The Synthesis and Biological Evaluation of N-Substituted 1H-Benzimidazol-2-yl-1H-pyrazole-3,5-diamines

Article abstract of DOI:10.1002/jhet.2315

The synthesis of 1H-benzimidazol-2-yl-1H-pyrazole-3,5-diamines has been developed. Synthesized bisheteroaryls contain two privileged medicinal scaffolds, aminopyrazole and benzimidazole, with two diversity positions at N1 of benzimidazole and C3 of pyrazo

Full text of DOI:10.1002/jhet.2315

Month 2015
The Synthesis and Biological Evaluation of N-Substituted
1H-Benzimidazol-2-yl-1H-pyrazole-3,5-diamines
a
b
a
*
Lukáš Jedinák, Vladimír Kryštof, and Petr Cankař
aDepartment of Organic Chemistry, Faculty of Science, Palacky University, 17. Listopadu 1192/12, 771 46 Olomouc,
Czech Republic
bLaboratory of Growth Regulators, Institute of Experimental Botany AS CR and Palacky University, Slechtitelu 11, 783
71 Olomouc, Czech Republic
*
E-mail: cankar@orgchem.upol.cz
Received April 17, 2014
DOI 10.1002/jhet.2315
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
The synthesis of 1H-benzimidazol-2-yl-1H-pyrazole-3,5-diamines has been developed. Synthesized
bisheteroaryls contain two privileged medicinal scaffolds, aminopyrazole and benzimidazole, with two
diversity positions at N1 of benzimidazole and C3 of pyrazole, respectively. The three-step synthesis
includes the Mitsunobu N-alkylation of benzimidazole and subsequent one-pot formation of aminopyrazole
involving substitution of methylthio groups with amine and hydrazine followed with final ring closure.
Inhibitory activity toward cyclin-dependent kinase 2/cyclin E and cytotoxicity against two cancer cell lines
were evaluated for all novel pyrazoles. Two compounds showed modest cyclin-dependent kinase inhibition
activity and cytotoxicity against cancer cell lines K562 and MCF7.
J. Heterocyclic Chem., 00, 00 (2015).
INTRODUCTION
applications in the development of chemosensors, lasers, and
molecular switches [6].
Pyrazole is a five-membered heterocycle, which exhibits
a wide range of biological activities [1]. Examples of
clinically used pyrazole-containing drugs include nonste-
roidal anti-inflammatory and analgesic celecoxib, antidote
in alcohol poisoning Fomepizole, and antibacterial
Sulfaphenazole. Additionally, metal complexes bearing
pyrazole ligands were studied as organic light-emitting
diodes, liquid crystals, and catalysts for the carbon–carbon
cross-coupling reactions [2].
The standard method for pyrazole preparation involves
cyclization of hydrazine or aryl/alkyl hydrazines with
1,3-dicarbonyl compounds such as carboxylic acid esters,
ketones, nitriles, enamines, and enolethers [3]. Other ap-
proaches toward pyrazoles include palladium-catalyzed
four-component coupling of arylhalide, terminal alkyne,
hydrazine, and carbon monoxide [4a]; copper-catalyzed
domino C-N coupling/hydroamination [4b]; 1,3-dipolar
cycloaddition of aryldiazo compounds with terminal
alkynes [4c]; base-mediated cycloaddition of hydrazones
with nitroolefines [4d]; and rhodium-catalyzed cyclization
of hydrazines with alkynes [4e] For recent review on the
synthesis of pyrazoles, see Reference [4f].
The common synthesis of benzimidazoles utilizes
condensation of ortho-aminoanilines with carboxylic acid
derivatives [7] or condensation with aldehydes under
oxidative conditions [8]. The key intermediate, ortho-
aminoanilines, are often synthesized in two steps from
ortho-halonitrobenzenes via nucleophilic substitution of
halogen with primary amine and subsequent reduction of
the nitro group. Some variations utilize ortho-haloanilines
instead; the second amino group is introduced by palla-
dium or copper-mediated C-N coupling [9]. In some cases,
introducing alkyl substituents at N1 of completed benz-
imidazole core can be advantageous. Alkylation of N1
using alkyl halides or triflates has been reported, but this
method suffers from the unwanted side reaction,
quarterization [10]. On the other hand, Mitsunobu alkyl-
ation of nitrogen heterocycles such as pyrimidinones,
1,2,3-triazoles, indoles, carbazoles, and imidazoles usually
proceeds under mild conditions and usually with high
chemoselectivity and regioselectivity [11]. Despite of this,
there are only few examples of Mitsunobu N-alkylation of
benzimidazoles in the literature [12].
During the course of our research program toward
inhibitors of cyclin-dependent kinases (CDK), we have
found diaminopyrazoles as potent and selective inhibitors
of CDK with strong antiproliferative activity in vitro
[13]. In this article, we disclose the three-step synthesis
of bisheteroaryls 1 containing diaminopyrazole and
Benzimidazole is one of the privileged scaffolds in the
medicinal chemistry. Compounds containing this motif dis-
play a wide range of pharmacological activities such as anti-
viral, antimicrobial, antiulcer, anticancer, anti-inflammatory,
and anticonvulsant [5]. Moreover, benzimidazoles have found
© 2015 HeteroCorporation

L. Jedinák, V. Kryštof, and P. Cankař
Vol 000
benzimidazole moieties connected through the C2 of
benzimidazole and C4 of pyrazole position, respectively.
Synthesized bisheteroaryls 1 were modified at N1 of benz-
imidazole and C3 of pyrazole position, respectively.
The model reaction of benzimidazole 2 and alcohol 4a
was examined under various conditions (Table 1). The
alkylation with two equivalents of diethyl azodicarboxylate
(DEAD) and triphenylphosphine in dioxane delivered
compound 5a in 69% yield (entry 1). When the reaction
was carried out in tetrahydrofuran (THF), the yield
increased to 74% (entry 2). Chlorinated solvents,
dichloromoethane and 1,2-dichloroethane, gave lower
yields of 56 and 54%, respectively (entries 3 and 4).
