Design, synthesis and bioactivity of catechin/epicatechin and 2-azetidinone derived chimeric molecules

Article abstract of DOI:10.1016/j.bmcl.2009.04.084

A new class of chimeric molecules have been developed. These are based on polyphenols like catechin and epicatechin and monocyclic β-lactams. The two units are joined via a triazole linker using the 'Click Chemistry' conditions. The compounds showed good

Full text of DOI:10.1016/j.bmcl.2009.04.084

Bioorganic & Medicinal Chemistry Letters 19 (2009) 7007–7010
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and bioactivity of catechin/epicatechin
and 2-azetidinone derived chimeric molecules
Basab Roy a, Arindam Chakraborty b, Sudip K. Ghosh b, Amit Basak a,
*
a Department of Chemistry, Indian Institute of Technology, Kharagpur 721 302, India
b Department of Biotechnolgy, Indian Institute of Technology, Kharagpur 721 302, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of chimeric molecules have been developed. These are based on polyphenols like catechin
and epicatechin and monocyclic b-lactams. The two units are joined via a triazole linker using the ‘Click
Chemistry’ conditions. The compounds showed good to weak antibacterial activity against Escherichia coli
as well as moderate inhibition of RNase A.
Received 16 February 2009
Revised 26 March 2009
Accepted 21 April 2009
Available online 24 April 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Chimeric
Catechin
Epicatechin
Beta lactam
Click reaction
Inhibition
Diseases caused by pathogenic bacteria still attract significant
attention of medicinal chemists and biologists because of growing
antibacterial resistance. For example, Staphylococcus aureus which
is one of the major causes of hospital- and community-acquired
infections worldwide like wound infections, bacteraemia and sep-
sis is associated with a high mortality rate.1 This is because Staph-
ylococcal resistance to wide spectrum b-lactam antibiotics, such as
methicillin, oxacillin and flucloxacillin, emerged soon after the
introduction of the first drug in this class and there has been a stea-
dy rise in the incidence of methicillin resistant S. aureus (MRSA)
clinical isolates.2 Staphylococci show a strong tendency to accumu-
late antibiotic resistant genes and the majority of MRSA isolates
are now resistant to a range of antibiotics.3 Literature survey
revealed that there has been an alternative approach to fight this
nightmare of bacterial resistance. It is based on the identification
of agents that have no intrinsic antibacterial activity but are able
to sensitize the pathogen to a previously ineffective antibiotic.
Such modification of the bacterial phenotype has been exploited
with Augmentin, an antibacterial combination that relies on the
co-administration of a b-lactamase inhibitor (clavulanate) and a
b-lactam antibiotic (amoxicillin). The polyphenolic compounds
(À)-epicatechin gallate (ECG) and (À)-epigallocatechin gallate
(EGCG), major constituents of Japanese green tea and to a smaller
extent other catechins have been shown to sensitize MRSA to
methicillin and other b-lactam antibiotics.4 However naturally
occurring catechin gallates, such as the potent modifier ECG, are
rapidly degraded in vivo to inactive products due to the presence
of esterase-susceptible linkage groups.5 Recently a couple of
amides were synthesized as ECG analogues6 in which the hydrolyt-
ically susceptible ester bond was replaced with an inherently more
stable amide linkage. These were found to reduce the oxacillin
resistance of the MRSA strain. These results prompted us to design
a chimera comprising catechin/epicatechin and the b-lactam moi-
ety (1–2) (Fig. 1).
Our inspiration has also come from two recent publications: one
fromour ownlaboratoryreporting7 theRNase Ainhibitionactivityof
nucleobase-polyphenol chimera and the other by Chen et al. report-
ing8 weak antibacterial activity of 6-trizolyl penicillin. These results
encouraged us to adopt the essentially chemically inert triazole lin-
ker to join the two pharmacophores, namely the polyphenol and the
monocyclic b-lactam. The resulting chimeric molecules were tested
against E. coli and also for inhibition of RNase A (Fig. 2).
Our first challenge was to suitably protect (+)-catechin and (À)-
epicatechin. Acid or base sensitive protecting groups needed to be
avoided because of possible racemization at C-2 of catechin and
epicatechin involving a quinine methide intermediate.9 Our previ-
ous experience of synthesis of chimeric molecules containing
nucleobase and polyphenol,7 guided us to protect the polyphenols
as their tetrabenzyl ethers 9 and 10 respectively.10 This is because
of possible deprotection of the benzyl ether under neutral condi-
tion by simple hydrogenolysis.
* Corresponding author. Tel.: +91 3222283300; fax: +91 3222282252.
E-mail address: absk@chem.iitkgp.ernet.in (A. Basak).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.084

