Toward the development of a cephalosporin-based dual-release prodrug for use in ADEPT

Article abstract of DOI:10.1021/jo048970i

In previous work we have shown that a cephalosporin structure bearing an S-aminosulfenimine at the 7-position behaved as a β-lactamase-dependent dual-release prodrug. Scission of the β-lactam ring of such a structure led to the rapid loss of the sulfur-at

Full text of DOI:10.1021/jo048970i

Toward the Development of a Cephalosporin-Based Dual-Release
Prodrug for Use in ADEPT
Jonathan W. Grant and Timothy P. Smyth*
Department of Chemical and Environmental Sciences, University of Limerick,
National Technological Park, County Limerick, Ireland
timothy.smyth@ul.ie
Received June 18, 2004
In previous work we have shown that a cephalosporin structure bearing an S-aminosulfenimine at
the 7-position behaved as a â-lactamase-dependent dual-release prodrug. Scission of the â-lactam
ring of such a structure led to the rapid loss of the sulfur-attached side chain moiety via an
intramolecular displacement, while the 3′-group was lost via the well-established elimination process
at that position. In the present work we report on an evaluation of the scope and limitations of
exploiting the S-aminosulfenimine functionality to generate a cephalosporin-based prodrug
incorporating two biologically active components. Starting from 7-ACA, a viable synthetic cycle
was put in place that avoided formation of the ∆2 isomer throughout and that allowed incorporation
of aminoglutethimide at the 3′-position and of a tosyl S-aminosulfenimine at the 7-position. The
direct incorporation of a biologically active sulfonamide (ethoxzolamide) or a sulfamate (coumate)
at this latter position was not achieved as a result of the difficulty of generating the corresponding
sulfur diimides. An indirect route for the formation of an S-aminosulfenimine was put in place, as
was a general method of alkylation (Mitsunobu reaction) of the tosyl S-aminosulfenimine following
its incorporation.
SCHEME 1
Introduction
The cephalosporin structure is well-established as a
mono-release prodrug nucleus owing to rapid elimination
of the 3′-substituent following enzyme-catalyzed scission
of the â-lactam ring. Examples are known where anti-
microbial (quinolones)1 and cytotoxic components (mel-
phalan, doxorubicin)2 have been incorporated at this
position. The cephalosporin-cytotoxic conjugates have
been shown to be valuable prodrugs in the ADEPT
(antibody-directed enzyme prodrug therapy) mode of drug
targeting.2 Previously we reported on a modification that
allowed the cephalosporin structure to behave as a dual-
release prodrug nucleus.3 This involved incorporation of
an unsaturated linker at the 7-position with a moderately
nucleofugic group attached via an electron-deficient
center. The generalized structure type and its associated
reaction pattern are shown in Scheme 1: elimination of
the 3′-substitutent is shown as path a, and displacement
of the second leaving group is shown as path b. The
efficacy of this latter process is due to the rapidity of an
intramolecular displacement occurring within a five-
membered acyclic unit of restricted conformational space
(due to the double bond). The specific prototypic structure
had N-methyl-p-toluenesulfonamide as the sulfur-at-
tached moiety and acetate at the 3′-position. Herein, we
report on our evaluation of the scope and limitations of
using the S-aminosulfenimine unit to generate a dual-
release cephalosporin prodrug bearing biologically active
components relevant to the ADEPT mode of anticancer
drug targeting.4
(1) Albrecht, H. A.; Beskid, G.; Christenson, J. G.; Dietcher, K. H.;
Georgopapadakou, H. H.; Keith, D. D.; Konzelmann, F. M.; Pruess, D.
L.; Wei, C. C. J. Med. Chem. 1994, 37, 400-407 and references therein.
(2) (a) Svensson, H. P.; Frank, I. S.; Berry, K. K.; Senter, P. D. J.
Med Chem. 1998, 41, 1507-1512 and references therein. (b) Hudyma,
T. W.; Bush, K.; Colson, K. L.; Firestone, R. A.; King, H. D. Bioorg.
Med. Chem. Lett. 1993, 3, 323-328.
(3) (a) Smyth, T. P.; O’Connor, M. J.; O’Donnell, M. E. J. Org. Chem.
1999, 64, 3132-3138. (b) Smyth, T. P.; O’Donnell, M. E.; O’Connor,
M. J.; St Ledger, J. O. Tetrahedron 2000, 56, 5699-5707. (c) Smyth,
T. P.; O’Donnell, M. E.; O’Connor, M. J.; St Ledger, J. O. J. Org. Chem.
1998, 63, 7600-7618.
(4) For details of an alternative dual-release prodrug nucleus, see:
Shamis, M.; Lode, H. L.; Shabat, D. J. Am. Chem. Soc. 2004, 126,
1726-1731.
10.1021/jo048970i CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/19/2004
J. Org. Chem. 2004, 69, 7965-7970
7965

Grant and Smyth
SCHEME 2a
a (i) (a) (Ph)2CN2, (b) allyloxychloroformate/Py in CH2Cl2, 58% from 7-ACA; (ii) (a) acetylesterase pH 6.5, 2 h at rt, (b) acidification,
extraction into ethyl acetate, (Ph)2CN2, 70% crude from 1b, ∆3 isomer; (iii) (a) ClC(O)OCH(Cl)CCl3/Py/DMAP in CH2Cl2, 78%, ∆3 isomer,
(b) (R)-(+)-aminoglutehimide/Py/DMAP in CH2Cl2, 73%, ∆3 Isomer; (iv) Pd(P(Ph)3)4/p-toluenesulfinic acid in CH2Cl2, 62%, ∆3 isomer (trace
impurity present); (v) (p-CH3C6H4SO2Nd)2S in CH2Cl2, 30%; (vi) AlCl3 in CH3NO2/CH2Cl2 -80 °C, aqueous extraction, 70%.
