Acyloxymethyl as a drug protecting group. Part 6: N-acyloxymethyl- and N-[(aminocarbonyloxy)methyl]sulfonamides as prodrugs of agents containing a secondary sulfonamide group

Article abstract of DOI:10.1016/S0968-0896(00)00015-8

Tertiary N-acyloxymethyl- and N-[(aminocarbonyloxy)methyl]sulfonamides were synthesised and evaluated as novel classes of potential prodrugs of agents containing a secondary sulfonamide group. The chemical and plasma hydrolyses of the title compounds were studied by HPLC. Tertiary N- acyloxymethylsulfonamides are slowly and quantitatively hydrolysed to the parent sulfonamide in pH 7.4 phosphate buffer, with half-lives ranging from 20 h, for 7d, to 30 days, for 7g. Quantitative formation of the parent sulfonamide also occurs in human plasma, the half-lives being within 0.2-2.0 min for some substrates. The rapid rate of hydrolysis can be ascribed to plasma cholinesterase, as indicated by the complete inhibition observed at [eserine]=0.10 mM. These results suggest that tertiary N- acyloxymethylsulfonamides are potentially useful prodrugs for agents containing a secondary sulfonamide group, especially with pK(a)<8, combining a high stability in aqueous media with a high rate of plasma activation. In contrast, N-[(aminocarbonyloxy)methyl]sulfonamides 7h-j do not liberate the parent sulfonamide either in aqueous buffers or in human plasma and thus appear to be unsuitable for development as sulfonamide prodrugs. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00015-8

Bioorganic & Medicinal Chemistry 8 (2000) 707±716
Acyloxymethyl as a Drug Protecting Group. Part 6:
N-Acyloxymethyl- and N-[(Aminocarbonyloxy)methyl]sulfonamides
as Prodrugs of Agents Containing a Secondary Sulfonamide Group
Francisca Lopes, a Rui Moreira a,* and Jim Iley b
aCECF, Faculty of Pharmacy, University of Lisbon, Av. das ForcËas Armadas, 1600-083 Lisbon, Portugal
bChemistry Department, The Open University, Milton Keynes, MK7 6AA, UK
Received 19 July 1999; accepted 12 October 1999
AbstractÐTertiary N-acyloxymethyl- and N-[(aminocarbonyloxy)methyl]sulfonamides were synthesised and evaluated as novel
classes of potential prodrugs of agents containing a secondary sulfonamide group. The chemical and plasma hydrolyses of the title
compounds were studied by HPLC. Tertiary N-acyloxymethylsulfonamides are slowly and quantitatively hydrolysed to the parent
sulfonamide in pH 7.4 phosphate bu€er, with half-lives ranging from 20 h, for 7d, to 30 days, for 7g. Quantitative formation of the
parent sulfonamide also occurs in human plasma, the half-lives being within 0.2±2.0 min for some substrates. The rapid rate of
hydrolysis can be ascribed to plasma cholinesterase, as indicated by the complete inhibition observed at [eserine]=0.10 mM. These
results suggest that tertiary N-acyloxymethylsulfonamides are potentially useful prodrugs for agents containing a secondary sulfon-
amide group, especially with pKa<8, combining a high stability in aqueous media with a high rate of plasma activation. In contrast,
N-[(aminocarbonyloxy)methyl]sulfonamides 7h±j do not liberate the parent sulfonamide either in aqueous bu€ers or in human
plasma and thus appear to be unsuitable for development as sulfonamide prodrugs. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
as indomethacin, nimesulide, 5, the lead compound of the
methanesulfonamide class of cyclooxygenase-2 (COX-2)
inhibitors, still reveals gastric damage comparable to
isoxicam.9±12 Such gastric damaging e€ect is not restricted
to nimesulide, and has also been reported with other,
more recent, COX-2 methanesulfonamide inhibitors.13
The use of a secondary sulfonamide group as a bioiso-
steric replacement for the amide and carboxylic acid
groups, as well as an isostere of the transition-state for
the hydrolysis of the peptide bond, is becoming an
increasingly important tool in drug design.1±3 In
many cases, sulfonamide analogues often exhibit only
poor-to-moderate oral activity as a result of unfavour-
able physicochemical properties such as extreme low or
high lipophilicity. Numerous examples have been
reported recently and include the HT1D antagonist
sumatriptan 1, the leukotriene D4 antagonists 2, the
human leukocyte elastase inhibitors 3 and the endothelin
antagonist 4.4±8
A useful approach to improve drug delivery is to trans-
form the drug molecule into a prodrug, which in this
case might be achieved by linking the sulfonamide to an
inactive carrier.14 The main requisite is the quantitative
release of the parent drug in vivo, either chemically or
enzymatically. Also in the case of nimesulide and related
COX inhibitors, the prodrug approach could be used in
order to minimise the potential for gastrointestinal irri-
tation due to topical COX inhibition and prostaglandin
depletion by the parent drug. However, only few pro-
drugs for secondary sulfonamide agents have been
reported.15±17 From these, only N-acylsulfonamides, e.g.
6, have shown to have some potential as prodrugs of
secondary sulfonamides as a result of their high rate of
in vivo activation.16,18
Several drugs containing a secondary sulfonamide
group also exhibit undesirable side e€ects that a€ect
their therapeutic e€ectiveness after oral administration.
