Novel 1H-pyrazole-3-carboxamide derivatives: Synthesis, anticancer evaluation and identification of their DNA-binding interaction

Article abstract of DOI:10.1248/cpb.c13-00676

Four novel 1H-pyrazole-3-carboxamide derivatives were synthesized, and their antiproliferative effect on cancer cells, kinase inhibition, and in particular, the DNA-binding interaction were investigated to interpret the antitumor mechanisms. A DNA minor g

Full text of DOI:10.1248/cpb.c13-00676

238
Regular Article
Chem. Pharm. Bull. 62(3) 238–246 (2014)
Vol. 62, No. 3
Novel 1H-Pyrazole-3-carboxamide Derivatives: Synthesis, Anticancer
Evaluation and Identification of Their DNA-Binding Interaction
Yi Lu,a Ting Ran,a,b Guowu Lin,c Qiaomei Jin,a Jianling Jin,a Hongmei Li,a Hao Guo,a
,a,b
,a,d
Tao Lu,* and Yue Wang*
a State Key Laboratory of Natural Medicines, China Pharmaceutical University; b Laboratory of Molecular Design and
Drug Discovery, School of Sciences, China Pharmaceutical University; Nanjing 210009, P. R. China: c Department
of Structural Biology, University of Pittsburgh School of Medicine; Pittsburgh, PA 1526, U.S.A.: and d State Key
Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University; Nanjing
210093, P.R. China.
Received August 29, 2013; accepted December 16, 2013; advance publication released online December 20, 2013
Four novel 1H-pyrazole-3-carboxamide derivatives were synthesized, and their antiproliferative effect on
cancer cells, kinase inhibition, and in particular, the DNA-binding interaction were investigated to interpret
the antitumor mechanisms. A DNA minor groove binding model was developed, and the binding energy was
predicted for the compounds. In consistence with the prediction, the binding ability was determined by the
electronic absorption spectroscopy under physiological conditions for the compounds, and further verified by
viscosity measurement. One compound 5-(3-cyclopropylureido)-N-[4-[(4-methylpiperazin-1-yl)methyl]phen-
yl]-1-H-pyrazole-3-carboxamide (pym-5) exerted the highest DNA-binding affinity (Kpym-5=1.06ꢀ105 Mꢁ1).
And it demonstrated more than 50% decrease of the emission intensity of the ethidium bromide–calf thymus
DNA (EB–CT-DNA) complex in fluorescence spectra, suggesting that pym-5 could strongly affect the DNA
conformation. Furthermore, pym-5 showed the cleavage activity upon the supercoiled plasmid pBR322 DNA
in the pBR322 DNA cleavage assay. Our study suggests that DNA may serve as a potential target to these
pyrazole derivatives.
Key words pyrazole derivative; synthesis; DNA interaction; molecular docking; spectroscopy
Off-target effects, commonly emerged in anticancer drug ing region, such as distamycin, netropsin and Lexitropsins.7,8)
discovery, often leads to undesired side effects. However, it Moreover, through non-bonded interactions, groove binders
may also lead to favorable pharmacological profiles in drug can slide into the narrow and long helical cavity formed by
re-purposing.1) In kinase-targeted drug discovery, it has been the DNA double strands, less agitating the DNA structure or
verified that small molecule inhibitors usually bind to multiple damaging the integrity of DNA. Therefore, the DNA groove
targets, including the rarely reported nucleic acid such as dou- binder represents an important class of anticancer agents.
ble-strand DNA exemplified by flavopiridol.2,3) The off-target
The pyrazole ring is a useful molecular fragment in
effects of kinase inhibitors have always focused on homolo- drug design, and the pyrazoles have demonstrated diverse
gous kinases in a dysregulated signaling pathway while rarely pharmacological effects, such as antitumor, analgesic, anti-
on nucleic acid targets at the preliminary stage, although DNA inflammatory, antipyretic, anti-arrhythmic and antibiosis
binding affinities, at the late-stage, may be revealed in multi- properties.9) With the aim of increasing the structural diversity
target small-molecule antibiotic and anticancer drug study. of substituted pyrazoles conventional preparation approaches
When small molecules bind to the targeted DNA, they alter have been developed.10–12) In addition to the creation of C–N
the normal activity of the DNA with consequent cytotoxicity, and C–C bonds by cross-coupling reactions of aryl electro-
cell death, and the abnormal expression of oncogenes.
