Design, synthesis, and biological evaluation of cyclic and acyclic nitrobenzylphosphoramide mustards for E. coli nitroreductase activation

Article abstract of DOI:10.1021/jm051246n

In efforts to obtain anticancer prodrugs for antibody-directed or gene-directed enzyme prodrug therapy using E. coli nitroreductase, a series of nitrobenzylphosphoramide mustards were designed and synthesized incorporating a strategically placed nitro gro

Full text of DOI:10.1021/jm051246n

J. Med. Chem. 2006, 49, 4333-4343
4333
Design, Synthesis, and Biological Evaluation of Cyclic and Acyclic Nitrobenzylphosphoramide
Mustards for E. coli Nitroreductase Activation
Yongying Jiang,†,| Jiye Han,† Chengzhi Yu,†, Simon O. Vass,‡ Peter F. Searle,‡ Patrick Browne,§ Richard J. Knox,§ and
Longqin Hu*,†,∞
Department of Pharmaceutical Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State UniVersity of New Jersey,
160 Frelinghuysen Road, Piscataway, New Jersey 08854, The Cancer Institute of New Jersey, New Brunswick, New Jersey 08901,
UniVersity of Birmingham, Edgbaston, Birmingham B15 2TT, U.K., and MorVus Technology Ltd., Porton Down Science Park,
Salisbury, Wiltshire SP4 0JQ, U.K.
ReceiVed December 14, 2005
In efforts to obtain anticancer prodrugs for antibody-directed or gene-directed enzyme prodrug therapy using
E. coli nitroreductase, a series of nitrobenzylphosphoramide mustards were designed and synthesized
incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive
activation. All analogues were good substrates of E. coli nitroreductase with half-lives between 2.9 and
11.9 min at pH 7.0 and 37 °C. Isomers of the 4-nitrophenylcyclophosphamide analogues 3 and 5 with a
benzylic oxygen para to the nitro group showed potent selective cytotoxicity in nitroreductase (NTR)
expressing cells, while analogues 4 and 6 with a benzylic nitrogen para to the nitro group showed little
selective cytotoxicity despite their good substrate activity. These results suggest that good substrate activity
and the benzylic oxygen are both required for reductive activation of 4-nitrophenylcyclophosphamide
analogues by E. coli nitroreductase. Isomers of analogue 3 showed 23000-29000× selective cytotoxicity
toward NTR-expressing V79 cells with an IC50 as low as 27 nM. They are about as active as and 3-4×
more selective than 5-aziridinyl-2,4-dinitrobenzamide (CB1954). The acyclic 4-nitrobenzylphosphoramide
mustard ((()-7) was found to be the most active and most selective compound for activation by NTR with
170000× selective cytotoxicity toward NTR-expressing V79 cells and an IC50 of 0.4 nM. Compound (()-7
also exhibited good bystander effect compared to 5-aziridinyl-2,4-dinitrobenzamide. The low IC50, high
selectivity, and good bystander effects of nitrobenzylphosphoramide mustards in NTR-expressing cells suggest
that they could be used in combination with E. coli nitroreductase in enzyme prodrug therapy.
Introduction
genetic fusion to a tumor-specific antibody or by enzyme gene
delivery systems. This is then followed by the administration
of a prodrug, which is selectively activated by the delivered
enzyme at tumor cells.
Most anticancer agents in clinical use are often associated
with debilitating side effects because of their lack of tumor
selectivity. The side effects limit the maximum dose that can
be given to effectively treat tumors. Recently, tumor-targeted
prodrug therapy has been extensively studied to increase the
selectivity of cytotoxic agents toward tumor cells.1-3 In this
strategy, the cytotoxic agent is given in its prodrug form, which
is safe to normal cells but is selectively activated by certain
biochemical mechanisms unique to tumor cells, resulting in
localized cytotoxicity in tumor tissues. The mechanisms ex-
plored for this purpose include hypoxic reduction in solid
tumors, activation by enzymes overexpressed in tumor tissues,
and targeting of antigens or receptors specifically expressed on
tumor cell surface.1,2 Furthermore, activating enzymes can also
be delivered to tumor cells through antibody and gene therapy
approaches called antibody-directed enzyme prodrug therapy
(ADEPT) or gene-directed enzyme prodrug therapy (GDEPT).
In ADEPT and GDEPT, an exogenous enzyme is delivered site-
specifically into tumor cells through chemical conjugation or
A number of enzyme/prodrug systems for ADEPT/GDEPT
are in development and have been reviewed.4 Among the en-
zymes under evaluation is the nfsB gene product of Escherichia
coli, an oxygen-insensitive flavin mononucleotide-containing
nitroreductase (NTR). This flavoprotein is capable of reducing
certain aromatic nitro groups to the corresponding hydroxyl-
amines in the presence of cofactor NADH or NADPH.5,6 This
reduction process represents a very large electronic change and
can provide an efficient electronic “switch” that can be exploited
to generate potent cytotoxins.7 Four classes of prodrugs for
activation by NTR have been described, including 5-aziridinyl-
2,4-dinitrobenzamide (CB1954), dinitrobenzamide mustards
(e.g., SN 23862), 4-nitrobenzylcarbamates, and nitroindolines.7-9
Of the four classes, the first two are considered most promising
when used in combination with NTR. 5-Aziridinyl-2,4-dinitro-
benzamide, currently under phase II clinical trial in conjunction
with the virally delivered NTR enzyme, has high selectivity
(>1000-fold) in cell lines transfected with NTR and potent and
long-lasting inhibition of nitroreductase-transfected tumors in
mice. The related mustard SN 23862 has similar selectivity and
good bystander effects in animal models.
* To whom correspondence should be addressed. Phone: 732-445-5291.
Fax: 732-445-6312. E-mail: LongHu@rutgers.edu.
† Rutgers, The State University of New Jersey.
| Current address: Department of Pharmaceutical Chemistry, School of
Pharmacy, University of California, 600 16th Avenue, San Francisco, CA
94143.
We were interested in converting cyclophosphamide (1) into
reductively activated prodrugs and previously reported analogues
of nitrobenzene-fused cyclophosphamide (2) for activation by
NTR.10 While these compounds showed good substrate activity
for NTR with half-lives between 7 and 24 min, only compound
Current address: TorreyPines Therapeutics Inc., 11085 N Torrey Pines
Road, La Jolla, CA 92037.
‡ University of Birmingham.
§ Morvus Technology Ltd.
∞ The Cancer Institute of New Jersey.
10.1021/jm051246n CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/10/2006

4334 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Jiang et al.
Chart 1. Development of 4-Nitrophenylphosphoramide Mustard
Analogues for Bioreductive Activation
electron-withdrawing group (Hammett σp electronic parameter
of 0.78) and reduces the oxidation potential of the phosphorinane
ring system toward hepatic cytochrome P-450 oxidation. The
electron-withdrawing nitro group is converted to an electron-
donating hydroxylamino group (σp ) -0.34) upon NTR
reduction. This large difference in electronic effect (∆σp ) 1.12)
has been shown to cause cleavage of the benzylic C-O bond
in 4-nitrobenzyl carbamates upon nitro reduction.7,8,27,28 It is
used here to effect the formation of highly cytotoxic phosphor-
amide mustard from compounds 3 and 7 as shown in Scheme
1. Upon nitro reduction by NTR, the resulting electron-donating
hydroxylamino group relays its electrons to the para position
and promotes the cleavage of benzylic C-O bond, leading to
the subsequent activation of the phosphoramide mustard portion
in 10 and 12. The intermediates 10 and 11 also possess
electrophilic centers that could potentially form cross-links with
functionally important macromolecules, providing yet an ad-
ditional mechanism for cytotoxicity. To confirm the requirement
of the presence of benzylic oxygen in 3 and 7 in fragmentation
and the subsequent activation of the phosphoramide mustard,
the corresponding regioisomer 4 and the dioxa and diaza
analogues 5 and 6 were also synthesized and evaluated for NTR
activation. Although the analogues 4 and 6 are not expected to
undergo cleavage upon NTR reduction to release the activated
phosphoramidate, they were also designed to test whether the
aromatic hydroxylamine contributes to cytotoxicity. It was
proposed that the bioactivation of 5-aziridinyl-2,4-dinitro-
benzamide upon NTR reduction went through further acylation
of the 5-(aziridinyl)-4-hydroxylamino-2-nitrobenzamide by co-
enzyme A, resulting in the formation of lethal DNA-DNA
interstrand cross-links in cells.29
2 and the dioxa analogue with an oxygen at the benzylic position
showed a modest >33- to 36-fold enhanced cytotoxicity toward
NTR-expressing cells. Our efforts then focused on a series of
related exocyclic cognates, 4-nitrophenyl-substituted cyclophos-
phamide analogues (3-6). Structure-activity relationship stud-
ies led to the conclusion that cytotoxicity was dependent on
not only good substrate activity toward NTR but also the
presence of a benzylic oxygen para to the nitro group. Compared
to the most active compound 2 in the nitrobenzene-fused
cyclophosphamide series, the exocyclic cognate 3 exhibited an
over 100-fold increase in cytotoxicity and nearly 1000-fold
increase in selectivity toward NTR-expressing cells. This
observation prompted us to synthesize the acyclic 4-nitro-
benzylphosphoramide mustard (7), which to our surprise turned
out to be the most active and most selective compound for
activation by NTR reported so far. Compound 7 showed
170000× selective cytotoxicity toward NTR-expressing V79
cells with an IC50 as low as 0.4 nM. Chart 1 shows the
development process of 4-nitrobenzylphosphoramide mustard
analogues for reductive activation by NTR enzyme. A com-
munication highlighting the selectivity and potency of the most
active compounds in each series appeared recently in this
journal.11 Here, we report in more detail the structure-activity
relationship studies of 4-nitrophenyl-substituted cyclophos-
phamide analogues and 4-nitrobenzylphosphoramide mustard
as well as their cytotoxic bystander effects in cell culture assays.
