8-Sulfonyl-substituted tetrahydro-1H-pyrido[4,3-b]indoles as 5-HT6 receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2009.10.035

A series of novel 8-sulfonyl-substituted 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indoles (THPI) has been synthesized and their ability to interact with 5-HT₆ receptors evaluated in cell-based and radioligand binding assays. Amongst evaluated THPIs, compounds 9.HCl and 20.HCl have been identified as the most potent 5-HT₆ receptor antagonists with K_i values equal to 2.1 nM and 5.7 nM and IC₅₀ values (functional assay) equal to 15 nM and 78 nM, respectively. Affinities of these two compounds for several serotonin receptors in the competitive radioligand binding assays as well as their specificity profiles against a panel of therapeutic targets have been determined.

Full text of DOI:10.1016/j.ejmech.2009.10.035

European Journal of Medicinal Chemistry 45 (2010) 782–789
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
8-Sulfonyl-substituted tetrahydro-1H-pyrido[4,3-b]indoles
as 5-HT6 receptor antagonists
,
b
,
a
b
b,c
Alexandre V. Ivachtchenko a , Oleg D. Mitkin , Sergey E. Tkachenko , Ilya M. Okun
,
*
,
Volodymyr M. Kysil b
*
a Department of Organic Chemistry, Chemical Diversity Research Institute, 114401 Khimki, Moscow Reg., Russia
b ChemDiv, Inc., 6605 Nancy Ridge Drive, San Diego, CA 92121, USA
c Department of Molecular Biology and High-Throughput Screening, Chemical Diversity Research Institute, 114401 Khimki, Moscow Reg., Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 8-sulfonyl-substituted 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indoles (THPI) has been
synthesized and their ability to interact with 5-HT6 receptors evaluated in cell-based and radioligand
binding assays. Amongst evaluated THPIs, compounds 9.HCl and 20.HCl have been identified as the most
potent 5-HT6 receptor antagonists with Ki values equal to 2.1 nM and 5.7 nM and IC50 values (functional
assay) equal to 15 nM and 78 nM, respectively. Affinities of these two compounds for several serotonin
receptors in the competitive radioligand binding assays as well as their specificity profiles against a panel
of therapeutic targets have been determined.
Received 31 July 2009
Received in revised form
22 October 2009
Accepted 23 October 2009
Available online 31 October 2009
Keywords:
Ó 2009 Elsevier Masson SAS. All rights reserved.
Serotonin receptors
5-HT6 receptor
Antagonist
Dimebolin
Dimebon
Pyrido[3,4-b]indole
1. Introduction
Recently, we hypothesized that this action of dimebolin could be
associated with its ability to interact with and block 5-HT6 recep-
First reported in 1962 [1] and launched in 1983 as an antihis-
taminic agent [2,3], dimebolin (DimebonÔ, 2,8-dimethyl-5-[2-(6-
methylpyridin-3-yl)ethyl]-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole
dihydrochloride) (Fig.1) has attracted a new wave of attention since
its efficiency for the treatment of various neurodegenerative
disorders was discovered [4–7]. It is being currently evaluated in
advanced clinical trials as a promising small molecule drug-
candidate for the treatment of Alzheimer’s (Phase III) and Hun-
tington’s (Phase II) diseases [7]. Various pharmacological properties
of dimebolin have been studied extensively [8–12]. Dimebolin
possesses a remarkably broad spectrum of molecular target activity
as well as a very complex mechanism of action, which allows one to
consider it as an excellent example of a drug within the specific
group of ‘magic shotgun’ drugs [13]. However, the specific mech-
anism of its anti-neurodegenerative activity is still elusive.
tors [14]. We have found that compared to earlier disclosed
potential targets [4–7] dimebolin exhibits rather high potency to
antagonize 5-HT6 receptors (IC50 ¼ 0.890
mM) [12]. Furthermore,
the most recent research by other authors [15] has demonstrated
that dimebolin binds with moderate affinity to both the human and
rat recombinant 5-HT6 receptors (Ki ¼ 26.0 ꢀ 2.5 nM and
119 ꢀ 14 nM respectively) as well as the native rat 5-HT6 receptor,
and acts as an antagonist in functional adenylyl cyclase assays.
It has been shown also that dimebolin produces an acute
enhancement of short-term social recognition memory.
In mammals, 5-HT6 receptors localize primarily in CNS and the
related signaling cascades are deeply implicated in the processes of
information perception, learning, and memory formation [16].
It has also been shown that 5-HT6 receptors regulate several
neurotransmitter pathways including cholinergic, noradrenergic,
glutamatergic, and dopaminergic systems [17]. Taking into
consideration the fundamental role of these systems in normal
cognitive processes and their dysfunctions due to neuro-
degeneration, the exceptional role of 5-HT6 receptors in both the
formation of normal memory and its impairment becomes obvious.
* Corresponding authors. Tel.: þ1 858 794 4860; fax: þ1 858 794 4931.
