Investigating the Anticancer Activity of Isatin/Dihydropyrazole Hybrids

Article abstract of DOI:10.1021/acsmedchemlett.8b00596

A series of isatin-dihydropyrazole hybrids have been synthesized in order to assess their potential as anticancer agents. In particular, 12 compounds were evaluated for their antiproliferative activity toward A549, IGR39, U87, MDA-MB-231, MCF-7, BT474, Bx

Full text of DOI:10.1021/acsmedchemlett.8b00596

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Letter
Investigating the Anticancer Activity of Isatin/Dihydropyrazole Hybrids
Rita Meleddu, Vilma Petrikaite, Simona Distinto, Antonella Arridu, Rossella Angius, Lorenzo
Serusi, Laura Skarnulyte, Ugn# Endriulaityt#, Migle Paskeviciute, Filippo Cottiglia, Marco
Gaspari, Domenico Taverna, Serenella Deplano, Benedetta Fois, and Elias Maccioni
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00596 • Publication Date (Web): 18 Dec 2018
Downloaded from http://pubs.acs.org on December 19, 2018
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Investigating the Anticancer Activity of Isatin/Dihydropyrazole
Hybrids
Rita Meleddua‡, Vilma Petrikaiteb,c‡, Simona Distintoa*, Antonella Arridua, Rossella Angiusd, Lorenzo
Serusia, Laura Škarnulytėb , Ugnė Endriulaitytėb, Miglė Paškevičiūtėb, Filippo Cottigliaa, Marco
Gasparie, Domenico Tavernae, Serenella Deplanoa, Benedetta Foisa and Elias Maccionia.
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aDepartment of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
bDepartment of Drug Chemistry, Faculty of Pharmacy, Lithuanian University of Health Sciences, 50162 Kaunas, Lithuania
c Institute of Biotechnology, Vilnius University, LT-10257 Vilnius, Lithuania
dLaboratorio NMR e Tecnologie Bioanalitiche, Sardegna Ricerche, 09010 Pula, Cagliari, Italy
eDepartment of Experimental and Clinical Medicine, “Magna Græcia” University of Catanzaro, Viale Europa, 88100
Catanzaro, Italy
KEYWORDS: Anticancer agents, isatin-dihydropyrazole hybrids, apoptosis inducers.
ABSTRACT: A series of isatin-dihydropyrazole hybrids have been synthesized in order to assess their potential as anticancer agents.
In particular, twelve compounds were evaluated for their anti-proliferative activity toward A549, IGR39, U87, MDA-MB-231, MCF-
7, BT474, BxPC-3, SKOV-3, H1299 cell lines, and human foreskin fibroblasts. Four compounds exhibited an interesting anti-
proliferative activity and were further examined to determine their EC50 values toward a panel of selected tumor cell lines. Best
compounds were then investigated for their induced mechanism of cell death. Preliminary structure-activity relationship indicates
that the presence of a substituent such as chlorine atom or a methyl moiety in the position 5 of the isatin nucleus is beneficial for the
antitumor activity. EMAC4001 resulted the most promising compound within the studied series with EC50 values ranging from 0.01
M to 0.38 M.
Several anticancer chemotherapeutic agents act by causing cell
death either by directly inhibiting the synthesis of DNA or by
interfering with its function. Unfortunately, they are generally
not specific for tumor cells and therefore associated with a high
toxicity. Not surprisingly, nowadays, the focus of the scientific
community is oriented toward the development of new target-
directed, more specific cytotoxic agents. Such agents must be
capable to inhibit or modulate identified molecular targets that
are involved in the control of cancer cells, such as signal
transduction, apoptosis, transcription regulation, matrix
invasion, and angiogenesis. Our research group is currently
involved in several projects regarding the design and synthesis
of anticancer agents directed toward several targets such as
human carbonic anhydrases (hCA),1-5 DNA G-quadruplex, and
hCA/COX2 dual inhibitors.6-9 However, it is commonly
recognized that cancer is a complex multifactorial disease and,
therefore, cannot be treated with a single drug therapy.