Diisopropyl azodicarboxylate (DIAD) is considered chem-
ically equivalent but a safer alternative to DEAD. In our
case, DIAD gave comparable result with DEAD (80 vs
74% yield, entry 5 vs entry 2). Thus, we continued further
experiments using safer DIAD. Reduced amount of
triphenylphosphine and DIAD to 1.6 equivalents still
RESULTS AND DISCUSSION
The general synthesis of bisheteroaryls 1 is depicted in
Scheme 1. Benzimidazole 2 was prepared according to
modified procedure described by Manfred [14]. Nitrile 3
reacted with carbon disulfide in the presence of sodium
hydroxide followed with the methylation of the thiol group
with methyl iodide. In the next step, various alkyl substit-
uents were introduced at N1 of benzimidazole 2 via
Mitsunobu alkylation.
Scheme 1. General synthesis of bisheteroaryls 1a–q.
Table 1
Optimization of the Mitsunobu reactiona.
Entry
Reagent (equivalent)
Solvent
Time (h)
Yieldb (%)
1
2
3
4
5
6
7
DEAD/PPh3 (2.0)
DEAD/PPh3 (2.0)
DEAD/PPh3 (2.0)
DEAD/PPh3 (2.0)
DIAD/PPh3 (2.0)
DIAD/PPh3 (1.6)
DIAD/PPh3 (1.2)
Dioxane
THF
DCM
DCE
THF
2
1
5
5
1
1
4
69
74
56
54
80
78
53c
THF
THF
THF, tetrahydrofuran; DCM, dichloromethane; DCE, dichloroethane.
aReactions were carried out with one equivalent of 2-(diethylamino)ethanol 4a and monitored by thin-layer
chromatography and liquid chromatography–mass spectrometry.
bIsolated yield after extraction to dichloromethane and chromatography.
cUnreacted starting material 2 was recovered (16%).
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1H-Benzimidazol-2-yl-1H-pyrazole-3,5-diamines: Synthesis and CDK
Inhibitory Activity
provided full conversion of starting material 2 resulting in
product 5a in 78% yield (entry 6). On the other hand,
further decrease of reagents to 1.2 equivalents led to in-
complete conversion. The title compound 5a was isolated
only in 53% yield together with 16% of unreacted starting
material 2 (entry 7). Thus, we used 1.6 equivalents of
DIAD/PPh3 in THF (entry 6, Table 1) as standard
conditions for the synthesis of N-alkylated benzimidazoles
5a–d with aminoalcohols 4a–d (Scheme 2).
reaction started with the substitution of methylthio group
with benzylamine leading to intermediate 6a, which subse-
quently underwent cyclization with hydrazine to yield the
desired bisheteroaryl 1a (Table 2). The substitution of
methylthio group with benzylamine, carried out at room
temperature, gave only low conversion in ethanol, acetoni-
trile, and THF (entries 1, 2, and 3). The reaction proceeded
smoothly at higher temperatures. More than 95% conver-
sion was achieved (entries 4, 5, and 6). Then, hydrazine
monohydrate was added, and the reaction mixture was
heated under reflux until the intermediate 6 was completely
transformed into bisheteroaryl 1 [monitored by liquid
chromatography–mass spectrometry (LC-MS)]. Good
yields of bisheteroaryl 1a were achieved in THF and etha-
nol (entries 4 and 6); however, the highest isolated yield
was observed in acetonitrile (entry 5).
Next, we examined the transformation of benzimidazole
5a into the final bisheteroaryl 1a derivative. The one-pot
Scheme 2. Mitsunobu N-alkylation with alcohols 4a–d.
Subsequently, optimized conditions were applied in the
synthesis of 17 bisheteroaryls 1a–q from intermediates
5a–d (Scheme 3). All compounds were prepared in good
to excellent isolated yields, 67–91%. The substitution of
methylthio group with aniline required longer reaction
time, 24 h, presumably because of lower nucleophilicity
of the amino group conjugated with aromatic system (entry
1h and 1n, Scheme 3).
Because of the similarity of 1H-benzimidazol-2-yl-1H-
pyrazole-3,5-diamines to previously discovered diami-
nopyrazoles [13], the biological activity of prepared com-
pounds was evaluated. The compounds have been tested in
kinase inhibition assays for their inhibitory potency toward
Table 2
Optimization of the one-pot aminopyrazole formation.
Entry
Solvent
T (°C)
Time (h)c
Conversion to 6a (%)a
Yield of 1a (%)b
1
2
3
4
5
6
EtOH
MeCN
THF
EtOH
MeCN
THF
r.t.
r.t.
r.t.
Reflux
Reflux
Reflux
22/—
22/—
22/—
8/14
8/14
8/14
44
56
49
>95
>95
>95
NI
NI
NI
72
80
71
NI, not isolated.
aEstimated by high-performance liquid chromatography.
bIsolated yield after column chromatography on silica gel.
cTime for the SMe substitution/cyclization.
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L. Jedinák, V. Kryštof, and P. Cankař
Vol 000
Scheme 3. The synthesis of bisheteroaryls 1a–q.
recombinant CDK2/cyclin E and for their cytotoxicity against
two cancer cell lines. None of the tested compounds was active,
with the exception of 1n and 1o (Table 3). These two com-
pounds displayed inhibitory activity toward CDK2 (IC50 values
24 and 62 μM, respectively) and also cytotoxicity in MCF7 and
K562 cell lines. Both 1n and 1o bear aromatic substituents,
while all inactive compounds have aliphatic side chains. How-
ever, aromatic substitution of the diaminopyrazole must be
combined with unsubstituted benzimidazole; any substitution
at nitrogen of the benzimidazole moiety is not compatible with
Table 3
Biological evaluation of bisheteroaryls 1.
