Synthesis and biological evaluation of 4-styrylcoumarin derivatives as inhibitors of TNF-α and IL-6 with anti-tubercular activity

Article abstract of DOI:10.1016/j.bmcl.2011.02.016

A series of 4-styrylcoumarin have been synthesized by Knoevenagel condensation between substituted 4-methylcoumarin-3-carbonitrile and different heterocyclic or aromatic aldehydes. 4-Methylcoumarin-3-carbonitrile has been synthesized by the base catalyzed reaction between substituted 2-hydroxyacetophenone and ethyl cyanoacetate. The structures of the newly synthesized compounds were confirmed by ¹H NMR, IR and mass spectral analysis. All the compounds were evaluated for their anti-inflammatory activity (against TNF-α and IL-6) and anti-tubercular activity. Compounds 6a, 6h and 6j exhibited promising activity against IL-6 with 72-87% inhibition and compound 6v showed potent activity against TNF-α with 73% inhibition at 10 μM concentration. Whereas compounds 6n, 6o, 6r and 6u showed very good anti-tubercular activity against Mycobacterium tuberculosis H37Rv strain at <6.25 μM.

Full text of DOI:10.1016/j.bmcl.2011.02.016

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2547–2549
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 4-styrylcoumarin derivatives
as inhibitors of TNF-a and IL-6 with anti-tubercular activity
Kuldip Upadhyay a, Abhay Bavishi b, Shailesh Thakrar b, Ashish Radadiya b, Hardevsinh Vala b, Shrey Parekh b,
Dhairya Bhavsar b, Mahesh Savant b, Manisha Parmar b, Priti Adlakha a, Anamik Shah b,
⇑
a R&D Centre, Torrent Research Centre, Ahmedabad 380 009, Gujarat, India
b Department of Chemistry, Saurashtra University, Rajkot 360 005, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-styrylcoumarin have been synthesized by Knoevenagel condensation between substituted
4-methylcoumarin-3-carbonitrile and different heterocyclic or aromatic aldehydes. 4-Methylcoumarin-
3-carbonitrile has been synthesized by the base catalyzed reaction between substituted 2-hydroxyaceto-
phenone and ethyl cyanoacetate. The structures of the newly synthesized compounds were confirmed by
1H NMR, IR and mass spectral analysis. All the compounds were evaluated for their anti-inflammatory
Received 29 November 2010
Revised 22 January 2011
Accepted 4 February 2011
Available online 15 February 2011
activity (against TNF-
promising activity against IL-6 with 72–87% inhibition and compound 6v showed potent activity against
TNF- with 73% inhibition at 10 M concentration. Whereas compounds 6n, 6o, 6r and 6u showed very
good anti-tubercular activity against Mycobacterium tuberculosis H37Rv strain at <6.25 M.
a and IL-6) and anti-tubercular activity. Compounds 6a, 6h and 6j exhibited
Keywords:
4-Styrylcoumarin
Anti-inflammatory activity
a
l
l
TNF-a
Ó 2011 Elsevier Ltd. All rights reserved.
IL-6
Anti-tubercular
Inflammation is the complex process due to which it causes a
large number of diseases, among this some commonest are rheu-
matoid arthritis (RA), inflammatory bowel disease, psoriasis and
including end-stage renal disease and rheumatoid arthritis.11 The
IL-6 inhibitors can be used in Alzheimer’s disease, psychiatric disor-
ders, cancer, diabetes, and depression.12–14 Till date, designing the
IL-6 inhibitory agents has remained a significant hope in the main-
stream of anti-inflammatory drug development.
Tuberculosis (TB) is one of the leading causes of death and suffer-
ing worldwide among the infectious diseases. The ever-increasing
drug resistance, toxicity and side effects of currently used anti-
tuberculosis drugs and the absence of their bactericidal activity
highlight the need for new, safer and more effective anti-tuberculo-
sis drugs.15–19 Generation of functional molecular diversity to probe
biological activity space requires robust molecular scaffolds that are
low in molecular weight and are easily modified to create a variety of
chemically diverse, biologically active potential drugs.20,21
The coumarins have long been recognized to possess anti-inflam-
matory, antioxidant, antiallergic, hepatoprotective, antithrombotic,
antiviral, and anticarcinogenic activities.22 A series of 6- or 7-styryl-
coumarin were synthesized by Song et al.23 in order to find
compounds of antitumor activities and several compounds showed
different inhibitory effects on L-1210, HL-60, HCT-8, KB and Bel-
7402 cell lines in vitro. In continuation with this study Su and
co-workers have synthesized a series of 4-styryl coumarin and re-
ported in vitro antitumor activity of on KB cell lines.24 As an attempt
to determine basic structural features required as antimicrobial
agents Kawase et al.25 have synthesized a series of polyfunctional-
ized 3-cyano-4-styrylcoumarin and suggested that five compounds
shows moderate activity against Staphylococcus aureus NCTC 8530,
multiple sclerosis.1,2 Tumor necrosis factor-alpha (TNF-
a) and
Interleukin-6 (IL-6) are two major multifunctional proinflamma-
tory mediators of a variety of autoimmune diseases such as pain
and joint destruction characteristics of RA.3 The inhibition of
release of cytokines becomes a major focus of current drug devel-
opment and an important method for evaluating the bioactivity of
drugs.4 It is a key cytokine in the inflammation cascade, causing
the production and/or release of other cytokines and agents.