E-mail addresses: av@chemdiv.com (A.V. Ivachtchenko), vkysil@chemdiv.com
(V.M. Kysil).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.035

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 45 (2010) 782–789
783
AR
HBA
PI
HYD
Key recognition elements
of 5-HT pharmacophore model
6
Cl
CH
N
3
H
N
H C
3
CH
3
O
NH
H C
3
N
O
HN
N
N
N
H C
3
O
S
O
S
H C
3
O
O
2HCl
N
CH
3
PRX-07034
SB-742457
Dimebolin
Fig. 1. Schematic representation of the pharmacophore model for 5-HT6 receptor antagonists and their examples: PRX-07034 (phase I, EPIX Pharm,), SB-742457 (phase II,
GlaxoSmithKline) and dimebolin (phase III, Medivation-Pfizer).
Therefore, 5-HT6 receptors represent an extremely attractive target
for the development of novel small molecule therapeutics for the
treatment of various neurodegenerative disorders [18–20].
Numerous selective antagonists of 5-HT6 receptors have been
disclosed during the last decade [21,22] and a pharmacophore
model for this type of receptor antagonists has been developed
based on known structurally diverse 5-HT6 receptor antagonists
[23–29]. In general, the model entails the following key elements:
a positive ionizable atom (PI, usually secondary or tertiary amino-
group), a hydrogen bond acceptor group (HBA, usually sulfone or
sulfonamide group), a hydrophobic site (HYD), and an aromatic-
ring hydrophobic site (AR). For example, these elements can clearly
be identified in the structures of PRX-07034 and SB-742457 (Fig. 1)
– the drug-candidates that are currently under active development
for the treatment of cognitive impairment associated with
Alzheimer’s disease or schizophrenia [30].
step in both cases. The synthesis of N(5)-unsubstituted sulfo-
derivatives 16–19 entailed employment of 2-methyl-2,3,4,5-tetra-
hydro-1H-pyrido[4,3-b]indole-8-sulfinic acid 11 as a sulfinic acid
component in the coupling. In turn, this acid was obtained by the
reduction of sulfonyl chloride 3 with Na2SO3 in the presence of
NaHCO3 as an auxiliary base (hydrolysis of sulfonyl chloride 3 was
a dominating process when stronger bases such as NaOH or Na2CO3
were used). The obtained crude sulfinic acid 11 of 80–85% purity by
LC-MS (ELSD) analysis (inorganic salts were the only impurities)
was used in the next step without additional purification. Its CuI-
catalyzed coupling with (het)aryl iodides 12–15, proceeded cleanly
to afford sulfones 16–19 whereas corresponding bromo-derivatives
as well as 5-bromopyrimidine gave no product (Scheme 2).
To obtain 5-methyl-substituted THPIs 20 and 21, the other
approach was used starting from readilyavailable [31,32] 8-bromo-2-
Though active as the 5-HT6 receptor antagonist, dimebolin
clearly does not match the pharmacophore model (Fig. 1). There-
fore, we hypothesized that by introducing the sulfone or sulfon-
amide group as the HBA site into tetrahydropyridoindole core, we
could improve affinity of dimebolin-like molecules toward the
5-HT6 receptors. We focused our effort on the synthesis and bio-
logical evaluation of several 8-sulfo-derivatives of 2,3,4,5-tetrahy-
dro-1H-pyrido[4,3-b]indole (THPI). To the best of our knowledge,
no data relating activity of sulfo-derivatives of THPIs with 5-HT6
receptors have been reported. Surprisingly, there have been a very
limited number of reports in relation to chemistry and biological
properties of 8-sulfo-THPIs published to date.
1
R
SO H
3
1
R
HO S
3
N
N
a
+
N
H
O
NHNH
2
1,2
1: R1 = CH3
2: R1 = PhCH2
3,4
;
3: R1 = CH3;
4: R1 = PhCH2
b
3
R
2. Chemistry
2
R
N
O
O
1
1
R
R
ClO S
2
S
c
N
N
Sulfonamides 7–10 were synthesized starting from 4-piper-
idones 1,2 and 4-hydrazinobenzenesulfonic acid (Scheme 1). The
Fischer indole synthesis proceeded cleanly when a solution of these
starting materials in a mixture of conc. sulfuric and acetic acids was
heated at 80–90 ꢁC. The obtained sulfonic acids 3,4 were allowed to
react with POCl3 in sulfolane to afford sulfonyl chlorides 5,6 that
were isolated and used for further transformation in the form of
their hydrosulfates. Their interaction with amines yielded sulfon-
amides 7–10 (see experimental part).
N
H
N
H
7 - 10
7: R1 = R2 = R3 = CH3;
5,6
5: R1 = CH3;
8: R1 = CH3; R2R3 = -(CH2)2N(CH3)(CH2)2-;
9: R1 = CH3; R2 = 3-F-C6H4; R3 = H;
10: R1 = PhCH2; R2 = 3-F-C6H4; R3 = H
6: R1 = PhCH2
Two alternative approaches were used to obtain sulfones 16–21
employing coupling of sulfinic acids with (het)aryl halides as a key
Scheme 1. Synthesis of THPI-8-sulfonamides. Reagents and conditions: (a) H2SO4,
AcOH, 80–90 ꢁC, 1 h; (b) POCl3, sulfolane, 75–80 ꢁC, 2 h; (c) R2R3NH, pyridine, 20 ꢁC, 1 h.

