Synthesis and biological evaluation of the suberoylanilide hydroxamic acid (SAHA) β-glucuronide and β-galactoside for application in selective prodrug chemotherapy

Article abstract of DOI:10.1016/j.bmcl.2006.11.042

The β-O-glucuronide and β-O-galactoside of SAHA have been prepared and evaluated as prodrugs for selective cancer chemotherapy (ADEPT, PMT). These new compounds are stable under physiological conditions and do not exhibit any antiproliferative activity co

Full text of DOI:10.1016/j.bmcl.2006.11.042

Bioorganic & Medicinal Chemistry Letters 17 (2007) 983–986
Synthesis and biological evaluation of the suberoylanilide
hydroxamic acid (SAHA) b-glucuronide and b-galactoside for
application in selective prodrug chemotherapy
Mickael Thomas,a Freddy Rivault,a Isabelle Tranoy-Opalinski,a Joelle Roche,b
¨
¨
a
a,
*
´
Jean-Pierre Gesson and Sebastien Papot
a
`
´
´
UMR-CNRS 6514, Synthese et Reactivite des Substances Naturelles, Universite de Poitiers, 40,
´
Av. du Recteur Pineau, 86022 Poitiers, France
bUMR-CNRS 6187, Institut de Physiologie et de Biologie Cellulaires, Universite´ de Poitiers, 40,
Av. du Recteur Pineau, 86022 Poitiers, France
Received 20 October 2006; revised 13 November 2006; accepted 13 November 2006
Available online 17 November 2006
Abstract—The b-O-glucuronide and b-O-galactoside of SAHA have been prepared and evaluated as prodrugs for selective cancer
chemotherapy (ADEPT, PMT). These new compounds are stable under physiological conditions and do not exhibit any antipro-
liferative activity compared to the parent drug after a 48-h treatment of H661 cells. The glucuronide derivative did not lead to
the release of the drug in the presence of either Escherichia coli or bovine liver b-glucuronidase. On the other hand, under enzymatic
cleavage of galactoside prodrug by the corresponding enzyme, a rapid release of SAHA was observed demonstrating that the b-O-
galactoside of SAHA is a promising candidate for in vivo investigations.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Although considerable effort has been invested in the
discovery of selective anticancer drugs, most pharmaco-
logical approaches for the treatment of solid tumours
suffer from poor selectivity. The development of more
selective therapeutic agents has consequently become a
major goal of current anticancer research. Within this
framework, the use of non-toxic prodrugs designed to
deliver the corresponding anticancer agent by specific
enzymatic activation in cancerous tissues is currently
being investigated.1 In this approach, the active enzyme
may be naturally present in high concentration in the
vicinity of the tumour (PMT strategy)2 or previously
targeted to the tumour site using various strategies such
as ADEPT,3 GDEPT,4 or LEAPT.5
ising in vivo profiles. For example, HMR 1826, a
glucuronide prodrug of doxorubicin, has shown superi-
or efficacy in the treatment of various human tumour
xenografts in mice compared to standard chemotherapy,
both in ADEPT6 and PMT2b strategies. Very recently,
Sun and co-workers have reported the evaluation of a
b-galactoside prodrug of geldanamycin in combination
with a humanized monoclonal antibody chemically con-
jugated to b-galactosidase (ADEPT strategy).7 In this
study, the authors demonstrated the high potential of
b-galactoside prodrugs since the monoclonal antibody
they used was found to be an ideal candidate for tumour
targeting in the course of a pilot clinical trial.8 Thus, in
the light of these results, the extension of the range of
prodrugs that could be activated by either b-glucuroni-
dase and b-galactosidase seems to be warranted.
Over the past 20 years, glycoside prodrugs, including
b-glucuronide and b-galactoside ones, have been exten-
sively studied and some of them have led to very prom-
Histone deacetylase inhibitors (HDACi) have recently
emerged as new potential drug targets. Several classes
of small-molecule HDACi have been discovered so far,
among which hydroxamic acid derivatives have received
the most attention.9–11 Suberoylanilide hydroxamic
acid (SAHA) 1 is a member of the hydroxamate class
of HDAC inhibitors (Scheme 1).12–14 SAHA has
Keywords: Prodrug; Glucuronide; Galactoside; SAHA; HDAC;
ADEPT.
*
Corresponding author. Tel.: +33 549453862; fax: +33 549453501;
e-mail: sebastien.papot@univ-poitiers.fr
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.042

