S-aminosulfeniminopenicillins: Multimode β-lactamase inhibitors and template structures for penicillin-based β-lactamase substrates as prodrugs

Article abstract of DOI:10.1021/jo970737f

A series of novel penicillins, bearing an S-aminosulfenimine (R'(R'')NSN=) side chain at the 6-position, have been prepared by direct reaction of a penicillin ester with sulfur diimides. A set of these structures, with the thiazolidine-ring sulfur in the sulfide oxidation state, exhibited a pattern of reactivity not previously encountered in penicillin chemistry, viz., cleavage of the β-lactam ring resulted in a rapid intramolecular displacement of the S-amino moiety as R'(R'')NH. This was found to be the exclusive reaction occurring consequent on cleavage of the β-lactam ring. The mechanism of this process was delineated in a detailed study in basic methanol. That a similar reactivity pattern held for a penicillin salt in aqueous solution was also verified. Thus the salt 5bm (R' = CH₃, R'' = p-CH₃C₆H₄SO₂) behaved as a moderate substrate for β- lactamase type I from Bacillus cereus (k(cat)/K(m) = 6.26 x 10⁵ M^-1 min^{- 1}). On enzyme-catalyzed hydrolysis of this compound, displacement of N- methyl-p-toluenesulfonamide (R'(R'')NH) was directly observed (¹H NMR) and found to occur faster than displacement of this group from (intact) 5bm in aqueous buffer, by a factor of at least 600. These findings identified the potential of the s-aminosulfeniminopenicillin structure type to be developed as β-lactamase substrates for use as site-specific-release prodrugs. A degree of enzyme inhibition was also observed with this set of thiazolidine- ring-sulfide structures with the most potent inhibitor having the most nucleofugic S-amino moiety p-ClC₆H₄SO₂N(CH₃), indicating that displacement of this group, at the enzyme active site, played a role in their mode of inhibition. Structures with the thiazolidine-ring sulfur in the sulfone oxidation state were considerably more potent as inhibitors, with the structure 5a₂ being the most active. As this compound bore the least nucleofugic S-amino moiety C₂H₅OC(O)NH, it indicated that the mode of inhibition of the sulfones was distinct from that of the thiazolidine-ring sulfides; it is probable that the sulfones reacted in a manner similar to that shown by sulbactam viz., rapid scission of the thiazolidine-sulfone ring after cleavage of the β-lactam ring. Synergy of action was observed with 5a₂ at high concentration (78 μg/mL) against Escherichia coli when combined 1:1 with penicillin G; no synergy was observed at low concentration (4 μg/mL) when combined with pipericillin, indicating poor permeation characteristics.

Full text of DOI:10.1021/jo970737f

7600
J . Org. Chem. 1998, 63, 7600-7618
Articles
S-Am in osu lfen im in op en icillin s: Mu ltim od e â-La cta m a se In h ibitor s
a n d Tem p la te Str u ctu r es for P en icillin -Ba sed â-La cta m a se
Su bstr a tes a s P r od r u gs
Timothy P. Smyth,* Michael E. O’Donnell, Michael J . O’Connor, and J ames O. St Ledger
Department of Chemical and Environmental Sciences, University of Limerick,
National Technological Park, County Limerick, Ireland
Received April 24, 1997 (Revised Manuscript Received May 18, 1998)
A series of novel penicillins, bearing an S-aminosulfenimine (R′(R′′)NSNd) side chain at the
6-position, have been prepared by direct reaction of a penicillin ester with sulfur diimides. A set
of these structures, with the thiazolidine-ring sulfur in the sulfide oxidation state, exhibited a pattern
of reactivity not previously encountered in penicillin chemistry, viz., cleavage of the â-lactam ring
resulted in a rapid intramolecular displacement of the S-amino moiety as R′(R′′)NH. This was
found to be the exclusive reaction occurring consequent on cleavage of the â-lactam ring. The
mechanism of this process was delineated in a detailed study in basic methanol. That a similar
reactivity pattern held for a penicillin salt in aqueous solution was also verified. Thus the salt
5bm (R′ ) CH3, R′′ ) p-CH3C6H4SO2) behaved as a moderate substrate for â-lactamase type I
from Bacillus cereus (kcat/Km ) 6.26 × 105 M-1 min-1). On enzyme-catalyzed hydrolysis of this
compound, displacement of N-methyl-p-toluenesulfonamide (R′(R′′)NH) was directly observed (1H
NMR) and found to occur faster than displacement of this group from (intact) 5bm in aqueous
buffer, by a factor of at least 600. These findings identified the potential of the S-aminosulfen-
iminopenicillin structure type to be developed as â-lactamase substrates for use as site-specific-
release prodrugs. A degree of enzyme inhibition was also observed with this set of thiazolidine-
ring-sulfide structures with the most potent inhibitor having the most nucleofugic S-amino moiety
p-ClC6H4SO2N(CH3), indicating that displacement of this group, at the enzyme active site, played
a role in their mode of inhibition. Structures with the thiazolidine-ring sulfur in the sulfone
oxidation state were considerably more potent as inhibitors, with the structure 5a 2 being the most
active. As this compound bore the least nucleofugic S-amino moiety C2H5OC(O)NH, it indicated
that the mode of inhibition of the sulfones was distinct from that of the thiazolidine-ring sulfides;
it is probable that the sulfones reacted in a manner similar to that shown by sulbactam viz., rapid
scission of the thiazolidine-sulfone ring after cleavage of the â-lactam ring. Synergy of action was
observed with 5a 2 at high concentration (78 µg/mL) against Escherichia coli when combined 1:1
with penicillin G; no synergy was observed at low concentration (4 µg/mL) when combined with
pipericillin, indicating poor permeation characteristics.
In tr od u ction
catalytic components for hydrolysis are dislocated from
their optimum position.1,2 These factors allow for con-
siderable variation of inhibitor structure particularly in
those which are â-lactam based. The mode of action of
inhibitors such as clavulanic acid and sulbactam is
primarily driven by the enhanced tendency of the non-
lactam ring to cleave after opening of the â-lactam
moiety. A feature common to a number of inhibitors
The increasing prevalence of bacterial strains resistant
to â-lactam antibiotics, due to the production of â-lac-
tamase enzymes, has driven a good deal of research on
mechanism-based inhibitors for these enzymes. A fea-
ture common to most such inhibitors is the formation of
an acylated enzyme at the active site, which is not readily
cleaved by hydrolysis. The route leading to the formation
of such an acylated species can be quite varied as can
the cause for slow hydrolytic cleavage.1 Passive covalent
inhibitors operate on the basis of forming an acylated
enzyme which is inherently slow to hydrolyze. Active
covalent inhibitors lead to an acylated enzyme which is
slow to hydrolyze primarily because, in reactions occur-
ring after initial acylation of the enzyme, some of the
(2) (a) Bulychev, A.; Massova, I.; Lerner S. A.; Mobashery, S. J . Am.
Chem. Soc. 1995, 117, 4797. (b) Miyashita, K.; Massova, I.; Taibi, P.;
Mobashery, S. J . Am. Chem. Soc. 1995, 117, 11055.
(3) (a) Arisawa, M.; Adam, S. Biochem. J . 1983, 211, 447. (b) Chen,
Y. L.; Chang, C.-W.; Hedberg, K. Tetrahedron Lett. 1986, 27, 3449. (c)
Chen, Y. L.; Chang, C.-W.; Hedberg, K.; Guarino, K.; Welch, W. M.;
Kiessling, L.; Retsema, J . A.; Haskell, S. L.; Anderson, M.; Manuosos,
M.; Barrett, J . F. J . Antibiot. 1987, 40, 803. (d) Bennett, I. S.; Brooks,
G.; Broom, N. J . P.; Calvert, S. N.; Coleman, K.; Grancois, I. J . Antibiot.
1994, 44, 969. (e) Buynak, J . D.; Khasnis, D.; Bachmann, B.; Wu, K.;
Lamb, G. J . Am. Chem. Soc. 1994, 116, 10955. (f) Buynak, J . D.; Geng,
B.; Bachmann, B.; Hua, L. Bioorg. Med. Chem. Lett. 1995, 5, 1513.
(1) Pratt, R. F. In The Chemistry of â-Lactams; Page, M. I., Ed.;
Blackie Academic and Professional: Glasgow, 1992; Chapter 7.
10.1021/jo970737f CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/09/1998

S-Aminosulfeniminopenicillins
Sch em e 1
J . Org. Chem., Vol. 63, No. 22, 1998 7601
previously reported reaction between amines and sulfur
diimides is one of transimidation with the equilibrium
lying on the side of the less basic amine5 which, in the
reaction above, would have led to 3a . (An S-amino-
sulfenimine structure has been reported as one of the
many products formed in the reaction of amines with
elemental sulfur6 and also as a minor product in the
reaction of a cephalosporin with thionyl chloride in base.7)
Spectroscopic data clearly characterized the experimental
reaction product as an S-aminosulfeniminopenicillin. The
1H NMR spectrum showed a singlet at 5.79 ppm corre-
sponding to H-5, no signal corresponding to H-6, and one
exchangeable hydrogen (N-H) at 6.61 ppm (this chemical
shift was concentration dependent). A key feature of the
13C NMR spectrum8 was the occurrence of four peaks at
166.8, 165.0, 155.5, and 154.0 ppm. These chemical shifts
indicated sp2 carbons, bearing no hydrogens as shown
by the off-resonance spectrum,8 corresponding to the two
ester carbonyls, the â-lactam carbonyl, and the carbon
of the exocyclic imine,9 respectively. The appearance of
a band at 1665 cm-1 in the IR spectrum was character-
istic of an imine. The change in the shape of the 1H NMR
peak associated with H-5 of 4bm , from a sharp singlet
at 6.20 ppm (CDCl3) at 30 °C to a broad peak at -4 °C
and to a baseline peak at -20 °C, indicated an isomer-
izable system, and one which was rapidly equilibrating
1
at room temperature.8 The H NMR spectrum at -60
°C clearly indicated the predominance of one isomer with
a singlet for H-5 at 6.57 ppm; the H-5 peak of the minor
isomer appeared to be coincident with that of H-3 at 4.70
ppm. Taking the chemical shift observed at room tem-
perature of 6.20 ppm as a weighted average of the cis
and trans isomers, an equilibrium constant of 4 for their
interconversion was obtained; in deuterioacetone, at
ambient temperature, H-5 appeared as a sharp singlet
at 6.31 ppm indicating that the equilibrium constant was
solvent dependent. The activation energies for isomer-
ization of sulfeniminesswhich have a carbon-based group
attached to sulfursare known10a and for a selected set
vary from 12 to 14 kcal/mol.10b A crystal structure of a
penicillin compound bearing a sulfenimine revealed that
the side chain sulfur had a trans geometry with respect
to the lactam carbonyl,11 indicating that this was the
more stable isomeric form, at least in the crystalline solid
state.
developed more recently is a side group of latent reactiv-
ity which drives further structural rearrangement after
cleavage of the â-lactam ring.3 The inhibitor strategy in
the long term must, however, be one of decreasing efficacy
as it involves application of a selective pressure for the
evolution of new variants of â-lactamase enzymes.
In this paper we report the first penicillin-based
structures which behave as â-lactamase-dependent pro-
drugs. Such structuresstogether with the dual-acting
cephalosporins4soffer the possibility of combating â-lac-
tamase-based resistance to antibiotics by exploiting the
presence of these enzymes: exploitation of these enzymess
including the metalloenzymessmay present the only
strategy which, in the long term, should redress the
balance in favor of non-â-lactamase producers in the
general population of pathogenic bacteria.
In Scheme 2 is shown a stepwise mechanism illustrat-
ing how the reaction between a sulfur diimide and amine
can lead to the formation of an S-aminosulfenimine (route
a ) or to a transimidation product (route b). A key step
in route a involves removal of H-6 on the â-lactam ring
Resu lts a n d Discu ssion
The reaction of 6-aminopenicillanic acid ester 1 with
the sulfur diimide 2a at room temperature led to the
rapid formation of a structure which we have identified
as the S-aminosulfeniminopenicillin 4a (Scheme 1). The
(5) Bussas, R.; Kresze, G.; Munsterer, H.; Schwobel, A. In Sulfur
Reports; Senning, A., Ed.; Harwood Academic Publishers: New York,
1983; Vol. 2 (6), p 215.
(6) Sasaki, Y.; Olsen, F. P. Can. J . Chem. 1971, 41, 283.
(7) Saito, T.; Hiraoka, T. Chem. Pharm. Bull. 1977, 25, 784.
(8) Included in Supporting Information.
(9) Levy, G. C.; Lichter, R. L.; Nelson, G. L.; Eds. Carbon-13 Nuclear
Magnetic Resonance Spectroscopy, 2nd ed.; J ohn Wiley & Sons: New
York, 1980; Chapter 5.
(10) (a) Claus, P. K. In The Chemistry of Sulphenic Acids and
Derivatives; Patai, S., Ed.; J ohn Wiley & Sons: Chichester, 1990;
Chapter 17. (b) The overall electron withdrawing character of the sulfur
moiety in our structures may be broadly comparable to that present
in sulfinamides XC6H4S(O)NdC(C6H4CH3)2 for which coalescence
temperatures of -10 and -27 °C have been reported for X ) H and
p-NO2, respectively: Davis, F. A.; Kluger, E. W. J . Am. Chem. Soc.
1976, 302.
(4) The inherent reactivity of cephalosporins, resulting in elimina-
tion of the 3′ substitutent after cleavage of the â-lactam ring, has been
harnessed in the development of dual-action structures producing
effectively, cephalosporin based, â-lactamase substrate prodrugs: (a)
O’Callaghan, C. H.; Sykes, R. B.; Staniforth, S. E. Antimicrob. Agents
Chemother. 1976, 245. (b) Mobashery, S.; Lerner, S. A.; J ohnston, M.;
J . Am. Chem. Soc. 1986, 108, 1685. (c) Albrecht, H. A.; Beskid, G.;
Christenson, J . G.; Durkin, J . W.; Fallat, V.; Georgopapadakou, N. H.;
Keith, D. D.; Konzelmann, F. M.; Lipshitz, E. R.; McGarry, D. H.;
Siebelist, J .-A, Wei, C.-C.; Weigele, M.; Yang, R. J . Med. Chem. 1991,
34, 669. (d) Albrecht, H. A.; Beskid, G.; Christenson, J . G.; Georgopa-
padakou, N. H.; Keith, D. D.; Konzelmann, F. M.; Pruess, D. L.;
Rossman, P. L.; Wei, C.-C. J . Med. Chem. 1991, 34, 2857. (e) Vrudhula,
V. M.; Svensson, H. P.; Senter, P. D. J . Med. Chem. 1995, 38, 1380.
(11) Gordon, E. M.; Chang, H. W.; Cimarusti, C. M.; Toeplitz, B.;
Gougoutas, J . Z. J . Am. Chem. Soc. 1980, 102, 1690.