philes with substituted pyrazoles, more promising alternative
Intercalation, alkylation and groove binding are three main methods have been used in further synthesis and chemical pu-
mechanisms for compounds targeting DNA.4) The intercala- rification with short reaction time and excellent yield, includ-
tion often causes obvious conformational changes of DNA and ing ultrasound depositing, using condensation reagents and
it occurs non-selectively in different DNA binding sites. For microwave synthesis and so on.13–17) All these greatly promote
the intercalator, improving molecular planarity would facili- the progress of research in pyrazole compounds and recently
tate its embedding to DNA base pairs. Alkylation of DNA is such scaffold is not only employed in developing various ki-
to transfer an alkyl group from exogenous ligand to DNA and nase inhibitors,10) but also in molecules that directly interact
form covalent alkyl lesions. So, alkylators can strongly affect with DNA stands, such as metal complexes, alkylating agents,
the integrity of DNA double strands by forming alkylation dimeric analogues, polyamide derivatives and some pyrazole-
cross-linking and preventing uncoiling of the DNA double he- fused derivatives.18–25) The unexpected interaction between
lices.5) Due to the lack of selectivity, both DNA intercalators DNA and pyrazole-derived kinase inhibitors can cause off-
and alkylators may cause a potential risk of serious dysfunc- target effects, including potential improvement of cell toxic-
tion of DNA replication, and thus, they are unsuitable as drug ity and anticancer activity. Pyrazole ring can form a rigid
candidates.6) Comparatively, the groove binder fit into the plane with electron-rich system chromophores and embed into
minor groove and may form more selective interaction with the DNA minor grooves to make a π–π stacking with DNA
a specific DNA sequence, as it possesses a larger DNA bind- bases,26–28) which is usually essential for the DNA-binding.
Additionally, linking flexible groups to pyrazole-contained
scaffold may acquire favorable binding affinities to DNA
The authors declare no conflict of interest.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: lut163@163.com; zwy_1115@126.com

March 2014
239
strands.29,30) As sequence-specific DNA binding ability has
Instruments All the electronic absorption spectra were
been reported in an increasing number of pyrazole analogs,31) recorded on a Shimadzu UV-1800 Visible spectrophotometer
more attention should be paid to DNA interaction of pyrazole after mixing for 5min at 25°C. And all fluorescence spectra
derivatives in the process of drug development.
were recorded on a RF-5301PC Spectrofluorimeter (Shimadzu,
In our previous study, we synthesized a series of novel Japan) equipped with 0.2cm quartz cells, the widths of both
pyrazole derivatives designed as kinase inhibitors to be the excitation and emission slit were set to 5nm at 25°C. The
potential antitumor drugs. Their biological activity evalua- gel was photographed on a capturing system gel printer plus
tions elucidated that these compounds exhibited relatively (TDI, Tecnologia para Diagnostico e Investigación, Madrid,
remarkable cell proliferation inhibition and weak multiple Spain). Bands were quantified using the Scion Image for
kinase activities.32,33) Hence, besides the inhibition of kinase Windows software program based on NHI Kinases and the
leading antitumor activity, another mechanism may exist for solution used in assays (cyclin-dependent kinase 2 (CDK2),
the compounds’ inhibition of cell proliferation. Meanwhile, epidermal growth factor receptor (EGFR) and Abelson tyro-
enlightened by the potential DNA-binding ability of pyrazole sine-protein kinase 2 (ABL2) were purchased from Invitrogen
compounds as mentioned above, we hypothesized that the (U.S.A.). KinEASE-TK for Homogeneous Time Resolved
DNA binding of these 1H-pyrazole-3-carboxamide deriva- Fluorescence (HTRF) was provided by Cisbio (U.S.A.). Reac-
tives may contribute to their antitumor activity. To verify our tions were conducted in a 384-well plate (Corning Part #3676).
hypothesis and discover possible off-target effects, four most
In Vitro Pharmacological Evaluation All the compounds
active compounds from the preliminary screen (Chart 1) were were evaluated for their in vitro cytotoxic activity against
synthesized and tested by DNA-binding experiments to reveal human hepatocellular liver carcinoma cell Hep2G and human
the mechanism of their antitumor activity.
colon cancer cells HCT116 by 3-(4,5-dimethylthiazol-2-yl)-2,5-
In this paper, we employed a computational method to diphenyltetrazolium bromide (MTT) assay.34) HepG2 and
simulate the binding of these compounds with a known DNA HCT116 cells were cultured in Dulbecco’s modified Eagle’s
fragment. Considering the energy feasibility on their interac- medium (DMEM, Invitrogen, U.S.A.) supplemented with 10%
tions with DNA predicted by molecular docking, we carried fetal bovine serum (FBS, Hyclone, U.S.A.), 2mM L-glutamine,
out electronic absorption spectra, fluorescence spectroscopy 100 µg/mL streptomycin and 100U/mL penicillin (Invitrogen).
