Synthesis of β-boswellic acid derivatives as cytotoxic and apoptotic agents

Article abstract of DOI:10.1016/j.bmcl.2015.11.027

A series of β-boswellic acid derivatives were synthesized and evaluated for anticancer activity. One of the lead analog 4f displayed significant anticancer activity against a panel of cancer cells as well as substantially inhibited colony formation in HCT

Full text of DOI:10.1016/j.bmcl.2015.11.027

Accepted Manuscript
Synthesis of β-Boswellic acid Derivatives as Cytotoxic and Apoptotic Agents
Arvind Kumar, Arem Qayum, Parduman Raj Sharma, Shashank Kumar Singh,
Bhahwal Ali Shah
PII:
S0960-894X(15)30236-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.11.027
BMCL 23283
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
29 July 2015
23 October 2015
9 November 2015
Please cite this article as: Kumar, A., Qayum, A., Sharma, P.R., Singh, S.K., Shah, B.A., Synthesis of β-Boswellic
acid Derivatives as Cytotoxic and Apoptotic Agents, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://
dx.doi.org/10.1016/j.bmcl.2015.11.027
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Synthesis of β-Boswellic acid Derivatives as Cytotoxic and Apoptotic Agents
Arvind Kumara,b,‡, Arem Qayuma,c,‡, Parduman Raj Sharmac, Shashank Kumar Singh*,c and
Bhahwal Ali Shah*,a,b
c
aAcademy of Scientific and Innovative Research, bNatural Product Microbes, Cancer
Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-
Tawi, Jammu and Kashmir, 180001, India.
*Corresponding authors e-mail address: bashah@iiim.ac.in (BAS), sksingh@iiim.ac.in (SKS).
Keywords. β-boswellic acid, triterpenoid, anticancer, apoptosis.
Abstract: A series of β-boswellic acid derivatives were synthesized and evaluated for anticancer
activity. One of the lead analogue 4f displayed significant anticancer activity against a panel of
cancer cells as well as substantially inhibited colony formation in HCT-116 cells. Furthermore,
4f was found to be a potent inducer of apoptosis confirmed by loss of mitochondrial membrane
potential, DAPI staining, western blotting and ROS generation.
The alcoholic extract of the gum resin of Boswellia serrata has been traditionally used for the
treatment of adjuvant arthritis.1 The gum resin comprises of four major triterpenic acids known
as boswellic acids i.e., β-boswellic acid (BA), 11-keto-β-boswellic acid (KBA), acetyl-β-
boswellic acid (ABA) and acetyl-11-keto-β-boswellic acid (AKBA).2 The boswellic acids are
known for their biological properties like inflammation,3,4 arthritis,5 ulcerative colitis,6 chronic
colitis,7 asthma8 and hepatitis.9 However it’s their anticancer activity which has garnered
considerable interest in recent past as they have been shown to inhibit the growth and induce
apoptosis in brain tumors,10 malignant glioma cells,11 colon cancer cells12 and leukemic cells.13
Thus, in continuation of our work regarding development of anticancer leads based on natural
products,14 and boswellic acids15 in particular, herein we report synthesis of a novel series of
semi-synthetic analogues of β-boswellic acid modified at C-2 position and their bio-evaluation
against a panel of human cancer cell lines. The synthesized analogues thereby had a α,β-
unsaturated ketone moiety, which is not only a significant precursor for synthetic manipulations
but also forms a major component of the natural products having anti-cancer activity.16 Notably,
1

the incorporation of a Michael-acceptor into a molecular structure has previously been revealed
to enhance the biological activity of natural products.17,18 The study lead to the identification of a
lead compound capable of inhibiting colony formation in HCT-116 cells as well as induces
apoptosis evidenced by loss of mitochondrial membrane potential, DAPI staining, western
blotting and ROS generation.
The synthesis of new semi-synthetic analogues of β-boswellic acid derivatives is outlined in
Scheme 1. The compound 2 was prepared by esterification of 1 with diazomethane, which on
oxidation at C-3 using pyridinium chlorochromate in dichloromethane lead to the synthesis of 3,
the target compound for carrying out aldol reaction with different aldehydes. To begin with we
chose reaction of 4-nitrobenzaldehyde with 3 as model reaction to establish the optimum
conditions. For optimization and standardization of reaction conditions, we tried different bases
such as NaOH, KOtBu, KOH, K2CO3, NaH, TEA, DIPEA and DMAP (Supporting information,
Table S1), and observed that only sodium hydride resulted in formation of aldol product, which
subsequently gets dehydrated to afford the desired product having an α,β-unsaturated ketone
system. The failure of other bases might be because of the steric hindrance caused by the cyclic
system, whereas dehydration is possibly facilitated by the extended conjugation with aromatic
system. Thus, use of NaH (1.5 equiv), 3 (1.0 equiv) and aldehyde (1.5 equiv), in toluene was
found to be condition of choice. It would be pertinent to mention here that we screened several
solvents to find toluene as most suitable. After optimizing the reaction conditions, we extended
the substrate scope to different aromatic aldehydes. The reaction proceeded well with a range of
electron deficient, electron rich as well as heterocyclic benzaldehydes to give a library of 20
analogues 4a-t in good yields (Scheme 1).
2

