Total synthesis, biological evaluation of dendrodolides A–D and their analogues

Article abstract of DOI:10.1016/j.tet.2016.06.042

A concise total synthesis of dendrodolides A–D (1–4) has been accomplished in 10 steps from commercially available (R)-propylene oxide and 3-buten-1-ol as starting materials. The key steps involved in the synthesis are Jacobsen hydrolytic kinetic resolution, epoxide ring opening with 2-allyl-1, 3-dithiane, Yamaguchi esterification and ring-closing metathesis (RCM). In addition, a series of ester derivatives were prepared utilizing Yamaguchi esterification at the C-3 position of the dendrodolide core and screened for their efficacy against cancer cell lines.

Full text of DOI:10.1016/j.tet.2016.06.042

Accepted Manuscript
Total synthesis, biological evaluation of dendrodolides A–D and their analogues
B. Poornima, A. Venkanna, B. Swetha, Karthik reddy Kamireddy, Bandi Siva, V.S.
Phani, Ramesh Ummanni, K. Suresh Babu
PII:
S0040-4020(16)30555-5
10.1016/j.tet.2016.06.042
TET 27856
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 4 February 2016
Revised Date: 14 June 2016
Accepted Date: 15 June 2016
Please cite this article as: Poornima B, Venkanna A, Swetha B, Kamireddy Kr, Siva B, Phani VS,
Ummanni R, Babu KS, Total synthesis, biological evaluation of dendrodolides A–D and their analogues,
Tetrahedron (2016), doi: 10.1016/j.tet.2016.06.042.
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ACCEPTED MANUSCRIPT
Graphical abstract
Total Synthesis, Biological Evaluation of Dendrodolides A-D and
their Analogues.
a
a
b
b
a
c
B. Poornima, A. Venkanna, B. Swetha, Karthik reddy Kamireddy, Bandi Siva, V.S.Phani,
b
a
Ramesh Ummanni * K. Suresh Babu*

ACCEPTED MANUSCRIPT
Total Synthesis, Biological Evaluation of Dendrodolides A-D and
†
their Analogues
a
a
b
b
a
c
B. Poornima, A. Venkanna, B. Swetha, Karthik reddy Kamireddy, Bandi Siva, V.S.Phani,
b
a
Ramesh Ummanni * K. Suresh Babu*
a
Division of Natural Products Chemistry, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500 007, India.
bCentre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad
5
00 007, India.
C
Centre NMR and Structural Chemistry, CSIR-Indian Institute of
Technology, Hyderabad-500 007, India
E-mail: suresh@iict.res.in; Fax: +91-40-27160512
†
1
13
Electronic supplementary information (ESI) available: Copies of H NMR and C NMR
spectra of all the new compounds.
*
Corresponding author: E-mail: suresh@iict.res.in
Tel: +91-40-27191881, Fax: +91-40-27160512.
1

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Abstract:
A concise total synthesis of dendrodolides A–D (1-4) has been accomplished in10 steps from
commercially available (R)-propylene oxide and 3-buten-1-ol as starting materials. The key steps
involved in the synthesis are Jacobsen hydrolytic kinetic resolution, epoxide ring opening with 2-
allyl-1, 3-dithiane, Yamaguchi esterification and ring-closing metathesis (RCM). In addition, a
series of ester derivatives were prepared utilizing Yamaguchi esterification at the C-3 position of
the dendrodolide core and screened for their efficacy against cancer cell lines.
Keywords: Macrolides, Yamaguchi esterification, RCM approach, Jacobsen hydrolytic kinetic
resolution, Analogues, Cytotoxic activity, SAR studies.
Introduction
Marine microorganisms have been proven to be a rich source of innumerable secondary
1
metabolites with novel structures and wide-range of biologically properties, particularly, half of
2
them were from fungal genera, such as Aspergillus, Penicillum, Alternaria, and Cladosporium,
3
-7
are a significant source of new secondary metabolites from marine-derived fungi.
Dendrodolides are a group of naturally occurring macrolides isolated from Dendrodochium sp., a
8
fungus associated with the sea cucumber Holothuria nobilis Selenka .The gross structures were
established by extensive, IR, mass spectroscopy and NMR (1D and 2D) studies. Structurally,
dendrodolides contains three chiral centers (C-3, C-9 and C-11) and unusual 11-membered
lactone ring that represent a prominent member of lactone ring family of natural products (Figure
1
).
2