study, viscosity measurement and DNA cleavage assays to The cells were incubated in a humidified atmosphere with
analyze the binding ability and binding mode. Finally, we 5% CO2 at 37°C. During logarithmic growth phase, the cells
confirmed that these pyrazole compounds did interact with were seeded into 96-well plates to achieve a density of 1×104
DNA and the interaction could be an alternative contribution cells/well, and cultured for 24h, followed by exposure to
to inhibit cancer cell proliferation by an off-target mechanism. various concentrations of compounds tested for 48h. Twenty
microliter of MTT (Genview, U.S.A.) solution (5mg/mL) was
subsequently added to each well and mixed, then the cells
Experimental
Reagents and Materials The compounds were synthe- were incubated for an additional 4h. Culture supernatant was
sized according to the following procedure. Ethidium bromide removed and 150µL of dimethyl sulfoxide (DMSO) (Sangon
(EB), calf thymus DNA (CT-DNA) and pBR322 plasmid DNA Biotech, China) was added into each well to make the MTT-
were purchased from Sigma Chemical Co. Tris–HCl/NaCl formazan crystals completely dissolved. Cell growth inhibition
(5 mM [Tris(hydroxymethyl)aminomethane] and 50mM NaCl, was determined by measuring the absorbance at 570nm using
and adjusted to pH7.4 with hydrochloric acid) buffer solution a microplate reader and calculated according to the equation:
was prepared using deionized sonicated triple-distilled water Growth inhibition=(1−optical density (OD) of treated cells/
and other chemicals used were of analytical grade. Solution of OD of control cells)×100%. The half maximal inhibitory con-
the compound and other agents used for strand scission were centrations (IC50) were obtained from liner regression analysis
prepared freshly in triple-distilled water before use. Solvents for each tested compound, using GraphPad Prism software.
used in this research were purified by standard procedures.
Kinase activity was measured using HTRF assay.35,36) On
The purity and concentration per nucleotide of CT-DNA solu- the Beckman Coulter detection platform, HTRF assays were
tion (ε=6600M−1 cm−1 at 260nm) in the buffer were checked performed in 384-well plates. The following were combined
by a UV-Vis spectrophotometer, and a ratio of UV absorbance in the reaction mixture: 4µL of different concentrations of
of about 1.8–1.9:1 at 260nm and 280nm indicated that the compound (diluted in 100% DMSO at 50μM), 8µL of enzyme,
CT-DNA was sufficiently free of protein.
and 8µL of ATP/substrate mix (final concentrations around
Chart 1. Structure of 1H-Pyrazole-3-carboxamide Derivatives

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Vol. 62, No. 3
Km values). Enzymes, ATP, and substrate were previously viscosity of DNA alone. Viscosity values were calculated from
diluted in kinase buffer to get final concentrations in the well: the observed flowing time of DNA-containing solutions (t)
50mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid corrected for that of the buffer alone (t0), η=(t−t0)/t0.
(Hepes)/NaOH (pH7.4), 40μM Na3VO4, 0.005% ween-20, 1mM
pBR322 DNA Cleavage Assays The plasmid DNA
dithiothreitol (DTT), and various concentrations of MnCl2 or cleavage experiments were performed using pBR322 DNA
MgCl2. The ATP/substrate mixture was added to initiate the in Tris–HCl buffer. Reactions were performed by incubating
assay. The reaction was stopped by addition revelation mix- DNA (0.05mM bp) at 37°C in the presence or absence of the
ture (55µL, ethylenediaminetetraacetic acid (EDTA), 0.33M), compound for the indicated time periods. All reactions were
specific europium-cryptate antibody antiphospho-ubstrate, and quenched by loading buffer. Agarose gel electrophoresis was
XL-665 with various tags (CisBio International) after being carried out on a 1% agarose gel in 0.5× TAE (Tris–acetate–
incubated for different times at room temperature. The final EDTA) buffer containing 0.5µg/mL EB at 80V for 1.5h, then
concentrations of mixture prepared in revelation buffer in the the gel image was captured by the Scion Image System.
well: 50mM Hepes/NaOH (pH7.4), 0.1% bovine serum albu-
min (BSA), and 150mM KF. The reaction was incubated more
than 1h at room temperature, and then detected by Beckman
Results and Discussion
Synthesis The synthetic pathways adopted for the
Coulter detection platform. Kinase activities were expressed preparation of title compounds are illustrated in Chart 2.
as a percentage of maximal activity (without compound), re- pym-5, pym-55 and pym-n all bearing a characteristic
sulting from two independent experiments. In all cases, IC50 functional group have a common intermediate (5), which
values were calculated from replicate curves, using GraphPad was prepared through alkylation, reduction and acylation.
