Design, synthesis and biological evaluations of chirally pure 1,2,3,4-tertrahydroisoquinoline analogs as anti-cancer agents

Article abstract of DOI:10.1016/j.ejmech.2015.01.030

A series of fifteen chiral 1,2,3,4-tetrahydroisoquinoline (THIQ) derivatives have been synthesized and their antiproliferative properties have been studied. The in vitro screening was performed against five cancer cell lines; MCF-7 (breast cancer), A549 (

Full text of DOI:10.1016/j.ejmech.2015.01.030

European Journal of Medicinal Chemistry 92 (2015) 608e618
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluations of chirally pure
1,2,3,4-tertrahydroisoquinoline analogs as anti-cancer agents
,
Triparagiri Ramanivas a b, Bottu Sushma c, V. Lakshma Nayak a, Kunta Chandra Shekar a,
, b, *
Ajay Kumar Srivastava a
a Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Tarnaka, Hyderabad 500 007, India
b Academy of Scientific and Innovative Research, New Delhi 110025, India
c National Institute of Pharmaceutical Education and Research, Balanagar, Hyderabad 500037, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of fifteen chiral 1,2,3,4-tetrahydroisoquinoline (THIQ) derivatives have been synthesized and
their antiproliferative properties have been studied. The in vitro screening was performed against five
cancer cell lines; MCF-7 (breast cancer), A549 (lung cancer), DU-145 (prostate cancer), Hela (cervical
cancer) and HepG2 (liver cancer). Most of the compounds showed promising activity with IC50 Values
Received 7 November 2014
Received in revised form
13 January 2015
Accepted 14 January 2015
Available online 15 January 2015
ranging from 0.72 to 92.6
m
M. Among them, compounds 9a and 9b have shown significant activity
M respectively. To
against human prostate cancer cell line, i.e., DU-145 with IC50 value 0.72 and 1.23
m
investigate the mechanism of action, detailed biological studies of compounds 9a and 9b were carried
out on the human prostate cancer cell line, DU-145. Flow cytometric analysis revealed that these com-
pounds induced cell cycle arrest at G2/M phase. Tubulin polymerization assay and immunofluorescence
analysis results suggested that these compounds effectively inhibit microtubule assembly formation in
DU-145. The apoptosis inducing properties were evaluated by DNA fragmentation analysis, Caspase-3
activity assay, Annexin V-FITC assay and Western blot analysis of proapoptotic protein, Bax and anti-
apoptotic protein Bcl-2.
Keywords:
1,2,3,4-Tetrahydroisoquinolines (THIQ)
Tubulin polymerization
Cell cycle analysis
Caspase-3
Annexin V-FITC assay and apoptosis
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
(lymphoblastic leukemia) while the compound II was found to be
active against HepG2 cells [11] [Fig.1]. Hatano et al. investigated the
1,2,3,4-tetrahydroisoquinoline (THIQ) is one of the most abun-
dant heterocyclic scaffolds, found in various pharmaceutically
important antitumor antibiotics [1,2]. Natural and synthetic tetra-
hydroisoquinoline containing alkaloids exhibit a variety of phar-
macological properties including anticancer [3e5], antidepressant
[6], antiepileptic [7], cardioprotective [8], antiviral [9], anti-
glaucoma [10] etc.
Its unique structure provides a biocompatible skeleton to
develop new agents for the drug discovery and therefore efforts
have been made to develop efficient synthetic routes for highly
functionalized THIQs that could be explored for drug development.
Pingaew et al. have synthesized 1-substituted-N-tosyl-1,2,3,4-
tetrahydroisoquinoline analogs using the modified PicteteSpen-
gler reaction and evaluated for cytotoxicity and found that com-
pound I showed potent cytotoxicity against MOLT-3 cell lines
tumor-specific cytotoxicity and the type of cell death induced by
tetrahydroisoquinoline derivatives in human oral squamous cell
carcinoma cell lines and found that compound III and IV showed
the highest tumor-specific cytotoxicity [Fig. 1] [12]. Recently we
have reported a synthetic strategy for densely functionalized chiral
THIQs from Garner's aldehyde which involves
a
3,4-
dihydroisoquinoline derived bridged oxazolidine intermediate
[13]. In order to explore the antitumor properties of the chiral THIQ
analogs, we hypothesized to synthesize the prototypes VeVII that
can easily be accessed from 3,4-dihydroisoquinoline intermediate 4
[Fig. 1].
2. Results and discussion
2.1. Chemistry
The 3,4-dihyroisoquinoline (DHIQ) 4 was synthesized from
serine derived Garner's aldehyde 1 by reported procedure [13].
L
-
* Corresponding author.
E-mail address: aksrivastava@iict.res.in (A.K. Srivastava).
http://dx.doi.org/10.1016/j.ejmech.2015.01.030
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

T. Ramanivas et al. / European Journal of Medicinal Chemistry 92 (2015) 608e618
609
MeO
MeO
OMe
O
S
N
MeO
MeO
OMe
N
O
O
R1
R2
O
N
O
O
R4
O
R3
III
IV
R1 = OH, R2=R3=R4=H; I
R1 = H, R2=R3=R4=OMe; II
O
O
O
S
O
R
OH
N
N
OH
O
N
N
R
O
O
O
THIQ containing chalconamides; V
THIQ fused Piperazinones; VII
THIQ containing Amides; VI
Fig. 1. Pharmaceutically important 1,2,3,4-tetrahydroisoquinoline derivatives (IeIV) and designed prototypes (VeVII).
Reduction of DHIQ 4 by using sodium borohydride afforded the
2.2.3. Effect of compounds on cell cycle
desired 1,2,3,4-tetrahydroisoquinoline (THIQ) 5 in good yield
[Scheme 1]. Compound 5 was then coupled with various acid de-
rivatives to obtain THIQ amide analogs. Compounds 6aef of pro-
totype V, 7aeb and 10 of prototype VI were synthesized by
amidation of THIQ 5 with substituted trans-cinnamic acids, benzoic
acids and 2-butynoic acid respectively.
Compounds 9aec of prototype VII, were synthesized by
coupling THIQ 5 with N-tosyl-L-aminoacids [14] to yield 8aed
followed by intramolecular Mitsunobu reaction [Scheme 2]. Amide
8d did not yield the cyclized product under the Mitsunobu condi-
tion, probably due to the steric crowding caused by the isopropyl
group.
Regulation of the cell cycle and apoptosis are considered to be
effective cancer therapeutic methods [16] and most of the cytotoxic
compounds exert their growth inhibitory effect either by arresting
the cell cycle at a particular checkpoint of cell cycle or by induction
of apoptosis [17,18]. The in vitro screening results revealed that
compounds, 9aeb showed significant antiproliferative activity
against prostate cancer cell line DU-145. Therefore, it was consid-
ered of interest to understand whether this inhibition of cell
growth was on account of cell cycle arrest. In this study DU-145
cells were treated with these compounds at concentrations of 0.5
and 1 mM for 48 h. The data obtained clearly indicated that these
compounds show G2/M cell cycle arrest in comparison with the
untreated cells. These compounds (9aeb) showed 17.68% and
16.96% of cell accumulation in G2/M phase at 0.5
whereas they exhibited 44.17% and 40.27% of cell accumulation at
M concentration, respectively [Fig. 2, Table 2].
mM concentration,
2.2. Biology
1
m
2.2.1. Anticancer activity
All the newly synthesized compounds (6aef, 7aeb, 8aed, 9aec
and 10) were evaluated for their anticancer activity against a panel
of five human cancer cell lines such as MCF-7 (Human breast
cancer), A549 (Human lung cancer), DU-145 (Human prostate
cancer), Hela (Human cervical cancer) and HepG2 (Human Liver
Cancer) by employing MTT assay [15]. The results are summarized
in Table 1 and expressed as IC50 values. The in vitro screening results
revealed that these compounds exhibited promising anticancer
2.2.4. Effect on inhibition of tubulin polymerization
Considering the cell cycle analysis of compounds 9aeb, it was
hypothesised to check the tubulin inhibition property of these
compounds as the compounds that alter cell-cycle parameters with
preferential G2/M blockade are known to exhibit effects on tubulin
assembly [19]. We have investigated the progression of tubulin
polymerization [20,21]. In comparison to control, compounds 9a,
9b and nocodazole (a known tubulin polymerization inhibitor)
inhibited tubulin polymerization by 63.64, 40.91 and 69.32%
respectively [Fig. 3].
activity with IC50 values ranging from 0.72 to 92.6
mM against
different cancer cell lines. Among all the fifteen analogs, 9a and 9b
exhibited potent/significant anticancer against human prostate
cancer cell line, DU-145 with IC50 values 0.72 and 1.23
mM
respectively. Therefore, the DU-145 cell line was chosen as a model
cell line for subsequent experiments.
