognition elements within the ligand binding domain
of the serotonin 5-HT2A receptor.16 Indeed, Westkaem-
per and Glennon17 have identified specific serine resi-
dues in transmembrane helices 4 and 5 of this receptor
that are hypothesized to interact with the 2- and
5-methoxy groups of 1 and 2. If the heterocyclic oxygen
* Author for correspondence. Phone: (317) 494-1461. Fax: (317) 494-
6790. E-mail: drdave@pharmacy.purdue.edu.
† Eli Lilly and Co.
‡ Current address: Department of Chemistry, Cowley Hall, Uni-
versity of Wisconsin-La Crosse, La Crosse, WI 54601.
X Abstract published in Advance ACS Abstracts, J une 15, 1996.
S0022-2623(96)00199-9 CCC: $12.00 © 1996 American Chemical Society

2954 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
Monte et al.
Sch em e 1a
atoms of compounds such as 3-5 undergo similar
H-bonding interactions, these rigid analogues of 1
should offer valuable insight into the location of the
putative H-bond donors and into the overall topography
of the 5-HT2 agonist binding site.
a
(a) BrCH2CH2Cl; (b) Br2, CCl4; (c) n-BuLi, THF, 0 °C.
intermediate 2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b′]difu-
ran (10) (Scheme 2). The synthesis of this simple
coumaran was reported to be difficult, and the existing
literature method, although it involved only three steps,
remained undesirable because it afforded very poor
overall yields of 10.21 Thus, our initial goal was to
develop an improved synthesis of 10 as a starting point
for the construction of the series of (tetrahydrobenzodi-
furanyl)phenethylamine derivatives.
We recently began a series of investigations involving
3-5 that would address the binding conformations of
the aromatic methoxy groups in this class of compounds.
Thus, dihydrobenzofurans 3 and 4 were synthesized and
evaluated in assays for hallucinogen-like activity.18-20
In these studies, compound 3, which has the lone pair
electrons on the oxygen atom meta to the side chain
directed syn to the alkylamine chain, had dramatically
attenuated LSD-like behavioral effects in rats.18 In 4,
these electron pairs are directed anti to the side chain,
and this compound was equipotent to the untethered
parent compound 1b, in both in vivo and in vitro
pharmacological assays.19 Additionally, the benzox-
epins 5a -c were prepared as rigid analogues of 1b-d
to evaluate the possible active binding conformations
of the 2-methoxy group at serotonin receptors.21 In that
study, 5a -c all had low affinities for 5-HT2A sites,
possibly indicating that the anti orientation of the lone
pair electrons on the oxygen adjacent to the side chain
in these compounds may not be complementary to the
agonist binding site and that compounds having the
opposite (syn) lone pair orientation might provide a
better fit to the receptor.
As an extension of those earlier efforts, we present
here a series of rigid analogues of 1 and 2 in which the
remaining possible 2-methoxy group conformation is
explored. Thus 6, which possesses the syn orientation
of lone pair electrons on the O adjacent to the side chain
(“opposite” to the analogous O2 conformation of com-
pounds 5a -c), was initially synthesized and shown in
preliminary pharmacological screens to be a potent
serotonin agonist (unpublished data). These results
prompted us to synthesize the highly rigidified difuranyl
compounds 7a -e, which have both aromatic methoxy
groups tethered into rotationally restricted dihydrofuran
rings. We describe here the synthesis, hallucinogen-
like behavioral effects, and rat and human serotonin
5-HT2A, 5-HT2C, and 5-HT1A receptor affinities for these
compounds.
Our synthesis of 10, shown in Scheme 1, is based on
the work of Parham et al.,22 who achieved efficient
syntheses of benzocyclobutene and indan by lithiation
of the appropriate 1-bromo-2-(ω-chloroalkyl)benzenes at
-100 °C followed by intramolecular cyclization upon
warming to room temperature. Thus, hydroquinone
was alkylated with 1-bromo-2-chloroethane and potas-
sium carbonate in acetone to give the bis(2-chloroethyl)
ether 8. This procedure was preferred to the most
recent literature preparation of this compound23 in
which isolation of product was hampered by the pres-
ence of large amounts of phase-transfer catalyst. Bro-
mination in carbon tetrachloride, a reaction that was
readily carried out on a preparative scale, gave the
dibromo compound 9. Cyclization of 9 with 2 equiv of
n-butyllithium in THF then directly gave the benzodi-
furan 10 in excellent yield. We also discovered that the
lithiation and cyclization could be performed at 0 °C
with good results if the n-butyllithium was added
quickly to a rapidly stirred solution of 9 in THF.
With an efficient synthesis of 10 established, this
tricyclic nucleus was readily elaborated using conven-
tional procedures described previously24 to give the
desired series of 1-(2,5-dimethoxyphenyl)-2-aminopro-
pane analogues 7a -e. As shown in Scheme 2, 10 was
formylated according to our previously described meth-
ods25 to give the 4-formyl compound 11 in good yield.
This aldehyde was then condensed with nitroethane,
and the intermediate nitropropene 12 was reduced with
lithium aluminum hydride to afford 7a . As there was
only one remaining aromatic position, 7a was easily
brominated by treatment with elemental bromine in
acetic acid to afford the target compound 7b.26 Simi-
larly, the aminoethane analogues 7c,d were also syn-
thesized as shown in Scheme 2. Thus, 11 was con-
densed with nitromethane to give the nitrostyrene
derivative 13 in excellent yield, and this was reduced
with lithium aluminum hydride to afford the amino-
ethane 7c. Bromination of 7c in acetic acid26 then gave
the desired target compound 7d in good yield.
Ch em istr y
In considering the development of the benzodifuran
derivatives 7a -e, it was apparent that each of these
target compounds could be prepared from the common
The 8-methyl analogue 7e was constructed as shown
in Scheme 3. In this case, the formyl group of 11 was

Dihydrobenzofuran Analogues of Hallucinogens
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2955
Sch em e 2a
cies were measured using ED50 values with 95% confi-
dence intervals (CI). Additionally, 7a -d were tested
for their ability to compete for radioligand binding to
5-HT1A and 5-HT2A receptor sites (Table 1).27 Briefly,
the ability of test compounds to compete for 0.75 nM
[3H]-8-OH-DPAT at rat hippocampal homogenate bind-
ing sites and 0.75 nM [3H]ketanserin in rat frontal
cortex homogenate was measured, with the affinities of
the compounds for the receptor sites expressed as Ki.
7b, which was found to be exceptionally potent in the
initial behavioral and in vitro assays, was also evaluated
in cells expressing cloned human 5-HT2A, 5-HT2B, and
5-HT2C receptors that were labeled with agonist or
antagonist radioligands (Table 2).
Resu lts a n d Discu ssion
a
(a) Cl2CHOCH3, SnCl4, CH2Cl2; (b) RCH2NO2, NH4OAc, ∆; (c)
LiAlH4, THF, reflux; (d) Br2, AcOH.
