Syntheses, cytotoxic activity evaluation and HQSAR study of 1,2,3-triazole-linked isosteviol derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2016.10.028

A series of novel 1,2,3-triazole-linked isosteviol derivatives were designed and synthesized via Huisgen-click reaction. Their cytotoxicities in vitro against HCT-116 and JEKO-1 cells were screened. The preliminary bioassays indicated that most of the title compounds exhibited noteworthy cytotoxic activities. Particularly, the compound 10b revealed the most potent inhibitory activities against HCT-116 cells with IC₅₀value of 2.987?±?0.098?μM, which was better than that (3.906?±?0.261?μM) of positive control cisplatin. On the basis of these bioactivity data, hologram quantitative structure–activity relationship (HQSAR) was performed, and a statistically reliable model with good predictive power (r²?=?0.848, q²?=?0.544 and R²_pred?=?0.982) was achieved. Additionally, the contribution maps derived from the optimal model explained the individual atomic contributions to the activity for each molecule.

Full text of DOI:10.1016/j.bmcl.2016.10.028

Accepted Manuscript
Syntheses, cytotoxic activity evaluation and HQSAR study of 1,2,3-triazole-
linked isosteviol derivatives as potential anticancer agents
Cong-Jun Liu, Yan-Ping Liu, Shu-Ling Yu, Xing-Jie Dai, Tao Zhang, Jing-Chao
Tao
PII:
S0960-894X(16)31056-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.028
BMCL 24331
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
20 August 2016
26 September 2016
11 October 2016
Please cite this article as: Liu, C-J., Liu, Y-P., Yu, S-L., Dai, X-J., Zhang, T., Tao, J-C., Syntheses, cytotoxic activity
evaluation and HQSAR study of 1,2,3-triazole-linked isosteviol derivatives as potential anticancer agents,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.10.028
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Syntheses, cytotoxic activity evaluation and HQSAR study of
1
,2,3-triazole-linked isosteviol derivatives as potential
anticancer agents
a,b
b
c
a
a
a,
Cong-Jun Liu , Yan-Ping Liu , Shu-Ling Yu , Xing-Jie Dai , Tao Zhang , Jing-Chao Tao *
a
College of Chemistry and Molecular Engineering, New Drug Research & Development Center,
Zhengzhou University, 75 Daxue Road, Zhengzhou, Henan 450052, China
b
College of Chemical Engineering and Food Technology, Zhongzhou University, Zhengzhou,
Henan 450044, China
c
Key Laboratory of Natural Medicine and Immune-Engineering of Henan Province, Henan
University, Kaifeng, Henan 475004, China
Abstract: A series of novel 1,2,3-triazole-linked isosteviol derivatives were designed and
synthesized via Huisgen-click reaction. Their cytotoxicities in vitro against HCT-116 and JEKO-1
cells were screened. The preliminary bioassays indicated that most of the title compounds
exhibited noteworthy cytotoxic activities. Particularly, the compound 10b revealed the most potent
inhibitory activities against HCT-116 cells with IC50 value of 2.987 ± 0.098 μM, which was better
than that (3.906 ± 0.261 μM) of positive control cisplatin. On the basis of these bioactivity data,
hologram quantitative structure–activity relationship (HQSAR) was performed, and a statistically
2
2
2
reliable model with good predictive power (r =0.848, q =0.544 and R pred= 0.982) was achieved.
Additionally, the contribution maps derived from the optimal model explained the individual
atomic contributions to the activity for each molecule.
Key words: Isosteviol; Synthesis; Cytotoxicity; Click chemistry; HQSAR.
*
Corresponding author. Tel/Fax: +86-371-67767200. Email address: jctao@zzu.edu.cn.
Cancer is becoming the second leading cause of death after cardiovascular disorders throughout
the world. For the treatment of cancer, many chemotherapeutic agents, such as 5-fluorouracil,
1-5
cisplatin, doxorubicin and cetuximab, have been developed over the past few decades. However,
the resistance of cancer cells to cytotoxic drugs can lead to treatment failure. Therefore, it is
utmost significant and urgent to develop remarkable chemotherapeutic agents having a limited
1