For example, although signi®cantly better tolerated
than typical cyclooxygenase-1 (COX-1) inhibitors such
Recently, we reported that tertiary N-acyloxymethyl-
sulfonamides, 7 (X=OCOR3) are potential prodrugs
for both secondary sulfonamides and carboxylic acid
*Corresponding author. Tel.: +351-1-794-6416; fax: +351-794-6470;
e-mail: rmoreira@€.ul.pt
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00015-8

708
F. Lopes et al. / Bioorg. Med. Chem. 8 (2000) 707±716
agents.19 Compounds 7 (X=OCOR3) undergo sponta-
neous hydrolysis at pH 7.4 by an SN1-type mechanism,
involving the formation of an N-sulfonyliminium ion in
the rate-determining step.19,20 Accordingly, reactivity of
7 increases sharply with electron donating substituents
in the sulfonamide moiety (both R1 and R2), with the
N-aryl derivatives presenting the highest stability.19,20
Considering the potential utility of N-acyloxymethyl-
sulfonamides 7 it was therefore necessary to assess the
applicability of this prodrug approach with a wider
range of compounds representative of therapeutic
agents that contain the secondary sulfonamide group.
We now report the synthesis of a series of N-acyloxy-
methylsulfonamides 7a±g and a kinetic study designed
with the objective of evaluating the in¯uence of pKa and
structure of the sulfonamide on the chemical reactivity
and stability in human plasma. Their N-aminocarbonyl-
oxymethyl counterparts 7h±j were also synthesised to
assess the ability of a carbamoyloxymethyl group (7,
X=OCONR3R4), based on amino acids, to be chemically
and enzymatically activated to the parent sulfonamide.
The data are compared with N1-acetylsul®soxazole, 6,
a well-known taste-masking prodrug.
Chemistry
Tertiary N-acyloxymethylsulfonamides 7a±g were syn-
thesised by reaction of the secondary sulfonamide with
the appropriate chloromethyl ester.19 The strategy for
the synthesis of N-aminocarbonyloxymethylsulfon-
amides 7h±j involves the preparation of N-[(chloro-
methyl)oxy]carbonyl derivatives 8 by reaction of the
appropriate amino acid with chloromethyl chloro-
formate (Scheme 1, for 7h). Compounds 8 were con-
verted quantitatively to their iodomethyl counterparts
9 with sodium iodide at room temperature. Finally,
reaction of intermediates 9 with the sodium salt of
nimesulide, 5, a€orded 7 in ca. 30% yield.
The structure of compounds 7a±j follows from their
spectroscopic and analytical data. The 1H NMR spectra
of N-acyloxymethylsulfonamides 7a±g and derivative 7j
exhibit a characteristic singlet at ca. d 5.8±6.6 ppm due
to the NCH2O group. The carbamoyloxymethyl deriva-
tives 7h±i, which contain a tertiary carbamate group,
appear to be a mixture of E and Z rotamers, as revealed
by their 13C and 1H NMR spectra. Interestingly, for the
proline derivative, 7h, the NCH2O signal appears as two
pairs of doublets, re¯ecting the diastereotopic nature of
the methylene protons (2J=11.7 and 11.4 Hz) as well as
the presence of two rotamers due to restricted rotation
about the carbamate C±N bond. The spectroscopic
detection of rotamers has been reported for other prolyl
carbamates and is consistent with a barrier to rotation
for the carbamate N±CˆO framework similar to the
barrier of acyclic tertiary amides.21
Results and Discussion
Chemical hydrolysis
Products of hydrolysis. As revealed by the HPLC analy-
sis of reaction mixtures, the hydrolyses of N-acyloxy-
methylsulfonamides 7a±g proceed with quantitative
formation of the parent sulfonamide at all pH values
studied. No lag time was observed for the formation of
the parent sulfonamide at any pH value, indicating that
the corresponding N-hydroxymethylsulfonamide (7,
X=OH) decomposes very rapidly to the sulfonamide. A
similar behaviour has been reported for other N-hydroxy-
methyl derivatives of NH-acidic drugs.22 The rate of
decomposition of N-hydroxymethylamides and imides
increases sharply with the pKa of the parent NH-com-
pound, as indicated by the Bronsted blg value of 0.8.23
Using this structure±reactivity relationship, a half-life of
0.01 s can be calculated for the decomposition of the
proposed N-hydroxymethyl derivative of nimesulide
(pKa 5.9) at pH 7.4. In contrast to the N-acyloxymethyl

F. Lopes et al. / Bioorg. Med. Chem. 8 (2000) 707±716
709
Table 1. Structure, melting points, elemental analyses and IR for compounds 7a±m
m.p. (ꢀC) C% (calc.) H% (calc.) N% (calc.)
