Synthesis and biological evaluation of glucuronide prodrugs of the histone deacetylase inhibitor CI-994 for application in selective cancer chemotherapy

Article abstract of DOI:10.1016/j.bmc.2008.07.048

Two glucuronide prodrugs of the histone deacetylase inhibitor CI-994 were synthesized. These compounds were found to be soluble in aqueous media and stable under physiological conditions. The carbamoyl derivatisation of CI-994 significantly decreased its toxicity towards NCI-H661 lung cancer cells. Prodrug incubation with β-glucuronidase in the culture media led efficiently to the release of the parent drug and thereby restoring its ability to decrease cell proliferation, to inhibit HDAC and to induce E-Cadherin expression.

Full text of DOI:10.1016/j.bmc.2008.07.048

Bioorganic & Medicinal Chemistry 16 (2008) 8109–8116
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of glucuronide prodrugs of the histone
deacetylase inhibitor CI-994 for application in selective cancer chemotherapy
Mickaël Thomas a, Jonathan Clarhaut b, Isabelle Tranoy-Opalinski a, Jean-Pierre Gesson a, Joëlle Roche b,
Sébastien Papot a,
*
a UMR-CNRS 6514, Synthèse et Réactivité des Substances Naturelles, Université de Poitiers, 40 Av. du Recteur Pineau, 86022 Poitiers Cedex, France
b UMR-CNRS 6187, Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, 40 Av. du Recteur Pineau, 86022 Poitiers, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Two glucuronide prodrugs of the histone deacetylase inhibitor CI-994 were synthesized. These com-
pounds were found to be soluble in aqueous media and stable under physiological conditions. The car-
bamoyl derivatisation of CI-994 significantly decreased its toxicity towards NCI-H661 lung cancer
cells. Prodrug incubation with b-glucuronidase in the culture media led efficiently to the release of the
parent drug and thereby restoring its ability to decrease cell proliferation, to inhibit HDAC and to induce
E-Cadherin expression.
Received 26 June 2008
Revised 11 July 2008
Accepted 18 July 2008
Available online 23 July 2008
Keywords:
Prodrugs
Ó 2008 Elsevier Ltd. All rights reserved.
HDAC inhibitors
Chemotherapy
b-Glucuronidase
1. Introduction
adverse events including thrombocytopenia, anemia and neutrope-
nia. Another major drawback linked to its use in cancer chemother-
Recently, the inhibition of histone deacetylases (HDACs), a fam-
ily of enzymes which play a fundamental role in the regulation of
gene expression,1 has emerged as a new strategy in cancer chemo-
therapy. During the last decade, an increasing number of structur-
ally diverse small molecule HDAC inhibitors including
hydroxamates, carboxylates, benzamides, thiol derivatives, cyclic
peptides and hydrophilic ketones, have been studied as potential
therapeutic agents.2–5 These investigations demonstrated that sev-
eral HDAC inhibitors exhibited potent antitumour activities in the
course of the treatment of solid and haematological malignancies.
As a consequence, there are well over 100 clinical trials ongoing
with at least 13 different HDAC inhibitors as monotherapy or in
combination with other anticancer drugs.6 In October 2006, SAHA
1 (ZolinzaTM) (Fig. 1) became the first HDAC inhibitor approved by
the Food and Drug Administration for the treatment of patients
with cutaneaous T-cell lymphoma.
CI-994 2 (Fig. 1) is a potent member of the benzamide class of
HDAC inhibitors that has demonstrated significant antitumour
activity against a broad spectrum of murine, rat and human tu-
mour models.7–9 This compound is currently progressing through
clinical trials in combination with other standard anticancer agents
such as carboplatin, paclitaxel,10 capecitabine11 or gemcitabine.12
Although CI-994 is a promising chemotherapeutic agent, it induces
apy is its lack of aqueous solubility. Within this framework the
design of non-toxic hydrophilic prodrugs that could deliver CI-
994 predominantly in the vicinity of the tumour seems to be an
interesting alternative in order to enhance the therapeutic index
of this HDAC inhibitor. Such an approach has recently been pro-
posed for the selective targeting of SAHA.13
Toward this end, we have undertaken the study of the two glu-
curonide prodrugs 3 and 4 (Fig. 2). Indeed, several glucuronide pro-
drugs have already been selectively activated by b-glucuronidase,
either present in high concentration in necrotic tumour areas
(PMT)14 or previously targeted to the tumour sites (ADEPT,15
GDEPT16), and consequently demonstrated superior efficacy
in vivo compared to standard chemotherapy.17 These results were
attributed to the increased drug deposition and retention in the tu-
mour connected with reduced anticancer agent concentration in
normal tissues, considerably lowering the destruction of normal
cells.
Prodrug 3 includes a nitrobenzylphenoxy carbamate linker18
which has been employed successfully to release either anticancer
drug such as doxorubicine19 or magnetic resonance imaging con-
trast agent.20 According to these results, this linker should allow
easy recognition of 3 by b-glucuronidase and, after enzymatic
cleavage of the glycosidic bond should rapidly liberate CI-994 via
an 1,6 elimination as depicted in Figure 2.
On the other hand, for prodrug 4 CI-994 is attached directly to
the glucuronide moiety through a carbamate functional group.
* Corresponding author. Tel.: +33 5 49 45 38 62; fax: +33 5 49 45 35 01.
E-mail address: sebastien.papot@univ-poitiers.fr (S. Papot).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.048

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M. Thomas et al. / Bioorg. Med. Chem. 16 (2008) 8109–8116
O
O
H
N
O
NH
NHOH
NH2
N
H
O
1 SAHA
2 CI-994
Figure 1. SAHA and CI-994.
O
OMe
OH
O
NO2
O
NO2
HN
O
O
AcO
H
O
O
O
O
HO
HO
AcO
O
O
N
OAc
N
O
OH
H
NO2
5
O
3
HO
a
O
H2O
β-Glucuronidase
OMe
O
NO2
HN
NO2
NO2
H
O
RO
RO
O
O
N
OH
O
O
OH
NHAc
OR
O
O
HO
HO
OH
CO2
+
+ CI-994
6: R=OAc
7: R=OH
OH
b
c
β-Glucuronidase
3
O
OH
O
Scheme 1. Reagents and conditions: (a) CI-994, pyridine, DMF, rt, 3 days, 60%; (b)
MeONa/MeOH (0.3 equiv.), 0 °C to rt, 14 h, 40%; (c) NaOH, acetone, ꢀ40 °C, 15 min,
quant.
HN
O
H
N
O
HO
O
N
HO
OH
H
O
4
available 2-nitrophenyl isocyanate in a very high b-diastereoselec-
tivity using the elegant method developed earlier by Leenders et al.
(86%, e.d. 97%).22 The compound 9 was then converted quantita-
tively to the amine 10 by reduction of the nitro group (H2, Pd/C).