toxicity profile.
Isosteviol (ent-16-ketobeyeran-19-oic acid 1), a tetracyclic diterpenoid with an ent-beyerane
skeleton that possesses multifarious bioactivities, can be readily obtained as a metabolite of
6,7
stevioside isolated from the leaves of the natural stevia plant. In recent years, isosteviol and a
number of its derivatives have been shown to exhibit a wide range of biological activities, such as
anticancer, antihypertension, antihypotension, antihyperglycaemic, cardio and neuro protective
8
-17
effects.
inhibitory activity against cancer cells. For example, isosteviol-fused pyrazole derivatives A (Fig.
), obtained by means of functional interconversions in ring D of isosteviol in our laboratory,
exhibited growth inhibitory effects comparable to cisplatin in vitro against gastric (SGC-7901),
Some special group hybridized isosteviol derivatives have shown the promising
1
1
8
lung (A549), lympha (Raji) as well as cervical (Hela) carcinoma cells, which prompted us to
further investigate new heterocycle-linked isosteviol derivatives to develop novel stronger
anticancer agents for therapeutic use.
1,2,3-Triazoles, highly nitrogen-rich heterocyclic compounds, have been regarded as an
interesting unit in terms of biological activity and some of them have shown significant anticancer
1
9-25
activity in many of the human cell lines such as colon, lung, prostate, breast cancers and so on.
2
6
Banday and co-workers reported that a series of 1,2,3-triazolyl derivatives of pregnenolone B
Fig. 1) showed significant cytotoxicities against colon (HCT-15, 502713), prostrate (DU-145,
PC-3), liver (HEP-2) and lung (A-549) cancer cell lines with IC50 values 0.03–8.63 μM. In
(
27
addition, Khaybullin et al. revealed that the inhibitory activity against breast (MDA-231), lung
A549), pancreas (ASPC-1), prostate (PC-3), colon (HCT-116) and cervical (HeLa) cancer cells
(
were greatly improved as 1,2,3-triazole ring was introduced onto 19-carboxyl group of isosteviol
C (Fig. 1). Meanwhile, these reports explicitly pointed out that substituted 1,2,3-triazole rings
played an important role in inhibiting the growth of cancer cells, thus allowing new possibilities
for quantitative structure-activity relationship studies associated with structure-based good
bioactivity molecule design strategies. However, few reports have been focused on the activity
relationship of 1,2,3-triazole-linked isosteviol derivatives. In view of these results, the objective of
the current work is to synthesize novel 1,2,3-triazole-linked isosteviol derivatives through
Huisgen-click reaction, and to investigate in vitro their cytotoxicities against colon (HCT-116),
mantle cell lymphoma (JEKO-1) cancer cell lines. On the basis of the novel molecule structures
2

and their cytotoxicities, the robust and predictive model will be developed by the HQSAR
technique, and be further taken advantage of designing new anticancer molecules with improved
potency.
Fig. 1. Chemical structures of compounds A-C
In an effort to discover new target molecules with special anticancer potency, some structural
modifications were made at the C15 and C16-positions of isosteviol by preparing different
substituted 1,2,3-triazole derivatives. It is well known that Huisgen-click reaction of terminal
alkynes and organic azides to give five-membered 1,2,3-triazoles, a class of ‘click’ chemistry, has
emerged as a powerful linking reaction and found widespread applications ranging from
combinatorial drug research and material science to bioconjugate chemistry. Therefore, our initial
efforts were focused on the synthesis of intermediates 5 and 8, with azido group at different places,
to be suitable for Huisgen-click reaction. The synthetic routes were outlined in Scheme 1.
Scheme 1. Reagents and conditions: (i) CH
3
CH
2
Br, DMSO, KOH, rt, 6h, 97%; (ii) NaBH
4
,
o
2
C H
5
OH, rt, 6h, 98%; (iii) HCHO, C
2
H
5
ONa, C
2
H
5
OH, 55 C, 8 h, 85%; (iv) TsCl, pyridine, rt,
o
36h; (v) NaN
3
, DMF, 85 C, 8h.
Isosteviol 1, obtained through acidic hydrolysis of stevioside, was treated with CH
3 2
CH Br and
KOH in DMSO to yield the corresponding isosteviol ethyl ester 2. The 16-α-hydroxyl isosteviol
4
derivative 3 was prepared as a white powder by reduction of compound 2 with NaBH in
3

