1,2-Benzisoxazole Phosphorodiamidates as Novel Anticancer Prodrugs Requiring Bioreductive Activation

Article abstract of DOI:10.1021/jm020581y

Several 1,2-benzisoxazole phosphorodiamidates have been designed as prodrugs of phosphoramide mustard requiring bioreductive activation. Enzymatic reduction of 1,2-benziosoxazole moiety is expected to result in the formation of imine intermediate due to t

Full text of DOI:10.1021/jm020581y

5428
J . Med. Chem. 2003, 46, 5428-5436
1,2-Ben zisoxa zole P h osp h or od ia m id a tes a s Novel An tica n cer P r od r u gs
Requ ir in g Bior ed u ctive Activa tion
Monish J ain and Chul-Hoon Kwon*
Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. J ohn’s University,
J amaica, New York 11439
Received December 27, 2002
Several 1,2-benzisoxazole phosphorodiamidates have been designed as prodrugs of phosphora-
mide mustard requiring bioreductive activation. Enzymatic reduction of 1,2-benziosoxazole
moiety is expected to result in the formation of imine intermediate due to the cleavage of the
N-O bond. The imine should then be spontaneously hydrolyzed to a ketone metabolite, thereby
facilitating base-catalyzed â-elimination of cytotoxic phosphoramide mustard. As expected, the
proposed prodrugs 4, 9, and 12 were at least 3-5-fold more potent cytotoxins than control
compounds 5 and 15, which lack in the phosphoramide mustard group. Upon incubation with
phenobarb-induced rat liver S-9 fraction, compounds 4, 9, and 12 underwent extensive NADPH-
dependent metabolism with concomitant generation of alkylating activity under both hypoxic
and oxic conditions. Corresponding ketone metabolites were detected for 9 and 15. NADPH-
dependent bioreduction of 15 to its ketone metabolite 16 was located in the microsomal fraction
and inhibited by SKF-525A and pCMBA. Compared with phenobarb-induced rat liver
microsomal fraction, incubation of 15 with rat or human P450 reductase microsomes showed
moderate generation of 16. Microsomal cytochrome P450 and/or P450 reductase appear to be
involved in the reductive metabolism of 1,2-benzisoxazole moiety under hypoxic as well as oxic
conditions.
In tr od u ction
(Scheme 1). Human and rat liver microsomes have been
shown to metabolize zonisamide to SMAP, preferentially
under hypoxic conditions, with the suggested involve-
ment of cytochrome P450.15-20 Additional studies have
also reported the ability of liver cytosolic aldehyde
oxidase from various strains of rats and other animals
to catalyze reduction of zonisamide under hypoxic
conditions.21 On the basis of reductive metabolism of
zonisamide to SMAP, 1,2-benzisoxazole phosphorodia-
midate prodrugs were designed as bioreducible hypoxia-
selective antitumor alkylating agents with the ability
to deliver cytotoxic species following reductive activa-
tion. The designed prodrugs are expected to undergo
bioreduction in a manner similar to zonisamide that
sequentially leads to formation of the corresponding
imine intermediate and ketone metabolite, thereby
facilitating â-elimination of cytotoxic phosphoramide
mustard (PDA) (Scheme 2).
Phosphoramide mustard is also derived as the ulti-
mate cytotoxic species following activation of cyclophos-
phamide (CP), a widely used alkylating anticancer
prodrug. Following initial hepatic mixed function oxi-
dase mediated activation of CP, the resulting 4-hydroxy
cyclophosphamide (4-OH CP) undergoes ring-opening to
aldophosphamide (Aldo), followed by base-catalyzed
â-elimination of cytotoxic PDA. Intramolecular cycliza-
tion of PDA results in formation of reactive aziridinium
ion that cross-links interstrand DNA.22-23
In contrast to normal tissues that are well-perfused,
solid tumors possess abnormal vasculature and as a
result contain subpopulation of hypoxic tumor cells.
Hypoxic tumor cells are resistant to radiation and
chemotherapy and may lead to increased metastasis and
accelerated malignant progression. However, the hy-
poxic environment of the tumor offers an attractive
difference between normal and tumor cells that may be
exploited with the use of bioreducible cytotoxins. These
compounds are prodrugs that undergo reductive activa-
tion to yield cytotoxic metabolites. The reduction is
facilitated by the appropriate reductases under the
environment of low oxygen tension.1,2 Many compounds
of diverse chemical structures including quinones,3,4
nitroimidazoles,5 nitrobenzyl derivatives,6 benzotriazine
di-N-oxides,7,8 and sulfoxide aniline mustards9 have
been designed as hypoxia-selective bioreducible cyto-
toxins. More recently, nitrobenzyl derivatives,10 quino-
nes,11 and nitroheterocyclic12 prodrugs that release
appended phosphoramidate mustard derivatives as
DNA-alkylating species after bioreduction have also
been explored. The reductases involved include DT
diaphorase, cytochrome P450, NADPH-dependent cy-
tochrome P450 reductase, xanthine oxidase, and alde-
hyde oxidase, among others.13,14
Zonisamide, an anti-epileptic agent, is a 1,2-benzisox-
azole derivative that undergoes reductive activation,
leading to cleavage of the heterocyclic N-O bond. The
resulting imine intermediate readily hydrolyzes to the
ketone metabolite 2-(sulfamoylacetyl)phenol (SMAP)
In case of 1,2-benzisoxazole phosphorodiamidates, the
ketone metabolite releases PDA via â-elimination reac-
tion in a fashion similar to Aldo, except that the
electron-withdrawing activating moiety is a substituted
phenyl ketone instead of an aldehyde. The utility of
phenyl ketone driven â-elimination of PDA derivatives
* Corresponding author. Office: (718)-990-5214. Fax: (718)-990-
6551. E-mail: kwonc@stjohns.edu.
10.1021/jm020581y CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/12/2003

1,2-Benzisoxazole Phosphorodiamidates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5429
Sch em e 1
phorochloridate to give 5. Synthesis of 15 was carried
out by reaction of 2-hydroxypropiophenone with hy-
droxylamine that yielded oxime, which was subse-
quently acetylated and then refluxed with pyridine to
obtained cyclized product (Scheme 5).
Resu lts a n d Discu ssion
In Vitr o Cytotoxicity Eva lu a tion . The in vitro
cytotoxicity potential of 1,2-benzisoxazole phosphorodia-
midates was evaluated against Chinese hamster lung
(V-79) fibroblasts in a 96-well microtiter plate assay
using the sulforhodamine B (SRB) assay procedure of
Skehan et al.30 with some modifications. The drug
exposure period was 48 h. The drug concentration that
resulted in 50% reduction in cell-growth (indicated by
the absorbance value of bound SRB dye) in drug-treated
cells as compared to untreated control cells following
incubation was calculated as growth inhibition of 50%
(IC50). Results are reported in Table 1. 2-(Methylsulfo-
nyl)ethyl phosphorodiamidate, an Aldo analogue known
to deliver PDA under model physiological conditions
without enzyme activation requirement,31 was chosen
as a positive control cytotoxin. Among phosphorodia-
midate prodrugs, 9 was the most potent cytotoxin, with
an IC50 value of 241 µM, while 4 and 12 showed slightly
weaker cytotoxicity with IC50 values of 411 and 311 µM,
respectively. A correlation between electron-donating/
withdrawing effects of substituents on 1,2-benzisoxazole
and their cytotoxic potential could not be drawn, since
both methoxy- and nitro-substituent-bearing analogues
were more toxic than unsubstituted compound. Ana-
logue 5, lacking in alkylating phosphorodiamidate mus-
tard, was found to be approximately 3-5-fold less potent
than 4, 9, and 12. This suggested that activation of
prodrugs leading to expulsion of PDA, an alkylating
species, seems to be the mechanism of cytotoxic action
for 1,2-benzisoxazole phosphorodiamidates. The weak
cytotoxic activity observed for 5 could be attributed to
the formation of phenyl vinyl ketone, a potentially
reactive Michael-acceptor type byproduct of â-elimina-
tion reaction in the reductive activation pathway. This
conclusion was also supported by the lack of appreciable
cytotoxic activity of 15, possibly due to the absence of
an alkylating PDA moiety as well as the inability of
has been exploited earlier in the design of phenylketo-
phosphamide, which was shown to be a promising
antitumor agent.24
Ch em istr y
Several 1,2-benzisoxazole phosphorodiamidates (4, 9,
and 12) with the potential to release PDA upon meta-
bolic reduction were synthesized (Scheme 4). Electron-
donating 6-methoxy and electron-withdrawing 5-nitro
substituents were placed on the heterocycle to explore
their influence on bioreduction as well as release of
PDA. Additionally, N,N,N′,N′-tetramethylphosphorodia-
midate (5) was prepared as a control compound lacking
in alkylating phosphorodiamidate for in vitro cytotox-
icity studies. Analogue 15 was chosen as a model test
compound for in vitro metabolism studies.
