Identification of human telomerase inhibitors having the core of N-acyl-4,5-dihydropyrazole with anticancer effects

Article abstract of DOI:10.1016/j.bmcl.2016.02.025

Eight human telomerase inhibitors (5a–5h) having the core of N-acyl-4,5-dihydropyrazole with anticancer effects were identified in this study. Biological results revealed that a few compounds had potent anticancer activities against three common tumor cell lines (SGC-7901, HepG2 and MGC-803). Among them, compound 5c, with a molecular weight of only 272.2?Da, had antiproliferative activities against SGC-7901 and MGC-803 with EC₅₀values of 2.06?±?0.17 and 2.89?±?0.62?μM, respectively, better than 5-Fluorouracil. Compound 5c inhibited the enzyme of telomerase with an IC₅₀value of 1.86?±?0.51?μM, surpassing the control compound, ethidium bromide. Modeling study showed that this compound can reside in the binding pocket of the telomerase/TNA:DNA hairpin complex. When the moiety of N-acyl was changed to N-sulfonyl, the gotten compounds (8a–8i) had deteriorative activities against both these three cancer cell lines and the enzyme of telomerase.

Full text of DOI:10.1016/j.bmcl.2016.02.025

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of human telomerase inhibitors having the core
of N-acyl-4,5-dihydropyrazole with anticancer effects
Xuan Xiao a, Yong Ni a, Ying-Ming Jia b, Min Zheng a, Han-Fei Xu a, Jun Xu c, Chenzhong Liao a,
⇑
a School of Medical Engineering, Hefei University of Technology, Hefei 230009, China
b School of Chemistry and Chemical Engineering, Anhui University of Technology, Maanshan 243002, China
c Research Center for Drug Discovery and Institute of Human Virology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight human telomerase inhibitors (5a–5h) having the core of N-acyl-4,5-dihydropyrazole with anti-
cancer effects were identified in this study. Biological results revealed that a few compounds had potent
anticancer activities against three common tumor cell lines (SGC-7901, HepG2 and MGC-803). Among
them, compound 5c, with a molecular weight of only 272.2 Da, had antiproliferative activities against
Received 23 December 2015
Revised 28 January 2016
Accepted 10 February 2016
Available online xxxx
SGC-7901 and MGC-803 with EC50 values of 2.06 0.17 and 2.89 0.62
5-Fluorouracil. Compound 5c inhibited the enzyme of telomerase with an IC50 value of 1.86 0.51
l
M, respectively, better than
M,
l
Keywords:
Telomerase
Telomerase inhibitor
Dihydropyrazole
Anticancer
surpassing the control compound, ethidium bromide. Modeling study showed that this compound can
reside in the binding pocket of the telomerase/TNA:DNA hairpin complex. When the moiety of N-acyl
was changed to N-sulfonyl, the gotten compounds (8a–8i) had deteriorative activities against both these
three cancer cell lines and the enzyme of telomerase.
Ó 2016 Elsevier Ltd. All rights reserved.
Molecular modeling
Telomerase has been validated as an anticancer drug target
because of the following facts: (1) telomerase is active in the early
stages of life to maintain telomere length and therefore the chro-
mosomal integrity of frequently dividing cells, and it becomes dor-
mant in most somatic cells during adulthood;1 (2) this enzyme is
up-regulated in 80–90% of various cancer cells isolated from prin-
cipal human tumors but it is absent in neighboring cells of healthy
tissue.2–4 Consequently, telomerase has gotten considerable atten-
tions for developing anticancer drugs.
identification, biological and modeling studies of novel human
telomerase inhibitors with the core of N-acyl-4,5-dihydropyrazole.
Among them, compound 5c had antiproliferative activities against
SGC-7901 and MGC-803 cell lines with EC50 values of 2.06 0.17
and 2.89 0.62 lM, respectively, better than the positive control
compound, 5-Fluorouracil (5-FU).18,19 Compound 5c showed inhi-
bitory activity against telomerase with an IC50 value of
1.86 0.51 lM, surpassing the positive control compound, ethid-
ium bromide (2, Fig. 1).20
The catalytic subunit of this enzyme is termed as human
telomerase reverse transcriptase (hTERT),5 which is expressed at
a high level in malignant cells, but at a very low level in normal
cells. Accumulating evidences about hTERT indicate that hTERT
might be a therapeutic target as well and its inhibitors have poten-
tial applications for cancer treatment.6,7 In addition, hTERT may
relate to other age-associated disorders.8 Many hTERT inhibitors
were identified,2,9 and some of them, including BIBR1532 (1,
Fig. 1),10–12 showed promising anticancer effects.
