Synthesis and structure-activity relationship (SAR) of (5,7-disubstituted 3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methylamines as potent serotonin 5-HT6 receptor (5-HT6R) antagonists

Article abstract of DOI:10.1021/jm201079g

Syntheses, biological evaluation as 5-HT₆ receptor (5-HT ₆R) antagonists, and structure-activity relationships for a series of novel 5,7-disubstituted (3-arylsulfonyl-pyrazolo[1,5-a]pyrimidins are disclosed. The molecule conformational flexibility in the series is restricted by formation of the intramolecular hydrogen bond between 3-sulfo and 2-methylamino groups, which renders high potency and high selectivity to block serotonin-induced responses in HEK-293 cells stably expressing human 5-HT ₆R. In this work, we tested the hypothesis if addition of a positively ionizable group (PI) to the pyrimidine ring of the scaffold members in positions 5, 6, or 7 could further increase their 5HT₆R blocking potency. We show that the presence of the PI group with small substituents does not substantially affect either potency or selectivity of the ligands while causing substantial changes in their cLogP values. This provides a possibility for designing of the 5HT₆R ligands with modified ADME characteristics without grossly affecting efficiency of their interaction with the receptor. In respect to the structure-activity relationship (SAR), among other physiochemical parameters, only the molecule size and shape (described by gyration radii) showed a clear tendency for more compact molecules to be more potent antagonists of this receptor.

Full text of DOI:10.1021/jm201079g

Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationship (SAR) of (5,7-
Disubstituted 3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidin-2-yl)-
methylamines as Potent Serotonin 5-HT6 Receptor (5-HT6R)
Antagonists
Alexandre V. Ivachtchenko,†,‡ Elena S. Golovina,§ Madina G. Kadieva,† Volodymyr M. Kysil,‡
Oleg D. Mitkin,† Sergey E. Tkachenko,‡ and Ilya M. Okun*,‡
†Department of Organic Chemistry, Chemical Diversity Research Institute, 114401 Khimki, Moscow Region, Russia
‡ChemDiv, Inc., 6605 Nancy Ridge Drive, San Diego, California 92121, United States
§Department of Molecular Pharmacology, Chemical Diversity Research Institute, 114401 Khimki, Moscow Region, Russia
S
* Supporting Information
ABSTRACT: Syntheses, biological evaluation as 5-HT6
receptor (5-HT6R) antagonists, and structure−activity rela-
tionships for a series of novel 5,7-disubstituted (3-arylsulfonyl-
pyrazolo[1,5-a]pyrimidins are disclosed. The molecule con-
formational flexibility in the series is restricted by formation of
the intramolecular hydrogen bond between 3-sulfo and 2-
methylamino groups, which renders high potency and high
selectivity to block serotonin-induced responses in HEK-293
cells stably expressing human 5-HT6R. In this work, we tested the hypothesis if addition of a positively ionizable group (PI) to
the pyrimidine ring of the scaffold members in positions 5, 6, or 7 could further increase their 5HT6R blocking potency. We show
that the presence of the PI group with small substituents does not substantially affect either potency or selectivity of the ligands
while causing substantial changes in their cLogP values. This provides a possibility for designing of the 5HT6R ligands with
modified ADME characteristics without grossly affecting efficiency of their interaction with the receptor. In respect to the
structure−activity relationship (SAR), among other physiochemical parameters, only the molecule size and shape (described by
gyration radii) showed a clear tendency for more compact molecules to be more potent antagonists of this receptor.
SB-742457 (for the treatment of Alzheimer’s disease11) and Lu-
INTRODUCTION
■
AE-58054 (for the treatment of schizophrenia12), have failed to
The 5-HT6 receptors (5-HT6R) were first described in 1993
produce significant improvements over the placebo.
(rat),11994 (mouse),2 and 1996 (human).3 Almost exclusive
The first potent 5-HT6R-selective antagonists were discov-
localization of these receptors in the central nervous system
ered through both synthetic design and random screening.16−18
(CNS),3 as well as their relatively high affinity (though not
selectivity) toward some tricyclic antipsychotics,1,3 made the 5-
Since then, a wealth of potent and selective 5-HT6R antagonists
have been discovered that possess a common feature, the
HT6R a particularly intriguing potential therapeutic target for
treatment of many CNS disorders.4 Currently, several 5-HT6R
sulfonamide or sulfonyl moiety. A broad range of sulfonamide/
sulfonyl derivatives containing aromatic and heterocyclic
antagonists, for example, N,N-dimethyl-3-[naphthalen-1-ylsul-
fonyl)-1H-indazol-5-yloxy]propan-1-amine (SAM-531,
systems with high affinity to the 5-HT6R have been described
as potential drug candidates.4,19−23 On the basis of analysis
of a few selective 5-HT6R ligands known at that time, Holenz
et al.24 suggested a conceptual framework model (Figure.2),
which they used for the design and synthesis of four classes
of novel series of sulfonamides of indoles with different
substitution patterns. This hypothetical model was confirmed
and further developed into a three-dimensional pharmacophore
model based on the structural analysis of 5-HT6R antagonists
from a diverse group of 45 sulfonamide/sulfonyl containing
compounds (Figure 2).25,26
PF-5212365, WAY-262531),5−7 [2-(6-fluoro-1H-indol-3-yl)-
ethyl]-[3-(2,2,3,3-tetrafluoro-propoxy)-benzyl]-amine (Lu-AE-
58054),6−8 3-benzenesulfonyl-8-piperazin-1-yl-quinoline (SB-
742457, GSK-742457) (Figure 1), as well as AVN-2116,7,9 and
AVN-322 (Avineuro Pharmaceuticals),6,7,10 for which struc-
tures are not disclosed, are being tested in phase II clinical trials
for treatment of different cognitive and mental disorders
including Alzheimer’s disease and schizophrenia. Phase IIa
double blind clinical trials performed with AVN-211 (Avineuro
Pharmaceuticals) on 50 patients stabilized on an atypical
antipsychotic therapy showed positive results as an augmenta-
tion therapy to improve cognition in schizophrenia patients.9 It
should also be noted, however, that two other drug candidates,
Received: August 18, 2011
Published: October 26, 2011
© 2011 American Chemical Society
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Recently, we have described several series of highly potent 5-
HT6R antagonists represented by substituted 3-phenylsulfonyl-
pyrazolo[1,5-a]pyrimidines.32−35 Unlike previously disclosed
analogues, for example, 3-benzenesulfonyl-2-methylsulfanyl-8-
piperazin-1-yl-1,5,6,7-tetrahydro-cyclopenta[d]pyrazolo[1,5-a]-
pyrimidine (Ro-65−7674, Figure 3A),26,27 which fit into the
conceptual model PhM1, the disclosed series lacked a PI group.
The most potent antagonists from the 3-phenylsulfonyl-
pyrazolo[1,5-a]pyrimidines series30−35 were those with 2-
methylamino substitution (Figure 3B), which could contribute
in stabilization of the molecule structure by forming intra-
molecular hydrogen bond (IHB) between the methyl-amine
hydrogen bond donor (HBD) group and oxygen of the sulfo
group, HBA. The likelihood of the hydrogen bond formation is
reasonable to suggest as the calculated with DS ViewerPro 6.0
(Accelrys, San Diego, CA) distance between the methyl-amine
hydrogen and one of the sulfo group oxygen in energy
minimized molecules 1 and 2 is 2.10 and 2.11 Å, respectively.
Examples of such constricted 5-HT6R antagonists, represented
by (3-benzenesulfonyl-7,8-dihydro-6H-cyclopenta[e]pyrazolo-
[1,5-a]pyrimidin-2-yl)-methyl-amine (1) and (3-benzenesul-
fonyl-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-
amine (2) are shown in Figure 3B.
Figure 1. Structures of 5-HT6 receptor antagonists, SAM-531,13 Lu-
AE-58054,14 and SB-742457,15 currently in phase II clinical trials for
multiple mental disorders.
In spite of the absence of PI group, 1 and 2 are more potent
5-HT6R antagonists than Ro-65−7674. Taking this into
consideration, a new conceptual pharmacophore model,
PhM2, can be hypothesized (Figure 4) which reflects the
Figure 2. Conceptual pharmacophore model, PhM1, adapted from
25
Holenz et al.24 and Lοpez-Rodrigues et al. The HYD1 and HYD2
́
groups (circles) represent aromatic or the heterocyclic hydrophobic
moieties. HBA (in square) is a hydrogen bond acceptor group25 or
double electron acceptor,24 represented by either a sulfonamide or
sulfonyl group. PI (oval) is a positive ionizable atom25 or proton
donor,21 mainly represented by an amine group.