Chemistry. The synthesis of 3-5 all started from 4-nitro-
benzaldehyde (16). As shown in Scheme 2, the synthesis of
the dioxa analogue 5 of 4-(4-nitrophenyl)cyclophosphamide was
accomplished in four steps starting from 4-nitrobenzaldehyde.
Grignard reaction of aldehyde 16 with vinylmagnesium bromide
in THF at -78 to -50 °C gave the racemic allylic alcohol 17.
The low temperature was necessary to prevent the nitro group
from being affected under the Grignard reaction conditions.
Hydroboration of the allylic alcohol 17 followed by basic
hydrogen peroxide oxidation afforded the racemic diol 18, which
reacted with bis(2-chloroethyl)phosphoramidic dichloride to give
two chromatographically separable diastereomers of the target
compound: cis-(()-5 and trans-(()-5. The combined yield of
the two isomers varied with the base used in the cyclization
step; it increased to 68% from 14% when n-BuLi was used
instead of TEA. This is most likely due to the higher nucleo-
philicity of hydroxyl group upon deprotonation by n-BuLi.
Results and Discussion
Design Principle. Cyclophosphamide (1) is one of the most
successful anticancer agents developed over the past few
decades.12 Because of its activity against both cycling and
noncycling cells, it is one of the few anticancer agents effective
in the treatment of slow-growing solid tumors.13,14 Cyclophos-
phamide is an anticancer prodrug that has to be activated by
cytochrome P-450 enzyme in the liver to release the activated
phosphoramide mustard and acrolein.15-18 Phosphoramide mus-
tard is the ultimate alkylating species that cross-links interstrand
DNA.19-21 Acrolein is a byproduct that is responsible for
hemorrhagic cystitis, a life-threatening side effect associated with
cyclophosphamide.22
The interest in developing more selective cyclophosphamide-
type anticancer agents led to the development of phosphor-
amidate prodrugs incorporating a variety of specific activation
mechanisms, including acid-sensitive hexenopyranoside of aldo-
phosphoramide,23 hypoxia-selective nitroheterocyclic phos-
phoramides,24,25 and indolequinone phosphoramidates for DT-
diaphorase activation.26 Our efforts have focused on the design
of cyclophosphoramide analogues incorporating site-specific
activation mechanisms by strategically placing a nitro group
for bioreductive activation in order to move the site of activation
from liver into the tumor tissues. The nitro group is a strong
4-Nitrophenylcyclophosphamide diaza analogue 6 was syn-
thesized from the 1,3-diol intermediate 18 as shown in Scheme
3. Compound 18 was converted to the corresponding diazide
19 using a PPh3/DEAD-mediated Mitsunobu reaction.30 Hy-
drazoic acid (HN3) solution in benzene was prepared from NaN3
and sulfuric acid according to a literature procedure.31 The two
azido groups were reduced to amino groups by 1,3-propane-
dithiol to afford 1,3-diamine 20. Treatment with PPh3-H2O
failed to reduce the secondary azido group to the amino group.32
Compound 20 was cyclized with bis(2-chloroethyl)phosphor-
amidic dichloride in the presence of TEA to give the target
product 6 as a pair of diastereomers (cis-(()-6 and trans-(()-
6) in a combined yield of 62%.
For the synthesis of 6-(4-nitrophenyl)cyclophosphamide (3)
shown in Scheme 4, the Grignard reaction product allylic alcohol
17 was protected by the methoxymethyl (MOM) group prior to

Nitrobenzylphosphoramide Mustards
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4335
Scheme 1. Proposed Mechanism of Activation of Nitrobenzylphosphoramide Mustards upon Bioreduction.
Scheme 2. Synthesis of 4-Nitrophenylcyclophosphamide Dioxa Analogue 5a
a Reagents and conditions: (i) vinylmagnesium bromide, THF, -78 to -50 °C, 4 h, 95%; (ii) B2H6/THF, 0 °C, 20 h, followed by 3 N NaOH, 30% H2O2,
30 min, 82%; (iii) n-BuLi, bis(2-chloroethyl)phosphoramidic dichloride, THF, -78 °C, 6 h, cis-(()-5, 28%, trans-(()-5, 40%.
Scheme 3. Synthesis of 4-Nitrophenylcyclophosphamide Diaza Analogue 6a
a Reagents and conditions: (i) PPh3, DEAD, HN3, THF, room temp, 12 h, 73%; (ii) Et3N, 1,3-propanedithiol, MeOH, room temp, 36 h, 87%; (iii)
bis(2-chloroethyl)phosphoramidic dichloride, Et3N, EtOAc, room temp, 40 h, cis-(()-6, 28%, trans-(()-6, 34%.
Scheme 4. Synthesis of 6-(4-Nitrophenyl)cyclophosphamide 3a
a Reagents and conditions: (i) MOMCl/DIEA, 0 °C to room temp, 24 h, 95%; (ii) B2H6/THF, 0 °C, 5 h, then 3 N NaOH, 30% H2O2, 30 min, 78%; (iii)
MsCl/ Et3N, then NaN3, 91%; (iv) PPh3, THF-H2O, room temp, 72%; (v) CBB followed by HOAc, 76%; (vi) bis(2-chloroethyl)phosphoramidic dichloride,
Et3N, EtOAc, 48 h, cis-(()-3, 34%, trans-(()-3, 33%.
hydroboration to give compound 21. A two-step sequence of
activation and SN2 displacement converted the hydroxyl group
to azido. This was then followed by PPh3-mediated reduction
and deprotection to give the corresponding amino alcohol 22.
The deprotection of MOM was accomplished using a combina-
tion of catechol boron bromide (CBB) with acetic acid, a
modified procedure developed specifically to avoid the problem
of cyclization and formation of cyclic formylacetal encountered
using other common MOM-deprotection conditions.33 The
amino alcohol 22 was cyclized upon treatment with bis(2-
chloroethyl)phosphoramidic dichloride in the presence of TEA
to give the target product 3 as a pair of diastereomers (cis-
(()-3 and trans-(()-3) in a combined yield of 67%.
Scheme 5 outlined the synthesis of 4-(4-nitrophenyl)cyclo-
phosphamide (4). The primary hydroxyl group of 18 was
selectively protected with TBDPS at low temperature to give
the monoprotected compound 23. The secondary hydroxyl group
was converted to the azido group using the Mitsunobu reaction
conditions. Other conditions including (CF3SO2)2O/Pyr-NaN3,
MsCl/NEt3-NaN3, PPh3/DEAD-(PhO)2PON3,34 and DBU-
(PhO)2PON3 failed to give the desired product. This difficulty
could be attributed to facile elimination of the activated ester
intermediate under these conditions. Reduction of the azido
group in 24 using 1,3-propanedithiol gave the amino product
25.35 The TBDPS group was removed using TBAF to afford
26, which was cyclized with bis(2-chloroethyl)phosphoramidic
dichloride to give the desired product 4 as a pair of diastereomers
(cis-(()-4 and trans-(()-4) in a combined yield of 31%.
