Synthesis and antibacterial activity of hydrolytically stable (-)-epicatechin gallate analogues for the modulation of β-lactam resistance in Staphylococcus aureus

Article abstract of DOI:10.1016/j.bmcl.2005.03.063

Hydrolytically more stable analogues of (-)-epicatechin gallate (ECg) have been synthesised from ECg where an amine or amide function has been substituted for the ester linkage that joins the C-ring with the galloyl D-ring. Sub-inhibitory concentrations (

Full text of DOI:10.1016/j.bmcl.2005.03.063

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2633–2635
Synthesis and antibacterial activity of hydrolytically stable
(ꢀ)-epicatechin gallate analogues for the modulation of
b-lactam resistance in Staphylococcus aureus
James C. Anderson,a,* Catherine Headley,a Paul D. Stapletonb and Peter W. Taylorb,*
aSchool of Chemistry, University of Nottingham, Nottingham NG7 2RD, UK
bMicrobiology Group, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1 1AX, UK
Received 7 February 2005; accepted 8 March 2005
Available online 11 April 2005
Abstract—Hydrolytically more stable analogues of (ꢀ)-epicatechin gallate (ECg) have been synthesised from ECg where an amine
or amide function has been substituted for the ester linkage that joins the C-ring with the galloyl D-ring. Sub-inhibitory concentra-
tions (25 mg/L) of the amide analogue 7, possessing the natural C-3 stereochemistry, were able to reduce the resistance to oxacillin of
three strains of methicillin resistant Staphylococcus aureus (BB 568, EMRSA-15 and EMRSA-16) comparable to levels achieved
with ECg.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
bacteria. The polyphenolic compounds (ꢀ)-epicatechin
gallate (ECg) and (ꢀ)-epigallocatechin gallate (EGCg)
are major constituents of green tea (Camellia sinensis)
and have been shown to sensitise MRSA isolates to a
wide spectrum of b-lactam antibiotics.5,6
The emergence of multi-drug resistance in pathogenic
bacteria has created an urgent need for new antibiotics
and new approaches to the treatment of bacterial infec-
tions.1 Staphylococcus aureus is one of the major causes
of hospital- and community-acquired infections world-
wide; serious infections with this pathogen include
wound infections, bacteraemia and sepsis and are asso-
ciated with a high mortality rate.2 Staphylococcal resis-
tance 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 steady rise in the incidence of methicil-
lin resistant S. aureus (MRSA) clinical isolates.3 Staph-
In particular, ECg, without exception, reduced the in
vitro oxacillin resistance of around forty MRSA clinical
isolates to levels compatible with therapeutic doses.6
Unfortunately, naturally occurring catechin gallates
such as ECg and EGCg are not suitable for in vivo thera-
peutic use as they are poorly absorbed from the intestine
and are susceptible to hydrolysis by bacterial and possi-
bly host esterases,7 properties that would almost cer-
tainly abrogate their b-lactam-modifying capacity in
human subjects. In order to prevent the esterase-medi-
ated removal of the galloyl moiety from catechin gal-
lates, the initial step in in vivo metabolism of catechin
gallates, we have designed, synthesised and evaluated
modified ECg derivatives in which the hydrolytically
susceptible ester bond has been replaced with an inher-
ently more stable amide linkage.
ylococci show
a strong tendency to accumulate
antibiotic resistant genes and the majority of MRSA
isolates are now resistant to a range of antibiotics.4
Agents that suppress the expression of b-lactam resis-
tance in MRSA would facilitate the treatment of serious
MRSA infections with generic, cost-effective agents that
are currently of little use against these multi-resistant
2. Chemical synthesis
Keywords: MRSA; (ꢀ)-Epicatechingallate analogues; b-Lactam
antibiotics.
Our strategy for the synthesis of potentially hydrolyti-
cally stable ECg analogues relied upon the substitution
of the C-3 oxygen with an amino function. This was
*
Corresponding authors. Fax: +44 (0) 1159513564 (J.C.A); fax: +44
(0) 2077535867 (P.W.T); e-mail addresses: j.anderson@nottingham.
ac.uk; peter.taylor@ulsop.ac.uk
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.063

