Synthesis and serotonin receptor affinities of a series of trans-2- (indol-3-yl)cyclopropylamine derivatives

Article abstract of DOI:10.1021/jm980318q

A series of four racemic ring-substituted trans-2-(indol-3- yl)cyclopropylamine derivatives was synthesized and tested for affinity at the 5-HT(1A) receptor, by competition with [³H]-8-OH-DPAT in rat hippocampal homogenates, and for affinity at

Full text of DOI:10.1021/jm980318q

J . Med. Chem. 1998, 41, 4995-5001
4995
Syn th esis a n d Ser oton in Recep tor Affin ities of a Ser ies of
tr a n s-2-(In d ol-3-yl)cyclop r op yla m in e Der iva tives
Suwanna Vangveravong, Arthi Kanthasamy, Virginia L. Lucaites,‡ 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 May 21, 1998
A series of four racemic ring-substituted trans-2-(indol-3-yl)cyclopropylamine derivatives was
synthesized and tested for affinity at the 5-HT1A receptor, by competition with [3H]-8-OH-
DPAT in rat hippocampal homogenates, and for affinity at the agonist-labeled cloned human
5-HT2A, 5-HT2B, and 5-HT2C receptor subtypes. None of the compounds had high affinity for
the 5-HT1A receptor, with the 5-methoxy substitution being most potent (40 nM). At the 5-HT2A
and 5-HT2B receptor isoforms, most of the compounds lacked high affinity. At the 5-HT2C
receptor, however, affinities were considerably higher. The 5-fluoro-substituted compound was
most potent, with a Ki at the 5-HT2C receptor of 1.9 nM. In addition, the 1R,2S-(-) and 1S,2R-
(+) enantiomers of the unsubstituted compound were also evaluated at the 5-HT2 isoforms.
While the 1R,2S enantiomer had higher affinity at the 5-HT2A and 5-HT2B sites, the 1S,2R
isomer had highest affinity at the 5-HT2C receptor. This reversal of stereoselectivity may offer
leads to the development of a selective 5-HT2C receptor agonist. The cyclopropylamine moiety
therefore appears to be a good strategy for rigidification of the ethylamine side chain only for
tryptamines that bind to the 5-HT2C receptor isoform.
In tr od u ction
to identify the binding conformation. Although many
types of strategies have been employed in designing
conformationally rigid molecules,8-14 probably one of
the simplest is to incorporate an ethylamine side chain
into a cyclopropyl ring. This approach introduces
only minimal additional molecular bulk, while the
degrees of conformational freedom are effectively re-
duced. Cyclopropane analogues have been used for
many years and have proven useful in obtaining infor-
mation regarding the active conformations of various
compounds.8-10,13,15 Thus, this report describes the
synthesis of trans-2-(indol-3-yl)cyclopropylamines 1a -
d . These compounds were evaluated for affinity at the
rat brain 5-HT1A receptor, as well as at the cloned
human 5-HT2A, 5-HT2B, and 5-HT2C receptors, known
to be important targets for a number of tryptamine
agonists.
A number of tryptamine derivatives have been iso-
lated from natural sources or prepared by chemical
synthesis and evaluated for their biological activity.1-6
One of the more important and well-studied of these,
5-hydroxytryptamine (serotonin, 5-HT), is a neuro-
transmitter with numerous physiological functions in
the central and peripheral nervous systems. Many
tryptamines, such as N,N-dimethyl- and N,N-diethyl-
tryptamine, 5-methoxy-N,N-dimethyltryptamine, and
4-hydroxy-N,N-dimethyltryptamine (psilocin), have psy-
choactive properties similar to those of LSD.7 Others,
such as R-methyl- and R-ethyltryptamine, have various
pharmacological properties, for example, as inhibitors
of 5-HT reuptake.3
Identification of the conformations of drugs at their
biological targets remains an important topic of re-
search. Little is known regarding the mutual confor-
mational changes induced in a ligand and its receptor
during their interaction. Although it is sometimes
appealing to speculate that the molecule may bind in
its preferred solution or solid-state conformation, the
structural demands of the receptor may lead to binding
of the molecule in a conformation that would not be
favored in its nonbound state. One way to circumvent
this uncertainty is to evaluate the pharmacological
potencies of conformationally rigid analogues. With
respect to tryptamines, which are representatives of the
arylethylamine class of neurotransmitters, the confor-
mational flexibility of the side chain makes it difficult
Ch em istr y
Although cyclopropyl analogues of tryptamines have
been obvious targets for structure-activity relationship
studies for many years, the unique chemistry of indoles
has precluded many of the usual chemical approaches
employed with more stable aromatic templates. Fur-
* Address correspondence to: Dr. David E. Nichols at Purdue
University. Phone: (765)-494-1461. Fax: (765)-494-1414. E-mail:
drdave@pharmacy.purdue.edu.
‡ Eli Lilly and Co.
10.1021/jm980318q CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/06/1998

4996 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Vangveravong et al.
Sch em e 1a
a
Reagents: (a) p-toluenesulfonyl chloride, Et3N; (b) malonic acid, pyridine, piperidine; (c) i. CH2N2, ii. CH2N2/Pd(OAc)2; (d) NaOH/
MeOH, then HCl; (e) i. Et3N, ClCOOC2H5, NaN3, ii. benzyl alcohol, heat; (f) H2, Pd-C, EtOH; (g) Na-Hg, Na2HPO4, MeOH.
Ta ble 1. Affinities of Compounds 1a -d at 5-HT1A Receptors
thermore, arylcyclopropylamines with electron-rich aro-
in Rat Hippocampal Homogenate Labeled with
[3H]-8-OH-DPATa
matic systems tend to be chemically unstable. These,
and other obstacles, impeded our work on indolylcyclo-
propylamines until we noted the synthesis of phenyl-
cyclopropylamines published by Arvidsson et al.13 It
was reasoned that their general approach offered a
relatively facile entry into the desired indole compounds.
Indeed, this proved to be the case, and we initially used
that precedent as a basis for an asymmetric synthesis
of the two enantiomers of 1a .16
compound
Ki (nM) 5-HT1A
(1R,2S)-(-)-1a 16
(1S,2R)-(+)-1a 16
(()-1b
258 ( 23
810 ( 52
1528 ( 61
40.1 ( 2.4
258 ( 15
2.1 ( 0.1
(()-1c
(()-1d
8-OH-DPAT
a
Data are expressed as mean Ki values (nM) ( SEM for 3-4
separate experiments.
