Binding of tetrahydrocarboline derivatives at human 5-HT5A receptors

Article abstract of DOI:10.1021/jm030080s

On the basis of an earlier finding that 5-methyl-5H-1,2,3,4-tetrahydropyrido[4,3-b]indole (5-methyl-1,2,3,4-tetrahydro-γ-carboline; 1) binds at murine 5-HT _5A receptors, preliminary structure-affinity studies were conducted. The present investigation extends these structure-affinity studies using human 5-HT_5A receptors and examined additional analogues of 1. It was found (a) that there is little interspecies difference for the affinities of these compounds, (b) that an intact 1,2,3,4-tetrahydro-γ-carboline ring system seems optimal and an N²-(3-(substituted-phenoxy)propyl) moiety results in high affinity, (c) that structurally related 1,2,3,4-tetrahydro-β-carbolines also bind at 5-HT_5A receptors, and (d) that all examined derivatives also possess affinity for 5-HT _2A receptors. Evidence is provided that 5-HT_5A and 5-HT_2A receptor affinities probably do not covary and that it might be possible, with continued investigation, to develop analogues with enhanced 5-HT_5A selectivity.

Full text of DOI:10.1021/jm030080s

3930
J . Med. Chem. 2003, 46, 3930-3937
Bin d in g of Tetr a h yd r oca r bolin e Der iva tives a t Hu m a n 5-HT5A Recep tor s
Nantaka Khorana,† Carol Smith,‡ Kathy Herrick-Davis,‡ Anil Purohit,‡ Milt Teitler,‡ Brian Grella,†
Małgorzata Dukat,† and Richard A. Glennon*,†
Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University,
Richmond, Virginia 23298-0540, and Center for Neuropharmacology and Neuroscience,
Albany Medical College, Albany, New York 12208
Received February 18, 2003
On the basis of an earlier finding that 5-methyl-5H-1,2,3,4-tetrahydropyrido[4,3-b]indole (5-
methyl-1,2,3,4-tetrahydro-γ-carboline; 1) binds at murine 5-HT5A receptors, preliminary
structure-affinity studies were conducted. The present investigation extends these structure-
affinity studies using human 5-HT5A receptors and examined additional analogues of 1. It was
found (a) that there is little interspecies difference for the affinities of these compounds, (b)
that an intact 1,2,3,4-tetrahydro-γ-carboline ring system seems optimal and an N2-(3-
(substituted-phenoxy)propyl) moiety results in high affinity, (c) that structurally related 1,2,3,4-
tetrahydro-â-carbolines also bind at 5-HT5A receptors, and (d) that all examined derivatives
also possess affinity for 5-HT2A receptors. Evidence is provided that 5-HT5A and 5-HT2A receptor
affinities probably do not covary and that it might be possible, with continued investigation,
to develop analogues with enhanced 5-HT5A selectivity.
Of the seven families of serotonin (5-hydroxy-
tryptamine, 5-HT) receptors (5-HT1-5-HT7), 5-HT5
receptors are perhaps the most poorly understood and
the least well investigated and have been referred to
as “orphan 5-HT receptors”.1-4 Because there is no
direct evidence concerning the existence of functional
native receptors, they are most correctly referred to as
5-ht5 receptors.2,4 Mouse 5-HT5 receptors were first
identified by Plassat et al.,5 in 1992. Shortly thereafter,
two murine subpopulations were identified: 5-HT5A and
behaviors.14 There also has been speculation, primarily
on the basis of receptor localization, that 5-HT5A recep-
tors could be involved in certain other CNS disorders
including Alzheimer’s disease,15 anxiety, and feeding
behavior.5,9
Relatively little is known about the binding of various
agents at 5-HT5A receptors, and no selective agents have
been identified. Typical nonselective “serotonin ago-
nists” that bind at human 5-HT5A receptors include
5-HT (Ki ≈ 125 nM), 5-carboxamidotryptamine (Ki ≈ 20
nM), and sumatriptan (Ki ≈ 5000 nM) (see Chart 1 for
chemical structures); typical nonselective “serotonin
antagonists” that bind include metergoline (Ki ≈ 630
nM), methysergide (Ki ≈ 100 nM), and methiothepin
(metitepine; Ki ≈ 1 nM).9 More information is available
about the binding of various agents at rodent than
human 5-HT5A receptors, but their pharmacologies, for
the few agents examined, appear to be in reasonable
agreement.9 However, this is not always the case;
methiothepin, for example, binds with 60-fold lower
affinity at mouse versus human 5-HT5A receptors.9
8
5-HT5B receptors.6 Since then, rat 5-HT5A7 and 5-HT5B
and human 5-HT5A9 receptors have been cloned; human
5-HT5B receptors have not yet been identified. A human
5-HT5B gene has been isolated but apparently does not
encode a functional protein because its coding sequence
is interrupted by stop codons.10 5-HT5A receptors are
found primarily in the forebrain, cerebellum, and spinal
cord.2,4 The deduced structure of 5-HT5A receptors is
consistent with that of other transmembrane-spanning
G-protein-coupled receptors, but the transduction mech-
anism associated with these receptors has not been
unequivocally defined.2,4 Indeed, Noda et al.,11 have
most recently reported that activation of human 5-HT5A
receptors elicits multiple signaling mechanisms and that
these receptors regulate neuronal function in a rather
complex manner.
It has been speculated that mutations in the gene
encoding 5-HT5A receptors might be detrimental to brain
development,6 and that genetic variation in human
5-HT5A receptors could be involved in susceptibility to
psychosis or depression.12,13 5-HT5A-receptor knock-out
mice displayed increased locomotor activity and explo-
ration, indicating their possible involvement in these
Five years ago we undertook a structure-affinity
investigation to better understand the binding require-
ments of various tryptamines at mouse 5-HT5A recep-
tors.16 Although several high-affinity tryptamines were
identifiedssome with much higher affinity than 5-HT
itselfsnone was selective for 5-HT5A receptors. More
recently, we screened >100 nontryptamines in an at-
tempt to identify a novel lead structure for subsequent
investigation. One of the compounds to emerge from this
screen was 5-methyl-5H-1,2,3,4-tetrahydropyrido[4,3-
b]indole (5-methyl-1,2,3,4-tetrahydro-γ-carboline; 1). Al-
though the affinity of 1 (Ki ) 5300 nM) at 5-HT5A
receptors was modest, γ-carbolines typically display
reduced affinity for most other populations of 5-HT
receptors (with the exception of 5-HT2 receptors), and
it was felt that the tricyclic structure might be success-
fully exploited to develop a reasonably selective ligand.
* Correspondence to: Richard A. Glennon, Ph.D., Department of
Medicinal Chemistry, School of Pharmacy, Box 980540, Virginia
Commonwealth University, Richmond, VA 23298-0540. Phone: 804-
828-8487. Fax: 804-828-7404. E-mail: glennon@hsc.vcu.edu.
† Virginia Commonwealth University.
‡ Albany Medical College.
10.1021/jm030080s CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/26/2003

Binding of Tetrahydrocarbolines at 5-HT5A Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3931
Ch a r t 1. Structures of Some Standard Serotonergic Agents Described in the Text
Initial structure-affinity studies resulted in the iden-
tification of 2 (m5-HT5A Ki ) 13 nM), a compound with
400-fold higher affinity than 1 at 5-HT5A receptors.17
Ta ble 1. Physicochemical Properties of Novel
Phenyl-Substituted Tetrahydro-γ-carboline Derivatives
%
recrystallization
solvent
empirical
formulaa
n
X
mp (°C) yield
During the course of our investigation human 5-HT5A
receptors became available. The purpose of the present
investigation was severalfold: (i) to obtain representa-
tive human 5-HT5A binding data for selected compounds
we had already examined at mouse 5-HT5A receptors,
(ii) to determine if the human binding data (Ki values)
were similar to the mouse 5-HT5A binding data we had
previously reported, (iii) to continue our structure-
affinity investigation employing human 5-HT5A recep-
tors, and (iv) to examine the 5-HT2A receptor affinities
of selected compounds to determine their 5-HT5A versus
5-HT2A selectivity.