Figure 4. Conceptual pharmacophore model, PhM2, which illustrates
known potent 5-HT6R antagonists lacking a positively ionizable group
but carrying a hydrogen bond donor group, HBD (round cornered
square), capable of forming intramolecular hydrogen bond (dotted
line) with a hydrogen bond acceptor group, HBA (square). The HYD1
and HYD2 (circles) are hydrophobic moieties separated by the HBA.
Many 5-HT6R antagonists with either sulfonamide15,26,27 or
sulfonyl25−30 group as a hydrogen bond acceptor (HBA), which
conform well to this model, were tested as potential cognitive
enhancers16 and as drugs for treating obesity16 and Alzheimer’s
disease.31
essential physiochemically distinct moieties in the active 5-
HT6R ligands. This model does not require the PI group as a
Figure 3. Structures of potent 5-HT6 receptor antagonists with outline shapes representing corresponding functional groups (see description in
caption to Figure 2). (A) Ro-65−7674 structurally conforms to the PhM1 model. (B) 1 and 2 (Avineuro Pharmaceticals) without positively
ionizable (PI) group of PhM 1 but with hydrogen bond donor group (HBD, square with rounded corners) capable of forming intramolecular
hydrogen bond with HBA (square) group in accordance with PhM 2.
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Figure 5. Potent and selective 5-HT6 receptor antagonists with restricted conformational flexibility due to intramolecular hydrogen bond formation
which conform to PhM 2. The hydrogen bond formation was confirmed upon energy minimization of the molecules using DS Viewer Pro 6.0
(Accelrys, San Diego).
necessary feature for the high potency. Instead, the model includes
the hydrogen bond donor group (HBD, a round-cornered square
in Figures 3 and 4) which can form intramolecular hydrogen bond
with the hydrogen bond acceptor group (HBA, square) and thus
serve as a “conformational restrictor”. One can also argue that
HBD as well as HBA groups of the discussed molecules could
alternately participate in formation of an intermolecular, ligand−
Figure 6. Conceptual pharmacophore model, PhM 3, which
receptor interaction and, hence, increase the affinity of the ligands
illustrates known and new potent 5-HT6R antagonists. The HYD1,
to the receptor.
HYD2, HBA, PI, and HBD are as defined in the captions to Figures 2
and 4.
Another potent 5-HT6R ligand, (E)-3 benzenesulfonyl-2-
methylsulfanyl-pyrido[1,2-a]pyrimidin-4-ylideneamine (3), with
the structure conforming to the model PhM 2, absence of PI
group, and presence of the intramolecular hydrogen bond, was
described in the literature33 (Figure 5). However, the authors did
not specifically investigate the role the intramolecular hydrogen
bond could play in the ligand receptor potency.
CHEMISTRY
■
3-(Arylsulfonyl)-N2,5-dimethylpyrazolo[1,5-a]pyrimidin-2,7-
diamines 6 and 7 were obtained with a yield of 22−35% by
interaction of 4-(arylsulfonyl)-N3-methyl-1H-pyrazole-3,5-dia-
mines 33 and 34 with 3-aminocrotononitrile 35 in acetic acid at
elevated temperature (Scheme 1).
Previously described37 ligands, (2-benzenesulfonyl-5-piper-
azin-1-yl-phenyl)-ethyl-amine (4) and [5-[1,4]diazepan-1-yl-2-
(3-fluoro-benzenesulfonyl)-phenyl]-methyl-amine (5), with
high affinity toward 5-HT6R (Figure 5) similar to 3, also
contain characteristic to the PhM 2 hydrogen bond donor−
acceptor pair, HBD (round-cornered square) and HBA
(square), as well as a positively ionizable group (oval) adjacent
to the HYD1 group (circle).
Taken together, this data indicates that the presence of the PI
group does not seem to significantly improve the potency and its
essentiality for the receptor−ligand interaction seems questionable.
In this work, we set out to investigate in more detail the
question if addition of the positively ionizable group to the
conformationally restricted ligands could further improve their
affinity to the receptor. We have synthesized and evaluated a
new series of the 5-HT6R ligands, substituted 3-phenylsulfonyl-
pyrazolo[1,5-a]pyrimidines. This set of molecules structurally
could be illustrated by a pharmacophore model PhM 3, which
combines features characteristic of both the PhM 1 and PhM 2
models (Figure 6).
Similar conditions were used for the synthesis of 3-
(arylsulfonyl)-N2-methylpyrazolo[1,5-a]pyrimidin-2-amines 23
and 36−45. The compounds were synthesized using, as starting
reagents, both the 1H-pyrazole-3,5-diamines 33, 34, and 46 and
β-dicarbonyl compounds 47−53 (Scheme 2).
With asymmetric β-diketone 50, we have predominantly
obtained N2,5-dimethyl-3-(arylsulfonyl)pyrazolo[1,5-a]-
pyrimidin-2-amine 41. However, with 2-(2,4-dioxopentyl)-
isoindole-1,3-dione 49, we observed formation of a mixture
of the isomeric products 39 and 40 with the ratio of 87:13. The
mixture was separated by fractional crystallization and then
transformed by a hydrazinolysis reaction into 7-(aminomethyl)-
N,5-dimethyl- (17) and 5-(aminomethyl)-N,7-dimethyl-3-
(phenylsulfonyl)pyrazolo[1,5-a]pyrimidin-2-amine (21).
3-(Arylsulfonyl)-5-methyl-2-(methylamino)pyrazolo[1,5-a]-
pyrimidin-7-ols 36−38 were converted to corresponding 7-
chloro derivatives 54−56 by treatment with POCl3. Subsequent
reaction in dioxane or in dioxane−methanol with either
ammonia or primary or secondary amines led to desired
compounds 8−16 with 58−86% yield (Scheme 3).
N-(5,7-Dimethyl-2-methylamino-3-(phenylsulfonyl)-
pyrazolo[1,5-a]pyrimidin-6-yl)-acetamide 23 was converted
into the compound 22 by alkaline hydrolysis, which then was
selectively methylated by a reductive amination to yield
compound 24 (Scheme 4).
7-(2-Aminoethyl)-N,5-dimethyl-3-(phenylsulfonyl)- (19), 6-
(aminomethyl)-3-(arylsulfonyl)-N,5,7-trimethyl- (25−27), and
Using this conceptual framework model, we have synthesized
a series of novel compounds 6−32 (Figure 7). The potencies of
the compounds were assessed in a cell-based assay by their
ability to block serotonin-induced stimulation of adenylate
cyclase in HEK-293 cells stably expressing human recombinant
5-HT6 receptor.
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Figure 7. A series of highly potent 5-HT6R antagonists synthesized and tested in this work. Outline shapes around the characteristic groups are as
defined in captions to Figures 2 and 4.
(Scheme 5) from the corresponding esters 41−45. The
compounds 41−45 were converted into acids 57−61 by
alkaline hydrolysis, which were then transformed by successive
treatment with ethyl chloroformate and sodium azide into
acylazides 62−66 and further, by heating, to isocyanates 67−
71. The latter were hydrolyzed to the desired ligands 19, 25−
27, and 31. The ligands 20, 28−30, and 32 were synthesized by
reductive amination of 19, 25−27, and 31 in the presence of
formalin and sodium triacetoxyborohydride.
Scheme 1. Synthesis of 3-(Arylsulfonyl)-N2,5-dimethylpyrazolo-
[1,5-a]pyrimidin-2,7-diamines 6 and 7
a
Similarly, ligand 18 was prepared by reductive amination of
amine 17 in the presence of formalin with NaBH(OAc)3 in
DCM (Scheme 6).
The structures of synthesized compounds were confirmed
with LC-MS and NMR spectra. MS and UV detectors indicated
a purity of 98% and higher. The NMR spectra of the
compounds were in a good agreement with their expected
structures and purity.
a
Reagents and conditions: (a) AcOH, 100 °C, 15 h.
6-(2-aminoethyl)-3-(phenylsulfonyl)-N,5,7-trimethylpyrazolo-
[1,5-a]pyrimidin-2-amine (31) were synthesized in five steps
a
Scheme 2. Synthesis of Substituted 3-Arylsulfonyl-pyrazolo[1,5-a]pyrimidine-2-amines 17, 21, 23, 36−45
a
Reagents and conditions: (a) AcOH, 80−100 °C, 12 h; (b) N2H4·H2O, EtOH, reflux, 4 h.
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a
Scheme 3. Synthesis of N7-Substituted 3-(Arylsulfonyl)-N2,5-dimethylpyrazolo[1,5-a]pyrimidine-2,7-diamines 8−16
a
Reagents and conditions: (a) POCl3, sulfolane, 70 °C, 3 h; (b) one of the following: ammonia, primary amine, secondary amine, dioxane or
dioxane−MeOH, 70 °C, 3 h.