Because of the presence of two chiral centers, one at the
4-nitrophenyl-substituted carbon and the other at the phosphorus
atom, analogues 3-6 exist as a pair of diastereomers that are
referred to as the cis and trans (cis ) RS/SR; trans ) RR/SS) as
shown in Chart 2. The relative configurations of cis and trans
are defined on the basis of the relative orientations of the aryl
substituent and the oxygen atom of the PdO bond.36 Because
of the greater equatorial preference of the bulky aryl group, cis
isomers have both groups in equatorial positions and trans
isomers have the aryl group in the equatorial position and the
phosphoxide group in the axial position.37 Assignment of cis
and trans configurations was based on their chromatographic
behavior, 1H and 31P NMR chemical shifts, and infrared spectral
data (Table 1).37 Compared to its trans diastereomer, the cis

4336 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Scheme 5. Synthesis of 4-(4-Nitrophenyl)cyclophosphamide 4a
Jiang et al.
a Reagents and conditions: (i) t-BuPh2SiCl, imidazole, DMF, -30 to -20 °C, 1.2 h, 95%; (ii) PPh3, DEAD, HN3/PhH, THF, 0 °C to room temp, 5 h,
87%; (iii) Et3N, 1,3-propanedithiol, MeOH, room temp, 36 h, 70%, (iv) TBAF, THF, 1 h, 82%; (v) bis(2-chloroethyl)phosphoramidic dichloride, Et3N,
EtOAc, room temp, 48 h, cis-(()-4, 14%, trans-(()-4, 17%.
Chart 2. Diastereomers of Nitrophenyl-Substituted Cyclophosphamides
Table 1. Analytical Data of Diastereomeric
4-Nitrophenylcyclophosphamide Analogues
(Figure 1). 5-Aziridinyl-2,4-dinitrobenzamide, an excellent
substrate of E. coli nitroreductase currently in phase II clinical
trials,4,6 was used as a control in our experiments. All
NMR δ (ppm)
compounds were found to be good substrates of E. coli
nitroreductase with half-lives between 2.3 and 11.9 min, while
the control 5-aziridinyl-2,4-dinitrobenzamide had a half-life of
5 min under the same assay conditions. The trans isomers were
better substrates of E. coli nitroreductase than the corresponding
cis isomers as indicated by their relatively shorter half-lives,
suggesting that configuration might affect the substrate binding
to and/or the catalytic activity of E. coli nitroreductase.
Compound (()-7 turned out to be the best substrate with a half-
life of 2.3 min.
compd
Rf (TLC)a IR (νPdO, cm-1
)
1Hb
31P
X ) O
cis-(()-3
Y ) NH trans-(()-3
X ) NH cis-(()-4
0.45
0.10
0.45
0.12
0.26
0.20
0.50
0.45
1330
1325
1350
1330
1259
1246
1330
1325
4.74
4.81
5.45
5.63
5.62
5.79
4.65
4.74
9.6
14.2
11.2
14.6
3.5
7.4
12.9
17.1
Y ) O
X ) O
Y ) O
trans-(()-4
cis-(()-5
trans-(()-5
X ) NH cis-(()-6
Y ) NH trans-(()-6
a TLC (on silica gel) developing solvents: hexanes/EtOAc (2:1) for 3-5,
CHCl3/CH3OH (20:1) for 6. b The 1H chemical shift refers to the proton
on the carbon with the 4-nitrophenyl substituent.
isomer elutes more quickly on silica gel, has a higher PdO
stretching frequency, a more downfield C-5 proton signal,and
a more upfield phosphorus signal. The two isomers were easily
separated by flash column chromatography on silica gel. The
synthesis of 4-nitrobenzylphosphoramide mustard ((()-7) was
accomplished in a three-step, one-pot process as reported
earlier.11
Chemical Stability. All four 4-nitrophenylcyclophosphamide
analogues and 4-nitrobenzylphosphoramide mustard were in-
cubated in 50 mM phosphate buffer, pH 7.4, at 37 °C. The
stability of each compound was monitored by reversed-phase
HPLC analysis of the incubation mixtures. The analogues were
all stable under these conditions with no significant changes
over a period of 72 h (<10%).
Substrate Activity for E. coli Nitroreductase. Isomers of
3-6 and compound 7 were evaluated as substrates of E. coli
nitroreductase by incubating each compound (0.2 mM) in 10
mM phosphate buffer, pH 7.0, at 37 °C in the presence of 1
mM NADH as the cofactor. The reaction was initiated by the
addition of 1.8 µg of E. coli nitroreductase. Aliquots were
withdrawn at various time intervals, quenched with acetonitrile,
and stored frozen prior to HPLC analysis. The half-lives were
calculated on the basis of the disappearance of the substrates
Figure 1. Disappearance of substrates 3, 4, 6, 7, and CB1954 during
reduction by E. coli nitroreductase as monitored by HPLC. Each
substrate (0.2 mM) was incubated with 1.8 µg of E. coli nitroreductase
in 10 mM phosphate buffer, pH 7.0, in the presence of 1 mM of NADH
at 37 °C in a total volume of 250 µL.

Nitrobenzylphosphoramide Mustards
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4337
Table 2. E. coli Nitroreductase Activation of 4-Nitrophenyl-Substituted Cyclophosphamide Analogues in Chinese Hamster V79 Cells and Human
Ovarian Cancer Cells
SKOV3 cells,i
virally infected,
SKOV3 cells,i
stable clones,
V79 cells,i stable clones,
72 h of drug exposureb
V79 cells,i stable clones,
1 h of drug exposureb
18 h of drug exposureb
18 h of drug exposureb
c
c
c
c
c
c
c
c
NR assay
IC50
,
IC50
,
IC50
,
IC50
,
IC50
,
IC50
,
IC50
,
IC50,
compd
cis-(()-3
t
1/2 (min)a
NTR-
NTR+
ratiod
NTR-
NTR+
ratiod
>290
NTR-
NTR+
ratiod
NTR-
NTR+
ratiod
5.2
3.9
11.9
2.9
7.3
4.8
6.4
4.2
2.3
832
608
0.029
0.027
45.3
51.5
NDe
29,000
23,000
>2.2
>1.9
NDe
>100
>100
NDe
0.34
0.17
NDe
NDe
NDe
NDe
NDe
NDe
0.01
680
4.7
5
150
>930
570
>1000
>1000
>1000
505
2.1g
2.1g
530
>440
270
trans-(()-3
cis-(()-4
trans-(()-4
cis-(()-5
trans-(()-5
cis-(()-6
trans-(()-6
(()-7
>590
>1000
>1000
>1000
NDe
>200
>1.9
>4.5
NDe
>100
>100
NDe
NDe
56.8
>100
67
NDe
530
220
NDe
NDe
NDe
NDe
1.2
>1.9
>4.8
>230
200
NDe
NDe
210
NDe
NDe
4.3g
2.5g
NDe
NDe
0.16g
NDe
4.6
NDe
12
>2.1
170,000
NDe
NDe
NDe
NDe
NDe
NDe
NDe
NDe
NDe
NDe
48.3
0.0004
NDe
NDe
NDe
NDe
NDe
NDe
>100
>10,000
>1000
>1000
>1000
625
>830
>160f
>210
12f
>890
>5600
6.2f
4.7
5-aziridinyl-
2,4-dinitrobenzamide
5.0
254
3.1h
0.036
2.7h
7,100
1.1
>100
0.31
>320
>790
2.0g
>400
52f
4-hydroperoxy-
cyclophosphamide
a Half-lives of reduction by E. coli nitroreductase were determined using 0.2 mM of substrate in 10 mM phosphate buffer (pH 7.0) in the presence of 1
mM NADH at 37 °C in a total volume of 250 µL. The reaction was initiated by the addition 1.8 µg of E. coli nitroreductase. Aliquots were withdrawn and
analyzed by HPLC. b Cells were exposed to each test compound, and a standard cell viability assay was performed at the end of indicated incubation period.
Initially, the maximum concentration used was 100 µM in the case of V79 cells and 1000 µM in the case of SKOV3 cells. When necessary, assays were
repeated at higher drug concentrations to obtain accurate IC50 values. c IC50 values are the concentration in µM required to reduce cell number to 50% of
control after the cells were exposed to the drug for the indicated time. The standard errors of all assays were within 10% of the mean between replicates at
a given concentration and 10-23% for the fitted IC50 values. d Ratio of IC50 values (NTR-/NTR+) as an indication of activation by E. coli nitroreductase.
e Not determined. f SKOV3 cells for these experiments were infected at a lower infection ratio of 10 pfu/cell, while the others in the same column were
infected at a higher infection ratio of 100 pfu/cell. g SKOV3 cells stably expressing NTR (SKOV NTR), compared with nonexpressing cells (SKOV3).
h 4-Hydroperoxycyclophosphamide was tested using sealed plates and HEPES medium. i Cell lines: V79 (Chinese hamster fibroblast) and SKOV3 (human
ovarian carcinoma).