The drug discrimination (DD) paradigm is routinely
used in our laboratory as an initial screen for evaluating
the behavioral activity or hallucinogenic potential of
newly synthesized molecules.12,28-30 This assay system
has been used extensively to model human hallucino-
genic effects by studying the LSD-like discriminative
stimulus properties produced in animals.31,32 Previous
DD studies have established that the discriminative
behavioral cue is mediated primarily by the stimulation
of 5-HT2 receptors.9,29 Thus, agents exhibiting in vivo
hallucinogenic activity typically can be shown to activate
serotonin 5-HT2A and 5-HT2C receptors in vitro, and no
agonists have yet been found that can significantly
differentiate between these subtypes.4,7,33
Sch em e 3a
Table 1 shows the results of the DD studies in LSD-
trained rats and the results of the radioligand competi-
tion experiments at [3H]ketanserin-labeled and [3H]-8-
OH-DPAT-labeled rat 5-HT2A and 5-HT1A receptors,
respectively. As expected, the in vivo ED50 values of
7a -d in the DD assay closely parallel the affinities of
these compounds for 5-HT2A receptors. None of the
compounds had significant affinity for 5-HT1A sites. In
agreement with the established hallucinogenic phenyl-
alkylamine SAR, the agents not having a hydrophobic
substituent para to the alkylamine side chain (7a ,c)
were much less potent than those that did, providing
evidence that the benzofuranyl compounds bind to the
same agonist site as the untethered parent compounds.
Also in line with established SAR requirements, the
isopropylamine 7b was considerably more potent in vivo
than the phenethylamine 7d , but these compounds were
of similar potencies in the in vitro binding assay at
5-HT2A sites.
The most significant findings of the present report are
related to the extremely high potencies of benzodifurans
7b,d , in both the in vivo and in vitro assays. These
results support the idea that the O2 lone pair electron
syn orientation with respect to the alkylamine side chain
is optimal in this class of compounds. Thus, while the
benzoxepins 5, having the anti orientation of O2 lone
pair electrons, were essentially inactive, analogous
compounds having the syn orientation of these electrons
were quite potent. Since the optimal orientation of the
5-alkoxy group had already been established in our
previous study,19 it was predicted that molecules in
which both the 2- and 5-methoxy groups were tethered
into “active” conformations using dihydrofuran rings
would also be potent serotonin agonists. Thus, 7a -e
were synthesized as novel, highly rigidified analogues
a
(a) H2 (60 psig), Pd-C, EtOH; (b) Cl2CHOCH3, SnCl4, CH2Cl2;
(c) RCH2NO2, NH4OAc, ∆; (d) LiAlH4, THF, HCl.
Ta ble 1. Results of the Drug Discrimination Studies in
LSD-Trained Rats and of the Radioligand Competition
Experiments at [3H]Ketanserin-Labeled Rat Cortical 5-HT2A
and [3H]-8-OH-DPAT-Labeled Rat Hippocampal 5-HT1A
Receptors (Ki ( SEM)
DD Studies
5-HT2A
sites
(nM)
5-HT1A
sites
(µM)
ED50
drug (mmol/kg)
95% CI
na
1b
4
1.12
0.86-1.46 8-12 22 ( 3
6.1 ( 0.5
c
0.57 b
0.29-1.09
-
-
-
7a
7b
7c
7d
7e
64% @ 4.0 PSd
10-13 2010 ( 83 29 ( 2
0.061
1.43
0.31
0.78
0.031-0.12 7-15 18 ( 1
4.3 ( 0.4
0.45-4.5 7-14 2300 ( 170 5.2 ( 0.4
0.08-1.14 8-12 34 ( 2
0.80 ( 0.06
NT
5.1 ( 0.4 nM
0.48-1.22 8-9
0.03-0.05 15
NTe
4.4 ( 0.2
LSD 0.040
a
b
Number of animals tested at each dose. Data taken from ref
19. c Affinity at [3H]ketanserin sites has not been measured.
Affinity at [125I]DOI-labeled sites was virtually identical with that
of compound 1b.19 PS ) partial substitution. e Not tested.
d
completely reduced to a methyl, and the resulting
benzodifuran 14 was reformylated to give the 4-formyl-
8-methyl compound 15. Elaboration of the side chain
as before afforded 7e via its nitropropene 16.
P h a r m a cology
Compounds 7a -e were initially evaluated in the two-
lever drug discrimination assay in a group of rats
trained to discriminate the effects of ip injections of
saline from those of LSD tartrate (0.08 mg/kg) according
to methods described previously (Table 1).27 For those
compounds that completely substituted for LSD, poten-

2956 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
Monte et al.
Ta ble 2. Results of the Radioligand Competition Studies of 7b at Cloned Human 5-HT2A, 5-HT2B, and 5-HT2C Receptors (Ki values in
nM ( SEM)
[3H]antagonist radioligands
agonist radioligands
[3H]-5-HT
ketanserin
(5-HT2A
rauwolscine
mesulergine
(5-HT2C
[
125I]DOI
(5-HT2A
[
125I]DOI
(5-HT2C
)
(5-HT2B
)
)
)
(5-HT2B
)
)
10.73 ( 0.95
1.15 ( 0.04
2.28 ( 0.38
0.48 ( 0.03
1.60 ( 0.25
0.30 ( 0.02
of general structure 1. Confirming our hypothesis, 7b
was found to be nearly equipotent to LSD in the rat
behavioral assay, a result unparalleled by existing
substituted phenylalkylamines. Additionally, 7b,d had
nanomolar affinity for rat cortical 5-HT2A receptors,
indicating that the benzodifuran structure must be
highly complementary to these antagonist-labeled bind-
ing sites.
Given the exceptional activity of the benzodifuranyl
analogues in the rat assays, the most potent compound
of the series (7b) was also evaluated in cells expressing
cloned human 5-HT2A, 5-HT2B, and 5-HT2C receptor
subtypes. The results of these studies are shown in
Table 2. At the agonist-labeled human receptors, 7b
had very high, subnanomolar affinities, while at the
antagonist-labeled 5-HT2A site, the Ki value was com-
parable to the value obtained at the rat receptor.
Consideration of all the pharmacological results pro-
vides some definite clues about the topography of the
serotonin 5-HT2 agonist binding site for this series of
compounds. If the protonated amine undergoes an
electrostatic binding interaction with the conserved
aspartate residue in transmembrane helix 3 (TM 3), as
suggested in the molecular modeling studies of several
G-protein-coupled receptors,16,34 the direction of ap-
proach of the putative H-bond donors that might
interact with O2 and O5 of the prototypical (dimethoxy-
phenyl)alkylamines can now be established with respect
to this aspartrate-amine interaction site. At O2 the
H-bond donor likely approaches from a direction syn to
the side chain, and at O5 the donor approaches from a
direction anti to the side chain, near the para substitu-
ent. Also, the importance of the hydrophobic para
substituent in activating the 5-HT2 receptor is further
emphasized by the present study. A comparison of the
5-HT2A receptor affinities of 7a with 7b and 7c with 7d
indicates that the brominated analogues are approxi-
mately 100 times more potent in this assay than the
compounds lacking the hydrophobic substituent. These
new SAR data are summarized in the schematic in
Figure 1, adapted from the model proposed by West-
kaemper and Glennon17 which illustrates the relative
location of proposed binding interactions within the
5-HT2 agonist site based on the generalized (benzodi-
furanyl)isopropylamine structure.
F igu r e 1. Schematic representation of the 5-HT2 agonist
binding site, modified from the proposal of Westkaemper and
Glennon.17
orientation of the residues within various receptors or
enzymes that bind molecules containing aromatic meth-
oxy groups.
