Benzopyridooxathiazepine derivatives as novel potent antimitotic agents

Article abstract of DOI:10.1016/j.bmc.2008.12.039

Herein, we describe the structure-activity relationship study of a new 1-(arylalkyl)-11H-benzo[f]-1,2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide series of antimitotic agents. The pharmacological results obtained from previous works allowed us to

Full text of DOI:10.1016/j.bmc.2008.12.039

Bioorganic & Medicinal Chemistry 17 (2009) 1132–1138
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Benzopyridooxathiazepine derivatives as novel potent antimitotic agents
Sebastien Gallet a, Nathalie Flouquet a, Pascal Carato a, Bruno Pfeiffer b, Pierre Renard b, Stéphane Léonce b,
Alain Pierré b, Pascal Berthelot a, Nicolas Lebegue a,
*
a Laboratoire de Chimie Thérapeutique EA1043, Faculté des Sciences Pharmaceutiques et Biologiques de Lille, 3 rue du Professeur Laguesse, B.P. 83 59006 LILLE Cedex, France
b Institut de Recherches Servier, Division Recherche Cancérologie, 125 Chemin de Ronde, 78290 Croissy sur Seine, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we describe the structure–activity relationship study of a new 1-(arylalkyl)-11H-benzo[f]-1,2-
dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide series of antimitotic agents. The pharmacological
results obtained from previous works allowed us to identify compound 1 as a new cytotoxic agent inhib-
iting tubulin polymerization. We have undertaken the synthesis of its non-methylated analogue 7 and
have extended our investigations to a novel, structurally related benzopyridooxathiazepine dioxide ser-
ies. Among all analogues synthesized in this study, compound 10b was the most promising, being 12-fold
more potent than compound 1. Its activity over a panel of five tumoral cell lines was in the nanomolar
range for all of the histological types tested and flow cytometric studies performed on L1210 cells showed
an accumulation of the cells in the G2/M phases of the cell cycle with a significant percentage of tetra-
ploid cells (8N DNA content). This interesting pharmacological profile, resulting from inhibition of tubulin
polymerization, encouraged us to perform preliminary in vivo studies.
Received 28 October 2008
Revised 12 December 2008
Accepted 16 December 2008
Available online 25 December 2008
Keywords:
Benzopyridooxathiazepine
Antimitotic
Tubulin polymerization
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
introduction of the arylalkyl chain at the N-11 position do not re-
sult in significantly cytotoxic compounds, while incorporation of
In our continuing search for antitumor agents, we synthesized a
large series of benzopyridothiadiazepine derivatives and found
that many of them were cytotoxics agent inhibiting tubulin poly-
merization.1,2 Pharmacomodulations performed within this class
of compounds led to the discovery of the potent compound 1,
exhibiting inhibitory activity against tubulin polymerization com-
parable to that of the potent antimitotic sulfonamides E7010 and
ER-678653–10 (Fig. 1). The structure–activity relationship (SAR)
we had established showed that the best activities were obtained
when the benzopyridothiadiazepine tricyclic moiety is substituted
on the pyridine core by a 4-methoxyphenylethyl chain and bears
no substitution on the benzenic part of the tricycle. Compound 1
exhibited good in vitro cytotoxicity with IC50 values in the submi-
cromolar range toward the L1210 leukemia cell line, but was dev-
oided of significant in vivo activity in a P388 murine leukemia
model (iv/ip) probably for ADME problems.
Encouraged by these promising results, we have undertaken the
synthesis of the non-methylated analogue 7 in order to mimic
more closely the potent antimitotic sulfonamides E7010 and ER-
67865. Our investigations of the benzopyridothiadiazepine dioxide
series were then extended to a novel, structurally related ben-
zopyridooxathiazepine dioxide11 in which the sulfonamide moiety
is replaced by a sulfonate group. Preliminary studies showed that
the arylalkyl chain at the N-1 position gave active agents at least
as potent as their corresponding benzopyridothiadiazepines ana-
logues. Compound 14b, which is the direct analogue of compound
1 in the benzopyridooxathiazepine series, was about 12 times
more cytotoxic than compound 1 (IC50 of 9.5 nM vs 110 nM) to-
ward the L1210 leukemia cell line (Table 1). This compound exhib-
its also cytotoxic effects on other cell lines (Table 2). Based on these
initial results, a systematic investigation of substituted ben-
zopyridooxathiazepine dioxide derivatives bearing various arylal-
kyl substituents, was undertaken, and is reported here.
2. Chemistry
Non-methylated thiadiazepine dioxide
7 was synthesized
according to the general method described in Scheme 1. Tricyclic
thiadiazepine 5, bearing a methoxyethoxymethyl substituent, as
protecting group, at the nitrogen sulfonamide position, was pre-
pared according to a modification of the previously described
procedure.1 Alkylation of 5 with 4-methoxyphenylethyl methane-
sulfonate proceeded either on the nitrogen in position 1 or on the
nitrogen in position 11 affording predominantly compound 6b and
to its less extended regioisomer 6a. The structures of these
compounds, that were separated by preparative HPLC, with respec-
tively 40% and 15% yield, were determined by NMR spectra, includ-
ing steady state NOE measurements. In a last step, compound 7 was
obtained in 78% yield by refluxing compound 6b in 6 N HCl .
* Corresponding author. Tel.: +33 03 20 96 49 77; fax: +33 03 20 96 49 13.
E-mail address: nicolas.lebegue@univ-lille2.fr (N. Lebegue).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.039

S. Gallet et al. / Bioorg. Med. Chem. 17 (2009) 1132–1138
1133
O
S
CH3
O
H
N
H
N
SO2
SO2
N
N
N
H3CO
HN
N
H3C
HN
O
1
OH
E7010
ER-67865
OCH3
O
S
O
O
S
H
n= 1-3
R= H, Cl, CH3, OCH3
Ar=
O
O
S
O
N
O
O
OCH
O
N
N
N
N
N
N
R
OH
R
(CH2)n
Ar
OCH
7
(CH2)n
Ar
9
10
OCH
OCH
OCH3
Figure 1. References and synthesized compounds.