Antiproliferative Activity in Cell Culture. Compounds 3-7
were assayed for their cytotoxicity against cells expressing or
not expressing E. coli nitroreductase. Three pairs of cell lines
were used that were derived from V79 Chinese hamster cells
and the SKOV3 human ovarian cancer cells. The NTR+ V79
cells had been transfected with a bicistronic vector encoding
for the E. coli nitroreductase and puromycin resistance protein
as the selective marker, while the NTR- V79 cells were
transfected with vector only and were used as the controls. The
SKOV3 human ovarian cancer cells were made to express E.
coli nitroreductase in two ways. In some experiments, the
SKOV3 cells were infected with an E1, E3-deleted replication-
defective adenovirus vector vPS1233 expressing the wild type
E. coli nitroreductase from the CMV promoter at infection ratios
of 10 or 100 plaque forming units (pfu) per cell. Other
experiments utilized SKOV3 cells that had been stably trans-
duced with the retroviral vector rv.RD18.LNC-nr,38 which
confers resistance to neomycin/G418 and expresses nitroreduc-
tase from the CMV promoter. These SKOV NTR cells were
compared with the parental, untransduced SKOV3 cells. The
IC50 values and the ratios of IC50 (NTR-/NTR+) of 3-7 are
presented in Table 2.
It is clear from the data presented in Table 2 that analogues
with benzylic oxygen para to the nitro group were consistently
much more cytotoxic to NTR+ cells than to NTR- cells while
analogues with benzylic nitrogens were only marginally more
cytotoxic to NTR+ cells than to NTR- cells. In stable clones
of Chinese hamster V79 cells, cis-(()-3 and trans-(()-3 were
23000-29000× more cytotoxic toward NTR-expressing cells.
This level of selectivity is about 3-4× that of 5-aziridinyl-2,4-
dinitrobenzamide while IC50 values of cis-(()-3 and trans-(()-3
in NTR+ V79 cells were comparable to that of 5-aziridinyl-
2,4-dinitrobenzamide. In virally infected as well as in stably
transfected clones of SKOV3 cells, cis-(()-3, trans-(()-3, and
5-aziridinyl-2,4-dinitrobenzamide showed similar activity and
selectivity. The dioxa analogues cis-(()-5 and trans-(()-5
showed somewhat reduced activity and selectivity in stably
transfected clones of SKOV3 cells compared to cis-(()-3, trans-
(()-3, and 5-aziridinyl-2,4-dinitrobenzamide. Replacement of
the benzylic oxygen with a nitrogen as in cis-(()-4, trans-(()-
4, cis-(()-6, and trans-(()-6 nearly completely eliminated the
selectivity toward NTR+ cells, suggesting that these analogues
were not activated even though they were reduced by NTR as
indicated in our enzyme assays. This apparently relates to the
ability of -NH-PO(NR2)(OR′) or -NH-PO(NR2)(NHR′) as
a much poorer leaving group than that of -O-PO(NR2)(NHR′)
and -O-PO(NR2)(OR′). Without cleavage of the benzylic C-N
bond and the negatively charged phosphate oxy anion under
near-physiological pH, these analogues were not cytotoxic.
These results indicate that nitroreductase reduction is an
important first step and the conversion of the nitro to hydroxyl-
amino group is not sufficient for enhanced cytotoxicity in
nitroreductase-expressing cells. This is consistent with the
mechanism proposed in Scheme 1 where cleavage of the
benzylic C-O bond upon reduction is required for the activation
of phosphoramide mustard.
The acyclic analogue, 4-nitrobenzylphosphoramide mustard
((()-7), was originally synthesized as a control compound to
explore the mechanism of activation of cyclic phosphoramide
analogues and has turned out to be the most active compound
in all our assays. The selectivity of compound (()-7 was a
remarkable 170000× in NTR+ V79 cells with an IC50 as low
as 0.4 nM. Thus, compound (()-7 was about 100× more active
and over 20× more selective toward NTR+ V79 cells than
5-aziridinyl-2,4-dinitrobenzamide. 4-Hydroperoxycyclophos-
phamide, a preactivated cyclophosphamide analogue, was used
as a control to exclude the possibility that V79 cells expressing
E. coli nitroreductase was sensitized to phosphoramide mustard-
type alkylating agents. Because it was known that 4-hydro-
peroxycyclophosphamide releases a volatile metabolite, the
control experiment was performed using sealed plates as
suggested in the literature.39 The results of the control experi-
ment shown in Table 2 clearly indicate that expression of E.
coli nitroreductase has no effect on the antiproliferative activity

4338 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Jiang et al.
in V79 cells of the active metabolites released from the
preactivated cyclophosphamide precursor. The extremely low
IC50 for compound (()-7 in NTR+ V79 cells suggests that
intracellular activation and release of cytotoxic phosphoramide
mustard are apparently more effective. Even for the 1 h drug
exposure that was used to better mimic the in vivo situation,
compound (()-7 still showed an IC50 of 10 nM, about 30×
lower than that of 5-aziridinyl-2,4-dinitrobenzamide, suggesting
that compound (()-7 was quickly activated and had long lasting
cytotoxic effects in E. coli nitroreductase-expressing Chinese
hamster V79 cells. The fast activation kinetics and the lasting
cytotoxic effects upon activation could potentially overcome
an important hurdle known in the development of the enzyme-
prodrug therapy using E. coli nitroreductase.4
icity can be quantitated in terms of the percentage of activator
(NTR+) cells in a mixed population of NTR+ and NTR- cells
to produce an IC50 midway between those in either NTR+ or
NTR- cell type alone.42 In this assay, compound (()-7 showed
the best bystander effect with a TE50 value of 3.3%, slightly
better than that of 5-aziridinyl-2,4-dinitrobenzamide (TE50
)
4.5%), while cyclic analogues, cis-(()-3, trans-(()-3, cis-(()-
5, and trans-(()-5, exhibited poor bystander effects (data not
shown). The excellent bystander effect and selectivity suggest
that compound (()-7 is a better drug candidate than 5-aziridinyl-
2,4-dinitrobenzamide for use in combination with nitroreductase
in ADEPT or GDEPT.
Conclusions
In clinical use, NTR is delivered using a viral vector.
Therefore, we confirmed the level of cell sensitization to
different prodrugs that could be achieved, using a replication-
defective adenovirus to express NTR in the SKOV3 ovarian
cancer cells. When SKOV3 cells were infected with 100 pfu/
cell of adenovirus expressing NTR, compound (()-7 showed
an IC50 that is about 4× lower than the IC50 of 5-aziridinyl-
2,4-dinitrobenzamide and compound 3 isomers. When the level
of NTR expression was made more limiting by infection with
just 10 pfu/cell of the adenovirus, although both IC50 values
were higher, the differential increased such that the IC50 for
compound (()-7 was about 9× lower than that for 5-aziridinyl-
2,4-dinitrobenzamide. In stably transduced SKOV3 cells, com-
pound (()-7 had a submicromolar IC50 that is 6× lower than
that for 5-aziridinyl-2,4-dinitrobenzamide. The higher IC50
values of all prodrugs in the SKOV3 cells compared to those
in V79 cells could theoretically be due in part to differences in
the level of NTR expression. Another contributory factor could
be a greater DNA repair capability, or resistance to DNA
damage-induced apoptosis, in the human ovarian tumor cells.
In summary, we have developed a novel and superior class
of nitroaryl phosphoramides as potential prodrugs for nitro-
reductase-mediated enzyme-prodrug therapy. All analogues
were stable in phosphate buffer at pH 7.4 and 37 °C and were
all good substrates of E. coli nitroreductase with half-lives
between 2.9 and 11.9 min, but only the analogues with a
benzylic oxygen para to the nitro group showed significant
selective cytotoxicity in NTR-expressing cells. These results
suggest that the good substrate activity and the benzylic oxygen
are required for reductive activation of 4-nitrobenzylphosphor-
amide mustard analogues by E. coli nitroreductase. The low
IC50 and the high selectivity of cis-(()-3, trans-(()-3, cis-(()-
5, trans-(()-5, and (()-7 in E. coli nitroreductase-expressing
cells indicated their potential to become a drug candidate in
enzyme-prodrug therapy. These nitrobenzylphosphoramide
mustards have low cytotoxicity before reduction and are
converted to phosphoramide mustard or like reactive species
upon bioreduction. In addition, compound (()-7 exhibited
excellent bystander effects with a TE50 of 3.3% compared to
4.5% for 5-aziridinyl-2,4-dinitrobenzamide. The excellent bio-
logical activity of these compounds correlates well with their
substrate activity for E. coli nitroreductase and is consistent with
the expected high cytotoxicity of the reactive species released
upon reduction. Work is in progress in our laboratories to further
evaluate the biological activity of these analogues as potential
prodrugs for nitroreductase activation.