19
25
26
27
28
2
3
3
3
3
4-F
223-225 42
MeOH/Et2O
MeOH/Et2O
MeOH/Et2O
MeOH/Et2O
MeOH/Et2O
C20H21FN2O‚HCl
C21H23N3O3‚HClb
C21H23Cl N2O‚HCl
C21H22Cl2N2O‚HCl
C22H26N2O2‚HClc
4-NO2 226-228 46
4-Cl
3,4-Cl2 217-219 19
4-OMe 215-217 58
234-236 39
a
Compounds analyzed within 0.4% of theory for C, H, N.
Crystallized with 0.25 mol of H2O. c Crystallized with 0.1 mol of
b
H2O.
Sch em e 2a
Ch em istr y
The synthesis of many of the γ-carboline derivatives
(i.e., those for which murine 5-HT5A Ki values have been
Sch em e 1a
a
Reagents: (a) (i) LiAlH4, (ii) HCOOEt; (b) POCl3; (c) NaBH4;
(d) (i) ArO(CH2)3Cl, K2CO3, (ii) oxalic acid.
reported) was previously described as shown in Scheme
1.17 Several additional derivatives were prepared using
the same general route by alkylation of 58 (R ) CH3)
with the appropriate alkyl halide (Scheme 1), and their
physicochemical properties are shown in Table 1. Com-
pounds 38 and 40 were prepared in like manner using
1,2,3,4-tetrahydroisoquinoline and 1-methyl-4,5,6,7-tet-
rahydro-1H-pyrrolo[3,2-c]pyridine, respectively, in place
of 58. Compound 40 was prepared from the appropri-
ately substituted pyrrole as shown in Scheme 2; cycliza-
tion of formamide 60 afforded pyrrolopyridine 61 which
a
Reagents: (a) NaH, RI, DMF; (b) KOH; (c) ClCH2-X-Y-Z,
NaI, K2CO3.

3932 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Khorana et al.
Sch em e 3a
Gramine analogue 39 was obtained by the condensa-
tion of indole-3-carboxaldehyde with 3-(4-fluorophen-
oxy)propylamine, followed by methylation. Carbazole
41, the deaza analogue of 29, was prepared via a
Fischer-type cyclization of 4-(3-hydroxypropyl)cyclohex-
anone; the hydroxy intermediate was mesylated to 64,
compound 64 was allowed to react with N-Boc-protected
4-hydroxyaniline, and the resulting product was depro-
tected to afford the desired product 41 (Scheme 3). The
â-carboline derivatives 53-55 were obtained by alky-
lation of the requisite 1,2,3,4-tetrahydro-â-carboline.
Resu lts a n d Discu ssion
Hu m a n ver su s Mou se 5-HT5A Bin d in g. Human
5-HT5A receptor radioligand binding data were obtained
for many of the same γ-carboline derivatives for which
we had previously reported mouse 5-HT5A data. The
results, along with the previously reported mouse
5-HT5A receptor affinities (Ki values), are shown in Table
2. In general, there was relatively little interspecies
difference between Ki values, and a significant correla-
tion (r ) 0.901) exists between human and murine
5-HT5A receptor affinities. Figure 1 shows a plot of
a
Reagents: (a) (i) MeHNNHPh, HCl, (ii) MsCl; (b) (i) tert-butyl
(4-hydroxyphenyl)carbamate, K2CO3, (ii) HCl/EtOAc.
was subsequently reduced with sodium borohydride to
62 and alkylated with the appropriate aryloxyalkyl
chloride. Oxime 6 was prepared by reaction of 4 with
hydroxylamine, and amine 29 was obtained by reduction
of nitro compound 25.
Ta ble 2. 5-HT5A and 5-HT2 Receptor Affinities of Tetrahydro-γ- and â-carboline Derivatives 3-35 and 53-55, Respectively
mouse 5-HT5A
human 5-HT5A
5-HT2A
5-HT2C
R
R′
X
Y
Z
Ki, nM ((SEM)a Ki, nM ((SEM)b Ki, nM ((SEM) Ki, nM ((SEM)
3
4
5
6
7
8
9
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2-
-CHdCH- (Z) -(CH2)2-
-CHdCH- (E) -(CH2)2-
-CtC-
-CH2CH2-
-CH2CH2-
-CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
CdO
4-F Ph
4-F Ph
Ph
4-F Ph
4-F Ph
Ph
4-OMe Ph
Ph
Ph
Ph
512
115
135
-
-
-
-
-
60c
-
CH3
CH3
CH3
CH3
CH3
CH3
CdO
6c
CdO
168 (10)
340 (25)
-
22 (4)
38 (2)
CdNOH
-CH2-
190 (15)
-
95
65 (20)
-
-CH2-
130
90
-
-
-CH2-
-
415 (80)
-
990 (180)
-
10 CH3
11 CH3
12 CH3
13 CH3
14 CH3
15 CH3
16 CH3
17 CH3
18
2
19 CH3
20 CH3
21
22 CH3
23 C2H5
24 CH2Ph
25 CH3
26 CH3
27 CH3
28 CH3
29 CH3
30 CH3
31 CH3
32 CH3
33 CH3
34 CH3
35 CH3
-CH2-
250
120
435
225
1100
330
350
1710
75
-
-(CH2)2-
-(CH2)3-
174 (6)
-
130c
1000 (60)
530 (20)
390 (10)
-
185 (30)
160 (30)
-
-CH(OH)- 4-F Ph
-CH2-
285 (10)
1120 (100)
208 (20)
185 (45)
1775 (100)
-
OH
Ph
120 (5)
60 (10)
-
285 (25)
245 (30)
-
Ph
-(CH2)2-
-O-
-O-
-O-
-O-
-O-
-O-
-O-
-O-
-O-
-O-
-O-
-O-
-O-
-O-
-O-
-O-
-O-
-O-
-O-
CdO
-O-
-O-
Ph
H
CH3
4-F Ph
4-F Ph
4-F Ph
CH2Ph
Ph
-
-
13
-
55 (5)
930 (100)
-
30c
75c
175 (15)
225 (20)
20 (2)
235 (15)
650 (80)
160 (15)
285c
295
600
25
H
-
Ph
100 (5)
100 (10)
-
70c
Ph
75
50 (5)
215 (10)
90 (20)
300 (60)
-
Ph
875
-
65 (15)
280 (90)
115 (20)
195 (15)
100 (10)
285 (50)
625 (220)
480 (180)
38 (5)
4-NO2 Ph
4-Cl Ph
3,4-Cl2 Ph
4-OMe Ph
4-NH2 Ph
1-naphthyl
2-naphthyl
4-F Ph
4-F Ph
4-F Ph
4-F Ph
Ph
250
-
200 (5)
200 (25)
350 (100)
190 (20)
-
-
-
-
-
-
300 (40)
400 (100)
300 (80)
150 (20)
225 (30)
300 (60)
225 (10)
115 (10)
310 (15)
400 (20)
650
655
98
-
8-OMe -CH2CH2-
6-OMe -CH2CH2-
8-Cl
6-Cl
H
H
H
165 (15)
1395 (50)
270 (50)
225 (10)
-
1220
180
590
550 (30)
335 (15)
360 (15)
120 (15)
130 (15)
210 (15)
16 (1)
50 (5)
150 (10)
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
53
54
55 CH3
H
H
Ph
Ph
-
620 (20)
a
b
Murine 5-HT5A Ki values were previously reported where SEM is not shown.17 For comparison, Ki ) 307 ((31) nM for 5-HT. c 5-
HT2 Ki values previously reported.17

Binding of Tetrahydrocarbolines at 5-HT5A Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3933
The role of an intact γ-carboline ring was next
examined. Compounds 36-40 were prepared and evalu-
ated. In compounds 36 and 37 (Ki > 10 000 nM), the
tetrahydrocarboline ring was abbreviated to a simpler
tertiary amine; lack of affinity indicated that the ring
system contributes to binding. With compound 38, the
pyrrole portion of the tetrahydro-γ-carboline ring was
excised; the affinity of 38 (Ki ) 400 ( 30 nM) was about
8-fold lower than that of its intact parent 2. The
tetrahydropyridyl portion of 2 was ring-opened to
provide 39; here too, affinity was reduced. The affinity
of 39 (Ki ) 1080 ( 200 nM) was about 20-fold lower
than that of 2. In compound 40 (Ki ) 1920 ( 370 nM)
the fused phenyl ring of 2 was eliminated, resulting in
about a 35-fold decrease in affinity. Taken together,
these results suggest that an intact ring system is
optimal for the binding of 2 at 5-HT5A receptors.