nmax (cm )
1
Compound
R1
R2
X
7a
OCOC6H5
122±124 57.1 (56.8)
184±187 53.0 (52.4)
4.7 (4.8)
10.4 (10.5) 3474, 3362, 1742
7b
7c
OCOC6H5
OCOC6H5
4.0 (3.9)
10.3 (10.8) 3460, 3360, 1725
184±186 56.6 (56.2)
132±134 58.3 (58.2)
158±161 55.3 (55.1)
120±122 57.1 (57.0)
4.6 (4.2)
4.9 (4.9)
6.2 (6.2)
4.1 (4.1)
14.2 (14.6) 3451, 3360, 1708
13.5 (13.6) 3474, 3375, 1726
14.0 (14.3) 3463, 3371, 1728
7d
7e
7f
OCOC6H5
OCOCMe3
OCOC6H5
Me
Me
Me
6.3 (6.3)
6.7 (6.6)
8.2 (8.5)
1719
1740
7g
7h
OCOCMe3
Oil
54.3 (54.0)
51.5 (51.1)
5.3 (5.2)
4.9 (4.7)
79±81
1754, 1719
7i
Me
Oil
50.0 (49.9)
4.3 (4.8)
9.2 (8.7)
1749, 1720
7j
Me
Me
Gum
Oil
48.9 (48.8)
49.9 (50.1)
4.2 (4.5)
4.5 (4.4)
9.2 (9.0)
9.0 (8.8)
3399, 1754, 1721
3605 (br),1722
7k
7l
Me
Me
Oil
Oil
48.0 (47.7)
46.7 (46.5)
4.2 (4.2)
4.3 (3.9)
9.2 (9.3)
9.9 (9.6)
3555 (br),1720
7m
3376, 2856, 1725

710
F. Lopes et al. / Bioorg. Med. Chem. 8 (2000) 707±716
Scheme 1. (i) ClCH2OCOCl/Et3N/CH2Cl2; (ii) NaI/acetone; (iii) 5
(sodium salt)/THF.
derivatives 7f±g, the carbamates 7h±m did not decom-
pose to nimesulide over the pH 7.4±13 range. Large-
scale hydrolyses of compounds 7h±j a€orded only the
product from the hydrolysis of the amino acid methyl/
ethyl ester functionality, as shown by comparison with
the standards 7k±m.
pH-Rate pro®les. The pseudo-®rst-order rate constants,
kobs, for the hydrolysis of derivatives 7 were determined
in HCl and NaOH solutions, as well as in aqueous buf-
fers using several acetate, phosphate and borate bu€er
concentrations. The rates of hydrolysis of the more
reactive compounds, e.g. 7d, were not subject to sig-
ni®cant bu€er catalysis over at least a 10-fold bu€er
concentration range. In contrast, the rates of decom-
position for the less reactive compounds, e.g. 7b, pre-
sented a slight dependence on bu€er concentration over
the same concentration range. Using the intercepts of
plots of kobs versus [bu€er], together with the kobs values
determined in HCl and NaOH solutions, the pH-rate
pro®les presented in Figure 1 were constructed. In
the acidic region, compounds 7b±d exhibit a sigmoidal
pH-rate pro®le, which re¯ects protonation of the 4-
amino group, rather than one of the heterocyclic nitro-
gen atoms (see below). The pro®le can be described by
eq (1)
Figure 1. pH-Rate pro®les for compounds 7b (*), 7c (&), 7d (&) and
7f (*) at 37 ꢀC. Insert: dependence of kobs with [HCl] for 7f, at 37 ꢀC.
È
Â
ÃÉ
Ka
‡
H
kobs ˆ ko ‡ kOH ‰OH Š ‡ kHNeut
Â
Ã
‡
‡
Ka ‡ H
Â
Ã
‡
The best computer ®t (solid lines) to the experimental
data (individual points) in Scheme 2 was achieved using
eqs (1) and (2), the experimentally determined values
for kobs and pH, and the values for kNeut, kHProt kOH , ko
Â
Ã
H
‡
‡ kPHrot
H
ꢀ1†
Â
Ã
‡
‡
Ka ‡ H
‡
‡
H
‡
‡
‡
where Ka=ꢁKa ‡ ‰H Š† and ‰H Š=ꢁKa ‡ ‰H Š† are the
fraction of the unionised and ionised forms of the
compound, respectively, and are the acid-catalysed
second-order rate constants for these species, ko is the
®rst-order rate constant for the pH-independent
decomposition of the unionised form, kOH the OH
catalysed second-order rate constant for the unionised
form and Ka is the apparent ionisation constant of the
compound (Scheme 2). In contrast, the pH-rate pro®le
of compound 7f, which does not contain any basic
function, displays a simple U-shape, indicative of the
presence of acid-catalysed, base-catalysed, and pH-
independent processes (eq. (2)). Similar pH-rate pro®les
have been previously described for other neutral N-acy-
loxymethyl derivatives of sulfonamides.20
and pKa listed in Table 2. For compound 7b, no repro-
ducible rate data was obtained between pH 3 and 5 and
thus kHNeut and kHProt were determined from eq. (1)
‡
‡
assuming that no spontaneous or alkaline hydrolyses
occur at pH<2.
pH-Independent pathway. The ko data presented in
Table 2 indicates that ester 7d is more reactive than 7c
by a factor of 4, suggesting that the pH-independent
pathway is accelerated by electron-donating sub-
stituents in the N-aryl moiety. This is consistent with an
SN1-type mechanism involving the formation of an N-
acyliminium ion in the rate-determining step.20 Further
evidence of an SN1-type mechanism for the pH-inde-
pendent hydrolysis of acyloxymethylsulfonamides 7c±d
is provided by the temperature dependence study with
ˆ
ˆ
‡
kobs ˆ ko ‡ kOH ‰OH Š ‡ kNHeut‰H Š
ꢀ2†
7d at pH 7.4, from which ÁS and ÁH values of
‡
25‹8 JK 1 mol 1 and 98.1‹0.5 kJ mol 1, respectively,

F. Lopes et al. / Bioorg. Med. Chem. 8 (2000) 707±716
711
7f is also most likely to hydrolyse by a SN1-type
mechanism as indicated by the monotonic increase of
kobs with [HCl] (insert in Fig. 1). A similar e€ect has
been reported for N-acyloxymethylamides.25
The calculated pKa values for compounds 7b±d are ca.
