Synthesis and evaluation of anti-thrombotic activity of benzocoumarin amide derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.03.059

A series of novel benzocoumarin amide derivatives have been synthesized and evaluated for their anti-thrombotic activity. Amongst these, compounds 5, 7 and 8 exhibited promising anti-thrombotic profile in an established model of mouse thrombosis. Hence, c

Full text of DOI:10.1016/j.bmcl.2012.03.059

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3115–3121
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of anti-thrombotic activity of benzocoumarin
amide derivatives q
Koneni V. Sashidhara a, , Gopala Reddy Palnati a, Srinivasa Rao Avula a, Surendra Singh b, Manish Jain b,
⇑
Madhu Dikshit b,
a Medicinal and Process Chemistry Division, Central Drug Research Institute, CSIR-CDRI, Lucknow 226 001, India
b Pharmacology Division, Central Drug Research Institute, CSIR-CDRI, Lucknow 226 001, 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 novel benzocoumarin amide derivatives have been synthesized and evaluated for their anti-
thrombotic activity. Amongst these, compounds 5, 7 and 8 exhibited promising anti-thrombotic profile
in an established model of mouse thrombosis. Hence, comprehensive profiling on platelet aggregation
and coagulation parameters was carried out to assess its potential as a lead candidate. In vitro treatment
of these compounds in mice plasma resulted into significant reduction in ADP (p < 0.01) and collagen
(p < 0.001) induced platelet aggregation. Moreover, Compounds 5, 7 and 8 also significantly increased
thrombin time (p < 0.05). Thus, in the present study, these benzocoumarin amide derivatives exhibited
anti-thrombotic profile via both anti-platelet as well as anti-coagulant action.
Received 23 December 2011
Revised 6 March 2012
Accepted 15 March 2012
Available online 21 March 2012
Keywords:
Pechmann condensation
1,5-Dihydroxy naphthalene
Benzocoumarins amide
Anti-thrombotic activity
Bleeding time
Ó 2012 Elsevier Ltd. All rights reserved.
Anti-platelet
Anti-coagulant
Thrombotic events are leading causes of cardiovascular-associ-
ated death in the developed world.1,2 Prophylaxis and treatment of
arterial thrombosis in patients with cardiovascular diseases are
achieved using anti-platelet drugs,3 whereas venous thrombosis
is treated with anti-coagulant drugs, which inhibit various prote-
ases in the blood coagulation cascade.4–6 The current therapies of
anti-platelet drugs include the most commonly used aspirin (a,
Fig. 1), the thienopyridine derivative clopidogrel (b, Fig. 1), and
its more potent analog prasugrel (c, Fig. 1), which are antagonists
of the cell-surface ADP receptor P2Y12,7 and furthermore, the
recently discovered antagonists of the thrombin receptor PAR-1
(protease-activated receptor-1) such as SCH 530348 (d, Fig. 1) is
currently under phase-III clinical trials.8,9 Anti-coagulant therapy,
used to treat a variety of conditions including cure and prevention
of venous thromboembolism and long-term prevention of ischemic
stroke in patients with arterial fibrillation, involves heparins and
the vitamin K antagonist, warfarin (e, Fig. 1).
clopidogrel) can entail a risk of recurrent thrombosis. On the other
hand, a number of drug-class-specific adverse reactions include, for
instance, aspirin-related gastrointestinal ulcers and bleeding,
thrombotic thrombocytopenic purpura that may be associated to
thienopyridines,10 heparin-induced thrombocytopenia,11 warfa-
rin-induced skin necrosis etc.12 Moreover, warfarin has a narrow
therapeutic window, and its activity is affected by diet and genetic
makeup, so requiring continuous monitoring for accurate dosing.13
Hence, the search for more potent and safer new chemical entities
for anti-platelet/anti-thrombotic activity is of current interest.
Based on our earlier laboratory experiences14 and the litera-
tures where various carboxamides (f and g Fig. 1)15 were reported
as an oral, factor Xa Inhibitors and coumarins were reported as
mechanism based inhibitors of thrombin (h, Fig. 1),16 selective
FXIIa inhibitors17 and anti-thrombotic activity.18 It appeared of
interest to explore the pharmacophoric character of this relatively
unexploited substituent for anti-thrombotic activity. Thus, instead
of chalcone moiety at position 6 and amide at position 3 (our
earlier lead, i as shown in Fig. 1), in our present approach, we have
placed the amide at position 7 and also introduced a methyl at
position 4 (since unsubstituted coumarins have been found to
be toxic since they undergo oxidative decarboxylation to form
very stable complexes with heavy metals inside the body). It ap-
peared of interest to explore these substructures along with
hydrophobic phenyl rings of the bioactive coumarin, for their
anti-thrombotic activity in mice model. Recently, we have
The main side effect of most of the available anti-thrombotic
drugs is bleeding, whereas therapy of insufficient intensity (aspirin,
q
Part XVIII in the series, ‘Advances in drug design and discovery’.
⇑
Corresponding author. Mobile: +91 9919317940; Tel.: +91 522 2625036;
fax: +91 522 2628493.
E-mail addresses: sashidhar123@gmail.com, kv_sashidhara@cdri.res.in (K.V.
Sashidhara), madhu_dikshit@cdri.res.in (M. Dikshit).
This author has been responsible for the biological activity, experimental design
and evaluations.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.059