2.2.5. Immunohistochemistry of tubulin
In order to corroborate the observed in vitro tubulin polymeri-
zation inhibition of compounds 9aeb, immunohistochemistry
studies were carried out to examine the in situ effects of 9aeb
cellular microtubules in DU-145 cells. DU-145 cells were treated
2.2.2. Structure activity relationship (SAR)
It was evident from the IC50 values that THIQ fused piper-
azinones 9a and 9b were the most promising analogs in the series
and were active against all the cancer cell lines. Apart from this,
amides containing OH, NO2, sulphonamides and alkyne were se-
lective towards A549, DU-145 and HepG2. Amino acid derivatives
of THIQ were more potent than cinnamide and benzamide
derivatives.
with test compounds at 0.5 mM concentration for 48 h. In this study,
untreated human prostate cancer cells displayed the normal dis-
tribution of microtubules [Fig. 4]. However, cells treated with
compounds 9a, 9b and nocodozole showed disrupted microtubule
organization as seen in Fig. 4, thus demonstrating the inhibition of
tubulin polymerization.

610
T. Ramanivas et al. / European Journal of Medicinal Chemistry 92 (2015) 608e618
Boc
OBn OH
BnO
OBn OH
NH
MgBr
O
N
O
N
b
+
a
c
O
N
CHO
O
Boc
O
4
5
1
2
O
anti-3 (major)
+
syn- 3 (minor)
Scheme 1. Reagents and conditions: a) i) THF, ꢀ78 ꢁC, DCM; ii) BnBr, NaH, DMF, rt, 4hr; b) 50% HCl, THF; c) NaBH4, THF.
R
OBn OH
HO
6d; R = 3-OMe, 4-OH
6e; R = 3-OH
6f; R = 4-NO2
6a; R = 2,3-OMe
6b; R = H
6c; R = 4-F
O
R
N
O
a
R
HO
OBn OH
NH
OBn OH
7a; R = 2-OH, 4-OMe
7b; R = 2-NHTs
O
a
R
N
O
5
Me
R
O
S
OBn
HO
OBn
O
OH
Intramoecular
Mitsunobu
Reaction
N
H
Me
S
N
O
O
R
O
O
N
N
a
b
R
N
S
Me
H
O
O
O
9a; R = H
8a; R = H
8b; R = CH3
9b; R = CH3
9c; R = CH2Ph
no reaction; R = CH(CH3)2
8c; R = CH2Ph
8d; R = CH(CH3)2
OBn OH
HO
O
10
N
a
O
Scheme 2. Reagents and conditions: a) EDCI, HOBT, DCM; b) PPh3, DIAD, THF.
2.2.6. DNA fragmentation analysis
these compounds at 0.5 and 1
mM concentrations for 48 h and
DNA laddering was carried out in order to elucidate the mode of
action of the compound especially for their ability to induce oli-
gonucleosomal DNA fragmentation (DNA ladder), which is a char-
acteristic feature of the programmed cell death or apoptosis
[22,23]. During apoptosis DNA is cleaved into small fragments
and fragmented DNA produces a series of bands which can be
observed by gel electrophoresis as ladders. DU-145 cells were
examined for the activation of caspase-3 activity. Results indicate
that there was nearly 3e9 fold induction in caspase-3 levels
compared to the control [Fig. 6].
2.2.8. Western blot analysis of Bcl-2 and Bax proteins
As the apoptotic regulation depends mainly on the internal
balance between anti-apoptotic protein Bcl-2 activity and pro-
apoptotic protein Bax activity, the intracellular balance between
proapoptotic Bax and antiapoptotic Bcl-2 family proteins is critical
for regulation between programmed cell death and cell survival
respectively [28]. In this context, DU-145 cells were treated with
treated with compounds, 9a and 9b at 0.5 mM concentration for
48 h and DNA was isolated from these cells. The DNA was run on 2%
agarose gel electrophoresis after staining with ethidium bromide
under UV illumination. It is observed that the test compounds
produced significant DNA fragmentation, which is indicative of
apoptosis [Fig. 5].
compounds 9a and 9b at 0.5 mM concentration for 48 h. After 48 h
of drug treatment, it was observed that the anti-apoptotic protein
Bcl-2 was down regulated and the pro-apoptotic protein Bax was
upregulated [Fig. 7]. This data reveals that the induction of
apoptosis by these new compounds is associated with Bcl-2 down-
regulation.
2.2.7. Effect on activation of caspase 3
From literature precedence, it is well established that molecules
affecting microtubule polymerization cause mitotic arrest and ul-
timately lead to apoptosis [24]. Caspases, are a family of cysteine-
aspartic proteases that are crucial mediators of apoptosis. Among
them, caspase-3 is the most studied mammalian caspases in terms
of its specificity role in apoptosis [25] and reports indicate that the
cell cycle arrest at G2/M phase takes place by the induction of
cellular apoptosis [26,27]. In the present study an investigation to
understand the correlation of cytotoxicity and apoptosis by com-
pounds 9a and 9b was undertaken. DU-145 cells were treated with
2.2.9. Annexin V-FITC assay for apoptosis
The caspase 3 activity assay, DNA fragmentation analysis and
Hoechst staining results suggest that these compounds induced
apoptosis in human prostate cancer cell line, DU-145. The apoptotic
effect of compounds 9a and 9b was further evaluated by Annexin V
FITC/PI (AV/PI) dual staining assay to examine the occurrence of
phosphatidylserine externalization and also to understand

T. Ramanivas et al. / European Journal of Medicinal Chemistry 92 (2015) 608e618
611
Table 1
distillation over P2O5 and was collected over activated 4 Å molec-
ular sieves. 1H NMR spectra were recorded on Avance 300 and
Avance 500 spectrometers using tetramethyl silane (TMS) as the
internal standard. Chemical shifts are reported in parts per million
(ppm) downfield from tetramethyl silane. Spin multiplicities are
described as s (singlet), bs (broad singlet), d (doublet), dd (double
douplet), t (triplet), q (quartet), and m (multiplet). Coupling con-
stant (J) values are reported in hertz (Hz). Analytical thin layer
chromatography (TLC) was performed on MERCK precoated silica
gel 60-F-254 (0.5 mm) aluminum plates. Visualization of the spots
on TLC plates was achieved either by exposure to iodine vapour or
UV light or by dipping the plates into ethanolic ninhydrin solution
or to ethanolic anisaldehyde solution and heating the plates to
120 ꢁC. Column chromatography was performed using silica gel of
100e200 mesh size. HPLC analysis was performed by using
Shimadzu-LC-2020, solvent system acetonitile and water (buffer
ammonium acetate pH 4.4) 75:25, flow rate 0.7, column; (Phe-
nomenex) Luna 5u C-18 size (250*4.60 mm), column oven temp
27 ꢁC, detector; PDA (wavelength254).