784
A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 45 (2010) 782–789
2
R
O
O
CH
HO S
2
3
CH
S
3
b
N
N
a
3
I-Ar
N
H
12 - 15
N
R
1
16 - 21
12 - 15
11
16: R1 = H; R2 = 3-F-C6H4;
12: Ar = 3-F-C6H4;
17: R1 = H; R2 = 3-Py;
18: R1 = H; R2 = 4-Py;
19: R1 = H; R2 = C6H5;
13: Ar = 3-Py;
14: Ar = 4-Py;
15: Ar = C6H5
20: R1 = CH3; R2 = C6H5;
21: R1 = R2 = CH3
b
CO Et
2
Br
CH
3
Br
CO Et
2
Br
N
N
c
N
d
N
N
N
H
CH
CH
3
3
22
23
24
Scheme 2. Synthesis of 8-(aryl- and alkyl-sulfonyl)-substituted THPIs. Reagents and conditions: (a) Na2SO3, NaHCO3, water, 1 h; (b) PhSO2Na or MeSO2Na, CuI,
CH3NHCH2CH2NHCH3, DMSO, DIPEA, 100 ꢁC, 12 h; (c) MeI, NaH, DMF, rt, 1 h; (d) LiAlH4, ꢂ20 to ꢂ25 ꢁC, 2 h.
O
O
CH
S
3
CH
S
3
N
O
N
O
2-CH =CH-Py
4-CH C H -SO Cl
2
3
6
4
2
19
N
S
N
a
b
O
O
N
25
26
CH
3
Scheme 3. Synthesis of N(6)-substituted 8-phenylsulfonyl-THPIs 25 and 26. Reagents and conditions: (a) 60% aq. KOH, DMSO, TBAC, 60 ꢁC, 12 h; (b) NaH, DMF.
aforementionedconditionsfurnishedtarget8-methylsulfonyl-and8-
Table 1
phenylsulfonyl-THPIs 20 and 21, respectively.
To synthesize 2-methyl-8-phenylsulfonyl-5-(2-pyridin-2-
Functional SAR data of 8-sulfo-derivatives of THPIs obtained in cell-based assay on
inhibition of 5-HT6 receptor-induced cAMP synthesis in the cells (Means of 2
independent experiments in duplicates ꢀ SE are presented).
ylethyl)-THPI 25 as a dimebolin analog, 8-phenylsulfonyl-THPI 19
underwent Michael addition with 2-vinylpyridine employing TBAC
as a phase transfer catalyst in a system of 60% aq. KOH – DMSO,
which provided target material in 76% yield (Scheme 3). Finally,
2-methyl-5-[(4-methylphenyl)sulfonyl]-8-(phenylsulfonyl)-
2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole 26 with two sulfonyl
groups on the periphery of THPI core was synthesized by the
reaction of 19 with p-toluenesulfonyl chloride in the presence of
NaH in DMF.
R1
R2
R3
5-HT6 receptor-
stimulated
cAMP production.
IC50 ꢀ SE (
mM)
Dimebolin
7
0.89
Me
Me
Me
PhCH2
H
H
Me
Me
Me
Me
26 ꢀ 6
8
–(CH2)2N(CH3)(CH2)2–
62 ꢀ 10
9.HCl
10
3-F–C6H4
3-F–C6H4
3-F–C6H4
3-Py
Ph
Me
H
H
0.015 ꢀ 0.004
3.9 ꢀ 1.3
16
0.085 ꢀ 0.015
3.4 ꢀ 0.9
17.HCl
20.HCl
21.HCl
25.HCl
26.HCl
0.078 ꢀ 0.018
No inh
3. Results and discussion
0.62 ꢀ 0.09
Synthesized 8-sulfosubstituted THPIs 7–10, 16, 17, 20, 21, 25, 26
were tested for their ability to interact with 5-HT6 receptors
(Table 1). In a functional assay testing for antagonism by measuring
decreasesin cyclic AMP levelsdue to inhibitionofreceptoractivation
by serotonin, we studied the ability of the THPIs to concentration-
dependently block serotonin-induced responses of HEK-293 cells
with heterologously expressed 5-HT6 human recombinant receptor.
28 ꢀ 7
ethoxycarbonyl-THPI 22. N(5)-Methylation of this compound with
methyl iodide in the presence of NaH gave 23. Then LAH-reduction of
the 2-ethoxycarbonyl group yielded 8-bromo-2,5-dimethyl-THIP 24.
Coupling of 24 with benzene- and methane- sulfinic acids under the

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 45 (2010) 782–789
785
Fig. 2. Specificity profile of THPI 9 (1
mM) measured by competitive displacement of corresponding radioligands (Mean ꢀ SE, n ¼ 2).
Addition of serotonin to the cells growing in 96-well micro titer
plates causes an increase in the cytoplasmic level of cAMP, which is
determined using cAMP LANCE technology [33].
All active compounds showed full antagonistic activity with Imax
close to 100%. As seen from data in Table 1, amongst the substituted
THPI-8-sulfonamides 7–10, the most potent antagonist of 5-HT6
receptors is THPI 9 bearing a hydrophobic N-(3-fluorophenyl)-
of magnitude reduction in the potency). These sulfonamides have
IC50 values of 26.4 M (7) and 62.0 M (8), respectively.
