Synthesis and biological evaluation of novel 4,7-dihydroxycoumarin derivatives as anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2019.05.008

A series of novel 4,7-dihydroxycoumarin based acryloylcyanohydrazone derivatives were synthesized and evaluated for antiproliferative activity against four different cancer cell lines (A549, HeLa, SKNSH, and MCF7). Most of the compounds displayed potent c

Full text of DOI:10.1016/j.bmcl.2019.05.008

Accepted Manuscript
Synthesis and biological evaluation of novel 4,7-dihydroxycoumarin derivatives
as anticancer agents
Pilli Govindaiah, Naresh Dumala, Paramjit Grover, M. Jaya Prakash
PII:
S0960-894X(19)30294-X
https://doi.org/10.1016/j.bmcl.2019.05.008
BMCL 26428
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
4 February 2019
3 May 2019
5 May 2019
Please cite this article as: Govindaiah, P., Dumala, N., Grover, P., Jaya Prakash, M., Synthesis and biological
evaluation of novel 4,7-dihydroxycoumarin derivatives as anticancer agents, Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.05.008
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Synthesis and biological evaluation of novel 4,7-dihydroxycoumarin derivatives as
anticancer agents
a,*
b
b
a
Pilli Govindaiah , Naresh Dumala , Paramjit Grover , M. Jaya Prakash
aDepartment of Chemistry, National Institute of Technology, Rourkela, Odisha-769008, India.
bToxicology Unit, Applied Biology Division,CSIR-Indian Institute Of Chemical Technology,
Hyderabad-500007,Telangana, India.
Abstarct:
A series of novel 4,7-dihydroxycoumarin based acryloylcyanohydazone derivatives were
synthesized and evaluated for antiproliferative activity against four different cancer cell lines
(A549, HeLa, SKNSH, and MCF7). Most of the compounds displayed potent cytotoxicity with
IC50 values ranging from 3.42 to 31.28 µM against all the tested cancer cell lines. The most
active compound, 8h was evaluated for pharmacological mechanistic studies on cell cycle
progression and tubulin polymerization inhibition assay. The results revealed that the compound
8
h induced the cell cycle arrest at G2/M phase and inhibited tubulin polymerization with IC50
=
6
.19 µM. Experimental data of the tubulin polymerization inhibition assay was validated by
molecular docking technique and the results exhibited strong hydrogen bonding interactions with
amino acids (ASN-101, TYR-224, ASN-228, LYS-254) of tubulin.
Key Words: 4,7-dihydoxycoumarin, acryloylcyanohydarzone, anticancer, cell cycle, tubulin,
molecular docking.
*
Corresponding author
Email id: govipharma@gmail.com (Pilli Govindaiah )
Postal Adress:
Department of chemistry
National Institute of Technology, Rourkela,
Odisha, India, 76900
1

Cancer is a group of diseases resulting in the uncontrolled division and involving in the
frequent spatiotemporal changes in the cell physiology. The main reasons for the manifestation
of cancer are an unhealthy diet, environmental pollution, overuse of drugs, hormonal imbalance
1
and some genetic mutations . No single therapeutic agent has been available for the 100%
successful treatment of cancer. The incomplete understanding of cancer causing mutations and
random errors in DNA replication renders difficulties to design novel anticancer agents for
2
treating cancer . Hence, the need of novel anticancer drugs is still persistent. Researchers are
trying hard to identify potential drug molecules that would successfully pass the preclinical
3
studies in the new drug development protocol and pave the way for treating cancer in a better
method. Recently, few novel organic compounds (coumarins, hydazones, benzthiazoles, etc)
have gained much attention in this regards 4–9
.
4
,7-Dihydroxycoumarins are one of the important class of benzopyrones having several
1
0
11
12
13
medicinal properties like antimicrobial , anti-tubercular , antileishmanial , antioxidant ,
1
4
15
antidiabetic and anticancer . These 4,7-dihydroxycoumarin can be prepared from the reaction
1
6
of resorcinol by reacting with malonic acid . The hydroxycoumarin based compounds could
exert good cytotoxic activity against different cancer cell lines like promyelocytelukemia-derived
(
HL-60), urinary bladder carcinoma-derived (EJ) 17, MCF-7 18 and Hela cell lines 19. The
coumarin-based chalcones, acryloylcyano hydrazide and pyridine derivatives effectively
6
inhibited liver cancer (Hep G2) and leukemia (K562) . Comarin based acryloylcyanohydrazones
were evaluated for cytotoxic activity against HaCaT (human keratinocytes) cells 20. These
compounds exhibit anticancer activity by several mechanisms:1) Inhibition of non-structural 5B
protein (NS5B) 21, 2) Inhibition of the enzyme Protein kinase CK2 22. 3)Target action with
estrogen 23 and COX-2 24 receptors. From through literature study we found that the major
targets for anticancer agents are tubulins, metabolites, DNA, topoisomerase etc.
Tubulin is a prominent target for the anticancer drugs, due to it’s involvement in wide
range of cellular processes i.e. cell proliferation, migration, signaling, and trafficking in
2
5
eukaryotic cells . The clinically available drugs such as vincristine, vinblastine, colchicine and
paclitaxel exhibit their anticancer activity by inhibiting the mitosis through interaction with
tubulin. These drugs alters the tubulin activity in different ways 26 for producing the activity. The
development of resistance and toxicity are major problems associated with the existing
2