2 5
anhydrous C H OH, whereas the 15-β-hydroxymethyl-16-α-hydroxyl isosteviol derivative 6 was
stereo-selectively synthesized via a “one-pot” two-steps Tollens’ reaction. The absolute
28
configurations of the compounds 3 and 6 were determined according to the literature. Treatment
of compound 3 or 6 with 1.1 equiv. of 4-Methylphenylsulfonyl chloride in pyridine readily
3
afforded the corresponding product 4 or 7 in good yield. Reaction of compound 7 with NaN in
dry DMF furnished the desired azide product 8 in high yield with excellent purity. However, at
the same reaction conditions, compound 5 was obtained with much lower yield, due to the
steric effects of the neighboring groups adjacent to C16. The absolute configuration of
compound 5 was indirectly confirmed by X-ray crystallographic analysis of its 1,2,3-triazole
derivative 9a, demonstrating a β orientation of the azido group at C-16.
Although procedures for Cu (0) and Cu (I)-catalyzed combination of organic azides and terminal
alkynes have been well developed, they were not suitable for our desired hybrids. After
investigating several Cu (II)-ligands catalyst, we found that 1,2,3-triazole-linked isosteviol
derivatives were exclusively obtained with no side products by employing copper (II) acetate and
sodium ascorbate as the catalytic system.
Scheme 2. Reagents and conditions: (i) the relative alkynes, sodium ascorbate, Cu(OAc)
tert-butanol/H O, rt; (ii) Jones reagent, CH COCH , rt, 1h, 97%.
2
,
2
3
3
Huisgen-click reaction of the azides (5 or 8) and appropriate substituted terminal alkynes, such as
alkyl-, phenyl-substituted alkynes as well as alkynes containing ortho-, meta-, para-aldehyde,
para-ketone and 2-methoxyl-4-aldehyde groups on the aromatic ring, readily took place at room
4

temperature in the presence of copper (II) acetate and sodium ascorbate as catalyst to afford the
corresponding 1,2,3-triazole derivatives 9a-9f in yields of 88-94% and 10a-10g in yields of
87-99%, respectively (Scheme 2). In order to further investigate the effect of stronger
electron-withdrawing substituent on the aromatic ring on cytotoxic activities, the aldehyde group
in compound 9d was converted to the carboxyl group via Jones reagent oxidation in acetone in
order to compare the effect of different groups at the benzene ring. Structures of these novel
compounds were elucidated on the basis of spectroscopic data. The IR spectrum of compound 9a,
as a representative example of the synthesized compounds, showed moderate absorbance peak at
-
1
3
145 cm due to the formation of 1,2,3-triazole ring leading to stretching vibration of associated
1
C=C–H. The H NMR spectrum of compound 9a displayed the characteristic 1,2,3-triazole ring
1
3
proton at δ 7.72 ppm in addition to the C16-H signal at δ 4.63 ppm. The C NMR spectrum of
compound 9a revealed two carbons of the 1,2,3-triazole ring appearing at δ 147.10 and 130.88
+
ppm, respectively. In accord with the molecular formula C30
H
42
N
3
O
2
, an [M + H] peak at m/z
476.3277 in the HRMS was observed. Furthermore, a conclusive evidence for the assigned
29
structure was provided by its X-ray crystallographic analysis (Fig. 2) .
Fig. 2. X-ray structure of compound 9a
Since the structural modifications of 19-carboxyl group could result in the bioactive improvement
8
in previous report, we intended to introduce the 1,2,3-triazole fragment to the C19-position of
isosteviol in order to construct a novel family of bioactive molecules for the discovery of
anticancer agent, and simultaneously to provide diverse molecule structures convenient for the
subsequent HQSAR study. Therefore, novel 1,2,3-triazole-linked C19-position of isosteviol
derivatives 12a-12c were designed and synthesized by Huisgen-click reaction. The synthetic
approaches employed to prepare the target derivatives were outlined in Scheme 3. Treatment of
5