Furthermore, compound (()-7 was confirmed to be a much
better substrate for NTR than 5-aziridinyl-2,4-dinitrobenzamide
in enzyme kinetic assays.11 Compound (()-7 has a Km of 195
( 14 µM and a kcat of 14.03 ( 0.35 s-1, while 5-aziridinyl-
2,4-dinitrobenzamide has a Km of 881 ( 42 µM and kcat of 6.60
( 0.11 s-1. Since 5-aziridinyl-2,4-dinitrobenzamide is reduced
to a 1:1 mixture of 2-hydroxylamino and 4-hydroxylamino
products by NTR and the 4-hydroxylamino is known to be the
Experimental Section
1
major cytotoxic product,40,41 the productive /2kcat of 3.30 s-1
General Methods. Moisture-sensitive reactions were performed
in flame-dried glassware under a positive pressure of nitrogen or
argon. Air- and moisture-sensitive materials were transferred by
syringe or cannula under an argon atmosphere. Except for redistil-
lation prior to use, solvents were either ACS reagent grade or HPLC
grade. Tetrahydrofuran was dried over sodium/benzophenone.
Triethylamine, dichloromethane, and ethyl acetate were dried over
calcium hydride. Methanol was distilled over sodium methoxide.
Pyridine was dried over potassium hydroxide and distilled over
calcium hydride. N,N-Dimethylformamide was dried over 4 Å
molecular sieves at least for 1 week prior to use. Unless otherwise
stated, all reactions were magnetically stirred and monitored by
thin-layer chromatography (TLC) using 0.25 mm Whatman pre-
coated silica gel plates. TLC plates were visualized using either
7% (w/w) ethanolic phosphomolybdic acid or 1% (w/w) aqueous
potassium permanganate containing 1% (w/w) NaHCO3. Flash
column chromatography was performed using silica gel (Merck
230-400 mesh). Yield refers to chromatographically and spectro-
scopically (1H NMR) homogeneous material, unless otherwise
noted. All reagents were purchased at the commercial quality and
used without further purification. n-BuLi was used freshly as
purchased and was not titrated.
is used for comparison purposes. This gave a specificity constant
of 71 900 M-1 s-1 (kcat/Km) for compound (()-7 and 3750 M-1
s-1 (1/2kcat/Km) for 5-aziridinyl-2,4-dinitrobenzamide. Thus,
compound (()-7 is 19 times better as a substrate of NTR than
5-aziridinyl-2,4-dinitrobenzamide. This at least partially con-
tributed to the better activity and superior selectivity in cell
culture assays of compound (()-7 in comparison with 5-aziri-
dinyl-2,4-dinitrobenzamide.
It should also be noted that compound (()-7 upon reductive
activation releases phosphoramide mustard, which is the active
metabolite of the clinical drug cylcophosphamide.15-17 The
excellent activity of compound (()-7 in nitroreductase-express-
ing cells was unexpected considering the fact that only a 2-fold
increase in cytotoxicity was observed for 4-nitrobenzyl N,N,N′,N′-
tetrakis(2-chloroethyl)phosporodiamidate toward cancer cells
under hypoxic conditions.25 This suggests that either these
compounds were poor substrates of the human reductase(s)
present or the expression of these reductase(s) was limited under
the hypoxic assay conditions used. For cancer treatment, the
extent of bystander effect will be critical to the success of any
gene-directed enzyme prodrug therapy. The bystander cytotox-
Melting points were determined on a Mel-Temp capillary
apparatus and are uncorrected. Infrared spectra were recorded with

Nitrobenzylphosphoramide Mustards
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4339
Perkin-Elmer model 1600 series FTIR spectrometer using poly-
styrene as an external standard. Infrared absorbance is reported in
reciprocal centimeters (cm-1) with broad signals denoted by br.
1H NMR spectra were recorded on Varian Gemini 200, 300, or
400 MHz spectrometers as indicated at ambient temperature and
calibrated using residual undeuterated solvents as the internal
reference. 13C NMR spectra were recorded at 50 MHz on a Varian
Gemini 200 MHz spectrometer or 75 MHz on a Varian Gemini
300 MHz spectrometer. 31P NMR spectra were recorded at 121
MHz on a Varian Gemini 300 MHz spectrometer or 162 MHz on
a Varian Gemini 400 MHz spectrometer using 5% H3PO4 in D2O
as an external standard. Chemical shifts are reported in parts per
million (δ) relative to CDCl3 (δ 7.27 ppm for 1H and 77.2 ppm for
washed with saturated NaCl, dried over Na2SO4, and evaporated
to dryness. The crude product was purified by flash column
chromatography (hexanes/ethyl acetate, 2:1 to 1:2) to afford the
desired product as two diastereomers.
cis-(()-5: yellow oil (100 mg, 28%); 1H NMR (200 MHz,
CDCl3) δ 8.26 (d, J ) 8.4 Hz, 2H), 7.68 (d, J ) 8.4 Hz, 2H),
5.68-5.57 (m, 1H), 4.57-4.46 (m, 2H), 3.70-3.63 (m, 4H), 3.53-
3.40 (m, 4H), 2.62-2.2.51 (m, 1H), 2.29-2.18 (m, 1H); 13C NMR
(50 MHz, CDCl3) δ 148.1, 146.5 (d, J ) 4.2 Hz), 126.9, 124.1,
80.1 (d, J ) 6.5 Hz), 66.6 (d, J ) 5.7 Hz), 49.2 (d, J ) 4.6 Hz),
41.9, 33.3 (d, J ) 12.6 Hz); 31P NMR (162 MHz, CDCl3) δ 3.5
(s); IR (film) 1607.0, 1521.5, 1348.4, 1258.6, 1081.2 cm-1; MS
(FAB, NBA) m/z (relative intensity) 383.0 (MH+, 3.6), 385.0 (MH+
+ 2, 1.6); HRMS (FAB) m/z calcd for C13H18N2O5PCl2 (MH+)
383.0330, found 383.0293.
1
13C) or CD3OD (δ 3.31 ppm for H and 49.2 for 13C). Coupling
constants (J values) are given in hertz (Hz). The following
abbreviations were used to explain the multiplicities: s ) singlet;
d ) doublet; t ) triplet; q ) quartet; m ) multiplet; br ) broad.
High-resolution mass spectral (HRMS) data were obtained from
the University of Kansas Mass Spectrometry Laboratory (Lawrence,
KS). HPLC analysis was performed on an HP 1090 system equipped
with a Phenomenex C18 column (5 µm, 4.6 mm × 250 mm) with
gradient elution of 5-80% CH3CN containing 0.1% TFA in 15
min at a flow rate of 1 mL/min and a detection wavelength at 220
nm.
trans-(()-5: yellow solid (145 mg, 40%); 1H NMR (300 MHz,
CDCl3) δ 8.28 (d, J ) 8.4 Hz, 2H), 7.53 (d, J ) 8.4 Hz, 2H), 5.79
(dd, J ) 10.9, 1.4 Hz, 1H), 4.84-4.71 (m, 1H), 4.54-4.33 (m,
1H), 3.76-3.46 (m, 8H), 2.28-1.95 (m, 2H); 13C NMR (50 MHz,
CDCl3) δ 148.1, 146.2 (d, J ) 9.5 Hz), 126.3, 124.2, 77.9 (d, J )
5.0 Hz), 66.3 (d, J ) 5.7 Hz), 49.3 (d, J ) 5.0 Hz), 42.0, 33.8 (d,
J ) 4.2 Hz); 31P NMR (162 MHz, CDCl3) δ 7.4 (s); IR (KBr)
1606.8, 1522.1, 1348.6, 1246.0, 1102.6, 1057.0 cm-1; MS (FAB,
NBA) m/z (relative intensity) 382.9 (MH+, 2.4), 385.0 (MH++2,
0.7); HRMS (FAB) m/z calcd for C13H18N2O5PCl2 (MH+) 383.0330,
found 383.0325.