1,2-Benzisoxazole phosphorodiamidates (4, 5, 9, and
12) were prepared as outlined in Schemes 3 and 4.
Initially, either unsubstituted or methoxy substituted
4-hydroxycoumarin was reacted with hydroxylamine to
yield corresponding 1,2-benzisoxazole-3-acetic acid in-
termediates 1 and 6, respectively, according to published
procedures.25,26 The 3-acetic acid intermediates were
sequentially esterified and reduced to afford corre-
sponding alcohols 327 and 8. For nitro-substituted
analogue, ester 2 was nitrated28 on the 5-position and
then reduced to alcohol 11. The obtained alcohols (3, 8,
and 11) were sequentially reacted with n-butyllithium,
N,N-bis(2-chloroethyl)phosphoramidic dichloride, and
ammonia to yield target phosphorodiamidates 4, 9, and
12, respectively. Alcohol 3 was sequentially reacted with
n-butyllithium and N,N,N′,N′-tetramethyldiamidophos-
Sch em e 2

5430 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
J ain and Kwon
Ta ble 1. In Vitro Cytotoxicity of 1,2-Benzisoxazole
Sch em e 3
Phosphorodiamidates against V-79 Cells
a
a
compd
IC50 (µM)
compd
IC50 (µM)
controlb
4
9
45 ( 2
411 ( 39
241 ( 10
12
5
15
311 ( 4
1238 ( 129
.2000
a
IC50: concentration (µM) of drug required to reduce cell growth
to 50% of the controls in a SRB assay obtained from the mean (
SD of three experiments. Each experiment comprised of triplicate
determinations of cell growth at eight concentrations of test com-
b
pound. Drug exposure period was 48 h. 2-(Methylsulfonyl)ethyl
phosphorodiamidate.
Ta ble 2. Metabolic Stability Profile of 1,2-Bemzisoxazole
Phosphorodiamidates upon Incubation with Phenobarb-Induced
Rat Liver S-9 Fractiona
% compound remainingb,c
incubation conditions
4
9
12
Hypoxic
Oxic
NADPH
no cofactor
34 ( 9
98 ( 7
45 ( 4
79 ( 4
1 ( 2
76 ( 5
Sch em e 4
NADPH
no cofactor
33 ( 4
97 ( 5
46 ( 2
78 ( 5
1 ( 1
79 ( 5
0.025 M phosphate buffer pH 7.4 97 ( 11 97 ( 6 102 ( 10
boiled S-9, NADPHd
96
a
b
Incubation time was 30 min. Mean ( SD from three experi-
ments. c The incubation mixtures of phosphorodiamidate prodrugs
were quantitatively analyzed for the unchanged starting com-
d
pounds by TLC densitometry. Mean from two experiments.
Ta ble 3. Generation of Alkylating Activity upon Incubation of
1,2-Bemzisoxazole Phosphorodiamidates with
Phenobarb-Induced Rat Liver S-9 Fractiona
absorbanceb,c
incubation conditions
controld
4
9
12
0.04
0.05
0.04
Hypoxic
NADPH
no Cofactor
2.02
0.38
2.35
0.42
2.23
0.85
Oxic
NADPH
no cofactor
0.025 M phosphate buffer pH 7.4
boiled S-9, NADPH
1.97
0.26
0.49
0.44
2.31
0.32
0.26
2.36
0.48
0.48
Sch em e 5
a
Incubation time was 30 min. Incubation samples from meta-
bolic stability experiments were utilized for alkylating activity
b
measurements. Mean of absorbance value at 542 nm from two
experiments. c Absorbance value is a measurement of NBP alkyl-
d
ation product, which is indicative of alkylating activity. Control
sample consisted of the S-9 fraction and NADPH without test
compound.
Preliminary experiments of 4 with uninduced rat liver
S-9 showed less than 10% metabolism. Upon incubation
with phenobarb-induced rat liver S-9 fraction, 4, 9, and
12 underwent extensive NADPH-dependent metabolism
(Table 2) under both hypoxic and oxic conditions and
concomitantly generated substantial alkylating activity
(Table 3). Under hypoxic as well as oxic conditions, 12
was almost quantitatively metabolized. In comparison
to 12, analogues 4 and 9 were metabolized to a lesser
extent, and 33-46% of the original amount was left
unchanged. Control incubations in the absence of NAD-
PH with aqueous buffer (0.025 M phosphate buffer, pH
7.4) or boiled S-9 supplemented with NADPH did not
show significant metabolism or alkylating activity.
These data provided initial indirect evidence in support
2-hydroxypropiophenone (16), a metabolite of 15, to
yield a reactive phenyl vinyl ketone byproduct as in 5.
In Vitr o Meta bolism of 1,2-Ben zisoxa zole P h os-
p h or od ia m id a tes. To determine whether 1,2-benzisox-
azole phosphorodiamidate prodrugs undergo reductive
metabolism and consequently liberate alkylating spe-
cies, metabolic stability studies of these prodrugs were
performed with rat liver S-9 fraction under hypoxic and
oxic conditions. Incubation mixtures were monitored for
disappearance of the parent compound and tested for
alkylating activity as determined by NBP assay.

1,2-Benzisoxazole Phosphorodiamidates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5431
Sch em e 6
of the proposed bioreductive pathway that 4, 9, and 12
undergo metabolism and, thereafter, liberate alkylating
species, most likely PDA. Analogue 12 was the most
susceptible compound toward metabolism by induced
rat S-9. If 12 is quantitatively metabolized to PDA, it
may be expected to have the greatest cytotoxicity among
tested compounds. However, cytotoxicity data showed
that 12 was less potent than 9. This suggested that,
besides reductive activation, some of 12 might be
vulnerable to other competing metabolic pathways, e.g.,
nitro group reduction. In contrast to the enhanced
bioreduction of zonisamide to SMAP under a hypoxic
environment,16-21 no significant differences were dis-
cernible between the extent of metabolism or alkylating
activities under hypoxic and oxic incubation conditions
for any of the tested 1,2-benzisoxazole phosphorodia-
midate prodrugs. Presumably, the replacement of the
sulfamoylmethyl side chain in zonisamide with eth-
ylphosphorodiamidate contributes toward loss of hy-
poxia-selective bioreduction in the tested prodrugs.
