Synthesis and β-glucuronidase mediated cleavage of an alcohol prodrug incorporating a double spacer moiety

Article abstract of DOI:10.1055/s-2002-19327

An alcohol prodrug has been prepared by incorporation of two spacer groups between the trigger (glucuronic acid) and nitroveratryl alcohol, used as a model. Release of the later has been observed after hydrolysis of the glycoside by β-glucuronidase. Such prodrugs may find application in ADEPT or PMT potocols in cancer chemotherapy.

Full text of DOI:10.1055/s-2002-19327

164
LETTER
Synthesis and -Glucuronidase Mediated Cleavage of an Alcohol Prodrug
Incorporating a Double Spacer Moiety
Synthesis and
é
-Glucuron
b
idase Media
a
ted Cleavag
s
e
of an A
t
lcohol
P
rodrug
e
Incorpora
n
ting a Double Spac
P
er
M
oiety apot, Freddy Rivault, Isabelle Tranoy, Jean-Pierre Gesson*
UMR 6514, Université de Poitiers et CNRS, 40 avenue du Recteur Pineau, 86022 Poitiers, France
E-mail: jean-pierre.gesson@univ-poitiers.fr
Received 12 November 2001
HOOC
NO2
O
Abstract: An alcohol prodrug has been prepared by incorporation
of two spacer groups between the trigger (glucuronic acid) and ni-
troveratryl alcohol, used as a model. Release of the latter has been
observed after hydrolysis of the glycoside by -glucuronidase. Such
prodrugs may find application in ADEPT or PMT protocols in can-
cer chemotherapy.
HO
HO
O
OH
H
O
N DOX
HMR 1826
O
Key words: prodrug, spacer, glycoside, cleavage, enzyme
O
R
HOOC
N
O
O
HO
HO
O
A major limitation in cancer chemotherapy is the poor se-
lectivity of cytotoxic agents for tumour cells. In order to
enhance the efficiency of this therapy, the use of non-toxic
prodrugs, which selectively liberate the corresponding ac-
tive drugs, after enzymatic cleavage, at the tumour site in
the course of ADEPT protocol have been widely stud-
ied.1–4
OH
NO2
A
Scheme 1
approaches in order to prepare glucuronide prodrugs suit-
able to target a wide variety of alcohol-containing drugs.
A glucuronylated prodrug of doxorubicin (DOX), HMR
18265 (Scheme 1), activated by a targeted fusion protein
(ADEPT strategy)6 or by -glucuronidases present in
higher concentration in necrotic areas (PMT strategy),7
has demonstrated superior efficacy in mice and monkeys
compared to standard chemotherapy.8 This amine prodrug
includes a self-immolative spacer between the drug and
the enzyme substrate. The 3’-amino group of DOX is
linked to the glucuronyl spacer moiety by a carbamate,
which is stable in vivo. However, most known anticancer
drugs do not have an amino group but one (or more) hy-
droxyl group (paclitaxel, etoposide…). Such a spacer can
not be used to prepare alcohol prodrugs since the required
plasma stability of prodrugs rules out a carbonate (instead
of a carbamate) as a connecting group between the drug
and the glucuronyl-spacer moiety.
We report here the synthesis and preliminary studies of a
model glucuronide prodrug, of type B, which includes two
spacer groups designed for targeting hydroxy compounds
(Scheme 2). In this way, the glycoside will be substantial-
ly far away from the drug in order to allow easy recogni-
tion by the enzyme. It is expected that cleavage of the first
spacer should proceed as for amine prodrugs5 to give both
methylene quinone 2 (which is rapidly trapped by water to
give 2-nitro-4-hydroxymethyl phenol)5–8 and the ami-
nocarbamate 1 which may then induce the release of a
leaving group R3OH from the aromatic carbamate by in-
tramolecular cyclization with concomitant formation of
the quinazolinone 3.
Previous studies in our laboratory13 have shown that N-
methylation of the N-aryl carbamate (R1 = CH3, R2 = H)
promotes cyclisation in CH2Cl2 or EtOH from aminocar-
bamate 1, while no cyclisation was observed with the un-
substituted compound (R1 = R2 = H).