As shown in Scheme 1, indole-3-carboxaldehydes
2a -d were first protected as the N(1)-p-toluenesulfonyl
derivatives 3a -d . This protection was key to the
success of the synthesis, not only because it enhanced
reactivity of the carboxaldehyde but also because at-
tempts to deprotect prior to the final step led to
intermediates that proved so unstable they could not
be isolated and purified. The protected indolecarbox-
aldehydes were condensed with malonic acid to afford
the trans-â-indol-3-ylacrylic acids 4a -d . The acids were
converted to their methyl esters, and cyclopropanation
with retention of trans stereochemistry was then ac-
complished using ethereal diazomethane in the presence
of catalytic palladium diacetate.13 Hydrolysis of the
cyclopropyl methyl esters provided acids 6a -d , which
were subjected to conditions of the Curtius rearrange-
ment. The intermediate isocyanates were trapped with
benzyl alcohol, followed by catalytic hydrogenation to
give the amines 8a -d . N-Detosylation as the final step
was accomplished using sodium amalgam under buff-
ered conditions to afford the desired indolecyclopropy-
lamines 1a -d .
Ta ble 2. Affinities of Compounds 1a -d at Cloned Human
5-HT2 Receptorsa
[
125I]DOI
5-HT2A
[3H]-5-HT
5-HT2B
[
125I]DOI
5-HT2C
compound
(()-1a
164 ( 37
98.2 ( 7. 5
132 ( 3
45.9 ( 7.6
30.9 ( 2.5
10.2 ( 3.2
41.0 ( 13.8
2.2 ( 0.3
58.3 ( 18.2
58.5 ( 4.0
93.5 ( 19.6
64.9 ( 5.7
38.1 ( 5.1
5.7 ( 0.3
29.9 ( 3.3
44.1 ( 5.5
21.3 ( 4.0
11.8 ( 1.9
17.8 ( 1.2
1.9 ( 0.5
9.3 ( 3.4
1.0 ( 0.1
(1R,2S)-(-)-1a 16
(1S,2R)-(+)-1a 16
(()-1b
(()-1c
(()-1d
R-methyltryptamine
5-methoxy-R-
methyltryptamine
87.0 ( 32.2
8.3 ( 0.5
a
Data are expressed as mean Ki values (nM) ( SEM for 3-4
separate experiments.
site, but the value obtained here is considerably lower
than the reported affinity for the flexible congener
5-methoxytryptamine.17 The order of affinity is consis-
tent, however, with the conclusion that 5-substituted
tryptamines are more potent at 5-HT1A receptors.18
Radioreceptor competition data at the agonist-labeled
cloned human 5-HT2 receptors are reported in Table 2.
While in general the affinities were low for these sites,
all the compounds had highest affinity at the 5-HT2C
receptor. The affinities of 1a ,c at the 5-HT2A receptor
are significantly lower than for their flexible analogues,
R-methyltryptamine and 5-methoxy-R-methyltryptamine.
This result contrasts sharply with the affinity at the
5-HT2A site of a ring-substituted phenylcyclopropy-
lamine, when compared to its R-methylphenethylamine
congener. For example, we have previously shown that
trans-2,5-dimethoxy-4-methylphenylcyclopropylamine has
very high affinity for 5-HT2A sites.19 It would appear
therefore, at least in the present tryptamine series, that
Ra d ioliga n d Com p etition Stu d ies
Serotonin receptor affinity data were obtained for the
four target compounds in rat brain homogenate at the
5-HT1A receptor labeled with [3H]-8-OH-DPAT. In
addition, affinities were measured for the new com-
pounds, as well as the enantiomers of 1a , at the cloned
human 5-HT2 receptor isoforms.
Resu lts a n d Discu ssion
As seen in Table 1, none of the compounds had
particularly high affinity at the 5-HT1A receptor. The
5-methoxy compound 1c had highest affinity at this

trans-2-(Indol-3-yl)cyclopropylamine Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 4997
chemical ionization mass spectra (CIMS) were determined on
the Purdue University Department of Medicinal Chemistry’s
Finnegan 4000 quadrupole mass spectrometer, using isobutane
as the reagent gas and are reported as m/z (relative intensity).
Fast-atom bombardment mass spectra (FABMS) were obtained
on a Kratos MS-50 spectrometer. Elemental analyses were
obtained from Purdue Microanalytical Laboratory and were
within 0.4% of the calculated values, unless otherwise noted.
A Parr apparatus was used for hydrogenations.
Gen er a l P r oced u r e for th e P r ep a r a tion of 1-(p-Tolyl-
su lfon yl)in d ole-3-ca r boxa ld eh yd es 3a -d . A mixture of
the appropriate indole-3-carboxaldehyde (10.3 mmol) and
p-toluenesulfonyl chloride (15.7 mmol) in triethylamine (25
mL) was heated at 90-95 °C for 1 h. The reaction mixture
was poured into ice-cold water (35 mL), stored in a refrigerator
for 1 h, and then filtered. The insoluble solid was washed with
water and air-dried. Recrystallization from ethyl acetate gave
the respective pure 1-(p-tolylsulfonyl)indole-3-carboxaldehydes.
1-(p-Tolylsu lfon yl)in d ole-3-ca r boxa ld eh yd e, 3a . This
material was obtained in 80% yield as a white powder: mp
148-150 °C, lit.22 mp 148-150 °C; 1H NMR δ 10.09 (s, 1H,
CHO), 8.26 (m, 1H, ArH), 8.22 (s, 1H, ArH), 7.96 (m, 1H, ArH),
7.85 (d, 2H, ArH, J ) 8.6 Hz), 7.38 (m, 2H, ArH), 7.30 (d, 2H,
ArH, J ) 8.6 Hz), 2.38 (s, 3H, ArCH3); CIMS 300 (MH+).
4-Me t h oxy-1-(p -t olylsu lfon yl)in d ole -3-ca r b oxa ld e -
h yd e, 3b. This material was obtained in 90% yield as off-
white crystals: mp 172-173 °C; 1H NMR δ 10.49 (s, 1H, CHO),
8.23 (s, 1H, ArH), 7.83 (d, 2H, ArH, J ) 8.0 Hz), 7.61 (d, 1H,
ArH, J ) 8.0 Hz), 7.31 (t, 1H, ArH, J ) 8.0 Hz), 7.27 (d, 2H,
ArH, J ) 8.0 Hz), 6.78 (d, 1H, ArH, J ) 8.0 Hz), 3.95 (s, 3H,
ArOCH3), 2.36 (s, 3H, ArCH3); CIMS 330 (MH+). Anal.