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B. Roy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 7007–7010
OH
OH
OH
OH
HO
O
HO
O
3
O
3
O
OH
OH
Cis
Cis
N
N
*
*
N
*
N
*
N
N
N
N
O
2a+2b
1a+1b
O
OH
OH
OH
OH
HO
O
HO
OMe
O
3
O
3
O
OMe
OH
OH
Cis
Cis
N
*
*
N
*
N
N
*
N
N
N
N
O
O
2c+2d
1c+1d
3S - (+) Catechin
3R - (-) Epicatechin
Figure 1. Target chimeric molecules.
Our next task was to introduce the alkyne and the azide func-
tionalities separately in protected polyphenol and b-lactam com-
ponents. For the alkyne component, tetrabenzyl catechin (TBC) or
epicatechin (TBEC)10 was converted to 3-O-propargyl catechin or
epicatechin by reacting with propargyl bromide in presence of
sodium hydride. The azido b-lactams (7 and 8) were prepared from
the corresponding alcohols via mesylation followed by nucleo-
philic displacement with azide. The alcohols were prepared by
the Kinugasa reaction11 between 3-butyn-1-ol and nitrones using
a published protocol12 from our laboratory (Scheme 1).
1
2
3
4
28s rRNA
18s rRNA
Figure 2. Gel electrophoresis experiment. Lane 1: RNA + water + Methanol, Lane 2:
RNA + RNase A + Methanol, Lane 3: RNA + RNase A + [1a+1b] (0.460 mM, MeOH),
Lane 4: RNA + RNase A + [2c+2d] (0.456 mM, MeOH).
R
R
H
HO
+
N
i
O
N
O
OH
Kinugasa Reaction
3 R = H
Cis :Trans = 10:1
Repeated
4 R = OMe
R
crystallization
from EtOAc-hexane
R
N3
HO
ii
N
O
N
O
7 R = H
8 R = OMe
5 R = H
6 R = OMe
[Only one enantiomer shown]
Scheme 1. Synthesis of azido b-lactams. Reagents and conditions: (i) CuI, Et3N, degassed acetonitrile, rt, (70%); (ii) (a) MsCl, Et3N, dry DCM, 0 °C, (90%); (b) NaN3, dry DMF,
50 °C, (80%).

