Synthesis and antitumor activity of 5-(5-halogenated-2-oxo-1H-pyrrolo[2,3-b]pyridin-(3Z)-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxamides

Article abstract of DOI:10.1016/j.bmcl.2015.05.017

We report herein the design and synthesis of a series of novel 5-halogenated-7-azaindolin-2-one derivatives containing a 2,4-dimethylpyrrole moiety. Nine target compounds with ≥70% inhibition against MCF-7 at 30 μM were further evaluated for their in vitro antitumor activity against seven human cancer cell lines by SRB assay. Results reveal that some compounds have potent antitumor activity, and the most active 13c7 (IC₅₀s: 4.49-15.39 μM) was found to be more active than Sunitinib (IC₅₀s: 4.70->30 μM) against all of the tested cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2015.05.017

Bioorganic & Medicinal Chemistry Letters 25 (2015) 2782–2787
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antitumor activity of 5-(5-halogenated-2-oxo-1H-
pyrrolo[2,3-b]pyridin-(3Z)-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-
3-carboxamides
Minghua Wang , Cheng Ye , Mingliang Liu a, , Zhaoyang Wu a,b, Linhu Li , Chunlan Wang ,
a,
a,
⇑
a
a
a
a
Xiujun Liu , Huiyuan Guo
a
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
New Drug Research & Development Center, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 February 2015
Revised 4 May 2015
Accepted 7 May 2015
Available online 15 May 2015
We report herein the design and synthesis of a series of novel 5-halogenated-7-azaindolin-2-one deriva-
tives containing a 2,4-dimethylpyrrole moiety. Nine target compounds with P70% inhibition against
MCF-7 at 30
cell lines by SRB assay. Results reveal that some compounds have potent antitumor activity, and the most
active 13c7 (IC50s: 4.49–15.39 M) was found to be more active than Sunitinib (IC50s: 4.70–>30 M)
against all of the tested cancer cell lines.
lM were further evaluated for their in vitro antitumor activity against seven human cancer
l
l
Keywords:
Ó 2015 Elsevier Ltd. All rights reserved.
7
-Azaindolin-2-ones
Synthesis
Antitumor activity
In the past decade, a numerous diverse scaffolds have been dis-
c-Kit both enzymatically and cellularly (<50 nM).8 In the above
studies, however, any group was not placed at the N-1 position
of the indolin-2-one scaffold. Therefore, we had focused our atten-
tion on introducing proper functional groups to this position in our
previous work. As novel lead compounds discovered in our lab, Z24
(Fig. 1) and LK-B030 (Fig. 1) bearing a (piperidin-1-yl)methyl and a
(3-dimethylamino)propyl group at the N-1 position, respectively,
were found to have a broad spectrum of antitumor activity by
1–3
covered as the multi-targeted tyrosine kinase inhibitors.
As an
important class of compounds with potent antitumor activity,
indolin-2-ones have been the subject of intense research recently.
Sunitinib (Su11248, Fig. 1), a small-molecule multi-targeted recep-
tor tyrosine kinase inhibitor with activity against many human
4
cancer cell lines, was approved for the treatment of advanced
renal cell carcinoma (RCC) and irresponsible or intolerant to
9
–11
Imatinib mesilate therapy on gastrointestinal stromal tumor
inhibiting angiogenesis in new blood vessels.
5
(
GIST) by the US Food and Drug Administration (FDA) in 2006.
Structural modifications mainly at the 3- and 5-positions of the
On the other hand, 7-azaindole (not indolin-2-one) scaffold
displays many pharmacological profiles. Beside the antitumor
activity, there are reports on antibacterial, antiviral, antihyperten-
sive, antiasthmatic, antiosteoporotic and antidepressant activities
indolin-2-one ring have made considerable progress in the ability
to increase antitumor activity through inhibition on different
receptors in recent years. It was reported that 5-carbamido-in-
dolin-2-one derivative BX-517 (Fig. 1) has single-digit nanomolar
activity against phosphoinositide-dependent kinase-1 (PDK1) and
excellent selectivity against protein kinase A (PKA).6 Sunitinib
analogs A and B (Fig. 1) containing a pyrrolo-fused-heterocycle
moiety at the 3-position of indolin-2-one core were reported to
be potent inhibitors of vascular endothelial growth factor receptor
1
2–16
of this class of compounds.
It is well-known that the replace-
ment of the quinoline core with 1,8-naphthyridine has attracted
great attention and led to the discovery of some new quinolone
drugs, such as Gemifloxacin (one of the fourth generation of quino-
lone antibacterials) and Vosaroxin (the first and only quinolone
,7
1
7,18
antitumor drug).
However, 7-azaindolin-2-one antitumor
agents, as we all know, have not been reported in the literature.
As part of our continuing modifications on Sunitinib as potential
antitumor drug candidates, we planned to take the place of the CH
at the 7-position of Sunitinib with N atom and meanwhile do
(
VEGFR), platelet-derived growth factor receptor (PDGFR), and
⇑
Corresponding author. Tel./fax: +86 10 63036965.
E-mail address: lmllyx@126.com (M. Liu).
These authors contributed equally to this work.
structural modifications at the 5-position of the 7-azaindolin-
0
2
-one core and the 3 -position of the pyrrole ring. Thus, a series
http://dx.doi.org/10.1016/j.bmcl.2015.05.017
960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
0

M. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2782–2787
2783
O
O
H
N
N
N
N
(
)n
N
N
N
H
H
N
H
H
H2N
F
F
O
O
O
O
N
H
N
H
N
H
Sunitinib
BX-517
A: n = 1 B: n = 2
O
H
N
N
N
H
N
H
F
O
O
N
N
N
N
Z-24
LK-B030
Figure 1. Structures of some indolin-2-ones.
of novel 5-halogenated-7-azaindolin-2-one derivatives containing
a 2,4-dimethylpyrrole moiety were designed, synthesized and
evaluated for their antitumor activity in this study. Our primary
objective was to optimize the potency of these compounds against
a set of solid tumors and contribute to the development of new
antitumor agents. A preliminary structure–activity relationship
(4b) respectively, by coupling with N-(2-hydroxyethyl)phthalim-
2
1
ide and hydrazinolysis successively. Condensation of the amines
6a,b with the acid 2 gave pyrrole-3-carboxamides 7a,b (Scheme 2).