(C17H15NO4S) C, H, N.
the cyclopropylamine fails to provide a satisfactory
replacement for the ethylamine side chain at the 5-HT2A
receptor.
The 5-fluoro compound 1d had high affinity at all
three 5-HT2 sites and was extremely potent at the
5-HT2C receptor. The recent report that fluorinated
tryptamines and isotryptamines are potent and selective
ligands for 5-HT2C receptors20 presents a parallel to
compound 1d . The present results reinforce the conclu-
sion of Bo¨s et al.20 that halogenated tryptamines have
higher affinities for 5-HT2C than for 5-HT2A receptors.
In addition, Bo¨s et al.20 note that compounds with the
S absolute configuration have higher affinity at the
5-HT2C receptor. Our previous study of enantiomeric
R-methyltryptamines showed that the S-(+) enanti-
omers of 5-oxygenated compounds had higher affinity
than their R isomers at the 5-HT2A site, while the order
of affinity was reversed for the unsubstituted and
4-hydroxy compounds.21 In that same study, we also
found that at the 5-HT1B receptor, R enantiomers
generally had higher affinity than their S isomers. The
order of affinities obtained at the 5-HT2A receptor for
the (+) and (-) isomers of 1a in the present study is in
agreement with our earlier study of the enantiomers of
unsubstituted R-methyltryptamine (1R,2S > 1S,2R; R
> S), but at the 5-HT2C receptor the stereoselectivity is
reversed.
This observation clearly merits further investigation
with other optically active compounds. The high degree
(79%) of structural homology between the transmem-
brane regions of the 5-HT2A and 5-HT2C receptors has
made it difficult to design agonist molecular probes
specific for one site or the other. To our knowledge
however, no one has so far exploited a possible reversal
of binding stereoselectivity to search for 5-HT2C ligands
among the various molecules known to possess high
affinity for both the 5-HT2A and 5-HT2C receptors.
These data, combined with our earlier studies,19,21 also
suggest the possibility of differences in binding orienta-
tions between the phenethylamines and the tryptamines
at various serotonin receptor isoforms.
5-Me t h oxy-1-(p -t olylsu lfon yl)in d ole -3-ca r b oxa ld e -
h yd e, 3c. This material was obtained in 90% yield as an off-
white powder: mp 124-126 °C, lit.23 mp 128-129 °C; 1H NMR
δ 10.06 (s, 1H, CHO), 8.17 (s, 1H, ArH), 7.82 (d, 3H, ArH, J )
8.8 Hz), 7.70 (d, 1H, ArH, J ) 2.3 Hz), 7.28 (d, 2H, ArH, J )
8.8 Hz), 7.00 (dd, 1H, ArH, J ) 9.0 and 2.6 Hz), 3.85 (s, 3H,
ArOCH3), 2.37 (s, 3H, ArCH3); CIMS 330 (MH+).
5-F lu or o-1-(p-t olylsu lfon yl)in d ole-3-ca r boxa ld eh yd e,
3d . This material was obtained in 82% yield as a white
powder: mp 226-227 °C; 1H NMR δ 10.06 (s, 1H, CHO), 8.25
(s, 1H, ArH), 7.93 (dd, 1H, ArH, J ) 8.7 and 2.5 Hz), 7.90 (dd,
1H, ArH, J ) 9.1 and 4.3 Hz), 7.84 (d, 2H, ArH, J ) 8.2 Hz),
7.32 (d, 2H, ArH, J ) 8.2 Hz), 7.14 (td, 1H, ArH, J ) 9.0 and
2.6 Hz), 2.39 (s, 3H, ArCH3); CIMS 318 (MH+). Anal. (C16H12
FNO3S) C, H, N.
-
In summary, constraining the ethylamine side chain
of tryptamines into a cyclopropyl ring does not provide
rigid analogues that are good models of the binding
conformations of flexible tryptamines, except perhaps
at the 5-HT2C receptor. This may not simply be the
consequence of increased steric demand and limited
conformational flexibility, but it could also be related
to amine pKa, which is lower for cyclopropylamines. The
binding data provide further support for the idea that
tryptamines bind to serotonin receptors in conforma-
tions that may differ from the binding conformations
of phenethylamine-derived molecules.
Gen er a l P r oced u r e for th e P r ep a r a tion of tr a n s-â-(1-
(p-Tolylsu lfon yl)in d ol-3-yl)a cr ylic Acid s 4a-d . Following
the method of Moffatt,24 a solution of the appropriate 1-(p-
tolylsulfonyl)indole-3-carboxaldehyde (6.68 mmol) and malonic
acid (21.62 mmol) in pyridine (8 mL) containing piperidine (8
drops) was heated at 75-80 °C under nitrogen for 3 h. The
solution was poured into ice-cold water (50 mL) and acidified
with 5 N hydrochloric acid. After the mixture had been stored
in the refrigerator overnight, the precipitate was filtered and
washed with water. Recrystallization from methanol gave the
respective trans-â-(1-(p-tolylsulfonyl)indol-3-yl)acrylic acids.
tr a n s-â-(1-(p-Tolylsu lfon yl)in d ol-3-yl)a cr ylic Acid , 4a .
This compound was obtained in 87% yield as a white powder:
mp 243-244 °C; 1H NMR (DMSO-d6) δ 12.40 (br s, 1H,
COOH), 8.43 (s, 1H, ArH), 7.96 (d, 1H, ArH, J ) 8.3 Hz), 7.92
(d, 1H, ArH, J ) 8.3 Hz), 7.90 (d, 2H, ArH, J ) 8.4 Hz), 7.74
(d, 1H, â-H, J ) 16.2 Hz), 7.41 (m, 1H, ArH), 7.39 (d, 2H, ArH,
J ) 8.4 Hz), 7.34 (m, 1H, ArH), 6.57 (d, 1H, R-H, J ) 16.2
Exp er im en ta l Section
Ch em istr y. Melting points, determined with a Thomas-
Hoover Meltemp apparatus, are uncorrected, except where
indicated. 1H NMR spectra were recorded on a Varian VXR-
500S 500-MHz instrument. Chemical shifts are reported in δ
values (parts per million, ppm) relative to an internal standard
of tetramethylsilane (TMS) in CDCl3, except where noted.