Dihydropyrazole derivatives are potential leads for drug
discovery,13 and they have shown biological activities against
cannabinoid receptor 1, monoamine oxidase, tumor necrosis,
among others. Many hTERT inhibitors with the core of dihydropy-
razole were reported recently.14–17 In this study, we report the
To carry out rational drug design, BIBR1532 (1) was docked into
a three-dimension human telomerase model to explore the binding
mode of this compound. We then designed drug-like hTERT inhibi-
tors which are easy to synthesize and incorporate the moiety of
dihydropyrazole to try to discover novel potent hTERT inhibitors.
The designed compounds were subsequently docked into the
model. The compounds which had similar interactions as BIBR1532
were then picked up for synthesis.
The synthesis of N-acyl-4,5-dihydropyrazole derivatives (5a–
5h) was presented in Scheme 1. The synthesis of compound 3
started from substituted-salicylaldehyde catalyzed by C2H5ONa.
Compounds 4a–4h were obtained from hydrazine monohydrate
and
a,b unsaturated ketone 3. The catalyst of DMAP was proved
to be an efficient alternative for the synthesis of the target com-
pounds 5a–5h. According to Scheme 2, compounds 8a–8i were
synthesized. These compounds were purified by column chro-
⇑
Corresponding author. Tel.: +86 551 62901254; fax: +86 551 62904675.
E-mail addresses: czliao@hfut.edu.cn, chenzhongliao@gmail.com (C. Liao).
http://dx.doi.org/10.1016/j.bmcl.2016.02.025
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
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2
X. Xiao et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
Antiproliferative activities of the synthesized compounds 5a–5h and 8a–8i against
MGC-803, SGC-7901 and Hep-G2 cell linesa
HO
O
Br
NH2
N
H
N
Compound
Antiproliferative activities (EC50
,
l
M)
H2N
O
SGC-7901
Hep-G2
MGC-803
5a
5b
5c
5d
5e
5f
5g
5h
8a
8b
8c
8d
8e
8f
14.01 0.58
8.60 1.11
2.06 0.17
4.18 0.69
45.70 1.98
50.28 2.20
9.21 1.02
7.00 0.77
55.6 2.44
48.07 1.93
27.33 1.28
>60
>60
>60
21.09 1.50
16.32 0.99
>60
20.29 1.22
9.41 0.97
4.80 0.81
14.40 1.22
40.33 1.87
39.20 2.41
12.29 1.08
4.21 0.41
>60
39.27 1.68
>60
50.11 1.75
>60
12.25 0.37
4.26 0.78
2.89 0.62
3.65 0.55
38.45 1.65
35.39 1.82
10.45 1.20
6.87 1.00
>60
1
2
Ethidium bromide ( )
BIBR1532 (
)
Figure 1. Chemical structures of BIBR1532 (1) and ethidium bromide (2).
matography, using acetone/petroleum ether as eluent to afford
title colorless solids 8a–8i.
All the target compounds were evaluated for their antiprolifer-
ative activities against three cancer cell lines: human gastric cancer
cell lines MGC-803 and SGC-7901 and human liver cancer cell line
Hep-G2. Compound 5-FU, a drug which is used in the treatment of
cancer in clinic, was used as the positive control. The cells were
allowed to proliferate in presence of tested compounds for 48 h,
and the results were reported with EC50 values and are shown in
Table 1. It is obvious from Table 1, compound 5c showed the most
potent antiproliferative activities against SGC-7901 and MGC-803
>60
22.21 0.99
15.45 1.33
>60
37.88 2.00
20.34 1.41
30.00 2.29
52.51 2.85
3.35 0.18
45.20 2.09
38.20 1.68
22.09 0.99
>60
8g
8h
8i
5-FU
7.03 0.29
2.21 0.20
a
with EC50 values of 2.06 0.17 and 2.89 0.62 lM, respectively,
Values are means of three experiments and are expressed as means SD.