7602 J . Org. Chem., Vol. 63, No. 22, 1998
Smyth et al.
Sch em e 2
Sch em e 3
Gordon for the sulfeniminopenicillins in that selective
oxidation of the thiazolidine-ring sulfur to the sulfoxide
was achieved with MCPBA; further oxidation to the
corresponding sulfones was not achieved with the sulfem-
inines using MCPBA and no other oxidation process was
reported by Gordon.11
Previously reported reactions of sulfur diimides with
amines5 do not include examples of amines analogous to
that present in penicillins. We have found that the
reaction of alanine diphenylmethyl ester and sulfur
diimide 2b led uniquely to the formation of the corre-
sponding S-aminosulfenimine 6 (Scheme 3) and not the
transimidation product, thus showing the generality of
this reaction with appropriate amines. The 1H NMR
spectrum of 6 had a singlet at 2.10 ppm due to the imine
methyl group while in the 13C NMR spectrum8 peaks
occurred at 160.0 and 143.8 ppm due to the conjugated
ester carbonyl and the imino carbon, respectively. The
variable temperature 1H NMR data8 obtained for 7
clearly showed an isomerizable structuresconsiderable
line broadening of the p-CH3 peak occurred at -21 °C.
In the following sections the chemical, enzymatic, and
bacteriological activity profiles of the S-aminosulfen-
iminopenicillins prepared in this work are presented and
discussed.
to form a zwitterionic intermediate, a process for which
there is good precedent in the literature.12 There may
be an element of kinetic control in the partitioning of
intermediate A (Scheme 2) in that H-6 may be more
acidic than the relevant N-H. The S-aminosulfenimine
may also be the thermodynamically favored product in
this case; semiempirical calculations (AM1, SAM1, PM3)13
showed that the S-aminosulfenimine structure type was
considerably more stable that the sulfur diimide form.
The range of S-aminosulfeniminopenicillins which we
have been able to produce directly by this method was
influenced by two factors. The first was the range of
substituted sulfur diimides which could be conveniently
prepared and purified, and the second factor was the
reactivity of some of the sulfur diimides themselves;
diphenyl sulfur diimide did not react with 1. Our present
work is limited to S-aminosulfenimines bearing carbeth-
oxy, p-toluenesulfonyl (tosyl), and p-chlorophenylsulfonyl
groups. Further structural variation was achieved by
oxidation of the thiazolidine-ring sulfur and also by
methylation of the side-chain nitrogen bearing the aryl-
sulfonyl group; this latter was cleanly effected using
Proton sponge and methyl iodide. In all cases selective
oxidation of the thiazolidine-ring sulfur to the sulfoxide
was achieved using 1 equiv of m-chloroperbenzoic acid
(MCPBA). Further selective oxidation of these sulfoxides
to the corresponding sulfones was achieved, except for
4b1, with KMnO4 in a two-phase system. The chemical
shift pattern of H-5 and of the R,â-methyl group clearly
indicated that the site of the successive oxidations was
the thiazolidine-ring sulfur (see Experimental Section for
details). This oxidation pattern paralleled that found by
Ch em ica l Rea ctivity. Cleavage of the â-lactam ring
of penicillin structures in methanol containing a low
concentration of base serves as a simple model for the
corresponding reaction by the â-lactamase-active-site
serine. As such, establishing the pattern of inherent
chemical reactivity of a structure in this system can
provide information which might be relevant to its
interaction with a â-lactamase enzyme. We carried out
a detailed analysis of the reaction end products, and
intermediate structures formed as a function of time, on
treatment of the thiazolidine-ring-sulfide esters 4a , 4b,
4bm , and 4cm in basic methanol (typically 35 mM unless
otherwise stated); samples were withdrawn from the
reaction solution at varying time intervals and extracted
1
and the H NMR spectra were recorded (see Experimen-
tal Section for other details). With 4b, 4bm , and 4cm a
common set of products was obtained. This set consisted
of the arylsulfonamide corresponding to the individual
S-amino moiety and one other structure in two epimeric
forms. The arylsulfonamides were identified by com-
1
parison with authentic samples while H and 13C NMR
data corroborated assignment of 9 (Scheme 4) as the
structure of the other product (these data are presented
later as the reaction sequences leading to its formation
were relevant to the structural assignment).
The overall results were analyzed in terms of the
reaction paths shown in Scheme 4: these provided a basis
(12) Grigg, R.; McMeekin, P.; Sridharan, V. Tetrahedron 1995, 48,
13347.
(13) Ampac 5.0, 1994, Semichem, 7204 Mullen, Shawnee, KS 66216.
For details of SAM1 see: Dewar, J . S.; J ie, C.; Yu, J . Tetrahedron 1993,
49, 5003.

S-Aminosulfeniminopenicillins
J . Org. Chem., Vol. 63, No. 22, 1998 7603
Sch em e 4a
a
Rate determing step for 4bm and 4cm ; trans indicates side-chain S away from original lactam carbonyl.
for proposing 9 as the end product and, as shown later,
the mechanistic work provided a set of results consistent
with this reaction pattern (12 was not observed as an
end productsno peak attributable to an aldimine hydro-
gen was apparent in the NMR spectra). The set of
reactions shown in Scheme 4 consist of methanolysis of
the â-lactam ring and both inter- and intramolecular
displacement of the S-amino moiety, all of which are well
precedented in the literature. Reactions involving dis-
placement of leaving groups from sulfur are well docu-
mented in the chemistry of sulfenyl chlorides and related
structures.14 Implicit in the reaction of piperidine-1-
sulfenyl chloride with diamines to form (cyclic) thia-
diazoles14a,15 is an intramolecular process which closely
parallels that shown leading to 9 from structure type 8.
The formation of five-membered rings via intramolecular
nucleophilic displacement enjoys a considerable kinetic
advantage over the corresponding intermolecular process
leading to acyclic products. This kinetic advantage is
quantified in terms of “effective molarity” (EM), and for
a variety of cyclizations, involving reaction of secondary
amines with a carbonyl group as the electron-deficient
center (nucleosite), EM values of the order of 103 M are
typical;16a the same should hold with sulfur as the
nucleosite. The efficacy of alkyl sulfides as nucleophiles
in intramolecular reactions is well documented in the
chemistry of ω-halogenoalkyl sulfides (sulfur mustards)
for which the formation of five-membered rings is par-
ticularly favored with alkyl as opposed to aryl sulfides.17
The sequence 8 f In ter -1 f 11 (Scheme 4) parallels the
chemistry of the sulfur mustards closely. The formation
of 9 from 11 is an intramolecular counterpart of the
established intermolecular reaction of methyl sulfenates
(RSOCH3) with primary and secondary amines to form
sulfenamides (RSNR′(R′′)).14b,18
The side-chain and thiazolidine-ring sulfur atoms are
held at 4.00 Åsi.e., just outside the sum of the van der
Waals radii for this atom pairsin a sulfeniminopenicillin
of which the crystal structure is known.11 Cleavage of
the lactam ring will release this constraint and could thus
trigger reaction between the thiazolidine-ring sulfur and
an appropriately functionalized sulfenimine side chain.
Conformational analysis (semiempirical methodology us-
ing SAM1) of the â-lactam-ring-cleaved structure 8bm
(as the free acid which is relevant to neutral structures
such as the corresponding ester, and also as the dicar-
boxylate dianion which is relevant to reaction in aqueous
solution detailed later) provided some substantiation for
this hypothesis. The conformational profiles (Figures 1
and 2) showed that, in conformers occupying minima on
the potential energy surface, the separation distances of
the side-chain sulfur atom to the thiazolidine-ring nitro-
gen and sulfur atoms, dNS and dSS, respectively, lay well
inside the sum of the van der Waals radii of the relevant
atom pairs. This is precisely the circumstance which has
(14) (a) Capozzi, C.; Modena, G.; Pasquato, L. In The Chemistry of
Sulphenic Acids and Derivatives; Patai, S., Ed.; J ohn Wiley & Sons:
Chichester, 1990; Chapter 10. (b) Hogg, D. R. In The Chemistry of
Sulphenic Acids and Derivatives; Patai, S., Ed.; J ohn Wiley & Sons:
Chichester, 1990; Chapter 9.
(17) (a) Bohme, H.; Sell, K. Chem. Ber. 1948, 81, 123. (b) Bird, R.;
(15) Bryce, M. R. J . Chem. Soc., Perkin Trans. 1 1984, 2591.
(16) (a) Kirby, A. J . Adv. Phys. Org. Chem. 1980, 183. (b) Kirby, A.
J .; Logan, C. J . J . Chem. Soc., Perkin Trans. 2 1978, 642. (c) Menger,
F. M. Acc. Chem. Res. 1985, 18, 128; 1993, 26, 206.
Stirling, J . M. J . Chem. Soc., Perkin Trans. 2 1973, 1221 and references
therein.
(18) Armitage, D. A.; Clark. M. J .; Kisey, A. C. J . Chem. Soc. C 1971,
3867.