Abbreviations used in NMR analysis are as follows: br s )
broad singlet, d ) doublet, dd ) doublet of doublets, m )
multiplet, q ) quartet, s ) singlet, t ) triplet, td ) triplet of
doublets. Analytical thin-layer chromatography (TLC) was
performed on Baker-flex silica gel 1B2-F plastic plates. The
Hz), 2.30 (s, 3H, ArCH3); CIMS 342 (MH+). Anal. (C18H15
NO4S) C, H, N.
-
tr a n s-â-(4-Meth oxy-1-(p-tolylsu lfon yl)in d ol-3-yl)a cr yl-
ic Acid , 4b. This compound was obtained in 89% yield as an
1
off-white solid: mp 255-256 °C; H NMR (DMSO-d6) δ 8.39
(s, 1H, ArH), 8.00 (d, 1H, â-H, J ) 16.0 Hz), 7.91 (d, 2H, ArH,
J ) 8.0 Hz), 7.54 (d, 1H, ArH, J ) 8.0 Hz), 7.40 (d, 2H, ArH,
J ) 8.0 Hz), 7.30 (t, 1H, ArH, J ) 8.0 Hz), 6.86 (d, 1H, ArH,

4998 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Vangveravong et al.
J ) 8 Hz), 6.63 (d, 1H, R-H, J ) 16.0 Hz), 3.88 (s, 3H, ArOCH3),
2.31 (s, 3H, ArCH3); CIMS 372 (MH+). Anal. (C19H17NO5S)
C, H, N.
ArOCH3), 3.75 (s, 3H, COOCH3), 2.44 (m, 1H, cyclopropyl H),
2.34 (s, 3H, ArCH3), 1.87 (m, 1H, cyclopropyl H), 1.58 (m, 1H,
cyclopropyl H), 1.26 (m, 1H, cyclopropyl H); CIMS 400 (MH+).
Anal. (C21H21NO5S) C, H, N.
tr a n s-â-(5-Meth oxy-1-(p-tolylsu lfon yl)in d ol-3-yl)a cr yl-
ic Acid , 4c. This compound was obtained in 76% yield as off-
Meth yl tr a n s-2-(5-F lu or o-1-(p-tolylsu lfon yl)in d ol-3-yl)-
cyclop r op a n eca r boxyla te, 5d . This compound was ob-
tained in 79% yield as a yellow oil that precipitated upon
1
white crystals: mp 244-245 °C; H NMR (DMSO-d6) δ 8.85
(br s, 1H, COOH), 8.38 (s, 1H, ArH), 7.86 (d, 2H, ArH, J ) 8.5
Hz), 7.84 (d, 1H, ArH, J ) 9.2 Hz), 7.73 (d, 1H, â-H, J ) 16.2
Hz), 7.38 (d, 2H, ArH, J ) 8.5 Hz), 7.32 (d, 1H, ArH, J ) 2.5
Hz), 7.00 (dd, 1H, ArH, J ) 9.2 and 2.5 Hz), 6.57 (d, 1H, R-H,
J ) 16.2 Hz), 3.80 (s, 3H, ArOCH3), 2.30 (s, 3H, ArCH3); CIMS
372 (MH+). Anal. (C19H17NO5S) C, H, N.
1
addition of methanol: mp 134-135 °C; H NMR δ 7.90 (dd,
1H, ArH, J ) 9.1 and 4.4 Hz), 7.72 (d, 2H, ArH, J ) 8.4 Hz),
7.29 (s, 1H, ArH), 7.23 (d, 2H, ArH, J ) 8.4 Hz), 7.20 (dd, 1H,
ArH, J ) 8.7 and 2.3 Hz), 7.05 (td, 1H, ArH, J ) 9.0 and 2.3
Hz), 3.75 (s, 3H, COOCH3), 2.45 (m, 1H, cyclopropyl H), 2.35
(s, 3H, ArCH3), 1.85 (m, 1H, cyclopropyl H), 1.59 (m, 1H,
cyclopropyl H), 1.27 (m, 1H, cyclopropyl H); CIMS 388 (MH+,
73.6), 356 (100). Anal. (C20H18FNO4S) C, H, N.
tr a n s-â-(5-F lu or o-1-(p -t olylsu lfon yl)in d ol-3-yl)a cr yl-
ic Acid , 4d . This compound was obtained in 65% yield as
1
white needles: mp 254-255 °C; H NMR (DMSO-d6) δ 12.35
tr a n s-2-(1-(p -Tolylsu lfon yl)in d ol-3-yl)cyclop r op a n e-
ca r boxylic Acid s 6a -d . Gen er a l P r oced u r e. The ap-
propriate methyl trans-2-(1-(p-tolylsulfonyl)indol-3-yl)cyclo-
propanecarboxylates (2.40 mmol) were subjected to hydrolysis
by stirring for 5 h in 2 mL of a 50% sodium hydroxide solution
in methanol at room temperature. The reaction mixture was
poured into a stirred mixture of water (10 mL), concentrated
HCl (2.3 mL), and ice. Extraction with dichloromethane (3 ×
10 mL), washing, drying (MgSO4), and evaporation gave the
respective trans-2-(1-(p-tolylsulfonyl)indol-3-yl)cyclopropan-
ecarboxylic acids which were used directly without further
purification.
tr a n s-2-(1-(p -Tolylsu lfon yl)in d ol-3-yl)cyclop r op a n e-
ca r boxylic Acid , 6a . This compound was obtained in 85%
yield as a brown oil: 1H NMR δ 7.97 (d, 1H, ArH, J ) 7.3 Hz),
7.75 (d, 2H, ArH, J ) 8.5 Hz), 7.58 (d, 1H, ArH, J ) 7.0 Hz),
7.29 (m, 3H, ArH), 7.27 (d, 2H, ArH, J ) 8.5 Hz), 2.58 (m, 1H,
cyclopropyl H), 2.34 (s, 3H, ArCH3), 1.88 (m, 1H, cyclopropyl
H), 1.67 (m, 1H, cyclopropyl H), 1.39 (m, 1H, cyclopropyl H);
CIMS 356 (MH+).
tr a n s-2-(4-Meth oxy-1-(p-tolylsu lfon yl)in d ol-3-yl)cyclo-
p r op a n eca r boxylic Acid , 6b. This compound was obtained
in 82% yield as a brown oil: 1H NMR δ 7.72 (d, 2H, ArH, J )
8.0 Hz), 7.54 (d, 1H, ArH, J ) 8.0 Hz), 7.21 (t, 1H, ArH, J )
8.0 Hz), 7.20 (d, 2H, ArH, J ) 8.0 Hz), 7.12 (s, 1H, ArH), 6.63
(d, 1H, ArH, J ) 8.0 Hz), 3.84 (s, 3H, ArOCH3), 2.85 (m, 1H,
cyclopropyl H), 2.34 (s, 3H, ArCH3), 1.75 (m, 1H, cyclopropyl
H), 1.63 (m, 1H, cyclopropyl H), 1.37 (m, 1H, cyclopropyl H);
CIMS 386 (MH+).
