2-Substituted 5,6-dimethyl-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidines: New series of highly potent and specific serotonin 5-HT6 receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2011.01.038

Syntheses, biological evaluation, and structure-activity relationships for a series of novel 2-substituted 3-benzenesulfonyl-5,6-dimethyl-pyrazolo[1,5-a] pyrimidines are disclosed. In spite of a wide, four orders of magnitude, SAR range (K_i var

Full text of DOI:10.1016/j.ejmech.2011.01.038

European Journal of Medicinal Chemistry 46 (2011) 1189e1197
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
2-Substituted 5,6-dimethyl-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidines: New
series of highly potent and specific serotonin 5-HT6 receptor antagonists
,
Alexandre V. Ivachtchenko a b, Elena S. Golovina c, Madina G. Kadieva a, Angela G. Koryakova c,
,
Oleg D. Mitkin a, Sergey E. Tkachenko b, Volodymyr M. Kysil b, Ilya Okun b
*
a Department of Organic Chemistry, Chemical Diversity Research Institute, 141400 Khimki, Moscow Reg, Russia
b ChemDiv, Inc., 6605 Nancy Ridge Drive, San Diego, CA 92121, USA
c Department of Molecular Pharmacology, Chemical Diversity Research Institute, 141400 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:
Syntheses, biological evaluation, and structureeactivity relationships for a series of novel 2-substituted
3-benzenesulfonyl-5,6-dimethyl-pyrazolo[1,5-a]pyrimidines are disclosed. In spite of a wide, four orders
Received 31 July 2010
Received in revised form
24 January 2011
Accepted 25 January 2011
Available online 1 February 2011
of magnitude, SAR range (Ki varied from 260 pM to 2.96
mM), no significant correlation of 5-HT6R
antagonistic potency was observed with major physiochemical characteristics, such as molecular weight,
surface polar area, cLogP, or number of rotatable bonds. Statistically significant trend was only observed
for size of substitute group, which was not enough to explain the deep SAR trend. Besides with the
substitute group size, another factor that presumably plays a role in defining the compound potencies is
a relative position of the heterocycle and sulfophenyl moieties. Among all synthesized derivatives,
(3-benzenesulfonyl-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-amine 18 is the most potent
(Ki ¼ 260 pM) and extremely selective, 5000 to >50,000-fold relative to 55 therapeutic targets, antag-
onist of the 5-HT6 receptor.
Keywords:
Phenylsulfonyl
Pyrazolopyrimidine
5-HT6 receptor antagonist
SAR
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
Unlike Ro-65-7674, the new highly potent and selective 5-HT6R
antagonists I and II either did not possess a positive ionizable group
Serotonin 5-HT6 receptors (5-HT6R) have attracted considerable
interest [1] due to high affinity to a wide range of psychiatric drugs
and its specific distribution in the brain. Development of highly
potent and selective antagonists would help clarifying the role of
the 5-HT6R in brain functions as well as in etiology of the central
neural system (CNS) diseases. At present, there are only few
selective antagonists that have been developed and which have
progressed to Phase I and Phase II clinical trials as potential drugs
for treatment of various CNS diseases [2e7].
(PI) at all [8] or the PI was located in R1 position [9]. Though in
accordance with the pharmacophore model suggested based on
analyses of 45 structurally diverse 5-HT6R antagonists, PI was
considered as an essential recognition group [10] our data indicated
that it might not be necessary determinant of the high antagonist
affinity. We have suggested [9] that R1 substitute group likely
served to constrain the phenylsulfo moiety in an appropriate
conformation that affected the binding affinity.
Recently, we have reported [8,9] on the synthesis and biological
activity of novel 5-HT6R antagonists, 3-phenylsulfonyl-cycloalkano
[e and d]pyrazolo[1,5-a]pyrimidines I and II (Fig. 1), which posses
both the picomolar range affinity and excellent selectivity profiles.
2. Chemistry
In this paper, we attempted to evaluate the role of the R1
substituent group in defining the 5-HT6R antagonistic activity
compounds. We have synthesized a series of new substituted 5,7-
dimethyl-3-phenylsulfonyl-pyrazolo [1,5-a] pyrimidines (DMPSPP)
13e23 (Scheme 1) and assessed their serotonin 5-HT6R activity. The
DMPSPPs 13e23 were synthesized by cyclocondensation of 3-ami-
nopyrazoles 1e11 with acetylacetone 12 in high yields (Scheme 1).
In addition, 2-{[5,7-dimethyl-3-(phenylsulfonyl)pyrazolo[1,5-a]
pyrimidin-2-yl]oxy}-N,N-dimethylethanamine 24 (Scheme 2) was
synthesized in 60% yield by one-pot procedure starting from
alcohol 15 that reacted with trifluoromethylsulfonic acid anhydride
Abbreviations: 5-HT6R, serotonin 5-HT6 receptor; 5-HT2BR, serotonin 5-HT2B
receptor; DMPSPP, 5,7-dimethyl-3-phenylsulfonyl-pyrazolo [1,5-a] pyrimidine;
TPSA, topological polar surface area; CNS, central neural system; PI, positive
ionizable group; DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, fetal bovine
serum; HBSS, Hank’s balanced salt solution; IBMX, 1-methyl-3-isobutylxanthine.
* Corresponding author. Tel.: þ1 858 974 4860; fax: þ1 858 794 4931.
E-mail address: iokun@chemdiv.com (I. Okun).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.038

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A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 46 (2011) 1189e1197
Scheme 2. Synthesis of [2-(5,7-dimethyl-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidin-
2-yloxy)-ethyl]-dimethyl-amine 24. Reagents and conditions: (a) Tf2O, CHCl3, -5e0 ꢁC,
1 h; (b) 40% aq. (CH3)2NH, 0e20 ꢁC, 2 h.
Fig. 1. 3-Phenylsulfonyl-sycloalkano[e and d]pyrazolo[1,5-a]pyrimidines I, II.
in CHCl3 for 1 h at a temperature between ꢀ5 ꢁC and 0 ꢁC followed
by treatment of intermediate triflate with excess of 40% aq. Me2NH
and stirring the resulted mixture at 0e20 ꢁC for 2 h.
