Short communication [624895]

Short communication
Novel pyrazole-5-carboxamide and pyrazole epyrimidine derivatives:
Synthesis and anticancer activity
Jing Bo Shia,*, Wen Jian Tanga, Xing Bao qia, Rong Lia, Xin Hua Liua,b,*
aSchool of Pharmacy, Anhui Medical University, Hefei 230032, PR China
bState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
article info
Article history:
Received 5 September 2014Received in revised form4 December 2014Accepted 8 December 2014Available online 9 December 2014
Keywords:
SynthesisPyrazole-5-carboxamideAnticancer activityTelomeraseabstract
A series of novel pyrazole-5-carboxamide and pyrazole epyrimidine 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,8cand8eexhibited
strong inhibitory activity against MGC-803 cell line. The flow cytometric analysis result showed that
compound 8ecould inhibit MGC-803 proliferation. Some title compounds were tested against telome-
rase, and compound 8eshowed the most potent inhibitory activity with IC
50value at 1.02 ±0.08mM. The
docking simulation of compound 8ewas 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.
©2014 Elsevier Masson SAS. All rights reserved.
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 rep-
resents one of the promising targets in drug discovery [1e4].
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 reversetranscriptase) 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-
speci ficity and thus non-selective toxicity and dose-limiting ef fi-
cacy. Therefore, the pursuit of novel telomerase inhibitors with
better antitumor effects and more safety pro file is still the main
issue.
The pyrazole epyrimidine and their bioisosteres are heterocyclic
compounds with important biological functions includingantitumor and other activities [6e8]. As is known to all that pyr-
azole epyrimidine derivatives PP242, PP30 and PDE5 ( Fig. 1 )h a v e
possessed good kinase selectivity pro file used as cyclin-dependent
kinase (CDK), ATP-competitive mTORC1/mTORC2 inhibitors [9e16].
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 eactivity re-
lationships (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 [17e20]. Furthermore, based on the
protein TERT structure of telomerase using LigandFit module, ourgroup 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 incor-
porate the moiety of pyrazole epyrimidine.
In our recent works [21,22] , several pyrazole derivatives were
designed, which had potent anticancer activity as potential telo-
merase inhibitors. Because moiety pyrazole-5-carboxamide is a
precursor of title pyrazole epyrimidine structure ( Fig. 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 pyr-
azole epyrimidine scaffolds were designed. We also will built the
relative SAR between pyrazole epyrimidines to their precursors as
antitumor agents, which maybe rationalize the experimental*Corresponding authors. School of Pharmacy, Anhui Medical University, Hefei
230032, PR China.
E-mail addresses: sjb0616@126.com (J.B. Shi), xhliuhx@163.com (X.H. Liu).
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
http://dx.doi.org/10.1016/j.ejmech.2014.12.013
0223-5234/ ©2014 Elsevier Masson SAS. All rights reserved.European Journal of Medicinal Chemistry 90 (2015) 889 e896

observations and guide us to screen good potency telomerase in-
hibitors in the further study.
2. Results and discussion
2.1. Chemistry
All derivatives were synthesized by acylation reaction starting
from 1-methyl-4-nitro-3- N-propylpyrazole-5-carboxamides 7,
which was prepared from 2-pentanone via claisen condensation,
hydrazinolytic, cyclization, methylation, hydrolysis, nitration, ami-
dation, reduction according to Scheme 1 . Compounds 8aeewere
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 7to form amide
compounds 8aee. Compounds 9aeewere obtained from com-
pounds 8aeevia cyclization. The reaction was carried out in the
presence of sodium ethoxide, the EtOH used as solvent at re flux
condition. All compounds were characterized by means of HR-MS,
1H NMR and13C NMR spectral analysis.
2.2. Crystal structure analysis
The structures of compounds 8dand9awere determined by X-
ray crystallography. Crystal data of 8d: Colorless crystals, yield, 77%;
mp 151 e152/C14C; C 18H24N4O5, Monoclinic, space group P121/c;
a¼14.4205(14), b¼17.7873(16), c¼8.8377(9) (Å); a¼90,
b¼100.983(10), g¼90 (/C14),V¼2225.4(4) nm3,T¼293(2) K, Z¼4,
Dc¼1.261 g/cm3,F(000) ¼904, Re flections collected/
unique ¼9519/4373, Data/restraints/parameters ¼4373/6/279,
Goodness of fito n F2¼1.087, Fine, R1¼0.0681, wR(F2)¼0.1722.