Results and Discussion
The synthetic plan that was implemented is given in
Scheme 2. A key step in the transformation was selective
removal of the 3′-acetate. Various protocols have been
described in the literature using hydroxide solutions at
low temperature;8 however, we did not find these to be
robust or reliable processes. The enzyme acetylesterase
from orange peel (commercially available) is known to
be selective and efficient9 (if somewhat expensive) for this
transformation and this gave reproducible results in our
work. The substrate used was 7-(allyloxycarbonyl)ami-
nocephalosporanic acid 1b; it was found that 5 mmol of
1b was efficiently processed using 1000 units of enzyme
activity within 2 h. The resulting crude desacetyl struc-
ture 2a was extracted and converted back to the benz-
hydryl ester 2b (70% from 1b). Chromatographic puri-
fication of 2b was avoided as this led to formation of some
lactone (via reaction of the 3′-OH and benzhydryl ester)
and to some of the ∆2 isomer. Treatment of crude 2b with
tetrachloroethyl chloroformate in the presence of pyridine
and DMAP gave the corresponding carbonate, 3a, which
readily reacted with (R)-(+)-aminoglutethimide to give
the carbamate 3b; both 3a and 3b were obtained as the
∆3 isomers exclusively following chromatographic puri-
fication.
The synthetic challenges addressed here (Scheme 2)
were to sequentially functionalize the 3′- and 7-positions
of 7-aminocephalosporanic acid (7-ACA) via procedures
that were reliable and robust in terms of removal of the
3′-acetate, incorporation of the S-aminosulfenimine, and
avoiding ∆3/∆2 isomerization throughout. It was envis-
aged to integrate a biologically active amine via a
carbamate linkage at the 3′-position and to incorporate
a biologically active sulfonamide or sulfamate as the
S-aminosulfenimine; in earlier work we had found that
direct formation of the S-aminosulfenimine occurred
readily on reaction of a sulfur diimide, derived from an
aryl sulfonamide, with the 7-amino group of a cepha-
losporin ester. Our synthetic endeavor initially focused
on A, which bears aminoglutethimide (an aromatase
inhibitor)5 at the 3′-position and coumate (a sulfatase
inhibitor)6 as the S-aminosulfenimine. Both inhibitors are
used clinically to reduce the production of estrogen in
hormone-dependent breast cancer. These estrogen-form-
ing enzymes are present in higher concentrations in
tumor tissue than elsewhere in the body7 and so may be
appropriate for the ADEPT mode of targeted drug deliver.
Removal of the allyl group from 3b to give 4 was
achieved with Pd(P(Ph)3)4 in the presence of p-toluene-
sulfinic acid; the standard additive for this process,
P(Ph)3, was omitted as its inclusion led to extensive
formation of the ∆2 isomer. It was also found that direct
chromatographic purification of crude 4 following removal
of solvent (entrainment with nitrogen) but without any
extraction step was optimal. The material obtained in
this manner contained a small amount of triphenylphos-
phine oxide, but this did not interfere with the next step
(use of N2 for the flash chromatography limited formation
of (Ph)3P(O)). The synthetic cycle was initially completed
by reaction of 4 with the sulfur diimide derived from
(5) Jones, C. D.; Winter, M. A.; Hirsch, K. S.; Stamm, N.; Taylor,
H. M.; Holden, H. E.; Davenport, J. D.; Krumkalns, E. V.; Suhr, R. G.
J. Med. Chem. 1990, 33, 416-429 and references therein.
(6) (a) Woo, L. W. L.; Howarth, N. M.; Purohit, A.; Hejaz, H. A. M.;
Reed, M. J.; Potter, B. V. L. J. Med. Chem. 1998, 41, 1068-1083. (b)
Woo, L. W.; Sutcliffe, O. B.; Bubert, C.; Grasso, A.; Chander, S. K.;
Purohit, A.; Reed, M. J.; Potter, B. V. L. J. Med. Chem. 2003, 46, 3193-
3196 and references therein.
(8) (a) Yamanaka, H.; Chiba, T.; Kawabata, K.; Takasugi, H.;
Masugi, T.; Takaya, T. J. Antibiot. 1985, 38, 1738-1751. (b) Mobash-
ery, S.; Johnson, M. J. Biol. Chem. 1986, 261, 7879-7887. (c) Lo´pez,
M.; Rodriguez, Z.; Valde´s, B., Velez, H.; Agu¨ero, J.; Fini, A.; Gonza´lez,
M. Farmaco 2001, 56, 629-631.
(9) Quante, J. M.; Hoke, R. A.; Mize, P. D.; Woodward, D. L.; Millner,
O. E. U.S. Patent 5,514,561, May 1996.
(7) Chetrite, G. S.; Phillipe, J. C.; Wright, F.; Pasqualini, J. R. J.
Steroid Biochem. Mol. Biol. 2000, 72, 23-27.