Ta ble 4. Formation of 16 upon Incubation of 15 with Rat
Liver Subcellular Fractions in the Presence of Various
Cofactorsa
% 16 formedb,c
hypoxic
oxic
Phenobarb-Induced Rat Liver
S-9 fraction
NADPH
NADH
benzaldehyde
2-hydroxypyrimidine
no cofactor
0.025 M phosphate buffer pH 7.4
boiled S-9, NADPH
microsomal fraction
NADPH
88.5 ( 3.8
28.5 ( 1.5
2.0 ( 0.3
0.5 ( 0.1
2.2 ( 0.5
80.9 ( 3.0
14.8 ( 1.6
0.7 ( 0.3
0.6 ( 0.1
0.5 ( 0.1
nd
Since the NBP assay does not discriminate between
alkylating species, it is conceivable that the alkylating
activity observed in the incubation mixtures may arise
from chloroacetaldehyde, the possible oxidative N-
dealkylation product of the chloroethylamine side chain.
To investigate if prodrugs generated alkylating species
as outlined in the proposed activation pathway (Scheme
2), incubation mixtures of 9 were subjected to LC/MS
analysis to detect the presence of the corresponding
ketone metabolite. Due to the electron-donating meth-
oxy substituent, it was postulated that the ketone
metabolite of 9 might be relatively stable to â-elimina-
tion in the incubation mixtures and thereby possible to
detect. Under reverse-phase chromatography, 9 eluted
at a retention time (tR) of 13.5 min. The parent com-
pound showed the expected (M + H)+ pseudomolecular
ion at m/z 396 and other characteristic ion-source
fragment ions at m/z 255 and 176 (Figure 1
0.1 ( 0.0
93.0 ( 9.1
73.0 ( 2.9
no cofactorc
cytosolic fraction
NADPHc
no cofactorc
Uninduced Rat Liver
S-9 fraction
0.4
0.6
3.6
1.4
0.7
0.4
NADPH
9.4 ( 0.1
9.1 ( 0.1
no cofactord
0.9
1.6
a
b
Incubation time was 30 min. Mean ( SD of three experi-
ments. c Metabolite 16 was analyzed by HPLC. The amount of 16
was calculated and is reported as percent of total, where the total
was based on an equimolar amount of substrate (15) used. Mean
from two experiments. nd, not detected.
d
unstable ketone metabolites that are difficult to mea-
sure, whereas stable 16 generated from 15 can be easily
quantitated against commercially available standard.
of the Supporting Information). A closely eluting
metabolite peak with tR ) 13.8 min showed a (M + H)+
ion with m/z 399. An increase of 3 amu for the later-
eluting peak indicated the presence of a ketone metabo-
lite. A similar shift of 3 amu was observed for ion-source
fragment ions with m/z values of 258 and 179. The
metabolite peak showed another distinct fragment ion
at m/z 151 that was not observed for 9. The metabolite
peak was detected in aerobic and hypoxic phenobarb-
induced rat liver S-9 incubation mixtures of 9 containing
NADPH, but not in the absence of NADPH or when
incubations were carried out in the presence of benzal-
dehyde or 2-hydroxypyrimidine, known cofactors of
enzyme aldehyde oxidase. The LC/MS data supported
the existence of the ketone metabolite of 9 and, in
conjunction with previous metabolism and alkylating
activity data, provided persuasive evidence in support
of the proposed reductive activation pathway.
Upon incubation of 15 with phenobarb-induced rat
liver S-9 fraction, 16 was formed under oxic (80.9%) as
well as hypoxic (88.5%) conditions with NADPH cofactor
(Table 4). Although formation of 16 was also observed
with NADH cofactor, the extent of NADH-dependent
formation of 16 was approximately 4-5-fold lower than
NADPH-dependent activity. Some degree of hypoxia-
selective metabolism was observed with NADH, but not
with NADPH cofactor. Incubation of 15 in the presence
of benzaldehyde or 2-hydroxypyrimidine, electron do-
nors for enzyme aldehyde oxidase, did not yield signifi-
cant amounts of 16. These data suggested the lack of
involvement of aldehyde oxidase in activation of 15. The
data were in disagreement with earlier report that
demonstrated aldehyde oxidase-mediated metabolism of
zonisamide to SMAP in some strains of rat liver cyto-
sol.21 These conflicting results may be explained due to
differences between strains of rats utilized in current
experiments and in the literature or most likely due to
15 being a poor substrate for enzyme aldehyde oxidase.
Uninduced rat liver S-9 fraction showed approximately
10-fold lower activity in generation of 16 as compared
to induced rat liver S-9.
In Vitr o Meta bolism of 15. Compound 15 was
selected as a model test compound to measure the rate
and extent of bioreduction of 1,2-benzisoxazoles and to
identify potential reductase enzyme(s). The ability of 15
to convert to 2-hydroxypropiophenone (16) (Scheme 6)
could provide additional supportive evidence for the
activation pathway of 1,2-benzisoxazole-based prodrugs.
Moreover, bioreduction of 4, 9, and 12 yields inherently

5432 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
J ain and Kwon
Ta ble 5. Effect of Inhibitors SKF-525A and PCMBA on
Formation of 16 upon Incubation of 15 with Phenobarb-Induced
Rat Liver Microsomal Fractiona
Ta ble 6. Formation of 16 upon Incubation of 15 with Human
or Rat P450 Reductase Microsomesa
% 16 formedb,c
concn of
inhibitor
(mM)
hypoxic
oxic
hypoxic
oxic
% 16 formedb,c % inhibn % 16 formedb,c % inhibn
Human P450 Reductase Microsomes
NADPH
no cofactor
NADPH, 2 mM pCMBA
Rat P450 Reductase Microsomes
NADPH
1.7
nd
0.6
nd
nd
nd
SKF-525A
0
1
2
4
70.8
54.6
50.3
45.0
0
23
29
36
44.9
36.9
38.5
26.9
0
18
14
40
1.7
nd
nd
1.5
nd
nd
no cofactor
NADPH, 2 mM pCMBA
pCMBA
0
1
2
4
93.2
0.7
1.2
0
99
99
99
68.8
0.5
0.5
0
99
99
99
a
b
Incubation time was 60 min. Mean of values obtained from
two experiments. nd, not detected. c See footnote c in Table 4.
0.5
0.7
Con clu sion s
a
b
Incubation time was 30 min. Mean of values obtained from
two experiments. c See footnote c in Table 4.
A series of 1,2-benzisoxazole phosphorodiamidate
prodrugs were designed and successfully synthesized.
The prodrugs demonstrated in vitro cytotoxicity against
V-79 cells. Consistent with the original hypothesis,
prodrugs underwent reductive metabolism and gener-
ated alkylating activity, however, with equal degree
under oxic and hypoxic conditions. Detection of ketone
metabolites of 9 and 15 provided additional convincing
evidence in support of the proposed metabolic pathway.
Model test compound 15 underwent NADPH-dependent
metabolic reduction in phenobarb-induced rat liver
microsomes under oxic and hypoxic conditions. On the
basis of inhibition of microsomal activity due to SKF-
525A and pCMBA as well as some metabolic activity
observed in human and rat P450 reductase microsomes,
cytochrome P450 and/or cytochrome P450 reductase
enzymes may be involved in bioreduction of 1,2-ben-
zisoxazoles.