Crystal data of 9a: Colorless crystals, yield, 85%; mp 200 e201/C14C;
C17H18N4O, Monoclinic, space group P121/c;a¼16.820(5),
b¼5.381(3), c¼18.370(6) (Å); a¼90,b¼112.73(4), g¼90 (/C14),
V¼1533.4(11) nm3,T¼293(2) K, Z¼4,Dc¼1.275 g/cm3,F(000) ¼624, Re flections collected/unique ¼3274/1312, Data/re-
straints/parameters ¼3274/0/202, Goodness of fito n F2¼0.880,
Fine, R1¼0.0741, wR(F2)¼0.1818. Their molecular structures were
shown in Fig. 3 . Crystallographic data (excluding structure factors)
for the structures had been deposited with the Cambridge Crys-
tallographic Data Center as supplementary publication No. CCDC-
981021 and 1021035.
2.3. In vitro anticancer activity
Recently, we focused on screening lead compounds with speci fic
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 50values ( Table 1 ).
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 8is the
precursor of compound 9. In general, series 5-propyl-1H-pyrazole-
3-carboxamide (compounds 8) leaded to increase in inhibitory
activity, among them, compound 8awas the most potent activity
against MGC-803 cell with IC 50value of 3.01 ±0.23mM, surpassing
that of the positive control 5- fluorouracil. Compared with the
compounds 8, compounds 9generally did not re flect the activity on
the tested cells ( 9b,9d). Therefore, for this kind of 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,
8band8cexhibited the strong inhibitory activity against the MGC-
803 cells (with IC 50sof 3.44 ±0.28, 4.57 ±0.88, 4.24 ±1.22mM,
respectively) and the values could compare with that of the potent
5-fluorouracil.
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 8eon induction of cell cycle. As shown in Fig. 4 ,
the cells in S phase in the MGC-803 control group accounted for
about 30.38%, while after cells treated with compound 8eMGC-803
for 48 h, the ratio was approximately 42.16%. This showed that the
cells were arrested in S phase.HNOH
N
NNN
NH2OH
N
SN
N
NN
N
NH2NHN
NNO
OEtSO
ON N
PP242 PP30PDE5
12 3
Fig. 1. Pyrazole epyrimidine scaffold based potential candidates and drugs.
NN
OH2N
HN
OR1NN
NHNO
R1
pyrazole-5-carboxamide pyrazole-pyrimidineCyclization
45
Fig. 2. Design of pyrazole epyrimidine and its precursor scaffold.J.B. Shi et al. / European Journal of Medicinal Chemistry 90 (2015) 889 e896 890

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 8aand8eshowed strong telomerase inhibitory activity
with IC 50values of 1.98 ±0.21, 1.02 ±0.08mM respectively, whichsurpassing that of the positive control ethidium bromide, compa-
rable 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-pyr-
azole-5-carboxamide showed higher inhibitory activity than the
others. This also pointed out the direction for us to further optimizeO+OO
O
OO ONaOO
NNHO
O
NNO
O
NNO
OH NNO
OHNO2
NNO
NH2NO2
NNO
NH2NH2(A)(B)
(C)(D)(E)
(G)12
34 5
6(F)
7NN
OH2N
HN
OR1
R1=NN
NHNO
R1
8a 8bO
O
OO
O
O8c
O
OO
8d O 8e9a 9b 9c
9d 9e8
9
R1= R1=
R1= R1=(H)
(I)
Scheme 1. Synthesis of title compounds 7~9.Reagent and conditions : (A) Na, EtOH, re flux, 6 ~ 8 h; (B) NH 2eNH 2$H2O, 60 e65/C14C, 2 h; (C) Dimethyl sulfate, 80/C14C, 2 h; (D) 6 N NaOH,
80/C14C, 2 h; (E) HNO 3/H2SO4,6 0/C14C, 4 h; (F) a: SOCl 2,C H C l 3, DMF, re flux, 3 h; b: NH 3$H2O, 0/C14C ; (G) Tin(II) dichloride dihydrate, EtOH, re flux, 2 h. (H) a: Substituted carboxylic acid,
(COCl) 2,C H 2Cl2, DMF (Cat), 25/C14C, 3 h; b: CH 2Cl2, TEA, 25/C14C, 12 h; (I) NaOEt, EtOH, re flux, 12 h.