Compound
CDK2/E (IC50
)
MCF7 (GI50
)
K-562 (GI50
)
Compound
CDK2/E (IC50
)
MCF7 (GI50
)
K-562 (GI50)
1a
1b
1c
1e
1h
1i
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1k
1l
>100
>100
>100
24.9
62.3
>100
>100
>100
>100
>100
33.32
63.35
>100
>100
>100
>100
>100
21.17
41.09
>100
>100
1m
1n
1o
1p
1q
1j
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1H-Benzimidazol-2-yl-1H-pyrazole-3,5-diamines: Synthesis and CDK
Inhibitory Activity
CDK2 inhibition, as seen for 1a, 1e, 1h, 1i, or 1k. These data
correlate with our previous findings, showing that small
aromatic substitution of 3,5-diaminopyrazole in position 4 is
important for CDK inhibition [13].
GENERAL PROCEDURE FOR MITSUNOBU
ALKYLATION
A mixture of 2-(1H-benzimidazol-2-yl)-3,3-bis(methylthio)-
prop-2-enenitrile 2 (0.26 g, 1.0 mmol), triphenylphosphine
(0.42 g, 1.6 mmol), DIAD (0.32 mL, 1.6 mmol), and appropri-
ate alcohol (1.0 mmol) in dry THF (8 mL) was stirred for 1 h
under nitrogen atmosphere. The resulting solution was
extracted to dichloromethane and washed with diluted hydro-
chloric acid. The aqueous layer of hydrochloride salt was
neutralized with 10% aqueous sodium hydroxide, and the
formed yellow oil was extracted to dichloromethane. The
crude material was purified with column chromatography on
silica gel, eluting with 0–10% methanol in dichloromethane
2-(1-(2-(Diethylamino)ethyl)-1H-benzo[d]imidazol-2-yl)-3,3-
CONCLUSION
In conclusion, a three-step protocol leading to 1H-
benzimidazol-2-yl-1H-pyrazole-3,5-diamines 1a–q from
readily available benzimidazole 2 was developed. The
Mitsunobu alkylation of benzimidazole 2 gave N-substituted
benzimidazoles 5a–d in high yields, 70–78%. Next,
the one-pot substitution of methylthio group and
cyclization with hydrazine afforded bisheteroaryls 1a–q
in good to high yields, 67–91%. As expected, the
substitution of methylthio group with aniline required
longer reaction time comparing with aliphatic amines, 24
versus 8 h, because of lower nucleophilicity of aromatic
amino group. Bisheteroaryls 1 were assayed toward
CDK2/cyclin E, but only compounds 1n and 1o showed
modest inhibition activity.
bis(methylthio)prop-2-enenitrile (5a).
Prepared according
to general procedure for Mitsunobu alkylation using
2-(diethylamino)ethanol 4a as pale yellow oil (0.28g,
78%), which solidified upon standing to low melting
1
almost white solid; H-NMR (400MHz, DMSO-d6) 0.73
(t, J = 7.0 Hz, 6H), 2.30 (s, 3H), 2.40 (q, J = 7.0 Hz, 4H),
2.64 (t, J = 6.1 Hz, 2H), 2.69 (s, 3H), 4.21 (t, J = 6.1 Hz,
2H), 7.25 (td, J = 7.4, 1.2 Hz, 1H), 7.32 (td, J = 7.4, 1.2 Hz,
1H), 7.62–7.69 (m, 2H); 13C-NMR (101MHz, DMSO-d6)
11.4, 17.4, 18.1, 43.3, 46.9, 51.1, 99.1, 111.1, 116.4,
EXPERIMENTAL SECTION
All starting materials, solvents, and reagents were purchased
from commercial suppliers and were used as received without
further purification. Melting points were determined on a Boetius
stage. The high-performance LC-MS (HPLC-MS) analyses were
carried out on an ultra-HPLC-MS system consisting of an Accela
ultra-HPLC chromatograph with a photodiode array detector and
a TSQ Quantum Access triple quadrupole mass spectrometer
(both Thermo Scientific, CA, USA), using a Nucleodur Gravity
C18 column (Macherey-Nagel, 1.8 μm, 2.1 × 50mm, Germany)
at 30°C and a flow rate of 800 μL/min. The atmospheric-pressure
chemical ionization source operated at a discharge current of
5 μA, vaporizer temperature of 400°C, and capillary temperature
119.5, 122.3, 123.2, 134.4, 142.3, 145.5, 167.1.
2-{1-[3-(Dimethylamino)propyl]-1H-benzimidazol-2-yl}-3,3-bis
(methylsulthio)prop-2-enenitrile (5b). Prepared according to
general procedure for Mitsunobu alkylation using
3-(dimethylamino)propanol 4b as pale yellow oil (0.27 g,
77%), which solidified upon standing to low melting almost
white solid; 1H-NMR (400 MHz, DMSO-d6) 1.87 (tt,
J = 7.0, 6.7 Hz, 2H), 2.10 (s, 6H), 2.17 (t, J= 6.7 Hz, 2H),
2.30 (s, 3H), 2.72 (s, 3H), 4.25 (t, J=7.0Hz, 2H), 7.23–7.30
(m, 1H), 7.34 (td, J =7.6, 1.2 Hz, 1H), 7.64–7.72 (m, 2H);
13C-NMR (101 MHz, DMSO-d6) 17.5, 18.4, 26.9, 42.0,
45.0, 55.7, 97.6, 111.1, 116.2, 119.6, 122.4, 123.4, 134.5,
142.3, 144.8, 169.2.
1
of 200°C. The H-NMR and 13C-NMR spectra were recorded
in DMSO-d6 at 20 °C on a Bruker 400 and 300 Fourier trans-
form NMR spectrometer, respectively. Elemental analyses
were performed on a Flash 2000 CHNS Elemental Analyzer
(Thermo Scientific).