A similar trend is found in the series of 8-sulfonyl-THPIs. 8-Phenyl-
sulfonyl-derivatives 16 (IC50 ¼ 0.085 M) and 20 (IC50 ¼ 0.078 M)
are the most potent 5-HT6 receptor antagonists whereas 8-pyridyl-
sulfonyl-THPI 17 (IC50 ¼ 3.45 M) is more than 40-fold less potent
and 8-methylsulfonyl- -carboline 21 is inactive in the cell-based
m
m
m
m
m
g
group (IC50 ¼ 0.015
a bulky and hydrophobic 2-benzyl group leads to 10 with 259-fold
M). A stronger effect in
potency reduction is observed upon substitution of arylamino-
moiety in sulfonamide 9 with dimethylamino-group in 7 or, even
more so, with hydrophilic N-methylpiperazino-group in 8 (3 orders
m
M). Replacement of 2-methyl group in 9 with
5-HT6 receptors functional assay.
Notably, all these observations are in a good agreement with the
above-mentioned pharmacophore model of 5-HT6 receptor
antagonists: potencies of both types of the evaluated compounds,
sulfamides and sulfones, correlate with lipophilicity of sub-
stitutents attached to the sulfone groups. Particularly, hydrophobic
reduction in its potency (IC50 ¼ 3.88
m
Fig. 3. Specificity profile of THPI 20 (10
mM) measured by competitive displacement of corresponding radioligands (Mean ꢀ SE, n ¼ 2).

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A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 45 (2010) 782–789
aryl substituents in the most potent antagonists 9, 16, and 20 match
well the HYD moiety in the pharmacophore model and their
substitution with either less hydrophobic or hydrophilic substitu-
ents reduces the antagonistic potency dramatically. Moreover,
these most potent antagonists demonstrate 10–60-fold higher
potency than dimebolin.
hydrochloride 20.HCl. These compounds have more than an order of
magnitude higher affinity to 5-HT6 receptors than that of dimebolin,
which is currently being tested in Phase III clinical trials as an anti
Alzheimer’s disease drug.
Comparing compounds 16 and 20, one can see that the intro-
duction of a methyl substituent at 5 position does not affect the
5-HT6 receptor blocking potency. In contrast, substitution of the
hydrogen or methyl groups in these compounds with bulky groups
causes significant potency reduction. Thus, 5-[2-(2-pyridyl)ethyl]-
5. Experimental protocols
Commercially available reagents and solvents were employed
without purification. NMR spectra at 400 MHz were recorded on
a Varian Unity þ spectrometer; chemical shifts reported in ppm
using TMS as internal standard in DMSO-d6.
Purity (more than 98% unless otherwise specified) of all
synthesized compounds was confirmed by LC-MS analysis per-
formed on Shimadzu HPLC equipped with PE SCIEX API 150EX
mass-, Alltech 2056 ELSD-, and Shimadzu UV- (254 and 215)
substituted THPI 25 (IC50 ¼ 0.615
mM) is 8-fold less potent and 5-
(4-methylphenyl)sulfonyl THPI 26 (IC50 ¼ 28
mM) is 360-fold less
potent than 5-methyl THPI 20.
The most potent 5-HT6 receptor antagonists, 9 and 20, were
further tested against a panel of therapeutic targets to characterize
their specificity profiles. Interaction of the 8-sulfonyl-THPIs 9 and
20 with the target proteins was assessed by their ability to compete
for the targets with corresponding radio-labeled ligands.
detectors. Separation was achieved with a Phenomenex Luna 3
m
C18 (4.6 ꢄ 150 mm) column using gradient 5–95% acetonitrile in
water both with 0.05% TFA over 12 min at 0.8 mL/min.
The target specificity profiles of 9 (measured at 1
mM) and 20
(measured at 10 M) are shown in Figs. 2 and 3, respectively. Except
m
for 5-HT1A receptor, compound 9, strongly interacts with serotonin
receptors (80–100% radioligand displacement), noticeably interacts
with adrenergic a2A and sigma s1 receptors (app. 60% radioligand
displacement) and exhibits minor competition for adrenergic a1A
(20%), a1B (37%), a1D (29%) receptors, L-type Ca2þ channel (benzo-
thiazepine site, 27%), dopamine D3 receptor (35%), and transporters
of glycine (20%) and norepinephrine (15%).
5.1. Synthesis
5.1.1. General procedure for 2,3,4,5-tetrahydro-1H-pyrido[4,3-
b]indole-8-sulfonyl chlorides 5,6
A mixture of 4-piperidone 1 or 2 (0.1 mol) and 4-hydrazino-
benzenesulfonic acid (18.8 g, 0.1 mol) was heated under reflux for
2 h, cooled, and the formed precipitate was filtered off, washed
with AcOH, i-PrOH, and hexane. A mixture of the obtained product,
AcOH (140 mL), and conc. H2SO4 (50 mL) was heated at 80–90 ꢁC
for 1 h, cooled, diluted with AcOH (200 mL), and poured into
vigorously stirred Et2O (1.5 L). Solvent layer was decanted, oily
residue was washed twice with Et2O, treated with hot MeOH, and
kept in a refrigerator overnight. The formed precipitate was filtered
off, washed with MeOH, Et2O, and dried. Yield 59–74% of sulfonic
acids 3, 4. LC-MS (M þ H)þ: 267 for 3 and 343 for 4.