Investigations with other phenobarb-induced rat liver
subcellular fractions showed that the NADPH-depend-
ent metabolic activity of S-9 was primarily located in
the microsomal fraction with limited contribution from
the cytosolic fraction. These data suggested the possible
involvement of NADPH-dependent enzyme(s) cyto-
chrome P450 and/or cytochrome P450 reductase, in the
bioreduction of 15, both of which have been shown to
catalyze reduction of a variety of compounds. To test
the involvement of these enzymes in the microsomal
formation of 16 from 15, inhibition studies were carried
out in phenobarb-induced rat liver microsomes and
NADPH with varying concentrations of SKF-525A or
p-chloromercuribenzoic acid (pCMBA), known inhibitors
of cytochrome P450 and cytochrome P450 reductase,
respectively (Table 5). SKF-525A inhibited microsomal
formation of 16 from 15 under oxic as well as hypoxic
conditions in a concentration-dependent manner with
40% (oxic) and 36% (hypoxic) inhibition at 4 mM
concentration. This implied possible involvement of
cytochrome P450 in the reductive activation of 15. On
the other hand, pCMBA almost quantitatively inhibited
formation of 16 from 15 at all tested concentrations
under both oxic as well as hypoxic conditions. Results
from pCMBA inhibition studies suggested that P450
reductase might also be involved in reduction of 15 to
16. Alternatively, since P450 reductase activity is es-
sential for cytochrome P450-mediated metabolism, the
data might suggest indirect inhibition of cytochrome
P450 activity due to inhibition of the P450 reductase.
Exp er im en ta l Section
Melting points were determined on a Thomas-Hoover capil-
lary melting point apparatus and were uncorrected. H NMR
1
spectra were recorded on a Bruker Avance 400 (400 MHz)
instrument, and chemical shifts are reported as δ values (ppm)
downfield from tetramethylsilane as an internal standard.
NMR abbreviations used are as follows: s (singlet), d (doublet),
t (triplet), q (quartet), m (multiplet). MS analyses were carried
out on an LCQ (Finnigan) ion trap LC/MS instrument.
Elemental analyses were performed by Atlantic Microlab, Inc.
Norcross, GA. Silica gel GF plates (Analtech) were used for
TLC (250 µm, 2.5 × 10 cm) and preparative TLC (1000 µm,
20 × 20 cm). Silica gel (40 µm, Baker) was used for flash
column chromatography. NBP spray was used for the detection
of potential alkylating compounds as follows: the plates were
sprayed with 5% 4-(p-nitrobenzyl)pyridine (NBP) in acetone,
heated at 100 °C for 5 min, and sprayed with 5% methanolic
KOH. Alkylating agents are indicated by the appearance of a
blue chromophore. Spectrophotometric analysis was performed
on a Milton Roy Spectronic 301 spectrophotometer. Quantita-
tive TLC analyses were done on an Analtech Uniscan Video
Densitometer. All organic reagents and solvents were reagent
grade and purchased from commercial vendors.
1,2-Ben zisoxa zole-3-a cetic a cid (1) was prepared accord-
ing to the published procedure25 in 46% yield: mp 120-125
°C (lit.25 mp 125 °C); TLC Rf ) 0.50 in EtOAc/hexane (1:1)
with 1% acetic acid; 1H NMR (CDCl3/DMSO-d6, 8:1) δ 3.93 (s,
2H), 7.22-7.26 (m, 1H), 7.44-7.49 (m, 2H), 7.67 (d, J ) 7.9
Hz, 1H).
Meth yl 1,2-ben zisoxa zol-3-yl a ceta te (2) was prepared
according to published procedure27 with some modifications.
To a solution of 1 (1.0 g, 60.0 mmol) in 20 mL of methanol
were added 8-10 drops of concentrated sulfuric acid. The
mixture was heated under reflux with continuous stirring for
a period of 1.5 h. Following reflux, the reaction mixture was
To investigate the role of P450 reductase, 15 was
incubated with rat or human P450 reductase mi-
crosomes. In the presence of NADPH, 16 was formed
e1.7% under hypoxic or oxic conditions and found
inhibited due to pCMBA (Table 6). Reduction of 15 due
to P450 reductase alone was markedly weaker in
comparison with microsomal activity. It has been re-
ported that V-79 fibroblasts contain DT-diaphorase,
cytochrome P450 reductase, and xanthine oxidase33
enzymes, whereas no detectable cytochrome P450 are
observed.34,35 Therefore, in the absence of cytochrome
P450, endogenous cytochrome P450 reductase activity
in V-79 cells assumes relevance due to its potential
ability of reductive activation of phosphorodiamidate
prodrugs leading to cytotoxicity.

1,2-Benzisoxazole Phosphorodiamidates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5433
cooled to room temperature and evaporated in vacuo to yield
a pale yellow, oily residue. The residue was added with a
saturated solution of NaHCO3 (50 mL) to neutralize acid, and
the resulting alkaline aqueous mixture was extracted with
EtOAc (3 × 50 mL). Organic extracts were pooled, dried over
anhydrous sodium sulfate, and filtered, and filtrate was
evaporated under reduced pressure to yield a pale yellow, oily
product (1.0 g) in 93% yield: TLC Rf ) 0.45 in EtOAc/hexane
added dropwise to a stirring solution of N,N,N′,N′-tetrameth-
yldiamidophosphorochloridate (0.34 g, 1.65 mmol) in anhy-
drous THF (12 mL) at ice-bath temperature. The mixture was
stirred at ice-bath temperature and then gradually allowed
to warm to room temperature and stirred overnight. Reaction
mixture was evaporated under vacuum to obtain a yellow-
colored oily residue as the crude product. This was dissolved
in EtOAc (10 mL) and purification by column chromatography
of the resulting solution eluting with EtOAc/EtOH (6:1)
afforded pure 5 as a pale yellow oil (0.26 g, 58% yield): TLC
Rf ) 0.60 in EtOAc/EtOH (10:1.5); 1H NMR (CDCl3) δ 2.57 (d,
J ) 9.8 Hz, 12H), 3.38 (q, J ) 6.6 Hz, 2H), 4.39 (q, J ) 7.4 Hz,
2H), 7.32-7.35 (m, 1H), 7.54-7.59 (m, 2H), 7.75 (d, J ) 7.9
Hz, 1H). Anal. (C12H20N3O3P) H, N; C: calcd, 52.52; found,
52.04.
6-Meth oxy 1,2-ben zisoxa zole-3-a cetic a cid (6) was pre-
pared according to published procedure26 and obtained as light-
tan-colored solids (0.4 g, 59% yield): mp 175-179 °C (lit.26 mp
174-175 °C); TLC Rf ) 0.22 in CH2Cl2/EtOH/acetic acid (100:
1.25:0.5); 1H NMR (CDCl3/DMSO-d6, 10:1) δ 3.73 (s, 3H), 3.78
(s, 2H), 6.76 (d, J ) 8.72 Hz, 1H), 6.83 (d, J ) 2.0 Hz, 1H),
7.42 (d, J ) 8.7 Hz, 1H).
1
(1:5); H NMR (CDCl3) δ 3.77 (s, 3H), 4.08 (s, 2H), 7.33-7.37
(m, 1H), 7.56-7.62 (m, 2H), 7.73 (d, J ) 8.0 Hz, 1H). The
obtained product was chromatographically pure and used
without additional purification.
2-(1,2-Ben zisoxa zol-3-yl)eth a n ol (3) was synthesized ac-
cording to published procedure27 with some modifications. A
solution of 2 (2.5 g, 13 mmol) in 50 mL of anhydrous THF was
added dropwise from a dropping funnel into a stirring suspen-
sion of LiAlH4 (0.98 g, 26 mmol) in anhydrous THF (30 mL)
in a three-neck flask over a period of 15 min at ice-bath
temperature under nitrogen atmosphere. The reaction was
allowed to proceed for 15 min and then terminated with
dropwise addition of EtOAc (50 mL) at ice-bath temperature
with vigorous stirring. The mixture was evaporated under
reduced pressure and the residue suspended in 100 mL of
EtOAc/hexane (1:1). The suspension was filtered through
Celite and filtrate evaporated in vacuo to yield a pale yellow,
oily residue. The crude product was dissolved in 10 mL of
EtOAc/hexane (1:1) and purified using flash column with
eluent EtOAc/hexane (1:1) to give pure 3 as a yellow, oily
product (1.0 g, 53% yield): TLC Rf ) 0.49 in EtOAc/hexane
(1:1); 1H NMR (CDCl3) δ 3.26 (t, J ) 6.0 Hz, 2H), 4.15 (q, J )
3.9 Hz, 2H), 7.30-7.37 (m, 1H), 7.55-7.60 (m, 2H), 7.71 (d, J
) 7.9 Hz, 1H).