3,3-Bis(methylthio)-2-(1-2-(pyridin-2-yl)ethyl)-1H-(benzo[d]
imidazol-2-yl)prop-2-enenitrile (5c). Prepared according to
general procedure for Mitsunobu alkylation using
2-(pyridin-2-yl)ethanol 4c as pale yellow oil (0.26 g,
70%), which solidified upon standing to low melting almost
2-(1H-Benzimidazol-2-yl)-3,3-bis(methylthio)prop-2-enenitrile
(2).
Compound 2 was prepared according modified procedure
described by Manfred et al.[14]
1
white solid; H-NMR (400 MHz, DMSO-d6) 2.26 (s, 3H),
To a solution of benzimidazole 3 (15.7 g, 100 mmol) in THF
(300 mL) was added aqueous solution of sodium hydroxide
(3.0 g, 120 mmol in 50 mL) and carbon disulfide (7.25 mL,
120 mmol). The mixture was stirred for 0.5 h; then, methyl iodide
(15.0 mL, 240 mmol) was added and stirring continued for
additional 2 h. Then, the mixture was extracted with ethyl acetate
(3× 40mL), and the solvent was removed under vacuum. The crude
material was crystallized from MeOH-CH2Cl2 to yield 2-(1H-
benzimidazol-2-yl)-3,3-bis(methylsulphanyl)prop-2-enenitrile as
yellow crystalline solid 10.38 g (39%); mp 196–200°C; 1H-
NMR (400 MHz, DMSO-d6) δ ppm 2.46 (s, 3H), 2.66 (s, 3H),
7.22 (dd, J = 5.7, 2.9 Hz, 2H), 7.59 (br. s., 2H), 12.73 (br. s.,
1H); calcd for C12H11N3S2 (261.37); C, 55.14; H, 4.24; N,
16.08; found: C, 55.36; H, 4.11; N, 15.78.
2.70 (s, 3H), 3.23 (t, J = 6.8 Hz, 2H), 4.66 (t, J = 6.8 Hz,
2H), 7.05 (d, J = 7.8 Hz, 1H), 7.20 (ddd, J = 7.5, 4.8, 1.0 Hz,
1H), 7.25 (td, J = 7.6, 1.2 Hz, 1H), 7.32 (td, J = 7.6, 1.2 Hz,
1H), 7.59 (td, J = 7.6, 1.9 Hz, 1H), 7.62–7.68 (m, 2H),
8.49–8.52 (m, 1H); 13C-NMR (101 MHz, DMSO-d6) 17.5,
18.5, 36.9, 43.6, 97.7, 111.2, 116.1, 119.5, 121.9, 122.4,
123.3, 123.4, 134.2, 136.2, 142.3, 144.9, 149.3, 157.2, 168.9.
3,3-Bis(methylthio)-2-(1-3-(pyridin-4-yl)propyl)-1H-(benzo[d]
imidazol-2-yl)prop-2-enenitrile (5d). Prepared according to
general procedure for Mitsunobu alkylation using
3-(pyridon-4-yl)propan-1-ol 4d as pale yellow oil (0.28g,
73%), which solidified upon standing to low melting
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L. Jedinák, V. Kryštof, and P. Cankař
Vol 000
almost white solid; 1H-NMR (400 MHz, DMSO-d6) 2.08 (tt,
J = 7.8, 7.4 Hz, 2H), 2.27 (s, 3H), 2.64 (t, J = 7.8 Hz, 2H),
2.67 (s, 3H), 4.25 (t, J = 7.4 Hz, 2H), 7.21 (dd, J = 4.3,
1.6 Hz, 2H), 7.28 (td, J = 7.0, 1.2 Hz, 1H), 7.34 (td, J = 7.0,
1.2 Hz, 1H), 7.65–7.71 (m, 2H), 8.45 (dd, J = 4.3, 1.6 Hz,
2H); 13C-NMR (101 MHz, DMSO-d6) 17.5, 18.4, 29.6,
31.3, 43.5, 97.2, 111.1, 116.2, 119.7, 122.5, 123.5, 123.7,
134.4, 142.3, 144.7, 149.4, 149.6, 169.7.
(m, 4H), 2.34 (q, J = 7.0 Hz, 4H), 2.40 (t, J = 7.0 Hz, 2H),
2.82–2.90 (m, 4H), 4.58 (t, J = 7.0 Hz, 2H), 5.41 (br. s.,
2H), 7.11–7.19 (m, 2H), 7.48–7.57 (m, 2H); 13C-NMR
(101MHz, DMSO-d6) 11.9, 41.3, 45.8, 46.7, 48.9, 50.9,
54.4, 82.6, 109.9, 118.0, 121.1, 121.2, 135.0, 143.4, 149.6,
150.2, 154.1; calcd for C21H32N8 (396.53); C, 63.61; H,
8.13; N, 28.26; found: C, 63.22; H, 8.26; N, 28.52.
N3-Propyl-4-(1-(2-(diethylamino)ethyl)-1H-benzo[d]imidazol-
2-yl)-1H-pyrazol-3,5-diamin (1d).
Prepared according to
general procedure using benzimidazole 5a and propylamine,
white needles 0.12 g (67%); mp 70–73°C (dichloromethane–
hexane); 1H-NMR (400 MHz, DMSO-d6) 0.75 (t, J=7.2Hz,
6H), 0.88 (t, J=7.4Hz, 3H), 1.52 (qt, J = 7.4, 7.0 Hz, 2H),
2.32 (q, J=7.2Hz, 4H), 2.56 (t, J= 6.8Hz, 2H), 3.04 (br. t,
J= 7.0Hz, 2H), 4.39 (t, J=6.8Hz, 2H), 5.27 (br. s, 2H),
5.49 (br. s, 1H), 7.10–7.16 (m, 2H), 7.47–7.55 (m, 2H);
13C-NMR (101 MHz, DMSO-d6) 11.5, 11.6, 22.6, 42.5,
44.8, 46.8, 50.9, 85.0, 109.9, 117.7, 120.7, 121.0, 135.1,
143.6, 149.3, 145.0, 152.2; calcd for C19H29N7 (355.48);
C, 64.20; H, 8.22; N, 27.58; found: C, 63.94; H, 8.57; N,
GENERAL PROCEDURE FOR THE
PREPARATION OF BIARYLS (1A–Q)
A mixture of benzimidazole 5 (0.5 mmol) and appropriate
amine (0.6 mmol) were stirred in dry boiling acetonitrile
(4 mL) for 8 h. Then, hydrazine monohydrate (0.05 mL,
1.0 mmol) was added, and the mixture was stirred at ~80°C
for an additional 14 h. Solvent was removed under vacuum,
and the residue was purified by column chromatography on
silica gel, eluting with 0–10% methanol in dichloromethane.