The synthesis of the oxathiazepinic compounds is reported in
Scheme 2. Reaction of the already described tricyclic derivatives11
8a–d with 4-methoxybenzyl chloride or arylalkyl methanesulfo-
nates1,18,19 afford the two regioisomers 9 and 10 as already de-
scribed in Scheme 1.
compound 7 was prepared and evaluated. Surprisingly, 7 was
slightly less cytotoxic than 1 (IC50 of 0.3 M vs 0.11 M for 1)
l
l
whose substitution with a methyl group seemed to be most tol-
erant of the steric bulk of substituents. As some papers12,13 re-
port that it is possible, and sometimes favorable to replace the
sulfonamide moiety of antimitotic compounds by a sulfonate
function, we have prepared and evaluated the oxathiazepine
analogues (compounds 9 and 10) of our previously published
thiadiazepine derivatives. In most of the cases, compounds
substituted on the pyridine moiety (10) were clearly more cyto-
toxic than their counterpart (9) substituted on the oxathiazepine
moiety. Four compounds, 10b, 10d, 10e, and 10f were found
markedly cytotoxic with IC50 on L1210 cell line in the nanomolar
range (respectively 9.5, 27, 37, and 43 nM). The perturbations of
the cell cycle induced by these four active compounds were
studied on the L1210 cell line by flow cytometry (Table 2). When
exposed to the cells for two doubling times (21 h), all these com-
pounds induced a marked accumulation of tetraploid cells (8N).
3. Results and discussion
The substituted azepine derivatives 7, 9a–g,i,k–m, and 10a–
m were evaluated for their antiproliferative activity against the
L1210 leukemia cell line. The results expressed as IC50, are re-
ported in Table 1. In a previous study, we demonstrated that
introduction of bulky substituent (compared to methyl) on the
sulfonamide nitrogen resulted in inactive compounds (IC50 on
L1210 > 100
1 (IC50 = 0.11
l
M) compared to the N-methylated thiadiazepine
M). In order to mimic more closely the potent
l
antimitotic sulfonamides E7010 and ER-67865, which were all
non N-substituted on the sulfonamide moiety, the unsubstituted
Table 1
In vitro antiproliferative activities of compounds 7, 9a–g,i,k–m, and 10a–m on L1210 cell line
O
O
O
O
S
O
S
O
N
N
N
N
R
R
(CH2)n
Ar
10
(CH2)n
Ar
9
a
N°
n
R
Ar
IC50
(lM)
N°
n
R
Ar
IC50 (lM)
1
7
0.11
0.3
9a
9b
9c
9d
9e
9f
1
2
3
2
2
2
2
H
H
H
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
2-Methoxyphenyl
0.77
26.5
15.4
n.t.b
22.5
11.2
>10
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
1
2
3
2
2
2
2
2
2
2
2
2
2
H
H
H
9-Cl
9-CH3
9-OCH3
H
H
H
H
H
H
H
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
2-Methoxyphenyl
3-Methoxyphenyl
4-Benzyloxyphenyl
4-Hydroxyphenyl
3,4,5-Trimethoxyphenyl
1-Naphthyl
3.2
0.0095
8.6
0.027
0.037
0.043
14.8
0.8
>100
0.6
27.7
9.6
9-Cl
9-CH3
9-OCH3
H
9g
9i
2
H
4-Benzyloxyphenyl
n.t.
9k
9l
9m
2
2
2
H
H
H
3,4,5-Trimethoxyphenyl
1-Naphthyl
4-Biphenyl
36.8
>100
n.t.
4-Biphenyl
5
a
Concentration inhibiting L1210 cell proliferation by 50% relative to untreated controls after 48 h of drug exposure.
Not tested.
b

1134
S. Gallet et al. / Bioorg. Med. Chem. 17 (2009) 1132–1138
Table 2
Antiproliferative activity of 10b,d,e,f on selected tumoral cell lines (nM) and effects on L1210 cell cycle
In vitro antiproliferative activities (nM)
% of L1210 Cells in each phase of the cell cycleb
Cell type
Cell line
Prostate
DU145
Colon
HT29
Lung
A549
Skin
KB 3-1
Skin
G1
S
G2 + M
8N
Concentration (nM)
KB-A1a
10b
10d
10e
10f
17
10
11.5
n.t.c
n.t.
16
13.2
n.t.
n.t.
n.t.
4
3
4
8
3
3
3
11
25
29
17
22
60
65
75
59
25
50
100
200
42.3
n.t.
116
27.1
50.2
n.t.
n.t.
n.t.
n.t.
99.1
a
KB-A1, adriamycine resistant.
Distribution of control cells in the cell cycle: 45% (G1), 31% G2, 23% (G2 + M), 1% (8N).
Not tested.
b
c
MEM
MEM
CH3
O
S
H
MEM
O
SO2
N
SO2
N
SO2 N
N
a
b
c
N
Cl O2N
N
Cl O2N
N
Cl HN
O
N
N
H
4
2
3
5
d
O
S
O
O
S
MEM
H
MEM
O
O
O
S
N
N
N
e
+
N
N
N
N
N
N
7
6a
6b
OCH3
OCH3
OCH3
Scheme 1. Reagents and conditions: (a) NaH, MEMCl, DMF, rt, 78%; (b) (i) H2, Ni-R, EtOH; (ii) Ac2O, rt, 95%; (c) K2CO3, DMF, reflux, 45%; (d) NaH, DMF, 4-methoxyphenylethyl
methanesulfonate, 60 °C, 15–40%; (e) EtOH 95%, 6 N HCl, reflux, 78%.
This accumulation, is generally observed for numerous tubulin
interacting drugs.
with (1-naphthyl)ethyl (10l) or (4-biphenyl)ethyl (10m) resulted
in clearly less active compounds (IC50 of respectively 9.6 and
As for the thiadiazepine derivatives, the best results were ob-
tained when the benzopyridooxathiazepine is substituted on the
pyridine moiety by a 4-methoxyphenylethyl chain (10b). Modifica-
tion of the length of this substituent (4-methoxybenzyl 10a, or 4-
methoxypropyl 10c) resulted in a clear decrease of the cytotoxicity.
The conclusion was the same when the 4-methoxyphenylethyl
side chain was replaced by a 2-methoxy (10g), 3-methoxy (10h),
4-hydroxy (10j), and 3,4,5-trimethoxy (10k) phenylethyl group.
