Synthesis of thioether andrographolide derivatives and their inhibitory effect against cancer cells

Article abstract of DOI:10.1039/c7md00169j

A series of novel thioether andrographolide derivatives were synthesized by incorporating various aromatic (or heteroaromatic) substituents into C-12 or 14-OH. A total of 38 andrographolide derivatives were prepared and evaluated for their in vitro inhibi

Full text of DOI:10.1039/c7md00169j

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2017, DOI: 10.1039/C7MD00169J.
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Synthesis of Thioether Andrographolide Derivatives and Their
Inhibitory Effect Against Cancer Cells‡
Yi Liua†, RenꢀMing Lianga†, QingꢀPing Maa, Kai Xua, XinꢀYong Lianga, Wei Huangb,c,
Robert Suttonc, Jie Dinga*, Paul M. O'Neilc, ChunꢀRu Chenga,c,d
*
aSchool of Chemical Engineering, Institute of Pharmaceutical Engineering
Technology and Application, Key Laboratory of Green Chemistry of Sichuan Institutes
of Higher Education, Sichuan University of Science & Engineering, Xueyuan Street
180, Huixing Road, Zigong, Sichuan 643000, People’s Republic of China
bDepartment of Integrated Traditional Chinese and Western Medicine, Sichuan
Provincial Pancreatitis Center, West China Hospital, Sichuan University, Chengdu,,
Sichuan, 643204 China
c
Department of Molecular and Clinical Cancer Medicine, Institute of Translational
Medicine, University of Liverpool, Prescot Street, Liverpool L69 8XP, United
Kingdom
d Department of Chemistry, University of Liverpool, Crown Street, Liverpool L69 7ZD
† Both authors contributed equally to the work.
‡The authors declare no competing interests.
*Corresponding author: Tel.: +86 813 5505601; Eꢀmail addresses:
pharmaceutics@163.com (C. Cheng), dingjie@suse.edu.com (J. Ding)
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Abstract
A series of novel thioether andrographolide derivatives were synthesized by
incorporating various aromatic (or heteroaromatic) substituents into the Cꢀ12 or
14ꢀOH. Totally 38 andrographolide derivatives were prepared and evaluated for their
in vitro inhibitory activity against cancer cells. All of the derivatives exhibited better
activity against the prostate cancer cells (PCꢀ3) than that of the parent compound.
Among them, compounds 6a, 8, 9, 17, 19, 31, 32 demonstrated good activity. These
compounds were further evaluated for their anticancer activities against other cancer
cell lines, including MCFꢀ7, MDAꢀMBꢀ231, A549. Compounds 31 and 32 showed
excellent activity against MCFꢀ7 with IC50 value of 0.7 and 0.6 ꢁM, respectively. The
absolute configuration of 15a was determined by using the singleꢀcrystal Xꢀray
diffraction. The activity of 6a (12S), which is the precursor of 15a, was better than
that of the diastereoisomer 6b (12R). The preliminary structureꢀactivity relationship
was summarized. The results are very important for the further optimization of
andrographolide.
Keywords: Andrographolide, Antitumor, Thioether, Absolute configuration
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Introduction
Andrographolide, a labdane diterpenoid, is the major constituent of Andrographis
paniculata, which has been widely used in Asia as herbal medicine to treat a variety
of diseases, including sore throat, flu, upper respiratory tract infections, bacterial
dysentery, malaria, herpes, fever, and the common cold.1,2,3 Modern research reveals
that andrographolide is responsible for the pharmacological effects mentioned
above.2,4,5 As a result, not only the herb, but also the pure andrographolide has already
been prepared as tablets or capsules in Eastern Asia to treat bacterial infections and
inflammatory related diseases, such as bronchitis and pneumonia.6 Every year, tons of
pure andrographolide are produced through extraction and purification from A.
paniculata. However due to the poor water solubility of andrographolide, the
bioavailability is low7 and the pharmacological effect has been affected. Therefore,
modification of the chemical structure of andrographolide has been carried out to
improve the solubility and the activity. Series of andrographolide derivatives have
been designed and synthesized.2,4,5,8,9 Among them, several compounds (such as
Lianbizhi, Chuanhuning and Yanhuning)10, which show better activities and drugꢀlike
properties, have been further developed and approved to be used in Chinese clinic.
