Accepted Manuscript

Synthesis of New Coumarin Tethered Isoxazolines as Potential Anticancer

Agents

Gejjalagere S. Lingaraju, Kyathegowdanadoddi S. Balaji, Shankar Jayarama,

Seegehalli M. Anil, Kuppalli R. Kiran, Maralinganadoddi P. Sadashiva

PII: S0960-894X(18)30848-5
DOI: https://doi.org/10.1016/j.bmcl.2018.10.046
Reference: BMCL 26105

To appear in: Bioorganic & Medicinal Chemistry Letters

Received Date: 11 August 2018

Revised Date: 15 October 2018
Accepted Date: 28 October 2018

Please cite this article as: Lingaraju, G.S., Balaji, K.S., Jayarama, S., Anil, S.M., Kiran, K.R., Sadashiva, M.P.,
Synthesis of New Coumarin Tethered Isoxazolines as Potential Anticancer Agents, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.10.046

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
 Synthesis of New Coumarin Tethered Isoxazolines as Potential Anticancer
Agents
Gejjalagere S. Lingaraju,a Kyathegowdanadoddi S. Balaji,b Shankar Jayarama,b Seegehalli M.
Anil,a Kuppalli R. Kiran,a Maralinganadoddi P. Sadashivaa*
a Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
bP.G. Department of Biotechnology, Teresian College, Mysore 570 011, India
* Corresponding author. Tel.: +91 0821-2419467

E-mail address: mpsadashiva@gmail.com

Abstract

A series of new coumarin tethered isoxazolines (7a-l) were synthesized and evaluated for

their cytotoxic potency against human melanoma cancer cell line (UACC 903) as well as

fibroblast normal cell line (FF2441). Preliminary results revealed that some of these

coumarin tethered isoxazolines 7b, 7c, 7f and 7j exhibited significant antiproliferative effect

against human melanoma cancer (UACC 903) with IC50 values of 8.8, 10.5, 9.2 and 4.5 μM

respectively. However, compound 7c was non-toxic to normal human cells at the tested

concentration. Further, we have chosen compound 7c to check its efficacy in Ehrlich Ascites

Carcinoma animal model in-vivo for its antitumor and antiangiogenic properties. Our lead

compound significantly reduced the cell viability, body weight, ascites volume and

downregulated the formation of neovasculature such as regression of tumor volume. The

present study indicates the scope of developing into potent anticancer drug in near future.

Keywords: Coumarin, Isoxazoline, 1,3-Dipolar Cycloaddition, Anticancer, Angiogenesis,

Antiproliferative.
Cancer is seriously threatening the healthy life of humans and one of the leading

diseases to cause the morbidity and mortality around the world. More than ten millions of

people are detected with different types of cancer in every year worldwide.1 Treatment of

cancer is a difficult task because of its diversity, versatility and high degree of molecular

heterogeneity leading to drug resistivity.2 Consistent efforts have been made since from the

last few decades to cure this disease by better understanding of biological processes

involved in cancer-cell survival, which in turn aided by advancements in cellular and

molecular biology leading to the development of potent anticancer agents.3 Until today,

medicinal chemists actively involving in the development of new anticancer agents with no

or minimum side effects. The ideal drug should be safe, well tolerable, biocompatible and

capable of targeting cancerous tissues without affecting the normal cells.4 To achieve

ideality, natural products are the choice of selection and are significantly contributed to the

development of efficient anticancer drugs5 and more than half of the anticancer drugs

approved globally are either natural products or their mimics.6

The coumarins (2H-1-benzopyran-2-one) and its derivatives are widely distributed

throughout the nature and display variety of pharmacological properties.7 In addition, its

stability and solubility has fascinated medicinal chemists to explore the coumarins analogs

for their medicinal applicability.8 The pattern of substitutions on coumarin framework has

major influence on pharmacological and biochemical properties, including the therapeutic

applications.9 Of particular interest in cancer chemotherapy, number of naturally occurring

coumarins recognized as an efficient anticancer agents.10-13 Some coumarin, 7-

hydroxycoumarin or geranylated coumarin analogs are better sulfatase and aromatase