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Figure 1.Structures of dendrodolides A-M (1-13).
It was further shown that dendrodolide A –D (1-4) exhibited in vitro cytotoxicity against the
9
tumor cell line SMMC-7721with IC50 value of 19.2 µg/mL . Taken together, these attributes
led to the considerable interest in the dendrodolides as target for the total synthesis. As part of
1
0
our continuing efforts for the synthesis of bio-active macrocyclic natural products, recently, we
1
1
have reported the first total synthesis of dendrodolide A. Thus, synthetically challenging
structures of these macrolides combined with their biological profiles have encouraged us to
continue our work for the exploration of new synthetic routes for these dendrodolide series.
Herein, we report efficient total synthesis of dendrodolides A–D (1-4) by utilizing Jacobsen
hydrolytic kinetic resolution, Yamaguchi esterification and ring-closing metathesis (RCM) as
key steps. In addition, we also prepared a series of ester derivatives by introducing the various
acids and assessed them for their cytotoxic activity. To the best of our knowledge this is the first
report on the synthesis of dendrodolide analogues.
3

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Results and discussion
As outlined in scheme-1, we envisioned retro synthetically that all the dendrodolides can be
obtained via RCM reaction (Scheme 1). Thus, disconnection of the C-C double bond revealed
the bis terminal olefins 32, 39, 32a, 39a as potential key intermediates. A subsequent
disconnection of olefinic intermediates through Yamaguchi esterification converts it into
common acid fragment 16 and alcohol fragments 15, 36, 34 and 42. These alcohol fragments
envisaged by regioselective ring opening of epoxide (17, 33) with 2-allyl-1, 3-dithiane (18), the
epoxide (17) could, in turn be accessed from ring opening of (R)-propylene oxide (20) with vinyl
magnesium bromide through epoxidation followed by Jacobsen hydrolytic kinetic resolution.
The epoxide (33) could, in turn be accessed from 17a, which is subunit of Jacobsen resolution
reaction. On the other hand, common acid fragment (16) could be obtained by stepwise
transformations from commercially available 3-buten-1-ol (23) through epoxidation and
Jacobsen resolution reactions. Thus, our present total synthesis will be highlighted by utilization
of Jacobsen hydrolytic kinetic resolution which directs the construction of the stereocentres at
C-3, C-9, C-11 and Grubb’s ring closing metathesis for the formation of 12-membered
lactone ring. Furthermore, the simplicity of the precursors of this route makes it attractive for
the library synthesis of the dendrodolides for the biological screening.
4

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Scheme 1: Retrosynthetic analysis of dendrodolides A-D (1-4).
Synthesis of common acid fragment (16):
Our synthesis commenced with the preparation of acid fragment 16 as a common key
intermediate, as iilustrated in scheme-2. The readily available starting material 3-buten-1-ol (23)
initially protected as PMB ether using PMBBr, NaH and a catalytic amount of TBAI in DMF to
1
2
afford 24 in 96% yield. The compound 24 was subjected to m-CPBA epoxidation to give
1
3
racemic epoxide 25 , which was further subjected to Jacobsen’s hydrolytic kinetic resolution
1
4
using the (S, S)-catalyst to give optically pure epoxide (S)-22 in 44% yield (determined by
5