F igu r e 1. Relationship between human and murine 5-HT5A
receptor affinities (pKi values; data from Table 2, r ) 0.901; n
) 15).
human versus mouse 5-HT5A pKi values. Rees et al.9 had
previously commented that there was good general
agreement between the pharmacology of a small series
of agents at mouse and human 5-HT5A receptors. The
present findings substantiate this concept for tetrahy-
drocarboline derivatives. On this basis, it was felt
justified in not obtaining human data on all the γ-car-
bolines previously examined and in making structure-
affinity generalizations using mouse data in the few
instances where human data were unavailable.
5-HT5A Str u ctu r e)Affin ity Stu d y. Radioligand
binding data are shown in Table 2. Our earlier study
had identified compound 3 as binding with 10-fold
higher affinity than 1, and showed that N5-methylation
(i.e., 4) resulted in further 5-fold enhancement of
affinity.17 Because the aryl fluoro group was found to
make a minimal contribution to affinity, many subse-
quently examined compounds lacked this functionality.
It had been shown that replacement of the carbonyl
group of 5 with an ethylene group (i.e., 11) had no effect
on murine 5-HT5A receptor affinity; the same was found
in comparing the human 5-HT5A affinity of 5 (Ki ) 168
nM) with 11 (Ki ) 174 nM; Table 2). Introduction of
unsaturation to afford the alkenes 15 and 16 (Ki ) 208
and 185 nM, respectively) also had little influence on
affinity whereas the alkyne 17 displayed about 10-fold
reduced affinity. As in the mouse assay, replacement
of the carbonyl group with an ether oxygen atom was
found to be optimal (e.g., compare 5, Ki ) 168, with 2,
Ki ) 55 nM); removal of the fluoro group halved affinity
(22; Ki ) 100 nM). Nevertheless, compound 2 displayed
4-fold reduced affinity for human versus murine 5-HT5A
receptors. Shortening the alkoxy chain length of 2 by a
single methylene group (i.e., 19; Ki ) 930 nM) reduced
affinity by >15-fold. In general, where comparisons
could be made, the effect of chain alteration on human
5-HT5A binding was very similar to what was found for
binding at mouse 5-HT5A receptors.
Even though the presence of an amine moiety might
be intuitively expected to be a requirement for binding,
the role of the basic γ-carboline nitrogen atom was
examined. Compound 41 is the deaza analogue of 29
(Ki ) 190 nM). Replacement of the basic ring-nitrogen
atom with a methylene group (i.e., racemic 41, Ki
>10 000 nM) demonstrates that the amine is required
for optimal affinity.
Having established that a tricyclic ring system and
an amine are important for binding, and knowing that
a chain length of four to five atoms is tolerated, the
possibility was explored that the tetrahydro-γ-carboline
ring might be replaced by a tetrahydro-â-carboline
system. A series of â-carboline derivatives (i.e., pyrido-
[3,4-b]indoles), on hand as a result of other studies,18
was examined (Chart 2). Compounds 42-51 lacked
affinity for 5-HT5A receptors (i.e., Ki > 10 000 nM). The
individual optical isomers of 48 were examined; both
lacked affinity (Ki >10 000 nM). However, removal of
all ring substituents to give 1,2,3,4-tetrahydro-â-carbo-
line (52) (Ki ) 3200 ( 400 nM) resulted in an affinity
that was not very different than that of 1. Consequently,
several novel 1-desmethyl tetrahydro-â-carboline de-
rivatives were prepared. The â-carboline derivative 53
(Ki ) 550 nM; Table 2) was found to bind at murine
5-HT5A receptors with an affinity comparable to that of
Introduction of aromatic electron donating and elec-
tron-withdrawing substituents to the appended phenyl
ring (e.g., 25-29) failed to result in enhanced affinity.
Introduction of electron-donating and -withdrawing
substituents to the γ-carboline ring (e.g., 32-35) had
varying effects but failed to significantly enhance hu-
man 5-HT5A receptor affinity much in the same manner
that it failed to enhance murine 5-HT5A receptor affin-
ity.17

3934 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Khorana et al.
Ch a r t 2. Structures of â-Carbolines Examined at 5-HT5A Receptors
3 (Ki ) 512 nM) and only severalfold lower than that of
5, leading to speculation that replacement of the tet-
rahydro-γ-carboline nucleus with a tetrahydro-â-carbo-
line might be permissible. As a result, tetrahydro-â-
carboline derivatives of 21 and 22 (i.e., 54 and 55; Table
2) were prepared and examined. Compound 54 (m5-
HT5A Ki ) 335 nM) displayed twice the affinity of its
tetrahydro-γ-carboline counterpart (21, Ki ) 600 nM).
Because N-methylation had been shown to enhance
affinity in the tetrahydro-γ-carboline series, it was
expected that the N-methyl analogue of 54 (i.e., 55)
might also bind with enhanced affinity. However, 55
(m5-HT5A Ki ) 360 nM, h5-HT5A Ki ) 620 nM) failed to
F igu r e 2. Relationship between 5-HT5A versus 5-HT2A recep-
tor affinities for the tetrahydro-γ-carboline derivatives shown
in Table 2 (r ) 0.520; n ) 29). Where human 5-HT5A data were
unavailable, murine data were used.
show this effect. Although tetrahydro-â-carboline de-
rivatives might represent useful templates for future
study, they could behave somewhat differently, from a
structure-affinity perspective, than their tetrahydro-
γ-carboline counterparts.
separate the two activities. Due to the reversal in
selectivity seen with 9 and 28 compared to most of the
other compounds, further exploration of the distal
phenyl group portion of these molecules seems war-
ranted and might lead to improved selectivity.
5-HT5A ver su s 5-HT2 Selectivity. Our earlier in-
vestigation showed that several tetrahydro-γ-carbolines
bind at 5-HT2A receptors and that some of them dis-
played higher affinity for 5-HT2A than 5-HT5A recep-
tors;17 however, only a few analogues were examined
at that time. In the present investigation it was found
that 1 binds equally well at rat 5-HT2A (Ki ) 3100 ( 5
nM) and murine 5-HT5A (Ki ) 5300 nM) receptors.