inhibitors.14 The in-vitro study of coumarin derivatives against renal cell carcinoma, gastric

carcinoma, colon carcinoma, hepatoma-derived cell line, lymphoblastic cell line and several

other human cancer cell lines showed potent cytotoxic and cytostatic effects.15 Several

reports demonstrate the activities against prostate cancer, malignant melanoma, and

metastatic renal cell carcinoma, which is in clinical trials.16 Furthermore, coumarins

demonstrated antitumor activities at various stages of cancer formation through different

mechanisms, for example blocking cell cycle, inducing cell apoptosis, modulating estrogen

receptor (ER) or inhibiting the DNA-associated enzymes.17

The added triumph is that the administration of coumarin to rats and rabbits shown

no evidence of significant tissue accumulation.18 Further, the mechanism of action on the

growth and metabolism of human tumor cells confirmed that coumarin itself is not
responsible for the observed in-vivo effects, but it act as a prodrug of other active

metabolites.19 Therefore, it is worthy to study coumarin derived hybrid molecules as anti-

cancer agents, since coumarin can easily permeate through cell wall and act as prodrug,

which helps in active drug delivery that could act on more than one target with high

selectivity, specificity and with minimum side effects.20 Towards this effort, several research

groups involved in the strategy of conjugation of different bioactive pharmacophores into

coumarin framework (catechol-coumarin, stilbene–coumarin, coumarin–chalcone etc), the

resulting hybrids have shown significant anticancer activities (Fig. 1).21 Thus, a molecule

containing more than one pharmacophore with different mode of action could be highly

effective for the treatment of cancer.22

O

O O O NO2 N O O O
O
O
O O O N
N N
O

O
Br O O O
O O F N
R S N
N
Cl N S O O NH N HN
H O
S O
O
O
O S
O N
Cl S N
O O O O R
Br N
N Our prototype
O
O O O O
N

Figure 1: Selected structures of some biologically active compounds containing coumarin

motifs
On the other hand, our research group interested in isoxazoline framework because

of its vast biological applications. ∆2-isoxazolines are an important class of nitrogen-oxygen

containing five-membered heterocycles with various chemotherapeutic properties. They

exhibit a wide spectrum of biological activities such as antimicrobial,23 anti-tubercular,24

insecticidal,25 anti-HIV,26 antistress,27 anticancer,28 antagonists29 and promising

antineoplastic properties.30 Considering the promising antiproliferative profile of

isoxazolines as well as coumarin, and based on our previous studies on isoxazoline-tethered

dibenzoazepine derivatives as potent anticancer agents.31 Accordingly, we have designed

single molecule with both coumarin and isoxazoline pharmacophores, with the anticipation

of synergetic effect, with improved affinity and efficacy in comparison to the parent

structural components. Wherein coumarin could act as prodrug by transporting and

delivering active isoxazoline, bioavailabilty of the active drug might be effectively inhibits

the growth of tumor. Thus, we herein report the synthesis, cytotoxicity and structure–activity

relationship (SAR) studies of 4-methyl-7-((3-aryl-4,5-dihydroisoxazol-5-yl)methoxy)-2H-

chromen-2-one 7(a-l) (Scheme 1). In addition, detailed biological investigations for lead

compound 7c, such as preliminary screening and Ehrlich Ascites Carcinoma model have

also been discussed.

Using the Pechmann reaction,32 the requisite starting material, 7-hydroxy-4-methyl-

2H-chromen-2-one (3) was synthesized in good yield through the sequential esterification or

transesterification followed by cyclization and dehydration of resorcinol (1) with ethyl aceto

acetate (2) using urea/choline chloride ionic liquid at 60 °C. Further, the obtained 7-hydroxy

coumarin (3) was subjected to nucleophilic substitution reaction with allyl bromide yielding

allyl derivative (4), which was used as dipolarophile in the next step. Another key precursor,

aldoximes (6(a-l)) were synthesized by condensation of aldehyde (5(a-l)) with

hydroxylamine hydrochloride in methanol. Further, in a typical reaction, aldoximes (6(a-l))

was chlorinated with N-chlorosuccinimide in dichloromethane and added a mixture of

allylated coumarin (4) and triethyl amine in dichloromethane dropwise manner at 0 °C. The

reaction proceeds forward with disappearance of oximes to yield target compounds (7a-l)

and were purified by column chromatography with good yield (61-78%). The chemical

structures of the synthesized molecules and their percentage yield are presented in Table 1.
 O O
H
O
HO OH
+ O
1 2
i