Cleavage of BOC group in compound 23 by treatment with 6 M
solution of AcCl in ethanol at room temperature afforded 2-
(piperazin-1-yl)-DMPSPP 25 in 95% yield (Scheme 3).
Scheme 3. Synthesis of 5,7-dimethyl-3-phenylsulfonyl-2-piperazin-1-yl-pyrazolo[1,5-
a]pyrimidine 25. Reagents and conditions: (a) 6 N AcCl in EtOH, ambient temp.
2-Methylsulfinyl-DMPSPP 26 was synthesized in the 87% yield
by oxidation of 2-methythio-derivative 16 with H2O2 in acetic acid
at 80 ꢁC (Scheme 4).
Starting 3-aminopyrazoles 1e6 were prepared according to
previously described procedures [9,11,12]. Diaminopyrazoles 7e11
were synthesized in high yields by reaction of 3,3-bis(methylthio)-
2-(phenylsulfonyl)acrylonitrile 27 [13] with corresponding amines
(Scheme 5) followed by cyclocondensation of intermediate 3-
(methylamino)-3-(methylthio)-2-(phenylsulfonyl)acrylonitriles
28e32 with hydrazine hydrate.
The structures of synthesized DMPSPPs were confirmed with
LC-MS, HRMS, and NMR spectra. According to the content of
molecular ions in LC-MS and HRMS spectra, all synthesized
compounds had a purity of 98% and higher. The NMR spectra of the
compounds were in a good agreement with their structures.
antagonize 5-HT6R. Ki values vary in a broad, four orders of
magnitude, range of potencies, from Ki ¼ 0.26 nN (18) to
Ki ¼ 2.96
mN (26). When relative potencies, K(H), are considered,
the negative K(H) values reflect increase, while positive K(H) values
reflect decrease in the substituted compound potencies relative to
the unsubstituted DMPSPP 13.
No significant correlation was evident between the 5-HT6R
relative inhibition potency, K(H) and either LogP, polar surface area,
or volume of the molecules (Fig. 2AeC). When studying a rela-
tionship between the compound potencies to block 5-HT6 receptor-
activated cell response and the same physiochemical parameters
calculated for the R-substitute groups, the group volume seemed to
have statistically significant effect on the compound potency
(Fig. 2F, P ¼ 0.01). Neither the group hydrophobicity, cLogP
(Fig. 2D), nor its polar surface area, TPSA (Fig. 2E), significantly
affected the potency. Molecule flexibility (function of number of
rotatable bonds) was not a factor determining their potency either.
For example, while both the 16 and 26 have equal number of
rotatable bonds, their respective 2-substitute groups affected the
compound potency relative to the 13 (R ¼ H) in opposite directions.
Thus 16 exhibited more than 450-fold higher potency over 13 while
26 was 5-fold less potent. Compound 13, which has 2 rotatable
bonds, showed Ki value similar to those of compounds 22 and 20,
which have six and seven rotatable bonds, correspondingly.
Energy minimization performed with DS ViewerPro 6.0 (Accel-
rys, San Diego CA) [15], revealed that the 2-substituent group
affects both the value and directionality of the molecule dipole as
well as angle at which the sulfophenyl moiety is positioned relative
to the heterocycle plane. Neither molecule polarizabilities nor
dipole moments seem to correlate with the 5-HT6 receptor antag-
onistic activity (data not shown). The DMPSPPs with minimized 3D
conformations could be separated into two groups, those with
potencies higher than that of 13 are on the left side and those with
lower potencies are on the right side from the DMPSPP 13 (Fig. 3). It
3. Results and discussion
Potencies of the synthesized 2-substituted DMPSPPs to block 5-
HT6R were assessed in a cell-based assay by their ability to diminish
serotonin-induced cAMP production in HEK-293 cells stably
expressing human recombinant 5-HT6R (Table 1). For a few of the
compounds, we have also measured affinity tothe receptor based on
competition with the radioactively labeled 5-HT6 receptor ligand,
[3H] lysergic acid diethylamide. The data are summarized in Table 1.
As one can see, the nature of the 2-substituted group attached to
the DMPSPP heterocycle, greatly affects ability of the compounds to
Scheme 1. Synthesis of 2-substituted 5,7-dimethyl-3-phenylsulfonyl-pyrazolo[1,5-a]
pyrimidines 13e23. Reagents and conditions: (a) AcOH, reflux, 3 h t e torsion angle
formed by bonds ab and bc.
Scheme 4. Synthesis of 5,7-dimethyl-2-methylsulfinyl-3-phenylsulfonyl-pyrazolo[1,5-
a]pyrimidine 26. Reagents and conditions: (a) H2O2, AcOH, 80 ꢁC, 7 h.

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 46 (2011) 1189e1197
1191
the compounds with lower relative to 13 potencies, positive K(H)
values, excluding 19, the characteristic torsion angles substantially
deviate from those of the first group (Fig. 4).
Hydrogen replacement in compound 13 with methyl group
(compound 14) led to 6.3-fold increase in the potency, whereas its
replacement with methylamino group (compound 18) led to more
than three order of magnitude increase in the potency (K
(H) ¼ ꢀ3.33). However, substitution with dimethylamino group
(21) was substantially less effective than that with methylamino
group (18) in increasing the compound potency (K(H) ¼ ꢀ1.59 and
ꢀ3.33, correspondingly). Almost 2 orders of magnitude difference
in the 5-HT6R antagonistic potencies between the DMPSPPs 21 and
18 is most probably due to the formation of intramolecular
hydrogen bond in 18 (Fig. 3), which restricts molecular flexibility
and secures the molecule in a conformation that better fits the
receptor binding site. Notably, with the bulky R-groups, formation
of the intramolecular H-bond does not improve the compound
potency. For example, though such bond forms in both 19 and 20
Scheme 5. Preparation of substituted 3,5-diaminopyrazoles 7e11. Reagents and
conditions: (a) Me2NCH2CH2NH2, Me2NCH2CH2CH2NH2, Me2NH, Me2NCH2CH2NHMe
or 1-Boc-piperazine; i-PrOH, reflux for
N2H4$H2O, i-PrOH, reflux, 0.5 h.
1 h then ambient temp overnight; (b)
Table 1
SAR of the 2-substituted DMPSPPs. Compound potencies to block serotonin-induced
cAMP production in HEK-293 cells expressing recombinant human 5-HT6R and
several physiochemical parameters of the molecules.