Treatment of 1-aminopropan-2-ol (8) with tert-butyldiphenyl-
chlorsilane (TBDPSCl) in the presence of imidazole yielded selec-
tively hydroxy-protected compound 9, the latter condensed with
the acid 2 and then the TBDPS protecting group was removed by
tetra-n-butylammonium fluoride (TBAF) in tetrahydrofuran (THF)
to give 5-formyl-N-(2-hydroxyl-propyl)-2,4-dimethyl-1H-pyrrole-
3-carboxamide (11) (Scheme 3).
(
SAR) study is also explored to facilitate the further development
of 7-azaindolin-2-ones.
Detailed synthetic pathways to pyrrole-3-carboxamides (3a–f,
7
a,b, 11) and 5-halogenated-7-azaindolin-2-one derivatives
(
13a–c) are depicted in Schemes 1–4, respectively. According to
Finally, the target compounds 13a–c were prepared via aldol
condensation of 5-formyl-2,4-dimethylpyrrole derivatives (1–3,
7a,b, 11) with 5-halogenated(F, Cl, Br)-7-azaindolin-2-ones
(12a–c) in the presence of piperidine (Scheme 4). All of the new
well-established literature procedures,1
available ethyl 5-formyl-2,4-dimethylpyrrole-3-carboxylate
was hydrolyzed to the corresponding carboxylic acid 2, which upon
0,11,19,20
commercially
1
1
amidation with different primary amines (RNH
1
2
) in the presence of
hydrochloride
synthetic compounds were well characterized by H NMR, MS,
1
3
22
-ethyl-3-(3-dimethylaminopropyl)carbodiimide
HRMS and C NMR in part. As expected, the pyrrole-2-methyli-
dene geometry at the 3-position of 7-azaindolin-2-one ring was
(
EDCꢀHCl) and N-hydroxybenzotriazole (HOBt) was converted to
pyrrole-3-carboxamides 3a–f (Scheme 1).
-Phenoxyethanamine (6a) and 2-(benzyloxy)ethanamine (6b)
1
0,11
confirmed to have the Z-configuration.
2
For preliminary screening of antitumor candidates, all the
were conveniently obtained from phenol (4a) and benzyl bromide
newly synthesized 7-azaindolin-2-one derivatives 13a–c were first
O
O
O
R
NH
N
OH
3a: R = (CH2)2NMe2
3b: R = (CH2)2NEt2
3d: R =
OEt
1
) KOH,MeOH
H2O, ref., 3h
RNH2
EDC HCl, HOBt, DMF
O
O
O
.
N
H
N
N
H
2) HCl, 94%
N
H
3
e: R =
f: R =
H
0
°C-rt, 12h, 50-60%
H
H
O
1
2
3a-f
3
c: R =
N
3
Scheme 1. Synthesis of pyrrole-3-carboxamides 3a–f.
O
DEAD,P(Ph)3, THF, rt,
O
R
OH
6
h, 94% (4a)
O
+
N
85% NH NH . H O
2 2 2
N
n
or NaH, THF, 4h, 90% (4b)
EtOH, 50r&, 4h, 90%
O
4
4
a: R = OH
b: R = CH2Br
O
5a,b
O
O
n
NH2
O
2, EDC•HCl, HOBt,DMF
°C-rt, 12h,50-60%
NH
4a-7a: n = 0
4b-7b: n = 1
0
O
6
a,b
N
H
H
7
a,b
Scheme 2. Synthesis of pyrrole-3-carboxamides 7a,b.

2
784
M. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2782–2787
O
N
O
N
H
imidazole, TBDPSCl
2, EDC•HCl, HOBt,DMF
H
OTBDPS
TBAF, THF
rt, 5.5h, 90%
OH
H2N
H2N
O
O
OH CH2Cl2, rt, 4.5h, 98%
OTBDPS
0-4°C, 12h, 60%
N
H
N
H
H
H
8
9
10
11
Scheme 3. Synthesis of pyrrole-3-carboxamide 11.
antitumor activity in seven human cancer cell lines, including
MCF-7 (breast cancer), HepG2 (liver carcinoma), HT-29 (colon ade-
nocarcinoma), A549 (lung adenocarcinoma), PANC-1 (pancreatic
carcinoma), Hela (cervical cancer) and Skov-3 (ovarian carcinoma)
by SRB assay. The IC50 values were compared with those of
Sunitinib (Table 2).
The selected target 7-azaindolin-2-ones have potent in vitro
antitumor activity against the tested human cancer cell lines.
Among them, compounds 13c3, 13c4 and 13c7 (IC50s:
O
Z
X
1
-3, 7a,b, 11
O
N
N
H
piperidine, EtOH
rt, 3h, 50-70%
X
H
13a1-11: X = F
3b1-8: X = Cl
13c1-8: X = Br
N
23
1
O
1
1
1
2a: X = F
2b: X = Cl
2c: X = Br
N
H
13a-c
N
See Table 1 for structures
Scheme 4. Synthesis of 7-azaindolin-2-one derivatives 13a–c.
3
.99–27.8
(IC50s: 4.70–>30
most active compound 13c7 (IC50s: 4.49–15.39
l
M) are more active than or comparable to Sunitinib
M) against all of the cell lines. In particular, the
M) was found to
investigated for cytotoxic activity in vitro against MCF-7 (human
breast cancer cell line), and the compounds having P70% inhibi-
l
l
tion at the concentration of 30
inhibition concentrations) determination. Nine target compounds
13a4, 13a6, 13b4, 13b6, 13b7, 13c3, 13c4, 13c6, 13c7) meeting
l
M were subjected to IC50s (50 %
be more potent than the reference drug against these cancer cell
lines.
