(3-Phenylsulfonylcycloalkano[e and d]pyrazolo[1,5- a]pyrimidin-2-yl)amines: Potent and selective antagonists of the serotonin 5-HT6 receptor

Article abstract of DOI:10.1021/jm100350r

5-HT₆ receptors are exclusively localized in the CNS and have high affinity with many psychotropic agents. Though the role of this receptor in many CNS diseases is widely anticipated, lack of definite progress in the development of 5-HT₆ receptor-oriented drugs indicates a need for further discoveries of novel chemotypes with high potency and high selectivity to the receptor. Here we present preparations and biological evaluation of a series of (3-phenylsulfonylcycloalkano[e and d]pyrazolo[1,5-a]pyrimidin-2-yl) amines. Phenylsulfonylcyclopentapyrazolopyrimidine 7 was found to be a highly selective 5-HT₆ receptor antagonist with high affinity (low picomolar range) and potency. 7 and a few of its analogues were further tested for biological effect on 5-HT_2B receptors and hERG potassium channels, potential liability targets. Such liability appears to be minimal, based on the in vitro data.

Full text of DOI:10.1021/jm100350r

5186 J. Med. Chem. 2010, 53, 5186–5196
DOI: 10.1021/jm100350r
(3-Phenylsulfonylcycloalkano[e and d]pyrazolo[1,5-a]pyrimidin-2-yl)amines: Potent and Selective
Antagonists of the Serotonin 5-HT6 Receptor
Alexandre V. Ivachtchenko,†,§ Dmitri E. Dmitriev,† Elena S. Golovina,‡ Madina G. Kadieva,† Angela G. Koryakova,‡
Volodymyr M. Kysil,§ Oleg D. Mitkin,† Ilya M. Okun,*,§ Sergey E. Tkachenko,§ and Anton A. Vorobiev†
†Department of Organic Chemistry, and ‡Department of Molecular Pharmacology, Chemical Diversity Research Institute,
114401 Khimki, Moscow Reg, Russia, and ChemDiv, Inc., 6605 Nancy Ridge Drive, San Diego, California 92121
§
Received April 1, 2010
5-HT6 receptors are exclusively localized in the CNS and have high affinity with many psychotropic
agents. Though the role of this receptor in many CNS diseases is widely anticipated, lack of definite
progress in the development of 5-HT6 receptor-oriented drugs indicates a need for further discoveries of
novel chemotypes with high potency and high selectivity to the receptor. Here we present preparations
and biological evaluation of a series of (3-phenylsulfonylcycloalkano[e and d]pyrazolo[1,5-a]pyrimidin-
2-yl)amines. Phenylsulfonylcyclopentapyrazolopyrimidine 7 was found to be a highly selective 5-HT6
receptor antagonist with high affinity (low picomolar range) and potency. 7 and a few of its analogues
were further tested for biological effect on 5-HT2B receptors and hERG potassium channels, potential
liability targets. Such liability appears to be minimal, based on the in vitro data.
Introduction
antagonists as therapeutic tools revealed that these receptors
are promising targets for the development of new drug
candidates for treatment of various CNS diseases such as
schizophrenia, Alzheimer’s disease, and other neurodegenera-
tive diseases and cognitive disorders.14-17 In particular, the
successful ongoing clinical trials of new highly selective and
potent 5-HT6R antagonists, AVN-211 and AVN-322, have
recently been announced for treatment of some CNS
diseases.18,19
Serotonin (5-hydroxytryptamine, 5-HT a ) receptor (5-HT6R)
belongs to a group of Gs-protein-coupled receptors controlling
cellular levels of cAMP. Rat and then human receptor were
reported to be cloned by Monsma et al.1 and Kochen et al.2 in
1993 and 1996, respectively. The receptor is almost exclusively
localized in the central neural system (CNS). The receptor
attracted a good deal of attention as a potential target for
medicinal chemistry for such indications as anxiety,3,4 cogni-
tion,5 learning and memory,6 and mood.3,7 It was shown8 that
5-HT6R can modulate cholinergic, noradrenergic, glutama-
tergic, and dopaminergic neurotransmitter systems. Given the
fundamental role of these systems in normal cognitive pro-
cesses and in neurodegeneration, it became an apparent
necessity to elucidate the role of 5-HT6R in the processes
controlling normal or “pathologic” memory. Development of
5-HT6R antagonists facilitated discovery that blockage of
these receptors led to a significant enhancement of memory
consolidation in various animal models of learning and me-
morization, reproduction,9-11 and improvement of cognitive
function in old rats.9
Interest in the development of 5-HT6R antagonists is
growing (Figure 1), and the role of the receptors in metabolic
diseases, such as obesity, is suggested.14 However, in spite of
the ever growing number ofarticles, proofthat the antagonists
actually work through the 5-HT6R antagonism is still elusive
and novel, more selective and potent molecules are needed.
Numerous selective antagonists of 5-HT6R have been dis-
closed, most of them representing the heterocyclic compounds
bearing a sulfonyl or sulfamide group.14-16,20-24 On the basis
of available structurally diverse 5-HT6R antagonists, phar-
macophore models for this type of receptor antagonist have
been suggested.25-29 In general, the models entail positive
ionizable atom (PI, usually secondary or tertiary amino group),
strong multiple hydrogen bond acceptor group (mHBA, usual-
ly sulfonyl or sulfamide group), a hydrophobic site (HYD, for
example, phenyl), and π-electron donor aromatic or hetero-
cyclic ring (AR). These important recognition elements are
represented in a simplified form of a model A in Figure 2.22
The most typical representatives of the 5-HT6R antagonists
conforming to the pharmacophore A model are 3-benzene-
sulfonyl-8-piperazin-1-ylquinoline (SB-742457) with Ki =
0.234 nM10,30 and 3-benzenesulfonyl-2-methylsulfanyl-8-pi-
perazin-1-yl-6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyri-
midine (Ro-65-7674) with Ki=0.85 nM (Figure 3).11,31 The SB-
742457 is currently in phase II clinical trials for the treatment of
Alzheimer’s disease.12,32 The Ro-65-7674, though a highly
In recent years, an understanding of the role of 5-HT6R in
cognitive processes has improved. Possible pharmacophore
properties of their ligands were formulated,12 which helped in
developing quite potent and selective antagonists. It was also
shown that many typical and atypical antipsychotic agents
bind to 5-HT6R with high affinity.13 Use of the 5-HT6R
*To whom correspondence should be addressed. Phone: 1-858-794-
4860. Fax: 858-794-4931. E-mail: iokun@chemdiv.com.
a Abbreviations: CNS, central nervous system; GSPCR, Gs-protein-
coupled receptor; 5HT, 5-hydroxytryptamine; 5-HT6R, serotonin type 6
receptor; PI, positive ionizable atom; mHBA, multiple hydrogen bond
acceptor group; HYD, a hydrophobic site; AR, π-electron donor
aromatic or heterocyclic ring; Ki, equilibrium antagonist binding con-
stant; IC50, half maximal inhibition concentration.
r
pubs.acs.org/jmc
Published on Web 06/18/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5187
Figure 1. Number of publications and patents in the 5-HT6R field between the years 1993 and 2009. Data taken from the SciFinder database
searching the key word 5-HT6R, December 01, 2009.
to produce 3-(dimethylamino)-3-(methylthio)-2-(phenylsulfo-
nyl)acrylonitrile 25. The 25 was then reacted with N2H4 H2O
3
in an i-PrOH/water mixture, step c, which yielded 1H-pyra-
zole-3,5-diamine 20.
