Kinetics and Mechanism of Hydrolysis of N-Acyloxymethyl Derivatives of Azetidin-2-one Emı´lia Valente,† Jose´ R. B. Gomes,‡ Rui Moreira,† and Jim Iley*,§ CECF, Faculdade de Farma´ cia, Universidade de Lisboa, Av. Forc¸ as Armadas, 1600-083 Lisboa, Portugal, CIQ, Departamento de Quı´mica, Faculdade de Cieˆ ncias, Universidade do Porto, R. Campo Alegre, 687, 4169-007 Porto, Portugal, and Department of Chemistry, The Open University, Milton Keynes MK7 6AA, U.K.
[email protected] Received December 12, 2003
The pH-independent, acid-catalyzed and base-catalyzed hydrolyses of N-acyloxymethylazetidin-2ones all occur at the ester function. The pH-independent hydrolysis involves rate-limiting alkyl C-O fission and formation of an exocyclic β-lactam iminum ion. This iminium ion is then trapped by water at the exocyclic iminium carbon atom, rather than at the β-lactam carbonyl carbon atom, to form the corresponding N-hydroxymethylazetidin-2-ones. Calculations carried out at the B3LYP/ 6-31+G(d) level of theory also support that nucleophilic attack by water takes place at the exocyclic carbon rather than at the β-lactam carbonyl carbon of the iminium ion. The mechanism for the acid-catalyzed pathway involves a preequilibrium protonation, probably at the β-lactam nitrogen, followed by rate-limiting alkyl C-O fission with formation of an exocyclic iminum ion. The basecatalyzed hydrolysis involves rate-limiting hydroxide attack at the ester carbonyl carbon. These results imply formation of a β-lactam system containing a positively charged amide nitrogen atom that hydrolyzes via a pathway that preserves the β-lactam structure in the product and provide further evidence that cleavage of the β-lactam C-N bond is not as facile as is commonly imagined. Introduction
SCHEME 1
β-Lactams are effective inhibitors of several bacterial enzymes such as D,D-transpeptidases1 and β-lactamases2 that contain serine as the catalytic residue. Though these are the most widely studied therapeutic targets for β-lactams, other serine proteases, such as mammalian3 or viral proteases,4 have received increased attention, giving rise to further optimization of the β-lactam lead structure to obtain potent and selective inhibitors.5 Recently, we became interested in N-acyloxymethyl (1, LG ) OCOR) and N-aminocarbonyloxymethylazetidin2-ones (1, LG ) OCONHR) (see Scheme 1) as potential mechanism-based or suicide inhibitors of human leukocyte elastase (HLE, EC 3.4.21.37).6 HLE is a serine protease that very efficiently degrades various tissue †
Faculdade de Farma´cia, Universidade de Lisboa. Faculdade de Cieˆncias, Universidade do Porto, Portugal. The Open University. (1) Newall, C. E.; Hallam, P. D. In Comprehensive Medicinal Chemistry; Hansch, C., Ed.; Pergamon Press: Oxford, 1990; pp 609653. (2) Brown, A. G.; Pearson, M. J.; Southgate, R. In Comprehensive Medicinal Chemistry; Hansch, C., Ed.; Pergamon Press: Oxford, 1990; pp 655-702 (3) (a) Babine, R. E.; Bender, S. L. Chem. Rev. 1997, 97, 1359. (b) Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem. 2000, 43, 305. (4) LaPlante, S. R.; Bonneau, P. R.; Aubry, N.; Cameron, D. R.; De´ziel, R.; Grand-Maıˆtre, C.; Plouffe, C.; Tong, L.; Kaway, S. H. J. Am. Chem. Soc. 1999, 121, 2974. (5) Firestone, R. A.; Barker, P. L.; Pisano, J. M.; Ashe, B. M.; Dahlgren, M. E. Tetrahedron 1990, 46, 2255. (6) Clemente, A.; Domingos, A.; Grancho, A. P.; Iley, J.; Moreira, R.; Neres, J.; Palma, N.; Santana, A. B.; Valente, E. Bioorg. Med. Chem. Lett. 2001, 11, 1065. ‡ §
matrix proteins such as elastin.7 The imbalance between HLE and its endogenous inhibitors and the subsequent excessive elastin proteolysis has been implicated in acute and chronic inflammatory diseases of the lungs.8,9 Thus, the use of specific inhibitors of HLE to restore the protease/antiprotease imbalance represents an important strategy to treat such pathologies.10,11 (7) Janoff, A.; Scherer, J. J. Exp. Med. 1968, 128, 1137. (8) Snider, G. L.; Ciccolella, D. F.; Morris, S. M. Ann. NY Acad. Sci. 1991, 624, 45. (9) Stockley, R. A.; Hill, S. L.; Burnett D. Ann. NY Acad. Sci. 1991, 624, 257.