According to the biological evaluation results showed in Tables
1 and 2, antitumor activity of the 7-azaindolin-2-one derivatives
(
this criterion (Table 1) were further evaluated for their in vitro
Table 1
Structures and in vitro activity of compounds 13a–c against MCF-7 (at 30
l
M)
O
Z
N
H
X
O
N
H
N
Compounds
X
F
Z
% inhibition
60.4
Compounds
X
Z
% inhibition
88.9
1
3a1
OEt
13b4
Cl
NH(CH
)
2 2
NEt
2
O
1
3a2
F
F
F
OH
20.5
61.1
70.7
13b5
13b6
13b7
Cl
Cl
Cl
N
6.0
88.4
91.3
HN
N
H
N
1
3a3
3a4
NH(CH
2
)
)
2
NMe
2
N
1
NH(CH
2
2
NEt
2
HN
HN
O
1
3a5
3a6
F
F
N
15.6
78.0
13b8
13c1
Cl
6.2
6.3
HN
N
H
N
1
Br
OEt
1
1
1
3a7
3a8
3a9
F
F
F
N
37.5
6.3
13c2
13c3
13c4
Br
Br
Br
OH
19.6
79.4
89.7
HN
HN
NH(CH
NH(CH
2
)
2
2
NMe
2
H
N
2.8
2
)
NEt
2
O
O
H
N
O
13a10
13a11
13b1
F
4.1
0
13c5
13c6
13c7
Br
Br
N
6.0
82.8
86.9
HN
H
N
N
H
N
F
OH
N
Cl
OEt
54.8
Br
Br
HN
HN
1
3b2
3b3
Cl
Cl
OH
15.8
45.7
13c8
5.5
1
NH(CH
2
)
2
NMe
2
Sunitinib
94.0

M. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2782–2787
2785
Table 2
References and notes
In vitro activity of selected compounds against seven cells
1
2
.
.
Li, X.; Gao, J. H.; Chen, G. L. J. Shenyang Pharm. Univ. 2011, 28, 1005.
Wang, Y.; Long, Y. Q. Chin. J. Org. Chem. 2011, 31, 1595.
Cell lines
IC50 (lM)
Compounds MCF-7 HepG2 HT-29 A549 PANC-1 Hela
Skov-3
3. Cao, X.; You, Q. D.; Li, Z. Y.; Wang, X. J.; Lu, X. Y.; Liu, X. R.; Xu, D.; Liu, B. Mini-
Rev. Med. Chem. 2008, 8, 1053.
4
5
1
1
1
1
1
1
1
1
1
3a4
3a6
3b4
3b6
3b7
3c3
3c4
3c6
3c7
>30
>30
14.99
21.16
12.41
12.69
7.87
6.27
5.72
6.93
5.57
12.51
5.04
3.99
5.57
4.49
4.70
—
—
>30
>30
—
—
—
—
.
.
Zhu, X. Q.; Ye, M. Chin. J. New Drugs Clin. Rem. 2007, 26, 474.
Morabito, A.; De Maio, E.; Di Maio, M.; Normanno, N.; Perrone, F. Oncologist
26.28
>30
15.58
14.09
27.8
>30
10.32 13.98
16.88 >30
11.43 10.35
4.57
6.46
15.17 26.94
29.35 8.28
>30 16.40
24.46 16.12
17.86 4.98
26.66 11.79
2006, 11, 753.
6
.
Islam, I.; Bryant, J.; Chou, Y. L.; Kochanny, M. J.; Lee, W.; Phillips, G. B.; Yu, H. Y.;
Adler, M.; Whitlow, M.; Ho, E.; Lentz, D.; Polokoff, M. A.; Subramanyam, B.; Wu,
J. M.; Zhu, D.; Feldman, R. I.; Arnaiz, D. O. Bioorg. Med. Chem. Lett. 2007, 17,
3814.
6.57
11.55
15.58
12.03
21.81
9.36
>30
15.39 5.15
>30 9.21
19.20
7. Islam, I.; Brown, G.; Bryant, J.; Hrvatin, P.; Kochanny, M. J.; Phillips, G. B.; Yuan,
S. D.; Adler, M.; Whitlow, M.; Lentz, D.; Polokoff, M. A.; Wu, J.; Shen, J.; Walters,
J.; Ho, E.; Subramanyam, B.; Zhu, D.; Feldman, R. I.; Arnaiz, D. O. Bioorg. Med.
Chem. Lett. 2007, 17, 3819.
15.09
25.41
4.82
7.93
6.20
14.94
Sunitinib
16.06
8.
Tang, P. C.; Su, Y. D.; Feng, J.; Fu, J. H.; Yang, J. L.; Xiao, L.; Peng, J. H.; Li, Y. L.;
Zhang, L.; Hu, B.; Zhou, Y.; Li, F. Q.; Fu, B. B.; Lou, L. G.; Gong, A. S.; She, G. H.;
Sun, W. H.; Mong, X. T. J. Med. Chem. 2010, 53, 8140.
1
7
3a–c depends on both of the halogen atom at the 5-position of the
-azaindolin-2-one ring and the side chain at the 3 -position of the
0
9. Lu, H.; Lin, C.; Zheng, Z.; Li, S.; Guo, S.; Zhang, X.; Fu, M.; Liang, X.; Wu, M. J.
Pharmacol. Sci. 2005, 97, 533.
pyrrole ring. In general, 5-bromo-7-azaindolin-2-ones are gener-
ally more active than the corresponding 5-chloro analogs, and 5-
fluoro analogs have the worst rating of the threes with a few
exceptions (Table 2). As expected, converting the ester (13a1,
Z = OEt) and acid (13a2, Z = OH) into carboxamides
10. Lv, K.; Wang, L. L.; Liu, M. L.; Zhou, X. B.; Fan, S. Y.; Liu, H. Y.; Zheng, Z. B.; Li, S.
Bioorg. Med. Chem. Lett. 2011, 21, 3062.
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Med. Chem. Res. 2013, 22, 1723.
1
1
2. Tan, X.; Zhou, Y.; Yao, Q. Z. World Notes Antibiot. 2010, 31, 207.
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Massuard, M. C. Bioorg. Med. Chem. Lett. 2007, 17, 1934.
[
2 2 2 2 2 2
Z = NH(CH ) NMe (13a3) or NH(CH ) NEt (13a4)], just like in
1
4. Echalier, A.; Bettayeb, K.; Ferandin, Y.; Lozach, O.; Clément, M.; Valette, A.;
Liger, F.; Marquet, B.; Morris, J. C.; Endicott, J. A.; Joseph, B.; Meijer, L. J. Med.
