A new linker for glucuronylated anticancer prodrugs

Article abstract of DOI:10.1016/j.bmc.2003.11.026

The synthesis, enzymatic hydrolysis and self decomposition of model glucuronylated prodrugs, incorporating a new linker with different aryl substituents, have been studied. Determination of kinetic parameters (V _max, K_m and t_1/2

Full text of DOI:10.1016/j.bmc.2003.11.026

Bioorganic & Medicinal Chemistry 12 (2004) 675–682
A new linker for glucuronylated anticancer prodrugs
Freddy Rivault, Isabelle Tranoy-Opalinski and Jean-Pierre Gesson*
` ´ ´ ´
UMR 6514, Laboratoire de Synthese et Reactivite des Substances Naturelles, Universite de Poitiers,
40 avenue du Recteur Pineau, 86022 Poitiers, France
Received 18 July 2003; accepted 21 November 2003
Abstract—The synthesis, enzymatic hydrolysis and self decomposition of model glucuronylated prodrugs, incorporating a new
linker with different aryl substituents, have been studied. Determination of kinetic parameters (Vmax, Km and t1/2) showed the
important role of aromatic substitution in enzymatic recognition and linker decomposition.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
1,6-eliminations of an unstable carbamic acid after
hydrolysis of a phenol glucuronide. This work led to the
preclinical studies of HMR 1826 (Fig. 1) which has
demonstrated a superior efficiency in vivo compared to
doxorubicin toward several cancer cell lines.8 Related
linkers have been based on aniline derivatives and simi-
lar 1,4- and 1,6-elimination processes.9 In this strategy,
a glycoside is linked to an appropriate 4-amino benzyl
alcohol via a carbamate function, the amino drug being
then introduced via another carbamate.
The design of produgs is currently an area of high
interest in anticancer research. The lack of selectivity
toward cancer cells may be reduced by site specific
delivery of clinically used drugs which may be adminis-
tered as non toxic derivatives. Several strategies to
achieve this goal have been implemented using targeted
(ADEPT,1 GDEPT2) or endogenuous (PMT3) enzymes
or via specific chemical activation (hypoxia selective
agents4). The prodrug must fullfil some requirements
such as a highly reduced toxicity compared to the par-
ent drug, a large volume of distribution and a plasma
good stability, an easy recognition by the enzyme and a
fast liberation of the drug after activation to avoid dif-
fusion away from the tumour site. Therefore, a large
number of prodrugs have been designed in three parts,
that is, trigger, linker and effector units, which are gen-
erally, though not always, distinct (Fig. 1).5 The trigger
and effector units respectively depend on the mechanism
of activation of the prodrug and the targeted tumour
cells. They are joined together by the linker unit which
should release the drug, as fast as possible, after pro-
drug activation via a chemical breakdown. Thus, a
careful design of the linker unit is crucial for prodrug
efficiency.
In this paper, the synthesis and enzymatic cleavage of
new linkers based on the elimination of a carbamic acid
from a para-substituted 3-carbamoyl-2-aryl-propenal
are described (Scheme 1). The latter could arise from the
enzymatic hydrolysis of a b-glucuronide. Substitution of
the aryl moiety should play a key role in the enzyme-
catalysed hydrolysis (Vmax and Km) as well as in the
b-elimination of the carbamic acid, which, after
decarboxylation, will generate the amino drug (such as
doxorubicin).
This mechanism is reminiscent of the biotransformation
of felbamate, an anti-epileptic drug which has been
withdrawn from clinical use due to associated toxicity
such as aplastic anemia and hepatotoxicity. The meta-
bolic pathway of this drug has been studied in detail by
Mcdonald et al.:10 after hydrolysis of one carbamate
group, felbamate is oxidised in the liver to aldehyde 1
which is unstable (t1/2 c.a. 30s) and undergoes elimina-
tion to 2-phenylpropenal (atropaldehyde) 2 and mainly
reversible cyclisation to oxazinone 3 (Scheme 2). The
latter has a rather long half-life of 5 h and the sub-
sequent slow release of the toxic atropaldehyde
Different types of linker have been designed based on
elimination or cyclization processes.6 Thus, Monneret et
al.7 have previously studied linkers based on 1,4- and
* Corresponding author. Tel.: +33-5-4945-3862; fax: +33-5-4945-
3501; e-mail: jean-pierre.gesson@univ-poitiers.fr
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.11.026

676
F. Rivault et al. / Bioorg. Med. Chem. 12 (2004) 675–682
Figure 1. General principle of drug release; doxorubicin prodrug: HMR 1826.
Scheme 2. Metabolism of felbamate.
Scheme 1. Prodrug decomposition.
activated glucuronide 612 was carried out in CH3CN in
presence of Ag2O to afford b-glycosides 7a–c (54–71%).
As observed previously by Lubineau13 in the case of 2-
methylmalonaldehyde, only E-enol glycosides were
obtained. Then, reduction with NaBH4 in MeOH, in
(GI50=4.1ꢀ1.1 mM for HA1 cells) was assumed to
result from this side reaction. It is also interesting to
notice that acrolein (GI50=4.9ꢀ0.9 mM for HA1 cells)
is released from the well known anticancer agent cyclo-
phosphamide after bioactivation.
.
presence of CeCl3 7H2O, afforded the corresponding
alcohols 8a–c. It should be noticed that the isolated
yields for the nitro and fluoro derivatives were con-
sistently higher (90–95%) than for the methyl derivative
(44%) and that reduction is greatly improved in pre-
sence of cerium salts.14 Then several activating groups
were tested in order to introduce the carbamate moiety
(Table 1). Variable results were obtained, both in the
formation of a mixed carbonate or a mixed carbamate
and in the displacement with the amine, depending on
the X group. N,N-Carbonyldiimidazole and phenyl
chloroformate turned out to be the reagents of choice
for the first step but the latter proved to be less efficient
than the former one for the second step of amine
coupling, except for X=Me.
Thus, it seems worthwhile to design an anticancer pro-
drug based on such a linker since two toxic species, the
drug (effector) and a 2-arylpropenal will be released
near the tumor site. The synthesis, enzymatic hydrolysis
and self decomposition of model compounds incorpor-
ating different aryl substituents have been first studied
and are now reported.