7604 J . Org. Chem., Vol. 63, No. 22, 1998
Smyth et al.
The â-lactam-ring-opened structure 8a was the sole
product of the reaction of 4a in basic methanol; notably,
the S-amino moiety here was a poor leaving group and
in addition bore an ionizable NH group, the relevance of
which is elaborated on below. The chemical shift of the
R-methyl group at 1.05 ppm and the upfield shift of H-3
to 3.81 ppm were diagnostic of the â-lactam-ring-opened
structure19 (Figure 3b). The coupling of H-5 with the
thiazolidine-ring nitrogen (J ) 4 Hz) was readily appar-
ent while the spectrum resultant on addition of D2O
allowed inference of the chemical shifts of this N-H and
H-3 as a coupled set of hydrogens (J ) 10 Hz) to be made
(Figure 3b and 3c); the differing coupling constants
implied a cis and trans geometry of this N-H with H-5
and H-3, respectively. This compound was found to be
quite stable when stored in CDCl3 for up to 10 h. In the
case of 4b , with p-toluenesulfonamide as the S-amino
moiety, the â-lactam-ring-opened structure 8b was iso-
lated but was found to undergo a further reaction in
1
CDCl3 solution. Figure 4b shows the H NMR spectrum
of a sample obtained from the reaction of 4b in basic
methanol in which the major product was 8b: this was
identifiable primarily from the chemical shift of the
R-methyl group at 1.05 ppm and the upfield shift of H-3.
Some unreacted starting material was also present
together with small amounts of the eventual end prod-
1
ucts. The H NMR spectrum of this sample after 6 h in
F igu r e 1. Conformational profile (rotation about C6-C5), and
internuclear separations, of 8bm (free-acid); hydrogen atoms
are not shown.
CDCl3 at room temperature, and in the absence of added
bases or nucleophiles, indicated a considerably altered
structure (Figure 4c). A fine precipitate which formed
during this time was unambiguously identified as p-
toluenesulfonamide by comparison with an authentic
sample; a good deal of this latter material remained in
solution, but the chemical shifts of the p-methyl group
in 4b and in this latter compound were very close, thus
1
hindering its identification from the H NMR data alone.
The downfield shift of the R-methyl group (Figure 4c) was
an initial indication of the thiazolidine ring being in a
ring-fused system,20 and this together with the formation
of p-toluenesulfonamide pointed to 9 as a possible
structure for the major coproduct. Chromatographic
purification (flash technique) of the reaction products
readily removed p-toluenesulfonamide and yielded the
major and minor products as a mixture, roughly in a ratio
of 3:1sthis corresponded to the ratio in which they were
1
observed in the crude reaction sample. The H and 13C
NMR spectra of such a purified product sample are
shown in Figures 5 and 6, and the assignments are
summarized in Tables 1 and 2, respectively, together with
data for other relevant structures. On the basis of the
chemical shift and integration data in the 1H NMR
spectrum, and taking into consideration the reaction in
which the products were formed, the presence of the
following components was inferred for both the major and
minor products: (a) a geminal dimethyl group originating
from the R,â-methyl groups, (b) a methyl and a diphenyl-
methyl ester, (c) single, uncoupled, hydrogens originating
from H-3 and H-5, (d) no readily exchangeable (NH)
hydrogens (on addition of D2O), and (e) the complete
F igu r e 2. Conformational profile (rotation about C6-C5), and
internuclear separations, of 8bm (dicarboxylate dianion -
8′bm ); hydrogen atoms are not shown.
(19) Davis, A. M.; J ones, M.; Page. M. I. J . Chem. Soc., Perkin Trans.
2 1991, 1219.
been identified as providing the impetus unique to
intramolecular processes which exhibit exceptionally high
EM values.16b,c
The experimental detail and its analysis, which pro-
vided substantiation of the reaction patterns shown in
Scheme 4, follow.
(20) For examples of a similar pattern of chemical shift changes on
opening the â-lactam ring of a penicillin followed by closure to yield
bicyclic structures with the thiazolidine ring fused to five-membered
rings, see: (a) Marchand-Brynaert, J .; Ghosez, L. Bull. Soc. Chim. Belg.
1985, 94, 1021. (b) Tanaka, R.; Nakatsuka, T.; Ishiguro, M. Bioorg.
Med. Chem. Lett. 1993, 3, 2299.

S-Aminosulfeniminopenicillins
J . Org. Chem., Vol. 63, No. 22, 1998 7605
F igu r e 3. 1H NMR spectra of (a) 4a , (b) 8a , and (c) 8a on addition of D2O.
absence of the S-amino side chain. The 13C NMR
spectrum corroborated the presence of the geminal di-
methyl group and both the methyl and diphenylmethyl
esters, and in the case of the major product the spectrum
clearly showed the presence of an imino carbon. In
addition peaks attributable to the other required carbons,
C-2, C-3, and C-5, were clearly observed.
the R,â-methyl groups in this product set. First, it has
been found that the chemical shifts of H-5/H-3 occurred
at 5.03/3.69 ppm and at 5.48/4.37 ppm for stereoisomers
of a given penicillin structure, with these hydrogens cis
and trans (to each other), respectively.23a,b This pattern
was matched by the chemical shifts of H-5/H-3 in the
major and minor products (Figure 5; Table 1) indicating
that these products were, most probably, similarly related
The finding that no change in the line shape of H-5
1
occurred from +30 to -60 °C in the H NMR spectrum8
of the major and minor product set indicated that the
imino function was not isomerizable which was consistent
with its being in a ring system. Furthermore very
pronounced line broadening was observed for the geminal
dimethyl group below -35 °C. Similar line broadening
was seen with 4bm and was associated with a slowing
down of the thiazolidine ring flipping between “open” and
“closed” conformations21 which is well established in ring-
fused systems such as penicillin structures.22 Overall the
spectroscopic data were consistent with a structure
containing a thiazolidine ring bearing a geminal dimethyl
group and fused to a second ring containing an endocyclic
imino functionality; sensibly the diphenylmethyl ester
must be on the thiazolidine ring and the methyl ester on
the ring fused to the thiazolidine ring.
as stereoisomers. Second, it has been shown by Dobson
and co-workers22 that the 13C chemical shifts of the R,â-
methyl groups are very much closer together in penicillin
structures which are predominantly in an open confor-
mation than in penicillins predominantly in a closed
conformation; a similar result should hold when the
thiazolidine ring is fused to a five-membered ring as in
9. The observation of just such a pattern for the geminal
methyl group in the major and minor product set (Figure
The major and minor products were assigned as
stereoisomers of 9 with H-5/H-3 being either cis or trans
to each other on the basis of the chemical shift pattern
of these hydrogens and the 13C chemical shift pattern of
(23) (a) Busson, R.; Vanderhaeghe, H. J . Org. Chem. 1976, 41, 2561.
(b) A number of other penicillin, and closely related, structures showing
this pattern of chemical shifts for H5/H3 are given in Topics in
Antibiotic Chemistry, Vol. 4; Sammes, P. G., Ed.; Ellis Horwood:
Chichester, 1980; Chapter 2. (c) Busson and Vanderhaeghe observed
no epimerization at C-3 in the penicillin structures using NEt3, but
were able to so using the stronger base DBN.
(21) Davern, P.; Sheehy, J .; Smyth, T. J . Chem. Soc., Perkin Trans.
2 1994, 381 and references therein.
(22) Claydon, N. J .; Dobson, C. M.; Lian, L.-Y.; Twyman, M. J . Chem.
Soc., Perkin Trans. 1 1986, 1933. Twyman, M.; Fattah, J .; Dobson, C.
M. J . Chem. Soc., Chem. Commun. 1991, 647.

7606 J . Org. Chem., Vol. 63, No. 22, 1998
Smyth et al.
F igu r e 4. 1H NMR spectra of (a) 4b, (b) sample isolated from reaction of 4b in basic methanol, and (c) this isolated sample after
6 h in CDCl3 at 25 °C.
F igu r e 5. 1H NMR spectra of purified reaction products: the major and minor products were assigned as (3S,5S)-9 and (3S,5R)-
9, respectively.
6; Table 2) was consistent with interpreting the major
and minor products as being bicyclic ring fused systems,
with the major product bearing the geminal methyl group
in a more open conformation than in the minor product.
This was supported by conformational analysis (semiem-
pirical methodology using SAM1) which showed that
(3S,5S)-9 (cis H-5/H-3) had a considerably more open
aspect than (3S,5R)-9, and in addition, was the more
stable of the two by a small margin (Figure 7). The
finding that a 3:1 ratio of major:minor product was
obtained from 4bm , 4cm , and 8b was consistent with
this product set being an equilibrated mixture of (stereo)-
isomers of marginally differing stability.
The chemical shift pattern of H-5/H-3, the 13C NMR
chemical shift pattern of the R/â-methyl group, and the
results of the molecular modeling collectively supported

S-Aminosulfeniminopenicillins
J . Org. Chem., Vol. 63, No. 22, 1998 7607
F igu r e 6. 13C NMR spectra (100 MHz) of purified reaction products: the major and minor products were assigned as (3S,5S)-9
and (3S,5R)-9, respectively.
Ta ble 1. 1H NMR Ch em ica l Sh ifts (p p m )
(3S,5S)-9
major
product
(3S,5R)-9
minor
product
4bm a/4cm a
10a
R,â CH3
H-3
CH3OSN
1.30; 1.57 (1.57); 1.70 1.30b; 1.57b (1.30; 1.57)c
3.82
4.30
4.70b
4.70
3.86
CH3OC(O) 3.89
H-5 5.72
CH(C6H5)2 7.00
3.88
6.23
7.05
6.20/6.12
6.95/6.94
5.88
6.95
a
b
The configuration here is (3S,5R). Identical chemical shift
for 4bm and 4 cm . c Close overlap occurred with other peaks.
Ta ble 2. 13C NMR Ch em ica l Sh ifts (p p m )
(3S,5S)-9
major product minor product
(3S,5R)-9
4a a
R,â CH3
CH3O
C-2
C-3
C-5
OCH(Ph)2
C-6 (imine)
aromatic
26.1, 26.3
53.1
54.2
74.8
78.5
81.8
151.9
126.7-128.8;
139.1-139.2
28.2, 29.8
(53.0)
55.5
75.5
79.1
79.6
b
25.3, 34.2
64.0
71.2
72.0
78.5
154.1
127.0-129.4;
139.1
c
lactam
155.5
ester (alkyl) 159.0
(conjugated)
167.2
b
b
166.5
(nonconjugated)
165.0
F igu r e 7. Conformational profile (open/closed aspect) of
(3S,5S)-9 and (3S,5R)-9; H-3 and H-5 are the only hydrogen
atoms shown.
ester
(diphenyl-
methyl)
(nonconjugated)
(nonconjugated)
and In ter -2 (Scheme 4), which would indicate that the
major product was (3S,5S)-9. The transannular interac-
tion of a sulfur atom with a carbonium ion center in cyclic
structures is well established;24a substantiation for the
reversibility of the equilibrium between In ter -1 and
In ter -2 is provided by the observed ready contraction of
seven- and eight-membered cyclic sulfides to form fused
bicyclic structures, by the nucleophilic addition of the
sulfur atom to the carbonium center.24b
a
b
The configuration here is (3S,5R). Not readily identifiable.
c Coincident with those of the major product.
assignment of the major product as the stereoisomer
bearing H-5/H-3 cis to each other, i.e., either (3S,5S)-9
or its enantiomer (3R,5R)-9. As the stereochemistry in
the starting penicillin structure was (3S,5R)strans H-5/
H-3sepimerization must have occurred at some stage in
1
the reaction process, at either C-5 or C-3. The H NMR
Having thus substantiated the structure, and basis of
formation, of the reaction products, the pattern of reac-
tivity is further delineated in the following sections.
data clearly indicated that epimerization at these posi-
tions had not occurred, neither in the ring-opened
structures such as 8a (Figure 3) and 8b (Figure 4) nor,
as seen later, in the intact penicillin structures23c such
as 4bm (Figure 9) and 10 (Figure 8). No exchange of
H-5 or of H-3 in the product set was observed when 4bm
was reacted in CD3OD/NEt3. This finding restricted the
epimerization option to the rapid equilibration of In ter -1
(24) (a) Tabushi, I.; Yoshinao, T.; Yoshida, Z.-i; Sugimoto, T. J . Am.
Chem. Soc. 1975, 97, 2886. Block, E. Reactions of Organosulfur
Compounds; Academic Press: New York, 1978 Chapter 4 and refer-
ences therein. (b) deGroot, A.; Boerma, J . A.; Wynberg, H. Tetrahedron
Lett, 1968, 2365. Cere`, V.; Peri, F.; Pollicino, S.; Antonio, A. J . Chem.
Soc., Perkin Trans. 2 1998, 977 and references therein.