Examination of an extended series of such compounds
(Table 2) shows that, indeed, all compounds bind at rat
5-HT2A receptors and, although generally with some-
what lower affinity, at rat 5-HT2C receptors. In general,
there is little to no 5-HT5A selectivity; the 4-methoxy-
substituted compound 9, and its ether counterpart 28,
displayed maximal (only about 4-fold) preference for
5-HT5A receptors over 5-HT2A receptors. Will it be
possible to divorce 5-HT2 character from 5-HT5A char-
acter? Are 5-HT5A and 5-HT2 structure-affinity rela-
tionships inextricably linked; that is, do 5-HT5A and
5-HT2 Ki values covary? Figure 2 shows the relationship
between 5-HT5A and 5-HT2A affinity for the tetrahydro-
γ-carbolines examined; although there is a trend toward
covariance, the correlation coefficient (r ) 0.520) is not
particularly impressive. Hence, it might be possible to
Su m m a r y. 5-HT5A receptors represent a poorly in-
vestigated family of serotonin receptors for which no
selective agent has yet been identified. Screening of
various compounds identified tetrahydro-γ-carboline 1
as a possible lead structure and a structure-affinity
investigation was undertaken. Our initial studies em-
ployed mouse 5-HT5A receptors and subsequent inves-
tigations used human 5-HT5A receptors once they be-
came available. For 15 tetrahydro-γ-carboline derivatives,
it was found that there is little interspecies difference
in binding affinities. A total of >50 tetrahydrocarboline-
related derivatives was prepared and examined in order
to formulate structure-affinity relationships. The gen-
eral conclusion is that compounds of type 2 bind with
nanomolar affinity at 5-HT5A receptors, that an intact
ring-system is important, that the tetrahydro-γ-carbo-
line ring might be replaced by a tetrahydro-â-carboline
ring (although few examples were explored), but that
most of the compounds also bind at 5-HT2A receptors.
While it seems that most structural modifications had

Binding of Tetrahydrocarbolines at 5-HT5A Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3935
OD): δ 2.30-2.42 (m, 2H, CH2), 3.20-3.30 (t, J ) 8.1 Hz, 2H,
CH2), 3.50-3.58 (t, J ) 7.8 Hz, 2H, CH2), 3.62-3.72 (bs, 2H,
CH2), 4.10-4.18 (t, J ) 6.0 Hz, 2H, CH2), 4.48-4.58 (bs, 2H,
CH2), 6.90-7.09 (m, 4H, Ar-H), 7.22-7.36 (m, 4H, Ar-H).
Anal. Calcd (C18H20FNO.HCl) C, H, N.
similar effect on 5-HT5A and 5-HT2A receptor affinity, a
comparison of Ki values showed only a modest correla-
tion. Hence, continued work with these compounds, with
emphasis on the terminal phenyl portion of the mol-
ecule, might result in improved selectivity.
N-[3-(4-F lu or op h en oxy)p r op yl]-N-m eth yl-N-(1-m eth yl-
1H-in d ol-3-yl)m eth yla m in e Oxa la te (39). A mixture of
indole-3-carboxaldehyde 97% (0.44 g, 3 mmol), 3-(4-fluorophen-
oxy)propylamine19 (0.51 g, 3 mmol), and NaBH3CN 95% (0.19
g, 3 mmol) in anhydrous MeOH (20 mL) was allowed to stir
at room temperature for 48 h. Solvent was evaporated under
vacuum, H2O (25 mL) was added, and the mixture was
basidified to pH 10 with 10% KOH and extracted with Et2O
(4 × 25 mL). The combined organic portion was dried (MgSO4),
solvent was evaporated under vacuum, and the crude product
was purified by column chromatography (silica gel; EtOAc/
hexane; 9:1) to give 0.35 g (39%) of the intermediate secondary
amine as an oil. IR (neat): 3411 cm-1. Sodium hydride 95%
(0.06 g, 2.2 mmol) was added to a stirred solution of the amine
(0.30 g, 1 mmol) in dry DMF (10 mL) under N2. Methyl iodide
(0.60 g, 4.2 mmol) was added, and the reaction mixture was
allowed to stir at room temperature for 30 min. The resultant
solution was poured into ice/H2O (50 mL) and extracted with
Et2O (3 × 25 mL). The combined organic portion was dried
(MgSO4), and solvent was evaporated under vacuum to afford
a crude product that was purified by column chromatography
(silica gel; EtOAc/MeOH; 9:1). The free base of 39 in anhydrous
CH2Cl2 was treated with oxalic acid in anhydrous Et2O.
Recrystallization from anhydrous MeOH/Et2O afforded 0.19
g (45%) of 39 as a pink powder; mp 141-143 °C. 1H NMR (CD3-
OD): δ 2.22-2.33 (bs, 2H, CH2), 2.87-2.94 (s, 3H, CH3), 3.33-
3.50 (bs, 2H, CH2), 3.84-3.90 (s, 3H, CH3), 4.13-4.20 (bs, 2H,
CH2), 4.55-4.63 (bs, 2H, CH2), 6.80-6.90 (m, 2H, Ar-H),
6.97-7.06 (t, J ) 8.7 Hz, 2H, Ar-H), 7.17-7.26 (t, J ) 9.3
Hz, 1H, Ar-H), 7.27-7.37 (t, J ) 7.8 Hz, 1H, Ar-H), 7.46-
7.53 (d, J ) 9.0 Hz, 2H, Ar-H), 7.70-7.78 (d, J ) 8.4 Hz, 1H,
Ar-H). Anal. Calcd (C20H23FN2O‚C2H2O4‚0.25H2O) C, H, N.
5-[3-(4-Flu or oph en oxy)pr opyl]-1-m eth yl-4,5,6,7-tetr ah y-
d r o-1H-p yr r olo[3,2-c]p yr id in e Oxa la te (40). A mixture of
1-methyl-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]pyridine (62) (0.15
g, 1.1 mmol), 3-(4-fluorophenoxy)propyl chloride (0.21 g, 1.1
mmol), K2CO3 (0.50 g, 3.5 mmol), and a catalytic amount of
NaI in DMF (10 mL) was heated at reflux for 10 h. The
reaction mixture was allowed to cool to room temperature,
concentrated under high vacuum, and diluted with water (25
mL). The mixture was extracted with CH2Cl2 (3 × 25 mL),
the combined organic extract was dried (MgSO4), solvent was
evaporated under reduced pressure, and the crude product was
purified by column chromatography (silica gel; CH2Cl2 followed
with EtOAc/MeOH; 9:1). The product was treated with oxalic
acid in an anhydrous CH2Cl2/Et2O mixture to form the salt.
Recrystallization from anhydrous MeOH/Et2O gave 0.15 g
(36%) of 40 as orange crystals; mp 130-132 °C. 1H NMR (CD3-
OD): δ 2.26-2.36 (m, 2H, CH2), 3.00-3.07 (t, J ) 6.0 Hz, 2H,
CH2), 3.45-3.53 (t, J ) 8.1 Hz, 2H, CH2), 3.55-3.59 (s, 3H,
NCH3), 3.60-3.72 (m, 2H, CH2), 4.08-4.15 (t, J ) 5.7 Hz, 2H,
CH2), 4.30-4.37 (bs, 2H, CH2), 5.95-5.90 (d, J ) 2.7 Hz, 1H,
CH), 6.67-6.70 (d, J ) 3.0 Hz, 1H, CH), 6.91-6.98 (m, 2H,
Ar-H), 7.00-7.07 (m, 2H, Ar-H). Anal. Calcd (C17H21FN2O‚
C2H2O4‚0.25H2O) C, H, N.