IC50 values (in mM) of THIQ analogs.
Compounda
MCF-7b
A549c
DU-145d
Helae
HepG2f
6a
6b
6c
6d
6e
6f
7a
7b
8a
8b
8c
8d
9a
9b
9c
10
55.1
39.8
57.3
37.3
19.2
24.4
21.8
79.4
11.48
14.8
8.71
60.4
2.51
5.49
17.6
18.6
2.69
0.99
39.8
25.1
32.3
63.0
4.36
5.62
6.46
29.9
12.9
29.3
41.3
2.08
4.07
9.77
39.8
3.801
4.67
1.26
31.6
0.72
1.23
7.41
6.16
1.98
1.30
32.76
27.7
58.3
67.6
15.8
26.8
12.5
56.3
15.07
21.0
12.3
92.6
3.16
7.31
20.3
11.6
3.23
2.29
31.0
20.7
24.6
79.4
2.82
5.37
11.4
57.5
5.046
5.37
2.24
32.0
0.87
1.70
10.8
7.94
2.45
1.61
37.1
6.025
7.37
6.91
79.4
1.26
2.45
13.7
9.33
3.01
1.90
Etoposide
Doxorubicin
a
50% Inhibitory concentration after 48 h of drug treatment and the values are
average of three individual experiments.
b
4.2. ((3S,4R)-4-phenoxy-1,2,3,4-tetrahydroisoquinolin-3-yl)
methanol (5)
Human breast cancer.
Human lung cancer.
Human prostate cancer.
Human cervical carcinoma.
c
d
e
To an ice cooled solution of DHIQ 4 (1g, 3.74 mmols) in ethanol
(5 mL), sodium borohydride (0.22 g, 5.61 mmols) was added and
reaction mixture was stirred for 1h. After completion of the reac-
tion, 50% sodium bicarbonate solution (10 mL) was added to the
reaction mixture and extracted by dichloromethane (10mLX3).
Combined organic layer was dried over anhydrous sodium sulphate
and concentrated under reduced pressure to obtain the THIQ 5
(0.96 g) as pale yellow solid. The crude was used for the next step
f
Human liver cancer.
whether it is due to physiological apoptosis or nonspecific necrosis
[22]. In this study DU-145 cells were treated with test compounds
at 0.5 and 1 mM concentrations for 48 h to examine the apoptotic
effect. After treatment it was observed that these compounds
showed significant apoptosis against DU-145 cells as shown in
Fig. 8. Results indicate that compounds 9a and 9b showed 9.26 and
without further purification. Yield ¼ 95.23%. Rf ¼ 0.10 (5% Methanol
25
in DCM) [
d
a
]
D
¼ ꢀ19.32 (c 0.33, CHCl3) 1H NMR: (300 MHZ, CDCl3)
6.33% of apoptosis at 0.5
m
M and 29.94 and 26.08% of apoptosis at
7.41e7.30 (m, 6H), 7.25e7.20 (m, 2H), 7.09e7.03 (m, 1H), 4.63 (q,
1
mM concentrations respectively, whereas 3.24% of apoptosis was
J ¼ 11.51 Hz, 2H), 4.38 (d, J ¼ 5.66 Hz, 1H), 4.01 (q, J ¼ 16.99 Hz, 2H),
observed in control (untreated cells). From this experiment it was
observed that these compounds significantly induce apoptosis in
DU-145 cells [Fig. 8, Table 4].
3.56 (q, J ¼ 8.49 Hz, 1H), 3.36 (m, 1H), 1.68 (s, 1H). 13C NMR:
(75 MHZ, CDCl3) d 138.0,136.3,134.1,128.7,128.4,127.6,126.4,125.7,
73.8, 70.6, 61.5, 57.5, 45.1. ESIMS (m/z): [MþH]þ
¼
270,
[MþNa]þ ¼ 292.
3. Conclusion
4.3. General procedure for coupling of acids with THIQ for the
preparation of compounds 6aef, 7aeb and 8aed
In conclusion, an efficient method for the asymmetric synthesis
of novel anticancer 1,2,3,4-tetrahydroisoquinoline (THIQ) analogs
have been developed and their cytotoxicity studies were per-
formed. These compounds showed significant anticancer proper-
ties against five cancer cell lines with IC50 values in submicromolar
range. THIQ fused piperazinones were found to be more potent
than cinnamic acid derivatives. The detailed biological studies such
as cell cycle analysis, tubulin polymerization assay, western blot,
DNA-fragmentation analysis, caspase-3 activation studies and
anexin V-FITC assay of compounds 9a and 9b suggested that they
inhibit tubulin polymerization and induce cell death by apoptosis.
Overall, the current study demonstrates the anticancer potential of
chirally pure 1,2,3,4-tertrahydroisoquinoline (THIQ) analogs and
provides future insights for developing chiral tubulin inhibitors.
To a solution of compound 5 (1 eq) and acid (1.2 eq) in
dichloromethane (3 mL for 0.1 g), were added 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide hydrochloride (EDCIꢂ HCl)
(1.2 eq) and HOBT (1.2 eq) at 0 ꢁC. The reaction mixture was stirred
for 2 h at the same temperature and progress of the reaction was
monitored by TLC. After consumption of starting material 5, reac-
tion mixture was diluted with dichloromethane (5 mL) and washed
with water (5 mL) followed by brine (5 mL). Organic layer was dried
over anhydrous s sodium sulphate and concentrated under reduced
pressure. The crude was purified by silica gel column chromatog-
raphy with gradient elution of ethyl acetate/petroleum ether to
obtain the desired amode.
4. Experimental section
4.3.1. (E)-1-((3S,4R)-4-(benzyloxy)-3-(hydroxymethyl)-3,4-dihydro
isoquinolin-2(1H)-yl)-3-(2,3-dimethoxyphenyl)prop-2-en-1-one
(6a)
4.1. General information
Yellowish sticky liquid, Yield ¼ 64.3%, Rf ¼ 0.2 (50% EtOAc in
25
Anhydrous reactions were performed in dried glassware under
nitrogen atmosphere. All reagents and solvents were obtained from
commercial suppliers and were used without further purification.
Anhydrous THF was obtained by distilling over sodium/benzo-
phenone under nitrogen. Anhydrous DCM was obtained by
hexane). [
a
]
¼ ꢀ23.2 (c 0.15, CHCl3). 1H NMR: (500 MHz, CDCl3)
D
mixture of rotamers (1: 0.7)
d
8.01 (d, J ¼ 15.6 Hz, 1H), 7.93 (d,
J ¼ 15.9 Hz, 0.7H), 7.79 (d, J ¼ 8.0 Hz, 0.7H), 7.72e7.74 (m, 0.6H),
7.42e7.35 (m, 3.4H), 7.33e7.27 (m, 8.3H), 7.24e7.21 (m, 3.9H), 7.19
(d, J ¼ 7.0 Hz, 1H), 7.13e7.06 (m, 3H), 6.94 (d, J ¼ 7.9 Hz, 1H), 6.91 (d,

612
T. Ramanivas et al. / European Journal of Medicinal Chemistry 92 (2015) 608e618
Fig. 2. Flow cytometric analysis in DU-145 prostate cancer cell lines after treatment with compounds, 9a and 9b at 0.5 and 1
B: Etoposide (1 M), C: Nocodazole (1 M), D: 9a (0.5 M), E: 9a (1 M), F: 9b (0.5 M) and G: 9b (1 M).
mM concentrations for 48 h; A: Control cells (DU-145),
m
m
m
m
m
m
J ¼ 7.9 Hz, 0.7H), 5.27e5.24 (m, 1H), 5.19 (d, J ¼ 18.0 Hz, 0.7H), 5.00
(d, J ¼ 18.0 Hz, 1H), 4.74 (t, J ¼ 6.9 Hz, 0.7H), 4.68 (d, J ¼ 14.5 Hz, 1H),
4.58 (bs, 1H), 4.56 (d, J ¼ 2.28 Hz, 1H), 4.53 (s, 1H), 4.52e4.49 (m,
2.7H), 3.89 (s, 3H), 3.87 (bs, 5.4H), 3.82 (bs, 1.8H), 3.50 (dd,
J ¼ 7.3 Hz, 11.1 Hz, 1H), 3.45e3.37 (m, 2.8H), 3.28 (dd, J ¼ 7.9 Hz,
4.3.2. (E)-1-((3S,4R)-4-(benzyloxy)-3-(hydroxymethyl)-3,4-
dihydro isoquinolin-2(1H)-yl)-3-phenylprop-2-en-1-one (6b)
Brown sticky solid, Yield ¼ 68%. Rf ¼ 0.3 (50% EtOAc in hexane).