7966 J. Org. Chem., Vol. 69, No. 23, 2004

Dual-Release Cephalosporin-Based Prodrug
SCHEME 3a
SCHEME 4a
a (i) SO2Cl2/pyridine in benzene; (ii) pyridine in benzene.
p-toluenesulfonamide to give 5. A feature of the 1H NMR
spectrum of 5 is the quite broad appearance of the H-6
singlet; this is a characteristic feature of S-aminosulfenim-
inocephalosporins3a and is associated with dynamic isomer-
ization about the imine bond. Removal of the benzhydryl
group using AlCl3 gave the free acid 6 in 70% yield.
The standard route used for sulfur diimide preparation
starting from a sulfonamide was via dimerization of the
corresponding N-sulfinyl derivative; this process worked
well with p-toluenesulfonamide and p-chlorobenzene-
sulfonamide (Scheme 3).3c Despite many attempts we
were unsuccessful in forming the sulfur diimide of
coumate (prepared from commercially available 7-hy-
droxy-4-methylcoumarin)6 via this route. Ethoxzolamide
(commercially available), which is a known inhibitor of
carbonic anhydrase (CA XI)10 and has application in
cancer chemotherapy, was subjected to the same pro-
cesses, but it too failed to generate the corresponding
sulfur diimide. Some other methods of sulfur diimide
synthesis, involving N-chlorinated sulfonamides,11 were
examined but these processes gave intractable product
mixtures. Clearly, the difficulty of forming a variety of
sulfur diimides is a limiting feature for direct incorpora-
tion of a biologically active sulfonamide/sulfamate as an
S-aminosulfenimine at the 7-position of cephalosporins.
In a final evaluation of the S-aminosulfenimine chemistry
two further processes were studied: (a) an indirect
method for its formation, and (ii) alkylation of the
sulfonamide nitrogen in a Mitsunobu reaction following
formation of the S-aminosulfenimine. Both of these
processes gave some success.
a (i) p-CH3C6H4SO2N(CH3)SCl; (ii) MgBr2/H2O/e-/CH2Cl2.
SCHEME 5a
a (i) DIAD, P(Ph)3, CH3OH in CH2Cl2, -23 °C to rt, 45 min.
essential for the selective oxidation process so this
approach cannot be applied to coumate or ethoxzolamide.
The process may be applicable to N-methyl coumate;
however, this structure is known to be a less potent
sulfatase inhibitor than coumate itself.7a
Finally, the Mitsunobu reaction was evaluated as a
general alkylation process of the sulfonamide nitrogen
(Scheme 5) of the S-aminosulfenimine 9 (in previous work
we had methylated this position using diazomethane;
however, this method lacks generality as an alkylation
route). Methanol was chosen as an example of a simple
alcohol and also because 8 is a known structure.3a With
2 equiv of DIAD/P(Ph)3/MeOH the N-methylated struc-
ture 8 was obtained in 52% yield after chromatography
exclusively as the ∆3 isomer (prolonged reaction times
and/or use of more that 2 equiv of reagents did not
improve the yield but did lead to ∆2 isomer formation).
The potential value of this modification of the S-amino-
sulfenimine lies in the fact that sulfonamides, the
component that is displaced from the S-aminosulfen-
imine, find wide use as biological templates;10 the sul-
fonamide derivatives of epipodophyllotoxin are particu-
larly relevant in this context.13
A two-phase, electrochemically driven process was
developed by Torii and co-workers for the selective
oxidation of a cephalosporin-based sulfenamide to the
corresponding sulfenimine.12 We were successful in ap-
plying this process to conversion of the S-aminosulfena-
mide 7, derived from p-toluenesulfonamide, to the cor-
responding S-aminosulfenimine 8 exclusively as the ∆3
isomer (Scheme 4). The usefulness of this route is limited,
however, by the stability of the N-sulfenyl chloride
required for formation of the S-aminosulfenamide and
to oxidize this latter structure. An N-alkyl group is
Conclusion
In this work we have evaluated the scope and limita-
tions of exploiting the S-aminosulfenimine functionality
at the 7-position of a cephalosporin to prepare a â-lac-
tamase-dependent, dual-release prodrug structure bear-
ing two distinct biologically active components. Starting
from 7-ACA, a viable synthetic cycle was put in place that
avoided formation of the ∆2 isomer throughout and that
allowed incorporation of a biologically active amine
(10) Winum, J.-Y.; Vullo, D.; Casini, A.; Montero, J.-L.; Scozzafava,
A.; Supuran, C. T. J. Med. Chem. 2003, 46, 2197-2204 and references
therein.
(11) (a) Kresze, G.; Wucherpfennig, W. Angew. Chem., Int. Ed. Engl.
1967, 6 149. (b) Levtschenko, E. S.; Kirsanov, A. V. Zh.. Org. Khim.
1962, 32, 161-165. (c) Levtschenko, E. S.; Kirsanov, A. V. Zh.. Org.
Khim. 1962, 32, 2256-2262.
(12) Torii, S.; Tanaka, H.; Hamano, S.; Tada, N.; Nokami, J.;
Sasaoka, M. Chem. Lett. 1984, 1823-1826. The mechanism involves
formation of Br2 by oxidation of Br- in the aqueous phase with the
co-formation of Mg2+(-OH)2 following which the sulfenamide NH is
brominated in the organic phase and elimination of HBr is driven by
-OH at the interface to generate the sulfenimine.
(13) (a) Caddick, S.; Wilden, J. D.; Judd, D. B. J. Am. Chem. Soc.
