Novel pyrazole-5-carboxamide and pyrazole-pyrimidine derivatives: Synthesis and anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2014.12.013

A series of novel pyrazole-5-carboxamide and pyrazole-pyrimidine derivatives were designed and synthesized. All compounds have been screened for their antiproliferative activity against MGC-803, SGC-7901 and Bcap-37 cell lines in vitro. The results revealed that compounds 8a, 8c and 8e exhibited strong inhibitory activity against MGC-803 cell line. The flow cytometric analysis result showed that compound 8e could inhibit MGC-803 proliferation. Some title compounds were tested against telomerase, and compound 8e showed the most potent inhibitory activity with IC₅₀ value at 1.02 ± 0.08 μM. The docking simulation of compound 8e was performed to get the probable binding model, among them, LYS 189, LYS 372, LYS 249 and ASP 254 may be the key residues for the telomerase activity.

Full text of DOI:10.1016/j.ejmech.2014.12.013

Accepted Manuscript
Novel pyrazole-5-carboxamide and pyrazole-pyrimidine derivatives: synthesis and
anticancer activity
Jing Bo Shi , Wen Jian Tang , Xing Bao qi , Jun Li , Rong Li , Xin Hua Liu
PII:
S0223-5234(14)01122-2
10.1016/j.ejmech.2014.12.013
EJMECH 7573
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 September 2014
Revised Date: 4 December 2014
Accepted Date: 8 December 2014
Please cite this article as: J.B. Shi, W.J. Tang, X. Bao qi, J. Li, R. Li, X.H. Liu, Novel pyrazole-5-
carboxamide and pyrazole-pyrimidine derivatives: synthesis and anticancer activity, European Journal of
Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2014.12.013.
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ACCEPTED MANUSCRIPT
Graphical abstract
Novel pyrazole-5-carboxamide and pyrazole-pyrimidine derivatives:
synthesis and anticancer activity
a,
a
a
a
a
a,b
Jing Bo Shi *, Wen Jian Tang , Xing Bao qi , Jun Li , Rong Li , Xin Hua Liu
*
a
School of Pharmacy, Anhui Medical University, Hefei, 230032, P. R. China
b
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, P. R. China
Novel pyrazole-5-carboxamide and pyrazolo[4,3-d]pyrimidine derivatives were
designed and synthesized. Compound 8e exhibited strong inhibitory activity against
MGC-803 cells, and showed the most potent telomerase inhibitory activity.

ACCEPTED MANUSCRIPT
Novel pyrazole-5-carboxamide and pyrazole-pyrimidine derivatives:
synthesis and anticancer activity
a,
a
a
a
a
a,b
Jing Bo Shi *, Wen Jian Tang , Xing Bao qi , Jun Li , Rong Li , Xin Hua Liu
*
a
b
School of Pharmacy, Anhui Medical University, Hefei, 230032, P. R. China
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
10093, P. R. China
2
Abstract: A series of novel pyrazole-5-carboxamide and pyrazole-pyrimidine derivatives
were designed and synthesized. All compounds have been screened for their
antiproliferative activity against MGC-803, SGC-7901 and Bcap-37 cell lines in vitro. The
results revealed that compounds 8a, 8c and 8e exhibited strong inhibitory activity against
MGC-803 cell line. The flow cytometric analysis result showed that compound 8e could
inhibit MGC-803 proliferation. Some title compounds were tested against telomerase, and
compound 8e showed the most potent inhibitory activity with IC value at 1.02±0.08 µM.
5
0
The docking simulation of compound 8e was performed to get the probable binding model,
among them, LYS 189, LYS 372, LYS 249 and ASP 254 may be the key residues for the
telomerase activity.
Keywords: Synthesis; pyrazole-5-carboxamide; anticancer activity; telomerase
_
________________
*
Corresponding author.
Tel.: +86 551 65161115; fax: +86 551 65161115. E-mail: sjb0616@126.com.
86 551 65161115; fax: +86 551 65161115. E-mail: xhliuhx@163.com.
+
1