compound 1 with propargyl bromide under alkaline conditions in DMSO offered the
corresponding alkyne 11, which further reacted with the substituted azides in the presence of
copper (II) acetate and sodium ascorbate as catalyst to give the target products 12a-12c in good
yield. In the IR, NMR and HRMS spectra of 12c, additional signals were distinctly observed at ν
-
1
3
H C
142 cm , δ 7.82 ppm, δ 143.58, 122.16 ppm and m/z 738.3216 (theoretical value: 738.3214)
indicating the formation of 1,2,3-triazole ring.
Scheme 3. Reagents and conditions: (i) 3-bromo-1-propyne, DMSO, KOH, rt, 1h, 84%; (ii) the
2 2
relative azides, sodium ascorbate, Cu(OAc) , tert-butanol/H O, rt.
These newly synthesized isosteviol derivatives were further utilized to screen their cytotoxic
activities against human colon (HCT-116) and mantle cell lymphoma (JEKO-1) cancer cell lines.
The inhibitory activities of the 22 compounds were in vitro determined by means of MTT assay
using cisplatin as reference drug. The IC50 values, demonstrating the growth inhibition in the
30
presence of tested compounds, were summarized in Table 1.
Table 1. IC50 values (in µM) of tested compounds against HCT-116 and JEKO-1 cell lines
a,b
a,b
IC50 (μM)
HCT-116
>150
IC50 (μM)
Compd
Compd
JEKO-1
>150
HCT-116
2.987 ± 0.098
4.637 ± 0.138
6.620 ± 0.178
4.040 ± 0.098
8.155 ± 0.246
19.573 ± 0.698
JEKO-1
1
4
5
8
10b
10c
10d
10e
10f
12.693 ± 0.370
15.610 ± 0.399
16.400 ± 0.773
7.087 ± 0.351
19.593 ± 1.083
32.313 ± 1.350
51.433 ± 1.292 9.750 ± 0.178
46.101 ± 0.359 55.167 ± 1.558
14.822 ± 0.334 31.190 ± 1.747
48.440 ± 2.396 95.917 ± 3.513
9
9
a
b
3.801 ± 0.228
12.160 ± 0.167
10g
6

9
9
9
9
9
c
6.502 ± 0.094
8.200 ± 0.242
7.183 ± 0.189
9.624 ± 0.372
9.497 ± 0.337
11.737 ± 0.619
10.863 ± 0.074
13.862 ± 0.768
11
27.120 ± 1.134
24.670 ± 1.688
d
e
12a
12b
12c
125.713 ± 6.964 15.330 ± 0.330
19.913 ± 1.970
19.310 ± 0.818
11.360 ± 0.324
26.307 ± 0.900
f
d'
0a
11.750 ± 0.860 54.357 ± 3.807
8.570 ± 0.179 18.790 ± 0.842
1
Cisplatin 3.906 ± 0.261
2.743 ± 0.235
a
Values are the mean of triplicate of three independent experiments
b
±SD
From the observed cytotoxic activity data, it has been noticed that all the derivatives of isosteviol
showed better cytotoxic activities than their corresponding precursor. More importantly, the
inhibitory activities of most compounds were markedly improved as the 1,2,3-triazole subunit was
introduced onto the skeleton of isosteviol, which indicated the 1,2,3-triazole fragment exactly
played a significant role in inhibiting cancer cell proliferation. Going even further, substituted
groups and positions on aromatic ring had a significant effect on cytotoxic activities. Compounds
with aldehyde group on aromatic ring exhibited better inhibitory activities than the unsubstituted
compounds (9b-9f vs 9a and 10b-10f vs 10a). Oxidation of aldehyde group caused weaker
activities to the cancer cell lines (9d vs 9d').
The colon carcinoma cells were more sensitive to the tested compounds, half of which were found
to possess IC50 values lower than 10 μM. Especially, the compound 10b exhibited the most potent
anticancer activity with IC50 values of 2.987 ± 0.098 μM, which was better than that (3.906 ±
0.261 μM) of positive control cisplatin. In addition, the compound 9b showed the second-best
inhibitory activity against HCT-116 cells with IC50 values of 3.801 ± 0.228 μM, which indicates
ortho-position on aromatic ring has an important effect on cytotoxic activity. To be noteworthiness,
the inhibitory activities of isosteviol simultaneously fusing hydroxyl and 1,2,3-triazole subunits
were better than that of compounds with only 1,2,3-triazole subunit (10a vs 9a, 10b vs 9b, 10c vs
9c, 10d vs 9d, 10e vs 9e and 10f vs 9f), which illustrates that introduction of hydroxyl group can
result in higher inhibitory activity against HCT-116 cells. This conclusion is consistent with
3
1
32
previous work by Wu and Zhang .
When these synthesized compounds were evaluated against JEKO-1 cells, only compounds 4 and
7