The latter reacted with 4-acetamidobenzoic acid in the presence
of EDC and HOBt in DMF to yield the protected prodrug 11 (63%).
In spite of several attempts, using various amounts of MeONa in
MeOH (from 0.1 to 1 equiv.) cleavage of the acetate groups always
failed and resulted mainly in the formation of the methyl 4-aceta-
midobenzoate 12 and the cyclic urea 13 along with unidentified
products. Other standard basic deprotection conditions including
KCN/MeOH, K2CO3/MeOH, Et3N/MeOH, Ba(OH)2/MeOH, NaOH/ace-
tone or LiOH/H2O led to similar results. Further attempts, using
acidic reagents such as HF–BF3/MeOH or TiCl4/MeOH, generated
complex mixtures. These results appeared surprising since such a
problem was not observed during the deprotection steps of pro-
drug 3. However, although the reasons of this failure remain un-
clear, we considered it preferable to turn our attention to other
protecting groups.
Figure 2. Mechanism of CI-994 release from either prodrug 3 and 4 in the presence
of b-glucuronidase.
With this design, we anticipated that 4 would be an excellent sub-
strate for the enzyme since N-phenyl b-glucuronyl carbamate was
proved to be hydrolyzed by b-glucuronidase at a rate comparable
to that of p-nitrophenyl b-glucuronide.21 Thus, enzymatic hydroly-
sis should conduct to the expulsion of the carbamic acid of the drug
that should lead rapidly to the free aniline after decarboxylation
(Fig. 2).
In both cases, we hypothesised that (1) the carbamoyl derivat-
isation of the amino group of CI-994 may prevent its ability to in-
hibit HDACs and therefore may decrease its toxicity toward normal
cells and, (2) the hydrophilic character of the glucuronide moiety
should enhance the water solubility of the drug allowing i.v.
administration.
At this stage, triethylsilyl (TES) ether appeared as an interesting
alternative since such protective groups can be removed under
mild acidic conditions.23 For that purpose, we have first prepared
the key intermediate 16 in two steps as illustrated in Scheme 3.
Thus, the protected glucuronide 15 was obtained in quantitative
yield by treating the hydroxyl free carbohydrate derivative 1424
with triethylsilyltriflate and pyridine in CH2Cl2. The deprotection
of the anomeric center was then performed with DDQ to afford
the 1-hydroxyl-free glucuronide 16 as a mixture of diastereoiso-
mers (90%). This compound was then coupled to the 2-nitrophenyl
isocyanate using the same methodology as described above.22 Un-
der these conditions the carbamate 17 was obtained in 88% yield.
Unfortunately, in this case no diastereoselectivity has been ob-
served. Nevertheless, a careful flash column chromatography per-
mitted to isolate a pure fraction of the b-anomer which allowed
2. Results and discussion
2.1. Chemistry
Prodrug 3 was prepared in three steps from the readily accessi-
ble activated carbonate 519 (Scheme 1). First, CI-994 was con-
densed with 5 in the presence of pyridine to give the carbamate
6 in 60% yield. The O-acetyl groups of 6 were then deprotected
with MeONa in MeOH to afford 7 (40%). Finally, saponification of
the methyl ester using NaOH produced the target prodrug 3 in
quantitative yield.
In order to access to the requested glucuronide prodrug 4, our
initial efforts focused upon the synthetic route illustrated in
Scheme 2. Thus, the b-O-glucuronyl carbamate 9 was synthesized
by coupling the well known glucuronide 8 with commercially

M. Thomas et al. / Bioorg. Med. Chem. 16 (2008) 8109–8116
8111
OMe
O
OMe
O
NO2
O
H
N
O
a
AcO
AcO
O
AcO
AcO
OH
OAc
OAc
O
8
9
NHAc
b
OMe
O
OMe
O
O
NH2
O
AcO
H
N
HN
O
AcO
AcO
H
O
c
O
N
OAc
AcO
OAc
O
O
d
11
10
O
H
N
O
+
O
N
H
NHAc
12
13
Scheme 2. Reagents and conditions: (a) 2-nitrophenyl isocyanate, Et3N, toluene, 0 °C, 2.5 h, 86%, e.d. 97%; (b) H2, Pd/C 10%, EtOAc/EtOH 1:1, rt, 1 h, quant.; (c) 4-
acetamidobenzoic acid, EDC, HOBt, DMF, rt, 24 h, 63%; (d) MeONa/MeOH (from 0.1 to 1 equiv.), 0 °C.
OMe
OMe
OMe
O
OMe
O
OMe
OMe
O
O
a
HO
HO
TESO
TESO
O
O
OH
OTES
14
15
b
OMe
O
OMe
O
O
NO2
O
c
TESO
O
NH
TESO
OH
TESO
TESO
OTES
OTES
O
17
16
d
O
OMe
O
OMe
O
O
N
NH2
H
O
TESO
TESO
TESO
TESO
O
NH
O
NH
e
NHAc
OTES
OTES
O
O
19
18
f
O
OMe
O
g
N
H
O
HO
HO
4
O
NH
NHAc
OH
20
O
Scheme 3. Reagents and conditions: (a) TESOTf, pyridine, CH2Cl2, 0 °C to rt, 72 h, quant.; (b) DDQ, H2O/CH2Cl2 (1: 10), rt, 4 h, 90%; (c) 2-nitrophenyl isocyanate, Et3N, toluene,
0 °C, 2.5 h, 88%; (d) H2, Pd/C 10%, EtOH, rt, 1 h, quant.; (e) 4-acetamidobenzoic acid, EDC, DMAP, DMF, rt, 12 h, 80%; (f) HCOOH, EtOAc, rt, 14 h, 77%; (g) NaOH, acetone, ꢀ40 °C,
80%.
us to pursue our investigations in this direction. Reduction of the
nitro group of 17 undertaken under H2 atmosphere in the presence
of a catalytic amount of Pd/C led to the aniline 18 in quantitative
yield. The coupling of 18 with 4-acetamidobenzoic acid was
achieved using EDC and DMAP in DMF to give the protected pro-
drug 19 in 80% yield. The crucial deprotection of the 2,3,4-hydroxyl
groups of 19 was carried out with HCOOH in ethyl acetate at room
temperature over a period of 14 h. As expected, under such condi-
tions the glucuronide 20 was isolated in good yield after purifica-
tion by flash column chromatography (77%). It is worth

8112
M. Thomas et al. / Bioorg. Med. Chem. 16 (2008) 8109–8116
mentioning that neither the acetamide 12 nor the cyclic urea 13
was formed in this case. Finally, the cleavage of the methyl ester
of compound 20 was realized with NaOH in acetone at ꢀ40 °C to
produce the prodrug 4 in 80% yield.
CI-994
120
100
80
-β-Glu
+β-Glu
2.2. Solubility and stability
60
40
20
0
The aqueous solubility of the glucuronide prodrugs 3 and 4 was
measured in phosphate buffer (0.02 M) at pH 7 and compared to
that of the corresponding drug. Under these conditions, CI-994
exhibited a poor solubility (0.08 mg/mL) whereas both prodrugs
were perfectly soluble at a concentration of 1 mg/mL which is
compatible with i.v. administration.