Scheme 4. Synthesis of 3-Benzenesulfonyl-5,7,N2-trimethyl-pyrazolo[1,5-a]pyrimidine-2,6-diamine 22 and 3-Benzenesulfonyl-
5,7,N2,N6,N6-pentamethyl-pyrazolo[1,5-a]pyrimidine-2,6-diamine 24
a
Scheme 5. Synthesis of target compounds 19, 20, 25−32
a
Reagents and conditions: (a) KOH, H2O, EtOH, 80 °C, 3 h; (b) Et3N, acetone, ClCO2Et, 0 °C, 0.5 h, then NaN3, H2O, 0 °C, 1 h; (c) DCM,
dioxane reflux, 1 h, then (d) 20% HCl, 70−80 °C, 0.5 h; (e) DCM or DCE, HCHO, NaBH(OAc)3, rt, 15 h.
values were transformed into pKi (negative logarithm of Ki) to
directly reflect the compound antagonist potencies.
Scheme 6. Synthesis of Target Compound 18
Using Schild experimental set up (Figure 8), we show that
the compound-mediated inhibition of the receptor activity is
competitive (a parallel rightword shift of the 5-HT-induced
activation curves in the presence of increasing antagonist con-
centrations, with Schild coefficient close to unity). Figure 8A
illustrates such an experiment for compound 6. pA2 values
were determined for compounds 6, 19, and 22 as an
intersection of the X-axis at a Log(A′/A − 1) value equal to
zero (Figure 8B).39 As one can see (Table 1), the pA2 from
Schild analysis and pKi values from Cheng−Prusoff analysis
were close to each other, which confirms usabality of a simpler
Cheng−Prussoff approach for assessment of the compound
potencies.
RESULTS AND DISCUSSION
■
Potencies of the compounds 6−32 to block serotonin-induced
responses were investigated in a cell-based assay using HEK
293 cells stably expressing recombinant human 5-HT6 receptor
(5-HT6R-HEK) and are shown in Table 1. The Ki were calculated
from IC50 values using the Cheng−Prusoff38 equation, and pA2
values were calculated from Schild transformation.39 The Ki
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Table 1. SAR Analysis of the 5-HT6R Antagonist Potencies
(pKi and pA2) of Substituted 5,N 2-Dimethyl-3-
phenylsulfonyl-pyrazolo[1,5-a]pyrimidine-2-amines 6−32
a
b
c
ligand R1
R2
R3 R4
n
pKi pA2 ALogP98
d
2
H
H
H
Cl
H
H
Cl
H
9.58
2.38
1.89
2.55
2.55
3.22
2.44
2.80
2.58
2.94
2.65
2.49
2.76
1.42
2.39
1.67
2.63
1.42
1.63
1.50
2.54
1.49
1.69
2.15
2.45
2.66
3.12
1.81
2.77
6
H
H
H
H
H
H
0
0
0
0
0
0
0
0
0
0
0
1
1
2
2
9.52 9.3
9.66
7
H
8
H
9.85
9
H
Cl Cl
7.73
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
9.24
Me Me
CH2CH2NMe2
Me CH2CH2NMe2
9.46
H
7.16
7.65
H
CH2CH2CH2NMe2
CH2CH2OCH2CH2
6.95
7.78
CH2CH2N(Me)CH2CH2
8.36
H
H
8.73
Me Me
8.74
H
H
8.51 8.4
7.47
Me Me
9.01
H
H
H
H
H
H
H
F
0
0
0
1
1
1
1
1
1
2
2
9.28 9.4
8.33
MeCO
Me Me
9.68
H
H
H
H
H
H
9.19
8.85
Cl
H
F
9.26
Me Me
Me Me
Me Me
9.15
9.05
Cl
H
H
9.38
H
H
8.86
Me Me
8.68
a
pKi values to block serotonin-induced cyclic adenosine mono-
phosphate (cAMP) production in 5-HT6R-HEK were calculated in
accordance with the Cheng−Prusoff equation.38 IC50 values (and,
hence pKi) are averages of at least two independent experiments
performed in duplicates with less than 2-fold difference in IC50
Figure 8. (A) Concentration-dependent serotonin-induced activation
of cAMP production in HEK-5-HT6R cells in the presence of different
concentrations of 6. (B) Schild plot of the panel (A) data expressed in
units of Log(A′/A − 1) as a function of the Log concentration of the
blocker,39 where A and A′ are, respectively, serotonin-induced EC50
values measured without and in the presence of specified antagonist
concentration. Each concentration point represents a mean of
triplicates ± SE.
b
between the two experiments. pA2 values were calculated from
c
Schild type experiments39 as shown in Figure 8. ALogP98 was
d
calculated using DS Viewer Pro 6 (Accelrys Software Inc., CA). The
data is from ref 34.
In the series of 5,N2-dimethyl-3-phenylsulfonyl-pyrazolo[1,5-a]-
pyrimidine-2,7-diamines (6−16), substituent groups greatly
affect the ability of the compounds to antagonize 5-HT6R.
Thus, depending on the substitute, the Ki values vary 3 orders
of magnitude from Ki = 0.14 nM (pKi = 9.85) (8) to Ki = 112
nM (pKi = 6.95) (14) (Table 1).
Addition of a positively ionizable group in position 7, 7-
amino (6), 7-methylamino (10), and 7-dimethylamino (11)
produced a negligible, if any, effect on the antagonistic potency
(pKi, correspondingly, are 9.52, 9.24, 9.46) as compared to the
prototype compound 2 (pKi = 9.58). The secondary amine in
the position 7 renders slightly lower potency than correspond-
ing tertiary amine (compare pKi values of 10 vs 11 and 12 vs 13
in Table 1).
Bulkier substituents of the 7-amino group, on the other hand,
cause substantial reduction in the 5-HT6R antagonistic
potencies. Thus, 7-(2-dimethylaminoethyl)-amino derivative
12 and 7-(3-dimethylaminopropyl)-amino derivative 14 are,
respectively, 120-fold and 190-fold less potent antagonists than
7-methylamino derivative 10.
Substitution of the 7-amino group in 6 with heterocyclic
derivatives 7-morpholyn-4-yl (15) or 7-(4-methylpiperidine-1-yl)
(16) leads to a substantially reduced antagonistic potency
(Table 1).
Introduction of a chlorine atom either in position 6 of the
pyrazolopyrimidine or in position 3 of the phenyl ring leads to a
small increase in the potency (pKi = 9.66 (7) and pKi = 9.85
(8)) relative to its unsubstituted analogue 6, 5,N2-dimethyl-3-
phenylsulfonyl-pyrazolo[1,5-a]pyrimidine-2,7-diamine (pKi =
9.52). Unexpectedly, introduction of the chlorine atoms into
both positions (9) led to 60-fold reduction of the antagonistic
potency relative to its unsubstituted analogue 6.
Moving the 7-amino group away from the pyrazolopyr-
imidine core produced compounds with substantially dimin-
ished 5-HT6R antagonistic potencies. Thus, transition from
3-benzenesulfonyl-5,N2-dimethyl-pyrazolo[1,5-a]pyrimidine-
2,7-diamine (6) to (7-aminomethyl-3-benzenesulfonyl-5-
methyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-amine (17) or
to [7-(2-amino-ethyl)-3-benzenesulfonyl-5-methyl-pyrazolo-
[1,5-a]pyrimidin-2-yl]-methyl-amine (19) led, respectively, to
a 6.2-fold and 10.3-fold increase in Ki values (decrease in pKi,
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Figure 9. Concentration-dependent effect of 19 (A) and 20 (B) on serotonin-induced (open squares) and basal (black squares) levels of cAMP in
the 5-HT6R-HEK cells. Mean values (± SD, triplicates) of a typical experiment are shown.
Figure 10. Topological overlay of 5-HT with 19 (A), 17 (B), and 31 (C). The molecules were aligned by tethering their cyclic systems together after
energy minimization performed with DS ViewerPro 6.0. The atoms with partial negative and positive charges are shown, respectively, in red and
blue.
Table 1). A similar trend was also observed for the 7-dimethyl-
aminomethyl group in 11 being moved away from the
pyrazolopyrimidin core (compare 11 with 18 and 20).
antagonistic activity. Compounds 31 and 32 with the amine- or
dimethyl amine- group also set apart two atoms from the core
but located in 6- position, showed full antagonistic activity
(data not shown). One could rationalize the appearance of the
partial agonism in 19 and 20 based on a similarity of their
molecular topology with that of 5-HT. In Figure 10, the overlay
of 5-HT with 19, 17, or 31 is shown. Only 19 has a good
topological correspondence of its 7-(2-dimethylamino-ethyl)
group with that of 5-HT.