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1
5
chiral HPLC). The regioselective ring opening of epoxide 22 with trimethylsulfonium
+
-
methylide (Me S I ) and n-BuLi in THF afforded the allylic alcohol 21 in 92% yield. Subsequent
3
1
6
protection of alcohol functionality in 21 with TBSCl and imidazole in DCM to afford 26 in
1
5
9
6% yield. Finally, deprotection of PMB group with DDQ followed by oxidation with
1
7,18
TEMPO/BAIB in CH Cl :H O (1:1) afforded acid 16
in 92% yield.
2
2
2
Scheme 2. Reagents and conditions: (a) PMBBr, NaH,TBAI cat., DMF, 0 °C to rt, 12 h, 96%;
III
(
b) m-CPBA, CH Cl , 0 °C to rt, 12 h, 89%; (c) (S,S)-(salen)-Co ·OAc (0.5 mol%), H O (0.55
2
2
2
+
−
equiv.), 0 °C to rt, 18 h, 44% for 22; (d) Me S I , n-BuLi, THF, −20 °C, 4 h, 92%; (e) TBSCl,
3
o
H
imidazole, DCM, 0 C to rt, 96%; (f) DDQ, CH
2 2
Cl : P 7 buffer (9 : 1), 0 °C to rt, 1 h, 92%; (g)
TEMPO, BAIB, CH Cl : H O (1:1), 0 °C to rt, 2 h, 92%.
2
2
2
Synthesis of alcohol fragment:
We next targeted for synthesis of alcohol fragment 15 as illustrated in scheme-3. The synthesis
was intiated with (R)-propylene oxide 20 as a chiral synthon, which was readily prepared from
1
9
racemic-propylene oxide through Jacobsen hydrolytic kinetic resolution (HKR) using (R, R)-
salen)-CoIII OAc. Thus, treatment of (R)-propylene oxide (20) with vinyl magnesium bromide
(
2
0
in the presence of CuI afforded homoallylic alcoholin 89% yield , in which the secondary
1
9
hydroxyl group was protected as its TBS ether using TBSCl, imidazole, in DCM to give 19 in
8% yield. Epoxidation of 19 with m-CPBA afforded a mixture of two diastereomers 28
9
(anti:syn/3:1), which was then subjected to Jacobsen hydrolytic kinetic resolution (HKR)
6

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method with (S, S)-(salen)-CoIII . OAc complex (0.5 mol %) and water (0.55 equiv) in THF
1
9
(0.55 equiv) to afford the diastereomerically pure epoxide 17
in 45% yield (>94% ee)
(determined by chiral HPLC) and diol 17a in 47% yield. Ring opening of epoxide 17 with 2-
2
1
22
allyl-1,3-dithiane (18)
yielded compound 29 in 87% yield. Subsequent methylation of
2
2
alcohol 29 using MeI/NaH afforded 30 in 98% yield. Finally, removal of TBS group with
2
3
TBAF furnished the required alcohol 15 in 96% yield.
Scheme 3. Reagents and conditions: (a) (i) vinyl magnesium bromide, CuI, THF, -78°C to -20
°
°
C, 12 h, 89%; (ii) TBDMSCl, imidazole, CH Cl , 0 °C to rt, 4 h, 98%; (b) m-CPBA, CH Cl , 0
2 2 2 2
III.
C to rt, 12 h, 92%, (c) (S,S)-(salen)-Co OAc (0.5 mol %), H
2
O (0.55 equiv.), THF, 0°C to rt,
1
8 h, 45% for 17, 47% for 17a; (d) 18, n-BuLi, dry THF,–78 °C to –20 °C, 2 h, 87%; (e) NaH,
o
MeI, THF, 0 °C to rt, 5 h, 98%; (f) TBAF, THF, 0 C to rt, 4 h, 96%.
Synthesis of dendrodolide A (1):
With the two fragments 15 and 16 in our hand, we next targeted for the connection of these
2
4
fragments (Scheme 4) and subjected the Yamaguchi esterification
protocol (2, 4, 6-
trichlorobenzoyl chloride, Et N, THF, DMAP, Toluene). The highly efficient reaction provided
3
the key intermediate 14 in good yields. We then planned for ring-closing metathesis (RCM)
reaction of 31 with the Grubbs second generation catalyst (G-II, 25 mol %) to access the
2
5
macrolide core. However, the reaction did not progressed and the starting material was
completely recovered. At this stage, we investigated the alternative strategy, viz., deprotection of
7

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TBS group and 1, 3 dithiane then followed by the RCM reaction to achieve the target compound
2
6
1
1
. Thus, deprotection of TBS group using TBAF gave 14 with 96% yield. Finally, removal of
2
7
, 3-dithiane group in 14 with CaCO , I , in THF:H O (4:1) gave 32 with 92% yield.
3
2
2
Subsequent RCM reaction using G-II (25 mol %) in dry DCM (deoxygenated) at reflux
2
5
temperature yielded dendrodolide A (1) in 78% yield and with good E-selectivity (>95%). All
1
the intermediate compounds including the dendrodolide A (1) were fully characterized by H
1
3
NMR, C NMR, and Mass spectral data. The spectral and analytical data of synthetic
8
dendrodolide A (1) were in agreement with the reported natural dendrodolide data.
Scheme 4. Reagents and conditions: (a) 16, 2,4,6-trichlorobenzoylchloride, Et N, THF, 0 °C to
3
rt, 2 h, then 15, DMAP, toluene, 0 °C, 2 h, 98%; (b) G-II (25 mol%), CH Cl , reflux, 20 h, (c)
2
2
o
o
TBAF, THF, 0 C to rt, 4 h, 96%; (d) CaCO
3
, I
2
, THF:H
2
O (4:1), 0 C, 20 min, 92%. (e) G-II (25
mol%), CH Cl , reflux, 12 h, 78%.
2
2
Synthesis of dendrodolide B (2):
The structure of dendrodolide B (2) is very similar to the dendrodolide A (1), except for
the variation of stereo center at C-9. Thus, our strategy for the synthesis dendrodolide-B (2) was
initiated from diol 17a, which is the subunit of Jacobsen hydrolytic kinetic resolution (HKR)
8