Some paper have shown that it was possible to replace the polyox-
ygenated rings of combretastatin A-4 with naphthalene, quinoline
or quinoxaline moieties without significant loss of cytotoxicity and
inhibition of tubulin polymerization potency.14–17 Transposed to
our series, the replacement of the 4-methoxyphenyl side chain
5.0 lM). These results suggest that aromatic hydrophobic rings
are not good surrogates for the 4-methoxyphenyl ring and that
the interaction with tubulin is not only hydrophobic in nature
but needs some polar interactions. In a second step, we investi-
gated the influence of some substituents on the benzenic part of
the tricycle (no substituent 10b, 9-Cl 10d, 9-Me 10e, and 9-MeO
10f). As for the benzopyridothiadiazepine series, compounds
substituted on the phenyl moiety of the oxathiazepine were
slightly less active than their unsubstituted derivative 10b.
The most active compound within this series (10b,
IC50(L1210) = 9.5 nM) was about 12 times more cytotoxic than its
corresponding sulfonamide direct counterpart 1 (IC50(L1210) =
110 nM). Compound 10b was evaluated on five other cancer cell
lines (DU145, HT29, A549, KB 3-1, KB-A-1) and showed potent
nanomolar cytotoxic effects with IC50 of respectively 17, 10, 11.5,
16, and 13.2 nM (Table 2). As compounds 10b induced a marked
accumulation of tetraploid cells (8N), this compound was evalu-
ated for its ability to inhibit the polymerization of tubulin and
was found to be potent inhibitor of tubulin polymerization with
O
O
S
O
N
N
R
(CH2)n
Ar
O
S
9
O
n= 1-3
R= H, Cl, CH3, OCH3
Ar=
O
IC50 of 2.2 lM compared to 2.4 lM for desoxypodophyllotoxin
a
+
O
O
OCH
used as a reference compound. No effect was observed on tubulin
depolymerization. In a last step, compound 10b was evaluated
in vivo on a P388 murine leukemia model. On day 0, 106 leukemic
cells were grafted in the peritoneal cavity of B6D2F1 mice as de-
scribed.23 Administered iv or ip on day 1, 10b proved to be moder-
ately but significantly active, inducing an increase in survival of
41% (T/C = 141%) for a 100 mg/kg iv dose and 61% (T/C = 161%)
for 50–100 mg/kg ip doses. The relatively low in vivo antitumor
activity of compound 10b considering its high in vitro antiprolifer-
N
O
N
H
R
O
S
OH
8a-d
OCH
OCH
OCH
N
N
R
10
(CH2)n
Ar
Scheme 2. Reagents and conditions: (a) NaH, DMF, 4-methoxybenzyl chloride or
arylalkyl methanesulfonates, 60 °C, 3–39%.

S. Gallet et al. / Bioorg. Med. Chem. 17 (2009) 1132–1138
1135
ative activity toward cancer cell lines, could be explained by its
weak metabolic bioavailability on mice and human liver micro-
somes (<12%) while its intestinal permeability on Caco2 cells is
nearly full.
water, extracted with DCM, dried over sodium sulfate, filtered
and concentrated under vacuo. The resulting oil was used for next
step without further purification. Brown oil, yield 99%.
5.1.3. 6,11-Dihydro-6-methoxyethoxymethylbenzo[c]
pyrido-[2,3,f][1,2,5] thiadiazepines 5,5-dioxide (5)
4. Conclusion
A solution of 4 (10 mmol) in DMF (40 mL) with potassium car-
bonate (20 mmol) is refluxed for 8 h. The reaction mixture is evap-
orated under reduced pressure. The residue is poured in water,
extracted with DCM, dried, filtered, concentrated under reduced
pressure, and purified by silica gel chromatography with petro-
leum ether/ethyl acetate (7:3). White solid; mp 119–121 °C (yield
45%). IR (KBr) cmÀ1: 1180, 1360, 3280. 1H NMR (CDCl3): d 3.34 (s,
3H), 3.49 (t, 2H), 3.78 (m, 2H), 4.93 (s, 2H), 6.90 (dd, 1H), 6.99 (dd,
1H), 7.13 (dd, 1H), 7.33 (dd, 1H), 7.38 (dd, 1H), 7.56 (s, 1H), 8.15
(dd, 1H), 8.33 (dd, 1H).
We have synthesized sulfonate analogues of the thiadiazepine
compound 1, and have found many of these compounds to be po-
tent in vitro inhibitors of the proliferation of the murine L1210 leu-
kemia cell line. Compound 10b, which is the strict analogue in the
benzopyridooxathiazepine series of the sulfonamide 1 is a nano-
molar cytotoxic agent clearly (12 times) more potent than 1. This
compound is a potent inhibitor of tubulin polymerization with
100% of inhibition at 10 lM. Surprisingly within the ben-
zopyridothiadiazepine series, compound 7, which is not methyl-
ated on the sulfonamide moiety, and therefore mimic more
closely than compound 1 does the potent antimitotic sulfonamides
E7010 and ER-67865, was found slightly less active on L1210 cell
line (IC50 of 300 nM vs 110 nM) than 1. Our lead compound 10b
was found moderately but significantly active in vivo on a P388
murine leukemia model. Its relatively low in vivo antitumor activ-
ity, considering its high in vitro antiproliferative activity toward
cancer cell lines, could be explained by poor metabolic stability.
The design of new compounds with improved in vivo antitumor
activity is currently under investigation.
5.1.4. Synthesis of (6a) and (6b)
A solution of 5 (10 mmol) in DMF (20 mL) is added dropwise to
a suspension of sodium hydride (20 mmol) in DMF (10 mL) and
stirred for 3 h at 60 °C. A solution of 4-methoxyphenylethyl meth-
anesulfonate1 (25 mmol) in DMF (15 mL) is then added dropwise
to the previous solution and stirred for 12 h at 60 °C. The resulting
solution is concentrated under reduced pressure, the residue is di-
luted in DCM, washed with water, dried, concentrated and the two
regioisomers separated by preparative HPLC (petroleum ether/
ethyl acetate 6:4).
