Antagonists of 5-HT6 receptors. Substituted 3-(phenylsulfonyl) pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines and 3-(phenylsulfonyl)pyrazolo[1,5-a] pyrido[4,3-d]pyrimidines - Synthesis and 'structure-activity' relationship

Article abstract of DOI:10.1016/j.bmcl.2012.05.036

Synthesis and biological evaluation of a new series of structurally unrestricted and intramolecular hydrogen bond restricted derivatives of 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines (angular tricyclics) and 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[4,3-d]pyrimidines (linear tricyclics) are described. Structurally restricted derivatives are highly potent and selective blockers of 5-HT₆ receptors with little difference between angular or linear shape of the tricyclic core, the angular species being only slightly more potent. The angular representative of 3-(phenylsulfonyl) pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines, 5, can be considered as more favorable candidate for further development as it shows only weak 5-HT_2B blocking activity (IC₅₀ = 6.16 μM as compared with IC₅₀ = 1.8 nM for 5-HT₆ receptors) and very low hERG potassium channel blocking potency (IC₅₀ = 54.2 μM). The linear analog, 11, is less favorable as while showing no binding to the 5-HT_2B receptor at concentrations of up to 10 μM, it exhibits quite a high potency to block the hERG channel (IC₅₀ = 0.5 μM).

Full text of DOI:10.1016/j.bmcl.2012.05.036

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4273–4280
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antagonists of 5-HT6 receptors. Substituted 3-(phenylsulfonyl)
pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines and 3-(phenylsulfonyl)
pyrazolo[1,5-a]pyrido[4,3-d]pyrimidines—Synthesis
and ‘structure–activity’ relationship
Alexandre V. Ivachtchenko a,b, Elena S. Golovina a, Madina G. Kadieva a, Volodymyr M. Kysil b,
Oleg D. Mitkin a, Anton A. Vorobiev a, Ilya Okun b,
⇑
a Chemical Diversity Research Institute, 141401 Khimki, Moscow Reg, Russia
b ChemDiv, Inc., 6605 Nancy Ridge Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and biological evaluation of a new series of structurally unrestricted and intramolecular hydro-
gen bond restricted derivatives of 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines (angular
tricyclics) and 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[4,3-d]pyrimidines (linear tricyclics) are
described. Structurally restricted derivatives are highly potent and selective blockers of 5-HT6 receptors
with little difference between angular or linear shape of the tricyclic core, the angular species being only
slightly more potent. The angular representative of 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]
pyrimidines, 5, can be considered as more favorable candidate for further development as it shows only
Received 12 March 2012
Revised 4 May 2012
Accepted 8 May 2012
Available online 17 May 2012
Keywords:
Serotonin receptor 5-HT6
Antagonists
Phenylsulfonyl-pyrazolo-pyrido-
pyrimidines
Specificity
weak 5-HT2B blocking activity (IC50 = 6.16
very low hERG potassium channel blocking potency (IC50 = 54.2
able as while showing no binding to the 5-HT2B receptor at concentrations of up to 10
quite a high potency to block the hERG channel (IC50 = 0.5 M).
l
M as compared with IC50 = 1.8 nM for 5-HT6 receptors) and
M). The linear analog, 11, is less favor-
M, it exhibits
l
l
l
hERG channel
Ó 2012 Elsevier Ltd. All rights reserved.
Serotonin receptors of 5-HT6 subtype (5-HT6R) are attractive
targets for development of new medicines to treat different dis-
eases and impairments of the central nervous system (CNS). The
impressive number of publications in scientific journals as well
as a growing number of patents covering new inhibitors of the
5-HT6R1–4 reflects the high interest in the research and pharma-
ceutical industry communities.
Currently, several 5-HT6R antagonists, SAM-531 (PF-5212365,
Pfizer),5,6 SGS-518 (Lu-AE-58054, Lundbeck, Lilly)7–9 and
AVN-322 (Avineuro Pharm.),10–13 are in different phases of clinical
trials for treatment of Alzheimer’s disease. Another antagonist,
AVN-211 (Avineuro Pharm.) has successfully finished phase IIa
clinical trials to treat schizophrenia.14–16
Earlier, we studied the synthesis and SAR of 5-HT6R ligands
embodied by a 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrimidine tem-
plate.17–20 In the current work, we describe the synthesis and evalu-
ation of 3-(phenylsulfonyl)-6,7,8,9-tetrahydropyrozolo[1,5-a]pyrido
[3,4-e]pyrimidines 1–6 and 3-(phenylsulfonyl)-5,6,7,8-tetrahydro-
pyrozolo[1,5-a]pyrido[4,3-d]pyrimidines 7–12 as potent and selec-
tive 5-HT6R antagonists.
⇑
Corresponding author. Tel.: +1 858 794 4860; fax: +1 858 794 4931.
E-mail address: iokun@chemdiv.com (I. Okun).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.036

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A. V. Ivachtchenko et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4273–4280
Scheme 1. Synthesis of compounds 1 and 7.21
Scheme 2. Synthesis of compounds 2 and 8.22
Scheme 3. Synthesis of compounds 3 and 9.23
Scheme 4. Synthesis of compounds 4 and 10, 5 and 11.24

A. V. Ivachtchenko et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4273–4280
4275
Scheme 5. Synthesis of compounds 6 and 12.25
The reaction of 2H-pyrazol-3-ylamines (13, 19, 21, 26) with so-
dium enolate of either 1-Boc-4-oxopiperidine-3-carbaldehyde (14)
or 3-acetyl-1-Boc-4-oxopiperidine (29) or 3-[1-hydroxy-meth-(E)-
ylidene]-1-methylpiperidin-4-one (20) in AcOH, yields mixtures of
angular (1–6) and corresponding linear (7–12) tricyclic isomers in
ratios ranging from 1:1 to 1:3.