THPI 20 shows similar to 9 specificity profile, the most sensitive
to 20 being serotonin receptors (5-HT2A ¼ 5-HT2B ¼ 5-HT2C ¼ 5-
HT6 ¼ 5-HT7 w 100%). Similar to 9, THPI 20 interacts with a group of
adrenergic receptors (a1B
ꢃ
a1D
ꢃ
a2A
ꢃ
a1A). Unlike 9, THPI 20
interacts with dopamine and norepinephrine transporters and has
lower displacement affinity for a2A, dopamine D3, and Sigma 1
receptors (similar displacement values but at an order of magni-
tude higher concentration).
Compound 9 concentration-dependently competes with corre-
sponding radioligands for the 5-HT receptors with affinity rank
order of 5-HT6 > 5-HT2A > 5-HT7 ¼ 5-HT2C while the affinity rank
order of dimebolin is 5-HT7 > 5-HT6 > 5-HT2C > >5-HT2A (Table 2).
Except for 5-HT7, dimebolin exhibits lower affinities (higher Ki
values) to all the studied 5-HT receptors.
A mixture of sulfonic acid 3 or 4 (0.02 mol), POCl3 (4 mL), and
tetramethylensulfone (30 mL) was stirred at 75–80 ꢁC for 2 h. The
obtained clear solution after cooling was poured into vigorously
stirred Et2O (0.5 L), the semisolid precipitate was separated by
decantation, washed twice with Et2O, and then treated with EtOAc.
The obtained suspension was stirred overnight until crystalline
compound formation was completed. The precipitate was filtered off,
washed with EtOAc, Et2O, and dried. Yield 62–72% of technically pure
compound1 was used for the next step without further purification.
4. Conclusion
We describe here the synthesis and preliminary SAR of novel 8-
sulfonyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indoles. In particular,
the potency of the synthesized compounds to antagonize 5-HT6
receptors is highly dependent on lipophilicity of sulfonyl containing
substituent at the position 8. The most potent 5-HT6 receptor
antagonists are N-(3-fluorophenyl)-2-methyl-2,3,4,5-tetrahydro-1H-
pyrido[4,3-b]indole-8-sulfonamide hydrochloride 9.HCl and 2,5-
dimethyl-8-(phenylsulfonyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole
5.1.2. General procedure for compounds 7,8
A stirred mixture of sulfonyl chloride 5 (383 mg, 1.0 mmol) and
THF (3.0 mL) was treated with 2 M solution of amine in THF
(2.0 mL), the reaction mixture was kept in ultrasonic bath for 1 h
and concentrated under reduced pressure on a rotary evaporator.
The residue treated with ice water, neutralized with excess of aq.
NaHCO3, the formed precipitate was separated by centrifugation,
washed twice with i-PrOH, hexane, and dried.
Table 2
5.1.2.1. N,N,2-Trimethyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-
Affinities of 9, 20, and dimebolin to several serotonin receptors assessed in
a competitive radioligand binding assays (data of one representative experiment in
duplicates is shown).
8-sulfonamide 7. Yield 58% of colorless solid. 1H NMR (DMSO-d6,
400 MHz),
d
: 2.40 (s, 3H), 2.52 (s, 6H), 2.69 (t, J ¼ 5.3 Hz, 2H), 2.80
(t, J ¼ 5.3 Hz, 2H); 3.54 (s, 2H), 7.35 (dd, J1 ¼8.4 Hz, J2 ¼1.6 Hz, 1H),
7.45 (d, J ¼ 8.4 Hz, 1H), 7.72 (d, J ¼ 1.6 Hz, 1H), 11.40 (s, 1H). MS-ESI
m/z 294 (M þ H). LC-MS (UV-254) purity: 99%.
Target
Dimebolin
9
20
Ki, nM
Ki, nM
Ki, nM
5-HT2A
5-HT2C
5-HT6
5-HT7
>100
76
34
7.0
39
2.1
42
5.7
1
Although these compounds were not analyzed because of hydrolytic lability, we
8
assumed that they were isolated in forms of their hydrosulfates.

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 45 (2010) 782–789
787
5.1.2.2. 2-Methyl-8-[(4-methylpiperazin-1-yl)sulfonyl]-2,3,4,5-tetra-
5.1.4.2. 2-Methyl-8-(pyridin-3-ylsulfonyl)-2,3,4,5-tetrahydro-1H-
hydro-1H-pyrido[4,3-b]indole 8. Yield 80% of colorless solid. 1H
pyrido[4,3-b]indole hydrochloride 17.HCl. A solution of the
compound 17 obtained accordingly to the general procedure was
dissolved in acetone and treated with excess of HCl solution in
dioxane. Formed precipitate was separated by centrifugation,
washed with acetone, EtOAc, and Et2O. Yield 18%. 1H NMR (DMSO-
NMR (DMSO-d6, 400 MHz), d: 2.08 (s, 3H), 2.27–2.33 (m, 4H), 2.40
(s 3H), 3.54 (s, 2H), 2.76–2.83 (m, 6H), 2.69 (t, J ¼ 5.5 Hz, 2H), 7.33
(dd, J1 ¼8.6 Hz, J2 ¼1.4 Hz, 1H), 7.45 (d, J ¼ 8.6 Hz, 1H), 7.70 (d,
J ¼ 1.4 Hz, 1H), 11.42 (s, 1H). MS-ESI m/z 349 (M þ H). LC-MS
(UV-254) purity: 99%.