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1
. Introduction
Telomerase is active in the early stages of life. In majority of adult somatic cells, it
turns to dormancy. However, in cancer cells, telomerase is reactivated to keep the telomere
length short in rapidly dividing cells, leading to proliferation. So, telomerase represents
one of the promising targets in drug discovery [1-4]. Telomere and telomerase closely
related to the occurrence and development of gastric cancer has been reported [5]. In recent
publications, one new hypothesis could be utilized to explain the association between
telomerase TERT (human telomerase reverse transcriptase) and cancers. The regulation of
telomerase TERT predominantly leads to cell proliferation or apoptosis, ultimately
resulting in anticancer activity. Unfortunately, these broad-spectrum telomerase TERT
inhibitors have been limited by non-specificity and thus non-selective toxicity and
dose-limiting efficacy. Therefore, the pursuit of novel telomerase inhibitors with better
antitumor effects and more safety profile is still the main issue.
The pyrazole-pyrimidine and their bioisosteres are heterocyclic compounds with
important biological functions including antitumor and other activities [6-8]. As is known
to all that pyrazole-pyrimidine derivatives PP242, PP30 and PDE5 (Figure 1) have
possessed good kinase selectivity profile used as cyclin-dependent kinase (CDK),
ATP-competitive mTORC1/mTORC2 inhibitors [9-16]. It was of our interest to utilize
rational chemical approaches to generate and identify novel compounds as potential
telomerase inhibitors for cancer therapy. Results of structure-activity relationships (SAR)
about these derivatives show that the alkyl of the position 1 or 3 should help the activity,
there is some motivations provided in the design idea [17-20]. Furthermore, based on the
protein TERT structure of telomerase using LigandFit module, our group has recently
reported a novel docking model and we found that ASP 254 was a key residue for activity.
Focusing on residue ASP 254 and the volume of the active site of hTERT, we therefore
designed some of synthesizable drug-like scaffolds which incorporate the moiety of
pyrazole-pyrimidine.
In our recent works [21,22], several pyrazole derivatives were designed, which had
potent anticancer activity as potential telomerase inhibitors. Because moiety
2

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pyrazole-5-carboxamide is a precursor of title pyrazole-pyrimidine structure (Figure 2), in
order to summarize the SAR, we have also carried out active screening against them. In
this study, a series of novel small molecules elaborated around pyrazole-5-carboxamide
and pyrazole-pyrimidine scaffolds were designed. We also will built the relative SAR
between pyrazole-pyrimidines to their precursors as antitumor agents, which maybe
rationalize the experimental observations and guide us to screen good potency telomerase
inhibitors in the further study.
(Figures 1-2)
2
. Results and discussion
2
.1 Chemistry
All derivatives were synthesized by acylation reaction starting from
-methyl-4-nitro-3-N-propylpyrazole-5-carboxamides 7, which was prepared from
-pentanone via claisen condensation, hydrazinolytic, cyclization, methylation, hydrolysis,
1
2
nitration, amidation, reduction according to Scheme 1. Compounds 8a-e were obtained
through two steps: First, the substituted carboxylic acids were converted into acyl chlorides
with oxalyl chloride at room temperature and excess oxalyl chloride was evaporated, then
the acyl chloride was directly reacted with compounds 7 to form amide compounds 8a-e.
Compounds 9a-e were obtained from compounds 8a-e via cyclization. The reaction was
carried out in the presence of sodium ethoxide, the EtOH used as solvent at reflux
1
13
condition. All compounds were characterized by means of HR-MS, H NMR and
C
NMR spectral analysis.
(Scheme 1)
2
.2 Crystal structure analysis
The structures of compounds 8d and 9a were determined by X-ray crystallography.
o
Crystal data of 8d: Colorless crystals, yield, 77%; mp 151-152 C; C H N O ,
1
8
24
4
5
Monoclinic, space group P 2 /c; a = 14.4205(14), b = 17.7873(16), c = 8.8377(9) (Å);
α
=
1
1
3

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3
3
9
0,
β
= 100.983(10),
γ
= 90 (°), V = 2225.4(4) nm , T =293(2) K, Z =4, Dc = 1.261 g/cm ,
F(000)= 904, Reflections collected / unique = 9519 / 4373, Data / restraints / parameters =
2
2
4
373 / 6 / 279, Goodness of fit on F =1.087, Fine, R = 0.0681, wR(F ) = 0.1722. Crystal
1
o
data of 9a: Colorless crystals, yield, 85%; mp 200-201 C; C H N O, Monoclinic, space
1
7
18
4
group P 2 /c; a = 16.820(5), b = 5.381(3), c = 18.370(6) (Å);
α
= 90,
β
= 112.73(4),
°), V = 1533.4(11) nm , T =293(2) K, Z =4, Dc = 1.275 g/cm , F(000)= 624, Reflections
collected / unique = 3274/ 1312, Data / restraints / parameters = 3274 / 0 / 202, Goodness
γ = 90
1
1
3
3
(
2
2
of fit on F =0.880, Fine, R = 0.0741, wR(F ) = 0.1818. Their molecular structures were
1
shown in Figure 3. Crystallographic data (excluding structure factors) for the structures
had been deposited with the Cambridge Crystallographic Data Center as supplementary
publication No. CCDC-981021 and 1021035.
(Figure 3)
2
.3 In vitro anticancer activity
Recently, we focused on screening lead compounds with specific activity against
gastric cancer cells, so gastric cancer cell SGC-7901 and MGC-803 were chosen. In this
screening assay studies, all the compounds were evaluated for their cytotoxic activity
against SGC-7901, MGC-803 cell lines. In order to examine activity with other cancer
cells, Bcap-37 (human breast cancer cell line) was chosen. The results were reported in
terms of IC values (Table 1).
5
0
In the initial study of SAR, our title compounds were divided into two series, one was
the 5-propyl-1H-pyrazole-3-carboxamide (compounds 8a~8e), the other was the
pyrazolo[4,3-d]pyrimidin-7(6H)-one (compounds 9a~9e). Among them, compound 8 is the
precursor of compound 9. In general, series 5-propyl-1H-pyrazole-3-carboxamide
(compounds 8) leded to increase in inhibitory activity, among them, compound 8a was the
most potent activity against MGC-803 cell with IC50 value of 3.01±0.23 ꢀM, surpassing
that of the positive control 5-fluorouracil. Compared with the compounds 8, compounds 9
generally did not reflect the activity on the tested cells (9b, 9d). Therefore, for this kind of
4