Accordingly, new agents, combining diverse pharmacophores
in a single hybrid molecule, might represent a goal for the
treatment of cancer and indeed a big effort has been put into the
identification of anticancer multi-target hybrids agents.10-18 In
reported.28 More in detail the relevant role of the isatin nitrogen
and of the carbonyl group in the position 1 and 2, as H-bond
donor and acceptor network, was outlined. However, with
respect to the structurally similar, multi-kinase inhibitor
Sunitinib, the isatin nucleus was, in this case, decorated by a
methoxycarbonyl group in the position 6, instead of a fluorine
atom in the position 5. On this basis, we have synthesized a new
series of dihydropyrazole isatin dihydrothiazole hybrids
EMAC4000, 4001, 4003, 4005, 4007, 4008, 4011, 4012, 4014,
4015, 4018, and 4019 to evaluate their activity toward diverse
cancer cell lines. Analogous compounds have been previously
reported and the most relevant structural features that are
essential and/or beneficial for the activity have been outlined.23
this respect, isatin is commonly recognized as a privileged
19-24
scaffold in drug design.2,
Moreover, it is a highly
represented structural motif in kinase inhibitors anticancer
drugs (Figure 1).25-28 The structure activity relationships of the
isatin based multi-kinase inhibitor Nintedanib, as well as its
drug development to phase III clinical trial has been recently
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O
H3C
were reacted with thiosemicarbazide in refluxing ethanol by
CH3
N
N
N
1
2
3
4
5
6
7
8
adding a freshly prepared KOH 5% ethanol solution. The
formation of the dihydrothiazole ring and the condensation of
the substituted isatin was accomplished in a single three
component step. The 3,5-diaryldihydropyrazole, ethyl
bromoacetate and the appropriate isatin derivative were
refluxed in acetic acid in the presence anhydrous sodium acetate
to give the desired products EMAC4000, -01, -03, -05, -07, -
08, -11, -12, -14, -15, -18, -19).
O
N
H3C
N
H
NH
O
CH3
N
F
H
O
H3CO
N
N
H
H
O
Sunitinib (FDA approved 2006)
Nintedanib (FDA approved 2014)
All compounds were characterized by means of analytical and
spectroscopic methods (SI, Tables S1, S2, and Figures S2-S36)
and then evaluated for their ability to inhibit tumor cell growth.
Firstly, the activity of the new derivatives was evaluated for the
anti-proliferative activity in the MTT assay at a fixed
concentration of 10 M ) toward a panel of nine human cancer
cell lines, namely A549 (lung carcinoma), IGR39 (melanoma),
U87 (glioblastoma), MDA-MB-231 (triple-negative breast
cancer), MCF-7 (breast adenocarcinoma), and BT474 (invasive
ductal carcinoma), H1299 (non-small cell lung carcinoma),
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S
N
N
N
N
N
N
O
O
S
S
R
O
R
O
O
O
CH3
CH3
N
N
H
H
EMAC4000, -01, -03, -05, -07, -08
EMAC4011, -12, -14, -15, -18, -19
SKOV-3 (ovarian
cancer)
,
BxPC-3 (pancreatic
Figure 1. Structurally related multi-kinase inhibitor Sunitinib
(VEGFR, PDGFR, KIT, RET), Nintedanib (VEGFR, EGFR,
PDGFR), and EMAC4000, 4001, 4003, 4005, 4007, 4008,
4011, 4012, 4014, 4015, 4018, and 4019 derivatives.
adenocarcinoma) cell lines, and human foreskin fibroblasts.
When tested toward cancer cell lines some of the compounds
exhibited anti-proliferative activity. In particular, compounds
bearing a 2-naphtyl substituent in the position 3 of the
dihydropyrazole ring were generally more active than their
corresponding 2-thiophenyl analogues. Although with some
differences, compounds EMAC4001, EMAC4007, and
EMAC4008 resulted as the most active toward the entire cell
panel.
Prompted by these observations, we aimed to further investigate
the effect of both the introduction of diverse substituents on the
isatin nucleus and of the replacement of the naphtalen-2-yl
group with a thiazole-2-yl ring on the biological activity.
