Synthesis of novel 3-carboxamide-benzocoumarin derivatives as orally active antithrombotic agents

Article abstract of DOI:10.1016/j.bmcl.2011.09.100

In an effort to develop potent antithrombotic agents, a series of novel 3-carboxamide-benzocoumarin derivatives were synthesized and screened for their in vivo antithrombotic activity. Among 20 compounds tested, the compound 12b showed the most promising

Full text of DOI:10.1016/j.bmcl.2011.09.100

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7034–7040
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Bioorganic & Medicinal Chemistry Letters
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Synthesis of novel 3-carboxamide-benzocoumarin derivatives as orally
active antithrombotic agents q
Koneni V. Sashidhara a, , Abdhesh Kumar a, Manoj Kumar a, S. Singh b, Manish Jain b, Madhu Dikshit b,
⇑
a Medicinal and Process Chemistry Division, Central Drug Research Institute (CDRI-CSIR), Lucknow 226 001, India
b Pharmacology Division, Central Drug Research Institute (CDRI-CSIR), 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:
In an effort to develop potent antithrombotic agents, a series of novel 3-carboxamide-benzocoumarin
derivatives were synthesized and screened for their in vivo antithrombotic activity. Among 20 com-
pounds tested, the compound 12b showed the most promising antithrombotic activity which was com-
parable with clinically used aspirin or warfarin, but, at variance with these standard drugs, 12b did not
exhibit the increased bleeding time, suggesting its potential as a novel antithrombotic agent.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 4 August 2011
Revised 16 September 2011
Accepted 26 September 2011
Available online 29 September 2011
Keywords:
Synthesis
3-Carboxamide-benzocoumarins
Antithrombotic activity
Bleeding time
Aspirin
Warfarin
Thrombosis is a major cause of morbidity and mortality in the
industrial world. Intravascular thrombosis leads to myocardial
infarction, stroke or pulmonary thromboembolism, which is one
of the most common causes of death worldwide.1 The current ther-
apies used for prophylactic and prevention have considerable lim-
itations because they require careful clinical monitoring and are
associated with incidence of cardiovascular events and complica-
tions associated with bleeding.2,3 The current therapies include
the use of anti-platelet agents; aspirin, ticlopidine, clopidogrel
and anticoagulants such as heparin and warfarin.4 Figure 1 shows
the chemical structures of some clinically important antiplatelet
and anticoagulant agents.
Few drugs have stood the test of time for as long as the couma-
rin anticoagulants. Warfarin, the most commonly used candidate,
has been in clinical use for several decades, despite well-known
difficulties in dose optimization and safety (e.g., high risk of bleed-
ing) concerns, its clinical usage has continued because of its proven
effectiveness in the treatment and prophylaxis of thromboembolic
disease together with the failure of more novel drugs to meet mod-
ern requirements for efficacy and safety. Warfarin, the most com-
monly used coumarin derivative, achieves its anticoagulant effect
Figure 1. Some clinically important antiplatelet and anticoagulant agents.
by modulating the synthesis of vitamin k-dependent proteins,
resulting in the synthesis of defective coagulation factors without
any coagulant activities.5 On the other hand, aspirin, is still the
standard reference compound for long term oral treatment for
antiplatelet activity, which has been attributed to its indirect
thromboxane A2 inhibition by acetylating the cyclooxygenase en-
zyme, but, in view of its non-selectivity, there is a need for more
efficacious agent devoid of above limitations.6,7 Hence, the search
q
Part XIII in the series, ‘Advances in drug design and discovery’.
⇑
Corresponding author. Tel.: +91 9919317940; fax: +91 522 2623405.
E-mail
addresses:
sashidhar123@gmail.com,
kv_sashidhara@cdri.res.in
(K.V. Sashidhara), madhu_dikshit@cdri.res.in (M. Dikshit).
For Biological activity.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.100