In recent years, scientists have revealed that andrographolide and its derivatives
possess anticancer activity both in vitro and in vivo.2,11ꢀ19 The mechanism of the
antitumor actions is mainly through inducing cell cycle arrest and promoting
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apoptosis in human cancer cells by attenuating various cell signaling pathways.12ꢀ14
Series of andrographolide derivatives have been synthesized previously to screen the
in vitro cytotoxicity against cancer cells4ꢀ7. Based on the previous studies, γꢀlactone
moiety, the conjugated double bonds, C=O, and Cꢀ14 hydroxyl group play important
roles in the cytotoxicity.4,5,7,11 Esterification of Cꢀ14 hydroxyl group would result in
the increase of the activity, while removal of Cꢀ14 hydroxyl group is detrimental to
the activity. Besides, Cꢀ12 substituted derivatives also significantly improve the
cytotoxicity against cancer cells.20 Recently, Sai et al introduced alkyl amines, benzyl
amines, and phenyl thio at Cꢀ12 of andrographolide, and found that derivatives
containing aryl amino, aryl thio and benzyl amino groups showed broad range of
activity against most of the cancer cell lines.21
Natural products are a great source of modern drugs. Among the 1562 new
approved drugs during the year from 1981 to 2014, 47% of them are from or derived
from natural products.22 Therefore, choosing an active natural product as the lead
compound may have better chances of displaying desirable biological and
pharmacological properties. In continuation of our studies on antitumor natural
products, we chose andrographolide as the lead compounds to design and synthesize
more potent and active antitumor derivatives. Based on the previous reported
structureꢀactivity relationship studies4ꢀ7,11,13ꢀ19, we focused our effort on the
development of 14ꢀOH and Cꢀ12 derivatives of andrographolide. By introducing
different aromatic (or heteroaromatic) thioether into Cꢀ12 or 14ꢀOH, we envisaged
that it might increase hydrogen bonding and πꢀπ stacking interaction and thereby had
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better effects on the hydrophilic and antitumor properties. In the present study, a series
of novel andrographolide analogues were synthesized and the analogues were
evaluated for the inhibitory effects against the cancer cells. Herein, we reported the
synthesis, cytotoxic activity and preliminary structureꢀactivity relationship study.
Results and Discussion
Chemistry
As the esterification of 14ꢀOH could increase the antitumor activity, we connected the
aromatic (or heteroaromatic) thioether moiety to 14ꢀOH by using an ester bond (as
depicted in scheme 1). There were three hydroxyl groups in the chemical structure of
andrographolide. In order to esterify the 14ꢀOH selectively, 3, 19 hydroxyl groups
were protected to afford the 3,19ꢀisopropylideneandrographolide (a), which served as
key intermediate in preparing the following analogues. We adopted Sirion's
procedure,23 and obtained the desired product (a) with high yield (94%)
(supplementary data). In order to prepare the thioether analogues,
2ꢀ(tosyloxy)acetyl chloride was used to react with a to afford the intermediate
2ꢀ(tosyloxy)acetate (b), which we envisaged to go through a nucleophilic reaction
with the aromatic thiolate to form the thioether (III) (scheme 1). However, to our
surprise, there was no desired ester (III) formed after the reaction, instead the
12ꢀaromatic thioether (I) was formed in good yield. The proposed reaction
mechanism was depicted in figure 1. The thiopheneꢀ2ꢀthiolate formed by the reaction
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of thiopheneꢀ2ꢀthiol with CH3ONa attacked Cꢀ12 of the andrographolide to afford I.