Relative positioning of the amine group on the pyrimidine
ring did not substantially affect the antagonistic potency. Thus
(5-aminomethyl-3-benzenesulfonyl-7-methyl-pyrazolo[1,5-a]-
pyrimidin-2-yl)-methyl-amine (21) and (6-aminomethyl-3-
benzenesulfonyl-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-2-yl)-
methyl-amine (25) were, respectively, only 1.9-fold and 2.9-fold
more potent than their analogue 7-aminomethyl-3-benzene-
sulfonyl-5- methyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-
amine (17) (Table 1). In the group of 6-substituted derivatives,
like with the 7-substituted ones, lengthening of the amino
group distance from the pyrazolo[1,5-a]pyrimidine core led to
Interestingly, compounds 19 and 20 (panels A and B of Figure 9,
respectively), which have the amino or dimethyl amino group in
the 7 position spaced by two carbon atoms from the core,
exhibited a partial blockage of the serotonin-induced cyclic
adenosine monophosphate (cAMP) production in the cells.
The partial inhibitory activity of 19 and 20 indicated a
possibility that those compounds could be partial agonists.
Indeed, when the cells were treated with 19 (Figure 9A) or 20
(Figure 9B) without serotonin present, we observed partial
agonism. The maximal compound-stimulated cAMP level was
fairly close to that of the maximal compound-inhibited
serotonin-stimulated level, which is in line with the mechanism
characteristic for partial agonists.
Contrary to 19 and 20, the compounds 17 and 18 with the
amino- or dimethyl amino- group set apart one atom from the
core, and compounds 6 and 11 with the amino- or dimethyl
amino- group directly attached to the core, showed full
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Figure 11. Correlation between compound potencies (pKi) to block 5-HT6R and their calculated octanol/water partition coefficients, ALogP98 (A)
and radii of gyration, RofG (B).
a decrease in the compound potencies (22 = 25 > 31 for 6-
aminomethyl derivatives and 24 ≥ 28 > 32 for 6-
dimethylaminomethyl derivatives). Acetylation of the 6-amino
group in 23 substantially reduced its potency relative to 22
(Table 1).
In the series of 6-aminomethyl and 6-dimethylaminomethyl
derivatives 25 and 28, substitution with 3-(3-chlorophenylsul-
fonylchlorphenylsulfonyl) in 27 and 30, respectively, led to a
small increase in pKi values. When a more negatively charged
fluorine atom was introduced, compounds 26 and 29, a small
decrease in the potency was observed.
As one can see, at a concentration of 1 μM, 6 (PI in the
position 7) exhibits quite a clean profile with 100% inhibition
of the 1.5 nM [3H]lysergic acid diethylamide binding to the
5-HT6R. Besides the 5-HT6R, out of 32 receptors tested,
only one other receptor, 5-HT2BR, weakly interacts with 6
(approximately 50% inhibition of the 1.2 nM [3H]lysergic acid
diethylamide binding at this concentration (IC50 ∼ 1 μM). Shift
of the amino group into position 5 (21) or 6 (22) seems to
even improve the selectivity by reducing the ability of these
compounds to interact with the 5-HT2BR. Interestingly,
substitution of the amino group in position 6 with the dimethyl
amino group (24) leads to a restoration of the weak interaction
with the 5-HT2BR.
As we reported earlier for the pyrazolopyrimidines,33 the
newly synthesized compounds in this series showed no cor-
relation between the potencies and ALogP98 values (Table 1)
(Figure 11A). At the same time, similar to 3-arylsulfonyl-
pyrazolo[1,5-a]pyrimidines,34 a statistically significant trend
(P = 0.033) of increase in potency with decrease in the radius
of gyration was observed (Figure 11B). At this moment, it is
still to be defined why smaller (more compact) molecules
exhibit higher potency to block the receptor. One of the
possibilities is the assumption of a rather narrow pocket in the
receptor that accommodates better fit, and hence tighter
binding, of the smaller ligands.
Independence of the compound potencies on the ALogP98
opens a possibility to design highly potent 5-HT6R antagonists
with different levels of lipophilicity. For example compounds 6,
7, 8, 11, 22, 24, 25, and 27−30 all have potencies in a
submicromolar range, while their lipophilicities, ALogP98,
varied from low 1.89 to high 3.12, which could substantially
affect pharmacokinetic behavior.
We have also tested selectivity/specificity of a few highly
potent 5-HT6R ligands with the PI moiety located in either fifth
(21), sixth, (22 and 24), or seventh (6) position. A panel of
32 GPCR targets belonging to six classes (adenosinergic,
adrenergic, dopaminergic, histaminergic, muscarinic, and
serotoninergic) was used for the assessment. Also, a potassium
hERG channel has been included in the panel for the
assessment of potential liability of the compounds. The data
are presented in Figure 12, where values of the equilibrium
displacement of corresponding radiolabeled ligands from
related receptors with the compounds 6, 21, 22, and 24 are
shown.
Neither compound of the series showed potential liability
associated with inhibition of hERG channel (Figure 12).
CONCLUSION
■
SAR studies of new 5-HT6R ligands 6−32 described by the
pharmacophore model PhM3 (Figure 6) showed that all of the
tested compounds have high antagonistic potencies ranging
from 141 pM to 112 nM. The compounds from this
conformationally restricted series seem to have high selectivity
toward 5-HT6 receptors. The potency of 7-aminosubstituted
compounds did not significantly depend on either basicity or
the hydrogen bond donor capability of the nitrogen atom
(similar pKi for primary, secondary, and tertiary amines in,
correspondingly, 6, 10, and 11) nor did it depend on the
presence of the basic nitrogen atom at all (compare pKi of 6,
10, and 11 with that of 2 (Table 1), which does not possess the
corresponding amino group and can be described with the
PhM2 model, Figure 4). The major negative effect on the
potency is caused by introduction of a bulky substituent into
the 7-amine group. In agreement with our earlier published
work,32,33,35 this data indicates that the positively ionizable
group in positions 5-, 6-, or 7- does not further improve the 5-
HT6R potency relative to the parent compounds lacking such a
group as long as the core contains 2-amino substitution capable
of forming intra- and/or intermolecular hydrogen bonding.
SAR analysis of the tryptamine derivatives as 5HT6R ligands led
Pullagurla et al. to the same conclusion of a nonessential
contribution of the positively ionizable amino group to the
binding energy.40 While the ligands of this series have a very
clean selectivity/specificity profile, presence of the well tolerated
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Figure 12. Specificity profiles of 5-HT6R antagonists 6, 21, 22, and 24 were measured on a panel of 33 receptors by competitive displacement of
corresponding radiolabeled probes specific to each of the receptors. The compounds were tested at a concentration of 1 μM in duplicates and
average values of radioligand displacement ± SD are plotted.
6 and 7 formed after cooling, were filtered, washed with a minimum
amount of a mixture of acetic acid and ether (1:1), and dried.
6: N2,5-Dimethyl-3-(phenylsulfonyl)pyrazolo[1,5-a]pyrimidine-
2,7-diamine (yield: 22%). 1H NMR (DMSO-d6) δ 8.00 (m, 2H),
7.55 (m, 5H), 6.16 (q, J = 4.8 Hz, 1H), 6.02 (s, 1H), 2.91 (d, J = 4.8
Hz, 3H), 2.30 (s, 3H). HRMS calculated for C14H15N5O2S (M + H)
318.102470, found 318.1021. ESI-MS calculated for C14H15N5O2S
(M + H) 318, found m/z 318. LC-MS (UV-254), purity: 98%.
7: N2,5-Dimethyl-3-(3-chlorophenylsulfonyl)pyrazolo[1,5-a ]-
pyrimidine-2,7-diamine (yield: 35%). 1H NMR (DMSO-d6) δ 8.14
(m, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.96 (brs, 2H), 7.68 (d, J = 7.2 Hz,
1H), 7.60 (t, J = 8.0 Hz, 1H), 6,27 (brm, 1H), 6.10 (s, 1H), 2.91 (s,
3H), 2.37 (s, 3H). MS-ESI calculated for C14H14ClN5O2S (M + H)
352, found m/z 352. LC-MS (UV-254), purity: 98%.
relatively small PI group at the 5-, 6-, or 7-postion of the
pyrazolopyrimidine system does not seem to substantially
affect the specificity and could be used to modulate other
physiochemical parameters, which play an important role in
defining the ligand ADME characteristics. Thus, the PhM3
model can be useful for the designing of 5-HT6R antagonists
with improved pharmacokinetic parameters.
EXPERIMENTAL SECTION
■
General and Analytical Chemistry. In all cases, the end of the
reaction was determined by conversion of the substrate (LC-MS
control). Evaporation of solvents from reaction masses and drying of
the products was carried out at reduced pressure. Separation of
reaction products was performed using HPLC system Shimadzu LC-
8A equipped with Reprosil-Pur C-18-AQ 10 μm 250 mm × 20 mm
chromatographic column and Reprosil-Pur C-18-AQ 10 μm 50 mm ×
20 mm as a precolumn, at a flow rate of 25 mL/min in a gradient
mode with mobile phase MeCN + water + 0.05% CF3COOH.