5. Experimental
5.1. Chemistry
5.1.5. 6,11-Dihydro-11-(4-methoxyphenylethyl)-6-methoxyetho-
xymethylbenzo[c]pyrido[2,3-f][1,2,5] thiadiazepine 5,
5-dioxide (6a)
Beige solid; mp 80–83 °C (yield 15%). IR (KBr) cmÀ1: 1170, 1340.
1H NMR (CDCl3): d 2.92 (t, 2H), 3.42 (s, 3H), 3.63 (t, 2H), 3.78 (s,
3H), 3.93 (t, 2H), 4.36 (t, 2H), 4.78 (s, 2H), 6.83 (d, 2H), 6.99–7.07
(m, 3H), 7.28–7.43 (m, 3H), 7.51 (dd, 1H), 8.12 (dd, 1H), 8.47 (dd,
1H).
Melting points were determined on a BÜCHI B-540 apparatus
and are uncorrected. Infrared spectra were recorded as thin films
on potassium bromide disks on a Beckman Acculab IV spectropho-
tometer. Mass spectra were performed on a Finnigan MAT SSQ 710
Advantage spectrometer. 1H NMR spectra (LARMN, Université de
Lille 2) were recorded on a Bruker AC 300 P and 2D NMR spectra
on a Bruker DPX 300, using tetramethylsilane as internal standard.
Elemental analyses were performed by C.N.R.S—Vernaison, and
were in agreement with the calculated values within 0.4%.
5.1.6. 1-(4-Methoxyphenylethyl)-6-methoxyethoxymethyl-
benzo[c]-1,2-dihydropyrido[2,3-f][1,2,5] thiadiazepine 5,
5-dioxide (6b)
Yellow oil (yield 40%). IR (KBr) cmÀ1: 1170, 1340. 1H NMR
(CDCl3): d 3.10 (t, 2H), 3.34 (s, 3H), 3.52 (t, 2H), 3.81 (s, 3H), 3.95
(t, 2H), 4.30 (t, 2H), 4.90 (t, 2H), 5.81 (dd, 1H), 6.86 (d, 2H), 7.0–
7.15 (m, 4H), 7.20–7.35 (m, 3H), 7.85 (dd, 1H).
5.1.1. 2-Chloro-N-(methoxyethoxymethyl)-N-(2-nitro-phenyl)-
3-pyridinesulfonamide (3)
2-Chloro-N-(2-nitrophenyl)-3-pyridinesulfonamide 2 (10 mmol)
dissolved in DMF (25 mL) is added dropwise to a suspension of so-
dium hydride (15 mmol) in DMF (5 mL) and stirred for 1 h at room
temperature. Methoxyethoxymethyl chloride (12 mmol) in DMF
(5 mL) is added dropwise to the previous solution and stirred for
12 h at room temperature. The resulting solution is concentrated
under reduced pressure, hydrolyzed, extracted twice with DCM,
dried over sodium sulfate, filtered and concentrated under vacuo.
The resulting residue is then recrystallized from diisopropylic
ether. Yellow powder; mp 72–74 °C (diisopropyl ether) (yield
78%). IR (KBr) cmÀ1: 1160, 1350, 1520. 1H NMR (CDCl3): d 3.40
(s, 3H), 3.62 (t, 2H), 3.95 (m, 2H), 5.50 (m, 2H), 7.29 (dd, 1H),
7.50–7.65 (m, 3H), 7.71 (dd, 1H), 8.04 (dd, 1H), 8.58 (dd, 1H).
5.1.7. 1-(4-Methoxyphenylethyl)-benzo[c]-1,2-dihydro-pyrido
[2,3-f][1,2,5] thiadiazepine 5,5-dioxide (7)
A solution of 6b (1 mmol) in ethanol 95% (10 mL) and 6 N
hydrochloric acid (10 mL) is refluxed for 1 h 30 min. The reaction
mixture is concentrated and the residue poured into water. Satu-
rated aqueous sodium bicarbonate is added until pH 5 and the
solution is extracted with ethyl acetate, washed with water, dried,
filtered and concentrated under reduced pressure. The resulting
powder is recrystallized from diisopropylic ether. Yellow solid;
mp 174–175 °C (yield 78%). IR (KBr) cmÀ1: 1170, 1315, 3210. 1H
NMR (CDCl3): d 3.10 (t, 2H), 3.81 (s, 3H), 4.26 (t, 2H), 5.76 (dd,
1H), 6.26 (s, 1H), 6.86 (d, 2H), 6.95–7.25 (m, 7H), 7.77 (d, 1H).
MS (EI) m/z 381 (M+). Anal. Calcd for C20H19N3O3S: C, 62.97; H,
5.02; N, 11.02. Found: C, 62.84; H, 5.11; N, 11.09.
5.1.2. 2-Chloro-N-(methoxyethoxymethyl)-N-(2-acetamido-
phenyl)-3-pyridinesulfonamide (4)
A solution of 3 (10 mmol) in ethanol was stirred in the presence
of 0.5 g of Raney Nickel under an atmosphere of hydrogen at room
temperature until reaction was completed as checked by TLC. The
solution was filtered through Celite, and the filtrate was concen-
trated under reduced pressure. The residue was dissolved in acetic
anhydride and stirred for 12 h. The solution was diluted with
5.1.8. General procedure for the synthesis of substituted 11-
(arylalkyl)-11H-benzo[f]pyrido[3,2,c][1,2,5]oxa-thiazepine 5,5-
dioxide(9a–m) and 1-(arylalkyl)-11H-benzo[f]-1,2-dihydropyrido
[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10a–m)
A solution of substituted 11H-benzo[f]pyrido[3,2-c][1,2,5]oxa-
thiazepine 5,5-dioxide 8a–d (4 mmol) in DMF (10 mL) is added

1136
S. Gallet et al. / Bioorg. Med. Chem. 17 (2009) 1132–1138
dropwise to a suspension of sodium hydride (8 mmol) in DMF
(5 mL) and stirred for 3 h at 60 °C. A solution of commercially avail-
able 4-methoxybenzyl chloride or synthesized arylalkyl methane-
sulfonate1,18,19 (12 mmol) in DMF (10 mL) is then added
dropwise to the previous solution and stirred for 24 h at 60 °C.