Exp er im en ta l Section
Ch em istr y. Melting points were determined using a
Thomas-Hoover apparatus and are uncorrected. 1H-NMR
spectra were recorded using either a 500 MHz Varian VXR-
500S or a 300 MHz Bruker ARX-300 NMR spectrometer.
Chemical shifts are reported in δ values ppm relative to
tetramethylsilane (TMS) as an internal reference (0.03%, v/v).
Abbreviations used in NMR analyses are as follows: s )
singlet, d ) doublet, t ) triplet, dd ) doublet of doublets, dt )
doublet of triplets, td ) triplet of doublets, q ) quartet, p )
pentet, m ) multiplet, b ) broad, Ar ) aromatic, cp )
cyclopropyl. Chemical ionization mass spectra (CIMS) using
methane as the carrier gas were obtained with a Finnigan
4000 spectrometer. IR measurements were taken with a
Perkin-Elmer 1600 Series FTIR spectrophotometer. Elemen-
tal analyses were performed by the Purdue University Mi-
croanalysis Laboratory and are within (0.4% of the calculated
values unless otherwise noted. Thin-layer chromatography
(TLC) was typically performed using Baker-flex silica gel IB2-
F, plastic-backed plates with fluorescent indicator (2.5 × 7.5
cm; J . T. Baker), eluting with CH2Cl2, and visualizing with
UV light at 254 nm and/or I2 vapor unless otherwise noted.
Plates used for radial centrifugal chromatography (Chroma-
totron; Harrison Research, Palo Alto, CA) were prepared from
silica gel 60 PF2-54 containing gypsum. Most reactions were
carried out under an inert atmosphere of dry nitrogen.
1,4-Bis(2-ch lor oeth oxy)ben zen e (8).23 A mixture of 50
g (0.455 mol) of hydroquinone, 196 g (1.37 mol) of 1-bromo-2-
chloroethane, 190 g (1.37 mol) of finely powdered anhydrous
potassium carbonate, and 300 mL of acetone was stirred and
heated at reflux under N2 for 24 h. Acetone and excess
dihaloethane were removed in vacuo, and the residue was
partitioned between Et2O and H2O. The Et2O phase was
extracted three times with 4 M NaOH, dried with anhydrous
MgSO4, and filtered. Solvent was removed in vacuo, yielding
In conclusion, we have effectively utilized the dihy-
drobenzofuran functionality as a rigid conformer of the
aromatic methoxy group in hallucinogenic phenylalkyl-
amines. By locking both methoxy groups of this chemi-
cal class into “active” conformations using the tetrahy-
drobenzodifuran nucleus, we have constructed the most
potent and selective phenylalkylamine-type serotonin
5-HT2 agonists yet reported. In so doing, a clearer
picture of the topography of the serotonin agonist
binding site is emerging. The use of the dihydroben-
zofuran functionality may have general applicability in
SAR investigations of other drug classes and should
serve as a powerful tool in exploring the relative
1
33.7 g (32%) of a white solid with a H-NMR spectrum identical
with that reported in ref 23.
1,4-Bis(2-ch lor oeth oxy)-2,5-d ibr om oben zen e (9). To a
stirred suspension of 1.0 g (4.26 mmol) of the bis-ether 8 in
20 mL of CCl4 was added 12 mg (0.21 mmol) of Fe granules.
Bromine (1.50 g, 9.37 mmol) in 5 mL of CCl4 was added
dropwise, and the reaction mixture was stirred for 4.5 h at
room temperature. The mixture was washed twice with H2O,

Dihydrobenzofuran Analogues of Hallucinogens
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2957
dried with MgSO4, and filtered. The solvent was removed in
vacuo, yielding 1.54 g (92%) of an off-white solid. An analytical
sample was recrystallized from EtOH: mp 119 °C; H NMR
of 5 N KOH were added, and the mixture was filtered through
Celite, rinsing the filter cake well with ether and CH2Cl2. The
volatiles were removed on the rotary evaporator, and the
residue was taken up in 100 mL of ether and extracted with
4 × 20 mL of 3 N HCl. The aqueous extracts were combined,
made strongly basic with 5 N KOH, and extracted with 5 ×
20 mL of CH2Cl2. The organic extracts were combined, washed
with brine, dried (MgSO4), and filtered through Celite. Re-
moval of solvent under reduced pressure gave 0.56 g (85%) of
the free amine 8 as a pale yellow oil. The hydrochloride salt
was formed by taking up the oil in ether and adding 1 N HCl
in anhydrous ethanol. Removal of solvent and crystallization
from ethanol-ethyl acetate afforded 0.49 g (64%) of 7a ‚HCl
1
(CDCl3) δ 3.83 (t, 4, ArOCH2CH2Cl, J ) 6 Hz), 4.23 (t, 4,
ArOCH2CH2Cl, J ) 6 Hz), 7.15 (s, 2, ArH).
2,3,6,7-Tetr a h yd r oben zo[1,2-b:4,5-b′]d ifu r a n (10).21
A
solution of 10 g (25.4 mmol) of the dibromo bis-ether 9 in 250
mL of dry THF was placed in a N2 atmosphere and cooled to
0 °C. A solution of 2.5 M n-butyllithium in hexanes (21.4 mL,
2.1 equiv) was added very quickly (addition time: 7 s) to the
rapidly stirred solution using a syringe with a large gauge
needle. (Fast addition of n-BuLi is critical. An otherwise
identical run in which this reagent was added over 40 s
resulted in a 25% reduction in yield.) The reaction mixture
was stirred for 10 min, and solvent was removed in vacuo. The
residue was partitioned between Et2O and H2O, and the
organic phase was dried with MgSO4 and filtered. The
resulting solution was evaporated in vacuo until crystallization
occurred. The crystals were filtered off, the filtrate was again
evaporated, and a second crop of crystals was collected. After
executing this procedure a third time, a total of 3.30 g (80%)
of off-white crystals was obtained. Recrystallization from ether
gave fluffy white crystals: mp 155-156 °C (lit.21 mp 153 °C);
1H NMR (CDCl3) δ 3.13 (t, 4, ArOCH2CH2, J ) 8.5 Hz), 4.52
(t, 4, ArOCH2CH2, J ) 8.5 Hz), 6.63 (s, 2, ArH); CIMS m/ z
163 (M + 1).
4-F or m yl-2,3,6,7-t e t r a h yd r ob e n zo[1,2-b:4,5-b′]d ifu -
r a n (11). A solution of 3.0 g (0.019 mol) of the tetrahydroben-
zodifuran 10 in 100 mL of dry CH2Cl2 was stirred under N2
and cooled over an ice bath. Tin(IV) chloride (2.81 mL, 0.024
mol) was added to the solution, and the mixture was stirred
for 5 min. Dichloromethyl methyl ether (1.67 mL, 0.019 mol)
in 5 mL of CH2Cl2 was then introduced into the mixture
dropwise over a 5 min period. After the mixture had stirred
for 30 min, the reaction was quenched by the addition of 50
mL of ice-H2O, and the layers were separated. The aqueous
phase was extracted with 2 × 30 mL of CH2Cl2, and the organic
fractions were combined. The organic extract was washed with
3 N HCl (3 × 100 mL), 100 mL of H2O, and 100 mL of brine,
dried over MgSO4, and filtered through a pad of Celite and
silica gel. Removal of solvent under reduced pressure and
drying under high vacuum gave 2.9 g (82%) of a yellow oil that
spontaneously crystallized upon standing. The solid was
recrystallized from chloroform-hexane to yield 2.51 g (71%)
of pure product 11 as light yellow crystals: mp 86-87 °C; 1H
NMR (CDCl3) δ 3.20 (td, 2, ArOCH2CH2, J ) 8.6 Hz, 1.1 Hz),
3.45 (t, 2, ArOCH2CH2, J ) 8.8 Hz), 4.60 (t, 2, ArOCH2CH2, J
) 8.8 Hz), 4.70 (t, 2, ArOCH2CH2, J ) 8.8 Hz), 6.80 (s, 1, ArH),
10.25 (s, 1, ArCHO); CIMS m/ z 191 (M + 1), Anal. (C11H10O3)
C, H.