Recently, phenol prodrug systems have been reported
with promising results.9–11 However, to date, only a few
studies concerning glucuronide-based prodrugs of alco-
hols have been published.12a,b Monneret and co-workers
designed quinine, dipyridamole and paclitaxel prodrugs
(A) using a N-(-2-hydroxy-5-nitrophenyl)-N-methylcar-
bamate spacer (Scheme 1). Although in vitro preliminary
tests showed the validity of this approach for the two
MDR reversal agent prodrugs, the paclitaxel prodrug was
poorly recognised by the enzyme due to steric hin-
drance.12a Thus, it seems worthwhile to investigate new
The required N-methyl-2-aminobenzylamine 5 was pre-
pared in 2 steps from anthranilonitrile (Scheme 3): N-me-
thylation was carried out by the method of Bergman14 to
give 4, which was then converted to 515 by reduction with
LiAlH4. Compound 5 was then condensed with the known
activated carbonate 65 to give selectively the mono-car-
bamate 7 (DMAP, CH3CN, r.t.). In order to introduce the
model alcohol, coupling of 7 with 6-nitroveratryl chloro-
formate was undertaken to afford bis-carbamate 816 (pyri-
dine, r.t.). Finally, the glucuronic acid moiety was
deprotected in two steps: first, transesterification with cat.
MeONa in MeOH afforded 917 after flash chromatogra-
Synlett 2002, No. 1, 28 12 2001. Article Identifier:
1437-2096,E;2002,0,01,0164,0166,ftx,en;G15101ST.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0936-5214

LETTER
Synthesis and -Glucuronidase Mediated Cleavage of an Alcohol Prodrug
165
with a half-life of 18 hours, to give quinazolinone 3 (R1 =
R1
N
O
O
CH3, R2 = H) and 6-nitroveratryl alcohol.
CO2
+
In conclusion, a new model for glucuronylated prodrugs
including a double spacer has been proposed. Such a pro-
drug was easily prepared in few steps and was found to be
stable under physiological conditions and easily recogn-
ised by -glucuronidase. Clean decomposition of both
spacers was observed leading to the release of the alcohol
moiety. However, in contrast with cyclisation rates mea-
sured in organic / aqueous media,14 the release of the alco-
hol is slower under aqueous conditions. Further studies
are underway to increase the rate of drug liberation.
R3
R2
HOOC
NO2
O
R2
HO
HO
O
O
NH
OH
β-glucuronidase
-β-Gluc-OH
O
NO2
B
2
O
+
R3
O
O
R2
R2
NH
N
R1
37°C, pH 7
R3OH
+
NH2
References
N
R1
O
R2 R2
1
3
(1) Bagshawe, K. D. Tumor Marker Update. 1997, 34, 220.
(2) Melton, R. G.; Sherwood, R. F. J. Natl. Cancer Inst. 1996,
21, 153.
Scheme 2
(3) Niculescu-Duvaz, I.; Springer, C. J. Adv. Drug Delivery Rev.
1997, 26, 151.
phy, then saponification using Ba(OH)2 8H2O in MeOH
gave the target prodrug 10 without further purification.
(4) Jungheim, L. N.; Shepherd, T. A. Chem. Rev. 1994, 94,
1553.
(5) (a) Jacquesy, J.-C.; Gesson, J.-P.; Monneret, C.; Mondon,
M.; Renoux, B.; Florent, J.-C.; Koch, M.; Tillequin, F.;
Sedlacek, H. H.; Gerken, M.; Kolar, C.; Gaudel, G.; Bosslet,
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1992, EP 511, 917; Chem. Abstr. 1994, 120, 271070S.