7608 J . Org. Chem., Vol. 63, No. 22, 1998
Smyth et al.
F igu r e 8. 1H NMR spectra of (a) 4cm and samples isolated at (b) 10, (c) 30, (d) 60, and (e) 120 min from reaction of 4 cm in basic
methanol.
As a neutral group p-toluenesulfonamide is a moderate
leaving group; however, ionization of the sulfonamide
hydrogen in 4b was found to occur quite readily in basic
methanol,25 thereby considerably reducing its nucleo-
fugacity. This would also occur for 8b in basic methanol.
Partial ionization of this acidic NH bond in CDCl3,
through formation of a strong hydrogen bond with the
basic thiazolidine-ring nitrogen in an inter- or intramo-
lecular fashion, would significantly retard the rate of
displacement of p-toluenesulfonamide from 8b in this
solvent system. These processes were blocked in the case
of the N-methylated structures, resulting in a modifica-
tion of the reaction kinetics leading to the same pattern
of end products, as discussed below.
1
A set of H NMR data obtained from reaction of the
N-methylated structures 4cm and 4bm in basic metha-
nol, at different time intervals, is shown in Figures 8 and
9, respectively. No â-lactam-ring-opened structures were
detected which implied that reactions occurred, after ring
opening, which were faster than the ring-opening step
itself. Displacement of the S-amino moiety, to give the
corresponding N-methylarylsulfonamide, was readily
identified by the change in the chemical shift of the
(25) (a) The structure 4b(sid e-ch a in
was readily observed (1H
a n ion )
NMR)8 by treatment of 4b in CDCl3 with Proton Sponge; it was quite
stable for an extended period of time (24 h). Cleavage of the â-lactam
ring in this structure was particularly slow and was effected using
0.8 M MeO- for a very short reaction time, see Experimental Section
for details. (b) The pKa of a related compound, TsNHBoc, has been
reported as 8.5 in DMSO: Koppel, I.; Koppel, J .; Degerbeck, F.; Grehn,
L.; Ragnarsson, U. J . Org. Chem. 1991, 56, 7172. (c) The ability of
sulfur to stabilize an adjacent anion is well documented: Wiberg, K.
B.; Castejon, H. J . Am. Chem. Soc. 1994, 116, 10489 and references
therein.

S-Aminosulfeniminopenicillins
J . Org. Chem., Vol. 63, No. 22, 1998 7609
F igu r e 9. 1H NMR spectra of (a) 4bm and samples isolated at (b) 10, (c) 30, (d) 60, and (e) 240 min from reaction of 4bm in basic
methanol; the quartet centered at 3.08 ppm in (c) and (d) is due to a trace of triethylamine hydrogen bonded to the sulfonamide.
N-methyl group. In 4cm this occurred as a singlet at
3.29 ppm which changed during the course of the reaction
to a doublet at 2.65 ppm (J ) 3.7 Hz; this collapsed to a
singlet on addition of D2O concomitant with the disap-
pearance of the broad peak at 4.55 ppm). Similarly, in
the reaction of 4bm the original singlet at 3.25 ppm (N-
methyl) changed progressively to a doublet at 2.63 ppm
which corresponded accurately with the chemical shift
of the N-methyl peak in N-methyl-p-toluenesulfonamide26
(J ) 3.7 Hz; this collapsed to a singlet on addition of D2O
concomitant with the disappearance of the broad peak
at 4.45 ppm). The buildup and decay of an intermediate
was clearly observed with both 4cm (Figure 8) and 4bm
(26) Pouchert, C. J . The Aldrich Library of NMR Spectra, 2nd ed.;
Aldrich Chemical Co., Inc.: Milwaukee, 1983; Vol. 2.

7610 J . Org. Chem., Vol. 63, No. 22, 1998
Smyth et al.
Ta ble 3. Va lu es of IC50 (µM), P a r tition Nu m ber , kca t/Km , a n d kIn a ct/Km (M-1 m in -1) a t p H 7.2, for th e
S-Am in osu lfen im in op en icillin Sod iu m Sa lts w ith â-La cta m a se Typ e I fr om B. cer eu s
sulfides
sulfoxides
sulfones
5b2m
structure
5a
5b
5bm
1 000
15 000
5cm
5a 1
5b1
5a 2
sulbactama
IC50
10 000
10 000
425
6800
1800
5600
1500
10 000
15
100
115
1000
80
partn. no.
>30 000
>30 000
500
k
cat/Km
6.26 × 105
kinact/Km
41.73b
a
Sulbactam was screened at the same time as the other compounds using the same enzyme stock solution (see Experimental Section
b
for details). Calcutated from the partition number and kcat/Km.
Sch em e 5
(Figure 9). A number of peaks were clearly assignable
to this intermediate: 6.95 (CH(Ph)2), 5.88 (H-5), 4.70 (H-
3), and 3.86 ppm (CH3OS) while the geminal dimethyl
set of peaks were coincident with other such peaks. The
coincidence of the chemical shift of H-3 in this structure
with that in 4bm and in 4cm was strongly indicative of
the intermediate being an intact penicillin structure. The
chemical shift at 3.86 ppm, assigned to the CH3OS
moiety, was similar to that found for the corresponding
hydrogens in closely related structures (ROSN(R′)R′′).27
That the buildup of this intermediate was more pro-
nounced in the case of 4cm (Figure 8), which contained
the better S-amino leaving group compared to 4bm
(Figure 9), was consistent with identification of the
intermediate as resulting from direct intermolecular
displacement of the S-amino moiety. That the same end
products resulted from the decay of this intermediate,
and at a slower rate than their formation directly from
4cm (see below), was also consistent with the identifica-
tion of this intermediate as 10.
It is evident from the data in Figure 8 (spectra of
reaction samples at 30, 60, and 120 min) that the rate of
disappearance of 10 to give 9 was a good deal slower than
molecular displacement of the S-amino moiety in 4bm .
The nondetection of a buildup of a â-lactam-ring-opened
structure indicated that cleavage of the â-lactam ring was
rate determining for formation of 9 via paths A and B
(Scheme 4). Thus 4bm and 10 define structure types in
which the predominant reaction pattern was rapid in-
tramolecular displacement of the sulfur-attached moiety
consequent on cleavage of the â-lactam ring. The finding
the initial reaction of 4cm itself (Figure 8, spectrum of
that 10 followed this pattern was significant in that it
reaction sample at 10 min) to generate the same end
showed the efficiency, and generality, of the intramo-
products. This was consistent with the greater electron-
lecular displacement process.
The relevance of this pattern of inherent chemical
reactivity to the observed interactions of these structures
withdrawing effect of the N-methyl-p-chlorophenylsul-
fonamido group over a methoxy group resulting in the
â-lactam ring of 4cm being more activated toward ring
with a â-lactamase enzyme is elaborated on in the next
opening28 compared to 10. On the basis of the combined
section.
integration of the peaks at 5.72 and at 6.23 ppm, it was
also evident that a significant amount of 9 had been
En zym a tic Activity. The interaction of the salts 5a -
5cm 29 with â-lactamase type I from Bacillus cereus was
formed quite rapidly (Figure 8, spectrum of reaction
studied in aqueous buffers: the results are summarized
sample at 10 min) and, therefore, could have arisen only
in Table 3. Evaluation of the their efficacy as mecha-
nism-based inhibitors was quantified in terms of IC50 (the
concentration of inhibitor required to lower enzyme
activity by 50%) and partition number. This latter is the
via path A (Scheme 4). The fact that at this reaction time
the amount of 10 formed was somewhat larger than that
of 9 (based on the combined integration of the peaks at
5.72 and at 6.23 ppm relative to that of the peak at 5.88
number of inhibitor molecules hydrolyzed by the enzyme
ppm) indicated that direct intermolecular displacement
per inactivation event30 and, on a practical basis, corre-
of the S-amino moiety in 4cm had outpaced â-lactam ring
sponds to the (minimum) inhibitor:enzyme molar ratio
at which complete inactivation of the enzyme occurs (see
also kinetic definition in Scheme 5). Benzylpenicillin was
used as the assay substrate, and measurement of the rate
opening by a small margin. With 4bm this situation was
reversed. The electron-withdrawing effect of the N-
methyl-p-toluenesulfonamido group was such that the
rates of both â-lactam ring opening and direct intermo-
of its enzyme catalyzed hydrolysis by samples of â-lac-
lecular displacement of this group were slower relative
tamase, which had been incubated (10 min) with varying
to that of 4cm . This is clear from the data in Figure 9
concentrations of inhibitor, was carried out in a standard
where after a 30 min reaction time a significantly larger
amount of starting material was still present compared
(27) (a) Bassindale, A. R.; Iley, J . In The Chemistry of Sulphenic
with that observed for 4cm at a 10 min reaction time
(Figure 8). On the basis of the combined integration of
the peaks at 5.72 and at 6.23 relative to that of the peak
at 5.88 ppm in Figure 9 (spectra of reaction samples at
10, 30 and 60 min) it was evident that cleavage of the
â-lactam ring, followed by rapid intramolecular displace-
ment of the S-amino moiety, had outpaced direct inter-
Acids and Derivatives; Patai, S., Ed.; J ohn Wiley & Sons: Chichester,
1990; Chapter 3. (b) Wenschuh, E.; Kuhne, U.; Mikolajczyk, M.;
Bujnicki, B. Z. Chem. 1981, 21, 217.
(28) Rao, S. N.; More O’Ferrall, R. A. J . Am. Chem. Soc. 1990, 112,
2729.
(29) 5cm was isolated as the free acid. For enzymatic studies 50
µL of a THF solution of 5cm -fr ee a cid was added to the aqueous buffer
containing the â-lactamase enzyme (see Experimental Section).
(30) Walsh, C. Tetrahedron 1982, 38, 871.

S-Aminosulfeniminopenicillins
J . Org. Chem., Vol. 63, No. 22, 1998 7611
Sch em e 6
manner using UV/vis spectroscopy. Using the same
assay procedure the minimum concentration of inhibitor
required for complete inactivation of a known concentra-
tion of enzyme, at the standard 10 min incubation time,
was determined and the inhibitor:enzyme molar ratio at
this point gave the partition number directly.31 In
addition, in the case of 5bm the degree to which it
behaved as an enzyme substrate was quantified in terms
of kcat/Km;32a this term is essentially a second-order rate
constant for hydrolysis of the substrate/inhibitor by this
enzyme32b,33 (Scheme 5). The values of kcat/Km were
determined using the expression kcat/Km ) v/[Eo][So] by
measuring v, the initial rate of hydrolysis (5%) of a known
concentration of substrate using a known concentration
of enzyme (see Experimental Section for details).
The value of kcat/Km for 5bm (Table 3) identified it as
a moderate substrate for this enzyme. This allowed us
to examine the intramolecular displacement reaction
from the carboxylate salt 8′bm (Scheme 6).34 We found
that the rate of displacement of N-methyl-p-toluene-
sulfonamide from 8′b m , generated by â-lactamase-
catalyzed hydrolysis of 5bm in aqueous buffer, was faster
that its direct displacement from 5bm in this medium,
by a factor of at least 600. This was verified by direct
observation of the reaction, when run on a small scale
in D2O buffer, using 1H NMR. To a sample of 5bm
(approximately 5 mg) in D2O buffer (pD 7.2, 0.1 M
phosphate) in an NMR tube was added a portion of
enzyme sufficient to effect immediate and complete
hydrolysis. A spectrum was recorded as quickly as
possible after addition of the enzyme solution, ap-
proximately 20 min was required for 200 accumulations.
This spectrum (Figure 10b) showed the complete absence
of the peak at 3.33 ppm corresponding to the side-chain
N-methyl (this chemical shift was characteristic of this
group while attached to the side-chain sulfur in both 5bm
and 8′bm ) but had a sharp singlet at 2.55 ppm corre-
sponding to the N-methyl group of N-methyl-p-toluene-
sulfonamide in this solvent; when this experiment was
run on a larger scale in H2O buffer a precipitate formed
and was recovered and identified unambiguously as
N-methyl-p-toluenesulfonamide. The experiment in D2O
buffer was rerun using a larger amount of 5bm (ap-
proximately 17 mg) which allowed a sufficiently detailed
spectrum to be obtained within 5 min of adding the
enzyme (Figure 10c). Although the spectral resolution
was not as good as that obtained at the lower concentra-
tion, due to the formation of a fine precipitate, both the
complete absence of the peak at 3.33 and the appearance
of the peak at 2.55 ppm were readily apparent. The half-
life for expulsion of N-methyl-p-toluenesulfonamide from
8′bm was thus estimated at <0.5 min, on the basis that
the 1H NMR observation made after 5 min, showing
complete loss of the side-chain N-methyl moiety, cor-
responded to 10 half-lives; this can only be taken as an
upper limit, however, in that the NMR technique did not
allow us to make observations at a shorter time interval
after addition of the enzyme. Significantly, the half-life
for the intermolecular displacement, by water, of N-
methyl-p-toluenesulfonamide from 5bm in the same D2O
buffer (no enzyme present) was found by direct observa-
(31) Enzyme inactivation was found to be time dependent. An
incubation time of 10 min proved to be convenient. The partition
number, when evaluated in this way, can vary with the incubation
time used: see ref 3e.
(32) (a) Fersht, A., Ed. Enzyme Structure and Mechanism, 2nd ed.;
W. H. Freeman and Company: New York, 1985; Chapter 3. (b) Strictly,
Km ) ((k-1 + k2)/k1) ((k3/(k2 + k3)) ≈ k-1/k1 ) Ks when k-1 . k2 and k3
. k2. An alternative condition under which Km ≈ Ks is when k-1 ) k3
which has been shown to be the case for a variety of â-lactamases (see
ref 33).
(33) Christensen, H.; Martin, M. T.; Waley, S. G. Biochem. J . 1990,
266, 853.
(34) The thiazolidine-ring in 8′bm is shown as being unprotonated
on nitrogen consistent with the fact that the pKa of the N-conjugate
acid of a closely related structure is 5.14: see ref 19.