0.8 units below the values reported for the parent
sulfonamides (Table 2). Such a di€erence might be
ascribed to the electron-withdrawing e€ect of the acy-
loxymethyl moiety on the sulfonamido group. The
range of pKa values for 7b±7d is 1.1±1.7, which is con-
sistent with protonation occurring at the 4-NH2 group
rather than at the heterocyclic nitrogen atom. Sub-
stantially lower pKa values would be expected if proto-
nation occurred in the heterocyclic moiety. For
example, the pKa of 2-(N-benzyl-N-p-tosylamino)thia-
zole is 0.53,27 while that estimated for compound 7c
is ca. 0.1.28 Not surprisingly, the protonated forms
of N-acyloxymethylsulfonamides 7b±7d are ca. 10- to
60-fold less reactive than the neutral forms in the acid-
catalysed pathway, re¯ecting the diculty of the
second, catalytic protonation. However, the acid-cata-
lysed hydrolysis of the ionised forms is also enhanced by
electron-donating substituents in the N-aryl group
(Table 2), suggesting that the ionised forms also
decompose via an SN1-type mechanism.
Scheme 2.
ˆ
were calculated. The slightly negative value of ÁS
is well within the range observed for unimolecular
reactions carried out in the presence of an organic co-
solvent.20,24 The SN1-type mechanism is also supported
by the absence of bu€er catalysis in the pH-independent
region of 7d. Ester 7f is much less reactive in this pH-
rate pro®le region, with a ko value closer to that
reported for N-acetoxymethyl-5-¯uorouracil.22 The
Base-catalysed pathway. Inspection of the kOH values
in Table 3 suggests that the alkaline hydrolysis of N-
acyloxymethylsulfonamides 7a±g is dependent on the
steric e€ect exerted by the sulfonamide and ester moi-
eties. Indeed, esters 7a and 7d, which contain two
methyl groups in the heterocyclic ring, hydrolyse ca. 2-
fold more slowly than unsubstituted compounds 7b and
7c. Also, the N-pivaloyloxymethylsulfonamides 7e and
7g are 3-fold less reactive toward OH than their N-
benzoyloxymethyl counterparts 7d and 7f, respectively.
The dependence of reactivity on the steric properties
suggests that the mechanism of alkaline hydrolysis of
N-acyloxymethylsulfonamides occurs via direct OH
attack at the carbonyl carbon atom.29 Unexpectedly,
both tertiary, 7h±i, and secondary, 7j, N-(aminocarbonyl-
oxy)methyl derivatives undergo hydrolysis exclusively at
the ester, rather than the carbamate, group. Secondary
carbamates with alcohol leaving groups with pKa<12.5
are known to hydrolyse via a facile elimination±addi-
tion mechanism (E1cB).30 We have recently reported
that the kinetically determined pKa value for N-hydro-
xymethyl-N-methylsulfonamides is ca. 12.5,20 and we
ˆ
value of ÁS for the hydrolysis of 7f at pH 7.4 is
85‹4 kJ mol 1, typical of a bimolecular process20
involving nucleophilic attack of the solvent at the ester
carbonyl carbon atom of 7f. Such a change in mechan-
ism might arise from highly unfavourable non-bonding
interactions between the ortho phenoxy group and the
NˆCH2 protons in the SN1 transition-state.
Acid-catalysed pathway. The kNHeut values in Table 2
‡
indicate that the acid-catalysed hydrolysis of the neutral
forms is also enhanced by electron-donating sub-
stituents in the N-aryl group, with ester 7d being more
reactive than 7c by a factor of 8. This is consistent with
a dissociative mechanism, identical to that for the pH-
independent pathway, involving protonation of the
substrate prior to iminium ion formation.20 Compound
Table 2. Second-order rate constants, kNHeut, kPHrot and kOH , for the acid- and base-catalysed, and pseudo-®rst-order rate constants, ko, for the pH-
‡
‡
independent hydrolyses of N-acyloxymethysulfonamides 7b±d and 7f, in aqueous bu€ers containing 10% acetonitrile (v/v), at 37 ꢀC
kNHeut (104 dm3 mol
s
)
kPHrot (10 dm3 mol
s )
pKa
1
1
7
1
1
1
7
1
1
Compound
kOH (dm3 mol
s
)
ko (10
s
)
‡
‡
a
7b
7c
7d
7f
0.562
0.152
0.095
0.134
0.115
9.00
75.0
5.52
130
1450
Ð
1.55 (2.36e)
1.07 (2.00e)
1.67 (2.36e)
Ð
25.0
96.9, 325b, 574c, 1400d
9.66, 15.9b, 31.8c, 91.6d
5.02
aNot determined.
b45 ꢀC.
c50 ꢀC.
d60 ꢀC.
eFrom ref 26.

712
F. Lopes et al. / Bioorg. Med. Chem. 8 (2000) 707±716
Table 3. Lipophilicity data, molar refractivities, rate data for base-catalysed pathway at 37 ꢀC and half-lives in human plasma at 37 ꢀC and pH 7.4
(phosphate saline bu€er) for compounds 6 and 7a±j
1
1
Compound
Log P
MR (cm3)
kOH (10 M
s
)
pH 7.4 bu€er
Human plasma
7
1
1
kpH 7.4 (10
s
)
t1/2 (min)
kplasma (10 5 s
)
t1/2 (min)
7a
7b
7c
7d
7e
7f
7g
7h
7i
2.83
1.47
2.15
3.59
3.07
3.97
3.96
2.85
2.20
2.28
0.82b
103.13
99.55
99.20
2.05
5.62
1.52
0.950
0.320
1.34
0.523
0.565
2.65
18.7
25.0
108
82.9
30.1
6.26Â103
4.65Â103
1.18Â103
1.40Â103
3.83Â103
13.5Â103
46.6Â103
47.1Â103
20.3Â103
7.03Â103
834
199
9580
833
5.78
0.12
1.38
41.4
244
108.45
102.27
112.39
106.20
119.14
116.57
111.71
76.68
28.1
4.71
596
11.3
NDa
0.390
1.20
3630
8.52
1.93
2.48
2.45
5.66
16.4
135
102
ND
2.95Â103
7j
6
9.60
56.5
954
0.3
aND, no decomposition during 72 h.