tr a n s-2-(5-Meth oxy-1-(p-tolylsu lfon yl)in d ol-3-yl)cyclo-
p r op a n eca r boxylic Acid , 6c. This compound was obtained
in 83% yield as a brown oil: 1H NMR δ 7.86 (d, 1H, ArH, J )
9.0 Hz), 7.71 (d, 2H, ArH, J ) 8.4 Hz), 7.24 (s, 1H, ArH), 7.21
(d, 2H, ArH, J ) 8.0 Hz), 6.99 (d, 1H, ArH, J ) 2.3 Hz), 6.94
(dd, 1H, ArH, J ) 9.0 and 2.4 Hz), 3.83 (s, 3H, ArOCH3), 2.51
(m, 1H, cyclopropyl H), 2.34 (s, 3H, ArCH3), 1.87 (m, 1H,
cyclopropyl H), 1.65 (m, 1H, cyclopropyl H), 1.35 (m, 1H,
cyclopropyl H); CIMS 386 (MH+).
tr a n s-2-(5-F lu or o-1-(p -t olylsu lfon yl)in d ol-3-yl)cyclo-
p r op a n eca r boxylic Acid , 6d . This compound was obtained
in 87% yield as a brown oil: 1H NMR δ 7.91 (dd, 1H, ArH, J
) 9.1 and 4.0 Hz), 7.72 (d, 2H, ArH, J ) 8.4 Hz), 7.32 (s, 1H,
ArH), 7.24 (d, 2H, ArH, J ) 8.4 Hz), 7.22 (dd, 1H, ArH, J )
8.6 and 2.3 Hz), 7.06 (td, 1H, ArH, J ) 9.0 and 2.3 Hz), 2.51
(m, 1H, cyclopropyl H), 2.36 (s, 3H, ArCH3), 1.86 (m, 1H,
cyclopropyl H), 1.66 (m, 1H, cyclopropyl H), 1.36 (m, 1H,
cyclopropyl H); CIMS 374 (MH+).
Gen er a l P r oced u r e for th e P r ep a r a tion of tr a n s-2-(1-
(p-Tolylsu lfon yl)in d ol-3-yl)ca r boben zoxa m id ocyclop r o-
p a n es 7a -d . Following the method of Weinstock,25 the
appropriate trans-2-(1-(p-tolylsulfonyl)indol-3-yl)cyclopropan-
ecarboxylic acid (3.38 mmol) was suspended in 0.5 mL of water,
and sufficient acetone was added to complete the solution. The
solution was cooled to 0 °C (ice-salt bath), and a solution of
triethylamine (4.30 mmol) in acetone (5 mL) was added. While
the temperature was maintained at 0 °C, a solution of ethyl
chloroformate (5.23 mmol) in acetone (2 mL) was added slowly.
(br s, 1H, COOH), 8.51 (s, 1H, ArH), 7.97 (dd, 1H, ArH, J )
9.1 and 4.4 Hz), 7.91 (d, 2H, ArH, J ) 8.4 Hz), 7.78 (dd, 1H,
ArH, J ) 9.4 and 2.4 Hz), 7.71 (d, 1H, â-H, J ) 16.2 Hz), 7.40
(d, 2H, ArH, J ) 8.4 Hz), 7.26 (td, 1H, ArH, J ) 9.1 and 2.4
Hz), 6.58 (d, 1H, R-H, J ) 16.2 Hz), 2.31 (s, 3H, ArCH3); CIMS
360 (MH+); Anal. (C18H14FNO4S) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of tr a n s-2-(1-
(p-Tolylsu lfon yl)in d ol-3-yl)cyclop r op a n eca r boxylic Acid
Meth yl Ester s 5a -d .13 Diazomethane was prepared as
previously described.25 A solution of 4.07 g (19.0 mmol) of
N-nitroso-N-methyl-4-toluenesulfonamide in ether (50 mL)
was slowly added to a heated mixture of potassium hydroxide
(1.23 g, 21.9 mmol), ether (3 mL), water (3 mL), and 2-(2-
ethoxyethoxy)ethane (6 mL). The ether solution of diaz-
omethane thus formed was continuously distilled into a cold
(ice-salt bath) stirring solution of the appropriate trans-â-(1-
(p-tolylsulfonyl)indol-3-yl)acrylic acid (4.39 mmol) in ether (15
mL) and dichloromethane (2 mL). The reaction mixture was
kept at -5 to 0 °C (bath temperature) until all diazomethane
had been distilled; stirring was continued for an additional 1
h. Palladium diacetate (10 mg) in a small amount of tetrahy-
drofuran was added, and an azeotrope of a second portion of
diazomethane and ether (prepared from the same amount of
reagents) was again distilled into the reaction mixture. After
all diazomethane had been distilled, the reaction mixture was
stirred at room temperature overnight and then filtered. The
solid on the filter was washed with dichloromethane. The
filtrate and washing were evaporated to give the crude product
which was then further purified by column chromatography
(silica gel, 5% ethyl acetate in methylene chloride).
Meth yl tr a n s-2-(1-(p-tolylsu lfon yl)in d ol-3-yl)cyclop r o-
p a n eca r boxyla te, 5a . This compound was obtained in 70%
yield as a yellow oil that precipitated upon addition of
1
methanol: mp 115-116 °C; H NMR δ 7.96 (d, 1H, ArH, J )
8.3 Hz), 7.74 (d, 2H, ArH, J ) 8.4 Hz), 7.56 (d, 1H, ArH, J )
7.8 Hz), 7.32 (m, 1H, ArH), 7.26 (s, 1H, ArH,), 7.25 (m, 1H,
ArH,), 7.22 (d, 2H, ArH, J ) 8.4 Hz), 3.74 (s, 3H, COOCH3),
2.50 (m, 1H, cyclopropyl H), 2.34 (s, 3H, ArCH3), 1.88 (m, 1H,
cyclopropyl H), 1.59 (m, 1H, cyclopropyl H), 1.29 (m, 1H,
cyclopropyl H); CIMS 370 (MH+). Anal. (C20H19NO4S) C, H,
N.