Meth yl (6-m eth oxy-1,2-ben zisoxa zol-3-yl)a ceta te (7)
was prepared from 6 (1.9 g, 9.0 mmol) as in the synthesis of 2
to give product 7 as a pale yellow, crusty solid (2.0 g,
quantitative yield): mp 70-74 °C (lit.26 mp 68-69 °C); TLC
Rf ) 0.63 in CH2Cl2/EtOH/acetic acid (100:1.25:0.5); 1H NMR
(CDCl3) δ 3.76 (s, 3H), 3.90 (s, 3H), 4.01 (s, 2H),), 6.95 (d, J )
8.7 Hz, 1H), 7.01 (d, J ) 2.0 Hz, 1H), 7.55 (d, J ) 8.0 Hz, 1H).
2-(6-Meth oxy-1,2-ben zisoxa zol-3-yl)eth a n ol (8) was pre-
pared by reduction of 7 (1.72 g, 8 mmol) with LiAlH4 as in the
procedure for preparation of 3. Crude product was obtained
as a dark yellow, oily residue. The residue was dissolved in
15 mL of EtOAc/hexane (1:1) solvent mixture and purified
using column chromatography with the use of EtOAc/hexane
(1:1) as the eluting solvent. Pure 8 was obtained as a pale
yellow, oily material (1.20 g, 81% yield): TLC Rf ) 0.43 in
2-(1,2-Ben zisoxa zol-3-yl)eth yl N,N-bis(2-ch lor oeth yl)-
p h osp h or od ia m id a te (4) was synthesized according to pub-
lished procedure24 with some modifications. A stirring solution
of 3 (0.25 g, 1.5 mmol) in anhydrous THF (10 mL) was added
to a n-butyllithium (0.6 mL, 1.5 mmol, 2.5 M in hexane)
solution in anhydrous THF (5 mL) from a dropping funnel at
ice-bath temperature in a two-neck round-bottom flask. After
complete addition of n-BuLi, the mixture was stirred at ice-
bath temperature for 15 min, then the ice bath was removed,
and stirring was continued at room temperature for additional
90 min. The resulting pale yellow suspension was aspirated
into a syringe, transferred into a dropping funnel, and added
dropwise to a stirring solution of N,N-bis(2-chloroethyl)-
phosphoramidic dichloride (0.4 g, 1.6 mmol) in anhydrous THF
(15 mL) at ice-bath temperature. The reaction mixture was
stirred at ice-bath temperature for 1 h and then at room
temperature for an additional 2.5 h. Ammonia was bubbled
into the reaction mixture with stirring at ice-bath temperature
for 45 min. Liquids in the reaction mixture were evaporated
in vacuo, and ether (50 mL) was added. Resulting solids were
filtered using Celite, and filtrate was evaporated under
vacuum to give viscous, pale yellow, oily residue. The crude
product was dissolved in EtOAc (10 mL) and purified using
flash column with eluent EtOAc/EtOH (10:1) to give pale
yellow, oily product that gave white crystals upon overnight
storage in freezer. The product was recrystallized using EtOAc/
hexane to give light, white-colored solids (0.12 g, 21% yield):
mp 80-85 °C; TLC Rf ) 0.56 in EtOAc/EtOH (10:1); APCI-
MS (M + H)+ m/z calcd 366.05, found 365.9; 1H NMR (CDCl3)
δ 3.23-3.61 (m, 10H), 4.39-4.60 (m, 2H), 7.33-7.38 (m, 1H),
1
EtOAc/hexane (1:1); H NMR (CDCl3) δ 3.20 (t, 5.9 Hz, 2H),
3.90 (s, 3H), 4.10 (q, J ) 4.6, 2H), 6.92-6.95 (d, J ) 8.7 Hz,
1H), 7.00 (d, J ) 2.0 Hz, 1H), 7.53 (d, J ) 8.8 Hz, 1H).
2-(6-Met h oxy-1,2-b en zisoxa zol-3-yl)et h yl N,N-b is(2-
ch lor oeth yl)p h osp h or od ia m id a te (9) was prepared from
8 (0.50 g, 2.6 mmol) as in the synthesis of 4 to give a pale
yellow, oily substance as a crude product. This was purified
using column chromatography with EtOAc/EtOH (10:1.5) as
the eluting solvent. Pure 9 was obtained as white-colored solids
(0.28 g, 27% yield): mp 86-90 °C; TLC Rf ) 0.69 in EtOAc/
EtOH (10:1); APCI-MS (M + H)+ m/z calcd 396.05, found 395.9;
1H NMR (CDCl3) δ 3.31-3.59 (m, 10H), 3.90 (s, 3H), 4.46 (d,
2H), 6.94 (d, J ) 8.6 Hz, 1H), 7.00 (d, J ) 2.0 Hz, 1H), 7.54 (d,
J ) 8.6, 1H). Anal. (C14H20Cl2N3O4P) C, H, Cl, N.
Meth yl (5-n itr o-1,2-ben zisoxa zol-3-yl)a ceta te (10) was
prepared from 2 (3.0 g, 15.7 mmol) according to published
procedure28 to obtain the crude product as a yellow oil. The
oil was dissolved in CH2Cl2 (15 mL) and purified using column
chromatography with the use of hexane/EtOAc (4:1) as the
eluting solvent. Appropriate fractions containing the product
were pooled and evaporated under reduced pressure to give a
pale yellow, oily residue that solidified upon addition of a few
drops of ether. Resulting solids were recrystallized with
methanol to give 10 as white solids (2.7 g, 74% yield): mp 65-
67 °C (lit.28 mp 66-67 °C); TLC Rf ) 0.26 in hexane/EtOAc
(4:1); 1H NMR (CDCl3) δ 3.82 (s, 3H); 4.14 (s, 2H), 7.54 (d, J )
9.2 Hz, 1H), 8.51 (d, J ) 6.9 Hz, 1H), 8.71 (d, J ) 2.2 Hz, 1H).
7.53-7.70 (m, 2H), 7.72 (d, J ) 7.2 Hz, 1H). Anal. (C13H18
Cl2N3O3P) H, Cl, N; C: calcd, 42.62; found, 43.29.
-
2-(1,2-Ben zisoxa zol-3-yl)et h yl N,N,N′,N′-Tet r a m et h -
ylp h osp h or od ia m id a te (5). To a stirring solution of 3 (0.25
g, 1.5 mmol) in anhydrous THF (10 mL) in a two-neck round-
bottom flask was added a solution of n-butyllithium (0.6 mL,
1.5 mmol, 2.5 M in hexane) in anhydrous THF (10 mL) from
a dropping funnel at ice-bath temperature. The reaction
mixture was stirred at ice-bath temperature for 15 min
followed by removal of the ice bath and continued stirring at
room temperature for 60 min. The mixture was aspirated into
a syringe, transferred quickly into a dropping funnel, and
2-(5-Nitr o-1,2-ben zisoxa zole-3-yl)eth a n ol (11) was syn-
thesized from 10 (0.50 g, 2 mmol) according to the procedure
utilized for preparation of 3. After quenching the reaction with
EtOAc, the liquids were evaporated under vacuum and the
dark residue partitioned between CH2Cl2 (100 mL) and 0.5 N
HCl (100 mL). The aqueous layer was further extracted with
EtOAc (2 × 75 mL). The CH2Cl2 and EtOAc extracts were
combined and dried under vacuum to obtain the crude product
as a dark-colored residue. The residue was subjected to
purification with column chromatography with the use of

5434 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
J ain and Kwon
hexane/EtOAc (2:1) eluting solvent. Fractions containing the
desired compound were pooled and the solvent evaporated
under vacuum to yield pure product as shiny, yellow crystals
(0.14 g, 32% yield): TLC Rf ) 0.69 in EtOAc/hexane (1:1); 1H
NMR (CDCl3) δ 3.31 (t, J ) 5.9 Hz, 2H), 4.11-4.18 (m, 2H),
7.69 (d, J ) 5.0 Hz, 1H), 8.49 (d, J ) 5.8 Hz, 1H), 8.73 (d, J )