This process, rather than attacking the carbon that connected to the TsOꢀ moiety, was
favored by the formation of the stable α,βꢀunsaturated carbonyl group within the
fiveꢀmember ring. The thiopheneꢀ2ꢀthiolate could attack the Cꢀ12 from both side of
the plane, and therefore generated two possible diastereoisomers. We successfully
separated the two diastereoisomers of 6a (320 mg) and 6b (38mg) (figure 1). The
1
chemical structures of 6a and 6b were unambiguously confirmed by MS, HꢀNMR,
13CꢀNMR, and HꢀHꢀCOSY spectra (see supplementary data). In order to determine
the configuration of 6a and 6b, the main product (6a) of the reaction was further
deprotected to give the product 15a, which was further recrystallized for three times
to get the single crystal. Then the singleꢀcrystal structure of 15a was determined by
Xꢀray crystallography (its molecular structure was illustrated in figure 2, deposition
number in Cambridge Crystallographic Data Centre: CCDC 1508339). As shown in
figure 2, the absolute configuration of the Cꢀ12 in the chemical structure of 15a was
determined to be S. Therefore the absolute configuration of the Cꢀ12 in the precursor
6a was S, while that of the diastereoisomer 6b was R. This reaction provided us with a
good method to prepare the Cꢀ12 thioether derivatives. In order to investigate the
influence of the aromatic and heteroaromatic rings, different Cꢀ12 thioether
andrographolide derivatives (compounds 1ꢀ18) substituted with benzene, thiophene,
pyridine, pyrimidine, thiazole were prepared in moderate to good yields (56%ꢀ80%,
see supplementary data). Furthermore, for the purpose of knowing that the effect of
electronꢀwithdrawing
and
electronꢀdonating
group,
different
thioether
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andrographolide derivatives substituted with fluorobenzene, nitrobenzene,
methylbenzene, and methoxylbenzene were synthesized using the same strategy. The
removal of isopropylidene group of I (1ꢀ9) was carried out by employing AcOH/H2O
to readily furnish the target compounds II (10ꢀ18).
As depicted in scheme 2, for the syntheses of Cꢀ14 thioether andrographolide
derivatives, the desired target compound III was smoothly achieved by treatment of
intermediate a with 2ꢀ(arythio) acetyl chloride in the presence of Et3N at room
temperature. The preparation of 2ꢀ(arythio) acetyl chloride was outlined in scheme 3.
Treatment of ethyl 2ꢀhydroxyacetate with TsCl/Et3N gave the ethyl
2ꢀ(tosyloxy)acetate, which was converted to ethyl 2ꢀ(arylthio)acetate via the
nucleophilic reaction with RSꢀNa+. Hydrolysis of ethyl 2ꢀ(arylthio)acetate afforded the
acid c, which was treated with oxalyl chloride to give the important intermediate
2ꢀ(arylthio) acetyl chloride. In order to preliminarily investigate the influence of the
length of the linker on the activity, we designed different lengths of the linker with
two or four carbons (scheme 2). The syntheses of compounds V and VI, which had a
linker of four carbons, were similar to those of III and IV.
Study of the andrographolide analogues on cancer cell lines
The andrographolide and the analogues were assayed for their inhibitory activity
against human prostate cancer cell line (PCꢀ3). Taxol, which has been used in clinic as
antiꢀcancer agent, was used as the positive control. The cell viability was performed
according to the MTT assay. The PCꢀ3 cells were exposed to all compounds at certain
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concentrations for four times repeat. The results of IC50 values were summarized in
Table 1. From the results obtained, it was clear that all of the derivatives (1ꢀ38)
showed better anticancer activity than that of the parent compound, andrographolide.
Among them, compounds 6a, 8, 9, 17, 19, 31, 32 showed equal or even better activity
than that of the reference compound, taxol. From analysis of the data, it was clear that
the 3,19ꢀisopropylideneandrographolide analogues exhibited better antitumor activity
than that of the C3, C19 hydroxyl groups unprotected andrographolide analogues. In
general, the heteroaromatic thioether analogues had better activity compared to those
aromatic thioether analogues. The diastereoisomers 6a (12S) and 6b (12R) possessed
different activity. The main product (6a) exhibited better activity (IC50 9 ꢁM) than that
of 6b (IC50 20 ꢁM).
For the Cꢀ12 andrographolide derivatives, introduction of the aromatic or
heteroaromatic thioether to the andrographolide skeleton obviously increased the
cytotoxity against the prostate cancer cells. Among them, the heteroaromatic thioether
andrographolide derivatives showed better activity. Compounds 8 and 17, bearing a
pyrimidine thioether in 12ꢀposition, showed good activity with IC50 value of 7 ꢁM. In
order to investigate the influence of the electron withdrawing and donating groups,
different substituted groups were attached to the aromatic ring. Comparing the
activities of compounds 2 with 1 (or 5), 11 with 10 (or 14), we concluded that it would
decrease the activity when connecting an electron donating group to the benzene ring.