Similarly, a mixture of 7-methyl-2-(methylthio)-3-(phenylsul-
fonyl)-6,7,8,9-tetrahydropyrazolo[1,5-a]pyrido[3,4-e]pyrimidine 2
and 7-methyl-2-(methylthio)-3-(phenylsulfonyl)-5,6,7,8-tetrahyd-
ropyrazolo[1,5-a]pyrido[4,3-d]pyrimidine 8 was obtained in the
reaction of 4-(phenylsulfonyl)-5-(methylthio)-2H-pyrazol-3-yla-
mine 19 with sodium enolate of 1-methyl-4-oxopiperidine-3-carb-
aldehyde 20 (Scheme 2). The compounds 2 and 8 were then
separated using HPLC.
3-(4-Fluorophenylsulfonyl)-7-methyl-2-(methylthio)-6,7,8,9-
tetr-ahydropyrazolo[1,5-a]pyrido[3,4-e]pyrimidine
3 and 3-(4-
fluorophenylsulfonyl)-7-methyl-2-(methylthio)-5,6,7,8-tetrahyd-
ropyrazolo[1,5-a]pyrido[4,3-d]pyrimidine 9 were obtained upon
reaction of 4-(4-fluorophenylsulfonyl)-5-(methylthio)-2H-pyra-
zol-3-ylamine 21 with sodium enolate of 1-Boc-4-oxopiperidine-
3-carbaldehyde 14. The synthetic conditions were analogous to
the ones used for synthesis of 1 and 7, also without separation of
the interim mixtures of isomers 22, 23 and 24, 25 (Scheme 3).
N-Methyl-3-(phenylsulfonyl)-6,7,8,9-tetrahydropyrazolo[1,5-a]
pyrido[3,4-e]pyrimidin-2-amine 4 and N-methyl-3-(phenylsulfonyl)-
5,6,7,8-tetrahydropyrazolo[1,5-a]pyrido[4,3-d]pyrimidin-2-amine 10,
as well as N,7-dimethyl-3-(phenylsulfonyl)-6,7,8,9-tetrahydropy-
razolo[1,5-a]pyrido[3,4-e]pyrimidin-2-amine 5 and N,7-dimethyl-
3-(phenylsulfonyl)-5,6,7,8-tetrahydropyrazolo[1,5-a]pyrido[4,3-d]
pyrimidin-2-amine 11 were obtained in the reaction of N3-methyl-
4-(phenylsulfonyl)-1H-pyrazole-3,5-diamine 26 with sodium eno-
late of 1-Boc-4-oxopiperidine-3-carbaldehyde 14, similar to the
synthesis of compounds 1 and 7 without separation of intermediate
isomers 27 and 28 (Scheme 4).
N,5,7-Trimethyl-3-(phenylsulfonyl)-6,7,8,9-tetrahydro-pyrazol-
o[1,5-a]pyrido[3,4-e]pyrimidine-2-amine 6 and N,7,9-trimethyl-
3-(phenylsulfonyl)-5,6,7,8-tetrahydropyrazolo[1,5-a]pyrido[4,3-d]
pyrimidine-2-amine 12 were obtained by employing the same se-
quence, but starting with N3-methyl-4-(phenylsulfonyl)-1H-pyra-
zole-3,5-diamine 26 with 3-acetyl-1-Boc-4-oxopiperidine 29 in
AcOH. The Boc group was removed without separation of isomers
34 and 35 and finally, 36 and 37 were transformed using condi-
tions of reductive amination, into a mixture of 6 and 12 (Scheme
5). The final compounds 6 and 12 were the separated by HPLC.
Structures of the synthesized compounds were confirmed with
LC/MS, HRMS, 1H and 13C NMR spectroscopy and by X-ray analysis
(see Experimental section).
Scheme 1 demonstrates the reaction of 4-(phenylsulfonyl)-5-
methyl-2H-pyrazol-3-ylamine 13 with sodium enolate of 1-Boc-
4-oxopiperidine-3-carbaldehyde 14 yielding a mixture of isomers
15 and 16, which were difficult to separate. The Boc group was
then removed from the compounds in the mixture, which led to
formation of a likewise inseparable mixture of isomers 17 and
18. Reductive amination of the mixture with formaldehyde led to
a mixture of isomers 1 and 7, which then were separated by HPLC
(Shimadzu LC-8A) using 50 mm  20 mm Reprosil-Pur-18-AQ
10
10
lm
pre-column and 250 mm  20 mm Reprosil-Pur-18-AQ
lm column at a flow rate 25 mL/min in a gradient mode with
mobile phase MeCN/water + 0.05% CF3COOH.
Conceptually, the compounds 1–3 and 7–9 correspond to a
pharmacophore model PhM1 while the compounds 4–6 and 10–
12 can be described by the pharmacophore model PhM2 both
shown in Figure 1.
The main difference between the two models is that in accor-
dance with PhM2, the hydrogen bond donor (HBD) represented
in these series by the methylamino group, forms an intramolecular
hydrogen bond with a hydrogen bond acceptor (HBA) represented
by the sulfonyl group.
