Penicillins as β-lactamase-dependent prodrugs: Enabling role of a vinyl ester exocyclic to the lactam ring

Article abstract of DOI:10.1039/b409517k

Incorporation of a vinyl ester exocyclic to the β-lactam ring of a penicillin nucleus enables this to act as a β-lactamase-dependent prodrug - rapid release of the (unactivated) alkoxy component of the vinyl ester is triggered by enzyme-catalysed hydrolys

Full text of DOI:10.1039/b409517k

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Penicillins as b-lactamase-dependent prodrugs: enabling role of a vinyl
ester exocyclic to the lactam ring{
Carol C. Ruddle and Timothy P. Smyth*
Department of Chemical and Environmental Sciences, University of Limerick, National Technological
Park, County Limerick, Ireland. E-mail: timothy.smyth@ul.ie
Received (in Cambridge, UK) 24th June 2004, Accepted 11th August 2004
First published as an Advance Article on the web 7th September 2004
Incorporation of a vinyl ester exocyclic to the b-lactam ring
(y37%) and c-lactam C1 (y13%). It is evident that conversion of
the ring-cleaved structures B2/B3 to a c-lactam is a facile reaction4
and, as neither allyl alcohol nor methanol is an activated leaving
group, the driving force lies in the kinetic and thermodynamic
aptness of these ring-cleaved structures to undergo an intramole-
cular nucleophilic displacement.5 Two reaction paths, distinguished
by the identity of the kinetically dominant nucleophile, are shown
for this in Scheme 2. In the case of the S-aminosulfeniminopeni-
cillin, stereochemical data of the co-product formed in a CH3OH/
NEt3 reaction system, identified the thiazolidine-ring sulfur as the
kinetically dominant nucleophile whilst the thermodynamically
stable end product involved the thiazolidine-ring nitrogen.1a,b In the
present work this issue was resolved in a study of the hydrolysis of
the salt A3’ (Scheme 3).
Progress of the hydrolysis in D2O buffer (0.2 M phosphate, pD
7.2, 18 uC) was monitored by 1H NMR spectroscopy. Changes in
the chemical shifts of the methyl ester singlet (3.81 in A3’ to
3.35 ppm in CH3OH) and of the thiazolidine-ring H-3 singlet (4.37
in A3’ to 4.20 ppm in the co-product) were diagnostic features.
After 120 h in this buffer the hydrolysis had progressed y30%
with the balance of the material being intact A3’. When a portion of
b-lactamase enzyme sufficient to generate immediate hydrolysis of
the b-lactam ring6 was added to a sample of A3’ in this buffer, the
of a penicillin nucleus enables this to act as a b-lactamase-
dependent prodrug – rapid release of the (unactivated) alkoxy
component of the vinyl ester is triggered by enzyme-catalysed
hydrolysis of the b-lactam ring, whilst buffer-catalysed
hydrolysis of the structure at neutral pH is particularly slow.
Enzyme-catalysed scission of a b-lactam ring can provide discrete
activation of prodrugs based on penicillin, cephalosporin and
closely related structures. Elimination of the 3’-substituent of
cephems and the 2’-substituent of penems is an inbuilt feature,
whereas, a structural modification is required to engender an
appropriate reactivity pattern in penams. Incorporation of an
unsaturated linker at the 6-position, that has a moderately
nucleofugic group attached via an electron deficient centre, has
been shown to provide such a pattern. The prototypic structure was
the S-aminosulfeniminopenicillin 1 (Scheme 1) for which it was
found that rapid loss of the sulfur-attached moiety (N-methyl-p-
toluenesulfonamide) occurred consequent on scission of the
b-lactam ring.1 The efficacy of this displacement is due to the
rapidity of an intramolecular nucleophilic substitution occurring
within an acyclic five-membered unit of restricted conformational
space (due to the unsaturated linker). Herein, we report that a vinyl
ester bearing a simple alcohol as the displaceable moiety, 2
(Scheme 1), is a viable alternative to the S-aminosulfenimine
construct. This finding augments the chemistry of the penam
structure and extends the scope of this to be configured as
b-lactamase-dependent prodrugs.
