Synthesis and SAR of 3-arylsulfonyl-pyrazolo[1,5-a]pyrimidines as potent serotonin 5-HT6 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2010.12.055

Syntheses of a series of novel 3-sulfonyl-pyrazolo[1,5-a]pyrimidines and their 5-HT₆ receptor antagonistic structure-activity relationship are disclosed. The nature and position of substituents, which affect their receptor antagonistic activity

Full text of DOI:10.1016/j.bmc.2010.12.055

Bioorganic & Medicinal Chemistry 19 (2011) 1482–1491
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and SAR of 3-arylsulfonyl-pyrazolo[1,5-a]pyrimidines as potent
serotonin 5-HT6 receptor antagonists
Alexandre V. Ivachtchenko a,b, Elena S. Golovina c, Madina G. Kadieva a, Volodymyr M. Kysil b,
Oleg D. Mitkin a, Sergey E. Tkachenko b, Ilya Okun b,
⇑
a Department of Organic Chemistry, Chemical Diversity Research Institute, 141401 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, 141401 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 of a series of novel 3-sulfonyl-pyrazolo[1,5-a]pyrimidines and their 5-HT6 receptor antagonistic
structure–activity relationship are disclosed. The nature and position of substituents, which affect their
receptor antagonistic activity, are analyzed. Among all synthesized derivatives, {3-(3-chlorophenylsulfo-
nyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-2-yl}-methyl-amine 33 (Ki = 190 pM), (3-phenylsulfonyl-7-
methyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-amine 44 (Ki = 240 pM), (3-phenylsulfonyl-5-metoxym-
ethyl-7-methyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-amine 50 (Ki = 270 pM), and (3-phenylsulfonyl-
5-methyl-7-metoxymethyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-amine 52 (Ki = 280 pM) are the most
potent antagonists of the 5-HT6 receptors.
Received 18 October 2010
Revised 22 December 2010
Accepted 27 December 2010
Available online 5 January 2011
Keywords:
Arylsulfonylpyrazolopyrimidines
5-HT6 receptor
Antagonist
Ó 2010 Elsevier Ltd. All rights reserved.
HEK 293 cells
cAMP stimulation
1. Introduction
an intramolecular hydrogen bond between the basic amino group
located next to the sulfonyl group, which may stabilize an advanta-
Exclusive localizationof 5-HT6 receptors (5-HT6R) in the CNS, and
high affinities of many psychiatric drugs to this receptor,1–3 have
attracted considerable attention of researchers in a quest for the
discovery of new drug candidates for treating diseases of the central
nervous system (CNS) including cognitive desorders.4–8 There are
only a few selective 5-HT6R antagonists that are being tested in
Phase I and Phase II clinical trials as drug candidates for treating
various CNS diseases.9–11 Recently, we reported on the synthesis
and structure–activity relationship (SAR) of 2-substituted 5,7-di-
methyl-12 and cycloalkane-annelated13,14 3-phenylsulfonyl-pyraz-
olo[1,5-a]pyrimidines, arylsulfonylpyrazolopyrimidines (ARSPP),
as exceptionally potent and selective 5-HT6R antagonists (Fig. 1).
We have concluded that at least three factors could be responsible
for the high potency of the compounds: (i) torsion angles between
the phenylsulfonyl moiety relative to the heterocycle core plane,
which define the conformation of the molecule, (ii) the nature and
size of a substituent in the 2-position,12 and (iii) the formation of
geous binding conformation.14
In particular, we have shown12–14 that in the corresponding
pairs of potent and selective 2-methylamino- and methylthio-
substituted ARSPPs A, B and C, D (Fig. 1), the methylamino-substi-
tuted B and D, which can form intramolecular hydrogen bonds,
exhibit substantially higher potencies as 5-HT6R antagonists.
In this paper, we further explore the SAR of 5,7-substituted
2-methylthio- and 2-methylamino-3-arylsulfonylpyrazolopyrimi-
dines for their ability to antagonize 5-HT6R (Table 1).
2. Results and discussion
2.1. Chemistry
2-Methylthio (9–12) and 2-methylamino (13–15) ARSPPs were
obtained in the reaction of 3-amino-4-arylsulfonylpyrazoles 1–7
with 1,1,3,3-tetramethoxypropane
(Scheme 1).
8 in acetic acid at 100 °C
5,7-Dimethyl-2-methylthio ARSPPs 23–28 and (5,7-dimethyl-2-
methylamino ARSPPs 29–33 were obtained in the reaction of cor-
responding 3-amino-4-arylsulfonylpirazoles 2–4, 6–7, and 16–21
with acetylacetone 22 in acetic acid (Scheme 2) in conditions sim-
ilar to ones described earlier.14
Abbreviations: 5-HT6R, serotonin 5-HT6 receptor; CNS, central nervous system;
SAR, structure–activity relationship; ARSPP, arylsulfonylpyrazolopyrimidines.
⇑
Corresponding author. Tel.: +1 858 794 4860; fax: +1 858 794 4931.
E-mail address: iokun@chemdiv.com (I. Okun).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.055

A. V. Ivachtchenko et al. / Bioorg. Med. Chem. 19 (2011) 1482–1491
1483
Figure 1. Substituted 3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidines A–D as potent 5-HT6R antagonists.
Table 1
Synthesized 5,7-substituted 2-methylthio- and 2-methylamino-ARSPPs 9–15, 23–36,
38–43, 45–55, and their 5-HT6R antagonistic activity
R1
O
S
O
R2
N
4
3
5
6
2
X
1
7
N
N
Me
R3
Compda
Substituent
R2
pKi
Scheme 1. Synthesis of 2-methylthio ARSPPs 9–12 and 2-methylamino ARSPPs
13–15. Reagents and conditions: (a) AcOH, 100 °C, 6 h.
R1
R3
X = S
X = NH
9, 13
54, 55b
38, 40
41, 43
46, 47
48, 49
50, 51
52, 53
10, 14
26, 31
25, 30
24
23, 29
11, 15
35, 36
28, 33
34
H
H
H
H
H
H
H
H
3-Cl
3-Cl
3-F
4-Cl
4-F
H
H
Me
H
6.94
8.92b
7.78
7.64
6.81
8.49
9.25
8.67
7.82
9.12
8.97
8.50
8.17
6.88
8.4
8.41
9.59b
8.84
9.62
8.12
9.57
9.55
9.32
8.93
9.73
9.30
NTc
Me
Me
H
Me
CH2OMe
Me
CH2OH
H
Me
Me
Me
Me
H
Me
Me
Me
H
1H NMR data showed that in the corresponding mixtures, prod-
ucts 38 and 39 are formed in a ratio of ꢀ1.4:1 relative to their cor-
responding counterparts 41 and 42. ARSPPs 40 and 43 were formed
in the ratio of ꢀ1:2.5. ARSPPs 40 and 43 were separated chromato-
graphically on a silica gel column with DCM as eluent. ARSPPs 39
and 42 were separated using HPLC. Using HPLC, we were able to
separate 41 with a purity of >95% from its mixture with 38, though
we failed to separate 38 with adequate purity. Instead, we obtained
ARSPP 38 by hydrogenation of 7-chloro-5-methyl-2-methylthio-3-
phenylsulfonyl-pyrazolo[1,5-a]pyrimidine 4415 (Scheme 6).
3-Amino-4-arylsulfonylpyrazoles 1 and 5 reacted with the
sodium salt of 1-methoxy-2,4-pentanedione16 45 in AcOH at
100 °C (Scheme 7) similar to the reaction described above in
Scheme 4. These reactions have also yielded approximately equi-
molar mixtures of, respectively, 46 and 48 or 47 and 49. Using
silica gel chromatography, we separated the mixtures and trans-
formed the individual compounds 46–49 into corresponding
3-phenylsulfonyl-heterocyclyl-methanols 50–53 using BBr3 in
DCM.
Me
CH2OMe
Me
CH2OH
Me
H
Me
Me
Me
Me
H
Me
Me
Me
H
Me
H
9.40
7.93
8.7
4-F
4-HO
4-MeO
4-Me2N
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
4-F, 3-Cl
7.43
7.43
7.35
8.42
8.1
8.02
NTc
12
27, 32
39
NTc
Me
Me
H
9.22
NTc
42
Me
NT
NT
a
First number in each pair is for the compounds with X = S and the second one is
for compounds with X = NH.