10e exhibited noteworthy inhibitory activity with IC50 values of 9.750 ± 0.178 and 7.087 ± 0.351
μM, respectively, and IC50 values of the remaining compounds were greater than 10 μM.
Interestingly, the cytotoxic activity against JEKO-1 cells was improved as 1,2,3-triazole ring was
introduced onto the 19-carboxyl group to give the derivative 12a, however, which was inactive to
HCT-116 cell line (11 vs 12a). It was very disappointing that the inhibitory activities against
JEKO-1 cells lacked of regularity, which would adversely influence on the development of
satisfactory HQSAR model in follow-up HQSAR study.
Newly synthesized 21 isosteviol derivatives (compounds 4-12c) were chosen for the HQSAR
study. Biological data (IC50) of the molecules in Table 1 were used as dependent variable in this
study. This whole dataset was segregated in such a way that both training (17 compounds) and test
set (4 compounds: 8, 9d, 9f and 10c) contain high active, moderately active, and low active
molecules. The IC50 values were converted into pIC50 values according to the formula (pIC50
log IC50).
HQSAR models were generated with seventeen training set compounds using three distinct
=
–
3
3
parameters: fragment distinction, fragment size and hologram length . When three distinct
parameters as independent variables were linearly correlated with the pIC50 values against JEKO-1
2
cells as dependent variables by the full cross validated (q ) partial least square (PLS)
leave-one-out (LOO) method, then no satisfactory HQSAR model was obtained due to the results
of anticancer activity lacking of regularity. However, predictable and statistical significant models
were developed using pIC50 values against HCT-116 cells in HQSAR analyses.
Initially, the effects of fragment distinction parameters on the statistical values of models were
investigated.
fragment
donor and acceptor
hologram length (53, 59, 61, 71, 83, 97, 151, 199, 257, 307, 353 and 401),
fragment size (4–7) and a combination of one or more fragment distinction parameters
Among these models model 9, which includes atoms,
2
connections,
and donor and acceptor (A/C/H/Ch/DA), was chosen as the best
8

2
2
HQSAR model to give significant q and r of 0.503 and 0.855 respectively with component 5 and
hologram length 53.
Table 2. HQSAR analysis for various fragment distinction using default fragment size (4–7)
2
2
model
Fragment distinction
A/B
q
SEcv
0.413
0.381
0.414
0.394
0.385
0.394
0.426
0.413
0.392
0.422
r
SEE
HL
61
N
5
4
5
4
4
3
5
5
5
1
2
3
4
5
6
7
8
9
0.450
0.489
0.446
0.453
0.477
0.407
0.413
0.448
0.503
0.423
0.820
0.845
0.795
0.832
0.835
0.725
0.840
0.813
0.855
0.821
0.236
0.210
0.252
0.218
0.217
0.268
0.222
0.241
0.211
0.235
A/B/C
97
A/C/H
53
A/B/Ch
71
A/B/C/Ch
A/C/H/Ch
A/C/Ch/DA
A/B/C/H/Ch
A/C/H/Ch/DA
A/B/C/H/Ch/DA
401
199
257
71
53
10
61
5
2
2
q : LOO cross-validated correlation coefficient. SECV: cross-validated standard error. r :
noncross-validated correlation coefficient. SEE: noncross-validated standard error. HL:
hologram length. N: optimal number of component. Fragment distinction: A-atoms, B-bonds,
C-connections, H-hydrogens, Ch-chirality, DA-donor and acceptor.
In order to optimize statistical results, various fragment sizes were applied with A/C/H/Ch/DA as
fragment distinction. The influence of distinct fragment sizes on the statistical parameters was
further investigated and the results were summarized in Table 3. As can be seen from it, although
2
2
their r values were lower than that of model 5, the q values of models 3, 4 and 6 were greater
2
than 0.503. Especially, the fragment size (5–6) led to the best statistical results with q value of
0.544. Therefore, the finally optimal HQSAR model 6 obtained from training set with 17
compounds was established using A/C/H/Ch/DA as fragment distinction, and 5–6 as fragment size
2
2
with component 5 showing cross-validated q value of 0.544 and conventional r value of 0.848.
Table 3. HQSAR analysis for the influence of fragment sizes using the best fragment
distinction (A/C/H/Ch/DA)
2
2
model
Fragment size
q
SEcv
0.424
0.433
0.382
0.386
r
SEE
HL
353
353
53
N
6
6
5
5
1
2
3
4
2-4
2-5
3-6
4-6
0.471
0.449
0.528
0.518
0.836
0.835
0.829
0.848
0.197
0.197
0.216
0.217
53
9