Fig. 3. ORTEP drawing of compounds 8dand9a.J.B. Shi et al. / European Journal of Medicinal Chemistry 90 (2015) 889 e896 891

the structure of 1H-pyrazole-5-carboxamide moiety with anti-
cancer activity as potential telomerase inhibitors.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 8einto active site containing key residue
ASP 254 was performed to simulate a binding model derived from
telomerase TERT (3DU6 pdb). In general, compound 8ecould 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 8ewith TERT was depicted in Fig. 5 . The 2D
and 3D pictures of binding were depicted in Fig. 5 A and B. In order
to reveal that the molecule was well filled in the active pocket, the
enzyme surface was shown in Fig. 5 C. There are six obvious bonding
interactions anchoring compound 8eto the active site tightly might
explain its good inhibitory activity. First, we found hydrogen bond
between the backbone amino groups of ASP 254 and Natom of
pyrazole moiety. Another two hydrogen bonds interaction were
between the LYS 189 and the Oatoms of amide. Second, three
PieCation interactions were formed (pyrazole ring with LYS 189;
furan ring with the LYS 372 and LYS 249, respectively).
3. Conclusions
In brief, based on rational design and reasonable analysis, we
designed some novel 1H-pyrazole-5-carboxamide derivatives.
Next, these compounds used as precursors due to exploringreasonable SAR, some new 1H-pyrazolo[4,3-d]pyrimidin-7(6H)
derivatives were designed, followed by chemical synthesis and
biological evaluated for them. The results revealed that some 1H-
pyrazole-5-carboxamide compounds re flected high activity against
gastric cancer cells. Among them, compound 8eexhibited strong
inhibitory activity against MGC-803 cells, and showed the most
potent telomerase inhibitory activity with IC
50value at
1.02±0.08mM. Flow cytometric analysis indicated that the cells
were arrested 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 ourTable 1
Cytotoxic activity of the synthesized compounds against SGC-7901, MGC-803, Bcap-37 cell lines.
a
Compound IC 50(mM)b
SGC-7901 MGC-803 Bcap-37
8a 8.11±0.40 3.44 ±0.28 46.70 ±1.77
8b 13.40 ±1.18 4.57 ±0.88 40.37 ±1.98
8c 12.77 ±1.25 4.24 ±1.22 55.70 ±2.21
8d 34.84 ±1.99 29.51 ±1.87 33.54 ±2.01
8e 8.30±1.07 3.01 ±0.23 10.50 ±0.87
9a 17.25 ±1.51 22.60 ±1.85 45.29 ±2.98
9b eee
9c 25.67 ±1.08 32.75 ±1.54 37.39 ±1.69
9d eee
9e 10.31 ±0.77 7.14 ±0.28 28.66 ±0.95
5-Fluorouracilc6.56±0.39 3.71 ±0.22 5.18 ±0.43
Negative control 0.1%DMSO, no activity.
aThe data represented the mean of three experiments in triplicate and were
expressed as means ±SD; only descriptive statistics were done in the text.
bThe IC 50value was de fined as the concentration at which 50% survival of cells
was observed.
cUsed as a positive control.
Fig. 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 8e(3mM),n¼3.
Results are the mean ±SD from three independent experiments. * p<0.05, ** p<0.01 vs control.
Table 2
Inhibitory effects of selected compounds against telomerase.