better than the positive control compound 5-FU. Regarding the cell
line of Hep-G2, compound 5h had the best antiproliferative activity
these three cancer cell lines. On the whole, the activities were
not as good as the N-acyl compounds (5a–5h), and several of them
did not show inhibitory activities even at 60 lM. The best one is
8h, which had antiproliferative activities against SGC-7901, Hep-
with an EC50 value of 4.21 0.41
antiproliferative activity against Hep-G2 with an EC50 values of
4.80 0.81 M, which, however, is inferior to 5-FU. Compound
5d and 5h showed anticancer activities against SGC-7901 with
EC50 values of 4.18 0.69 and 7.00 0.77 M, respectively, compa-
lM. This compound also had
l
G2 and MGC-803 with EC50 values of 16.32 0.99, 22.09 0.99
l
and 30.00 2.29 lM, respectively.
rable to that of positive control compound 5-FU. From the data pre-
sented in Table 1, it is obvious that derivatives of N-acyl-4,5-
dihydropyrazole (5b, 5c, 5d and 5h) exhibited better activities
against SGC-7901, Hep-G2 and MGC-803 cell lines than derivatives
of N-benzoyl-4,5-dihydropyrazole compounds (5e and 5f).
To confirm if the compounds discussed herein performed anti-
cancer activities via telomerase inhibition, five compounds (5b, 5c,
5e, 5f and 8h) were selected and assayed for their enzyme inhibi-
tion against the target of telomerase by a modified TRAP assay21–23
using an extraction from MGC-803 cells. Modified TRAP is a pow-
erful technique to determine small molecules inhibiting telomere
N-Sulfonyl-4,5-dihydropyrazole derivatives 8a–8i were synthe-
sized and evaluated for their antiproliferative activities against
OH
O
OH
O
OH
OH
N
N
A
B
C
N
H
H
N
R
R
R
R
R1
O
3
4a 4h
-
5a 5h
-
5e
5a
: R= 5-Cl; R1= -CH2-Cl
: R= H; R1=
OMe
F
5b
5f
: R= H; R1= -CH2-F
: R= H; R1=
5c
5d
5g
5h
: R= H; R1= -CF3
: R=5-Br; R1= -CH2-Cl
: R=5-F; R1= -CH2-Cl
: R= H; R1= -CH2-Br
Scheme 1. Synthesis of compounds 5a–5h. Reagent and conditions: (A) CH3COCH3, C2H5ONa, 20–30 °C, 10 h; (B) NH2–NH2ꢀH2O, C2H5OH, reflux, 3 h. (C) R0COOH, DMAP,
60 °C, 4 h.
OH
Br
O
OH
Br
O
OH
Br
OH
Br
N
N
A
B
C
N
H
H
N
S
O
R
O
6
7a 7i
-
8a 8i
-
8a
8b
8c
: R= H
: R= 2-Me
: R= 4-NO2
8f: R= 2,4-diCl
8i
8d: R= 4-F
8g
8e: R= 4-Cl
8h
: R= 4-OMe
: R= 4-CF3
: R= 2-I
Scheme 2. Synthesis of compounds 8a–8i. Reagent and conditions: (A) CH3COCH3, C2H5ONa, 20–30 °C, 12 h; (B) NH2–NH2ꢀH2O, C2H5OH, reflux, 1 h; (C) substituted-
benzenesulfonyl chloride, CHCl3, 10 °C, DMAP, 3 h.
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X. Xiao et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 2
The enzyme inhibitory activities of five selected compounds against telomerasea
Compound
Enzyme inhibitory activities (IC50, l
M)a,b
5b
5c
5e
5f
4.00 0.48
1.86 0.51
24.95 1.28
29.81 1.59
>60
8h
Ethidium bromide
2.18 0.36
a
Values are means of three experiments and are expressed as means SD.