(b) Florent, J.-C.; Dong, X.; Gaudel, G.; Mitaku, S.;
Monneret, C.; Gesson, J.-P.; Jacquesy, J.-C.; Mondon, M.;
Renoux, B.; Andrianomenjanahary, S.; Michel, S.; Koch,
M.; Tillequin, F.; Gerken, M.; Czech, J.; Straub, R.; Bossley,
K. J. Med. Chem. 1998, 41, 3572.
Stability and enzymatic hydrolysis of 10 were carried out
at 37 °C in a 0.02 M phosphate buffer (pH = 7.2) and mon-
itored by HPLC.18 As expected, no decomposition of the
prodrug was detected after 24 hours under these condi-
tions. After addition of E. Coli -glucuronidase ( -glucu-
ronidase: 750 U mL-1; 10: 65 g mL-1), rapid
disappearance of the prodrug was observed (t < 2 min) to-
gether with formation of two compounds. The first one
was identified as 4-hydroxy-3-nitrobenzyl alcohol result-
ing from nucleophilic addition of water to the intermedi-
ate methylene quinone 2.19 The second one (1, R1 = CH3,
R2 = H, R3 = 6-nitroveratryl alcohol) slowly disappeared,
(6) Bosslet, K.; Czech, J.; Lorenz, P.; Sedlacek, H. H.;
Schuermann, M.; Seemann, G. Br. J. Cancer 1992, 65, 234.
(7) Bosslet, K.; Czech, J.; Hoffmann, D. Tumor Targeting 1995,
1, 45.
CN
CN
i
ii
NH2
NH
NH2
NH
5
4
anthranilonitrile
NO2
MeOOC
AcO
AcO
NO2
O
iii
O
OAc
6
O
O
O
O2N
OMe
NH
NH
OMe
MeOOC
AcO
NO2
O
O
O
ROOC
R'O
NO2
O
O
O
iv
AcO
N
O
OAc
R'O
OR'
O
H
N
7
O
8: R=Me, R'=Ac
9: R=Me, R'=H
10: R=H, R'=H
v
O
vi
Scheme 3 Reagents and conditions: i, dimethyl oxalate (1.5 equiv), t-BuOK (1.25 equiv), DMF, reflux, 16 h, (85%); ii, LiAlH4 (12 equiv),
Et2O, r.t., 1.5 h, (95%); iii, DMAP (1 equiv), CH3CN, 6 (0.8 equiv), r.t., 1.5 h, (67%); iv, pyridine, 6-nitroveratryl chloroformate (2 equiv),
CH2Cl2, r.t., 3 h, (93%); v, MeOH/MeONa (1 equiv), 0 °C, 3 h, (60%); vi, Ba(OH)2 8H2O (1.2 equiv), MeOH, r.t., 3.5 h, (quant.).
Synlett 2002, No. 1, 164–166 ISSN 0936-5214 © Thieme Stuttgart · New York

166
S. Papot et al.
LETTER
(8) Bosslet, K.; Czech, J.; Hoffmann, D. Cancer Res. 1994, 54,
2151.
(9) Schmidt, F.; Florent, J.-C.; Monneret, C.; Straub, R.; Czech,
J.; Gerken, M.; Bosslet, K. Bioorg. Med. Chem. Lett. 1997,
7, 1071.
(10) Lougerstay-Madec, R.; Florent, J.-C.; Monneret, C.; Nemati,
F.; Poupon, M. F. Anti-Cancer Drug Design. 1998, 13, 995.
(11) Tietze, L. F.; Lieb, M.; Herzig, T.; Haunert, F.; Schuberth, I.
Bioorg. Med. Chem. 2001, 9, 1929.
(12) (a) Schmidt, F.; Ugureanu, I.; Duval, R.; Poupon, A.;
Monneret, C. Eur. J. Org. Chem. 2001, 2129. (b) Desbene,
S.; Van Dufat-Trinh, H.; Michel, S.; Tillequin, F.; Koch, M.;
Schmidt, F.; Florent, J.-C.; Monneret, C.; Straub, R.; Czech,
M.; Gerken, M.; Bosslet, K. Anti-cancer Drug Design 1999,
14, 93.
(d, 1 H, J = 9.5 Hz, H-5), 5.00 (s, 2 H, CH2O), 5.18–5.69 (m,
6 H, H-1,2,3,4, CH2O), 6.48 (s, 1 H, Harom), 7.02 (s, 1 H,
NH), 7.30–7.84 (m, 8 H, Harom) ppm; 13C NMR (75 MHz,
CD3COCD3), : 37.9 (CH3N), 41.3 (CH2N), 53.0
(COOCH3), 56.6 (OCH3), 65.0–65.1 (CH2O), 69.9, 71.1,
72.1, 72.8 (C-2,3,4,5), 100.0 (C-1), 121.2–149.6 (Carom),
155.0–156.1 (Carom), 167.7 (COOMe), 169.5, 170.0, 170.2
(CH3CO) ppm; mp 100 °C.