Compounds 54 and 55 correspond to compounds 3 and 4, respectively, in 12.
The data is taken from Ref. 12.
b
c
NT—Not tested.
Structures of synthesized ARSPPs were confirmed by LC–MS and
NMR spectra, including 2D NMR NOESY experiments. According to
the LC–MS spectra, all of the synthesized materials had purity
above 98%. The NMR spectra are in good agreement with the struc-
tures illustrated (see Section 4).
5,7-Dimetyl-3-(4-dimethylaminophenylsulfonyl)-2-methylthi-
opyrazolo[1,5-a]pyrimidine 34 was synthesized from the corre-
sponding 4-chlorophenylsulfonyl derivative 24 under reaction
with dimethylamine (Scheme 3).
3-(4-Methoxyphenyl) ARSPPs 28 and 33 were further
transformed into 4-(5,7-dimethyl-2-methylthio- (35) and 4-(5,7-
dimethyl-2-methylamino- (36) pyrazolo[1,5-a]pyrimidine-3-sul-
fonyl)-phenols under treatment with HBr (Scheme 4).
5-Methyl- (38–40) and 7-methyl- (41–43) substituted ARSPPs
were obtained from reaction of corresponding 3-amino-4-aryl-
sulfonylpyrazoles 1, 4, and 5 with 4,4-dimethoxy-butan-2-one 37
(Scheme 5), similar to the synthesis of ARSPPs 9–15. In these reac-
tions, mixtures of 5-methyl- and 7-methyl-substituted ARSPPs, 38
and 41, 39 and 42, and 40 and 43, were formed.
2.2. Inhibition of 5-HT6 receptors
Experiments were performed as was described earlier14 in a
functional assay using recombinant human 5-HT6 receptor exoge-
nously expressed into HEK 293 cells, which responded to serotonin
with increase in cAMP synthesis. Interaction of the ARSPPs with
the receptor was competitive with serotonin (data not shown)
and the potencies (pKi) of the compounds to block serotonin-in-
duced cAMP accumulation 5-HT6R are summarized in Table 1. It
can be seen that the novel ARSPPs synthesized are very potent

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Scheme 2. Synthesis of 5,7-dimethyl-2-methylthio ARSPPs 23–28 and 5,7-dimethyl-2-methylamino ARSPPs 29–33. Reagents and conditions: (a) AcOH, 100 °C, 6 h.
Scheme 6. Synthesis of 5-methyl-2-methylthio-ARSPP 38. Reagents and condi-
tions: (a) H2, 10% Pd/C, benzene, MeOH, rt, 2 h.
Scheme 3. Synthesis of 5,7-dimetyl-3-(4-dimethylaminophenylsulfonyl)-2-meth-
ylthiopyrazolo[1,5-a]pyrimidine (34). Reagents and conditions: (a) 40% Me2NH,
DMF, 140 °C, 48 h.
compounds (X = S) have higher Ki values (lower potency) than their
corresponding analogs in the 2-aminomethyl (X = NH) series.
When all the compounds were analyzed as a whole set, no signif-
icant correlation was observed between their potencies to block
5-HT6R and the most common physicochemical molecular descrip-
tors, such as MW, molecular volume, polar surface area, lipophilic-
ity (A Log P98), polarizability, dipole moment, and radius of
gyration (data not shown) calculated using DS ViewerPro 6.0
(Accelrys Software Inc., San Diego).
This is in agreement with our earlier data obtained with 2-
substituted 5,7-dimethyl-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimi-
dine analogs.12 However, when the compounds were separated
into two groups, namely, the 2-thiomethyl (2-SMe) and the 2-ami-
nomethyl (2-NHMe)-substituted ARSPPs, there was a significant
Scheme 4. Synthesis of 4-(5,7-dimethyl-2-methylthio- (35) and 4-(5,7-dimethyl-2-
correlation between pKi and the radius of gyration within each
group (Fig. 2A and B, respectively).
methylamino-(36) pyrazolo[1,5-a]pyrimidine-3-sulfonyl)-phenols. Reagents and
conditions: (a) 40% HBr, reflux, 24 h.
In both series of compounds, the larger gyration radii of the
molecules are associated with lower compound potencies, pKi. As
the radius of gyration reflects hydrodynamic size and shape of
the molecule,17 it seems reasonable to assume from the reciprocal
correlation that the binding pocket of the 5-HT6 receptor is more
suitable for accommodation of smaller molecules.
5-HT6R antagonists with Ki values ranging from 0.19 nM (com-
pound 31) to 155.7 nM (compound 46). The nature of the substitu-
ent groups in positions 2, 5, and 7 determines a SAR range of more
than two orders of magnitude. It is noticeable that 2-thiomethyl
Scheme 5. Synthesis of 5-methyl- (38, 39), 7-methyl- (41, 42) 2-methylthio-ARSPPs and 5-methyl- (40), and 7-methyl- (43) 2-methyl-amine ARSPPs. Reagents and
conditions: (a) AcOH, 100 °C, 6 h.

A. V. Ivachtchenko et al. / Bioorg. Med. Chem. 19 (2011) 1482–1491
1485
Scheme 7. Synthesis of methoxymethyl-3-phenylsulfonyl-pyrazolopyrimidines 46–49 and methylmethanol-3-phenylsulfonyl-pyrazolopyrimidines 50–53. Reagents and
conditions: (a) AcOH, 100 °C, 12 h. (b) DCM, BBr3, rt, 12 h.
Figure 2. Correlation between radius of gyration (calculated using DS ViewerPro 6.0) and pKi values of the compounds in 2-SMe (A) or 2-NHMe (B) subgroups. Dotted lines
represent confidence interval of 95%.
The 5,7-substituents as well as those on the phenyl moiety (R1)
affect compound potencies in a similar rank-order independently
of the nature of the 2-substituent group, 2-SMe or 2-NHMe.
However, the latter substituents significantly affect overall potency
of the compounds in each of the two series, compounds in the
2-NHMe series being approximately an order of magnitude more
potent than their 2-SMe counterparts (Fig. 3).
being repeated until the convergence occured at a convergence cri-
terion of 0.00001) showed that all of the 2-NHMe-substituted mol-
ecules have two stable conformations, as exemplified in Figure 4 by
compound 55. Depending on the oxygen atom participating in the
hydrogen bond formation, O(1) or O(2), the two conformations
differ in their torsion angles between the planes formed by the
ab and bc bonds.
A plausible explanation of this increase in 5-HT6R blocking
potency attributable to the 2-NHMe group could be that it forms
an intramolecular hydrogen bond between the proton of the
2-methylamine group and one of the two oxygens of the 3-aryl-
sulfonyl group. This restricts the molecule mobility in a conforma-
tion advantageous for the binding. The energy minimization
(Accelrys DS ViewerPro 6.0 software; minimization cycles were
The 2-SMe-substituted compounds, exemplified in Figure 5 by
compound 54, unlike 2-NHMe-substituted ones, accept at least
three major conformations defined by the torsion angle between
the planes formed by the same ab and bc bonds (see Fig. 4). With
symmetrical derivatization of the phenyl ring, there are three addi-
tional conformations with 2-SMe facing in opposite to shown
direction (total of six conformations). With asymmetric derivatiza-

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provides an additional binding energy by forming intermolecular
hydrogen bonding with Ser193 and/or Thr196, which are located
in the transmembrane region V of the 5-HT6R receptor.18
The compound potencies are substantially affected by the ature
and position of the substituents in the 5- and 7-positions. As
compared with previously described12 5,7-dimethyl substituted
compounds 54 and 55, removal of both methyl moieties in com-
pounds 9 and 13 leads to a substantial (one to two orders of mag-
nitude) increase in Ki (see Table 1). 5-Me (38) or 7-Me (41)
substitution in the 2-SMe series leads to, respectively, 4.9-fold
and 6.9-fold decrease in Ki (compared with 9). Similar substitutions
in 2-NHMe series lead to 2.7-fold (40) and 16.2-fold (43) decrease
in Ki (compared with 13). In both series, methyl substitution of the
7-position is more effective than that of 5-position in increasing
the compound potencies.