B. Roy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 7007–7010
7009
With the alkyne and the azide components in hand, the ‘click’
methodology13 was utilized to join the two arms together to gen-
erate the desired chimera. The click reaction was performed in
assay. The diameter of inhibition zone is directly proportional to
the degree of sensitivity of bacterial strain and the concentration
of compound under test. The results of antibacterial activity testing
of the chimeric molecules are given in Table 1. It is interesting to
note that the isolated monocyclic azido b-lactams 7 and 8 as well
as the alcohols 5 and 6, were found to be ineffective as antibacte-
rial agents. Moreover free catechin and epicatechin do not possess
any antibacterial activity. Thus the earlier ineffective monobac-
tams have been successfully sensitized towards E. coli after conju-
gating with catechin/epicatechin.
presence of Cu (I) made in situ by reduction of CuSO4 with L-ascor-
bate in acetonitrile–water (1:1) solvent systems (Scheme 2). The
protected chimeric molecules were finally deprotected by hydrog-
enolysis in presence of Pearlman catalyst.14 The free phenols were
isolated in about 80% yield (Scheme 3).
The antibacterial activity was measured against E. coli by disc-
diffusion assay.15 In this assay, a colony of the organism was inoc-
ulated in 2–5 mL nutrient broth and grown for 2.5 h. The agar
plates were dried and inoculated by spreading the bacterial sus-
pension evenly over it. The sterile paper discs (6 mm) impregnated
with fixed dose viz., 1 mmol of compound were placed on the pre-
inoculated surface. The disc-bearing plates were incubated at 37 °C
and examined at 48 h for zone of inhibition, if any, around the disc.
Ampicillin was used in assay as a standard control drug. An addi-
tional negative control disc without any sample, but impregnated
with equivalent amount of solvent (DMSO), was also used in the
The RNase A inhibition study16 was done qualitatively by an
agarose gel based assay, wherein the degradation of the 28s and
18s rRNA was studied. The ribonuclease A of concentration
0.011
bated for 1 h at 37 °C. To these preincubated enzyme solutions,
the RNA (0.22 g/ l) was added as substrate and the degradation
lM was mixed with the synthesized compounds and incu-
l
l
was allowed for 15 minutes. The resulting solution was mixed with
40% sucrose solution (made from RNase free water) and 0.25% bro-
mophenol blue (dissolved in 1Â TAE buffer) and loaded onto a 1.1%
OBn
OBn
BnO
O
OBn
OH
OBn
N3
BnO
O
OBn
N
a
OBn
OBn
O
O
7
OBn
11a+11b
BnO
O
b
N
N
N
(73%)
O
N
O
OBn
OMe
9
OBn
N3
OBn
N
O
BnO
O
8
OMe
O
OBn
b
(75%)
N
N
N
N
OBn
11c+11d
O
OBn
BnO
O
OBn
OBn
OH
BnO
O
OBn
OBn
a
OBn
7
O
BnO
O
OBn
(70%)
b
O
N
N
N
OBn
12a+12b
N
O
10
OBn
OBn
8
BnO
O
b
(74%)
OMe
O
OBn
N
N
N
N
O
12c+12d
Scheme 2. The click reaction. Reagents and conditions: (a) NaH, propargyl bromide, dry THF, 0 °C–rt, 10 h, (75%); (b) CuSO4Á5H2O, Na-ascorbate, MeCN–H2O (1:1), rt, 80 h.

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B. Roy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 7007–7010
OH
OBn
OH
OBn
HO
O
BnO
O
3
O
3
O
R
R
OBn
OH
N
N
N
N
(78%)
N
N
N
N
O
O
11
1a+1b R = H
1c+1d R = OMe
OH
OBn
OH
OBn
HO
O
BnO
O
3
O
3
O
R
R
OH
OBn
(75%)
N
N
N
N
N
N
N
N
O
O
2a+2b R = H
12
2c+2d R = OMe
Scheme 3. Deprotection by hydrogenolysis. Reagents and conditions: H2 [2 bar], 20% Pd(OH)2-C, THF/MeOH/H2O = 20:1:1, rt.
References and notes
Table 1
Results of antibacterial activity testing by disc diffusion assay
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Compd 2a+2b
Compd 1c+1d
Compd 2c+2d
+++
++
++
+
+
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+++
Highly active (inhibition zone diameter >12 mm).
Moderately active (inhibition zone diameter 9–12 mm).
Slightly active (inhibition zone diameter 6–9 mm); Control (inhibition zone
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++
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lanes indicated that these catechin and epicatechin and 2-azetidi-
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of the chimeric molecules as a whole in RNase A inhibition. The
other important observation is that the inhibition activity appears
to be valid for the 28s rRNA. The specific reason for this differential
behaviour is not known at present.
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Acknowledgements
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.04.084.