Compound IC 50(mM) telomerasea
8a 98±0.21
8b 87±0.69
8c 49±0.55
8d 12.20 ±0.98
8e 1.02±0.08
9c 18.99 ±1.44
9e 15.17 ±1.80
Ethidium bromideb2.10±0.11
BIBR1532b0.28±0.09
aTelomerase supercoiling activity.
bEthidium bromide and BIBR1532 are reported as a control. The inhi-
bition constant of ethidium toward telomerase has been reportedpreviously.J.B. Shi et al. / European Journal of Medicinal Chemistry 90 (2015) 889 e896 892

Fig. 5. The binding mode between the active conformation of compound 8eand the protein TERT (PDB code: 3DU6). Panel is a view into the active site cavity.J.B. Shi et al. / European Journal of Medicinal Chemistry 90 (2015) 889 e896 893

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 section4.1. 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., Beijing, China), and
are uncorrected.
1H and13C NMR spectra were recorded on a
Brucker AM-300 (300 MHz) spectrometer with CDCl 3or DMSO- d6
as the solvent and TMS as the internal standard. Chemical shifts are
reported in d(parts per million) values. Coupling constants Jare
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 wayand redistilled.
4.2. General procedure for the synthesis of title compounds 8
To a methylene chloride (10 mL) solution of substituted Car-
boxylic acid 3 (5 mmol) in an ice bath was added oxalyl chloride
(10 mmol), and catalytic N,N-dimethylformamide, while the reac-
tion contents were stirred continuously. The reaction mixture was
allowed to stand at 25 e30
/C14C 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 saturated sodium
bicarbonate (20 mL) and then with water (20 mL). Organic solvent
was dried over anhydrous Na 2SO4,filtrated, and then evaporated to
give solid. Crude solid product was recrystallized from suitable
solvent to give title compounds 8aee(Scheme 1 ) as solids.
4.2.1. 8a: 4-Cinnamamido-1-methyl-3-propyl-1H-pyrazole-5-
carboxamide
Off-white powder solid, yield, 75%; mp: 225 e226/C14C (recryst.
from ethanol);1H NMR (300 MHz, CDCl 3țDMSO- d6)d(ppm): 9.03
(Brs, 1H, NH), 7.56 (d, J¼15.52, 1H, Ar eCH), 7.36 (s, 2H, ArH), 7.21
(m, 3H, ArH), 6.54 (d, J¼15.72, 1H, CO eCH), 6.13 (Brs, 2H, NH 2),
3.86 (s, 3H, N eCH3), 2.35 (t, J¼7.6 Hz, 2H, N eCH2), 1.45 (m, 2H,
CH2), 0.78 (t, J¼7.0 Hz, 3H, CH 3). HR-MS: calcd for C 17H20N4O2
[MțNa]ț, 335.1478; found 335.1479.
4.2.2. 8b: 1-Methyl-3-propyl-4-(3,4,5-trimethoxybenzamido)-1H-
pyrazole-5-carboxamide
Off-white powder solid, yield, 73%; mp: 190 e191/C14C (recryst.
from ethanol);1H NMR (300 MHz, CDCl 3)d(ppm): 7.98 ț7.74 (Brs,
1H, NH), 7.12 (d, 2H, ArH), 5.69 (Brs, 2H, NH 2), 3.92 (s, 12H, 3O eCH3,
NeCH3), 2.53 (m, 2H, N eCH2), 1.63 (m, 2H, CH 2), 0.92 (m, 3H, CH 3).
HR-MS: calcd for C 18H24N4O5[MțNa]ț, 399.1639; found 399.1638.
4.2.3. 8c: (E)-1-Methyl-3-propyl-4-(3-(3,4,5-trimethoxyphenyl)
acrylamido)-1H-pyrazole-5-carboxamide
Off-white powder solid, yield, 80%; mp: 203 e204/C14C (recryst.
from ethanol);1H NMR (300 MHz, CDCl 3țDMSO- d6)d(ppm): 9.62
(Brs, 1H, NH), 7.62 (d, J¼15.63, 1H, Ar eCH), 6.81 (s, 2H, ArH), 6.69
(d,J¼15.63, 1H, CO eCH), 4.14 (s, 3H, N eCH3), 3.90 (s, 6H, 2O eCH3),
3.85 (s, 3H, O eCH3), 2.51 (t, J¼7.6 Hz, 2H, N eCH2), 1.62 (m, 2H,CH2), 0.91 (t, J¼7.3 Hz, 3H, CH 3). HR-MS: calcd for C 20H26N4O5
[MțNa]ț, 425.1795; found 425.1794.
