A b-galactoside phosphoramide mustard prodrug for use in conjunction with
gene-directed enzyme prodrug therapy
Ajit K. Ghosh, Saeed Khan and David Farquhar*
Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
77030, USA. E-mail: dfarquha@mdanderson.org
Received (in Corvallis, OR, USA) 22nd September 1999, Accepted 2nd November 1999
4-(b-d-Galactopyranosyl)benzyl
N,N,NA,NA-tetrakis(2-
chloroethyl)phosphorodiamidate 1 was facilely biotrans-
formed to the alkylating antitumor agent, N,N,NA,NA-
tetrakis(2-chloroethyl) phosphorodiamidic acid, 4, when
incubated with E. coli b-galactosidase, and therefore has
potential for use in conjunction with gene-directed enzyme
prodrug therapy.
Gene-directed enzyme prodrug therapy (GDEPT) has found
increasing application in cancer therapy. In this approach, a
foreign gene is introduced into the target tumor, usually with the
aid of a viral or liposomal vector.1–3 When the gene is
expressed, it encodes for an enzyme that can convert a nontoxic
prodrug to a cytotoxic product. Most GDEPT approaches
reported to date have used ganciclovir and fluorocytosine as
prodrugs, with HSV thymidine kinase (HSV tk-) and cytosine
deaminase (CD), respectively, as the activating enzymes.
Although promising results have been described with both
prodrug/enzyme combinations, a limitation of HSV tk- and CD
as drug-activating enzymes is that they only accept as substrates
compounds that closely resemble the natural substrates. In an
effort to broaden the scope of the GDEPT approach, we
investigated the potential of E. coli b-galactosidase as a drug-
activating enzyme. Because of its permissive nature, b-
galactosidase can cleave a wide structural variety of b-
galactosides. Here we describe the preparation of
4-(b-d-galactopyranosyl)benzyl-N,N,NA,NA-tetrakis(2-chloro-
ethyl) phosphorodiamidate 1, a prodrug of the antitumor
alkylating phosphoramide mustard N,N,NA,NA-tetrakis(2-chloro-
ethyl)phosphorodiamidic acid, 4.
The anticipated mechanism of activation of 1 is shown in
Scheme 1. Under neutral aqueous conditions, the prodrug is
expected to be stable and non-reactive. In the presence of b-
galactosidase, however, it should be cleaved to the correspond-
ing phenol 2. This intermediate should undergo spontaneous
1,6-elimination to release the cytotoxic mustard 4 and quinone
methide 3. The latter should have only a transient existence
before being converted to 4-hydroxybenzyl alcohol 5.
Compound 1 was prepared as shown in Scheme 2.
2,3,4,6-Tetra-O-acetyl-a-d-galactopyranosyl bromide 6 was
reacted with 4-hydroxybenzaldehyde 7 in the presence of
freshly prepared anhydrous Ag2O (2 equiv.) in MeCN at room
Scheme 2 Reagents and conditions: i, Ag2O, MeCN; ii, NaBH4, CHCl3–
PriOH; iii, PCl3, Et3N; iv, HN(CH2CH2Cl)2; v, tert-butyl hydroperoxide; vi,
NaOMe, MeOH.
temperature for 8 h to give 8 (73% yield). Reduction of 8 with
NaBH4 in anhydrous CHCl3–PriOH (4+1) gave the correspond-
ing benzyl alcohol 9 in 72% yield. Compound 9 was reacted
successively with PCl3 (1 equiv.) and bis(2-chloroethyl)amine
(2 equiv.), and the intermediate phosphoramidite was oxidized
in situ with tert-butyl hydroperoxide to afford 10† as a white
solid (47% yield) after purification over silica gel. Compound
10 was converted to the free galactoside 1‡ in 87% yield by
treatment with NaOMe in anhydrous MeOH.
The stability and enzyme activation of 1 was then investi-
gated. A solution of 1 (1024 M) in 0.05 M phosphate buffer, pH
7.4, was incubated at 37 °C in the absence or presence of E. coli
b-galactosidase (2 units per mmole of 1). At selected time
intervals, 100 ml aliquots were withdrawn and analyzed by
HPLC.§ In the absence of enzyme, the peak intensity of 1
decreased with a half life of 9.4 h. In presence of enzyme, the
half-life was reduced to 7.6 min. A new peak, chromato-
graphically identical to 4-hydroxybenzyl alcohol, appeared
soon after incubation started and progressively increased with
time. Since the phosphoramide mustard 4, does not contain a
strong chromophore, it was not evident in the reaction mixture
using a UV detector. To establish the formation of 4, the
incubation mixture was further analyzed by LC/MS.¶ The peak
suspected to be 4-hydroxybenzyl alcohol gave rise to a
molecular ion of mass 107. This is consistent with the
hydroxytropylium ion formed by rearrangement of the 4-hy-
droxybenzyl cation ion derived from 7. Another peak, not
evident when the mixture was analyzed using a UV detector,
gave rise to a four-chlorine molecular ion cluster with m/z 345
characteristic of the free mustard 4. These findings strongly
support the rationale inherent in the design of 1. Biological
studies of this compound are in progress and will be reported in
the future.
In summary, 4-(b-d-galactopyranosyl)benzyloxy N,N,NA,NA-
tetrakis(2-chloroethyl) phosphorodiamidate 1 was prepared and
shown to be converted to the free phosphoramide 4 in the
presence of b-galactosidase. Compounds such as 1 might have
good potential in conjunction with GDEPT to increase anti-
tumor selectivity in cancer chemotherapy. The synthesis of
other b-galactoside anticancer prodrugs are in progress.
This research was supported by NIH grant CA 71527.
Scheme 1
Chem. Commun., 1999, 2527–2528
This journal is © The Royal Society of Chemistry 1999
2527
Ghosh, Ajit K.