25
[a
]
¼ ꢀ87.6 (c 0.17, CHCl3). 1H NMR (300 MHz, CDCl3): mixture of
D
rotamers (1: 0.7)
d
7.77 (d, J ¼ 15.1 Hz, 1H), 7.74 (d, J ¼ 15.8 Hz, 1H),
10.8 Hz, 1.6H). 13C NMR: (125 MHz, CDCl3)
d
166.5, 152.9, 148.4,
7.67 (s, 0.7H), 7.58e7.49 (m, 6.3H), 7.40e7.29 (m, 16.9H), 7.25e7.21
(m, 4.4H), 7.09 (d, J ¼ 15.1 Hz, 0.7H), 7.00 (d, J ¼ 15.1 Hz, 0.7H), 5.23
(d, J ¼ 18.1 Hz, 1H), 5.11 (pseudo t, J ¼ 5.3 Hz, 0.7H), 5.02 (d,
J ¼ 14.3 Hz, 1H), 4.76 (bs, 0.7H), 4.70 (d, J ¼ 15.10 Hz, 1H), 4.57 (d,
J ¼ 2.26 Hz, 2.4H), 4.51 (s, 3H), 3.52e3.37 (m, 2.8H), 3.29 (dd,
148.1, 140.6, 139.8, 137.5, 133.4, 130.3, 129.9, 129.2, 128.3, 127.8,
127.6, 127.7, 124.0, 119.3, 118.0, 114.1, 73.7, 69.9, 61.0, 55.7, 55.1, 43.0.
HRMS: m/z calculated for C28H30NO5 [MþH]þ: 458.19514; Found:
458.19528.
J ¼ 7.5 Hz, 10.5 Hz, 1.3H). 13C NMR: (125 MHz, CDCl3)
d 168.3, 145.4,

T. Ramanivas et al. / European Journal of Medicinal Chemistry 92 (2015) 608e618
613
Table 2
FACS Analysis of compounds.
146.6, 145.8, 137.6, 135.1, 130.5, 129.5, 129.4, 129.1, 129.8, 128.4,
128.0, 127.9, 127.7, 127.5, 127.3, 73.8, 69.9, 63.1, 55.3, 43.5. HRMS: m/
z calculated for C26H24FNO3 [M]þ: 417.18890; found [MꢀH]þ:
416.18548.
Compound
A: Control
B: Etoposide (1
C:Nocodazole(1
D: 9a (0.5
E: 9a (1 M)
F: 9b (0.5
G: 9b (1
Sub G1%
G0/G1%
S %
G2/M %
9.36
7.13
4.52
8.22
5.02
8.25
6.74
79.15
48.84
48.19
67.34
46.74
68.53
46.67
1.65
1.45
0.86
2.64
0.90
2.80
1.83
6.75
38.75
43.40
17.68
44.17
16.96
40.27
m
m
M)
M)
4.3.4. (E)-1-((3S,4R)-4-(benzyloxy)-3-(hydroxymethyl)-3,4-
dihydroisoquinolin-2(1H)-yl)-3-(4-hydroxy-3-methoxyphenyl)
prop-2-en-1-one (6d)
mM)
m
m
M)
m
M)
Brown sticky solid, Yield ¼ 63%; Rf ¼ 0.2 (50% EtOAc in hexane).
25
[a
]
¼ ꢀ55.6 (c 0.19, CHCl3). 1H NMR (500 MHz, CDCl3): mixture
D
of rotamers (1: 0.6)
d
7.73 (d, J ¼ 15.4 Hz, 1H), 7.65 (t, J ¼ 15.4 Hz,
1H), 7.37e7.27 (m, 13H), 7.13e7.12 (m, 2H), 7.10e7.07 (m, 1.6H), 7.04
(s, 1.2H), 6.97e6.87 (m, 3H), 6.84 (d, J ¼ 15.4 Hz, 1H), 5.27 (bs, 1.4H),
5.03 (d, J ¼ 14.9 Hz, 1H), 4.75 (bs, 0.7H), 4.70 (d, J ¼ 14.8 Hz, 1H),
4.56 (t, J ¼ 14.0 Hz, 3H), 4.51 (s, 2.7H), 3.95 (s, 3H), 3.93 (s, 0.7H),
3.87 (s, 1.8H), 3.52e3.47 (m, 1H), 3.44e3.39 (m, 1.7H), 3.26 (t,
Table 3
Inhibition of tubulin polymerization (IC50
)
of compounds,
9aeb.
a
Compound
IC50
SD (in mM)
9a
9b
1.68 0.08
3.55 0.12
1.64 0.03
J ¼ 8.2 Hz, 1H). 13C NMR: (125 MHz, CDCl3)
d 168.6, 147.7, 147.3,
146.7, 144.1, 142.8, 139.1, 137.7, 132.2, 128.9, 128.3, 127.7, 127.3, 126.9,
126.7, 126.5, 122.1120.5, 115.5, 110.2, 73.8, 69.8, 61.7, 58.5, 55.8, 45.7.
HRMS: m/z calculated for C27H28NO5 [MþH]þ: 446.19449; found:
446.19476.
Nocodazole
a
Concentration of drug to inhibit 50% of tubulin assembly.
143.6, 142.4, 137.8, 135.1, 133.2, 130.8, 129.9, 128.6, 128.2, 127.7,
127.3, 129.3, 126.8, 126.5, 124.9, 118.3, 73.9, 69.8, 61.5, 58.5, 45.6.
HRMS: m/z calculated for C26H26NO3 [MþH]þ: 400.18993; found:
400.19002.
4.3.5. (E)-1-((3S,4R)-4-(benzyloxy)-3-(hydroxymethyl)-3,4-
dihydroisoquinolin-2(1H)-yl)-3-(3-hydroxyphenyl)prop-2-en-1-
one (6e)
Brown solid, Yield ¼ 54%; Rf ¼ 0.2 (50% EtOAc in hexane).