Substitution of the 5-Me group in 54 and 55 with methoxy-
methyl (correspondingly, 48 and 49), engendered a minimal, if
any, effect on the compound potencies. On the other hand, similar
substitution in position 7 (46 and 47) led to a substantial, 130-fold
and 32-fold, decrease in the compound potencies. Interestingly, the
methyl substitution in either position with a more hydrophilic
moiety, hydroxymethyl, does not cause substantial changes in
the compound Ki values (compare 50 and 52 with 54 (2-SMe ser-
ies) and 51 and 53 with 55 (2-NHMe series)).
Figure 3. Corellation between pKi values for blocking serotonin-induced cAMP
production in HEK cells expressing 5-HT6 receptor by 2-aminomethyl- and
2-thiomethyl-substituted ARSPPs. Dotted lines represent confidence interval of
95%.
3-Cl-phenyl derivatized compounds (R1 in Table 1) show the
same Ki trend as with the compounds having unsubstituted phe-
nyl. 5,7-dimethyl substituted compounds 26 (2-SMe series) and
31 (2-NHMe series) are 20-fold and 6-fold more potent than their
5,7-unsubstituted analogs, 10 and 14, respectively. 3-F-phenyl
substitution for 3-Cl-phenyl, 25 and 30, leads to a moderate, 1.4-
fold to 2.6-fold, reduction in the compound potencies (compare,
correspondingly, with 26 and 31).
Derivatizing the para-position in the phenyl ring with a chlorine
atom (in 24) instead of the meta-position (in 26) leads to 4.2-fold
increase in Ki value. Substitution of the 4-Cl with 4-F leads to fur-
ther reduction in the potency (compare 23 and 24). 4-F-phenyl-
substituted 5,7 dimethyl ARSPPs 23 and 29 were 5.7-fold and
1.5-fold less potent than their corresponding unsubstituted ana-
logs 54 and 55. 4-Fluoro substitution of 5,7-unsubstituted 2-
SMe-ARSPP 11 does not affect its potency (compare with 9),
whereas the same substitution of 2-NHMe-ARSPP (13) with 4-F
(15) leads to a threefold decrease in the potency (increase in Ki).
In a series of 5,7-dimethyl-substituted compounds, there is a
clear trend in decreasing potency (pKi values) with increase in a
partial negative charge, d, on the 4-phenyl substituent (Fig. 6). The
d values were calculated with the DS ViewerPro 6.0 using method
of Gasteiger. The potency rank order of compounds in the 5,7-di-
methyl-2-SMe series is 54 (d = +0.062) > 24 (d = À0.084) > 23
(d = À0.207) > 28 (d = À0.349) = 34 (d = À0.378). The trend indicates
that the binding site of the receptor could have a preference to a
hydrophobic moiety in this position. However, almost one order of
Figure 4. Calculated stable conformations (DS ViewerPro 6.0) of the compound 55.
The torsion angle between planes formed by bonds ab and bc are: (A) À52.65°
(E = 58.876635 kcal/mol) and (B) +52.47° (E = 58.875965 kcal/mol).
tion of the phenyl ring, there are up to six additional conformations
with local minima. However, the global free energy minimum
calculated with the DS ViewerPro 6.0, corresponds to the confor-
mation with the ab/bc torsion angle close to zero. This conforma-
tion is characteristic for all of the 2-SMe-substituted compounds
(Fig. 5C). Thus, the generally lower receptor affinity of the
compounds within the 2-SMe-substituted series compared with
2-NHMe-substituted one could result from the entropy factor,
higher conformational freedom, which effectively reduces the con-
centration of the compounds in an appropriate ‘active’ binding
conformation.
Alternative explanation of the higher potencies of compounds
with the 2-NHMe substitution could be that the 2-NHMe group
Figure 5. Calculated stable conformations (DS ViewerPro 6.0) of the compound 54. The characteristic torsion angles are: (A) À85.21° (E = 60.91859 kcal/mol), (B) +85.23°
(E = 60.91838 kcal/mol), and (C) À0.35° (E = 60.132609 kcal/mol).

A. V. Ivachtchenko et al. / Bioorg. Med. Chem. 19 (2011) 1482–1491
1487
400 (400 MHz, 27 °C), 13C NMR and two-dimensional spectra uti-
lized a Bruker DPX-300 (300 MHz, 27 °C).
LC–MS analyses were obtained with a Shimadzu HPLC, equipped
with a column Waters XBridge C18 3.5 mm (4.6 Â 150 mm), mass
detector PE SCIEX API 150 EX and spectrophotometric detector
Shimadzu (kmax 220 and 254 nm).
In all cases, the end of the reaction was determined by conver-
sion of the substrate (LC–MS control). Evaporation of solvents and
drying of products were carried out at reduced pressure. Separa-
tion of reaction products was performed using a HPLC system
using Shimadzu LC-8A on the chromatographic column Repro-
sil-Pur C-18-AQ 10 micron, 20 Â 250 mm (precolumn Reprosil-
Pur C-18-AQ 10 micron, 20 Â 50 mm) at a flow rate of 25 mL/
min in gradient mode with mobile phase MeCN/water + 0.05%
CF3COOH.
According to the LC–MS, the purity of the obtained compounds
exceeded 98.0%.
Figure 6. Dependence of ARSPPs potencies (pKi) on a partial charge of the 4-
phenyl-substitute atom (H, Cl, F, O, or N). Compound 6 with R1 = 4-OH is shown as
open symbol. Numbers denote compound IDs.
4.1.1. Aminopyrazoles 1–7, 16–21
Aminopyrazoles 1–7, 16–21 were synthesized from correspond-
ing arylsulfonylacetonitriles according to the procedures described
previously.14
magnitude higher potency than the one expected from the partial
atom charge trend (Fig. 6) was observed for compound 35 having
4-OH substituent. This substantial deviation could indicate a possi-
bility of the hydroxyl group forming a hydrogen bond in the receptor
binding pocket and thus increasing binding energy. Though a fewer
number of 4-substituted 5,7-dimethyl-2-NHMe ARSPPs was tested
(Table 1), the same rank-order trend in pKi values (see Table 1)
was observed: 55 (4-H, d = +0.062) > 29 (4-F, d = À0.207) > 33 (4-
MeO, d = À0.349).
4.1.1.1. 3-(Methylthio)-4-(phenylsulfonyl)-1H-pyrazol-5-amine
1. 1H NMR (DMSO-d6, 400 MHz), d 12.00 (br s, 1H), 7.91 (m, 2H),
7.63 (m, 1H), 7.57 (m, 2H), 6.10 (s, 2H), 2.32 (s, 3H). MS-ESI m/z
270 (M+H).
4.1.1.2. 4-(3-Chlorophenylsulfonyl)-3-(methylthio)-1H-pyrazol-
5-amine 2. 1H NMR (DMSO-d6, 400 MHz), d 12.04 (s, 1H), 7.94 (s,
1H), 7.88 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.62 (t,
J = 7.6 Hz, 1H), 6.24 (s, 2H), 2.34 (s, 3H). MS-ESI m/z 304, 306
(M+H).
3-Cl-phenyl substitution leads to increased compound potency
(pKi): 10 > 9 and 26 > 54 in 2-SMe series; and 14 > 13 and 31 > 55
in 2-NHMe series. However, additional 4-phenyl substitution with
partially negative fluorine atom leads to a decrease in pKi values for
both the 2-SMe- and 2-NHMe-substituted series: 12 < 10, 27 < 26,
and 32 < 31 (see Table 1).
4.1.1.3. 4-(4-Fluorophenylsulfonyl)-3-(methylthio)-1H-pyrazol-
5-amine 3. MS-ESI m/z 288 (M+H).
3. Conclusions
4.1.1.4.