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structure moiety, activity of precursor compounds was superior to that cyclization
compounds.
The SAR also indicated that all title compounds showed good activity against gastric
cancer cells but poor activity against Bcap-37 cells. Scanning from Table 1, it is obvious
that compounds 8a, 8b and 8c exhibited the strong inhibitory activity against the MGC-803
cells (with IC50s of 3.44±0.28, 4.57±0.88, 4.24±1.22 µM, respectively) and the values
could compare with that of the potent 5-fluorouracil.
(Table 1)
2
.4 Cell Cycle Analysis
To understand whether the cell cycle arrest lead to decrease cell proliferation [23], we
used flow cytometric analysis to measure the effect of compound 8e on induction of cell
cycle. As shown in Figure 4, the cells in S phase in the MGC-803 control group accounted
for about 30.38%, while after cells treated with compound 8e MGC-803 for 48 h, the ratio
was approximately 42.16%. This showed that the cells were arrested in S phase.
(Figure 4)
2
.5 Telomerase activity
Some compounds were assayed for telomerase inhibition, using MGC-803 cells
extract, ethidium bromide and BIBR1532 were used as the reference compounds. The
results (Table 2) suggested that compounds 8a and 8e showed strong telomerase inhibitory
activity with IC values of 1.98±0.21, 1.02±0.08 µM respectively, which surpassing that
5
0
of the positive control ethidium bromide, comparable to that of positive control BIBR1532,
furthermore, there was a good correlation between antiproliferative activity and telomerase
activity of compound 8e (Tables 1 and 2). Among them, compound 8e
(E)-4-(3-(furan-2-yl)acrylamido)-1-methyl-3-propyl-1H-pyrazole-5-carboxamide showed
higher inhibitory activity than the others. This also pointed out the direction for us to
5

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further optimize the structure of 1H-pyrazole-5-carboxamide moiety with anticancer
activity as potential telomerase inhibitors.
(Table 2)
2
.6 Molecular docking
In an effort to elucidate the mechanism by which the title compound exhibited strong
inhibitory activity against telomerase and to establish SAR based on our experimental
studies, molecule docking of the compound 8e into active site containing key residue ASP
2
54 was performed to simulate a binding model derived from telomerase TERT (3DU6.
pdb). In general, compound 8e could fit well into the catalytic subunit of telomerase
TERT) and inhibit telomerase activity as a substitute of substrate nucleotides. There are
(
six obvious interactions diagram were shown. The binding model of compound 8e with
TERT was depicted in Figure 5. The 2D and 3D pictures of binding were depicted in
Figure 5A and Figure 5B. In order to reveal that the molecule was well filled in the active
pocket, the enzyme surface was shown in Figure 5C. There are six obvious bonding
interactions anchoring compound 8e to the active site tightly might explain its good
inhibitory activity. First, we found hydrogen bond between the backbone amino groups of
ASP 254 and N atom of pyrazole moiety. Another two hydrogen bonds interaction were
between the LYS 189 and the O atoms of amide. Second, three Pi-Cation interactions were
formed (pyrazole ring with LYS 189; furan ring with the LYS 372 and LYS 249,
respectively).
(Figure 5)
3
. Conclusions
In brief, based on rational design and reasonable analysis, we designed some novel
H-pyrazole-5-carboxamide derivatives. Next, these compounds used as precursors due to
1
exploring reasonable SAR, some new 1H-pyrazolo[4,3-d]pyrimidin-7(6H) derivatives
were designed, followed by chemical synthesis and biological evaluated for them. The
6