ACCEPTED MANUSCRIPT
2
0
reaction of 28 as depicted in scheme 3. The treatment of diol 17a with tosylimidazole/NaH in
2
8
THF at 0 °C provided required epoxide (33)
in 89% yield. The epoxide 33 was further
subjected to sequential reactions (opening of epoxide 33 with 2-allyl-1, 3-dithiane (18),
2
1-24
methylation and TBS ndeprotection)
as shown in scheme-3, via 29a, 30a. Finally, the
required alcohol 34 was obtained by deprotection of TBS group in 30a using TBAF in THF
afforded 34 in 89% yield. Having both alcohol 34 and acid 16 in hand, construction of the
macrocyclic framework was achieved similar to as shown in scheme 6, via 31a, 14a, 32a (which
is similar showed in scheme-4). The spectral and analytical data of synthetic dendrodolide B (2)
8
were in agreement with the reported natural dendrodolide data.
Scheme 5. Reagents and conditions: (a) tosylimidazole, NaH, 0 °C to rt, 1 h, 89%.
Scheme 6. Reagents and conditions: Reagents and conditions: (a) 16, 2,4,6-
trichlorobenzoylchloride, Et N, THF, 0 °C to rt, 2 h, then 34, DMAP, toluene, 0 °C, 2 h, 95%;
3
o
(b) G-II (25 mol%), CH
2
Cl
2
, reflux, 20 h, (c) TBAF, THF, 0 C to rt, 4 h, 88%; (d) CaCO
3
, I
2
,
o
THF:H O (4:1), 0 C, 20 min, 89%. (e) G-II (25 mol%), CH Cl , reflux, 12 h, 74%.
2
2
2
Synthesis of dendrodolide C (3):
9

ACCEPTED MANUSCRIPT
The dendrodolide C (3) is also similar to the dendrodolide A (1), with the
variation at C-9. Dendrodolide A (1) having methoxy group at C-9, whereas dendrodolide C (3)
has –OH group at C-9. Thus, our strategy for the synthesis of dendrodolide C (3) was started
from 29, in which hydroxyl group was protected with MOMCl, in dry DCM to give its
2
9
26
corresponding MOM ether 35 in 86% yield. Finally deprotection of TBS group in 35 with
TBAF gave alchol 36 in 94% yield (Scheme-7). Having both the alcohol 36 and the acid 16 in
hand, construction of the macrocyclic framework was achieved as shown in Scheme 8. The
2
4
esterification reaction of 36 with acid 16 was carried out under Yamaguchi conditions (2, 4, 6-
trichlorobenzoyl chloride, Et3N, THF, DMAP, toluene) to furnish the RCM precursor 37 in 96%
yield. The TBS group in compound 37 was deproteced using TBAF in THF to obtain 38 in 97%
2
7
yield, further removal of 1,3 dithiane group by using CaCO3, I2, THF:H2O (4:1) obtained 39
in 92% yield. The bis olefin 39 was then subjected to the RCM reaction using G-II (25 mol%). in
dry DCM (deoxygenated), at reflux temperature yielded macrolide 40 in 68% yield with good E-
3
0
selectivity (>95%). Finally, removal of MOM in 40 furnished the dendrodolide C (3) (Scheme-
).
8
Scheme 7. Reagents and conditions: (a) MOMCl, DIPEA, dry DCM, 0 °C- rt, 12 h, 86%; (b)
o
TBAF, THF, 0 C to rt, 4 h, 94%.
10