2. Results
Three compounds have been prepared using 4-methoxy-
benzylamine as the model effector: 4a (X=NO2), 4b
(X=F) and 4c (X=Me) (Scheme 3). The starting aryl-
malonaldehydes 5a–c were either commercially available
(5c) or prepared in two steps from the corresponding
arylacetic acid according to the method of Arnold11 (46-
52% over yield). Glycosidation of 5a–c with the known
Finally, glucuronides 4a–c were obtained in two steps:
hydrolysis of acetate groups by the method of Zemplen
(cat. MeONa in MeOH) to afford 13a–c and then of the
methyl ester with Ba(OH)2 (61–66% overall).

F. Rivault et al. / Bioorg. Med. Chem. 12 (2004) 675–682
677
Scheme 3. Synthesis of compounds 4a–c.
Table 1. Results of alcohol activation and carbamate formation
Table 2. Kinetic results of enzymatic hydrolysis
Alcohol
Carbonate or
carbamate (%)
Carbamate
(%)
Glucuronides
Km
(mM)
Vmax
(mmol/min)
kcat
(sꢂ1
kcat/Km
(Mꢂ1.sꢂ1
)
)
8
a
(89)
9a 12a (83)
12a (47)
(n.r.)a
61077
46
0 75,40
10a (99)
11a (76)
4a
4b
4c
120
1140126
504
17.5
10.5
066,20
2.5
87,500
4,950
8
8
b
(94)
10b (71)
9b 12b (44)
12b (9)
75.5
11b (n.r.) a
(n.r.)a
4
c
(81)
9c 12c (50)
12c (65)
(n.r.)a
10c (78)
11c (38)
3. Discussion
a No reaction was observed.
Prodrugs 4a and 4b are excellent substrates of the
enzyme (high kcat/Km), comparable to the reference
compound (p-nitrophenyl glucuronide) and much more
better than 4c (Table 2). These results are consistent
with those obtained for phenol glucuronides: affinity
and hydrolysis rate are enhanced with electron with-
drawing group substitution on the aromatic ring.15 The
2.1. Enzymatic hydrolysis
First, model prodrugs 4a–c were incubated in 0.02 M
phosphate buffer (pH=7) at 37 ꢁC. After 48 h no
decomposition of 4a–c was detected. Then, enzymatic
studies were conducted, in the presence of Escherichia
coli b-glucuronidase to determinate kinetic parameters,
Vmax and Km, of the glycosidic bounds hydrolysis (see
Experimental). Results are reported in Table 2 with
para-nitrophenol b-glucuronide as reference.
highest ratio kcat/Km (87,500 Mꢂ1 ꢂ1) of 4a is due to a
s
low Km (120 mM) resulting from a strong enzyme affi-
nity. Thereby, and despite a lower kcat (10.5 sꢂ1), 4a will
undergo a relative fast hydrolysis at low concentrations
of substrate and enzyme. Thus, 4a can be considered as
a good candidate for an ADEPT protocol. The inter-
esting kcat/Km ratio (66,200 Mꢂ1.Sꢂ1) for 4b is due to a
high kcat (75.5 sꢂ1), but at low concentration (like
nearby tumours), 4b is expected to be less efficient than
4a. Introduction of a weak electron donating group in
4c did not give any satisfactory results.
In a second step, we investigated the rate of the linker
decomposition (Scheme 4). After addition of an excess
of enzyme, the reaction was followed by HPLC. The
intermediate compounds 14a–c were immediately
observed followed by the simultaneous formation of the
expected amine and of a 2-arylacrolein. The half-life of
the methyl intermediate 14c was found to be much
higher (t1/2=13.5 h) than for the fluoro 14b (t1/2=5.3 h)
and the nitro 14a (t1/2=1.6 h) intermediates.
Determination of t1/2 is also of great importance to
evaluate the potential of such linkers for prodrug use.
The slow half-life observed for intermediates 14a–c

678
F. Rivault et al. / Bioorg. Med. Chem. 12 (2004) 675–682
Scheme 4. Postulated decomposition of the linker.
should allow undesired diffusion of the drug from the
tumour site. Results are similar to the reported half-life
of felbamate metabolite 3 (t1/2=5 h)10 which exists as an
equilibrium with aldehyde 1 (Scheme 2). In our case, for-
mation of oxazinones 15a–c is also possible (Scheme 4).
However, the 10times acceleration of amine release on
going from the methyl intermediate to the nitro one is
consistent with the increase of acidity of the benzylic
proton for 14a (compared to 14b and 14c). The form-
ation of N-substituted oxazinones 15a–c may lower the
concentration of the intermediate aldehyde 14a–c (this
process should be rather insensitive to the para sub-
stituent of the aryl group), but the rate limiting step may
be formation of the enolate 16a–c. On the one hand, the
lower rate of elimination for such linkers, compared to
aryl ones such as in HMR 1826, is thus mainly due (as
shown in the aryl series) to the lower concentration of
the enolate (compared to the phenate). On the other
hand, a more hindered effector such as doxorubicin,
could prevent oxazinone formation and thus higher rate
of elimination may be observed.
carried out using a Waters HPLC system with UV
detection at 254 nm. The separation was performed on a
reversed phase column (Discovery RP amide C16,
150ꢃ4.6 mm) using isocratic conditions (1 mL/min),
eluent: 0.02 M phosphate buffer (pH=3)–acetonitrile
60:40.
5.2. Stability of compounds in a buffer solution
A solution of 100 mg/mL of prodrug in 0.02 M phos-
phate buffer (pH=7.2) was incubated at 37 ꢁC. Aliquots
were taken at various times and analysed by HPLC.
5.3. Enzymatic cleavage by E. coli ꢀ-D-glucuronidase
Hydrolysis was investigated by incubating a solution of
62.5 mg/mL of prodrug and 100 U/mL of E. coli b-d-
glucuronidase in 0.02 M phosphate buffer (pH=7.2)
at 37 ꢁC. Aliquots were taken at various times and
analysed by HPLC.
5.4. Determination of Km and Vmax values
Experiments were run at different prodrug concentra-
tions [80to 160 mg/mL in 0.02 M phosphate buffer
(pH=7.2)] in presence of E. coli b-glucuronidase (100
U/mL). After incubation for 8 min at 37 ꢁC, an aliquot
was taken and analysed by HPLC and Km and Vmax
values were calculated from the Lineweaver–Burk plot.