Exp er im en ta l Section
Ch em istr y. Melting points were taken in glass capillary
tubes on a Thomas-Hoover melting point apparatus and are
uncorrected. 1H NMR spectra were recorded with a Varian
EM-390 spectrometer, and peak positions are given in parts
per million (δ) downfield from tetramethylsilane as the
internal standard. Microanalyses were performed by Atlantic
Microlab (GA) for the indicated elements, and the results are
within 0.4% of the calculated values. Chromatographic separa-
tions were performed on silica gel columns (Kieselgel 40,
0.040-0.063 mm, Merck) by flash chromatography. Reactions
and product mixtures were routinely monitored by thin-layer
chromatography (TLC) on silica gel precoated F254 Merck
plates.
5-Meth yl-2-(4-oxim in o-(4-flu or oph en yl)bu tyl)-5H-1,2,3,4-
tetr a h yd r op yr id o[4,3-b]in d ole Hyd r och lor id e (6). A solu-
tion of compound 4 (free base)17 (0.10 g, 0.28 mmol) in a
mixture of EtOH (1 mL) and H2O (0.5 mL), in which NH2OH‚
HCl (0.04 g, 0.60 mmol) and NaOH (0.06 g) had been dissolved,
was allowed to stir at room temperature for 30 min. The
mixture was evaporated to dryness, H2O (25 mL) was added,
and the whole was extracted with CH2Cl2 (2 × 25 mL). The
combined organic portion was dried (MgSO4), and solvent was
evaporated under vacuum to yield a crude product. Purification
was achieved using column chromatography (silica gel; EtOAc/
MeOH; 9:1). A solution of the free base in anhydrous Et2O was
treated with ethereal HCl to give 0.08 g (70%) of 6 as a white
powder following recrystallization from an anhydrous MeOH/
Et2O mixture; mp 215-217 °C. 1H NMR (CD3CN): δ 1.78-
1.90 (m, 2H, CH2), 2.58-2.67 (t, J ) 7.8 Hz, 2H, CH2), 2.80-
2.92 (m, 6H, 3CH2), 3.60-3.70 (bs, 5H, CH2 and NCH3), 6.98-
7.19 (m, 4H, Ar-H), 7.30-7.40 (m, 2H, Ar-H), 7.68-7.76 (m,
2H, Ar-H). Anal. Calcd (C22H24FN3O‚HCl‚0.25H2O) C, H, N.
2-((4-Am in op h en oxy)p r op yl)-5-m et h yl-5H -1,2,3,4-t et -
r a h yd r op yr id o[4,3-b]in d ole Hyd r och lor id e (29). A mix-
ture of 25 (free base) (0.32 g, 0.89 mmol) and SnCl2‚2H2O (2.40
g, 10 mmol) in absolute EtOH (10 mL) was heated at 70 °C
for 3 h under N2. The reaction mixture was allowed to cool to
room temperature and poured into ice-water. The slurry was
extracted with EtOAc (2 × 30 mL). The combined organic
portion was dried (MgSO4), solvent was evaporated under
vacuum, and the crude product was purified by column
chromatography (silica gel; EtOAc/MeOH; 9:1). The solid
material in anhydrous MeOH was treated with methanolic
HCl. Recrystallization from an anhydrous MeOH/Et2O mixture
gave 0.17 (45%) of 29 as a white powder; mp 227 °C (decom-
1
poses). H NMR (CD3OD): δ 2.40-2.51 (m, 2H, CH2), 3.30-
3.36 (m, 2H, CH2), 3.60-3.68 (m, 2H, CH2), 3.72-3.76 (s, 3H,
NCH3), 3.90-4.10 (bs, 2H, CH2), 4.19-4.26 (t, J ) 5.7 Hz, 2H,
CH2), 4.40-4.52 (bs, 2H, CH2), 7.08-7.23 (m, 6H, Ar-H),
7.40-7.46 (d, J ) 7.5 Hz, 1H, Ar-H), 7.48-7.52 (d, J ) 7.5
Hz, 1H, Ar-H). Anal. Calcd (C21H25N3O‚2HCl‚1.25H2O) C, H,
N.
2-(3-(4-F lu or op h en oxy)p r op yl)-1,2,3,4-t et r a h yd r oiso-
qu in olin e Hyd r och lor id e (38). A mixture of 1,2,3,4-tetrahy-
droisoquinoline (0.14 g, 1 mmol), 3-(4-fluorophenoxy)propyl
chloride (0.19 g, 1 mmol), K2CO3 (0.50 g, 3.7 mmol), and a
catalytic amount of NaI in MeCN (10 mL) was heated at reflux
for 24 h. The reaction mixture was filtered, and the filtrate
was evaporated to dryness under reduced pressure. The
residue was suspended in H2O (25 mL) and extracted twice
with Et2O (25 mL). The combined ethereal extract was
evaporated under vacuum, and the crude product was purified
by column chromatography (silica gel; CH2Cl2 followed with
EtOAc/MeOH; 1:1). The free base was dissolved in anhydrous
MeOH and treated with methanolic HCl. Recrystallization
from anhydrous MeOH/Et2O afforded 0.14 g (43%) of the title
(()3-[3-(4-Am in op h en oxy)p r op yl]-9-m eth yl-1,2,3,4-tet-
r a h yd r oca r ba zole Hyd r och lor id e (41). A saturated solu-
tion of HCl (g) in EtOAc (1 mL) was added to 65 (0.2 g, 0.46
mmol) in EtOAc (3 mL), and the reaction mixture was allowed
to stir at room temperature for 18 h. The precipitated material
was collected by filtration and recrystallized from anhydrous
MeOH/Et2O to give 0.15 g (87%) of 41 as a white powder; mp
176-178 °C. 1H NMR (DMSO): δ 1.48-1.62 (m, 4H, 2CH2),
1.74-1.84 (m, 2H, CH2), 1.98-2.09 (m, 1H, CH), 2.18-2.30
(m, 1H, CH), 2.66-2.89 (m, 3H, CH2, and CH), 3.57-3.62 (s,
3H, N-CH3), 3.97-4.05 (t, J ) 6.3 Hz, 2H, CH2), 6.91-7.09
(m, 4H, Ar-H), 7.18-7.25 (m, 2H, Ar-H), 7.30-7.39 (t, J )
6.0 Hz, 2H, Ar-H). Anal. Calcd (C22H26N2O‚HCl‚0.25H2O) C,
H, N.
1
compound as white crystals; mp 195-196 °C. H NMR (CD3-

3936 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Khorana et al.
2-((4-Oxo-4-ph en yl)bu tyl)-1,2,3,4-tetr ah ydr o-9H-pyr ido-
[3,2-b]in d ole Hyd r och lor id e (53). Sodium carbonate (0.80
g, 5.8 mmol) and KI (0.01 g, 0.06 mmol) were added to a
solution of 1,2,3,4-tetrahydro-9H-pyrido[3,2-b]indole (1.0 g, 5.8
mmol) in DMF (10 mL), 4-chloro-1-phenyl-1-butanone (0.93
mL, 5.8 mmol) was added to the stirred reaction mixture in a
dropwise manner, and stirring was allowed to continue at 65
°C for 3.5 h under an N2 atmosphere. The reaction mixture
was allowed to cool to room temperature and poured into H2O
(50 mL), extracted with EtOAc (3 × 25 mL), and the combined
EtOAc portion was combined and evaporated to afford a yellow
oil. The oil was purified by column chromatography with CH2-
Cl2:MeOH (0% MeOH increasing to 2% MeOH) as the eluent
to afford 0.02 g (11%) of an off-white solid. The HCl salt was
vinyl compound as pale-yellow crystals; mp 99-100 °C (lit.21
mp 101-102 °C).