1H NMR and 13C NMR spectra of the investigated compounds
were recorded in solutions of DMSO-d6 or CDCl3, respectively, using
N 2,5-Dimethyl-3-(phenylsulfonyl)-pyrazolo[1,5-a]-
pyrimidine-2,7-diamines 8 and 9 (Scheme 3). Suspensions of 7-
chloro-N,5-dimethyl-3-(phenylsulfonyl)pyrazolo[1,5-a]pyrimidin-2-
amines 55 and 56 (0.54 mmol each) in 2 mL of THF were burbled
with a flow of ammonia. The corresponding mixtures were stirred for
12 h at room temperature. The formed precipitates of 8 and 9 were
filtered, washed with THF, hot acetone, and dried.
8: 6-Chloro-N2,5-dimethyl-3-(phenylsulfonyl)pyrazolo[1,5-a ]-
pyrimidine-2,7-diamine (yield: 66%). 1H NMR (DMSO-d6) δ 7.99
(d, J = 6.8 Hz, 2H), 7.91 (s, 2H), 7.56 (m, 3H), 6.22 (q, J = 4.8 Hz,
1H), 2.93 (d, J = 4.8 Hz, 3H), 2.43 (s, 3H). MS-ESI calculated for
C14H14ClN5O2S (M + H) 352, found m/z 352. LC-MS (UV-254),
purity: 99%.
1
a Bruker DPX-400 spectrometer (400 MHz, 27 °C) for collecting H
NMR spectra and Bruker DPX-300 spectrometer (300 75 MHz, 27 °C)
to collect 13C NMR and two-dimensional spectra.
LC-MS spectra were obtained using Shimadzu HPLC liquid
chromatograph, equipped with Waters XBridge C18 3.5 mm column
(4.6 mm × 150 mm), PE SCIEX API 150 EX mass detector, and
Shimadzu spectrophotometric detector (λ max 220 and 254 nm).
Purity of the synthesized compounds was determined using the LC-
MS method with UV detector at the absorption wavelength of 254 nm.
Purity of the compounds was ≥95%.
9: 6-Chloro-3-(3-chlorophenylsulfonyl)-N2,5-dimethylpyrazolo-
1
[1,5-a]pyrimidine-2,7-diamine (yield: 64%). H NMR (DMSO-d6) δ
8.10 (s, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 7.2 Hz, 1H), 7.53 (t,
J = 7.6 Hz, 1H), 7.28 (s, 2H), 5.68 (m, 1H), 2.81 (d, J = 4.8 Hz, 3H),
2.29 (s, 3H). MS-ESI calculated for C14H13Cl2N5O2S (M + H) 487,
found m/z 487. LC-MS (UV-254), purity: 98%.
Synthesis of Tested Compounds. N 2,5-Dimethyl-3-
(phenylsulfonyl)pyrazolo[1,5-a]pyrimidine-2,7-diamines 6 and 7
(Scheme 1).
N 2,5-Dimethyl-3-(phenylsulfonyl)-pyrazolo[1,5-a]-
pyrimidine-2,7-diamines 10−16 (Scheme 3). Compounds 54−
56 (1 mmol each) were stirred separately in a mixture with dioxane
(1 mL) and were treated with primary or secondary amines (5 mmol)
(or solutions of methylamine or dimethylamine in MeOH). The mixtures
were stirred at 70 °C for 1 h (reaction monitored by LC-MS) and
To a suspension of N5-methyl-4-(phenylsulfonyl)-1H-pyrazole-3,5-
diamines 33 and 34 (3.17 mmol each) in acetic acid (3 mL) and
3-aminocrotononitrile, 35 (520 mg, 6.34 mmol) was added. The
reaction mixtures were heated at 100 °C for 15 h. The precipitates of
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dispersed into ice/water (6 mL). Resulting mixtures were treated with
0.5 mL of 5N aq NaOH and stirred for 0.5 h. Formed precipitates
were separated by centrifugation, twice washed with water, i-PrOH
(twice), and dried. To obtain HCl salts, the products 10−16 were
dissolved in CHCl3 and treated with an excess of 6N solution of HCl
(as AcCl in EtOH) and then diluted with EtOAc. The formed
precipitates were separated by centrifugation, washed with EtOAc
(twice), acetone (twice), and dried. Compounds 10−16 were thus
obtained as hydrochlorides.
obtained with a corresponding ratio of 87:13 (ratio was determined by
1H NMR analysis). After cooling, the formed precipitate was filtered,
washed with ethanol and ether, and dried. The yield of obtained
isomer 40 was 65%. The mother liquor was evaporated in vacuo, 2-
propanol was added and the formed precipitate was filtered, washed
with hexane, and dried. The other isomer was isolated by
recrystallization from MeCN. The yield of obtained isomer 39 was
9%. The phthalimido-derivatives 39 and 40 (1 mmol each) were
refluxed separately with 4 mmol of hydrazine hydrate in 5 mL of
EtOH for 4 h. After cooling, each mixture was filtered and evaporated
in vacuo. The residues were treated with DCM, filtered, and
evaporated in vacuo. The 7-aminomethyl-5-methyl-3-phenylsulfonyl-
pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-amine (17) and 5-aminometh-
yl-7-methyl-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-
amine (21) were converted to hydrochlorides 17·HCl and 21·HCl by
an equivalent amount of 6 M ethanolic HCl.
10·HCl: 3-Phenylsulfonyl-5,N 2,N7-trimethyl-pyrazolo[1,5-a ]-
1
pyrimidine-2,7-diamine hydrochloride (yield: 76%). H NMR (DMSO-
d6) δ 8.00 (d, J = 6.4 Hz, 2H), 7.55 (m, 4H), 6.15 (m, 1H), 6.13 (s, 1H),
2.90 (m, 6H), 2.36 (s, 3H). HRMS calculated for C15H17N5O2S (M +
H) 332.118120, found 332.1186. MS-ESI calculated for C15H17N5O2S
(M + H) 332, found m/z 332. LC-MS (UV-254), purity: 98%.
11·HCl: 3-Phenylsulfonyl-5,N2,N7,N7-tetramethyl-pyrazolo[1,5-a]-
pyrimidine-2,7-diamine hydrochloride (yield: 82%). 1H NMR
(DMSO-d6) δ 8.02 (d, J = 68 Hz, 2H), 7.55 (m, 3H), 6.22 (s, 1H),
6.16 (m, 1H), 3.24 (s, 6H), 2.88 (d, J = 5.2 Hz, 3H), 2.37 (s, 3H).
HRMS calculated for C16H19N5O2S (M + H) 346.133770, found
346.1326. MS-ESI calculated for C16H19N5O2S (M + H) 346, found
m/z 346. LC-MS (UV-254), purity: 98%.
17·HCl: (7-Aminomethyl-5-methyl-3-phenylsulfonylpyrazolo-
1
[1,5-a]pyrimidin-2-yl)-methyl-amine hydrochloride (yield: 37%). H
NMR (DMSO-d6), δ 8.76 (brs, 3H), 8.03 (m, 2H), 7.59 (m, 3H), 7.15
(s, 1H), 6.51 (brm, 1H), 4.39 (s, 2H), 2.95 (d, J = 2.4 Hz, 3H), 2.56
(s, 3H). HRMS calculated for C15H17N5O2S (M + H) 332.118120,
found 332.1176. MS-ESI calculated for C15H17N5O2S (M + H) 332,
found m/z 332. LC-MS (UV-254), purity: 98%.
12·HCl: N7-(2-Dimethylamino-ethyl)-5,N2-dimethyl-3-phenylsul-
fonyl-pyrazolo[1,5-a]pyrimidine-2,7-diamine hydrochloride (yield:
21·HCl: (5-Aminomethyl-7-methyl-3-phenylsulfonylpyrazolo-
1
1
65%). H NMR (DMSO-d6), δ: 10.07 (brs, 1H), 8.02 (d, J = 8.0
[1,5-a]pyrimidin-2-yl)-methyl-amine hydrochloride (yield: 32%). H
Hz, 2H), 7.52−7.62 (m, 3H), 6.95 (s, 1H), 6.72 (brt, J = 5.7 Hz, 1H),
3.69−3.76 (m, 2H), 3.29−3.35 (m, 2H), 2.80 (s, 6H), 2.54 (s, 3H),
2.47 (s, 3H). HRMS calculated for C18H24N6O2S (M + H)
389.175969, found 389.1765. MS-ESI calculated for C18H24N6O2S
(M + H) 389, found m/z 389. LC-MS (UV-254), purity: 98%.