The resulting solution is concentrated under reduced pressure,
the residue is diluted in DCM, washed with water, dried, concen-
trated, and the two regioisomers are separated by preparative
HPLC.
(M+). Anal. Calcd for C21H20N2O5S: C, 61.15; H, 4.89; N, 6.79.
Found: C, 61.05; H, 5.01; N, 6.46.
5.1.15. 11-(2-Methoxyphenylethyl)-11H-benzo[f]pyrido-[3,2,c]
[1,2,5]oxathiazepine 5,5-dioxide (9g)
White solid; purified by chromatography with petroleum ether/
ethyl acetate (9:1); mp 132–133 °C (ethanol) (yield 11%). IR (KBr)
cmÀ1: 1180, 1350. 1H NMR (CDCl3): d 3.04 (t, 2H), 3.77 (s, 3H),
4.44 (t, 2H), 6.81 (d, 2H), 6.87 (dd, 1H), 6.96 (dd, 1H), 7.10–7.40
(m, 5H), 7.50 (d, 1H), 8.10 (dd, 1H), 8.49 (dd, 1H). MS (EI) m/z
382 (M+). Anal. Calcd for C20H18N2O4S: C, 62.81; H, 4.74; N, 7.32.
Found: C, 62.92; H, 4.70; N, 7.32.
5.1.9. 11-(4-Methoxybenzyl)-11H-benzo[f]pyrido[3,2,c]
[1,2,5]oxathiazepine 5,5-dioxide (9a)
Beige solid; purified by chromatography with petroleum ether/
ethyl acetate (7:3); mp 126–127 °C (ethanol) (yield 26%). IR (KBr)
cmÀ1: 1180, 1370. 1H NMR (CDCl3): d 3.76 (s, 3H), 5.38 (s, 2H),
6.81 (d, 2H), 6.98 (dd, 1H), 7.20–7.40 (m, 6H), 8.13 (dd, 1H), 8.43
(dd, 1H). MS (EI) m/z 368 (M+). Anal. Calcd for C19H16N2O4S: C,
61.94; H, 4.34; N, 7.60. Found: C, 61.97; H, 4.37; N, 7.67.
5.1.16. 11-[2-(4-Benzyloxyphenyl)ethyl]-11H-benzo[f]
pyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (9i)
White solid; purified by chromatography with petroleum ether/
ethyl acetate (75:25); mp 92–94 °C (methanol) (yield 14%). IR (KBr)
cmÀ1: 1190, 1360. 1H NMR (CDCl3): d 2.95 (t, 2H), 4.33 (t, 2H), 5.11
(s, 2H), 6.99 (dd, 1H), 6.80–7.55 (m, 13H), 8.11 (dd, 1H), 8.49 (dd,
1H). MS (EI) m/z 458 (M+). Anal. Calcd for C26H22N2O4S: C, 68.11;
H, 4.84; N, 6.11. Found: C, 68.21; H, 4.76; N, 6.14.
5.1.10. 11-(4-Methoxyphenylethyl)-11H-benzo[f]pyrido[3,2,c]
[1,2,5]oxathiazepine 5,5-dioxide (9b)
White solid; purified by chromatography with petroleum ether/
ethyl acetate (75:25); mp 93–94 °C (ethanol) (yield 12%). IR (KBr)
cmÀ1: 1180, 1370. 1H NMR (CDCl3): d 2.95 (t, 2H), 3.79 (s, 3H),
4.44 (t, 2H), 6.83 (d, 2H), 6.99 (dd, 1H), 7.12 (d, 2H), 7.25–7.50
(m, 4H), 8.11 (dd, 1H), 8.50 (dd, 1H). MS (EI) m/z 382 (M+). MS
(EI); Anal. Calcd for C20H18N2O4S: C, 62.81; H, 4.74; N, 7.32. Found:
C, 62.91; H, 4.68; N, 7.46.
5.1.17. 11-[2-(3,4,5-Trimethoxyphenyl)ethyl]-11H-benzo-[f]
pyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (9k)
White solid; purified by chromatography with petroleum ether/
ethyl acetate (6:4); mp 108–110°C (ethanol 95%) (yield 18%). IR
(KBr) cmÀ1: 1180, 1360. 1H NMR (CDCl3): d 2.94 (t, 2H), 3.81 (s,
9H), 4.51 (t, 2H), 6.37 (s, 2H), 7.00 (dd, 1H), 7.20–7.50 (m, 4H),
8.11 (dd, 1H), 8.51 (dd, 1H). MS (EI) m/z 442 (M+). Anal. Calcd for
C22H22N2O6S: C, 59.72; H, 5.01; N, 6.33. Found: C, 59.97; H, 5.02;
N, 6.46.
5.1.11. 11-[3-(4-Methoxyphenyl)propyl]-11H-benzo[f]pyrido-[3,
2,c][1,2,5]oxathiazepine 5,5-dioxide (9c)
White solid; purified by chromatography with petroleum ether/
ethyl acetate (75:25); mp 86–87 °C (ethanol) (yield 31%). IR (KBr)
cmÀ1: 1170, 1370. 1H NMR (CDCl3): d 1.90 (m, 2H), 2.62 (t, 2H),
3.77 (s, 3H), 4.23 (t, 2H), 6.77 (d, 2H), 6.90–7.60 (m, 7H), 8.11 (d,
1H), 8.50 (d, 1H). MS (EI) m/z 396 (M+). Anal. Calcd for
C21H20N2O4S: C, 63.62; H, 5.08; N, 7.07. Found: C, 63.47; H, 5.17;
N, 7.19.
5.1.18. 11-[2-(1-Naphthyl)ethyl]-11H-benzo[f]pyrido-[3,2,c]
[1,2,5]oxathiazepine 5,5-dioxide (9l)
White solid; purified by chromatography with petroleum ether/
ethyl acetate (7:3); mp 59–61 °C (ethanol 95%) (yield 10%). IR (KBr)
cmÀ1: 1180, 1360. 1H NMR (CDCl3): d 3.52 (t, 2H), 4.57 (t, 2H), 7.03
(dd, 1H), 7.25–7.45 (m, 6H), 7.45–7.60 (m, 2H), 7.75 (d, 1H), 7.87
(dd, 1H), 8.15 (dd, 1H), 8.26 (d, 1H), 8.57 (dd, 1H). MS (EI) m/z
402 (M+). Anal. Calcd for C23H18N2O3S: C, 68.64; H, 4.51; N, 6.96.