Data from compounds 3, 4, 12, 13 indicated that introducing the nitro group and F
into the benzene ring had less influence on the activity.
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For the 14ꢀOH andrographolide derivatives, connecting the aromatic or the
heteroaromatic thioether to the Cꢀ14 hydroxyl group through a linker could increase
the antitumor activity. Similarly, the heteroaromatic thioether derivatives had better
activities. Among them, compounds 31 and 32, which embraced a moiety of
thiophene and pyridine, respectively, had good activities (IC50 9 ꢁM). From the data,
the length of the linker ( 2 and 4 carbon atoms) appeared to have little influence on the
activity. Besides, the substitution on the benzene ring didn't have significant influence
on the activity.
Compounds 6a, 8, 9, 17, 19, 31, and 32 were further chose to evaluate the in vitro
inhibitory effects against other three different cancer cells. The results were
summarized in Table 2. From the data, we could find out that most of these
compounds also showed good activity against the breast cancer cells (MCFꢀ7 and
MDAꢀMBꢀ231). Among them, compounds 31 and 32 showed excellent activity
against MCFꢀ7 cells with IC50 value of 0.7 ꢁM and 0.6 ꢁM, respectively. However,
the lung cancer cells (A549) were not significantly sensitive to these compounds.
Only compound 17 showed good activity with an IC50 value of 9 ꢁM. We also tested
the toxicity of these compounds against normal cells. No obvious toxicity of these
compounds was observed against normal cells (Vero cell) at a concentration of 100
ꢁM. These results indicated that these compounds may be regarded as promising
potential anticancer candidates.
Conclusions
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In summary, the present study preliminarily investigated the modification of the Cꢀ12
and 14ꢀOH of andrographolide by introducing the novel aromatic or heteroaromatic
thioether into the skeleton. Totally 38 andrographolide derivatives were successfully
synthesized, and further evaluated for their inhibitory effect against cancer cells. All
the synthetic compounds exhibited better activity than that of the parent compound.
Among them, compounds 6a, 8, 9, 17, 19, 31, 32 showed excellent activity against
prostate cancer cells (PCꢀ3) and breast cancer cells (MCFꢀ7 and MDAꢀMBꢀ231). The
absolute configuration of 15a was determined to be S by using singleꢀcrystal Xꢀray
crystallography. The activity of 6a (12S) was better than that of the diastereoisomer
6b (12R).
This study also provided us with preliminary structureꢀactivity relationship. The
3,19ꢀisopropylideneandrographolide analogues exhibited better antitumor activity
than that of the C3, C19 hydroxyl groups unprotected andrographolide analogues. The
heteroaromatic thioether showed better activity compared to the aromatic thioether.
For the Cꢀ12 analogues, the substitution of the electron donating group on the benzene
ring will decrease the activity, while the electron withdrawing group on the benzene
ring appeared to have little influence on the activity. For the Cꢀ14 analogues, the
length of the linker, and the substitution pattern on the aromatic ring had little
influence on the activity. Inspired by the results, further investigation of the biological
mechanism and optimization of the Cꢀ12 and 14ꢀOH of andrographolide analogues
are ongoing.
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Acknowledgement
This work was supported by the Discipline Construction Project of Sichuan
University of Science & Engineering, the National Natural Science Foundation of
China (No. 81373964), Introduction Program of Scientific Researchist of Sichuan
University of Science & Engineering (2016RCL07, 2012RC15), Graduate Innovation
Fund (y2016009), the Opening Project of Key Laboratory of Green Chemistry of
Sichuan Institutes of Higher Education (LYJ1302, LYJ1505), the Scientific Research
Fund of the Sichuan Provincial Education Department (13ZB0133, 15ZB0211), the
Science and Technology Programme Project of Sichuan (2015JY0024), and the
Science and Technology Programme Project of Zigong (2015SF01).
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Figure 1. Proposed mechanism of the reaction between thiolate and intermediate b.
Figure 2. The Xꢀray crystal structure of compound 15a.