Figure 1. Conceptual pharmacophore models, PhM1 (adapted from Holenz et al.32
and Lo´pez-Rodrigues et al.33) and PhM2.20 The HYD1 and HYD2 groups (circles)
represent aromatic and/or heterocyclic hydrophobic moieties. HBA (square) is a
hydrogen bond acceptor33 or double electron acceptor21 group, represented by
either a sulfonamide or sulfonyl group. HBD (round cornered square) is a hydrogen
bond donor capable of forming intramolecular hydrogen bond (dotted line) with the
HBA.20 PI (oval) is either a positively ionizable atom33 or proton donor,32 mainly
represented by an amine group.

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A. V. Ivachtchenko et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4273–4280
Figure 2. 3D X-ray structures of isomers 5 (CCDC 880442) and 11 (CCDC 880441) were plotted with software package Mercury 3.0 (CCDC 880441 and CCDC 880442 contain the
supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif).
Table 1
Functional potencies (Ki antagonism) and binding affinities (Ki binding) of 3-(phenylsulfonyl)-6,7,8,9-tetrahydropyrazolo[1,5-a]pyrido[3,4-e]pyrimidines
3-(phenylsulfonyl)-5,6,7,8-tetrahydropyrazolo[1,5-a]pyrido[4,3-d]pyrimidines 7–12
1
– 6 and
R1
R2
R3
R4
5-HT6R, Kia (nM)
IC50
(lM)
a
Compds
Tricyclic shape
Bindingb
Antagonismc
5-HT2B
hERGe
54.2
d
1
2
3
4
5
6
7
8
Angular
Angular
Angular
Angular
Angular
Angular
Linear
Linear
Linear
Linear
Linear
Me
SMe
SMe
NHMe
NHMe
NHMe
Me
SMe
SMe
H
H
F
H
H
H
H
H
F
Me
Me
Me
H
Me
Me
Me
Me
Me
H
H
H
H
H
H
Me
H
H
F
60.4
7.7
35.7
14.8
1.9
1.8
0.39
6.16
0.68
>10,000
372
492
1155
2.1
9
10
11
12
NHMe
NHMe
NHMe
H
H
H
H
H
Me
Me
Me
0.82
0.66
14.3
NA
0.5
Linear
NA—not active.
a
Values are geometric means of three independent experiments. Variations of Ki and IC50 values were less than twofold from the mean values. Ki values were calculated as
indicated.26
b
c
Radioligand competitive binding with 5-HT6R was performed by Ricerca Biosciences (Taiwan) as described.27
5-HT6 receptor functional activity was determined as described.28
5-HT2B receptor functional activity was determined as described.29
d
e
hERG functional activity was determined as described.30
X-ray analysis (Fig. 2) confirms formation of the intramolecular
methylthio R1-substituted compounds, 2 and 8 (PhM1), exhibit
hydrogen bond between methylamine and sulfonyl moieties of
both the angular 5 and linear 11 isomers (Fig. 2).
substantially lower affinities than those of their structurally re-
stricted methylamino analogs, 5 and 11 (PhM2). Thus, in the
group of angular tricyclic 5-HT6R ligands, a 20-fold increase in
the Ki (binding) value is observed upon transition from 5 to 2.
In the group of linear tricyclic compounds, such transition from
methylamino, 11, to methylthio-substituted compound, 8, led to
even higher, 450-fold increase in the binding Ki. Methyl substitu-
tion in the R1 position, led to further decrease in the affinity
(compare 1 with 2 and 7 with 8). Addition of a methyl group in
the R4 position has no substantial effect on the binding affinity
(compare Ki values for 5 and 6 in angular and 11 and 12 in linear
tricyclic groups).
The ligand affinities to the receptors were measured by concen-
tration-dependent competition with radiolabled ligand.27 Poten-
cies of the compounds 1–12 to block serotonin-induced
responses were investigated in a cell-based assay using HEK 293
cells stably expressing recombinant human 5-HT6 receptor.28 The
inhibition constants, Ki, were calculated from IC50 values26 using
the Cheng–Prusoff equation.34 The data are shown in Table 1.
Among both series of the derivatives, angular and linear, the
highest affinity is observed for compounds 4–6 and 10–12, irre-
spective of the tricyclic shape (PhM2). These compounds are
capable of forming intramolecular hydrogen bonds between the
methylamino group (R1) and sulfonyl group (Fig. 2). The
To determine the functional consequences of the ligand interac-
tion with the 5-HT6R in cells, we tested selected compounds in a

A. V. Ivachtchenko et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4273–4280
4277
Figure 3. Specificity profiles of (A) angular 5 and (B) linear 11 antagonists of 5-HT6R. Shown are percentages ( SD) of a radio labeled ligand displacements from
corresponding targets at the antagonist concentration of 1
M. Measurements were performed by Ricerca Biosciences (Taiwan) in duplicates.31
l
functional cell-based assay by their ability to either stimulate or
block serotonin-induced accumulation of intracellular cAMP in
cells exogenously expressing 5-HT6R.28 All of the tested
compounds showed antagonistic activity with potencies (Ki antag-
onism) roughly corresponding to their affinities (Ki binding) mea-
sured in the equilibrium radioligand binding assay27 (Table 1).