Found: C, 68.41; H, 4.23; N, 7.17.
5.1.12. 9-Chloro-11-(4-methoxyphenylethyl)-11H-benzo-[f]
pyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (9d)
White solid; purified by chromatography with petroleum ether/
ethyl acetate (7:3); mp 133–135 °C (ethanol 95%) (yield 3%). IR
(KBr) cmÀ1: 1170, 1360. 1H NMR (CDCl3): d 2.96 (t, 2H), 3.79 (s,
3H), 4.41 (t, 2H), 6.83 (d, 2H), 7.03 (dd, 1H), 7.11 (d, 2H), 7.20–
7.45 (m, 4H), 8.12 (dd, 1H), 8.52 (dd, 1H). MS (EI) m/z 416 (M+).
Anal. Calcd for C20H17ClN2O4S: C, 57.62; H, 4.11; N, 6.72. Found:
C, 57.54; H, 4.03; N, 6.98.
5.1.19. 11-[2-(4-Biphenyl)ethyl]-11H-benzo[f]pyrido-[3,2,c]
[1,2,5]oxathiazepine 5,5-dioxide (9m)
White solid; purified by chromatography with petroleum ether/
ethyl acetate (75:25); mp 55–58 °C (propanol) (yield 6%). IR (KBr)
cmÀ1: 1180, 1360. 1H NMR (CDCl3): d 3.06 (t, 2H), 4.52 (t, 2H),
6.83 (d, 2H), 7.00 (dd, 1H), 7.25–7.45 (m, 9H), 7.50–7.60 (m, 4H),
8.12 (dd, 1H), 8.51 (dd, 1H). MS (EI) m/z 428 (M+). Anal. Calcd for
C25H20N2O3S: C, 70.07; H, 4.70; N, 6.54. Found: C, 70.12; H, 4.59;
N, 6.38.
5.1.13. 11-(4-Methoxyphenylethyl)-9-methyl-11H-benzo-[f]
pyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (9e)
White solid; purified by chromatography with petroleum ether/
ethyl acetate (8:2); mp 137–138 °C (ethanol 95%) (yield 25%). IR
(KBr) cmÀ1: 1170, 1350. 1H NMR (CDCl3): d 2.37 (s, 3H), 2.95 (t,
2H), 3.79 (s, 3H), 4.41 (t, 2H), 6.83 (d, 2H), 6.97 (dd, 1H), 7.05–
7.19 (m, 3H), 7.13–7.32 (m, 2H), 8.10 (dd, 1H), 8.50 (dd, 1H). MS
(EI) m/z 396 (M+). Anal. Calcd for C21H20N2O4S: C, 63.62; H, 5.08;
N, 7.07. Found: C, 63.58; H, 5.09; N, 7.13.
5.1.20. 1-(4-Methoxybenzyl)-11H-benzo[f]-1,2-dihydropyrido-
[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10a)
Yellow solid; purified by chromatography with petroleum
ether/ethyl acetate (7:3); mp 162–163 °C (ethanol) (yield 29%). IR
(KBr) cmÀ1: 1180, 1360. 1H NMR (CDCl3): d 3.82 (s, 3H), 5.28 (s,
2H), 5.98 (dd, 1H), 6.91 (d, 2H), 7.00–7.30 (m, 4H), 7.33 (d, 2H),
7.50 (dd, 1H), 7.90 (dd, 1H). MS (EI) m/z 368 (M+). Anal. Calcd for
C19H16N2O4S: C, 61.94; H, 4.34; N, 7.60. Found: C, 61.92; H, 4.38;
N, 7.62.
5.1.14. 9-Methoxy-11-(4-methoxyphenylethyl)-11H-benzo-[f]
pyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (9f)
White solid; purified by chromatography with petroleum ether/
ethyl acetate (7:3); mp 110–114 °C (ethanol 95%) (yield 13%). IR
(KBr) cmÀ1: 1170, 1360. 1H NMR (CDCl3): d 3.01 (t, 2H), 3.80 (s,
6H), 4.39 (t, 2H), 6.80–6.90 (m, 3H), 6.99 (dd, 1H), 7.12–7.20 (m,
3H), 7.31 (d, 1H), 8.10 (dd, 1H), 8.49 (dd, 1H). MS (EI) m/z 412
5.1.21. 1-(4-Methoxyphenylethyl)-11H-benzo[f]-1,2-dihydro-
pyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10b)
Yellow solid; purified by chromatography with petroleum
ether/ethyl acetate (75:25); mp 115–116 °C (ethanol) (yield

S. Gallet et al. / Bioorg. Med. Chem. 17 (2009) 1132–1138
1137
15%). IR (KBr) cmÀ1: 1180, 1370. 1H NMR (CDCl3): d 3.11 (t, 2H),
5.1.28. 1-(4-Benzyloxyphenylethyl)-11H-benzo[f]-1,
3.81 (s, 3H), 4.43 (t, 2H), 5.84 (dd, 1H), 6.87 (d, 2H), 7.00–7.35
(m, 7H), 7.89 (dd, 1H). MS (EI) m/z 382 (M+). Anal. Calcd for
C20H18N2O4S: C, 62.81; H, 4.74; N, 7.32. Found: C, 62.62; H,
4.74; N, 7.39.
2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10i)
Yellow solid; purified by chromatography with petroleum
ether/ethyl acetate (75:25); mp 142–143 °C (ethanol) (yield
23%). IR (KBr) cmÀ1: 1190, 1360. 1H NMR (CDCl3): d 3.11 (t,
2H), 4.31 (t, 2H), 5.07 (s, 2H), 5.82 (dd, 1H), 6.85–7.55 (m,
14H), 7.89 (dd, 1H). MS (EI) m/z 458 (M+). Anal. Calcd for
C26H22N2O4S: C, 68.11; H, 4.84; N, 6.11. Found: C, 68.08; H,
5.04; N, 5.72.