2.2 Hz, 1H).
well microtiter plates with some modifications. One hundred
microliters of cell suspension (approximately 15 × 103 cells
per 100 µL) was transferred to a well and preincubated for 24
h. Fifty microliter of media containing variable concentrations
of drugs as well as 50 µL of fresh media was added to each
well. Plates were incubated under aerobic conditions for a 48-h
period. Cultures were fixed with 50 µL of 50% cold trichloro-
acetic acid and incubated at 4 °C for 1 h. Plates were then
washed five times with tap water to remove trichloroacetic
acid, growth medium, and serum protein and then air-dried.
The fixed cells were stained for 30 min with 100 µL of 0.4%
sulforhodamine B (SRB) solution in 1% acetic acid. At the end
of the staining period, plates were rinsed five times with 1%
acetic acid to remove unbound dye. The bound dye was
solublized with 200 µL of 10 mM Tris buffer for 5 min. A 100-
µL aliquot was diluted with 2 mL of 10 mM Tris buffer and
absorbance of the resulting solution was measured at 564 nm.
Absorbance is directly proportional to cell-growth after
incubation. Percent cell growth following drug treatment was
calculated as (absorbance from drug-treated well/absorbance
from untreated control well) × 100. Drug concentrations were
plotted semilogarithmically versus percent cell-growth values,
data were curve-fitted to a first-order decay equation, and IC50
values were calculated from the slope (k) of the equation. Each
experiment consisted of controls and varying concentrations
of drug treatments in triplicates, and three experiments were
carried out to obtain the mean IC50 value for a compound.
In Vitr o Meta bolism Stu d ies. P r ep a r a tion of Ra t Liver
F r a ction s. Rat liver S-9 cytosolic and microsomal fractions
were prepared as described previously.32 Protein concentra-
tions were determined by the method of Bradford (Protein
Assay Kit, Bio-Rad Laboratories, Inc.) using bovine serum
albumin as standard. Protein concentrations for various rat
liver fractions were obtained as follows: Uninduced rat liver
S-9 fraction, 22.5 mg/mL; phenobarb-induced rat liver S-9
fraction, 17.9 mg/mL (preparation I) and 18.2 mg/mL (prepa-
ration II); phenobarb-induced rat liver cytosolic fraction, 12.7
mg/mL; phenobarb-induced rat liver microsomal fraction, 6.3
mg/mL. Cytochrome c reductase activity of P450 reductase
microsomes was carried out in the following manner: A 2.5
mL reaction mixture containing 0.04 mg/mL protein, 1 mg/
mL cytochrome c, and 1.6 mM NADPH in 0.025 M phosphate
buffer, pH 7.4 was incubated. Incubations were initiated with
addition of NADPH, and absorbance was monitored at 550 nm
as a function of time. Activity was based on the extinction
coefficient of cytochrome c (21 cm2/mmol), the rate of increase
in absorbance per minute of incubation, and the amount of
protein added to the incubation. Cytochrome c reductase
activity was obtained as follows: human P450 reductase
microsomes, 436.8 nmol/(min × mg protein); rat P450 reduc-
tase microsomes, 401.2 nmol/(min × mg protein); phenobarb-
induced rat liver microsomal fraction, 202.4 nmol/(min × mg
protein).
2-(5-Nitr o-1,2-ben zisoxa zol-3-yl)eth yl N,N-bis(2-ch lo-
r oeth yl)p h op sh or od ia m id a te (12) was synthesized with the
use of 11 (0.21 g) according to the procedure in preparation of
4 on a 1.0 mmol scale. Crude product was obtained as an
orange-colored oily residue. The residue was dissolved in
EtOAc (15 mL) and purified using column chromatography
with EtOAc used as the eluting solvent. Appropriate fractions
containing the desired product were pooled and dried under
vacuum to give pure 12 as viscous, sticky, yellow oil (0.1 g,
24% yield): TLC Rf ) 0.34 in EtOAc; ESI-MS (M + H)+ m/z
calcd. 411.03. Found 410.9; 1H NMR (CDCl3) δ 3.34-3.65 (m,
10H), 4.40-4.57 (m, 2H), 7.72 (d, J ) 9.1 Hz, 1H), 8.49-8.51
(d, J ) 9.1 Hz, 1H), 8.74 (d, J ) 2.1 Hz, 1H). Anal. (C13H17
Cl2N4O5P) H, Cl, N; C: calcd, 37.97; found, 38.68.
-
1-(2-Hyd r oxyp h en yl)p r op a n -1-on e oxim e (13) was syn-
thesized according to a published procedure29 with some
modifications. A solution of hydroxylamine hydrochloride (1.5
g, 22 mmol) and sodium acetate trihydrate (3.13 g, 23 mmol)
dissolved in 50 mL of EtOH/H2O (7:3) was added to a solution
of 2-hydroxypropiophenone (2.89 g, 19 mmol) in 25 mL of
EtOH/H2O (7:3) solvent. The resulting mixture was heated to
reflux. After 2 h, additional hydroxylamine hydrochloride
(0.675 g, 9.5 mmol) and sodium acetate trihydrate (9.5 mmol)
were dissolved in water (15 mL) and added, and the reflux
was continued for additional 30 min. The reaction mixture was
cooled to room temperature, concentrated by evaporation
under vacuum, and white solids obtained were filtered, washed
with water, and dried to give sufficiently pure 13 (2.7 g, 85%
yield): mp 90-94 °C; TLC Rf ) 0.46 in hexane/EtOAc (5:1);
1H NMR (CDCl3) δ 1.25 (t, J ) 7.7 Hz, 3H), 2.92 (q, J ) 7.6
Hz, 2H), 6.92-6.96 (m, 1H), 7.01 (d, J ) 8.2 Hz, 1H), 7.27-
7.32 (m, 1H), 7.47 (d, J ) 8.0 Hz, 1H).
1-(2-Hyd r oxyp h en yl)p r op a n -1-on e O-a cetyloxim e (14)
was synthesized according to a published procedure29 with
some modifications. Oxime intermediate 13 (3.0 g, 18 mmol)
was dissolved in acetic anhydride (6 mL) in a round-bottom
flask at room temperature with gentle swirling. The flask
became warm to the touch. Swirling was continued for 10 min,
following which white precipitates separated out. Water (50
mL) was added to the flask, and the mixture was filtered to
obtain white-colored solid residues. The solids were washed
with water and dried to give product 14 in 3.35 g yield (89%):
1
mp 87-92 °C; TLC Rf ) 0.66 in CH2Cl2; H NMR (CDCl3) δ
1.27 (t, J ) 9.5 Hz, 3H), 2.28 (s, 3H), 2.94 (q, J ) 9.5 Hz, 2H),
6.93-6.97 (m, 1H,), 7.12 (d, J ) 8.0 Hz, 1H), 7.34-7.39 (m,
2H), 7.50 (d, J ) 8.0 Hz, 1H).