antitubulin drugs. The discovery of antimitotic agents has led to new approaches in cancer
chemotherapy. The mitosis is a vital phase in the cell cycle for many critical proteins. Tubulin,
kinetochore and some other essential proteins are synthesized in this phase 27,28. These proteins
are involved in the formation of a chromatid during cell division which assists in the attachment
with the spindle fiber on chromosome. There are few reports on novel class of coumarin
derivatives being tested an antimitotic drugs with G2/M phase arrest 29–31
.
Based on the above reported studies we understood the potential of hydroxycoumarin
derivatives in treatment of cancer. Some reasearchers reported the cyotoxic properties of
cyanohydarazone moiety. To the best of our knowledge, no literature is available on 4’7-
dihydroxycoumarin based cyanohydrazone moieties with cytotoxic property. All these factors
encouraged us to design coumarin based acryloylcyanohydrazone derivatives (8a-m) as
anticancer agents. The target coumarin based acryloylcyanohydrazone derivatives were
synthesized from the starting material, resorcinol in a sequence of chemical reactions, depicted in
1
13
scheme 1. The compounds were characterized by FT-IR, H NMR, C NMR and ESI-MS
spectral techniques. The newly synthesized compounds were evaluated for their biological
activity by both computational as well as experimental works. The most active compound (8h)
was subjected to cell cycle analysis and tubulin polymerization inhibitory activity to identify the
pharmacological mechanism studies. To find out the type of binding interactions between
compounds and tubulin protein, simulation was performed using molecular docking technique.
All the target compounds (8a-m) were synthesized according to Scheme-1. The coumarin
based acryloylcyanohydrazones (8a-m) were synthesized from resorcinol (1) in a sequence of
reactions. First, resorcinol reacted with malonic acid (2) in presence of POCl
dihydroxycoumarin (3).
3
that gives 4,7–
3

Scheme 1: Synthesis of coumarin based acryloylcyanohydrazones (8a-m). Reagents and
ο
conditions: a) POCl
3
, ZnCl
2
, 60 C, 12h b) CH
3
COOH, POCl , reflux, 3h; c) ethanol, reflux, 4h;
3
d) ethanol, piperidine, reflux, 3-4h.
Acetylation of compound 3 with glacial acetic acid in the presence of phosphorus oxychloride
yields compound 4 32,33. The compound 4 on condensation with the cyanoacetohydrazide (5)
produced the key intermediate 6 finally, the compound 6 condensed with appropriate aromatic
aldehydes (7) in presence of piperidine base. The reaction the mixture was poured into ice cold
dilute HCl which afforded the title compounds (8a-m) in good yield. The newly synthesized
4

compounds were purified by recrystallization method with DMF/ethanol (1:5) solvent mixture.
The purity was determined by Thin Layer chromatography (TLC) method. Further, devoid of
1
13
additional absorption band/devoid of additional signal in FT-IR/ H NMR, C NMR reflects the
good purity of the synthesized compounds. The structures of the compounds were confirmed by
1
13
FT-IR, H NMR, C NMR, and ESI-MS spectral techniques.
All the synthesized coumarin based acryloylcyanohydrazone derivatives were evaluated for
antiproliferatve activity using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] assay against four human cancer cell lines (A549, HeLa, SK-N-SH, MCF7) and
normal rat kidney cell line (NRK-49F) using doxorubicin as reference drug. The results are
summarised in Table-1. Thes synthesized compounds were showed good to excellent
cytotoxicity with IC50 values ranging from 3.42 to 31.28 µM against all the tested cancer cell
lines. The potency of the compounds varies with respect to substitution on the simple phenyl
ring. Hydrazide-hydrazone backbone is more potent when cyano group is attached to hydrazone
moiety, which tends to increases the activity of compounds. From the figure 3B it was noticed
that cyano group was involved in the formation of hydrogen bond with the target. The same was
performed with the compound without cyano group that exhibited less interactions (Figure 4B)
with the target compared to compound having cyano group. The para-substituted compounds
(8b, 8e, 8g, 8h and 8j) and ortho-substituted compounds (8k and 8m) exhibits high potency
compared to meta-substituted compounds (8i and 8l). Among the para-substituted compounds,
compounds (8b, 8e and 8h) with an electron releasing group have more potency than the
compounds (8g and 8j) with an electron withdrawing group. Introduction of additional electron
releasing group(s) in para-substituted compounds gives compounds 8c, 8d and 8f. These
compounds retain the cytotoxic activity but less potent than monosubstituted compounds.
5