4.2.4. 8d: 1-Methyl-3-propyl-4-(2,4,5-trimethoxybenzamido)-1H-
pyrazole-5-carboxamide
Off-white powder solid, yield, 77%; mp: 150 e151/C14C (recryst.
from ethanol);1H NMR (300 MHz, CDCl 3)d(ppm): 9.30 (Brs, 1H,
NH), 8.0 ț5.60 (Brs, 2H, NH 2), 7.83 (s, 1H, ArH), 6.60 (s, 1H, ArH),
4.08 (s, 3H, N eCH3), 3.93 e4.05 (s, 9H, 3O eCH3), 2.61 (t, J¼7.5 Hz,
2H, N eCH2), 1.63 (m, 2H, CH 2), 0.94 (t, J¼7.3 Hz, 3H, CH 3). HR-MS:
calcd for C 18H25N4O5[MțH]ț, 377.1819, found 377.1824.
4.2.5. 8e: (E)-4-(3-(Furan-2-yl)acrylamido)-1-methyl-3-propyl-
1H-pyrazole-5-carboxamide
Light brown powder solid, yield, 65%; mp: 249 e252/C14C (recryst.
from 95% ethanol);1H NMR (300 MHz, DMSO- d6)d(ppm): d9.57
(Brs, 1H, NH), 7.83 (d, J¼1.3 Hz, 1H, Furan-H), 7.73 (s, 1H, Furan-H),
7.41 (d, J¼15.6 Hz, 1H, Ar eCH), 6.86 (d, J¼3.4 Hz, 1H, Furan-H),
6.63 (d, J¼15.6 Hz, 1H, CO eCH), 3.88 (s, 3H, N eCH3), 2.37 (t,
J¼7.5 Hz, 2H, N eCH2), 1.59 e1.46 (m, 2H, CH 2), 0.86 (t, J¼7.4 Hz,
3H, CH 3).
4.3. General procedure for the synthesis of title compounds 9
Compound 8(1 mmol) and sodium ethoxide (5 mol) were dis-
solved in ethanol (15 mL), and re fluxed for 12 h. After completion of
the reaction, the solvent was concentrated to a low volume; the
residue was added HCl (1 N), the precipitated product was granu-
lated at pH 7 and 10/C14C for a further hour. The title compounds
9ae9ewas collected by filtration, washed with water, and dried to
give as solid.
4.3.1. 9a: (E)-1-Methyl-3-propyl-5-styryl-1H-pyrazolo[4,3-d]
pyrimidin-7(6H)-one
Off-white powder solid, yield, 85%; mp: 200 e201/C14C;1H NMR
(300 MHz, CDCl 3)d(ppm): 11.60 (Brs, 1H, NH), 7.80 (d, J¼16.5, 1H,
AreCH), 7.60 (d, J¼7.1 Hz, 2H, ArH), 7.39 (m, J¼6.8 Hz, 3H, ArH),
6.96 (d, J¼16.5, 1H, CO eCH), 4.28 (s, 3H, N eCH3), 2.92 (t, J¼7.5 Hz,
2H, N eCH2), 1.84 (m, 2H, CH 2), 1.03 (t, J¼7.3 Hz, 3H, CH 3).13C NMR
(75 MHz, CDCl 3): 156.27, 149.17, 146.78, 139.63, 136.99, 135.57,
129.65, 129.07, 127.54, 124.35, 121.54, 38.34, 27.85, 22.56, 14.24.