Over-expression of TNF-a is responsible for a number of patholog-
ical conditions like Crohn’s disease, ulcerative colitis,5 diabetes,6
multiple sclerosis,7 atherosclerosis,8 and stroke.9 In spite of enor-
mous efforts, no small molecule has yet been approved to specifi-
cally inhibit TNF-a activity. TNF-a inhibitor drugs in clinics are
proteins (Etanercept, Infliximab, Adalimumab, and Anakinra) that
display adverse effects such as aplastic anemia, pancytopenia, vas-
culitis, demyelination and congestive heart failure.10
Among pro-inflammatory cytokines, the IL-6 is a multifunctional
cytokine produced by a variety of cells in response to infection, trau-
ma, or immunological challenge. It appears to be the central media-
tor of anemia of chronic disease in a range of inflammatory diseases,
⇑
Corresponding author.
E-mail address: anamik_shah@hotmail.com (A. Shah).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.016

2548
K. Upadhyay et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2547–2549
Table 1
S. aureus NCTC 8531, S. aureus ML 36, Shigella dysenteriae 1,
Escherichia coli Raw, Vibrio cholerae945, V. cholerae946, E. coli Raw
and Shigella Sonnei at MIC value of 25 lg/mL.
Physical constants of 6-chloro-2-oxo-4-[-2-subst. ethenyl]-2H-chromene-3-
carbonitriles
The 50-chloro-20-hydroxy acetophenone were synthesized by
Fries rearrangement of o-acetyl ester derivative of 4-chlorophenol
gave only single isomer as para position was already occupied with
the substitution. This on Claisen condensation with ethyl cyanoac-
etate gave 6-chloro-3-cyano-4-methylcoumarin. The method, we
earlier practiced suggested by Kendall and Axford method71 was
very drastic and time consuming. The current work presented a
direct, yield efficient and operationally convenient approach to
the synthesis of some styryl derivatives of the substituted 3-
cyano-4-methyl coumarins as during the course of cyclization
the rate of both the Aldol condensation and intramolecular Claisen
condensation proceeds very smoothly and speedily at mild reac-
tion conditions and the reaction outcome as high purity of the
product. Thus, the 6-chloro-3-cyano-4-methylcoumarin obtained
on Knoevenagel condensation with different substituted aromatic
and hetero (aromatic) aldehydes, afforded the corresponding
6-chloro-3-cyano-4-styrylcoumarin derivatives (6a–6v) as per
Scheme 1. All the newly synthesized compounds were well charac-
terized by IR, NMR and mass spectral and elemental analysis. The
physical constant and elemental analysis data of all the synthe-
sized compounds were shown in Table 1.
Entry
Substitution
Mp
6a
6b
6c
6d
6e
6f
6g
6h
6i
Phenyl
4-F Phenyl
2-Cl Phenyl
3-Cl Phenyl
4-Cl Phenyl
3-Br Phenyl
2-NO2 Phenyl
3-NO2 Phenyl
3-OH Phenyl
4-OH Phenyl
2-OCH3 Phenyl
4-OCH3 Phenyl
3,4-DiOCH3 phenyl
3,4,5-TriOCh3 phenyl
3-OC6H5 Phenyl
4-SCH3 Phenyl
2-Ethenyl phenyl
9-Anthracyl
193–195
246–248
257–259
239–241
275–277
235–237
139–141
236–238
229–231
160–162
157–159
>300
203–205
261–263
221–223
205–207
257–259
115–117
173–175
158–160
156–158
202–204
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
2-Furyl
3-Indolyl
4-N,N-DiCh3 amine-phenyl
3- OCH3Phenyl
6u
6v
All the synthesized compounds were evaluated for anti-
inflammatory and anti-tubercular activity and the results are
summarized in Table 2. All molecules of the series show moderate
Table 2
Anti-inflammatory (TNF-
chloro-2-oxo-4-[-2-subst. ethenyl]-2H-chromene-3-carbonitriles
a and IL-6 Inhibition) and anti-tubercular activity of 6-
to good TNF-
Compound 6v possessing methoxy group on phenyl ring at
m-position shows good TNF- inhibitory activity, while phenyl
a and IL-6% inhibition and anti-tubercular activity.