The penicillin structures A1–A3 (Z-isomers) were prepared by
the method of Buynak,2 and the behaviour of these in weakly basic
methanol was examined in terms of the reaction patterns shown in
Scheme 2. The question of interest was to what extent displacement
of the alcohol moiety from the vinyl ester would be influenced by
scission of the b-lactam ring? The t-butyl derivative A1 was
quantitatively converted to the ring-cleaved structure B1 within 1 h
at room temperature with y0.2 M NEt3. In contrast, both the allyl
and methyl derivatives A2 and A3 were completely converted into
the ring-fused c-lactam C1 and the corresponding alcohol after
40 min under the same reaction conditions.3 In the case of A2, a
sample isolated after a 15 min reaction time (using y0.02 M NEt3)
was found to contain A2 (y50%), ring-cleaved structure B2
Scheme 1
{ Electronic supplementary information (ESI) available: experimental
details of the syntheses and characterisation data on all structures. See
http://www.rsc.org/suppdata/cc/b4/b409517k/
Scheme 2
2 3 3 2
C h e m . C o m m u n . , 2 0 0 4 , 2 3 3 2 – 2 3 3 3
T h i s j o u r n a l i s ß T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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mediated coupling of the resulting free acid with 7-hydroxy-4-
methylcoumarin to give the (non-fluorescent) ester A4. Treatment
of A4 with CH3OH/NEt3, as described above, led to the rapid
release of 7-hydroxy-4-methylcoumarin (as a fluorescent compo-
nent) with the concomitant formation of c-lactam C1.
In conclusion, we have shown that incorporation of a vinyl ester
exocyclic to the b-lactam ring of a penicillin nucleus enables this to
act as a b-lactamase-dependent prodrug – rapid release of the
(unactivated) alkoxy component of the vinyl ester is triggered by
enzyme-catalysed hydrolysis of the b-lactam ring. The vinyl ester
sidechain is more amenable to synthetic variation than the
S-aminosulfenimine unit, with the result that the scope to configure
penams as b-lactamase-dependent prodrugs is extended consider-
ably. Potential applications of such materials arise in combating
certain forms of antibiotic resistance (via release of an antimicrobial
entity – e.g. triclosan8 – on contact with a b-lactamase enzyme), in
the ADEPT mode of drug targeting9 (e.g. via site-specific release
and activation of DNA-minor-groove alkylating agents related to
Carzelesin10), and in the development of chromogenic and
fluorogenic substrates11 as diagnostics for certain classes of
b-lactamase enzymes.
We are grateful to N. Hurley, T. Greene, J. Grant and L. Kirby
for technical assistance, to Dr D. McCarthy (UCC) for high-
resolution NMR spectra, to Ken Harris (Scripps Center for Mass
Spectrometry) and Dr D. O’Shea (UCD) for high-resolution mass
spectra, and to IRCSET for financial support (C. C. R).
Notes and references
Scheme 3
1 (a) T. P. Smyth, M. E. O’Donnell, M. J. O’Connor and J. O. St Ledger,
Tetrahedron, 2000, 56, 5699–5707; (b) T. P. Smyth, M. E. O’Donnell,
M. J. O’Connor and J. O. St Ledger, J. Org. Chem., 1998, 63, 7600–7618;
(c) T. P. Smyth, M. J. O’Connor and M. E. O’Donnell, J. Org. Chem.,
1999, 64, 3132–3138.
2 (a) J. D. Buynak, A. S. Rao and S. D. Nidamarthy, Tetrahedron Lett.,
1998, 39, 4945–4946; (b) J. D. Buynak, Patent number WO 99/33837,
1999; (c) J. D. Buynak, K. Wu, B. Bachmann, D. Khasnis, L. Hua,
H. K. Nguyen and C. L. Carver, J. Med. Chem., 1995, 38, 1022–1034;
(d) J. D. Buynak, V. R. Doppalapudi and G. Adam, Bioorg. Med. Chem.