Scheme-1. Reagents and conditions: (i) CH2N2, diethyl ether (94%); (ii) PCC, DCM, RT (82%); (iii) NaH, Toluene,
RCHO, 0oC to RT (78%-90%).
3

All the synthesized analogues were evaluated for their anticancer activity against a panel of
human cancer cell lines viz., A549 (Lung cancer), PC-3 (Prostate cancer), HCT-116 (Colon
cancer) and HT-1080 (Fibro sarcoma) cells (Table 1). The results of preliminary screening
shown that aromatic aldehydes with halo-substitutions (mono- and disubstituted) were not
favourable groups to improve antitumor activity, as these compounds lead to lower cell growth
inhibition compared to parent compound 3 (Table 1). However, the derivative having 2, 4-
chloro-substitution (4b) displayed better growth inhibition values in various cancer cell lines.
Furthermore, results also disclosed that aromatic aldehydes having electron withdrawing groups
(-NO2, -CF3 and –OCF3) as well as electron donating groups (-CH3 and -OCH3) were not suitable
substitutions, which was clearly indicated by lower growth inhibition values of respective
compounds in various cancer cell lines (Table 1). Based on the growth inhibition data of these
compounds in various cell lines, it had been find out that products having pyridine ring (4b and
4f) were found to be more active as compare to the products having other heterocyclic rings
(quinoline, furan and thiophene) (Table 1). Therefore, preliminary screening led to the
identification of three lead compounds 4b, 4f and 4r, displaying promising growth inhibition.
These compounds were then further taken up for IC50 value determination.
19
Table 1. In vitro cytotoxicity of compounds (4a-t) against different cancer cells by SRB assay
Cell line
A549 (Lung
cancer)
PC-3 (Prostate
cancer)
HCT-116 (Colon
cancer)
HT-1080
(Fibrosarcoma cancer)
S.no.
1.
2.
3.
4.
5.
6.
7.
8.
Code
3
Conc.(µM)
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
% growth inhibition
56
47
97
47
48
55
99
47
5
14
14
0
28
7
44
44
79
40
44
41
100
52
7
58
0
21
23
100
43
16
17
95
36
0
78
15
13
26
6
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
49
27
18
21
100
30
10
0
8
6
20
0
0
32
37
21
100
57
40
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
35
6
10
11
10
25
20
44
11
100
24
19
0
21
0
33
37
42
100
74
42
43
100
95
99
37
4t
4

Among the three lead molecules, 4f was found to be most cytotoxic against various cancer cell
lines viz., A549, PC-3 HCT-116 and T47D having IC50 values of 2.13, 1.96, 1.84 and 1.26 µM
respectively (Table 2 and Figure 1). The other selected molecule 4b was found to be
comparatively less toxic in all the four cancer cell lines, whereas 4r displayed moderate activities
against various cancer cell lines having IC50 values of 26.1, 24.4, 7.2 and 15.6 µM respectively
against A549, PC-3, HCT-116 and T47D cell lines (Table 2). It would be pertinent to mention
here that IC50 values of compounds 4b, 4f and 4r were considerably high in normal human breast
epithelial cells (FR-2) (Table 2). Thus, based on IC50 values we chose 4f for further studies to
determine if the cell death is caused by apoptosis or necrosis.
Table 2. In vitro cytotoxicity of lead compounds against different human cancer cell lines19
Cell line
A549
PC-3
HCT-116
T47D
FR-2
(Lung cancer)
(Prostate cancer) (Colon cancer) (breast cancer)
(Normal epithelial)
S. No.
Code
4b
4f
4r
AKBA
IC50(µM)
1
2
3
4
56.1
2.13
26.1
25.8
69.5
1.96
24.4
32.7
>100
1.84
7.2
>100
1.26
15.6
43.4
>100
21.4
59
13.8
67.2
100
90
80
70
60
50
40
30
20
10
0
A549
PC-3
HCT-116
T47D
0
10 20 30 40 50 60 70 80 90 100
4f (uM)
Figure 1. Graphical representation of cell inhibition on different concentrations of 4f against various cancer cell
lines (A549, PC-3, HCT-116, T47D) determined by SRB assay. The inhibitory values were calculated by using
Graph PAD Prism software version 5.0.The results represent mean ± SD of three experiments.
To gain further insight into the anti-proliferative activity, we investigated the effect of compound
4f on HCT-116 cells by clonogenic assay. Colony formation assay is a cell survival assay based
5