R H
+ HCl. H2N OH

5(a-l) HO O O
iii 3
ii
OH
N Br

R H
+ O O O
6(a-l) 4

iv

R O O O
N O
7(a-l)
Scheme 1: Synthesis of coumarin tethered isoxazoline derivatives
Reagents and reaction conditions: (i) 20 mol% choline chloride-urea ionic liquid, heated to
60 °C; (ii) K2CO3, DMF, 0 °C – rt; (iii) CH3COONa, MeOH, powdered ice; (iv) N-
chlorosuccinamide, NEt3, CH2Cl2.
Further, all the synthesized compounds were characterized by various spectral

techniques such as IR, 1H NMR, 13C NMR, and mass analyses. Analytical and spectral data

of all synthesized compounds were in good agreement with the proposed structure. For

example, 1H NMR spectra of compounds 7(a-l) exhibited, besides the expected peaks of

aromatic protons, the -CH3 protons and olefinic proton of pyron ring observed as singlet at δ

2.34-2.36 and δ 6.1-6.2 respectively. Further, -OCH2- protons resonates at δ 4.2-4.3 as

doublet, -CH- proton at 5th position on isoxazoline ring as multiplet δ 5.0-5.2 and -CH2-

proton at 4th position on isoxazoline ring as doublet of doublet at δ 3.3-3.7. An additional

singlet peak was observed at δ 3.8-4.1 in case of compounds bearing –OCH3 group. The

detailed spectral data and elemental analyses of all the compounds are presented in

supplementary material.
Table 1: Structure and yield of synthesized coumarin tethered isoxazoline derivatives
7(a-l)
Compound % of isolated
Entry Structure of the compound
Number Yield

7a 63
O O O
N O

7b O 72
O O O
N O

7c O 61
O O O
O N O

7d O 72
O O O
O N O

7e 73
O O O
N O

7f O O O
78
N O
Cl

7g Cl 76
O O O
N O

7h Br 68
O O O
N O

7i F 66
O O O
N O

F
7j 67
O O O
N O
Cl
F

7k 72
O O O
N O
 7l F3C 75
O O O
N O
The novel coumarin tethered isoxazolines (7a-l) were screened for their cytotoxic

activity against melanoma cancer cell line UACC 903 and fibroblast normal cell line

FF2441 through in vitro MTS assay. Results unveiled that coumarin-isoxazoline conjugates

7(a-l) affected the viability of melanoma cells over human fibroblast normal cells (Fig 1).

Interestingly, results implied that the activity was dose and time dependent, among the tested

compounds 7b, 7c, 7f and 7j induced the highest cytotoxicity against the melanoma cells.

The compound 7j was found to exhibit the maximum cytotoxicity on melanoma cells with

an IC50 value of 4.5 μM, which could be attributed due to the presence of chloro and fluoro

substitution at ortho positions on phenyl ring of isoxazoline. The molecule 7b with methoxy

substitution at para position on phenyl ring of isoxazoline shown IC50 value of 8.8 μM.

Whereas, compound 7c emerged as a prime candidate among the series, inhibiting

selectively melanoma cells with an IC50 value of 10.5 μM without affecting the normal cells,

could be due to the presence of 3,4-dimethoxy group on the phenyl ring of isoxazoline ring.

The 3,4,5-trimethoxyphenyl substituted isoxazoline (7d) is not active, reason for which is

uncertain. The coumarin derived isoxazoline 7f with an IC50 value of 9.2 μM, which could

be due to the presence of chloro substitution at ortho position on phenyl ring of isoxazoline.