Meth yl tr a n s-2-(4-Meth oxy-1-(p-tolylsu lfon yl)in d ol-3-
yl)cyclop r op a n eca r boxyla te, 5b. This compound was ob-
tained in 65% yield as a yellow oil that precipitated upon
addition of methanol: mp 146-147 °C; 1H NMR δ 7.72 (d, 2H,
ArH, J ) 8.4 Hz), 7.54 (d, 1H, ArH, J ) 7.8 Hz), 7.21 (d, 2H,
ArH, J ) 8.2 Hz), 7.20 (t, 1H, ArH, J ) 7.8 Hz), 7.10 (s, 1H,
ArH), 6.62 (d, 1H, ArH, J ) 7.8 Hz), 3.82 (s, 3H, ArOCH3),
3.74 (s, 3H, COOCH3), 2.80 (m, 1H, cyclopropyl H), 2.34 (s,
3H, ArCH3), 1.75 (m, 1H, cyclopropyl H), 1.56 (m, 1H,
cyclopropyl H), 1.28 (m, 1H, cyclopropyl H); CIMS 400 (MH+).
Anal. (C21H21N5S) C, H, N.
Meth yl tr a n s-2-(5-Meth oxy-1-(p-tolylsu lfon yl)in d ol-3-
yl)cyclop r op a n eca r boxyla te, 5c. This compound was ob-
tained in 60% yield as a pale yellow oil that solidified upon
addition of methanol: mp 137-139 °C; 1H NMR δ 7.85 (d, 1H,
ArH, J ) 9.0 Hz), 7.70 (d, 2H, ArH, J ) 8.4 Hz), 7.21 (d, 2H,
ArH, J ) 8.4 Hz), 7.20 (s, 1H, ArH), 6.97 (d, 1H, ArH, J ) 2.5
Hz), 6.93 (dd, 1H, ArH, J ) 9.0 and 2.5 Hz), 3.83 (s, 3H,
The mixture was stirred for 1 h at this temperature.
A
solution of sodium azide (12.3 mmol) in water (3 mL) was

trans-2-(Indol-3-yl)cyclopropylamine Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 4999
added dropwise, and the mixture was stirred for 2 h. The
reaction mixture was poured into ice water (15 mL) and
extracted with toluene (4 × 10 mL). The toluene solution was
dried (MgSO4) and filtered. The filtrate was heated to gentle
reflux with dry benzyl alcohol (24.0 mmol) for 10 h. The
solvent and excess benzyl alcohol were removed in vacuo.
Purification of the crude product by column chromatography
(silica gel, 1% ethyl acetate in methylene chloride) provided
the respective N-carbobenzoxy derivatives.
1.70 (m, 1H, cyclopropyl H), 0.92 (m, 2H, cyclopropyl CH2);
CIMS 357 (MH+). Anal. (C19H20N2O3S) C, H, N.
tr a n s-2-(5-Meth oxy-1-(p-tolylsu lfon yl)in d ol-3-yl)cyclo-
p r op yla m in e, 8c. This compound was obtained in 50% yield
as a yellow oil: 1H NMR δ 7.84 (d, 1H, ArH, J ) 9.0 Hz), 7.68
(d, 2H, ArH, J ) 8.5 Hz), 7.19 (d, 2H, ArH, J ) 8.5 Hz), 7.05
(s, 1H, ArH), 7.01 (d, 1H, ArH, J ) 2.3 Hz), 6.92 (dd, 1H, ArH,
J ) 9.0 and 2.3 Hz), 3.83 (s, 3H, ArOCH3), 2.49 (m, 1H,
cyclopropyl H), 2.33 (s, 3H, ArCH3), 1.78 (m, 1H, cyclopropyl
H), 1.70 (br s, 2H, NH2), 1.02 (m, 1H, cyclopropyl H), 0.93 (m,
1H, cyclopropyl H); CIMS 357 (MH+). Anal. (C19H20N2O3S)
C, H, N.
tr a n s-2-(1-(p -Tolylsu lfon yl)in d ol-3-yl)ca r b ob en zoxa -
m id ocyclop r op a n e, 7a . This was obtained in 70% yield as
1
a yellow oil that solidified upon standing: mp 86-88 °C; H
tr a n s-(5-F lu or o-1-(p-tolylsu lfon yl)in d ol-3-yl)cyclop r o-
p yla m in e, 8d . This compound was obtained in 50% yield as
a yellow oil: 1H NMR δ 7.89 (dd, 1H, ArH, J ) 9.1 and 4.4
Hz), 7.69 (d, 2H, ArH, J ) 8.3 Hz), 7.22 (dd, 1H, ArH, J ) 8.8
and 2.6 Hz), 7.21 (d, 2H, ArH, J ) 8.3 Hz), 7.14 (s, 1H, ArH),
7.03 (td, 1H, ArH, J ) 9.1 and 2.6 Hz), 2.49 (m, 1H, cyclopropyl
H), 2.34 (s, 3H, ArCH3), 1.85 (br s, 2H, NH2), 1.76 (m, 1H,
cyclopropyl H), 1.02 (m, 1H, cyclopropyl H), 0.93 (m, 1H,
cyclopropyl H); CIMS 345 (MH+). Anal. (C18H17FN2O2S) C,
H, N.
Gen er a l P r oced u r e for th e N(1)-Detosyla tion , 1a -d .28
The appropriate trans-2-(1-(p-tolylsulfonyl)indol-3-yl)cyclopro-
pylamine 8a -d (0.45 mmol) and anhydrous disodium hydro-
gen phosphate (1.87 mmol) were stirred magnetically in dry
methanol (8 mL) at room temperature under nitrogen. To the
suspension was added pulverized 6% sodium amalgam (904
mg). Stirring was continued until the reaction was complete
as indicated by TLC (about 45 min). The mixture was poured
into water (10 mL), extracted with ether (4 × 10 mL), dried
(MgSO4), filtered, and concentrated to afford a yellow oil that
was converted to its oxalate salt and recrystallized from
ethanol-petroleum ether.
tr a n s-2-(In d ol-3-yl)cyclop r op yla m in e Oxa la te, 1a . This
was obtained in 60% yield as an off-white powder: mp 153-
154 °C dec; 1H NMR (CD3OD) δ 7.66 (td, 1H, ArH, J ) 7.9
and 0.9 Hz), 7.32 (td, 1H, ArH, J ) 8.1 and 0.8 Hz), 7.10 (m,
1H, ArH), 7.03 (m, 1H, ArH), 7.02 (s, 1H, ArH), 2.73 (m, 1H,
cyclopropyl H), 2.40 (m, 1H, cyclopropyl H), 1.35 (m, 1H,
cyclopropyl H), 1.26 (m, 1H, cyclopropyl H); FABMS 173
(MH+); high-resolution FABMS calcd for C11H12N2 173.1079,
found 173.1080.