on the ability of a single cell to grow into a colony. It primarily tests each cell in the population
for its ability to undergo extensive division and monitors all cells that have retained the capacity
for producing a large number of progeny after various treatments that can cause cell death as
well as evaluate the reproductive integrity of different cells. This assay is used to determine the
effect of cytotoxic compounds on colony forming ability of cancer cell lines. The HCT- 116 cells
were treated with compound 4f at different concentrations of 1, 2.5 and 5 µM. After treatment,
cells were seeded in appropriate dilution to form colonies in 1-3 weeks.20,21 The results of the
colony formation assay revealed that 4f significantly inhibited colony formation in HCT-116
cells (Figure 2) and the inhibitory effect was found to be increased with increasing doses of 4f.
Figure 2. Clonogenic assay was performed in six well plate, with clones produced by HCT-116 colon cancer cells
for 48h. (a) Untreated control with 600 clones, respectively, formed after seeding 500 cells. Treated HCT-116 cells
with compound 4f at different concentrations of 1µM, 2.5µM, 5µM (B, C, D). Paclitaxel was taken as a positive
control (E); (b) bar diagram representation of survival fraction of HCT-116 cells treated with 4f compound in a dose
dependent manner for each analysis versus control. The inhibitory effect of 4f compound on the relative
clonogenicity of the control and 4f treated HCT-116 colon cancer cell line.*P value<0.05 Vs control.
Apoptotic bodies’ formation is a distinguishing feature of cells undergoing apoptosis.22 Induction
of apoptotic bodies by chemotherapeutic agents has always been a preferred choice in
developing anti-cancer therapeutics. 4',6-Diamidino-2-phenylindole (DAPI) is a fluorescent
nuclear stain which binds to minor groove of A-T regions of DNA. Characteristic apoptotic
morphology like chromatin condensation, nuclear shrinkage and formation of apoptotic bodies
can easily be observed under fluorescence microscopy, after staining of nuclei with DNA
6

specific DAPI.23,24 The HCT-116 cells were treated with test compound 4f at 1, 2.5 and 5 µM for
48 h, stained with DAPI and investigated for morphological changes. It was observed that nuclei
of HCT-116 cells treated with 4f compound increased formation of apoptotic bodies and
chromatin condensation in concentration dependent manners compared to untreated control
(Figure 3). This data suggests that 4f inhibits growth of HCT-116 cells by arresting the
proliferation of cells and subsequent induction of apoptosis.
Figure 3. DAPI staining in HCT-116 cells. The cells were treated with test compound 4f at 1, 2.5 and 5µM for 48 h,
stained with DAPI and observed to determine morphological changes. Untreated HCT-116 cells showed normal
nuclear morphology while Paclitaxel showed typical apoptotic bodies. Cells treated with test compound 4f showed
concentration dependent effect. The arrow in each case showed the appearance of apoptotic bodies.
The initiation of apoptosis is investigated by the mitochondria compartmentalization. The
changes in mitochondrial membrane potential were determined by using RH-123 in a population
of apoptotic cells. RH-123 is a fluorescent dye and the electrochemical potential of the proton
gradient across the mitochondrial membrane was assessed by monitoring fluorescence quenching
of RH-123. Its rate of fluorescence decay is proportional to the mitochondrial membrane
potential. The loss of mitochondrial integrity results in leakage of the RH-123 from mitochondria
results in consequent decrease in fluorescence.25 HCT-116 cells were treated with compound 4f
at different concentrations of 1, 2.5 and 5 µM. RH-123 staining results revealed loss of
mitochondrial membrane potential in 4f treated HCT-116 cells. HCT-116 treated cells with 4f
displayed a remarkable increase in mitochondrial membrane potential loss with increase in
7