The reaction of 19 and 20 with 2-formylcyclopentanone 21
in acetic acid at room temperature, condition d, or at 100 °C,
Figure 2. Simplified recognition elements representation of phar-
macophores A and B characteristic for 5-HT6 receptor anta-
gonists.22,28
condition e, or in acetic acid in the presence of HCl at room
temperature, condition f, exclusively yielded (3-phenylsulfo-
nyl-7,8-dihydro-6H-cyclopenta[e]pyrazolo[1,5-a]pyrimidin-2-
yl)methylamine7 and N2,N2-dimethyl-3-phenylsulfonyl-7,8-
dihydro-6H-cyclopenta[e]pyrazolo[1,5-a]pyrimidin-2-amine 11.
When sodium 2-formylcyclohexanone 22 was used in con-
dition d, the reaction produced (3-phenylsulfonyl-5,6,7,8-
tetrahydropyrazolo[5,1-b]quinazolin-2-yl)methylamine 1. The
same reaction performed in condition f produced (3-phenyl-
sulfonyl-6,7,8,9-tetrahydropyrazolo[1,5-a]quinazolin-2-yl)methyl-
amine 8. Our data agree well with those of the synthesis of
3-cyanocycloalkano[e and d]pyrazolo[1,5-a]pyrimidines.34
Reaction of the 3,5-diamino-1H-pyrazole 19 with 2-ace-
tylcycloalkanones 23 and 24 (Scheme 1) was less selective than
that with 2-formylcycloalkanones 21 and 22. The reaction of
19 with 2-acetylcyclopentanone 23 in condition a, b, or c
yielded a 1:9 mixture of linear 3 and angular 9 products
(Figure 5). Reaction of 19 with 2-acetylcyclohexanone 24
yielded a mixtureof products4 and 10. When being performed
in condition a þ c, this reaction produced the mixture of 4 and
10 with a ratio of ∼7:3. In the condition b þ c, the 4/10 ratio
was 6:5.
potent and selective 5-HT6R antagonist with good DMPK
properties, did not reach the clinical trials stage because of its
high affinity to the hERG channel.31
Recently, we have described the synthesis of cycloalkane-
annelated 3-phenylsulfonylpyrazoles (C and D in Figure 3),
which are highly potent and highly specific antagonists of the
5-HT6R.33 In spite of their high affinity (IC50 in a single-digit
nanomolar range), the C and D models do not include the
positive ionizable group, PI, and could be described by the
model B of the recognition elements pharmacophore
(Figure 2). The most active 5-HT6R antagonist in this series
of cycloalkane-annelated 3-phenylsulfonylpyrazolo[1,5-a]pyri-
midines (C and D, Figure 3) was 5-methyl-2-methylsulfanyl-3-
phenylsulfonyl-6,7,8,9-tetrahydropyrazolo[1,5-a]quinazoline
C (R1 = SMe, R2 = Me, n = 2) with 5-HT6R antagonistic
IC50 = 6 nM.
Results
In this paper, we describe further development of the
5-HT6R antagonists based on pharmacophore D containing
a pyrazolo[1,5-a]pyrimidine template. The new synthesized
5-HT6R antagonists, (3-phenylsulfonyl-cycloalkano[d]pyra-
zolo[1,5-a]pyrimidin-2-yl)amines 1-6 and (3-phenylsulfo-
nylcycloalkano[e]pyrazolo[1,5-a]pyrimidin-2-yl)amines 7-16,
are shown in Figure 4.
The synthesis was carried out as shown in Scheme 1 with
benzenesulfonylacetonitrile 17 as a starting material. Reac-
tion of 17 with CH3NCS in KOH/dioxane for 2 h at room
temperature, step a, and with MeI for 1 h at room tempera-
ture, step b, resulted in formation of 2-phenylsulfonyl-3-
methylamino-3-methylsulfanylacrylonitrile 18, which then
led, in step c, to formation of the N3-methyl-4-phenylsulfo-
nyl-1H-pyrazole-3,5-diamine 19. N3,N3-Dimethyl-4-(phenyl-
sulfonyl)-1H-pyrazole-3,5-diamine 20 was obtained by react-
ing 17 with CS2 in a mixture of KOH/dioxane at room tem-
perature for 2 h in step g. The reaction product, 3,3-bis-
(methylthio)-2-(phenylsulfonyl)acrylonitrile, was then reacted
with dimethylamine in ethanol for an additional 12 h in step h
5-Methyl-3-phenylsulfonyl-2-piperazin-1-yl derivatives 15
and 16 were obtained from 2-phenylsulfonyl-3,3-bis-methyl-
sulfanylacrylonitrile 26.35 The latter was transformed into
2-phenylsulfonyl-3-mercapto-3-piperazin-1-ylacrylonitrile 27
by reaction with 1-Boc-piperazine. Subsequent reaction of 27
with N2H4 H2O in H2O yielded 4-(5-amino-4-phenylsulfonyl-
3
1H-pyrazol-3-yl)piperazine-1-carboxylic acid tert-butyl ester
28. The reaction of the 5-amino-1H-pyrazol 28 with dike-
tones 23 proceeded to yield a mixture of both the linear 5
and angular 13 products in a mass ratio of 15:85. In the
reaction with 2-acetylcyclohexanone 24, angular product 14
was obtained without impurity of the linear product 6. As a
result of removing the Boc protection from 13 and 14, we
obtained the individual 3-piperazin-1-yl derivatives 15 and 16
(Scheme 2).
The structures of the synthesized compounds 9 and 12 were
confirmed with X-ray crystallography and with LC/MS and
NMR analyses.

5188 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Ivachtchenko et al.
Figure 3. Antagonists of 5-HT6R with structures conforming either to model A (SB-742457 and Ro-65-7674) or to model B (C and D) in
Figure 2.
Figure 4. Novel synthesized 5-HT6R antagonists 1-16.
Discussion
bond between the oxygen atom O(2) and the amine proton
HN(4).
In Figure 6, we show the 3D structures of cyclopenta-
[e]pyrazolo[1,5-a]pyrimidine derivatives 9 and 12, which were
minimized (convergence criterion of 0.000 01) using Ac-
celrys DS ViewerPro 6.0 software. The molecules have three
rotatable bonds, and the energy minimization showed ex-
istence of at least two local energy minima defined by
position of the oxygen atoms O(1) and O(2) relative to the
nitrogen atom N(4), to which either a methyl (in 9) or
dimethyl (in 12) moiety is attached. The distances between
the oxygen and nitrogen atoms, torsion angles on the
rotatable bond, S-C(10) for 9 or S-C(8) for 12, and mini-
mized energies of the molecule conformation are shown in
Table 1.
As one can see, the minimal free energies of the two 9
conformations, O(2)N(4) and O(1)N(4), are practically the
same. The major difference between the two conformers is the
torsion angle around C(10)-S bond, which defines the benzene
ring position relative to the plane of the cyclopenta[e]pyrazolo-
[1,5-a]pyrimidine core. In the O(1)N(4) conformation, the ring
is directed toward the viewer, while in the O(2)N(4) conforma-
tion, the ring is directed away from the viewer (Figure 6). It
is important to note that in both conformations, the distance
between one of the two oxygen atoms and a proton of N(4)
comprises only 2.1 A, which indicates formation of the hydro-
gen bond and, hence, stabilization of the molecule in either
conformation. The X-ray data indicate that in the crystal state, 9
exists in a conformation closer to the O(2)N(4) conformation
(Figure 7A), with the benzene ring directed away from the
viewer. These data also support formation of the hydrogen
N,N,5-Trimethyl-3-(phenylsulfonyl)-7,8-dihydro-6H-cy-
clopenta[e]pyrazolo[1,5-a]pyrimidin-2-amine 12 could also
have at least two conformations, O(2)N(4) and O(1)N(4)
(Figure 6 and Table 1), though with the local free energy
minima much higher than those of 9 conformations. This can
be explained by the absence of the stabilizing hydrogen bond
in 12. The two 12 conformations differ from each other at a
substantially higher degree than those of the 9. As shown in
Figure 6, the benzene ring occupies a drastically different
position relative to the cyclopenta[e]pyrazolo[1,5-a]pyrimi-
dine core in the two conformations and the energy of the
O(2)N(4) is 1.2 kcal/mol as low as that of O(1)N(4). X-ray
data confirm that in the crystal form, 12 exists in a conforma-
tion closer to the O(2)N(4) conformation (Figure 7B) with the
benzene ring facing up and away from the cyclopenta-
[e]pyrazolo[1,5-a]pyrimidine core.