0
50
100
150
200
250
300
350
Concentration (µM)
The stability of both prodrugs was examined by incubating
these compounds in a 0.02 M phosphate buffer (pH 2.1 or 7) or
in cell culture media supplemented with 10% foetal calf serum at
37 °C. The evolution of each solution was followed by HPLC (UV
detection) over a period of 72 h. As expected, no detectable decom-
position of 3 or 4 has been observed in the course of these
experiments.
3
120
100
80
-β-Glu
+β-Glu
60
40
20
0
2.3. Enzymatic hydrolysis
Enzymatic hydrolysis of each compound was first conducted in
the presence of an excess of enzyme which is always the case in the
course of an ADEPT protocol (3 or 4: 0.1 mg/mL; Escherichia coli b-
glucuronidase: 133 U/mL). Both prodrugs were readily cleaved by
b-glucuronidase and quantitative conversion of either 3 or 4 to
CI-994 was achieved in less than 5 min. Further experiments were
undertaken using a lower concentration of the E. coli enzyme (5 U/
mL) in order to compare kinetics of drug release from glucuronides
3 or 4 (Fig. 3).
These experiments showed that CI-994 was liberated slightly
faster starting from prodrug 4 than prodrug 3. However, kinetic
profiles were very similar and both compatible with either an
ADEPT or a PMT strategy. The absence of major differences be-
tween the kinetics for 3 and 4 seems to indicate that the linker
decomposition step of prodrug 3 is not rate-determining.
0
50
100
150
200
250
300
350
Concentration (µM)
4
120
100
80
60
40
20
0
-β-Glu
+β-Glu
0
50
100
150
200
250
300
350
Concentration (µM)
2.4. Biological evaluations
Figure 4. Proliferative activity of NCI-H661 cells treated during 48 h with the
indicated compounds with (black dot) or without (white dot) b-glucuronidase.
Three experiments were done in triplicate. Standard deviation is indicated.
Compounds 3 and 4 were then tested for their anti-proliferative
activity on NCI-H661 non-small cell lung cancer cells after 48 h
treatment. IC50 value was 20 lM for CI-994 (Fig. 4). In contrast,
prodrugs 3 and 4 did not exhibit any anti-proliferative activity
(300 lM) for 3. This demonstrated that the carbamoyl derivatisa-
tion of the amino group of CI-994 reduces significantly its toxicity.
Thus, the use of glucuronide prodrugs such as 3 or 4 in the course
of a tumour targeting strategy may prevent the adverse events re-
corded with the free drug. However, addition of b-glucuronidase
(40 U/mL) in the culture media induced a dramatic anti-prolifera-
tive effect with 3 and 4 showing that these two glucuronide pro-
drugs behaved with similar activity than CI-994 in the presence
on these cells, except a slight decrease of proliferation at high dose
100
90
80
of the enzyme (IC50 = 20 lM). These latter results can be unambig-
uously attributed to an efficient release of the drug in the culture
media therefore restoring its initial activity.
70
CI-994 released from 3
60
Prodrug 3
50
The HDAC inhibition was further estimated by Western blot for
histone H4 acetylation (Fig. 5). As previously shown, Trichostatin A
(TSA), a well known HDAC inhibitor, induced histone H4 acetyla-
tion.25 This HDAC inhibition was also detected for CI-994 but not
for 3 or 4 when incubated alone. Again, after b-glucuronidase treat-
ment, 3 and 4 were able to induce histone H4 acetylation to similar
level obtained with the same concentration of CI-994.
Next, we tested the ability of the compounds to induce E-Cad-
herin expression in NCI-H661 cells. E-Cadherin is a cell surface
transmembrane protein that plays a major role in epithelial cell
adhesion and connects the extracellular environment to the con-
CI-994 released from 4
Prodrug 4
40
30
20
10
0
0
20
40
60
80
100
120
Time (min)
Figure 3. Enzymatic cleavage of prodrugs
3 and 4. Prodrug concentration:
0.1
l
mol mLꢀ1, enzyme concentration 5 U mLꢀ1
.

M. Thomas et al. / Bioorg. Med. Chem. 16 (2008) 8109–8116
8113
grade. 1H and 13C NMR were performed on an Avance 300 DPX
Bruker. The chemical shifts are expressed in part per million
(ppm) relative to TMS (d = 0 ppm) and the coupling constant J
in hertz (Hz). NMR multiplicities are reported using the following
abbreviations: b, broad; s, singulet; d, doublet; t, triplet; q, qua-
druplet; m, multiplet.
16KDa
acetyl-histone H4
Optical rotations were measured on a Schmidt + Haensch Polar-
tronic HH-8 polarimeter, in a 1 dm cell. Melting points were mea-
sured on a Büchi Melting Point B-545 instrument and were
uncorrected. The reaction progress was monitored on precoated
silica gel TLC plates Macherey-Nagel ALUGRAMÒ SIL G/UV254
(0.2 mm silica gel 60 Å). Spots were visualized under 254 nm UV
light and/or by dipping the TLC plate into a solution of 3 g of
phosphomolibdic acid in 100 mL of ethanol followed by heating
with a hot gun. Flash column chromatography was performed
6KDa
64KDa
α-tubulin
50KDa
Figure 5. Western blot analysis for histone H4 acetylation. NCI-H661 cells were
treated with the indicated compounds for 6 h. -tubulin is used as loading controls.
a
Representative of three independent experiments.
tractile cytoskeleton. E-Cadherin loss is one of the hallmark of the
epithelial–mesenchymal transition (EMT) that plays a critical role
in tumor progression and invasion, leading to distant metasta-
sis.26,27 E-Cadherin loss is also associated with resistance to EGFR
inhibitors and poor prognosis in lung cancer.28,29 Therefore, restor-
ing E-Cadherin expression has therapeutical interest. This induc-
tion can be achieved by TSA treatment in NCI-H661 cells.30 In
our experiment, we found E-Cadherin expression with CI994 and
3 and 4 after b-glucuronidase treatment, whereas prodrugs alone
did not show any activity on E-Cadherin expression (Fig. 6).
using Macherey-Nagel silica gel 60 (15–40 lm).