1-Meth yl-6,7-d ih yd r o-1H-p yr r olo[3,2-c]p yr id in e (61).
Nitrovinyl compound 59 (1.00 g, 6.5 mmol) was added por-
tionwise to a stirred suspension of LiAlH4 (1.00 g, 26 mmol)
in dry THF (40 mL) under N2. The reaction mixture was
allowed to stir at room temperature for 5 h and chilled to 0
°C, and the excess LiAlH4 was destroyed by the dropwise
addition of water. The mixture was purified by column
chromatography (silica gel; EtOAc:MeOH; 9:1) to give 0.55 g
(69%) of the amine as an oil. Employing the general method
of Herz et al.,22 a mixture of the amine (0.7 g, 5.6 mmol) and
ethyl formate (30 mL) was heated at reflux for 7 h. Solvent
was evaporated under reduced pressure, and the crude product
was purified by column chromatography (silica gel; EtOAc:
1
prepared and recrystallized from MeOH; mp 254-255 °C. H
1
NMR (DMSO-d6) δ 3.01-3.06 (m, 2H), 3.23-3.28 (m, 2H),
3.34-3.39 (m, 2H), 3.41-3.50 (bs, 1H), 3.79-3.83 (bs, 1H),
4.41-4.63 (m, 2H), 7.01-7.15 (m, 2H), 7.37-7.40 (d, 1H, J )
8.3 Hz), 7.47-7.58 (m, 3H), 7.64-7.69 (m, 1H), 7.98-8.01 (m,
2H), 11.05 (s, 1H). Anal. Calcd (C21H22N2O‚HCl‚0.1MeOH) C,
H, N.
MeOH; 5:1) to give 0.52 g (60%) of formamide 60 as an oil. H
NMR (CDCl3): δ 2.81-2.89 (t, J ) 6.6 Hz, 2H, CH2), 3.54-
3.64 (m, 5H, CH2 and CH3), 5.74-5.86 (bs, 1H, NH, D2O
exchangeable), 5.94-6.02 (m, 1H, CH), 6.08-6.14 (m, 1H, CH),
6.56-6.66 (m, 1H, CH), 8.16-8.22 (s, 1H, CH). Formamide
60 (0.5 g, 3.3 mmol) in refluxing toluene (15 mL) was treated
in a dropwise manner with a solution of POCl3 (0.5 mL) in
toluene. After heating at reflux for a total of 3 h, the reaction
mixture was allowed to cool to room temperature and was
washed several times with hot H2O (3 × 25 mL). The combined
aqueous portion was washed with CH2Cl2 (3 × 15 mL) and
basified by the addition of KOH. Extraction with CH2Cl2 (3 ×
25 mL) gave 0.16 g (36%) of 61 an oil. The compound was used
in the preparation of 62 without further purification. 1H NMR
(CDCl3): δ 2.78-2.87 (t, J ) 8.4 Hz, 2H, CH2), 3.59-3.63 (s,
3H, CH3), 3.86-3.95 (t, J ) 8.7 Hz, 2H, CH2), 6.29-6.33 (d, J
) 3 Hz, 1H, CH), 6.57-6.62 (d, J ) 3 Hz, 1H, CH), 8.29-8.36
(s, 1H, CH).
1-Me t h yl-4,5,6,7-t e t r a h yd r o-1H -p yr r olo[3,2-c]p yr i-
d in e (62). A mixture of NaBH4 (0.05 g, 1.3 mmol) in H2O (2.5
mL) was added in a dropwise manner to a stirred solution of
1-methyl-6,7-dihydro-1H-pyrrolo[3,2-c]pyridine (61) (0.15 g, 1.1
mmol) in MeOH (5 mL) at 0 °C. After 1 h, the mixture was
washed with brine and extracted with CH2Cl2 (3 × 20 mL).
The combined organic portion was dried (MgSO4) and solvent
evaporated under reduced pressure to give 0.23 g of a solid
product that was used in the synthesis of 40 without further
purification. 1H NMR (CDCl3): δ 2.54-2.62 (t, J ) 6.0 Hz, 2H,
CH2), 3.14-3.22 (t, J ) 6.0 Hz, 2H, CH2), 3.48-3.54 (s, 3H,
CH3), 3.84-3.89 (s, 2H, CH2), 5.90-5.95 (d, J ) 3 Hz, 1H, CH),
6.50-6.54 (d, J ) 3 Hz, 1H, CH).
(()3-[3-(Meth a n esu lfon yl)p r op yl]-9-m eth yl-1,2,3,4-tet-
r a h yd r oca r ba zole (64). Fischer-type cyclization of 4-(3-
hydroxypropyl)cyclohexanone23 using N-methyl-N-phenylhy-
drazine was used to obtain the corresponding carbazole
derivative. A mixture of N-methyl-N-phenylhydrazine (0.85 g,
6.5 mmol), 75% EtOH (60 mL), and 3 N HCl (1.2 mL) was
heated at reflux. 4-(3-Hydroxypropyl)cyclohexanone (63) (1.04
g, 6.5 mmol) was added in a dropwise manner, and the reaction
mixture was heated at reflux for 3 h. Heat was removed, and
the reaction was allowed to stir at room-temperature over-
night. Solvent was removed under reduced pressure, and the
crude product was extracted with CH2Cl2 (3 × 25 mL). The
combined organic portion was dried (Na2SO4), solvent was
evaporated under reduced pressure, and the crude product
purified by column chromatography (silica gel; CH2Cl2:MeOH;
20:1) to give 1.40 g (88%) of the tetrahydrocarbazole interme-
diate. Methansulfonyl chloride (0.42 mL) and Et3N (0.95 mL)
were added to a solution of this intermediate (1.1 g, 4.5 mmol)
in anhydrous CH2Cl2 (25 mL) at -10 °C, and the mixture was
allowed to stir at -10 °C for 15 min. The mixture was washed
with H2O (2 × 25 mL), the organic layer was separated and
dried (MgSO4), and solvent was evaporated under reduced
pressure. The crude product was purified by column chroma-
tography (silica gel; CH2Cl2) to give 1.42 g (98%) of the
mesylate which was used without further characterization in
the synthesis of 65.
2-(3-P h en oxyp r op yl)-1,2,3,4-tetr a h yd r o-9H-p yr id o[3,2-
b]in d ole Hyd r och lor id e (54). Solid sodium carbonate (0.80
g, 5.8 mmol) and KI (0.01 g, 0.06 mmol) were added to a
solution of 1,2,3,4-tetrahydro-9H-pyrido[3,2-b]indole (1.0 g, 5.8
mmol) in DMF (10 mL), 1-bromo-3-phenoxypropane (0.91 mL,
5.8 mmol) was added to the stirred mixture in a dropwise
manner, and stirring was allowed to continue at 65 °C for 3.5
h under an N2 atmosphere. The reaction mixture was cooled
to room temperature, poured into H2O (50 mL), and extracted
with EtOAc (3 × 25 mL), and the combined EtOAc portion
was evaporated to afford a yellow oil. The oil was purified by
column chromatography with EtOAc:hexanes (8:2) as the
eluent to afford 1.29 g (72%) of an off-white solid; mp 121-
123 °C. The HCl salt was prepared and recrystallized from
MeOH; mp 252-253 °C. 1H NMR (DMSO-d6) δ 2.29-2.34 (m,
2H), 2.99-3.07 (m, 2H), 3.39-3.45 (m, 3H), 3.80 (bs, 1H),
4.08-4.12 (t, 2H, J ) 6 Hz), 4.44-4.58 (m, 2H), 6.93-6.97 (m,
3H), 7.00-7.05 (m, 1H), 7.09-7.l4 (m, 1H), 7.28-7.39 (m, 3H),
7.46-7.49 (d, 1H, J ) 7.7 Hz), 11.14 (s, 1H). Anal. Calcd
(C20H22N2O‚HCl) C, H, N.