The rest of the tested compounds 7a, 7d, 7e, 7g, 7h, 7i, 7k and 7l bearing 3-methoxy, 3,4,5-

trimethoxy, naphthyl, 4-chloro, 4-bromo, 4-fluoro, 3-fluoro and 4-trifluoromethyl groups on

the phenyl ring of isoxazoline respectively were not cytotoxic against both the cell lines

tested. The In-vitro screening of compounds with cytotoxic effect on cancer and normal cell

lines depicted in Fig-1. It was observed from structural activity relationship studies that the

nature of substitution on the phenyl ring of isoxazoline could play a vital role in the

inhibition of melanoma cancer cell line. It appears that electron withdrawing halogen

substituents at o-position and electron releasing methoxy groups at m and p-positions are
essential for good activity. However, compounds 7b, 7f and 7j were shown little

cytotoxicity to human fibroblast normal cells at the tested concentrations. However, the

compound 7c with 3,4-dimethoxy substitution was selectively toxic to melanoma cancer

cells than normal cells. Therefore, compound 7c is worth studying further in-vivo to evaluate

anti-proliferative and anti-angiogenic efficacy.

FF2441 UACC 903

100
Half maximal inhibitory

80
concentration (μM)

60

40

20

0
3 4 7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l
Test compounds
Fig 1: In-vitro screening of coumarin-tethered isoxazolines for their cytotoxic effect on
melanoma UACC 903 and normal FF2441 cell lines by MTS assay.

Ehrlich Ascites Carcinoma (EAC) murine model is an appropriate system to evaluate

the antitumor effect of leading compound 7c. Six to eight weeks old Swiss albino mice of

either sex were used for establishing EAC model. Mice were obtained and maintained in the

animal house, Barathi College of Pharmacy, Barathinagara, Mandya, India

(BCP/IACE/EXPT/05/2018). The Compound 7c was administrated intraperitoneally at the

concentration of 100 mg/kg body weight into EAC bearing mice on every alternative day

from 7th day of tumor implantation. After the treatment, there was gradual decrease in the

body weight of the mice indicating the dose dependent regression and resulted in 85%

suppression in tumor volume after three doses of compound administration. Whereas,

control mice has shown an increased body weight signifying the growth of the tumor (Fig

2A). Further, ascites volume (Ascites is a fluid accumulates in the region of peritoneum, it
serves as a nutritional source for tumor volume) and total cell count of mice were

determined to interpret the activity of the compound directed. The results illustrated that

there was a decrease in the ascites volume in 7c treated mice when compared to control mice

(Fig 2B). Thus, compound 7c has proficient inhibitory effect on the EAC by shrinking the

level of ascites fluid leading to suppression of tumor volume. Trypan blue dye exclusion

method was used to determine the cell viability. Upon treatment with compound 7c, a

significant decrease in the number of viable cells were observed (Fig 2C).

Fig 2: Anti-tumour effect of compound 7c on EAC tumor model in-vivo

(A) Dose dependent decrease in tumor volume. (B) Dose dependent decrease in ascites volume (C)
Dose dependent decreased cell count (D) Dose dependent increase in survivability
Survivability assay was done to determine mean life span of the treated and control

mice. The mean survival time of the 7c treated mice displayed an increase in the survival

time whereas in control mice mean survival time was very less (17 days). There was two
fold increase in survival span of the treated mice and the studies suggested that compound

7c actively suppressing the tumor volume, where regression in the tumor burden is directly

correlated with increase in the survivability of mice (Fig. 2D). Further, the elucidation of

apoptosis was confirmed by the use of light microscopy and morphological changes of

treated mice cells were assessed using Giemsa stain. Apoptotic morphology such as plasma

membrane degradation, shrinkage of cells, formation of small blebs on the membrane and

apoptotic body formation (Fig 3) were observed which leading to regression in tumor

volume.

Fig 3: Apoptotic morphology of EAC cells upon 7c compound treatment

In order to evaluate the antiangiogenic activity of the lead compound 7c, subjected

for peritoneal angiogenesis and CAM assays in-vivo. In the peritoneal angiogenesis assay

the mice was dissected on the 12th day of the 7c administration to observe the extent of

neovascularization. The result showed limited blood vessels formation in the peritoneal wall

of 7c treated mice compared with the extensive neovascularization of untreated one. The

formation of blood vessels is due to the involvement of growth factor VEGF and secretion

of angiogenic stimulating factors in the ascites fluid. The mice bearing EAC treated with 7c

showed significant suppression in peritoneal angiogenesis indicated the inhibition of the

secretion of angiogenic stimulator and thereby preventing the formation of new unwanted

blood vessels. Treatment with 7c showed beneficial suppression in the peritoneum blood
vessels in contrast with control mice (Fig. 4A).