4.3.2. 9b: 1-Methyl-3-propyl-5-(3,4,5-trimethoxyphenyl)-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one
Off-white powder solid, yield, 84%; mp: 204 e206/C14C;1H NMR
(300 MHz, CDCl 3)d(ppm): d11.05 (Brs, 1H, NH), 7.29 (s, 2H, ArH),
4.24 (s, 3H, N eCH3), 4.00 (s, 6H, 2 /C2OeCH3), 3.88 (s, 3H, O eCH3),
2.95 (t, J¼7.6 Hz, 2H, N eCH2), 1.95 e1.78 (m, 2H, CH 2), 1.04 (t,
J¼7.3 Hz, 3H, CH 3).
4.3.3. 9c: (E)-1-Methyl-3-propyl-5-(3,4,5-trimethoxystyryl)-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one
Off-white powder solid, yield, 81%; mp: 266 e270/C14C;1H NMR
(300 MHz, CDCl 3)d(ppm): d11.61 (Brs, 1H, NH), 7.67 (d, J¼16.4 Hz,
1H, Ar eCH), 6.91 (d, J¼16.4 Hz, 1H, CO eCH), 6.82 (s, 2H, ArH), 4.29
(s, 3H, N eCH3), 3.91 (d, 9H, 3O eCH3), 2.92 (t, J¼7.6 Hz, 2H,
NeCH2), 1.93 e1.77 (m, 2H, CH 2), 1.04 (t, J¼7.3 Hz, 3H, CH 3). HR-MS:
calcd for C 20H25N4O4[MțH]ț, 385.187; found 385.1874.
4.3.4. 9d: 1-Methyl-3-propyl-5-(2,4,5-trimethoxyphenyl)-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one
Off-white powder solid, yield, 82%; mp: 184 e187/C14C;1H NMR
(300 MHz, CDCl 3)d(ppm): d10.99 (Brs, 1H, NH), 8.04 (s, 1H, ArH),
6.59 (s, 1H, ArH), 4.27 (s, 3H, N eCH3), 4.05 (s, 3H, O eCH3), 3.98 (s,
6H, 2O eCH3), 2.94 (t, J¼7.71, 2H, N eCH2), 1.95 e1.81 (m, 2H, CH 2),J.B. Shi et al. / European Journal of Medicinal Chemistry 90 (2015) 889 e896 894

1.04 (t, J¼7.4 Hz, 3H, CH 3). HR-MS: calcd for C 18H23N4O4[MțH]ț,
359.1716; found 359.1716.
4.3.5. 9e: (E)-5-(2-(Furan-2-yl)vinyl)-1-methyl-3-propyl-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one
Light brown powder solid, yield, 79%; mp: 270 e272/C14C;1H NMR
(300 MHz, DMSO- d6)d(ppm): d12.29 (Brs, 1H, NH), 7.82 (d,
J¼1.5 Hz, 1H, Ar eCH), 7.64 (d, J¼15.84 Hz, 1H, CO eCH), 6.85 (d,
J¼3.3 Hz, 1H, Furan-H), 6.80 (d, J¼15.84 Hz, 1H, Furan-H), 6.63
(dd, J¼3.4, 1.8 Hz, 1H, Furan-H), 4.12 (s, 3H, N eCH3), 2.77 (t,
J¼7.5 Hz, 2H, N eCH2), 1.81 e1.69 (m, 2H, CH 2), 0.95 (t, J¼7.4 Hz,
3H, CH 3).
4.4. Crystallographic studies
A colorless single crystal of title compounds 8dand 9awere
chosen for X-ray diffraction analysis performed on a BRUCKER
SMART APEX-CCD diffractometer equipped with a graphite
monochromatic MoKa radiation ( l¼0.71073 Å) radiation at
296(2) K. A total re flections were collected in the range of
2.30<q<25.30/C14by using a j-uscan mode with independent ones,
of which I>2s(I) were observed and used in the succeeding re-
finements. 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 method on F2with SHELXL-97
[24]. The non-hydrogen atoms were re fined anisotropically, and the
hydrogen atoms were added according to theoretical models. The
structures were re fined by full-matrix least-squares method on F2
with SHELXT-97.