Cpd
R
Ki, nM
K(H)c
Functionala
Bindingb
18
16
17
21
14
15
22
13
20
25
24
19
26
MeNH
MeS
EtS
Me2N
0.26
1.20
1.91
14.5
88.8
283
489
561
764
1380
2200
2600
2960
0.2
2.0
ꢀ3.33
ꢀ2.67
ꢀ2.47
ꢀ1.59
ꢀ0.80
ꢀ0.30
ꢀ0.06
0.00
þ0.13
þ0.39
þ0.59
þ0.67
þ0.72
(Fig. 3), their potencies are 3.4-fold apart (Ki values are 2.6
mM and
0.76 M), which
m
M, respectively) and the potency of 22 (Ki ¼ 0.49
m
does not have such bond, is 5.3 times as high as that of its corre-
sponding homolog 19.
Me
68.4
22.2
HOCH2CH2O
Me2NCH2CH2NMe
H
Me2NCH2CH2CH2NH
Piperazin-1-yl
Me2NCH2CH2O
Me2NCH2CH2NH
MeS(O)
Alkylthio-derivatives 16 and 17 were 4-folde7-fold less potent
than the 18. Comparable potencies of the DMPSPPs 16 and 17 imply
some limited tolerance of the 5-HT6R binding site to the size of the
R-group. However, hydrogen substitution with more bulky 2-
hydroxyethoxy group (15), N-(2-dimethylamino-ethyl)-N-methyl-
amino group (22) or (3-dimethylamino-propyl)-amino group (20)
practically led to no change in the compound potency (K(H) ¼ ꢀ0.3,
ꢀ0.06, and þ0.13, correspondingly). This seemingly controversial
behavior could be due to a presence of a rather narrow recognition
pocket in the receptor, which accommodates R-groups of molecules
18, 16, 17, 21, and 14 and orients the molecule to form a tighter
binding. Absence of the 2-substitute group in 13 or too bulky
substitutes in 15, 22, and 20, do not fit the pocket and thus prevent
this “orientation constriction”. The relatively low potency of
DMPSPPs 26, K(H) ¼ þ0.72, seems to indicate that the receptor
small binding pocket does not tolerate electron-withdrawing entity
in the group. Alternative explanation could be that this group
237.0
a
Potency to antagonize serotonin-induced cAMP production in HEK cell heter-
ologously expressing 5-HT6R.
b
Affinity by competitive displacement of [3H]LSD from its complex with 5-HT6R.
c
K(H) ¼ Log(KRi (i)/KRi (H)) reflects change in potency (functional assay) of the R(i)-
substituted compounds relative to the DMPSPP 13 with R ¼ H.
is interesting to note that for the compounds with R-groups sup-
porting higher than 13 potencies, negative K(H) values, the torsion
angles, t (Scheme 1), between the heterocycle plane and the plane
formed by two bonds, b and c, connecting serum with the pyrozol
and phenyl rings, respectively, are between þ0.31ꢁ and ꢀ52.5ꢁ. For
Fig. 2. Correlation between relative potencies of the compounds to block 5-HT6 receptors in cell-based assay and their physiochemical parameters. cLogP, TPSA, and molecule
volume values were calculated using MolInspiration on-line calculator [14] for whole molecules (A, B, and C) and for R-substitute groups (D, E, and F). Dotted lines represent 95%
confidence intervals. Dotted arrows show position of 13 on the K(H) scale.

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A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 46 (2011) 1189e1197
Fig. 3. Free energy minimized conformations of the 5-HT6R antagonists. Atoms are colored in accordance with their partial charges (red/reddish e negative, blue/bluish-positive).
Energy minimization was performed using DS ViewerPro 6.0. Top center panel shows unsubstituted molecule 13. Compounds with increased potencies relative to the compound 13
(negative K(H) values) are grouped on the left side and those with decreased potencies (positive K(H) values) on the right side. K(H) ¼ Log(KRi (i)/KRi (H)); where Ki is a functional inhibition
constant, superscript R denotes specific substitute group (i), and RH reflects R ¼ H (compound 13). For each compound, the conformation with the lowest local free energy minimum is
shown. The torsion angles between the heterocyle plane and the plane formed by two bonds connecting serum with the pyrozol and phenyl rings, respectively, are shown in yellow and
in green, the H-bond distance is shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Relation between relative 5-HT6R blocking potency, K(H), of the R-substituted
compounds and the torsion angles between the heterocycle plane and the plane
formed by two bonds connecting serum with the pyrozol and phenyl rings.
Fig. 5. Correlation between DMPSPP potencies (Ki, functional) to inhibit 5-HT6R-
induced cell response and their affinities (Ki, binding) measured in a competitive
radioligand binding assay.

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 46 (2011) 1189e1197
1193
Fig. 6. Target-specific profile determined for DMPSPP 18 (10
presented.
mM) using competitive radioligand binding assays. Measurements were performed in duplicates. Mean ꢂ SD values are
causes the torsion angle, t (see Scheme 1), of the sulfophenyl
moiety (ꢀ87.75ꢁ) that is incompatible with proper accommodation
of this part of the molecule by the binding site of the receptor.
Indeed the other two low potency compounds 24 and 25 both have
torsion angles, þ145.71ꢁ and ꢀ131.09ꢁ, respectively, which subs-
tantially deviate from those characteristic of compounds with the
higher potencies (Figs. 3 and 4). It seems plausible to suggest that
orientation of the sulfophenyl moiety relative to the core hetero-
cyclic plane of the molecules acts concurrently with the presence of
rather small 2-substitute group (presumably with some electron-
donor characteristics) to define their 5-HT6 receptor antagonistic
potency. Further work to determine crystal structures of these
molecules is underway to confirm this preliminary conclusion.