1
as a white, crystalline solid: mp 267-269 °C; H NMR (free
base in CDCl3) δ 1.16 (d, 3, ArCH2CHCH3, J ) 6.3 Hz), 1.55
(bs, 2, NH2), 2.58 (m, 2, ArCH2CHCH3), 3.13 (q, 4, ArOCH2CH2),
3.22 (m, 1, ArCH2CHCH3), 4.50 (q, 4, ArOCH2CH2), 6.52 (s, 1,
ArH); CIMS m/ z 220 (M + 1), 203, 176. Anal. (C13H17
NO2‚HCl) C, H, N.
-
1-(8-Br om o-2,3,6,7-tetr ah ydr oben zo[1,2-b:4,5-b′]difu r an -
4-yl)-2-a m in op r op a n e Hyd r och lor id e (7b). A mixture of
200 mg (0.91 mmol) of 7a ‚HCl in 4 mL of glacial acetic acid
was stirred under N2, and 0.51 mL of 1.79 N Br2 in HOAc
solution was added dropwise. After 5 min the salt had
completely dissolved to give a clear orange solution. Subse-
quently, a precipitate began to form, and after 1.5 h the
volatiles were removed on the rotary evaporator. The solid
residue was taken up in 3 N HCl and washed with 2 × 20 mL
of ether. The aqueous phase was made strongly basic by the
addition of 5 N KOH and extracted with 5 × 15 mL of CH2-
Cl2. The organic fractions were combined, washed with brine,
dried (MgSO4), and filtered through Celite. Removal of solvent
under reduced pressure gave 199 mg (67%) of the free amine
as a pale yellow oil. The hydrochloride salt was formed by
the addition of 1 N HCl in anhydrous ethanol, and, after
solvent removal, this was crystallized from ethanol-ethyl
acetate to yield 7b‚HCl as a white crystalline solid: mp 244-
245 °C; 1H NMR (free base in CDCl3) δ 1.16 (d, 3, ArCH2-
CHCH3, J ) 6.3 Hz), 1.78 (bs, 2, NH2), 2.51 (t, 2, ArCH2-
CHCH3), 3.23 (m, 4, ArOCH2CH2), superimposed upon 3.25
(m, 1, ArCH2CHCH3), 4.60 (t, 2, ArOCH2CH2, J ) 8.8 Hz), 4.65
(t, 2, ArOCH2CH2, J ) 8.8 Hz); CIMS m/ z 298 (M + 1), 281,
254, 218. Anal. (C13H16NO2Br‚HCl) C, H, N.
4-(2-Nitr o-1-eth en yl)-2,3,6,7-tetr a h yd r oben zo[1,2-b:4,5-
b′]d ifu r a n (13). A mixture of 3.2 g (0.017 mol) of the aldehyde
11, 1.3 g (0.017 mol) of ammonium acetate, and 8 mL of
nitromethane was stirred under nitrogen while heating over
a 96 °C oil bath for 1 h. The nitromethane was then removed
under reduced pressure, and the solid residue was partitioned
between CH2Cl2 and water. The layers were separated, and
the organic phase was washed with 3 × 50 mL of 3 N HCl, 2
× 50 mL of water, and 50 mL of brine. The ether solution
4-(2-Nit r o-1-p r op en yl)-2,3,6,7-t et r a h yd r ob en zo[1,2-b:
4,5-b′]d ifu r a n (12). The aldehyde 11 (2.3 g, 0.012 mol) and
ammonium acetate (0.93 g, 0.012 mol) were dissolved in 10
mL of nitroethane and stirred under a nitrogen atmosphere
at 90 °C for 15 h. The volatiles were removed in vacuo, and
the residue was taken up in ether-ethyl acetate and washed
with 3 × 50 mL of 3 M HCl, 3 × 50 mL of 5% NaHCO3, and
50 mL of brine. The organic phase was dried over MgSO4 and
filtered through Celite-silica gel, and the solvent was removed
under reduced pressure to yield a thick red oil. The oil was
purified on the Chromatotron (4 mm silica plate, CH2Cl2) to
give 1.74 g (60%) of the desired product 12 as an orange oil.
The oil was crystallized from methanol to give yellow-orange
was dried over MgSO4
and filtered through Celite, and the
solvent was removed on the rotary evaporator to give 3.7 g
(94%) of a bright red-orange solid. The solid was recrystallized
from methanol to give 3.5 g (91%) of the nitroethene 13 as
pumpkin-orange needles: mp 134 °C; 1H NMR (CDCl3) δ 3.25
(t, 2, ArOCH2CH2, J ) 8.7 Hz), 3.28 (t, 2, ArOCH2CH2, J )
8.7 Hz), 4.73 (t, 2, ArOCH2CH2, J ) 8.7 Hz), 4.75 (t, 2, ArOCH2-
CH2
, J ) 8.7 Hz), 6.76 (s, 1, ArH), 7.85 (d, 1, ArCHdCH, J )
13.4 Hz), 8.05 (d, 1, ArCHDCHNO2, J ) 13.5 Hz); CIMS m/ z
234 (M + 1), 216. Anal. (C12H11NO4) C, H, N.
1-(2,3,6,7-Tetr a h yd r oben zo[1,2-b:4,5-b′]d ifu r a n -4-yl)-2-
a m in oeth a n e h yd r och lor id e (7c). In a manner identical
with that for the reduction of compound 12 above, 3.0 g (0.013
mol) of nitroethene 13 in 100 mL of dry THF was added to a
stirred suspension of 1.31 g (0.032 mol) of LiAlH4 in 200 mL
of THF and heated at reflux for 2 h. After standard workup,
1.7 g (69%) of the free amine 7c was obtained as a yellow oil.
This was precipitated as its hydrochloride salt with the
addition of 1 N HCl in anhydrous ethanol and recrystallized
from ethanol to yield 1.68 g (54%) of 7c‚HCl as a white
1
crystals: mp 94 °C; H NMR (CDCl3) δ 2.24 (s, 3, CH3), 3.10
(t, 2, ArOCH2CH2, J ) 8.6 Hz), 3.20 (t, 2, ArOCH2CH2, J )
8.7 Hz), 4.60 (t, 4, ArOCH2CH2, J ) 8.6 Hz), 6.73 (s, 1, ArH),
7.80 (s, 1, ArCHdC); CIMS m/ z 248 (M + 1), 230, 191. Anal.
(C13H13NO4) C, H, N.