4.5. Anticancer assay
The cytotoxicity evaluation was conducted by using a modi fied
procedure as described in the literature. Brie fly, target tumor cells
were grown to log phase in RPMI 1640 medium supplemented with
10% fetal bovine serum. After diluting to 3 /C2104cells/mL with the
complete medium, 100 mL of the obtained cell suspension was
added to each well of 96-well culture plates. The subsequent in-
cubation was performed at 37/C14C, 5% CO 2atmosphere 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. After 48 h exposure period, 25 mL
of PBS containing 2.5 mg/mL of MTT (3-(4, 5-dimethylthiazol-2-yl)-
2, 5-diphenyltetrazolium bromide) was added to each well. After
4 h, the medium was replaced by 150 mL 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 repre-
sented the mean of three experiments in triplicate and were
expressed as means ±SD using Student t test. The IC 50value was
defined as the concentration at which 50% 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 /C020/C14C for 12 h. After fix-
ation, 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 mg/mL PI, at 37/C14C 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 moleculeinhibitors 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/C14C in a humidi fied atmosphere
containing 5% CO 2. After trypsinization, 5 /C2104cultured 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 0.082 l 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 mL 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/C14C and stored at /C080/C14C. The TRAP-PCR-ELISA assay was
performed using a telomerase detection kit (Roche, Basel,
Switzerland) according to the manufacturer's protocol. In brief, 2 mL
of cell extracts were mixed with 48 mL TRAP reaction mixtures. PCR
was then initiated at 94/C14C, 120 s for predenaturation and per-
formed using 35 cycles each consisting of 94/C14C for 30 s, 50/C14C for
30 s, 72/C14C for 90 s. Then 20 mL of PCR products were hybridized to a
digoxigenin (DIG)-labeled telomeric repeat speci fic detection
probe. And the PCR products were immobilized via the biotin-
labeled primer to a streptavidin-coated microtiter plate subse-
quently. 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 In finite 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 de fined and sphere of 5 Åwas
generated around the active site pocket, with the active site pocket
of BSAI model using C-DOCKER, a molecular dynamics (MD)
simulated-annealing based 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 pro-
tein, substrate were subjected to energy minimization using
CHARMm force field as implemented in DS 3.1. A full potential final
minimization was then used to re fine 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 Foun-
dation of China (No. 21272008), Anhui Provincial Natural Science
Foundation, (1308085MH137), Science and Technological Fund of
Anhui Province for Outstanding Youth (1408085J04).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.12.013 .J.B. Shi et al. / European Journal of Medicinal Chemistry 90 (2015) 889 e896 895

References
[1]D.R. Corey, Chem. Biol. 16 (2009) 1219 e1223 .
[2]Z. Ding, C.J. Wu, M. Jaskelioff, E. Ivanova, M. Kost-Alimova, A. Protopopov,
G.C. Chu, G. Wang, X. Lu, E.S. Labrot, J. Hu, W. Wang, Y. Xiao, H. Zhang, J. Zhang,
B. Gan, S.R. Perry, S. Jiang, L. Li, J.W. Horner, Y.A. Wang, L. Chin, R.A. DePinho,Cell 148 (2012) 896 e907.
[3]J.W. Shay, W.E. Wright, Nat. Rev. Drug Discov. 5 (2006) 577 e584.
[4]S.A. Stewart, A.A. Bertuch, Cancer Res. 70 (2010) 7365 e7371 .
[5]A.J. Gillis, A.P. Schuller, E. Skordalakes, Nature 455 (2008) 633 e637.
[6]M. Chauhan, R. Kumar, Bioorg. Med. Chem. Lett. 21 (2013) 5657 e5668 .
[7]Y.L. Li, J.M. Fevig, J. Cacciola, J. Buriak, K.A. Rossi Jr., J. Jona, R.M. Knabb,
J.M. Luettgen, P.C. Wong, S.A. Bai, R.R. Wexler, P.Y.S. Lam, Bioorg. Med. Chem.Lett. 16 (2006) 5176 e5182 .
[8]V.N. Devegowda, J.H. Kim, K.C. Han, E.G. Yang, H. Choo, A.N. Pae, G. Nama,
K.I. Choi, Bioorg. Med. Chem. Lett. 20 (2010) 1630 e1633 .
[9]D. Geffken, R. Soliman, F.S.G. Soliman, M.M. Abdel-Khalek, D.A.E. Issa, Med.