9-Met h yl-2-(3-p h en oxyp r op yl)-1,2,3,4-t et r a h yd r o-9H -
p yr id o[3,2-b]in d ole Hyd r och lor id e (55). Sodium hydride
(60% dispersion, 0.04 g, 1.1 mmol) was added to a solution of
2-(3-phenoxypropyl)-2,3,4,9-tetrahydro-1H-â-carboline (54; 0.30
g, 1.0 mmol) in DMF (5 mL) at 0 °C. The reaction mixture
was allowed to stir at 0 °C for 15 min under an N2 atmosphere.
Methyl iodide (0.15 g, 1.1 mmol) in DMF (2 mL) was added to
the mixture in a dropwise manner, and stirring was continued
at room temperature under an N2 atmosphere for 1.5 h. The
reaction mixture was poured into H2O (25 mL) and extracted
with EtOAc (3 × 25 mL); the combined EtOAc portion was
evaporated to afford an oil that was purified by column
chromatography with CH2Cl2:MeOH (99:1) as the eluent to
afford 0.21 g (67%) of an yellow oil. The HCl salt was prepared
and recrystallized from MeOH; mp 221-224 °C. 1H NMR
(DMDO-d6) 2.35-2.39(m, 2H), 2.97-3.16(m, 2H), 3.45(s, 3H),
3.67-3.68(m, 3H), 3.76-3.84(m, 1H), 4.10-4.14(m, 2H), 4.45-
4.50(d, 1H, J ) 14.07), 4.78-4.83(d, 1H, J ) 14.07), 6.93-
6.98(m, 3H), 7.03-7.09(m, 1H), 7.15-7.21(m, 1H), 7.27-
7.34(m, 2H), 7.44-7.52(m, 2H). Anal. Calcd (C21H24N2O‚HCl)
C, H, N.
1-Meth yl-2-(2-n itr ovin yl)-1H-p yr r ole (59). The title com-
pound was synthesized according to the general procedure
described by Spadoni et al.20 A solution of 1-methyl-2-pyrrole-
carboxaldehyde (1.00 g, 9 mmol) and NaOAc (0.34 g, 4 mmol)
in CH3NO2 (10 mL) was heated at reflux for 1.5 h under N2.
The reaction mixture was allowed to cool to room temperature,
EtOAc (25 mL) was added, and the organic phase was
decanted. The organic phase was washed with H2O (2 × 25
mL) and dried (Na2SO4), solvent was evaporated under
reduced pressure, and the crude product was purified by
column chromatography (silica gel; CH2Cl2). Recrystallization
from anhydrous CH2Cl2/Et2O gave 1.02 g (75%) of the nitro-
(()3-[3-(4-ter t-Bu tylca r ba m ylp h en oxy)p r op yl]-9-m eth -
yl-1,2,3,4-tetr a h yd r oca r ba zole (65). A mixture of mesylate

Binding of Tetrahydrocarbolines at 5-HT5A Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3937
(6) Matthes, H.; Boschert, U.; Amlaiky, N.; Grailhe, R.; Plassat, J .
L.; Muscatelli, F.; Mattei, M. G.; Hen, R. Mouse 5-hydroxytrypt-
amine5A and 5-hydroxytryptamine5B receptors define a new
family of serotonin receptors: cloning, functional expression, and
chromosomal localization. Mol. Pharmacol. 1993, 43, 313-319.
(7) Erlander, M. G.; Lovenberg, T. W.; Baron, B. M.; deLeccea, L.;
Danielson, P. E.; Racke, M.; Slone, A. L.; Siegel, B. W.; Foye, P.
E.; Cannon, K.; Burns, J . E.; Sutcliffe, J . G. Two members of a
distinct subfamily of 5-hydroxytryptamine receptors differen-
tially expressed in rat brain. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 3452-3456.
(8) Wisden, W.; Parker, E. M.; Mahle, C. D.; Grisel, D. A.; Nowak,
H. P.; Yocca, F. D.; Felder, C. C.; Seeburg, P. H.; Voight, M. M.
Cloning and characterization of the rat 5-HT5B receptor. FEBS
Lett. 1993, 333, 25-31.
(9) Rees, S.; den Daas, I.; Foord, S.; Goodson, S.; Bull, D.; Kilpatrick,
G.; Lee, M. Cloning and characterization of the human 5-HT5A
serotonin receptor. FEBS Lett. 1994, 355, 242-246.
(10) Grailhe, R.; Grabtree, G. W.; Hen, R. Human 5-HT5 receptors:
the 5-HT5A receptor is functional but the 5-HT5B receptor was
lost during mammalian evolution. Eur. J . Pharmacol. 2001, 418,
157-167.
64 (0.64 g, 2.0 mmol), (4-hydroxyphenyl)carbamic acid tert-
butyl ester24 (0.42 g, 2.0 mmol), and K2CO3 (1.00 g, 7.0 mmol)
in MeCN (30 mL) was heated at reflux for 18 h. The reaction
mixture was allowed to cool to room temperature, concentrated
under vacuum, and extracted twice with CH2Cl2 (30 mL). The
combined organic portion was dried (MgSO4), solvent was
evaporated under reduced pressure, and the crude product was
purified by column chromatography (silica gel; CH2Cl2:MeOH;
20:1). Recrystallization from anhydrous MeOH give 0.45 g
(52%) of 65 as a white powder; mp 139-141 °C. 1H NMR
(CDCl3): δ 1.42-1.46 (s, 9H, 3CH3), 1.48-1.58 (m, 4H, 2CH2),
1.70-1.92 (m, 2H, CH2), 1.98-2.07 (m, 1H, CH), 2.22-2.34
(m, 1H, CH), 2.63-2.73 (m, 2H, CH2), 2.82-2.92 (m, 1H, CH),
3.53-3.57 (s, 3H, N-CH3), 3.86-3.94 (t, J ) 6.6 Hz, 2H, CH2),
6.21-6.27 (bs, 1H, NH), 6.74-6.81 (m, 2H, Ar-H), 6.96-7.13
(t, J ) 7.3 Hz, 1H, Ar-H), 7.04-7.12 (t, J ) 6.9 Hz, 1H, Ar-
H), 7.14-7.22 (m, 3H, Ar-H), 7.36-7.41 (d, J ) 7.5 Hz, 1H,
Ar-H). Anal. Calcd (C27H34N2O3‚HCl) C, H, N.
Ra d ioliga n d Bin d in g Assa y. The 5-HT5A radioligand
binding studies were performed as previously described.17
Tritiated (+)lysergic acid diethylamide (LSD) was used to label
murine 5-HT5A receptors (0.5 nM) and human 5-HT5A receptors
(1 nM) (cell line generously donated by Dr. R. Hen) and 1 µM
LSD was used to determine nonspecific binding. The 5-HT2
binding assays were also conducted according to published
procedures.25 In the 5-HT2 binding assays, either 0.5 nM [3H]-
ketanserin (5-HT2A) or 2.0 nM [3H]mesulergine (5-HT2C) were
used as radioligand; ketanserin (10 µM, 5-HT2A) and me-
sulergine (1 µM, 5-HT2C) were used to determine nonspecific
binding. The general procedure is as follows: NIH-3T3 cells
stably transfected with rat 5-HT2A receptors (donated by Dr.