Prism software. Staurosporine and Roscovitine were used as The reduction step of the nitro group to amide employed
positive controls in the kinase assay.
hydrazine hydrate in the presence of an iron(III) oxide
Molecular Modeling and Docking Molecular model- hydroxide (FeO(OH)/C) as a catalyst38) in the refluxing
ing was performed using SYBYL7.3 on a Hewlett-Packard 95% ethanol to get a high yield product within 2h.39–41)
XW4100 PC workstation with a Red Hat Enterprise 4 Linux Furthermore, the catalyst can be recycled after the reac-
operating system. All molecules except the DNA were built tion through washing with anhydrous ethanol. pym-5 bear-
using SYBYL. The DNA structure was downloaded from the ing a urea group was synthesized from compound 5 with
PDB database and prepared by removing both the bound mol- cyclopropylamine employed CDI. pym-55 was prepared
ecules and all waters to avoid potential interference with the using N-hydroxybenzotriazole (HOBT) and 1-ethyl-(3-(3-
docking. Hydrogen was added to the DNA by SYBYL. The dimethylamino)propyl)carbodiimide hydrochloride (EDCI) as
compounds were docked into the solution structure of B-DNA condensing agent to acylate compound 5 by benzonic acid.
dodecamer d(AATCTTTAAAGATT)2 (PDB ID:2K4L) with pym-n was gained through catalyzed hydrogenation with
DOCK5.4.37)
NaBH4 from the intermediate of Schiff base which was gener-
Electronic Absorption and Fluorescence Spectra The ated by mixing compound 5 with pyridine formaldehyde. The
DNA-binding experiments were performed at 25°C. Using the preparation of pyz-5 was similar to pym-5 with the starting
electronic absorption spectra, the relative binding of the four reactant morphine instead of N-methylpiperazine.
complexes to CT-DNA was studied in 5mM Tris–HCl/50mM
4-Amino-N-[4-[(4-methylpiperazin-1-yl)methyl]phenyl]-1H-
NaCl buffer (pH=7.4). The compounds were dissolved in pyrazole-3-carboxamide (5): The mixture of N-methyl
a solvent of Tris–HCl buffer (5mM Tris–HCl, 50mM NaCl, piperazine (4.9mL, 44.2mmol) and triethylamine (12mL,
pH7.4) at the concentration 1.0×10−5 M. Absorption titration 86.3mmol) in dichloromethane (20mL) was added dropwise
experiments were performed with fixed concentration drugs to a stirred solution of Nitro-benzyl bromide 1 (10.0mg,
(10 mM) while gradually increasing the concentration of CT- 46.3mmol) in dichloromethane (100mL) under the ice-water
DNA. The ratio of CDNA/Ccompound in each curve decreased as bath and was refluxed for 1h. The reaction mixture was ex-
follows: 6/100, 13/100, 25/100, 38/100, 51/100, 76/100, 102/100, tracted with chloroform (100mL×3), washed with water and
127/100, 152/100, 230/100, 254/100. In order to eliminate the saturated sodium chloride each time (100mL×1). The organic
absorbance of CT-DNA itself, an equal amount of CT-DNA phase was dried with anhydrous magnesium sulfate, filtered,
was added to both the compound solution and the reference evaporated under vacuum to give pale yellow solid 2. Without
solution during measuring the absorption spectra. By the fluo- further purification, 2 mixed with FeO(OH)/C (catalyst 2.0g)
rescence spectral method, the relative binding of compounds and 95% ethanol (100mL) were kept refluxing and dropwise
to CT-DNA were studied with an EB-bound CT-DNA solution added the mixture of hydrazine hydrate (25mL) and 95%
in 5mM Tris–HCl/50mM NaCl buffer (pH=7.4). The ratio of ethanol (20mL), then filtrated when the solution was hot. The
CEB-CT-DNA/Ccompound in each curve decreased as follows: 0, residue was washed with hot ethanol (30mL×2). The solvent
1/10, 1/5, 3/10, 2/5, 1/2, 3/5, 4/5 and 1/1 (Fig. 6). The excitation was distilled under vacuum to give white solid 3 (6.7g), then
wavelength was 520nm for all cases.
mixed with 4-nitro-1H-pyrazole-3-acid (6.3g, 40.0mmol),
Viscosity Experiments Viscosity measurements were EDCI (8.4g, 43.8mmol) and HOBT (6.0g, 44.4mmol) in an-
carried on an Ostwald’s viscometer at 30 0.1°C in a con- hydrous DMF (100mL), stirred for 24h at room temperature.
stant temperature bath. The DNA concentration was fixed at The reaction mixture was poured into ice water (200mL).
1.5×10−4 M and the flow time were measured with a digital A large amount of yellow solid precipitation was acquired.
stopwatch; the mean values of three measurements were used The pure product 4 was got from recrystallizing with mixed
to evaluate the viscosity of each sample. Data are presented as solvent of methanol and ethyl acetate, then the same process
(η/η0)1/3 versus r (r=[compound]/[DNA]=0–1), where η is the of hydrazine hydrate reduction was done with the catalyst of
viscosity of DNA in the presence of compound and η0 is the FeO(OH)/C. The solvent was distilled under vacuum to give

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Reagents and conditions: (a) N-Methylpiperazine, N(C2H5)3, DCM, 0°C in an ice bath; (b) FeO(OH)/C, hydrazine hydrate, 95% ethanol, reflux; (c) 4-nitropyrazole-
3-carboxylic acid, hydroxybenzotriazole (HOBT), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N-dimethylformamide (DMF); (d) 1,1′-carbon-
yldiimidazole (CDI), DMF, cyclopropylamine; (e) benzonic acid, HOBT, EDCI, DMF; (f) pyridine formaldehyde, sodium borohydride (NaBH4); (g) morpholine, N (C2H5)3,
DCM, 0°C in an ice bath.