10.1021/jo0358123 CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/20/2004
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Compounds 1, in addition to the ethyl groups at C-3 required for molecular recognition by the enzyme,12 incorporate the following structural features. First, a good leaving group LG (e.g., a carboxylic or carbamic acid) that unmasks an electrophilic Schiff base, 2, following the initial serine attack at the β-lactam carbonyl atom (Scheme 1). This reactive functionality has the potential of reacting with a second amino acid residue within the active site, leading to a doubly modified inactivated enzyme. Second, an electron-withdrawing substituent, X, at C-4 was required for increasing the acylating power of the β-lactam. Strong evidence has been put forward suggesting that the driving force for β-lactam reactivity toward nucleophiles lies on the leaving group ability of the amine formed from the decomposition of the tetrahedral intermediate rather than on the strain energy in the four-membered ring or on the reduced amide resonance.13,14 The C-4 substituent and the acyloxymethyl moiety reduce the pKa of the amine leaving group to ca. 2,15 a value in the region of those reported for other β-lactam inhibitors (e.g., penicillins 3 and cephalosporins 416). An approach similar to that depicted in Scheme 1 has been used to design inhibitors 5 of β-lactamases using other leaving groups such as sulfinic acids, thiols, and fluoride.17
nitrogen finds parallel in the chemistry of N-acyloxymethylamides, which undergo an SN1-type hydrolysis in neutral buffers.18,19 To obtain further insight to the mechanism of hydrolysis of N-acyloxymethylazetidin-2ones and its implication in the design of more potent inhibitors of this class, herein we report a kinetic study on the reactivity of simple N-acyloxymethylazetidin-2ones 6.
Both N-acyloxymethyl- and N-aminocarbonyloxymethylazetidin-2-ones, 1, are time-dependent irreversible inhibitors of HLE but, remarkably, are very stable at 37 °C and pH 7.4, with pseudo-first-order rate constants for hydrolysis ranging from 10-8 to 10-7 s-1.6 In contrast, the C-4-unsubstituted analogue is hydrolyzed at least 103 times more rapidly than any C-4-substituted N-acyloxymethylazetidin-2-one. Such a dramatic effect on chemical reactivity exerted by substituents close to the lactam
Results and Discussion
(10) Hastla, D. J.; Pagani, E. D. Ann. Rep. Med. Chem. 1994, 29, 195. (11) Bernstein, P. R.; Edwards, P. D.; Williams, J. C. Prog. Med. Chem. 1994, 31, 59. (12) Shah, S. K.; Dorn, C. P.; Finke, P. E.; Hale, J. J.; Hagmann, W. K.; Brause, K. A.; Chandler, G. O.; Kissinger, A. L.; Ashe, B. M.; Weston, H.; Knight, W. B.; Maycock, A. L.; Dellea, P. S.; Fletcher, D. S.; Hand, K. M.; Mumford, R. A.; Underwood, D. J.; Doherty, J. B. J. Med. Chem. 1992, 35, 3745. (13) Page, M. I. Adv. Phys. Org. Chem. 1987, 23, 165. (14) Page, M. I.; Laws, A. P. Tetrahedron 2000, 56, 5631. (15) Perrin, D. D.; Dempsey, B.; Serjeant, E. P. pKa Prediction for Organic Acids and Bases; Chapman and Hall: London, 1981. A pKa value of 2.03 for the conjugated acid of HO2C(CH2)CH(SO2Ph)NHCH2OCOMe was calculated from the equation pKa ) 10.59-3.23Σσ*, derived for the dissociation of R′R′′NH2+ and using the assumptions set out on pp 38-40 of the above reference. (16) Proctor, P.; Gensmantel, N. P.; Page, M. I. J. Chem. Soc., Perkin Trans. 2 1982, 1185. (17) Beauve, C.; Bouchet, M.; Touillaux, R.; Fastrez, J.; MarchandBrynaer, J. Tetrahedron 1999, 55, 13301.