(17) Data for 9: [ D] = +6,4 (c 0.95, CHCl3); 1H NMR (300 MHz,
CD3COCD3), : 3.51 (s, 3 H, CH3N), 3.59–3.62 (m, 3 H,
H-2,3,4), 3.71 (s, 3 H, COOCH3), 3.87 (s, 6 H, OCH3), 4.20
(d, 1 H, J = 9.5 Hz, H-5), 4.37 (d, 2 H, J = 6 Hz, CH2N), 5.00
(s, 2 H, CH2O), 5.33 (s, 2 H, CH2O), 5.51 (d, 1 H, J = 16 Hz,
H-1), 7.00 (broad s, 1 H, HNH), 7.31–7.83 (m, 9 H, Harom
)
ppm; 13C NMR (75 MHz, CD3COCD3), : 37.9 (CH3N),
(13) Papot, S.; Bachmann, C.; Combaud, D.; Gesson, J.-P.
Tetrahedron 1999, 55, 4699.
(14) (a) Bergman, J.; Brynolf, A.; Vuorinen, E. Tetrahedron
1986, 42, 3689. (b) Bergman, J.; Norrby, P. O.; Sand, P.
Tetrahedron 1990, 46, 6113.
(15) Coyne, W. E.; Cusic, J. W. J. Med. Chem. 1968, 11, 1208.
(16) Preparation of 8: 6-Nitroveratryl chloroformate (85 mg, 0.30
mmol) and pyridine (0.075 mL, 0.9 mmol) were added to a
stirred solution of 7 (100 mg, 0.15 mmol) in CH2Cl2 (0.65
mL) at r.t. After 3 h the mixture was worked up with a
saturated solution of NaHCO3 and the aqueous layer was
washed with CH2Cl2. The combined organic layers were
dried (MgSO4), filtered, concentrated to dryness and the
remaining yellow solid was purified by flash
41.3 (CH2N), 52.5 (COOCH3), 56.5, 56.6 (OCH3), 65.0, 65.1
(CH2O), 72.3, 74.1, 76.4, 76.9 (C-2,3,4,5), 101.9 (C-1),
118.1–150.0 (Carom), 155.2, 157.1 (Ccarbamate), 169.5
(COOMe) ppm; mp 90 °C. The highly polar compound 10
does not lead to well resolved NMR spectra (this is observed
for all glucuronylated prodrugs).
(18) HPLC conditions : C-16 reverse phase column : Discovery
RP amide, 5 m (15 cm 4.6 mm) and pre-column Discovery,
5 m, UV detection ( 254 nm), mobile phase (acetonitrile–
0.065 M acetate ammonium solution, 30:70, 1 mL min–1).
Retention time: 10: 7.8 min; 4-hydroxy-3-nitrobenzyl
alcohol: 3.6 min; 3 (R1 = CH3, R2 = H): 4.1 min; 6-
nitroveratryl alcohol: 5.2 min; 1 (R1 = CH3, R2 = H, R3 = 6-
nitroveratryl alcohol): 6.3 min.
chromatography (petroleum ether–EtOAc 1:1) to give 8
(116 mg, 0.13 mmol, 87%). 1H NMR (300 MHz,
CD3COCD3), : 2.04, 2.05, 2.07 (3 s, 9 H, CH3COO), 3.26
(s, 3 H, CH3N), 3.50 (s, 3 H, OCH3), 3.70 (s, 3 H, COOCH3),
3.87 (s, 6 H, OCH3), 4.37 (d, 2 H, J = 6 Hz, CH2N), 4.65
(19) We have recently shown that 2-nitro-quinone-methides are
not irreversible inhibitors of bovine -glucuronidase:
Azoulay, M.; Chalard, F.; Gesson, J.-P.; Florent, J.-C.;
Monneret, C. Carbohydr. Res. 2001, 332, 151.
Synlett 2002, No. 1, 164–166 ISSN 0936-5214 © Thieme Stuttgart · New York