Fig 4: (A) The reduction in micro vessel density count in mice peritoneum treated with
7c compared to the control and normal. (B) Reduction in new vasculature induced by
rVEGF165 after 7c treatment by CAM assay.

The CAM (chorioallantoic membrane) provides an ideal model for the in-vivo

evaluation of angiogenesis. A potent angiogenic stimulator rVEGF165 was used to induce the

growth of new blood vessels. In-vivo angio-suppressive effect of compound 7c on CAM

displayed usual vascular zone formation not only at the site of introduction but also in

around the disc compared with the extensive vascularization in the rVEGF165 stimulated

CAM (Fig 4B). The study clearly portrays that 7c effectively inhibits the angiogenesis and

may limit the expression of VEGF-like factors or inhibit the secretion of such factors,

thereby inhibiting the formation of new unwanted blood vessels.

In conclusion, a new series of twelve novel coumarin tethered isoxazoline derivatives

were synthesized and well characterized with advanced spectroscopic techniques.

Preliminary studies revealed that the compound 7c emerged as lead anticancer agent among
the series examined, which was selectively cytotoxic to cancerous tissues without affecting

normal cells. Further, administration of compound 7c showed reduction in the tumor

volume, ascites volume, cell number and increased in the life span of the mice. The

avascular zone in peritoneum and CAM assay clearly indicates that compound 7c has strong

antiangiogenic effect. The interpretation of results clearly suggest that the compound 7c can

be considered as good antitumor and antiangiogenic agent which could be useful starting

point in drug development to combat various types of cancer.

Acknowledgment

LGS thank to UGC for financial support (Grant F. No. 37-456/2009 [SR]) and IOE,

University of Mysore, Karnataka for NMR and Mass spectrometry facilities. MPS thank

UGC-SAP-DRS-Phase III, New Delhi for funding.