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Kinetics. Typical pH-rate profiles for the hydrolysis of N-acyloxymethylazetidin-2-ones 6a, 6h, and 6i are presented in Figure 1. For comparison purposes, the pHrate profile for an acyclic counterpart, N-benzoyloxymethyl-N-methylacetamide, 7, is also included. Both
N-acyloxymethylazetidin-2-ones6andtheN-acyloxymethylamide 7 undergo acid- and base-catalyzed hydrolysis, as well as pH-independent hydrolysis. These three processes conform to eq 1. The corresponding pseudo-first-order rate constants for the pH-independent hydrolytic pathway, k0, and the second-order rate constants for the acid, kH+, and alkaline, kOH-, hydrolyses are presented in Table 1.
kobs ) k0 + kH+ [H+] + kOH- [OH-]
(1)
pH-Independent Reaction. The most striking feature of the pH-rate profiles of N-acyloxymethylazetidin-
Hydrolysis of N-Acyloxymethyl Derivatives of Azetidin-2-one TABLE 2. Dependence of the Pseudo-First-Order Rate Constants, ko, on the Buffer Concentration for the Hydrolysis of 6a at 25 °C and µ ) 0.5 mol dm-1 compd
buffer
6a
CH2ClCO2-/CH2ClCO2H
AcO-/AcOH
HPO42-/H2PO4-
6h
FIGURE 1. pH-rate profiles for compounds 6a (b), 6h (9), 6i (O), and 7 (2) at 25 °C in aqueous buffers containing 20% (v/v) acetonitrile.
TABLE 1. Pseudo-First-Order Rate Constants, ko, for the pH-Independent Hydrolysis and Second-Order Rate Constants, kH+ and kOH-, for the Acid- and Base-Catalyzed Hydrolyses of N-Acyloxymethylazetidin-2-ones 6 and Compound 7 at 25 °C and in Aqueous Media Containing 20% (v/v) Acetonitrile compd
105ko (s-1)
6a
2.35; 2.25;a 5.11;b 7.88;c 15.1;d 28.1;e 49.1f 0.954 8.94 18.0 1.46 1.71 4.83 0.121; 0.0906;a 0276;b 0.626;g 1.30;e 3.09f 0.0226h 0.0120h 0.0197h 77.3
6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 7 h
103kH+ (L mol-1 s-1)
102kOH(L mol-1 s-1)
1.93; 2.86;a 3.77;b 10.3; 12.0;a 14.2;b 6.50;c 10.5;d 21.5e 18.7;c 22.9d 1.95 1.48 1.57 8.67 10.7 15.7 0.0888; 0.111;a 0.147;b 0.244;c 0.488;d 0.763e 0.0204 0.000598 0.000419 35.2
1.84 31.2 193 3.32 61.2 7.83 971; 1196a
AcO-/AcOH
[buffer]/10-2 M pH 1.0 2.0 5.0 10.0 1.0 2.0 5.0 10.0 1.0 2.0 5.0 10.0 1.0 2.0 5.0 10.0
2.59 2.39 2.17 2.06 5.06 5.01 4.98 4.98 6.85 6.87 6.87 6.90 4.83 4.84 4.80 4.80
kobs/10-5 s-1 2.87 2.88 3.17 3.36 2.22 2.38 1.85 2.10 2.53 2.35 2.43 2.35 0.134 0.121 0.122 0.135
Cephalosporins also exhibit a significant pH-independent hydrolysis, usually ranging from pH 2 to pH 7.21,22 However, the spontaneous hydrolysis of cephalosporins has been ascribed to the intramolecular general-basecatalyzed attack of water at the β-lactam carbonyl carbon atom by the amide side chain,21 a neighboring group effect that cannot be present in the N-acyloxymethylazetidin-2-ones 6. The pH-rate profiles of 6a-g also differ significantly from those for simple ester hydrolysis, which occurs via an addition-elimination mechanism and usually exhibits the incursion of specific base catalysis at pH 6-7.23 In contrast, N-acyloxymethylazetidin-2-ones 6i-k, which contain strongly electron-withdrawing substituents in C-4, are at least 102 less reactive than their unsubstituted counterparts (Table 1). Four potential mechanisms could be considered for the pH-independent hydrolysis of N-acyloxymethylazetidin-2-ones 6: nucleophilic attack of water to the ester carbonyl atom (A, path a), SN1 ionization with departure of the carboxylate anion (A, path b), SN2 nucleophilic attack of water to the acyloxymethyl carbon atom (A, path c), and β-lactam ring opening via nucleophilic attack of water to the lactam carbonyl atom (A, path d).