4-(3-Chloro-4-fluorophenylsulfonyl)-3-(methylthio)-
Here, we have described syntheses of novel 3-arylsulfonyl-pyr-
azolo[1,5-a]pyrimidines, which are competitive 5-HT6R antago-
nists with a range of potency (pKi) between 9.72 (compound 31)
and 6.81 (compound 46). SAR of the synthesized compounds estab-
lished several structural trends affecting the compounds’ potencies
towards blocking 5-HT6 receptors. First, structurally restricted, due
to intramolecular hydrogen bonding, 2-aminomethyl substituted
compounds show substantially increased 5-HT6R antagonistic po-
tency compared with less restricted 2-thiomethyl substituted ones.
Secondly, derivatization of both the 5- and 7-positions in the
pyrimidine cycle as well as that of 3- and 4-positions in the phenyl
ring leads to similar potency trends for both the 2-aminomethyl
and 2-thiomethyl substituted series of compounds (Fig. 3). Thirdly,
the compound potencies (in both the 2-aminomethyl and 2-thio-
methyl substituted series) reversely depend on a radius of mole-
cule gyration, which reflects better compatibility of the receptor
binding site with more compact molecules (Fig. 2). Finally, in both
the 2-aminomethyl-5,7-dimethyl and 2-thiomethyl-5,7-dimethyl
substituted series of compounds, a clear reciprocal correlation be-
tween pKi and partial negative charge of the 4-phenyl substituent
was observed (Fig. 6).
1H-pyrazol-5-amine 4. MS-ESI m/z 322, 324 (M+H).
4.1.1.5. N3-Methyl-4-(phenylsulfonyl)-1H-pyrazole-3,5-diamine
5. 1H NMR (DMSO-d6, 400 MHz), d 10.94 (br m, 1H), 7.90 (m, 2H),
7.60 (m, 1H), 7.54 (m, 2H), 5.83 (br m, 1.5H), 5.00 (br s, 1.3H), 2.68
(d, J = 2.8 Hz, 3H). MS-ESI m/z 253 (M+H).
4.1.1.6. 4-(3-Chlorophenylsulfonyl)-N3-methyl-1H-pyrazole-3,5-
diamine 6. 1H NMR (DMSO-d6, 400 MHz), d 10.97 (br s, 1H), 7.96
(s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.67 (d, J = 7.6 Hz, 1H), 7.58 (t,
J = 8.0 Hz, 1H), 5.82 (br s, 2H), 5.25 (br m, 1H), 2.68 (d, J = 4.4 Hz,
3H). MS-ESI m/z 287, 289 (M+H).
4.1.1.7. 4-(4-Fluorophenylsulfonyl)-N3-methyl-1H-pyrazole-3,5-
diamine 7. MS-ESI m/z 271 (M+H).
4.1.1.8. 4-(4-Chlorophenylsulfonyl)-3-(methylthio)-1H-pyrazol-
5-amine 16. MS-ESI m/z 304, 306 (M+H).
4.1.1.9. 4-(3-Fluorophenylsulfonyl)-3-(methylthio)-1H-pyrazol-
5-amine 17. 1H NMR (DMSO-d6, 400 MHz), d 12.06 (br s, 1H), 7.76
(d, J = 7.6 Hz, 1H), 7. 72 (m, 1H), 7. 65 (td, J1 = 8.0 Hz, J2 = 5.6 Hz,
1H), 7.51 (td, J1 = 8.8 Hz, J2 = 2.4 Hz, 1H), 6.19 (s, 2H), 2.34 (s, 3H).
MS-ESI m/z 288 (M+H).
4. Experimental section
4.1. Chemistry
1H NMR spectra of solutions of the investigated compounds in
DMSO-d6 or CDCl3 were recorded on spectrometer Bruker DPX-
4.1.1.10. 4-(4-Methoxyphenylsulfonyl)-3-(methylthio)-1H-pyra-
zol-5-amine 18. MS-ESI m/z 300 (M+H).

1488
A. V. Ivachtchenko et al. / Bioorg. Med. Chem. 19 (2011) 1482–1491
4.1.1.11. 4-(3-Fluorophenylsulfonyl)-N3 -methyl-1H-pyrazole-
3,5-diamine 19. 1H NMR (DMSO-d6, 400 MHz), d 10.99 (br s,
1H), 7.75 (m, 2H), 7.62 (td, J1 = 8.8 Hz, J2 = 6.4 Hz, 1H), 7.47 (td,
J1 = 8.4 Hz, J2 = 1.2 Hz, 1H), 5.89 (s, 2H), 5.06 (m, 1H), 2.49 (d,
J = 3.6 Hz, 3H). MS-ESI m/z 271 (M+H).
MS (UV-254) purity: 98%. Anal. Calcd for C13H12N4O2S: C, 54.15; H,
4.20; N, 19.43. Found: C, 54.19; H, 4.11; N, 19.44.
4.1.2.6. [3-(3-Chloro-phenylsulfonyl)-pyrazolo[1,5-a]pyrimidin-
2-yl]-methyl-amine 14. 1H NMR (CDCl3, 400 MHz), d: 8.53 (dd,
J1 = 1.6 Hz, J2 = 4.4 Hz, 1H), 8.46 (dd, J1 = 1.6 Hz, J2 = 6.4 Hz, 1H),
8.13 (s, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.43
(t, J = 8.0 Hz, 1H), 6.87 (dd, J1 = 4.4 Hz, J2 = 6.4 Hz, 1H), 6.07 (q,
J = 5.2 Hz, 1H), 3.06 (d, J = 5.2 Hz, 3H). MS-ESI m/z 323 (M+H).
LC–MS (UV-254) purity: 98%. Anal. Calcd for C13H11ClN4O2S: C,
48.37; H, 3.44; N, 17.36. Found: C, 48.44; H, 3.46; N, 17.41.
4.1.1.12. 4-(3-Chloro-4-fluorophenylsulfonyl)-N3 -methyl-1H-
pyrazole-3,5-diamine 20. MS-ESI m/z 305, 307 (M+H).
4.1.1.13. 4-(4-Methoxyphenylsulfonyl)-N3-methyl-1H-pyrazole-
3,5-diamine 21. MS-ESI m/z 283 (M+H).
4.1.2. General procedure for synthesis of pyrazolo[1,5-
a]pyrimidines 9–15, 23–33, 38–43, 46–49
4.1.2.7. [3-(4-Fluoro-phenylsulfonyl)-pyrazolo[1,5-a]pyrimidin-
2-yl]-methyl-amine 15. 1H NMR (CDCl3, 400 MHz), d: 8.50 (dd,
J1 = 4.4 Hz, J2 = 1.6 Hz, 1H), 8.45 (dd, J1 = 6.8 Hz, J2 = 1.6 Hz, 1H),
8.14–8.18 (m, 2H), 7.13–7.18 (m, 2H), 6.85 (dd, J1 = 6.8 Hz,
J2 = 4.4 Hz, 1H), 6.08 (q, J = 4.8 Hz, 1H), 3.06 (d, J = 4.8 Hz, 3H).
MS-ESI m/z 307 (M+H). LC–MS (UV-254) purity: 98%. Anal. Calcd
for C13H11FN4O2S: C, 50.97; H, 3.62; N, 18.29. Found: C, 50.81; H,
3.67; N, 18.22.
A solution of 10 mmol of an aminopyrazole 1–7, 16–21 and
11 mmol of either 1,1,3,3-tetramethoxypropane 8, acetylacetone
22, 4,4-dimethoxybutan-2-one 38 or sodium 1-methoxy-2,4-pen-
tanedionate16 45 in 10–15 mL of AcOH was heated for 0.5–6 h at
100 °C. After reaction completion (LC–MS control), 5 mL of water
was added and the heating continued for 1 h. The reaction mixture
was cooled, poured into 150 mL of ice water and stirred for 1 h. The
formed precipitate was filtered, washed with water, i-PrOH, hex-
ane, and dried to obtain ARSPPs 9–15, 23–33, 38–43, 46–49 with
60–91% yield. The formed mixtures of ARSPPs 39 and 42, 40 and
43, 46 and 48, 47 and 49 were separated by column chromatogra-
phy on silica gel (eluent—hexane/AcOEt = 2:1–0:1) or by HPLC.