Chem. 2008, 51, 737.
the case of Sunitinib, enhances the antitumor activity (Table 1).
For heterocyclic groups, pyrrolidine (equivalent to the closed-ring
one of diethylamine) makes more contribution to antitumor activ-
ity than piperidine (13b7 vs 13b6, 13c7 vs 13c6, Table 2), while
morpholine is the lowest (Table 1). Surprisingly, replacing the
diethylamine in 13a4 by either 2-phenoxy (13a9) or benzyloxy
15. Liddlea, J.; Bamborougha, P.; Barkera, M. D.; Camposa, S.; Cousinsa, R. P. C.;
Cutlerb, G. J.; Hobbsa, H.; Holmesa, D. S.; Ioannoua, C.; Mellorb, G. W.; Morsea,
M. A.; Paynea, J. J.; Pritcharda, J. M.; Smitha, K. J.; Tapea, D. T.; Whitwortha, C.;
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(13a10) markedly reduces activity (although O atom is a isostere
of N), and introduction of a free hydroxyl group (13a11) leads to
the loss of antitumor activity. In addition, N-cyclopropylcarboxam-
ide at the 3 -position of the pyrrole ring (13a8, 13b8, 13c8) is not
preferred (Table 1). The above SARs suggest that candidates with
better activity than Sunitinib, may be obtained from a good
combination of the two substituents at the 5-position of the
0
18. Tsuzuki, Y.; Tomita, K.; Shibamori, K.; Sato, Y.; Kashimoto, S.; Chiba, K. J. Med.
Chem. 2004, 47, 2097.
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0
7
-azaindolin-2-one ring and the side chain at the 3 -position of
20. Zou, H. B.; Zhang, L.; Ouyang, J. F.; Giulianotti, M. A.; Yu, Y. P. Eur. J. Med. Chem.
the pyrrole ring. Based on this, further studies on structural mod-
ifications and related mechanism of action are currently in
progress.
2
011, 46, 5970.
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2. To a stirring solution of 1(19.4 g, 100 mmol) in methanol (100 mL) and water
2
In summary, a series of novel 5-halogenated-7-azaindolin-2-one
(
400 mL) was added 5 N KOH solution (200 mL). The reaction mixture was
derivatives containing
a
2,4-dimethylpyrrole moiety were
heated to refluxing and stirred for 3 h. Cooled to room temperature, the
mixture was adjusted to pH 3 with 5 N HCl, and then filtered. The precipitate
was washed with water and dried in vacuo to give the title compound 2
1
designed, synthesized and characterized by H NMR, MS, HRMS
1
3
and
C NMR in part. Nine target compounds having P70%
M were further evaluated for their
(
15.5 g, 94 %) as an yellowish brown solid. A solution of compound 2 (167 mg,
1 mmol), EDCꢀHCl (230 mg, 1.2 mmol) and HOBt (162 mg, 1.2 mmol) in DMF
15 mL) was stirred at 0–4 °C under the atmosphere of nitrogen for 20 min. The
inhibition against MCF-7 at 30
l
(
in vitro antitumor activity against seven human cancer cell lines by
SRB assay. Results reveal that some compounds show potent
antitumor activity, and the most active compound 13c7 is
found to have better activity than Sunitinib against all of the tested
cancer cell lines. The results reported here provide a foundation for
further improvement of the potency of these compounds to
discover more potent novel 7-azaindolinone antitumor agents in
future studies.
substituted amine a–f (2 mmol) was added to the mixture and stirred at the
same temperature for 30 min and then overnight at room temperature. The
mixture was diluted with water and adjusted to pH 9 with saturated Na CO3
2
solution and then extracted with dichloromethane and methanol (9:1, v/v).
The combined extracts were dried over anhydrous Na SO , and concentrated
2 4
under reduced pressure to provide crude product 3a–f (50–60%) as red oils.
To a solution of 4a (1.88 g, 20 mmol), N-(2-hydroxyethyl)phthalimide (3.82 g,
20 mmol) and triphenylphosphine(7.87 g, 30 mmol) in tetrahydrofuran
(
3
30 mL) was added dropwise a solution of diethyl azodicarboxylate (4.7 mL,
0 mmol) in tetrahydrofuran (10 mL) at 0 °C over 0.5 h. The mixture was
stirred at the same temperature for 6 h and concentrated under reduced
Acknowledgments
pressure. The residue was diluted in diethyl ether and stirred at ꢁ5 to 0 °C for
30 min and filtered. The filtrate was concentrated under reduced pressure. The
residue was purified by column chromatography (silica gel), eluting with
petroleum ether and ethyl acetate (6:1, v/v) to give 5a (5.02 g, 94 %) as a off-
white solid. To a solution of N-(2-hydroxyethyl)phthalimide (2 g, 10.5 mmol)
in anhydrous tetrahydrofuran (30 mL) was added 60% NaH (0.84 g, 21 mmol)
at ꢁ5 °C. The mixture was stirred at the same temperature for 20 min.
Commercially available 4b (1.5 mL, 12.6 mmol)was added to the mixture and
was stirred at the same temperature for 30 min and then stirred at room
temperature for 4 h and filtered. The filtrate was concentrated under reduced
pressure to give 5b (2.65 g, 90%) as a off-white solid. To a solution of 5a,b
This work was supported by the National S&T Major Special
Project on Major New Drug Innovations (2012ZX09301002-001-
0
17/023, 2014ZX09507009-003) and NSFC 81373267-003.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.05.
(
3
3 mmol) in ethanol (30 mL) was added 85% hydrazine hydrate (0.19 mL,
.3 mmol) at 50 °C. The reaction mixture was stirred at the same temperature
0
17. These data include MOL files and InChiKeys of the most
for 4 h, cooled to room temperature and filtered. The filtrate was concentrated
important compounds described in this article.
under reduced pressure to give crude 6a,b (90%). 7a,b were obtained as red oils

2
786
M. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2782–2787
+
+
in a similar manner as for the preparation of 3a–f, yield 50–60%.
To solution of (5 g, 66.6 mmol) and imidazole (18 g, 266 mmol) in
methylene chloride (40 mL) was added dropwise solution of tert-
318.42 (M+H) . HRMS-ESI (m/z): Calcd for C15
H
13
O
3 3
N
Cl (M+H) : 318.06400;
1
a
8
Found: 318.06403. Compound 13b3, mp 275–277 °C.