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7034–7040
7035
for new strategies and novel chemical entities for antiplatelet/anti-
thrombotic activity is of current interest.8,9
which in turn was condensed with various amines to cleanly
furnish the coumarinic amides (7a–7j) (Scheme 1). Alternatively,
the reaction of dialdehyde 2 with p-methyl acetophenone in reflux-
ing dioxane with concd HCl as a catalyst resulted in formation of
regioselective para-condensed chalcone 8 in an impressive 70%
yield.15 Compound 8 was then subjected to same series of above
mentioned transformations resulting in coumarinic amides (12a–
12j) (Scheme 2) having chalcone appended at position 4. In all the
chalcones synthesized, the trans double bond was obtained exclu-
sively. All compounds were characterized using 1H NMR, 13C
NMR, and Mass spectrometry and IR spectroscopy. The purity of
these compounds was ascertained by TLC and spectral analysis
(see Supplementary data).
The objective of the study was to identify anti-thrombotic
efficacy of the synthesized compounds, since the compounds syn-
thesized were expected to possess both anti-platelet and anti-coag-
ulant activity, it was better to assess anti-thrombotic efficacy after
oral administration of the compounds, which will be the overall ef-
fect on both platelet, and coagulation cascade after their bio-
availability. In vitro assessment of anti-thrombotic activity will
require a sample/compound to be tested against many screening
systems such as anti-platelet activity (ADP, AA, PMA, A23187, colla-
gen and thrombin are used to activate platelets, it also requires that
Based on our earlier laboratory experiences and the literatures
where various coumarins were reported as mechanism based
inhibitors of thrombin,10 selective FXIIa inhibitors11 and to posses
antithrombotic activity,12 it appeared of interest to explore phar-
macophoric character of this relatively unexploited substituent
for antithrombotic activity. Moreover, the structure of our deriva-
tives possesses chalcone moiety that is reported to have antiplate-
let activity.13
In the present study, we synthesized a series of new 3-carbox-
amide-benzocoumarin derivatives and also varied the substituent
at position 4 by incorporating the chalcone moiety instead of
methyl group. The route followed for the preparation of 3-carbox-
amide-benzocoumarin derivatives is illustrated in Scheme 1. The
Duff reaction on naphthalen-1-ol 1 resulted in expected compound
2 and unexpected compound 3 that was previously unambiguously
characterized by us.14 The compound 3 was engaged in a Knoevena-
gel-type reaction with diethyl malonate and piperidine as
catalyst and resulted in the formation of ethyl 6-methyl-2-
oxo-2H-benzo[h]chromene-3-carboxylate (4). Furthermore, com-
pound 4 on alkaline hydrolysis, furnished the acid derivative 5,
which was converted to the acid chloride 6 with thionyl chloride,
Scheme 1. Synthesis of novel 3-carboxamide-benzocoumarins 7a–7j. Reagents and conditions: (a) (1) HMTA/TFA, 120 °C, 3 h, (2) 10% H2SO4, 90–100 °C, 2 h; (b)
CH2(COOC2H5)2, EtOH, piperidine, reflux, 30 min, (c) 10% KOH, EtOH, rt, 30 min; (d) benzene, SOCl2, refulx, 2 h; (e) benzene, amine, rt, 10 min.

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K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7034–7040
Scheme 2. Synthesis of chalcone appended novel 3-carboxamide-benzocoumarins 12a–12j. Reagents and conditions: (a) Concd HCl/dioxane, 80–90 °C; (b) CH2(COOC2H5)2,
EtOH, piperidine, reflux, 30 min, (c) 10% KOH, EtOH, rt, 30 min; (d) benzene, SOCl2, refulx, 2 h; (e) benzene, amine, rt, 10 min.
the test agent should be soluble in water/buffers, hydrophobic sam-
ples are very difficult to test against platelets, as DMSO/methanol
are very potent inhibitors of platelet activation), anti-coagulant
activity (many serine proteases are involved in the activation of
coagulation cascade, which finally lead to the formation of fibrin
from soluble fibrinogen) and thrombolytic activity (plasmin and
PAI activity assessments). Realizing the complexity of thrombus for-
mation in the present study we used a very simple, reliable, and
reproducible animal model to assess the anti-thrombotic activity.
Also, the bleeding time was assessed to have an idea of their effect
on hemostasis at the particular dose.
Since the synthesized compounds had coumarin core in their
molecular makeup, warfarin was used as a reference compound.
Thus experiments were conducted to evaluate the in vivo effects
of the new synthesized compounds on antithrombotic activity,16
bleeding time,17 and prothrombin time,18 in mice, warfarin the
reference compound was tested under the same conditions and
the results are presented in Table 1. Out of all the compounds
evaluated, eight compounds showed activity of 40% or above,
while remaining compounds exhibited activity between 20%
and 30%. The most promising was the compound 12b, showed
comparable antithrombotic protection of 44% (warfarin under
the similar conditions showed 50% protection) but at variance
with warfarin, 12b exhibited low prothrombin time of 11.1 min
as against 116.18 min for warfarin, suggesting that the mecha-
nism of the newly synthesized compounds might be different.
Next, we evaluated these compounds with aspirin as standard
and the results are presented in Table 2. Five compounds (7a,
12b, 12c, 12e, and 12j) showing comparable antithrombotic
activity with the standard drug aspirin, four belonged (12b,
12c, 12e, and 12j) to the second series of compounds having
chalcone moiety in their molecular makeup. Tolerability of the
subacute treatment with the compounds was examined by eval-
uating their adverse pro-harmorrhagic effect on tail bleeding
time. Again the compound 12b, emerged as the most promising
one, as in addition to showing 40% protection, it did not substan-
tially increase the bleeding tendency in comparison to aspirin at
its optimal antithrombotic dose. While the other active com-
pounds though exhibited the percent protection also exhibited
increased bleeding time, which was their drawback.
It seemed that the replacement of the methyl group (7a–7j) at
position 4 of 3-carboxamide-benzocoumarin with chalcone moiety
(12a–12j) slightly increased the antithrombotic activity. The above
results indicated that hybrid of 3-carboxamide-benzocoumarin
and chalcone moiety compounds are more potent than 3-carbox-
amide-benzocoumarin. Also, in terms of SAR, compounds having
amide group (–CO–NH) are more potent. Furthermore, since the
new synthesized derivative 12b did not increase bleeding at vari-
ance with aspirin or warfarin at antithrombotic doses, the initial
observation reveals good therapeutic index and thus the interest
in these compounds with potent antithrombotic activity deserves
further investigations.
A series of novel 3-carboxamide-benzocoumarin derivatives
have synthesized and some of them exhibited even better potency
than the reference drugs. Interestingly, the most potent compound
12b seems to possess a potentially good benefit-risk profile,