2004, 126, 1024-1025 and references therein. (b) Guianvarc’h, D.;
Duca, M.; Boukarim, C.; Kraus-Berthier, L.; Le´once, S.; Pierre´, A.;
Pfeiffer, B.; Renard, P.; Arimondo, P. B.; Monneret, C.; Dauzonne, D.
J. Med. Chem. 2004, 47, 2365-2374.
J. Org. Chem, Vol. 69, No. 23, 2004 7967

Grant and Smyth
(1.32 g, 3.70 mmol, 74%): δH NMR (90 MHz, CDCl3) 2.01 (s,
3H), 3.46 (d, J ) 18.16 Hz, 1H), 3.71 (d, J ) 18.16 Hz, 1H),
4.57 (d, J ) 4.32 Hz, 2H), 4.78 (d, J ) 12.1 Hz, 1H), 5.09 (d,
J ) 12.1 Hz, 1H), 5.14 (d, J ) 5.19 Hz, 1H), 5.34-6.10 (m,
4H), 7.30 (br d, J ) 7.78 Hz, 1H).
(aminoglutethimide) at the 3′-position and a tosyl S-
aminosulfenimine at the 7-position. The direct incorpora-
tion of a biologically active sulfonamide or sulfamate at
this latter position was not achieved as a result of the
difficulty of forming the required sulfur diimide reagent.
An indirect route for the formation of an S-aminosulfen-
imine was put in place, as was a general method of
alkylating the sulfonamide nitrogen of the tosyl S-
aminosulfenimine following its incorporation. Given that
sulfonamides find varied uses as biological templates this
latter process offers the possibility of forming a biologi-
cally active sulfonamide13 (as the displaceable component)
following formation of the S-aminosulfenimine.14
Benzhydryl 7-(Allyloxycarbonyl)amino-3-hydroxymeth-
yl Cephalosporinate (2b). Compound 1b (2 g, 5.61 mmol)
was suspended in distilled water (30 mL), and the pH was
adjusted to pH 6.5 using 35% ammonia solution. Acetyl
esterase (Sigma; 1000 units of activity in 72 mL of ammonium
sulfate solution) was added to the cephalosporin solution in
one portion with constant stirring. The pH immediately
decreased and was maintained between pH 6.7 and 6.4 over
the course of the reaction using ammonia solution. After
approximately 1.5-2 h the pH of the solution changed at a
very slow rate. The pH was then adjusted to 2 with 2 M HCl,
and the solution was extracted with ethyl acetate (3 × 75 mL).
The organic layer was washed with brine (2 × 60 mL) and
distilled water (60 mL), separated, dried over anhydrous
MgSO4, filtered, and concentrated under reduced pressure to
∼20 mL. A solution of diphenyldiazomethane was added
dropwise with stirring until the characteristic color of the
diphenyldiazomethane persisted. The reaction mixture was left
stirring overnight and then concentrated under reduced pres-
sure to yield crude 2b as a pale yellow solid (2.08 g): νmax (KBr)
cm-1 3298, 1785 (lactam), 1717; δH (300 MHz, CDCl3) 2.62 (br
s), 3.59 (s, 2H), 3.97 (d, J ) 12.66 Hz, 1H), 4.41 (d, J ) 13.03
Hz, 1H), 4.62 (d, J ) 4.65 Hz, 2H), 4.95 (d, J ) 4.86 Hz, 1H),
5.25 (app dd, J ) 1.35, 10.35 Hz, 1H), 5.33 (app dd, J ) 1.35,
17.4 Hz, 1H), 5.59 (d, J ) 9.9 Hz, 1H), 5.71 (dd, J ) 4.8, 9.9
Hz, 1H), 5.92 (ddt, J ) 5.7, 6.61, 10.5 Hz, 1H), 6.92 (s, 1H),
7.26-7.46 (m, 10H). This material was used without further
purification in the following step.
Benzhydryl 7-(Allyloxycarbonyl)amino-3-[[(1′,2′,2′,2′-
tetrachloroethoxycarbonyl)oxy] methyl] Cephalospori-
nate (3a). To a solution of 2b (1.14 g, 2.38 mmol) in
dichloromethane (30 mL) were added tetrachloroethyl chloro-
formate (384 µL, 2.5 mmol), freshly distilled pyridine (202 µL,
2.5 mmol), and DMAP (11.65 mg, 0.095 mmol). The reaction
mixture was stirred for 4.5 h. Dichloromethane (20 mL) was
added, and the organic layer was washed with brine (2 × 40
mL) and distilled water (2 × 40 mL), separated, dried over
anhydrous MgSO4, filtered, and removed under reduced pres-
sure. Purification by flash chromatography (silica gel, 60/40
hexanes/ethyl acetate) gave 2b as a pale yellow solid (1.28 g,
1.86 mmol, 78%): mp 69-71 °C (dec); δH (300 MHz, CDCl3)
3.43 (d, J ) 18.66 Hz, 1H), 3.61 (d, J ) 18.58 Hz, 1H), 4.62 (d,
J ) 5.58 Hz, 2H), 5.0 (m, 2H), 5.29 (app dd, J ) 1.2, 10.2 Hz,
1H), 5.33 (app dd, J ) 1.5, 17.4 Hz, 1H), 5.34 (d, J ) 7.8 Hz,
1H), 5.48 (d, J ) 9.6 Hz, 1H), 5.73 (dd, J ) 5.1, 9.6 Hz, 1H),
5.93 (ddt, J ) 5.7, 6.9, 10.2 Hz, 1H), 6.65 (s, 1H), 6.94 (s, 1H),
7.27-7.44 (m, 10H); δC (75.47 MHz, CDCl3) 26.2, 57.6, 61.2,
66.7, 68.3, 80.2, 91.1, 96.9, 118.6, 124.5, 126.5, 127.0, 127.7,
128.2, 128.3, 128.5, 128.6, 131.9, 138.8, 138.9, 151.6, 155.2,
160.5, 165.0. Anal. Calcd for C28H24Cl4N2O8S: C, 48.71; H,
3.50; N, 4.06. Found: C, 48.46; H, 3.44; N, 3.82.