1-(2,3,6,7-Tetr a h yd r oben zo[1,2-b:4,5-b′]d ifu r a n -4-yl)-2-
a m in op r op a n e Hyd r och lor id e (7a ). A solution of 0.75 g
(3.03 mmol) of the nitropropene 12 in 25 mL of dry THF was
added dropwise to a stirred suspension of 0.31 g (7.58 mmol)
of LiAlH4 in 50 mL of dry THF under N2. The reaction mixture
was heated at reflux over an oil bath for 3.5 h and then cooled
on an ice bath and the reaction quenched by the cautious
addition of 2 mL of water in 10 mL of THF. Celite and 5 mL
1
crystalline solid: mp 295-296 °C dec; H NMR (free base in
CDCl3) δ 1.48 (bs, 2, NH2), 2.75 (t, 2, ArCH2CH2N, J ) 6.95
Hz), 2.95 (t, 2, ArCH2CH2N, J ) 6.95 Hz), 3.15 (q, 4,
ArOCH2CH2, J ) 7.4 Hz), 4.58 (q, 4, ArOCH2CH2, J ) 8.5 Hz),

2958 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
6.58 (s, 1, ArH); CIMS m/ z 206 (M + 1), 189, 176. Anal.
Monte et al.
(t, 2, ArOCH2CH2, J ) 8.6 Hz), 7.83 (s, 1, ArCHdC); CIMS
m/ z 262 (M + 1), 244, 220, 205. Anal. (C14H15NO4) C, H, N.
1-(8-Meth yl-2,3,6,7-tetr ah ydr oben zo[1,2-b:4,5-b′]difu r an -
4-yl)-2-a m in op r op a n e Hyd r och lor id e (7e). The nitropro-
pene 16 (0.50 g, 1.90 mmol) was dissolved in 40 mL of dry
THF and added dropwise over 40 min to a stirred slurry of
0.20 g (4.80 mmol) of LiAlH4 in 75 mL of dry THF. The
mixture was heated at reflux over an oil bath for 3 h, and the
reaction was quenched by the cautious addition of 10 mL of 5
N KOH while cooling over an ice bath. The reaction mixture
was filtered through a Celite pad, rinsing the filter cake well
with dichloromethane. The volatiles were removed on the
rotary evaporator, and the aqueous residue was taken up in
ether and extracted with 4 × 20 mL of 3 N HCl. The extracts
were combined and made strongly basic by the addition of 5
N KOH while cooling the mixture on an ice bath. The basic
phase was extracted with 4 × 20 mL of CH2Cl2, and the
combined extracts were washed with brine, dried (MgSO4), and
filtered through Celite. Removal of the solvent in vacuo
afforded 0.39 g (88%) of the free base 7e as a yellow oil. The
base was converted to its hydrochloride salt by adding exactly
1 equiv of HCl as an anhydrous 1 M solution in ethanol. The
salt was recrystallized from ethanol-ethyl acetate to give
7e‚HCl as very fluffy, white crystals: mp 275-276 °C; 1H NMR
(free base in CDCl3) δ 1.14 (d, 3, ArCH2CHCH3, J ) 6.3 Hz),
1.48 (bs, 2, NH2), 2.10 (s, 3, ArCH3), 2.52 (m, 2, ArCH2CHCH3),
3.19 (m, 4, ArOCH2CH2), 3.23 (m, 1, ArCH2CHCH3), 4.55 (pair
of nearly superimposed triplets, 4, ArOCH2CH2, J ) 8.7 Hz);
CIMs m/ z 234 (M + 1), 217, 190. Anal. (C14H19NO2‚HCl) C,
H, N.
P h a r m a cology Meth od s: Dr u g Discr im in a tion Stu d -
ies. The procedures for the drug discrimination assays were
essentially as described in previous reports.10,27 Twenty male
Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN)
weighing 200-220 g at the beginning of the drug discrimina-
tion study were used as subjects trained to discriminate LSD
tartrate from saline. None of the rats had previously received
drugs or behavioral training. Water was freely available in
the individual home cages, and a rationed amount of supple-
mental feed (Purina Lab Blox) was made available after
experimental sessions to maintain approximately 80% of free-
feeding weight. Lights were on from 0700 to 1900. The
laboratory and animal facility temperature was 22-24 °C, and
the relative humidity was 40-50%. Experiments were per-
formed between 0830 and 1700 each day, Monday-Friday.
Six standard operant chambers (Model E10-10RF; Coul-
bourn Instruments, Lehigh Valley, PA) consisted of modular
test cages enclosed within sound-attenuated cubicles with fans
for ventilation and background white noise. A white house
light was centered near the top of the front panel of the cage,
which was also equipped with two response levers, separated
by a food hopper (combination dipper-pellet trough, Model
E14-06, module size 1/2), all positioned 2.5 cm above the floor.
Solid state logic in an adjacent room, interfaced through a Med
Associates interface to a 486-based microcomputer, controlled
reinforcement and data acquisition with a locally written
program.
.
(C12H15NO2 HCl) C, H, N.
1-(8-Br om o-2,3,6,7-tetr ah ydr oben zo[1,2-b:4,5-b′]difu r an -
4-yl)-2-a m in oeth a n e Hyd r och lor id e (7d ). A solution of
0.51 g (2.48 mmol) of the free amine 7c in 6 mL of glacial acetic
acid was treated with 2.80 mL of a 0.885 N Br2 in HOAc
solution. The bromine color rapidly dissipated, and the
hydrobromide salt precipitated after 15 min. The mixture was
stirred for 2 h and then diluted with dry ether. The precipitate
was collected by suction filtration, rinsed well with ether on
the filter, and dissolved in 75 mL of 3 N HCl. The acidic
solution was washed with 3 × 50 mL of CH2Cl2 and then made
strongly basic with the addition of 5 N KOH. The basic phase
was extracted with 4 × 30 mL of CH2Cl2, and the extracts were
combined and washed with brine. After drying (MgSO4) and
filtration through Celite, the solvent was removed under
reduced pressure to afford 0.52 g (73%) of the free amine 7d
as a yellow oil. The hydrochloride salt was precipitated from
an ether solution by adding 1 N HCl in anhydrous ethanol.
The salt was freed of solvent under reduced pressure and
recrystallized from ethanol-ethyl acetate to give the desired
product 7d ‚HCl as a white crystalline solid: mp >310 °C; 1H
NMR (free base in CDCl3) δ 1.52 (bs, 2, NH2), 2.65 (t, 2, ArCH2-
CH2N, J ) 6.9 Hz), 2.90 (t, 2, ArCH2CH2N, J ) 6.9 Hz), 3.20
(m, 4, ArOCH2CH2), 4.60 (pair of superimposed triplets, 4,
ArOCH2CH2); CIMS m/ z 284, 286 (M + 1), 267, 269, 254, 256,
247. Anal. (C12H14NO2Br‚HCl) C, H, N.
4-Met h yl-2,3,6,7-t et r a h yd r ob en zo[1,2-b:4,5-b′]d ifu r a n
(14). To a suspension of 1.0 g of 10% Pd-C in 250 mL of
absolute ethanol in a Parr hydrogenation flask was added 7.0
g (0.037 mol) of the benzaldehyde 11. The mixture was shaken
under 60 psi of H2 for 24 h and then filtered through Celite to
remove the catalyst. Evaporation of the solvent under reduced
pressure and recrystallization of the white, solid residue from
ethyl acetate-hexane gave 6.18 g (95%) of pure product 14 as
fluffy, white crystals: mp 78 °C; 1H NMR (CDCl3) δ 2.12 (s, 3,
ArCH3), 3.07 (t, 2, ArOCH2CH2, J ) 8.7 Hz), 3.13 (t, 2,
ArOCH2CH2, J ) 8.7 Hz), 4.53 (m, 4, ArOCH2CH2), 6.62 (s, 1,
ArH); CIMS m/ z 177 (M + 1). Anal. (C11H12O2) C, H.