The ratio of the corresponding compound pairs 1-6 and
7-16 in the reaction mixtures was determined by the ratio of
integral signal intensities in pairs of protons of methylene
groups, 2.64-2.79 (m) and2.58-2.63(m), and methylgroups,
2.57 (s) and 2.50 (s).
The structural assignments of 1-6 and 7-16 were made on
the basis of 2D NMR experiment analyses (NOESY and
HMBC) for all synthesized compounds 1-16 and confirmed
for compounds 9 and 12 based on the X-ray data (Figures S1
and S2 ofSupportingInformation). The dataobtained areina
good agreement with the published NMR spectra and quan-
tum chemical calculations for known analogues.34,36,37

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5189
Scheme 1. Preparation of Compounds 2, 4, 7-12a
a Reagents and conditions: (a) CH3NCS, KOH, dioxane, room temp 2 h; (b) MeI, room temp 12 h; (c) N2H4 H2O, i-PrOH, room temp 1 h, then reflux
3
2 h; (d) AcOH, room temp 12 h; (e) AcOH, 100 °C, 3 h; (f) AcOH, HCl, room temp 12 h; (g) KOH, dioxane, CS2, room temp 2 h; (h) Me2NH, EtOH,
room temp 12 h.
Figure 5. Products 3, 9 and 4, 10 of the reaction of 3,5-diamino-1H-pyrazole 19 with 2-acetylcycloalkanones 23 and 24.
Scheme 2. Preparation of Compounds 15 and 16a
a Reagents and conditions: (a) 1-Boc-piperazine, i-PrOH, reflux 1 h, then room temp for 12 h; (b) N2H4 H2O, i-PrOH, room temp for 0.5 h; (c) AcOH,
3
100°C, 3 h; (d) 6 N AcCl in EtOH, room temp 1 h.
The synthesized compounds 1-16 were tested in a 5-HT6R
radioligand binding assay for their ability to compete with
[3H]lysergic acid diethylamide and in functional cell-based
assays for their ability to block serotonin-induced cAMP
production in HEK293 cells expressing human 5-HT6R
(Table 2). For comparison, some of the compounds were also
assessed for their ability to interact with targets with potential
liability: 5-HT2B receptor (blockage of RMe-5-HT-induced
[Ca2þ]i mobilization in HEK293 cells expressing 5-HT2B
receptor) and hERG potassium channels (whole cell patch
clamp).
The synthesized compounds 1-16 showed very high affi-
nity to the 5-HT6R, with Ki values in the picomolar range and
functional IC50 values in a single-digit nanomolar range. In
the pair of linear molecules 2 and 4, substitution of a proton
with Me in R3 position led to a 2-fold increase in the affinity.
Similar substitution in the angular molecules, 8 and 10,
exhibited even more profound effect, 4-fold increase in the

5190 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Ivachtchenko et al.
Figure 6. Free energy minimized 3D structures for the compounds 9 and 12. For each compound, the energy minimization was performed
for each of the two conformations with shortest distances between N(4) and either O(1), O(1)N(4), or O(2), O(2)N(4), atoms shown with solid
green lines. The green dotted line shows the hydrogen bond (distance less than 2.5 A) between either O(2) or O(1) and a proton of N(4).
Table 1. Parameters of the Energy Minimized 3D Structures of (5-Methyl-3-phenylsulfonyl-7,8-dihydro-6H-cyclopenta[e]pyrazolo[1,5-a]pyrimidin-
2-yl)methylamine, 9, and (5-Dimethyl-3-phenylsulfonyl-7,8-dihydro-6H-cyclopenta[e]pyrazolo[1,5-a]pyrimidin-2-yl)methylamine, 12
9 conformation
12 conformation
O(2)N(4)
O(1)N(4)
crystala
O(2)N(4)
O(1)N(4)
crystala
distance,
A
angle,
deg
distance,
A
angle,
deg
distance,
A
angle,
deg
distance,
A
angle,
deg
distance,
A
angle,
deg
distance,
A
angle,
deg
parameter
O(2) HN(4)
O(1) HN(4)
2.104
3.924
2.926
4.313
3.923
2.104
4.312
2.926
2.218
4.23
3 3 3
3 3 3
O(2) HN(4)
O(1) N(4)
2.882
4.561
3.244
3.937
4.808
3.211
4.383
3.129
3 3 3
3 3 3
S-O HN(4)
96.6
52.25
96.6
-52.22
103.3
86.05
3 3 3
torsion S-C
energy
-154.03
95.09
-125.39
68.499 706
68.498 966
209.097 534
79.507 404
80.773 939
271.264 518
a Parameters were calculated on the basis of X-ray-determined positions of the atoms.
was also more potent than its linear 2 counterpart. In the R3
proton-substituted compounds, the cycloalkane ring size does
not seem to play a substantial role in either the affinity or
potency of the compounds to bind to and block 5-HT6R res-
ponse (compare 7 with 8). In R3 Me-substituted compounds, 9
and 10, cyclopentane derivative shows slightly (2.5-fold) high-
er affinity than the cyclohexane derivative.
To the best of our knowledge, the 3-phenylsulfonyl-2-
methylamino-7,8-dihydro-6H-cyclopenta[e]pyrazolo[1,5-a]-
pyrimidine, 7, and 3-phenylsulfonyl-2-methylamino-7,8-dihy-
dro-6H-cyclohexa[e]pyrazolo[1,5-a]pyrimidine, 8, have the
highest affinity to the 5-HT6R known among all 5-HT6R
antagonists measured.
Noteworthy, dimethyl-substituted cyclopenta[e]pyrazolo-
[1,5-a]pyrimidines 11 and 12 exhibit 40- to 70-fold lesser po-
tency to antagonize the 5-HT6R (Table 2) than their respective
2-methylamino-substituted counterparts 7 and 9. This agrees
well with the above-discussed ability of the 2-methylamino-
substituted molecules to form an intramolecular hydrogen
bond that can limit the molecular rotational freedom in an
advantageous for the receptor binding conformation. The
2-(piperazin-1-yl)-substituted derivatives 15 and 16 were
even less potent (IC50 in the micromolar range) relative to
Figure 7. 3D structures deduced from the X-ray data, of (A)
(5-methyl-3-phenylsulfonyl-7,8-dihydro-6H-cyclopenta[e]pyrazolo-
[1,5-a]pyrimidin-2-yl)methylamine, 9, and (B) (5-dimethyl-3-phenyl-
sulfonyl-7,8-dihydro-6H-cyclopenta[e]pyrazolo[1,5-a]pyrimidin-2-yl)-
methylamine, 12.