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A. V. Ivachtchenko et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4273–4280
sudden death. The data are shown in Figure 5. Remarkably, while
exhibiting practically equal affinities to 5-HT6R (Table 1), the angu-
lar 5 and linear 11 methylamino-substituted compounds showed a
starkly different ability to inhibit human hERG potassium channel,
5 (IC50 = 54.2
lM) being 100-fold less potent than its analog 11
(IC50 = 0.5 M). In this regard, while both compounds block the
l
hERG channel at much higher concentrations than those necessary
to block 5-HT6R, 5 as the 5-HT6R antagonist, can be considered
substantially safer than 11. The effective concentration of 5 to
block hERG is four orders of magnitude higher than that to block
5-HT6R. 11 exhibits the ‘safety window’ of only two orders of mag-
nitude between the 5-HT6R and hERG channel efficacy.
In the conclusion, we have shown that structurally restricted
derivatives are highly potent and selective blockers of 5-HT6 recep-
tors with little difference between angular or linear shape of the
tricyclic core. The angular species though not substantially, are
slightly more potent than their linear analogs. The most potent
angular representative of 3-(phenylsulfonyl)pyrazolo[1,5-a]pyr-
ido[3,4-e]pyrimidines, 5 (IC50 = 1.8 nM, Table 1), can be considered
as more favorable candidate for further development as a drug can-
didate for treatment of such mental disorders as Alzheimer’s dis-
ease and schizophrenia. 5 is highly potent and very selective
against a panel of 68 other therapeutic targets. Though it shows
a weak interaction with 5-HT2B receptor (Fig. 3A), functionally, it
Fig. 4. Concentration-dependent inhibition of agonist-induced [Ca2+]i mobilization
in HEK-293 cells expressing h5-HT2B receptor with 5. Intracellular concentration of
free calcium ions was measured using calcium sensitive dye Fluoro-4. The calcium
ion concentration spike in the presence of different concentrations of 5 is expressed
as a percentage of the spike induced by 5 nM a
Me-5-HT.29
is an antagonist with low potency (IC50 = 6.16
is not associated with any therapeutic liability. 5 also exhibits very
low hERG potassium channel blocking potency (IC50 = 54.2 M,
lM, Fig. 4), which
l
Fig. 5). Compound 11, the linear analog of 5, while showing better
specificity (no binding to the 5-HT2B receptor at concentrations of
up to 10
magnitude less potent to block 5-HT6R (14.3 nM, Table 1) and
exhibits quite high potency to block the hERG channel
(IC50 = 0.5 M), which could potentially be associated with QT
lM, data not shown) is less favorable as it is one order of
a
l
Fig. 5. Blockade of hERG potassium channel in patch clamp assay with angular (5)
and linear (11) 5-HT6R antagonists.30
elongation syndrome.
Supplementary data
Representative angular and linear tricyclic 5-HT6R antagonists,
5 and 11, were evaluated on a panel consisting of 69 related (other
serotonergic) and unrelated (non-serotonergic) therapeutic
targets, by their ability to compete with corresponding radio-la-
beled ligands31 (Fig. 3).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05.
036. These data include MOL files and InChiKeys of the most
important compounds described in this article.
The data in Figure 3 shows that both the angular 5 and linear 11
analogs are highly selective and specific 5-HT6R ligands. 11 dis-
plays no substantial interaction with all but 5-HT6R tested targets.
compound 5 exhibits a low affinity competition with the radioli-
gand for the 5-HT2B receptor (ꢀ50% [3H]lysergic acid diethylamide
References and notes
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2. Pharmacology of 5-HT6 receptors, Part I, (International Review of Neurobiology);
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3. Pharmacology of 5-HT6 receptors, Part II, (International Review of Neurobiology);
Borsini, F., Ed.; Academic Press, 2010; Vol. 96, p 242.
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2010, 20, 1247.
5. Macauley, D. Abstracts of American Chemical Society. 239th national meeting-
Investigating new therapeutic candidates: part 1. 21-25 March 2010, San
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gov Web Site 2009
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(June 6–10, Hong Kong) 2010, P-04.003.
10. http://www.avineuro.com/avineuro-pharmaceuticals-inc-reports-positive-
phase-i-clinical-trial-results-on-avn-322-potent-small-molecule-for-treat-
ment-of-alzheimer%e2%80%99s-disease/.
11. Tkachenko, S.; Ivachtchenko, A.; Khvat, A.; Okun, I. Abstracts of 9th Int Conf.
Alzheimer Parkinson Dis. (AD/PD), (March 11–15, Prague) 2009.
12. Ivachtchenko, A. V.; Kysil V. M., Ivachtchenko, A. A.; Savchuk, N. P. RU Patent
239134.
13. Ivachtchenko, A. V.; Savchuk, N. P.; Ivachtchenko, A. A. WO Patent, 136814,
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14. http://www.avineuro.com/avineuro-pharmaceuticals-inc-reports-positive-phase-
2a-clinical-proof-of-concept-trial-results-on-avn-211-potent-small-molecule-for-
treatment-of-schizophrenia.
displacement at 1 l
M). It has been shown35 that activation of 5-
HT2BR with fenfluramine and other serotonergic medications can
lead to valvular heart disease and hence the agonistic activity
could represent a potential liability. On the other hand, antagonists
of the receptor are being tested for treatment of chronic heart dis-
ease.36,37 Therefore, it was important to clarify if the interaction of
5 with the 5-HT2BR as measured in the radio-ligand binding assay,
exhibits an agonistic or antagonistic mode of action. We studied
the effect of
5 on 5-HT2B-induced calcium mobilization in
HEK293 cells with stably expressed human recombinant 5-HT2B
receptor.29 When added directly to the cells, 5 caused no effect
on the intracellular free calcium concentration but concentra-
tion-dependently suppressed the calcium spike induced by subse-
quent addition of the agonist, aMe-5-HT, with IC50 of 6 lM (Fig. 4).