13·HCl: N7-(2-Dimethylamino-ethyl)-5,N2,N7-trimethyl 3-phenyl-
sulfonyl-pyrazolo[1,5-a]pyrimidine-2,7-diamine hydrochloride (yield:
NMR (DMSO-d6), δ 8.50 (s, 3H), 8.14 (d, J = 7.6 Hz, 2H), 7.62 (t, J =
7.2 Hz, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.12 (s, 1H), 6.50 (d, J = 4.0 Hz,
1H), 4.24 (s, 2H), 2.93 (d, J = 4.4 Hz, 3H), 2.62 (s, 3H). HRMS
calculated for C15H17N5O2S (M + H) 332.118120, found 332.1191.
MS-ESI calculated for C15H17N5O2S (M + H) 332, found m/z 332.
LC-MS (UV-254), purity: 98%.
[7-Dimethylaminomethyl-5-methyl-3-phenylsulfonyl-
pyrazolo[1,5-a]pyrimidin-2-yl]-methyl-amine Hydrochloride
(18) (Scheme 6) and [7-(2-Dimethylamino-ethyl)-5-methyl-3-
phenylsulfonyl-pyrazolo[1,5-a]pyrimidin-2-yl]-methyl-amine
Hydrochloride (20) (Scheme 5). Formalin (83 mL of 37% , 91 mg,
1.12 mmol) was added to solutions of amines 17 and 19 (the synthesis
procedure of 19 is described below) (0.28 mmol each) in 3 mL of
DCM, followed by the addition of NaBH(OAc)3 (159 mg, (0.84 mmol).
The mixture was stirred for 4 h at ambient temperature, treated with 10%
K2CO3 solution, and extracted with DCM. The organic layer was dried
over Na2SO4 and evaporated in vacuo. The residue was dissolved in
acetone and treated with an excess of 6 M ethanolic HCl. Subsequent
evaporation and recrystallization from EtOH yielded compounds 18·HCl
and 20·HCl, correspondingly.
1
69%). H NMR (DMSO-d6), δ: 10.46 (brs, 1H), 8.01 (d, J = 8.0 Hz,
2H), 7.52−7.62 (m, 3H), 7.04 (s, 1H), 3.73 (t, J = 6.6 Hz, 2H), 3.36
(t, J = 6.6 Hz, 2H), 3.03 (s, 3H), 2.78 (s, 6H), 2.58 (s, 3H), 2. 48 (s,
3H). HRMS calculated for C19H26N6O2S (M + H) 403.191619, found
403.1906. MS-ESI calculated for C19H26N6O2S (M + H) 403, found
m/z 403. LC-MS (UV-254), purity: 99%.
14·HCl: N7-(3-Dimethylamino-propyl)-5,N2-dimethyl-3-phenylsul-
fonyl-pyrazolo[1,5-a]pyrimidine-2,7-diamine hydrochloride (yield:
1
58%). H NMR (DMSO-d6), δ: 10.52 (s, 1H), 8.29 (s, 1H), 8.06 (d,
J = 6.8 Hz, 2H), 7.60 (m, 3H), 6.53 (m, 1H), 6.44−6.17 (brm, 1H),
3.51 (m, 2H), 3.06 (m, 2H), 2.93 (s, 3H), 2.69 (d, J = 4.8 Hz, 5H), 2.45
(s, 3H), 2.01 (m, 2H). HRMS calculated for C19H26N6O2S (M + H)
403.191619, found 403.1920. MS-ESI calculated for C19H26N6O2S
(M + H) 403, found m/z 403. LC-MS (UV-254), purity: 98%.
15·HCl: (5-Methyl-7-morpholin-4-yl-3-phenylsulfonyl-pyrazolo-
18·HCl: (7-Dimethylaminomethyl-5-methyl-3-phenylsulfonyl-
pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-amine hydrochloride (yield:
1
1
27%). ESI-MS 360. H NMR (DMSO-d6), δ: 10.94 (brs, 1H), 8.04
[1,5-a]pyrimidin-2-yl)-methyl-amine hydrochloride (yield: 84%). H
(m, 2H), 7.55−7.64 (m, 3H), 7.37 (s, 1H), 6.51 (brm, 1H), 4.69 (s,
2H), 2.95 (brm, 3H), 2.86 (s, 6H), 2.56 (s, 3H). HRMS calculated for
C17H21N5O2S (M + H) 360.14942, found 360.1489. MS-ESI
calculated for C17H21N5O2S (M + H) 360, found m/z 360. LC-MS
(UV-254), purity: 99%.
20·HCl: [7-(2-Dimethylamino-ethyl)-5-methyl-3-phenylsulfonyl-
pyrazolo[1,5-a]pyrimidin-2-yl]-methyl-amine hydrochloride (yield:
18%). 1H NMR (DMSO-d6), δ: 10.40 (brs, 1H), 8.03 (m, 2H),
7.54−7.63 (m, 3H), 7.01 (s, 1H), 6.44 (q, J = 4.8 Hz, 1H), 3.49 (t, J =
7.0 Hz, 2H), 3.41 (t, J = 7.0 Hz, 2H), 2.93 (d, J = 4.8 Hz, 3H), 2.81 (s,
6H), 2.51 (s, 3H). HRMS calculated for C18H23N5O2S (M + H)
374.16507, found 374.165. MS-ESI calculated for C18H23N5O2S (M +
H) 374, found m/z 374. LC-MS (UV-254), purity: 98%.
NMR (DMSO-d6), δ: 8.00 (d, J = 6.4 Hz, 2H), 7.56 (m, 3H), 6.40 (s,
1H), 6.24 (m, 1H), 3.73 (s, 4H), 3.71 (s, 4H), 2.87 (d, J = 5.2 Hz,
3H), 2.41 (s, 3H). MS-ESI calculated for C18H21N5O3S (M + H) 388,
found m/z 388. LC-MS (UV-254), purity: 98%.
16·HCl: N,5-Dimethyl-7-(4-methylpiperazin-1-yl)-3-phenylsulfonyl-
1
pyrazolo[1,5-a]pyrimidin-2-amine hydrochloride (yield: 86%). H
NMR (DMSO-d6), δ: 11.24 (brs, 1H), 7.99 (d, J = 8.0 Hz, 2H), 7.50−
7.60 (m, 3H), 6.54 (s, 1H), 6.24 (brs, 1H), 4.50−4.60 (m, 4H), 3.40−
3.50 (m, 4H), 2.87 (s, 3H), 2.75 (d, J = 4.3 Hz, 3H), 2.41 (s, 3H).
HRMS calculated for C19H17N5O2S (M + H) 380.118120, found
380.1175. MS-ESI calculated for C19H17N5O2S (M + H) 380, found
m/z 380. LC-MS (UV-254), purity: 98%.
7-Aminomethyl-5-methyl-3-phenylsulfonyl-pyrazolo[1,5-a]-
pyrimidin-2-yl)-methyl-amine (17) and 5-Aminomethyl-7-
methyl-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-
amine (21) (Scheme 2). A solution of N5-methyl-4-phenylsulfonyl-1H-
pyrazole-3,5-diamine (33) (0.60 g, 2.38 mM) and 2-(2,4-dioxopentyl)-
isoindol-1,3-dione (49) (1.17 g, 4.76 mM) in acetic acid (20 mL) was
heated at 80 °C for 3 h. A mixture of isomers 2-(3-phenylsulfonyl-5-
methyl-2-methylamino-pyrazolo[1,5-a]pyrimidin-7-ylmethyl)-isoindole-
1,3-dione (39) and 2-(3-phenylsulfonyl-7-methyl-2-methylamino-
pyrazolo[1,5-a]pyrimidin-5-ylmethyl)-isoindole-1,3-dione (40) was
N 2,5,7-Trimethyl-3-(phenylsulfonyl)pyrazolo[1,5-a]-
pyrimidine-2,6-diamine (22) (Scheme 4). To a suspension of N-
(5,7-dimethyl-2-methylamino)-3-phenylsulphonyl)-pyrazolo[1,5-a]-
pyrimidine-6-yl)acetamide (23) (100 g, 0.37 M) in 1350 mL of 3:1
methanol−water mixture, KOH (135 g, 2.4 M) was added. The
reaction mixture was refluxed for 72 h. After completion of the
reaction (LC-MS control), the formed precipitate was thoroughly
dispersed with rotary dispergator or ultrasound, filtered, and first
washed with an alkaline solution and then with water.
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22: N 2,5,7-Trimethyl-3-(phenylsulfonyl)pyrazolo[1,5-a ]-
pyrimidine-2,6-diamine (yield 94%). H NMR (DMSO-d6), δ: 7.98
19 HCl: 7-(2-Aminoethyl)-5-methyl-3-phenylsulfonyl-pyrazolo[1,5-
a]pyrimidin-2-yl]-methylamine hydrochloride (yield: 46%). H NMR
1
1
(d, J = 8.4 Hz, 2H), 7.51−7.59 (m, 3H), 4.59 (brm, 3H), 2.89 (s, 3H),
2.55 (s, 3H), 2.47 (s, 3H). HRMS calculated for C15H17N5O2S (M +
H) 332.11812, found 332.1175. MS-ESI calculated for C15H17N5O2S
(M + H) 332, found m/z 332. LC-MS (UV-254), purity: 99%.