bCLOGP from ref 26.
would expect a pKa<12.5 for the N-hydroxymethyl
derivative of 5. Thus, we expected compound 7j to
undergo hydrolysis of the carbamate group. The pre-
ferred ester hydrolysis observed with 7j may be the
result of a strong electron-withdrawing e€ect of the
carbamoylmethyl moiety on the ester function. Further
evidence in support of a common pathway for the
alkaline hydrolysis of 7h±j is provided by the good cor-
relation between log kOH values and the corresponding
molar refractivities (Table 3), MR (r2=0.95), indicating
that for these compounds reactivity depends on the
steric properties of the amino acid residue.31
7e and 7g. Similar dependence of plasma hydrolysis on
the steric hindrance of the ester moiety has also been
reported for N-acyloxymethyl derivatives of other NH-
acidic drugs.22,32,33 The reactivity of N1-acetylsul®sox-
azole, 6, in pH 7.4 bu€er and in human plasma is also
presented in Table 3. The most remarkable feature is the
7-fold greater reactivity of 6 at pH 7.4 when compared
with 7a. Such a di€erence cannot be ascribed to the
di€erent acyl moieties, as N-benzoyl-N-methyl-p-tol-
uenesulfonamide is ca. 1.5 times more reactive than its
N-acetyl analogue both at pH 4.0 and pH 7.4.16 Thus,
N-acyloxymethylsulfonamides appear to be more stable
than their N-acyl counterparts, and therefore are more
amenable to liquid pharmaceutical formulations.
Hydrolysis in human plasma
The hydrolysis in human plasma followed strict ®rst-
order kinetics for at least four half-lives, with r2>0.95
in all cases where a reaction could be monitored. Pro-
duct analysis by HPLC reveals that the plasma cata-
lysed hydrolysis of esters 7a±g leads to the quantitative
formation of the corresponding sulfonamide (Fig. 2),
which is consistent with the instantaneous decomposi-
tion of the N-hydroxymethyl intermediate (7, X=OH) to
the parent sulfonamide. In contrast, N-(aminocarbonyl-
oxy)methyl derivatives 7i,j undergo decomposition in
plasma exclusively via ester hydrolysis while, unexpect-
edly, 7h is stable to cleavage of either functionality.
The rate data in Table 3 were analysed for quantitative
relationships with several physicochemical parameters,
including the molar refractivity (MR) of the prodrug,
the pKa of the parent sulfonamide and the log P values.
No clear correlation between the rate data encompass-
ing all N-acyloxymethylsulfonamides 7a±g and the
The susceptibility of compounds 7 to undergo enzy-
matic plasma activation was assessed in vitro by com-
paring the rates of hydrolysis in 80% human plasma
and in pH 7.4 phosphate bu€er at 37 ꢀC. From the rate
data presented in Table 3, it is clear that human plasma
enzymes markedly accelerate the rate of hydrolysis of
N-acyloxymethylsulfonamides 7a±g, with half-lives ran-
ging from 0.1 to ca. 240 min. The plasma hydrolysis of
N-acyloxymethylsulfonamides 7a±g appears to be
markedly a€ected by the steric properties of the sulfo-
namide and ester moieties. Indeed, compounds 7b,c
hydrolyse 40 times faster than the more sterically hin-
dered esters 7a,d. Similarly, the N-benzoyloxymethyl
derivatives 7d and 7f are more susceptible to plasma
hydrolysis than their N-pivaloyloxymethyl counterparts
Figure 2. Time-courses for N-benzoyloxymethylnimesulide 7f (*) and
nimesulide 5 (*) in the hydrolysis of 7f in 80% human plasma at
37 ꢀC.

F. Lopes et al. / Bioorg. Med. Chem. 8 (2000) 707±716
713
corresponding physicochemical parameters emerged.
However, a good correlation (r2=0.89) was observed
between log kOH and log kplasma, suggesting that both
alkaline and plasma hydrolyses follow similar pathways
and thus have the same structural requirements (Fig. 3).