984
M. Thomas et al. / Bioorg. Med. Chem. Lett. 17 (2007) 983–986
R3
R1
O
O
R3
H
N
R1
NH
HN
O
R2
NHOH
O
R2
HO
+
OH
O
HO
O
O
β-glu
or
OH
1: SAHA
OH
β-gal
2: R1=COOH, R2=OH, R3=H
3: R1=CH2OH, R2=H, R3=OH
Scheme 1.
demonstrated antitumour activity in several in vivo
models of cancer, including a xenograft of human pros-
tate tumour implanted in mice,15 a transgenic mouse
model of leukaemia and a carcinogen-induced tumour
model in rodents.16 Phase I clinical trials have estab-
lished that SAHA displayed antitumour activity in pa-
tients with solid tumours and haematological
malignancies.17 SAHA is currently progressing through
phase III clinical trials.18
drug since crystallographic studies demonstrated that
SAHA acted by binding the zinc ion in the active site
of HDAC in a bidentate fashion, through its CO and
OH groups.19–21 Furthermore, we expected that such
prodrugs would be readily activated by either b-glucu-
ronidase or b-galactosidase to release the corresponding
active drug under physiological conditions.
Despite the renewal of interest for hydroxamic acids
over the past decade, the development of synthetic
methods leading to the corresponding O-glycosides has
received little attention. To the best of our knowledge
only one method has been reported in the literature.22
In this study, the authors have prepared the glucuronide
adduct of TrocadeTM, a selective inhibitor of MMP colla-
genase, by coupling the protected O-glucuronsylhydr-
oxylamine 6a with the corresponding carboxylic acid
followed by suitable deprotection (Scheme 2). Recently,
a similar strategy has been used with success by Hosok-
awa and co-workers for the first synthesis of Trichosta-
tin D, the a-D-glucopyranoside of Trichostatin A.23
Thus, in the light of these results the preparations of
the requested prodrugs 2 and 3 were achieved employing
the strategy depicted in Scheme 2.
Although it appeared to be a promising chemotherapeu-
tic agent, SAHA, like other hydroxamate-based inhibi-
tors, exhibits poor pharmacodynamic properties
mainly due to instability and rapid elimination (half-life
ranged from 21 to 58 min).17 As a consequence, SAHA
needs daily administration to sustain its in vivo drug le-
vel. Moreover, adverse events such as anorexia, anaemia
and thrombocytopenia have been reported in the course
of phase I clinical trials.17
Thus, with the aim to enhance the therapeutic index of
this promising new anticancer agent, we have undertak-
en the synthesis and preliminary evaluations of the two
glycosyl prodrugs of SAHA 2 and 3 depicted in Scheme
1. We hypothesised that the glycosyl derivatization of
the hydroxamate moiety may prevent the rapid in vivo
degradation of SAHA. We also anticipated that these
compounds would be less toxic than the corresponding
The two O-glycosylhydroxylamines 6a and 6b were syn-
thesised in two steps from the corresponding activated
bromo-glycosides 4a and 4b. First, stereoselective gly-
R3
R3
R1
R3
R1
R1
O
R2
NH2
O
R2
O
R2
AcO
ii
O
O
i
O
AcO
N
AcO
OAc
AcO
OAc
Br
5a, 5b
6a, 6b
O
4a: R1=COOMe, R2=OAc, R3=H
4b: R1=CH2OAc, R2=H, R3=OAc
O
iii
COOH
(
)
6
N
H
7
O
O
H
N
R3
R2
AcO
H
N
R3
R1
R1
HN
O
HN
O
iv
R2
O
O
O
O
HO
8a, 8b
OAc
3: R1=CH2OH, R2=H, R3=OH
9: R1=COOMe, R2=OH, R3=H
OH
v
2: R1=COOH, R2=OH, R3=H
Scheme 2. Reagents and conditions: (i) HONFt, TBAHS, CH2Cl2/Na2CO3 (1 M), rt, 4 h (5a: 60%, 5b: 55%); (ii) N2H4, MeOH, rt, 5 min (6a: quant.,
6b: quant.); (iii) 7, EDC, HOBt, Et3N, CH2Cl2, rt, 12 h (8a: 73%, 8b: 62%); (iv) MeONa/MeOH, 0 ꢁC, 1 h then Amberlist IRC 50 (3: quant, 9: 67%);
(v) acetone/NaOH (1 N), À20 ꢁC, 5 min then Amberlist IRC 50 (2: quant.).