Entry
Anti-inflammatory activity
(%Inhibition at 10 M concn)
Anti-tubercular
activity (%Inhibition at
l
a
<6.25 lM Concn) Mycobacterium
tuberculosis H37Rv strain
ring having electron withdrawing group as substitution makes
molecules inactive (6b–6h). Molecule 6a with unsubstituted
phenyl group acquires good inhibitory activity of IL-6, while sub-
stitution weakens the inhibitory activity. Though, molecule 6j con-
taining hydroxyl group at p-position in phenyl ring induces the
potency while bulky group makes molecule inactive.
We observed that molecules having bulky group shows good
anti-tubercular activity, as 6n and 6r having 3,4,5 tri-OCH3 phenyl
and 9-anthracyl group improves the potency, respectively. More-
over, electron withdrawing groups attached at m-position of phe-
nyl ring (6d and 6h) demonstrates induced activity.
TNF-
a
IL-6
6a
6b
6c
6d
6e
6f
6g
6h
6i
32
0
0
0
0
0
0
0
23
0
19
ND
0
38
0
87
61
19
21
4
55
ND
ND
69
ND
23
11
69
ND
ND
24
33
63
87
72
ND
ND
81
63
ND
79
31
2
14
72
39
73
17
ND
40
8
11
15
0
ND
0
6j
6k
6l
Pro-inflammatory cytokine production by lipopolysaccharide
(LPS) in THP-1 cells was measured according to the method
described by Hwang et al., 1933.26 Briefly, THP-1 cells were cul-
tured in RPMI 1640 culture medium (Gibco BRL, Pasley, UK) con-
6m
6n
6o
6p
6q
6r
6s
6t
0
52
ND
9
31
33
73
taining 100
l
/mL penicillin and 100 mg/mL streptomycin (100ꢀ
solution), Sigma Chemical Co. St. Louis, MO) containing 10% fetal
bovine serum (FBS, JRH). Cells were differentiated with phorbol
myristate acetate (PMA, Sigma). Following cell plating, the test
compounds or vehicle (0.5% DMSO) was added to each well and
the plate was incubated for 30 min at 37 °C. Finally, LPS (E. coli
0
0
0
6u
6v
0127: B8, Sigma Chemical Co., St. Louis, MO) was added, at a final
concentration of 1 mg/mL. Plates were incubated at 37 °C for 24 h,
O
CH3
OH
5% CO2. Supernatants were harvested and assayed for TNF-
a and
Cl
Cl
ii
Cl
Cl
i
O
IL-6 by ELISA as described by the manufacturer (BD Biosciences).
The cells were simultaneously evaluated for cytotoxicity using
CCK-8 from Dojindo Laboratories. Percent inhibition of cytokine re-
lease compared to the control was calculated.
O
CH3
OH
3
1
2
iii
R
CH3
The primary screen is conducted at 6.25 lg/mL (or molar equiv-
N
N
Cl
O
iv
alents of highest molecular weight compound in a series of cong-
eners) against Mycobacterium tuberculosis H37Rv (ATCC 27294) in
BACTET 12B medium using Microplate Almar Blue Assay (MABA).27
Compounds exhibiting fluorescence are tested in the BACTET 460-
radiometric system.27 Compounds effecting >99% inhibition in the
R
O
4
O
O
O
5
6(a-v)
Scheme 1. Reagents and conditions: (i) acetic anhydride, cat. sulfuric acid, reflux,
2 h; (ii) anhyd AlCl3, fusion 160° C, 6 h; (iii) ethyl cyanoacetate, sodium ethoxide,
ethanol, reflux, 2 h; (iv) cat. piperidine, dichloromethane or chloroform, 1.5–3 h.
primary screen (MIC <6.25 lg/mL) are generally evaluated further.

K. Upadhyay et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2547–2549
2549
In summary, we have synthesized a new series of 4-styrylcoum-
References
arin derivatives and evaluated for their anti-inflammatory and
anti-tubercular activity. Among them, molecules 6a, 6h and 6j
exhibited promising activity against IL-6 with 72–87% inhibition
1. Marc, F.; Brennan, F. M.; Brian, F.; Maini, R. N. Curr. Dir. Autoimmun. 2001, 3, 188.
2. Daniel, T.; Lars, K.; Eric, S. H.; Jochen, S. G.; Paul, T. P. Pharmacol. Ther. 2008, 117,
244.