4. Conclusion
Three glucuronylated prodrugs, incorporating a new
linker, have been synthetised and evaluated with a
model effector. Our synthetic strategy is suitable for any
amine compound and connection with the linker is
made by a stable (in vivo) carbamate. After enzymatic
hydrolysis, drug release is observed via elimination of a
carbamic acid from a substituted 3-carbamoyl-2-aryl-
propenal. Kinetic studies in phosphate buffer have
pointed out the important role of the aromatic sub-
stitution in enzymatic recognition and linker decom-
position. Our linker is of special interest as it behave as
a dual-release drug. Further studies are now needed to
improve the self decomposition process and evaluate (in
vitro and in vivo) the potential of such linker in a drug
targeting strategy.
5.5. General procedure for preparation of Z-2-aryl-3-hy-
droxypropenal 5a,b11
5.5.1. Z-2-aryl-3-N,N-dimethylaminopropenal. To a vig-
orously stired solution of DMF (2.9 mL) at 0 ꢁC, POCl3
(2.8 mL) was added dropwise. After 5 min, the arylace-
tic acid (0.01 mol) in solution in DMF (5 mL) was
carefully added. Then the reaction mixture was warm
up to 70 ꢁC overnight and then poured on ice.
For X=NO2: after neutralisation by K2CO3, a solution
of NaOH 50% (12 mL) was added and a precipitate was
obtained upon cooling at 0 ꢁC. The filtrate was dis-
solved in CH2Cl2 and recrystallized from Et2O to give a
yellow powder (65%).
5. Experimental
5.1. General methods
All solvents used were of HPLC quality and chemicals
were of analytical grade. E. coli b-glucuronidase was
purchased from Sigma-Aldrich.16 Analytical HPLC was
For X=F: the solution was basified with K2CO3, 10 mL
of toluene was added and the mixture was allowed to
reflux during 1 h 30min. The remaining solution was

F. Rivault et al. / Bioorg. Med. Chem. 12 (2004) 675–682
679
00
00
extracted with toluene and the organic phases were
washed with water. The combinated organic extracts
were dried and concentrated in vacuo. The crude pro-
duct was dissolved in a minimal amount of CH2Cl2 and
recrystallized from petroleum ether to afford a white
powder (68%).
H2 ); 8.23 (d, J=9 Hz, 2H, H3 ); 9.50(s, 1H, H CHO);
13C NMR (CDCl3) d(ppm): 20.3–20.4–20.5 (3CCH CO);
53.2 (COCH ); 68.4–70.2–70.6–73.0 (C2,3,4,5); 100.83(C1);
3
0
123.1–123.3–130.3–130.5 (C2 ,1
00
(C1 ); 166.5–168.8–169.3–169.7 (CCH
(CCHO); [a]D ꢂ22 (c 0.10, CHCl3); HRMS (ESI) calcd
for C22H23NO13Na (M+Na+) 532.1067, found
532.1070.
00
); 136.0(C ); 147.0
000200,300
4
OOCH ); 189.3
C
C
O,
3
3
5.5.2. Z-2-(40-Nitrophenyl)-3-N,N-dimethylaminoprope-
nal. Mp 131 ꢁC; H NMR (CDCl3) d (ppm): 2.89 (sl,
1
6H, HN(CH )2); 6.97 (sl, 1H, H3); 7.38 (d, J=9 Hz, 2H,
0
5.6.2. 2,3,4-Tri-O-acetyl-(20-(40-fluorophenyl)-30-oxo-10-
E-propenyloxy)-ꢀ-D-methyl glucopyranosiduronate (7b).
White solid (60%); mp 163 ꢁC; 1H NMR (CDCl3) d
(ppm): 2.02 to 2.07 (3s, 9H, HCH CO); 3.76 (s, 3H,
3
0
H2 ); 8.21 (d, J=9 Hz, 2H, H3 ); 9.12 (s, 1H, HCHO);
[a]D=ꢂ16 (c=0.09; CHCl3).
5.5.3.
Z-2-(40-fluorophenyl)-3-N,N-dimethylaminopro-
HOCH ); 4.24 (d, J=9 Hz, 1H, H5); 35.17–5.32 (2m, 4H,
3
penal. Mp 80 ꢁC; H NMR (CDCl3) d (ppm): 2.82 (sl,
H1,2,3,4); 7.08 (t, J=9 Hz, 2H, H3 ); 7.29 (s, 1H, H1 );
00
1
00
0
6H, HN(CH )2); 6.81 (sl, 1H, H3); 7.03–7.13 (m, 4H,
Harom); 9.10(s, 1H, H CHO); [a]D=ꢂ1 (c=0.30; CHCl3).
7.42 (d, J=9 Hz, 2H, H2 ); 9.48 (s, 1H, HCHO). 13C
3
NMR (CDCl3) d (ppm): 20.4-20.5–20.6 (3CCH CO);
53.2 (COCH ); 68.5–70.1–70.7–73.0 (C2,3,4,5); 100.53(C1);
3
0
00
00
00 00
114.9–115.2 (C2 ,3 ); 124.6 (C2 ); 131.2–131.3 (C1 ,4 );
5.5.4. Z-2-aryl-3-hydroxypropenal (5a,b). 25% NaOH
(20mL) was added to a solution of Z-2-aryl-3-N,N-
dimethylaminopropenal (0.01 mmol) in ethanol (15 mL)
and the reaction mixture was stirred at reflux for 3 h.
Ethanol was removed under reduce presure and the
residue was cooled down with ice to give a powder
which was filtered and washed with CH2Cl2, then taken up
in water and acidified by 6 N HCl to give a precipitate.
0
162.3 (C1 ); 166.5–168.8–169.3–169.8 (CCH
OOCH );
C
C
O,
3
3
190.3 (CCHO); [a]Dꢂ3 (c 0.17, CHCl3); HRMS (ESI)
calcd for C22H23O11FNa (M+Na+) 505.1122, found
505.1122.
5.6.3. 2,3,4-tri-O-acetyl-(20-(40-methylphenyl)-30-oxo-10-
E-propenyloxy)-ꢀ-D-methyl glucopyranosiduronate (7c).
White solid (54%); mp 126 ꢁC; 1H NMR (CDCl3) d
X=NO2: the solid was disolved in acetone and recrys-
tallized from ether to give 5a as a light brown powder
(71%).
(ppm): 2.00–2.04 (3s, 9H, HCH CO); 2.34 (s, 3H,
3
HPhCH3); 3.73 (s, 3H, HOCH ); 4.26 (d, J=8.8 Hz, 1H,
H5); 5.15–5.21–5.29–5.35 (2m, 4H, H1,2,3,4); 7.17 (d,
3
00
10,200
J=8.1 Hz, 2H, H3 ); 7.28–7.30(m, 3H, H
); 9.44 (s,
X=F: 5b was obtained as a white solid (77%).