affinity. When comparing affinities of linear analogues with
their angular counterparts, 2 vs 8 and 4 vs 10, one can see that
in the pair of R3 proton-substituted compounds, the angular
molecule has more than 4-fold higher affinity whereas in the
pair of R3 methyl-substituted, the linear molecule has slightly
higher affinity. In a cell-based functional assay, the angular 8

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5191
Table 2. Biological Activity of (3-Phenylsulfonylcycloalkano[e and d]pyrazolo[1,5-a]pyrimidin-2-yl)amines 2, 4, 7-12, 15, 16
Ki, nM
(binding)a
Ki, nM (functional)b
% inhibition ( SEM (conc)c
compd
n
NR1R2
R3
5-HT6
5-HT6
5-HT2B
169
HERG
2
2
2
1
2
1
2
1
1
1
2
NHMe
NHMe
H
0.499
0.256
0.088
0.112
0.202
0.490
0.542
4
Me
H
12.5 ( 10.1 (3 μM)
29.5 ( 3.9 (90 μM)
7
NHMe
NHMe
0.375
0.259
0.413
464
153
188
8
H
9
NHMe
NHMe
Me
Me
H
4.3 ( 2.4 (3 μM)
52.4 ( 2.8 (10 μM)
10
11
12
15
16
NMe2
NMe2
28.8
25.5
869
Me
Me
Me
piperazin-1-yl
piperazin-1-yl
1764
a Ki was determined in radioligand receptor binding assay (see Supporting Information)by concentration-dependent displacement of [3H]lysergic acid
diethylamide. Each curve was measured in duplicate. b Ki was determined in cell-based functional assays (see Experimental Section) by concentration-
dependent inhibition of serotonin-induced (5-HT6R) or RMe-serotonin-induced (5-HT2BR) responses. The values are the geometric mean of three to
four independent experiments. Each concentration curve was measured in duplicate. c Inhibition of hERG channel was determined in triplicate at several
concentrations. The mean values ( SEM for the highest concentration tested for each compound (shown in parentheses) are presented.
Figure 8. Specificity profile of (3-phenylsulfonyl-7,8-dihydro-6H-cyclopenta[e]pyrazolo[1,5-a]pyrimidin-2-yl)methylamine 7. Displacement
of target-specific radiolabeled ligands was measured at a compound concentration of 1 μM.38 Shown is a typical experiment performed in
duplicate; mean values ( SD are presented.
their analogues 9 and 10. The most plausible explanation
of much stronger binding of the 2-methylamino-substituted
cyclopenta[e]pyrazolo[1,5-a]pyrimidines with the 5-HT6R
compared to their 2-dimethylamino or 2-(piperazin-1-yl)
analogues 11, 12, 15, 16 can be attributed to the stabilization
of the 7, 9, 10 molecules in an advantageous binding con-
formation assisted by the intramolecular hydrogen bond. The
bulky 2-(piperazin-1-yl) group may additionally impose a
steric hindrance for binding within the 5-HT6R site.
The antagonist 7, one of the compounds with the highest
affinity (Table 1), was then tested on a panel of 55 therapeutic
targets consisting of GPCRs, ion channels, and neurotrans-
mittertransporters. Thespecificityprofile of7 was determined
by its ability to compete (at 1 μM) with radiolabeled ligands
for the targets studied (Figure 8).
The data of Figure 8 show that the (3-phenylsulfonyl-7,8-
dihydro-6H-cyclopenta[e]pyrazolo[1,5-a]pyrimidin-2-yl)me-
thylamine, 7, has a very good profile with high specificity and
selectivity toward 5-HT6R. Some interaction of 7 with the
5-HT2B receptor known to be responsible for drug-induced
valvular heart disease (VHD)39 prompted us to test 7 for its
potential 5-HT2B receptor agonistic and antagonistic activity
(for details, see Experimental Section). At concentrations
of up to 10 μM tested, 7 had no agonistic activity (data not
shown) and exhibited rather weak antagonistic activity (IC50
=
3.5 μM) (Figure 9). These data suggest the safety of 1 in relation
to potentially causing VHD. Besides, 7 has 3 orders of magni-
tude selectivity index between the 5-HT6R and 5-HT2BR
(Figure 9; also see Table 1).

5192 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Ivachtchenko et al.
solvents and drying of products were carried out only at reduced
pressure. Separation of reaction products was performed using a
HPLC system using Shimadzu LC-8A on the chromatographic
column Reprosil-Pur C-18-AQ 10 mm, 250 mm ꢀ 20 mm
(column Reprosil-Pur C-18-AQ 10 mm, 50 mm ꢀ 20 mm) at a
flow rate of 25 mL/min in gradient mode with mobile phase
MeCN/water þ 0.05% CF3COOH.
According to LC-MS, the purity of the obtained compounds
1 and 2 exceeded 98.0%.
Preparation of (3-Phenylsulfonylcycloalkano[e and d]pyra-
zolo[1,5-a]pyrimidin-2-yl)amines (1-16 and Their Mixtures).
Procedure A. Stirred for 1 h at 0 °C were N(3)-methyl-4-
(phenylsulfonyl)-1H-pyrazole-3,5-diamine 19, 20 (1 mmol)
and sodium 2-oxocycloalkylidenmethylate 21, 22 (2 mmol) or
2-acetylcycloalkanone 23, 24 (2 mmol) and 3.5 mL of glacial
AcOH. The mixture was allowed to stir overnight at room
temperature. The resulting precipitate was filtered, washed with
AcOH, hexane, and dried in a vacuum. Product 2 was obtained
in 63% yield, or a mixture of products 3 and 9 (ratio 1:9) or
products 4 and 10 (ratio 1:3) was obtained.
Procedure B. Stirred for 3 h at 100 °C were aminopirazole 19,
20 (0.5 mmol) and sodium 2-oxocycloalkylidenmethylate 21, 22
(2 mmol) or 2-acetylcycloalkanone 23, 24 (2 mmol) in 1 mL of
AcOH. Evaporation of solvent was done in a vacuum. The
residue was mixed with 20 mL of i-PrOH at 0 °C, and the
resulting precipitate was separated, washed twice with i-PrOH,
hexane, and dried in a vacuum. Products 9 and 13 were obtained
in a yield of 80-90%, or a mixture of products was obtained: 4
and 10 (ratio ∼6: 5), which were separated by using HPLC; a
mixture of products 3 and 9 (ratio ∼1:10)
Figure 9. Compound 7 concentration-dependently inhibits both
the 5-HT2BR and 5-HT6R-induced functional responses in HEK293
cells exogenously expressingcorresponding receptors. Each point is an
average of two replicates ( SD.
7 interacted with hERG (human ether-a-go-go-related
gene) potassium channel neither in binding38 (Figure 8) nor
in functional (Table 1) assays. The hERG is another target
with potential liability associated with QT prolongation and
torsade de pointes,40 which was implicated in withdrawal of
previously approved drugs and in preventing others from
gaining FDA approval. Our data clearly indicate the safety
of 7 and make it an ideal candidate as a therapeutic tool in
probing the role of the 5-HT6R in different diseases.
Procedure C. To a solution of 0.3 g (2 mmol) of sodium
2-oxocyclohexylidenmethylate in 3.5 mL of AcOH was added
1 mL of 6 M HCl solution in EtOAc-EtOH (prepared by
dissolving AcCl in EtOH). The reaction mixture was cooled in
a water-ice bath to ∼0 °C, and 0.252 g (1 mmol) of N(3)-methyl-
4-(phenylsulfonyl)-1H-pyrazole-3,5-diamine 19 was added.
Stirring continued for 1 h at this temperature and then 12 h at
room temperature. The precipitate was filtered, washed on the
filter with AcOH, hexane, and dried in a vacuum. Target pro-
ducts were purified by column chromatography (eluent,
CHCl3). Product 8 was obtained in 67% yield, or product
mixtures of 3 and 9 (ratio 1: 4) or of 4 and 10 (ratio ∼2:1; but
when the reaction was carried out in 2 weeks at room tempera-
ture, the ratio was ∼4:1) were obtained.