25
4.3.3. (E)-1-((3S,4R)-4-(benzyloxy)-3-(hydroxymethyl)-3,4-
dihydro isoquinolin-2(1H)-yl)-3-(4-fluorophenyl)prop-2-en-1-one
(6c)
[a
]
¼ ꢀ105.7 (c 0.16, CHCl3). 1H NMR (500 MHz, CDCl3): mixture
D
of rotamers (~1: 0.8)
d
7.65 (d, J ¼ 15.1 Hz, 1H), 7.49 (d, J ¼ 15.7 Hz,
1H), 7.36e7.32 (m, 3.4H), 7.27e7.22 (m, 6.8H), 7.20e7.16 (m, 10H),
7.04 (s, 1.6H), 7.02 (t, J ¼ 7.9 Hz, 1.7H), 7.89 (d, J ¼ 15.7 Hz, 1.2H), 6.82
(t, J ¼ 7.8 Hz, 1.7H), 6.71 (dd, J ¼ 1.9 Hz, 8.0 Hz, 1H), 5.27e5.25 (m,
1.4H), 4.95 (d, J ¼ 14.8 Hz, 1H), 4.77e4.74 (m, 1.4H), 4.64 (d,
J ¼ 14.7 Hz, 1H), 4.57 (bs, 1.4H), 4.51 (d, J ¼ 5.03 Hz, 1.9H), 4.48e4.47
(m,1.4H), 4.45 (d, J ¼ 5.1 Hz,1.7H), 3.48e3.41 (m, 1H), 3.39e3.31 (m,
Yellowish brown sticky liquid, Yield ¼ 48%. Rf ¼ 0.2 (50% EtOAc
25
in hexane). [
a
]
¼ ꢀ7.19 (c 0.50, CHCl3). 1H NMR (300 MHz,
D
CDCl3): mixture of rotamers (1: 0.7)
d
7.75 (dd, J ¼ 15.2 Hz, 1H), 7.72
(d, J ¼ 15.6 Hz, 1H), 7.63e7.54 (m, 4H), 7.51e7.45 (m, 3H), 7.40e7.28
(m, 11H), 7.09 (t, J ¼ 8.3 Hz, 3.9H), 7.02 (d, J ¼ 8.3 Hz, 1.2H), 6.93 (d,
J ¼ 15.1 Hz, 0.7H), 5.25 (bs, 0.7H), 5.02 (d, J ¼ 14.3 Hz,1H), 4.73e4.68
(m, 2H), 4.58e4.54 (bs, 3.8H), 4.51 (s, 3.5H), 3.48e3.40 (m, 3H), 3.27
3.5H). 13C NMR: (125 MHz, CDCl3)
d 168.9, 156.8, 144.1, 143.0, 137.6,
136.2,135.1,132.9,132.0,130.6,130.0,129.1,128.3,127.5,127.8,126.7,
120.6, 118.1, 115.1, 73.7, 69.9, 61.4, 58.6, 45.7. HRMS: m/z calculated
(dd, J ¼ 8.3 Hz, 11.3 Hz, 1H). 13C NMR: (125 MHz, CDCl3)
d
167.8,
Fig. 3. Effect of compounds on tubulin polymerization: tubulin polymerization was monitored by the increase in fluorescence at 360 nm (excitation) and 420 nm (emission) for 1 h
at 37 ꢁC. All the compounds were included at a final concentration of 3
M. Nocodazole was used as a positive control. Values indicated are the mean SD of two different
experiments performed in triplicates. The IC50 value for tubulin polymerization was found to be 1.68 and 3.55 M for compounds 9a and 9b respectively in comparison to the IC50
value of nocodazole that was found to be 1.64 M [Table 3].
m
m
m

614
T. Ramanivas et al. / European Journal of Medicinal Chemistry 92 (2015) 608e618
Fig. 4. Immuno histochemistry (IHC) analysis of compounds on the microtubule network: DU-145 cells were treated with compounds 9a, 9b and nocodazole at a concentration of
50 nM for 48 h followed by staining with -tubulin antibody.
a
for C26H26NO4 [MþH]þ: 416.18441; found: 416.18442.
J ¼ 15.1 Hz, 1H), 4.73e4.68 (m, 2.2H), 4.58e4.45 (m, 8H), 3.46e3.29
(m, 3.6H). 13C NMR: (125 MHz, CDCl3)
167.3, 141.5, 140.7, 139.3,
d
133.0,130.7,129.2,128.5,128.4,128.2,127.7,127.6,127.5,127.4,127.0,
126.7, 121.6, 123.8, 73.1, 74.0, 61.8, 59.0, 42.9. HRMS: m/z calculated
for C26H25N2O5 [MþH]þ: 445.17420; found: 445.17433.
4.3.6. (E)-1-((3S,4R)-4-(benzyloxy)-3-(hydroxymethyl)-3,4-
dihydroisoquinolin-2(1H)-yl)-3-(4-nitrophenyl)prop-2-en-1-one
(6f)
Yellow solid, Yield ¼ 65%; Rf ¼ 0.2 (50% EtOAc in hexane).
25
[
a]
¼ ꢀ91.6 (c 0.27, CHCl3). 1H NMR: (300 MHz, CDCl3) mixture
4.3.7. ((3S,4R)-4-(benzyloxy)-3-(hydroxymethyl)-3,4-
dihydroisoquinolin-2(1H)-yl) (2-hydroxy-4-methoxyphenyl)
methanone (7a)
D
of rotamers (~1: 0.8)
d
8.24 (d, J ¼ 8.68 Hz, 1.5H), 8.14 (d, J ¼ 8.68 Hz,
2H), 7.72e7.65 (m, 2.7H), 7.60e7.53 (m, 3H), 7.37e7.28 (m, 9H),
7.26e7.20 (m, 6H), 5.25 (bs, 1H), 5.20 (d, J ¼ 12.1 Hz, 1H), 5.00 (d,
Yellowish sticky solid, Yield ¼ 50%; Rf ¼ 0.3 (50% EtOAc in
25
hexane). [
d
a]
¼ ꢀ7.5 (c 0.09, CHCl3). 1H NMR (500 MHz, CDCl3):
D
7.53 (d, J ¼ 7.32 Hz, 1H), 7.41e7.31 (m, 7H), 7.12 (d, J ¼ 6.71 Hz, 1H),
7.04 (d, J ¼ 7.17 Hz, 1H), 6.92e6.84 (m, 2H), 5.20 (bs, 1H), 4.74e4.68
(m, 1H), 4.64e4.59 (m, 1H), 4.58e4.50 (m, 3H), 4.32e4.27 (m, 1H),
3.81 (s, 3H), 3.56e3.44 (m, 2H). HRMS: m/z calculated for
C
25H26O5N [MþH]þ: 420.18308; found: 420.18318.
4.3.8. N-(2-((3S,4R)-4-(benzyloxy)-3-(hydroxymethyl)-1,2,3,4-
tetrahydroisoquinoline-2-carbonyl)phenyl)-4-
methylbenzenesulfonamide (7b)
Yellow solid, Yield ¼ 54%. Rf ¼ 0.2 (50% EtOAc in hexane).
25
[a
]
¼ ꢀ7.26 (c 0.12, CHCl3); 1H NMR (300 MHz, CDCl3): mixture
D
of rotamers
d 8.43 (bs, 1H), 7.75e7.68 (m, 4H), 7.61e7.53 (m, 4.8H),
7.39e7.27 (m, 16H), 6.96e6.81 (m, 3H), 5.56e5.46 (m, 0.7H),
5.33e5.27 (m, 1H), 4.74 (d, J ¼ 9.8 Hz, 2H), 4.47 (bs, 4H), 4.25 (t,
J ¼ 13.5 Hz, 5H), 3.62e3.57 (m, 1H), 3.43e3.29 (m, 3.5H), 2.37 (s,
3H), 2.33 (s, 2.1H). HRMS: m/z calculated for C31H31O5 N2 S [MþH]þ:
543.19849; found: 543.19838.
4.3.9. N-(2-((3S,4R)-4-(benzyloxy)-3-(hydroxymethyl)-3,4-
dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)-4-
methylbenzenesulfonamide (8a)
Yellowish sticky solid, 63%yield. Rf ¼ 0.5 (10% Methanol in DCM).