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7034–7040
7037
Table 1
Ex-vivo assay of novel 3-carboxamide-benzocoumarins for antithrombotic activity, bleeding time and prothrombin time after three day treatment at dose of 30
l
M/kg
Compounds
Control
Structure
Antithrombotic activity (% protection)
—
Bleeding time (% increase)
—
Prothrombin time
11.9
7a
7b
7c
7d
40
30
20
40
65
68
8.5
10.2
10.2
10.0
55
100
7e
40
82
10.2
7f
29
20
33
33
91
55
9.4
8.1
7g
7h
7i
100
11.6
10.2
91
7j
40
63
9.3
(continued on next page)

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K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7034–7040
Table 1 (continued)
Compounds
Structure
Antithrombotic activity (% protection)
Bleeding time (% increase)
Prothrombin time
12a
12b
12c
12d
22
55
100
127
82
9.9
44
33
33
11.1
10.3
10.5
12e
44
109
10.6
12f
42
45
9.7
12g
22
36
8.7

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7034–7040
7039
Table 1 (continued)
Compounds
Structure
Antithrombotic activity (% protection)
Bleeding time (% increase)
Prothrombin time
12h
30
55
11.4
12i
33
45
11.0
12j
44
50
109
46
9.5
13
Warfarin
116.18
Singh for technical support, SAIF for NMR, IR, and Mass spectral
data. A.K., M.K. & M.J. are thankful to CSIR, New Delhi, India for
financial support. This is CDRI communication number 8134.
Table 2
Ex-vivo assay of novel 3-carboxamide-benzocoumarins for antithrombotic activity as
well as bleeding time after one hour treatment at dose of 30 M/kg
l
Compounds Antithrombotic activity (%
protection)
Bleeding time (%
increase)
Supplementary data
7a
7b
7c
7d
7e
7f
7g
7h
35
30
10
20
35
20
10
20
30
30
20
40
40
30
40
30
20
20
30
40
37
48
45
17
58
53
59
17
33
50
58
17
33
100
59
84
25
34
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.100.
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K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7034–7040
16. Mouse thrombosis model: Pulmonary thromboembolism was induced by a
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
method as described by DiMinno and Silver (1983). The compounds to be
tested (30 lmol/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 each test compound, while a group of five mice was
used to evaluate the effect of standard drugs or vehicle. A mixture of collagen
(1.5 mg/kg) and adrenaline (0.5 mg/kg) was injected into the tail vein to induce
hind limb paralysis or death. Results have been reported as% protection, which
represents the protection against hind limb paralysis or death. The test
compounds exhibiting protection equivalent to standard drugs (30 lmol/kg)
were considered as active molecules (DiMinno, G.; Silver, M. J. J. Pharmacol. Exp.
Ther. 1983, 225, 57).
tested compounds (30 lmol/kg), standard drugs (30 lmol/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).
18. Coagulation parameters: Animals were anaesthetized under anesthetic ether
and blood was drawn from the heart into a plastic syringe containing 2.5% tri-
sodium citrate. The blood was centrifuged at 2500g for 20 min 20 °C to plasma.
Prothrombin time (PT), were assayed in plasma samples of all the groups
within 2 h of sample collection. Assay was performed using commercial kits
as per manufacturer’s instructions and measured by using a Coagulometer
(Start4 Semi automated, Young Instruments, Stago, France. Essler, M.; Retzer,
M.; Bauer, M.; Zangl, K. J.; Tigyi, G.; Siess, W. Ann. N.Y. Acad. Sci. 2000, 905,
282).
17. 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 antithrombotic dose. The tail 2 mm from tip of mice was incised