Conclusion
(3-Phenylsulfonylcycloalkano[e and d]pyrazolo[1,5-a]pyri-
midin-2-yl)amines 1-16 represent a new type of highly potent
(Ki < 1 nM) 5-HT6R antagonists with the basic secondary
amino group located next to (rather than apart from) the
sulfonyl group. This closeness of the basic amine and sulfonyl
groups makes it possible for formation of an intramolecular
hydrogen bond, which stabilizes the molecule in an appro-
priate conformation and thus provides gain in the receptor
binding energy. The 2-methylamino-3-phenylsulfonyl-7,8-di-
hydro-6H-cyclopenta[e]pyrazolo[1,5-a]pyrimidine, 7, to the
best of our knowledge possesses the highest affinity (Ki =88
pM) to the 5-HT6R and is the most selective, based on a panel
of 55 therapeutic targets, among known antagonists of the
receptor. The data also indicate the absence of potential
liability coupled with either 5-HT2B receptor or hERG chan-
nel. This makes it an excellent candidate for development of a
therapeutic tool to probe the role of 5-HT6R in different
diseases of the CNS.
N-Methyl-3-(phenylsulfonyl)-5,6,7,8-tetrahydropyrazolo[5,1-
b]quinazolin-2-amine (2). 1H NMR (400 MHz, DMSO-d6) δ 8.71
(s, 1H, 9-CH), 8.00 (m, 2H, 2-CH-Ph), 7.59 (m, 1H, 4-CH-Ph),
7.55 (m, 2H, 3-CH-Ph), 6.32 (q, J = 4.8 Hz, 1H, NH), 2.88 (d,
J = 4.8 Hz, 3H, NCH3), 2.86 (t, J = 6.4 Hz, 2H, 5-CH2), 2.69 (t,
J = 6.2 Hz, 2H, 8-CH2), 1.82 (m, 2H, 6-CH2), 1.72 (m, 2H,
7-CH2). 13C NMR (75 MHz, DMSO-d6) δ 161.82 (4a-C), 158.06
(2-C), 145.67 (3a-C), 144.14 (1-Ph-C), 133.80 (9-C), 132.69 (4-
Ph-C), 129.13 (2-Ph-C), 125.62 (3-Ph-C), 118.98 (8a-C), 88.75
(3-C), 32.43 (5-C), 29.05 (NCH3), 25.09 (8-C), 21.93 (6-C), 21.56
(7-C). HRMS calculated for C17H17N3O2S2 (M þ H) 343.1229,
found 343.1230. MS-ESI calculated for C17H17N3O2S2 (M þ H)
343, found m/z 343. LC-MS (UV-254) purity: 98%.
N,9-Dimethyl-3-(phenylsulfonyl)-5,6,7,8-tetrahydropyrazolo-
[5,1-b]quinazolin-2-amine (4). 1H NMR (400 MHz, DMSO-d6) δ
8.00 (m, 2H, 2-CH-Ph), 7.55 (m, 3H, 3,4-CH-Ph), 6.31 (br m,
1H, NH), 2.91 (s, 3H, NCH3), 2.84 (t, J = 5.2 Hz, 2H, 5 or
8-CH2), 2.64 (t, J = 5.2 Hz, 2H, 5 or 8-CH2), 2.53 (s, 3H,
9-CH3), 1.77 (m, 4H, 6,8-CH2). 13C NMR (75 MHz, DMSO-d6)
δ 160.32 (4a-C), 157.63 (2-C), 145.47 (3a-C), 144.43 (9-C),
144.05 (1-Ph-C), 132.46 (4-Ph-C), 128.92 (2-Ph-C), 125.73 (3-
Ph-C), 116.76 (8a-C), 89.12 (3-C), 33.07 (5-C), 28.96 (NCH3),
24.02 (8-C), 21.88, 21.70 (6-C, 7-C), 12.81 (9-CH3). HRMS cal-
culated for C18H20N4O2S (M þ H) 357.1380, found 357.1377.
MS-ESI calculated for C18H20N4O2S (M þ H) 357, found m/z
357. LC-MS (UV-254) purity: 98%.
Experimental Section
Chemistry. General Methods. 1H NMR spectra of solutions
of the investigated compounds in DMSO-d6 or CDCl3 were
recorded on a Bruker DPX-400 spectrometer (400 MHz, 27 °C),
and 13C NMR and two-dimensional spectra were obtained on a
Bruker DPX-300 (75 MHz, 27 °C).
LC-MS data were obtained using a Shimadzu HPLC
equipped with a Waters XBridge C18 3.5 mm column (4.6 mm ꢀ
150 mm), PE SCIEX API 150 EX mass detector, and Shimadzu
spectrophotometric detector (λ, 220 and 254 nm).
HR mass spectra (ESI-TOF, positive) were obtained on a
Waters Qtof API US instrument in the positive mode.
In all cases, the end of the reaction was determined by
conversion of the substrate (LC-MS control). Evaporation of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5193
N,N-Dimethyl-3-(phenylsulfonyl)-7,8-dihydro-6H-cyclopenta-
[e]pyrazolo[1,5-a]pyrimidin-2-amine (11). H NMR (400 MHz,
Mixture (∼6:5) of N,9-Dimethyl-3-(phenylsulfonyl)-5,6,7,8-
1
tetrahydropyrazolo[5,1-b]quinazolin-2-amine (4) and N,5-Dime-
thyl-3-(phenylsulfonyl)-6,7,8,9-tetrahydropyrazolo[5,1-a]quinazolin-
2-amine (10). 1H NMR (400 MHz, CDCl3) δ 8.11-8.17 (m, 2H),
7.39-7.49 (m, 3H), 5.91-6.00 (br m, 1H), 2.94-3.05 (m, 5H),
2.64-2.79 (m, 0.8H), 2.58-2.63 (m, 1.2H), 2.57 (s, 0.96H), 2.50 (s,
1.60H), 1.79-1.90 (m, 4H). MS-ESI calculated for C18H20N4O2S
(M þ H) 357, found m/z 357.
CDCl3) δ 8.50 (s, 1H, 5-H), 8.14 (m, 2H, 2-CH-Ph), 7.50 (m, 1H,
4-CH-Ph), 7.45 (m, 2H, 3-CH-Ph), 3.29 (t, J = 7.6 Hz, 2H,
8-CH2), 3.10 (s, 6H, N(CH3)2), 3.09 (t, J = 8.0 Hz, 2H, 6-CH2),
2.31 (p, J = 7.6 Hz, 2H, 7-CH2). 13C NMR (75 MHz, CDCl3) δ
161.45 (2-C), 149.55 (8a-C), 148.48 (3a-C), 147.32 (5-C), 144.29
(1-Ph-C), 131.63 (4-Ph-C), 128.06 (2-Ph-C), 125.92 (3-Ph-C),
124.03 (5a-C), 95.25 (3-C), 42.03 (N(CH3)2), 29.19, 28.27 (6-C,
8-C), 22.03 (7-C). HRMS calculated for C17H18N4O2S (M þ H)
343.1380, found 357.1223. MS-ESI calculated for C17H18N4O2S
(M þ H) 343, found 343. LC-MS (UV-254) purity: 98.8%.
N,N,5-Trimethyl-3-(phenylsulfonyl)-7,8-dihydro-6H-cyclopen-
ta[e]pyrazolo[1,5-a]pyrimidin-2-amine (12). 1H NMR (400 MHz,
CDCl3) δ 8.18 (m, 2H, 2-CH-Ph), 7.50 (m, 1H, 4-CH-Ph), 7.45
(m, 2H, 3-CH-Ph), 3.26 (t, J = 7.6 Hz, 2H, 8-CH2), 3.10 (s, 6H,
N(CH3)2), 2.98 (t, J = 7.6 Hz, 2H, 6-CH2), 2.53 (s, 3H, 5-CH3),
2.28 (p, J = 7.6 Hz, 2H, 7-CH2). 13C NMR (75 MHz, CDCl3) δ
161.39 (2-C), 157.77 (5-C), 148.29, 148.27 (3a-C, 8a-C), 144.18
(1-Ph-C), 131.66 (4-Ph-C), 127.96 (2-Ph-C), 126.52 (3-Ph-C),
123.14 (5a-C), 94.86 (3-C), 42.35 (N(CH3)2), 29.37, 28.92 (6-C,
8-C), 22.61, 21.59 (7-C, 5-CH3). HRMS calculated for
C18H20N4O2S (M þ H) 357.1380, found 357.1379. MS-ESI cal-
culated for C18H20N4O2S (M þ H) 357, found 357. LC-MS
(UV-254) purity: 99%.