Chart 2. Synthesis of the 1H-Pyrazole-3-carboxamide Derivatives
white solid 5 (3.5g). Yield=63.9%; mp 199–201°C; IR (KBr) methanol–chloroform 1:50) to get pym-55 (200.3mg). Yield=
cm−1: 3369, 3227 (NH2), 1346 (C=C), 1456 (C=N) pyrazole, 48.7%; mp 236–238°C; IR (KBr) cm−1: 3369 (–NH–) Amie,
1
646 (ArH); H-NMR (300MHz, DMSO-d6) δ (ppm): 2.1 (3H, 3335 (NH), 1446 (C=N), 1377 (C=C) pyrazole, 1593 (ArH),
1
s, –CH3), 2.3–2.5 (8H, m, –CH2–), 3.3 (2H, s, –CH2–), 4.7 1656 (O=C–NH), 1174 (C–N); H-NMR (300MHz, DMSO-
(1H, s, –NH2), 7.1–7.2 (3H, m, ArH), 7.7 (2H, d, J=10.5Hz, d6) δ: 2.1 (3H, s, –CH3), 2.4–2.6 (8H, m, –NCH2–), 4.1 (2H,
ArH), 9.7 (1H, s, –NHCO–), 12.7 (1H, s, Pyrazole); MS m/z: s, –CH2–), 6.9–7.0 (3H, m, ArH), 7.1 (2H, d, J=8.7Hz, ArH),
315.82 (M+); Anal. Calcd for C16H22N6O: C, 61.13; H, 7.05; N, 7.2 (1H, m, ArH), 7.7 (2H, d, J=8.7Hz, ArH), 8.0 (1H, s,
26.73; O, 5.09. Found: C, 61.31; H, 6.84; N, 26.52.
ArH), 8.2 (1H, m, ArH), 8.9 (1H, s, –NHCO–), 10.0 (1H, s,
5-(3-Cyclopropylureido)-N-[4-[(4-methylpiperazinyl)- –NHCO–), 13.2 (1H, s, pyrazole). MS m/z: 419.30 (M+); Anal.
methyl]phenyl]-1H-pyrazole-3-carboxamide (pym-5): A mix- Calcd for C23H26N6O2: C, 66.01; H, 6.26; N, 20.08; O, 7.65.
ture of 5 (314.0mg, 1.0mmol), CDI (210.6mg, 1.3mmol) in an- Found: C, 65.98; H, 6.43; N, 20.35.
hydrous DMF (20mL) was kept refluxing. Cyclopropylamine
N-[4-[(4-Methylpiperazin-1-yl)methyl]phenyl]-5-(pyridin-4-
(0.5mL) was added to the reaction solution after cooling down yl-methyl)-1H-pyrazole-3-carboxamide (pym-n): The mixture
to room temperature. The solution was stirred at room tem- of 5 (314.0mg, 1.0mmol), pyridine formaldehyde (0.5mL) and
perature for 3h, evaporated under vacuum to give pale yellow methanol (30mL) was heated to reflux, then cooled to room
oily substance. The crude product was separated by column temperature. An excess sodium borohydride (500.0mg) was
chromatography (developing solvent: methanol–chloroform added, then the reaction mixture was kept refluxing for 2h an-
1:30) to get pym-5 (222.0mg). Yield=56.1%; mp 156–158°C; devaporated under vacuum to give pale yellow oily substance
IR (KBr) cm−1: 2995 (CH2) cycloproyl, 1656 (C=O), 1280 and separated by column chromatograph (developing solvent:
(C–N) uera, 3371 (NH), 1344 (C=C), 1454 (C=N) pyrazole; methanol–chloroform 1:20). Yield=48.6%; mp 262–264°C;
1H-NMR (300MHz, DMSO-d6) δ: 0.4–0.6 (4H, m, –CH2–), IR (KBr) cm−1: 3369 (–NH–), 3335 (NH), 1456 (C=N), 1348
2.1 (3H, s, –CH3), 2.3–2.7 (9H, m, –CH2–, –CHN–), 3.3 (2H, (C=C), pyrazole, 1593 (ArH), 1666 (O=C–NH), 1130 (C–N).