References:
a. Jemal, A., Bray, F., Center, M. M., Ferlay, J., Ward, E., Forman, D., Global cancer statistics, CA:
a cancer journal for clinicians, 2011, 61(2), 69-90.
b. World Health Organization, Global status report on noncommunicable diseases, 2014
a. Longley, D. B., Johnston, P. G., Molecular mechanisms of drug resistance, The Journal of
Pathology: A Journal of the Pathological Society of Great Britain and Ireland, 2005, 205(2), 275-
292.
b. Swanton, C., Intratumor heterogeneity: evolution through space and time, Cancer research,
2012, 72(19), OF1-OF8
1. Holohan, C., Van Schaeybroeck, S., Longley, D. B., Johnston, P. G., Cancer drug resistance: an
evolving paradigm, Nature Reviews Cancer, 2013,13(10), 714.
2. Giannakakou, P., Sackett, D., Fojo, T., Tubulin/microtubules: still a promising target for new
chemotherapeutic agents, Journal of the National Cancer Institute, 2000, 92(3), 182-183.
3.
a. Ram, V. J., Kumari, S., Natural products of plant origin as anticancer agents, Drug News
Perspect, 2001, 14(8), 465.
b. Petit, K., Biard, J. F., Marine natural products and related compounds as anticancer agents: an
overview of their clinical status, Anti-Cancer Agents in Medicinal Chemistry (Formerly Current
Medicinal Chemistry-Anti-Cancer Agents), 2013, 13(4), 603-631.
c. Martin-Cordero, C., Jose Leon-Gonzalez, A., Manuel Calderon-Montano, J., Burgos-Moron, E.,
Lopez-Lazaro, M., Pro-oxidant natural products as anticancer agents, Current drug targets,
2012, 13(8), 1006-1028.
d. Newman, D. J., Cragg, G. M., Natural products as sources of new drugs over the last 25 years,
Journal of natural products, 2007, 70(3), 461-477.
4. Bhanot, A., Sharma, R., Noolvi, M. N., Natural sources as potential anti-cancer agents: A
review, International journal of phytomedicine, 2011, 3(1), 09-26.
5.
a. Egan, D., O'kennedy, R., Moran, E., Cox, D., Prosser, E., Thornes, R. D., The pharmacology,
metabolism, analysis, and applications of coumarin and coumarin-related compounds, Drug
metabolism reviews, 1990, 22(5), 503-529.
b. Borges, F., Roleira, F., Milhazes, N., Santana, L., & Uriarte, E., Simple coumarins and analogues
in medicinal chemistry: occurrence, synthesis and biological activity, Current medicinal
chemistry, 2005, 12(8), 887-916.
6.
a. Kaur, M., Kohli, S., Sandhu, S., Bansal, Y., Bansal, G., Coumarin: A promising scaffold for
anticancer agents, Anti-Cancer Agents in Medicinal Chemistry, 2015, 15(8), 1032-1048.
b. Emami, S., Dadashpour, S., Current developments of coumarin-based anti-cancer agents in
medicinal chemistry, European journal of medicinal chemistry, 2015,102, 611-630.
7.
a. Murray, R. D., The naturally occurring coumarins, In Fortschritte der Chemie organischer
Naturstoffe/Progress in the Chemistry of Organic Natural Products, 2002, Springer, Vienna.
b. Hoult, J. R. S., Paya, M., Pharmacological and biochemical actions of simple coumarins: natural
products with therapeutic potential. General Pharmacology: The Vascular System, 1996, 27(4),
713-722.
c. Carotti, A., Carrieri, A., Chimichi, S., Boccalini, M., Cosimelli, B., Gnerre, C., Testa, B., Natural
and synthetic geiparvarins are strong and selective MAO-B inhibitors. Synthesis and SAR
studies, Bioorganic & medicinal chemistry letters, 2002,12(24), 3551-3555.
8. Luo, K. W., Sun, J. G., Chan, J. Y. W., Yang, L., Wu, S. H., Fung, K. P., Liu, F. Y., Anticancer
effects of imperatorin isolated from Angelica dahurica: induction of apoptosis in HepG2 cells
through both death-receptor and mitochondria-mediated pathways, Chemotherapy, 2011, 57(6),
449-459.
9. Yang, D., Gu, T., Wang, T., Tang, Q., Ma, C. Effects of osthole on migration and invasion in
breast cancer cells, Bioscience, biotechnology and biochemistry, 2010, 74(7), 1430-1434.
10. Lee, C. R., Shin, E. J., Kim, H. C., Choi, Y. S., Shin, T., Wie, M. B., Esculetin inhibits N-methyl-D-