3116
K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3115–3121
Figure 1. Chemical structures of some important anti-platelet and anti-coagulant agents and our designed prototypes.
reported 3-carboxamide-benzocoumarin derivatives as orally ac-
tive anti-thrombotic agents.14 Our initial studies towards the
identification of a new lead for anti-thrombotic agents indicated
compound (E)-N-ethyl-2-oxo-6-(3-oxo-3-p-tolylprop-1-enyl)-2H-
benzo[h]chromene-3-carboxamide (i, Fig. 1) as biologically impor-
tant scaffold. This compound showed 40% protection in mouse
thrombosis model, thus a new series of novel benzocoumarin
amide derivatives were synthesized and assessed for the
anti-thrombotic activity against collagen epinephrine induced
thrombosis in mice, an established methodology for preliminary
screening of anti-thrombotic agents. The details of these studies
are discussed next.
The reaction sequences employed for synthesis of title com-
pounds are shown in Scheme 1. The Pechmann condensation on
1,5-dihydroxy naphthalene resulted in formation of 7-hydroxy-4-
methyl-2H-benzo[h]chromen-2-one (1) which on reaction with
ethylbromoacetate in presence of K2CO3 and acetonitrile as solvent
under reflux conditions cleanly produced ethyl 2-(4-methyl-2-oxo-
2H-benzo[h]chromen-7-yloxy)acetate (2). Furthermore, the com-
pound 2 on alkaline hydrolysis furnished the acid derivative 3,
which was converted to its respective acid chloride 4 using thionyl
chloride. This acid chloride 4 was then condensed with various
amines to cleanly furnish the benzocoumarin amides (5–18)19
(Scheme 1). All compounds were characterized using 1H NMR,
13C NMR, mass spectrometry and IR spectroscopy. The purity of
these compounds was ascertained by TLC and spectral analysis
(See Supplementary data).
The compounds (5–18) were administered po (30 lM), 1 h prior to
the thrombotic challenge. The anti-thrombotic effect of these syn-
thesizedcompoundswas assessed by the percent protection (% P) of-
fered by these agents to mice from death or paralysis following
thrombotic challenge. Aspirin, the reference compound was tested
under the same conditions and the results are presented in
Table 1. Out of 14 compounds evaluated, three compounds (5, 7,
and 8) showed 40%, 40%, and 45% protection for the thrombosis,
respectively, while remaining compounds (9, 10, 14, and 18)
exhibited activity ranging in between 20% and 30%. From this study,
compound 8 was found to be most promising, which exhibited 45%
anti-thrombotic protection (Aspirin under the similar conditions
showed 37% protection). The % increase in bleeding time was less
for the compounds 5 (91.5%) and 7 (96.5%) but for the most active
compound 8 the bleeding time (112.5%) was comparable with
Aspirin (100%). It is important to mention that due to presence of
coumarin moiety in the synthesized compounds, these were also
evaluated with warfarin as standard, in a separate set of experi-
ments (data not shown). Interestingly, there were no significant
differences in the results, in terms of anti-thrombotic protection of-
fered by these compounds. With these promising activities in hand,
platelet aggregation and coagulation parameters of compounds 5, 7,
and 8 were also studied, which are discussed next. Platelet aggrega-
tion22 studies were carried out to analyze the anti-plateleteffect of 5,
7, and 8. In this study, significant reduction in ADP (Adenosine 50-
diphosphate) (p < 0.01) and collagen (p < 0.001) induced aggrega-
tion was observed by these compounds at 30
comparison to the vehicle treated control as shown in Figure 2.
However at 10 M concentrations of these compounds had no
lM concentration in
Present study was undertaken to assess the anti-thrombotic and
anti-coagulant efficacy of benzocoumarin amide derivatives.20,21
l