4-F or m yl-8-m et h yl-2,3,6,7-t et r a h yd r ob en zo[1,2-b:4,5-
b′]d ifu r a n (15). To an ice-salt bath-cooled solution of 2.60
g (0.015 mol) of 14 in 30 mL of dry CH2Cl2 was added 2.24
mL (0.019 mol) of tin(IV) chloride via syringe through septum.
The mixture was stirred under N2 for 5 min, and 1.33 mL
(0.015 mol) of R,R-dichloromethyl methyl ether was added
dropwise over 5 min. The mixture was stirred for 5 min more
and then the reaction quenched with the addition of 50 mL of
ice-water. The layers were separated, and the aqueous phase
was extracted with 3 × 20 mL of CH2Cl2. The combined
organic extracts were washed with 3 × 50 mL of 6 N HCl, 2 ×
50 mL of 5% sodium bicarbonate, and brine, dried over MgSO4,
and filtered through a pad of Celite and silica gel. Removal
of solvent under reduced pressure gave a bright yellow solid
that weighed 2.99 g (99%). The solid was recrystallized from
ethyl acetate-hexane to give 2.82 g (94%) of the product 15
1
as yellow needles: mp 152 °C; H NMR (CDCl3) δ 2.18 (s, 3,
ArCH3), 3.09 (t, 2, ArOCH2CH2, J ) 8.6 Hz), 3.45 (t, 2,
ArOCH2CH2, J ) 8.7 Hz), 4.57 (t, 2, ArOCH2CH2, J ) 8.7 Hz),
4.70 (t, 2, ArOCH2CH2, J ) 8.7 Hz), 10.21 (s, 1, ArCHO); CIMS
m/ z 205 (M + 1). Anal. (C12H12O3) C, H.
A fixed ratio (FR) 50 schedule of food reinforcement (Bioserv
45 mg dustless pellets) in a two-lever paradigm was used. The
drug discrimination procedure details have been described
elsewhere.35,36 After habituation to the experimental condi-
tions (1 week after isolation in the individual home cages and
at the beginning of the food deprivation), the rats’ initial
shaping was started. During the first two to three sessions,
rats were trained only to associate a characteristic noise (click)
after lever pressing with a delivered food pellet (without drug
injections). Initially, rats were shaped to lever press on an
FR1 schedule so that one food pellet was dispensed for each
press. One-half of the rats were trained on drug-L (left),
saline-R (right) and the other one-half on drug-R, saline-L to
avoid positional preference. Training sessions lasted 15 min
and were conducted at the same time each day. Levers were
cleaned between animals with 10% ethanol solution to avoid
olfactory cues. Only one appropriate lever was present during
the first 10 sessions of initial learning (after beginning to
administer saline or training drug ip 30 min before sessions).
8-Meth yl-4-(2-n itr o-1-pr open yl)-2,3,6,7-tetr ah ydr oben zo-
[1,2-b:4,5-b′]d ifu r a n (16). The aldehyde 15 (1.0 g, 4.90 mmol)
was stirred with 0.40 g (4.90 mmol) of ammonium acetate in
5 mL of nitroethane over an 80 °C oil bath for 19 h. The
volatiles were removed on the rotary evaporator, and the
orange residue was taken up in ether and washed with 4 ×
30 mL of 3 N HCl, 2 × 30 mL of 5% NaHCO3, and brine. The
organic phase was dried over MgSO4, filtered through a pad
of Celite-silica gel, and concentrated on the rotary evaporator
to give 1.2 g of an impure (by TLC) orange gum. The crude
material was purified on the Chromatotron (4 mm silica plate,
CH2Cl2) to afford 0.60 g (46%) of pure nitropropene 16 as a
bright, pumpkin-orange solid that was crystallized from
methanol: mp 105 °C; 1H NMR (CDCl3) δ 2.12 (s, 3,
ArCHdCCH3), 2.22 (s, 3, ArCH3), 3.11 (m, 4, ArOCH2CH2),
4.59 (t, 2, ArOCH2CH2, J ) 8.6 Hz) superimposed upon 4.60

Dihydrobenzofuran Analogues of Hallucinogens
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2959
Afterwards both levers were present during all following
phases of training, but reinforcements were delivered only
after responses on the appropriate lever. Presses on the
incorrect lever had no programmed consequences. As respond-
ing rates stabilized (during the next 15 sessions), the schedule
of reinforcement was gradually increased to an FR50. Once
at the FR50, training continued until an accuracy of at least
85% (no. of correct presses × 100/no. of total presses) was
attained for 8 of 10 consecutive sessions (approximately 40-
60 sessions). Once criterion performance was attained, test
sessions were interspersed between training sessions, either
one or two times per week. At least one drug and one saline
session separated each test session. Rats were required to
maintain the 85% correct responding criterion on training days
in order to be tested. In addition, test data were discarded
when the accuracy criterion of 85% was not achieved on the
two training sessions following a test session. Test drugs were
administered ip 30 min prior to the sessions; test sessions were
run under conditions of extinction, with rats removed from the
operant chamber when 50 presses were emitted on one lever.
If 50 presses on one lever were not completed within 5 min,
the session was ended and scored as a disruption. Treatments
were randomized at the beginning of the study.
All experiments were performed with triplicate determina-
tions using the appropriate buffer to which 200-400 µg of
protein was added, giving a final volume of 1 mL. The tubes
were allowed to equilibrate for 15 min at 37 °C before filtering
through Whatman GF/C filters using a cell harvester (Brandel,
Gaithersburg, MD) followed by two 5 mL washes using ice-
cold Tris buffer. Specific binding was defined as that displace-
able with 10 µM cinanserin in the [3H]ketanserin binding study
and with 10 µM 5-HT in the [3H]-8-OH-DPAT binding study.
Filters were air-dried, placed into scintillation vials with 10
mL of Ecolite scintillation cocktail, allowed to sit overnight
before counting at an efficiency of 37% for tritium, and directly
counted in a gamma counter for [125I]ligand at an efficiency of
79.4%.
Ra d ioliga n d Com p etition Exp er im en ts Usin g Clon ed
Hu m a n Recep tor s. All chemicals were obtained from the
sources previously described.39 [3H]-5-HT was purchased from
DuPont-NEN (Wilmington, DE) or Amersham Corp. (Arlington
Heights, IL) at 22.8-26.7 or 81-91 Ci/mmol, respectively. [125I]-
DOI (2200 Ci/mmol), [3H]rauwolscine (70-90 Ci/mmol), and
[3H]ketanserin (60-78.7 Ci/mmol) were purchased from Du-
pont-NEN (Wilmington, DE).
Mem br a n e P r ep a r a tion fr om Tr a n sfor m ed Cell Lin es.