Thus, 5 is unlikely to cause problematic effect through the interac-
tion with the 5-HT2B receptor.
We have also tested 5 and 11 in cell-based patch clamp exper-
iments30 to assess their ability to functionally block exogenously
expressed hERG (human Ether-à-go-go-Related Gene) potassium
channel associated with a long QT syndrome, which can lead to

A. V. Ivachtchenko et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4273–4280
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2009.
17. Ivachtchenko, A. V.; Dmitriev, D. D.; Golovina, E. S.; Dubrovskaya, E. S.; Kadieva,
M. G.; Koryakova, A. G.; Kysil, V. M.; Mitkin, O. D.; Tkachenko, S. E.; Okun, I. M.;
Vorobiov, A. A. Bioorg. Med. Chem. Lett. 2010, 20, 2133.
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A. G.; Kysil, V. M.; Mitkin, O. D.; Okun, I. M.; Tkachenko, S. E.; Vorobiov, A. A. J.
Med. Chem. 2010, 53, 5186.
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D.; Tkachenko, S. E.; Kysil, V. M.; Okun, I. M. Eur. J. Med. Chem. 2011, 46, 1189.
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3.45 (t, J = 6.1 Hz, 2H); 3.22 (t, J = 6.6 Hz, 2H); 2.90 (s, 3H). 13C NMR (DMSO-d6,
75 MHz) d 157.79, 149.35, 146.20, 143.57, 140.72, 132.90, 129.17, 125.58,
111.79, 91.24, 40.04, 38.97, 29.01, 21.08. HRMS calculated for C16H17N5O2S
(M+H) 344.11812, found 344.1179. MS-ESI calculated for C16H17N5O2S (M+H)
344, found m/z 344. LC–MS (UV-254) purity: 99%. Compound 10: 1H NMR
(DMSO-d6): 9.68 (br.s, 2H); 8.95 (s, 1H); 7.98 (d, J = 8.0 Hz, 2H); 7.47–7.63 (m,
3H); 6.49 (br.s, 1H); 4.22 (s, 2H); 3.45 (t, J = 6.1 Hz, 2H); 3.13 (t, J = 6.6 Hz, 2H);
2.89 (s, 3H). 13C NMR (DMSO-d6, 75 MHz) d 158,35, 156,07, 145,98, 143,61,
133,23, 132,87, 129,15, 125,61, 112,07, 89,73, 41,07, 40,20, 28,97, 28,53. HRMS
calcd for C16H17N5O2S (M+H) 344.11812, found 344.1183. MS-ESI calculated
for C16H17N5O2S (M+H) 344, found m/z 344. LC–MS (UV-254) purity: 98%.
Compound 5: 1H NMR (400 MHz, DMSO-d6), d: 11.73 (br.s, 1H), 8.44 (s, 1H),
8.00 (m, 2H), 7.61 (m, 1H), 7.56 (m, 2H), 6.54 (q, J = 4.8 Hz, 1H), 4.37 (br.m, 2H),
3.55 (br.m, 2H), 3.36 (br. m, 2H), 2.94 (d, J = 4.8 Hz, 3H), 2.89 (s, 3H). 13C NMR
21. Synthesis of 2,7-dimethyl-3-(phenylsulfonyl)-6,7,8,9-tetrahydropyrazolo[1,5-
a]pyrido[3,4-e]pyrimidine
1
and 2,7-dimethyl-3-(phenylsulfonyl)-5,6,7,8-
(D2O, 75 MHz) d 158.08, 148.89, 146.34, 141.33, 140.26, 133.28, 128.94,
tetrahydropyrazolo[1,5-a]pyrido[4,3-d]pyrimidine 7. A mixture of 3-methyl-
4-(phenylsulfonyl)-1H-pyrazole-5-amine 13 (1 mMol), sodium enolate of 1-
Boc-4-oxopiperidine-3-carbaldehyde 14 (1.4 mMol) and AcOH (3 mL) was
stirred at 80–90 °C for 3 h (or at ambient temperature overnight), precipitate
(mixture of 15 and 16) was filtered, washed with i-PrOH, dried, and dissolved
in CHCl3. This solution was treated with excess of 6 M HCl in EtOAc, stirred at
ambient temperature for 1 h, and treated with Et2O. The precipitate was
separated and washed sequentially with Et2O, EtOAc, and then EtOH. Once
dried, analysis revealed a mixture of isomers 17ÁHCl and 18ÁHCl. The free base
was obtained by treating an aqueous solution with 2 N NaOH, filtering the
precipitate, washing with water, and re-crystallizing from i-PrOH. The solid
mixture (89 mg) of isomers 17 and 18 was mixed with NaBH(OAc)3 (200 mg),
CHCl3 (4 mL), and 40% aq CH2O (0.4 mL), stirred at 50 oC for 0.5 h, cooled,
washed with 10% aq NaOH, water, dried, and concentrated. The obtained
mixture of isomers 1 and 7 was purified by silica gel flash chromatography and
separated by HPLC into individual isomers 1 and 7. The compounds 1 and 7
were dissolved in CHCl3 (2 mL) and treated with 6 M solution of AcCl in EtOH
(0.5 mL). The compounds were concentrated, filtered off, washed twice with i-
PrOH, hexane, and dried. Compound 1: 1H NMR (400 MHz, CDCl3), d: 8.45 (s,
1H); 8.12 (d, J = 8.0 Hz, 2H); 7.40–7.53 (m, 3H); 3.62 (s, 2H); 3.22 (t, J = 5.9 Hz,
2H); 2.83 (t, J = 5.9 Hz, 2H); 2.73 (s, 3H); 2.52 (s, 3H). MS-ESI calculated for
124.81, 110.50, 89.81, 50.10, 48.97, 42.02, 28.14, 21.24. 13C NMR (DMSO-d6, 75
MHz) d 157.99 (2-C), 149.25 (5-C), 146.48 (3a-C), 143.69 (ipso-Ph), 140.20 (9a-
C), 133.05 (g-Ph), 129.29 (v-Ph), 125.74 (o-Ph), 111.11 (5a-C), 91.53 (3-C),
49.47 (6-C), 48.35 (8-C), 41.30 (2-NHCH3), 29.14 (7-NCH3), 21.35 (9-C). HRMS
calcd for C17H19N5O2S (M+H) 358.13377, found 358.1336. MS-ESI calcd for
C17H19N5O2S (M+H) 358, found m/z 358. LC–MS (UV-254) purity: 99%.