Experimental Section
Benzhydryl 7-Aminocephalosporinate (1). To a suspen-
sion of 7-ACA (2 g, 7.35 mmol) in methanol (7 mL) was added
a solution of diphenyldiazomethane (14.58 mmol) in dichlo-
romethane (40 mL), and the mixture was stirred for 3 days at
room temperature over which time the characteristic purple
color of the diphenyldiazomethane disappeared. The solution
was filtered and concentrated under reduced pressure to yield
a brown oily material that precipitated as a fine solid on
addition of diethyl ether. The ether was decanted to remove
diphenylmethyl methyl ether (a side product); this suspension/
decantation step was repeated twice. Purification by flash
chromatography (silica gel, 50/50 ethyl acetate/dichloromethane)
gave 1 as a pale yellow solid (2.02 g, 4.77 mmol, 65%): mp
122-123 °C (dec) (lit.3a 120-121 °C); νmax (KBr) cm-1 3419,
1770 (lactam), 1727; δH (90 MHz, CDCl3) 1.84 (br s, 2H), 2.03
(s, 3H), 3.33 (d, J ) 18.16 Hz, 1H), 3.60 (d, J ) 18.16 Hz, 1H),
4.66-5.09 (m, 4H), 6.98 (s, 1H), 7.34 (s, 10H). Anal. Calcd for
C23H22N2O5S: C, 63.00; H, 5.06; N, 6.39. Found: C, 62.70; H,
4.98; N, 6.28.
Benzhydryl 7-(Allyloxycarbonyl)aminocephalospori-
nate (1a). To a solution of 1 (2 g, 4.56 mmol) in dichlo-
romethane (30 mL) were added allyl chloroformate (727 µL,
6.84 mmol), freshly distilled pyridine (369 µL, 4.56 mmol), and
DMAP (22.28 mg, 0.18 mmol), and the solution was stirred
for 4.5 h. Dichloromethane (20 mL) was added, and the organic
layer was washed with brine (2 × 40 mL) and distilled water
(2 × 40 mL), separated, dried over anhydrous MgSO4, filtered,
and removed under reduced pressure to leave a pale yellow
solid (2.27 g). Purification by flash chromatography (silica gel,
60/40 ethyl acetate/hexanes) yielded 1a as a pale yellow solid
(2.12 g, 4.07 mmol, 89%): mp 68-70 °C (dec); νmax (KBr) cm-1
1790 (lactam), 1726, 1684; δH (300 MHz, CDCl3) 2.02 (s, 3H),
3.39 (d, J ) 18.6 Hz, 1H), 3.56 (d, J ) 18.6 Hz, 1H), 4.62 (d,
J ) 4.68 Hz, 2H), 4.80 (d, J ) 13.65 Hz, 1H), 4.98 (d, J ) 4.92
Hz, 1H), 5.05 (d, J ) 13.65 Hz, 1H), 5.25 (app dd, J ) 1.11,
11.40 Hz, 1H), 5.33 (app dd, J ) 1.41, 17.19 Hz, 1H), 5.50 (d,
J ) 11.40 Hz, 1H), 5.70 (dd, J ) 5.1, 10 Hz, 1H), 5.92 (ddt, J
) 5.7, 7.2, 9.9 Hz, 1H), 6.94 (s, 1H), 7.26-7.45 (m, 10H). Anal.
Calcd for C27H26N2O7S: C, 62.06; H, 5.01; N, 5.36. Found: C,
61.8; H, 5.04; N, 5.24.
Benzhydryl 7-(Allyloxycarbonyl)amino-3-[[3′-[[(4-ami-
nocarbonyl)phenyl]-3′-ethyl-2′,6′-piperidinedione]oxy]-
methyl] Cephalosporinate (3b). To a solution of 3a (0.67
g, 0.97 mmol) in dichloromethane (15 mL) were added ami-
noglutethimide (231.9 mg, 1.0 mmol), freshly distilled pyridine
(80.88 µL, 1.0 mmol), and DMAP (4.75 mg, 0.039 mmol), and
the mixture was stirred for 5 h. Dichloromethane (30 mL) was
added, and the organic layer was washed with brine and
distilled water, separated, dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. Purification
by gravity chromatography (silica gel, 70/30 dichloromethane/
ethyl acetate) gave 3b as a pale yellow solid (0.5207 g, 0.71
mmol, 73%): mp 116-118 °C (dec); νmax (KBr) cm-1 3315, 1787
(lactam), 1722, 1701, 1647; δH (500 MHz, CDCl3) 0.85 (t, J )
7.5 Hz, 3H), 1.90 (m, J ) 7.5 Hz, 1H), 2.02 (m, J ) 7.5 Hz,
1H), 2.20 (m, 1H), 2.33 (m, 1H), 2.40 (m, 1H), 2.61 (m, 1H),
7-(Allyloxycarbonyl)aminocephalosporanic Acid (1b).