To assess whether the rate-enhancement observed with
human plasma, compared to hydrolysis in pH 7.4 buf-
fer, could be ascribed to the action of plasma esterases,
the e€ect of eserine on the rate of hydrolysis was also
studied. Eserine is a speci®c inhibitor of the serine
hydrolase cholinesterase (E.C. 3.1.1.8, also called
pseudocholinesterase, plasma cholinesterase or butyryl-
cholinesterase).34 Most signi®cantly, a 220-fold decrease
in reactivity was observed upon incubation with 10 mM
eserine, and a complete inhibition of hydrolysis was
observed with 100 mM eserine (Table 4). This strongly
suggests that plasma cholinesterase is the major enzyme
responsible for the rapid hydrolysis of the N-acylox-
ymethylsulfonamides 7a±g. A similar inhibition pro®le
by eserine has been reported for the esterase-catalysed
hydrolysis of glycolamide esters in 50% human
plasma.35
moiety bearing electron-withdrawing substituents. The
N-acyloxymethyl derivatives of sulfonamides combine a
high stability in aqueous bu€ers together with a high
susceptibility to enzyme-catalysed hydrolysis. Thus, N-
acyloxymethylation is a more attractive approach than
N-acylation to obtain potentially useful prodrug sys-
tems for agents containing the secondary sulfonamide
function. Hydrolysis of this new prodrug type in human
plasma is reduced by the steric hindrance exerted by
substituents in the sulfonamide and ester moieties. In
contrast, the N-aminocarbonyloxymethylsulfonamides
do not hydrolyse to the parent sulfonamide in aqueous
bu€ers or in human plasma, rendering them unsuitable
as sulfonamide prodrugs. Studies are in progress to
examine the utility of N-acyloxymethylsulfonamides as
potential prodrug systems for carboxylic acid agents.
Experimental
Elemental analyses were obtained from ITQB (Oeiras,
Portugal) and from Medac (Egham, UK) laboratories.
1H and 13C NMR spectra were recorded as CDCl3
solutions on a General Electric QE-300 or Jeol JNM-
EX400 spectrometers using Me4Si as internal standard;
coupling constants, J, are quoted in Hz. FTIR spectra
were recorded on a Nicolet Impact 400 spectro-
photometer. Electron-impact ionisation (EI) and fast-
atom bombardment (FAB) mass spectra were recorded
on a VG Mass Lab 25±250 spectrometer. Melting points
were determined using a Buchi 510 instrument and are
uncorrected. HPLC was performed using a system
comprising a Shimadzu LC-9A pump coupled to a
variable wavelength Shimadzu SPD-6AV UV±vis
detector, a 20 ml loop injection valve and a Merck
LiChrospher1 100 RP-8 5 mm 125Â4 mm column. The
sulfonamides, amino acids, chloromethyl pivalate and
chloromethyl chloroformate were purchased commer-
cially. All chemicals were reagent grade except those for
kinetic studies and HPLC, which were analytical or
LiChrosolv (Merck) grade. Chloromethyl benzoate was
prepared according to a literature method.36 N1-Acetyl-
sul®soxazole was a kind gift of Professor Maria J. Trigo
(University of Oporto, Portugal).
Conclusions
The rate data for the pH-independent pathway clearly
indicate that highly stable N-acyloxymethylsulfon-
amides can be prepared when the parent sulfonamide
contains either an N-heterocyclic ring or an N-aryl
4-Amino-N-benzoyloxymethyl-N-(3,4-dimethyl-3-isoxaz-
olyl)benzenesulfonamide 7a. A solution of chloromethyl
benzoate (1 mol equiv) in dry THF (1 mL) was added
dropwise to a suspension of the sul®soxazole, sodium
salt (1 mol equiv) in dry THF (5 mL). When the reac-
tion was complete, as indicated by TLC, the solvent was
removed under vacuum and CH2Cl2 (20 mL) was added
to the residue. The resulting solution was washed with
sodium hydrogen carbonate, water and then evaporated
to a€ord the crude 7a, which was puri®ed by column
chromatography on silica gel (SiO2) using diethyl ether
as eluent; 44%; dH (ppm): 1.95 (3H, s, isox-Me), 2.23
(3H, s, isox-Me), 4.22 (2H, s, NH2), 5.78 (2H, s,
NCH2O), 6.59±7.83 (9H, m, ArH); m/z 401 (M+).
Figure 3. Plot of log kOH versus log kplasma for N-acyloxymethyl-
sulfonamides 7a±g. The data are from Table 3.
Table 4. E€ect of eserine on the pseudo-®rst-order rate constants,
kobs, and half-lives, t1/2, for the hydrolysis of 7f in 80% human plasma
at 37 ꢀC
1
[Eserine] (mM)
kplasma (10 5 s
)
t1/2 (min)
0
5
10
100
596
1.93
282
423
4.08
2.72
NDa
4-Amino-N-benzoyloxymethyl-N-(2-thiazolyl)benzesulfon-
amide 7b. Puri®ed by column chromatography (SiO2,
diethyl ether:ethyl acetate (9:1)); crystallized from ethyl
ND
aND, no decomposition during 48 h.

714
F. Lopes et al. / Bioorg. Med. Chem. 8 (2000) 707±716
ether; 52%; dH (ppm): 4.02 (2H, s, NH2), 6.06 (2H, s,
NCH2O), 6.61±7.43 (11H, m, ArH); m/z 389 (M+), 268
(M C6H5CO2).