4.2. Synthesis and characterization of described compounds
4.2.1. 4-Acetamido-N-(2-aminophenyl)-benzamide or CI-994
(2)
To a solution of 4-acetamidobenzoic acid (500 mg, 2.79 mmol, 1
equiv.) in dry DMF (10 mL) was added benzene-1,2-diamine
(904 mg, 8.37 mmol,
3 equiv.) followed by EDC (695 mg,
6.63 mmol, 1.3 equiv.) and a catalytic amount of DMAP (34 mg,
0.28 mmol, 0.1 equiv.). After stirring overnight, the mixture was
concentrated under vacuum. Dichloromethane was added and
the reaction mixture was placed in the fridge overnight. The solu-
tion was filtered and washed with hot dichloromethane to afford 2
as a white solid (609 mg, 2.26 mmol, 81%): m.p. 216.1 °C; 1H NMR
(DMSO, d) 10.21 (bs, 1H), 9.57 (bs, 1H), 7.94 (d, 2H, J = 8.5 Hz), 7.69
(d, 2H, J = 8.6 Hz), 7.15 (d, 1H, J = 7.5 Hz), 6.96 (t, 1H, J = 7.1 Hz),
6.78 (d, 1H, J = 7.4 Hz), 6.59 (t, 1H, J = 7.3 Hz), 4.89 (bs, 2H), 2.09
3. Conclusion
In summary, we have reported the synthesis of the first CI-994
glucuronide prodrugs to date aimed at b-glucuronidase activation
in ADEPT or PMT strategies. Such prodrugs were at least 12.5 times
more soluble in aqueous media compared to the parent drug and
exhibited good stability under physiological conditions. In contrast
with CI-994, prodrugs 3 and 4 have no activity on cell proliferation,
histone acetylation and E-Cadherin expression in our cellular mod-
el. On the other hand, when incubated in the presence of b-glucu-
ronidase, CI-994 was efficiently release and consequently its anti-
tumour properties were restored. All these results suggested that
both glucuronide prodrugs 3 and 4 possess the necessary prerequi-
sites for further in vivo investigation in the course of a tumour tar-
geting strategy such as ADEPT or PMT.
13
(s, 3H); C NMR (DMSO, d) 169.1, 165.1, 143.5, 142.5, 129.1 (*2),
129.0, 127.0, 126.7, 123.8, 118.4 (*2), 116.6, 116.5, 24.2.
4.2.2. 3,4,5-Triacetoxy-6-{4-[2-(4-acetylamino-benzoylamino)-
phenylcarbamoyloxymethyl]-2-nitro-phenoxy}-tetrahydro-
pyran-2-carboxylic acid methyl ester (6)
Carbonate 5 (1 g, 1.54 mmol, 1 equiv.) and CI-994 2 (830 mg,
3.08 mmol, 2 equiv.) were dissolved in DMF (20 mL). Pyridine
(0.31 mL, 3.85 mmol, 2.5 equiv.) was added and the solution was
stirred at room temperature for 72 h. The mixture was then hydro-
lysed with distilled water and extracted three times with chloro-
form. The organic layer was washed with HCl (1 M) twice, dried
over MgSO4 and evaporated to dryness. The residue was purified
by flash chromatography (90/10 AcOEt/PE and 100% AcOEt). Prod-
uct 6 was isolated as a white powder with a yield of 60% (720 mg,
4. Experimental
4.1. General chemistry methods
All reactions were performed under N2 atmosphere. Solvents
used were of HPLC quality and chemicals were of analytical
0.92 mmol): m.p. 112.5 °C; ½a D20
ꢁ
: +8 (c 0.11, CHCl3); 1H NMR
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
(CD3COCD3, d) 9.52 (bs, 1H), 9.48 (bs, 1H), 8.58 (bs, 1H), 7.95 (m,
3H), 7.78–7.52 (m, 6H), 7.21 (m, 2H), 5.67 (d, 1H, J = 7.7 Hz), 5.46
(t, 1H, J = 9.4 Hz), 5.25 (m, 4H), 4.68 (d, 1H, J = 9.7 Hz), 3.69 (s,
3H), 2.15 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H); 13C NMR
(CD3COCD3, d) 170.2, 169.9, 169.6, 169.5, 167.7, 166.1, 155.5,
149.8, 144.1, 142.2, 134.7, 134.1, 132.9, 131.8, 129.9, 129.8 (*2),
127.1, 126.8, 126.1, 125.6, 125.4, 119.8 (*2), 119.7, 100.2, 73.2,
72.5, 71.5, 70.3, 66.1, 53.4, 24.8, 20.8 (*3); MS (ESI) [M+Na]+ for
C36H36O16N4Na = 803.8.
4.2.3. 6-{4-[2-(4-Acetylamino-benzoylamino)-phenylcarba-
moyloxymethyl]-2-nitro-phenoxy}-3,4,5-trihydroxy-tetrahydro-
pyran-2-carboxylic acid methyl ester (7)
NT
TSA
CI-994
3
3+β-Glu
4
4+β-Glu
Compound 6 (987 mg, 1.26 mmol, 1.0 equiv.) was dissolved in
distilled MeOH (40 mL). The solution was cooled to 0 °C and MeO-
Na (20 mg, 0.37 mmol, 0.3 equiv.) was added. The reaction mixture
was stirred during 2 h and then allowed to stand to room temper-
Figure 6. E-Cadherin mRNA level was measured by quantitative real-time RT-PCR
on NCI-H661 cells grown 16 h with the indicated compounds. This experiment was
done 2 times and each RT PCR in duplicate. Values are expressed in % of GAPDH
expression. Bars, SD.

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M. Thomas et al. / Bioorg. Med. Chem. 16 (2008) 8109–8116
ature during 14 h. The reaction was neutralised with IRC 50 during
20 min. The mixture was then filtered and evaporated to dryness.
Flash chromatogtraphy (6/94, 8/92 and 10/90 MeOH/CH2Cl2) gave
compound 7 (330 mg, 0.5 mmol) as a white solid with a yield of
4.2.7. 3,4,5-Triacetoxy-6-[2-(4-acetylamino-benzoylamino)-
phenyl-carbamoyloxy]-tetrahydro-pyran-2-carboxylic acid
methyl ester (11)
To a solution of 4-acetylamino-benzoic acid (717 mg, 4.0 mmol,
2.0 equiv.) in dry DMF (4 mL) was added compound 10 (940 mg,
2.0 mmol, 1.0 equiv.), followed by EDC (997 mg, 5.2 mmol, 2.6
equiv.) and hydroxybenzotriazole hydrate (703 mg, 5.2 mmol, 2.6
equiv.). After stirring during 72 h at room temperature, the mixture
was quenched by addition of a saturated NH4Cl solution (10 mL)
and extract with AcOEt/benzene (v/v, 2/1, 20 mL) twice. The organ-
ic phase was washed consecutively with water and saturated brine
(10 mL each). The organic layer was dried over Na2SO4 and concen-
trated under vacuum. The crude product was purified by flash
chromatography (80/20, AcOEt/EP), yielding the coupling com-
pound 11 as a white solid (800 mg, 1.27 mmol): m.p. 125.5 °C;
40%: m.p. 144.1 °C; ½a D20
ꢁ
: +0.17 (c 0.1, MeOH); 1H NMR (MeOD, d)
7.81 (m, 3H), 7.64 (d, 2H, J = 8.7 Hz), 7.49 (m, 3 H), 7.29 (d, 1H,
J = 8.7 Hz), 7.22 (m, 2H), 5.15 (m, 3H), 4.07 (d, 1H, J = 9.6 Hz),
3.60 (s, 3H), 3.51–3.47 (m, 3H), 2.15 (s, 3H); 13C NMR (MeOD, d)
170.8, 169.6, 166.8, 155.3, 149.4, 142.4, 140.8, 133.4, 131.9,
131.7, 129.0, 128.4 (*2), 126.5, 126.2, 125.5, 124.6, 121.6, 121.3,
119.1 (*2), 117.5, 101.0, 75.9, 75.6, 73.2, 71.5, 65.2, 51.8, 22.9; MS
(ESI) [M+Na]+ for C30H31O13N4Na = 678.5.