s, –CH2–), 7.2 (2H, d, J=8.4Hz, ArH), 7.3 (1H, s, ArH), 7.7 1H-NMR (300MHz, DMSO-d6) δ: 2.2 (3H, s, –CH3), 2.3–2.5
(2H, d, J=8.4Hz, ArH), 8.0 (1H, s, –NHCONH–), 8.78 (1H, (8H, m, –CH2–), 3.4 (2H, s, –CH2–), 4.4 (2H, s, –CH2–),
s, –NHCONH–), 10.0 (1H, s, –NHCO–), 13.1 (1H, s, pyrazole); 5.6 (1H, m, –NH–), 6.9 (2H, m, ArH), 7.2 (2H, d, J=8.4Hz,
MSm/z: 396.94 (M−); Anal. Calcd for C20H27N7O2: C, 60.44; ArH), 7.3 (1H, s, ArH), 7.4 (2H, d, J=8.7Hz, ArH), 7.7 (2H, d,
H, 6.85; N, 24.67; O, 8.05. Found: C, 60.32; H, 7.06; N, 24.45. J=8.4Hz, ArH), 9.7 (1H, s, –NHCO–), 12.8 (1H, s, pyrazole);
N-[4-[(4-Methylpiperazin-1-yl)methyl]phenyl]-5- MS m/z: 406 (M+); Anal. Calcd for C22H27N7O: C, 65.16; H,
(phenylamino)-1H-pyrazole-3-carboxamide (pym-55): Com- 6.71; N, 24.1; O, 3.95. Found: C, 65.32; H, 6.90; N, 24.25.
pound
5
(314.0mg, 1.0mmol), benzoic acid (112.0mg,
5-(3-Cyclopropylureido)-N-[4-(morpholinomethyl)phenyl]-
1.3mmol), EDCI (249.2mg, 1.3mmol), HOBT (175.7mg, 1H-pyrazole-3-carboxamide (pyz-5): The preparation process
1.3mmol) were mixed in anhydrous DMF (30mL), stirred of pyz-5 which began with morphine was the same as pym-5.
24h at room temperature. The solvent was evaporated under Yield=56.1%; mp 121–123°C; IR (KBr) cm−1: 3335 (NH),
vacuum to give pale oily substance, the crude product was 1344 (C=C), 1454 (C=N) pyrazole, 2995 (CH2) cycloproyl,
separated by column chromatography (developing solvent: 1666 (O=C–NH), 1533, 1265 (amide), 1066 (C–O) ether;

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Table 1. Antiproliferative Activity and Kinase Inhibition of Four Derivatives
a)
a)
Cell line inhibition IC50 (µM)
Kinase inhibition IC50 (µM)
Compd.
HepG2
HCT116
CDK2
EGFR
ABL2
pym-5
32.23
89.5
15.94
10.93
NT
7.23
9.5
3.31
13.51
32.67
7.92
NT
2.05
3.98
12.24
2.34
NT
pym-n
pym-55
pyz-5
15.96
244.18
NT
10.96
6.18
NT
NT
Iressa
9.34
NT
Roscovitine
Staurosporine
9.87
0.01
NT
NT
NT
NT
NT
0.11
0.054
a) Values are the mean from two independent dose–response curves; variation was generally ( 25%) with the reference compounds with Iressa and Roscovitine. NT, not
tested.
1H-NMR (300MHz, DMSO-d6) δ: 0.7–1.0 (4H, m, –CH2–),
2.5 (4H, m, –NCH2–), 2.7 (1H, m, –CH–), 3.3 (2H, s, –CH2–),
3.6 (4H, m, –OCH2–), 7.0 (1H, m, ArH), 7.2 (2H, d, J=8.7Hz,
ArH), 7.7 (2H, d, J=8.7Hz, ArH), 10.0 (1H, s, –NHCO–), 11.3
(1H, s, –NHCONH–), 12.0 (1H, s, –NHCONH–), 14.0 (1H, s,
pyrazole); MS m/z: 383.90 (M−); Anal. Calcd for C19H24N6O3:
C, 59.36; H, 6.29; N, 21.86; O, 12.49%. Found: C, 59.65; H,
6.63; N, 21.55.
In Vitro Pharmacological Evaluation The antiprolifera-
tive activity of the four selected compounds was evaluated
by MTT tetrazolium dye assays on HCT116 cells and HepG2
cells (Table 1). Among the four compounds, the inhibition IC50
values of pym-5 and pym-55 are similar to the positive drug
Iressa (IC50=9.34 µM, HCT116) and Roscovtine (IC50=9.87 µM,
HepG2) respectively. However, the targeted kinase inhibition
IC50 values for CDK2, EGFR and ABL2 of four compounds
were not comparable to the positive drug.