concentration as compared to the untreated cells which exhibited intact mitochondria (Figure 4).
Compound 4f induced concentration dependent depolarization of the mitochondrial membrane
potential (low ∆Ψmt).
Figure 4. (a) Mitochondrial membrane potential of 4f compound treated HCT-116 cells were measured by
Rhodamine-123 staining and visualized by confocal microscopy. Untreated cells showed intact mitochondria as
compared to treated cells which induced a remarkable increase in mitochondrial membrane potential loss with
increase in concentration. Treated HCT-116 cells with compound 4f at different concentrations of 1µM, 2.5µM,
5µM (B, C, D). Paclitaxel (50 nM) was taken as a positive control (E); (b) effect of 4f on Δψm measured with laser
scanning confocal microscope. Fluorescence intensity (FI) indicates membrane potential of mitochondria in the
cells. FI decreased in a concentration-dependent manner. Data are presented as mean ± SD, statistical analysis was
done with **P<0.01 and ***P<0.001.
Our preliminary data indicated the role of mitochondrial membrane potential loss in the cell
death induced by 4f in HCT-116 cells. PARP cleavage is considered as a sign of apoptosis and is
associated with mitochondrial cell death. PARP is a nuclear enzyme involved in DNA repair
whose cleavage is considered to be a hallmark of the apoptotic mode of cell death. The PARP is
cleaved into 89 kDa fragment upon the induction of apoptosis. The cleavage inactivates the
enzyme by destroying its ability to respond to DNA strand breaks.26 Therefore, we tried to
investigate if PARP cleavage has any role to play in the apoptosis induced by 4f, by western blot
analysis. HCT-116 cells were incubated with 4f at 1, 2.5 and 5µM concentrations for 48 h.
Results demonstrated that 4f treatment significantly induced PARP cleavage in HCT-116 cells;
and was comparable to positive control paclitaxel (Figure 5). All these data indicated that 4f
induced apoptosis in HCT-116 cells.
8

-
+
4f
4f
4f
(1 µM)
(2.5 µM) (5 µM)
Intact PARP(116KDa)
Cl-PARP(84KDa)
β-Actin
Figure 5. Western blot analysis revealed the expression levels of intact and cleaved PARP-1 in HCT-116 colon
cancer cells on treatment with varying concentrations (1, 2.5 and 5µM) of 4f compound for 48 h. Paclitaxel was
used as positive control. The housekeeping protein β–actin served as a loading control.
Reactive Oxygen Species (ROS) are the common mediator of apoptotic signalling. It is generated
as a by-product of cellular metabolism through leakage of electrons by mitochondrial electron
transport. The total cellular ROS level was measured using 2', 7'-dichlorofluorescien diacetate
(DCFDA) dye which is oxidized in the presence of free radicals to form a green fluorescent by
product dichlorofluorescein (DCF).27 Numerous anticancer agents have confirmed to put forth
their cytotoxic effects by the generation of reactive oxygen species (ROS).28 Therefore, we
examined the role of compound 4f in inducing the production of ROS that could potentially lead
to the cytotoxic effect in the HCT-116 cells. After treatment with 4f at 1 and 2.5 μM
concentrations, fluorescence intensity of DCF was increased in a dose dependent manner after
48h exposure to the test compound. The increase of fluorescent product dichlorofluorescein
(DCF) was observed in treated cells as compared to untreated cells (Figure 6). The dose
dependent increase in fluorescent product dichlorofluorescein (DCF) confirmed the ROS
generation.
9

Figure 6. (a) Detection of ROS in HCT-116 cells. The cells were treated with the indicated concentrations of 4f for
48h. H2O2 taken as a positive control. The fluorescence intensity of DCF was increased in a dose dependent manner
after 48h exposure to the test compound (*P<0.05 vs. control); (b) bar diagram representation of ROS generation
following 48h exposure of various concentrations (1 and 2.5µM) of 4f in HCT-116 cells assessed by
spectrofluorometirc analysis.
In conclusion, we synthesized a series of β-boswellic acid derivatives using aldol condensation
approach. The synthesized analogues were evaluated for their anticancer activities using growth
inhibition assay. The lead compound 4f displayed significant anti-proliferative activity against
various cancer cell lines viz., A549, PC-3 HCT-116 and T47D having IC50 values of 2.13, 1.96,
1.84 and 1.26 µM respectively. The lead analogue 4f substantially inhibited colony formation in
HCT-116 cells. The induction of apoptosis was further confirmed by loss of mitochondrial
membrane potential, DAPI staining, western blotting and ROS generation in HCT-116 cells.
These data suggest that compound 4f can be transformed into promising lead molecule and
warrant further detailed investigation and optimization.
Supporting Information:
Experimental procedures, biological materials and methods, compound characterization data,
NMR spectra (1H and 13C) and HRMS spectra.
Author Contribution
‡These authors contributed equally
10

Acknowledgment
We thank the Department of Science and Technology (DST), India (SB/S1/OC-07/2013) and
CSIR, India (BSC-0108) for financial assistance. IIIM Communication No. 1821.
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Graphical abstract
13