Table-1: In vitro cytotoxicity results of target compounds (8a-m) against human cancer cell line
A549, HeLa, SKNSH, MCF7 along with normal rat kidney cell line NRK 49F.
Sample
Code
R
IC50 (in µMol)
A549
HeLa
SKNSH
MCF7
NRK 49F
8
8
8
a
b
c
H
1
6
4.82 ± 0.61
.23 ± 0.18
15.75 ± 0.86
6.02 ± 0.28
13.52 ± 0.71
8.12 ± 0.35
19.22 ± 0.48
7.32 ± 0.58
55.45 ± 0.62
61.31 ± 0.55
4-OH
3-OCH
3
,
1
2.63 ± 0.23
10.46 ± 0.32
14.35 ± 0.56
13.65 ± 0.54
55.42 ± 0.14
4
-OH
8
8
8
8
8
8
8
8
d
e
f
3,4-OCH
3
1
8
1
1
4
2
9
4.32 ± 0.65
.15 ± 0.48
5.32 ± 0.35
1.43 ± 0.13
.31 ± 0.04
1.9 ± 0.41
.64 ± 0.26
13.52 ± 0.26
7.85 ± 0.26
18.46 ± 0.35
10.56 ± 0.26
5.14 ± 0.16
24.64 ± 0.29
21.53 ± 0.45
9.46 ± 0.39
14.61 ± 0.18
12.05 ± 0.14
6.09 ± 0.32
28.26 ± 0.09
14.35 ± 0.42
8.94 ± 0.73
11.34 ± 0.05
9.85 ± 0.06
3.42 ± 0.52
16.35 ± 0.24
45.28 ± 0.36
96.10± 0.89
55.34 ± 0.36
49.64 ± 0.11
53.41 ± 0.26
46.53 ± 0.09
4-OCH
3
3,4,5-OCH
3
g
h
i
4-Br
3 2
4-N(CH )
3-NO
4-NO
2-NO
2
2
2
j
8.36 ± 0.05
10.26 ± 0. 15
12.26 ± 0.18
8.20 ± 0.21
9.18 ± 0.39
43.22 ± 0.10
68.59 ± 0.57
k
11.35 ± 0.26
10.31 ± 0.36
8
8
l
3-Br
2-Cl
22.15± 0.54
14.23 ± 0.18
24.28 ± 0.82
11.78 ± 0.41
31.28 ± 0.36
14.02 ± 0.34
19.64 ± 0.67
8.88 ± 1.41
59.39 ± 0.38
55.89 ± 0.17
m
Doxo
6. 22 ± 0.03
7.01 ± 0.015
6.42 ± 0.56
9.86 ± 0.12
24.22 ± 0.18
IC50 is the 50% inhibitory concentration of the samples, and the results were represented as
average values ± standard deviation.
Doxo = Doxorubicin used as positive control (Standard)
From the screening results we are concluded that the compounds 8b, 8e, 8h and 8j were
showed excellent cytotoxicity against all cancer cell lines. The compound 8h has exhibited
highest cytotoxicity than standard drug doxorubicin and further pharmacological mechanistic
studies were evaluated.
6

To uncover the pharmacological mechanism, the highly potent compound 8h was
selected against MCF-7 cell lines by flow cytometry method. After exposure of MCF-7 cells to
compound 8h at 4μM for 24 h, there was a significant increase in the percentage of cells at Sub
0
G phase as compared to control (Fig.1A). In addition, accumulation of cells was detected at
Figure 1: Flow cytometric analysis of MCF-7 cell lines treated with synthesized compound (8h)
and standard drug nocodazole. A) Exposure with negative control DMSO B) Cell cycle
disruption of compound 8h for 24h treatment at indicated concentration analysis of MCF-7 cell
line C) Cell cycle disruption of Reference drug nocodazole treatment at indicated concentration
analysis of MCF-7 cell line D) Bar graph represents mean ± in SD at least three independent
experiments.
G2/M phase by almost 4 folds compared to the control [from 13.9% in the vehicle group
(
Fig.1A) to 45.3% after 24h (Fig. 1B). On the other hand, the reference drug nocodazole showed
9.3% of G2/M arrest (Fig. 1C)] These results suggested that the compound 8h inhibited MCF-7
cells proliferation through cell cycle arrest at G2/M phase followed by cell death. These results
3
3
4
were in good agreement with the previously reported findings .
7