After column chromatography, title product was crystallized
27.37.
from dichloromethane–hexane or acetonitrile.
N3-Benzyl-4-(1-(3-(dimethylamino)propyl)-1H-benzo[d]imidazol-
N3-Benzyl-4-(1-(2-(diethylamino)ethyl)-1H-benzo[d]imidazol-
2-yl)-1H-pyrazol-3,5-diamine (1e).
Prepared according to
2-yl)-1H-pyrazol-3,5-diamine (1a).
Prepared according to
general procedure using benzimidazole 5b and benzylamine,
white needles 0.18 g (89%), mp 116–118°C (dichloromethane–
hexane); 1H-NMR (400 MHz, DMSO-d6) 1.66 (quin,
J=7.0Hz, 2H), 2.00 (s, 6H), 2.02 (t, J=7.0Hz, 2H), 4.30
(d, J=6.3Hz, 2H), 4.35 (t, J=7.0Hz, 2H), 5.34 (br. s, 2H),
5.56 (br. s, 1H), 7.10–7.17 (m, 2H), 7.19 (t, J=7.4Hz,
1H), 7.27 (dd, J=7.4, 7.0Hz, 2H), 7.34 (d, J=7.0Hz, 2H),
7.47–7.51 (m, 1H), 7.53–7.56 (m, 1H), 10.58 (br. s, 1H);
13C-NMR (101 MHz, DMSO-d6) 26.9, 41.9, 45.0, 46.4,
56.0, 80.6, 110.0, 117.9, 120.8, 121.0, 126.4, 127.3, 128.0,
135.0, 141.3, 143.6, 149.4, 150.0, 154.1; calcd for
C22H27N7 (389.50); C, 67.84; H, 6.99; N, 25.17. found: C,
general procedure using benzimidazole 5a and benzylamine,
white needles 0.16 g (80%), mp 76–78°C (dichloromethane–
hexane); 1H-NMR (400 MHz, DMSO-d6) 0.74 (t, J= 7.0 Hz,
6H), 2.31 (q, J =7.0 Hz, 4H), 2.57 (t, J =6.7 Hz, 2H), 4.31
(d, J =5.9Hz, 2H), 4.39 (t, J =6.7 Hz, 2H), 5.40 (br. s., 2H),
5.92 (br. s., 1H), 7.09–7.16 (m, 2H), 7.19 (t, J = 7.2Hz, 1H),
7.27 (t, J= 7.2Hz, 2H), 7.33 (d, J= 7.2Hz, 2H), 7.47–7.55
(m, 2H), 10.68 (br. s., 1H); 13C-NMR (101 MHz,
DMSO-d6) ppm 11.6, 42.6, 46.5, 46.8, 50.9, 80.6, 110.0,
117.7, 120.8, 121.0, 126.4, 127.3, 128.0, 135.1, 141.0,
143.6, 148.7, 149.8, 152.7; calcd for C23H29N7 (403.52); C,
68.46; H, 7.24; N, 24.30; found: C, 68.13; H, 7.29; N, 24.58.
4-(1-(2-(Diethylamino)ethyl)-1H-benzo[d]imidazol-2-yl)-3-
morpholino-1H-pyrazol-5-amine (1b). Prepared according
67.57; H, 7.08; N, 25.35.
4-(1-(3-(Dimethylamino)propyl)-1H-benzo[d]imidazol-2-yl)-3-
morpholino-1H-pyrazol-5-amine (1f).
Prepared according to
to general procedure using benzimidazole 5a and morpholine,
white needles (0.16 g, 83%); mp 74–76°C (dichloromethane–
general procedure using benzimidazole 5b and morpholine,
white needles 0.15 g (81%), mp 90–92°C (dichloromethane–
1
hexane); H-NMR (400 MHz, DMSO-d6) 0.75 (t, J=7.0Hz,
1
hexane); H-NMR (400 MHz, DMSO-d6) 1.63 (tt, J=7.4,
6H), 2.35 (q, J=7.0Hz, 4H), 2.44 (t, J=7.2Hz, 2H), 2.85
(dd, J= 4.7, 4.3 Hz, 4H), 3.63 (dd, J=4.7, 4.3Hz, 4H), 4.58
(t, J=7.0Hz, 2H), 5.44 (br. s., 2H), 7.11–7.20 (m, 2H), 7.50
(dd, J=6.8, 1.8Hz, 1H), 7.55 (dd, J=6.8, 1.6Hz, 1H), 11.15
(br. s, 1H); 13C-NMR (101 MHz, DMSO-d6) 11.8, 41.3,
46.8, 49.5, 50.9, 65.9, 82.4, 110.0, 118.1, 121.1, 121.2,
135.0, 143.4, 149.4, 150.0, 154.3; calcd for C20H29N7O
(383.49); C, 62.64; H, 7.62; N, 25.57; found: C, 62.29; H,
7.0 Hz, 2H), 1.99 (s, 6H), 2.04 (t, J=7.0Hz, 2H), 2.84 (dd,
J= 4.7, 4.3 Hz, 4H), 3.61 (dd, J= 4.7, 4.3 Hz, 4H), 4.47 (t,
J= 7.4, 7.0 Hz, 2H), 5.37 (br. s, 2H), 7.11–7.20 (m, 2H),
7.49–7.59 (m, 2H), 11.12 (br. s, 1H); 13C-NMR (101 MHz,
DMSO-d6) 27.0, 41.5, 44.9, 49.4, 56.0, 65.8, 82.2, 110.2,
118.2, 121.2, 135.0, 143.4, 149.0, 149.7, 154.7, 162.3; calcd
for C19H27N7O (369.46); C, 61.77; H, 7.37; N, 26.54; found:
61.42; H, 7.45; N, 26.83.