1-(4-Nitrophenyl)prop-2-en-1-ol (17). To a solution of 4-nitro-
benzaldehyde (855 mg, 5.66 mmol) in freshly distilled THF (20
mL) was added dropwise vinylmagnesium bromide solution (1 M
in THF, 6.8 mL) at -78 °C. The mixture was stirred at -50 °C
for 40 min, and the reaction was then quenched by saturated
aqueous ammonium chloride (10 mL). After the addition of ethyl
acetate (100 mL), the organic phase was washed with saturated
NaCl, dried over Na2SO4, and evaporated in vacuo. The crude
product was purified by flash column chromatography (petroleum
ether/ethyl acetate, 6:1 to 4:1) to afford the desired product 17 as
a yellow solid (968 mg, 95%): mp (EtOAc) 54-55.5 °C; 1H NMR
(300 MHz, CDCl3) δ 8.15 (d, J ) 8.1 Hz, 2H), 7.51 (d, J ) 8.2
Hz, 2H), 6.02-5.90 (m, 1H), 5.40-5.20 (m, 3H), 2.80 (br, s, 1H);
IR (KBr) 3300 (br), 1580, 1500, 1330, 1250, 1030, 920, 840, 730
1-(4-Nitrophenyl)propane-1,3-diazide (19). Hydrazoic acid
solution (1.2 M in benzene) was prepared according to the reported
procedure.31 (Warning! Hydrazoic acid is a highly toxic and volatile
compound. The following operations must be carried out under a
well-ventilated hood.) To a solution of 1-(4-nitrophenyl)propane-
1,3-diol (709 mg, 3.6 mmol) and triphenylphosphine (2.83 g, 10.8
mmol) in anhydrous THF (50 mL) was added, at room temperature,
a hydrazoic acid solution (1.2 M benzene solution, 7.7 mL) and
then a solution of diethyl azodicarboxylate (1.68 mL, 10.8 mmol)
in THF (5 mL). The reaction solution was stirred at room
temperature for 12 h and poured into ethyl acetate (100 mL) and
brine (30 mL). The organic phase was washed with saturated
NaHCO3 and brine, dried over Na2SO4, and evaporated in vacuo.
The residue was purified by flash column chromatography (petro-
leum ether/ethyl acetate, 5:1) to give the desired product 19 as an
oil (653 mg, 73%): 1H NMR (300 MHz, CDCl3) δ 8.27 (d, J )
8.1 Hz, 2H), 7.52 (d, J ) 8.4 Hz, 2H), 4.81-4.70 (m, 1H), 3.57-
3.42 (m, 1H), 2.05-1.82 (m, 2H); IR (film) 2980, 2080, 1720, 1510,
cm-1 13C NMR (50 MHz, CDCl3) δ 149.9, 147.4, 139.3, 127.1,
;
123.8, 116.8, 74.6; MS (FAB, NBA) m/z (relative intensity) 180.1
(M + 1, 18.9), 162.0 (M - OH, 18.8); HRMS (FAB) m/z calcd
for C9H10NO3 (MH+) 180.0661, found 180.0670.
1-(4-Nitrophenyl)propane-1,3-diol (18). To a solution of 1-(4-
nitrophenyl)prop-2-en-1-ol (3.1 g, 17.3 mmol) in freshly distilled
THF (150 mL) at 0 °C was added dropwise a solution of borane in
THF (1 M, 18 mL). After the reaction solution was stirred at 0 °C
overnight, 3 N NaOH (19 mL) was added, followed by the dropwise
addition of 30% hydrogen peroxide (19 mL). The turbid solution
was stirred at 0 °C for 30 min, and the remaining hydrogen peroxide
was destroyed by the addition of a solution of sodium bisulfite.
Ethyl acetate (150 mL) was then added, and the organic phase was
washed with saturated aqueous NaHCO3 and saturated NaCl, dried
over Na2SO4, and evaporated in vacuo. The crude product was
purified by flash column chromatography (petroleum ether/ethyl
acetate, 4:1 to 1:2) to afford the desired product 18 as a yellow oil
(2.8 g, 82%): 1H NMR (300 MHz, CDCl3) δ 8.20 (d, J ) 8 Hz,
2H), 7.55 (d, J ) 8 Hz, 2H), 5.10 (t, J ) 7 Hz, 1H), 3.90 (m, 2H),
3.65 (br s, 1H), 2.40 (br, s, 1H), 1.96 (m, 2H); 13C NMR (50 MHz,
CDCl3) δ 152.0, 147.3, 126.5, 123.8, 73.1, 61.1, 40.4; IR (KBr)
3400 (br), 1500, 1320 cm-1; MS (FAB, NBA) m/z (relative
intensity) 198.1 (MH+, 11.0), 180.1 (MH+ - OH, 13.6); HRMS
(FAB) m/z calcd for C9H12NO4 (MH+) 198.0766, found 198.0788.
2-[Bis(2-chloroethyl)amino]-4-(4-nitrophenyl)-2H-1,3,2-dioxa-
phosphorinane 2-Oxide (5). To a stirred solution of 1-(4-
nitrophenyl)propane-1,3-diol (186 mg, 0.944 mmol) in anhydrous
THF (100 mL) at -78 °C under argon was added a solution of
n-BuLi in hexane (2.5 M, 760 µL). After 15 min, a solution of
bis(2-chloroethyl)phosphoramidic dichloride (252 mg, 0.944 mmol)
in anhydrous THF (20 mL) was added. The reaction mixture was
stirred at <-70 °C for 6 h and then at room temperature for 18 h.
After removal of the white precipitate by filtration, the filtrate was
1470, 1425, 1335, 1220, 1170, 1110, 1050, 980, 700, 680 cm-1
;
MS (FAB, 3NBA) m/z (relative intensity) 248.1 (MH+, 7.9), 219.2
(M - 28, 30.8), 177.1 (31.5).
1-(4-Nitrophenyl)propane-1,3-diamine (20). To a solution of
1-(4-nitrophenyl)propane-1,3-diazide (625 mg, 2.5 mmol) in an-
hydrous methanol was added propane-1,3-dithiol (1.0 mL, 10.1
mmol) and triethylamine (1.4 mL, 10.1 mmol). The reaction solution
was stirred at room temperature for 36 h and filtered to remove
the precipitate. The filtrate was evaporated in vacuo, and the residue
was purified by flash column chromatography (chloroform/
methanol, 5:1 to 2:1, the chloroform was saturated with ammonium
hydroxide) to give the desired product 20 as a yellow oil (428 mg,
87%): 1H NMR (300 MHz, CDCl3) δ 8.21 (d, J ) 9.0 Hz, 2H),
7.51 (d, J ) 9.0 Hz, 2H), 4.19 (t, J ) 6.8, 1H), 2.75 (t, J ) 6.75
Hz, 2H), 1.81-1.76 (m, 2H), 1.40 (br s, 4H); 13C NMR (50 MHz,
CDCl3, 5% CD3OD) δ 153.3, 147.2, 127.2, 124.0, 53.9, 41.4, 38.8;
IR (film) 3300, 2950, 1590, 1500, 1500, 1330, 840 cm-1; MS (FAB,
3NBA) m/z (relative intensity) 196.1 (MH+, 72.8), 165.1 (M -
30, 8.4), 151.1 (14.2); HRMS (FAB) m/z calcd for C9H14N3O2
(MH+) 196.1086, found 196.1124.
2-[Bis(2-chloroethyl)amino]-4-(4-nitrophenyl)-2H-1,3,2-diaza-
phosphorinane 2-Oxide (6). To a solution of 1-(4-nitrophenyl)-
propane-1,3-diamine (50 mg, 0.26 mmol) in anhydrous ethyl acetate
(40 mL) at 0 °C was added a solution of bis(2-chloroethyl)-
phosphoramidic dichloride (77.7 mg, 0.3 mmol) and triethylamine
(86 µL, 0.6 mmol). The reaction solution was stirred at room
temperature for 48 h and filtered to remove the precipitate. The

4340 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Jiang et al.
filtrate was washed with saturated NaCl, dried over Na2SO4, and
evaporated in vacuo. The residue was purified by flash column
chromatography (chloroform/methanol, 30:1 to 24:1) to give the
desired product as two diastereoisomers.
MS (FAB, 3NBA) m/z (relative intensity) 242.1 (MH+, 19.6), 210.1
(M - 31, 25.8), 224.1 (MH+ - 18, 5.6); HRMS (FAB) m/z calcd
for C11H16NO5 (MH+) 242.1028, found 242.1030.