The antagonistic potency values of the compounds were very
similar to and correlated well (r ¼ 0.96) with their affinities
measured in a competitive radioligand binding assay (Fig. 5). This
allows one to conclude that the effect of the 2-substituted group on
the compounds’ functional potency to block serotonin-evoked
Fig. 7. The DMPSPP 18 concentration-dependent displacement of radio-labeled ligand (1.2 nM [3H]LSD) from its complex with 5-HT2BR (A) and blockage of 5
serotonin-induced calcium ion mobilization in HEK cells heterologously expressing 5-HT2BR (B). Shown are mean ꢂ SD values of typical experiments performed in duplicates.
mM a-methyl

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A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 46 (2011) 1189e1197
either LogP or polar surface area of the molecules, though statis-
tically significant correlation was observed with the volume of
substitute group. The 2-substituents affect relative topology
between the heterocycle plane and the sulfophenyl ring, which is
presumably one of defining factors of the compound affinities. The
affinity of most potent antagonist of the 5-HT6R, 2-methylamino-
DMPSPP 18, is in a mid picomolar range (Ki ¼ 0.2 nM) and the
compound has shown remarkable specificity/selectivity character-
istics on a panel consisting of 56 therapeutic targets. Based on the
large “safety window” between its effect on 5-HT6R (Ki ¼ 260 pM)
and that on 5-HT2B
R
(IC50
¼
5.13 mM) and hERG channel
(IC50 > 30.0 M), we conclude that 18 is a perspective molecule for
m
further development as clinical candidate to treat associated with
5-HT6R CNS diseases.
5. Experimental protocols
Fig. 8. The DMPSPP 18 is extremely week blocker of the hERG potassium channels.
Patch clamp measurements were performed in triplicates (mean ꢂ SD).
5.1. General
1H and 13C NMR spectra of the investigated compounds were
recorded in DMSO-d6 or CDCl3, respectively, with spectrometer
Bruker DPX-400 (400 MHz, 27 ꢁC). HRMS spectra (ESI-TOF, positive
mode) were obtained with a Waters Qt of API US instrument.
Purities of all synthesized compounds (more than 98%) were
assessed by LC-MS spectra with Shimadzu HPLC instrument
equipped with PE SCIEX API 150EX mass-, Alltech 2056 ELS-, and
Shimadzu UV- (254 and 215 nm) detectors. Separation was per-
cAMP synthesis in 5-HT6-HEK cells, directly relates to their binding
affinities to the receptor.
We then studied a specificity profile of the most potent DMPSPP,
18, using a panel of 56 therapeutic targets, including GPCRs, ion
channels, and transporters. The interaction of 18 with the targets
was assessed by its ability to compete with corresponding target-
specific radio-labeled ligands (Fig. 6). Except for the 5-HT6 and 5-
HT2B receptors, 18 does not practically bind to any other targets
formed with a Phenomenex Luna 3
a gradient flow of 5e95% acetonitrile in water (both with 0.05%
TFA) over 12 min at 0.8 mL minꢀ1
m
C18 (4.6 ꢃ 150 mm) column in
tested (the radioligand displacement at 10 mM is significantly lower
than 50%). Except for 5-HT2B receptors, this represents more than
.
50,000-fold specificity of 18 over all the other targets studied.
We have assessed the affinity of DMPSPP 18 to the 5-HT2B
(Fig. 7A) and its potency to inhibit the receptor-induced intracel-
Free local minima energies of 3D conformations were calculated
using DS ViewerPro 6.0 (Accelrys, San Diego CA) [19] using
convergence criterion of 0.00001 and 50,000 to 200,000 iterations
of energy minimizing. For each structure, energy minimization
cycles were performed starting with different initial conformations.
Competitive radioligand displacement experiments were per-
formed by MDS Pharma (currently Ricerca) in accordance with their
internally developed protocols briefly described elsewhere [20].
R
lular calcium ion mobilization (Fig. 7B). The data shows that affinity
of 18 to bind to 5-HT2BR (Ki ¼ 1.04
for 5-HT6R (Table 1). Potency to inhibit 5-HT2BR-induced cell
response (IC50 ¼ 5.13 M) in 5-HT2BR-HEK cells was 1000-fold
mM) is 5000 times as low as that
m
lower than that for 5-HT6R-HEK cells (Table 1). No agonistic activity
of 18 towards 5-HT2BR was detected (data not shown). 5-HT2B
receptor is considered as a potential liability target as its agonists
were linked to cardiac valvulopathy [16]. On the other hand, the
antagonists of the receptor are considered as possible candidates
for treatment of chronic heart diseases [17,18]. Fig. 6 manifests
exceptionally high selectivity of 18 towards 5-HT6R with practically
no potential cardiac liability pertinent to activation of 5-HT2B
receptors (Fig. 7).
5.2. Chemistry
5.2.1. 5,7-Dimethyl-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidines
13e23 (general procedure)
A mixture of aminopyrazole 1e11 (1 mmol), acetylacetone 12
(0.5 g, 5 mmol), and AcOH (2 mL) was heated under reflux for 3 h,
the solvent was removed under reduced pressure, the residue was
treated with i-PrOH, the obtained mixture was kept in ultrasonic
bath for 0.5 h at 0 ꢁC. Formed precipitate was separated by
centrifugation, washed twice with fresh portions of cooled i-PrOH,
hexane, and dried to provide 65e95% of colorless compound
13e23.
We then assessed if 18 could inhibit potassium channel, hERG
(product of human analog of Drosophila ether-a-go-go gene).
Blockage of this channel by a diverse group of drugs leads to
a prolongation of QT interval in a cardiac electric cycle and
a concomitant risk of sudden death [19]. In the patch clamp
experiments (Fig. 8) performed for us by MDS Pharma (currently
Ricerca), we have found that 18 showed a very weak antagonistic
5.2.1.1. 5,7-Dimethyl-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidine
activity with IS50 > 30.0
mM, that was more than six orders of
13. 1H NMR (DMSO-d6, 400 MHz),
d
: 8.60 (s, 1H); 8.02e8.06 (m,
2H); 7.53e7.63 (m, 3H); 7.18 (q, J ¼ 1 Hz, 1H); 2.66 (d, J ¼ 1 Hz, 3H);
2.57 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
163.38, 147.43, 145.11,
magnitude higher than the IC50 of the 5-HT6R blockage with the
DMPSPP 18.
d
144.40, 142.94, 133.13, 129.27, 126.56, 111.61, 109.15, 24.69, 16.54.
4. Conclusion
MS-ESI m/z 288 (M þ H). LC-MS (UV-254) purity: 98%.