5-Methyl-2-methylamino-3-phenylsulfonyl-pyrazolo[1,5-a]-
pyrimidine (23) (Scheme 2). N3-Methyl-4-(phenylsulfonyl)-1H-
pyrazole-3,5-diamine 33 (27.8 mmol) was mixed with a 1,3-dicarbonyl
compound, 51 (83.4 mmol), and AcOH (20 mL). The mixture was
stirred overnight at ambient temperature with subsequent heating for
1−2 h at 80−100 °C and cooling down. Precipitate was filtered,
washed with AcOH and i-PrOH, and dried to produce 5-methyl-2-
methylamino-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidine (23) as col-
orless solid compound (yields: 63−75%).
(DMSO-d6), δ: 8.03 (brs, 3H), 8.03 (m, 2H), 7.59 (m, 3H), 6.96 (s,
1H), 6.41 (m, 1H), 3.29 (brm, 4H), 2.92 (d, J = 3.6 Hz, 3H), 2.51 (s,
3H). HRMS calculated for C16H19N5O2S (M + H) 346.13377, found
346.1340. MS-ESI calculated for C16H19N5O2S (M + H) 346, found
m/z 346. LC-MS (UV-254), purity: 98%.
25 HCl: 6-Aminomethyl-5,7-dimethyl-3-phenylsulfonyl-pyrazolo-
[1,5-a]pyrimidin-2-yl]-methylamine hydrochloride (yield: 52%). 1H
NMR (400 MHz, DMSO-d6), δ: 8.36 (brs, 3H), 8.03 (m, 2H), 7.54−
7.63 (m, 3H), 6.43 (q, J = 4.8 Hz, 1H), 2.93 (d, J = 4.8 Hz, 3H), 2.74
(s, 3H), 2.66 (s, 3HH). HRMS calculated for C16H19N5O2S (M + H)
346.13377, found 346.1339. MS-ESI calculated for C16H19N5O2S
(M + H) 346, found m/z 346. LC-MS (UV-254), purity: 98%.
26 HCl: 6-Aminomethyl-5,7-dimethyl-3-(3-fluorophenylsulfonyl)-
pyrazolo[1,5-a]pyrimidin-2-yl]-methylamine hydrochloride (yield:
23: N-(5,7-Dimethyl-2-methylamino-3-phenylsulfonyl-pyrazolo-
1
1
[1,5-a]pyrimidin-6-yl)-acetamide (yield 83%). H NMR (DMSO-d6),
54%). H NMR (400 MHz, DMSO-d6), δ: 8.06 (brs, 3H), 7.85 (m,
δ: 9.66 (s, 1H), 8.02 (d, J = 6.8 Hz, 2H), 7.56 (m, 3H), 6.39 (q, J = 4.8
Hz, 1H), 2.92 (d, J = 4.8 Hz, 3H), 2.45 (s, 3H), 2.39 (s, 3H), 2.08 (s,
3H). HRMS calculated for C17H19N5O3S (M + H) 374.128685, found
374.1291. MS-ESI calculated for C17H19N5O3S (M + H) 374, found
m/z 374. LC-MS (UV-254), purity: 98%.
2H), 7.63 (dt, J1 = 8 Hz, J2 = 5.6 Hz, 1H), 7.48 (dt, J1 = 8.8 Hz, J2 =
2.4 Hz, 1H), 6.47 (q, J = 4.8 Hz, 1H), 4.17 (s, 2H), 2.92 (d, J = 5.2 Hz,
3H), 2.72 (s, 3H), 2.65 (s, 3H). HRMS calculated for C16H18FN5O2S
(M + H) 364.124348, found 364.1244. MS-ESI calculated for
C16H18FN5O2S (M + H) 364, found m/z 364. LC-MS (UV-254),
purity: 98%.
N2,N6,N6,5,7-Pentamethyl-3-(phenylsulfonyl)pyrazolo[1,5-
a]pyrimidine-2,6-diamine (24) (Scheme 4). To a suspension of
N2,5,7-trimethyl-3-(phenylsulphonyl)pyrazolo[1,5-a]pyrimidine-2,6-
diamine (22) (75 g, 0.23 mol;) in 3750 mL of ethanol, a 3 M solution
of 370 mL of H2SO4 was added. The mixture was heated to 90 °C and
then cooled to a room temperature. Formalin solution (35%, 116.5 g,
1.4 mol) was added, the mixture was stirred for 30 min, and NaBH4
(56 g, 1.5 mol) was added portionwise to maintain the temperature
below 25 °C. After the reaction was complete (LC-MS control), the
precipitate was filtered, thoroughly washed with water, and dried.
Ethanol from the mother liquor was evaporated in vacuo, and the
additional precipitate of 24 was filtered, washed with water, and dried
(yield: 97%).
27 HCl: 6-Aminomethyl-5,7-dimethyl-3-(3-chlorophenylsulfonyl)-
pyrazolo[1,5-a]pyrimidin-2-yl]-methylamine hydrochloride (yield:
1
55%). H NMR (400 MHz, DMSO-d6), δ: 8.34 (brs, 3H), 8.10 (t,
J = 1.6 Hz, 1H), 7.97 (ddd, J1 = 7.6 Hz, J2 = 1.6 Hz, J3 = 1.2 Hz, 1H),
7.69 (ddd, J1 = 8.0 Hz, J2 = 2.0 Hz, J3 = 1.2 Hz, 1H), 7.61(t, J = 8.0 Hz,
1H), 6.48 (q, J = 4.8 Hz, 1H), 4.14 (s, 2H), 2.92 (d, J = 4.8 Hz, 3H),
2.75 (s, 3H), 2.67 (s, 3H). HRMS calculated for C16H18ClN5O2S (M + H)
380.094797, found 380.0948. MS-ESI calculated for C16H18ClN5O2S
(M + H) 380, found m/z 380. LC-MS (UV-254), purity: 99%.
31 HCl: 6-(2-Aminoethyl)-5,7-dimethyl-3-phenylsulfonyl-pyrazolo-
[1,5-a]pyrimidin-2-yl]-methylamine hydrochloride (yield: 68%). 1H
NMR (400 MHz, DMSO-d6), δ: 8.17 (brs, 3H), 8.01 (d, J = 7.6 Hz,
2H), 7.53−7.61 (m, 3H), 2.98 (m, 2H), 2.92 (s, 3H), 2.89 (brs, 2H),
2.64 (s, 3H), 2.56 (s, 3H). HRMS calculated for C17H21N5O3S (M +
H) 360.14942, found 360.1501. MS-ESI calculated for C17H21N5O3S
(M + H) 360, found m/z 360. LC-MS (UV-254), purity: 98%.
[6-(2-Dimethylaminomethyl)-5,7-dimethyl-3-phenylsulfon-
yl-pyrazolo[1,5-a]pyrimidin-2-yl]-methylamines 28−30 and [6-
Dimethylaminoethyl)-5,7-dimethyl-3-phenylsulfonyl-
pyrazolo[1,5-a]pyrimidin-2-yl]-methylamine (32) (Scheme 5).
To a solution of amine 25−27 and 31 (1 mmol each) in 10 mL of
DCM, formalin (3 mmol) and sodium triacetoxyborohydride (0.53 g,
2.5 mmol) were added. The mixture was stirred at room temperature for
3 h, and then more formalin (3 mmol) and sodium triacetoxyborhydride
(0.53 g, 2.5 mmol) were added and the stirring continued for 12 h.
Mixtures were diluted with water, extracted with DCM, washed with
10% potassium carbonate solution, dried over Na2SO4, and evaporated
in vacuo. The product was isolated by column chromatography (eluent:
hexane/ethylacetate/triethylamine in ratio of 30:10:1). Hydrochlorides
were obtained by addition of 3 M HCl solution in dioxane to a solution
of amine in acetone.
1
24: H NMR (DMSO-d6), δ: 8.02 (m, 2H), 7.53−7.61 (m, 3H),
6.29 (br, 1H), 2.91 (s, 3H), 2.74 (s, 6H), 2.57 (s, 3H), 2.49 (s, 3H).
HRMS calculated for C17H21N5O2S (M + H) 360.14942, found
360.1493. MS-ESI calculated for C17H21N5O2S (M + H) 360, found
m/z 360. LC-MS (UV-254), purity: 98%.