aspartate neurotoxicity via glutathione preservation in primary cortical cultures. Laboratory
animal research, 2011, 27(3), 259-263.
11. Whang, W. K., Park, H. S., Ham, I., Oh, M., Namkoong, H., Kim, H. K., Kim, J. R., Natural
compounds, fraxin and chemicals structurally related to fraxin protect cells from oxidative
stress, Experimental & molecular medicine, 2005, 37(5), 436-446.
12.
a. Musa, M. A., Cooperwood, J. S., Khan, M. O. F., A review of coumarin derivatives in
pharmacotherapy of breast cancer, Current medicinal chemistry, 2008,15(26), 2664-2679.
b. Devji, T., Reddy, C., Woo, C., Awale, S., Kadota, S., Carrico-Moniz, D., Pancreatic anticancer
activity of a novel geranylgeranylated coumarin derivative, Bioorganic & medicinal chemistry
 letters, 2011, 21(19), 5770-5773.
13.
a. Egan, D., James, P., Cooke, D., O'Kennedy, R., Studies on the cytostatic and cytotoxic effects
and mode of action of 8-nitro-7-hydroxycoumarin, Cancer letters, 1997,118(2), 201-211.
b. Finn, G., Creaven, B., Egan, D., Modulation of mitogen-activated protein kinases by 6-nitro-7-
hydroxycoumarin mediates apoptosis in renal carcinoma cells, European journal of
pharmacology, 2003, 481(2-3), 159-167.
c. Elinos‐Báez, C. M., Leon, F., Santos, E., Effects of coumarin and 7OH‐coumarin on bcl‐2 and
Bax expression in two human lung cancer cell lines in-vitro, Cell biology international, 2005,
29(8), 703-708.
14.
a. Stanchev, S., Momekov, G., Jensen, F., Manolov, I., Synthesis, computational study and
cytotoxic activity of new 4-hydroxycoumarin derivatives, European journal of medicinal chemistry,
2008, 43(4), 694-706.
b. Thornes, R. D., Daly, L., Lynch, G., Breslin, B., Browne, H., Browne, H. Y., Henley, J., Treatment
with coumarin to prevent or delay recurrence of malignant melanoma, Journal of cancer research
and clinical oncology, 1994, 120(1), S32-S34.
c. Mohler, J. L., Gomella, L. G., Crawford, E. D., Glode, L. M., Zippe, C. D., Fair, W. R., Marshall,
M. E., Phase II evaluation of coumarin (1, 2‐benzopyrone) in metastatic prostatic carcinoma, The
Prostate, 1992, 20(2), 123-131.
15.
a. Wang, C. J., Hsieh, Y. J., Chu, C. Y., Lin, Y. L., Tseng, T. H., Inhibition of cell cycle progression
in human leukemia HL-60 cells by esculetin, Cancer letters, 2002, 183(2), 163-168.
b. Nasr, T., Bondock, S., Youns, M., Anticancer activity of new coumarin substituted hydrazide–
hydrazone derivatives, European journal of medicinal chemistry, 2014, 76, 539-548.
16. Cohen, A. J., Critical review of the toxicology of coumarin with special reference to interspecies
differences in metabolism and hepatotoxic response and their significance to man, Food and
cosmetics toxicology, 1979, 17(3), 277-289.
17. Lacy, A., O'kennedy, R., Studies on coumarins and coumarin-related compounds to determine
their therapeutic role in the treatment of cancer, Current pharmaceutical design, 2004, 10(30),
3797-3811.
18. Shan, D., Nicolaou, M. G., Borchardt, R. T., Wang, B., Prodrug strategies based on
intramolecular cyclization reactions, Journal of pharmaceutical sciences, 1997, 86(7), 765-767
19.
a. Kolodziej, H., Kayser, O., Woerdenbag, H. J., van Uden, W., Pras, N., Structure-cytotoxicity
relationships of a series of natural and semi-synthetic simple coumarins as assessed in two
human tumour cell lines, Zeitschrift für Naturforschung C, 1997, 52(3-4), 240-244.
b. Belluti, F., Fontana, G., Dal Bo, L., Carenini, N., Giommarelli, C., Zunino, F., Design, synthesis
and anticancer activities of stilbene-coumarin hybrid compounds: Identification of novel
proapoptotic agents, Bioorganic & medicinal chemistry, 2010,18(10), 3543-3550.
c. Sashidhara, K. V., Kumar, A., Kumar, M., Sarkar, J., & Sinha, S., Synthesis and in-vitro
evaluation of novel coumarin–chalcone hybrids as potential anticancer agents, Bioorganic &
 medicinal chemistry letters, 2010, 20(24), 7205-7211
20.
a. Mayur, Y. C., Peters, G. J., Rajendra Prasad, V. V. S., Lemos, C., & Sathish, N. K., Design of