We have described syntheses and biological evaluations of
small-molecule DMPSPPs 13e26, representing novel chemotype of
the serotonin 5-HT6R antagonists. The antagonistic potency
dramatically depends on the nature of a substituent group in
position 2 of the heterocycle with a SAR in the series having depth
of 4 orders of magnitude. The potency does not correlate with
5.2.1.2. 2,5,7-Triimethyl-phenylsulfonyl-pyrazolo[1,5-a]pyrimidine
14. 1H NMR (DMSO-d6, 400 MHz),
d
: 8.00e8.04 (m, 2H); 7.52e7.62
(m, 3H); 7.10 (s, 1H); 2.62 (s, 3H); 2.61 (s, 3H); 2.55 (s, 3H). 13C NMR
(75 MHz, DMSO-d6) 162.79, 153.30, 146.60, 146.50, 143.59, 132.99,
d
129.22, 126.29, 111.18, 105.86, 24.65, 16.48, 14.09. MS-ESI m/z 302
(M þ H). LC-MS (UV-254) purity: 99%.

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 46 (2011) 1189e1197
1195
5.2.1.3. 2-{[5,7-Dimethyl-3-(phenylsulfonyl)pyrazolo[1,5-a]pyr-
NMR (75 MHz, DMSO-d6)
d
161.64, 159.13, 147.92, 145.87, 143.84,
imidin-2-yl]oxy}ethanol 15. 1H NMR (DMSO-d6, 400 MHz),
d:
132.84, 129.02, 126.44, 110.64, 95.55, 53.05, 48.54, 42.54, 41.02,
24.48,16.51. MS-ESI m/z 388 (M þ H). LC-MS (UV-254) purity: 98.5%.
HRMS calculated for C19H25N5O2S (M þ H) 388.1807, found 388.1811.
7.97e8.01 (m, 2H); 7.50e7.61 (m, 3H); 7.05 (s, 1H); 4.91 (t,
J ¼ 5.3 Hz, 1H, exch. with D2O); 4.36 (t, J ¼ 4.9 Hz, 2H); 3.74 (m, 2H);
2.55 (s, 3H); 2.52 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
d 162.72,
162.67, 146.70, 146.43, 143.93, 132.82, 129.09, 126.24, 110.73, 92.91,
71.00, 59.19, 24.59, 16.53. MS-ESI m/z 348 (M þ H). LC-MS (UV-254)
purity: 98%.
5.2.1.11. tert-Butyl 4-[5,7-dimethyl-3-(phenylsulfonyl)pyrazolo[1,5-a]
pyrimidin-2-yl]piperazine-1-carboxylate 23. 1H NMR (DMSO-d6,
400 MHz),
d
: 8.14 (d, J ¼ 7.7 Hz, 2H); 7.42e7.54 (m, 3H); 6.64 (s, 1H);
3.59e3.65 (m, 4H); 3.43e3.49 (m, 4H); 2.63 (s, 3H); 2.58 (s, 3H);
1.49 (s, 9H). MS-ESI m/z 472 (M þ H). LC-MS (UV-254) purity: 98%.
5.2.1.4. 5,7-Dimethyl-2-(methylthio)-3-(phenylsulfonyl)pyrazolo[1,5-
a]pyrimidine 16. 1H NMR (DMSO-d6, 400 MHz),
d
: 8.21e8.23 (m,
2O), 7.45e7.54 (m, 3O), 6.67 (s, 1H), 2.68 (s, 3O), 2.64 (s, 3O), 2.62
(s, 3O). 13C NMR (75 MHz, DMSO-d6)
162.92, 154.74, 147.08,
5.2.2. (2-{[5,7-Dimethyl-3-(phenylsulfonyl)pyrazolo[1,5-a]
pyrimidin-2-yl]oxy}ethyl) dimethylamine 24
d
146.33, 143.11, 133.05, 129.13, 125.94, 110.73, 104.76, 24.53, 16.26,
12.62. MS-ESI m/z 334 (M þ H). LC-MS (UV-254) purity: 98.5%.
5.2.2.1. 24$HCl. Trifluoromethanesulfonic anhydride (0.33 mL,
1.8 mmol) was added to a stirred solution of alcohol 15 (520 mg.
1.5 mmol) in CHCl3 (2 mL) at 0 ꢁC, the mixture was stirred for 1 h,
then 40% aq. solution of Me2NH (2 mL) was added. The mixture was
allowed to warm to ambient temperature and stirred for additional
2 h. Organic layer was separated, washed with water three times,
and extracted with 6 N aq. HCl, aq. layer was washed with CHCl3,
neutralized with 20% aq. NaOH, and extracted with CHCl3. Organic
layer was washed with H2O, brine, dried (Na2SO4) and concen-
trated. The residue was dissolved in CHCl3 (2 mL) and treated with
6 M solution of AcCl in EtOH (0.5 mL). The mixture was concen-
trated, the residue was treated with i-PrOH, formed precipitate was
filtered off, washed twice with i-PrOH, hexane, and dried to afford
369 mg (60%) of compound 24$HCl.
5.2.1.5. 5,7-Dimethyl-2-(ethylthio)-3-phenylsulfonyl-pyrazolo[1,5-a]
pyrimidine 17. 1H NMR (DMSO-d6, 400 MHz),
d: 8.01 (m, 2O), 7.60
(m, 3O), 7.11 (s,1O), 3.18 (q, J ¼ 7.6 Hz, 2O), 2.64 (s, 3O), 2.57 (s, 3O),
1.38 (t, J ¼ 7.6 Hz, 3O). 13C NMR (75 MHz, DMSO-d6)
d 162.49,
153.73, 146.56, 145.91, 142.79, 132.70, 128.78, 125.62, 110.41, 104.40,
24.18, 23.65, 15.92, 13.86. MS-ESI m/z 347 (M þ H). LC-MS (UV-254)
purity: 98%.
5.2.1.6. N,5,7-Trimethyl-3-(phenylsulfonyl)pyrazolo[1,5-a]pyrimidin-
2-amine 18. 1H NMR (DMSO-d6, 400 MHz),
d
: 8.14 (d, J ¼ 8.5 Hz,
2H); 7.40e7.51 (m, 3H); 6.51 (s, 1H); 5.99 (br. q, J ¼ 5.1 Hz, 1H); 3.02
(d, J ¼ 5.1 Hz, 3H); 2.57 (s, 3H); 2.53 (s, 3H). 13C NMR (CDCl3,
75 MHz),
d: 161.2; 160.3; 158.2147.8; 145.2; 144.0; 132.3; 128.6;
5.2.2.2. 24 (free base). 1H NMR (DMSO-d6, 400 MHz),
d: 7.95e7.99
128.4; 109.1; 29.0; 24.8; 17.0. MS-ESI m/z 317 (M þ H). LC-MS (UV-
(m, 2H); 7.50e7.61 (m, 3H); 7.04 (s, 1H); 4.42 (t, J ¼ 5.5 Hz, 2H); 2.63
254) purity: 99%.