David J ulius) and A-9 cells stably transfected with rat 5-HT2C
receptors (donated by Dr. Beth Hoffman) were grown to
confluence, suspended in 50 mM Tris-HCl buffer, and cen-
trifuged at 12 000g for 30 min. The pellet was resuspended in
buffer and centrifuged for an additional 20 min. Assay buffer
used in the experiments consisted of 50 mM Tris-HCl, 0.5 mM
EDTA, 10 mM MgCl2, and 0.1% ascorbate (pH 7.4). After
resuspension in assay buffer, 1-mL membrane aliquots (∼10
µg protein measured by bicinchoninic assay) were added to
each tube containing 1 mL of assay buffer, radioligand, and
test compound. Competition experiments were performed in
triplicate in a 2.0-mL volume. Typically, 11 concentrations of
test agent (10-10 to 10-5 M) were evaluated, except where the
compound displayed <30% inhibition at 10 000 nM in which
case it was not further examined. Membranes were incubated
for 30 min at 37 °C, filtered on Schleicher and Schuell (Keene,
NH) glass fiber filters (presoaked in 0.1% polyethyleneimine),
and washed with 10 mL of buffer. The filters were counted in
an Ecoscint liquid scintillation counter at 40% efficiency.
Competition experiments were plotted and analyzed using
GraphPad Prism. Ki values were determined from the Cheng-
Prusoff equation: Ki ) IC50/(1 + [D]/KD).26 The results reflect
a minimum of three replicate determinations.
(11) Noda, M.; Yasuda, S.; Okada, M.; Higashida, H.; Shimada, A.;
Iwata, N.; Ozaki, N.; Nishikawa, K.; Shirasawa, S.; Uchida, M.;
Aoki, S.; Wada, K. Recombinant human serotonin5A receptors
stably expressed in C6 glioma cells couple to multiple signal
transduction pathways. J . Neurochem. 2003, 84, 222-232.
(12) Birkett, J . T.; Arranz, M. J .; Munro, J .; Osbourn, S.; Kerwin, R.
W.; Collier, D. A. Association analysis of the 5-HT5A gene in
depression, psychosis and antipsychotic response. Neuroreport
2000, 11, 2017-2020.
(13) Arias, B.; Collier, D. A.; Gasto, C.; Pintor, L.; Gutierrez, B.;
Valles, V.; Fananas, L. Genetic variation in the 5-HT5A receptor
gene in patients with bipolar disorder and major depression.
Neurosci. Lett. 2001, 303, 111-114.
(14) Grailhe, R.; Waeber, C.; Dulawa, S. C.; Hornung, J . P.; Zhuang,
X.; Brunner, D.; Geyer, M. A.; Hen, R. Increased exploratory
activity and altered response to LSD in mice lacking the 5-HT5A
receptor. Neuron 1999, 22, 581-591.
(15) Carson, M. J .; Thomas, E. J .; Danielson, P. A.; Sutcliffe, J . G.
The 5-HT5A serotonin receptor is expressed predominantly by
astrocytes in which it inhibits cAMP accumulation: A mecha-
nism for neuronal suppression of reactive astrocytes. Glia 1996,
17, 317-326.
(16) Teitler, M.; Scheick, C.; Howard, P.; Sullivan, J . E.; Iwamura,
T.; Glennon, R. A. 5-HT5A serotonin receptor binding: A pre-
liminary structure-affinity investigation. Med. Chem. Res. 1997,
7, 207-218.
(17) Khorana, N.; Purohit, A.; Herrick-Davis, K.; Teitler, M.; Glennon,
R. A. γ-Carbolines: Binding at 5-HT5A serotonin receptors.
Bioorg. Med. Chem. 2003, 11, 717-722.
(18) Glennon, R. A.; Dukat, M.; Grella, B.; Hong, S.; Costantino, L.;
Teitler, M.; Smith, C.; Egan, C.; Davis, K.; Mattson, M. V.
Binding of â-carbolines and related agents at serotonin (5-HT2
and 5-HT1A), dopamine (D2) and benzodiazepine receptors. Drug
Alcohol Depend. 2000, 60, 121-132.
(19) Ismaiel, A. M.; De Los Angeles, J .; Teitler, M.; Ingher, S.;
Glennon, R. A. Antagonism of 1-(2,5-dimethoxy-4-methylphenyl)-
2-aminopropane stimulus with a newly identified 5-HT2 versus
5-HT1C-selective antagonist. J . Med. Chem. 1993, 36, 2519-
2525.
(20) Spadoni, G.; Balsamini, C.; Bedini, A.; Diamantini, G.; Giacomo,
B.; Tontini, A.; Tarzia, G. 2-[N-Acylamino(C1-C3)alkyl]indoles
as MT1 melatonin receptor partial agonists, antagonists, putative
inverse agonists. J . Med. Chem. 1998, 41, 3624-3634.
(21) Clitheroe, A.; Green, D.; J ansen, A. B. A.; Phillips, P. C.; Rule,
A. W. Nitroethylenes and related compounds as trichomonacides
and candidacides. J . Pharm. Pharmacol. 1965, 17, 167-172.
(22) Herz, W.; Tocker, S. Pyrrolo[3,2-c]pyridines. J . Am. Chem. Soc.
1955, 77, 6353-6355.
(23) Marvell, E. N.; Sturmer, D.; Rowell, C. Bicyclo[3.3.1]nonanes.
A new synthesis of 2-bicyclo[3.3.1]nonanone. Tetrahedron 1966,
22, 861-866.
(24) Vigroux, A.; Bergon, M.; Zedde, C. Cyclization-activated pro-
drugs: N-(Substituted 2-hydroxyphenyl and 2-hydroxypropyl)-
carbamates based on ring-opened derivatives of active benzox-
azolones and oxazolidinones as mutual prodrugs of acetamino-
phen. J . Med. Chem. 1995, 38, 3983-3994.
(25) Chang-Fong, J .; Addo, J .; Dukat, M.; Smith, C.; Mitchell, N. A.;
Herrick-Davis, K.; Teitler, M.; Glennon, R. A. Evaluation of
isotryptamine derivatives at 5-HT2 serotonin receptors. Bioorg.
Med. Chem. Lett. 2002, 12, 155-158.
(26) Cheng, Y.-C.; Prusoff, W. H. Relationship between inhibitory
constant (Ki) and the concentration of inhibitor which causes
50% inhibition (IC50) of an enzyme reaction. Biochem. Phar-
macol. 1973, 22, 3099-3108.
Ack n ow led gm en t. The authors are grateful to Dr.
Rene Hen for providing the human 5-HT5A-expressing
cell line, and Dr. David J ulius and Dr. Beth Hoffman
for the 5-HT2 cell lines.
Refer en ces
(1) Glennon, R. A.; Dukat, M. Serotonin receptors and drugs
affecting serotonergic neurotransmission. In Foye’s Principles of
Medicinal Chemistry; Williams, D. A., Lemke, T. M., Eds.,
Lippincott Williams & Wilkins, Philadelphia, 2002; pp 315-337.
(2) Hoyer, D.; Hannon, J . P.; Martin, G. R. Molecular, pharmaco-
logical and functional diversity of 5-HT receptors. Pharmacol.
Biochem. Behav. 2002, 71, 533-554.
(3) Humphrey, P. P. A.; Hartig, P. R.; Hoyer, D. A new nomenclature
for 5-HT receptors. Trends Pharmacol. Sci. 1993, 14, 233-236.
(4) Barnes, N. M.; Sharp, T. A review of central 5-HT receptors and
their function. Neuropharmacology 1999, 38, 1083-1152.
(5) Plassat, J .-L.; Boschert, U.; Amlaiky, N.; Hen, R. The mouse
5-HT5 receptor reveals a remarkable heterogeneity within the
5-HT1D family. EMBO J . 1992, 11, 4779-4786.
J M030080S