4.2.4. 6-{4-[2-(4-Acetylamino-benzoylamino)-phenylcar-
bamoyloxymethyl]-2-nitro-phenoxy}-3,4,5-trihydroxy-
tetrahydro-pyran-2-carboxylic acid (3)
½
a 2D0: +4 (c 0.21, CHCl3); 1H NMR (CDCl3, d) 8.99 (bs, 1H), 8.34
ꢁ
(bs, 1H), 8.03 (bs, 1H), 7.90 (d, 2H, J = 8.4 Hz), 7.60 (d, 2H,
J = 8.7 Hz), 7.55 (m, 1H), 7.42 (dd, 1H, J = 2.9 Hz, J0 = 7.0 Hz), 7.07
(m, 2H), 5.76 (d, 1H, J = 8.0 Hz), 5.30 (t, 1H, J = 9.2 Hz), 5.03 (t,
1H, J = 6.5 Hz), 4.94 (t, 1H, J = 9.1 Hz), 4.20 (d, 1H, J = 9.7 Hz), 3.67
(s, 3H), 2.11 (s, 3H), 1.95–2.05 (s, 9H); 13C NMR (CDCl3, d) 170.2,
170.0, 169.9, 169.5, 167.8, 166.8, 152.0, 149.3, 142.0, 130.8, 129.3
(*2), 129.2, 126.9, 126.5, 125.6, 123.3, 119.8 (*2), 92.9, 72.7, 71.9,
70.0, 69.2, 53.6, 24.9, 20.9 (*3); MS (ESI) [M+Na]+ for
To a solution of methyl ester 7 (52 mg, 0.08 mmol, 1.0 equiv.) in
acetone (3 mL) cooled to ꢀ30 °C was added NaOH (0.8 mL, 1 M,
0.8 mmol, 10 equiv.). After stirring for 15 min, the starting material
has totally disappeared. The solution was neutralised with IRC 50
for 20 min. Evaporation of the filtrate under vacuum afforded com-
pound 3 in quantitative yield as a white solid (51 mg, 0.079 mmol):
m.p. 161.6 °C; ½a D20
ꢁ
: +0.21 (c 0.1, MeOH); 1H NMR (D2O, d) 7.81 (m,
3H), 7.64 (d, 2H, J = 8.7 Hz), 7.49 (m, 3H), 7.29 (d, 1H, J = 8.7 Hz),
7.22 (m, 2H), 5.15 (m, 3H), 4.07 (d, 1H, J = 9.6 Hz), 3.60 (s, 3H),
3.51–3.47 (m, 3H), 2.15 (s, 3H); 13C NMR (MeOD, d) 170.8, 169.6,
166.8, 155.3, 149.4, 142.4, 140.8, 133.4, 131.9, 131.7, 129.0,
128.4, 126.5, 126.2, 125.5, 124.6, 119.1, 117.5, 101.0, 75.9, 75.6,
73.2, 71.5, 65.2, 51.8, 22.9; HRMS Calcd for C29H27O13N4Na2
[MꢀH+2Na]+ = 685.1370, Found: [MꢀH+2Na]+ = 685.1380. Anal
Calcd for C29H28N4O13 ꢂ1.5H2O: C 52.18, H 4.68, N, 8.39; Found:
52.10, H 4.75, N 8.14.
C29H31N3O13Na = 652.5.
4.2.8. 6-(3,4-Dimethoxy-benzoyloxy)-3,4,5-tris-triethylsilanol-
oxy-tetrahydro-pyran-2-carboxylic acid methyl ester (15)
TESOTf (15.3 mL, 67.2 mmol, 6.0 equiv.) was added to
a
stirred solution of the triol 14 (4 g, 11.2 mmol, 1.0 equiv.), anhy-
drous pyridine (50 mL, 618 mmol) and anhydrous dichlorometh-
ane (285 mL) at 0 °C. At the end of the addition, the reaction was
allowed to reach to room temperature. After complete conver-
sion of starting material (72 h), the solvents were removed in
vacuo and the residue was poured into a mixture of dichloro-
methane and saturated Na2CO3 solution (25 mL). The organic
phase was washed with distilled water (25 mL), dried, filtered
and concentrated in vacuo. The resulting residue was purified
by flash chromatography (15/85, AcOEt/PE) to yield the fully
protected glucuronide 15 (7.85 g, 11.2 mmol) as a clear oil:
4.2.5. 3,4,5-Triacetoxy-6-(2-nitro-phenyl-carbamoyloxy)-
tetrahydro-pyran-2-carboxylic acid methyl ester (9)
To a solution of glucuronide 8 (2.5 g, 7.49 mmol) in anhydrous
toluene (150 mL) and Et3N (3.2 mL, 22.5 mmol, 3 equiv.) cooled
to 0 °C was added slowly (over a period of 1 h) a solution of 2-
nitrophenyl isocyanate (7.49 mmol, 1 equiv.) in toluene (60 mL).
After 2 h, the mixture was evaporated and subjected to flash col-
umn chromatography (60/40, EP/AcOEt) to furnish 9 as a yellow
½
a 2D0
ꢁ
: ꢀ21 (c 0.19, CHCl3); 1H NMR (CDCl3, d) 6.89 (d, 1H,
J = 1.7 Hz), 6.79 (dd, 1H, J = 8.2 Hz, J0 = 1.7 Hz), 6.71 (d, 1H,
J = 8.2 Hz), 4.84 (d, 1H, J = 11.2 Hz), 4.67 (d, 1H, J = 6.1 Hz), 4.34
(d, 1H, J = 11.2 Hz), 4.13 (m, 2H), 3.79 (s, 3H), 3.78 (s, 3H),
3.67 (s, 3H), 3.60 (m, 2H), 0.81 (m, 27H), 0.51 (m, 18H); 13C
NMR (CDCl3, d) 170.7, 149.1, 148.8, 130.4, 121.0, 112.0, 110.9,
101.6, 78.8, 78.1, 77.9, 73.4, 72.1, 56.2, 56.1, 52.5, 7.3 (*3), 7.2
(*3), 7.1 (*3), 5.4 (*3), 5.2 (*3), 5.1 (*3); MS (ESI) [M+Na]+ for
solid (3.2 g, 86%, d.e. b = 97%, 6.4 mmol): m.p. 154.2 °C; ½a D20
ꢁ : ꢀ22
(c 0.1, CHCl3); 1H NMR (CDCl3, d) 9.97 (bs, 1H), 8.50 (d, 1H,
J = 7.4 Hz), 8.23 (d, 1H, J = 8.5 Hz), 7.67 (t, 1H, J = 7.2 Hz), 7.22 (t,
1H, J = 7.4 Hz), 5.86 (d, 1H, J = 7.5 Hz), 5.30 (m, 3H), 4.26 (d, 1H,
J = 9.0 Hz), 3.75 (s, 3H), 2.06 (s, 9H); 13C NMR (CDCl3, d) 170.3,
169.8, 169.5, 167.2, 150.9, 136.9, 136.4, 134.3, 126.3, 123.8,
121.3, 93.0, 73.2, 71.8, 70.0, 69.1, 53.4, 20.9, 20.8, 20.7; MS (ESI)
[M+Na]+ for C20H22N2O13Na = 521.6.
