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A. Kathuria et al. / Bioorg. Med. Chem. 17 (2009) 1550–1556
1551
ities of both the mono/diacetoxy 3-alkyl coumarins are lower than
the corresponding non alkylated analogs, that is, 7-acetoxy-4-
methyl coumarin (MAMC) and 7,8-diacetoxy-4-methyl coumarin
(DAMC). It can also be concluded that the the increase in chain
length of the alkyl substituent at the pyran nucleus lead to de-
crease in CRTAase activity.
The acetylation of NOS by acetoxy coumarins is an effective ap-
proach to activate NOS thereby enhancing NO levels in human
platelets. We have in this report demonstrated the effect of substi-
tution at C-3 position of acetoxy coumarins on the enhancement of
NO levels in platelets. From the present investigations it can be in-
ferred that alkyl substitution at C-3 position of the coumarin nu-
cleus led to inhibition of NOS as compared to the corresponding
unsubstituted coumarins, that is, MAMC/DAMC.
better substrate for the CRTAase in comparison to other C-3 alkyl
diacetoxycoumarins. Further, it is clearly evident from Figures 1
and 2 that activities with diacetoxy 3-alkyl-4-methylcourmarins
are twice as high as those with the corresponding monoacetoxy
coumarins.
CRTAase mediated inhibition of glutathione S-transferase (GST)
by alkylated diacetoxy coumarins 5a–c is almost twice as com-
pared to the corresponding monoacetoxy analogs, that is, 7-acet-
oxy alkylcoumarins 4a–c. Acetoxycoumarins 5a, 5b and 5c when
compared with DAMC showed a gradual decline with the increase
in chain length of the alkyl substituent at the C-3 position in the
coumarin moiety. Similar trend was observed with 7-acetoxy-4-
methyl-3-alkylcoumarins 4a, 4b and 4c.
Similarly, in case of CRTAase catalyzed activation of NADPH
Cytochrome c reductase (Fig. 2), the results revealed that 3-alkyl-
4-methyl-7,8-diacetoxycoumarin 5a–c upon incubation with liver
microsomes resulted in the activation of the reductase to the max-
imum extent. The monoacetoxy 3-alkyl-4-methylcoumarins 4a, 4b
and 4c caused significantly lesser activation of the reductase as
compared to their diacetoxy coumarin analogs. Also, smaller the
alkyl group present at the C-3 position, the higher was the CRTAase
catalyzed activation of NADPH Cytochrome c reductase. However,
among the mono and di acetoxycoumarins, it is the C-3 unsubsti-
tuted coumarins, viz. MAMC and DAMC that have the maximum
substrate specificity for CRTAase.
2. Results
In our earlier work we elucidated the role of acetoxy groups on
the benzenoid ring of coumarin moiety in facilitating the acetyla-
tion of the receptor protein catalyzed by Calreticulin Transacety-
lase. In this regard we also studied the factors, such as the
proximity of the acetoxy group to the oxygen heteroatom, the role
of carbonyl group on the benzopyran nucleus, and the effect of
substituents on the coumarin molecule in controlling the protein
acetylation.7–9
Studies were extended with the view to examine the influence
of acetoxy coumarins (4a–c, 5a–c) on activation of NOS. The plate-
However, the effect of substituent at the C-3 position of couma-
rin has not been thoroughly studied earlier. In this report, we have
meticulously compared the specificities of acetoxy derivatives of a
number of 3-alkyl-4-methylcoumarins as substrate to Calreticulin
Transacetylase by the replacement of C-3 hydrogen with different
alkyl groups (ethyl, hexyl and decyl). As alkyl groups are hydro-
phobic in nature, these may enhance hydrophobic interactions be-
tween the substrate (acetoxycoumarins) and the target protein
thus influencing the CRTAase catalytic activity. Such a study would
allow us to study the effect thereof on the rate of catalytic activity
of CRTAase and the efficacy of these acetoxy coumarins to activate
platelet NOS. The C-3 alkyl coumarins (2a–c and 3a–c) were syn-
thesized via Pechmann condensation of 2-alkyl ethyl acetoacetates
(1a–c) with resorcinol and pyragallol. The 2-alkyl ethyl acetoace-
tates in turn were prepared from ethyl acetoacetate by its alkyl-
ation using alkyl bromide and sodium hydride. The mono/
dihydroxy coumarins thus obtained were acetylated using DMAP
(N,N-dimethyl aminopyridine) and acetic anhydride to give the
corresponding monoacetoxy (4a–c) and diacetoxy coumarins
(5a–c) bearing different alkyl substituents at the C-3 position and
a methyl group at C-4 position (Schemes 1 and 2). All the com-
pounds were fully characterized on the basis of their physical
and spectral data, and of these coumarins, 2c, 3c, 4c, 5b and 5c
are novel.
lets upon incubation with
mono and diacetoxy coumarins exhibited enhanced DCF fluores-
cence. It is worth noting that the inclusion of -NAME failed to re-
L-arginine and ethyl/hexyl substituted
L
duce the fluorescence to the control level indicating the possible
formation of oxygen radicals apart from NO. The decyl substituted
acetoxy coumarins virtually produced no enhancement of NO lev-
els in platelets.
3. Discussion
The existence of the unique enzyme acetoxy drug: protein
Transacetylase (TAase) now identified as Calreticulin Transacety-
lase (CRTAase) in rat liver microsomes catalyzing transfer of the
acetyl group from acetoxy polyphenols to specific proteins was evi-
denced from the studies published earlier.6–8 As a part of the quan-
titative structure–activity relationship (QSAR) studies on CRTAase,
we have demonstrated earlier (a) positional specificity of acetoxy
groups on the benzenoid ring of certain polyphenols and (b) abso-
lute requirement of carbonyl group on pyran ring.6,7 We have in
this report highlighted the outcome of the replacement of the C-
3 hydrogen by an alkyl group on the catalytic activity of CRTAase.
For this task, a number of acetoxy coumarins and diacetoxy cou-
marins were synthesized and their specificities to CRTAase were
determined. The compounds 4a–c and 5a–c apart from bearing
mono acetoxy and diacetoxy groups also differ in substitution at
the C-3 position of pyran ring. The compounds with ethyl group
at the C-3 position yielded higher CRTAase activities (Figs. 1 and
2), in comparison to their hexyl or decyl analogs. However, the
highest catalytic activity was found with C-3 unsubstituted acet-
oxy coumarins MAMC and DAMC. These results convincingly sug-
gested that substitution at C-3 by (larger) alkyl groups accounted
for significant decrease in CRTAase activity. The presence of bulky
alkyl groups at C-3 position in the pyran ring of coumarins reduces
their abilities to transfer acetyl group to the functional proteins,
suggesting that bulkier alkyl groups might be causing steric hin-
drance for the acetoxy substrates from accessing the active site
of CRTAase. This results in the decreased rate of transfer of the
acetyl group to the receptor protein in the case of hexyl or decyl
The CRTAase activities with these compounds as the substrate
were compared with DAMC and MAMC. The results clearly indi-
cated that among all the acetoxy coumarins DAMC caused highest
inhibition of GST (Fig. 1), and as well the activation of NADPH Cyto-
chrome c reductase (Fig. 2). However, among the alkylated couma-
rins it is the 3-ethyl-4-methyl-7,8-diacetoxycoumarin (5a) is the
O
O
i) NaH / THF
ii) RBr
O
O
OC2H5
OC2H5
R
1a R = C2H5
1b R = C6H13
1c R = C10H21
Scheme 1. Alkylation of ethyl acetoacetate.