5.1.22. 1-[3-(4-Methoxyphenyl)propyl]-11H-benzo[f]-1,
2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10c)
Yellow solid; purified by chromatography with petroleum
ether/ethyl acetate (75:25); mp 99–100 °C (ethanol) (yield 38%).
IR (KBr) cmÀ1: 1170, 1370. 1H NMR (CDCl3): d 2.15 (m, 2H), 2.70
(t, 2H), 3.82 (s, 3H), 4.12 (t, 2H), 5.96 (dd, 1H), 6.87 (d, 2H), 6.95–
7.25 (m, 7H), 7.39 (dd, 1H), 7.90 (dd, 1H). MS (EI) m/z 396 (M+).
Anal. Calcd for C21H20N2O4S: C, 63.62; H, 5.08; N, 7.07. Found: C,
63.39; H, 5.23; N, 7.26.
5.1.29. 1-(4-Hydroxyphenylethyl)-11H-benzo[f]-1,2-dihydro-
pyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10j)
A solution of 14i (4 mmol) in acetic acid (6 mL) and 33% hydro-
bromic acid (4 mL) is heated at 35 °C for five days. The reaction
mixture is hydrolyzed with ice, extracted with ethyl acetate,
washed with aqueous saturated sodium bicarbonate solution.
The organic layer is dried, filtered, evaporated and recrystallized
in ethanol 95%. Red solid; mp 58–61 °C (ethanol 95%) (yield 53%).
IR (KBr) cmÀ1: 1180, 1350, 3400. 1H NMR (CDCl3): d 3.11 (t, 2H),
4.32 (t, 2H), 5.20 (m, 1H), 5.84 (dd, 1H), 6.79 (d, 2H), 7.00–7.35
(m, 7H), 7.89 (dd, 1H). MS (EI) m/z 368 (M+). Anal. Calcd for
C19H16N2O4S: C, 61.94; H, 4.38; N, 7.60. Found: C, 62.03; H, 4.35;
N, 7.54.
5.1.23. 9-Chloro-1-(4-methoxyphenylethyl)-11H-benzo[f]-1,
2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10d)
Yellow solid; purified by chromatography with petroleum
ether/ethyl acetate (7:3); mp 126–127 °C (ethanol 95%) (yield
18%). IR (KBr) cmÀ1: 1190, 1360. 1H NMR (CDCl3): d 3.10 (t, 2H),
3.81 (s, 3H), 4.33 (t, 2H), 5.89 (dd, 1H), 6.86 (d, 2H), 7.00–7.35
(m, 7H), 7.92 (dd, 1H). MS (EI) m/z 416 (M+). Anal. Calcd for
C20H17ClN2O4S: C, 57.62; H, 4.11; N, 6.72. Found: C, 57.94; H,
4.16; N, 6.81.
5.1.30. 1-(3,4,5-Trimethoxyphenylethyl)-11H-benzo[f]-1,
2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10k)
Yellow solid; purified by chromatography with petroleum
ether/ethyl acetate (6:4); mp 165–166 °C (ethanol 95%) (yield
39%). IR (KBr) cmÀ1: 1170, 1380. 1H NMR (CDCl3): d 3.12 (t, 2H),
3.82 (s, 9H), 4.36 (t, 2H), 5.84 (dd, 1H), 6.37 (s, 2H), 6.95–7.40
(m, 5H), 7.90 (dd, 1H). MS (EI) m/z 442 (M+). Anal. Calcd for
C22H22N2O6S: C, 59.72; H, 5.01; N, 6.33. Found: C, 59.67; H, 4.99;
N, 6.41.
5.1.24. 1-(4-Methoxyphenylethyl)-9-methyl-11H-benzo[f]-1,
2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10e)
Yellow solid; purified by chromatography with petroleum
ether/ethyl acetate (8:2); mp 114–115 °C (ethanol 95%) (yield
31%). IR (KBr) cmÀ1: 1170, 1350. 1H NMR (CDCl3): d 2.36 (s, 3H),
3.12 (t, 2H), 3.81 (s, 3H), 4.31 (t, 2H), 5.80 (dd, 1H), 6.80–6.91
(m, 3H), 7.01–7.16 (m, 5H), 7.87 (dd, 1H). MS (EI) m/z 396 (M+).
Anal. Calcd for C21H20N2O4S: C, 63.62; H, 5.08; N, 7.07. Found: C,
63.68; H, 5.13; N, 7.01.
5.1.31. 1-[2-(1-Naphthyl)ethyl]-11H-benzo[f]-1,2-dihydro-pyri-
do[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10l)
5.1.25. 9-Methoxy-1-(4-methoxyphenylethyl)-11H-benzo[f]-1,
2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10f)
Yellow solid; purified by chromatography with petroleum
ether/ethyl acetate (7:3); mp 115–116 °C (ethanol 95%) (yield
21%). IR (KBr) cmÀ1: 1150, 1360. 1H NMR (CDCl3): d 3.11 (t, 2H),
3.81 (s, 3H), 3.84 (s, 3H), 4.32 (t, 2H), 5.84 (dd, 1H), 6.60 (dd,
1H), 6.80 (d, 1H), 6.86 (d, 2H), 7.05–7.15 (m, 4H), 7.89 (dd, 1H).
MS (EI) m/z 412 (M+). Anal. Calcd for C21H20N2O5S: C, 61.15; H,
4.89; N, 6.79. Found: C, 61.40; H, 5.08; N, 6.51.
Yellow solid; purified by chromatography with petroleum
ether/ethyl acetate (7:3); mp 201–203 °C (ethanol 95%) (yield
16%;). IR (KBr) cmÀ1: 1160, 1350. 1H NMR (CDCl3): d 3.63 (t, 2H),
4.50 (t, 2H), 5.76 (dd, 1H), 7.01 (dd, 1H), 7.09 (dd, 1H), 7.23 (dd,
1H), 7.25–7.35 (m, 2H), 7.40–7.45 (m, 2H), 7.50–7.60 (m, 2H),
7.79 (d, 1H), 7.80–7.90 (m, 2H), 8.24 (d, 1H). MS (EI) m/z 402
(M+). Anal. Calcd for C23H18N2O3S: C, 68.64; H, 4.51; N, 6.96.
Found: C, 68.91; H, 4.48; N, 7.01.