EMAC compounds were synthesized slightly modifying
previously reported methods (Scheme 1).2, 29
Interestingly, EMAC4012 and EMAC4019 were the most
active within the 2-thiophenyl series when tested on IGR39,
U87 and IGR39 respectively, with anti-proliferative activity
comparable with 2-naphtyl analogues. Nevertheless, it should
be noted that these derivatives are the analogues of the two most
potent compounds of the 2-naphtyl series EMAC4001 and
EMAC4008, indicating that the presence of the 5-chloro, or of
its isoster 5-CH3 substituent is optimal for the anti-proliferative
activity within this class of compounds. Prompted by these
encouraging results we measured the EC50 values of the most
active compounds of the 2-naphtyl series EMAC4001,
EMAC4007, EMAC4008, and of the best performing
derivative within the 2-thiophenyl series EMAC4012 on a
panel of selected cancer cell lines (Table 2).
Scheme 1. Synthetic Pathway to Compounds EMAC4000,
4001, 4003, 4005, 4007, 4008, 4011, 4012, 4014, 4015, 4018,
and 4019a
CHO
Ar
OCH3
i
ii
N
Ar
N
OCH3
C
OCH3
O
NH2
S
iii
OCH3
All compounds exhibited EC50 values in the low
micromolar/high nanomolar range. EMAC4001 resulted as the
most potent within all the tested compounds with EC50 values
ranging from 0.01 M against H1299 to 0.38 M against U87
cells (Table 2). The substitution of the 2-naphtyl moiety with
the 2-thiophenyl group in the position 3 of the dihydropyrazole
ring in EMAC4012, led to an evident decrease of the potency
and to EC50 values of 2.97 M and 5.76 M toward IGR39 and
U87 cell lines respectively. Interestingly, when tested on A549,
IGR39, and U87, EMAC4008 exhibited the highest activity
with respect to compounds EMAC4001, EMAC4007, and
EMAC4012, with EC50 values of 0.18 M, 0.14 M, and
0.23 M, respectively. On the basis of these results it can be
observed that the 5-Cl-isatin is generally the most efficient, but,
at least in some cases, its isosteric replacement with the 5-CH3-
isatin is well tolerated or even more advantageous.
Furthermore, to better characterize the biological behavior of
these derivatives we investigated the mechanism of cellular
death when the cells are treated with half of the EC50
O
O
NH
S
N
Ar
N
N
Ar = naphtalen-2-yl; thiophene-2-yl
R = 7-Br; 5-Cl; 5,7-CH3;7-F; 5-OCH3; 5-CH3
R
aReagents and conditions: (i) 2-acetylnaphtalene or 2-
acetylthiophene, ethanol, NaOH 10% water solution, 0°C; (ii),
thiosemicarbazide, ethanol, KOH 5%, reflux; (iii) ethyl
bromoacetate, R-isatin, dry sodium acetate, acetic acid, reflux.
Briefly, an ethanol solution of 2-acetylnaphthalene (for the
synthesis of EMAC4000, -01, -03, -05, -07, -08) or 2-
acetylthiophene (for the synthesis of EMAC4011, -12, -14, -15,
-18, -19) was reacted at 0°C with an equimolar amount of 4-
methoxybenzaldehyde in the presence of 1,2 equivalent of
sodium hydroxide 10% water solution. The obtained solids
were crystallized from ethanol. The obtained diarylpropenones
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concentration of compounds EMAC4001 and EMAC4008.
Results are presented in Figure 1, and the percentage of
apoptotic and necrotic cells is reported in Figure 2.
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2
3
4
5
6
7
8
9
Figure 2. a) Percentage of necrotic cells after treatment with ½
EC50 of EMAC4001 and EMAC4008; b) Percentage of
apoptotic cells after treatment with ½ EC50 of EMAC4001 and
EMAC4008.
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Although further studies are needed to further clarify and
identify the exact mechanism of action of such derivative, our
data indicates that the hybridization of 5-chloroisatin with 3,5-
diaryldihydropyrazoles by the interposition of a dihydrothiazole
spacer is a promising approach to the identification of
anticancer agents. With this information in our hands, we are
encouraged to further investigate these scaffolds in order to
optimize their activity and pharmacokinetic properties.
Figure 1. Visualization of apoptotic (bright blue) and necrotic
(red) cells after treatment with ½ EC50 of EMAC4001 and
EMAC4008.
Results were more than encouraging. In all the three considered
cell lines the percentage of apoptotic cells ranges between 13.5
and 27%. Conversely, when the number of necrotic cells is
considered, either EMAC4001 or EMAC4008 induced the
necrosis of less than 1% of the cell population. Results show
that tested compounds induce cell death mostly through
apoptosis. Overall these results indicate that a specific
mechanism, such as the inhibition of a signaling pathway, might
be the target of EMAC derivatives.