d6, 400 MHz),
d
: 2.92 (d, J ¼ 4.8 Hz, 3H), 3.07 (br. m, 1H), 3.24 (m,
1H), 3.44 (m, 1H), 3.67 (br. m, 1H), 4.29 (dd, J1 ¼14.4 Hz, J2 ¼ 7.6 Hz,
1H), 4.66 (d, J ¼ 14.4 Hz, 1H), 7.55 (d, J ¼ 8.4 Hz, 1H), 7.62 (dd,
J1 ¼8.4 Hz, J2 ¼ 4.0 Hz, 1H), 7.69 (dd, J1 ¼8.4 Hz, J2 ¼ 2.0 Hz, 1H),
8.25 (d, J ¼ 1.6 Hz, 1H), 8.31 (dt, J1 ¼8.0 Hz, J2 ¼ 1.6 Hz, 1H), 8.79 (d,
J ¼ 4.8 Hz, 1H), 9.11 (d, J ¼ 1.6 Hz, 1H), 11.34 (br. m, 1H), 12.00 (s, 1H).
MS-ESI m/z 328 (M þ H). LC-MS (UV-254) purity: 98%.
5.1.3. General procedure for compounds 9, 10
To a stirred solution of m-fluoroaniline (1.05 mmol) in pyridine
(2 mL), sulfonyl chloride 5 or 6 (1 mmol) was added. The reaction
mixture was kept in ultrasonic bath for 1 h, concentrated under
reduced pressure on a rotary evaporator, the residue treated with
ice water, neutralized with excess of aq. NaHCO3, the formed
precipitate was separated by centrifugation, washed twice with i-
PrOH, hexane, and dried.
5.1.4.3. 8-(Phenylsulfonyl)-2-methyl-2,3,4,5-tetrahydro-1H-pyr-
ido[4,3-b]indole 19. This compound was contaminated with non-
identified non-removable impurity (about 5%) and therefore was
not characterized. However, it was successfully used for the
synthesis of compounds 25 and 26.
5.1.3.1. N-(3-Fluorophenyl)-2-methyl-2,3,4,5-tetrahydro-1H-pyr-
ido[4,3-b]indole-8-sulfonamide hydrochloride 9.HCl. A solution of
the compound obtained according to the general procedure was
dissolved in acetone and treated with excess of HCl solution in
dioxane. Formed precipitate was separated by centrifugation,
washed with acetone, EtOAc, and Et2O. Yield 80%. 1H NMR (DMSO-
5.1.4.4. 2-Methyl-8-(pyridin-4-ylsulfonyl)-2,3,4,5-tetrahydro-1H-
pyrido[4,3-b]indole hydrochloride 18.HCl. 1H NMR (DMSO-d6,
400 MHz),
d
: 2.93 (d, J ¼ 4.4 Hz, 3H), 3.08 (m, 1H), 3.24 (m, 1H), 3.45
d6, 400 MHz),
d
: 2.90 (d, J ¼ 4.5 Hz, 3H), 2.98–3.08 (m,1H), 3.14–3.25
(m, 1H), 3.69 (m, 1H), 4.30 (dd, J1 ¼14.4 Hz, J2 ¼ 7.6 Hz, 1H), 4.68 (d,
J ¼ 14.4 Hz, 1H), 7.58 (d, J ¼ 8.4 Hz, 1H), 7.67 (dd, J1 ¼8.4 Hz,
J2 ¼ 1.8 Hz, 1H), 7.86 (dd, J1 ¼4.4 Hz, J2 ¼ 1.6 Hz, 2H), 8.24 (d,
J ¼ 1.8 Hz, 1H), 8.83 (dd, J1 ¼4.4 Hz, J2 ¼1.6 Hz, 2H), 11.17 (br. s, 1H),
12.00 (br. s, 1H). MS-ESI m/z 328 (M þ H). LC-MS (UV-254)
purity: 98%.
(m, 1H), 3.39–3.44 (m, 1H), 3.62–3.70 (m, 1H), 4.22–4.27 (m, 1H),
4.58–4.66 (m, 1H), 6.72–6.77 (m, 1H), 6.87–6.90 (m, 2H), 7.15–7.21
(m, 1H), 7.46–7.52 (m, 2H), 7.97 (s, 1H), 10.49 (s, 1H), 11.09 (br. s, 1H),
11.86 (s, 1H). MS-ESI m/z 360 (M þ H). LC-MS (UV-254) purity: 98%.