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results revealed that some 1H-pyrazole-5-carboxamide compounds reflected high activity
against gastric cancer cells. Among them, compound 8e exhibited strong inhibitory activity
against MGC-803 cells, and showed the most potent telomerase inhibitory activity with
IC value at 1.02±0.08 µM. Flow cytometric analysis indicated that the cells were arrested
5
0
in S phase by compound 8e. The docking simulation was performed to get the probable
binding models poses and the key residues ASP 254, LYS 189, LYS 372, LYS 249.
Compared with our previous work, the advantage of this study was that new skeleton
structure targeting telomerase was found. These results are of help in the rational design of
higher selectivity telomerase inhibitors in further.
4
. Experimental section
4
.1 Chemistry
The reactions were monitored by thin layer chromatography (TLC) on pre-coated
silica GF254 plates. Melting points were determined on a XT4MP apparatus (Taike Corp.,
1
13
Beijing, China), and are uncorrected. H and C NMR spectra were recorded on a Brucker
AM-300 (300 MHz) spectrometer with CDCl or DMSO-d as the solvent and TMS as the
3
6
internal standard. Chemical shifts are reported in δ (parts per million) values. Coupling
constants J are reported in Hz. High-resolution electron impact mass spectra (HR-MS)
were recorded under electron impact (70 eV) condition using a MicroMass GCT CA 055
instrument. All chemicals or reagents were purchased from standard commercial suppliers
and treated with standard methods before use. Solvents were dried in a routine way and
redistilled.
4
.2. General procedure for the synthesis of title compounds 8
To a methylene chloride (10mL) solution of substituted Carboxylic acid 3 (5 mmol)
in an ice bath was added oxalyl chloride (10 mmol), and catalytic N,N-Dimethylformamide,
while the reaction contents were stirred continuously. The reaction mixture was allowed to
o
stand at 25-30 C for 3 h. Solvent distilled under reduced pressure and dried under high
vacuum to remove oxalyl chloride. Reaction mass was dissolved in dichloromethane (10
ml). The solution was added to the mixture of amine 7 (4.5 mmol) and TEA (10 mmol) and
then allowed to stir at room temperature for overnight. Reaction mixture was washed with
7

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saturated sodium bicarbonate (20 ml) and then with water (20 ml). Organic solvent was
dried over anhydrous Na SO , filtrated, and then evaporated to give solid. Crude solid
2
4
product was recrystallied from suitable solvent to give title compounds 8a-e (Scheme 1) as
solids.
8
a: 4-cinnamamido-1-methyl-3-propyl-1H-pyrazole-5-carboxamide
o
1
Off-white powder solid, yeild, 75%; mp: 225-226 C (recryst. from ethanol); H NMR
(
300 MHz, CDCl +DMSO-d ) δ (ppm): 9.03 (Brs, 1H, NH), 7.56 (d, J=15.52, 1H, Ar-CH),
3 6
7
3
3
8
.36 (s, 2H, ArH), 7.21 (m, 3H, ArH), 6.54 (d, J=15.72, 1H, CO-CH), 6.13 (Brs, 2H, NH2),
.86 (s, 3H, N-CH ), 2.35 (t, J = 7.6 Hz 2H, N-CH ), 1.45 (m, 2H, CH ), 0.78 (t, J = 7.0 Hz,
3
2
2
+
H, CH ). HR-MS: calcd for C H N O [M+Na] , 335.1478; found 335.1479.
3
17 20
4
2
b: 1-methyl-3-propyl-4-(3,4,5-trimethoxybenzamido)-1H-pyrazole-5-carboxamide
o
1
Off-white powder solid, yeild, 73%; mp: 190-191 C (recryst. from ethanol); H NMR
300 MHz, CDCl ) δ (ppm): 7.98+7.74 (Brs, 1H, NH), 7.12 (d, 2H, ArH), 5.69 (Brs, 2H,
(
3
NH ), 3.92 (s, 12H, 3O-CH , N-CH ), 2.53 (m, 2H, N-CH ), 1.63 (m, 2H, CH ), 0.92 (m,
2
3
3
2
2
+
3
H, CH ). HR-MS: calcd for C H N O [M+Na] , 399.1639; found 399.1638.
3 18 24 4 5
8
c: (E)-1-methyl-3-propyl-4-(3-(3,4,5-trimethoxyphenyl)acrylamido)-1H-pyrazole-5-
carboxamide
o
1
Off-whit powder solid, yeild, 80%; mp: 203-204 C (recryst. from ethanol); HNMR
(
300 MHz, CDCl +DMSO-d ) δ (ppm): 9.62 (Brs, 1H, NH), 7.62 (d, J=15.63, 1H, Ar-CH),
3
6
6
.81 (s, 2H, ArH), 6.69 (d, J=15.63, 1H, CO-CH), 4.14 (s, 3H, N-CH ), 3.90 (s, 6H, 2 O-
3
CH ), 3.85 (s, 3H, O-CH ), 2.51 (t, J = 7.6 Hz, 2H, N-CH ), 1.62 (m, 2H, CH ), 0.91 (t, J =
3
3
2
2
+
7
.3 Hz, 3H, CH ). HR-MS: calcd for C H N O [M+Na] , 425.1795; found 425.1794.
3 20 26 4 5
8
d: 1-methyl-3-propyl-4-(2,4,5-trimethoxybenzamido)-1H-pyrazole-5-carboxamide
o
1
Off-whit powder solid, yeild, 77%; mp: 150-151 C (recryst. from ethanol); H NMR
300 MHz, CDCl ) δ (ppm): 9.30 (Brs, 1H, NH), 8.0+5.60 (Brs, 2H, NH ), 7.83 (s, 1H,
(
3
2
ArH), 6.60 (s, 1H, ArH), 4.08 (s, 3H, N-CH ), 3.93~4.05 (s, 9H, 3O-CH ), 2.61(t, J = 7.5
3
3
Hz, 2H, N-CH ), 1.63 (m, 2H, CH ), 0.94 (t, J = 7.3 Hz, 3H, CH ). HR-MS: calcd for
2
2
3
+
C H N O [M+H] , 377.1819, found 377.1824.
18
25
4
5
8
e: (E)-4-(3-(furan-2-yl)acrylamido)-1-methyl-3-propyl-1H-pyrazole-5-carboxamide
o
Light brown powder solid, yeild, 65%; mp: 249-252 C (recryst. from 95% ethanol);
8