7612 J . Org. Chem., Vol. 63, No. 22, 1998
Smyth et al.
F igu r e 10. 1H NMR spectra of (a) 5bm in D2O buffer (pD 7.2) at 25 °C, (b) reaction mixture of 5bm (5 mg) 20 min after addition
of â-lactamase enzyme, and (c) reaction mixture of 5bm (17 mg) 5 min after enzyme addition; DSS was used as external reference.
tion (1H NMR) to be approximately 300 min at 25 °C
(Figure 11). The ratio of these two half-lives gave a value
of 600 for kintra/(kinter[D2O]); as [D2O] ) 55.3 M an
apparent EM value (kintra/kinter) of 3.32 × 104 M was
obtained for the intramolecular displacement of N-
methyl-p-toluenesulfonamide from 8′bm sfor the reasons
mentioned above this must be taken only as a lower limit
(this value is labeled as an apparent one as the inter-
and intramolecular nucleophiles are not the same, water
vs the thiazolidine-ring sulfur; however, this EM value
gives a practical measure of the kinetic advantage
pertaining to the intramolecular process in aqueous
solution). The identity of the coproduct formed from 5bm
substrates and as poor inhibitors. In the context of
chemical reactivity the intramolecular displacement of
the sulfur-attached moiety must be characterized by a
high EM value. There must be a far greater propensity
for intra- over intermolecular displacement in aqueous
solution: the actual rate of this displacement would
depend on the nature of the leaving group (Scheme 7).
Thus 5bm has a set of kinetic characteristics which
match the required pattern shown in Scheme 7: the
partition number gave k3/k4 directly as 1.5 × 104, while
kintra/kinter ) EMapp > 3.32 × 104 M. The value of kcat/Km
(6.26 × 105 M-1 min-1) is not optimal, and neither is the
S-amino moiety of 5bm a toxic entity or precursor.
Nonetheless this compound constitutes a valuable lead
structure, and the delineation of its pattern of intrinsic
reactivity is significant.36
It is apparent that â-lactamases are ancient enzymess
certainly they predate human usage of â-lactam antibiot-
ics by eonssand there is evidence that they share a
common ancestry with the transpeptidase domain of
penicillin binding proteins (PBPs).37 It has been shown33
that a variety of â-lactamases are “fully efficient” en-
zymes, which means that they have evolved to become
“perfect” catalysts38 for hydrolysis of their natural
1
in aqueous solution was not deducible from the H NMR
data. Although resonances attributable to the R,â-methyl
group were obvious (Figure 10b), there were no peaks
present which could have been attributed to H-5 or H-3
(albeit the residual water peak could have masked some
detail), and neither was the presence of an aldimine
hydrogen apparent. The exploitable potential of the
S-amino sulfeniminopenicillin structure type, detailed in
the following section, is dependent only on a rapid
intramolecular displacement of the sulfur-attached moi-
ety but not on the coproduct formed from the penicillin
part of the molecule.
The above results clearly identified 5bm as a prototypic
penicillin-based â-lactamase substrate with potential for
development as a site-specific-release prodrug.35 Such
substrates, bearing an appropriate sulfur-attached moi-
ety, would allow exploitation of â-lactamase enzymes,
present in many pathogenic bacteria, to catalyse the
release of a bactericidal agent, or precursor, within the
bacterial periplasmic space. In the context of enzymatic
behavior such structures should have high kcat/Km and
partition number values; they should behave as good
(36) We are currently preparing a variant of 5bm designed to
directly establish (UV/vis spectroscopy, see ref 45b) if the release of
the sulfur-attached moiety inside a â-lactamase-producing strain of a
bacterium is faster that its release inside the corresponding nonpro-
ducing strain. Also, we are investigating how reduction of the imino
group might influence kcat/Km and EM values. In the context of
enhancing the value of kcat/Km it is relevant to note than all penicillin-
based structures which are good substrates of â-lactamase enzymes
have a saturated carbon, with an R configuration and bearing a
hydrogen atom, at C-6.
(37) (a) Bush, K. In Antibiotic Resistance: Origins, Evolution,
Selection and Spread; Wiley: Chichester (Ciba Foundation Symposium
207): 1997; p 152 and references therein. (b) Neuhaus, F. C.; Geor-
gopapadakou, N. H. In Emerging Targets in Antibacterial and Anti-
fungal Chemotherapy; Sutcliffe, J ., Georgopapadakou, N. H., Eds.;
Chapman Hall: New York, 1992; Chapter 9 and references therein.
(38) (a) Albery, W. J .; Knowles, J . R. Biochemistry 1976, 25, 5631.
(b) Albery, W. J .; Knowles, J . R. Angew. Chem., Int. Ed. Engl. 1977,
16, 285.
(35) For a definition and examples of site-specific-release prodrugs,
see: Wermuth, C. G.; Gaignault, J .-C.; Marchandeau, C. In The
Practice of Medicinal Chemistry; Wermuth, C. G., Ed.; Academic
Press: London, 1996; Chapter 31.

S-Aminosulfeniminopenicillins
J . Org. Chem., Vol. 63, No. 22, 1998 7613
F igu r e 11. 1H NMR spectra of (a) 5bm in D2O buffer (pD 7.2), and this reaction mixture after (b) 4, (c) 6, and (d) 20 h at 25 °C;
DSS was used as external reference.
substratessthe naturally occurring inhibitors of transpep-
tidase enzymes. Therefore, some structures elaborated
to be good substrates for â-lactamase enzymes must also
behave as transpeptidase inhibitors: such structures,
embodying â-lactamase catalyzed release of a bactericidal
entity or precursor, would realize a checkmate stratagem
with respect to the problem of â-lactamase-based resis-
tance to antibiotics. The fact that a variety of semisyn-
thetic penicillin structures, known to be transpeptidase
inhibitors, are hydrolyzed at close to the diffusion-
controlled rate by a number of â-lactamases33 buttresses
the strategy of developing penicillin-based â-lactamase
substrates as site-specific-release prodrugs. The S-
aminosulfeniminopenicillin structure constitutes a robust
template for the elaboration of such materials. Synthesis
of 4bm and 4cm is efficient, and variation of the sulfur-
attached moiety is readily achievable by activation of the
S-amino moiety toward direct displacement: cyano-
methylation of the side-chain nitrogen in these structures
allows for facile displacement of this whole group by a
wide variety of nucleophiles.39
In the context of â-lactamase inhibition the thiazoli-
dine-ring sulfides were much less active as inhibitors
than the corresponding thiazolidine-ring sulfones.40 No-
table, however, was the finding that the inhibitory
potency (Table 3) of 5b, 5bm , and 5cm mirrored the
nucleofugacity of the S-amino moiety as observed in their
reactions in basic methanol. This correlation, although
qualitative, indicated that displacement of the S-amino
moiety at the enzyme active site was probably a key
factor in the mode of inhibition operating for these
structures; this is consistent with the overall reaction
pattern shown in Scheme 7. There is a limit on the
extent to which inhibitory potency could be enhanced by
attachement of better leaving groups, in that displace-
(39) Smyth, T. P.; O’Donnell, M. E.; O’Connor, M. J .; St Ledger, J .
O. Unpublished results.
(40) In all cases no recovery of enzyme activity was detected after
up to 19 h.

7614 J . Org. Chem., Vol. 63, No. 22, 1998
Smyth et al.
Sch em e 7
and over sulbactam was primarily due to the presence
of a polar, potentially ionizable, hydrogen on the side
chain of 5a 2. The more efficient partitioning of the initial
acylated enzyme derived from 5a 2 along an inhibitory
route could be due to either slower hydrolytic cleavage
(path a, Scheme 8) or more rapid sulfone elimination
(path c, Scheme 8). A glutamic acid residue (Glu166) has
been implicated as one of the catalytic components in the
active site of class A â-lactamases, and it is evident from
a combination of X-ray data and molecular modeling42
that the 6-amino side chain of penicillin structures
occupies a position proximate to Glu166. The formation
of a strong hydrogen bond between Glu166 and the polar
hydrogen on the side chain of 5a 2 (b, Scheme 8) would
disrupt its role as a general base in hydrolysis of the
acylated enzyme.
Ba cter iologica l Activity. In a preliminary biological
screening against three â-lactamase-producing E. coli
strains (NCIMB 12091, 9517, and 9472),43 5a 2 showed
synergy of action when combined 1:1 with penicillin G.
Thus penicillin G showed no halo of inhibition up to 100
µg/mL whereas at 78 µg/mL 5a 2 plus penicillin G gave
inhibition halos of 11-18 mm in diameter: these halo
sizes were comparable to those obtained with a 1:1
combination of sulbactam and penicillin G at 78 µg/mL
run at the same time. On its own 5a 2 showed no
antibiotic activity against these E. coli strains (weak
antibiotic activity was detected at high concentrations
against S. aureus (NCIMB 8625 (ATCC 6538P)). In a
more stringent screening 5a 2 was held at a fixed concen-
tration of 4 µg/mL and combined, in turn, with varying
concentrations of pipericillin (0.06-128 µg/mL) and
tested against E. coli strains producing nine different
â-lactamase types (see Experimental Section).44 Under
these circumstances no synergy of action was observed.
The observation of synergy at high but not at low
concentration was indicative of slow permeation of the
S-aminosulfeniminopenicillin structures through the por-
ins of the outer membrane of Gram-negative bacteria;
in general the permeation rate of any given solute
decreases in proportion to its external concentration
when the concentration is low.45a The problem of poor
permeation of â-lactamase inhibitors has been identified
in a number of instances and constitutes a major barrier
to the exploitation of many novel inhibitor structures. The
combination of a â-lactamase inhibitor and an antibiotic
involves competition between the two for diffusion through
the porins, and this competition can reduce the thera-
peutic usefulness of effective â-lactamase inhibitors
which have poor permeation characteristics. The mono-
lactam structures synthesized by Miller and co-workers46
while very effective enzyme inhibitors failed to show any
synergy even at high concentration (100 µg/mL). Like-
wise the penicillin-based structures prepared by Buynak
Sch em e 8
ment of such groups by opportunistic nucleophiles would
occur prior to binding of the intact structure at the
enzyme active site.
The sulfone 5a 2 was clearly the most active of all the
structures prepared here, with values of IC50 and parti-
tion number lower than that of sulbactam by approxi-
mately a factor of 5. Significantly 5a 2 contained the
poorest S-amino leaving group which implied that the
mode of action of this sulfone was distinct from that of
the thiazolidine-ring sulfides; notably, the thiazolidine-
ring sulfur in the sulfone structures cannot behave as a
nucleophile. We viewed the mode of action of 5a 2 as
being similar to that of sulbactam41 involving rapid
scission of the thiazolidine sulfone ring following cleavage
of the â-lactam ring (Scheme 8). It is possible that the
greater â-lactamase inhibitory activity of 5a 2 over 5b2m ,
(42) Ishiguru, M.; Imajo, S. J . Am. Chem. Soc. 1996, 39, 2207 and
references therein.
(43) (a) These tests were carried out by CPA Laboratories, Birken-
head, Wirral, U.K. (b) NCIMB (National Collection of Industrial and
Marine Bacteria), Aberdeen, Scotland.
(44) These tests were carried out at the Centre for Research in Anti-
Infectives and Biotechnology, Creighton University, Omaha, NB.
(45) (a) Nikaido, H.; Rosenberg, E. Y. J . Bacteriol. 1983, 153, 241.
(b) Nikaido, H.; Rosenberg, E. Y.; Foulds, J . J . Bacteriol. 1983, 153,
232. (c) Nikaido, H.; Pharmacol. Ther. 1985, 27, 231.
(46) Teng, M.; Miller, M. J .; Nicas, T. I.; Grissom-Arnold, J .; Cooper,
R. D. G. Biorg. Med. Chem. Lett. 1993, 1, 151.
(41) Knowles, J . R. Acc. Chem. Res. 1985, 18, 97.