4.1.2.8. 3-(4-Fluoro-phenylsulfonyl)-5,7-dimethyl-2-methylthio-
pyrazolo[1,5-a]pyrimidine 23. 1H NMR (CDCl3, 400 MHz), d: 8.23
(m, 2H), 7.13 (t, J = 4.6 Hz, 3H), 6.69 (s, 1H), 2.69 (s, 3H), 2.64 (s,
3H), 2.62 (s, 3H) MS-ESI m/z 352 (M+H). LC–MS (UV-254) purity:
98%. Anal. Calcd for C15H14FN3O2S2: C, 51.27; H, 4.02; N, 11.96.
Found: C, 51.10; H, 4.15; N, 11.93.
4.1.2.1. 2-Methylthio-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimi-
dine 9. 1H NMR (DMSO-d6, 400 MHz), d: 8.70 (dd, J1 = 1.6 Hz,
J2 = 4.4 Hz, 1H), 8.59 (dd, J1 = 1.6 Hz, J2 = 6.8 Hz, 1H), 8.20 (m, 2H),
7.49–7.55 (m, 3H), 6.97 (dd, J1 = 4.4 Hz, J2 = 6.8 Hz, 1H), 2.62 (s,
3H). MS-ESI m/z 306 (M+H). LC–MS (UV-254) purity: 98%. Anal.
Calcd for C13H11N3O2S2: C, 51.13; H, 3.53; N, 13.76. Found: C,
50.91; H, 3.62; N, 13.70.
4.1.2.9. 3-(4-Chloro-phenylsulfonyl)-5,7-dimethyl-2-methylthio-
pyrazolo[1,5-a]pyrimidine 24. 1H NMR (DMSO-d6, 400 MHz), d:
8.01 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.8 Hz, 2H), 7.13 (s, 1H), 2.66 (s,
3H), 2.58 (m, 6H). MS-ESI m/z 368 (M+H). LC–MS (UV-254) purity:
98%. Anal. Calcd for C15H14ClN3O2S2: C, 48.97; H, 3.84; N, 11.42.
Found: C, 49.02; H, 3.80; N, 11.43.
4.1.2.2. 3-(3-Chloro-phenylsulfonyl)-2-methylthio-pyrazolo[1,5-
a]pyrimidine 10. 1H NMR (CDCl3, 400 MHz), d: 8.73 (dd,
J1 = 1.2 Hz, J2 = 4.4 Hz, 1H), 8.62 (dd, J1 = 1.2 Hz, J2 = 6.8 Hz, 1H),
8.19 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.43 (t,
J = 8.0 Hz, 1H), 7.01 (dd, J1 = 4.4 Hz, J2 = 6.8 Hz, 1H), 2.63 (s, 3H).
MS-ESI m/z 340 (M+H). LC–MS (UV-254) purity: 98%. Anal. Calcd
for C13H10ClN3O2S2: C, 45.95; H, 2.97; N, 12.37. Found: C, 45.88; H,
2.99; N, 12.31.
4.1.2.10. 3-(3-Fluoro-phenylsulfonyl)-5,7-dimethyl-2-methyl-
thio-pyrazolo[1,5-a]pyrimidine 25. 1H NMR (CDCl3, 400 MHz),
d: 8.00 (d, J = 8.0 Hz, 1H), 7.96 (dt, J1 = 8.0 Hz, J2 = 2.0 Hz, 1H),
7.45 (td, J1 = 8.0 Hz, J2 = 5.2 Hz, 1H), 7.21 (tdd, J1 = 8.4 Hz,
J2 = 2.4 Hz, J3 = 0.4 Hz, 1H), 6.70 (s, 1H), 2.70 (s, 3H), 2.66 (s, 3H),
2.63 (s, 3H). MS-ESI m/z 352 (M+H). LC–MS (UV-254) purity:
98%. Anal. Calcd for C15H14FN3O2S2: C, 51.27; H, 4.02; N, 11.96.
Found: C, 51.13; H, 4.09; N, 11.89.
4.1.2.3. 3-(4-Fluoro-phenylsulfonyl)-2-methylthio-pyrazolo[1,5-
a]pyrimidine 11. 1H NMR (CDCl3, 400 MHz), d: 8.71 (dd,
J1 = 4.4 Hz, J2 = 2.0 Hz, 1H), 8.61 (dd, J1 = 6.8 Hz, J2 = 2.0 Hz, 1H),
8.22 (m, 2H), 7.16 (m, 2H), 7.00 (dd, J1 = 6.8 Hz, J2 = 4.4 Hz, 1H),
2.63 (s, 3H). MS-ESI m/z 324 (M+H). LC–MS (UV-254) purity: 98%.
Anal. Calcd for C13H10FN3O2S2: C, 48.29; H, 3.12; N, 12.99. Found:
C, 48.36; H, 3.08; N, 13.04.
4.1.2.11. 3-(3-Chloro-phenylsulfonyl)-5,7-dimethyl-2-methyl-
thio-pyrazolo[1,5-a]pyrimidine
26. 1H
NMR
(DMSO-d6,
400 MHz), d: 8.26 (m, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.48 (m, 1H),
7.41 (t, J = 7.6 Hz, 1H), 6.70 (s, 1H), 2.70 (s, 3H), 2.66 (s, 3H), 2.63
(s, 3H). MS-ESI m/z 368 (M+H). LC–MS (UV-254) purity: 98%.
4.1.2.12. 3-(3-Chloro-4-fluoro–phenylsulfonyl)-5,7-dimethyl-2-
methylthio-pyrazolo[1,5-a]pyrimidine 27. 1H NMR (CDCl3,
400 MHz), d: 8.35 (dd, J1 = 6.8 Hz, J2 = 2.0 Hz, 1H), 8.11 (ddd,
J1 = 6.8 Hz, J2 = 4.4 Hz, J3 = 2.0 Hz, 1H), 7.22 (t, J = 8.8 Hz, 1H), 6.71
(d, J = 0.8 Hz, 1H), 2.71 (d, J = 0.8 Hz, 3H), 2.66 (s, 3H), 2.63 (s,
3H). MS-ESI m/z 386 (M+H). LC–MS (UV-254) purity: 98%.
4.1.2.4. 3-(3-Chloro-4-fluoro-phenylsulfonyl)-2-methylthio-pyr-
azolo[1,5-a]pyrimidine 12. 1H NMR (CDCl3, 400 MHz), d: 8.73 (m,
1H), 8.63 (d, J = 7.2 Hz, 1H), 8.29 (dd, J1 = 6.8 Hz, J2 = 2.0 Hz, 1H),
8.11 (ddd, J1 = 6.8 Hz, J2 = 4.4 Hz, J3 = 2.0 Hz, 1H), 7.25 (t,
J = 8.8 Hz, 1H), 7.02 (dd, J1 = 6.0 Hz, J2 = 4.4 Hz, 1H), 2.63 (s, 3H).
MS-ESI m/z 358 (M+H). LC–MS (UV-254) purity: 98%. Anal. Calcd
for C13H10ClFN3O2S2: C, 43.64; H, 2.54; N, 11.74. Found: C, 43.47;
H, 2.61; N, 11.68.
4.1.2.13. [4-(5,7-Dimethyl-2-methylthio-pyrazolo[1,5-a]pyrimi-
dine-3-sulfonyl)-phenyl]-dimethyl-amine 28. 1H NMR (DMSO-
d6, 400 MHz), d: 7.77 (d, J = 9.2 Hz, 2H), 7.06 (s, 1H), 6.73 (d,
J = 8.8 Hz, 2H), 2.95 (s, 6H), 2.64 (s, 3H), 2.56 (m, 6H). MS-ESI m/z
377 (M+H). LC–MS (UV-254) purity: 98%.