5.1.3.2. 2-Benzyl-N-(3-fluorophenyl)-2,3,4,5-tetrahydro-1H-pyr-
ido[4,3-b]indole-8-sulfonamide 10. Yield 76%. 1H NMR (DMSO-d6,
5.1.5. General procedure for compounds 20, 21
400 MHz),
d
: 2.79 (s, 4H), 3.57 (s, 2H), 3.74 (s, 2H), 6.75 (dd,
Anhydrous DMF (10 mL) was added to preliminarily washed
with hexane NaH (60% suspension in mineral oil, 3.85 mmol), the
stirred mixture was cooled to 0 ꢁC, 8-bromo-2-ethoxycarbonyl-
2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole 22 (1.13 g, 3.5 mmol)
was added, and stirring was continued for 30 min. Methyl iodide
(0.54 g, 3.8 mmol) was added in one portion, and the mixture was
allowed to warm to ambient temperature. After stirring for addi-
tional 1 h, the mixture was poured into ice water. The formed
precipitate was separated by filtration, washed with water, and
dried to afford 1.12 g (yield 95%) of 8-bromo-2,5-dimethyl-2,3,4,5-
tetrahydro-1H-pyrido[4,3-b]indole 23 [95% purity by LC-MS, UV-
254 detection; ESI-MS m/z 337 (M þ H)]. The crude product was
used at the next step without further purification.
To a stirred at ꢂ20 to ꢂ25 ꢁC, suspension of LiAlH4 (125 mg,
3.3 mmol) in Et2O (50 mL), a solution of the crude product 23
(1.08 g, 3.20 mmol) was added dropwise. The mixture was stirred at
this temperature for 2 h, and water (30 mL) was added dropwise.
The organic layer was separated, aqueous layer was extracted with
Et2O, combined organic layers was washed with 10% aq. K2CO3,
dried under Na2SO4, and concentrated to dryness on a rotary
evaporator. Recrystallization of the residue with EtOAc furnished
0.57 g (64%) of 8-bromo-2,5-dimethyl-2,3,4,5-tetrahydro-1H-pyr-
ido[4,3-b]indole 24 contaminated with 5–10% of 8-unsubstituted
compound as a product of 8-Br-reduction. The crude product was
added to a stirred mixture prepared in inert atmosphere by
consecutive mixing DMSO (2 mL), N,N0-dimethylethylenediamine
(24 mg, 0.27 mmol), CuI (24 mg, 0.14 mmol), water (0.7 mL),
benzene or methane sulfinic acid (1.5 mmol), and diisopropyle-
thylamine (0.26 mL, 1.50 mmol). The resulted mixture was stirred
in a sealed tube at 100 ꢁC for 24 h and poured into water. Formed
precipitate was separated by centrifugation, washed with water,
saturated aq. NaHCO3, 10% aq. NH3, and water. Silica purification
(hexane – EtOAc – Et3N) of the dried crude product furnished pure
compound that was dissolved in acetone and treated with 10%
J1 ¼8.4 Hz, J2 ¼1.6 Hz, 1H), 6.87 (m, 2H), 7.18 (m, 1H), 7.28 (m, 1H),
7.33–7.42 (m, 6H), 7.76 (s,1H),10.26 (s,1H),11.38 (s,1H). MS-ESI m/z
436 (M þ H). LC-MS (UV-254) purity: 98%.
5.1.4. General procedure for compounds 16–19
Sulfonyl chloride 5 (1.28 g, 3.35 mmol) was added to a vigor-
ously stirred saturated solution of Na2SO3 (20 mL) and then
NaHCO3 (3.36 g, 40 mmol) was added in three portions. The reac-
tion mixture was stirred at 80–85 ꢁC for 1 h, neutralized with AcOH,
evaporated under reduced pressure to dryness; the solid residue
was treated with MeOH, filtered off, washed with ice water, MeOH,
and dried. Accordingly to LC-MS (ELSD-detection), the crude
product was 2-methyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-
8-sulfinic acid 11 of 85% purity contaminated with inorganic salts.
It was used at the next step without additional purification. To a
stirred solution of N,N0-dimethylethylenediamine (24 mg,
0.27 mmol) in DMSO (2 mL), CuI (24 mg, 0.14 mmol) was added.
The mixture was stirred at ambient temperature for 10 min, and
then water (0.7 mL), sulfinic acid 11 (375 g, 1.5 mmol), diisopropy-
lethylamine (0.26 mL, 1.50 mmol), and aryliodide 12–15 (1.5 mmol)
were added in consecutive order. The resulted mixture was purged
with argon, heated with stirring at 100 ꢁC overnight, cooled, and
diluted with water. Formed precipitate was separated by centrifu-
gation, washed with water, saturated aq. NaHCO3, 10% aq. NH3,
water, and recrystallized with i-PrOH.
5.1.4.1. 8-[(3-Fluorophenyl)sulfonyl]-2-methyl-2,3,4,5-tetrahydro-
1H-pyrido[4,3-b]indole 16. Yield 23%. 1H NMR (DMSO-d6,
400 MHz),
d
: 2.42 (s, 3H), 2.71 (t, J ¼ 5.2 Hz, 2H), 2.80 (t, J ¼ 5.2 Hz,
2H), 3.58 (s, 2H), 7.44 (d, J ¼ 8.4 Hz, 1H), 7.47 (td, J1 ¼8.4 Hz,
J2 ¼ 2.0 Hz, 1H), 7.56 (dd, J1 ¼8.4 Hz, J2 ¼ 2.0 Hz, 1H), 7.62 (dt,
J1 ¼9.2 Hz, J2 ¼ 8.0 Hz, 1H), 7.76 (m, 2H), 8.05 (d, J ¼ 1.2 Hz, 1H),
11.46 (s, 1H). MS-ESI m/z 345 (M þ H). LC-MS (UV-254) purity: 98%.