5
6
7
8
9
1
4-7
5-6
5-7
5-8
6-9
8-9
0.503
0.544
0.456
0.242
0.248
0.273
0.392
0.376
0.410
0.429
0.428
0.420
0.855
0.848
0.834
0.498
0.486
0.501
0.211
0.217
0.212
0.349
0.354
0.348
53
53
5
5
5
2
2
2
53
307
61
0
61
Since the structure encoded with in a two-dimensional fingerprint is directly related to bioactivity
of molecule, the HQSAR model is able to predict the activity of new related molecule according
34
2
to its fingerprint. In virtue of the finally optimal HQSAR model revealing noncross-validated (r
2
=
0.848) and cross-validated (q = 0.544) correlation coefficients, these parameters indicated that
the model was provided with good internal robustness and predictive power, the predicted pIC50
values of training set compounds are summarized in Table 4. In order to further investigate
external predictive power of this model, it was validated by an external test set of four compounds
2
with satisfactory predictive r value of 0.982. The predicted results of test set compounds also
listed in Table 4. As shown in it, all the pIC50 residuals of test set compounds were not greater than
0.225 log unit, which is a strong evidence that highly predictive QSAR model was obtained. The
correlation between the experimental and predicted activities of both the training set and the test
set was graphically displayed in Fig. 3.
Table 4. Experimental and predicted activities (pIC50) with residual values for 21 compounds
Compd
Exp
Pred
Res
Compd
10f
Exp
Pred
Res
Training
-0.911
-1.292
-1.433
-2.099
-1.299
-1.286
-0.677
-1.161
-1.308
-1.866
-1.514
-1.312
-0.234
-0.131
-0.125
-0.233
0.215
0.026
4
5
-1.711
-1.664
-1.685
-0.580
-0.813
-0.856
-1.070
-0.933
-0.475
-0.821
-1.792
-1.757
-1.459
-0.947
-0.996
-0.903
-0.947
-1.107
-0.595
-0.648
0.081
0.093
-0.226
0.367
0.183
0.047
-0.123
0.174
0.120
-0.173
10g
11
9
9
9
9
9
1
1
1
a
12a
b
12b
12c
c
e
d'
0a
0b
0d
Test
8
-1.171
-0.914
-0.983
-1.396
-1.000
-1.029
0.225
0.086
0.046
9d
9f
10

10e
-0.606
-0.551
-0.055
10c
-0.666
-0.644
-0.022
Fig. 3. Plot of experimental values versus predicted values of pIC50 of the training and test set
An attractive property of HQSAR technique is that it provides straightforward clues about the
3
5
individual atomic contributions to the bioactivities using different color codes. The HQSAR
analysis results can be graphically displayed in the form of contribution maps, where the color
coding of each atom reflects its contribution to the activity of the whole molecule. The colors (red,
red orange and orange) at end of the spectrum indicate the negative contribution (NC) to the
activity, while ones (yellow, green blue and green) of the other end signify positive contribution
(
PC) to the activity. The white in the middle domain denotes intermediate contribution (IC) to the
activity.
Fig. 4. The HQSAR contribution maps of compounds 8 and 10b
11