H NMR (400 MHz,
a
DMSO-d
6
) d: 13.44 (s, 1H), 11.61 (s, 1H), 8.35 (d, J = 2.2 Hz, 1H), 8.02 (d,
butylchlorodiphenylsilane (22 g, 80 mmol) for 10 min. The mixture was
stirred at the same temperature for 4.5 h. The mixture was washed with
J = 2.2 Hz, 1H), 7.84 (s, 1H), 7.55 (t, J = 5.6 Hz, 1H), 3.34–3.28 (m, 2H), 2.45 (s,
3H), 2.42 (s, 3H), 2.39 (t, J = 6.8 Hz, 2H), 2.19 (s, 6H). MS-ESI (m/z): 388.79
+
+
water , saturated brine, dried over anhydrous Na
2
SO
4
, and concentrated under
(M+H) . HRMS-ESI (m/z): Calcd for C19
388.15355. Compound 13b4, mp 225–226 °C. H NMR (400 MHz, DMSO-d ) d:
H O N Cl (M+H) : 388.15348; Found:
23 2 5
1
reduced pressure to give crude 9 (20.46 g, 98%) as a colorless oil. 10 was
obtained as a red oil from 9 (6.27 g, 20 mmol) in a similar manner as for the
preparation of 3a–f, yield 60%. To a solution of 10 (13.78 g, 30 mmol) in
6
13.45 (s, 1H), 11.61 (s, 1H), 8.35 (d, J = 2.2 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.84
(s, 1H), 7.48 (t, J = 5.2 Hz, 1H), 3.28 (q, J = 6.2 Hz, 2H), 2.55 – 2.50 (m, 6H), 2.46
+
tetrahydrofuran (40 mL) was added
a
solution of tetrabutylammonium
(s, 3H), 2.43 (s, 3H), 0.98 (t, J = 7.1 Hz, 6H). MS-ESI (m/z): 416.31 (M+H) .
+
fluoride (1 M in tetrahydrofuran) (36 mL, 36 mmol) and stirred at room
temperature for 5.5 h and concentrated under reduced pressure. The residue
was diluted with ethyl acetate (20 mL), washed with water, saturated brine,
dried over anhydrous Na SO , and concentrated under reduced pressure to
2 4
give crude 11 (6.06 g, 90%) as a red solid.
A solution of 1–3, 7a,b, 11 (1 mmol) and 12a–c (0.7 mmol) in anhydrous
ethanol (10 mL) and piperidine (6 drops) was stirred at room temperature
under atmosphere of nitrogen for 2–3 h. The yellow precipitate was collected
by suction and washed with ethanol and then dried under vacuum to afford the
HRMS-ESI (m/z): Calcd for
C
21
H
27
O
N
2 5
Cl (M+H) : 416.18478; Found:
1
6
416.18492. Compound 13b5, mp 274–275 °C. H NMR (400 MHz, DMSO-d )
d: 13.46 (s, 1H), 11.57 (s, 1H), 8.37 (d, J = 2.2 Hz, 1H), 8.03 (d, J = 2.2 Hz, 1H),
7.86 (s, 1H), 7.55 (t, J = 5.5 Hz, 1H), 3.59 (t, J = 4.6 Hz, 4H), 3.36 (q, J = 6.4 Hz,
+
2H), 2.47 (s, 3H), 2.45 (s, 3H), 2.46–2.41 (m, 6H). MS-ESI (m/z): 430.87 (M+H) .
+
HRMS-ESI (m/z): Calcd for
C
21
H
25
O
3
N
5
Cl (M+H) : 430.16404; Found:
1
6
430.16419. Compound 13b6, mp 276–277 °C. H NMR (400 MHz, DMSO-d )
d: 13.45 (s, 1H), 11.60 (s, 1H), 8.35 (d, J = 2.2 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H),
7.84 (s, 1H), 7.71 (t, J = 5.8 Hz, 1H), 3.11 (t, J = 6.2 Hz, 2H), 2.72–2.78 (m, 2H),
2.44 (s, 3H), 2.41 (s, 3H), 2.15 (s, 3H), 1.83 (t, J = 10.8 Hz, 2H), 1.67–1.62 (m,
targeted compounds 13a1–11, 13b1–8, 13c1–8 (50–70%) as yellow solids.
1
+
Compound 13a1: mp 275–276 °C. H NMR (400 MHz, DMSO-d
6
) d: 13.72 (s,
2H), 1.53–1.40 (m, 1H), 1.26–1.13 (m, 2H). MS-ESI (m/z): 428.88 (M+H) .
+
1
7
3
H), 11.60 (s, 1H), 8.23 (dd, J = 9.2, 2.7 Hz, 1H), 8.00 (dd, J = 2.4, 2.0 Hz, 1H),
HRMS-ESI (m/z): Calcd for
C
22
H
27
O
N
2 5
Cl (M+H) : 428.18478; Found:
1
.85 (s, 1H), 4.22 (q, J = 7.1 Hz, 2H), 2.55 (s, 3H), 2.52 (s, 3H), 1.30 (t, J = 7.1 Hz,
6
428.18502. Compound 13b7, mp 265–267 °C. H NMR (400 MHz, DMSO-d )
+
H). MS-ESI (m/z): 330.23 (M+H) . HRMS-ESI (m/z): Calcd for C17
H
17
O
3 3
N F
d: 13.44 (s, 1H), 11.55 (s, 1H), 8.35 (d, J = 2.2 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H),
7.85 (s, 1H), 7.58 (t, J = 5.6 Hz, 1H), 3.30–3.34 (m, 2H), 2.45 (s, 3H), 2.42 (s, 3H),
2.39 (t, J = 6.8 Hz, 2H), 3.33 (q, J = 6.8 Hz, 2H), 2.56 (t, J = 6.9 Hz, 3H), 2.49–2.46
(m, 4H), 2.45 (s, 3H), 2.42 (s, 3H), 1.72–1.65 (m, 4H). 13C NMR (126 MHz,
+
1
(
M+H) : 330.12485; Found: 330.12481. Compound 13a2, mp >300 °C. H NMR
(
400 MHz, DMSO-d ) d: 13.68 (s, 1H), 12.25 (s, 1H), 11.59 (s, 1H), 8.23 (dd,
6
J = 9.2, 2.7 Hz, 1H), 8.01 (dd, J = 2.5, 1.8 Hz, 1H), 7.86 (s, 1H), 2.56 (s, 3H), 2.53
+
(
(
s, 3H). MS-ESI (m/z): 302.29 (M+H) . HRMS-ESI (m/z): Calcd for C15
H
13
O
3
N
3
F
H
6
DMSO-d ) d: 169.30, 164.34, 150.95, 142.10, 137.89, 131.87, 126.79, 126.04,
+
1
M+H) : 302.09355; Found: 302.09350. Compound 13a3, mp 272–274 °C.