S-Aminosulfeniminopenicillins
J . Org. Chem., Vol. 63, No. 22, 1998 7615
23 °C); 1H NMR (90 MHz, CDCl3) δ 1.25 (t, J ) 8 Hz, 3H), 4.4
(q, J ) 8 Hz, 2H).
and co-workers47 showed either none or only weak
synergy which was not commensurate with their high
level of enzyme inhibition. Permeation rates have been
well studied by Nikaido and co-workers for both penicillin
and cephalosporin antibiotic structures.45 Hydrophobic-
ity in particular seems to be critical in determining
permeation rates for â-lactam-based structures where
molecular weight is not a problem. Cephalosporin zwit-
terions with proximate opposing charges were found to
have higher rates of permeation than either cepha-
losporin monoanions or zwitterions with remote opposing
charges.45 This identifies a structural feature, the in-
corporation of which on some of the compounds reported
here, may merit evaluation.
P r ep a r a tion of N-Su lfin ylsu lfon a m id e. Thionyl chloride
(60 mL, 0.82 mol) was added to p-toluenesulfonamide (30 g,
0.175 mol) in a 100 mL round-bottomed flask, and the mixture
was heated to reflux for 8 h. Following this unreacted thionyl
chloride was distilled off under reduced pressure, and further
residual traces were removed by entrainment with benzene
(3 × 50 mL) under reduced pressure at 80 °C. Upon cooling
N-sulfinylsulfonamide solidified as a yellow solid50 (30.4 g, 0.14
mol, 80%): 1H NMR (90 MHz, CDCl3) δ 2.47 (s, 3H), 7.40 (d,
J ) 10 Hz, 2H), 7.94 (d, J ) 10 Hz, 2H). This product was
sufficiently pure for direct use in further reactions.
P r ep a r a t ion of Bis(et h oxyca r b on yl)su lfu r Diim id e
(2a ).51 N-Sulfinylcarbamate (6 g, 0.044 mol) and pyridine (0.2
mL, 2.5 mmol) were mixed in a small flask and a CaCl2 drying
tube attached. The mixture was allowed to stand at ambient
temperature overnight. Crude 2a was obtained as a light
orange liquid (3.65 g, 0.019 mol, 85%): 1H NMR (90 MHz,
CDCl3) δ 1.22 (t, J ) 8 Hz, 6H), 4.30 (q, J ) 8 Hz, 4H). This
material was used without further purification but was freshly
prepared from the N-sulfinyl precursor as required.
P r ep a r a tion of Bis(p-tolu en esu lfon yl)su lfu r Diim id e
(2b).52 N-Sulfinylsulfonamide (3.0 g, 13.8 mmol) was added
to benzene (4 mL) together with pyridine (100 µL, 1.3 mmol),
and the solution was allowed to stand at ambient temperature
overnight by which time the reaction mixture had turned
bright yellow and 2b had crystallized out as a yellow solid (2.43
g, 6.55 mmol, 95%): 1H NMR (90 MHz, CDCl3) δ 2.46 (s, 6H),
7.35 (d, J ) 10 Hz, 4H), 7.86 (d, J ) 10 Hz, 4H). This material
was used without further purification but was freshly prepared
from the N-sulfinyl precursor as required.
P r ep a r a tion of Bis(p-ch lor op h en ylsu lfon yl)su lfu r Di-
im id e (2c). Thionyl chloride (20 mL, 280 mmol) was added
to p-chlorophenylsulfonamide (10 g, 52 mmol) in a 100 mL
round-bottomed flask, and the mixture was heated to reflux
for 20 h. Following this, unreacted thionyl chloride was
distilled off under reduced pressure and further residual traces
were removed by entrainment with benzene (3 × 50 mL) under
reduced pressure at 80 °C. Upon cooling, N-sulfinyl-p-chlo-
rophenylsulfonamide solidified as a tan solid (11.9 g, 50.2
mmol, 96.6%): 1H NMR (90 MHz, CDCl3) δ 7.45 (d, J ) 9 Hz,
2H), 7.83 (d, J ) 9 Hz, 2H). This product was sufficiently pure
for direct use in further reactions. N-Sulfinyl-p-chlorophenyl-
sulfonamide (1.46 g, 6.2 mmol) was added to benzene (1.6 mL)
and pyridine (23 µL, 0.3 mmol) and the mixture was stirred
at room temperature for 3 h by which time 2c had crystallized
out as a bright yellow solid (1.19 g, 2.89 mmol, 94%): 1H NMR
(90 MHz, CDCl3) δ 7.55 (d, J ) 9 Hz, 4H), 8.00 (d, J ) 9 Hz,
4H). This material was used without further purification but
was freshly prepared from the N-sulfinyl precursor as required.
P r ep a r a tion of Dip h en ylm eth ylp en icilla n a te (1). This
compound was prepared, as the p-toluenesulfonate salt, ac-
cording to the method of Petursson and Waley53 for the free
amine: 1H NMR (90 MHz, CDCl3) δ 1.25 (s, 3H), 1.60 (s, 3H),
4.5 (d, J ) 4 Hz, 1H), 4.5 (s, 1H), 5.5 (d, J ) 4 Hz, 1H), 6.95
(s, 1H), 7.3 (s, 10H).
Con clu sion s
An efficient and direct route to the preparation of a
novel functionalitysan S-aminosulfenimineshas been
developed involving the reaction of selected amines with
sulfur diimides. When incorporated as the side chain of
a penicillin nucleus, the S-aminosulfenimine moiety has
been shown to engender a pattern of reactivity not
previously encountered in penicillin chemistry: expulsion
of the S-amino moiety was found to occur, consequent
on cleavage of the â-lactam ring, via a rapid intramo-
lecular nucleophilic displacement reaction. This pattern
held for cleavage of the ester derivatives in basic metha-
nol and, significantly, on cleavage of a carboxylate salt
structure by a â-lactamase enzyme in aqueous solution.
This pattern of inherent chemical and enzymatic reactiv-
ity has identified the potential of these materials to be
developed as penicillin-based â-lactamase substrates for
use as site-specific-release prodrugs. Such materials
should allow a strategy of combating â-lactamase-based
resistance to antibiotics to be pursued, which is based
on exploitation of the presence of these enzymes in
pathogenic bacteria.
Exp er im en ta l Section
Gen er a l P r oced u r es. Melting points were determined on
an electrothermal melting point apparatus and are uncor-
rected. Column chromatography (dry flash technique)48 was
carried out using Kieselgel S (32-63 µm) with light petroleum
ether (bp 60-80 °C) and ethyl acetate in various ratios as
eluants; the specific solvent ratio in which a pure product was
obtained is given below. â-Lactamase enzyme was obtained
from Sigma, Type I ex. Bacillus cereus, EC 3.5.2.6. Elemental
analyses were performed by the Microanalytical Laboratory,
U.C.D., Dublin.
P r ep a r a tion of N-Su lfin ylca r ba m a te. Thionyl chloride
(18.9 mL, 0.26 mol) in benzene (30 mL) and pyridine (38 mL,
0.42 mol) in benzene (20 mL) were added, with stirring, to
ethyl carbamate (22.2 g, 0.25 mol) in benzene (60 mL) over 20
min. After an initial exotherm the reaction mixture was
stirred for 1 h after which time the temperature had returned
to ambient. Precipitated pyridium hydrochloride was filtered
off and washed with benzene (50 mL) and the filtrate collected.
The solvent was removed under reduced pressure and the light
orange viscous residue distilled to yield pure N-sulfinylcar-
bamate (12.14 g, 0.90 mmol, 36% yield): bp20 48 °C (lit.49 bp0.5
P r ep a r a tion of Dip h en ylm eth yl N-Ca r beth oxy-S-a m i-
n osu lfen im in op en icilla n a te (4a ). 1 (as the free amine) (1
g, 2.6 mmol) was dissolved in dichloromethane (25 mL). 2a
(0.56 g, 2.9 mmol) was added dropwise, and the reaction
mixture was stirred at ambient temperature for 10 min. The
solution was then washed with distilled water (2 × 60 mL),
and the organic layer was separated, dried, and concentrated
under reduced pressure. Crude 4a was obtained as a yellow
solid. Using dry flash chromatography, the product eluted in
the 60/40 fraction. Removal of the solvent under reduced
pressure yielded 4a as a light yellow solid (625 mg, 1.25 mmol,
(47) Buynak, J . D.; Geng, B.; Bachmann, B.; Hua, L. Biorg. Med.
Chem. Lett. 1995, 5, 1513.
(48) Casey, M.; Leonard, J .; Lygo B.; Procter, G. Advanced Practical
Organic Chemistry, Blackie: Glasgow, 1990; Chapter 9.
(49) Hanson. P.; Stockburn, W. A. J . Chem. Soc., Perkin Trans. 2
1985, 589.
(50) Kresze, G.; Wucherpfennig, W. Angew. Chem., Int. Ed. Engl.
1967, 6, 149.
(51) This is a modification of the procedure reported in Katz, T. J .;
Shi, S. J . Org. Chem. 1994, 59, 8297.
(52) This is a modification of the procedure reported in ref 50.
(53) Petrusson, S.; Walley, S. G. Tetrahedron 1983, 39, 2465.