Compound 11: 1H NMR (400 MHz, DMSO-d6), d: 11.60 (br.s, 1H); 8.97 (s,
1H); 8.00 (d, J = 8.0 Hz, 2H); 7.50–7.62 (m, 3H); 6.51 (br.q, J = 4.7 Hz, 1H); 4.35–
4.49 (br.m, 1H); 4.16–4.30 (br.m, 1H); 3.62–3.76 (br.m, 1H); 3.40–3.52 (br.m,
1H); 2.90 (s, 3H); 2.86 (d, J = 4.7 Hz, 3H). 13C NMR (DMSO-d6, 75 MHz) d 158.58,
155.39, 146.28, 143.72, 133.33, 132.99, 129.28, 125.76, 111.62, 89.96, 50.39,
49.69, 41.56, 29.11, 28.67. HRMS calcd for C17H19N5O2S (M+H) 358.13377,
found 358.1335. MS-ESI calculated for C17H19N5O2S (M+H) 358, found m/z 358.
LC–MS (UV-254) purity: 98%.
25. Synthesis
of
N,5,7-trimethyl-3-(phenylsulfonyl)-6,7,8,9-
and N,7,9-
tetrahydropyrazolo[1,5-a]pyrido[3,4-e]pyrimidine-2-amine
6
trimethyl-3-(phenylsulfonyl)-5,6,7,8-tetrahydro-pyrazolo[1,5-a]pyrido[4,3-
d]pyrimidine-2-amine 12. Reactions were performed similar to those
providing
1
and 7, but starting with N3-methyl-4-(phenylsulfonyl)-1H-
pyrazole-3,5-diamine 26 and 3-acetyl-1-Boc-4-oxopiperidine 29. Compound
6: 1H NMR (400 MHz, CDCl3), d: 8.12 (d, J = 8.4 Hz, 2H); 7.40–7.50 (m, 3H); 5.96
(br. q, J = 5.1 Hz); 3.44 (c, 2H); 3.11 (t, J = 6.0 Hz, 2H); 3.01 (d, J = 5.1 Hz, 3H);
2.76 (d, J = 6.0 Hz, 2H); 2.51 (s, 3H); 2.46 (c, 3H). MS-ESI calculated for
C
17H18N4O2S (M+H) 343, found m/z 343. LC–MS (UV-254) purity: 98%. 7: 1H
NMR (400 MHz, DMSO-d6), d: 8.25 (s, 1H); 8.13 (d, J = 8.0 Hz, 2H); 7.38–7.54
(m, 3H); 3.59 (s, 2H); 3.21 (t, J = 5.9 Hz, 2H); 2.82 (t, J = 5.9 Hz, 2H); 2.69 (s, 3H);
2.48 (s, 3H). 13C NMR (DMSO-d6, 75 MHz) d 155,90, 151,50, 146,42, 142,83,
141,16, 133,49, 129,44, 126,05, 112,58, 106,62, 49,02, 47,90, 41,08, 21,10,
12,87. MS-ESI calculated for C17H18N4O2S (M+H) 343, found m/z 343. LC–MS
(UV-254) purity: 98%.
C18H21N5O2S (M+H) 372, found m/z 372. LC–MS (UV-254) purity: 98%.
26. Curve fitting and statistical analysis: The concentration curves were fitted with
Prism 5 (GraphPad, 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’s34
modified equation, Ki = IC50/(1 + [Ag]/EC50). Where IC50 is the concentration
of antagonist causing 50% inhibition of serotonin-induced cell response; [Ag] is
a concentration of serotonin (10 nM), at which inhibition was measured and
EC50 is the 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-
22. Synthesis of 7-methyl-2-(methylthio)-3-(phenylsulfonyl)-6,7,8,9-tetrahydro-
pyrazolo[1,5-a]pyrido[3,4-e]pyrimidine
2 and 7-methyl-2-(methylthio)-3-
(phenylsulfonyl)-5,6,7,8-tetrahydropyrazolo[1,5-a]pyrido[4,3-d]pyrimidine 8.