In cu ba tion s. A typical incubation mixture consisted of 2
mL of rat liver fraction containing 5 mM cofactors and 2.5 mM
substrates. The test compound was dissolved in methanol/
water (50:50) and a 100-µL volume added to the incubation
mixture to obtain a final concentration of 2.5 mM. In case of
anaerobic incubations, rat liver fraction was transferred to a
vial, and the vial was capped with rubber septa with a
provision for inlet and outlet needles for nitrogen gas. The
fraction was purged with nitrogen for 15-20 min, and septa
were sealed. Incubations were initiated with addition of test
compound, carried out for 30 min at 37 °C, and terminated
with addition of 2 mL of ice-cold methanol. The contents were
transferred into a glass test tube and then centrifuged (3000
rpm, 15 min) to allow separation of precipitated proteins. The
supernatant was used for either TLC or HPLC analysis. For
TLC analysis, 64-80 µL was spotted on a TLC plate and the
plates eluted with ethyl acetate/ethanol (10:1) (for compounds
4 and 12) or with ethyl acetate/ethanol 10:2 (for compound 9).
3-Eth yl-1,2-ben zisoxa zole (15) was prepared according to
the published procedure29 with some modifications. Oxime
acetate (14) (2.5 g, 12.0 mmol) was heated under reflux with
anhydrous pyridine (25 mL) for 3.5 h. Following reflux, the
dark yellow reaction mixture was allowed to cool to room
temperature and acidified with 5 N HCl (approximately 100
mL). The mixture was extracted with ether (3 × 75 mL). The
ether layers were pooled, washed with 1 N HCl (75 mL), dried
over anhydrous sodium sulfate, and evaporated under vacuum
to give yellow-colored, oily residue as crude product. The crude
product was purified using column chromatography with
hexane/EtOAc (100:7) as the eluting solvent. Fractions con-
taining the desired compound were combined and dried under
vacuum to give 15 as colorless oil (1.5 g, 85% yield): TLC Rf
) 0.74 in hexane/EtOAc (5:1); ESI-MS (M + H)+ m/z calcd
1
148.07, found 147.9; H NMR (CDCl3) δ 1.48 (t, J ) 7.5 Hz,
3H), 3.04 (q, J ) 7.5 Hz, 2H), 7.31 (m, 1H), 7.53-7.58 (m, 2H),
7.68 (d, J ) 7.1 Hz, 1H). Anal. (C9H9NO) H, N; C: calcd, 73.45;
found, 72.49.
For LC/MS analysis of incubation mixtures of 9, a HP1050
liquid chromatographic system was used for chromatographic
separation of ketone metabolite from 9. A 50 µM aliquot of
In Vitr o Cytotoxicity. In vitro cytotoxicity assays were
carried out according to a procedure by Skehan et al.30 in 96-

1,2-Benzisoxazole Phosphorodiamidates
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5435
(7) Zeman, E. M.; Brown, J . M.; Lemmon, M. J .; Hirst, V. K.; Lee,
W. W. SR4233: A new bioreductive agent with high selective
toxicity for hypoxic mammalian cells. Int. J . Radiat. Oncol. Biol.
Phys. 1986, 12, 1239-1242.
(8) Denny WA, Wilson WR. Tirapazamine: A bioreductive antican-
cer drug that exploits tumour hypoxia. Expert Opin. Investig.
Drugs 2000, 9 (12), 2889-901.
(9) Sun, Z.-Y.; Botros, E.; Su, A.-D.; Kim, Y.; Wang, E.; Baturay,
N.; Kwon, C.-H. Sulfoxide-containing aromatic nitrogen mus-
tards as hypoxia-directed bioreductive cytotoxins. J . Med. Chem.
2000, 43 (22), 4160-4168.
(10) Mulcahy, R. T.; Gipp, J . J .; Schmidt, J . P.; J oswig, C.; Borch, R.
F. Nitrobenzyl phosphorodiamidates as potential hypoxia-selec-
tive agents. J . Med. Chem. 1994, 37 (11), 1610-1615.
(11) Flader, C.; Liu, J .; Borch, R. F.; Development of novel quinone
phosphorodiamidate prodrugs targeted to DT-diaphorase. J .
Med. Chem. 2000, 43, 3157-3167.
(12) Borch, R. F.; Liu, J .; Schmidt, J . P.; Marakowits, J . T.; J oswig,
C.; Gipp, J . J .; Mulcahy, R. T. Synthesis and evaluation of
nitroheterocyclic phosphoramidates as hypoxia-selective alkyl-
ating agents. J . Med. Chem. 2000, 43, 2258-2265.
(13) Workman, P.; Stratford, I. J . The experimental development of
bioreductive drugs and their role in cancer therapy. Cancer and
Metastasis Reviews 1993, 12, 73-82.
(14) Workman, P. Bioreductive mechanisms. Int. J . Radiat. Oncol.
Biol. Phys. 1992, 22 (4), 631-637.
(15) Stiff, D. D.; Zemaitis, M. A.; Metabolism of the anticonvulsant
agent zonisamide in the rat. Drug Metab. Dispos. 1990, 18 (6),
888-894.
(16) Stiff, D. D.; Robicheau, J . T.; Zemaitis, M. A. Reductive metabo-
lism of the anticonvulsant agent zonisamide, a 1,2-benzisoxazole
derivative. Xenobiotica 1992, 22 (1), 1-11.
(17) Nakasa, H.; Komiya, M.; Ohmori, S.; Rikihisa, T.; Kiuchi, M.;
Kitada, M. Characterization of human liver microsomal cyto-
chrome P450 involved in the reductive metabolism of zonisa-
mide. Mol. Pharmacol. 1993, 44, 216-221.
(18) Nakasa, H.; Komiya, M.; Ohmori, S.; Rikisha, T.; Kitada, M. Rat
liver microsomal cytochrome P450 responsible for reductive
metabolism of zonisamide. Drug Metab. Dispos. 1993, 21 (5),
777-781.
(19) Nakasa, H.; Ohmori, S.; Kitada, M. Formation of 2-sulphamoyl-
acetylphenol from zonisamide under aerobic conditions in rat
liver microsomes. Xenobiotica 1996, 26 (5), 495-501.
(20) Nakasa, H.; Nakamura, H.; Ono, S.; Tsutsui, M.; Kiuchi, M.;
Ohmori, S.; Kitada, M. Prediction of drug-drug interactions of
zonisamide metabolism in humans from in vitro data. Eur. J .
Clin. Pharmacol. 1998, 54, 177-183.
(21) Sugihara, K.; Kitamura, S.; Tatsumi, K. Involvement of mam-
malian liver cytosols and aldehyde oxidase in reductive metabo-
lism of zonisamide. Drug Metab. Dispos. 1996, 24 (2), 199-202.
(22) Sladek, N. E. Metabolism of oxazaphosphorines. Pharm. Ther.
1988, 37, 301-355
(23) Ludeman, S. M. The chemistry of the metabolites of cyclophos-
phamide. Curr. Pharm. Des. 1999, 5, 627-643.
supernatant obtained after protein precipitation, as described
above, was injected onto a reverse phase C-18 column (Zorbax
XDB-C18; 75 × 4.6 mm; 3.5 µm particle size) and eluted with
mobile phase consisting of two solvents, (A) 90:10 H2O/
methanol with 10 mM ammonium formate, (B) 10:90 H2O/
methanol with 10 mM ammonium formate. For the first 3 min,
isocratic elution at A/B 80:20 was used; thereafter, a linear
gradient for 15 min from A/B 80:20 to A/B 20:80 was used.
Mobile phase composition was returned to its original value
over the next 5 min and allowed to equilibrate for the next
analysis. The flow rate throughout the gradient was 0.8 mL/
min. For MS analysis, the HPLC effluent was introduced into
a Finnigan ion trap (LCQ) mass spectrometer. Electrospray
positive ionization in the full scan mode from 100 to 600 m/z
was used for detection. The heated capillary temperature was
maintained at 150 °C with a spray voltage at 4.5 kV.