7.74; N, 25.80.
4-(1-(2-(Diethylamino)ethyl)-1H-benzo[d]imidazol-2-yl)-3-
4-(1-(3-(Dimethylamino)propyl)-1H-benzo[d]imidazol-2-yl)-3-
(4-methylpiperazin-1-yl)-1H-pyrazol-5-amine (1g).
Prepared
(4-methylpiperazin-1-yl)-1H-pyrazol-5-amine (1c).
Prepared
according to general procedure using benzimidazole 5b
according to general procedure using benzimidazole 5a and
and 1-methylpiperazine, white needles 0.14g (76%), mp
89–91°C (dichloromethane–hexane); H-NMR (400MHz,
1-methylpiperazine, white needles 0.16 g (79%); mp
69–71°C (dichloromethane–hexane); H-NMR (400MHz,
1
1
DMSO-d6) 1.61 (ddd, J = 7.4, 7.0, 6.7 Hz, 2H), 1.98 (s,
DMSO-d6) 0.75 (t, J = 7.0 Hz, 6H), 2.16 (s, 3H), 2.33–2.38
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
1H-Benzimidazol-2-yl-1H-pyrazole-3,5-diamines: Synthesis and CDK
Inhibitory Activity
N3-Benzyl-4-(1-(3-(pyridin-4-yl)propyl)-1H-benzo[d]imidazol-2-
yl)-1H-pyrazol-5-amine (1k). Prepared according to general
6H), 2.02 (dd, J = 7.0, 6.7 Hz, 2H), 2.12 (s, 3H), 2.27–2.37
(m, 4H), 2.79–2.91 (m, 4H), 4.47 (dd, J = 7.9, 7.4 Hz, 2H),
5.34 (br. s., 2H), 7.06–7.24 (m, 2H), 7.45–7.61 (m, 2H),
11.09 (br. s, 1H); 13C-NMR (101 MHz, DMSO-d6) 27.0,
41.5, 45.0, 45.9, 48.8, 54.3, 56.0, 82.4, 110.1, 118.2,
121.2, 135.0, 143.4, 149.2, 149.8, 154.8, 162.4; calcd for
C20H30N8 (382.51); C, 62.80; H, 7.91; N, 29.29; found: C,
procedure using benzimidazole 5d and benzylamine, white
needles 0.18 g (87%), mp 83–85°C (dichloromethane–
hexane); 1H-NMR (400 MHz, DMSO-d6) 1.91 (tt,
J =7.4Hz, 2H), 2.37 (t, J= 7.4Hz, 2H), 4.29 (d, J =6.7Hz,
2H), 4.34 (t, J= 7.0 Hz, 2H), 5.20 (br. s, 2H), 7.02 (dd,
J =4.7, 1.6 Hz, 2H), 7.12–7.16 (m, 2H), 7.17 (td, J =7.0,
1.6 Hz, 1H), 7.22 (ddd, J= 7.0, 6.8, 1.9 Hz, 2H), 7.31 (dd,
J = 6.8, 1.6 Hz, 2H), 7.47–7.51 (m, 1H), 7.54–7.58 (m, 1H),
8.37 (dd, J= 4.7, 1.6Hz, 2H), 10.69 (br. s, 1H); 13C-NMR
(101 MHz, DMSO-d6) 29.1, 31.2, 43.2, 46.4, 80.7, 110.2,
118.0, 120.9, 121.1, 123.6, 126.4, 127.3, 128.0, 134.9,
140.1, 143.7, 149.3, 149.4, 149.9, 150.6, 154.4; calcd for
C25H25N7 (423.51); C, 70.90; H, 5.95; N, 23.15; found: C,
62.47; H, 8.05; N, 29.48.
N3-Fenyl-4-(1-(2-(pyridin-2-yl)ethyl)-1H-benzo[d]imidazol-2-yl)-
1H-pyrazol-3,5-diamine (1h). Prepared according to general
procedure using benzimidazole 5c and aniline. The mixture
was heated for 24 h instead of 8 h, white needles 0.16g
1
(81%), mp 68–71°C (dichloromethane–hexane); H-NMR
(400MHz, DMSO-d6) 3.02 (t, J = 7.4 Hz, 2H), 4.63 (t,
J = 7.4 Hz, 2H), 5.49 (br. s., 2H), 6.70 (td, J = 7.4, 1.2 Hz,
1H), 6.87 (d, J = 7.8 Hz, 1H), 7.03 (ddd, J = 7.5, 4.8,
1.0 Hz, 1H), 7.11–7.20 (m, 4H), 7.33 (d, J = 7.8 Hz, 2H),
7.46 (td, J = 7.6, 2.0 Hz, 1H), 7.49–7.53 (m, 1H), 7.60–
7.65 (m, 1H), 8.12 (br. s., 1H), 8.35 (ddd, J = 4.7, 1.6,
0.8 Hz, 1H), 11.16 (br. s., 1H); 13C-NMR (101MHz,
DMSO-d6) 36.9, 43.8, 82.8, 110.2, 115.4, 118.1, 118.3,
121.1, 121.2, 121.5, 122.9, 128.6, 134.9, 136.2, 143.2,
143.5, 147.3, 148.4, 148.6, 148.7, 157.8; calcd for
C23H21N7 (395.46); C, 69.85; H, 5.35; N, 24.79; found: C,
0.77; H, 6.31; N, 22.92.