Fig. 5. DNA fragmentation of compounds, 9a and 9b in DU-145 cells: Lane-1: 9a
¼ ꢀ15.6 (c), 1H NMR: (500 MHz, CDCl3) mixture of rotamers
25
[a
]
D
(0.5
bp).
mM), Lane-2: 9b (0.5 mM), Lane-3: Untreated control DNA and Lane-4: Marker (100
(~1: 0.5) d 7.80e7.68 (m, 4H), 7.38e7.27 (m, 8H), 7.26e7.13 (m, 6H),

T. Ramanivas et al. / European Journal of Medicinal Chemistry 92 (2015) 608e618
615
3.28e3.20 (m, 1H), 3.12e3.06 (m, 1H), 3.01 (d, J ¼ 6.3 Hz, 2H),
2.92e2.72 (m, 4H), 2.40 (s, 3H), 2.31 (s, 3H). 13C NMR: (75 MHz,
CDCl3)
d 172.3, 143.4, 137.9, 137.1, 135.0, 133.1, 129.5, 129.3, 128.9,
128.4, 127.6, 127.0, 126.5, 74.0, 70.4, 61.9, 59.4, 54.7, 42.2, 37.4, 21.3.
HRMS: m/z calculated for C33H36N2O5S [MþH]þ: 571.22904; found:
571.22939.
4.3.12. N-((S)-1-((3S,4R)-4-(benzyloxy)-3-(hydroxymethyl)-3,4-
dihydroisoquinolin-2(1H)-yl)-1-oxopropan-2-yl)-4-
methylbenzenesulfonamide (8d)
Yellowish sticky liquid, 60% yield; Rf ¼ 0.5 (10%Methanol in
¼ þ4.0 (c 0.11, CHCl3). 1H NMR: (300 MHz, CDCl3)
Fig. 6. Caspase-3 actvation studies.
DCM). [
a]
D
mixture of rotamers (~1: 0.2) signals mentioned for major product
only;
d
7.70 (d, J ¼ 8.30 Hz, 2H), 7.40e7.37 (m, 2H), 7.36 (d,
5.76 (bs, 1H), 5.15 (bs, 0.7H), 5.08 (d, J ¼ 17.8 Hz, 0.5H), 4.58 (d,
J ¼ 14.9 Hz, 1H), 4.46e4.40 (m, 2H), 4.37 (d, J ¼ 12.9 Hz 1H), 4,23 (d,
J ¼ 18.0 Hz, 1H), 4.12 (q, J ¼ 7.17 Hz, 1H), 4.00e3.93 (m, 1H),
3.89e3.71 (m, 3H), 3.69 (t, J ¼ 6.10 Hz, 1H), 3.51 (dd, J ¼ 6.1 Hz,
17.7 Hz, 1H), 3.42 (dd, J ¼ 5.5 Hz, 17.7 Hz, 1H), 2.40 (s, 1.6H), 2.37 (s,
3H). 13C NMR: (125 MHz, CDCl3): 168.4, 167.6, 143.8, 143.7, 143.6,
129.7, 128.5, 128.5, 127.9, 127.8, 127.5, 127.1, 127.1, 73.1, 69.8, 63.3,
52.0, 44.0, 43.9, 29.6, 21.5. ESIMS (m/z): 481 [MþH]þ.
J ¼ 3.5 Hz, 2H), 7.33e7.29 (m, 3H), 7.24e7.23 (m, 2H), 7.22 (s, 1H),
7.19 (d, J ¼ 8.3 Hz, 1H), 7.06 (d, J ¼ 7.3 Hz,1H), 4.65 (bs, 1H), 4.54 (dd,
J ¼ 11.1 Hz, 25.8 Hz, 2H), 4.32 (d, J ¼ 5.8 Hz, 1H), 4.06e4.02 (m, 2H),
3.94e3.90 (m, 2H), 3.77 (d, J ¼ 4.9 Hz, 1H), 3.31 (dd, J ¼ 5.8 Hz,
10.8 Hz, 1H), 2.32 (s, 3H), 2.04e1.98 (m, 1H), 0.94 (d, J ¼ 6.8 Hz, 3H),
0.83 (d, J ¼ 6.8 Hz, 3H). 13C NMR: (125 MHz, CDCl3)
d 173.2, 143.7,
142.8, 137.7, 137.2, 132.0, 131.4, 130.0, 129.6, 129.0, 128.5, 128.3,
127.5, 126.4, 73.8, 69.9, 61.8, 56.1, 49.4, 44.9, 21.4, 19.5. HRMS: m/z
calculated for C29H35N2O5S [MþH]þ: 523.2267; found: 523.2280.
4.3.10. N-((S)-1-((3S,4R)-4-(benzyloxy)-3-(hydroxymethyl)-3,4-
dihydroisoquinolin-2(1H)-yl)-3-methyl-1-oxobutan-2-yl)-4-
methylbenzenesulfonamide (8b)
4.4. General procedure for Mitsunobu reaction to prepare
compounds 9aec
Yellow solid, Yield ¼ 60%; Rf ¼ 0.5 (10% Methanol in DCM).
25
[
a]
¼ ꢀ10.11 (c 0.13, CHCl3). 1H NMR (300 MHz, CDCl3): mixture
D
To a cooled solution of amide (1eq), and triphenylphosphine
(TPP) (1.5 eq) in anhydrous tetrahydrofuran (THF), was added a
solution of diisopropylazodicarboxylate (DIAD) (1.5 eq in THF)
dropwise. Progress of the reaction was monitored by TLC. After
complete consumption of the starting material, solvent was evap-
orated and crude was purified by silica gel chromatography to yield
the cyclized products.
of rotamers (~1: 0.5)
d 7.78e7.70 (m, 4H), 7.39e7.29 (m, 10H),
7.22e7.14 (m, 5.8H), 4.93e4.87 (m, 1H), 4.63 (d, J ¼ 15.2 Hz, 1.7H),
4.57e4.54 (m, 2H), 4.45 (d, J ¼ 6.0 Hz, 2H), 4.39 (d, J ¼ 6.7 Hz, 2H),
4.15e3.98 (m, 2H), 3.42e3.36 (m, 1H), 3.05e2.93 (m, 3.8H), 2.33 (s,
1.5H), 2.30 (s, 3H), 1.34 (d, J ¼ 6.9 Hz, 1.5H), 1.27 (d, J ¼ 8.9 Hz, 3H).
13C NMR: (75 MHz, CDCl3)
d 171.1, 143.5, 137.9, 136.9, 135.7, 133.3,
129.5, 128.5, 128.0, 127.8, 127.8, 127.2, 126.6, 125.9, 73.4, 70.5, 64.5,
61.2, 54.9, 45.2, 29.6, 21.4, 19.0, 17.4. HRMS: m/z calculated for
C
29H35N2O5S [MþH]þ: 523.22808; found: 523.22837.
4.4.1. (11R)-11-(benzyloxy)-2-tosyl-2,3,11,11a-tetrahydro-1H-
pyrazino[1,2-b]isoquinolin-4(6H)-one (9a)
4.3.11. N-((S)-1-((3S,4R)-4-(benzyloxy)-3-(hydroxymethyl)-3,4-
dihydroisoquinolin-2(1H)-yl)-1-oxo-3-phenylpropan-2-yl)-4-
methylbenzenesulfonamide (8c)
Yellow solid, Yield ¼ 40%. Rf ¼ 0.7 (40% EtOAc in hexane).