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3115–3121
3117
Scheme 1. Synthesis of novel benzocoumarin amide derivatives (5–18). Reagents and conditions: (a) Ethylacetoacetate, PTSA, ethanol, reflux, 3 h; (b) ethylbromoacetate,
CH3CN, K2CO3, reflux, 4 h; (c) 20% aq KOH, ethanol, rt,1–3 h; (d) SOCl2, benzene, reflux, 2 h; (e) different amines, benzene, reflux, 50–90 min.
significant effect on ADP or collagen induced platelet aggregation.
The standard anti-platelet agent, aspirin exerted significant inhibi-
tory effect against ADP and collagen induced platelet aggregation
aggregation (in vitro), in which significant platelet inhibition was
observed against both ADP and collagen. The anti-platelet effects
shown by these derivatives led us to conclude that these three
compounds exhibited significant anti-thrombotic due to inhibi-
tory effect on platelet activation. We also evaluated efficacy of
compounds 5, 7, and 8 on blood coagulability and compared with
direct thrombin inhibitor that is, Hirudin. Pre-incubation with
these compounds resulted into significant increase in TT, while
other coagulation parameters namely PT and APTT remained unal-
tered. These results seem to be in agreement to the previous pub-
lished reports on amide derivatives such as iodoacetamide, which
was responsible for non-specific inhibition of factor, XIIIa.23 Ex
vivo studies in the mice plasma after feeding with compounds
5, 7, and 8 for three days, resulted in enhanced TT, whereas no
change in PT and APTT was observed, suggesting that the mecha-
nism of action of these compounds might be different than that of
standard coumarin derivative that is, Warfarin.24 These results
suggest that benzocoumarin amide derivatives 5, 7, and 8 have
anti-thrombotic activity, and their mode of anti-thrombotic action
seems to be due to anti-platelet and by anti-coagulant
mechanisms.
at 100
effect on ADP and collagen induced aggregation, which was reduced
by 38% and 90%, respectively at 30 M/kg dose, which was also
lM concentration. Compound 7 was tested for its ex vivo
l
exhibiting anti-thrombotic effect against collagen and epinephrine
induced thrombosis. These active compounds 5, 7, and 8 were fur-
ther tested for their effect on coagulation cascade,22 a significant
increase in the TT (Thrombin time) was observed with compounds
5, 7, and 8 (p < 0.05) in comparison to vehicle treated control, while,
no significant effect was observed on PT (prothrombin time) and
aPTT (activated partial thromboplastin time) in presence of these
compounds. Clotting parameters were not affected at 10 lM con-
centration of these compounds. Whereas, the standard direct
thrombin inhibitor that is, Hirudin exhibited significant increase
on TT, PT and APTT (p < 0.001) results are shown in Figure 3. Ex vivo
coagulation studies revealed around 74% increase in TT in mice fed
with compound 7 (30 lM/kg dose) as compared to control. Thus,
in the present study, these benzocoumarin amide derivatives exhib-
ited anti-thrombotic profile via both anti-platelet as well as anti-
coagulant action.
In terms of structure–activity relationship compounds having
an aliphatic amide group (–CO–NH) at the position 7 were found
to be more potent (5, 7, and 8), while the presence of cyclic
amides (9–12) showed moderate activity and a complete loss of
activity was observed in case of aromatic amides (13–18). The ac-
tive compounds 5, 7, and 8 were further explored on mice platelet
In conclusion, current study has led to new developments and
insights about benzocoumarin amide based anti-thrombotic
agents. The advantage of these compounds over aspirin lies in
the fact that these compounds exhibited both anti-platelet and
anti-coagulant activity. Overall, our current results establish a
new scaffold as a promising structure for the development of orally
active anti-thrombotic agents.

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K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3115–3121
Table 1
In vivo assay of novel benzocoumarins amide derivatives for anti-thrombotic activity
Compounds
Structure
Molecular weight
Anti-thrombotic activity20
(% protection)
Bleeding time21 (% increase)
LogPb
5
325.5
40
10
40
45
91.5
2.55
6
7
8
325.5
339.5
341.5
66.5
96.5
112.5
2.38
2.6
1.31
9
353.5
30
66.8
1.56
10
351.5
30
58.5
2.69
11
366.5
10
8.5
1.71

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3115–3121
3119
Table 1 (continued)
Compounds
Structure
Molecular weight
Anti-thrombotic activity20
(% protection)
Bleeding time21 (% increase)
LogPb
12
365.5
10
0.0
3.27
13
403.5
NAa
8.5
2.94
14
449.5
20
41.7
3.01
15
438.5
NAa
16.5
2.13
16
373.5
10
8.5
3.46
17
403.5
10
8.2
3.33
(continued on next page)