M. Thomas et al. / Bioorg. Med. Chem. Lett. 17 (2007) 983–986
985
cosidations were carried out in the presence of N-
hydroxyphthalimide (HONFt) and tetrabutylammoni-
um hydrogenosulfate (TBAHS) to afford 5a22 and 5b
exclusively as the b-anomers in 60% and 55% yields,
respectively. Second, treatment of 5a and 5b with hydra-
zine in methanol gave O-glycosylhydroxylamines 6a22
and 6b, both in quantitative yields. Condensations of
6a and 6b with the well-known carboxylic acid 724 were
next undertaken by using 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide hydrochloride (EDC), 1-hydrox-
ybenzotriazole (HOBt) and triethylamine to yield the
corresponding protected b-glycosides of SAHA (8a:
73%, 8b: 62%). The b-galactoside 8b was deprotected
in the presence of 10 equiv of MeONa in MeOH at
0 ꢁC to give prodrug 3 in quantitative yield.25 Deprotec-
tion of the glucuronide 8a was achieved using a two-step
procedure. Thus, the acetyl groups were first cleaved by
treatment with 1 equivalent of MeONa in MeOH and
the methyl ester was subsequently saponified at
À20 ꢁC (acetone/NaOH (1 N) 5 equiv) to afford prodrug
2 (67% over 2 steps).25
not observed under these conditions. Thus, further
experiments were undertaken using higher concentra-
tions of the enzyme. However, in these cases 2 was
found to be perfectly stable even at a b-glucuronidase
concentration of 700 U mLÀ1. Further enzymatic assays
were then carried out with prodrug 2 (100 lg mLÀ1) in
the presence of various concentrations of bovine liver
b-glucuronidase (from 200 to 1000 U mLÀ1). In the
course of these experiments, 2 was again found to be sta-
ble and release of SAHA was not detected.
Prodrug 3 (430 lg mLÀ1) was incubated for 7 min in the
presence of E. coli b-galactosidase (40 U mLÀ1) and the
mixture was then analysed by HPLC. Total disappear-
ance of 3 was observed together with the release of
SAHA (Fig. 1).
The antiproliferative activities of prodrugs 2 and 3 were
evaluated after a 48-h treatment of non-small cell lung
cancer H661 cells and compared to SAHA
(IC50 = 1 lM). In the course of these experiments, both
compounds 2 and 3 did not present any detectable anti-
proliferative activities when tested from 1 to 25 lM.
Stability and enzymatic hydrolysis of prodrugs 2 and 3
were carried out in 0.02 M phosphate buffer (pH 7) at
37 ꢁC and monitored by HPLC.26 As expected, no
decomposition of these two compounds was detected
after 72 h under these conditions.
The two b-glycoside prodrugs of SAHA 2 and 3 were
stable under physiological conditions and exhibited a re-
duced cytotoxicity compared to the parent drug against
H661 cells. The prodrug 2 did not lead to the release of
SAHA in the presence of a large excess of either E. coli
or bovine liver b-glucuronidase. This result was surpris-
ing taking into account that b-glucuronidase is known
to lack structural specificity for the aglycone moiety
and the results obtained by Mitchell et al. who have
shown that the TrocadeTM glucuronide is a substrate
for this enzyme.22 Nevertheless, our result clearly dem-
onstrated that such a glucuronide is not useful as a pro-
drug for b-glucuronidase-mediated PMT or ADEPT
strategies. On the other hand, the b-galactoside 3 was
totally and rapidly converted to SAHA in the presence
of an excess of the corresponding enzyme (which is al-
ways the case in the course of an ADEPT protocol).
Enzymatic hydrolysis of 2 and 3 was conducted in the
presence of an excess of the corresponding enzyme.26
First, prodrug 2 (100 lg mLÀ1) was incubated with
Escherichia coli b-glucuronidase (100 U mLÀ1). Aliquots
were taken at 7 and 30 min. The release of SAHA was
a
b
In summary, we have prepared the non-toxic b-glucuro-
nide 2 and b-galactoside 3 of SAHA. The compound 2
was found to be inappropriate for both ADEPT and
PMT strategies. However, it should be mentioned that
this derivative may be of biological interest since
SAHA-glucuronide was recently found to be one of
the drug metabolites.27 Under enzymatic cleavage of
the galactosyl moiety by E. coli b-galactosidase, a rapid
and efficient release of the drug was observed. Thus, as
these data are compatible for an ADEPT strategy, the
b-galactoside prodrug of SAHA 3 seems to be a good
candidate for further in vivo investigations.
c
Acknowledgments
The authors thank MENESR, CNRS, La Ligue Natio-
3.0
3.5
4.0
4.5
5.0
5.5
6.0
´
nale contre le Cancer, Comite de Charente-Maritime
and the Region Poitou-Charentes for financial support
Minutes
´
´
of this study. The authors also thank B. Tetaud for tech-
nical help during antiproliferative assays.
Figure 1. HPLC analysis (for HPLC conditions, see Ref. 26). (a)
prodrug 3; (b) prodrug 3 + E. coli b-galactosidase; (c) SAHA.