Table 1. Anti-proliferative activity of compounds EMAC4000, -01, -03, -05, -07, -08, -11, -12, -14, and -15, -18, -19 at 10 M
concentration.
Ar
N
N
N
O
S
O
R
OCH3
N
H
% growth
EMAC
Ar
R
Compound
MDA-
MB-231
A549 IGR39 U87
MCF-7 BT474 BxPC-3 SKOV-3 H1299 Fibroblasts
101.82 88.24 55.31 89.86 81.02 47.28
4000
4001
4003
4005
4007
7-Br
5-Cl
94.18 98.64
96.71 68.15
3.84
1.30
1.59
6.87
3.81
24.22 5.62
6.69
9.76
14.40
5,7-CH3 43.8
7-F
5-OCH3 3.93
63.40
64.20 72.98
120.38 62.18 44.87 26.55 87.83 55.56
118.14 112.38 73.07 67.65 60.49 89.44
86.55 109.13 86.19 87.29
1.20 1.13 9.67
3.64
49.12 5.13
41.43 9.85
13.31
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4008
4011
4012
4014
4015
4018
4019
5-CH3 6.21
7-Br 86.11 100.94 57.01 73.90
5-Cl 22.38 0.90 4.35 80.40
0.46
14.20 10.77
4.81
40.25 5.02
66.61 19.34 11.84
1
2
3
4
5
6
7
8
125.55 94.35 53.68 87.98 89.15 46.28
112.02 86.78 59.64 56.58 116.69 47.54
S
S
5,7-CH3 103.73 116.76 126.25 110.92 114.44 103.19 72.26 79.92 119.87 80.32
S
S
S
S
9
5-F
5-OCH3 62.52 98.60
5-CH3 22.74 1.37
63.59 28.15
14.40 81.47
44.85 99.30
12.08 76.91
112.42 102.48 77.24 94.06 68.35 20.03
119.79 98.43 67.58 49.54 85.88 64.36
112.02 94.97 62.44 38.35 83.97 48.31
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Table 2. EC50 values of EMAC4001, EMAC4007, EMAC4008, and EMAC4012 toward a panel of selected tumor cells.
Ar
N
N
N
O
S
O
R
OCH3
N
H
EC50 values (M)
EMAC
Ar
R
SKOV-
3
Fibro
blasts
Compounds
A549
0.34
IGR39
U87
MCF-7
BT474
BxPC-3
H1299
//
4001
4007
4008
4012
5-Cl
0.33
0.50
0.14
2.97
0.38
0.67
0.23
5.76
0.07
0.27
0.31
//
0.09
0.24
//
0.06
0.10
0.10
//
0.06
//
0.01
0.15
//
5-OCH3 0.73
0.27
0.15
//
5-CH3
5-Cl
0.18
//
//
//
//
//
S
Sunitinib
//
//
//
0.96
0.90
2.5
1.36
1.54
0.30
Pula (CA) – Italy. The authors also thank the COST action
CA15135 (Multitarget Paradigm for Innovative Ligand
Identification in the Drug Discovery Process MuTaLig) for
support.
ASSOCIATED CONTENT
Supporting Information
(Experimental procedures and compounds characterization are
reported (PDF). The Supporting Information is available free of
charge on the ACS Publications website at DOI:
ABBREVIATIONS
hCA, human carbonic anhydrase; COX2, cyclooxygenase2;
VEGFR, vascular endothelial growth factor; PDGFR, platelet-
derived growth factor receptors; KIT, Mast/stem cell growth factor
receptor; RET, proto-oncogene tyrosine-protein kinase receptor;
MTT,
bromide.
AUTHOR INFORMATION
Corresponding Author
3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium
*E-mail: s.distinto@unica.it.
Author Contributions
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‡These authors contributed equally. The manuscript was written
through contributions of all authors. All authors have given
approval to the final version of the manuscript.
Melis, C.; Distinto, S.; Bianco, G.; Meleddu, R.;
ACKNOWLEDGMENT
The authors wish to acknowledge the “Ufficio Valorizzazione dei
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