25
[a
]
¼ þ39.8 (c 0.14,CHCl3). 1H NMR: (500 MHz, CDCl3)
d 7.70 (d,
D
J ¼ 8.24 Hz, 2H), 7.53 (t, J ¼ 7.78 Hz, 3H), 7.42 (t, J ¼ 7.3 Hz, 2H), 7.38
(d, J ¼ 8.0 Hz, 2H), 7.38e7.35 (m, 1H), 7.30e7.27 (m, 2H), 7.11 (d,
J ¼ 8.1 Hz, 1H), 5.37 (d, J ¼ 16.93 Hz, 1H), 5.10 (d, J ¼ 10.9 Hz, 1H),
5.03 (d, J ¼ 10.9 Hz, 1H), 4.88 (d, J ¼ 10.46 Hz, 1H), 4.14e4.02 (m,
3H), 3.63e3.59 (m, 1H), 3.34 (d, J ¼ 16.17 Hz, 1H), 2.95 (dd,
J ¼ 3.96 Hz, 12.35 Hz, 1H), 2.46 (s, 3H). 13C NMR: (125 MHz, CDCl3)
Brown sticky liquid, 60%yield; Rf ¼ 0.5 (10% Methanol in DCM).
25
[
a]
¼ ꢀ46.6 (c 0.20, CHCl3). 1H NMR: (300 MHz, CDCl3) mixture
D
of rotamers (~1: 1)
d 7.63e7.58 (m, 3H), 7.42e7.27 (m, 20H),
7.23e7.14 (m, 15H), 6.86 (d, J ¼ 9.8 Hz, 2H), 5.33e5.26 (m, 1H),
4.52e4.50 (m, 5H), 4.39 (d, J ¼ 4.5 Hz, 2H), 4.29e4.22 (m, 3H),
4.12e4.02 (m, 2H), 3.96e3.85 (m, 2H), 3.54 (d, J ¼ 6.0 Hz, 1H),
d
163.1, 144.7, 137.7, 136.0, 131.7, 131.1, 130.0, 128.6, 128.0, 127.9,
127.2, 126.6, 125.8, 74.9, 74.7, 57.1, 49.4, 44.5, 43.6, 21.6. HRMS: m/z
calculated for C26H27N2O4S [MþH]þ: 463.1692; found 463.17182.
4.4.2. (3S,11R)-11-(benzyloxy)-3-methyl-2-tosyl-2,3,11,11a-
tetrahydro-1H-pyrazino[1,2-b]isoquinolin-4(6H)-one (9b)
Yellowish sticky solid, 40% yield. Rf ¼ 0.7 (40% EtOAc in hexane).
25
[a
]
¼ þ28.6 (c 0.20, CHCl3). 1H NMR: (500 MHz, CDCl3)
d 7.70 (d,
D
J ¼ 8.24 Hz, 2H), 7.51e7.43 (m, 3H), 7.42e7.33 (m, 3H), 7.32e7.26
(m, 4H), 7.14e7.11 (m,1H), 5.39 (d, J ¼ 16.78 Hz, 1H), 4.95 (dd,
J ¼ 11.29 Hz, 30.21 Hz, 2H), 4.81 (d, J ¼ 9.15 Hz, 1H), 4.54 (q,
J ¼ 7.01 Hz, 1H), 4.15e4.08 (m, 2H), 3.63 (q, J ¼ 9.30 Hz, 1H), 3.47
(dd, J ¼ 4.12 Hz, 13.42 Hz, 1H), 2.43 (s, 3H), 1.28 (d, J ¼ 4.42 Hz, 3H).
13C NMR: (125 MHz, CDCl3)
d 167.3, 144.0, 138.0, 136.5, 135.8, 132.8,
Fig. 7. The effect of compounds on Bcl-2 and Bax levels: DU-145 cells were treated
132.5, 129.9, 128.5, 127.9, 127.8, 127.6, 127.3, 127.2, 127.0, 125.9, 74.4,
with compounds 9a and 9b at 0.5
collected, and the expression levels of Bcl-2 and Bax were determined by western blot
analysis. -Actin was used as a loading control.
mM concentration for 48 h. Cell lysates were
73.5, 57.0, 54.1, 44.1, 21.5, 15.4. HRMS: m/z calculated for
b
C
27H29N2O4S [MþH]þ: 477.1848; found: 477.1873.

616
T. Ramanivas et al. / European Journal of Medicinal Chemistry 92 (2015) 608e618
Fig. 8. Annexin V-FITC staining. DU-145 cells were treated with compounds 9a and 9b at 1 mM concentration for 48 h.
4.4.3. (3S,11R)-3-benzyl-11-(benzyloxy)-2-tosyl-2,3,11,11a-
tetrahydro-1H-pyrazino[1,2-b]isoquinolin-4(6H)-one (9c)
128.26, 127.8, 127.5, 127.2, 126.6, 126.3, 91.3, 73.7, 73.4, 70.1, 61.3,
58.9, 46.3, 4.0. HRMS: m/z calculated for C21H22NO3 [MþH]þ:
336.15854; found: 336.15876.
Yellow sticky solid, Yield ¼ 41%; Rf ¼ 0.7 (40% EtOAc in hexane).
25
[
a]
¼ ꢀ3.8 (c 0.18, CHCl3). 1H NMR: (500 MHz, CDCl3)
d 7.60 (d,
D
J ¼ 8.08 Hz, 2H), 7.39e7.31 (m, 6H), 7.26e7.23 (m, 4H), 7.15e7.10 (m,
4H), 7.02e6.97 (m, 2H), 5.12 (d, J ¼ 16.47 Hz, 1H), 4.67 (dd,
J ¼ 11.74 Hz, 4.73 Hz, 2H), 4.54 (t, J ¼ 4.88 Hz, 1H), 4.44 (d,
J ¼ 8.08 Hz, 1H), 4.15 (d, J ¼ 16.32 Hz, 1H), 3.34 (dd, J ¼ 4.73 Hz,
12.81 Hz, 1H), 3.27e3.21 (m, 3H), 3.02e2.97 (m, 1H), 2.41 (s, 3H).
HRMS: m/z calculated for C33H33N2O4S [MþH]þ: 553.21859; found:
553.21884.
4.5. Cell culture
Cells were obtained from National Centre for Cell Science (NCCS)
Pune, India and stocks were maintained in the laboratory. MCF-7
(Human breast cancer), A549 (Human lung cancer), DU-145 (Hu-
man prostate cancer), Hela (Human cervical cancer) and HepG2
(Human Liver Cancer) cells were grown in tissue culture flasks in
DMEM (Dulbecco modified Eagle medium, Sigma) or MEM (Mini-
mum Essential Medium, Sigma) supplemented with 10% fetal
bovine serum with 1X stabilized antibiotic-antimycotic solution
(Sigma) in a CO2 incubator at 37 ꢁC with 5% CO2 and 90% relative
humidity. The cells at sub confluent stage were harvested with 1X
porcine pancreatic trypsin (Hi media) and seeded in required
density in tissue culture plates for the assay.
4.4.4. 1-((3S,4R)-4-(benzyloxy)-3-(hydroxymethyl)-3,4-dihydroiso-
quinolin-2(1H)-yl)but-2-yn-1-one (10)
Brown solid, Yield ¼ 62%; Rf ¼ 0.3 (50% EtOAc in hexane).
25
[
a]
¼ ꢀ61.4 (c 0.19, CHCl3). 1H NMR: (300 MHz, CDCl3) mixture of
D
rotamers (1: 0.7)
d 7.27e7.12 (m, 15H), 5.12e4.99 (m, 3H), 4.65 (d,
J ¼ 15.8 Hz, 0.7H), 4.57e4.51 (m, 4.5H), 4.29 (d, J ¼ 18.1 Hz, 1H),
3.51e3.45 (m, 0.8H), 3.37e3.23 (m, 2H), 2.00 (s, 2.1H), 1.96 (s, 3H).