3-Amino-1-(4-nitrophenyl)propan-1-ol (22). To a stirred solu-
tion of 3-methoxymethoxy-3-(4-nitrophenyl)propane-1-ol (128 mg,
0.53 mmol) in dry dichloromethane (10 mL) at 0 °C was added
TEA (0.22 mL, 1.59 mmol) and methane sulfonyl chloride (80 µL,
1.06 mmol). After being stirred for 15 min, the reaction solution
was diluted with ethyl ether (100 mL). The organic solution was
washed with saturated aqueous NaHCO3, saturated NaCl, dried over
Na2SO4, and evaporated in vacuo. The crude product was dissolved
in dry DMF (10 mL) and was treated with NaN3 (207 mg, 3.18
mmol) and 15-crown-5 (cat.). The reaction solution was stirred at
room temperature for 4.5 h and partitioned between ethyl ether and
water. The organic phase was washed with saturated aqueous
NaHCO3 and saturated NaCl, dried over Na2SO4, and evaporated
in vacuo. The crude product was purified by flash column
chromatography (petroleum ether/ethyl acetate, 4:1 to 3:1) to afford
3-methoxymethoxy-3-(4-nitrophenyl)-1-propyl azide as a yellow oil
(129 mg, 91%): 1H NMR (300 MHz, CDCl3) δ 8.22 (dd, J ) 1.8,
6.8 Hz, 2H), 7.52 (dd, J ) 0.3, 6.9 Hz, 2H), 4.83 (dd, J ) 4.5, 9.0
Hz, 1H), 4.60 (d, J ) 6.9 Hz, 1H), 4.51 (dd, J ) 1.2, 6.8 Hz, 1H),
3.51-3.40 (m, 2H), 3.37 (s, 3H), 2.07-2.02 (m, 1H), 1.93-1.89
(m, 1H); 13C NMR (50 MHz, CDCl3) δ 149.1, 147.8, 127.5, 124.0,
95.0, 74.5, 56.0, 47.8, 37.1; IR (film) 2955, 2070, 1580, 1500, 1330,
1135, 1080, 1020 cm-1; MS (FAB, 3NBA) m/z (relative intensity)
267.2 (MH+, 4.8), 207.1 (3.6), 198.1 (5.6); HRMS (FAB) m/z calcd
for C11H15N4O4 (MH+) 267.1093, found 267.1082.
cis-(()-6: yellow solid (27 mg, 28%); mp (CHCl3-MeOH)
119-120 °C; 1H NMR (300 MHz, CDCl3) δ 8.22 (d, J ) 9.0 Hz,
2H), 7.69 (d, J ) 8.7 Hz, 2H), 4.70-4.60 (m, 1H), 3.69 (t, J ) 6.3
Hz, 4H), 3.60-3.20 (m, 8H), 2.15-2.00 (m, 1H), 1.90-1.80 (m
1H); 13C NMR (50 MHz, CDCl3, 5% CD3OD) δ 151.3 (d, J ) 3.8
Hz), 127.2, 123.9, 57.3 (d, J ) 2.3 Hz), 48.5 (d, J ) 4.6 Hz), 42.2
(d, J ) 1.6 Hz), 40.1, 34.8 (d, J ) 8.8 Hz); 31P NMR (121 MHz,
CDCl3) δ 12.9 (s); IR (KBr) 3140, 2940, 2900, 2830, 1580, 1495,
1440, 1330, 1190, 1155, 1100, 960, 890, 710 cm-1; MS (FAB,
3NBA) m/z (relative intensity) 381.1 (MH+, 72.4), 383.1 (MH+
+
2, 43.8), 385.1 (MH+ + 4, 9.4); HRMS (FAB+) m/z calcd for
C13H20N4O3PCl2 (MH+) 381.0650, found 381.0626; HRMS (FAB+)
m/z calcd for C13H20N4O3P35Cl37Cl (MH+ + 2) 383.0621, found
383.0630.
trans-(()-6: yellow solid (33 mg, 34%); mp (CHCl3-MeOH)
1
148.5-149.2 °C; H NMR (300 MHz, CDCl3) δ 8.24 (d, J ) 8.7
Hz, 2H), 7.54 (d, J ) 8.8 Hz, 2H), 4.78-4.70 (m, 1H), 3.70-3.50
(m, 12H), 2.10-1.75 (m, 2H), 1.90-1.80 (m 1H); 13C NMR (50
MHz, CDCl3) δ 151.1 (d, J ) 11.8 Hz), 147.6, 127.0, 124.2, 56.8,
48.0 (d, J ) 5.0 Hz), 42.5, 40.7, 34.8; 31P NMR (121 MHz, CDCl3)
δ 17.1 (s); IR (KBr) 3140, 2900, 1570, 1500, 1450, 1430, 1410,
1325, 1150, 1090, 980, 850, 720 cm-1; MS (FAB, 3NBA) m/z
(relative intensity) 381.1 (MH+, 10.1), 383.1 (MH+ + 2, 2.6), 385.1
(MH+ + 4, 1.0); HRMS (FAB) m/z calcd for C13H20N4O3PCl2
(MH+) 381.0650, found 381.0643; HRMS (FAB) m/z calcd for
C13H20N4O3P35Cl37Cl (MH+ + 2) 383.0621, found 383.0612.
3-Methoxymethoxy-3-(4-nitrophenyl)propan-1-ol (21). A solu-
tion of 1-(4-nitrophenyl)prop-2-en-1-ol (1.94 g, 10.8 mmol) in dry
dichloromethane (40 mL) at 0 °C was treated sequentially with
DIEA (11.33 mL, 64.8 mmol) and chloromethyl methyl ether (4.94
mL, 60.8 mmol). The reaction mixture was stirred at room
temperature for 24 h and quenched with 5% NaHCO3. Dichloro-
methane was evaporated in vacuo, and the aqueous solution was
extracted with ethyl ether (40 mL × 3). The extractions were
combined, washed with brine, dried over Na2SO4, and evaporated
in vacuo. The residue was purified by flash column chromatography
(hexanes/ethyl acetate, 8:1 to 6:1) to give 1-methoxymethoxy-1-
(4-nitrophenyl)-2-propene as a yellow oil (2.31 g, 95%): 1H NMR
(300 MHz, CDCl3) δ 8.10 (dd, J ) 1.8, 6.9 Hz, 2H), 7.54-7.51
(m, 2H), 5.90-5.78 (m, 1H), 5.39-5.27 (m, 2H), 5.18 (d, J ) 6.6.
Hz, 1H), 4.78 (d, J ) 4.8 Hz, 1H), 4.61 (d, J ) 5.7 Hz, 1H), 3.36
(s, 3H); 13C NMR (50 MHz, CDCl3) δ 148.3, 147.6, 137.1, 127.7,
123.8, 118.2, 94.0, 55.7; IR (film) 3020, 2920, 2880, 1580, 1500,
1330, 1130, 1080, 1020, 900, 835 cm-1; MS (FAB, 3NBA) m/z
(relative intensity) 224.1 (MH+, 22.4), 194.1 (MH+ - 30, 1.5),
To a solution of 3-methoxymethoxy-3-(4-nitrophenyl)-1-propyl
azide (4.13 g, 15.45 mmol) in THF (80 mL, 0.5% water) was added
triphenylphosphine (4.12 g, 15.45 mmol). The reaction solution was
stirred at room temperature for 24 h before being concentrated in
vacuo. The crude product was purified by flash column chroma-
tography to afford 3-methoxymethoxy-3-(4-nitrophenyl)-1-propyl-
amine as a yellow oil (2.69 g, 72%): 1H NMR (300 MHz, CDCl3)
δ 8.21 (dd, J ) 2.1, 6.9 Hz, 2H), 7.50 (d, J ) 8.7 Hz, 2H), 4.83
(dd, J ) 4.8, 8.4 Hz, 1H), 4.59 (d, J ) 6.9 Hz, 1H), 4.50 (dd, J )
0.3, 6.9 Hz, 1H), 3.37 (s, 3H), 2.83 (t, J ) 6.9 Hz, 2 H), 2.00-
1.93 (m, 1H), 1.82-1.75 (m, 1H), 1.31 (br s, 2H); IR (film) 2900,
1630, 1580, 1500, 1330, 1130, 1080, 1000, 900, 830, 680 cm-1
;
MS (FAB, 3NBA) m/z (relative intensity) 241.1 (MH+, 100.0),
225.1 (1.9), 209.1 (1.5); HRMS (FAB) m/z calcd for C11H17N2O4
(MH+) 241.1188, found 241.1182.
To a solution of 3-methoxymethoxy-3-(4-nitrophenyl)-1-propyl-
amine (1.0 g, 4.17 mmol) in dry dichloromethane (50 mL) at -50
°C was added a solution of B-bromocatecholborane in dichloro-
methane (0.245 N, 17 mL). The reaction mixture was allowed to
warm to -20 °C for 2 h and treated with glacial acetic acid (0.24
mL, 4.17 mmol). After the mixture was stirred at room temperature
for another 7 h, the reaction was quenched with 3 N NaOH (15
mL). The organic phase was separated, and the aqueous phase was
extracted with dichloromethane (30 mL × 3). The organic phases
were combined, washed with saturated NaCl, dried over Na2SO4,
and evaporated in vacuo. The crude product was purified by flash
column chromatography (chloroform/methanol, 9:1 to 8:1) to afford
the desired product 22 as a yellow solid (629 mg, 77%): mp
208.1 (MH+ - 15, 3.1), 192.1 (MH+ - 31, 1.3), 162.1 (MH+
-
OMOM, 77.5); HRMS (FAB) m/z calcd for C11H14NO4 (MH+)
224.0923, found 224.0924.