5.84 23.2 8.62 7.75
a In D O. b 30 °C. c 35 °C. d 40 °C. e 45 °C. f 50 °C. g 36.9 °C. 2 Reaction followed up to ca. 30%.
2-ones 6a-g (exemplified in Figure 1 by that for 6a) is the unusually broad plateau extending from pH ca. 2 to pH ca. 10 corresponding to the pH-independent pathway, which is characterized by the absence of both generalbase and nucleophilic catalysis (Table 2). This contrasts markedly with the hydrolysis of penicillin β-lactam antibiotics, which have no significant spontaneous reaction (e.g., benzylpenicillin)13 or have only small plateaus around neutral pH (e.g., cyclacillin or ampicillin).20,21 (18) Iley, J.; Moreira, R.; Rosa, E. J. Chem. Soc., Perkin Trans. 2 1991, 563. (19) Iley, J.; Moreira, R.; Calheiros, T.; Mendes, E. Pharm. Res. 1997, 14, 1634.
The analysis of reaction mixtures of 6a-k by HPLC and by HPLC-MS, together with spiking experiments with authentic samples, shows that at pH 3-5 the corresponding N-hydroxymethylazetidin-2-one, 8, and (20) Yamana, T.; Tsuji, A.; Mizukami, Y. Chem. Pharm. Bull. 1974, 22, 1186. (21) Llina´s, A.; Vilanova, B.; Frau, J.; Mun˜oz, F.; Donoso, J.; Page, M. I. J. Org. Chem. 1998, 63, 9052. (22) Yamana, T.; Tsuji, A. J. Pharm. Sci. 1976, 65, 1563. (23) Euranto, E. K. In The Chemistry of Carboxylic Acids and Esters; Patai, S., Ed.; Wiley: Chichester, 1969; p 505.
J. Org. Chem, Vol. 69, No. 10, 2004 3361
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FIGURE 2. Brønsted plot for the pH-independent hydrolysis of compounds 6a-g at 25 °C.
SCHEME 2
azetidin-2-one, 9 (Scheme 2), are the products of these reactions and that the two products together account for all the substrate lost. This implies that pH-independent hydrolysis of N-acyloxymethylazetidin-2-ones does not involve β-lactam ring opening (A, path d). Further evidence that enables us to discount this pathway is the observation that compound 6k, which is 3,3-diethyl substituted, is slightly more reactive than its unsubstituted counterpart 6j. If β-lactam hydrolysis were occurring then such substitution should hinder the reaction, as suggested by the 2-fold reduction in the HO--induced hydrolysis of 1,3-dimethylazetidin-2-one when compared with 1-methylazetidin-2-one.24 A plot of the apparent first-order rate constants of N-acyloxymethylazetidin-2-ones 6a-g versus the pKa of the carboxylic acid leaving group generates a Brønsted βlg value of -1.20 (r2 ) 0.96) (Figure 2). Significantly, the 2-methoxybenzoate 6e is included in this plot, indicating that the 2-MeO substituent does not exert any retarding effect on reactivity and that the source of reactivity of N-acyloxymethylazetidin-2-ones 6a-g is not conventional ester hydrolysis (A, path a). Similar Brønsted plots, also including sterically hindered carboxylate groups, have been reported for the pH-independent hydrolysis of N-acyloxymethylamides19 and N-acyloxymethylsulfonamides,25 which undergo SN1 ionization with departure of the carboxylate anion. Compound 6h, which contains a CO2Et substituent in the linker between the β-lactam and ester moieties, is hydrolyzed 25 times slower than its unsubstituted counterpart 6a. This rate retardation is consistent with an apparent Taft F* value for R-substitution of ca. -1. This is inconsistent with path c, as electron-withdrawing groups would enhance the electrophilic nature of the (24) Washkuhn, R. J.; Robinson, J. R. J. Pharm. Sci. 1971, 60, 1168. (25) Lopes, F.; Moreira, R.; Iley, J. J. Chem. Soc., Perkin Trans. 2 1999, 431.