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1
H NMR (300 MHz, DMSO-d ) δ (ppm): δ 9.57 (Brs, 1H, NH), 7.83 (d, J = 1.3 Hz, 1H,
6
Furan-H), 7.73 (s, 1H, Furan-H), 7.41 (d, J = 15.6 Hz, 1H, Ar-CH), 6.86 (d, J = 3.4 Hz, 1H,
Furan-H), 6.63 (d, J = 15.6 Hz, 1H, CO-CH), 3.88 (s, 3H, N-CH ), 2.37 (t, J = 7.5 Hz, 2H,
3
N-CH ), 1.59-1.46 (m, 2H, CH ), 0.86 (t, J = 7.4 Hz, 3H, CH ).
2
2
3
4
.3. General procedure for the synthesis of title compounds 9
Compound 8 (1 mmol) and sodium ethoxide (5 mol) were dissolved in ethanol (15
mL), and refluxed for 12 h. After completion of the reaction, the solvent was concentrated
to a low volume; the residue was added HCl (1N), the precipitated product was granulated
o
at pH 7 and 10 C for a further hour. The title compounds 9a-9e was collected by filtration,
washed with water, and dried to give as solid.
9
a: (E)-1-methyl-3-propyl-5-styryl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one
o
1
Off-whit powder solid, yeild, 85%; mp: 200-201 C; H NMR (300 MHz, CDCl ) δ
3
(ppm): 11.60 (Brs, 1H, NH), 7.80 (d, J=16.5, 1H, Ar-CH), 7.60 (d, J = 7.1 Hz, 2H, ArH),
7
.39(m, J = 6.8 Hz, 3H, ArH), 6.96 (d, J=16.5, 1H, CO-CH), 4.28( s, 3H, N-CH ), 2.92 (t,
3
13
J = 7.5 Hz, 2H, N-CH ), 1.84 (m, 2H, CH ), 1.03 (t, J = 7.3 Hz, 3H, CH ). CNMR (75
2
2
3
MHz, CDCl ): 156.27, 149.17, 146.78, 139.63, 136.99, 135.57, 129.65, 129.07, 127.54,
3
1
24.35, 121.54, 38.34, 27.85, 22.56, 14.24.
9
b: 1-methyl-3-propyl-5-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)
-one
o
1
Off-whit powder solid, yeild, 84%; mp: 204-206 C; H NMR (300 MHz, CDCl ) δ
3
(
ppm): δ 11.05 (Brs, 1H, NH), 7.29 (s, 2H, ArH), 4.24 (s, 3H, N-CH ), 4.00 (s, 6H, 2 x
3
O-CH ), 3.88 (s, 3H, O-CH ), 2.95 (t, J = 7.6 Hz, 2H, N-CH ), 1.95–1.78 (m, 2H, CH ),
3
3
2
2
1
.04 (t, J = 7.3 Hz, 3H, CH3).
9
c: (E)-1-methyl-3-propyl-5-(3,4,5-trimethoxystyryl)-1H-pyrazolo[4,3-d]pyrimidin-7
(6H)-one
o
1
Off-whit powder solid, yeild, 81%; mp: 266-270 C; H NMR (300 MHz, CDCl ) δ
3
(ppm): δ 11.61 (Brs, 1H, NH), 7.67 (d, J = 16.4 Hz, 1H, Ar-CH), 6.91 (d, J = 16.4 Hz, 1H,
CO-CH), 6.82 (s, 2H, ArH), 4.29 (s, 3H, N-CH ), 3.91 (d, 9H, 3O-CH ), 2.92 (t, J = 7.6 Hz,
3
3
2
H, N-CH ), 1.93 – 1.77 (m, 2H, CH ), 1.04 (t, J = 7.3 Hz, 3H, CH ). HR-MS: calcd for
2 2 3
+
C H N O [M+H] , 385.187; found 385.1874.
20
25
4
4
9