(t, J ¼ 5.5 Hz, 2H); 2.55 (s, 3H); 2.52 (s, 3H); 2.20 (s, 6H). 24$HCl: 1H
NMR (DMSO-d6, 400 MHz), d: 11.01 (br.s, 1H); 7.96e8.00 (m, 2H);
5.2.1.7. N0-[5,7-Dimethyl-3-(phenylsulfonyl)pyrazolo[1,5-a]pyr-
7.52e7.63 (m, 3H); 7.12 (s, 1H); 4.74e4.79 (m, 2H); 3.55e3.60 (m,
2H); 2.88 (s, 6H); 2.59 (s, 3H); 2.54 (s, 3H). MS-ESI m/z 375 (M þ H).
LC-MS (UV-254) purity: 98%.
imidin-2-yl]-N,N-dimethylethane-1,2-diamine
(DMSO-d6, 400 MHz),
19$HCl. 1H NMR
: 10.07 (br.s, 1H); 8.02 (d, J ¼ 8.0 Hz, 2H);
d
7.52e7.62 (m, 3H); 6.95 (s, 1H); 6.72 (br.t, J ¼ 5.7 Hz, 1H); 3.69e3.76
(m, 2H); 3.29e3.35 (m, 2H); 2.80 (s, 6H); 2.54 (s, 3H); 2.47 (s, 3H).13C
5.2.3. 5,7-Dimethyl-3-(phenylsulfonyl)-2-piperazin-1-ylpyrazolo
[1,5-a]pyrimidine hydrochloride 25$HCl
NMR (75 MHz, DMSO-d6)
d 161.36, 156.29, 146.94, 145.94, 143.79,
132.89,129.14,125.96,109.93, 90.74, 55.50, 42.54, 37.36, 24.37,16.57.
MS-ESI m/z 374 (M þ H). LC-MS (UV-254) purity: 98%. HRMS
calculated for C18H23N5O2S (M þ H) 374.165070, found 374.165.
A solution of compound 23 (141 mg, 0.3 mmol) in CHCl3 (1 mL)
was treated 6 M solution of AcCl in EtOH (1 mL), reaction mixture
was stirred at ambient temperature for 1 h, and treated with Et2O.
Formed precipitate was separated by centrifugation, washed three
times with EtOAc, twice with acetone, hexane, and dried. Yield
111 mg (91%) of colorless solid compound 23$HCl.
5.2.1.8. N0-[5,7-Dimethyl-3-(phenylsulfonyl)pyrazolo[1,5-a]pyr-
imidin-2-yl]-N,N-dimethylpropane-1,3-diamine 20$HCl. 1H NMR
(DMSO-d6, 400 MHz),
d
: 10.30 (br.s, 1H); 8.01 (d, J ¼ 8.0 Hz, 2H);
1H NMR (DMSO-d6, 400 MHz),
d: 9.57 (br.s, 2H); 8.01 (d,
7.52e7.62 (m, 3H); 6.92 (s, 1H); 6.52 (br.t, J ¼ 6.1 Hz, 1H); 3.38e3.44
(m, 2H); 3.02e3.08 (m, 2H); 2.72 (s, 6H); 2.52 (s, 3H); 2.46 (s, 3H);
J ¼ 8.0 Hz, 2H); 7.49e7.62 (m, 3H); 7.06 (s, 1H); 3.57e3.65 (m, 4H);
3.16e3.26 (m, 4H); 2.57 (s, 3H); 2.50 (s, 3H). MS-ESI m/z 372
(M þ H). LC-MS (UV-254) purity: 98.5%. HRMS calculated for
C18H21N5O2S$HCl (M þ H) 372.14942, found 372.1491.
1.92e2.06 (m, 2H). 13C NMR (75 MHz, DMSO-d6)
d 161.08, 156.61,
146.95, 145.81, 143.91, 132.84, 129.19, 125.80, 109.80, 90.28, 54.53,
41.97, 24.33, 23.89, 16.63. MS-ESI m/z 388 (M þ H). LC-MS (UV-254)
purity: 99%. HRMS calculated for C19H25N5O2S (M þ H) 388.18072,
found 388.1813.
5.2.4. 5,7-Dimethyl-2-(methylsulfinyl)-3-(phenylsulfonyl)pyrazolo
[1,5-a]pyrimidine 26
A mixture of compound 16 (333 mg,1 mmol), AcOH (10 mL), and
5.2.1.9. N,N,5,7-Tetramethyl-3-(phenylsulfonyl)pyrazolo[1,5-a]pyr-
35% aq. solution of H2O2 (88 m
L) was stirred at 80 ꢁC for 7 h,
imidin-2-amine 21. 1H NMR (CDCl3, 400 MHz),
d
: 8.15 (dd, J ¼ 8.0 Hz,
J ¼ 1.8 Hz, 2H); 7.41e7.51 (m, 3H); 6.60 (s, 1H); 3.07 (s, 6H); 2.60 (s,
3H); 2.55 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
161.14, 160.29,
concentrated under reduced pressure, and residue was purified by
silica column chromatography (eluent CHCl3/EtOAc 5:1). Yield
304 mg (87%) of compound 26.
d
148.20, 145.60, 144.25, 132.64, 128.94, 126.29, 110.31, 94.93, 42.14,
1H NMR (DMSO-d6, 400 MHz),
d: 8.22 (m, 2H), 7.58 (m, 1H), 7.51
24.43, 16.50. MS-ESI m/z 331 (M þ H). LC-MS (UV-254) purity: 99%.
(m, 2H), 6.88 (s, 1O), 3.24 (s, 3O), 2.83 (s, 3O), 2.71 (s, 3O). MS-ESI
m/z 350 (M þ H). LC-MS (UV-254) purity: 99%.