1H, HCHO); 13C NMR (CDCl3)
d (ppm): 20.4
(CCH CO); 21.1 (CPhCH3); 52.9 (COCH ); 68.4–69.9–
3
5.5.5. Z-2-(40-nitrophenyl)-3-hydroxypropenals (5a). Mp
70.6–372.7 (C2,3,4,5); 100.3 (C1); 125.1–126.0–128.5–129.0
223 ꢁC; H NMR (CDCl3) d (ppm): 7.45 (d, J=9 Hz,
(C2 ,1 ,2 ,3 ); 137.7 (C4 ); 162.4 (C1 ); 166.5–168.7–169.1–
169.8 (CCH OOCH ); 190.8 (CCHO); [a]D +0( c 0.23,
1
0
00 00 00
00
0
0
0
2H, H2 ); 8.28 (d, J=9 Hz, 2H, H3 ); 8.76 (s, 2H, H3,
3CO,
C
3
CHO); 14,67 (sl, 1H, OH); 13C NMR (CDCl3) d (ppm):
CHCl3); HRMS (ESI) calcd for C23H26O11Li
(M+Li+) 485.1635, found 485.1653.
0
113.2 (C-2); 124.5 (C1 , C3 ); 126.4 (C2 ); 149.2 (C3);
0
0
0
164.3 (C4 ); 187.5 (CHO).
5.7. General procedure of reduction
5.5.6. Z-2-(40-fluorophenyl)-3-hydroxypropenals (5b).
ꢁ
Hz, 2H, H3 ); 7,24 (d, J=6.5 Hz, 2H, H2 ); 8,60(s,
1
Mp 163 C; H NMR (CDCl3) d (ppm): 7.10(t, J=8.5
To a stirred solution of aldehyde 7 (1 equiv) and
CeCl3.7H2O (1 equiv) in CH3OH (7.5 mL/mmol) was
added NaBH4 (1 equiv). After 5 min, the reaction mix-
ture was neutralised with 1 N HCl and extracted with
CH2Cl2. The organic layer was dried over MgSO4, fil-
tered and concentrated in vacuo. Recrystallisation from
AcOEt/Pet. ether gave alcohols 8.
0
0
2H, H3, CHO); 14,29 (sl, 1H, OH); 13C NMR
0
(CDCl3) d (ppm): 116,1 (d, J=21 Hz, C3 ); 117.5 (C2);
0
0
128 4 (d, J=8 Hz, C2 ); 129.5 (C1 ); 160.7 (C3); 164.0
0
0
(C4 ); 180.9 (CHO).
5.6. General procedure for glycosidation
5.7.1. 2,3,4-Tri-O-acetyl-(20-(40-nitrophenyl)-30-hydroxy-
10-E-propenyloxy)-ꢀ-D-methyl glucopyranosiduronate (8a).
Yellow powder (95%); mp 206 C, H NMR (CDCl3) d
A solution of Z-2-aryl-3-hydroxypropenal 5 (0.01 mol),
1 equiv of bromoglucuronide 6 (3.97 g) and 1.5 equiv of
freshly prepared Ag2O in CH3CN (20mL) was stirred at
0 ꢁC for 3 h. The reaction mixture was filtered through
Celite and concentrated in vacuo. The residue was dis-
solved in CH2Cl2 and recrystallized from CH3OH.
ꢁ
(ppm): 2.00–2.04 (3s, 9H, HCH CO); 3.75 (s, 3H,
1
3
0
HOCH ); 4.18 (d, J=8.8 Hz, 1H, H5); 4.47 (s, 2H, H3 );
3
4.92 (d, J=7.4 Hz, 1H, H1); 5.28–5.32 (2m, 3H, H2,3,4);
0
00
6.76 (s, 1H, H1 ); 7.79 (d, J=9 Hz, 2H, H2 ); 8.19 (d,
00
J=9 Hz, 2H, H3 ); 13C NMR (CDCl3) d (ppm): 20.4
5.6.1. 2,3,4-Tri-O-acetyl-(20-(40-nitrophenyl)-30-oxo-10-E-
propenyloxy)-ꢀ-D-methyl glucopyranosiduronate (7a).
(CCH3CO); 53.1 (COCH ); 63.6 (C3 ); 68.8–70.3–71.2–72.8
0
3
0
00 00 00
(C2,3,4,5); 100.1 (C1); 118.0(C 2 ); 123.3–128.8 (C1 ,2 ,3 );
Yellow solid (71%); mp 166 ꢁC; H NMR (CDCl3) d
1
00
141.4 (C4 ); 144.2 (C1 ); 166.6–168.9–169.3–169.9
0
(ppm): 2.02 to 2.06 (3s, 9H, HCH3CO); 3.75 (s, 3H,
HOCH ); 4.29 (d, J=9 Hz, 1H, H5); 5.17 to 5.34 (2m,
0
4H, H1,2,3,4); 7.47 (s, 1H, H1 ); 7.64 (d, J=9 Hz, 2H,
(CCH
OOCH ); [a]D ꢂ21 (c 0.18, CHCl3); HRMS
C
C
O,
3
(ESI)3 calcd for C22H25NO13Na (M+Na+) 534.1224,
found 534.1224.
3

680
F. Rivault et al. / Bioorg. Med. Chem. 12 (2004) 675–682
5.7.2. 2,3,4-tri-O-acetyl-(20-(40-fluorophenyl)-30-hydroxy-
10-E-propenyloxy)-ꢀ-D-methyl glucopyranosiduronate (8b).