2634
J. C. Anderson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2633–2635
OTBS
B
OH
OH
OR
OR
OTBS
TBSO
O
C
HO
O
OTBS
2
B
A
RO
O
C
TBSO
O
3
OTBS
ii-iv
OH
NH
3
3
A
ii
OTBS
OH
OH
OH
i
O
3 NH
3
O
9
+
OR
OR
OR
OTBS
OTBS
OTBS
O
HO
D
D
7 3R
8 3S
10 3R
11 3S
OH
OR
OTBS
4
Scheme 3. Reagents and conditions: (i) BnNNH2, AcOH, NaCNBH3,
THF, rt 14 h, 10 45% and 11 26%; (ii) H2, 5% Pd–C, EtOH, rt, 16 h, 3R
100% and 3S 91%; (iii) tri-O-TBS protected gallic acid, DCC, DMAP,
CH2Cl2, rt, 16 h, 3R 83% and 3S 59%; (iv) HFÆPy, THF/Py (4:1), 0 °C,
2 h, 7 41% and 8 69%.
R=H (-)-epicatechingallate (1)
R=t-BuMe2Si- 2
i
Scheme 1. Reagents and conditions: (i) TBSCl, imidazole, DMF, rt,
14 h, 91%; (ii) LiAlH4, THF, 0 °C, 30 min, 3 80%, 4 67%.
favouring the natural C-3 (3R) stereochemistry of EC
and ECg.15 These could each individually be taken
through to the desired amides 7 and 8 by hydrogenolysis
of the N-benzyl group, DCC coupling with the carboxy-
lic acid derived from 416 and global cleavage of the silyl
ethers with HFÆpyridine complex in good overall yield.
achieved firstly by global protection of the seven hydr-
oxyl groups as their tert-butyldimethylsilyl ethers to give
2 and cleavage of the ester linkage with LiALH4 to give
3 and 4 (Scheme 1). Sufficiently mild conditions for
hydrolysis of the ester function could not be found
due to the lability of the protecting groups and sensitivity
of the ACB segment to strong acid or base. The
stereochemical integrity at C-2 of the ACB fragment 3
was verified by analysis of pertinent coupling constants
3. Microbiological evaluation
1
in the H NMR spectrum with those of ECg and (ꢀ)-
epicatechin (EC).8 This position is prone to epimerisa-
tion in basic systems9 through a quinone methide inter-
mediate.10 Fragment 3 is a pivotal compound to us in
the synthesis of other C-3 analogues where we have
explored ester,6 ether, sulfonate ester and carbamate
analogues at this position. Synthesis of the selectively
protected 3 from EC would be very difficult in terms
of selectivity and the use of synthetically useful protect-
ing groups11 and this route from 1 represents the most
efficient to date.
With the amide derivatives (7 and 8) in hand, their effi-
cacy as modulators for b-lactam resistance in S. aureus
was evaluated by determining their capacity to reduce
the minimum inhibitory concentration (MIC) of oxacil-
lin against MRSA strains BB 568, EMRSA-15 and
EMRSA-16 (Table 1).17
The galloyl amides 7 and 8 possessed extremely weak
intrinsic antibacterial activity against three MRSA
strains: BB 568 and the two epidemic strains EMRSA-
15 and EMRSA-16 (MIC mg/L, Table 1). This level of
activity was comparable to that found with ECg (this
study and Ref. 6). Sub-inhibitory concentrations
(25 mg/L) of 7 were able to reduce the resistance to oxa-
cillin of all three strains examined (oxacillin MIC mg/L,
Table 1). In particular, oxacillin MICs for EMRSA-15
and EMRSA-16 were comparable to those obtained
when these strains were cultured in the presence of
ECg. The MIC of oxacillin against BB 568 by 7 (from
256 mg/L to 4–8 mg/L) was less than that observed with
ECg, but represented a very large diminution of sensitiv-
ity. Compound 8 was less effective that 7 with regard to
its capacity to modify the sensitivity to oxacillin of BB
568 and EMRSA-16, although a significant degree of
sensitisation was observed with MRSA-15 (Table 1).
Substitution of the naked C-3 hydroxyl group of 3 for
an amino function was performed by reductive amina-
tion of the corresponding ketone with the amine 512
and sodium cyanoborohydride (Scheme 2). Global
deprotection led to the amino analogue of ECg 6.13
With conditions found for reductive amination that did
not affect the C-1 stereocentre this methodology was
then used to synthesise the epimerically pure amide ana-
logues 7 and 8 (Scheme 3). Treatment of ketone 9,
formed from the Dess–Martin periodinane14 oxidation
of 3 in 91% yield (Scheme 2), with benzylamine and so-
dium cyanoborohydride gave a 2:1 mixture of separable
amines 10 and 11 in 45% and 26% yield, respectively,
OH
4. Conclusion
HO
O
OH
2
The results show that sub-inhibitory concentrations
(25 mg/L) of the amide analogue 7, possessing the natu-
ral C-3 stereochemistry, was able to reduce the resis-
tance of three strains of methicillin resistant S. aureus
(BB 568, EMRSA-15 and EMRSA-16) to oxacillin com-
parable to levels achieved with ECg. The higher activity
of amide 7, compared to amide 8 indicates that carbonyl
derived linkers demonstrating the natural 3R stereo-
chemistry may provide other compounds for improved
i, ii, iii
3
NH
3
OTBS
OTBS
OH
OH
OH
H2N
6
5
OTBS
OH
Scheme 2. Reagents and conditions: (i) DMP, CH2Cl2, 0 °C, 9 h, 91%;
(ii) 5, AcOH, NaCNBH3, THF, rt 14 h, 30%; (iii) HFÆPy, THF/Py
(4:1), 0 °C, 2 h, 51%.