tr a n s-2-(4-Met h oxyin d ol-3-yl)cyclop r op yla m in e Ox-
a la te, 1b. This was obtained in 60% yield as a yellow-brown
powder: mp >200 °C dec; 1H NMR (CD3OD) δ 7.00 (t, 1H,
ArH, J ) 8.0 Hz), 6.92 (d, 1H, ArH, J ) 8.1 Hz), 6.82 (s, 1H,
ArH), 6.49 (d, 1H, ArH, J ) 7.6 Hz), 3.93 (s, 3H, ArOCH3),
2.76 (m, 1H, cyclopropyl H), 2.64 (m, 1H, cyclopropyl H), 1.25
(m, 2H, cyclopropyl CH2); FABMS 203 (MH+); high-resolution
FABMS calcd for C12H14N2O 203.1184, found 203.1186.
tr a n s-2-(5-Met h oxyin d ol-3-yl)cyclop r op yla m in e Ox-
a la te, 1c. This was obtained in 60% yield as a yellow-brown
powder: mp >200 °C dec; 1H NMR (CD3OD) δ 7.21 (d, 1H,
ArH, J ) 8.8 Hz), 7.15 (d, 1H, ArH, J ) 2.5 Hz), 6.98 (s, 1H,
ArH), 6.77 (dd, 1H, ArH, J ) 8.8 and 2.5 Hz), 3.83 (s, 3H,
ArOCH3), 2.70 (m, 1H, cyclopropyl H), 2.36 (m, 1H, cyclopropyl
H), 0.97 (m, 1H, cyclopropyl H), 0.92 (m, 1H, cyclopropyl H);
FABMS 203 (MH+). Anal. (C14H16N2O5) C, H, N.
NMR δ 7.95 (d, 1H, ArH, J ) 8.5 Hz), 7.72 (d, 2H, ArH, J )
8.4 Hz), 7.32 (m, 9H, ArH), 7.20 (d, 2H, ArH, J ) 8.1 Hz), 5.15
(s, 2H, ArCH2O), 5.13 (br s, 1H, NH), 2.73 (m, 1H, cyclopropyl
H), 2.33 (s, 3H, ArCH3), 2.06 (m, 1H, cyclopropyl H), 1.18 (m,
2H, cyclopropyl CH2); CIMS 461 (MH+). Anal. (C26H24N2O4S)
C, H, N.
tr a n s-2-(4-Met h oxy-1-(p -t olylsu lfon yl)in d ol-3-yl)ca r -
boben zoxa m id ocyclop r op a n e, 7b . This was obtained in
65% yield as a yellow oil that solidified upon standing: mp
1
55-57 °C; H NMR δ 7.70 (d, 2H, ArH, J ) 8.4 Hz), 7.56 (d,
1H, ArH, J ) 8.1 Hz), 7.36 (m, 5H, ArH), 7.21 (d, 2H, ArH, J
) 8.4 Hz), 7.20 (t, 1H, ArH, J ) 8.1 Hz), 7.04 (s, 1H, ArH),
6.63 (d, 1H, ArH, J ) 8.1 Hz), 5.12 (s, 2H, ArCH2O), 5.08 (br
s, 1H, NH), 3.85 (s, 3H, ArOCH3), 2.65 (m, 1H, cyclopropyl
H), 2.35 (m, 1H, cyclopropyl H), 1.20 (m, 2H, cyclopropyl CH2);
CIMS 491 (MH+). Anal. (C27H26N2O5S) C, H, N.
tr a n s-2-(5-Met h oxy-1-(p -t olylsu lfon yl)in d ol-3-yl)ca r -
boben zoxa m id ocyclop r op a n e, 7c. This was obtained in
60% yield as a yellow oil that solidified upon standing: mp
1
54-55 °C; H NMR δ 7.82 (d, 1H, ArH, J ) 9.0 Hz), 7.68 (d,
2H, ArH, J ) 8.2 Hz), 7.35 (m, 6H, ArH), 7.18 (d, 2H, ArH, J
) 8.0 Hz), 7.17 (s, 1H, ArH), 6.92 (dd, 1H, ArH, J ) 9.0 and
2.5 Hz), 5.13 (s, 2H, ArCH2O), 5.09 (br s, 1H, NH), 3.86 (s,
3H, ArOCH3), 2.64 (m, 1H, cyclopropyl H), 2.32 (s, 3H, ArCH3),
2.00 (m, 1H, cyclopropyl H), 1.14 (m, 2H, cyclopropyl CH2);
CIMS 491 (MH+). Anal. (C27H26N2O5S) C, H, N.
tr a n s-2-(5-Flu or o-1-(p-tolylsu lfon yl)in dol-3-yl)car boben -
zoxa m id ocyclop r op a n e, 7d . This was obtained in 70% yield
as a yellow oil that solidified upon standing: mp 119-120 °C;
1H NMR δ 7.88 (dd, 1H, ArH, J ) 9.0 and 4.0 Hz), 7.70 (d,
2H, ArH, J ) 8.2 Hz), 7.36 (m, 7H, ArH), 7.21 (d, 2H, ArH, J
) 8.2 Hz), 7.04 (td, 1H, ArH, J ) 9.0 and 2.6 Hz), 5.15 (s, 2H,
ArCH2O), 5.08 (br s, 1H, NH), 2.67 (m, 1H, cyclopropyl H),
2.35 (s, 3H, ArCH3), 2.00 (m, 1H, cyclopropyl H), 1.15 (m, 2H,
cyclopropyl CH2); CIMS 479 (MH+). Anal. (C26H23FN2O4S) C,
H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of tr a n s-2-(1-
(p-Tolylsu lfon yl)in d ol-3-yl)cyclop r op yla m in es 8a-d .27
A
solution of the appropriate N-carbobenzoxy derivative (2.39
mmol) in absolute ethanol (50 mL) containing 10% palladium
on charcoal (250 mg) was shaken on a Parr apparatus for 7 h
under 50 psi of hydrogen. The catalyst was removed by
filtration and washed with ethanol. The combined filtrate and
washing were evaporated to obtain a residue, which was
purified via centrifugal rotary chromatography (Chromatotron)
to give the respective amines.
t r a n s-2-(1-(p -Tolylsu lfon yl)in d ol-3-yl)c yc lop r op y-
la m in e, 8a . This compound was obtained in 53% yield as a
yellow oil: 1H NMR δ 7.95 (d, 1H, ArH, J ) 7.5 Hz), 7.72 (d,
2H, ArH, J ) 8.3 Hz), 7.59 (d, 1H, ArH, J ) 7.7 Hz), 7.31 (m,
1H, ArH), 7.25 (m, 1H, ArH), 7.20 (d, 2H, ArH, J ) 8.3 Hz),
7.11 (s, 1H, ArH), 2.51 (m, 1H, cyclopropyl H), 2.33 (s, 3H,
ArCH3), 1.85 (br s, 3H, NH2 and cyclopropyl H), 1.03 (m, 1H,
cyclopropyl H), 0.95 (m, 1H, cyclopropyl H); CIMS 327 (MH+).