Membranes were prepared essentially as previously de-
scribed39 using AV12 cell lines (Syrian hamster fibroblast,
ATCC no. CRL 9595) stably transformed with the human
5-HT2A, 5-HT2B, or 5-HT2C receptor. In brief, cells expressing
the receptor of interest were grown in suspension and har-
vested by centrifugation. The cell pellets were then resus-
pended in a minimal volume of a hypotonic buffer, 50 mM Tris
HCl, pH 7.4, and frozen at -70 °C until needed. On the day
of the assay, the membrane suspension was thawed and
diluted to 35 mL/0.5 × 109 cells, original cell number, with 50
mM Tris HCl, pH 7.4. The combination of hypotonic buffer
and vortexing was sufficient to lyse the cells for the membrane
preparation. After vortexing, the preparation was centrifuged
at 39000g for 10 min at 4 °C, and the resulting membrane
pellet was resuspended and incubated at 37 °C for 10 min and
then centrifuged at 39000g for 10 min at 4 °C. This pellet
was resuspended and centrifuged one more time, and the final
membrane pellet was resupended (using a Tissumizer, setting
65 for 15 s) in Tris HCl, pH 7.4, for cells expressing the human
5-HT2B receptor, in Tris HCl, pH 7.4, containing MgCl2 and
EDTA for [125I]DOI binding to 5-HT2A or 5-HT2C receptors, or
in Tris HCl, pH 7.6, for [3H]ketanserin and [3H]mesulergine
binding to 5-HT2A and 5-HT2C receptors respectively.
5-HT2B [3H]-5-HT Bin d in g Stu d ies. Human 5-HT2B re-
ceptor binding assays using [3H]-5-HT were performed as
previously described.39 The assay was automated using a
Biomek 1000 instrument (Beckman Instruments, Fullerton,
CA). [3H]-5-HT in Tris HCl containing CaCl2, pargyline, and
L-ascorbic acid, adjusted to pH 7.4, was added to drug
dilutions, spanning 6 log units, in water. Then 200 µL of
membrane suspension (approximately 100-150 µg of protein)
was added with mixing and incubated for 15 min at 37 °C.
The total incubation volume was 800 µL, and all incubations
were performed in triplicate. The final concentration of CaCl2,
pargyline, Tris, and L-ascorbic acid was 3 mM, 10 µM, 50 mM,
and 0.1% resptively. The assay was terminated by vacuum
filtration through Whatman GF/B filters which had been
presoaked with 0.5% poly(ethylenimine) (w/v) and precooled
with 4 mL of ice-cold wash buffer (50 mM Tris HCl, pH 7.4),
using a Brandel cell harvester (Model MB-48R, Brandel,
Gaithersburg, MD). The filters were then washed rapidly four
times with 1 mL of ice-cold wash buffer. The amount of [3H]-
5-HT trapped on the filters was determined by liquid scintil-
lation spectrometry (Ready Protein, LS 6000IC, Beckman
Instruments, Fullerton, CA). The final [3H]-5-HT concentra-
tion for competition studies was approximately 2 nM (range
) 1.7-2.5 nM). The actual free radioligand concentration was
determined by sampling the supernatant of identical tubes
where bound ligand was removed by centrifugation. Nonspe-
cific binding was defined with 10 µM 5-HT or 10 µM 1-naph-
thylpiperazine (1-NP). The amount of protein was determined
by the method of Bradford,40 with bovine serum albumin as
the standard.
The training drug was (+)-lysergic acid diethylamide tar-
trate (LSD; 0.08 mg/kg, 186 nmol/kg; NIDA). All drugs were
dissolved in 0.9% saline and were injected intraperitoneally
in a volume of 1 mL/kg 30 min before the sessions.
Data from the drug discrimination study were scored in a
quantal fashion, with the lever on which the rat first emitted
50 presses in a test session scored as the “selected” lever. The
percentage of rats selecting the drug lever (% SDL) for each
dose of test compound was determined. The degree of sub-
stitution was determined by the maximum % SDL for all doses
of the test drug. “No substitution” is defined as 59% SDL or
less, and “partial” substitution is 60-79% SDL. If the drug
was one which completely substituted for the training drug
(at least one dose resulted in a % SDL ) 80% or higher), the
method of Litchfield and Wilcoxon37 was used to determine
the ED50 (log-probit analysis as the dose producing 50 percent
drug-lever responding) and 95% confidence interval (95% CI).
This method also allowed for tests of parallelism between
dose-response curves of the drug and the training drug. If
50% or more of the animals tested were disrupted at a dose
where the nondisrupted rats gave 80% SDL, no ED50 was
calculated.
Ra d ioliga n d Com p etition Assa ys in Ra t Br a in Hom o-
gen a te. Male Sprague-Dawley rats (Harlan Laboratories,
Indianapolis, IN) weighing 175-199 g were used. The animals
were kept in groups of 5 rats/cage, at the same conditions
described above, but with free access to food and water.
[3H]Ketanserin and [3H]-8-OH-DPAT were purchased from
New England Nuclear (Boston, MA) at specific activities of 61
and 135.5-216 Ci/mmol, respectively. (+)-LSD tartrate was
obtained from the National Institute on Drug Abuse. Cin-
anserin was a gift from the SQUIBB Institute for Medical
Research, and 5-HT was purchased from Sigma (St. Louis,
MO).
The procedures of J ohnson et al.38 were employed. Briefly,
the frontal cortex or hippocampal brain regions from 20 to 40
rats were pooled and homogenized (Brinkman Polytron, setting
6 for 2 × 20 s) in 4 or 8 vol of 0.32 M sucrose for frontal cortex
or hippocampus, respectively. The homogenates were centri-
fuged at 36000g for 10 min, and the resulting pellets were
resuspended in the same volume of sucrose. Separate aliquots
of tissue suspension were then frozen at -70 °C until assay.
For each separate experiment, a tissue aliquot was thawed
slowly and diluted 1:25 with 50 mM Tris HCl (pH ) 7.4). The
homogenate was then incubated at 37 °C for 10 min and
centrifuged twice at 36500g for 20 min with an intermittent
wash. The resulting pellet was resuspended in 50 mM Tris
HCl with 0.5 mM Na2EDTA, 0.1% Na ascorbate, and 10 mM
pargyline HCl (pH ) 7.4). In experiments with [3H]ketanserin,
either 10 mM MgCl2 or 5.7 mM CaCl2 was included, respec-
tively. A second preincubation for 10 min at 37 °C was
conducted, and the tissues were then cooled in an ice bath.

2960 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
5-HT2A a n d 5-HT2C
125I]DOI Bin d in g Stu d ies. Human
Monte et al.
(6) Sanders-Bush, E.; Burris, K. D.; Knoth, K. Lysergic Acid
Diethylamide and 2,5-Dimethoxy-4-methylamphetamine are
Partial Agonists at Serotonin Receptors Linked to Phospho-
inositide Hydrolysis. J . Pharmacol. Exp. Ther. 1988, 246, 924-
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(7) Sanders-Bush, E. Neurochemical Evidence That Hallucinogenic
Drugs Are 5-HT1C Receptor Agonists: What Next? In NIDA
Research Monograph 146, Hallucinogens: An Update; Lin, G.
C., Glennon, R. A., Eds.; NIH Publication No. 94-3872; NIDA:
Rockville, 1994; pp 203-213.
(8) Seggel, M. R.; Yousif, M. Y.; Lyon, R. A.; Titeler, M.; Roth, B.
L.; Suba, E. A.; Glennon, R. A. A Structure-Affinity Study of
the Binding of 4-Substituted Analogs of 1-(2,5-Dimethoxyphen-
yl)-2-aminopropane at 5-HT2 Serotonin Receptors. J . Med. Chem.