new drug molecules to be used in reversing multidrug resistance in cancer cells, Current cancer
drug targets, 2009, 9(3), 298-306.
b. Solomon, V. R., Hu, C., Lee, H., Hybrid pharmacophore design and synthesis of isatin–
benzothiazole analogs for their anti-breast cancer activity, Bioorganic & medicinal
chemistry, 2009,17(21), 7585-7592.
a. Milinkevich, K. A., Yoo, C. L., Sparks, T. C., Lorsbach, B. A., Kurth, M. J., Synthesis and
biological activity of 2-(4, 5-dihydroisoxazol-5-yl)-1, 3, 4-oxadiazoles, Bioorganic & medicinal
chemistry letters, 2009,19(19), 5796-5798.
b. Jayashankara, B., & Rai, K. L., Synthesis and evaluation of antimicrobial activity of a new series
of bis (isoxazoline) derivatives, Arkivoc, 2008,11, 75-85.
21. Al Houari, G., Kerbal, A., Bennani, B., Baba, M. F., Daoudi, M., Hadda, T. B., Drug design of new
antitubercular agents: 1, 3-dipolar cycloaddition reaction of para-substituted-benzadoximes and
3-para-methoxy-benzyliden-isochroman-4-ones, Arkivoc, 2008,12, 42-50.
22. Lahm, G. P., Cordova, D., Barry, J. D., Pahutski, T. F., Smith, B. K., Long, J. K. Schroeder, M.
E., 4-Azolylphenyl isoxazoline insecticides acting at the GABA gated chloride
channel, Bioorganic & medicinal chemistry letters, 2013, 23(10), 3001-3006.
23. Zhang, P., Wei, C., Wang, E., Wang, W., Liu, M., Yin, Q., Zhang, J., Synthesis and biological
activities of novel isoxazoline-linked pseudodisaccharide derivatives, Carbohydrate
research, 2012, 351, 7-16.
24. Maurya, R., Ahmad, A., Gupta, P., Chand, K., Kumar, M., Rawat, P., Palit, G., Synthesis of novel
isoxazolines via 1, 3-dipolar cycloaddition and evaluation of anti-stress activity, Medicinal
chemistry research, 2011, 20(2), 139-145.
25.
a. Puttaswamy, N., Kumar, G. P., Al-Ghorbani, M., Vigneshwaran, V., Prabhakar, B. T., Khanum, S.
A., Synthesis and biological evaluation of salicylic acid conjugated isoxazoline analogues on
immune cell proliferation and angiogenesis, European journal of medicinal chemistry, 2016, 114,
153-161.
b. Shi, L., Hu, R., Wei, Y., Liang, Y., Yang, Z., & Ke, S., Anthranilic acid-based diamides derivatives
incorporating aryl-isoxazoline pharmacophore as potential anticancer agents: design, synthesis
and biological evaluation, European journal of medicinal chemistry, 2012, 54, 549-556.
c. Kamal, A., Bharathi, E. V., Reddy, J. S., Ramaiah, M. J., Dastagiri, D., Reddy, M. K., Bhadra, M.
P., Synthesis and biological evaluation of 3, 5-diaryl isoxazoline/isoxazole linked 2, 3-
dihydroquinazolinone hybrids as anticancer agents, European Journal of Medicinal
Chemistry, 2011, 46(2), 691-703.
26. Conti, P., Dallanoce, C., De Amici, M., De Micheli, C. & Klotz, K. N., Synthesis of new Δ2-
isoxazoline derivatives and their pharmacological characterization as β-adrenergic receptor
antagonists, Bioorganic & medicinal chemistry, 1998, 6(4), 401-408.
27. Kaur, K., Kumar, V., Sharma, A. K., & Gupta, G. K., Isoxazoline containing natural products as
 anticancer agents: a review, European journal of medicinal chemistry, 2014, 77, 121-133.
28. Sadashiva, M. P., NanjundaSwamy, S., Li, F., Manu, K. A., Sengottuvelan, M., Prasanna, D. S.,
Rangappa, K. S., Anti-cancer activity of novel dibenzo [b, f] azepine tethered isoxazoline
derivatives, BMC chemical biology, 2012,12(1), 5.
29. Potdar, M. K., Mohile, S. S., Salunkhe, M. M., Coumarin syntheses via Pechmann condensation
in Lewis acidic chloroaluminate ionic liquid, Tetrahedron Letters, 2001,42(52), 9285-9287.

Synthesis of New Coumarin Tethered Isoxazolines as Potential Anticancer

Agents

Highlights:
 A novel series of new coumarin tethered isoxazolines were synthesized via 1,3-cyclo
addition reaction.
 Compound 7c is active against human melanoma cancer line without toxic to normal
human cells at tested concentration.
 Compound 7c is effective in Ehrlich Ascites Carcinoma animal model in in-vivo.