Compound 1a (2.60 g, 4.99 mmol) was dissolved in dichlo-
romethane (60 mL), and the solution was cooled to 0 °C under
nitrogen. Trifluoroacetic acid (23 mL, 0.30 mol) and trifluoro-
acetic anhydride (7 µL, 0.05 mmol) were added. After 10 min,
the solution was washed with aqueous acid (pH 2.5), and the
organic layer was separated and extracted with 10% NaHCO3
(2 × 50 mL). This aqueous extract was acidified (2 M HCl) to
pH 2 and extracted with ethyl acetate (3 × 50 mL). The organic
layer was dried over anhydrous MgSO4, filtered, and concen-
trated under reduced pressure to yield 1b as a cream solid
(14) For details of an alternative side chain linkage, see: Ruddle,
C. C.; Smyth, T. P. Chem. Commun. 2004, 2332-2333.
7968 J. Org. Chem., Vol. 69, No. 23, 2004

Dual-Release Cephalosporin-Based Prodrug
3.44 (d, J ) 18.5 Hz, 1H), 3.58 (d, J ) 18.5 Hz, 1H), 4.61 (d,
J ) 4 Hz, 2H), 4.85 (d, J ) 13.5 Hz, 1H), 4.97 (d, J ) 5 Hz,
1H), 5.14 (d, 1H, J ) 13.5 Hz, 1H), 5.24 (app d, 10.5 Hz, 1H),
5.32 (app dd, J ) 0.75, 17.25 Hz, 1H), 5.65 (d, J ) 9.5 Hz,
1H), 5.68 (dd, J ) 5, 9.5 Hz, 1H), 5.91 (ddt, J ) 5.5, 6.5, 10.5
Hz, 1H), 6.78 (s, 1H), 6.96 (s, 1H), 7.19-7.44 (m, 14H), 8.05
(s, 1H); δC NMR (125 MHz, CDCl3) 9.0, 26.6, 27.0, 29.3, 32.9,
50.6, 57.6, 61.3, 63.6, 66.7, 79.3, 118.5, 119.2, 125.9, 127.0,
127.2, 127.8, 128.2, 128.3, 128.5, 128.6, 132.1, 133.9, 136.9,
139.0, 139.2, 152.9, 155.9, 160.8, 165.1, 172.1, 175.1; MALDIFT-
HRMS (DHB) m/z 761.2275 (M + Na+, C39H38N4O9S requires
761.2252).
Benzhydryl 7-Amino-3-[[3′-[[(4-aminocarbonyl)phenyl]-
3′-ethyl-2′,6′-piperidinedione]oxy] methyl] Cephalospori-
nate (4). To a stirred solution of p-toluenesulfinic acid (167.7
mg, 1.07 mmol) and 3b (198 mg, 0.27 mmol) in dichlo-
romethane (4 mL) was added Pd(PPh3)4 (93.06 mg, 0.081
mmol) in six portions. After indication by TLC that the reaction
was complete, the solution was concentrated to a volume of 1
mL by entrainment with a current of nitrogen. Purification of
the residue by flash chromatography (silica gel, 90/10 ethyl
acetate/hexanes, under nitrogen) gave a yellow solid (130.8 mg)
containing 4 (116 mg, 0.177, mmol, 66% as determined by
integration from the 1H NMR spectrum) contaminated by a
small amount of triphenylphosphine oxide: δH (300 MHz,
CDCl3) 0.86 (t, J ) 7.5 Hz, 3H), 1.89 (m, 1H), 2.01 (m, 1H),
2.19 (m, 1H), 2.34 (m, 1H), 2.44 (m, 1H), 2.59 (m, 1H), 3.40 (d,
J ) 18.6 Hz, 1H), 3.58 (d, J ) 18.6 Hz, 1H), 4.78 (d, J ) 4.5
Hz, 1H) overlapping with 4.80 (d, J ) 13.2 Hz, 1H), 4.92 (d, J
) 5.1 Hz, 1H), 5.09 (d, J ) 13.2 Hz, 1H), 6.86 (s, 1H), 6.98 (s,
1H), 7.18-7.70 (m, 14H), 8.02 (s, 1H); δC (125 MHz, CDCl3)
9.0, 26.3, 26.9, 29.3, 32.9, 50.6, 58.8, 60.4, 63.7, 79.7, 119.1,
125.7, 126.2, 126.9, 127.2, 127.8, 128.2, 128.5, 128.53, 128.6,
132.0, 132.2, 133.7, 137.0, 139.0, 139.2, 152.9, 161.2, 169.0,
172.2, 175.1; ESI-HRMS m/z 655.2218 (M + H+, C35H34N4O7S
requires 655.2221).