White powder (90%); mp 148 C; H NMR (CDCl3) d
0
H1 ); 7.02–7.07 (m, 3H, H2
000
); 7.35 (s, 1H, H3 ); 7.50
000,300
(t, J=6.4 Hz, 2H, H2 ); 8.06 (s, 1H, H1 ); 13C NMR
00
000
ꢁ
(ppm): 1.92 to 2.03 (3s, 9H, HCH CO); 3.73 (s, 3H,
1
(CDCl3) d(ppm): 20.6 (CCH CO); 55.4 (COCH ); 64.7
0
(C3 ); 69.0–70.4–71.4–71.8 (C2,3,4,5); 100.2 (C1); 114.4–
3
3
3
0
HOCH ); 4.14 (d, J=8.8 Hz, 1H, H5); 4.31 (s, 2H, H3 );
000,300,2000,3000
); 130.8 (C);
122.1–125.2–129.2–129.3 (C2,2
0
3
00 00 000
4.85 (d, J=7.4 Hz, 1H, H1); 5.13–5.29 (2m, 3H, H2,3,4);
00
144.8 (C); 153.2 (C); 156.0(C
166.7–169.0–169.4–170.0 (CCH
); 159.4 (CO ON);
C
1 ,1 ,4 ,1
0
6.57 (s, 1H, H1 ); 7.00 (t, J=8.8 Hz, 2H, H3 ); 7.53 (dd,
00
OOCH ); [a]D ꢂ17 (c
C
C
O,
3
3
J=5.5–8.8 Hz, 2H, H2 ); 13C NMR (CDCl3) d (ppm):
0.26, CHCl3); HRMS (ESI) calcd for C26H27N2O12FNa
(M+Na+) 601.1446, found 601.1449.
0
20.3 (CCH CO); 53.0(C OCH ); 63.6 (C3 ); 69.0–70.2–
3
71.4–72.5 (C2,3,4,5); 99.9 (C1); 3114.7–115.0–119.2–129.8–
0
00 00 00 00
0
129.9 (C2 ,1 ,2 ,3 ,4 ); 141.2 (C1 ); 166.8–169.0–169.4–
170.0 (CCH OOCH ); [a]D +40( c 0.12, CHCl3);
5.8.3. Preparation of 10c: 2,3,4-tri-O-acetyl-(20-(40-me-
thylphenyl)-30-(O-phenylcarbonyloxy)-10-E-propenyloxy)-
ꢀ-D-methyl glucopyranosiduronate. Alcohol 8c (2.6
mmol, 1.26 g) was disolved in dichloromethane with
pyridine (3 equiv 0.6 mL). The mixture was stirred at
0 ꢁC and phenyl chloroformate (3 equiv, 0.98 mL) was
added dropwise, the colorless solution became yellow
and precipitated. After 30min at 0 ꢁC, the reaction was
quenched with a saturated solution of NaHCO3 and
extracted with dichloromethane. After filtration with
Na2SO4 and solvent evaporation, a yellow oil was
obtained. Compound 10c was obtained as a white pow-
der after recrystallisation with AcOEt/pet. ether (1.225
g, 78%).
C
C
O,
3
3
HRMS (ESI) calcd for C22H25O11FNa (M+Na+)
507.0750, found 507.0770.
5.7.3. 2,3,4-tri-O-acetyl-(20-(40-methyophenyl)-30-hydroxy-
10-E-propenyloxy)-ꢀ-D-methyl glucopyranosiduronate (8c).
White powder (44%); mp 124 ꢁC, H NMR (CDCl3) d
(ppm): 1.92–2.03 (3s, 9H, HCH CO); 2.33 (s, 3H,
HPhCH3); 3.74 (s, 3H, HOCH ); 4.12 (d, J=8.8 Hz, 1H,
0
H5); 4.36 (s, 2H, H3 ); 4.83 (d, J=7.4 Hz, 1H, H1); 5.13–
1
3
3
0
5.30(m, 3H, H 2,3,4); 6.58 (s, 1H, H1 ); 7.14 (d, J=8.2
00
Hz, 2H, H3 ); 7.44 (d, J=8.2 Hz, 2H, H2 ); 13C NMR
00
(CDCl3) d (ppm): 20.5 (CCH CO); 21.2 (CPhCH3); 53.0
0
(COCH ); 63.9 (C3 ); 69.1–70.4–71.6–72.8 (C2,3,4,5);
0
100.0 (C1); 120.3 (C2 ); 128.0–128.9 (C2 , ); 131.3–
00 00
136.9 (C1 ,4 ); 140.9 (C1 ); 166.8–169.0–169.3–170.0
3
3
1H NMR (CDCl3): 1.95–2.05 (3s, 9H, HCH CO); 2.35 (s,
3H, HPhCH3); 3.73 (s, 3H, HOCH ); 4.14 (m, 1H, H5);
00
300
3
0
3
0
4.88 (d, J=7.3 Hz, H, H1); 4.98 (s, 2H, H3 ); 5.18–5.30
(CCH
OOCH ); [a]D ꢂ34 (c 0.12, CHCl3); HRMS
C
C
O,
(ESI)3calcd for C323H28O11Li (M+Li+) 487.1792, found
487.1793.
(m, 3H, H2,3,4); 6.75 (s, 1H, H1 ); 7.11–7.46 (m, 9H,
0
Harom); 13C NMR (CDCl3) d (ppm): 20.5 (CCH CO);
3
0
21.2 (CPhCH3); 53.0(C OCH ); 68.9 (C3 ); 69.7–70.3–
3
0
71.4–72.8 (C2,3,4,5); 100.1 (C1); 115.3 (C2 ); 121.5–126.5–
127.8–128.3–129.4–129.9–130.9–137.6 153.62 (Carom);
0
144.4 (C1 ); 151 (COCOO); 166.7-168.9-169.3-170.0
5.8. General procedure for preparation of compounds
9a,b
To a solution of alcohol 8 (1 mmol) in dry dichloro-
methane (10mL), carbonyldiimidazole (2 equiv) was
added and the mixture was stirred for 30min at room
temperature. The solvent was removed in vacuo and the
desired compounds were obtained after recrystallisation
from CH2Cl2/Et2O.
(CCH
OOCH ).
C
O,
3
C
3
5.9. General procedure for p-methoxybenzyl amine
coupling
To a stirred solution of activated alcohols 9a,b–10c
(0.32 mmol) in CH3CN (4 mL), para-methox-
ybenzylamine (1 equiv) and DMAP (1 equiv) were
added at room temperature and the solution was stirred
for 24 h. The reaction was hydrolysed with a saturated
solution of NaHCO3 and extracted with CH2Cl2. The
organic layers were dried with MgSO4 and the solvents
were removed in vacuo. The crude reaction mixture was
purified by flash chromatography (eluent: MeOH/
CH2Cl2 1:99).