Reactions were performed similar to those providing 1 and 7 but starting
with 3-(methylthio)-4-(phenylsulfonyl)-1H-pyrazole-5-amine 19 and sodium
enolate of 1-methyl-4-oxopiperidine-3-carbaldehyde 20. Compound 2: 1H
NMR (400 MHz, DMSO-d6), d: 11.76 (br.s, 1H), 8.69 (s, 1H), 8.01 (m, 2H), 7.64
(m, 1H), 7.58 (m, 2H), 4.60 (br.m, 1H), 4.37 (br.m, 1H), 3.76 (br.m, 1H), 3.47
(br.s, 2H), 3.34 (br.m, 1H), 2.94 (s, 3H), 2.61 (s, 3H). 13C NMR (DMSO-d6,
75 MHz) d 155.90, 151.50, 146.42, 142.83, 141.16, 133.49, 129.44, 126.05,
112.58, 106.62, 49.02, 47.90, 41.08, 21.10, 12.87. MS-ESI calculated for
HT6R-HEK cells was 1.91 0.13 nM as determined from
4 independent
experiments with three to five repeats (separate plates), each in
quadruplicates.
27. Competitive radioligand binding with 5-HT6 receptor: The assays were performed
by Ricerca Biosciences in accordance with their internal protocols. In short, the
membranes of HeLa cells expressing human recombinant 5-HT6R were used to
determine competitive displacement of the radio-labeled [3H]lysergic acid
diethylamide with the tested compounds. The membrane samples were
C
17H18N4O2S2 (M+H) 375, found m/z 375. LC–MS (UV-254) purity: 98%.
Compound 8: 1H NMR (400 MHz, DMSO-d6), d: 11.59 (br.s, 1H), 9.25 (s, 1H),
8.01 (m, 2H), 7.65 (m, 1H), 7.59 (m, 2H), 4.51 (br.m, 1H), 4.34 (br.m, 1H), 3.75
(br.m, 1H), 3.54 (br.m, 1H), 3.35 (br.m, 2H), 2.95 (s, 3H), 2.57 (s, 3H). 13C NMR
(DMSO-d6, 75 MHz) d 157.89, 156.89, 146.41, 143.27, 133.84, 133.17, 129.19,
126.22, 113.36, 105.44, 50.65, 49.83, 41.78, 29.02, 12.95. MS-ESI calculated for
incubated with the mixture of the label (1.5 nM) and
a compound at
specified concentrations for 120 min at 37 °C in the buffer consisting of (in
mM): Tris–HCl (50, pH 7.4), NaCl (150), ascorbic acid (2), BSA (0.001%). After
the incubation, the samples were vacuum-filtered through WhatmanÒ grade
GF/F glass microfiber filters with subsequent 3Â wash with cold media. For
C
17H18N4O2S2 (M+H) 375, found m/z 375. LC–MS (UV-254) purity: 98%.
23. Synthesis of 3-(4-Fluorophenylsulfonyl)-7-methyl-2-(methylthio)-6,7,8,9-
tetrahydropyrazolo[1,5-a]pyrido[3,4-e]pyrimidine hydrochloride 3 and 3-(4-
fluorophenylsulfonyl)-7-methyl-2-(methylthio)-5,6,7,8-
determination of non-specific binding, 5
wells.
lM serotonin was added into control
tetrahydropyrazolo[1,5-a]pyrido[4,3-d]pyrimidine hydrochloride 9. Reactions
were performed similar to those providing 1 and 7 but starting with 4-(4-
fluorophenylsulfonyl)-3-(methylthio)-1H-pyrazole-5-amine 21 and sodium
enolate of 1-Boc-4-oxopiperidine-3-carbaldehyde 14. Compound 3: 1H NMR
(400 MHz, DMSO-d6), d: 11.69 (br.s, 1H), 8.69 (s, 1H), 8.07 (m, 2H), 7.43 (m,
2H), 4.60 (br.m, 1H), 4.38 (br.m, 1H), 3.75 (br.m, 1H), 3.47 (br.s, 2H), 3.29 (br.m,
1H), 2.94 (s, 3H), 2.61 (s, 3H). MS-ESI calculated for C17H17FN4O2S2 (M+H) 393,
found m/z 393. LC–MS (UV-254) purity: 98%. Compound 9: 1H NMR (400 MHz,
DMSO-d6), d: 11.59 (br.s, 1H), 9.26 (s, 1H), 8.07 (m, 2H), 7.44 (m, 2H), 4.52
(br.m, 1H), 4.34 (br.m, 1H), 3.75 (br.m, 1H), 3.53 (br.m, 1H), 3.40 (br.s, 2H), 2.95