Telomerase supercoiling activity.
b
elongation qualitatively and quantitatively.24 The results are sum-
marized in Table 2. Not surprisingly, compound 5c had the most
potent activity against telomerase with an IC50 value of
1.86 0.51
ium bromide (IC50 = 2.18 0.36
4,5-dihydropyrazole derivative, did not show inhibitory activity
even at 60 M.
lM, better than the positive control compound, ethid-
lM). Compound 8h, a N-sulfonyl-
l
It is worth noting that the chemical structure of compound 5c is
simple, with a molecular weight less than 300 Da, and can be con-
sidered as a fragment, hence, techniques of fragment-based drug
discovery25 may apply for to get novel and more potent telomerase
inhibitors.
The single-crystal X-ray diffraction data for compound 5e was
determined on a Bruker SMART APEX CCD diffractometer using
MoKa radiation (k = 0.71073 Å) by the x scan mode. The structure
(Fig. 2) was solved by direct methods using the SHELXS program.
Crystallographic data (excluding structure factors) for the structure
of 5e were deposited into the Cambridge Crystallographic Data
Center with a Registered No. of CCDC-814974.
In this study, a three-dimension human telomerase model26
and an advanced docking method—Induced Fit Docking of the
Schrödinger Suite were employed to explore the binding mode of
BIBR1532. The docked complex was then done a 10 ns MD simula-
tion using the program of Desmond. Our designed compounds
were then docked into the active site of the modeled hTERT–
BIBR1532 complex to see if these designed compounds have simi-
lar interactions with the hTERT as BIBR1532.
One docking pose of compound 5c may can account for the SAR
of the compounds (see Fig. 3A): the phenolic hydroxyl group forms
a hydrogen bond with the DNA; another hydrogen bond can be
observed between one nitrogen atom of the moiety of 4,5-dihy-
dropyrazole and Lys 710. The benzene ring may have weak
hydrophobic interaction with the residue of Pro 929; the trifluo-
romethyl group of 5c also has hydrophobic interactions with Pro
627, Lys 902 and Val 904. When the –CF3 group was replaced by
a bulkier group, such as a substituted phenyl group (5e and 5f),
the enzyme inhibitory activities dropped because these bulkier
groups may conflict with the protein.
Figure 3. (A) Putative binding pose of compound 5c (green) to a human telomerase
model; (B) the superimposition of 5c (green) to BIBR1532 (yellow) in the three-
dimension human telomerase model. The surface of the binding pocket is presented
and colored by lipophilicity: hydrophilic and lipophilic surfaces are shown in
orange and blue, respectively.
BIBR1532 is much bulkier than compound 5c, but these two
compounds share similar hydrophobic interactions with the
enzyme of telomerase in the model (Fig. 3B). Moreover, BIBR1532
can reach Lys 710 and use its carboxylic group to interact with it.
This hydrogen bond also can be observed for compound 5c.
Whereas compound 5c uses its phenolic hydroxyl group to form
another hydrogen bond with the DNA. Because BIBR1532 can form
more interactions with telomerase/TNA:DNA hairpin than 5c, it is
no wonder that BIBR1532 (IC50 value of 0.093
lM) had more
potent inhibitory activity than 5c against the target.
All in all, in this study, several human telomerase inhibitors
having the core of N-acyl-4,5-dihydropyrazole with anticancer
effects were identified. Biological results demonstrated that a few
compounds had potent anticancer activities against three common
tumor cell lines (SGC-7901, HepG2 and MGC-803). Among them,
compound 5c had antiproliferative activities against SGC-7901
and MGC-803 with EC50 values of 2.06 0.17 and 2.89 0.62 lM,
respectively, better than the positive control compound, 5-FU.
Compound 5c could inhibit the enzyme of telomerase with an
IC50 value of 1.86 0.51 lM, better than the control compound,
ethidium bromide. Modeling study showed that this compound
can reside in the binding pocket of the telomerase/TNA:DNA hair-
pin complex. When the moiety of N-acyl was changed to N-sul-
fonyl, the gotten compounds (8a–8i) had deteriorative activities
against both the three cancer cell lines and the enzyme of telom-
erase, indicating the importance of the moiety of N-acyl. Since
the chemical structure of compound 5c is simple, and its molecular
Figure 2. The 3D chemical structure of compound 5e gotten by single-crystal X-ray
diffraction (Registered No.: CCDC-814974).
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4
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.02.
025.
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