C34H64O9Si3Na = 723.8.
4.2.9. 6-Hydroxy-3,4,5-tris-triethylsilanyloxy-tetrahydro-
pyran-2-carboxylic acid methyl ester (16)
4.2.6. 3,4,5-Triacetoxy-6-(2-amino-phenyl-carbamoyloxy)-
tetrahydro-pyran-2-carboxylic acid methyl ester (10)
To a solution of the fully protected glucuronide 15 (7.8 g,
11.1 mmol, 1.0 equiv.) in dichloromethane/water (v/v, 10/1,
200 mL/20 mL) was added DDQ (5.05 g, 22.3 mmol, 2.0 equiv.).
The resulting mixture was stirred at room temperature for 4 h.
A solution of compound 9 (1.0 g, 2.0 mmol) in EtOH/AcOEt (v/
v, 1/1, 30 mL/30 mL) was hydrogenated over 10% palladium/car-
bon (30 mg) at room temperature for 1 h. The catalyst was fil-
tered through a pad of celite and the filtrate was evaporated in
vacuo to give the desired product 10 as a white solid in quantita-
The reaction was poured into
a saturated NaHCO3 solution
tive yield (936 mg, 2.0 mmol): m.p. 145.1 °C; ½a D20
ꢁ
: +21 (c 0.1,
(100 mL) and extracted with dichloromethane (3 ꢃ 100 mL). The
organic phases were dried over MgSO4 and concentrated under
vacuum. Purification by silica gel chromatography (5/95 and 10/
90 AcOEt/EP) afforded glucuronide 16 (5.5 g, 10.0 mmol, 90%) as
CHCl3); 1H NMR (CDCl3, d) 7.32 (d, 1H, J = 7.2 Hz), 7.03 (t, 1H,
J = 7.5 Hz), 6.97 (s, 1H), 6.79 (m, 2H, J = 7.8 Hz), 5.81 (d, 1H,
J = 7.7 Hz), 5.28 (m, 3H), 4.23 (d, 1H, J = 9.5 Hz), 3.75 (s, 2H),
3.73 (s, 3H), 2.05 (s, 9H), 13C NMR (CDCl3, d) 170.3, 170.2,
170.0, 167.5, 152.4, 140.6, 127.2, 125.3, 123.5, 119.5, 117.7,
93.0, 72.9, 72.1, 70.4, 69.4, 53.4, 21.0, 20.9, 20.8; MS (ESI)
[M+Na]+ for C20H24N2O11Na = 491.3.
a colourless oil: ½a D20
ꢁ
: +24 (c 0.15, CHCl3); 1H NMR (CDCl3, d) 5.24
(dd, 1H, J = 10.9 Hz, J0 = 2.7 Hz), 4.28 (d, 1H, J = 3.0 Hz), 3.90 (t,
1H, J = 3,3 Hz), 3.72 (t, 1H, J = 4.1 Hz), 3.59 (s, 3H), 3.45 (t, 1H),
0.81 (m, 27H), 0.51 (m, 18H); 13C NMR (CDCl3, d) 170.8, 89.8,

M. Thomas et al. / Bioorg. Med. Chem. 16 (2008) 8109–8116
8115
75.8, 73.2, 71.8, 71.4, 52.4, 7.3 (*3), 7.2 (*3), 7.1 (*3), 5.4 (*3), 5.2 (*3),
5.1 (*3); MS (ESI) [M+Na]+ for C25H54O7Si3Na = 573.9.
in vacuo and the residue was purified by flash column chromatog-
raphy (10/90, 13/87 and 15/85, MeOH/CH2Cl2) to yield the triol 20
(190 mg, 3.8 mmol, 78%) as a white solid: m.p. 178.1 °C; ½a D20
ꢁ
: +2 (c
4.2.10. 6-(2-Nitro-phenylcarbamoyloxy)-3,4,5-tris-triethyl-
silanoloxy-tetrahydropyran-2-carboxylic acid methyl ester (17)
To a solution of glucuronide 16 (5.5 g, 10.0 mmol, 1.0 equiv.) in
anhydrous toluene (165 mL) and Et3N (7 mL, 50 mmol, 5 equiv.)
cooled to 0 °C was added slowly (over a period of 1 h) a solution
of 2-nitrophenyl isocyanate (1.8 g, 11.1 mmol, 1.1 equiv.) in toluene
(60 mL). After stirring overnight at room temperature, the mixture
was evaporated and subjected to flash column chromatography (4/
96, 5/95, 7/93, 9/91 and 10/90 AcOEt/PE) to furnish compound 17
0.1, MeOH); 1H NMR (MeOD, d) 7.94 (d, 2H, J = 8.7 Hz), 7.72 (d, 2H,
J = 8.7 Hz), 7.59 (d, 1H, J = 7.5 Hz), 7.54 (dd, 1H, J = 7.2 Hz,
J0 = 1.7 Hz); 7.29–7.20 (m, 2H), 5.55 (d, 1H, J = 7.9 Hz), 4.01 (d,
1H, J = 9.2 Hz), 3.74 (s, 3H), 3.60–3,44 (m, 3H), 2.16 (s, 3H); 13C
NMR (MeOD, d) 172.4, 171.3, 168.8, 155.2, 144.1, 144.0, 133.0,
131.9, 130.3, 130.2, 128.1, 127.8, 127.2, 126.0, 120.9, 120.8, 97.3,
77.6, 77.5, 73.9, 73.3, 53.4, 24.5; MS (ESI) [M+Na]+ for
C23H25O10N3Na = 526.3.