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carbon atom that is attached, but it conforms with path b, i.e. the unimolecular ionization presented in Scheme 3, in which the developing positive charge on nitrogen is destabilized by electron-withdrawing substituents. In contrast, conventional ester hydrolysis would be enhanced by electron-withdrawing substituents in the alcohol leaving group, which further implies that path a is not followed by the compounds under study.23,26 The proposal of an SN1-type mechanism in the pH-independent region receives additional support from the substituent effect at C-4 of the azetidin-2-one moiety. The electron-withdrawing substituents SPh, 6i, and SO2Ph, 6j, lead to a dramatic decrease in reactivity, with less than 30% of both compounds being hydrolyzed after 20 days in pH 5.0 acetate buffer (Table 1). This decrease in reactivity is inconsistent with A, path c, in which electron-withdrawing groups would be expected to increase the rate of reaction. The order of reactivity is 6a . 6i > 6j, which suggests the development of a positive charge in the transition state is consistent with path b above. According to the mechanism presented in Scheme 3 for the pH-independent process, the hydrolysis of compounds 6 involves the formation of an iminium ion, 10, and a carboxylate anion; the corresponding rate law, as derived using the steady-state assumption for intermediate 10, is given by eqs 2 and 3.
kobs )
k1kH2O kH2O + k-1[RCO2-]
k-1 1 1 ) [RCO2-] + kobs k1kH2O k1
(2)
(3)
Accordingly, the rate of hydrolysis of compound 6a was found to decrease in the presence of increasing concentrations of added benzoate anion (Figure 3). This is consistent with generation of a free, solvent-equilibrated iminium ion with a lifetime that is long enough to allow trapping by added benzoate. The plot of 1/kobs versus [PhCO2-] for these data (insert in Figure 3) gives a slope, k-1/k1kH2O, of 6.37 × 107 s L mol-1 and an intercept, 1/k1, 3.25 × 104 s, from which a value for k-1/kH2O of 1960 L mol-1 is obtained. That is, the benzoate anion is ca. 2000 times more effective at trapping the iminium ion than water. Assuming that the value of k-1 is diffusion controlled, i.e., k-1 ≈ 5 × 109 L mol-1 s-1,27 a lifetime for the iminium ion, 1/kH2O, of 3.9 × 10-7 s can be estimated from the k-1/kH2O ratio. The assumption that the rate constant for the reaction of the iminium ion with the (26) Nielsen, N. M.; Bundgaard, H. Int. J. Pharm. 1987, 39, 75. (27) Eldin, S.; Jencks, W. P. J. Am. Chem. Soc. 1995, 117, 4851.
Hydrolysis of N-Acyloxymethyl Derivatives of Azetidin-2-one
FIGURE 3. Inhibition of the pH-independent hydrolysis of
6a at 25 °C as a function of [PhCO2-]. The solid line shows the least-squares fit of the data to eq 4. The inset is a plot of 1/kobs vs [PhCO2-].
benzoate ion is diffusion controlled is supported by the dependence of log kobs values on the pKa of the benzoate leaving group. The βlg value of -1.20 for the hydrolysis of 6a-g indicates that the reaction has a very late transition state in which the C-O bond is almost completely broken. By the principle of microscopic reversibility,28 the reverse reaction, i.e., the combination of the benzoate anion with the iminium ion, must proceed via a transition state with almost no bond formation. Additional evidence for the SN1-type mechanism depicted in Scheme 3 comes from the following observations. First, the solvolysis of compounds 6a and 6h proceeds slightly faster in H2O than in D2O, giving a kinetic solvent isotope effect (KSIE), k0H2O/k0D2O, of 1.2. This value is consistent with an unimolecular ionization process being the rate-limiting step29,30 and is similar to the KSIE reported for the pH-independent hydrolyses of N-acyloxymethyl derivatives of amides18,19 and sulfonamides,25 which also hydrolyze via a SN1 mechanism. The k0H2O/k0D2O value of 1.2 for compounds 6 contrasts sharply with the primary KSIE of 4.5 reported for the pHindependent hydrolysis of penicillins, which involves β-lactam hydrolysis.13 Second, the temperature dependence for the reaction of compounds 6a and 6h gave rise to entropies of activation, ∆Sq, of -20.1 ((9.2) and -38.9 ((18.9) J K-1 mol-1, respectively. These values are slightly negative but are well within the range observed for unimolecular ionization reactions carried out in the presence of organic cosolvents.31 Compound 6a is ca. 25 times less reactive than its acyclic counterpart 7. The decreased reactivity of 6a, when compared to 7, may be ascribed to an increase in ring strain resulting from the presence of two exocyclic double bonds in the iminium ion 10. This observation is supported by theoretical calculations as described in the section “Fate of β-Lactam Iminium Ion”. Acid-Catalyzed Hydrolysis. N-Acyloxymethylazetidin-2-ones unsubstituted in C-4 (6a-h) undergo acid(28) Page, M. I.; Williams, A. Organic and Bio-organic Mechanisms; Longman: Harlow, 1997; p 42. (29) Thornton, E. K.; Thornton, E. R. In Isotope Effects In Chemical Reactions; Collins, C. J., Bowman, N. S., Eds.; Van Nostrand Reinhold: New York, 1970; p 213. (30) Johnson, S. L. Adv. Phys. Org. Chem. 1967, 5, 273. (31) Winstein, S.; Fainberg, A. H. J. Am. Chem. Soc. 1957, 79, 5937.