Mixture (15:85) of tert-Butyl 4-[8-Methyl-3-(phenylsulfonyl)-
6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidin-2-yl]pipe-
razine-1-carboxylate (5) and tert-Butyl 4-[5-Methyl-3-(phenyl-
sulfonyl)-7,8-dihydro-6H-cyclopenta[e]pyrazolo[1,5-a]pyrimidin-
2-yl]piperazine-1-carboxylate (13). 1H NMR (400 MHz, CDCl3)
δ 8.07-8.18 (m, 2H), 7.39-7.53 (m, 3H), 3.57-3.64 (m, 4H),
3.39-3.47 (m, 4H), 3.24 (t, J = 7.6 Hz, 1.7H), 3.10 (t, J = 7.8
Hz, 0.3H), 2.07 (t, J = 7.6 Hz, 1.7H), 2.93 (t, J = 7.8 Hz, 0.3H),
2.58 (s, 0.45H), 2.54 (s, 2.55H), 2.18-2.32 (m, 2H), 1.48 (s, 9H).
MS-ESI calculated for C25H31N5O4S (M þ H) 498, found m/z 498.
N-Methyl-(3-phenylsulfonyl)-7,8-dihydro-6H-cyclopenta[e]-
pyrazolo[1,5-a]pyrimidin-2-amine (7). 1H NMR (400 MHz,
DMSO-d6) δ 8.44 (s, 1H, 5-CH), 8.00 (m, 2H, 2-CH-Ph), 7.59
(m, 1H, 4-CH-Ph), 7.54 (m, 2H, 3-CH-Ph), 6.44 (q, J = 4.8 Hz,
1H, NH), 3.17 (t, J = 7.6 Hz, 2H, 8-CH2), 2.96 (t, J = 7.6 Hz,
2H, 6-CH2), 2.92 (d, J = 4.8 Hz, 3H, NCH3), 2.15 (p, J = 7.6
Hz, 2H, 7-CH2). 13C NMR (75 MHz, DMSO-d6) δ 158.67 (2-C),
150.77 (8a-C), 147.58 (5-C), 146.92 (3a-C), 144.25 (1-Ph-C),
132.61 (4-Ph-C), 129.03 (2-Ph-C), 125.67 (3-Ph-C), 124.53 (5a-
C), 90.23 (3-C), 29.49, 29.11, 28.32 (NCH3, 6-C, 8-C), 22.19 (7-
C). HRMS calculated for C16H16N4O2S (M þ H) 329.1072,
found 329.1077. MS-ESI calculated for C16H16N4O2S (M þ H)
329, found m/z 329. LC-MS (UV-254) purity: 99%.
tert-Butyl 4-[5-Methyl-3-(phenylsulfonyl)-6,7,8,9-tetrahydro-
pyrazolo[1,5-a]quinazolin-2-yl]piperazine-1-carboxylate (14). 1H
NMR (400 MHz, CDCl3) δ 8.14 (d, J = 7.7 Hz, 2H), 7.40-
7.52 (m, 3H), 3.58-3.63 (m, 4H), 3.41-3.46 (m, 4H), 2.99 (t, J =
5.7 Hz, 2H), 2.64 (t, J = 5.9 Hz, 2H), 2.53 (s, 3H), 1.82-1.95 (m,
4H), 1.49 (s, 9H). MS-ESI calculated for C26H33N5O4S (M þ H)
512, found 512. LC-MS (UV-254) purity: 99%.
N-Methyl-(3-phenylsulfonyl)-6,7,8,9-tetrahydropyrazolo[1,5-
a]quinazolin-2-amine (8). 1H NMR (400 MHz, DMSO-d6) δ 8.32
(s, 1H, 5-CH), 7.99 (m, 2H, 2-CH-Ph), 7.59 (m, 1H, 4-CH-Ph),
7.54 (m, 2H, 3-CH-Ph), 6.36 (q, J = 4.8 Hz, 1H, NH), 2.92 (m,
5H, NCH3, 9-CH2), 2.67 (t, J = 5.8 Hz, 2H, 6-CH2), 1.83 (m,
2H, 8-CH2), 1.72 (m, 2H, 7-CH2). 13C NMR (75 MHz, DMSO-
d6) δ 157.51 (2-C), 151.71 (5-C), 146.04 (3a-C), 145.01 (9-C),
144.09 (1-Ph-C), 132.73 (4-Ph-C), 129.16 (2-Ph-C), 125.60 (3-
Ph-C), 118.52 (5a-C), 90.14 (3-C), 29.07 (NCH3), 23.89, 23.65
(6-C, 9-C), 21.20 (7-C), 20.51 (8-C). HRMS calculated for
C17H18N4O2S (M þ H) 343.1229, found 343.1227. MS-ESI
calculated for C17H18N4O2S (M þ H) 343, found m/z 343.
LC-MS (UV-254) purity: 98%.
N,5-Dimethyl-3-(phenylsulfonyl)-7,8-dihydro-6H-cyclopenta-
[e]pyrazolo[1,5-a]pyrimidin-2-amine (9). 1H NMR (400 MHz,
DMSO-d6) δ 8.02 (m, 2H, 2-CH-Ph), 7.54 (m, 3H, 3,4-CH-Ph),
6.19 (q, J = 6.4 Hz, 1H, NH), 3.18 (t, J = 6.0 Hz, 2H, 6 or
8-CH2), 2.95 (d, J = 6.4 Hz, 3H, NCH3), 2.92 (t, J = 6.0 Hz, 2H,
6 or 8-CH2), 2.45 (s, 3H, 5-CH3), 2.18 (p, J = 6.0 Hz, 2H,
7-CH2). 13C NMR (75 MHz, DMSO-d6) δ 157.92 (2-C), 157.11
(5-C), 149.01, 146.43 (3a-C, 8a-C), 143.90 (1-Ph-C), 132.04 (4-
Ph-C), 128.47 (2-Ph-C), 125.27 (3-Ph-C), 122.91 (5a-C), 89.68
(3-C), 29.16 (NCH3), 28.62, 28.33 (6-C, 8-C), 21.93, 21.19 (7-C,
5-CH3). HRMS calculated for C17H18N4O2S (M þ H) 343.1229,
found 343.1234. MS-ESI calculated for C17H18N4O2S (M þ H)
343, found m/z 343. LC-MS (UV-254) purity: 99%.
5-Methyl-3-phenylsulfonyl-2-piperazin-1-yl-7,8-dihydro-6H-
cycloalkane[e]pyrazolo[1,5-a]pyrimidines, Hydrochlorides (15, 16).
An amount of 0.3 mmol of Boc derivative mixture 5 and 13 or Boc
derivative 15 was added to 1.5 mL of 6 N HCl solution of AcOH in
EtOH. The reaction mixture was stirred for 1 h at room tempera-
ture before an excess of ether was added. Then hydrochloride
precipitates of 15 and 16 were separated, successively washed with
AcOEt (twice), acetone (twice), and hexane (once), and dried in a
vacuum to provide colorless crystalline products 15 and 16.