Fig. 1. The Docking Mode of Compound pym-5 with DNA in the
Minor Groove
Study of the Interaction with DNA. Molecular Model- tial hydrogen bonds could be formed between urea group and
ing of Compound–DNA Interactions Molecular docking DNA. Both interactions may influence the conformation of the
was applied to investigate the molecular mechanism of DNA- phosphate-deoxyribose backbone. Due to the factors stated
ligand binding. The four compounds all interact with the base above, pym-5 would penetrate deeply into the minor groove
pairs of DNA via groove binding mode. Docking modes of the of DNA and exhibit a better DNA binding affinity.
four compounds are similar, and the detailed view of the DNA
Electronic Absorption Spectral Studies The potential
binding mode of pym-5 is shown in Fig. 1. The molecule is binding ability of four pyrazole compounds (pym-5, pyz-5,
shaped as a chain by two amide bonds on the pyrazole ring, pym-55 and pym-n) to calf thymus DNA (CT-DNA) was stud-
which enables the molecule to properly stretch along the DNA ied by electronic absorption spectroscopy. The UV-Vis spectra
minor groove. Furthermore, acetyl groups are conjugated with were measured in the way: Fixing the concentration of com-
the whole aromatic heterocycle, leading this part of the mol- pounds (Ccompound=3.00×10−5 M) while increasing the concen-
ecule to be a planar rigid system which could exactly extend tration of CT-DNA. The tendency of hypochromism without
into the space shaped by the adjacent base pairs and benefit obvious red shift occurred in four compounds can be observed
the binding through van der Waals. The benzene ring stacks in Fig. 2, which suggests that the interaction of compounds to
with two adenine bases in a parallel displaced conformation DNA is not from a classic intercalation mechanism but prob-
for π stacking interactions.
ably groove binding mode.42,43) The absorption relationship
The binding energy values for pym-5, pyz-5, pym-55 and between the compounds and DNA can be expressed by double
pym-n were scored as −49.67, −44.56, −30.91, −28.80kcal/ reciprocal equation44)
mol, respectively. Among them, pym-5 had the strongest
:
binding affinity with the lowest binding energy. Compared
(1)
(2)
(3)
1/ (A0 − A)=1/ A0 +1/ (Kb × A0 ×CDNA
)
with pym-n and pym-55, the cyclopropyl group of pym-5
formed a smaller steric-negative interaction with DNA, so
that pym-5 fit the minor groove of DNA more snugly and
exhibited a higher binding affinity. Also, at physiological
pH, the protonation of N atom on the piperazine ring is more
favorable than the morpholine of pyz-5 to interact with the
electronegative phosphorous group. In addition, the pyrazole
plane of pym-5 formed a weak σ–π stacking with the C4′ of
ΔA=A − A0
1/ΔA=−1/ (Kb × A0 ×CDNA ) −1/A0
Where A0 and A are the absorbance of compounds without
sugar residues compared to others, and meanwhile, the poten- and in the presences of DNA, respectively. Kb is the binding

March 2014
243
Fig. 2. Absorbance Changes of Compounds (3.00×10−5 M) in Tris–HCl Solution at 25°C in the Presence of Increasing Amount of CT-DNA
(0–1.27×10−4 M)
The arrow indicates the changes in absorbance upon increasing the concentration of DNA.
−1
Fig. 3. The Double Reciprocal Curve of DNA to the Four Compounds, Plot ∆A versus 1/CDNAM
−1
−1
Kpym-5=1.06×105
M
−1, Kpyz-5=8.83×104
M
−1, Kpym-55=6.85×104
M
and Kpym-n=1.64×104
M
.
constant between the compounds and DNA, and CDNA is the evaluated by electronic spectra that is pym-5>pyz-5>pym-
concentration of DNA. In the plot of ∆A versus 1/CDNA (Fig. 55>pym-n.
3), Kb is then given by the ratio of the intercept to slope inter-
Fluorescence Spectroscopic Studies With higher sen-
cept listed in Table 2, which indicates that the docking result sitivity and accuracy, fluorescence spectroscopic study was
of the DNA binding order is in accordance with the result carried out to further clarify the interaction mode of four

244
Vol. 62, No. 3
Table 2. The Docking Results and DNA-Binding Constants
Compound
pym-5
pyz-58
pym-55
pym-n
Binding energy (kcal/mol)
Binding constant K (L/mol)
−49.67
−44.56
−30.91
−28.80
1.06×105
8.83×104
6.85×104
1.64×104
Fig. 4. Representative Fluorescence Emission Spectra of DNA-EB in Tris–HCl Buffer (pH 7.4) with the Increasing in Molar Ratio of pym-5 to DNA
(0, 1/10, 1/5, 3/10, 2/5, 1/2, 3/5, 4/5, 1/1) at 25°C
Fig. 5. (a) Stern–Volmer Plots for the Fluorescence Quenching of the EB–CT-DNA Complex with the Compound pym-5 and (b) log((F0−F)/F) of
log[Q] for the Linear
compounds with DNA. No luminescence was observed at quencher compound) is linear for either simple dynamic
room temperature in the aqueous solution, neither in any quenching or static quenching. Hence, a broken line shown
organic solvent examined. Thus, the experiment was carried in Fig. 5a reveals that two dominant quenching processes
out in the EB background. EB molecule can make a strong exist in the system possibly.46) The turning point was found
interaction with the adjacent DNA base pairs and emit intense at the concentration of 4×10−5 M. That means the mechanism
fluorescence at about 550–650nm in the hydrophobic envi- of the quenching process changed at this concentration. Kq
ronment provided by the groove of DNA. This fluorescence (1.8×1012
−1/s) given by the ratio of the slope to intercept
M
would be quenched when the compound squeezed out of the in the second stage of this plot is greater than other kinds
EB molecule from the hydrophobic region and exposed the EB of quencher (the maximum diffusion of biological macro-
molecule to the polarity micro-environment by inserting into molecules sudden collision off constant Kq is 2×1010
−1/s),
M
the groove or the internal structure of the DNA. Therefore, suggesting that the second quenching is a static quenching
the quenching degree can be used to measure the binding process.47) The binding constant K and the site number of n
−1
propensity of the compound to DNA. As the concentration were calculated by Eq. (5),48) K and n were 8.57×105 M and
of the compound was increased, all the compounds showed 1.38, respectively shown in Fig. 5b.