4.1.2.5. (3-Phenylsulfonyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-
amine 13. 1H NMR (DMSO-d6, 400 MHz), d: 8.96 (dd, J1 = 0.8 Hz,
J2 = 6.8 Hz, 1H), 8.55 (dd, J1 = 1.2 Hz, J2 = 4.4 Hz, 1H), 8.02 (m, 2H),
7.54–7.64 (m, 3H), 7.07 (dd, J1 = 4.4 Hz, J2 = 6.8 Hz, 1H), 6.47 (q,
J = 4.8 Hz, 1H), 2.91 (d, J = 4.8 Hz, 3H). MS-ESI m/z 289 (M+H). LC–
4.1.2.14. 5,7-Dimethyl-2-methylthio-3-(4-methoxy-phenylsul-
fonyl)-pyrazolo[1,5-a]pyrimidine 30. 1H NMR (DMSO-d6,
400 MHz), d: 7.94 (d, J = 8.8 Hz, 2H), 7.09 (m, 3H), 3.79 (s, 3H),

A. V. Ivachtchenko et al. / Bioorg. Med. Chem. 19 (2011) 1482–1491
1489
2.65 (m, 3H), 2.57 (s, 6H). MS-ESI m/z 364 (M+H). LC–MS (UV-254)
purity: 98%.
J2 = 2.0 Hz, 1H), 8.11 (ddd, J1 = 6.4 Hz, J2 = 4.0 Hz, J3 = 2.0 Hz, 1H),
7.24 (t, J = 8.8 Hz, 1H), 6.85 (d, J = 7.2 Hz, 1H), 2.71 (s, 3H), 2.61
(s, 3H). MS-ESI m/z 372 (M+H). LC–MS (UV-254) purity: 98%.
4.1.2.15. 5,7-Dimethyl-[3-(4-fluoro-phenylsulfonyl)-pyrazolo[1,5-
a]pyrimidin-2-yl]-methyl-amine 29. 1H NMR (CDCl3, 400 MHz), d:
8.18 (m, 2H), 7.13 (m, 2H), 6.56 (s, 1H), 5.99 (q, J = 4.8 Hz, 1H), 3.05
(d, J = 4.8 Hz, 3H), 2.61 (s, 1H), 2.56 (s, 1H). MS-ESI m/z 335 (M+H).
LC–MS (UV-254) purity: 98%.
4.1.2.24. (7-Methyl-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidin-
2-yl)-methyl-amine 43. 1H NMR (CDCl3, 400 MHz), d: 8.26 (d,
J = 7.2 Hz, 1H), 8.16–8.18 (m, 2H), 7.46–7.56 (m, 3H), 6.67 (d,
J = 6.8 Hz, 1H), 6.06 (q, J = 4.4 Hz, 1H), 3.03 (d, J = 5.2 Hz, 3H),
2.61 (s, 3H). MS-ESI m/z 303 (M+H). LC–MS (UV-254) purity: 98%.
4.1.2.16.
5,7-Dimethyl-[3-(3-fluoro-phenylsulfonyl)-pyrazol-
o[1,5-a]pyrimidin-2-yl]-methyl-amine 30. 1H NMR (DMSO-d6,
400 MHz), d: 7.85 (m, 2H), 7.63 (dt, J1 = 7.6 Hz, J2 = 6.0 Hz, 1H),
7.47 (dt, J1 = 8.4 Hz, J2 = 2.4 Hz, 1H), 6.94 (s, 1H), 6.37 (q,
J = 4.4 Hz, 1H), 2.91 (d, J = 4.4 Hz, 3H), 2.55 (s, 3H), 2.49 (s, 3H).
MS-ESI m/z 335 (M+H). LC–MS (UV-254) purity: 98%.
4.1.2.25. 7-Methoxymethyl-5-methyl-2-methylthio-3-phenyl-
sulfonyl-pyrazolo[1,5-a]pyrimidine
46. 1H
NMR
(CDCl3,
400 MHz), d: 8.22 (m, 2H), 7.45–7.55 (m, 3H), 6.96 (s, 1H), 4.85
(s, 2H), 3.59 (s, 3H), 2.70 (s, 3H), 2.59 (s, 3H). 13C NMR (CDCl3,
75 MHz), d: 162.89, 156.15, 147.27, 145.14, 142.98, 132.30,
128.30, 126.45, 106.39, 105, 81, 66.88, 59.35, 25.08, 12.88. MS-
ESI m/z 364 (M+H). LC–MS (UV-254) purity: 98%.
4.1.2.17.
[3-(3-Chloro-phenylsulfonyl)-5,7-dimethyl-pyrazol-
o[1,5-a]pyrimidin-2-yl]-methyl-amine 31. 1H NMR (DMSO-d6,
400 MHz), d: 8.22 (s, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.44 (m, 1H),
7.40 (t, J = 7.6 Hz, 1H), 6.57 (s, 1H), 5.97 (q, J = 5.2 Hz, 1H), 3.05
(d, J = 5.2 Hz, 3H), 2.61 (s, 3H), 2.58 (s, 3H). MS-ESI m/z 351
(M+H). LC–MS (UV-254) purity: 98%.
4.1.2.26. (7-Methoxymethyl-5-methyl-3-phenylsulfonyl-pyraz-
olo[1,5-a]pyrimidin-2-yl)-methyl-amine 47. 1H NMR (CDCl3,
400 MHz), d: 8.17 (m, 2H), 7.45–7.55 (m, 3H), 6.84 (s, 1H), 6.05
(d, J = 4.8 Hz, 1H), 4.78 (s, 2H), 3.57 (s, 3H), 3.03 (d, J = 4.8 Hz,
3H), 2.62 (s, 3H). 13C NMR (CDCl3, 75 MHz), d: 161.41, 158.09,
147.29, 144.78, 143.55, 132.01, 128.26, 126.04, 105.17, 90.58,
67.16, 59.25, 28.68, 24.78. MS-ESI m/z 347 (M+H). LC–MS (UV-
254) purity: 98%.
4.1.2.18. [3-(3-Chloro-4-fluoro-phenylsulfonyl)-5,7-dimethyl-
pyrazolo[1,5-a]pyrimidin-2-yl]-methyl-amine 32. 1H NMR
(CDCl3, 400 MHz), d: 8.32 (dd, J1 = 6.8 Hz, J2 = 2.4 Hz, 1H), 8.11
(ddd, J1 = 6.8 Hz, J2 = 4.4 Hz, J3 = 2.4 Hz, 1H), 7.21 (t, J = 8.8 Hz,
1H), 6.58 (s, 1H), 5.95 (q, J = 5.2 Hz, 1H), 3.05 (d, J = 5.2 Hz, 3H),
2.62 (s, 3H), 2.57 (s, 3H). MS-ESI m/z 369 (M+H). LC–MS (UV-
254) purity: 98%.
4.1.2.27. 5-Methoxymethyl-7-methyl-2-methylthio-3-phenyl-
sulfonyl-pyrazolo[1,5-a]pyrimidine
48. 1H
NMR
(CDCl3,
400 MHz), d: 8.20 (d, J = 7.2 Hz, 2H), 7.45–7.55 (m, 3H), 7.05 (s,
1H), 4.66 (s, 2H), 3.52 (s, 3H), 2.75 (s, 3H), 2.63 (s, 3H). 13C NMR
(CDCl3, 75 MHz), d: 162.37, 156.32, 147.11, 146.25, 142.99,
132.27, 128.29, 126.51, 106.43, 106.15, 74.42, 58.70, 16.74, 12.87.
MS-ESI m/z 364 (M+H). LC–MS (UV-254) purity: 98%.
4.1.2.19. [5,7-Dimethyl-3-(4-methoxy-phenylsulfonyl)-pyrazol-
o[1,5-a]pyrimidin-2-yl]-methyl-amine 33. 1H NMR (DMSO-d6,
400 MHz), d: 7.94 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 8.8 Hz, 2H), 6.89
(s, 1H), 6.31 (q, J = 4.8 Hz, 1H), 3.79 (s, 3H), 2.91 (d, J = 4.8 Hz,
3H), 2.54 (s, 3H), 2.47 (s, 3H). MS-ESI m/z 347 (M+H). LC–MS
(UV-254) purity: 98%.