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3
Scheme 8. Reagents and conditions: (a):16, 2,4,6-trichlorobenzoylchloride, Et N, THF, 0 °C to
o
rt, 2 h, then 36, DMAP, toluene, 0 °C, 2 h, 96%; (b) TBAF, THF, 0 C to rt, 4 h, 97%; (c) CaCO ,
3
o
I , THF:H O (4:1), 0 C, 20 min, 92%; (d) G-II (25 mol%), CH Cl , reflux, 20 h, 68%; (e) SMe ,
2
2
2
2
2
.
BF
3
Et
2
O, 0 °C, 1 h, 79%.
Synthesis of dendrodolide D (4):
The synthesis of dendrodolide D (4) was achived from 29a, using similar
reactions as mentioned in synthesis of dendrodolide C (3), the synthetic route is shown in
schemes 9 and 10. All the intermediate compounds including the dendrodolide D (4) were fully
1
13
characterized by H NMR, C NMR, and Mass spectral data. The spectral and analytical data of
8
synthetic dendrodolide D (4) were in agreement with the reported natural dendrodolide data.
Scheme 9. Reagents and conditions: (a) MOMCl, DIPEA, dry DCM, 0 °C- rt, 12 h, 86%; (b)
o
TBAF, THF, 0 C to rt, 4 h, 94%.
MOM
MOM
MOM
OH
O
O
MOM
O
O
O
O
O
O
O
O
O
O
S
S
S
S
42
+
16
O
OH
O
OTBS
O
OH
O
OH
O
OH
4
3
44
45
46
Dendrodolide D (4)
Scheme 10. Reagents and conditions: . Reagents and conditions: (a):16, 2,4,6-
trichlorobenzoylchloride, Et N, THF, 0 °C to rt, 2 h, then 42, DMAP, toluene, 0 °C, 2 h, 90%;
3
o
o
(
b) TBAF, THF, 0 C to rt, 4 h, 87%; (c) CaCO
3
, I
2
2
, THF:H O (4:1), 0 C, 20 min, 82%; (d) G-II
.
(
25 mol%), CH Cl , reflux, 20 h, 68%; (e) SMe , BF Et O, 0 °C, 1 h, 72%.
2
2
2
3
2
Synthesis of Debdrodolide A-D analogues
Further, in order to improve the activity of Dendrodolides A-D, we planned for the synthesis of
derivatives and for an investigation of their structure activity relationships (SARs). It is well
11

ACCEPTED MANUSCRIPT
known that introduction of aromatic /aliphatic functionality into organic molecules can often
1
3
dramatically change their physical and chemical properties. In addition; extension and
branching of the hydrocarbon chain may affect their bioactivity. Thus, we synthesized a series of
2
4
ester derivatives utilizing Yamaguchi esterification (2, 4, 6-trichlorobenzoyl chloride, Et N,
3
THF, DMAP, Toluene) at the C-3 position of the dendrodolide core (Scheme-10).
3
Scheme 11. Reagents and conditions: (a) 2,4,6-trichlorobenzoylchloride, Et N, THF, 0 °C-rt 2 h,
then acid, DMAP, toluene, 0 °C-rt, 2-4 h, 98%.
12

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COOH
COOH
1
a =
COOH
COOH
4
0a =
O
NO2
COOH
46c =
46d =
NO2
COOH
OMe O
OMe O
O
Br
1b =
40b =
40c =
O
S
R1
R2
NO2
COOH
O
O
O
O
NO2
O
COOH
Br
2
1
1c =
COOH
R = a, b, c, d
R2 =a
1
CHOOH
NO2
46e =
MOM
MOM
O
O
O
O
O
O
Cl
1
d =
4
6a =
COOH
S
NO2
COOH
COOH
COOH
R3
R4
O
O
O
O
2a
=
46b =
46f =
40
46
a, b, c
R = a, b, c, d, e, f
4
R =
3
Biological activity:
Cytotoxicity evaluation against different cancer cell lines
The ability of Dendrodolide A-D (1-40 and their ester derivatives in inhibiting cancer cell growth
were examined against cancer cell lines [prostate cancer cell line (DU-145), Liver carcinoma cell
line (HePG2), cervical cancer (HeLa) and Breast cancer cell line (MCF7)] by SRB assay using
3
1
DMSO and Doxorubicin as negative and positive controls, respectively. Compounds showing
showing more than 30% growth inhibition at standard concentration of 10µM were considered as
active and those comppounds were further tested to ascertain their IC values against the cell
5
0
lines DU145, HeLa, HepG2, MCF7. Table-1 summarizes their IC50 values against panel of
human cancer cell lines. Of all compounds analysed, 46b and 46c displayed more cytotoxic
effect against DU145 with an IC50 value of 7.7±0.1µM and 7.1±0.4 µM, respectively. The effect
of these compounds on DU145 was further tested to study the mode of action. These selected
compounds were found to be more cytotoxic against DU145 cell line, the change in morphology
13