5-Methyl-3-(phenylsulfonyl)-2-piperazin-1-yl-7,8-dihydro-6H-
1
cyclopenta[e]pyrazolo[1,5-a]pyrimidine, Hydrochloride (15). H
NMR (400 MHz, DMSO-d6) δ 9.68 (br m, 2H, NH2þ), 8.03 (m,
2H, 2-CH-Ph), 7.55 (m, 3H, 3,4-CH-Ph), 3.64 (br m, 4H,
2-piperazin-CH2), 3.24 (br m, 4H, 3-piperazin-CH2), 3.20 (t,
J = 10.4 Hz, 2H, 8-CH2), 2.92 (t, J = 9.6 Hz, 2H, 6-CH2), 2.47
(s, 3H, 5-CH3), 2.16 (m, 2H, 7-CH2). 13C NMR (75 MHz,
DMSO-d6) δ 159.57, 159.20 (2-C, 5-C), 149.77 (8a-C), 147.22
(3a-C), 143.65 (1-Ph-C), 132.85 (4-Ph-C), 128.96 (4-Ph-C),
126.43 (3-Ph-C), 124.92 (5a-C), 95.85 (3-C), 47.48 (2-piperazin-
CH2), 42.52 (3-piperazin-CH2), 29.56, 28.82 (6-C, 8-C), 22.75 (5-
CH3), 21,56 (7-C). HRMS calculated for C20H23N5O2S
(M þ H) 398.1651, found 398.1646. MS-ESI calculated for C20-
H23N5O2S (M þ H) 398, found 398. LC-MS (UV-254) purity:
99%.
5-Methyl-3-(phenylsulfonyl)-2-piperazin-1-yl-6,7,8,9-tetrahy-
dropyrazolo[1,5-a]quinazoline, Hydrochloride (16). 1H NMR
(400 MHz, DMSO-d6) δ 9.34 (br m, 2H, NH2þ), 8.03 (m, 2H,
2-CH-Ph), 7.57 (m, 3H, 3,4-CH-Ph), 3.63 (br m, 4H, 2-piper-
azin-CH2), 3.26 (br m, 4H, 3-piperazin-CH2), 2.95 (br m, 2H,
9-CH2), 2.63 (br m, 2H, 6-CH2), 2.47 (s, 3H, 5-CH3), 1.79 (m,
4H, 7,8-CH2). 13C NMR (75 MHz, DMSO-d6) δ 161.81 (5-C),
158.47 (2-C), 145.47, 144.22, 143.71 (3a-C, 9a-C, 1-Ph-C),
132.86 (4-Ph-C), 128.98 (2-Ph-C), 126.44 (3-Ph-C), 118.76 (5a-
C), 95.88 (3-C), 47.48 (2-piperazin-CH2), 42.57 (3-piperazin-
CH2), 23.94, 23.68 (6-C, 9-C), 22.84 (5-CH3), 21.18 (8-C), 20.12
(7-C). HRMS calculated for C21H25N5O2S (M þ H) 412.1807,
found 412.1811. MS-ESI calculated for C21H25N5O2S (M þ H)
412, found 412. LC-MS (UV-254) purity: 99%.
N,5-Dimethyl-3-(phenylsulfonyl)-6,7,8,9-tetrahydropyrazolo-
[1,5-a]quinazolin-2-amine (10). 1H NMR (400 MHz, DMSO-d6)
δ 8.01 (m, 2H, 2-CH-Ph), 7.54 (m, 3H, 3,4-CH-Ph), 6.20 (q, J =
5.6 Hz, 1H, NH), 2.91 (d, J = 5.6 Hz, 3H, NCH3), 2.90 (br m,
2H, 9-CH2), 2.59 (br m, 2H, 6-CH2), 2.44 (s, 3H, 5-CH3), 1.78
(br m, 4H, 7-CH2, 8-CH2). 13C NMR (75 MHz, DMSO-d6) δ
160.29 (5-C), 157.28 (2-C), 145.07, 144.26 (3a-C, 9a-C), 143.98
(1-Ph-C), 132.58 (4-Ph-C), 129.03 (2-Ph-C), 125.67 (3-Ph-C),
117.24 (5a-C), 89.57 (3-C), 29.05 (NCH3), 24.16, 23.73 (6-C,
9-C), 22.56 (5-CH3), 21.31, 20.23 (7-C, 8-C). HRMS calculated
for C18H20N4O2S (M þ H) 357.1380, found 357.1382. MS-ESI
calculated for C18H20N4O2S (M þ H) 357, found m/z 357.
LC-MS (UV-254) purity: 98%.

5194 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Ivachtchenko et al.
N3-Methyl-4-(phenylsulfonyl)-1H-pyrazole-3,5-diamine (19).
With vigorous stirring at room temperature, 6.16 g (110 mmol)
of finely triturated KOH was added to a solution of 9 g (50
mmol) of sulfonylacetonitrile 17 and 4.0 g (55 mmol) of MeNCS
in 80 mL of dioxane. The reaction mixture was stirred for 2 h
before adding 7.1 g (50 mmol) of MeI, and stirring continued at
room temperature for 12 h. The resultant mixture was poured
onto 400 mL of a mixture of ice and H2O and stirred for 1 h. The
precipitate was filtered, washed with H2O, and freeze-dried.
Obtained was 4.1 g of 2-phenylsulfonyl-3-methylamino-3-meth-
ylsulfanylacrylonitrile 18. In addition, 4.74 g of the acrylonitrile
18, identical according to LC-MS and NMR with the first
portion, was obtained after neutralization of the filtrate with
AcOH, followed by filtering of the precipitate, washing with
H2O, and drying. The overall yield of 18 was 67%, considering
the source acetonitrile 17. When using an excess of MeI (>50
mmol), one gets the product 18 containing, according to
LC-MS, 2-phenylsulfonyl-3-dimethylamino-3-methylsulfany-
lacrylonitrile 26 in the amount of the corresponding excess of
MeI. To 8.84 g (0.033 mol) of acrylonitrile 18 in 70 mL of i-PrOH
dried. Product obtained was 3.32 g (84%) of 4-(2-phenylsulfo-
nyl-2-cyano-1-methylsulfanylvinyl)piperazine-1-carboxylic acid
tert-butyl ester 10. An amount of 3.32 g (7.85 mmol) of this
product in 15 mL of i-PrOH was stirred with 0.48 g (9.5 mmol) of
N2H2 H2O with heating. The reaction mixture was poured into a
3
mixture of H2O with ice, and the precipitated oil quickly solidi-
fied. The solid was filtered and washed with H2O, cold i-PrOH,
and hexane to provide 2.65 g (83%) of the target product 28. MS-
ESI calculated for C18H25N5O4S (M þ1) 408, found 408.
Biological Assays. Cell-Based Functional Assays. 5-HT2B
Receptor Functional Assay. The 5-HT2B-HEK cells were grown
in T-175 flasks at 37 °C in an atmosphere of air/CO2 (95%:5%)
in DMEM (Sigma, MO) supplemented with 10% FBS, 1%
AAS, blasticidine S, and phleomycin (Invitrogen, Carlsbad,
CA). The T-Rex/5-HT2B receptor expression was activated by
addition of tetracycline, as recommended by the manufacturer,
a day before the experiments. The cells were dissociated with
TrypLE Express (Invitrogen, Carlsbad, CA), washed twice with
PBS, and loaded at room temperature with 4 μM calcium-sen-
sitive dye, Fura-2AM (Invitrogen, Carlsbad, CA), for 30 min.
After the loading, the cells were washed once with PBS, resus-
pended into protein free hybridoma media without phenol red
(Sigma, St. Louis, MO), and allowed to incubate for an addi-
tional 30 min with gentle shaking at room temperature. All
loading procedures were performed in dark conditions. The
loaded cells were washed twice with PBS and resuspended into
the hybridoma media at a cell density of (3-4) ꢀ 106 cells/mL
for subsequent experiments. Fura-2 ratiometric fluorescence
signal was registered at 510 nm upon alternate excitation at 340
and 380 nm using spectrofluorometer RF5301PC (Shimadzu,
Columbia, MD). In a square (1 cm) optical cuvette with a mag-
netic stirring bar, 100 μL aliquots of the loaded cells were diluted
into 2.4 mL of buffer containing (mM) NaCl (145), KCl (5.4),
MgSO4 (0.8), CaCl2 (1.8), HEPES (30), and D-glucose (11.2).