5.2.1.10. N-[5,7-Dimethyl-3-(phenylsulfonyl)pyrazolo[1,5-a]pyr-
imidin-2-yl]-N,N0,N0-trimethylethane-1,2-diamine 22$HCl. 1H NMR
5.2.5. 4-(Phenylsulfonyl)-1H-pyrazole-3,5-diamines 7e11 (general
procedure)
A mixture of 2-phenylsulfonyl-3,3-bis-methylsulfanyl-acrylo-
nitrile 27 (2.67 g, 9.35 mmol), a corresponding amine (9.35 mmol),
(DMSO-d6, 400 MHz),
d
: 10.46 (br.s, 1H); 8.01 (d, J ¼ 8.0 Hz, 2H);
7.52e7.62 (m, 3H); 7.04 (s, 1H); 3.73 (t, J ¼ 6.6 Hz, 2H); 3.36 (t,
J ¼ 6.6 Hz, 2H); 3.03 (s, 3H); 2.78 (s, 6H); 2.58 (s, 3H); 2.48 (s, 3H).13C

1196
A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 46 (2011) 1189e1197
and i-PrOH (15 mL) was stirred and heated under reflux for 1 h and
then stirred overnight at ambient temperature. Formed precipitate
was filtered off, washed with i-PrOH, suspended in i-PrOH (15 mL),
and N2H2$H2O (0.48 g, 9.5 mmol) was added into the suspension.
The resulted mixture was heated under reflux for 0.5 h, cooled, and
poured into a mixture of ice and H2O (150 mL). Formed precipitate
was filtered off, washed with ice water, cooled i-PrOH, and dried.
Obtained in yields 85e92% compounds 7e11 were used for further
reactions without additional purification.
serotonin, cells were treated as described in the cAMP LANCE assay
kit protocol (PerkinElmer, Waltham, MA). The LANCE signal was
measured using multimode plate reader VICTOR 3 (PerkinElmer,
Waltham, MA) with built-in settings for the LANCE detection.
5.3.1.3. Radioligand binding assays. The assays were performed by
Ricerca (former MDS Pharma) in accordance with their optimized
protocols for each target. The procedures are described in Ref. [20].
5.4. Curve fitting and statistical analysis
5.3. Biological assays
The concentration curve data were fitted with Prism 5 (Graph-
Pad, CA) using built-in 4-parametric equation to calculate IC50
values. All experiments were performed in duplicate. Standard
deviations (SD) were calculated with Prism built-in statistical
package. Ki values for functional 5-HT6 receptor inhibition assays
were calculated using Cheng-Prusoff’s [21] modified equation,
Ki ¼ IC50/(1þ[Ag]/EC50). Where IC50 is the concentration of antag-
onist causing 50% inhibition of serotonin-induced cell response;
[Ag] is a concentration of serotonin (10 nM), at which inhibition
was measured and EC50 is serotonin concentration causing 50%
stimulation of the cell response, measured simultaneously with the
test compounds on the same plates. The mean EC50 value for
serotonin-induced cAMP production in 5-HT6R-HEK cells was
1.91 ꢂ 0.13 nM as determined from 4 independent experiments
with three to five repeats (separate plates), each in quadruplicates.
5.3.1. Cell-based functional assays
5.3.1.1. 5-HT2B receptor functional assay. The 5-HT2BR was sub-
cloned into T-Rex system (Invitrogen, Carlsbad, CA) and expressed
into HEK (5-HT2BR-HEK) cells. The cells were grown in T-175 flasks at
37 ꢁC in 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 disso-
ciated with TrypLEÔ Express (Invitrogen, Carlsbad, CA), washed
twice with PBS and loaded at room temperature with 4 mM calcium-
sensitive dye, Fura-2AM (Invitrogen, Carlsbad, CA) for 30 min. After
loading, the cells were washed once with PBS, re-suspended into
protein free Hybridoma media without phenol red (Sigma, St. Louis,
MO) and allowed to incubate for additional 30 min with gentle
shaking at room temperature. All loading procedures were per-
formed in dark conditions. The loaded cells were washed twice with
PBS and re-suspended into the Hybridoma media at a cell density of
3e4 ꢃ 106 cells/mL for subsequent experiments. Fura-2 ratiometric
fluorescence signal was registered at 510 nm upon alternate exci-
tation at 340 nm and 380 nm using spectrofluorometer RF5301PC
(Shimadzu, Columbia, MD). In a square (1 cm) optical cuvette with
Acknowledgment
Authors thank Dr. Norman Lee from Chemical Instrumentation
Center, Boston University for HRMS measurements. We also want
to express our gratitude to Dr. F. Lakner for his language and
proofreading help during the manuscript preparation.
a magnetic stirring bar, 100
diluted into 2.4 mL buffer containing (mM): NaCl (145), KCl (5.4),
MgSO4 (0.8), CaCl2 (1.8), HEPES (30), -glucose (11.2). The fluores-
cence signal was allowed to stabilize for 20e30 s before addition of
a test compound or vehicle to assess potential agonistic activity of
mL aliquots of the loaded cells were
References
[1] T.A. Branchek, T.P. Blackburn, 5-ht6 receptors as emerging targets for drug
discovery, Annu. Rev. Pharmacol. Toxicol. 40 (2000) 319e334.
D
[2] J. Holenz, P.J. Pauwels, J.L. Diaz, R. Merce, X. Codony, H. Buschmann, Medicinal
chemistry strategies to 5-HT6 receptor ligands as potential cognitive
enhancers and antiobesity agents, Drug Discov. Today 11 (2006) 283e299.
[3] D.J. Heal, S.L. Smith, A. Fisas, X. Codony, H. Buschmann, Selective 5-HT6
receptor ligands: progress in the development of a novel pharmacological
approach to the treatment of obesity and related metabolic disorders, Phar-
macol. Ther. 117 (2008) 207e231.
the compounds, with subsequent addition of serotonin (2.5
mL,
10 mM) to assess the compounds’ blocking activity.