7616 J . Org. Chem., Vol. 63, No. 22, 1998
Smyth et al.
48%): mp 53-56 °C; 1H NMR (90 MHz, CDCl3) δ 1.3 (m, 6H),
1.55 (s, 3H), 4.23 (q, J ) 8 Hz, 2H), 4.7 (s, 1H), 5.79 (s, 1H),
6.61 (s, 1H, D2O exchangeable), 6.95 (s, 1H), 7.3 (s, 10H); 13C
NMR (90 MHz, CDCl3) δ 14.5, 25.2, 34.1, 63.9, 65.0, 71.5, 72.0,
78.9, 127.2, 127.5, 128.5-129.0, 139.5, 154.0, 155.5, 165.0,
65/35 and 60/40 light petroleum ether/ethyl acetate fractions
and on removal of the solvent under reduced pressure the
product was obtained as a light yellow solid (700 mg, 1.17 mol,
68%): mp 85-89 °C; 1H NMR (90 MHz, CDCl3) δ 1.30 (s, 3H),
1.57 (s, 3H), 2.42 (s, 3H), 3.27 (s, 3H), 4.7 (s, 1H), 6.2 (s, 1H),
6.97 (s, 1H), 7.25-7.85 (m, 14H); IR (KBr) 1775, 1740, 1660
cm-1; UV log ꢀ ) 4.10, λ290 (MeOH). Anal. Calcd for
166.8; IR (KBr) 1785, 1740, 1660 cm-1
. Anal. Calcd for
C24H25N3O5S2: C, 57.71; H, 5.01; N, 8.41. Found: C, 57.99;
H, 5.24; N, 8.05.
C
29H29N3O5S3: C, 58.47; H, 4.90; N, 7.05. Found: C, 58.45;
P r ep a r a tion of Dip h en ylm eth yl N-Ca r beth oxy-S-a m i-
n osu lfen im in o p en icilla n a t e-1-su lfoxid e (4a 1). 4a (200
mg, 0.40 mmol) was dissolved in chloroform (5 mL) at 0 °C.
To this was added a solution of m-chloroperbenzoic acid (130
mg, 0.75 mmol) in chloroform (5 mL). The mixture was stirred
for 20 min, while allowed the temperature to rise to ambient.
The solution was washed successively with 1 M sodium sulfite
(50 mL), 5% sodium hydrogen carbonate (50 mL), and distilled
water (50 mL). The organic layer was separated and dried
and the solvent removed under reduced pressure to leave crude
4a 1 as a yellow solid. Using dry flash column chromatography
the product eluted in the 25/75 light petroleum ether/ethyl
acetate fraction. Removal of the solvent under reduced
pressure yielded 4a 1 as a white solid (150 mg, 0.29 mmol,
H, 4.93; N, 6.81.
P r ep a r a t ion of Dip h en ylm et h yl N-Tosyl-S-a m in o-
su lfen im in op en icilla n a te-1- su lfoxid e (4b1). This was
prepared in a manner similar to that given for 4a 1 above.
Using dry flash column chromatography the product eluted
in the 30/70 light petroleum ether/ethyl acetate fraction.
Removal of the solvent under reduced pressure gave 4b1 as a
foamy white solid (290 mg, 0.48 mmol, 68%): mp 145 °C (dec);
1H NMR (90 MHz, CDCl3) δ 1.1 (s, 3H), 1.7 (s, 3H), 2.43 (s,
3H), 4.82 (s, 1H), 6.30 (s, 1H), 7.02 (s, 1H), 7.25-7.85 (m, 15
H); IR (KBr) 1805, 1745, 1660 cm-1
.
P r ep a r a tion of Dip h en ylm eth yl N-Meth yl-N-tosyl-S-
a m in osu lfen im in op en icilla n a te-1-su lfoxid e (4b 1m ). This
was prepared in a manner similar to that given for 4a 1 above.
Using dry flash column chromatography the product eluted
in the 60/40 light petroleum ether/ethyl acetate fraction.
Removal of the solvent under reduced pressure gave 4b1m as
a foamy white solid (650 mg, 1.06 mmol, 62%): 1H NMR (90
MHz, CDCl3) δ 1.04 (s, 3H), 1.70 (s, 3H), 2.45 (s, 3H), 3.30 (s,
3H), 4.88 (s, 1H), 6.03 (s, 1H), 7.35 (s, 1H), 7.25-7.85 (m, 14
H).
1
73%): mp 107-110 °C; H NMR (90 MHz, CDCl3) δ 0.98 (s,
3H), 1.30 (t, J ) 7.5 Hz, 3H), 1.68 (s, 3H), 4.24 (q, J ) 7.5 Hz,
2H), 4.81 (s, 1H), 5.4 (s, 1H), 7.00 (s, 1H), 7.2 (br s, 1H, D2O
exchangeable), 7.35-7.4 (s, 10H); IR (KBr) 1800, 1750, 1665
cm-1
. Anal. Calcd for C24H25N3O6S2: C, 55.91; H, 4.88; N,
8.15. Found: C, 56.02; H, 5.09; N, 7.71.
P r ep a r a tion of Dip h en ylm eth yl N-Ca r beth oxy-S-a m i-
n osu lfen im in op en icilla n a te-1-su lfon e (4a2). A solution of
4a 1 (1 g, 1.9 mmol) in dichloromethane (25 mL) was added to
an aqueous solution of KMnO4 (300 mg, 1.9 mmol) and
tetrabutylammonium hydrogen sulfate (1 mg, 2.9 µmol) as
phase transfer catalyst and the mixture vigorously stirred at
room temperature for 1 h. The organic layer was separated,
and the precipitated manganese dioxide was filtered off. The
organic layer was dried and concentrated under reduced
pressure to leave 4a 2 as a colorless solid. Using dry flash
column chromatography 4a 2 eluted in the 30/70 light petro-
leum ether/ethyl acetate fraction. The solvent was removed
under reduced pressure to leave 4a 2 as a white foamy solid
(700 mg, 1.32 mmol, 69%): mp 135 °C (dec); 1H NMR (90 MHz,
CD3OD) δ 1.15 (s, 3H), 1.30 (t, J ) 7.5 Hz, 3H), 1.58 (s, 3H),
4.24 (q, J ) 7.5 Hz, 2H), 4.62 (s, 1H), 5.1 (s, 1H), 6.98 (s, 1H),
7.29-7.40 (s, 11H); IR (KBr) 1795, 1750 cm-1. Anal. Calcd
for C24H25N3O7S2: C, 54.24; H, 4.71; N, 7.91. Found: C, 54.37;
H, 4.84; N, 7.57.
P r ep a r a tion of Dip h en ylm eth yl N-Meth yl-N-tosyl-S-
a m in osu lfen im in op en icilla n a t e-1-su lfon e (4b 2m ). This
was prepared from 4b1m in a manner similar to that given
for 4a 2 above. Using dry flash column chromatography the
product eluted in the 50/50 light petroleum ether/ethyl acetate
fraction. Removal of the solvent under reduced pressure gave
4b2m as a foamy white solid (110 mg, 0.18 mmol, 63%): mp
105 °C (dec); 1H NMR (90 MHz, CDCl3) δ 1.21 (s, 3H), 1.60 (s,
3H), 2.45 (s, 3H), 3.28 (s, 1H), 4.68 (s, 1H), 5.72 (s, 1H), 7.02
(s, 1H), 7.25-7.87 (m, 14 H); IR (KBr) 1800, 1750, 1660 cm-1
.
P r ep a r a tion of Dip h en ylm eth yl N-Meth yl-N-(p-ch lor o-
ph en ylsu lfon yl)-S-am in osu lfen im in open icillan ate (4cm ).
1 (as the free amine) (1 g, 2.7 mmol) was dissolved in
dichloromethane (35 mL), and to this was added 2c (1.28 g, 3
mmol). The mixture was stirred overnight at room tempera-
ture. Material which had precipitated at that point was
removed by filtration. Distilled water (2 mL) was added to
the supernatant (to decompose unreacted sulfur diimide)
followed by the slow addition of light petroleum ether, and the
mixture was stirred for 1 h at room temperature by which time
a white precipitate had formed. This was removed by filtration
and dried under vacuum at ambient temperature to yield a
white solid (800 mg) which was found to be a mixture of
diphenylmethyl N-(p-chlorophenylsulfonyl)-S-aminosulfenimi-
nopenicillanate and p-chlorophenylsulfonamide in a ratio of
1:2 by 1H NMR. This material was methylated, without
further purification, in a manner similar to that used to
prepare 4bm . Using dry flash chromatography 4cm eluted
in the 80/20 and 90/10 dichloromethane/light petroleum ether
fractions. On removal of the solvent under reduced pressure
4cm (460 mg, 0.75 mmol, 26% starting from 1) was obtained
P r ep a r a t ion of Dip h en ylm et h yl N-Tosyl-S-a m in o-
su lfen im in op en icilla n a te (4b). 1 (as the free amine) (2 g,
5.2 mmol) was dissolved in dichloromethane (25 mL). 2b (2.2
g, 6 mmol) was added, and the mixture was stirred for 5 min,
followed by the slow addition of light petroleum ether (20 mL).
The precipitated sulfonamide was filtered off, and the filtrate
was washed with distilled water (3 × 200 mL). On standing
4b (900 mg) precipitated from the organic layer and was
filtered and dried. Further crude 4b was obtained on concen-
tration of the filtrate. Using dry flash chromatography 4b
eluted in the 55/45 and 50/50 light petroleum ether/ethyl
acetate fractions to yield an additional 600 mg (total yield was
1
1.5 g, 2.58 mmol, 49%): mp 177-179 °C; H NMR (90 MHz,
1
CDCl3) δ 1.29 (s, 3H), 1.55 (s, 3H), 2.4 (s, 3H), 4.7 (s, 1H), 6.05
(s, 1H), 6.95 (s,1H), 7.05 (s, 1H, D2O exchangeable), 7.25-7.87
(m, 14 H); IR (film) 3207, 1779, 1758, 1667 cm-1; UV log ꢀ )
4.09, λ289 (MeOH). Anal. Calcd for C28H27N3O5S3: C, 57.83;
H, 4.65; N, 7.23. Found: C, 57.59; H, 4.64; N, 7.20.
as a yellow solid: mp 126-129 C; H NMR (CDCl3, 90 MHz)
δ 1.30 (s, 3H), 1.57 (s, 3H), 3.20 (s, 3H), 4.70 (s, 1H), 6.12 (s,
1H), 6.94 (s, 1H), 7.20-7.92 (m, 14 H); IR (KBr) 1781, 1745,
1644 cm-1. Anal. Calcd for C28H26N3O5ClS3: C, 54.58; H, 4.25;
N, 6.82. Found: C, 54.90; H, 3.90; N, 6.46.
P r ep a r a t ion of Dip h en ylm et h yl N-Met h yl-n -t osyl-S-
am in osu lfen im in open icillan ate (4bm ). 4b (1 g, 1.72 mmol)
was dissolved in chloroform (10 mL). Proton Sponge (400 mg,
1.87 mmol) was added and the mixture stirred until the
solution was clear. Methyl iodide (0.25 mL, 4.0 mmol) was
added and the mixture stirred briefly. The solution was
allowed to stand at ambient temperature for 12 h. The Proton
Sponge-HI salt which had crystallized out of solution was
filtered off. The filtrate was concentrated, and the product
was purified by dry flash chromatography. 4bm eluted in the
R em ova l of t h e P r ot ect in g Gr ou p a n d Isola t ion of
Sa lts.54 Aluminum trichloride (133.5 mg, 1 mmol) in ni-
tromethane (3 mL) was added to dichloromethane (15 mL)
containing the required diphenylmethyl S-aminosulfenimi-
nopenicillin ester (0.5 mmol) at -80 °C and the mixture stirred
for 15 min. The was followed by the addition of sodium
bicarbonate (5%, 50 mL) and ethyl acetate or dichloromethane
(30 mL), and the mixture was vigorously stirred while allowing
the temperature to rise to ambient. The mixture was further
diluted by the addition of distilled water (100 mL). The