(6.2 g, 8.7 mmol, d.e. b = 4%) with 88% of yield: ½a D20
ꢁ : ꢀ15 (c 0.22,
4.2.14. 6-[2-(4-Acetylamino-benzoylamino)-phenylcar-
bamoyloxy]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic
acid (4)
To a solution of compound 20 (190 mg, 0.38 mmol, 1 equiv.) in
methanol (14 mL) cooled to ꢀ40 °C was added NaOH 1 M (1.9 mL,
1.9 mmol, 5 equiv.). After 15 min of stirring, the starting material
has totally disappeared. The solution was neutralised with IRC50
for 20 min. The resin was removed by filtration and the filtrate
was evaporated to dryness. The compound 4 was isolated as a white
CHCl3); 1H NMR (CDCl3, d) 9.98 (s, 1H), 8.60 (t, 1H, J = 8.6 Hz),
8.22 (d, 1H, J = 8.5 Hz), 7.64 (t, 1H, J = 8.5 Hz), 7.16 (t, 1H,
J = 8.4 Hz), 6.06 (d, 1H, J = 6.1 Hz), 4.50 (d, 1H, J = 2.2 Hz), 4.34 (bs,
1H), 3.86 (dd, 1H, J = 6.1 Hz, J0 = 1.8 Hz), 3.83 (dd, 1H, J = 4.1 Hz
J0 = 2.0 Hz), 3.75 (s, 3H), 0.81 (m, 27H), 0.51 (m, 18H); 13C NMR
(CDCl3, d) 169.6, 151.6, 136.4, 136.0, 135.0, 126.0, 121.8, 120.9,
95.2, 78.7, 76.3, 74.1, 72.6, 52.3, 7.3 (*3), 7.2 (*3), 7.1 (*3), 5.4 (*3),
5.2 (*3), 5.1 (*3); MS (ESI) [M+Na]+ for C32H58O10N2Si3Na = 737.7.
solid (149 mg, 0.30 mmol) with a yield of 80%: m.p. 194 °C; ½a D20
ꢁ
: +9
(c 0.1, MeOH); 1H NMR (D2O, d) 7.65 (d, 2H), 7.40 (bs, 3H), 7.28–7.18
(m, 3H), 5.37 (d, 1H, J = 7.7 Hz), 3.71 (d, 1H, J = 9.0 Hz), 3.51–3.40
(m, 3H), 2.03 (s, 3H); 13C NMR (D2O, d) 177.9, 175.6, 171.7, 157.3,
143.8, 133.9, 132.5, 132.3, 131.4 (*2), 130.6, 129.8, 129.5, 127.9,
123.2 (*2), 97.8, 79.3, 78.0, 74.5, 74.4, 26.0; HMRS Calcd for
4.2.11. 6-(2-Amino-phenylcarbamoyloxy)-3,4,5-tris-triethyl-
silanoloxy-tetrahydropyran-2-carboxylic acid methyl ester (18)
A solution of compound 17 (580 mg, 0.81 mmol) in EtOH (25 mL)
was hydrogenated over 10% palladium/carbon (50 mg) at room
temperature for 50 min. The catalyst was filtered through a pad of
celite and the filtrate was evaporated to dryness. The residue was
purified by flash column chromatography (10/90, 15/85 and 20/80
AcOEt/PE) to yield the desired product 18 (465 mg, 0.68 mmol,
C
22H22O10N3Na2 [MꢀH+2Na]+ = 534.1101, Found: [MꢀH+2Na]+ =
534.1098. Anal Calcd for C22H23N3O10 ꢂ2H2O: C 50.29, H 5.18, N,
8.00; Found: 50.24, H 4.68, N 7.72.
84%) as a colourless oil: ½a D20
ꢁ
: +18 (c 0.12, CHCl3); 1H NMR (CDCl3,
4.3. Biological assays
d) 7.31 (d, 1H, J = 7.7 Hz), 7.05 (t, 1H, J = 7.6 Hz), 6.79 (t, 2H,
J = 7.9 Hz), 6.43 (bs, 1H), 6.02 (d, 1H, J = 6.2 Hz), 4.48 (bs, 1H), 4.27
(bs, 1H), 3.80 (m, 2H), 3.75 (s, 3H), 0.95 (m, 27H), 0.59 (m, 18H);
13C NMR (CDCl3, d) 170.3, 153.2, 140.8, 127.1, 125.5, 124.0, 119.8,
117.8, 95.1, 79.0, 77.2, 75.3, 72.9, 52.6, 7.1 (*3), 6.9 (*3), 6.8 (*3), 5.3
(*3), 5.0 (*3), 4.9 (*3); MS (ESI) [M+Na]+ for C32H60O8N2Si3Na = 707.5.
4.3.1. HPLC analysis
Analytical HPLC was carried out using a Dionex Ultimate 3000
System with UV variable wavelength detector. Compounds 2–4
analysis and enzymatic hydrolysis analysis were performed on a
reverse phase column chromatography (Acclaim(R) 120, C18,
250 ꢃ 4.6 mm, 5
lm, 120 Å) using a mobile phase (1 mL/min) of
4.2.12. 6-[2-(4-Acetylamino-benzoylamino)-phenylcar-
bamoyloxy]-3,4,5-tris-triethylsilanoxy-tetrahydropyran-2-
carboxylic acid methyl ester (19)
CH3CN/H2O(0.2%TFA) 3:7. Retention time for compounds 3, 4 and
2 were 9, 3.88 and 3.68 min , respectively. Peak area and calibra-
tion curves were obtained with Dionex Chromeleon software.
To a solution of 4-acetylamino benzoic acid (1.57 g, 8.77 mmol,
2.0 equiv.) in dry DMF (15 mL) was added the amine 18 (3.0 g,
4.39 mmol, 1.0 equiv.) followed by EDC (2.19 g, 11.4 mmol, 2.6
equiv.) and DMAP (50 mg, 0.41 mmol, 0.1 equiv.) at room tempera-
ture. After stirring overnight, the mixture was quenched by addi-
tion of a saturated NH4Cl solution (25 mL) and extracted with
AcOEt/benzene (v/v, 2/1, 40 mL) twice. The organic layer was dried
over Na2SO4 and concentrated under vacuum. The crude product
was purified by flash column chromatography (50/50, AcOEt/PE)
yielding the coupling compound 19 (3.0 g, 3.55 mmol, 81%) as a col-
4.3.2. Compound solubility
Ten milligrams of 2 were dissolved in 1 mL of DMSO and several
dilutions with phosphate buffer (0.02 M, pH 7) were realized to ob-
tain a calibration curve in a range of 0.1–0.025 mg/mL. Then, the
solubility of compound 2 was determined in phosphate buffer
(0.02 M, pH 7). The sample was filtered through a 0.45
filter, diluted in HPLC phase, and analyzed by HPLC.
lm Milipore
4.3.3. Compound stability
ourless oil: ½a 2D0
ꢁ
: ꢀ18 (c 0.1, CHCl3); 1H NMR (CDCl3, d) 9.05 (bs, 1H),
Prodrugs 3 and 4 were placed in phosphate buffer (0.02 M, pH
2.1 or 7) and 10% foetal calf serum at 37 °C for a period of 72 h.
HPLC analysis showed no detectable degradation of these com-
pounds in these conditions.