) d: 13.50 (s, 1H), 11.52 (s, 1H), 8.19 (dd, J = 9.2,
.7 Hz, 1H), 7.98 (dd, J = 2.7, 1.7 Hz, 1H), 7.80 (s, 1H), 7.53–7.56 (t, J = 5.6 Hz,
H), 3.30-3.34 (m, 2H), 2.45 (s, 3H), 2.42 (s, 3H), 2.39 (t, J = 6.8 Hz, 2H), 2.20 (s,
H). MS-ESI (m/z): 372.23 (M+H) . HRMS-ESI (m/z): Calcd for C19
M+H) : 372.18303; Found: 372.18296. Compound 13a4, mp 220–221 °C.
125.28, 124.36, 121.72, 121.47, 111.17, 54.78 (2C), 53.54, 38.06, 23.22 (2C),
+
NMR (400 MHz, DMSO-d
6
13.31, 10.50. MS-ESI (m/z): 414.84 (M+H) . HRMS-ESI (m/z): Calcd for
+
2
1
6
C
21
H
25
O
2
N
5
Cl (M+H) : 414.16913; Found: 414.16917. Compound 13b8, mp
1
6
>300 °C. H NMR (400 MHz, DMSO-d ) d: 13.44 (s, 1H), 11.61 (s, 1H), 8.35 (d,
+
H
23
O
N
2 5
F
H
J = 2.2 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.84 (s, 1H), 7.77 (d, J = 4.1 Hz, 1H), 2.82–
+
1
(
2.77 (m, 1H), 2.41 (s, 3H), 2.39 (s, 3H), 0.71–0.66 (m, 2H), 0.54–0.50 (m, 2H).
+
NMR (400 MHz, DMSO-d
6
) d: 13.50 (s, 1H), 11.52 (s, 1H), 8.19 (dd, J = 9.2,
MS-ESI (m/z): 357.07 (M+H) . HRMS-ESI (m/z): Calcd for C18
H
18
O
N
2 4
Cl (M+H)+:
1
2
3
.7 Hz, 1H), 7.98 (dd, J = 2.7, 1.7 Hz, 1H), 7.81 (s, 1H), 7.48 (t, J = 5.6 Hz, 1H),
.28 (q, J = 6.4 Hz, 2H), 2.57–2.51 (m, 6H), 2.46 (s, 3H), 2.43 (s, 3H), 0.97 (t,
357.11128; Found: 357.11123. Compound 13c1, mp 296–298 °C. H NMR
(400 MHz, DMSO-d ) d: 13.66 (s, 1H), 11.69 (s, 1H), 8.51 (d, J = 2.1 Hz, 1H), 8.12
6
+
J = 7.1 Hz, 6H). MS-ESI (m/z): 400.69 (M+H) . HRMS-ESI (m/z): Calcd for
C
2
(d, J = 2.1 Hz, 1H), 7.90 (s, 1H), 4.23 (q, J = 7.1 Hz, 2H), 2.56 (s, 3H), 2.52 (s, 3H),
+
+
21
H
27
O
2
N
5
F (M+H) : 400.21433; Found: 400.21404. Compound 13a5, mp
1.31 (t, J = 7.1 Hz, 3H). MS-ESI (m/z): 390.20 (M+H) . HRMS-ESI (m/z): Calcd for
1
+
6
49–251 °C. H NMR (400 MHz, DMSO-d ) d: 13.50 (s, 1H), 11.52 (s, 1H), 8.19
C
17
H
17
O
N
3 3
Br (M+H) : 390.04478; Found: 390.04491. Compound 13c2, mp
1
(
dd, J = 9.2, 2.7 Hz, 1H), 7.98 (dd, J = 2.8, 1.7 Hz, 1H), 7.80 (s, 1H), 7.53 (t,
6
285–286 °C. H NMR (400 MHz, DMSO-d ) d: 13.61 (s, 1H), 12.25 (s, 1H), 11.67
J = 5.3 Hz, 1H), 3.59–3.56 (t, J = 4.4 Hz, 4H), 3.35 (q, J = 6.4z Hz, 2H), 2.45–2.46
(s, 1H), 8.50 (d, J = 2.0 Hz, 1H), 8.11 (d, J = 2.0 Hz, 1H), 7.90 (s, 1H), 2.55 (s, 3H),
2.52 (s, 3H). MS-ESI (m/z): 362.12 (M+H) . HRMS-ESI (m/z): Calcd for
+
+
(
m, 5H), 2.44–2.39 (m, 7H). MS-ESI (m/z): 414.85 (M+H) . HRMS-ESI (m/z):
+
Calcd for C21
H
25
O
3 5
N
F (M+H) : 414.19359; Found: 414.19351. Compound
C H O N Br (M+H)+: 362.01348; Found: 362.01347. Compound 13c3, mp
15 13 3 3
1
1
1
3a6, mp 246–247 °C. H NMR (400 MHz, DMSO-d
6
) d: 13.50 (s, 1H), 11.52 (s,
6
272–273 °C. H NMR (400 MHz, DMSO-d ) d: 13.44 (s, 1H), 11.60 (s, 1H), 8.46
1
H), 8.19 (dd, J = 9.2, 2.7 Hz, 1H), 7.98 (dd, J = 2.7, 1.7 Hz, 1H), 7.80 (s, 1H), 7.70
(d, J = 2.1 Hz, 1H), 8.10 (d, J = 2.1 Hz, 1H), 7.85 (s, 1H), 7.55 (t, J = 5.6 Hz, 1H),
(
t, J = 5.8 Hz, 1H), 3.11 (t, J = 6.3 Hz, 2H), 2.77–2.72 (m, 2H), 2.44 (s, 3H), 2.41 (s,
3.31 (q, J = 5.6 Hz, 2H), 2.45 (s, 3H), 2.42 (s, 3H), 2.39 (t, J = 6.8 Hz, 2H), 2.19 (s,
+
3
1
C
2
H), 2.14 (s, 3H), 1.84–1.77 (m, 2H), 1.67–1.62 (m, 2H), 1.49–1.43 (m, 1H),
6H). MS-ESI (m/z): 432.15 (M+H) . HRMS-ESI (m/z): Calcd for C19
(M+H) : 432.10296; Found: 432.10328. Compound 13c4, mp 242–244 °C.