Benzhydryl 7-(N-Tosyl-S-aminosulfenimino)-3-[[3′-[[(4-
aminocarbonyl)phenyl]-3′-ethyl-2′,6′-piperidinedione]-
oxy]methyl] Cephalosporinate (5). To a solution of 4 (198
mg, 0.30 mmol) in dichloromethane (10 mL) was added bis-
(p-toluenesulfonyl) sulfur dimide3a (133.5 mg, 0.36 mmol), and
the mixture was stirred for 1 h. Water (2 mL) was added
followed by the slow addition of light petroleum ether (8 mL),
the solution was stirred for 10 min, and the precipitated
p-toluenesulfonamide was filtered off. Dichloromethane (10
mL) and light petroleum ether (8 mL) were added to the
filtrate, which was washed with distilled water (2 × 30 mL)
and brine (2 × 30 mL), separated, dried over anhydrous
MgSO4, filtered, and concentrated under reduced pressure to
leave a yellow solid. Purification by gravity chromatography
(silica gel, 75/25 dichloromethane/ethyl acetate) gave 5 as a
yellow solid (78.2 mg, 0.092 mmol, 30%): mp 125-127 °C; νmax
(KBr) cm-1 3306, 1780 (lactam), 1725, 1700, 1637; δH (500
MHz, CDCl3) 0.85 (t, J ) 7.25 Hz, 3H), 1.89 (m, 1H), 2.02 (m,
1H), 2.19 (m, 1H), 2.35 (m, 1H), 2.41 (s, 3H), 2.42 (m, 1H),
2.57 (m, 1H), 3.39 (d, J ) 18 Hz, 1H), 3.62 (d, J ) 18 Hz, 1H),
4.82 (d, J ) 13.25 Hz, 1H), 5.11 (d, 2H, J ) 13.25 Hz, 1H),
5.39 (br s, 1H), 6.86 (s, 1H),7.01 (s, 1H), 7.19-7.52 (m, 16H),
7.75 (d, J ) 8 Hz, 2H), 8.08 (s, 1H); δC (125 MHz, CDCl3) 9.0,
21.7, 27.0, 28.2, 29.3, 32.9, 50.7, 60.7, 63.5, 80.2, 119.3, 126.8,
127.0, 127.2, 127.7, 127.9, 128.2, 128.3, 128.6, 128.63, 130.0,
136.0, 136.9, 138.8, 139.2, 145.0, 152.1, 152.9, 158.1, 160.7,
172.4, 175.2; ESI-HRMS m/z 876.1795 (M + Na+, C42H39N5O9S3
requires 876.1802).
was vigorously stirred while allowing the temperature to rise
to ambient. The mixture was further diluted by the addition
of water (40 mL). The organic layer was separated and
discarded. The aqueous layer was filtered through a bed of
Celite until clear, acidified to pH 2.2 with 2 M HCl, extracted
with dichloromethane (2 × 25 mL), separated, dried over
anhydrous MgSO4, filtered, and concentrated under reduced
pressure to yield the free acid as a yellow solid (45.5 mg, 0.068
mmol, 70%): mp 123-127 °C (dec); ESI-HRMS (MeOH) m/z
[M - H]- calcd for C29H28N5O9S3 686.1055, found 686.1056;
νmax (KBr) cm-1 3440, 1776, 1719, 1701; δH (300 MHz, CDCl3/
5% DMSO-d6) 0.85 (t, J ) 7.05 Hz, 3H), 1.90 (m, 1H), 2.01 (m,
1H), 2.19 (m, 1H), 2.34 (m, 1H), 2.41 (s, 3H), 2.42 (m, 1H),
2.57 (m, 1H), 3.47, (d, J ) 16.5 Hz, 1H), 3.69 (d, J ) 18.6 Hz,
1H), 4.99 (d, J ) 12.9 Hz, 1H), 5.18 (d, J ) 13.2 Hz, 1H), 5.51
(br s, 1H), 5.84 (s, 1H), 7.19 (d, J ) 8.4 Hz, 2H), 7.32 (d, J )
8 Hz, 2H), 7.45 (d, J ) 8.1 Hz, 2H), 7.79 (d, J ) 7.8 Hz, 2H),
8.35 (br s, 1H), 8.85 (br s, 1H); δC (75.47 MHz, CDCl3/5%
DMSO-d6) 9.0, 21.1, 27.0, 28.2, 29.3, 32.8, 50.5, 60.6, 63.3,
119.1, 123.6, 126.3, 127.4, 129.5, 129.8, 136.7, 137.6, 144.5,
151.7, 153.5, 156.6, 163.1, 172.8, 175.6.
N-Methyl-p-toluenesulfonamide Sulfenyl Chloride. Sul-
fur dichloride was distilled under nitrogen at 58-60 °C with
0.1% w/v phosphorus pentachloride.15 Sulfur dichloride (0.12
mL, ∼1.5 mmol) was added to N-methyl-p-toluenesulfonamide
(193 mg, 1 mmol) in chloroform (0.5 mL) under nitrogen, and
this mixture was left overnight in a stoppered glass vial at
room temperature. The solvent and other volatile components
were removed under a stream of nitrogen to leave a yellow
solid: δH (90 MHz, CDCl3) 2.48 (s, 3H), 3.43 (s, 3H), 8.64 (dd,
J ) 9.5, 45 Hz, 2H). This material was used directly in the
following reaction.
Benzhydryl N-Methyl-N-tosyl-S-aminosulfenamidoce-
phalosporinate (7). To a solution of 8 (0.5 g, 1.14 mmol) in
chloroform (15 mL) was added to N-methyl-p-toluenesulfon-
amide sulfenyl chloride (0.36 g, 1.43 mmol) in chloroform (15
mL). The reaction mixture was washed immediately with 0.25
M sodium bicarbonate (2 Iˆ 30 mL) and distilled water (2 × 20
mL), separated, dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure to yield a yellow solid.