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9
d: 1-methyl-3-propyl-5-(2,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7
(6H)-one
o
1
Off-whit powder solid, yeild, 82%; mp: 184-187 C; H NMR (300 MHz, CDCl ) δ
3
(ppm): δ 10.99 (Brs, 1H, NH), 8.04 (s, 1H, ArH), 6.59 (s, 1H, ArH), 4.27 (s, 3H, N-CH3),
4
.05 (s, 3H, O-CH ), 3.98 (s, 6H, 2O-CH ), 2.94 (t, J=7.71, 2H, N-CH ), 1.95–1.81 (m, 2H,
3 3 2
+
CH ), 1.04 (t, J = 7.4 Hz, 3H, CH ). HR-MS: calcd for C H N O [M+H] , 359.1716;
2
3
18 23
4
4
found 359.1716.
9
e: (E)-5-(2-(furan-2-yl)vinyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one
o
1
Light brown powder solid, yeild, 79%; mp: 270-272 C; H NMR (300 MHz,
DMSO-d ) δ (ppm): δ 12.29 (Brs, 1H, NH), 7.82 (d, J = 1.5 Hz, 1H, Ar-CH), 7.64 (d, J =
6
1
5.84 Hz, 1H, CO-CH), 6.85 (d, J = 3.3 Hz, 1H, Furan-H), 6.80 (d, J = 15.84 Hz , 1H,
Furan-H), 6.63 (dd, J = 3.4, 1.8 Hz, 1H, Furan-H), 4.12 (s, 3H, N-CH ), 2.77 (t, J = 7.5 Hz,
3
2
H, N-CH ), 1.81–1.69 (m, 2H, CH ), 0.95 (t, J = 7.4 Hz, 3H, CH ).
2
2
3
4
.4 Crystallographic Studies
A colorless single crystal of title compounds 8d and 9a were chosen for X-ray
diffraction analysis performed on a BRUCKER SMART APEX-CCD diffractometer
equipped with a graphite monochromatic MoKa radiation (λ = 0.71073 A) radiation at
o
2
96(2) K. A total reflections were collected in the range of 2.30<θ<25.30 by using a ψ-ω
scan mode with independent ones, of which I >2σ(I) were observed and used in the
succeeding refinements. The data set was corrected by SADABS program; the structure
were solved by direct methods with SHELXS-97 and refined by full-matrix least-squares
2
method on F with SHELXL-97 [24]. The non-hydrogen atoms were refined
anisotropically, and the hydrogen atoms were added according to theoretical models. The
2
structures were refined by full-matrix least-squares method on F with SHELXT-97.
4
.5 Anticancer assay
The cytotoxicity evaluation was conducted by using a modified procedure as
described in the literature. Briefly, target tumor cells were grown to log phase in RPMI
4
1
640 medium supplemented with 10% fetal bovine serum. After diluting to 3×10 cells
-1
mL with the complete medium, 100 ꢀL of the obtained cell suspension was added to each
well of 96-well culture plates. The subsequent incubation was performed at 37 ˚C, 5% CO2
1
0

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atmosphere for 24 h before subjecting to cytotoxicity assessment. Tested samples at pre-set
concentrations were added to 6 wells with 5-fluorouracil co-assayed as a positive reference.
-1
After 48 h exposure period, 25 ꢀL of PBS containing 2.5 mg mL of MTT (3-(4,
5
4
-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) was added to each well. After
h, the medium was replaced by 150 ꢀL DMSO to dissolve the purple formazan crystals
produced. The absorbance at 570 nm of each well was measured on an ELISA plate reader.
The data represented the mean of three experiments in triplicate and were expressed as
means ±SD using Student t test. The IC value was defined as the concentration at which
5
0
5
0% of the cells could survive.
4
.6 Cell cycle analysis
For cell cycle analysis, we performed cell cycle kit (Beyotime, China). MGC-803
cells were washed three times by cold PBS, and then cells were fixed in 70% ethanol at -20
C for 12 h. After fixation, cells were washed with cold PBS and stained with 0.5 mL of
˚
propidium iodide (PI) staining buffer, which contain 200 mg/mL RNase A, 50 ꢀg/mL PI, at
◦
3
7 C for 30 min in the dark. Analyses were performed on FACScan flow cytometer. The
experiments were repeated three times.
4
.7 Telomerase activity assay
Compounds were tested in a search for small molecule inhibitors of telomerase
activity by using the TRAP-PCR-ELISA assay. In detail, the MGC-803 cells were firstly
maintained in DMEM medium (GIBCO, New York, USA) supplemented with 10% fetal
bovineserum (GIBCO, NewYork, USA), streptomycin (0.1 mg/mL) and penicillin (100
IU/mL) at 37 °C in a humidified atmosphere containing 5% CO . After trypsinization,
2
4
5
×10 cultured cells in logarithmic growth were seeded into T25 flasks (Corning, New
York, USA) and cultured to allow to adherence. The cells were then incubated with
Staurosporine (Santa Cruz, Santa Cruz, USA) and the drugs with a series of concentration
as 60, 20, 6.67, 2.22, 0.74, 0.25 and 0.082l g/mL, respectively. After 24 h treatment, the
cells were harvested by cell scraper orderly following by washed once with PBS. The cells
were lysed in 150 ꢀL RIPA cell lysis buffer (Santa Cruz, Santa Cruz, USA), and incubated
on ice for 30 min. The cellular supernatants were obtained via centrifugation at 12,000 g
for 20 min at 4 °C and stored at -80 °C. The TRAP-PCR-ELISA assay was performed
11