986
M. Thomas et al. / Bioorg. Med. Chem. Lett. 17 (2007) 983–986
Cordo, C.; Chiao, J. H.; Rifkind, R.; Marks, P. A.; Scher,
H. Clin. Cancer Res. 2003, 9, 3578.
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25. Compound 2: 1H NMR (D2O) d (ppm): 1.25–1.35 (m,
4H), 1.50–1.70 (m, 4H), 2.16 (t, 2H, J = 7.1 Hz), 2.38 (t,
2H, J = 7.3 Hz), 3.40–3.60 (m, 3H), 3.70 (d, 1H,
J = 9.3 Hz), 4.65 (d, 1H, J = 7.8 Hz), 7.23 (dd, 1H,
J = 8.7 and 3.8 Hz), 7.35–7.40 (m, 4H); 13C NMR (D2O)
d (ppm): 25.0, 25.5, 28.0, 28.1, 32.7, 36.7, 71.6, 71.8, 75.7,
75.9, 105.0, 122.6, 126.0, 129.6, 137.1, 173.7, 175.9, 176.4.
Compound 3: 1H NMR (CD3OD) d (ppm): 1.30–1.40 (m,
4H), 1.60–1.70 (m, 4H), 2.12 (m, 2H), 2.33 (t, 2H,
J = 7.3 Hz), 3.40–3.80 (m, 6H), 4.42 (d, 1H, J = 7.8 Hz),
7.03 (t, 1H, J = 8.4 Hz), 7.25 (t, 2H, J = 8.4 Hz), 7.51 (d,
2H, J = 8.4 Hz); 13C NMR (CD3OD) d (ppm): 25.2, 25.6,
28.6, 28.7, 32.4, 36.7, 61.4, 68.9, 69.3, 73.4, 76.1, 107.0,
120.1, 124.0, 128.6, 138.7, 172.2, 173.5.
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26. HPLC conditions: Analytical HPLC was carried out using
a WatersTM HPLC system with UV detection at 254 nm.
The separation was performed on a reversed phase column
chromatography (Discoveryꢂ RP amide 16, 150 · 4.6 mm,
5 lm) using isocratic conditions (1 mL/min), eluent
CH3CN/H2O/TFA 3:7:0.028. Retention times for com-
pounds 2, 3 and SAHA, respectively, were 3.40, 3.31 and
4.15 min. E. coli b-glucuronidase, bovine liver b-glucu-
ronidase and b-galactosidase were purchased from Sigma–
Aldrich (reference Sigma: G2513-1KU, G0501-100KU
and G7896-2KU, respectively).
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