(s, 3H), 2.57 (s, 3H). MS-ESI calculated for C17H17FN4O2S2 (M+H) 393, found m/z
393. LC-MS (UV-254) purity: 98%.
28. Determination of 5-HT6 receptor functional activity: 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 lg/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 Â 106 cells/mL in SB2 buffer, HBSS,
supplemented with 5 mM HEPES, pH 7.4, 0.05% BSA, and 1 mM IBMX (Sigma–
Aldrich, St. Louis, MO) containing Alexa Fluor 647-anti cAMP antibody (from
24. Synthesis
of
N-methyl-3-(phenylsulfonyl)-6,7,8,9-tetrahydropyrazolo[1,
5-a]pyrido[3,4-e]pyrimidin-2-amine 4, N-methyl-3-(phenylsulfonyl)-5,6,7,8-
tetrahydropyrazolo[1,5-a]pyrido[4,3-d]pyrimidin-2-amine 10, N,7-dimethyl-
3-(phenylsulfonyl)-6,7,8,9-tetrahydropyrazolo[1,5-a]pyrido[3,4-e]pyrimidin-
2-amine 5, and N,7-dimethyl-3-(phenylsulfonyl)-5,6,7,8-tetrahydropyrazolo
[1,5-a]pyrido[4,3-d]pyrimidin-2-amine 11. Reactions were performed similar
to those providing 1 and 7, but starting with N3-methyl-4-(phenylsulfonyl)-
1H-pyrazole-3,5-diamine 26 and sodium enolate of 1-Boc-4-oxopiperidine-3-
carbaldehyde 14. Compound 4: 1H NMR (DMSO-d6): 9.81 (br.s, 2H); 8.45 (s,
1H); 7.98 (d, J = 8.0 Hz, 2H); 7.45–7.63 (m, 3H); 6.53 (br.s, 1H); 4.28 (s, 2H);
LANCE cAMP 384 kit, PerkinElmer, Waltham, MA). 6
lL (ꢀ4000 cells/well)
aliquots were then transferred into 384-well assay plates (PerkinElmer White
OptiPlates). The test compounds at different concentrations were premixed
with serotonin hydrochloride (Sigma, MO) and added to the cells (final
serotonin concentration-10 nM, final DMSO concentration-0.32%, final IBMX
concentration-500 mM). Each assay plate contained serotonin (to define 100%
activation signal) and cAMP standard calibration curves (to transform
fluorescent signal into cAMP concentrations). After 2 h of incubation with

4280
A. V. Ivachtchenko et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4273–4280
the mixture of compound/serotonin, cells were treated as described in the
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 +20 mV
from a holding potential of À80 mV, and peak tail currents were recorded upon
re-polarization to À50 mV. This voltage-clamp pulse protocol was performed
continuously during the experiment (vehicle control, test compound, washout,
and positive control additions). An inter pulse interval of 15 s allowed recovery
from any residual inactivation. Test compounds were incubated with cells until
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.
29. Determination of 5-HT2B receptor functional activity: 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 dissociated
with TrypLE™ Express (Invitrogen, Carlsbad, CA), washed twice with PBS and
loaded with 4 mM calcium-sensitive dye, Fura-2AM (Invitrogen, Carlsbad, CA)
at room temperature 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 an additional 30 min
with gentle shaking at room temperature. All loading procedures were
performed in the dark. The loaded cells were washed twice with PBS and re-
suspended into the Hybridoma media at a cell density of 3–4 Â 106 cells/mL for
subsequent experiments. Fura-2 ratiometric fluorescence signal was registered
at 510 nm upon alternate excitation at 340 nm and 380 nm using
spectrofluorometer RF5301PC (Shimadzu, Columbia, MD). In a square (1 cm)
optical cuvette with a magnetic stirring bar, 100 mL aliquots of the loaded cells
were diluted into 2.4 mL buffer containing (mM): NaCl (145), KCl (5.4), MgSO4
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 perfusion of extracellular solution for 3 min, followed by
application of positive control (10 lM 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.
31. Target specificity. The assays were performed by Ricerca Biosciences in
accordance with their internally optimized protocols for each target. The
radioligand displacement was assessed in the presence of 1
compounds in duplicates.
lM of the tested
´
32. Holenz, J.; Merce, R.; Dia z, J. L.; Guitart, X.; Codony, X.; Dordal, A.; Romero, G.;
Torrens, A.; Mas, J.; Las Andaluz, B.; Herna´ndez, S.; Monroy, X.; Sa´nchez, E.;
Herna´ndez, E.; Pe´rez, R.; Cubi, R.; Sanfeliu, O.; Buschmann, H. J. Med. Chem.
2005, 48, 1781.
(0.8), CaCl2 (1.8), HEPES (30),
D-glucose (11.2). The fluorescence signal was
33. Lo´pez-Rodrigues, M. L.; Benhamu, B.; De la Fuente, T.; Sanz, A.; Pardo, L.;
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 mL, 10 mM) to assess the compounds blocking
activity.
Campillo, M. J. Med. Chem. 2005, 48, 4216.
34. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
35. Rothman, R. B.; Baumann, M. H.; Savage, J. E.; Rauser, L.; McBride, A.; Hufeisen,
S. J.; Roth, B. L. Circulation 2000, 102, 2836.
30. Determination of hERG channel functional activity: The experiments were
performed by AVIVA Biosciences (San Diego, CA). Human recombinant hERG
channel was stably expressed in HEK-297 cells. Intracellular solution consisted
of 130 mM KCl, 5 mM EGTA, 5 mM MgCl2, 10 mM HEPES, 5 mM Na-ATP, pH 7.2.
36. Shyu, K. G. Circ. Res. 2009, 104, 1.
37. Moss, N.; Choi, Y.; Cogan, D.; Flegg, A.; Kahrs, A.; Loke, P.; Meyn, O.; Nagaraja,
R.; Napier, S.; Parker, A.; Peterson, T., Jr.; Ramsden, P.; Sarko, C.; Skow, D.;
Tomlinson, J.; Tye, H.; Whitaker, M. Bioorg. Med. Chem. Lett. 2009, 19, 2206.