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using a telomerase detection kit (Roche, Basel, Switzerland) according to the
manufacturer’s protocol. In brief, 2 ꢀL of cell extracts were mixed with 48 ꢀL TRAP
reaction mixtures. PCR was then initiated at 94 °C, 120 s for predenaturation and
performed using 35 cycles each consisting of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 90 s.
Then 20 ꢀL of PCR products were hybridized to a digoxigenin (DIG)-labeled telomeric
repeat specific detection probe. And the PCR products were immobilized via the
biotin-labeled primer to a streptavidin-coated microtiter plate subsequently. The
immobilized DNA fragment were detected with a peroxidase-conjugated anti-DIG
antibody and visualized following addition of the stop regent. The microtitre plate was
assessed on TECAN Infinite M200 microplate reader (Mannedorf, Switzerland) at a
wavelength of 490 nm, and the final value were presented as mean ±SD.
4
.8 General procedure for molecular docking
Discovery Studio 3.1 (DS 3.1, Accelrys Software Inc., San Diego, California, USA).
Crystal structure of telomerase (PDB entry 3DU6) was used as template. Hydrogen atoms
were added to protein model. The added hydrogen atoms were minimized to have stable
energy conformation and to also relax the conformation from close contacts. The active
site was defined and sphere of 5 Å was generated around the active site pocket, with the
active site pocket of BSAI model using C-DOCKER, a molecular dynamics (MD)
simulated-annealingbased algorithm module from DS 3.1. Random substrate
conformations are generated using high-temperature MD. Candidate poses are then created
using random rigid-body rotations followed by simulated annealing. The structure of
protein, substrate were subjected to energy minimization using CHARMm forcefield as
implemented in DS 3.1. A full potential final minimization was then used to refine the
substrate poses. Based on C-DOCKER, energy docked conformation of the substrate was
retrieved for postdocking analysis.
Acknowledgments
The authors wish to thank the National Natural Science Foundation of China (No.
2
1272008), Anhui Provincial Natural Science Foundation, (1308085MH137), Science and
Technological Fund of Anhui Province for Outstanding Youth (1408085J04).
1
2

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Supporting information
CCDC-981021 (compound 8d) and CCDC-1021035 (compound 9a) contain the
supplementary crystallographic data for this paper. These data can be obtained free of
charge via the URL http:// www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC,
1
2 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223 336033; e-mail:
deposit@ccdc.cam.ac.uk).
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Figure Captions
Table 1. Cytotoxic activity of title compounds against SGC-7901, MGC-803, Bcap-37
Table 2. Inhibitory effects of slected compounds against telomerase
Figure 1. Pyrazole-pyrimidine scaffold based potential candidates and drugs
Figure 2. Design of pyrazole-pyrimidine and its precursor scaffold
Figure 3. ORTEP drawing of compounds 8d and 9a
Figure 4. Cells cycle analysis by flow cytometry with MGC-803 cells
Figure 5. The binding mode between the active conformation of compound 8e and the
protein TERT (PDB code: 3DU6)
Scheme 1. Synthesis of title compounds 7~9
1
5