4.1.2.28. (5-Methoxymethyl-7-methyl-3-phenylsulfonyl-pyraz-
olo[1,5-a]pyrimidin-2-yl)-methyl-amine 49. 1H NMR (CDCl3,
400 MHz), d: 8.14 (m, 2H), 7.44–7.54 (m, 3H), 6.90 (s, 1H), 6.06
(q, J = 5.2 Hz, 1H), 4.59 (s, 2H), 3.49 (s, 3H), 3.07 (d, J = 5.2 Hz,
3H), 2.66 (s, 3H). 13C NMR (CDCl3, 75 MHz), d: 160.74, 158.05,
147.13, 145.86, 143.49, 132.03, 128.25, 126.01, 105.66, 90.74,
74.40, 58.61, 28.66, 16.94. MS-ESI m/z 347 (M+H). LC–MS (UV-
254) purity: 98%.
4.1.2.20. Mixture of 5-methyl-2-methylthio-3-phenylsulfonyl-
pyrazolo[1,5-a]pyrimidine 38 and of 7-methyl-2-methylthio-3-
1
phenylsulfonyl-pyrazolo[1,5-a]pyrimidine 41H. NMR (CDCl3, 400
MHz), d: 8.58 (d, J = 4.8 Hz, 1H), 8.41 (d, J = 7.2 Hz, 1.4H), 8.21 (m,
4.8H), 7.50 (m, 7.2H), 6.82 (d, J = 4.8 Hz, 1H), 6.80 (d, J = 7.2 Hz,
1.4H), 2.76 (s, 3H), 2.69 (s, 4.2H), 2.64 (s, 3H), 2.59 (s, 4.2H). The
most distinguished are the doublet signals at 8.58 and 8.41 ppm
corresponding to the protons in position 5 and 7 of ARSPPs 38 and
41. Their integrals ratio is 1:1.4. At the same time, the chemical
shift of the doublet at 8.41 ppm is identical to those in the
spectrum of independently synthesized ARSPP 38.
4.1.3. Synthesis of [4-(5,7-dimethyl-2-methylthio-pyrazolo[1,5-
a]pyrimidine-3-sulfonyl)-phenyl]-dimethyl-amine 34
To a solution of 87 mg (0.24 mmol) of 3-[(4-chlorophenyl)sulfo-
nyl]-5,7-dimethyl-2-(methylthio)pyrazolo[1,5-a]pyrimidine (24)
in 0.5 mL of DMF was added 1 mL 40% aqueous dimethylamine,
and the mixture was stirred in a closed tube at 140 °C for 48 h.
After the cooling the formed precipitate was separated by centrifu-
gation, suspended in hot MeOH, cooled and centrifuged again,
washed with i-PrOH, hexane and dried in vacuum. Yield 72%. 1H
NMR (DMSO-d6, 400 MHz), d: 7.77 (d, J = 9.2 Hz, 2H), 7.06 (s, 1H),
6.73 (d, J = 8.8 Hz, 2H), 2.95 (s, 6H), 2.64 (s, 3H), 2.56 (m, 6H).
MS-ESI m/z 377 (M+H). LC–MS (UV-254) purity: 98%.
4.1.2.21. 3-(3-Chloro-4-fluoro-phenylsulfonyl)-5-methyl-2-me-
thylthio-pyrazolo[1,5-a]pyrimidine
39. 1H
NMR
(CDCl3,
400 MHz), d: 8.60 (d, J = 4.4 Hz, 1H), 8.28 (dd, J1 = 6.4 Hz,
J2 = 2.0 Hz, 1H), 8.11 (ddd, J1 = 6.4 Hz, J2 = 4.4 Hz, J3 = 2.0 Hz, 1H),
7.23 (t, J = 8.8 Hz, 1H), 6.85 (d, J = 4.4 Hz, 1H), 2.79 (s, 3H), 2.66
(s, 3H). MS-ESI m/z 372 (M+H). LC–MS (UV-254) purity: 98%.
4.1.2.22. (5-Methyl-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidin-
2-yl)-methyl-amine 40. 1H NMR (CDCl3, 400 MHz), d: 8.38 (d,
J = 4.4 Hz, 1H), 8.13–8.15 (m, 2H), 7.45–7.54 (m, 3H), 6.69 (d,
J = 4.4 Hz, 1H), 6.08 (q, J = 3.6 Hz, 1H), 3.08 (d, J = 5.2 Hz, 3H),
2.67 (s, 3H). MS-ESI m/z 303 (M+H). LC–MS (UV-254) purity: 98%.
4.1.4. General procedure for synthesis of 5,7-dimethyl-3-(4-
hydroxyphenyl)-ARSPP 35, 36
A solution of 0.5 mmol of methoxyderivative 28 or 33 in 10 mL
of 40% HBr was refluxed for 24 h. After reaction completion (LC–MS
control), the solvent was stripped off in vacuo and the residue was
separated by column chromatography on silica gel (eluent—hex-
ane/AcOEt = 1:1 for ARSPP 35, 1:2 for ARSPP 36). Yield 52–61%.
4.1.2.23. 3-(3-Chloro-4-fluoro-phenylsulfonyl)-7-methyl-2-me-
thylthio-pyrazolo[1,5-a]pyrimidine
42. 1H
NMR
(CDCl3,
400 MHz), d: 8.44 (d, J = 7.2 Hz, 1H), 8.35 (dd, J1 = 6.8 Hz,

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A. V. Ivachtchenko et al. / Bioorg. Med. Chem. 19 (2011) 1482–1491
4.1.4.1. 4-(5,7-Dimethyl-2-methylthio-pyrazolo[1,5-a]pyrimi-
dine-3-sulfonyl)-phenol 35. 1H NMR (DMSO-d6, 400 MHz), d:
10.46 (br, 1H), 7.84 (d, J = 8.0 Hz, 2H), 7.36 (s, 1H), 7.07 (s, 1H),
6.87 (d, J = 8.0 Hz, 2H), 2.64 (s, 3H), 2.56 (s, 6H). MS-ESI m/z 350
(M+H). LC–MS (UV-254) purity: 98%.
4.2. Biology
4.2.1. Cell handling
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, blastic-
idine 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.
4.1.4.2. 4-(5,7-Dimethyl-2-methylamino-pyrazolo[1,5-a]pyrimi-
dine-3-sulfonyl)-phenol 36. 1H NMR (DMSO-d6, 400 MHz), d:
10.37 (s, 1H), 7.83 (d, J = 8.8 Hz, 2H), 6.87 (s, 1H), 6.85 (d,
J = 8.8 Hz, 2H), 6.28 (q, J = 4.8 Hz, 1H), 2.91 (d, J = 4.8 Hz, 3H),
2.54 (s, 3H), 2.47 (s, 3H). MS-ESI m/z 333 (M+H). LC–MS (UV-
254) purity: 98%.
4.2.2. Measurements of the 5-HT6R-HEK cell responses
4.1.5. Synthesis of 5-methyl-2-methylthio-3-phenylsulfonyl-
pyrazolo[1,5-a]pyrimidine 38
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 an HBSS buffer 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 anti-
A solution of 354 mg (1 mmol) of 7-chloro-5-methyl-2-methyl-
thio-3-phenylsulfonylpyrazolo[1,5-a]pyrimidine 44 in 15 mL of
methanol and 5 mL of benzene was stirred under hydrogen with
100 mg of 10% Pd/C. The mixture was filtered through Celite and
solvent was removed by roto-evaporation. ARSPP 38 was isolated
by column chromatography on silica gel (eluent—DCM) with a
yield of 22%. 1H NMR (CDCl3, 400 MHz), d: 8.41 (d, J = 7.2 Hz, 1H),
8.20–8.23 (m, 2H), 7.54 (m, 1H), 7.49 (m, 2H), 6.80 (d, J = 7.2 Hz,
1H), 2.69 (s, 3H), 2.59 (s, 3H). MS-ESI m/z 320 (M+H). LC–MS
(UV-254) purity: 98%.
body (from 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 hydro-
chloride (Sigma, MO) and added to the cells (final serotonin con-
centration 10 nM, final DMSO concentration 0.32%, final IBMX
4.1.6. General procedure for synthesis of (3-phenyl-ARSPP-yl)-
methanols 50–53
concentration 500 lM). Each assay plate contained serotonin and
cAMP standard concentration curves for the assay quality control.
After 2 h of incubation with the mixture of compound/serotonin,
cells were treated as described in the cAMP LANCE assay kit proto-
col (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.