corresponding N-[(iodomethyloxy)carbonyl]proline meth-
yl ester 9h. The sodium salt of nimesulide (5, 1 mmol)
was added to
a solution of N-[(iodomethyloxy)-
carbonyl]proline methyl ester 9h (1.1 mmol) in dry THF
(3 mL) at room temperature. When the reaction was
complete, as indicated by TLC, the solvent was removed
under vacuum and CH2Cl2 (20 mL) was added to the
residue. The resulting solution was washed with sodium
hydrogen carbonate, water and then evaporated to
a€ord the crude product 7h which was puri®ed by
column chromatography on SiO2 using diethyl ether:
petroleum b.p. 40±60 (7:3) as eluent. Crystallized from
ethyl ether:petroleum; 42%; dH (ppm): 1.79±2.31 (4H,
m, ProCH2), 3.22 and 3.16 (3H, 2Âs, MeS), 3.32 and
3.60 (2H, m, CH2N), 3.69 and 3.72 (3H, 2Âs, MeO),
4.27±4.31 (1H, m, a-CH), 5.62 and 5.76 (1H, 2Âd,
NCH2O, J=11.7 Hz), 5.68 and 5.79 (1H, 2Âd, NCH2O,
J=11.4 Hz), 7.15±7.96 (8H, m, ArH); dC (ppm): 23.31
and 24.09 (ProCH2), 29.67 and 30.78 (ProCH2), 41.80
and 42.05 (MeS), 46.47 and 46.93 (CH2N), 52.27 and
52.39 (MeO), 58.76 and 59.05 (CH), 74.12 and 74.13
(NCH2O), 112.30 and 112.38 (ArCH), 117.78 and
117.84 (ArCH), 120.09 (ArCH), 125.78 (ArCH), 130.57
(ArCH), 133.04 and 133.23 (ArCH), 134.38 (ArC),
148.56 (ArC), 153.18 and 153.62 (NCO), 154.47 (ArC),
155.54 and 155.72 (ArC), 172.53 and 172.90 (OCO); m/z
493 (M+), 463 (M NO), 321 (M O2CProOMe).
4-Amino-N-benzoyloxymethyl-N-(2-pyrimidinyl)benzene-
sulfonamide 7c. Puri®ed by column chromatography
(SiO2, diethyl ether:ethyl acetate (9:1)); 30%; dH (ppm)
(d6-DMSO): 6.21 (2H, s, NH2), 6.44 (2H, s, NCH2O),
7.18±8.64 (12H, m, ArH); m/z 369 (M NH3), 263
(M C6H5CO2).
4-Amino-N-benzoyloxymethyl-N-(4,6-dimethyl-2-pyrimi-
dinyl)benzenesulfonamide 7d. Puri®ed by column chro-
matography (SiO2, diethyl ether:ethyl acetate (9:1));
58%; dH (ppm): 2.32 (6H, s, pyr-Me), 4.18 (2H, s, NH2),
6.60 (2H, s, NCH2O), 6.65±8.01 (10H, m, ArH); m/z:
291 (M C6H5CO2).
4-Amino-N-pivaloyloxymethyl-N-(4,6-dimethyl-2-pyrimi-
dinyl)benzenesulfonamide 7e. Puri®ed by column chro-
matography (SiO2, diethyl ether:ethyl acetate); 47%; dH
(ppm): 1.15 (9H, s, CMe3), 2.31 (6H, s, pyr-Me), 4.14
(2H, s, NH2), 6.31 (2H, s, NCH2O), 6.61±7.96 (5H, m,
ArH); m/z 291 (M-ButCO2).
N-benzoyloxymethyl-N-(2-phenoxy-4-nitrophenyl)methane-
sulfonamide 7f. Puri®ed by column chromatography
(SiO2, diethyl ether:petroleum b.p. 40±60 (7:3)); 29%;
dH (ppm): 3.26 (3H, s, CH3S), 5.98 (2H, s, NCH2O),
6.98±8.01 (13H, m, ArH); m/z: 442 (M+), 412 (M NO),
321 (M C6H5CO2).
N-{[(N0-Ethoxycarbonylmethyl-N0-methyl)aminocarbonyl-
oxy]methyl}-N-(2-phenoxy-4-nitrophenyl)methanesulfon-
amide 7i. Puri®ed by column chromatography (SiO2,
diethyl ether:petroleum b.p. 40±60 (7:3)); 24%; dH
(ppm): 1.26 and 1.27 (3H, 2Ât, CH3CH2, J=7.2 Hz),
2.93 and 2.96 (3H, 2Âs, N-Me), 3.15 and 3.21 (3H, 2Âs,
CH3S), 3.93 and 3.94 (2H, 2Âs, CH2N), 4.17 and 4.21
(2H, 2Âq, CH3CH2, J=7.2 Hz), 5.71 and 5.75 (2H,
2Âs, NCH2O), 7.12±7.98 (8H, m, ArH); dC (ppm): 14.15
and 14.16 (CH3CH2), 35.45 and 36.05 (N±Me), 41.86
and 42.05 (MeS), 50.48 and 50.64 (CH2N), 61.35 and
61.49 (CH2O), 74.49 and 74.70 (NCH2O), 112.36 and
112.42 (ArCH), 117.79 and 117.89 (ArCH), 120.16
(ArCH), 125.84 (ArCH), 130.62 (ArCH), 133.00 and
133.12 (ArCH), 134.28 and 134.32 (ArC), 148.46 (ArC),
153.45 and 153.47 (NCO), 155.02 (ArC), 155.58 and
155.71 (ArC), 168.98 and 169.12 (OCO); m/z 481 (M+),
321 (M EtOCOCH2N(Me)CO2).
N-Pivaloyloxymethyl-N-(2-phenoxy-4-nitrophenyl)methane-
sulfonamide 7g. Puri®ed by column chromatography
(SiO2, diethyl ether:petroleum b.p. 40±60 (7:3)); 23%;
dH (ppm): 1.16 (9H, s, CMe3), 3.20 (3H, s, CH3S), 5.73
(2H, s, NCH2O), 7.12±7.96 (8H, m, ArH); m/z 422
(M+), 392 (M NO), 321 (M-ButCO2).