5.1.32. 1-[2-(4-Biphenyl)ethyl]-11H-benzo[f]-1,
5.1.26. 1-(2-Methoxyphenylethyl)-11H-benzo[f]-1,2-dihydro-
pyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10g)
2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10m)
Yellow solid; purified by chromatography with petroleum
ether/ethyl acetate (75:25); mp 163–165 °C (ethanol) (yield
23%;). IR (KBr) cmÀ1: 1190, 1360. 1H NMR (CDCl3): d 3.22 (t, 2H),
4.39 (t, 2H), 5.86 (dd, 1H), 7.06 (dd, 1H), 7.15–7.20 (m, 2H),
7.20–7.30 (m, 3H), 7.30–7.40 (m, 2H), 7.46 (dd, 2H), 7.55–7.65
(m, 4H), 7.91 (dd, 1H). MS (EI) m/z 428 (M+). Anal. Calcd for
C25H20N2O3S: C, 70.07; H, 4.70; N, 6.54. Found: C, 70.27; H, 4.73;
N, 6.40.
Yellow solid; purified by chromatography with petroleum
ether/ethyl acetate (9:1); mp 138–140 °C (propan-2-ol/water 9:1)
(yield 27%). IR (KBr) cmÀ1: 1180, 1370. 1H NMR (CDCl3): d 3.20
(t, 2H), 3.81 (t, 3H), 4.37 (t, 2H), 5.78 (dd, 1H), 6.80–6.90 (m, 2H),
7.00–7.35 (m, 7H), 7.86 (dd, 1H). MS (EI) m/z 382 (M+). Anal. Calcd
for C20H18N2O4S: C, 62.81; H, 4.74; N, 7.32. Found: C, 62.57; H,
4.69; N, 7.25.
5.1.27. 1-(3-Methoxyphenylethyl)-11H-benzo[f]-1,2-dihydro-
pyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide (10h)
5.2. Cell culture and cytotoxicity assays
Yellow solid; purified by chromatography with petroleum
ether/ethyl acetate (75:25); mp 133–134 °C (ethanol 95%) (yield
18%). IR (KBr) cmÀ1: 1180, 1370. 1H NMR (CDCl3): d 3.15 (t, 2H),
3.81 (s, 3H), 4.36 (t, 2H), 5.85 (dd, 1H), 6.72–6.83 (m, 3H), 7.05
(td, 1H), 7.12 (dd, 1H), 7.18 (dd, 1H), 7.21–7.28 (m, 3H), 7.32 (dd,
1H), 7.89 (dd, 1H). MS (EI) m/z 382 (M+). Anal. Calcd for
C20H18N2O4S: C, 62.81; H, 4.74; N, 7.32. Found: C, 62.64; H, 4.78;
N, 7.36.
Cells were cultivated in RPMI 1640 medium (Invitrogen Inc.)
supplemented with 10% fetal calf serum, 2 mM
L-glutamine,
100 units/mL penicillin, 100 g/mL streptomycin, and 10 mM
l
HEPES buffer (pH 7.4). Cytotoxicity was measured by the micro-
culture tetrazolium assay (MTA). Adherent cells (DU145, HT29,
A549, KB 3-1 and KB-A1)were seeded in 96 well microplates
and incubated for 2 days. Tested compounds were then added
and plates were incubated for 96 h (about 4 doubling times).

1138
S. Gallet et al. / Bioorg. Med. Chem. 17 (2009) 1132–1138
The murine L1210 cells were exposed for 48 h to graded concen-
trations of drug. At the end of this period, 15 L of 5 mg/mL of
A. Supplementary data
l
3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT,
Sigma) were added to each well and the plates were incubated
for 4 h at 37 °C. The medium was aspirated and the formazan
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.12.039.
solubilized by 100 lL of DMSO. The IC50, concentration reducing
References and notes
by 50% the optical density at 540 nm, was calculated by a linear
regression performed on the linear zone of the dose–response
curve. All the measurements were performed in triplicate.
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5.3. Cell cycle analysis20,21
L1210 cells (2.5 Â 105 cells/mL) were incubated for 21 h with
various concentrations of the compounds. Cells were then fixed
in 70% ethanol (v/v), washed and incubated in Dulbecco’s phos-
phate buffered saline (D-PBS) containing 100 mg/mL RNAse and
25 mg/mL propidium iodide for 30 min. at 20 °C. For each sample,
104 cells were analyzed on a Epics XL/MCL flow cytometer (Beck-
man Coulter, France).
5.4. Tubulin test
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Effects of the compounds on tubulin polymerization were eval-
uated as previously reported.22
12. Gwaltney, S. L., II; Imade, H. M.; Barr, K. J.; Li, Q.; Gehrke, L.; Credo, R. B.;
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5.5. In vivo antitumor activity23
The in vivo antitumor activity of compound 14b was evaluated
on the P388 leukemia murine model. P388 cells (NCI, Frederik)
were inoculated ip (106 cells/mouse) into B6D2F1 mice (Iffa credo)
on day 0. Compound 14b was administered ip or iv on day one. The
results are expressed in term of percent T/C (median survival time
of treated animals divided by median survival time of control, X
100).
17. Maya, A. B.; Perez-Melero, C.; Mateo, C.; Alonso, D.; Fernandez, J. L.; Gajate, C.;
Mollinedo, F.; Pelaez, R.; Caballero, E.; Medarde, M. J. Med. Chem. 2005, 48, 556.
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5.6. Determination of metabolic bioavailability24
19. Hardy, G. W.; Lowe, L. A.; Mills, G.; Sang, P. Y.; Simpkin, D. S. A.; Follenfant, R.
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2445.
Metabolic bioavailability predictions were based on in vitro
metabolic stability measurements with hepatic microsomes
assuming total absorption.
5.7. Determination of intestinal absorption25
Assuming no possible limitation due to solubility, absorbed
fractions (Abs%) were estimated using permeability measurements
through Caco2 cell monolayers.
24. Bertrand, M.; Jackson, P.; Walther, B. Eur. J. Pharm. Sci. 2000, 11, S61.
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Acknowledgments
We thank Laboratoire d’Application de Résonnance Magnétique
Nucléaire de l’Université de Lille 2 for its help in the interpetation
of the 1H NMR spectra.