3120
K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3115–3121
Table 1 (continued)
Compounds
Structure
Molecular weight
Anti-thrombotic activity20
(% protection)
Bleeding time21 (% increase)
LogPb
18
401.5
20
58.5
4.43
19
Aspirin
37
3
100 20
a
Compounds exhibited below 10% inhibition was considered as NA (not active).
LogP values were calculated using Cambridge Soft Chem office Ultra version 10.0.
b
References and notes
Control
5 (30µM)
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80
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6. Vacca, J. P. Curr. Opin. Chem. Biol. 2000, 43, 94.
7 (30µM)
8 (30µM)
60
Aspirin (100µM)
**
**
40
***
7. Gachet, C. Pharmacol. Ther. 2005, 108, 180.
20
8. Andronati, S. A.; Karaseva, T. L.; Krysko, A. A. Curr. Med. Chem. 2004, 11, 1183.
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Boykow, G.; Hsieh, Y.; Palamanda, J.; Agans-Fantuzzi, J.; Kurowski, S.; Graziano,
M.; Chintala, M. J. Med. Chem. 2008, 51, 3061.
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13. Krynetskly, E. J. Pharmacol. Exp. Ther. 2007, 322, 427.
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Med. Chem. Lett. 2011, 21, 7034.
0
ADP
Collagen
Figure 2. Bar diagram representing ADP and collagen induced mice platelet
aggregation in mice plasma pre-incubated with vehicle, 5, 7, 8, and aspirin. Results
are expressed as mean SEM. (⁄p < 0.05, ⁄⁄p < 0.01 & ⁄⁄⁄p < 0.001 vs control) (n = 3).
15. Nazare, M.; Will, D. W.; Matter, H.; Schreuder, H.; Ritter, K.; Urmann, M.;
Essrich, M.; Bauer, A.; Wagner, M.; Czech, J.; Lorenz, M.; Laux, V.; Wehner, V. J.
Med. Chem. 2005, 48, 4511.
16. Frédérick, R.; Robert, S.; Charlier, C.; de Ruyck, J.; Wouters, J.; Pirotte, B.;
Masereel, B.; Pochet, L. J. Med. Chem. 2005, 48, 7592.
17. Robert, S.; Bertolla, C.; Masereel, B.; Dogné, J. M.; Pochet, L. J. Med. Chem. 2008,
51, 3077.
18. Jeanneret, V.; Vogel, P.; Renaut, P.; Millet, J.; Theveniaux, J.; Barberousse, V.
Bioorg. Med. Chem. Lett. 1998, 13, 1687.
Control
160
µ
5 (30 M)
***
140
7 (30µM)
8 (30µM)
120
Hirudin (20 ng/ml)
100
60
19. General procedure for synthesis of benzocoumarin amides (5–18): To the
suspension of 2-(4-methyl-2-oxo-2H-benzo[h]chromen-7-yloxy)acetic acid
3
*
(200 mg, 0.79 mmol) in benzene (10 mL), thionyl chloride (1.0 mL), was
added and the mixture was heated at refluxed for 2 h. The resulting solution
was evaporated to dryness under reduced pressure and excess thionyl chloride
was removed from the residue of crude 4 by co evaporation with dry benzene
under reduced pressure. The residue was then dissolved in benzene (10 mL)
and to it was added appropriate amine in excess and the reaction mixture was
refluxed for 50–90 min. After completion of the reaction (TLC monitoring) the
solvent was removed by evaporation under reduced pressure and extracted
threefold with CHCl3 (30 mL). The combined organic layer was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The crude
product thus obtained was purified by column chromatography (60–120
40
***
***
20
0
TT
PT
aPTT
Figure 3. Thrombin time, prothrombin time and activated partial thromboplastin
time in mice plasma pre-incubated with vehicle, 5, 7, 8, and Hirudin. Results are
expressed as mean SEM. (⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 vs control) (n = 3).
mesh)
to
furnish
corresponding
amides,
2-(4-methyl-2-oxo-2H-
benzo[h]chromen-7-yloxy)-N-propylacetamide (5), as white solid with yield
70%; mp 192–194 °C; IR (KBr,cmꢀ1): 3431, 3020, 2925,1718, 1645, 1566, 1504,
1217, 1073; 1H NMR (CDCl3, 300 MHz) d: 8.10 (d, J = 8.4 Hz, 1H) 8.01 (d,
J = 8.91 Hz, 1H) 7.57 (d, J = 8.9 Hz, 1H) 7.53–7.47 (m, 1H) 6.50 (d, J = 7.6 Hz, 1H)
6.51 (bs, NH) 6.3 (s, 1H) 4.73 (s, 2H) 3.38 (q, J = 6.0 Hz, 2H) 2.49 (s, 3H) 1.67–
1.55 (m, 2H) .96 (t, J = 7.4 Hz, 3H);13C NMR (CDCl3, 75 MHz) d: 167.7, 160.5,
153.0, 127.3, 126.3, 124.4, 120.1, 117.5 116.2, 115.7, 114.8, 108.5, 68.1, 40.8,
22.8, 19.1, 11.2; ESI-MS: (m/z): 326 (M+H)+.
Acknowledgments
The authors are grateful to the Director, CDRI, Lucknow, India
for constant encouragement in drug development program, S.P.
Singh for technical support, SAIF for NMR, IR, and Mass spectral
data. G.R.P. and S.R.A. are thankful to CSIR, and M.J. is thankful to
ICMR, New Delhi, India for financial support. This is CSIR-CDRI
communication number 8219.
20. Mouse thrombosis model: Pulmonary thromboembolism was induced by a
method as described by Dimnno and Silver (1983). The compounds to be tested
(30 lM/kg), standard drugs or the vehicle were administered by oral route
60 min prior to the thrombotic challenge. Ten mice were used for evaluating
the effect of test compound, while a group of five mice was used to evaluate the
effect of aspirin or vehicle. A mixture of collagen and adrenaline was injected
into the tail vein to induce hind limb paralysis or death. Results have been
Supplementary data
reported as
% protection, which represent protection against hind limb
paralysis or death. The test compounds exhibiting protection equivalent to
Aspirin (30 mg/kg) were considered as active molecules (DiMinno, G.; Silver,
M. J. J. Pharmacol. Exp. Therap. 1983, 225, 57.).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.059.