5.8.1. 2,3,4-Tri-O-acetyl-(20-(40-nitrophenyl)-30-(N-imido-
carbonyloxy)-10-E-propenyloxy)-ꢀ-D-methyl glucopyra-
nosiduronate (9a). Yellow powder (65%); mp 130 ꢁC;
1H NMR (CDCl3): 2.00–2.04 (3s, 9H, HCH CO); 3.76 (s,
3H, HOCH ); 4.19 (d, J=9 Hz, 1H, H5); 5.300 (d, J=7.4
3
0
Hz, 1H, H1); 5.19–5.37 (m, 5H, H2,3,4,3 ); 7.00–7.11 (m,
0
000,3000
2H, H2
00
); 7.35 (s, 1H, H1 ); 7.73 (d, J=9 Hz, 2H,
000
H2 ); 8.07 (s, 1H, H1 ); 8.23 (d, J=9 Hz, 2H, H3 ); 13C
00
NMR (CDCl3) d (ppm): 20.7 (CCH CO); 53.3 (COCH3);
3
5.9.1. 2,3,4-Tri-O-acetyl-20-(400-nitrophenyl)-30-(4000-meth-
oxybenzylaminocarbonyloxy) -10 -E-propenyloxy)-ꢀ-D -
0
68.6–68.8–70.4–71.2–73.0 (C2,3,4,5,3 ); 100.4 (C1); 112.5
300,2000,3000
0
(C2 ); 117.1–123.8–128.6 (C2 ,
00
); 131.0–137.2–
140.4 (C1 ,4 ,1 ); 146.6 (C1 ); 148.1 (COO N); 166.6–
1
00 00 000
0
methyl glucopyranosiduronate (12a). Red oil (83%); H
NMR (CD3COCD3) d (ppm): 1.82–2.11 (3s, 9H,
HCH CO); 3.74–3.78 (2s, 6H, HOCH ); 4.18 (d, J=9 Hz,
C
169.0–169.4–170.0 (CCH
MeOH); HRMS (ESI) calcd for C26H27N3O14Na
(M+Na+) 628.1391, found 628.1387.
OOCH ); [a]D ꢂ3 (c 0.15,
C
C
O,
3
3
3
3
000
1H, H5); 4.25 (d, J=5.8 Hz, 2H, H1 ); 4.80–5.00 (m, 3H,
0
H1,3 ); 5.18–5.36 (m, 3H, H2,3,4); 7.16 (d, J=8.4 Hz, 2H,
5.8.2. 2,3,4-Tri-O-acetyl-(20-(40-methylphenyl)-30-(N-imi-
docarbonyloxy)-10-E-propenyloxy)-ꢀ-D-methyl glucopyr-
anosiduronate (9b). White powder (94%); mp 131 ꢁC;
1H NMR (CDCl3): 1.95–2.04 (3s, 9H, HCH CO); 3.75 (s,
H3 ); 7.70(d, J=8.7 Hz, 2H, H2 ); 8.15 (d, J=8.7 Hz, 2H,
00
000
00
H3 ); 13C NMR (CD3COCD3) d (ppm): 20.5 (3CCH CO);
0
44.4 (C1 ); 52.7–55.2 (2COCH ); 64.8 (C3 ); 68.8–70.2–71.1–
3
000
3
0
72.6 (C2,3,4,5); 100.1 (C6); 113.7 (C2 ); 123.0–130.2
3H, HOCH ); 4.17 (d, J=9 Hz, 1H, H5); 4.392 (d, J=7.3
,NCOO); 166.5–
00 00
(C2 ,3 ,3
0 00 00
000,5000
); 141.0–158.0 (C1 ,1 ,4 ,2
000,4000
3
0
Hz, H, H1); 5.07–5.30 (m, 5H, H2,3,4,3 ); 6.82 (s, 1H,
169.1–169.4–169.7 (CCH
OOCH ); [a]D ꢂ16 (c 0.09,
C
C
O,
3
3

F. Rivault et al. / Bioorg. Med. Chem. 12 (2004) 675–682
681
0
(C1); 112.8 (C2 ); 114.5 (C4 ); 123.8 (C3 ); 129.4–129.7
000
00
CHCl3); HRMS (ESI) calcd for C31H34N2O15Na
(M+Na+) 697.1857, found 697.1857.
00 000
(C2 ,3 ); 132.5 (C2 ); 143.4 (C1 ); 146.7 (C1 ); 150.6
000
0
00
00
000
(C4 ); 157.2 (C5 ); 159.7 (CNCOO); 169.6 (CCOOCH );
3
5.9.2.
2,3,4-Tri-O-acetyl-20-(400-fluorophenyl)-30-(4000-
[aD] +6 (c 0.16, MeOH); HRMS (ESI) calcd for
C25H28N2O12Na (M+Na+) 571.1127, found 571.1190.
methoxybenzylaminocarbonyloxy)-10-E-propenyloxy)-ꢀ-
D-methyl glucopyranosiduronate (12b). Yellow powder
5.10.2. [20-(400-fluorophenyl)-30-(4000-methoxybenzylami-
1
(44%); H NMR (CDCl3) d (ppm): 1.94–2.03 (3s, 9H,
HCH CO); 3.73–3.77 (2s, 6H, HOCH ); 4.13 (d, J=9 Hz,
nocarbonyloxy)-10-E-propenyloxy]-ꢀ-D-methyl glucopyr-
3
1H, H5); 4.25 (d, J=5.4 Hz, 2H, HC3 H2NH); 4.74 to 5.29
anosiduronate (13b). H NMR (CD3OD) d (ppm): 3.33–
3.60(m, 3H, H 2,3,4); 3.71–3.72 (2s, 6H, HOCH ); 3.92 (d,
1
00
(m, 7H, H1,2,3,4,3 , NH); 6.67 (s, 1H, H1 ); 6.83 (d, J=8.4
0
3
000
00
Hz, 2H, H3 ); 6.99 (d, J=8.4 Hz, 2H, H3 ); 7.15 (d,
000
J=9 Hz, 1H, H5); 4.12 (s, 2H, HC NH); 4.69–4.77 (m,
H2
J=8.4 Hz, 2H, H2 ); 7.48 (d, J=8.4 Hz, 2H, H2 ); 13C
00
0
3H, H3 ,1); 6.72 (s, 1H, H1 ); 6.79 (d, J=8.5 Hz,
0
000 00
2H,H3 ); 6.97 (t, J=8.8 Hz, 2H, H3 ); 7.09 (d, J=8.4
NMR (CDCl3) d (ppm): 20.3–20.4–20.5 (3CCH CO);
3
Hz, 2H,H2 ); 7.60(t, J=8.8 Hz, 2H, H2 ); 13C NMR
(CD CD) d(ppm): 44.8 (CCH NH); 52.9 (CCOO H3); 55.6
00
44.5 (CCH NH); 52.9–55.3 (2COCH ); 65.6 (C3 ); 69.4–
000
00
3
70.5–71.5–272.6 (C2,3,4,5); 100.2 (C1); 114.0(C ); 114.2
3000
3
C
2
0
00
000
(C2 ); 115.0(d, J=21 Hz, 2C, C3 ); 128.9 (C2 ); 129.8
0
(CO H3); 67.4 (C3 ); 72.8–74.2–77.0–77.2 (C2,3,4,5); 105.0
C
00
(d, J=8 Hz, 2C, C2 ); 130.3 (d, J=3 Hz, 1C, C1 ); 130.5
00
000 0 00
(C1); 114.8 (C3 ); 114.9 (C2 ); 115.5 (d, J=21 Hz, C3 );
000
(C1 ); 143.6 (C1 ); 156.3–160.1–163.3 (C4 ,4 ,NCOO);
0
000 00
000 00
129.5 (C2 ); 131.2 (d, J=8 Hz, C2 ); 132.6–132.8
00 000
(C1 ,1 );
0
(C1 );
167.0–169.0–169.4–170.0 (CCH
0.09, CHCl3); HRMS (ESI) calcd for C31H34NO13FNa
(M+Na+) 670.1912, found 670.1905.