Tubulin is one of the major targets for chemotherapeutic agents. These agents are called
as antimitotic agents since they act at the mitotic phase and stop the mitosis process by inhibiting
tubulin action. These drugs attack the cancer cells at both primary and metastatic phase. These
therapeutic agents are primarily bound to the beta-tubulin protein in the mitotic spindle.
Figure 2: Effect of tubulin polymerization inhibition activity of compound (8h) and colchicine at
different concentrations.
Since compound 8h showed good G2/M arrest or mitotic arrest, we have tested this compound
for tubulin polymerization inhibition property and compared with colchicine, a reference drug.
The test compound 8h exhibited significant tubulin polymerization inhibitory potency with IC50
6
.19 μM in comparison to the reference drug, colchicine, IC50 7.60 μM (figure-2).
Docking studies were carried out in order to find plausible binding modes of the novel
inhibitor with tubulin. From Figure 3 of docking picture it can be observed that deep green color
8

(A)
(B)
9

(C)
(D)
Figure 3: Plausible binding mode of the compound 8h with tubulin protein (PDB ID: 4YJ3). A)
Stick model of the proposed binding mode B) 2D representation of binding interactions the green
colour indicates hydrogen bonding. C) 3D representation of binding interactions. D) 3D
representation of binding interactions of colchicine with tubulin protein.
1
0

interactions designate the covalent hydrogen bonding interactions. The cyano group and two
hydroxyl groups of the coumarin ring are involved in formation of hydrogen bonds with target
tubulin protein. Figure 3, it can be observed that the compound 8h was formed hydrogen bond
interaction with amino acids with ASN-101, TYR-224, ASN-228, LYS-254, amino acids of
tubulin protein with -63.66 CDOCKER interaction energy. To study the involvement of cyano
group in the hydrogen bond formation, the docking result of compound 8h has compared the
results with compound without cyano moiety. Further we have also compared with standard
tubulin targeted anticancer drug colchicine (Figure 3D).
A)
1
1

B)
Figure 4: Docking results of the two examples without cyano group A) Docking result of the
compound 8a without cyano group B) Docking result of the compound 8h without cyano group
Figure 4 depicted that docking interactions of compounds without cyano group shown less
interactions with tubulin. The results pointed out to the fact that the cyano group has the ability
to form hydrogen bonding interactions with the target. Further to validate these results we also
performed docking study on the standard drug doxorubicin. The results revealed that it also
formed strong hydrogen bonding interactions with the tubulin protein and same can be noticed in
figure-5.
1
2

Figure 5: Docking result of the doxorubicin with the tubulin protein.
The hydrogen bonding interactions occurred with GLN-11, ALA-12, GLU-71, ASN-101 and
GLN-247. The obtained results suggested that these compounds may show the activity against
pancreatic, prostate, ovarian, and GBM cancers. The most active compound 8h can be taken up
for cancer treatment drug delivery, labeling for imaging and act at the direct on the targeted cell
specific site.
In summary, we have successfully synthesized novel coumarin-based
acryloylcyanohydrazone compounds by a facile method. These chemical entities were well
characterized by FT-IR, NMR and, ESI-MS spectroscopic methods. Furthermore, these
compounds were screened for antiproliferative activity against A-549, Hela, SKNSH, MCF-7
human cancer cell lines and normal rat kidney cell line NRK 49F by using MTT assay. Among
the all compounds 8b, 8e, 8h, and 8j were showed good antiproliferative activity. The most
active compound 8h was subjected to further pharmacological evaluation studies; cell cycle
analysis was carried out on MCF-7 cell lines which possess G2/M phase arrest. Further, 8h was
evaluated for the tubulin polymerization inhibition and the obtained result (IC50 6.19 μM) were
1
3

promising than colchicine standard tubulin inhibitory drug. The molecular docking studies were
done to validate the experimental results of the tubulin inhibition assay which exhibited strong
hydrogen bonding interactions between compound 8h and aminoacids ASN-101, TYR-224,
ASN-228 and LYS-254, amino acids of tubulin. In the present study, we found 8h act as
antiproliferative agent which arrest the cell cycle at G2/M phase and inhibits the tubulin
polymerization. Hence, we can conclude that the molecule 8h has the potential to act as
antimitotic agent by targeting tubulin protein for treating cancer.
ACKNOWLEDGMENTS:
We would like to thank the National Institute of Technology, Rourkela for providing the
necessary infrastructural and instrumentation facilities and TEQIP-II for partial funding. We
would like to convey my gratitude towards Dr. Thangaraj Devadoss and Mr. Tuhin Subhra Dash
for their valuable suggestions.
SUPPORTING INFORMATION:
1H, and 13C NMR of the compounds, antiproliferation assay (MTT assay), tubulin
polymerization inhibition assay, molecular docking protocols were included in the supporting
information.
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