3-Morpholino-4-(1-(3-(pyridin-4-yl)propyl)-1H-benzo[d]imidazol-
2-yl)-1H-pyrazol-5-amine (1l). Prepared according to general
procedure using benzimidazole 5d and morpholine, white
needles 0.16 g (77%), mp 97–99°C (dichloromethane–
1
hexane); H-NMR (400 MHz, DMSO-d6) 1.88 (tt, J = 7.4,
7.0 Hz, 2H), 2.43 (t, J = 7.4 Hz, 2H), 2.75 (t, J=4.3Hz, 4H),
3.52 (t, J=4.3Hz, 4H), 4.45 (t, J=7.0Hz, 2H), 5.39 (br. s,
2H), 7.04 (d, J=5.1Hz, 2H), 7.12–7.21 (m, 2H), 7.52 (d,
J=7.8Hz, 1H), 7.58 (d, J=7.0Hz, 1H), 8.37 (d, J=5.1Hz,
2H), 11.15 (br. s, 1H); 13C-NMR (101 MHz, DMSO-d6)
28.8, 31.1, 42.6, 49.3, 65.8, 82.2, 110.2, 118.3, 121.2, 121.3,
123.6, 134.8, 143.5, 149.1, 149.4, 149.8, 150.3, 154.7; calcd
for C22H25N7O (403.48); C, 65.49; H, 6.25; N, 24.30; found:
69.52; H, 5.43; N, 25.05.
N3-Benzyl-4-(1-(2-(pyridin-2-yl)ethyl)-1H-benzo[d]imidazol-2-
yl)-1H-pyrazol-3,5-diamine (1i).
Prepared according to
general procedure using benzimidazole 5c and benzylamine,
white needles 0.18 g (90%), mp 68–70°C (dichloromethane–
hexane); 1H-NMR (400 MHz, DMSO-d6) 3.02 (t, J =7.6Hz,
2H), 4.29 (d, J = 5.9 Hz, 2H), 4.71 (t, J = 7.6 Hz, 2H), 5.25
(br. s., 2H), 5.77 (br. s., 1H), 7.01 (d, J = 7.4 Hz, 1H),
7.11–7.14 (m, 2H), 7.15–7.21 (m, 2H), 7.25 (td, J = 7.2,
1.6 Hz, 2H), 7.33 (dd, J = 7.2, 1.6 Hz, 2H), 7.45–7.50 (m,
1H), 7.52–7.56 (m, 1H), 7.57–7.60 (m, 1H), 8.43–8.48
(m, 1H); 13C-NMR (101 MHz, DMSO-d6) 37.0, 43.6,
46.5, 80.6, 110.1, 117.8, 120.9, 121.1, 121.6, 123.1,
126.4, 127.3, 128.0, 134.9, 136.4, 141.0, 143.6, 148.9,
149.0, 149.4, 152.6, 158.0; calcd for C24H23N7 (409.49);
C, 65.61; H, 6.46; N, 23.97.
N3-Propyl-4-(1-(3-(pyridin-4-yl)propyl)-1H-benzo[d]imidazol-2-
yl)-1H-pyrazol-5-amine (1m). Prepared according to general
procedure using benzimidazole 5d and propylamine, white
needles 0.16 g (86%), mp 92–94°C (dichloromethane–
hexane); 1H-NMR (400 MHz, DMSO-d6) 0.83 (t, J =7.4Hz,
2H), 1.48 (qt, J= 7.4, 7.0Hz, 2H), 1.92 (tt, J= 7.4, 7.0Hz,
2H), 2.39 (t, J=7.4Hz, 2H), 3.01 (td, J = 7.0, 5.2 Hz, 2H),
4.33 (t, J =7.2Hz, 2H), 5.09 (br. s, 2H), 5.30 (br. s, 1H),
7.04 (dd, J= 4.3, 1.6 Hz, 2H), 7.12–7.18 (m, 2H), 7.47–7.51
(m, 1H), 7.54–7.58 (m, 1H), 8.37 (dd, J= 4.3, 1.6Hz, 2H),
10.72 (br. s, 1H); 13C-NMR (101 MHz, DMSO-d6) 11.5,
22.5, 29.0, 31.2, 43.1, 44.8, 80.5, 110.1, 117.9, 120.9,
121.1, 123.6, 134.9, 143.7, 149.4, 149.6, 149.9, 150.3,
154.3; calcd for C21H25N7 (375.47); C, 67.18; H, 6.71; N,
C, 70.39; H, 5.66; N, 23.94; found: C, 70.14; H, 5.58;
N, 24.27.
N3-Propyl-4-(1-(2-(pyridin-2-yl)ethyl)-1H-benzo[d]imidazol-2-
yl)-1H-pyrazol-3,5-diamine (1j).
Prepared according to
general procedure using benzimidazole 5c and propylamine,
white needles 0.15 g (84%), mp 70–72°C (dichloromethane–
hexane); 1H-NMR (400 MHz, DMSO-d6) 0.85 (t, J =7.4Hz,
3H), 1.50 (qt, J = 7.4, 7.0 Hz, 2H), 2.97–3.07 (m, 4H), 4.68
(t, J =7.8Hz, 2H), 5.12 (br. s, 2H), 5.34 (br. s, 1H), 7.03 (d,
J= 7.8 Hz, 1H), 7.09–7.18 (m, 3H), 7.43–7.49 (m, 1H),
7.51–7.55 (m, 1H), 7.58 (td, J=7.6, 2.0 Hz, 1H), 8.44 (ddd,
J= 4.9, 2.0, 0.8Hz, 1H); 13C-NMR (101 MHz, DMSO-d6)
11.6, 22.6, 37.0, 43.6, 44.8, 80.5, 110.1, 117.8, 120.9,
121.1, 121.6, 123.1, 134.9, 136.4, 143.6, 149.0, 149.5,
149.6, 152.3, 158.0; calcd for C20H23N7 (361.44); C, 66.46;
H, 6.41; N, 27.13; found: C, 66.15; H, 6.36; N, 27.49.