13C NMR: (125 MHz, CDCl3)
d 155.4, 137.9, 132.4, 130.3, 130.1, 128.8,

T. Ramanivas et al. / European Journal of Medicinal Chemistry 92 (2015) 608e618
617
Table 4
slips were blocked with a 1% bovine serum albumin (BSA) solution
for 60 min and then incubated with anti- -tubulin antibody
Annexin V-FITC analysis after treatment with compound 9a and 9b.
a
(1:1000) at room temperature for 2 h. The slides were washed three
times for 5 min each with PBST. Next, the cover slips were incu-
bated with FITC-conjugated anti-mouse secondary antibody (Cell
signaling technology) for 1 h and then washed three times with
PBST solution. Finally, the cells were observed under a fluorescence
microscope (Leica, Germany), and the pictures were analyzed for
the integrity of the microtubule network [21].
Sample
UL %
LL %
UR %
LR %
A: Control
0.31
0.63
4.48
0.82
4.70
96.45
90.12
65.57
92.85
69.22
2.47
6.47
25.79
4.45
0.77
2.79
4.15
1.88
4.94
B: 9a (0.5
m
M)
M)
D: 9b (0.5 M)
E: 9b (1 M)
C: 9a (1
m
m
m
21.14
4.6. Anticancer activity
4.10. Caspase 3 activity
The cytotoxic activity of the compounds was determined using
To determine the caspase-3 activity of 9a and 9b for detection of
apoptosis in human prostate cancer cell line (DU-145), the
commercially available apoptosis detection kit (Sigma-Caspase 3
Assay kit, Florometric) was used. DU-145 cells were treated with
MTT assay [15]. 1 ꢃ 104 cells/well were seeded in 200
ml DMEM,
supplemented with 10% FBS in each well of 96-well microculture
plates and incubated for 24 h at 37 ꢁC in a CO2 incubator. Com-
pounds, diluted to the desired concentrations in culture medium,
were added to the wells with respective vehicle control. After 48 h
compounds 9a and 9b at 0.5 and 1 mM concentrations for 48 h. Here
the substrate used is Ac-LEHD-AFC to the cell lysate and incubation
was carried out at 37 ꢁC for 1 h. Readings were taken at excitation
wavelength 400 nm and emission wavelength 505 nm.
of incubation, 10
ml MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-
diphenyl tetrazolium bromide) (5 mg/mL) was added to each well
and the plates were further incubated for 4 h. Then the supernatant
from each well was carefully removed, formazon crystals were
4.11. DNA fragmentation analysis
dissolved in 100
was recorded.
ml of DMSO and absorbance at 570 nm wavelength
DU-145 Cells were seeded (1 ꢃ 106) in six-well plates. After
incubation of 24 h cells were treated with compound 9a and 9b at
4.7. Cell cycle analysis
0.5 mM concentration. After 48 h of treatment, cells were collected
and centrifuged at 2500 rpm for 5 min at 4 ꢁC. Pellet was collected
and washed with Phosphate buffered saline (PBS). Lysis buffer was
added, the pellet was collected centrifuged at 3000 rpm for 5 min at
4 ꢁC and the supernant was collected. Sodium dodecyl sulfate (SDS,
Flow cytometric analysis (FACS) was performed to evaluate the
distribution of the cells through the cell cycle phases. DU-145,
human prostate cancer cells were incubated with compounds (9a
and 9b) at 0.5 and 1 mM concentrations for 48 h. Untreated and
10%, 10
mixture was incubated for 2 h at 56 0C. After incubation, 10
proteinase K (25 mg/mL) was added and the mixture was further
incubated at 37 ꢁC for 2 h. Ammonium acetate (10 m, 65
l) and ice-
cold ethanol (500 l) were then added, and the reaction was mixed
ml) and 50 mg/mL RNase A (10
ml) were then added, and the
treated cells were harvested, washed with PBS, fixed in ice-cold 70%
ethanol and stained with propidium iodide (Sigma Aldrich). Cell
cycle was performed by flow cytometry (Becton Dickinson FACS
Caliber) as earlier described [29].
ml of
m
m
well. These samples were incubated at ꢀ80 ꢁC for 1 h. After incu-
bation, the samples were centrifuged at 12,000 rpm for 20 min at
4 ꢁC. After centrifugation, the pellet was washed with 80% ethanol
and air dried for 10 min at room temperature. The pellet was dis-
4.8. Tubulin polymerization assay
A fluorescence based in vitro tubulin polymerization assay was
performed according to the manufacturer's protocol (BK011, Cyto-
skeleton, Inc.). Briefly, the reaction mixture in a total volume of
solved in 50 ml TE buffer, and DNA laddering was determined by
using 2% agarose gel electrophoresis in TE buffer.
4.12. Protein extraction and western blot analysis
DU-145 cells were treated with compounds 9a and 9b at 0.5
10
mL contained PEM buffer, GTP (1
m
M) in the presence or absence
of test compounds (final concentration of 3
m
M). Tubulin poly-
merization was followed by a time dependent increase in fluores-
cence due to the incorporation of a fluorescence reporter into
microtubules as polymerization proceeds. Fluorescence emission at
420 nm (excitation wavelength is 360 nm) was measured by using a
Varioscan multimode plate reader (Thermo scientific Inc.). Noco-
dazole was used as positive control in each assay. The IC50 value was
defined as the drug concentration required inhibiting 50% of
tubulin assembly compared to control. The reaction mixture for
these experiments include: tubulin (3 mg/ml) in PEM buffer, GTP
mM
concentration for 48 h. The cell lysates were obtained by lysing the
cells in ice-cold radioimmunoprecipitation assay (RIPA) buffer
(1 ꢃ PBS, 1% NP-40 detergent, 0.5% sodium deoxycholate, and 0.1%
SDS) containing 100 mg/mL phenylmethanesulfonyl fluoride
(PMSF), 5 mg/mL aprotinin, 5 mg/mL leupeptin, 5 mg/mL pepstatin,
and 100 mg/mL NaF. After centrifugation at 12,000 rpm for 10 min,
the protein in the supernatant was quantified by the Bradford
method (BIO-RAD) by using a Multimode Varioskan instrument
(Thermo Fischer Scientifics Ltd.). Protein (50 mg per lane) was
applied in 12% SDS polyacrylamide gel. After electrophoresis, the
protein was transferred to a polyvinylidine difluoride (PVDF)
membrane (Thermo Scientific Inc.). The membrane was blocked at
room temperature for 2 h in Tris-buffered saline (TBS) with 0.1%
Tween 20 (TBST) containing 5% blocking powder (Santa Cruz). The
membrane was washed with TBST for 5 min, then primary antibody
was added and the membrane was incubated at 480C overnight.
(1
mM), in the presence or absence of test compounds at 2.5, 5, 10,
and 15
m
M concentrations. Polymerization was monitored by in-
crease in the Fluorescence as mentioned above at 37 ꢁC [20,21].
4.9. Immunohistochemistry
DU-145 cells were seeded on glass cover slips and incubated for
48 h in the presence or absence of the test compounds 9a and 9b
(50 mM). After treatment, the cover slips were fixed with a para-
formaldehyde solution (4% in 1 ꢃ PBS) for 20 min at room tem-
perature. Cell permeabilization was achieved by administration of a
Triton X-100 solution (0.2% in 1 ꢃ PBS) for 5 min. The cover slips
were left in 100% MeOH overnight at 4 ꢁC. Subsequently, the cover
Bcl-2, Bax and b-actin antibodies were purchased from Cell
Signaling Technology (CST). The membrane was incubated with the
corresponding horseradish peroxidase labeled secondary antibody
(1:2000; CST) at room temperature for 1 h. Membranes were

618
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Acknowledgements
This work was funded by CSIR, New Delhi under the XIIth Five
year plan project ‘Affordable Cancer Therapeutics-ACT’ (CSC-0301).
RT thanks CSIR, New Delhi and SB thanks Ministry of Chemicals and
Fertilizers for providing fellowships.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.01.030.
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