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induced by these compounds was also observed. Upon exposure of the cells to the compounds,
the inherent morphology of cells was changed and the cells eventually detached from the surface
(Fig 2). To study the long term effect of the selected compounds on the anchorage-independent
proliferative capacity of the DU145, colony formation assay (soft agar assay) mimicking the in
3
2
vivo environment was performed . From the results of the colony formation assay it was
indicated that the selected compounds have an inhibitory effect on proliferating cancer cells. 46b
inhibited proliferation of cells and initiated decrease in the size of individual colonies at 15µM
concentration while 46c showed similar effect at 15µM concentration and at higher
concentrations there was significant decrease in the size of the colony (Fig 3). A dose dependent
inhibition of colony formation ability suggests that these selected compounds affect anchorage
independent growth of DU145 cells. As the compounds caused inhibition of proliferation of
cells, it was further tested whether the compounds cause any cell death. In order to do so, cell
cycle analysis was performed using a FACS flow cytometry. The cells were seeded and treated
as above and incubated for 48hrs, upon completion of incubation, FACS for the cells was
conducted The resultant histograms from the FACS analysis for the respective compounds show
that with increasing concentration of compound, cell population accumulates in the subG1 phase
while there is negligible cell population in the G2/M phase. As is shown in the table 3 46b and
4
6c show a similar effect at higher concentrations of 20µM respectively (Fig 4). The results
showing increased growth arrest in sub G1 indicates cell death upon exposure to compound in a
3
3
concentration dependent manner (Table 2). It has been reported in literature that caspase-3
plays a major role in causing apoptosis and eventual cell death thus taking into consideration the
3
4
result obtained in the FACS analysis, the activation of caspase-3 was analysed . Caspase-3 is an
3
4
effective caspase whose activity is influenced by caspase-9 , thus it was of interest to analyse
14

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the activity of caspase-9. Therefore to determine whether the observed cell death is due to
activation of caspases, we have measured activation of key caspases by these selected
compounds. This was done using fluorogenic substrate Ac-DEVD-AMC and Ac-DEVD-AFC
for caspase -3 and caspase-9, respectively. The results have confirmed the activation of both
caspase-3 and caspase-9 in DU145 cells treated with the selected compounds for 48 h
continuously. The activation of procaspase-3 is a distinguishing feature of apoptotic cells. A
dose-dependent increase in the activity of caspase was observed with different concentrations of
the selected compounds as compared to those DU145 cells which were treated with DMSO (Fig.
5
a and 5b). Activation of caspase-9 is mitochondrial dependant which in turn activates caspase-
executing apoptotic cell death in cancer (Fig. 5c and 5d). Doxorubicin was used as positive
3
control for measurement of caspase activation.
Conclusions
In conclusion, we have accomplished the total synthesis of four dendrodolide A-D (1-4) in 10
steps, featuring the Jacobsen catalyst hydrolytic kinetic resolution (HKR), Yamaguchi
esterification and ring-closing metathesis reactions (RCM) as key reactions and synthesized their
novel analogues. Evaluation for their anticancer activity toward cultured HepG2, DU145, HELA,
and MCF7 were carried out. From the series of compounds synthesized in the present study,
compounds 46b and 46c affecting prostate cancer cell proliferation by direct targeting cell cycle
progression. This from all above experiments it is evident that these two compounds showed
more promising anti cancer potential than the other substitutions. We hypothesize that the
compounds 46b and 46c may target many pro-survival pathways in cancer cells shows aptness to
consider this scaffold as basis to develop novel anticancer agent.
Cell Based Assays
15