1990, 33, 1032-1036.
(9) Titeler, M.; Lyon, R. A.; Glennon, R. A. Radioligand Binding
Evidence Implicates the Brain 5-HT2 Receptor as a Site of Action
for LSD and Phenylisopropylamine Hallucinogens. Psychophar-
macology 1988, 94, 213-216.
(10) Nichols, D. E.; Frescas, S.; Marona-Lewicka, D.; Huang, X.; Roth,
B. L.; Gudelsky, G. A.; Nash, J . F. 1-(2,5-Dimethoxy-4-(trifluo-
romethyl)phenyl)-2-aminopropane: A Potent Serotonin 5-HT2A/2C
Agonist. J . Med. Chem. 1994, 37, 4346-4351.
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A Correlation with Serotonin Receptor Affinities. Psychophar-
macology 1980, 68, 155-158.
(12) Colpaert, F. C.; Niemegeers, C. J . E.; J anssen, P. A. J . A Drug
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tryptamine Antagonists and of Pirenpirone, a LSD-Antagonist.
J . Pharmacol. Exp. Ther. 1982, 16, 206-214.
(13) Nichols, D. E.; Oberlender, R.; McKenna, D. J . Stereochemical
Aspects of Hallucinogenesis. In Biochemistry and Physiology of
Substance Abuse; Watson, R., Ed.; CRC Press: Boca Raton, FL,
1991; pp 1-39.
[
5-HT2A or 5-HT2C binding studies were performed essentially
as described for [3H]-5-HT binding to the 5-HT2B receptor with
the following exceptions. The assay buffer contained, in final
concentration, 10 µM pargyline, 9.75 mM MgCl2, 0.5 mM
(ethylenedinitrilo)tetraacetic acid, disodium salt (EDTA), 0.1%
sodium ascorbate and 50 mM Tris HCl, pH 7.4. Incubations
were performed at 37 °C for 30 min with approximately 40
and 30 µg of protein for the 5-HT2A and 5-HT2C receptors,
respectively, and then filtered and washed as described above.
The amount of [125I]DOI trapped on the filters was determined
using a gamma counter. Nonspecific binding was determined
with 10 µM mianserin for 5-HT2C and 1 µM ketanserin for
5-HT2A receptors. The final concentration of [125I]DOI was
approximately 0.07-0.15 nM.
[3H]Keta n ser in Bin d in g to th e Hu m a n 5-HT2A Recep -
tor . Membranes were prepared as described above, and the
assay conditions were essentially as previously described.41
Assays consisted of 0.8 mL total volume containing 50 mM
Tris HCl, 100 nM prazosin (to block potential binding of [3H]-
ketanserin to R1-adrenergic receptors), 0.4-0.5 nM [3H]ket-
anserin, and varying concentrations of the competing com-
pound of interest (final pH 7.6). Mianserin, 3 µM, was used
to define the level of nonspecitic binding. Tubes were incu-
bated at 37 °C for 15 min and then rapidly filtered and washed
as described above. The amount of [3H]ketanserin trapped on
the filters was determined by liquid scintillation spectrometry.
[3H]Ra u w olscin e Bin d in g to th e Hu m a n 5-HT2B Re-
cep tor . This assay is based on
a previously described
procedure.42 Membrane preparation and the filtration binding
assay were essentially as described above. Conditions specific
to this assay were as follows (all concentrations given as final
concentrations): 2 nM [3H]rauwolscine, 500 nM efaroxan (to
mask rauwolscine binding to R2-adrenergic receptors), and 50
mM Tris HCl, pH 7.4. Tubes were incubated at 37 °C for 20
min and then rapidly filtered as described above. Nonspecific
binding was defined in the presence of 10 µM 1-naphthylpip-
erazine.
(14) Nichols, D. E. Medicinal Chemistry and Structure-Activity
Relationships. In amphetamine and Its Analogs; Cho, A. K.,
Segal, D. S., Eds.; Academic Press, Inc.: San Diego, 1994; pp
3-41.
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Howell, H. G.; Partyka, R. A.; Dyer, D. C. Potential Psychoto-
mimetics. 2. Rigid Analogs of 2,5-Dimethoxy-4-methylphenyl-
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mine, Adrenaline, Serotonin, Acetylcholine, and Mammalian
Opsin Receptors. J . Med. Chem. 1992, 35, 3448-3462.
(17) Westkaemper, R. B.; Glennon, R. A. Molecular Modeling of the
Interaction of LSD and Other Hallucinogens With 5-HT2 Recep-
tors. In NIDA Research Monograph 146, Hallucinogens: An
Update; Lin, G. C., Glennon, R. A., Eds.; NIH Publication No.
94-3872; NIDA: Rockville, 1994; pp 263-283.
(18) Nichols, D. E.; Hoffman, A. J .; Oberlender, R. A.; Riggs, R. M.
Synthesis and Evaluation of 2,3-Dihydrobenzofuran Analogues
of the Hallucinogen 1-(2,5-Dimethoxy-4-methylphenyl)-2-ami-
nopropane: Drug Discrimination Studies in Rats. J . Med. Chem.
1986, 29, 302-304.
(19) Nichols, D. E.; Snyder, S. E.; Oberlender, R.; J ohnson, M. P.;
Huang, X. 2,3-Dihydrobenzofuran Analogues of Hallucinogenic
phenethylamines. J . Med. Chem. 1991, 34, 276-281.
(20) Monte, A. P.; Marona-Lewicka, D.; Cozzi, N. V.; Nelson, D. L.;
Nichols, D. E. Conformationally Restricted Tetrahydro-1-ben-
zoxepin Analogues of Hallucinogenic Phenethylamines. Med.
Chem. Res. 1995, 5, 651-663.
[3H]Mesu ler gin e Bin d in g to th e Hu m a n 5-HT2C Recep -
tor . This assay was adapted from that described by Pazos et
al.43 Membranes were prepared as described above. Final
concentrations for the 0.8 mL assays were 0.74-0.82 nM [3H]-
mesulergine, varying concentrations of competing compound,
and 50 nM Tris HCl, final pH 7.6. Nonspecific binding was
determined using 3 mM mianserin. Assay tubes were incu-
bated for 30 min at 37 °C, after which the samples were filtered
and washed, and radioactivity was determined as for the [3H]-
ketanserin binding assay described above.
Sta tistica l An a lysis. Nonlinear regression analysis for the
competition curves was performed as previously described39
to determine IC50 values. These were converted to Ki values
by the method of Cheng and Prusoff.44
(21) Blade´-Font, A.; Rocabayera, T. M. Synthesis of Dihydrobenzo-
furans from Phenolic Mannich Bases and their Quaternized
Derivatives. J . Chem. Soc., Perkin Trans. I 1982, 841-848.
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1976, 41, 1184-1186.
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Ack n ow led gm en t. A. Monte was supported in part
by a National Institutes of Health-National Institute of
General Medical Sciences Predoctoral Training Grant
in Chemical Pharmacology. This work was supported
by U.S. Public Health Service grant DA02189.
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Synthesis and Pharmacological Examination of Benzofuran,
Indan, and Tetralin Analogues of 3,4-(Methylenedioxy)amphet-
amine. J . Med. Chem. 1993, 36, 3700-3706.
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F.; Morin, R. D. Asymmetric Synthesis of Psychotomimetic
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J . Med. Chem. 1995, 38, 958-966.
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