H
23
O
2
N Br
H
5
+
+
1
.24–1.14 (m, 2H). MS-ESI (m/z): 412.89 (M+H) . HRMS-ESI (m/z): Calcd for
+
22
H
27
O
2
N
5
F (M+H) : 412.21433; Found: 412.21410. Compound 13a7, mp
NMR (400 MHz, DMSO-d ) d: 13.44 (s, 1H), 11.61 (s, 1H), 8.47 (d, J = 2.1 Hz,
6
1
49–250 °C. H NMR (400 MHz, DMSO-d
6
) d: 13.49 (s, 1H), 11.52 (s, 1H), 8.19
1H), 8.10 (d, J = 2.1 Hz, 1H), 7.85 (s, 1H), 7.49 (t, J = 5.5 Hz, 1H), 3.28 (q,
J = 6.4 Hz, 2H), 2.53 (q, J = 7.6 Hz, 6H), 2.46(s, 3H), 2.43 (s, 3H), 0.98 (s, 6H). MS-
(
dd, J = 9.2, 2.7 Hz, 1H), 7.98 (dd, J = 2.7, 1.7 Hz, 1H), 7.80 (s, 1H), 7.58 (t,
+
+
J = 5.6 Hz, 1H), 3.34 (q, J = 6.4 Hz, 2H), 2.57 (t, J = 6.9 Hz, 2H), 2.45 (s, 3H), 2.42
(
1
1
ESI (m/z): 460.20 (M+H) . HRMS-ESI (m/z): Calcd for C21
460.13426; Found: 460.13460. Compound 13c5, mp >300 °C.
(400 MHz, DMSO-d
H
27
O
2
N
5
Br (M+H) :
1
3
1
s, 3H), 1.71–1.67 (m, 4H). C NMR (126 MHz, DMSO-d
6
) d: 169.61, 164.59,
55.52, 148.89, 137.47, 131.59, 130.88(d, J = 27.3 Hz), 126.62, 125.89, 121.66,
21.37(d, J = 6.4 Hz), 113.72(d, J = 23.7 Hz), 111.87(d, J = 2.4 Hz), 56.06, 55.16
H NMR
6
) d: 13.44 (s, 1H), 11.62 (s, 1H), 8.47 (d, J = 1.7 Hz, 1H),
8.10 (d, J = 1.7 Hz, 1H), 7.85 (s, 1H), 7.57 (s, 1H), 3.63–3.58 (m, 4H), 3.40–3.32
(m, 2H), 2.47 (s, 3H), 2.44 (s, 3H), 2.48–2.36 (m, 6H). MS-ESI (m/z): 474.17
+
(
(
2C), 35.41, 18.60 (2C), 13.41, 10.57. MS-ESI (m/z): 398.63 (M+H) . HRMS-ESI
+
+
+
m/z): Calcd for
C
21
H
25
O
2
N
5
F
(M+H) : 398.19868; Found: 398.19890.
) d: 13.49 (s, 1H),
1.53 (s, 1H), 8.20 (dd, J = 9.2, 2.7 Hz, 1H), 7.99 (dd, J = 2.6, 1.8Z Hz, 1H), 7.80 (s,
(M+H) . HRMS-ESI (m/z): Calcd for C21
H O N Br (M+H) : 474.11353; Found:
25 3 5
1
1
Compound 13a8, mp >300 °C. H NMR (400 MHz, DMSO-d
6
3474.11374. Compound 13c6, mp 283–285 °C. H NMR (400 MHz, DMSO-d ) d:
6
1
1
0
13.44 (s, 1H), 11.58 (s, 1H), 8.46 (d, J = 2.1 Hz, 1H), 8.10 (d, J = 2.1 Hz, 1H), 7.84
(s, 1H), 7.70 (t, J = 5.8 Hz, 1H), 3.11 (t, J = 6.3 Hz, 2H), 2.78–2.72 (m, 2H), 2.44 (s,
3H), 2.41 (s, 3H), 2.14 (s, 3H), 1.84–1.77 (m, 2H), 1.68–1.62 (m, 2H), 1.50–1.40
H), 7.78 (d, J = 4.0 Hz, 1H), 2.80–2.77 (m, 1H), 2.42 (s, 3H), 2.39 (s, 3H), 0.72–
+
.66 (m, 2H), 0.55–0.50 (m, 2H). MS-ESI (m/z): 341.14 (M+H) . HRMS-ESI (m/z):
+
+
Calcd for C18
1
1
H
18
O
2
N
4
F (M+H) : 341.14083; Found: 341.14074. Compound
(m, 1H), 1.24–1.14 (m, 2H). MS-ESI (m/z): 472.22 (M+H) . HRMS-ESI (m/z):
1
+
3a9, mp 243–244 °C. H NMR (400 MHz, DMSO-d
6
) d: 13.50 (s, 1H), 11.52 (s,
Calcd for C22
H
27
O
N
2 5
Br (M+H) : 472.13426; Found: 472.13457. Compound
1
H), 8.19 (dd, J = 9.2, 2.7 Hz, 1H), 8.00–7.96 (m, 1H), 7.86 (t, J = 5.6 Hz, 1H), 7.80
6
13c7, mp 282–283 °C. H NMR (400 MHz, DMSO-d ) d: 13.43 (s, 1H), 11.59 (s,
(
s, 1H), 7.32–7.27 (m, 2H), 6.99–6.92 (m, 3H), 4.10 (t, J = 5.8 Hz, 2H), 3.60 (q,
1H), 8.46 (d, J = 2.1 Hz, 1H), 8.10 (d, J = 2.1 Hz, 1H), 7.85 (s, 1H), 7.58 (t,
+
J = 5.7 Hz, 2H), 2.44 (s, 3H), 2.42 (s, 3H). MS-ESI (m/z): 421.26 (M+H) . HRMS-
ESI (m/z): Calcd for
Compound 13a10, mp 216–217 °C. H NMR (400 MHz, DMSO-d
1
7
3
J = 5.6 Hz, 1H), 3.33 (q, J = 5.2 Hz, 2H), 2.56 (t, J = 6.9 Hz, 2H), 2.45 (s, 3H), 2.42
+
13
C
23
H
22
O
3
N
4
F 1(M+H) : 421.16705; Found: 421.16697.
) d: 13.50 (s,
H), 11.52 (s, 1H), 8.19 (dd, J = 9.2, 2.7 Hz, 1H), 8.00–7.96 (m, 1H), 7.80 (s, 1H),
6
(s, 3H), 1.68 (m, 4H). C NMR (126 MHz, DMSO-d ) d: 169.15, 164.34, 151.17,
6
144.22, 137.91, 131.92, 127.83, 126.83, 126.06, 122.29, 121.51, 112.59, 111.06,
+
54.78(2C), 53.55, 38.04, 23.21(2C), 13.32, 10.50. MS-ESI (m/z): 458.26 (M+H) .
+
.73 (t, J = 5.7 Hz, 1H), 7.35 (d, J = 4.4 Hz, 4H),7.32–7.26 (m, 1H), 4.52 (s, 2H),
.57 (t, J = 5.8 Hz, 2H), 3.44 (q, J = 5.7 Hz, 2H), 2.44 (s, 3H), 2.41 (s, 3H). MS-ESI
HRMS-ESI (m/z): Calcd for
C
21
H
25
O
2
N
5
Br (M+H) : 458.11861; Found:
1
6
458.11868. Compound 13c8, mp >300 °C. H NMR (400 MHz, DMSO-d ) d:
+
+
(
(
m/z): 435.25 (M+H) , 457.10 (M+Na) . HRMS-ESI (m/z): Calcd for C24
M+H)+: 435.18270; Found: 435.18290. Compound 13a11, mp 281–282 °C.
) d: 13.51 (s, 1H), 11.52 (s, 1H), 8.20 (dd, J = 9.2,
.7 Hz, 1H), 7.98 (dd, J = 2.7, 1.7 Hz, 1H), 7.81 (s, 1H), 7.58 (t, J = 5.8 Hz, 1H),
.72 (q, J = 4.8 Hz, 2H), 3.81–3.73 (m, 1H), 3.22–3.17 (m, 2H), 2.47 (s, 3H), 2.43
H
24
O
3
N
4
F
H
13.43 (s, 1H), 11.60 (s, 1H), 8.46 (d, J = 2.2 Hz, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.84
(s, 1H), 7.77 (d, J = 4.2 Hz, 1H), 2.82–2.76 (m, 1H), 2.41 (s, 3H), 2.39 (s, 3H),
1
+
NMR (400 MHz, DMSO-d
2
4
6
0.71–0.65 (m, 2H), 0.54–0.49 (m, 2H). MS-ESI (m/z): 401.08 (M+H) . HRMS-ESI
+
(m/z): Calcd for C18
H
18
O
2
N
4
Br (M+H) : 401.06076; Found: 401.06073.
23. SRB assay: Various human cancer cell lines were cultured in minimum
+
(
s, 3H), 1.10 (d, J = 6.4 Hz, 3H). MS-ESI (m/z): 359.23 (M+H) . HRMS-ESI (m/z):
essential medium (MEM), supplemented with 10% fetal calf serum,
+
Calcd for C18
1
H
20
O
3
N
4
F (M+H) : 359.31539; Found: 359.31540. Compound
100 units/mL penicillin and 100 mg/mL streptomycin in
a humidified
1
3b1, mp 294–295 °C. H NMR (400 MHz, DMSO-d
6
) d: 13.66 (s, 1H), 11.69z (s,
atmosphere in 5% CO at 37 °C. Cell culture media were renewed every three
2
1
H), 8.40 (d, J = 2.2 Hz, 1H), 8.05 (d, J = 2.2 Hz, 1H), 7.90 (s, 1H), 4.23 (q,
days, up to the confluence of the monolayer. Cell culture was washed upon
formation of confluent cultures, using trypsine-EDTA to detach the cells from
their culture flasks or dishes. Test compounds were stored at ꢁ18 °C and
solubilized in 100% dimethyl sulfoxide. Cells were seeded in 96-well plates at a
density of 104 for 24 h. After 24 h, cells were cultured with test compounds.
Drug concentrations were based on preliminary experiments and were
J = 7.1 Hz, 2H), 2.56 (s, 3H), 2.52 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H). MS-ESI (m/z):
3
+
+
46.23 (M+H) . HRMS-ESI (m/z): Calcd for C17
H
17
O
3
N
3
Cl (M+H) : 346.09530;
1
Found: 346.09519. Compound 13b2, mp 274–276 °C.
DMSO-d
.04 (d, J = 2.2 Hz, 1H), 7.89 (s, 1H), 2.55 (s, 3H), 2.52 (s, 3H). MS-ESI (m/z):
H NMR (400 MHz,
6
) d: 13.61 (s, 1H), 12.24 (s, 1H), 11.67 (s, 1H), 8.39 (d, J = 2.2 Hz, 1H),
8

M. Wang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2782–2787
2787
adjusted experimentally. After additional incubation of test compounds for
8 h, cells were fixed with 50% trichloroacetic acid(TCA) and SRB
sulforhodamine B) at 0.4% (w/v) in 1% acetic acid was added to stain cells.
Unbound SRB was washed out by 1% acetic acid and SRB-bound cells were
solubilized with 10 mM trizma base. The absorbance was read at a wavelength
of 570 nm. The percentage of cell survival was calculated, and IC50 for the
compounds could be derived using curve-fitting methods with statistical
analysis software SigmaPlot 10.0.
4
(