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Cytotoxicity evaluation against different cancer cell lines
Cellular viability in presence of the compound was tested by performing a Sulforhodamine B
3
1
Assay (SRB) . Briefly, the cell line of interest was seeded in flat bottom 96- well plate (5000
cells/well) in a medium containing 10% FBS followed by incubating the plate for 20 h in an
incubator continuously supplied with 5 % CO2, allowing cells to adhere to the surface of the
wells. After 20 hrs the cells were treated with the compound at desired concentration. Working
dilutions of 500 µM concentration of the compounds were prepared, of which 2µL aliquot was
added to the each well, thereby making the final concentration of compound 0 to 100 µM.DMSO
and Doxorubicin (as standard control anti cancer drug) were taken as vehicle and positive
controls, respectively. Further, the plates were incubated for another 48 h in the incubator. After
4
8 h of treatment cells were fixed using 10% TCA for 1 h at 4ºC. The plates were rinsed
carefully with MQ water removing TCA and air dried. Then 0.057% SRB solution was added to
plates and incubated for 30 mins before washing to remove unbound SRB using 1% acetic acid
and the plates were then air dried. 100µL of 10mM Tris Base was added to each well to
solubilise the SRB. The absorbance was measured using Tecan Multimode Reader at 510nm.
The measure of absorbance is directly proportional to cell growth and is thus used to calculate
the IC50 values. In the present study, different types of cancer cell lines i.e., breast cancer (MCF-
7
), prostate cancer (DU145), Hepatocellular carcinoma (HepG2) and cervical cancer (HeLa) cell
lines were tested for the cytotoxic effect of the compounds. Based on the IC50 values obtained,
the selected compounds were further assayed to ascertain its effect on prostate cancer cell Line
(DU145).
Change in Morphology
16

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As the compounds 46b and 46c were found to be cytotoxic to DU145 cells showing promising
IC50 values, its effect on the morphology of cells was further ascertained. Cells were seeded in
2
4 well plate and incubated for 20 h. Then the cells were treated with increasing concentrations
of selected two compounds. After 48 h of incubation, the experiment was terminated and the
cells were observed under the microscope and images were captured using Olympus Xi71
microscope.
Colony formation assay
The long term effect of selected compounds on the anchorage-independent growth of DU145
3
2
cells was analyzed by soft agar assays as reported previously with minor modifications . In the
soft agar assay, base agar was prepared by mixing 1% of agarose (Bacto Agar: Becton,
Dickinson, Sparks, MD) with 2 × DMEM with 20% FBS and 2X antibiotics in 6-well plates in
order to achieve final concentration of 0.5% of agar in 1X growth medium with 10% serum
concentration. After the solidification of the base agar, 2.5 × 104cells were mixed with
cultivation medium containing compound at varying concentrations and agar solution to obtain a
final concentration of 0.35% agar. This was spread on top of the base agar previously solidified.
The plate was incubated for 10 days with periodic replacement of medium and compounds for
every 3 days. Over period of time plates were monitored regularly for appearance of colonies.
After 10 days of incubation the plates were stained with 0.005% crystal violet solution until
colonies turned purple in colour. The excess stain was removed and washed off using MQ water
and the colonies were photographed using a Olympus Xi71 microscope
Determination of caspase activity
Caspase-3 and -9 activities were measured by treating the DU145 cells with selected compounds
using specific fluorogenic substrates Ac-DEVD-AMC (substrate for caspase-3) and Ac-LEHD-
AFC (substrate for caspase-9). DMSO and Doxorubicin were taken as negative and positive
17

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controls, respectively. After 48 h of treatment with selected compounds, harvested cells were
lysed directly in caspase lysis buffer (50 mM HEPES, 5mM CHAPS, 5 mM DTT, pH 7.5). The
lysates were incubated with the respective substrate (Ac-DEVD-AFC/Ac-LEHD-AMC) in
2
0mM HEPES (pH 7.5), 0.1% CHAPS, 2mM EDTA and 5 mM DTT at 37 °C for 2 h. The
release of AFC and AMC was analyzed by a fluorimeter using an excitation/emission
wavelength of 400/505 nm (for AFC) and 380/460 nm (for AMC) which is directly proportional
to caspase-9 and -3 activity, respectively. The observed fluorescence values were normalized
with total protein concentration estimated by Bradford method
Cell Cycle analysis
5
DU145 Cells (1X10 cells per well) were seeded in six-well dishes and incubated for 20 hrs and
treated without (Control) or with selected compounds at desired concentrations for 48 hr. The
0
cells of each well were then harvested and fixed with 1 mL 70% ice-cold ethanol at -20 C
overnight. After fixation, the cells were washed in PBS and stained with 500 µL propidium
iodide (PI) solution (50µg/mL in PBS) containing 100 µg/mL RNase. The cells were incubated
at room temperature for 1 hr in the dark. The resultant PI uptake was analyzed by fluorescence
activated cell sorting (FACS Calibur System; BD Bio- science, Erembodegem, Belgium) in a
FL-2 fluorescence detector (10000 events were recorded for each condition). Flow cytometry
data was analyzed using FCS express 4 software (De Novo Software, Los Angeles, CA).
18