Anal. (C18H18N2O2S) C, H, N.
tr a n s-2-(5-F lu or oin d ol-3-yl)cyclop r op yla m in e Oxa la te,
1d . This was obtained in 50% yield as a yellow-brown
powder: mp 141-144 °C; H NMR (CD3OD) δ 7.35 (dd, 1H,
ArH, J ) 9.8 and 2.5 Hz), 7.28 (dd, 1H, ArH, J ) 9.0 and 4.4
Hz), 7.09 (s, 1H, ArH), 6.88 (td, 1H, ArH, J ) 9.1 and 2.5 Hz),
2.71 (m, 1H, cyclopropyl H), 2.33 (m, 1H, cyclopropyl H), 1.34
(m, 1H, cyclopropyl H), 1.24 (m, 1H, cyclopropyl H); FABMS
191 (MH+); high-resolution FABMS calcd for C11H11FN2
191.0985, found 191.0977.
1
tr a n s-2-(4-Meth oxy-1-(p-tolylsu lfon yl)in d ol-3-yl)cyclo-
p r op yla m in e, 8b. This compound was obtained in 50% yield
as a yellow oil: 1H NMR δ 7.68 (d, 2H, ArH, J ) 8.5 Hz), 7.53
(d, 1H, ArH, J ) 8.0 Hz), 7.30 (m, 3H, ArH), 6.94 (s, 1H, ArH),
6.60 (d, 1H, ArH, J ) 8.0 Hz), 3.86 (s, 3H, ArOCH3), 2.31 (s,
3H, ArCH3), 2.24 (m, 1H, cyclopropyl H), 1.80 (br s, 2H, NH2),
P h a r m a cology Meth od s. Ra d ior ecep tor Com p etition
Assa ys in Ra t Br a in Hom ogen 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]-8-OH-DPAT was purchased

5000 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Vangveravong et al.
from New England Nuclear (Boston, MA) at a specific activity
of 216 Ci/mmol. 5-HT was purchased from Sigma (St. Louis,
MO).
The procedures of Huang et al.29 were employed. Briefly,
the hippocampal brain regions from 20-40 rats were pooled
and homogenized (Brinkman polytron, setting 6 for 2 × 20 s)
in 8 volumes of 0.32 M sucrose. The homogenate was
centrifuged at 36000g for 10 min, and the resulting pellet was
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). A second preincubation for 10 min at
37 °C was conducted, and the tissues were then cooled in an
ice bath.
All experiments were performed with triplicate determina-
tions using 200-400 µg of protein, in a final incubation 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 displaceable with 10 µM 5-HT. Filters were
air-dried, placed into scintillation vials with 10 mL of Ecolite
scintillation cocktail, and allowed to sit overnight before
counting at an efficiency of 37% for tritium.
Gaithersburg, MD). The filters then were 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, with bovine serum albumin as the
standard.31
5-HT2A/2C [
125I]DOI Bin d in g Stu d ies. Human 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 disodium EDTA,
0.1% sodium ascorbate, and 50 mM Tris HCl, pH 7.4. Incuba-
tions 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, followed by filtration and washing 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 receptors and 1
µM ketanserin for 5-HT2A receptors. The final concentration
of [125I]DOI was approximately 0.07-0.15 nM.
Da ta An a lysis. Radioligand competition data for the
5-HT1A receptor were analyzed using the computer programs
EBDA and Ligand as described by McPherson.32 The values
from three separate experiments were combined.
For the competition curves run against the 5-HT2 family of
receptors, the IC50 values were determined by nonlinear
regression analysis using a four-parameter logistic equation
described by De Lean et al.33 After verification that the curves
best fit a one-site model, the IC50 values were then converted
to Ki values using the Cheng-Prusoff equation.34
Ra d ioliga n d Com p etition Exp er im en ts Usin g Clon ed
Hu m a n 5-HT2 Recep tor s. All chemicals were obtained from
the sources previously described.30 [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, and [125I]DOI (2200 Ci/mmol) was purchased from
DuPont-NEN (Wilmington, DE).
Membranes were prepared essentially as previously de-
scribed using AV12 cell lines (Syrian hamster fibroblast, ATCC
Ack n ow led gm en t. This work was supported by
NIH Grant DA02189 from the National Institute on
Drug Abuse.
no. CRL 9595) stably transformed with the human 5-HT2A
,
5-HT2B, or 5-HT2C receptors.30 In brief, cells expressing the
receptor of interest were grown in suspension and harvested
by centrifugation. The cell pellets were then resuspended 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 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 vortex-
ing, the preparation was centrifuged at 39000g for 10 min at
4 °C, and the resulting membrane pellet was resuspended,
incubated at 37 °C for 10 min, and then centrifuged at 39000g
for 10 min at 4 °C. This pellet was resuspended and centri-
fuged 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
and in Tris HCl, pH 7.4, containing MgCl2 and EDTA for [125I]-
DOI binding to 5-HT2A or 5-HT2C receptors.
Refer en ces
(1) Lessin, A. W.; Long, R. F.; Parkes, M. W. The Central Stimulant
Properties of some Substituted Indolylalkylamines and â-Car-
bolines and their Activities as Inhibitors of Monoamine Oxidase
and the Uptake of 5-Hydroxytryptamine. Br. J . Pharmacol.
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(2) Ho, B. T.; McIsaac, W. M.; An, R.; Harris, R. T.; Walker, K. E.;
Kralik, P. M.; Airaksinen, M. M. Biological Activities of some
5-Substituted N,N-Dimethyltryptamines, R-Methyltryptamines,
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(3) Horn, A. S. Structure Activity Relations for the Inhibition of
5-HT Uptake into Rat Hypothalamic Homogenates by Serotonin
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