26.11; found: C, 66.91; H, 6.75; N, 28.34.
4-(1H-Benzimidazol-2-yl)-N3-phenyl-1H-pyrazol-3,5-diamine
(1n).
Prepared according to general procedure using
benzimidazole 2 and aniline. The mixture was heated for
24 h instead of 8 h, white solid 0.11 g (73%), mp 272–274°C
1
(acetonitrile); H-NMR (400 MHz, DMSO-d6) 6.03 (br. s.,
2H), 6.79 (t, J=7.2Hz, 1H), 7.08–7.17 (m, 2H), 7.24 (t,
J= 7.8Hz, 2H), 7.46 (d, J=7.0Hz, 1H), 7.55 (d, J=7.0 Hz,
2H), 7.59 (d, J=7.4Hz, 1H), 9.79 (br. s., 1H), 11.15 (br. s.,
1H), 11.76 (s, 1H); 13C-NMR (75 MHz, DMSO-d6) 82.0,
110.4, 115.9, 116.9, 118.7, 120.9, 121.2, 128.8, 133.6,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

L. Jedinák, V. Kryštof, and P. Cankař
Vol 000
142.5, 142.8, 146.2, 148.6, 150.0; calcd for C16H14N6
(290.32); C, 66.19; H, 4.86; N, 28.95; found: C, 65.93; H,
(the drug concentration that reduced the number of viable
cells to 50%) values were determined from the dose–
response curves.
5.03; N, 29.04.
4-(1H-Benzimidazol-2-yl)-N3-benzyl-1H-pyrazol-3,5-diamine
(1o).
Prepared according to general procedure using
Acknowledgments. This work was supported by the Operational
Program Research and Development for Innovations CZ.1.07/
2.4.00/31.0130, CZ1.07/2.3.00/20.0009, CZ1.07/2.4.00/17.0015,
IGA_Prf 2012 027, and IGA_Prf 2013 036.
benzimidazole 2 and benzylamine, white solid 0.12 g
(78%), mp 258–260°C (acetonitrile); H-NMR (400 MHz,
1
DMSO-d6) 4.46 (d, J = 5.9 Hz, 2H), 5.88 (br. s., 2H), 6.73
(br. s., 1H), 7.03–7.11 (m, 2H), 7.23 (d, J = 7.4 Hz, 1H),
7.32 (dd, J = 7.4, 7.0 Hz, 2H), 7.40 (d, J = 7.0 Hz, 2H),
7.43–7.48 (m, 2H), 10.76 (br. s., 1H), 11.57 (br. s., 1H);
13C-NMR (101 MHz, DMSO-d6) 46.3, 81.1, 110.1, 116.6,
120.4, 120.9, 126.6, 127.3, 128.2, 133.6, 140.7, 143.0,
148.6, 149.2, 153.2; calcd for C17H16N6 (304.35); C,
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67.09; H, 5.30; N, 27.61; found: C, 66.85; H, 5.43; N, 27.72.
4-(1H-Benzo[d]imidazol-2-yl)-3-(4-methylpiperazin-1-yl)-1H-
pyrazol-5-amine (1p).
Prepared according to general
procedure using benzimidazole 2 and 1-methylpiperazine,
white solid 0.14 g (91%), mp 214–216°C (acetonitrile);
1H-NMR (400 MHz, DMSO-d6) 2.26 (s, 3H), 2.55–2.61
(m, 4H), 2.97–3.05 (m, 4H), 6.11 (br. s, 2H), 7.04–7.10
(m, 2H), 7.46–7.54 (m, 2H), 10.99 (br. s, 1H), 11.24
(br. s, 1H); 13C-NMR (101 MHz, DMSO-d6) 45.9, 50.6,
53.9, 83.9, 111.1, 116.9, 120.6, 120.9, 133.7, 143.1, 148.6,
149.6, 156.0; calcd for C15H19N7 (297.34); C, 60.59; H
6.44; N, 32.97; found: C, 60.21; H, 6.56; N, 33.15.
4-(1H-Benzimidazol-2-yl)-N3-propyl-1H-pyrazol-3,5-diamine
(1q).
Prepared according to general procedure using
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benzimidazole 2 and propylamine, white solid 0.11 g
(84%), mp 244–246°C (acetonitrile); H-NMR (400 MHz,
1
DMSO-d6) 0.95 (t, J = 7.4 Hz, 3H), 1.61 (qt, J = 7.4,
7.0 Hz, 2H), 3.17 (t, J = 7.0 Hz, 2H), 5.60 (br. s., 2H),
6.51 (br. s, 1H), 7.02–7.10 (m, 2H), 7.40–7.45 (m, 2H),
10.80 (br. s., 1H), 11.47 (br. s., 1H); 13C-NMR
(101 MHz, DMSO-d6) 11.6, 22.7, 44.6, 80.9, 110.1,
116.4, 120.54, 120.63, 133.6, 142.9, 148.9, 149.4, 152.1;
calcd for C13H16N6 (256.31); C, 60.92; H, 6.29; N,
32.79; found: C, 60.61; H, 6.36; N, 33.03.
BIOLOGICAL ASSAYS
Biological activity of prepared compounds was assayed
as described previously 13a. The compounds have been
tested in kinase inhibition assays for their inhibitory potency
toward human CDK2/cyclin E produced through a
baculoviral expression system and using histone H1 as a sub-
strate. The concentration of the test compounds required to
decrease the CDK2 activity by 50% was determined from
dose–response curves and designated as IC50. The compounds
were also assayed for cytotoxicity against MCF7 and K562
cancer cell lines. Briefly, cells were incubated for 72 h with
compounds using threefold dilutions in triplicate. After
this period, live cells were labeled by Calcein AM and
quantified by measurement of their fluorescence with a
Fluoroskan Ascent microplate reader (Labsystems). IC50
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
1H-Benzimidazol-2-yl-1H-pyrazole-3,5-diamines: Synthesis and CDK
Inhibitory Activity
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet