Design, synthesis and in vitro cytotoxicity evaluation of 5-(2-carboxyethenyl)isatin derivatives as anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2013.12.001

Forty four di- or trisubstituted novel isatin derivatives were designed and synthesized in 5-6 steps in 25-45% overall yields. Their structures were confirmed by ¹H NMR and ¹³C NMR as well as LC-MS. The anticancer activity of these new isatin derivatives against three human tumor cell lines, K562, HepG2 and HT-29, were evaluated by MTT assay in vitro. SAR studies suggested that the combination of 1-benzyl and 5-[trans-2- (methoxycarbonyl)ethen-1-yl] substitution greatly enhance their cytotoxic activity, whereas an intact carbonyl functionality on C-3 as present in the parent ring is required to such a potency. This study leads to the identification of two highly active molecules, compounds 2h (IC₅₀ = 3 nM) and 2k (IC₅₀ = 6 nM), against human leukemia K562 cells.

Full text of DOI:10.1016/j.bmcl.2013.12.001

Bioorganic & Medicinal Chemistry Letters 24 (2014) 591–594
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and in vitro cytotoxicity evaluation of
5-(2-carboxyethenyl)isatin derivatives as anticancer agents
Kailin Han a,b, Yao Zhou a,b, Fengxi Liu a,b, Qiannan Guo a,b, Pengfei Wang a,b, Yao Yang a,b
,
Binbin Song a,b, Wei Liu c, Qingwei Yao d, Yuou Teng a,b, , Peng Yu a,b,
⇑
⇑
a Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China
b Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China
c College of Sciences, Tianjin University of Science and Technology, Tianjin 300457, PR China
d Chemo Dynamics, Inc., 3 Crossman Road South, Sayreville, NJ 08872, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Forty four di- or trisubstituted novel isatin derivatives were designed and synthesized in 5–6 steps in 25–
45% overall yields. Their structures were confirmed by 1H NMR and 13C NMR as well as LC–MS. The
anticancer activity of these new isatin derivatives against three human tumor cell lines, K562, HepG2
and HT-29, were evaluated by MTT assay in vitro. SAR studies suggested that the combination of 1-benzyl
and 5-[trans-2-(methoxycarbonyl)ethen-1-yl] substitution greatly enhance their cytotoxic activity,
whereas an intact carbonyl functionality on C-3 as present in the parent ring is required to such a potency.
This study leads to the identification of two highly active molecules, compounds 2h (IC50 = 3 nM) and 2k
(IC50 = 6 nM), against human leukemia K562 cells.
Received 21 October 2013
Revised 14 November 2013
Accepted 2 December 2013
Available online 8 December 2013
Keywords:
Isatin derivatives
In vitro cytotoxicity
Antitumor activity
Ó 2014 Published by Elsevier Ltd.
Isatin is an indole derivative widely present endogenously in
both human and other mammalian tissues and fluids likely as a re-
sult of the tryptophan metabolic pathway. The versatility of isatin’s
molecular architecture makes it an ideal platform for structural
modification and derivatization as evidenced by the fact that many
isatin derivatives exhibit a broad range of biological activities such
as anticancer,1,2 antidepressant,3 anticonvulsant,4 antifungal,5
anti-HIV6 and anti-inflammatory,7 etc. In the last several decades,
increasing numbers of researchers from both industry and academia
have embarked on the development of new isatin-based anticancer
agents.8–16 Eshba’s group found that 5-bromoisatin 3-(2-nitro-
phenyl)hydrazone and a series of 5-[5-bromo-(2-oxo-3-indolinylid-
ene)]amino thiazolidine-2,4-diones substituted by various Mannich
bases exhibit antileukemic activity against P388 lymphocytic
leukemia in mice, respectively.12 Popp et al. has reported that some
3-hydroxy-3-substituted oxindoles obtained from condensation of
substituted isatins with cyclic ketones possess anticonvulsant
activity.13 SU11248 (Sutent), a 5-fluoro-3-substituted isatin deriva-
tive, was approved by the FDA in 2006 for the treatment of advanced
renal carcinoma and gastrointestinal stromal tumors.14,15 C5- and
C6-substituted isatin analogues were shown to be selective MAO B
inhibitors with 5-(4-phenylbutyl)isatin exhibiting the highest activ-
ity and being 18,500-fold more potent than isatin.16 Recently, the
groups of Mach17,18 and Reichert19 synthesized several N alkyl and
C-5 sulfonamido isatin analogues as small molecule caspase-3 and
caspase-7 inhibitors.
As part of our research program on the SAR study of isatin
derivatives’ anticancer property, herein we wish to report the syn-
thesis and antitumor activity of a series of N-benzylated isatins
possessing an acrylate moiety at C5 against three human cancer
cell lines, including human leukemia K562, human liver cancer
HepG2, and human colon HT-29.
As shown in Scheme 1, 5-substituted isatins 1a–1g were pre-
pared in two steps in 55–80% yield by following literature proce-
dures.20 Microwave-assisted Heck coupling reaction was
employed to converted 5-bromo isatin (1d) 5-trans-(2-methoxy-
carbonylethenyl)isatin (1h) in 70%. Further derivatization led to
the synthesis of compounds 2a–2p in good to excellent yield by
N-alkylation with alkyl halides in the presence of K2CO3.
Based on the identification of several major structure motifs
that main potentially improve isatin’s antitumor activity as
illustrated in Figure 1, the investigation was started with phenyl
portion’s substitution pattern which was followed by N-derivatization
and then C-3 variation.
The in vitro antitumor activities of the 5-substituted isatins
1a–1h against three human tumor cells, K562, HepG2 and HT-29
were evaluated by MTT assay with Camptothecin (CPT) as the
positive control.21,22 As can be seen from the data in Table 1, isatin
derivatives 1a–1f possessing simple substituent groups on the C5
position with different sizes and varing electronic properties do
not have significant impact on their activities. Interestingly, when
⇑
Corresponding authors. Tel.: +86 22 60601268; fax: +86 22 60602298.
E-mail addresses: tyo201485@tust.edu.cn (Y. Teng), yupeng@tust.edu.cn (P. Yu).
0960-894X/$ - see front matter Ó 2014 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2013.12.001

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K. Han et al. / Bioorg. Med. Chem. Lett. 24 (2014) 591–594
Table 1
NH2
R
O
Na2SO4
NH2OH
R
NOH
O
R
Antitumor activities of compounds 1a–1h in vitro
conc. H2SO4
55-80%
O
CCl3CH(OH)2
N
N
H
H
O
R1
1a-1g
O
O
O
O
OCH3
N
H
MeO
1d
O
PdCl2(PPh3)2, KOAc, DMF
(R = Br)
N
145 oC, MW, 70%
O
Compd
R1
IC50
(lM)
H
1h
O
K562
HepG2
HT-29
R2X, K2CO3
MeO
2a-2p
1h
CPT
1a
1b
1c
1d
1e
1f
—
H
F
Cl
Br
CH3
OCH3
0.040 0.02
>10
0.050 0.02
>10
0.060 0.01
>10
O
DMF, 75-90%
N
R2
>10
>10
>10
>10
>10
>10
>10
>10
Scheme 1. Synthesis of isatin derivatives (1a–1h, 2a–2p).
>10
>10
>10
>10
>10
>10
>10
1g
1h
CH2CH2CH2CH3
CH@CHCOOCH3
>10
0.22 0.06
>10
0.37 0.07
>10
0.27 0.01
a methyl acrylate group was introduced at C5, the resulting com-
pound 1h has a potency that is at least 130–230 times stronger
than isatin itself. However, compound 1g, which contains an n-
butyl group at C-5, has only a very minimal improvement in
potency. This observation suggests that the good inhibitory activity
of 1h does not simply correlate with the size of the acrylate moiety
and the limited spatial orientation due to its conjugation with the
aromatic ring may play a role.
With the identification of compound 1h as a potential lead, our
SAR study was then focused on the N-derivatized analogues. Re-
cently, some N-benzyl isatin derivatives have been reported to exhi-
bit a broad range of biological activities, including activity related to
the treatment of Neuropathic Pain,23 reverse transcriptase inhibi-
tor,24 caspase inhibitory activity17,25 and in vitro cytotoxicity.2,8,26
Therefore, it seemed highly likely that the combination of C-5 olef-
ination with an acrylate moiety and N-substitution may further
enhance their potency.
Table 2
Antitumor activities of compounds 2a–2p in vitro
O
O
H3CO
O
N
R2
Compd R2
IC50
(lM)
K562
HepG2
HT-29
CPT
1h
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
—
H
CH3
0.040 0.02
0.22 0.06
0.34 0.09
0.17 0.11
0.040 0.02
0.030 0.03
0.030 0.001 0.050 0.01 0.050 0.001
0.040 0.02
0.040 0.01
0.0030 0.001 0.030 0.02 0.030 0.01
0.030 0.01
0.060 0.03
0.0060 0.001 0.040 0.05 0.030 0.01
0.030 0.01
0.070 0.05
0.050 0.02 0.060 0.01
0.37 0.07
0.53 0.19
0.40 0.01
0.040 0.01 0.070 0.02
0.040 0.02 0.060 0.02
0.27 0.01
0.30 0.05
0.21 0.05
As shown in Table 2, simple alkyl groups such as the N-methyl
and N-ethyl (compounds 2a and 2b) do not result in a significant
change in the cell growth inhibition compared to 1h. This observa-
tion also suggests that the N–H group is not a necessary structure
unit for the activity of 1h. Recognizing that the N-methyl and
N-ethyl groups maybe too small to cause a significant effect in po-
tency, a benzyl group was introduced to the N-1 position. Pleas-
ingly, compound 2c was found to exhibit a potency with a 5–9
fold improvement, which suggests that larger groups at the N-1
position could increase the potency. Further test on compounds
2d–2f (with an electron withdrawing group) and 2g, 2p (with an
electron donating group) showed that these derivatives do not dif-
fer significantly with the benzyl 2c in their potency.
CH2CH3
CH2C6H5
CH2C6H4Fa
CH2C6H4CNa
a
CH2C6H4CF3
CH2C6H4CH3
0.070 0.04 0.040 0.001
0.040 0.01 0.030 0.01
a
a
CH2C6H4OCH3
CH2C6H4Cla
0.040 0.01 0.060 0.03
0.16 0.09 0.21 0.08
CH2C6H3ClbCla
CH2C6H4Bra
CH2C6H4Brb
CH2C6H4Brc
0.17 0.08
0.26 0.07
0.24 0.02
0.24 0.11
0.10 0.06
0.20 0.02
2m
2n
2o
2p
CH2C10H7
0.040 0.001 0.090 0.08
0.060 0.02
0.040 0.03
CH2CH2OC6H5
0.21 0.22
0.15 0.10 0.070 0.03
b
CH2C6H3CH3bCH3
However, compounds 2h27 and 2k28 containing a 4-methoxy
and 4-bromo benzyl group, respectively, exhibit excellent activity
against human cancer cell lines. For K562, their potency reached
a
b
c
Substitution at the para position.
Substitution at the meta position.
Substitution at the ortho position.
R2 = H, F, Cl, Br, CH3,
O
X,Y: =O; H, H; H, OH; =N-OH;
O
OCH3, But,
O
OR
NH2
N
X
Y
OPh
Br
Me
Et
O
O
R2
O
N
Cl
Cl
R1
Br
O
O
X
N
(X=Br, CF3, CN, H, CH3, F, Cl)
Figure 1. Structure modifications of isatin derivatives for SAR study.

K. Han et al. / Bioorg. Med. Chem. Lett. 24 (2014) 591–594
593
Table 3
procedure for the synthesis of 2h (Table 3). The 6- and 7-substi-
tuted 2r and 2s were found to be much less potent than 2h
(1000 folds) and the 4-substituted counterpart 2q (10 folds). Along
with the inferior potency of 2q as compared to 2h, these data
suggests that the presence of a methyl acrylate group at the
para-position found in 2h is vital to maintain a high level of
potency, probably as the result of electronic and/or geometric
preference.
In vitro antitumor activities of compounds 2q–2s
O
H3COOC
O
N
OCH3
After the N-substitution and C-substitution patterns were
established, target compounds 3a–3o were synthesized by micro-
wave-assisted Heck coupling reaction in a similar way for the syn-
thesis of 2h and their potency evaluated (Table 4). The results
indicated that the potency generally decreases with the increase
in the size of the alkyl group (3a, 3c, 3g and 3e vs 2h; 3b, 3d, 3h
and 3f vs 2k in going from Me to Et, Bu, 2-hydroxyethyl and t-
Bu). The presence of a free acrylic acid (3i) brought about a drastic
diminishment in potency, pointing to a detrimental effect of the
highly polar and readily ionizable free carboxyl group. All the
amide analogues examined (3j–3o) were found to possess low
activities relative to the esters either duo to the high polarity of
these groups or an increase in their sized in the case of 3l/3m
and 3n/3o.
To examine the importance of the C-3 carbonyl functionality,
eight new isatin derivatives (3p–3w) were synthesized from 2h
and 2k through an appropriate carbonyl group derivatization reac-
tion (Scheme 2). All these derivatives proved to be inferior candi-
dates as inhibitors of K562, HepG2, HT-29 (Table 5), suggesting
that the carbonyl functionality at C-3 is essential in order to main-
tain the observed high antitumor activity.
Compd
Position
IC50
(lM)
K562
HepG2
HT-29
CPT
2h
2q
2r
—
5
4
6
7
0.040 0.02
0.050 0.02
0.030 0.02
5.49 2.23
>10
0.060 0.01
0.030 0.01
3.12 0.96
>10
0.0030 0.001
0.35 0.05
4.86 0.56
4.13 1.27
2s
>10
>10
3 and 6 nM, respectively. It was also noticed that the potency of the
meta and ortho bromo-substituted benzyl compounds 2l
(0.030
l
M) and 2m (0.070
l
M) dropped by 5–11 folds when com-
pared to their para counterpart 2k (0.0060
l
M). Moreover, when
the N-1 substitution was changed to the more bulky 2-naphthyl-
methyl (2n) or the phenoxyethyl (2o) groups, the potency also
dinimished. The above results illustrated the importance of the size
and orientation for N-substitution in the acrylate-containing isatin
analogues for their anticancer activity.
To clarify the importance for the position of the acrylate moiety
in 2h, regioisomers 2q–2s were synthesized by using a similar
Preliminary results from a flow cytometric analysis conducted
in our laboratory revealed that compounds 2h and 2k could signif-
icantly induce the levels of apoptosis in K562 cells in vitro at low
micro molar concentrations (Fig. 2).29
Table 4
In conclusion, a series of 1,5-disubstituted and 1,3,5-trisubsti-
tuted isatin derivatives were synthesized and tested for their
in vitro antitumor activity against three strains of cancer cell lines
K562, HepG2, HT-29. The SAR study of these compounds led to the
identification of two new isatins, 2h and 2k, as highly potent anti-
cancer compounds with IC50 = 3 nM, IC50 = 6 nM, respectively,
against human leukemia K562 cells. Further chemo-biological
study of these two compounds with regards to their antitumor
pathway as well as their enzymatic targets and in vivo investiga-
tion are ongoing in this laboratory.
In vitro antitumor activities of compounds 3a–3q
O
R1
O
N
R2
Compd R1
R2
—
IC50
HepG2
0.050 0.02 0.060 0.01
(lM)
K562
HT-29
CPT
—
0.040 0.02
O
2h
2k
OCH3 0.0030 0.001 0.030 0.02 0.030 0.01
Br 0.0060 0.001 0.040 0.05 0.030 0.01
CH3O
O
3a
3b
OCH3 0.010 0.002 0.090 0.01 0.060 0.01
OH
H3COOC
Br
0.030 0.01
0.29 0.31 0.050 0.04
H3COOC
EtO
O
O
N
O
3c
OCH3 0.040 0.02
Br 0.15 0.01
0.63 0.17 0.42 0.04
0.65 0.21 0.60 0.06
N
3d
BuO
tBuO
R
3p
3t
:
: R = O CH3
R = OCH3
R
a
O
3e
3f
OCH3 0.050 0.04
Br 0.29 0.04
0.62 0.02 0.48 0.02
2.05 0.21 0.73 0.01
3q: R = Br
3u: R = Br
c
O
3g
3h
OCH3 0.040 0.01
Br 0.050 0.03
0.86 0.13 0.55 0.16
3.66 1.51 0.68 0.16
2h 2k
/
HO(CH2)2O
O
b
d
3i
OCH3 >10
>10
>10
HO
O
OH
O
N
O
O
3j
3k
OCH3 0.20 0.02
0.89 0.18 0.56 0.16
>10 0.55 0.15
H3COOC
H3COOC
Br
0.30 0.01
O
H2N
O
N
N
3l
3m
OCH3 0.72 0.24
Br 1.38 0.40
7.38 2.26 4.24 0.74
7.79 1.66 4.59 0.55
3v: R = OCH3
3w
3r: R = OCH3
3s
R
R
: R = Br
: R = Br
N
O
O
3n
3o
OCH3 0.46 0.11
Br 1.71 0.53
7.81 0.55 5.23 0.34
6.30 0.90 4.42 0.58
Scheme 2. C-3 modification of isatin derivatives (3p–3w). Reagents and conditions:
(a) NaBH4, MeOH, rt,50–55%; (b) ethylene glycol, pTSA, reflux, 85–90%; (c)
hydrazine monohydrate, EtOH, reflux, 60–65%; (d) NH2OHÁÁHCl, EtOH, reflux, 80–
90%.
N

594
K. Han et al. / Bioorg. Med. Chem. Lett. 24 (2014) 591–594
Table 5
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.12.
001.
Antitumor activity of the C-3 derivatized compounds 3p-3w
O
O
References and notes
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O
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C-3
—
IC50
HepG2
0.050 0.02 0.060 0.01
(lM)
K562
HT-29
CPT
2h
2k
3p
3q
3r
—
0.040 0.02
OCH3 @O
Br
0.0030 0.010 0.030 0.02 0.030 0.01
0.0060 0.001 0.040 0.05 0.030 0.01
OCH3 –OHÁH
0.030 0.01
0.26 0.03
2.11 1.13
2.92 0.34
0.99 0.56
3.16 0.21
0.38 0.07
0.67 0.12
3.56 1.54
9.96
9.17
>10
0.32 0.03
0.66 0.17
4.94 2.54
3.64 1.19
3.72 0.03
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3v
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2.63 0.03
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7.47 2.15
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5.11 1.38
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Figure 2. Compounds 2h and 2k induced apoptosis in K562 cells. (A) Phase contrast
images of K562 cells after culture with 30 nM 2 h or 2k for 12 h. (B) Apoptosis was
evaluated by Annexin-V and PI staining of K562 cells cultured in the absence or
presence of 30 nM 2h or 2k for 12 h.
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27. Analytical data of 2h: 1H NMR (400 MHz, CDCl3) d 3.78 (s, 3H), 3.80 (s, 3H), 4.88
(s, 2H), 6.33–6.37 (d, 1H, J = 16.0 Hz), 6.83 (d, 1H, J = 8.4 Hz), 6.87 (d, 2H,
J = 8.4 Hz), 7.26 (d, 2H, J = 8.8 Hz), 7.58 (d, 1H, J = 16.0 Hz), 7.62 (m, 1H), 7.77 (s,
1H). 13C NMR (100 MHz, CDCl3) d 43.78, 51.85, 55.31, 111.43, 114.52, 114.52,
118.08, 118.16, 124.10, 126.10, 128.95, 128.95, 130.49, 137.93, 142.43, 151.56,
158.11, 159.60, 166.92, 182.77. ESI-MS: 352.1 [M+H]+, 374.1 [M+Na]+.
28. Analytical data of 2k: 1H NMR (400 MHz, CDCl3) d 3.80 (s, 3H), 4.90 (s, 2H),
6.34–6.38 (d, 1H, J = 16.0 Hz), 6.77–6.79 (d, 1H, J = 8.0 Hz), 7.20–7.22 (d, 2H,
J = 8.4 Hz), 7.49 (d, 2H, J = 8.4 Hz), 7.59 (d, 1H, J = 16.0 Hz), 7.63 (m, 1H), 7.79 (s,
1H). 13C NMR (100 MHz, CDCl3) d 43.69, 51.88, 111.22, 118.10, 118.43, 122.42,
124.26, 129.14, 129.14, 130.80, 132.35, 132.35, 133.20, 138.00, 142.26, 151.11,
158.09, 166.85, 182.34. ESI-MS: 400.1 [M+H]+.
Acknowledgments
The authors sincerely thank the financial support from the Na-
tional Natural Science Foundation of China (81241104, 31301142),
the International Science & Technology Cooperation Program of
China (2013DFA31160) and the Ministry of Education Changjiang
Scholars and Innovative Research Team Development Plan
(IRT1166).
29. For flow cytometric analysis of apoptosis, phosphatidylserine exposure on the
outer leaflet of the plasma membrane was detected using the TACS Annexin V
Kit (Trevigen, Gaithersburg, MD, USA) according to the manufacturer’s
Supplementary data
instructions. In brief, 1 Â 106 cells were resuspended in 100
lL of binding
Supplementary data (synthetic procedures and analytical data
of all compounds and the 1H NMR spectra, 13C NMR spectra and
LC–MS data of 2h and 2k) associated with this article can be found,
buffer containing of annexin V-FITC and propidium iodide (PI) and incubated
for 15 min. After 400
lL of binding buffer was added, the cells were analysed
using a Becton Dickinson FACSscan flow cytometer (Mountain View, CA, USA).