A solution of 1.2 mmol of BBr3 in 10 mL of DCM was added to a
solution of 0.4 mmol of ARSPP 46–49 in 10 mL of DCM and the
mixture was stirred for 12 h at ambient temperature. The mixture
was quenched by the addition of 20 mL of water and stirred vigor-
ously for 30 min. The mixture was extracted three times with DCM,
the combined extracts were washed with water, dried over Na2SO4
and evaporated in vacuo. The residue was separated by column
chromatography on silica gel (eluent—CHCl3/AcOEt = 10:1) to ob-
tain ARSPPs 50–53 with a yield of 42–61%.
4.3. Curve fitting and statistical analysis
The concentration curve data were fitted with Prism 5 (Graph-
Pad, CA) using built-in 4-parametric equation to calculate IC50 val-
ues. 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’s19 modified equation,
Ki = IC50/(1 + [Ag]/EC50). Where IC50 is the concentration of antago-
nist 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 0.13 nM as determined from 4 independent experiments
with three to five repeats (separate plates), each in quadruplicates.
4.1.6.1. (5-Methyl-2-methylthio-3-phenylsulfonyl-pyrazolo[1,5-
a]pyrimidin-7-yl)-methanol 50. 1H NMR (DMSO-d6, 400 MHz),
d: 8.02 (m, 2H), 7.56–7.65 (m, 3H), 7.18 (s, 1H), 5.98 (t, J = 5.6 Hz,
1H), 4.90 (d, J = 5.6 Hz, 2H), 2.64 (s, 3H), 2.55 (s, 3H). 1H NMR
(CDCl3, 400 MHz), d: 8.16 (m, 2H), 7.53 (m, 1H), 7.47 (m, 23H),
6.88 (s, 1H), 5.00 (d, J = 6.8 Hz, 2H), 3.70 (t, J = 6.8 Hz, 1H), 2.63
(s, 3H), 2.58 (s, 3H). MS-ESI m/z 350 (M+H). LC–MS (UV-254) pur-
ity: 98%.
4.1.6.2. (5-Methyl-2-methylamino-3-phenylsulfonyl-pyrazolo[1,5-
a]pyrimidin-7-yl)-methanol 51. 1H NMR (CDCl3, 400 MHz), d: 8.15
(d, J = 7.2 Hz, 2H), 7.54 (m, 1H), 7.48 (m, 2H), 6.68 (s, 1H), 6.11 (q,
J = 4.8 Hz, 1H), 4.89 (d, J = 6.8 Hz, 2H), 3.92 (t, J = 6.8 Hz, 1H), 3.03
(d, J = 4.8 Hz, 3H), 2.59 (s, 3H). MS-ESI m/z 333 (M+H). LC–MS (UV-
254) purity: 98%.
Acknowledgments
4.1.6.3. (7-Methyl-2-methylthio-3-phenylsulfonyl-pyrazolo[1,5-
a]pyrimidin-5-yl)-methanol 52. 1H NMR (CDCl3, 400 MHz), d:
8.16 (m, 2H), 7.46–7.56 (m, 3H), 6.81 (s, 1H), 4.83 (d, J = 4.8 Hz,
2H), 3.73 (t, J = 4.8 Hz, 1H), 2.73 (s, 3H), 2.64 (s, 3H). MS-ESI m/z
350 (M+H). LC–MS (UV-254) purity: 98%.
Authors extend their gratitude to Dr. F. Lakner for his valuable
suggestions and help in manuscript preparation. We also are
grateful to Dr. V. Tseitin for his help in small molecule structure
modeling.
Supplementary data
4.1.6.4.
(7-Methyl-2-methylamino-3-phenylsulfonyl-pyrazol-
o[1,5-a]pyrimidin-5-yl)-methanol 53. 1H NMR (CDCl3, 400
MHz), d: 8.11 (m, 2H), 7.53 (m, 1H), 7.48 (m, 2H), 6.64 (s, 1H),
6.05 (q, J = 4.8 Hz, 1H), 4.74 (d, J = 5.2 Hz, 2H), 3.49 (t, J = 5.2 Hz,
1H), 3.07 (d, J = 4.8 Hz, 3H), 2.65 (s, 3H). MS-ESI m/z 333 (M+H).
LC–MS (UV-254) purity: 98%.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.055. These data
include MOL files and InChiKeys of the most important compounds
described in this article.

A. V. Ivachtchenko et al. / Bioorg. Med. Chem. 19 (2011) 1482–1491
1491
HT6 Antagonist for Cognition Enhancement in Treating Neurodegenerative
Diseases, Abstract of Papers, 9th International Conference AD/PD 2009; Prague,
Czech Republic; Abstracts, 2009; 945.
References and notes
1. Branchekm, T. A.; Blackburn, T. P. Annu. Rev. Pharmacol. Toxicol. 2000, 40, 319.
2. Roth, B. L.; Hanizavareh, S. M.; Blum, A. E. Psychopharmacology 2004, 174, 17.
3. Glennon, R. A.; Lee, M.; Rangisetty, J. B.; Dukat, M.; Roth, B. L.; Savage, J. E.;
McBride, A.; Rauser, L.; Hufeisen, S.; Lee, D. K. J. Med. Chem. 2000, 43, 1011.
4. Holenz, J.; Pauwels, P. J.; Diaz, J. L.; Merce, R.; Codony, X.; Buschmann, H. Drug
Discovery Today 2006, 11, 283.
5. Heal, D. J.; Smith, S. L.; Fisas, A.; Codony, X.; Buschmann, H. Pharmacol. Ther.
2008, 117, 207.
6. Mitchell, E. S.; Neumaier, J. F. Pharmacol. Ther. 2005, 108, 320.
7. Liu, K. G.; Robicaud, A. J. Drug Dev. Res. 2009, 70, 145.
8. Johnson, C. N.; Ahmed, M.; Miller, N. D. Curr. Opin. Drug Discovery Dev. 2008, 11,
642.
9. SB-742457 and Donepezil in Alzheimer’s Disease, http://clinicaltrials.gov/ct2/
show/NCT00348192.
10. AVN-211, Potent Small Molecule for Treatment of Schizophrenia, http://
www.biospace.com/news_story.aspx?NewsEntityId=141148.
11. Tkachenko, S.; Ivachtchenko, A.; Khvat, A.; Okun, I.; Lavrovsky, Y.; Salimov, R.
Discovery and Preclinical Studies of AVN-322 Highly Selective and Potent 5-
12. Ivachtchenko, A. V.; Golovina, E. S.; Kadieva, M. G.; Koryakova, A. G.; Mitkin, O.
D.; Tkachenko, S. E.; Kysil, V. M.; Okun, I. M. Eur. J. Med. Chem. 2011, in press.
13. Ivachtchenko, A. V.; Dmitriev, D. D.; Golovina, E. S.; Kadieva, M. G.; Koryakova,
A. G.; Kysil, V. M.; Mitkin, O. D.; Okun, I. M.; Tkachenko, S. E.; Vorobiov, A. A.
Bioorg. Med. Chem. Lett. 2010, 20, 2133.
14. Ivachtchenko, A. V.; Dmitriev, D. D.; Golovina, E. S.; Kadieva, M. G.; Koryakova,
A. G.; Kysil, V. M.; Mitkin, O. D.; Okun, I. M.; Tkachenko, S. E.; Vorobiov, A. A. J.
Med. Chem. 2010, 53, 5186.
15. Bos, M.; Riemer, C.; Stadler, H. US 6194410, 2001.
16. Bruce, W. F.; Coover, H. W., Jr. J. Am. Chem. Soc. 1944, 66, 2092.
17. Khokhlov, A. R.; Grosberg, A. Y.; Pande, V. S. Statistical Physics of
Macromolecules (Polymers and Complex Materials). In AIP Series in Polymers
and Complex Materials; Larson, R., Pincus, P. A., Eds.; American Institute of
Physics, 2002.
18. Boess, F. G.; Monsma, F. J., Jr.; Meyer, V.; Zwingelstein, C.; Sleight, A. J. Mol.
Pharmacol. 1997, 52, 515.
19. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.