Scheme 1. Reagents and conditions: (i) DMP/PPTS/acetone, rt, 2h; (ii)
2ꢀ(tosyloxy)acetyl chloride, CH2Cl2, Et3N, 0˚C, 1h; (iii) CH3ONa/MeOH; reflux; (iv)
70 % HOAc, rt, 45 min.
Scheme 2. Reagents and conditions: (i) Et3N, CH2Cl2, 0˚C, 1 h; (ii) 70 % HOAc, rt,
45 min.
Scheme 3. Reagents and conditions: (i) TsCl/Et3N, 0 ˚C, 2 h; (ii) CH3ONa/MeOH;
reflux; (iii) 5 % NaOH/EtOH, 40 ˚C, 2 h; (iv) SOCl2 or (COCl)2, reflux, 6 h.
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Table 1. Evaluation of in vitro cell growth inhibitory effects of andrographolide analogues (1ꢀ38) in prostate cancer cell (PCꢀ3).a,b
compound
1
analogue
R Group
IC50 (µM)
compound
20
analogue
R1,or R2, or R3 Group
IC50 (µM)
IꢀR1
23
IIIꢀR2
11
2
3
IꢀR1
IꢀR1
IꢀR1
IꢀR1
IꢀR1
12
12
13
17
9
21
22
23
24
25
IIIꢀR2
IIIꢀR2
IVꢀR2
IVꢀR2
IVꢀR2
13
15
17
27
14
4
5
6ac
7
8
9
IꢀR1
IꢀR1
IꢀR1
14
7
26
27
28
IVꢀR2
VꢀR3
VꢀR3
18
18
17
10
15

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10
11
12
IIꢀR1
IIꢀR1
IIꢀR1
29
29
30
31
VꢀR3
VꢀR3
VꢀR3
14
13
9
14
17
13
IIꢀR1
16
32
VꢀR3
9
14
IIꢀR1
IIꢀR1
15
15
33
34
VIꢀR3
VIꢀR3
19
17
15a
16
17
18
19
IIꢀR1
IIꢀR1
IIꢀR1
IIIꢀR2
18
7
35
36
37
38
VIꢀR3
VIꢀR3
VIꢀR3
VIꢀR3
16
12
16
14
14
9
16

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a. Results were expressed as the mean value of four data points. Taxol was used as a positive control ( IC50=7 ꢁM).
b. IC50 of andrographolide is 30 ꢁM.
c. IC50 of 6b, which is the diastereoisomer of 6a, is 20 ꢁM.
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Table 2. Evaluation of in vitro cell growth inhibitory effects of andrographolide
analogues in different cancer cells (PCꢀ3, MCFꢀ7, MDAꢀMBꢀ231, A549) .a
Cell growth inhibition in IC50 (ꢁM)
compound
PC-3
MCF-7
MDA-MB-231
A549
6a
8
9
7
5
8
9
20
20
31
9
7
9
10
7
3
12
16
19
10
6
17
19
31
32
23
10
0.7
0.6
9
11
25
23
9
9
a. Results were expressed as the mean value of four data points.
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Figure 1. Proposed mechanism of the reaction between thiolate and intermediate b.
197x56mm (300 x 300 DPI)

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Figure 2. The X-ray crystal structure of compound 15a.
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Scheme 1. Reagents and conditions: (i) DMP/PPTS/acetone, rt, 2h; (ii) 2-(tosyloxy)acetyl chloride, CH2Cl2,
Et3N, 0˚C, 1h; (iii) CH3ONa/MeOH; reflux; (iv) 70 % HOAc, rt, 45 min.
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Scheme 2. Reagents and conditions: (i) Et3N, CH2Cl2, 0˚C, 1 h; (ii) 70 % HOAc, rt, 45 min.
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Scheme 3. Reagents and conditions: (i) TsCl/Et3N, 0 ˚C, 2 h; (ii) CH3ONa/MeOH; reflux; (iii) 5 %
NaOH/EtOH, 40 ˚C, 2 h; (iv) SOCl2 or (COCl)2, reflux, 6 h.
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Table of content
Andrographolide
6a IC50 = 5 µM
31 IC50 = 0.7 µM
32 IC50 = 0.6 µM
Novel thioether andrographolide derivatives were designed and synthesized, and some
of them exhibited excellent anticancer activity.