J. C. Anderson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2633–2635
2635
Table 1. Antibacterial activity of ECg, 6–8 and in combination with oxacillin against methicillin resistant Staphylococcus aureus (MRSA) strains
MRSA strain
MIC (mg/L)a
Oxacillin MIC (mg/L)a
ECg
7b,c
b
—
ECg
7
8
8b,c
64/64
BB 568
EMRSA 15
EMRSA 16
256
256
128
256
256
128
256
256
128
256/256
32/32
512/512
60.5
60.5
60.5
4/8
60.5/60.5
1/1
2/4
256/512
a MICꢀs were determined in Mueller–Hinton broth + 2% salt at 35 °C after 24 h incubation.
b Data for two separate experiments are shown.
c Fixed concentration of 25 mg/L.
(d 4.63, d, J = 8.5 Hz) in Tuckmantel, W.; Kozikowski, A.
P.; Romanczyk, L. J. J. Am. Chem. Soc. 1999, 121, 12073.
9. (a) Freudenberg, K.; Bo¨hme, O.; Purrman, L. Ber. Dutsch.
Chem. Ges. B 1922, 55, 1734; (b) Freudenberg, K.;
Purrman, L. Justus Liebigs Ann. Chem. 1924, 437, 274.
10. Mehta, P. P.; Whalley, W. E. J. Chem. Soc. 1963, 5327.
11. Selective protection of the hydroxyl groups of catechins
involves multistep procedures. See Ref. 8 and Cren-Olive,
C.; Lebrun, S.; Rolando, C. J. Chem. Soc., Perkin Trans. 1
2002, 821, and references therein.
12. Derived from 4 by treatment with PPh3, phthalimide,
DEAD, rt, 1 h then NH2NH2ÆH2O, EtOH, rt, 14 h, in 60%
yield over two-steps.
13. The natural C-3 stereochemistry (R) was confirmed based
on the small coupling constant for the C-2H (d 5.11, d,
J = 2.6 Hz) indicating the relative cis-stereochemistry
between C-2 and C-3 as before (Ref. 8).
sensitisation of MRSA isolates to a wide spectrum of b-
lactam antibiotics.
Acknowledgements
This work was supported by MRC Strategic Grant
G0000996. We thank S. Shah for undertaking the anti-
bacterial assays, Dr. S. Pih for preliminary studies, Dr.
S. Brocchini and Professor J. M. T. Hamilton-Miller
for helpful discussions, Mr. T. Hollingworth and Mr.
D. Hooper for providing mass spectra and Mr. T. J.
Spencer for micro analytical data.
References and notes
14. (a) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155;
(b) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113,
7277.
15. These were assigned based on the coupling constants their
C-2H protons as before (Ref. 8), 10 (natural 3R stereo-
chemistry) C-2H (d 5.09, d, J = 2.2 Hz) and 11 (3S) C-2H
(d 4.70, m, J = 7.6 Hz).
16. Prepared by Dess–Martin oxidation (Ref. 14) of 4 to give
the corresponding aldehyde which was oxidised with
NaO2Cl in the presence of 2-methyl-2-butene in t-BuOH
and pH 4 buffer in 58% yield over two-steps.
1. Taylor, P. W.; Stapleton, P. D.; Luzio, J. P. Drug Discov.
Today 2002, 7, 1086.
2. Romero-Vivas, J.; Rubio, M.; Fernandez, C.; Picazo, J. J.
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Cuevas, C. F., Ed.; Horizon Scientific: Wymondham,
2003, pp 33–60.
17. MIC determinations: the capacity of the various com-
pounds to modulate b-lactam resistance was evaluated by
determination of the MIC at a fixed concentration in
combination with oxacillin. Assays were performed in 96-
well microtitre trays with an inoculum of about 104
colony-forming units (cfu) in 100 lL of Mueller–Hinton
broth (Oxoid, Basingstoke, UK) supplemented with 2% w/
v NaCl. Doubling dilutions of oxacillin were employed.
MIC values were recorded after incubation of the trays at
35 °C for 24 h; S. aureus ATCC29213 was used as the
standard. The intrinsic anti-staphylococcal activity of
compounds was also evaluated using these methods.
5. Yam, T. S.; Hamilton-Miller, J. M. T.; Shah, S. J.
Antimicrob. Chemother. 1998, 42, 211.
6. Stapleton, P. D.; Shah, S.; Anderson, J. C.; Hara, Y.;
Hamilton-Miller, J. M. T.; Taylor, P. W. Int. J. Antimic-
rob. Agents 2004, 23, 462.
7. Kohri, T.; Matsumoto, N.; Yamakawa, M.; Suzuki, M.;
Nanjo, F.; Hara, Y.; Oku, N. J. Agric. Food Chem. 2001,
49, 4102.
8. The 1H NMR signal of C-2H (d 4.90, s) compares
favourably with the analogous signal in 5,7,30,40-tetra-O-
benzyl-(ꢀ)-epicatechin (d 4.91, s) and is in stark contrast
to the same signal in 5,7,30,40-tetra-O-benzyl-(+)-catechin