Purification by wet flash chromatography (silica gel, dichlo-
romethane/ethyl acetate 70/30) gave 7 as a yellow solid (0.51
g, 0.8 mmol, 68%): mp 76-78 °C; δH (300 MHz, CDCl3) 1.64
(br s, 1H), 2.02 (s, 3H), 2.42 (s, 3H), 3.28 (s, 3H), 3.39 (d, J )
18.6 Hz, 1H), 3.64 (d, J ) 18.6 Hz, 1H), 4.79 (d, J ) 13.53 Hz,
1H), 5.02 (d, J ) 13.8 Hz, 1H), 5.10 (d, J ) 4.50 Hz, 1H), 5.29
(dd, J ) 4.5, 10.2 Hz, 1H), 6.95 (s, 1H), 7.26-7.45 (m, 12H),
7.75 (d, J ) 8.19 Hz, 2H); δC NMR (75.47 MHz, CDCl3) 20.7,
21.6, 26.7, 40.9, 58.6, 63.1, 73.1, 79.7, 125.8, 126.3, 127.1, 127.3,
127.7, 128.1, 128.2, 128.5, 128.6, 129.8, 136.2, 139.1, 139.2,
144.0, 160.8, 165.1, 170.5. Anal. Calcd for C31H31N3O7S3: C,
56.95; H, 4.78; N, 6.43. Found: C, 56.77; H, 4.73; N, 6.27.
Benzhydryl N-Methyl-N-tosyl-S-aminosulfeniminoce-
phalosporinate (8). Electrochemical Method. The elec-
trolysis was carried out in a biphasic cell system in a beaker-
type undivided cell, with two Pt electrodes (2 × 2 cm2) held in
the upper aqueous layer (25 mL) containing MgBr2 (0.5 g, 2.71
mmol) and with sulfenamide 7 (150 mg, 0.23 mmol) in
dichloromethane (25 mL) as the (lower) organic layer. Direct
current (20-25 mA) was applied, and the mixture was stirred
at a rate that was moderate enough to allow the electrodes to
remain immersed in the aqueous medium. After 4.5 h the
organic layer was separated, washed with brine (2 × 25 mL)
and distilled water (25 mL), separated, dried over anhydrous
MgSO4, filtered, and concentrated under reduced pressure to
yield a dark brown solid. Purification by gravity chromatog-
raphy (silica gel, 90/10 dichloromethane/ethyl acetate) yielded
8 as a light yellow solid (113.4 mg, 0.17 mmol, 76%): mp 86-
89 °C (lit.3a 87-92); δH NMR3a (90 MHz, CDCl3) 2.03 (s, 3H),
2.45 (s, 3H), 3.28 (d, J ) 7.95 Hz, 1H) overlapping with (s,
7-(N-Tosyl-S-aminosulfenimino)-3-[[3′-[[(4-aminocar-
bonyl)phenyl]-3′-ethyl-2′,6′-piperidinedione]oxy]meth-
yl] Cephalosporanic Acid (6). A solution of aluminum
trichloride (25.95 mg, 0.19 mmol) in nitromethane (0.5 mL)
was added to dichloromethane (8 mL) containing the benzhy-
dryl ester 24 (83.1 mg, 0.097 mmol) at -84 °C (bath), and the
mixture was stirred for 20 min. Next, 5% sodium bicarbonate
(25 mL) and ethyl acetate (25 mL) were added, and the mixture
(15) Tardif, S. L.; Harpp, D. N. J. Org. Chem. 2000, 65, 4791-4795.
J. Org. Chem, Vol. 69, No. 23, 2004 7969

Grant and Smyth
3H), 3.50 (d, J ) 7.95 Hz, 1H), 4.76 (d, J ) 12.54 Hz, 1H),
5.03 (d, J ) 12.54 Hz, 1H), 5.49 (s, 1H), 6.99 (s, 1H), 7.22-
7.55 (m, 12H), 7.79 (d, J ) 7.98 Hz, 2H).
dichloromethane) gave 8 as a yellow solid (134.5 mg, 0.21
mmol, 53%). The 1H NMR spectrum was identical to that given
above.
Benzhydryl N-Methyl-N-tosyl-S-aminosulfeniminoce-
phalosporinate (8). Mitsunobu Process. Triphenylphos-
phine (205.3 mg, 0.78 mmol) was dissolved in a solution of
methanol (32 µL, 0.78 mmol) and dichloromethane (1.5 mL)
under N2. The solution was cooled to -23 °C (bath), and di-
isopropyldiazidodicarboxylate (154.5 µL, 0.78 mmol) in dichlo-
romethane (3 mL) was added dropwise with stirring. DIAD
(yellow) decolorized almost instantaneously. 93a (250 mg, 0.39
mmol) in dichloromethane (2 mL) was added, and the reaction
mixture was stirred for 10 min at -23 °C (bath) and then
allowed to warm to ambient temperature (total reaction time
of 45 min). Dichloromethane (15 mL) was added, and the
organic layer was washed with brine (2 × 30 mL) and distilled
water (2 × 30 mL), separated, dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure to yield a
yellow solid. Purification by flash chromatography (silica gel,
Acknowledgment. We are grateful to B. Kennedy
and L. Kirby for technical assistance, to D. McCarthy
(University College Cork) for high-resolution NMR
spectra, to Ken Harris (Scripps Center for Mass Spec-
trometry) for high-resolution mass spectra, to M. Karner
(Sandoz GmbH) for a generous gift of 7-ACA, and to
Enterprise Ireland for financial assistance (J.W.G.).
Supporting Information Available: Copies of 1H and 13
C
NMR spectra for 3a, 3b, 4, 5, and 6. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO048970I
7970 J. Org. Chem., Vol. 69, No. 23, 2004