OOCH ); [a]D ꢂ30( c
143.6
158.7–160.0–161.3–162.4
C
C
O,
3
3
000,400,4000
,
(C1
OONH); 170.9 (CCOOCH ); [a]D +36 (c 0.08,
C
3
MeOH); HRMS (ESI) calcd for C25H28NO10FNa
(M+Na+) 544.1595 found 544.1591.
5.9.3.
2,3,4-tri-O-acetyl-20-(400-methylphenyl)-30-(4000-
methoxybenzylaminocarbonyloxy)-10 -E-propenyloxy)-ꢀ-
D-methyl glucopyranosiduronate (12c). White powder
5.10.3. [20-(400-Methylphenyl)-30-(4000-methoxybenzylami-
nocarbonyloxy)-10-E-propenyloxy]-ꢀ-D-methyl glucopyr-
anosiduronate (13c). H NMR (CD3COCD3) d (ppm):
2.29 (s, 3H, HPhCH ); 3.47–3.59 (m, 3H, H2,3,4); 3.70–
3.74 (2s, 6H, HOCH ); 4.01 (d, J=9.6 Hz, 1H, H5); 4.21
1
1
(65%); H NMR (CDCl3) d (ppm): 1.93–2.04 (3s, 9H,
HCH CO); 2.32 (s, 3H, HPhCH ); 3.73–3.78 (2s, 6H,
HOCH ); 4.13 (d, J=9 Hz, 1H, H5); 4.25 (d, J=5.4 Hz,
3
3
3
3
3
0
2H, HCH NH); 4.82–5.28 (m, 7H, H1,2,3,4,3 ,NH); 6.65 (s,
0
(d, J=6 Hz, 2H, HC NH); 4.78–4.86 (m, 3H, H3 ,1);
H2
2
0
000
0 000
6.60(s, 1H, H NH); 6.80–6.85 (m, 3H, H1 ,3 ); 6.97 (d,
1H, H1 ); 6.83 (d, J=8.4 Hz, 2H, H3 ); 7.10–7.18 (m,
00 000
4H, H3 ,2 ); 7.40(d, J=8.4 Hz, 2H, H2 ); 13C NMR
(CDCl3) d(ppm): 20.3 (3CCH CO); 21.0(C PhCH3); 44.3
00
(CCH2NH); 52.8–55.1 (2COCH3); 65.5 (C3 ); 68.9–70.3–
J=8 Hz, 2H, H3 ); 7.10(d, J=8.2 Hz, 2H, H2 ); 7.55
00
00
00
000
(d, J=8.1 Hz, 2H, H2 ); 13C NMR (CD3COCD3) d
3
(ppm): 21.1 (CPhCH ); 44.5 (CCH NH); 52.4 (CCOO H );
C
3
2
3
000
71.4–72.6 (C2,3,4,5); 99.9 (C1); 113.8 (C3 ); 115.6 (C2 );
0
0
55.4 (CO H ); 66.2 (C3 ); 72.5–73.9–76.9–77.0(C 2,3,4,5);
C
3
00 00 000
127.7–128.6–128.7 (C2 ,3 ,2 ); 130.5 (C1 ); 131.2 (C1 );
000
00
0 000
105.0 (C1); 114.3–114.5 (C2 ,3 ); 128.8–129.3–129.4
00
0
000
136.7 (C4 ); 143.2 (C1 ); 156.3–158.7 (C4 ,NCOO); 166.6–
00 00 000 000 00 00
(C2 ,3 ,2 ); 132.6 (C1 ); 133.4 (C1 ); 136.6 (C4 ); 147.0
0
(C1 ); 157.4 (C4 ); 159.6 (CCOONH); 169.6 (CCOOCH );
[a]D +48 (c 0.11, MeOH); HRMS (ESI) calcd for
C26H31NO10Na (M+Na+) 540.1846, found 540.1843.
000
168.9–169.2–169.8 (CCH
OOCH ); [a]D ꢂ28 (c 0.03,
C
C
O,
3
3
CHCl3); HRMS (ESI3) calcd for C32H38NO13Na
(M+Na+) 666.2163, found 666.2166.
5.10. General procedure for deacylation
5.11. General procedure for methyl ester hydrolysis
To a solution of fully protected glucuronide 12a–c (0.35
mmol) in an anhydrous mixture of CH2Cl2/MeOH (8/
16: v/v), a 0.32 M solution of MeONa in MeOH (1.08
mL) was added at 0 ꢁC and the mixture was stirred for 2
h. Thereafter, the solution was neutralised by addition
of Amberlyst IRC-50ion-exchange resin. Filtration fol-
lowed by evaporation under reduced pressure of the fil-
trate afforded a crude residue which was purified by
flash chromatography.
To a solution of methyl ester 13a–c in methanol was
.
added Ba(OH)2 8H2O (1.2 equiv) and the mixture was
stirred for 4 h at room temperature. A white precipitate
was formed and filtered. Purity and structure were con-
firmed by HPLC and SM analysis. 1H NMR analysis of
4a,b gave poorly resolved spectra.