788
A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 45 (2010) 782–789
excess of 6 N HCl in dioxane. The precipitate of HCl-salt was
separated by centrifugation, washed with acetone, and dried.
was substituted with a buffer comprising: HBSS supplemented with
1 mM MgCl2, 1 mM CaCl2 100 M IBMX, 5 mM HEPES, pH 7.4. Test
m
compounds were added at different concentrations while main-
taining constant final DMSO concentration of 0.1%. After 15 min
incubation with the compounds at room temperature, serotonin
hydrochloride (Sigma, MO) was added to a final concentration of
10 nM and incubation continued for additional 30 min. Cells were
treatedforcAMPcontentas describedin theprotocolofcAMP LANCE
assay kit (PerkinElmer, MA) recommended by the manufacturer. The
LANCE signal was measured in white 384-well plates (Corning, MA)
using multimode plate reader VICTOR2V (PerkinElmer, MA) with
built-in settings for LANCE detection.
5.1.5.1. 2,5-Dimethyl-8-(phenylsulfonyl)-2,3,4,5-tetrahydro-1H-pyr-
ido[4,3-b]indole hydrochloride 20.HCl. Yield 45%. 1H NMR (DMSO-
d6, 400 MHz), d: 2.93 (s, 3H), 3.18 (m, 2H), 3.47 (m, 1H), 3.71 (s, 3H),
3.72 (m, 1H), 4.32 (m, 1H), 4.67 (m, 1H), 7.55–7.64 (m, 3H), 7.68 (m,
2H), 7.92 (m, 2H), 8.19 (s, 1H), 11.18 (br. s, 1H). MS-ESI m/z 341
(M þ H). LC-MS (UV-254) purity: 99%.
5.1.5.2. 2,5-Dimethyl-8-(methylsulfonyl)-2,3,4,5-tetrahydro-1H-pyr-
.
ido[4,3-b]indole hydrochloride 21HCl. Yield 54%. 1H NMR (DMSO-d6,
400 MHz),
d
: 2.98 (s, 3H), 3.13 (s, 3H), 3.23 (t, J ¼ 5.8 Hz, 2H), 3.65
(m, 2H), 3.75 (s, 3H), 4.52 (m, 2H), 7.69 (m, 2H), 8.10 (s, 1H). MS-ESI
5.2.2. Receptor binding assays
m/z 279 (M þ H). LC-MS (UV-254) purity: 98%.
Assessments of competitive displacement of radio-labeled
ligands with tested compounds from the receptors utilized in this
manuscript, were performed by MDSPharma Services (Taiwan) in
accordance with their internal protocols.
5.1.6. 2-Methyl-8-(phenylsulfonyl)-5-(2-pyridin-2-ylethyl)-2,3,4,5-
tetrahydro-1H-pyrido[4,3-b]indole hydrochloride 25.HCl
A mixture of 19 (0.68 g, 2 mmol), DMSO (2 mL), TBAC (0.1 mL of
50% solution), fresh distilled 2-vinylpyridine (0.32 g, 3 mmol), and
KOH (8 mL of 60% aq. solution) was vigorously stirred under argon
at 60 ꢁC for 12 h, cooled and extracted with CH2Cl2. The organic
layer was washed with water, brine, dried (Na2SO4), and concen-
trated on a rotary evaporator. The pure product obtained after silica
purification of residue was dissolved in acetone and treated with an
excess of 6 N solution of HCl in dioxane. Centrifugation of the
formed precipitate followed by washing with acetone furnished
5.2.3. Statistical analysis
All experiments were performed in duplicates. The data were
fitted with Prism 4 (GraphPad, CA) using 4 parametric equation
built-in into the software.
Acknowledgements
The authors thank Dr. Frederick Lakner for critical reading and
correction of the manuscript.
76% of colorless solid compound. 1H NMR (DMSO-d6, 400 MHz),
d:
2.92 (d, J ¼ 2.8 Hz, 3H), 3.19 (m, 2H), 3.40 (t, J ¼ 6.4 Hz, 2H), 3.46 (m,
1H), 3.73 (m, 1H), 4.29 (dd, J1 ¼14.4 Hz, J2 ¼ 6.8, 1H), 4.63–4.69 (m,
3H), 7.68 (t, J ¼ 6.4 Hz, 1H), 7.57–7.64 (m, 4H), 7.73 (d, J ¼ 8.8 Hz,
1H), 7.78 (d, J ¼ 7.2 Hz, 1H), 7.92 (d, J ¼ 7.2 Hz, 2H), 8.18 (s, 1H), 8.20
(t, J ¼ 6.4 Hz, 1H), 8.70 (d, J ¼ 5.2 Hz, 1H), 11.40 (br. s, 1H). MS-ESI m/
z 432 (M þ H). LC-MS (UV-254) purity: 98%.
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