6-Substituted ω-[5,7-Dimethyl-2-methylamino-3-arylsulfon-
yl-pyrazolo[1,5-a]pyrimidin-2 or 6-yl]alkylamines 19, 25−27,
and 31 (Scheme 5). To a suspension of a corresponding ester 41−
45 (0.73 mmol each) in ethanol (20 mL), a solution of potassium
hydroxide (96 mg, 1.5 mmol) in water (2 mL) was added. The
mixtures were stirred for 3 h at 80 °C. Then ethanol was evaporated in
vacuo, and water (10 mL) was added. The obtained solutions were
acidified with 20% HCl (270 mL). The residues were filtered, washed
with water, and dried in air to obtain acids 57−61 (yield: 95−97%).
To a suspension of each starting acid 57−61 (1.6 mmol each) in
acetone (12 mL), a solution of Et3N (263 mL, 1.9 mmol) in acetone
(1 mL) was added at 0 °C. After dissolution of the acid, a solution of
ethyl chloroformate (197 mL, 2.1 mmol) in acetone (1 mL) was
added. The mixture was stirred for 30 min at 0 °C, and then a solution
of sodium azide (0.16 g, 2.45 mmol) in water (450 mL) was added,
maintaining the same temperature. The mixture was stirred for 1 h at
0 °C, and then water (20 mL) was added. The obtained acylazides
62−66 were extracted with DCM, the organic layer was dried with
sodium sulfate, and the solvent was evaporated in vacuo at 30 °C to
obtain acylazides 62−66, which were used in the next step without
further purification. Solutions of each acylazide 62−66 in a mixture of
DCM and dioxane (5 mL:5 mL) were added dropwise to a boiled
dioxane (30 mL). The reaction mixture was refluxed for 1 h, cooled to
70−80 °C, and 20% solution of hydrochloric acid (450 mL) was
added. In 20−30 min, hydrochlorides of the desired compounds,
19·HCl, 25·HCl−27·HCl, 31·HCl, began to precipitate. The reaction
mixture was stirred for 2 h at 70−80 °C, cooled to room temperature,
the residue was filtered and washed with dioxane and dried in vacuo
to obtain compounds 19·HCl, 25·HCl−27·HCl, 31·HCl (yield: 46−
68%).
28 HCl: [6-Dimethylaminomethyl)-5,7-dimethyl-3-phenylsulfonyl-
pyrazolo[1,5-a]pyrimidin-2-yl]-methylamine hydrochloride (yield:
57%). 1H NMR (DMSO-d6), δ: 10.10 (brs, 1H), 8.04 (m, 2H),
7.54−7.63 (m, 3H), 6.47 (q, J = 4.4 Hz, 1H), 2.93 (d, J = 4.4 Hz, 3H),
2.80 (s, 6H), 2.76 (s, 3H), 2.72 (s, 3H). MS-ESI calculated for
C18H23N5O2S (M + H) 374, found m/z 374. LC-MS (UV-254),
purity: 98%.
29 HCl: [6-Dimethylaminomethyl)-5,7-dimethyl-3(3-fluorophenyl-
sulfonyl)-pyrazolo[1,5-a]pyrimidin-2-yl]-methylamine hydrochloride
1
(yield: 37%). H NMR (DMSO-d6), δ: 10.3 (brs, 1H), 7.87 (t, J =
7.6 Hz, 2H), 7.64 (m, 1H), 7.49 (m, 1H), 6.48 (m, 1H), 4.46 (d, J =
5.6 Hz, 2H), 2.93 (d, J = 4.4 Hz, 3H), 2.80 (d, J = 2.8 Hz, 6H), 2.77 (s,
3H), 2.73 (s, 3H). HRMS calculated for C18H22FN5O2S (M + H)
392.155648, found 392.1555. MS-ESI calculated for C18H22FN5O2S
(M + H) 392, found m/z 392. LC-MS (UV-254), purity: 98%.
8171
dx.doi.org/10.1021/jm201079g|J. Med. Chem. 2011, 54, 8161−8173

Journal of Medicinal Chemistry
Article
30 HCl: [6-Dimethylaminomethyl)-5,7-dimethyl-3-(3-chlorophe-
nylsulfonyl)-pyrazolo[1,5-a]pyrimidin-2-yl]-methylamine hydrochlor-
ide (yield: 56%). 1H NMR (DMSO-d6), δ: 10.1 (brs, 1H), 8.10 (t, J =
2.0 Hz, 1H), 7.99 (dt, J1 = 7.6 Hz, J2 = 1.6 Hz, 1H), 7.70 (ddd, J1 = 8.0
Hz, J2 = 2.0 Hz, J3 = 0.8 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 6.50 (q, J =
4,8 Hz, 1H), 4.47 (d, J = 5.6 Hz, 2H), 2.93 (d, J = 4.8 Hz, 3H), 2.82 (s,
3H), 2.81 (s, 3H), 2.77 (s, 3H), 2.73 (s, 3H). MS-ESI calculated for
C18H22ClN5O2S (M + H) 409, found m/z 409. LC-MS (UV-254),
purity: 98%.
have been plotted. This yielded the Schild plots for the tested
compounds, from which pA2 values were determined as an intersection
of the straight line with the X-axis.
Receptor Selectivity/Specificity Profiling. The panel of 33
different receptors expressed in cell membranes was used for the
profiling. The compounds were tested at a concentration of 1 μM in
duplicates at equilibrium binding conditions with radiolabeled ligands
specific to each of the receptor. The testing was performed by Ricerka
(Taiwan) in accordance with their internally optimized protocols.
32 HCl: [6-(2-Dimethylaminoethyl)-5,7-dimethyl-3-phenylsulfonyl-
pyrazolo[1,5-a]pyrimidin-2-yl]-methylamine hydrochloride (yield:
61%). H NMR (DMSO-d6), δ: 10.78 (brs, 1H), 8.02 (d, J = 13.2
Hz, 2H), 7.53−7.62 (m, 3H), 6.34 (q, J = 4.8 Hz, 1H), 3.09 (brm,
4H), 2.92 (d, J = 4.8 Hz, 3H), 2.82 (s, 6H), 2.67 (s, 3H), 2.60 (s, 3H).
MS-ESI calculated for C19H25N5O2S (M + H) 388, found m/z 388.
LC-MS (UV-254), purity: 99%.
ASSOCIATED CONTENT
* Supporting Information
Synthesis procedures, analytical and spectral data of the
synthesized compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
1
S
Cell-Based 5-HT6 Receptor Functional Assay. The 5-HT6R
was subcloned 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 μg/mL), as recommended by the T-Rex system
manufacturer (Invitrogen, Carlsbad, CA), a day before the experi-
ments. 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 pipet tip several times to break down cell aggregates, washed
with Serum Free Medium, and counted. The cells were resuspended to
0.67 × 106 cells/mL in SB2 buffer, HBSS, supplemented with 5 mM
HEPES, pH 7.4, 0.05% BSA, and 1 mM IBMX (SigmaAldrich, St.
Louis, MO) containing Alexa Fluor 647-anti cAMP antibody (from
LANCE cAMP 384 kit, PerkinElmer, Waltham, MA). Then 6 μL of
the cell suspension (∼4000 cells) were aliquoted into the wells of 384-
well assay plates (PerkinElmer White OptiPlates). The test
compounds were premixed at different concentrations with serotonin
hydrochloride (Sigma, MO), and the mixtures were added to the assay
plates with the cells (final concentrations: serotonin, 10 nM; DMSO,
0.32%; IBMX, 500 mM). Each assay plate contained serotonin and
cAMP standard concentration curves for quality assurance. Cells, after
2 h of incubation with the compound/serotonin mixtures, were treated
as described in the cAMP LANCE assay kit protocol (PerkinElmer,
Waltham, MA). The LANCE signal was measured using the
multimode plate reader, VICTOR 3 (PerkinElmer, Waltham, MA),
with built-in settings for the LANCE detection.
AUTHOR INFORMATION
Corresponding Author
*Phone: 1-858-794-4860. Fax: 1-858-794-4931. E-mail:
iokun@chemdiv.com.
■
ACKNOWLEDGMENTS
■
We thank Dr. Norman Lee from Chemical Instrumentation
Center, Boston University, for HRMS measurements. We also
extend our gratitude to Dr. F. Lakner for his valuable
suggestions in improving the manuscript and help with
language and proofreading during the manuscript preparation.
ABBREVIATIONS USED
■
5-HT6R, serotonin 5-HT6 receptor; cAMP, cyclic adenosine
monophpsphate; CNS, central nervous system; HBA, hydrogen
bond acceptor; HBD, hydrogen bond donor; HEK 293, human
embryonic kidney cell line; HYD, hydrophobic region; IHB,
intramolecular hydrogen bond; IC50, concentration of half-
maximal inhibition; IHB, intramolecular hydrogen bond; Ki,
activity constant; PD, proton donor; PhM, pharmacophore
model; PI, positively ionizable group; SAR, structure−activity
relationship
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