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O
O
O
O
(B)
(A)
+
O
O
O
O
NH
O
ONa
N
O
1
2
O
NO2
(D)
O
(E)
O
(C)
O
N
N
N
OH
N
N
N
OH
N
3
4
5
H N
2
NO2
NH2
(F)
O
(G)
O
N
O
N
(H)
N
NH2
NH2
N
N
HN
R1
6
7
O
8
O
HN
(I)
N
R1
N
N
9
O
O
O
8
a 9a R1=
8b 9b R1=
8c 9c R1=
O
O
O
O
O
8
d
9d R1=
8e
R1=
9e
O
O
Scheme 1. Synthesis of title compounds 7~9
o
Reagent and conditions: (A) Na, EtOH, reflux, 6~8 h; (B) NH -NH •H O, 60-65 C, 2 h;
2
2
2
o
o
o
(
C) Dimethyl sulfate, 80 C, 2 h; (D) 6 N NaOH, 80 C, 2 h; (E) HNO /H SO , 60 C, 4 h;
3 2 4
o
(
F) a: SOCl , CHCl , DMF, reflux, 3 h; b: NH3•H O, 0 C ; (G) Tin(II) dichloride dihydrate,
2
3
2
o
EtOH, reflux, 2 h. (H) a: Substituted carboxylic acid, (COCl) , CH Cl , DMF (Cat), 25 C,
2
2
2
o
3
h; b: CH Cl , TEA, 25 C, 12 h; (I) NaOEt, EtOH, reflux, 12 h.
2 2
1
6

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Table 1. Cytotoxic activity of the synthesized compounds against SGC-7901, MGC-803,
a
Bcap-37 cell lines
b
Compound
IC (µM)
50
SGC-7901
8.11±0.40
13.40±1.18
12.77±1.25
34.84±1.99
8.30±1.07
17.25±1.51
__
MGC-803
3.44±0.28
4.57±0.88
4.24±1.22
29.51±1.87
3.01±0.23
22.60±1.85
__
Bcap-37
46.70±1.77
40.37±1.98
55.70±2.21
33.54±2.01
10.50±0.87
45.29±2.98
__
8
8
8
8
8
9
9
9
9
9
a
b
c
d
e
a
b
c
25.67±1.08
__
32.75±1.54
__
37.39±1.69
__
d
e
10.31±0.77
6.56±0.39
7.14±0.28
3.71±0.22
28.66±0.95
5.18±0.43
c
5
-Fluorouracil
a
The data represented the mean of three experiments in triplicate and were expressed as
means ±SD; Only descriptive statistics were done in the text.
b
c
The IC value was defined as the concentration at which 50% survival of cells was
50
observed.
Used as a positive control.
Negative control 0.1%DMSO, no activity.
1

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Table 2. Inhibitory effects of slected compounds against telomerase
a
Compound
IC (µM) telomerase
50
8
8
8
8
8
9
9
a
b
c
d
e
c
e
1.98±0.21
7.87±0.69
3.49±0.55
12.20±0.98
1.02±0.08
18.99±1.44
15.17±1.80
2.10±0.11
0.28±0.09
b
Ethidium bromide
b
BIBR1532
a
Telomerase supercoiling activity
b
Ethidium bromide and BIBR1532 are reported as a control. The inhibition constant
of ethidium toward telomerase has been reported previously.

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O
N
N
N
N
N
O
S
HN
OH
N
H
N
N
N
N
N
HN
N
O
O
N
NH2
N
NH2
S
OEt
N
PDE5
PP242
PP30
1
2
3
Figure 1. Pyrazole-pyrimidine scaffold based potential candidates and drugs

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H N
2
Cyclization
O
N
HN
O
N
N
R1
N
HN
N
R1
O
pyrazole-5-carboxamide
pyrazole-pyrimidine
4
5
Figure 2. Design of pyrazole-pyrimidine and its precursor scaffold

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Figure 3A. ORTEP drawing of compound 8d
8
d

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Figure 3B. ORTEP drawing of compound 9a
9
a

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N
8e
Figure 4. Cells cycle analysis by flow cytometry with MGC-803 cells. MGC-803
cells were incubated with PI and examined by flow cytometry. N, normal; compound
8
e (3 µM), n = 3. Results are the mean ± SD from three independent experiments. *
p<0.05, ** p<0.01 vs control.

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Figure 5A. The binding mode between the active conformation of compound 8e and
the protein TERT (PDB code: 3DU6). Panel is a view into the active site cavity
(A). The 2D picture of binding was depicted

ACCEPTED MANUSCRIPT
Figure 5B. The binding mode between the active conformation of compound 8e and
the protein TERT (PDB code: 3DU6). Panel is a view into the active site cavity
(B). The 3D picture of binding was depicted

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Figure 5C. The binding mode between the active conformation of compound 8e and
the protein TERT (PDB code: 3DU6). Panel is a view into the active site cavity
(C). In order to reveal that the molecule was well filled in the active pocket, the
enzyme surface was shown

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Highlights
►
►
►
Novel pyrazole-5-carboxamides as potential telomerase inhibitors were designed.
Compound 8e exhibited strong inhibitory activity against MGC-803 cells.
Results also revealed 8e exhibited the most telomerase inhibitory activity.

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