5.11.1. [20-(400-Nitrophenyl)-30-(4000-methoxybenzylamino-
carbonyloxy)-10-E-propenyloxy]-ꢀ-D-glucopyranosiduronic
acid (4a). [a]D ꢂ6 (c 0.9, MeOH); HRMS (ESI) calcd for
C24H26N2O12Na (M+Na+) 557.1278, found 557.1272.
5.10.1. [20-(400-Nitrophenyl)-30-(4000-methoxybenzylamino-
carbonyloxy)-10-E-propenyloxy]-ꢀ-D-methyl glucopyra-
nosiduronate (13a). 1H NMR (CD3COCD3) d (ppm):
5.11.2. [20-(400-Fluorophenyl)-30-(4000-methoxybenzylamino-
carbonyloxy)-10-E-propenyloxy]-ꢀ-D-glucopyranosiduro-
nic acid (4b). [a]D ꢂ5 (c 0.9, MeOH); HRMS (ESI)
calcd for C24H26NO10FNa (M+Na+) 530.1018, found
530.1023.
3.51–3.67 (m, 3H, H2,3,4); 3.72–3.75 (2s, 6H, HOCH );
3
4.04 (d, J=9.5 Hz, 1H, H5); 4.22 (d, J=6.2 Hz, 1H,
000
H1 ); 4.90(s, 2H, H 3 ); 4.98 (d, J=7.4 Hz, 1H, H1); 6.70
0
000
(s, 1H, HNH); 6.84 (d, J=8.6 Hz, 2H, H4 ); 7.13 (s, 1H,
0
000
H1 ); 7.19 (d, J=8.6 Hz, 2H, H3 ); 7.96 (d, J=9 Hz,
00
2H, H2 ); 8.17 (d, J=9 Hz, 2H, H3 ); 13C NMR
5.11.3. [20-(400-Methylphenyl)-30-(4000-methoxybenzylami-
00
000
(CD3COCD3) d (ppm): 44.6 (C1 ); 52.5 (CCOO H ); 55.4
nocarbonyloxy)-10 -E-propenyloxy]-ꢀ-D-glucopyranosi-
duronic acid (4c). H NMR (D2O) d (ppm): 2.35 (s, 3H,
C
3
1
0
(CO H ); 65.4 (C3 ); 72.5–73.9–76.8–76.9 (C2,3,4,5); 105.1
C
3

682
F. Rivault et al. / Bioorg. Med. Chem. 12 (2004) 675–682
HPhCH ); 3.36–3.60(m, 3H, H 2,3,4); 3.83 (s, 3H, HOCH3);
0
4.18 (s, 2H, HC NH); 4.76 (s, 2H, H ); 6.82 (s, 1H,
8. (a) Florent, J.-C.; Dong, X.; Gaudel, G.; Mitaku, S.;
Monneret, C.; Gesson, J.-P.; Jacquesy, J.-C.; Mondon,
M.; Renoux, B.; Andrianomenjanahary, S.; Michel, S.;
Koch, M.; Tillequin, F.; Gerken, M.; Czech, J.; Straub,
R.; Bosslet, K. J. Med. Chem. 1998, 41, 3572. (b) Bosslet,
K.; Straub, R.; Blumrich, M.; Czech, J.; Gerken, M.;
Sperker, B.; Kroemer, H. K.; Gesson, J.-P.; Koch, M.;
Monneret, C. Cancer Res. 1998, 58, 1195.
3
H2
3
0
H1 ); 6.95 (sl, 2H, H3 ); 7.10(sl, 2H, H ); 7.24 (sl, 2H,
000
00
3
000
00
H2 ); 7.39 (sl, 2H, H2 ); [a]D 0(c 0.2, MeOH); HRMS
(ESI) calcd for C25H29NO10Na (M+Na+) 548.1509,
found 548.1494.
9. (a) Leenders, R. G. G.; Gerrits, K. A. A.; Ruijtenbeek,
R.; Scheeren, H. W.; Haisma, H. J.; Boven, E. Tetra-
hedron Lett. 1995, 36, 1701. (b) Leenders, R. G. G.;
Scheeren, H. W.; Houba, P. H. J.; Boven, E.; Haisma,
H. J. Bioorg. Med. Chem. Lett. 1995, 5, 2975. (c) Leen-
ders, R. G. G.; Damen, E. W. P.; Bijsterveld, E. J. A.;
Scheeren, J. W.; Houba, P. H. J.; van der Meulen-Muile-
man, I. H.; Boven, E.; Haisma, H. J. Bioorg. Med. Chem.
1999, 7, 1597.
Acknowledgements
We thank MRT, CNRS and Region Poitou-Charentes
for financial support and La Ligue Nationale Contre le
Cancer Comite des Deux Sevres for a research grant
(F.R.).
10. (a) Thompson, C. D.; Kinter, M. T.; Macdonald, T. L.
Chem. Res. Toxicol. 1996, 9. (b) Thompson, C. D.; Gul-
den, P. H.; Macdonald, T. L. Chem. Res. Toxicol. 1997,
10, 457. (c) Dieckhaus, C. M.; Santos, W. L.; Sofia, R. D.;
Macdonald, T. L. Chem. Res. Toxicol. 2001, 14, 958. (d)
Dieckhaus, C. M.; Miller, T. A.; Sofia, R. D.; Macdonald,
T. L. Drug Metab. Dispos. 2000, 28, 814. (e) Kapetanovic,
I. M.; Torchin, C. D.; Thompson, C. D.; Miller, T. A.;
Mcneilly, P. J.; Macdonald, T. L.; Kupferberg, H. J.;
Perhach, J. L.; Sofia, R. D.; Strong, J. M. Drug Metab.
Dispos. 1998, 26, 1089. (f) Thompson, C. D.; Miller, T. A.;
Barthen, M. T.; Dieckhaus, C. M.; Sofia, R. D.; Macdo-
nald, T. L. Drug Metab. Dispos. 2000, 28, 434. (g) Diec-
khaus, C. M.; Roller, S. G.; Santos, W. L.; Sofia, R. D.;
Macdonald, T. L. Chem. Res. Toxicol. 2001, 14, 511. (h)
Roller, S. G.; Dieckhaus, C. M.; Santos, W. L.; Sofia,
R. D.; Macdonald, T. L. Chem. Res. Toxicol. 2002, 15,
815.
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