The individual atomic contribution of the most potent molecule 10b and its precursor molecule 8
to its molecular bioactivity, resulting from the best HQSAR model, was shown in Fig. 4. From Fig.
4
3
, it can be seen that the C–1 and C10-CH of compound 8 were colored the red and orange
indicating their negative contributions to the activity, and the remaining structural units were
colored the white as intermediate contribution. After introduction of substituted 1,2,3-triazole ring
subunit onto the skeleton of isosteviol, although two nitrogen atoms of 1,2,3-triazolering were full
of the red and orange colors as unfavorable contribution, A and B rings of compound 10b were
wholly covered by the green and yellow colors reflecting its positive contribution to the activity.
This result reinforced the importance of the 1,2,3-triazole ring moiety in establishing the
pharmacophore of these inhibitors, and it was deduced that the introduction of substituted
1,2,3-triazole ring may indirectly enhance the interaction between the isosteviol skeleton and the
active site of target HCT-116 cell. What’s more, regions with intermediate or negative
contributions can be identified as the potential targets for the synthesis as well as
structure–activity relationship studies.
In conclusion, a series of novel 1,2,3-triazole-linked isosteviol derivatives have been rationally
designed and successfully synthesized via a Huisgen-click reaction in high yields. Inhibitory
activities against HCT-116 and JEKO-1 cancer cell lines of the newly synthesized analogs were
investigated in vitro using cisplatin as positive control according to the standard procedure. Most
of the tested compounds exhibited significant anticancer properties. Especially, compound 10b
(
IC50 = 2.987 ± 0.098 μM) revealed remarkable anticancer potency compared to that of positive
control cisplatin (IC50 = 3.906 ± 0.261 μM). On the basis of novel molecular structures and
bioactivity data, the HQSAR technique was employed to investigate further structure-activity
2
2
relationship of these compounds and afforded the robust and good predictive model (q = 0.544, r
2
=
0.848 and r pred = 0.982). The results will provide a practical tool for guiding the design and
synthesis of novel and more potent isosteviol derivatives containing 1,2,3-triazole moiety in due
course.
Acknowledgments
We look forward to financial support from the National Natural Science Foundation of China
(
Project No. 20772113), Scientific Research Fund of Xinxiang Medical University (2014QN148).
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1464.
2
3
9. Crystallographic data (excluding structure factors) has been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication numbers CCDC 1419389 (compound 9a). Copies of the data can
be obtained, free of charge, on applications to CCDC, 12 Union Road, Cambridge CB2 1EZ. UK (fax: +44
(
0) 1223 336033 or E-mail: deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
0. Cytotoxicity assay in vitro: HCT-116 and JEKO-1 cells were cultured in RPMI-1640 medium (GIBCO：
o
3
1800-022) supplemented with 10% FBS, 100 U/mL of penicillin and 100 mg/mL of streptomycin at 37 C
in a 5% CO
2
humidified atmosphere. Cell cytotoxicity was assayed by MTT method. Briefly, cells were
o
seeded in 96-well tissue culture plates. After 24 h incubation at 37 C, 5% CO
2
, removed the culture medium
and replaced with fresh medium containing the studied compounds in different concentrations to the wells,
and the cells were incubated for another 72 h. Afterwards, the MTT (MP: 102227) solution (0.5 mg/mL) was
added and incubated for an additional 4 h. Two hundred microliters of DMSO was added to each well to
dissolve the reduced MTT crystals. Optical density of each well was measured at 492/630 nm with enzyme
immunoassay instrument (TECAN: Infinite 200 Pro). Then the inhibitory percentage of each compound at
various concentrations to the cell proliferation was calculated, and the IC50 value was determined.
3
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3
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2
and max = 7) were used. The best model was selected on the basis of best crossvalideted r . Two options
dialog boxes, which are show statistics for model ensemble and retain fragment information for preditiction
2
2
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3
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14

Highlights
►
►
►
►
A series of novel 1,2,3-triazole-linked isosteviol derivatives were synthesized.
Their cytotoxicities were assessed against HCT-116 and JEKO-1cell lines.
Compound 10b is promising anticancer agent against HCT-116 (IC50 = 2.987 ± 0.098 μM).
HQSAR studies revealed good predictive model.
15

Graphical Abstract:
Syntheses, cytotoxic activity evaluation and HQSAR study of
1
,2,3-triazole-linked isosteviol derivatives as potential anticancer agents
Cong-Jun Liu, Yan-Ping Liu, Shu-Ling Yu, Xing-Jie Dai, Tao Zhang, Jing-Chao Tao*
16