FIGURE 4. Hammett plots for the acid-catalyzed hydrolysis of 6a-d (b) and base-catalyzed hydrolysis of 6a-e (9) at 25 °C.
catalyzed hydrolysis with kH+ values ca. 2 orders of magnitude higher than those reported for the acidcatalyzed hydrolysis of other azetidin-2-ones (e.g., for nocardicin A, 11, kH+ is 7.1 × 10-5 L mol-1 s-1 at 30 °C16), which undergo β-lactam ring opening via an A-1 mechanism. The products of degradation of 6a-h are those resulting from ester hydrolysis, i.e., N-hydroxymethylazetidin-2-one and the corresponding carboxylic acid. In contrast, penicillins16 are more reactive toward acid than N-acyloxymethylazetidin-2-ones 6a-h by a factor of 102, but this high reactivity as been ascribed to the neighboring group participation by the 6-acylamido moiety,16,21 a pathway not available to compounds 6. Moreover, N-acyloxymethylazetidin-2-ones 6a-h are at least 103 times more reactive than simple alkyl benzoates, which undergo acid-catalyzed ester hydrolysis via an A-2 mechanism.23 The acid-catalyzed pathway is very sensitive to the electronic effects of the substituent X at C-4 of the β-lactam. The kH+ value for compound 6j (X ) PhSO2) is 3000-fold less than that for its counterpart 6a (X ) H), while 6i (X ) PhS) has a 100-fold reduction in the kH+ value when compared with the unsubstituted derivative 6a. Compounds 6a, 6i, and 6j conform to the Taft equation (eq 4). The Taft F* value of -1.3 is indicative of the development of a positive charge (in the lactam) in the transition state. The second-order rate constant for the acid-catalyzed hydrolysis of the N-acyloxymethylazetidin-2-one 6h is 20-fold less than that for the unsubstituted compound 6a, indicative of an apparent Taft F* value of -0.7 for the substituent at the C-1′ in these compounds. This value is similar to that observed for the pH-independent hydrolysis and is also consistent with the development of a positive charge in the transitionstate. In contrast with these Taft F* values, the Hammett plot for the esters 6a-d gives rise to a F value of almost zero (Figure 4).
log kH+ ) -1.3σ* - 2.2 (n ) 3, r2 ) 1.0)
(4)
Activation parameters (∆Sq) for the acid-catalyzed hydrolyses of 6a and 6h were determined to be +3.6 ((11.7) and -40.7 ((11.3) J K-1 mol-1, respectively, from the kH+ values in the range of 25-45 °C (Table 1). These J. Org. Chem, Vol. 69, No. 10, 2004 3363
Valente et al. SCHEME 4
values provide strong evidence for a dissociative mechanism,32 rather than one involving nucleophilic attack by solvent, and are similar to those of N-acyloxymethylamides18 and N-acyloxymethylsulfonamides.25 Taken together, these results suggest that N-acyloxymethylazetidin-2-ones 6 undergo acid-catalyzed hydrolysis via a dissociative mechanism identical to that for the pH-independent pathway, but involving preequilibrium protonation of the substrate prior to the dissociative step (Scheme 4). Additional evidence comes from the absence of general-acid catalysis (Table 2) and from the KSIE, kH+/kD+, of 0.7 and 0.8 for compounds 6a and 6h, respectively (Table 1). Moreover, the relative rates of hydrolysis of 6a and its acyclic counterpart, 7, differ by less than a factor of 2 between the acid-catalyzed (k7/k6a ) 18) and pH-independent (k7/k6a ) 33) pathways, suggesting that the two reactions follow similar pathways. Unfortunately, is not possible to determine the exact site of protonation that leads to hydrolysis. Despite amides being well-known oxygen bases,33 Page has suggested that the acid-catalyzed hydrolysis of bicyclic β-lactams involves N-protonation and that the pKa for such protonated species must be