5.3.1.2. 5-HT6 receptor functional assay. The 5-HT6R was sub-cloned
into T-Rex system (Invitrogen, Carlsbad, CA) and expressed into HEK
(5-HT6R-HEK) cells. The cells were grown in DMEM supplemented
with 10% FBS, 1%AAS, blasticidine S, and zeocin (all from Invitrogen,
Carlsbad, CA) in a T-175 cell culture flask. T-Rex/5-HT6 receptor
expression was activated by addition of tetracycline (1 ug/mL), as
recommended by the T-Rex system manufacturer (Invitrogen,
Carlsbad, CA), a day before the experiments. On the day of the
experiment, the cells were harvested from the flask using 6 mM
EDTA/HBSS solution, gently triturated by passing through a pipette
tip several times to break down cell aggregates, washed with Serum
Free Medium, and counted. The cells were re-suspended to
0.67 ꢃ 106 cells/mL in SB2 buffer, HBSS, supplemented with 5 mM
HEPES, pH 7.4, 0.05% BSA, and 1 mM IBMX (SigmaeAldrich, St. Louis,
MO) containing Alexa Fluor 647-anti cAMP antibody (from LANCE
cAMP 384 kit, PerkinElmer, Waltham, MA). 6 uL (w4000 cells/well)
aliquots were then transferred into 384-well assay plates (Perki-
nElmer White OptiPlates). The test compounds at different concen-
trations were premixed with serotonin hydrochloride (Sigma, MO)
and added to the cells (final serotonin concentration 10 nM, final
[4] M.L. Woolley, C.A. Marsden, K.C. Fone, 5-HT6 receptors, Curr. Drug Targets
CNS Neurol. Disord. 3 (2004) 59e79.
[5] http://clinicaltrials.gov/ct2/show/NCT00348192.
[6] http://www.biospace.com/news_story.aspx?NewsEntityId¼141148.
[7] S. Tkachenko, A. Ivachtchenko, A. Khvat, I. Okun, Y. Lavrovsky, R. Salimov,
Discovery and preclinical Studies of AVN-101, a novel “balanced” molecule for
treatment of Alzheimer disease (AD), in: 9th Intern. conf. AD/PD 2009, Prague,
Czech Republic, Abstracts, 2009, p. 945.
[8] A.V. Ivachtchenko, D.E. Dmitriev, E.S. Golovina, E.S. Dubrovskaya,
M.G. Kadieva, A.G. Koryakova, V.M. Kysil, O.D. Mitkin, S.E. Tkachenko,
I.M. Okun, A.A. Vorobiov, Synthesis of cycloalkane-annelated 3-phenyl-
sulfonyl-pyrazolo[1,5-a]pyrimidines and their evaluation as 5-HT6 receptor
antagonists, Bioorg. Med. Chem. Lett. 20 (2010) 2133e2136.
[9] A.V. Ivachtchenko, D.E. Dmitriev, E.S. Golovina, M.G. Kadieva, A.G. Koryakova,
V.M. Kysil, O.D. Mitkin, I.M. Okun, S.E. Tkachenko, A.A. Vorobiev, (3-Phenyl-
sulfonyl-sycloalkano[e and d]pyrazolo[1,5-a]pyrimidin-2-yl)-amines: potent
and selective 5-HT6 receptor antagonists, J. Med. Chem. 53 (2010)
5186e5196.
[10] M.L. López-Rodríguez, B. Benhamu, T. de la Fuente, A. Sanz, L. Pardo, M.A. Campillo,
Three-Dimensional pharmacophore model for 5-Hydroxytryptamine6 (5-HT6)
receptor antagonists, J. Med. Chem. 48 (2005) 4216e4219.
[11] A.V. Ivachtchenko, O.D. Mitkin, I.M. Okun, S.E. Tkachenko, V.M. Kysil, 8-
Sulfonyl-substituted tetrahydro-1H-pyrido[4,3-b]indoles as 5-HT6 receptor
antagonists, Eur. J. Med. Chem. 45 (2010) 782e789.
[12] M. Bous, C. Riemer, H. Stadler, Pyrazolopyrimidines and Pyrazolotriazines with
5-HT6 Receptor Affinity, EP 0941994 B1 (2003).
[13] M. Augustin, R. Schmidt, W.-D. Rudors, Synthese und Reaktivitat von Phe-
nylsulfonyl-cyan-keten-S, S-acetalen, Z. Chem. 17 (1977) 289e290.
[14] http://www.molinspiration.com.
DMSO concentration 0.32%, final IBMX concentration 500 mM). Each
assay plate contained serotonin and cAMP standard concentration
curves. After 2 h of incubation with the mixture of compound/

A.V. Ivachtchenko et al. / European Journal of Medicinal Chemistry 46 (2011) 1189e1197
1197
[15] Accelrys Software Inc. http://www.accelrys.com.
potency, selectivity, and rat pharmacokinetic properties, Bioorg. Med. Chem.
Lett. 19 (2009) 2206e2210.
[19] M.C. Sanguinetti, M. Tristani-Firouzi, hERG Potassium channels and cardiac
arrhythmia, Nature 440 (2006) 463e469.
[20] I. Okun, S. Tkachenko, A. Khvat, O. Mitkin, V. Kazey, A. Ivachtchenko, From
anti-allergic to anti-Alzheimer’s: molecular pharmacology of Dimebon, Curr.
Alzheimer Res. 7 (2010) 97e112.
[21] Y. Cheng, W.H. Prusoff, Relationship between the inhibition constant (K1) and
the concentration of inhibitor which causes 50 per cent inhibition (I50) of an
enzymatic reaction, Biochem. Pharmacol. 22 (1973) 3099e3108.
[16] R.B. Rothman, M.H. Baumann, J.E. Savage, L. Rauser, A. McBride, S.J. Hufeisen,
B.L. Roth, Evidence for possible Involvement of 5-HT2B receptors in the cardiac
valvulopathy associated with fenfluramine and other serotonergic medica-
tions, Circulation 102 (2000) 2836e2841.
[17] K.G. Shyu, Serotonin 5-HT2B receptor in cardiac fibroblast contributes to cardiac
hypertrophy:anewtherapeutictargetforheartfailure?Circ. Res. 104 (2009)1e3.
[18] N. Moss, Y. Choi, D. Cogan, A. Flegg, A. Kahrs, P. Loke, O. Meyn, R. Nagaraja,
S. Napier, A. Parker, T.J. Peterson, P. Ramsden, C. Sarko, D. Skow, J. Tomlinson,
H. Tye, M. Whitaker, A new class of 5-HT2B antagonists possesses favorable