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Table 1: Table shows the IC50 values of the selected compounds against the cancer cell lines.
DU145
STD
MCF7
STD
Hela
HepG2
STD
Compound
STD
DEV
4.6
IC50
IC50
IC50
IC50
DEV
0.1
1.0
1.1
1.9
0.1
0.4
3.9
0.8
0.9
0.2
DEV
0.1
3.3
2.5
2.0
0.6
0.6
2.1
0.4
0.2
0.4
DEV
1.3
5.1
1.0
1.6
0.4
0.3
1.1
0.6
1.1
0.1
1
b
33.7
34.2
11.5
29.4
7.7
28.0
43.3
15.4
23.1
9.2
27.3
94.5
17.4
46.2
13.5
18.0
63.8
44.8
37.8
5.7
32.8
78.9
41.0
49.8
5.8
27.5
35.6
58.9
38.3
5.7
2
a
3.5
1.8
1.9
1.4
0.1
2.0
4.0
0.9
4
0a
0c
6b
6c
6d
6e
6f
DOXO
4
4
4
4
7.1
4.5
22.6
22.5
16.6
5.6
29.7
40.9
36.7
4.0
4
4
0.1
Table 2: Selected compounds induced subG1 phase cell cycle arrest in DU145 cells. Cells were
treated with varying concentrations of selected compounds (0-20 µM) for 48 h and cell cycle
progression was examined by flow cytometry. Table shows the percentage cell fractions in
subG1, G0/G1, S and G2/M phases of treated DU145 cells.
SubG1
G0/G1
S
G2/M
DMSO
µM
2.69
19.10
35.74
32.62
1.26
2.76
6.16
4.14
5
10 µM
25.85
30.99
1.66
3.29
1
2
5 µM
0 µM
24.30
32.29
2.69
23.62
26.16
34.69
39.34
26.07
32.45
35.74
33.08
22.40
23.59
15.82
2.51
2.11
1.26
2.26
1.62
1.60
1.32
2.51
3.05
6.16
3.66
3.23
2.82
2.02
DMSO
µM
5
10 µM
15 µM
20 µM
19

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Figure 2: DU145 cell were treated with selected compound at indicated concentration and
DMSO. Upon exposure of these compounds to DU145 cells the extent of change in cell
morphology is observed with increasing concentration.
Figure 3: Long term effect of selected compound on the number of colony-forming DU145
cells. DU145 cells were treated with desired concentration of selected compounds(0− 20 ꢀM)
and allowed to grow for 10 days to form colonies. Representative images of the colony-forming
assay are shown here. Number of colonies and their size formed by DU145 in soft agar is
decreased on exposure to these selected compounds.
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Figure 4: Cell cycle analysis of DU145 cells treated with selected compounds. Cells were
treated with compounds and DMSO and the DNA content was measured by propidium iodide
staining to determine the distribution of cells in various phases of cell cycle. The percentage of
cells increased in subG1 phase up on exposure to these selected compounds suggesting its role in
G0/G1 arrest.
Figure 5: Effect of selected compounds on activation of caspases inducing apoptosis in DU145
cells. Treatment of DU145 cells with different concentration of selected compounds for 48 h
induced activation of caspases-3 (a & b) and caspase-9 (c & d) significantly in concentration
dependent manner. Doxorubicin was used as control for activation of both caspases.
21

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Aknowledgement:
The authors gratefully acknowledge keen interest shown by Dr. S. Chandrasekhar, Director,
IICT, Hyderabad. A.V.K thanks CSIR and B.P thanks UGU, New Delhi for financial support.
This work was supported by the Grant CSIR XII FYP Network project grant "NaPAHA" (CSC-
1
30).
Supplementary data:
Supplementary data associated with this article
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