Chem. Res. 20 (2011) 408 e420.
[10] M.E. Feldman, B. Apsel, A. Uotila, R. Loewith, Z.A. Knight, D. Ruggero,
K.M. Shokat, PLoS Biol. 7 (2009) e38 .
[11] M.R. Janes, J.J. Limon, L. So, J. Chen, R.J. Lim, M.A. Chavez, C. Vu, M.B. Lilly,
S. Mallya, S.T. Ong, M. Konopleva, M.B. Martin, P. Ren, Y. Liu, C. Rommel,
D.A. Fruman, Nat. Med. 16 (2010) 205 e213.
[12] J.A. Menendez, L. Vellon, C. Oliveras-Ferraros, S. Cufí, A. Vazquez-Martin, Cell
Cycle 10 (2011) 3658 e3677 .
[13] D.J. Richard, J.C. Verheijen, K. Curran, J. Kaplan, L. Toral-Barza, I. Hollander,J. Lucas, K. Yu, A. Zask, Bioorg. Med. Chem. Lett. 19 (2009) 6830 e6835 .
[14] A. Zask, J.C. Verheijen, K. Curran, J. Kaplan, D.J. Richard, P. Nowak, D.J. Malwitz,
N. Brooijmans, J. Bard, K. Svenson, J. Lucas, L. Toral-Barza, W.G. Zhang,
I. Hollander, J.J. Gibbons, R.T. Abraham, S. Ayral-Kaloustian, T.S. Mansour,
K. Yu, J. Med. Chem. 52 (2009) 5013 e5016 .
[15] A. Zask, J. Kaplan, K. Curran, J.C. Verheijen, D.J. Richard, N. Brooijmans,
E. Bennett, J. Lucas, L. Toral-Barza, I. Hollander, J.J. Gibbons, R.T. Abraham,
S. Ayral-Kaloustian, T.S. Mansour, K. Yu, Mol. Cancer Ther. 8 (2009) B145 .
[16] E. Strocchi, F. Fornari, M. Minguzzi, L. Gramantieri, M. Milazzo, V. Rebuttini,
S. Breviglieri, C.M. Camaggi, E. Locatelli, L. Bolondi, M. Comes-Franchini, Eur. J.
Med. Chem. 48 (2012) 391 e401.
[17] H. Kumar, D. Saini, S. Jain, N. Jain, Eur. J. Med. Chem. 70 (2013) 248 e258.
[18] A. Balbi, M. Anzaldi, C. Maccio, C. Aiello, M. Mazzei, R. Gangemi, P. Castagnola,
M. Miele, C. Rosano, M. Viale, Eur. J. Med. Chem. 46 (2011) 5293
e5309 .
[19] L. Yuan, C. Song, C. Li, Y. Li, L. Dong, S. Yin, Eur. J. Med. Chem. 67 (2013)
152e157.
[20] J. Sun, X.H. Lv, H.Y. Qiu, Y.T. Wang, Q.R. Du, D.D. Li, Y.H. Yang, H.L. Zhu, Eur. J.
Med. Chem. 68 (2013) 1 e9.
[21] X.H. Liu, B.F. Ruan, J.X. Liu, B.A. Song, L.H. Jing, J. Li, Y. Yang, H.L. Zhu, X.B. Qi,
Bioorg. Med. Chem. Lett. 21 (2011) 2916 e2920 .
[22] X.H. Liu, H.F. Liu, J. Chen, Y. Yang, B.A. Song, L.S. Bai, J.X. Liu, H.L. Zhu, X.B. Qi,
Bioorg. Med. Chem. Lett. 20 (2010) 5705 e5708 .
[23] W.J. Tang, Y.A. Yang, X. He, J.B. Shi, X.H. Liu, Sci. Rep. 4 (2014) 7106 .
[24] G.M. Sheldrick, SHELXTL-97, Program for Crystal Structure Solution and
Refinement, University of G €ottingen, G €ottingen, Germany, 1997 .J.B. Shi et al. / European Journal of Medicinal Chemistry 90 (2015) 889 e896 896