N-{[(S)-(2-Carbomethoxypyrrolidin-1-yl)carbonyloxy]-
methyl}-N-(2-phenoxy-4-nitrophenyl)methanesulfonamide
7h. Step A. A solution of the (S)-proline methyl ester
hydrochloride (11.9 mmol) and triethylamine (21.6
mmol) in CH2Cl2 (20 mL) was added to a stirred solu-
tion of chloromethyl chloroformate (10.8 mmol) in
CH2Cl2 (25 mL) at 10 ꢀC. After 25 min at 10 ꢀC, the
reaction was allowed to reach room temperature. The
precipitate was ®ltered and the ®ltrate washed with
water, then brine, and dried with magnesium sulfate.
Evaporation of the solvent gave the corresponding pure
N-[(chloromethyloxy)carbonyl]proline methyl ester 8h
(1.64 g, 68%); dH (ppm): 1.92±2.38 (4H, m, ProCH2),
3.47±3.70 (2H, m, CH2N), 3.74 and 3.75 (3H, 2Âs,
MeO), 4.36±4.43 (1H, m, a-CH), 5.69 and 5.88 (1H,
2Âd, ClCH2O, J=6.0), 5.74 (1H, s, ClCH2O).
N-{[(N0-Ethoxycarbonylmethyl)aminocarbonyloxy]methyl}-
N-(2-phenoxy-4-nitrophenyl)methanesulfonamide 7j. Pur-
i®ed by column chromatography (SiO2, diethyl ether:
petroleum b.p. 40±60 (7:3)); 7%; dH (ppm): 1.28 (3H, t,
CH3CH2, J=7.2 Hz), 3.21 (3H, s, CH3S), 3.86 (2H, d,
CH2N, J=5.4 Hz), 4.20 (2H, q, CH3CH2, J=7.2 Hz),
5.27 (1H, t, NH, J=5.4 Hz), 5.75 (2H, s, NCH2O),
7.14±7.96 (8H, m, ArH); m/z 467 (M+), 321
(M EtOCOCH2 NHCO2).
Step B. A solution of N-[(chloromethyloxy)carbonyl]-
proline methyl ester 8h (2 mmol) and NaI (4 mmol) in
dry acetone (2 mL) was stirred at room temperature.
This reaction was monitored by 1H NMR, following the
disappearance of the OCH2Cl signals at d 5.7 ppm and
the appearance of the OCH2I signals at d 5.8 ppm (ca.
1 h). Filtration and evaporation of the solvent gave the
N-{[(S)-(2-Carboxypyrrolidin-1-yl)carbonyloxy]methyl}-
N-(2-phenoxy-4-nitrophenyl)methanesulfonamide 7k. A
solution of 7h (50 mg) and NaOH 1 M (1 mL) in ace-
tonitrile±water (1:1 mixture, 2 mL) was stirred at room
temperature for 1 h. The reaction mixture was acidi®ed
with HCl 1 M until pH 1 and then extracted with ethyl

F. Lopes et al. / Bioorg. Med. Chem. 8 (2000) 707±716
715
acetate (2Â10 mL). Evaporation of the solvent gave the
pure 7k; dH (ppm): 1.78±2.29 (4H, m, ProCH2), 3.10
and 3.18 (3H, 2Âs, MeS), 3.23±3.63 (2H, m, CH2N),
4.22±4.29 (1H, m, a-CH), 5.60 and 5.73 (1H, 2Âd,
NCH2O, J=11.5), 5.65 and 5.76 (1H, 2Âd, NCH2O,
J=11.2), 7.10±7.95 (8H, m, ArH).
analysed separately by the HPLC method described
above. The partition coecients, P, were calculated
from the ratio of the peak area in octanol to the peak
area in bu€er.
Computations
N-{[(N0 -Carboxymethyl-N0 -methyl)aminocarbonyloxy]-
methyl}-N-(2-phenoxy-4-nitrophenyl)methanesulfonamide
7l. Prepared from 7i; dH (ppm): 2.86 and 2.90 (3H, 2Âs,
N±Me), 3.09 and 3.14 (3H, 2Âs, CH3S), 3.87 and 3.88
(2H, 2Âs, CH2N), 5.66 and 5.70 (2H, 2Âs, NCH2O),
7.01±7.92 (8H, m, ArH); m/z (FAB ): 452 (M H), 307
(5-H).
Molecular refractivities, MR, were calculated with the
software ChemSketch v.3.0 from Advanced Chemistry
Development Inc. Calculation of the kinetic parameters
from eq (1) was performed using Excel1 (Microsoft)
and the Solver option.
Acknowledgements
N-{[(N0 -Carboxymethyl)aminocarbonyloxy]methyl}-N-
(2-phenoxy-4-nitrophenyl)methanesulfonamide 7m. Pre-
pared from 7j; dH (ppm): 3.19 (3H, s, MeS), 3.89 (2H, d,
glyCH2), 5.10 (1H, brs, NH), 5.72 (2H, s, NCH2O),
7.06±7.92 (8H, m, ArH); m/z (FAB-) 438 (M H), 307
(5 H).
This work was supported by Fundacao para a Ciencia e
Tecnologia (Portugal) under the contract PBIC/SAU/
1084/92. We are grateful to Dr. Luis Gouveia for
assisting in the computation of the kinetic parameters.
Kinetic measurements
References and Notes
Reactions were monitored using HPLC, following
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The ionic strength was maintained at 0.5 M with
NaClO4. Reactions were initiated by injecting ca. 50 mL
of the appropriate substrate stock solution to 5 mL of
the bu€er solution. At regular intervals, samples of the
reaction mixture were analysed using acetonitrile±water
containing 0.2 M sodium acetate bu€er (55:45 to
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