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3115–3121
3121
21. Bleeding time: Bleeding time in mice was evaluated by the method of Dejana
et al. (1979) to assess the increase in bleeding time in comparison to aspirin at
its optimal anti-thrombotic dose. The tail 2 mm from tip of mice was incised
and the blood oozed was soaked on a filter paper, which was monitored at an
interval of 10–15 s till the bleeding stopped. The time elapsed from the tail tip
incision to the stoppage of bleeding was recorded as the bleeding time. The
vehicle for three days and PRP as well as PPP were isolated. Platelet
aggregation was induced by adenosine-50-diphosphate (ADP) (5
M) or
collagen (2 g/ml) and was monitored on dual channel aggregometer
(Chrono log Corp., US) (Prakash, P.; Khanna, V.; Singh, V.; Jyoti, A.; Jain, M.;
Keshari, R. S.; Barthwal, M. K.; Dikshit, M. Cardiovasc. Drugs. Ther. 2011, 25,
285.). Coagulation parameters namely thrombin time (TT), prothrombin time
(PT) and activated partial thromboplastin time (APTT), were assayed in plasma
samples using commercial kits as per manufacturer’s instructions and
l
l
a
tested compounds (30 lM/kg), aspirin (30 mg/kg) or vehicle was given orally
60 min prior to the tail incision in a group of five mice each. (Dejana, E.;
Callioni, A.; Quintana, A. Giovanni G. Thromb. Res. 1979, 15, 191.).
measured by using
a Coagulometer (Start4 Semi automated, Young
22. Platelet aggregation and coagulation parameters: Mice were anaesthetized under
anesthetic ether and blood was drawn by cardiac puncture. Platelet rich
plasma (PRP) was collected for aggregation studies. Remaining blood was used
for isolating platelet poor plasma (PPP) to study coagulation parameters.
Plasma samples were pooled and then divided into sub-pools and spiked with
Instruments, Stago, France) (Singh, V.; Jain, M.; Prakash, P.; Misra, A.;
Khanna, V.; Tiwari, R. L.; Keshari, R. S.; Singh, S.; Dikshit, M.; Barthwal, M. K.
J. Physiol. Biochem. 2011, 67, 205.). At least three similar experiments were
conducted with each compound.
23. Finney, S.; Seale, L.; Sawyer, R. T.; Wallis, R. B. Biochem. J. 1997, 324, 797.
24. Sato, K.; Taniuchi, Y.; Kawasaki, T.; Hirayama, F.; Koshio, H.; Matsumoto, Y.;
Iizumi, Y. Jpn. J. Pharmacol. 1998, 78, 191.
5 (30
lM), 7 (30
lM), 8 (30
lM), Aspirin (100
lM), Hirudin (20 ng/ml) or
vehicle. For ex vivo studies mice were treated with test agent (30
lM) or