8.50 (bs, 1H), 7.94 (d, 2H, J = 8.5 Hz), 7.73 (bs, 2H), 7.64 (d, 2H,
J = 8.7 Hz), 7.41 (d, 1H, J = 7.3 Hz), 7.22 (t, 1H, J = 6.7 Hz), 7.13 (t,
1H, J = 7.04 Hz), 6.08 (d, 1H, J = 7.2 Hz), 4.51 (s, 1H), 4.28 (s, 1H),
3.80 (s, 1H), 3.78 (s, 1H), 3.66 (s, 3H), 1.99 (s, 3H), 1.0–0.80 (m,
27H), 0.65–0.54 (m, 18H); 13C NMR (CDCl3, d) 170.4, 170.1, 166.4,
153.2, 142.0, 131.7, 129.6, 129.1, 128.9, 127.1, 126.7, 125.6, 124.0,
119.8, 95.3, 79.4, 77.0, 75.4, 72.9, 52.6, 24.7, 7.2 (*6), 7.1 (*3), 5.3
(*3), 5.1 (*6); MS (ESI) [M+Na]+ for C41H67O10N3Na = 868.1.
4.3.4. Enzymatic cleavage of prodrugs
Escherichia coli b-glucuronidase was purchased from Sigma–Al-
drich (reference: G8162). Prodrugs 3 and 4 (0.1 mg/mL) were incu-
bated with E. coli b-glucuronidase (133 U/mL) in phosphate buffer
(0.02 M, pH 7) at 37 °C and sample were analyzed by HPLC after
5 min.
4.2.13. 6-[2-(4-Acetylamino-benzoylamino)-phenylcar-
bamoyloxy]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic
acid methyl ester (20)
4.3.5. Kinetic of drug release
The glucuronide 19 (415 mg, 0.49 mmol) was stirred overnight
in ethyl acetate/formic acid (3/2, 6 mL). The solvents were removed
Prodrugs 3 and 4 (1.5
glucuronidase (5 U/mL) in phosphate buffer (0.02 M, pH 7) at
lmol/mL) were incubated with E. coli b-

8116
M. Thomas et al. / Bioorg. Med. Chem. 16 (2008) 8109–8116
37 °C. Aliquots of 50 lL were taken at 0, 1, 5, 15 min and then every
Acknowledgements
15 min and diluted in 450
lL of HPLC mobile phase and analyzed
by HPLC.
The authors thank CNRS, La Ligue Nationale contre le Cancer
(Comité de Charentes-Maritime) and the Région Poitou-Charentes
for financial support of this study.
4.3.6. Cell culture
The NCI-H661 non-small cell lung cancer cells were grown in
RPMI 1640 plus 10% foetal calf serum (Invitrogen) at 37 °C and un-
der 5% CO2. When indicated, b-glucuronidase (Sigma, G8162) was
added at 40 U/ml in the culture media.
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4.3.7. Cell proliferation
The Cell Proliferation Kit II (XTT; Roche) was used to assess
cell proliferation. This assay is based on the cleavage of XTT by
metabolic active cells resulting in the production of an orange
formazan dye that is quantified by spectrophotometry. Assays
were carried out essentially as described by the manufacturer.
Briefly, 4 ꢃ 103 cells/well were plated in 100
ll of media in a
96-well plate. Cells were grown for 24 h before adding the com-
pound at the indicated concentration. After 48 h treatment with
or without b-glucuronidase in the media, 50 ll of the XTT label-
ing mixture were added per well. Cells were further incubated
for additional 4 h at 37 °C before reading the absorbance at
480 nm.
4.3.8. Western blot analysis
Cells (5 ꢃ 105) were treated with the compounds for 6 h
with or without b-glucuronidase in the media, for histone H4
acetylation analysis. Cells were lysed in 200 ll of the electro-
phoresis loading buffer from Laemmli and sonicated. Protein ly-
sates were resolved by SDS–PAGE, followed by Western blot.
Primary antibodies were rabbit polyclonal anti-acetylated his-
tone H4 antibody (1/500; Upstate) or mouse monoclonal anti-
a-tubulin antibody (1/2000; Sigma). HRP-conjugated anti-mouse
and anti-rabbit secondary antibodies (Amersham) were used at
1/5000 dilution. Detection was performed with ECL (Perkin–
Elmer).
20. Duimstra, J. A.; Femia, F. J.; Meade, T. J. J. Am. Chem. Soc. 2005, 127, 12847–12855.
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8509.
22. Leenders, R. G. G.; Ruytenbeek, R.; Damen, E. W. P.; Scheeren, H. W. Synthesis
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3776.
4.3.9. E-Cadherin expression
Cells were treated during 16 h with the indicated compounds
(at 100 nM for TSA, and 100 lM for CI994, 3 and 4). Total RNA
25. Loudni, L.; Roche, J.; Potiron, V.; Clarhaut, J.; Bachmann, C.; Gesson, J. P.;
Tranoy-Opalinski, I. Bioorg. Med. Chem. Lett. 2007, 17, 4819–4823.
26. Thiery, J. P. Nat. Rev. Cancer 2002, 2, 442–454.
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28. Bremnes, R. M.; Veve, R.; Gabrielson, E.; Hirsch, F. R.; Baron, A.; Bemis, L.;
Gemmill, R. M.; Drabkin, H. A.; Franklin, W. A. J. Clin. Oncol. 2002, 20, 2417–2428.
29. Witta, S. E.; Gemmill, R. M.; Hirsch, F. R.; Coldren, C. D.; Hedman, K.; Ravdel, L.;
Helfrich, B.; Dziadziuszko, R.; Chan, D. C.; Sugita, M.; Chan, Z.; Baron, A.;
Franklin, W.; Drabkin, H. A.; Girard, L.; Gazdar, A. F.; Minna, J. D.; Bunn, P. A., Jr.
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was extracted using the RNA Total Isolation Kit (Promega). Reverse
transcription-PCR (RT-PCR) was done with SuperScript II (Invitro-
gen) using the procedure supplied by the manufacturer. Gene
expression was assessed relative to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) expression by quantitative real-time
PCR with the GeneAmp 5700 Sequence Detection System and SYBR
Green chemistry (Applied Biosystems). Primer sequences were as
follows: GAPDH, 50-TGCACCACCAACTGCTTAGC-30 and 50-GGCATG
GACTGTGGTCATGAG-30 and E-Cadherin, 50-CGGGAATGCAGTTGA
GGATC-30 and 50-AGGATGGTGTAAGCGATGGC-30.
30. Ohira, T.; Gemmill, R. M.; Ferguson, K.; Kusy, S.; Roche, J.; Brambilla, E.; Zeng,
C.; Baron, A.; Bemis, L.; Erickson, P.; Wilder, E.; Rustgi, A.; Kitajewski, J.;
Gabrielson, E.; Bremnes, R.; Franklin, W.; Drabkin, H. A. Proc. Natl. Acad. Sci. USA
2003, 100, 10429–10434.