Lett., 2000, 10, 853–857.
liberation of methanol and formation of the co-product were
complete in less than 20 min. The co-product that was generated in
the buffer- and enzyme-catalysed processes was identical; the H,
1
13C NMR and HRMS data{ of this material support its
identification as the c-lactam C’. Thus, the kinetically dominant
nucleophile toward
a carbonyl group is the thiazolidine-
ring nitrogen whilst this role falls to the thiazolidine-ring sulfur
in the the S-aminosulfenimine which has a sulfur atom as the
electron-deficient centre.
The role of the unsaturated linker in facilitating the intramo-
lecular displacement within the ring-cleaved structure was assessed
by studying carbamate E. This was completely converted to the
ring-opened structure F after 45 min in methanol containing
y0.2 M NEt3; no ring-fused imidazolidinone structure7 was
formed. It is clear that the unsaturated linker is essential for
rapid release of the simple alkoxy component of the ester in the
b-lactam-ring-cleaved structures such as B3’.
3 Structure C1 was shown by semiempirical modelling (AM1) to be
approximately 14 kcal mol21 more stable than its isomeric form C.
4 In the case of B1, steric hindrance by the t-butyl group blocks addition of
the nucleophile to the ester carbonyl, which is the rate-determining step.
5 A parallel for this is found in the intramolecular nucleophilic addition
that occurs within the b-lactam-ring-cleaved structure derived from 6(Z)-
acetylmethylenepenicillanic acid (a potent b-lactamase inhibitor):
M. Arisawa and S. Adam, Biochem. J., 1983, 211, 447–454.
6 6(Z)-t-butoxycarbonylmethylenepenicillanate and 6(Z)-methoxycarbo-
nylmethylenepenicillanate have been found to be b-lactamase substrates
(the corresponding sulfones are inhibitors), however, the fate of the
b-lactam-ring-cleaved structures was not reported on: (a) J. D. Buynak,
B. Geng, B. Bachmann and L. Hua, Bioorg. Med. Chem. Lett., 1995, 5,
1513–1518; (b) D. Habich and K. Metzger, Heterocycles, 1986, 24, 289–
296.
7 J. Marchand-Brynaert, H. Vanlierde and L. Ghosez, Bull. Soc. Chim.
Belg., 1988, 97, 1081–1093.
8 Q. Li, J. Y. Lee, R. Castillo, M. S. Hixon, C. Pujol, V. R. Doppalapudi,
H. M. Shepard, G. M. Wahl, T. J. Lobl and M. F. Chan, Antimicrob.
Agents Chemother., 2002, 46, 1262–1268.
9 (a) H. P. Svensson, I. S. Frank, K. K. Berry and P. D. Senter, J. Med.
Chem., 1998, 41, 1507–1512 and references therein; (b) T. W. Hudyma,
K. Bush, K. L. Colson, R. A. Firestone and H. D. King, Bioorg. Med.
Chem. Lett., 1993, 3, 323–328.
10 A. Awada, C. J. A. Punt, M. J. Piccart, O. van Tellingen, L. van Manen,
J. Kerger, Y. Groot, J. Wanders, J. Verweij and D. J. Wagener, Br.
J. Cancer, 1999, 79, 1454–1461 and references therein.
Variation of the displaceable moiety was achieved by selective
removal (Pd(PPh3)4) of the allyl group of A2 followed by DCC
11 W. Gao, B. Xing, R. Y. Tsien and J. Rao, J. Am. Chem. Soc., 2003, 125,
11146–11147 and references therein.
C h e m . C o m m u n . , 2 0 0 4 , 2 3 3 2 – 2 3 3 3
2 3 3 3