3. Josef, S. S.; Gunter, S. Nat.Rev.Drug Discov. 2003, 2, 473; Daniel, F. E. Clin. Ther.
2004, 26, 1960.
4. Papadakis, K. A.; Targan, S. R. Inflamm. Bowel. Dis. 2000, 6, 303.
5. Newton, R. C.; Decicco, C. P. J. Med. Chem. 1999, 42, 2295.
6. Hotamisligil, G. S.; Arner, P.; Caro, J. F.; Atkinson, R. L.; Spiegelman, B. M. J. Clin.
Invest. 1995, 95, 2409.
and molecule 6v showed significant TNF-
73% inhibition at 10 M concentration. In addition, molecules 6n,
6o, 6r and 6u showed 87%, 72%, 81% and 79% inhibition respec-
tively against M. tuberculosis H37Rv strain at <6.25 M. However,
a inhibitory activity with
l
l
this structure activity relationship (SAR) might provide these
7. Selmaj, K.; Raine, C. S.; Cannella, B.; Brosnan, C. F. J. Clin. Invest. 1991, 87, 949.
8. Rus, H. G.; Niculescu, F.; Vlaicu, R. Atherosclerosis 1991, 89, 247.
9. Lovering, F.; Zhang, Y. Curr. Drug Targets 2005, 4, 161.
10. Desai, B. S.; Furst, D. E. Best Pract. Res. Clin. Rheumatol. 2006, 20, 757.
11. Dominic, S. C. R. Semin. Arthritis Rheum. 2009, 38, 382.
12. Handraskar, B.; Mitchell, D. H. Hepato-Gastroenterology 2005, 45, 1807.
13. Rosler, N.; Wichart, I.; Jollinger, K. A. Acta Neurol. Scand. 2001, 103, 126.
14. Jahromi, M. M.; Millward, B. A.; Demaine, A. G. J. Interferon Cytokine Res. 2000,
20, 885.
compounds a potential suitable tool for designing new molecules
having specific selectivity for TNF-
a and IL-6 inhibition and anti-
tubercular activity. Furthermore, the 4-styrylcoumarin moiety
can be optimized as a lead molecule for anti-inflammatory or
anti-tubercular agents.
Acknowledgments
15. Bodiang, C. K. Scott. Med. J. 2000, 45, 25.
16. Van Scoy, R. E.; Wilkowske, C. J. Mayo Clin. Proc. 1999, 74, 1038.
17. Long, R. Can. Med. Assoc. J. 2000, 163, 425.
18. Gazdic, A. Med. Arch. 1998, 52, 207.
19. Pozniak, A. Int. J. Tuberc. Lung Dis. 2000, 4, 993.
20. Dimoglo, A. S.; Sim, E.; Shvets, N. M.; Ahsen, V. Mini-Rev. Med. Chem. 2003, 3,
293.
The authors are thankful to Mrs. Asha Almeida, Piramal Life Sci-
ences Ltd., Mumbai for screening of the compounds against TNF-
a
and IL-6 and Dr.Cecil Kwong, Antimicrobial Acquisition and Coor-
dinating Facility (TAACF), Alabama, USA, for anti-tubercular activ-
ity screening and also to National Facility for Drug Discovery
supported by DPRS at Saurashtra University, Rajkot.
21. Dimoglo, A. S.; Shvets, N. M.; Tetko, I. V.; Livingstone, D. J. Quant. Struct.-Act.
Relat. 2001, 20, 31.
22. Kostava, I. Curr. Med. Chem.-Anti Cancer Agents 2005, 5, 29.
23. Song, X.; Shiping, X.; Lanmin, L. Acta Pharm. Sin. 2000, 35(2), 103.
24. Xu, S.; Su, S.; Li, L. Yaoxue Xuebao 2001, 36(4), 273.
25. Kawase, M.; Varu, B.; Shah, A.; Motohashi, N.; Tani, S.; Saito, S.; Debnath, S.;
Mahapatra, S.; Dastidar, S.; Chakrabarty, A. Arzneim.-Forsch. Drug Res. 2001, 51, 67.
26. Hwang, C.; Catanaga, M.; Gale, A.; Gatanaga, T. J. Immunol. 1993, 151, 5631.
27. Collins, L.; Franzblau, S. Antimicrob. Agents Chemother. 1997, 41, 1004.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.016.