The fluorescence signal was allowed to stabilize for 20-30 s
before addition of a test compound or vehicle to assess potential
agonistic activity of the compounds, with subsequent addition
of serotonin (2.5 μL, 10 mM) to assess the compounds’ blocking
activity.
was added 2.5 g (0.05 mol) of N2H2 H2O, and the mixture was
3
stirredfor 1 hat room temperature, thenboiledfor2 h, and diluted
with 70 mL of H2O. The precipitate was filtered and dried in a
vacuum. Obtained was 7.5 g (90%) of pyrazole 19. LC-MS purity
was 97%þ. MS-ESI calculated for C10H12N4O2S (M þ1) 253,
found 253. When using 10-15% excess MeI in the synthesis of
acrylonitrile 18, pyrazole 19 was obtained containing, according
to LC-MS, a corresponding excess of 4-benzenesulfonyl-N(3),
N(3)-dimethyl-1H-pyrazole-3,5-diamine 20. MS-ESI calculated
for C11H14N4O2S (M þ1) 267, found 267.
N3,N3-Dimethyl-4-(phenylsulfonyl)-1H-pyrazole-3,5-diamine
(20). To a solution of 5.00 g (27 mmol) of phenylsulfonylaceto-
nitrile 17 and 2.31 g (30 mmol) of CS2 in 50 mL of dioxane, 3.99 g
(55 mmol) of finely grounded KOH was added with vigorous
stirring. The mixture was stirred under Ar for 2 h. Then 8.61 g
(55 mmol) of MeI was added and the stirring was continued for
3 h. The resultant mixture was poured into 100 mL of ice-water
and stirred for 1 h. The precipitate was filtered, washed with
H2O, hexane, and freeze-dried to give 6.24 g (80%) of 3,3-
1
5-HT6 Receptor Functional Assay. The 5-HT6-HEK cells were
grown in Corning 384-well plates (Lowell, MA) at 37 °C in an
atmosphere of air/CO2 (95%:5%) in DMEM supplemented
with 10% FBS, 1% AAS, blasticidine S, and phleomycin
(Invitrogen, Carlsbad, CA). T-Rex/5-HT6 receptor expression
was activated by addition of tetracycline, as recommended by
the manufacturer, a day before the experiments. On the day of
the experiment, the medium in the wells was substituted with
phenol red-, calcium-, and magnesium-free HBSS (Invitrogen,
Carlsbad, CA), supplemented with 1 mM MgCl2, 1 mM CaCl2,
5 mM HEPES, pH 7.4, and 100 μM IBMX. The test compounds
were added at different concentrations while maintaining con-
stant final DMSO concentration of 0.1%. After 15 min of incu-
bation, serotonin hydrochloride (Sigma, MO) was added to a
final concentration of 10 nM and incubation continued for
additional 30 min at room temperature. The cells were treated
as described in the cAMP LANCE assay kit protocol (Perkin-
Elmer, Waltham, MA) as recommended by the manufacturer.
The LANCE signal was measured in white 384-well plates
(Corning, MA) using multimode plate reader VICTOR2V
(PerkinElmer, Waltham, MA) with built-in settings for the
LANCE detection.
hERG Channel Functional Assay. Human recombinant
hERG channel was stably expressed in HEK-297 cells. Intra-
cellular solution consisted of 130 mM KCl, 5 mM EGTA, 5 mM
Mg Cl2, 10 mM HEPES, 5 mM Na-ATP, pH 7.2. Extracellular
solution consisted of 137 mM NaCl, 4 mM KCl, 1.8 mM CaCl2,
1.0 mM MgCl2, 11 mM dextrose, 10 mM HEPES, pH 7.4.
Voltage clamp measurements were performed at 25 °C using
PatchXpress 7000A (Molecular Devices, Sunnyvale, CA).
hERG channels were activated by 2 s pulses to þ20 mV from
bis(methylthio)-2-(phenylsulfonyl)acrylonitrile. H NMR (400
MHz, CDCl3) δ 8.06 (m, 2H), 7.70 (m, 1H), 7.59 (m, 2H), 2.72 (s,
3H), 2.57 (s, 3H). To a solution of 571 mg (2.0 mmol) of the
obtained 3,3-bis(methylthio)-2-(phenylsulfonyl)acrylonitrile in
10 mL of MeOH, 0.32 mL (2.5 mmol) of 40% aqueous Me2NH
was added. The mixture was stirred at ambient temperature for
12 h and then concentrated and dried under vacuum to afford
565 mg (100%) of 3-(dimethylamino)-3-(methylthio)-2-(phenyl-
sulfonyl)acrylonitrile 25, configuration undetermined. 1H
NMR (400 MHz, CDCl3) δ 7.99 (m, 2H), 7.60 (m, 1H), 7.54
(m, 2H), 3.37 (s, 6H), 2.49 (s, 3H). To 565 mg (2.0 mmol) of
acrylonitrile 25 in 10 mL of i-PrOH was added 200 mg (4.0
mmol) of N2H2 H2O. The mixture was stirred for 1 h at room
3
temperature, then refluxed for 2 h, concentrated under vacuum,
treated with water, and extracted with CH2Cl2. The organic
phase was washed with 10% K2CO3 solution, dried over Na2-
SO4, and concentrated under vacuum. The resulting mixture
(450 mg) consisted of 20 and 3-(methylthio)-4-(phenylsulfonyl)-
1H-pyrazol-5-amine, 2:1 ratio according to LC-MS. The pyr-
azole 20 was isolated by HPLC. Isolated yield was 175 mg
1
(33%). H NMR (400 MHz, DMSO-d6) δ 11.30 (br s, 0.6H),
7.81 (m, 2H), 7.60 (m, 1H), 7.55 (m, 2H), 5.93 (br s, 2H), 2.65 (s,
6H). MS-ESI calculated for C11H14N4O2S (M þ1) 267, found
267.
4-(5-Amino-4-phenylsulfonyl-1H-pyrazol-3-yl)piperazine-1-
carboxylic Acid tert-Butyl Ester (28). An amount of 1.74 g (9.35
mmol) of 1-Boc-piperazine was added to a mixture of 2.67 g
(9.35 mmol) of 2-phenylsulfonyl-3,3-bis-methylsulfanylacrylo-
nitrile 27 in 15 mL of i-PrOH. The resulting mixture was heated
with stirring for 1 h and sat for 12 h at room temperature. Then
the precipitate was filtered, washed with i-PrOH, hexane, and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5195
a holding potential of -80 mV, and peak tail currents were
recorded upon repolarization to -50 mV. This voltage-clamp
pulse protocol was performed continuously during the experi-
ment (vehicle control, test compound, washout, and positive
control additions). An interpulse interval of 15 s allowed
recovery from any residual inactivation. Test compounds were
incubated with cells until the current reached a steady state level
(3-8 min). After the final test compound concentration was
tested, test compound was washed out with continuous perfu-
sion of extracellular solution for 3 min, followed by application
of positive control (10 μM cisapride). Data were analyzed using
DataXpress software. Percent of control values were calculated
on the basis of current peak at each compound concentration
relative to maximal tail current in the presence of vehicle
control.
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Supporting Information Available: X-ray crystallographic
data for compounds 9 and 12 (including two files in cif format)
and a description of the competitive radioligand displacement
assays. This material is available free of charge via the Internet
at http://pubs.acs.org. CCDC-769754 and CCDC-769755 con-
tain supplementary crystallographic data of 9 and 12 for this
paper, and these data can be obtained free of charge via www.
ccdc.cam.ac.uk/data_request/cif by emailing data_request@
ccdc.cam.ac.uk or by contacting The Cambridge Crystallo-
graphic Data Centre, 12, Union Road, Cambridge CB2 1EZ,
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