distinctive decrease happened to the intensity of EB–CT-DNA
system. The most interesting spectrum was pym-5, in Fig.
4, more than 50% decrease of the emission intensity of the
EB–CT-DNA (1×10−4 M) complex with the compound pym-5
(5)
log[(F0 − F) / F]=log+n log[Q]
Viscosity Measurement Spectra probes generally provide
(3×10−5 M) being added, which indicates a strong interaction necessary, but not more persuasive evidence than viscos-
between pym-5 and DNA. Based on the classical theory of ity measurement. The value of viscosity is sensitive to the
fluorescence quenching: Sterm–Volmer equation45)
length change of DNA. The distance between adjacent base
pair will be larger, when the molecule intercalates into DNA,
causing obviously lengthening. In other way of interaction
model (electrostatic attraction or groove binding), due to no
(4)
F /F0 =1+Kqτ [Q]
0
The plot of F0/F versus [Q] ([Q] is the concentration of increase in the DNA length the viscosity of the sample solu-

March 2014
245
cleavage activity of pym-5 may be attributed to a hydrolysis
of pBR322DNA based on the nucleophile attacking phospho-
nates by the uramido group. Also, cationic protonated tertiary
amine under the pH7.4 may stabilize the negatively charged
nuclear phosphorus penta-coordinated transition state.50)
Conclusion
In this study, four compounds with the skeleton of 1H-pyr-
azole-3-carboxamide were synthesized in moderate reaction
condition and high yield. In the in vitro pharmacological eval-
uation, they exhibited significant inhibition against two can-
cer cell lines (HCT116, HepG2) and weak kinase inhibition.
Then molecular docking combined with spectra experiments
and viscosity measurements were employed to investigate
their potential DNA-damaging ability and DNA interaction
mode. The concentration dependence of hypochromism in the
electronic absorption spectra demonstrated the validity and
reliability of mode predicted by the docking. Meanwhile, the
result of binding constants calculated from the UV absorption
experiment was consistent with the rank order of binding en-
ergy obtained from docking. Besides, the efficient competitive
binding ability in fluorescence spectra and chemical nuclease
activity for DNA strand scission in Agarose gel electrophore-
sis were observed in pym-5. In summary, compound pym-5
was identified to have a significant off-target interaction with
DNA, which may contribute to inhibiting the proliferation of
cancer cells by DNA damage mechanism. Furthermore, the
study emphasized the possible off-target effects based on the
specific characteristics of the pyrazole group in the kinase
inhibitor discovery.
Fig. 6. Effect of Increasing Amounts of Compound on the Viscosity of
CT-DNA (1.5×10−4 M) in Tris–HCl Buffer (r=0.0, 0.05, 0.10, 0.25, 0.3,
0.45, 0.6, 0.8, 1)
Acknowledgments We are grateful to Huifang Li for her
valuable and constructive suggestions during the revising and
development of this research work. We also acknowledge the
financial support from the National Nature Science Founda-
tion of China (No. 81172933), Project Program of State Key
Laboratory of Natural Medicines, China Pharmaceutical Uni-
versity (Program No. JKGP201105) and Specialized Research
Fund for the Doctoral Program of Higher Education (No.
20100096110007).
Fig. 7. Agarose Gel Electrophoresis Patterns for the Cleavage of pBR
322 Plasmid DNA (0.25µg/µL) with the Addition of Four Compounds
Separately in Different Concentration under Dark for 1.5h, the Result
Shown in a (pym-5), b (pym-55), c (pym-n), d (pyz-5) and Lane 1: DNA
Control; Lane 2: (1×10−11 M); Lane 3: (5 ×10−10 M); Lane 4: (5×10−9 M);
Lane 5: (5×10−8 M); Lane 6: pym-5 (5×10−7 M); Lane 7: (5×10−6 M); Lane
8: (5×10−5 M); Lane 9: (5×10−4 M); Lane 10: (5×10−3 M)
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