S-Aminosulfeniminopenicillins
J . Org. Chem., Vol. 63, No. 22, 1998 7617
organic layer was separated and discarded. The aqueous layer
was repeatedly filtered (4-6 times) until clear, acidified to pH
2.2 with 1 M HCl, and extracted with ethyl acetate (20 mL).
The organic layer was separated and concentrated to yield the
free acid of the S-aminosulfeniminopenicillin. The sodium
salts were obtained by extracting an ethyl acetate solution (20
mL) of an accurately weighed amount the free acid (typically
0.25 mmol) with aqueous sodium bicarbonate (0.20 mmol), i.e.,
with a deficiency of bicarbonate. The aqueous layer was
separated and freeze-dried to yield the sodium salts as yellow
solids.
N-Meth yl-N-tosyl-S-a m in osu lfen im in oa la n in e Dip h en -
ylm eth yl Ester (7). 6 (338 mg, 0.75 mmol) was added to
chloroform (10 mL). To this was added methyl iodide (154 mg,
1.09 mmol) and Proton Sponge (0.179 g, 0.75 mmol), and the
solution was stirred at room temperature for 30 min. On
standing overnight Proton Sponge-HI salt had precipitated
from solution and was filtered off and the solution concentrated
under reduced vacuum to leave crude 7 as a brown liquid.
Using (wet) flash chromatography the product was eluted with
light petroleum ether/ethyl acetate 20/80. A white solid
crystallized on standing, and this was filtered and dried to
1
Sod iu m N-ca r beth oxy-S-a m in osu lfen im in op en icilla n -
a te (5a ) (84 mg, 0.236 mmol, 32%): 1H NMR (90 MHz, D2O)
δ 1.32 (t, J ) 7.5 Hz, 3H), 1.53 (s, 3H), 1.57 (s, 3H), 4.27 (q, J
) 7.5 Hz, 2H), 4.4 (s, 1H), 5.95 (s, 1H); IR (KBr) 1770, 1615
yield pure 7 (216 mg, 0.46 mmol, 62%): mp 125-127 °C; H
NMR (90 MHz, CDCl3) δ 2.10 (s, 3H), 2.38 (s, 3H), 3.29 (s,
3H), 6.92 (s, 1H), 7.22 (d, J ) 6 Hz, 2H), 7.37 (s, 10 H), 7.77
(d, J ) 6 Hz, 2H). Anal. Calcd for C24H24N2O4S2: C. 61.52;
H, 5.16; N, 5.98. Found: C, 61.79; H,5.14; N, 6.04.
cm-1
. Anal. Calcd for C11H14N3O5S2Na‚2H2O: C, 33.75; H,
4.60; N, 10.73. Found: C, 33.65; H, 4.36; N, 10.37.
P r ep a r a tion of 8a . To a solution of 4a (50 mg, 0.1 mmol)
in methanol (5 mL) was added Et3N (15 µL). After the solution
was briefly stirred, the flask was sealed and allowed to stand
at room temperature for 90 min. The reaction mixture was
diluted with dichloromethane (20 mL) and washed with
distilled water (2 × 50 mL). The organic layer was separated,
dried, and concentrated under reduced pressure to yield 8a
as a gel-like solid (42 mg, 0.079 mmol, 79%): 1H NMR (90
MHz, CDCl3) δ 1.09 (s, 3H), 1.25 (t, J ) 7.5 Hz, 3H), 1.55 (s,
3H), 3.83 (s, 3H), 3.88 (s, 1H), 4.20 (q, J ) 7.5 Hz, 2H), 5.71
(s, 1H), 6.65 (br s, 1H, D2O exchangeable), 6.98 (s, 1H), 7.35
(s, 10H) (see Figure 3b).
P r ep a r a tion of 8b. To a solution of 4b (50 mg, 0.09 mmol)
in methanol (5 mL) was added 0.8 M sodium methoxide in
methanol (0.5 mL). (Stronger base was required here com-
pared to 8a above as ionization of the side chain of 4b reduced
the susceptibility of the â-lactam ring to cleavage by nucleo-
philes.) The solution was briefly stirred and allowed to stand
at room temperature for approximately 1.5 min, or until the
reaction mixture turned an orange color. The reaction mixture
was diluted with dichloromethane (20 mL) and washed with
distilled water (3 × 50 mL). The organic layer was separated,
dried, and concentrated under reduced pressure to yield 9b
as a white solid (35 mg): 1H NMR (90 MHz, CDCl3), see Figure
4b.
F or m a tion of 10 fr om 8b. A solution of 8b in CDCl3 in
an NMR tube was monitored after 6 h. A new set of peaks
was observed to appear: δ 1.31 (s, 3H), 1.57 (s, 3H), 3.87 (s,
1H), 3.88 (s, 3H), 5.72 (s, 1H), 7.00 (s, 1H), 7.25-7.38 (m, 11H),
see Figure 4c. A fine precipitate which had formed was filtered
off and dried: mp 137-139 °C (lit.55 mp 138-139 °C p-
toluenesulfonamide); 1H NMR (90 MHz, DMSO/CDCl3) δ 2.41
(s, 3H), 6.95 (br s, 2H, D2O exchangeable), 7.32 (d, J ) 14 Hz),
7.82 (d, J ) 14 Hz, 2H). 9 was purified chromatographically
as indicated below.
F or m a tion a n d isola tion of 9 fr om 4bm . To a solution
of 4bm (250 mg, 0.47 mmol) in methanol (15 mL) was added
Et3N (25 µL). The solution was briefly stirred, sealed, and
maintained at 25 °C for 4 h. The reaction mixture was diluted
with dichloromethane (50 mL) and washed with distilled water
(2 × 150 mL). The organic layer was separated, dried, and
concentrated under reduced pressure. Using dry flash column
chromatography the product eluted in the 50/50 light petro-
leum ether/ethyl acetate fraction. Removal of the solvent
under reduced pressure yielded 9 as a yellow gel-like solid (190
mg, 0.43 mmol, 91%). This sample was further purified by
another stage of (wet) flash chromatography using dichlo-
romethane as eluant. Removal of the solvent under reduced
pressure yielded a yellow solid which was further dried under
vacuum at ambient temperature (40 mg, 0.09 mmol): 1H NMR
(CDCl3, 90 MHz), see Figure 5 and Table 1; 13C NMR (100
MHz, CDCl3), see Figure 6 and Table 2. Anal. Calcd for
Sod iu m N-ca r beth oxy-S-a m in osu lfen im in op en icilla n -
a te-1-su lfoxid e (5a 1) (110 mg, 0.296 mmol, 26%): 1H NMR
(90 MHz, D2O) δ 1.30 (t, J ) 7.5 Hz, 3H), 1.33 (s, 3H), 1.68 (s,
3H), 4.28 (q, J ) 7.5 Hz 2H), 4.50 (s, 1H), 6.00 (s, 1H); IR (KBr)
1780, 1660, 1620 cm-1
.
Sod iu m N-ca r beth oxy-S-a m in osu lfen im in op en icilla n -
a te-1-su lfon e (5a 2) (70 mg, 0.18 mmol, 24%): 1H NMR (90
MHz, D2O) δ 1.30 (t, J ) 7.5 Hz, 3H), 1.43 (s, 3H), 1.55 (s,
3H), 4.2 (q, J ) 7.5 Hz, 2H), 4.25 (s, 1H), 5.36 (s, 1H); IR (KBr)
1785, 1620 cm-1
. Anal. Calcd for C11H14N3O7S2Na‚2.5H2O:
C, 30.55; H, 4.42; N, 9.71. Found: C, 30.19; H, 4.10; N, 9.88.
Sod iu m N-tosyl-S-a m in osu lfen im in op en icilla n a te (5b)
(100 mg, 0.23 mmol, 16%): 1H NMR (90 MHz, D2O) δ 1.5 (s,
6H), 2.37, (s, 3H), 4.34 (s, 1H), 6.2 (s, 1H), 7.3-7.8 (m, 4H); IR
(KBr) 1775, 1615 cm-1
.
Sod iu m N-tosyl-S-a m in osu lfen im in op en icilla n a te-1-
su lfoxid e (5b1) (205 mg, 0.45 mmol, 33%): 1H NMR (90 MHz,
D2O) δ 1.32 (s, 3H), 1.65 (s, 3H), 2.40 (s, 3H), 4.48 (s, 1H),
6.17 (s, 1H), 7.3-7.8 (m, 4H); IR (KBr) 1785, 1625 cm-1. Anal.
Calcd for C15H16N3O6S3Na‚3.5H2O: C, 34.84; H, 4.45; N, 8.13.
Found: C, 34.41; H, 3.99; N, 7.90.
Sod iu m N-m eth yl-N-tosyl-S-a m in osu lfen im in op en icil-
la n a te (5bm ) (84 mg, 0.186 mmol, 16%): 1H NMR (90 MHz,
D2O) δ 1.55 (s, 6H), 2.43 (s, 3H), 3.33 (s, 3H), 4.45, (s, 1H),
5.97 (s, 1H), 7.40-7.85 (m, 4H); IR (KBr) 1770, 1620 cm-1
;
corresponding free acid 1H NMR (90 MHz, CDCl3) δ 1.52 (s,
6H), 2.45 (s, 3H), 3.27 (s, 3H), 4.50, (s, 1H), 6.10 (s, 1H), 7.30-
7.85 (m, 4H). Anal. Calcd for C16H19N3O5S3: C, 44.74; H, 4.45;
N, 9.78. Found: C, 44.96; H, 4.42; N, 9.87.
Sod iu m N-m eth yl-N-tosyl-S-a m in osu lfen im in op en icil-
la n a te-1-su lfon e (5b2m ) (25 mg, 0.052 mmol, 28%): 1H NMR
(90 MHz, D2O) δ 1.45 (s, 6H), 2.37 (s, 3H), 3.30 (s, 3H), 4.44
(s, 1H) 5.85 (s, 1H), 7.3-7.8 (m, 4H); IR (KBr) 1785, 1625 cm-1
.
N-Meth yl-N-(p-ch lor op h en ylsu lfon yl)-S-a m in osu lfen -
im in op en icilla n ic a cid (5cm -fr ee a cid ) (215 mg, 0.455
mmol, 65%): 1H NMR (90 MHz, CDCl3) δ 1.61 (s, 6H), 3.31 (s,
3H), 3.70 (br s, exchangeable with D2O), 4.65, (s, 1H), 6.03 (s,
1H), 7.55 (d, J ) 6 Hz, 2H), 7.86 (d, J ) 6 Hz, 2H).
N-Tosyl-S-a m in osu lfen im in oa la n in e Dip h en ylm eth yl
Ester (6). Alanine diphenylmethyl ester (1.79 g, 7.04 mmol)
was added to 2b (3.12 g, 8.45 mol) in dichloromethane (60 mL)
and stirred at room temperature overnight. Distilled water
(2 mL) was added (to decompose unreacted sulfur diimide)
followed by the addition of light petroleum ether (60 mL) and
the solution stirred for 20 m to complete the precipitation of
p-toluenesulfonamide which was removed by filtration. The
filtrate was washed with water (3 × 50 mL), separated, and
dried (MgSO4) and the solvent removed under reduced pres-
sure to yield crude 6 as a pale yellow solid (1.56 g, 3.43 mmol,
49%). This was purified by (wet) flash chromatography using
80/20 light petroleum ether/ethyl acetate to yield pure 6 as a
pale yellow solid: mp 121-123 °C; 1H NMR (90 MHz, CDCl3)
δ 2.10 (s, 3H), 2.38 (s, 3H), 6.90 (s, 1H), 7.21 (d, J ) 6 Hz,
2H), 7.32 (s, 10H), 7.79 (d, J ) 6 Hz, 2H); 13C NMR (90 MHz,
CDCl3) δ 21.36 (CH3CdN and p-CH3), 79.11, 126.94-129.86,
138.96, 143.78, 160.0; UV log ꢀ ) 3.94 λ283 (MeOH); FAB-MS
m/z 587 (4.6%) (M + Cs+). Anal. Calcd for C23H22N2O4S2: C.
60.79; H, 4.84; N, 6.16. Found: C, 60.65; H, 4.76; N, 6.03.
C
22H22N2S2O4: C, 59.65; H, 4.97; N, 6.32. Found: C, 58.06;
H, 5.11; N, 5.90. For the kinetic profiles shown in Figures 8
(54) Buynak, J . D.; Khasnis, D.; Bachmann, B.; Wu, K.; Lamb, G.
J . Am. Chem. Soc. 1994, 166, 10955.
(55) J ames, A. M.; Lord, M. P. Macmillan’s Chemical and Physical
Data; The Macmillan Press: London, 1992.

7618 J . Org. Chem., Vol. 63, No. 22, 1998
Smyth et al.
and 9 the above procedure, omitting the chromatographic
purification step, was repeated for each reaction time shown
using 25-30 mg samples of 4bm and 4cm in MeOH (3 mL)
and Et3N (15 µL).
15 to 60 µM. The decrease in absorbance at 290 nm was
monitored as a function of time; the initial rate was determined
from the linear absorbance change within the first 10 s (0.3
absorbance units/m with 60 µM 5bm ); ∆e290 was 11 400 M-1
cm-1. Under these conditions the initial rate varied with the
substrate concentration; however, as the variation in rate was
found to be directly proportional to the substrate concentration
over this range, it was not possible to determine accurately a
value for Km.
Ba cter iologica l Scr een in g. Test organisms included nine
strains of E. coli (C600N) involving producers of seven different
â-lactamases58 (TEM-1, OSBL; TEM-10; SHV-1; SHV-4; MIR-
1; AmpC (Ecl-c); AmpC (Ecl-i)).
En zym e In h ibition Stu d ies. Phosphate buffer (0.1 M, pH
7.2) was used for all solutions. EDTA (50 µM) was present to
suppress activity of type II â-lactamase (a zinc dependent
metalloenzyme)56 present at low levels in the Sigma type I
â-lactamase preparations. The specific activity for the hy-
drolysis of benzylpenicillin at pH 7.2, 25 °C was 2.71 mmol
min-1 per mg of protein. The temperature was held constant
at 25 ( 0.1 °C for the kinetic runs. A stock solution was made
up by dissolving a portion of enzyme (0.25 mg of protein based
on log ꢀ ) 4.41 at 280 nm for B. cereus Type I)57 in 2.5 mL of
buffer. From this 250 µL was withdrawn and made up to 5
mL with buffer to provide the working enzyme solution. The
incubation mixture was made up by adding the inhibitor to
the working enzyme solution to give the desired inhibitor
concentration (20-2000 µM). After a standard 10 min incuba-
tion time, 50 µL aliquots were withdrawn from this solution
and injected into benzylpenicillin solution (0.5 mM) in a
cuvette, and the rate of hydrolysis of the benzylpenicillin
measured by following the zero order decrease in absorbance
at 232 nm with time. Note that 5cm was isolated as the free
acid and for all enzyme experiments 50 µL of a THF solution
of 5cm was added to the aqueous buffer containing the
enzyme; this volume of THF had no effect on its own on
enzyme activity.
Ack n ow led gm en t. We are grateful to Dr. Angela
Savage (NUI, Galway) for the 100 MHz 13C spectrum,
to Dr. J ohn Briody (NUI, Maynooth) for the FAB-MS of
6, and to Professor Rory More O’Ferrall (UCD) for some
helpful discussions.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra are
available for the esters 4b1, 4b2m , 4cm -fr ee a cid , and the
salts 5a 1, 5b, and 5b2m for which satisfactory elemental
analysis was not obtained. The following are also included:
1H NMR spectrum of 4b(sid e-ch a in
and variable temper-
a n ion )
ature spectra of 4bm , 7, and the product set (3S,5S)-9 and
(3S,5R)-9, 13C NMR of 4a (decoupled and off resonance) and 6
(decoupled) (10 pages). This material is contained in libraries
on microfiche, immediately follows this article in the microfilm
version of the journal, and can be ordered from the ACS; see
any current masthead page for ordering information.
For the measurement of kcat/Km the enzyme concentration
was 0.7 µM, and the concentration of 5bm was varied from
J O970737F
(56) Buckwell, S. C.; Page, M. I. J . Chem. Soc., Perkin Trans. 2 1988,
1809.
(57) Kiener, P. A.; Knott-Hunziker, V.; Petursson, S.; Waley, S. G.
Eur. J . Biochem. 1980, 109, 575.
(58) Bush, K.; J acoby, G. A.; Medeiros, A. A. Antimicrob. Agents
Chemother. 1995, 39, 1211.