For incubation mixtures of 15, analyses were carried out
by HPLC with UV detection at 254 nm. Chromatography was
performed by injecting a 25 µL sample volume on a reverse
phase C-18 column (Zorbax XDB-C18; 75 × 4.6 mm; 3.5 µm
particle size). Compounds 15 (tR ) 6.2 min) and 16 (tR ) 8.2
min) were separated at a flow rate of 1.0 mL/min with isocratic
elution of with a mobile phase composition of methanol/water
(50:50). The HPLC system consisted of a 501 HPLC pump
(Waters), a Lambda-Max Model 481 LC spectrophotometer
(Waters) detector, D-2500 (Hitachi) integrator, and Model U6K
(Waters) injector.
For inhibition experiments, 50 µL solutions of SKF-525A
in methanol or pCMBA in DMF were added to the microsomal
fraction to give the desired inhibitor concentration in 2 mL of
incubation mixture. The microsomal fractions were preincu-
bated for 30 min with pCMBA and then added with NADPH.
SKF-525A was incubated with microsomes and NADPH for
15 min. Thereafter, oxic samples were added with test
compound, and hypoxic samples were purged with nitrogen
prior to addition of test compound.
Incubations of 15 with P450 reductase microsomes were
carried out at
a protein concentration of 1 mg/mL and
substrate and cofactor concentrations of 0.25 and 0.5 mM,
respectively. Incubations were performed in an identical
fashion as above with rat liver fractions, except that the
duration of incubation at 37 °C was 60 min.
Deter m in a tion of Alk yla tin g Activity in th e In Vitr o
In cu ba tion Mixtu r es by NBP Assa y. Following incubation
with test compound, the rat liver fraction (2 mL) was added
to 2 mL of ice-cold methanol to precipitate proteins. The
mixture was centrifuged and 1 mL of supernatant was used
for alkylating activity test as described earlier.32
(24) Ludeman, S. M.; Boyd, V. L.; Regan, J . B.; Gallo, K. A.; Zon, G.;
Ishii, K. Synthesis and antitumor activity of cyclophosphamide
analogues. 4. Preparation, kinetic studies, and anticancer
screening of “phenylketophosphamide” and similar compounds
related to the cyclophosphamide metabolite aldophosphamide.
J . Med. Chem. 1986, 29, 716-727.
Su p p or tin g In for m a tion Ava ila ble: LC/MS spectra of
9 and its ketone metabolite.This material is available free of
charge via the Internet at http://pubs.acs.org.
(25) Mustafa, A.; Hsihmat, O. H.; Zayed, S. M. A. D.; Nawar, A. A.
Experiments with substituted (3,2-c)-pyranyl-2,10-diones and
benzopyranyl-(3,2-c)pyran-2,8-diones. Tetrahedron 1963, 19,
1831-1839.
(26) Gianella, M.; Gualtieri, F.; Melchiorre, C. Benzisoxazole and
benisothiazole analogues of auxin. Phytochemistry 1971, 10,
539-544.
(27) Uno, H.; Kurokawa, M.; Natsuka, K.; Yamato, Y.; Nishimura,
H. Studies on 3-substituted 1,2-benzisoxazole derivatives. I.
Chem. Pharm. Bull. 1976, 24 (4), 632-643.
(28) Uno, H.; Kurokawa, M. Studies on 3-substituted 1,2-benzisox-
azole derivatives V. Electrophilic substitution of 1,2-benzisox-
azole-3-acetic acid. Chem. Pharm. Bull. 1978, 26 (11), 3498-
3503.
(29) Villalobos, A.; Blake, J . F.; Biggers, C. K.; Butler, T. W.; Chapin,
D. S.; Chen, Y. L.; Ives, J . L.; J ones, S. B.; Liston, D. R.; Nagel,
A. A.; Nason, D. M.; Nielsen, J . A.; Shalaby, I. A.; White, W. F.
Novel benzisoxazole derivatives as potent and selective inhibitors
of acetylcholinesterase. J . Med. Chem. 1994, 37, 2721-2734.
(30) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J .;
Vistica, D.; Warren, J . T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
New colorimetric cytotoxicity assay for anticancer-drug screen-
ing. J . Natl. Cancer Inst. 1990, 82 (13), 1107-1112.
(31) J ain, M. Design, synthesis and evaluation of mechanistic
analogues of aldophosphamide as novel anticancer prodrugs.
Master’s Thesis, St. J ohn’s University, New York, 1994.
Refer en ces
(1) Brown, J . M. The hypoxic cell: A target for selective cancer
therapy-Eighteenth Bruce F. Cain Memorial Award Lecture.
Cancer Res. 1999, 59, 5863-5870.
(2) Brown, J . M.; Giaccia, A. J . The unique physiology of solid
tumors: Opportunities (and Problems) for cancer therapy.
Cancer Res. 1998, 58, 1408-1416.
(3) Oostween, E. A.; Speckamp, W. N. Mitomycin analogues I.
Indolequinones as (potential) bisalkylating compounds. Tetra-
hedron 1987, 43, 255-262.
(4) Naylor, M. A.; Swann, E.; Everett, S. A.; J affar, M.; Nolan, J .;
Robertson, N.; Lockyer, S. D.; Patel, K. B.; Dennis, M. F.;
Stratford, M. R. L.; Wardman, P.; Adams, G. E.; Moody, C. J .;
Stratford, I. J . Indolequinone antitumor agents: Reductive
activation and elimination from (5-methoxy-1-methyl-4,7-diox-
oindol-3-yl)methyl derivatives and hypoxia-selective cytotoxicity
in vitro. J . Med. Chem. 1998, 41, 2720-2731.
(5) Brenner, J . C. M. Assessing the bioreductive effectiveness of the
nitroimidazole RSU1069 and its prodrug RB6145: With par-
ticular reference to in vivo methods of evaluation. Cancer
Metastasis Rev. 1993, 12, 177-193.
(6) Tercel, M.; Wilson, W. R.; Anderson, R. F.; Denny, W. A.
Hypoxia-selective antitumor agents. 12. Nitrobenzyl quaternary
salts as bioreductive prodrugs of the alkylating agent mechlo-
rethamine. J . Med. Chem. 1996, 39, 1084-1094.

5436 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
J ain and Kwon
(32) Kwon, C.-H.; Moon, K.-Y.; Baturay, N.; Shirota, F. Chemically
stable, lipophilic prodrugs of phosphoramide mustard as poten-
tial anticancer agents. J . Med. Chem. 1991, 34 (2), 588-592.
(33) Keyes, S. R.; Francasso, P. M.; Heimbrook, D. C.; Rockwell S.;
Sliger, S. G.; Sartorelli, A. C. Role of NADPH: Cytochrome c
reductase and DT-diaphorase in the biotransformation of mito-
mycin C. Cancer Res. 1984, 44, 5638-5643.
N. P. The use of macroporous microcarriers for the large-scale
growth of V79 cells genetically designed to express single human
cytochrome P450 isoenzymes and for the characterization of the
expressed cytochrome P450. Protein Expr. Purif. 1996, 8 (4),
439-46.
(35) McGregor, D. B.; Edwards, I.; Wolf, C. R.; Forrester, L. M.;
Caspary, W. J . Endogenous xenobiotic enzyme levels in mam-
malian cells. Mutat. Res. 1991, 261 (1), 29-39.
(34) Onderwater, R. C.; Goeptar, A. R.; Levering, P. R.; Vos, R. M.;
Konings, P. N.; Doehmer, J .; Commandeur, J . N.; Vermeulen,
J M020581Y