To a solution of 1-methoxymethoxy-1-(4-nitrophenyl)-2-propene
(742 mg, 3.3 mmol) in freshly distilled THF (15 mL) at 0 °C was
added dropwise a solution of borane in THF (1 M, 3.3 mL). After
the reaction solution was stirred at 0 °C overnight, 3 N NaOH (3.5
mL) was added, followed by the dropwise addition of 30%
hydrogen peroxide (3.5 mL). The turbid reaction mixture was stirred
at 0 °C for 30 min, and the remaining hydrogen peroxide was
destroyed by the addition of a solution of sodium bisulfite. Ethyl
acetate (100 mL) was added, and the organic phase was washed
with saturated aqueous NaHCO3 and saturated NaCl, dried over
Na2SO4, and evaporated in vacuo. The crude product was purified
by flash column chromatography (petroleum ether/ethyl acetate,
2:1 to 1:1) to afford the desired product 21 as a yellow oil (625
mg, 78%): 1H NMR (300 MHz, CDCl3) δ 8.21 (dd, J ) 1.8, 6.8
Hz, 2H), 7.53-7.50 (m, 2H), 4.95 (dd, J ) 4.5, 8.9 Hz, 1H), 4.62
(d, J ) 6.6 Hz, 1H), 4.52 (d, J ) 6.9 Hz, 1H), 3.83-3.77 (m, 1H),
3.75-3.71 (m, 1H), 3.38 (s, 3H), 2.21 (br s, 1H); 13C NMR (50
MHz, CDCl3) δ 149.5, 147.7, 127.4, 123.9, 95.1, 75.9, 59.7, 56.0,
1
(CH3Cl-MeOH) 126-127.5 °C; H NMR (300 MHz, CDCl3) δ
8.13 (dd, J ) 2.0, 6.9 Hz, 2H), 7.52-7.47 (m, 2H), 5.03 (dd, J )
2.7, 8.7 Hz, 1H), 3.12-3.06 (m, 1H), 3.07-2.92 (m, 1H), 1.99-
1.81 (m, 1H), 1.67-1.41 (m, 1H); 13C NMR (50 MHz, CDCl3) δ
152.9, 147.1, 126.5, 123.6, 75.2, 40.8, 39.1; IR (film) 3330, 3260,
3100, 2880, 2850, 1575, 1490, 1400, 1330, 1300, 1275, 1075, 1085,
1050, 1000, 935, 810, 730, 680 cm-1; MS (FAB, 3NBA) m/z
(relative intensity) 197.1 (MH+, 30.5), 181.0 (1.8); HRMS (FAB)
m/z calcd for C9H13N2O3 (MH+) 197.0926, found 197.0939.
2-[Bis(2-chloroethyl)amino]-6-(p-nitrophenyl)-2H-1,3,2-oxaza-
phosphorinane 2-Oxide (3). To a solution of 3-amino-1-(4-
nitrophenyl)-1-propanol (131 mg, 0.67 mmol) in anhydrous ethyl
acetate (20 mL) at 0 °C was added a solution of bis(2-chloroethyl)-
phosphoramidic dichloride (173 mg, 0.67 mmol) and triethylamine
(185 µL, 1.34 mmol) in ethyl acetate (5 mL). The reaction solution
40.4; IR (film) 3400, 2950, 1500, 1330, 1130, 1080, 1010 cm-1
;

Nitrobenzylphosphoramide Mustards
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4341
was stirred at room temperature for 48 h and filtered to remove
the precipitate. The filtrate was washed with saturated NaCl, dried
over Na2SO4, and evaporated in vacuo. The residue was purified
by flash column chromatography (chloroform/methanol, 30:1 to 15:
1) to give the desired product as two diastereoisomers.
1235, 1080, 775, 675 cm-1; MS (FAB, 3NBA) m/z (relative
intensity) 461.3 (MH+, 2.1), 419.3 (MH+ - 42, 3.6), 403.2 (MH+
- 56, 17.1).
3-(tert-Butyldiphenylsilyloxy)-1-(4-nitrophenyl)propylamine
(25). To a solution of 3-(tert-butyldiphenylsilyloxy)-1-(4-nitro-
phenyl)propyl azide (300 mg, 0.655 mmol) in methanol (6 mL)
were added propane-1,3-dithiol (0.33 mL, 3.28 mmol) and triethyl-
amine (0.46 mL, 3.28 mmol). The reaction solution was stirred at
room temperature for 12 h and concentrated in vacuo. The residue
was purified by flash column chromatography (chloroform/
methanol, 30:1) to afford the desired product 25 as a yellow oil
(198 mg, 70%): 1H NMR (300 MHz, CDCl3) δ 8.15 (dd, J ) 2.1,
6.6 Hz, 2H), 7.68-7.36 (m, 12H), 4.33 (t, J ) 6.8 Hz, 1H), 3.76-
3.64 (m, 2H), 1.95-1.80 (m, 2H), 1.72 (br s, 2H), 1.08 (s, 9H);
13C NMR (50 MHz, CDCl3) δ 153.7, 135.6, 133.6, 129.9, 127.8,
127.4, 123.8, 61.2, 53.1, 41.9, 27.0, 19.3; IR (film) 3040, 2920,
2840, 1650, 1585, 1500, 1410, 1325, 1080, 835, 805, 720, 680
cm-1; MS (FAB, 3NBA) m/z (relative intensity) 435.2 (MH+, 35.3),
377.1 (MH+ - 56, 13.1), 257.1 (MH+ - 78, 5.8); HRMS (FAB)
m/z calcd for C25H31N2O3Si (MH+) 435.2104, found 435.2119.
3-(4-Nitrophenyl)-3-amino-1-propanol (26). To a solution of
3-(tert-butyldiphenylsilyloxy)-1-(4-nitrophenyl)propylamine (200
mg, 0.46 mmol) in THF (25 mL) at 0 °C was added dropwise a 1
M solution of tetrabutylammonium fluoride in THF (2.3 mL). The
solution was stirred at room temperature for 1 h, after which a
saturated KHSO4 was added to acidify the solution to pH 6. After
extraction with ethyl ether, the aqueous solution was basified with
3 N NaOH and extracted with dichloromethane (40 mL × 3). The
organic phase was dried over Na2SO4 and evaporated in vacuo,
and the residue was purified by flash column chromatography
(chloroform/methanol, 50:1 to 40:1; the chloroform was saturated
with ammonium hydroxide) to give the desired product 26 as a
yellow solid (74 mg, 82%): 1H NMR (300 MHz, CDCl3) δ 8.23
(d, J ) 9.0 Hz, 2H), 7.51 (d, J ) 9.0 Hz, 2H), 4.34-4.25 (m, 1H),
cis-(()-3: yellow solid (79 mg, 34%); mp (CHCl3-MeOH)
125-127 °C; 1H NMR (300 MHz, CDCl3) δ 8.21 (d, J ) 6.9 Hz,
2H), 7.62 (d, J ) 8.7 Hz, 2H), 5.50-5.40 (m, 1H), 1H), 3.80-
3.60 (m, 6H), 3.52-3.35 (m, 5H), 2.20-1.95 (m, 2H); 13C NMR
(50 MHz, CDCl3) δ 147.9, 147.3 (d, J ) 6.5 Hz), 126.6, 124.0,
80.3 (d, J ) 6.8 Hz), 48.6 (d, J ) 3.8 Hz), 42.1 (d, J ) 2.7 Hz),
40.6, 34.4 (d, J ) 9.2 Hz); 31P NMR (121 MHz, CDCl3) δ 11.2
(s); IR (film) 3400, 3140, 2920, 2820, 1580, 1490, 1420, 1325,
1220, 1195, 1095, 1075, 1020, 965, 890, 840, 830, 790, 725 cm-1
;
MS (FAB, 3NBA) m/z (relative intensity) 382.2 (MH+, 4.1), 384.2
(MH+ + 2, 2.9), 386.0 (MH+ + 4, 3.9); HRMS (FAB) m/z calcd
for C13H19N3O4PCl2 (MH+) 382.0490, found 382.0479; HRMS
(FAB) m/z calcd for C13H21N3O4P35Cl37Cl (MH+ + 2) 384.0461,
found 384.0459.
trans-(()-3: yellow solid (77 mg, 33%); mp (CHCl3-MeOH)
1
138-140 °C; H NMR (300 MHz, CDCl3) δ 8.20 (dd, J ) 1.8,
6.8 Hz, 2H), 7.62 (d, J ) 9.6 Hz, 2H), 5.63 (d, J ) 11.1 Hz, 1H),
1H), 3.65-3.30 (m, 10H), 3.10 (br s, 1H), 2.10-1.80 (m, 2H); 13
C
NMR (50 MHz, CDCl3) δ 147.8, 147.3 (d, J ) 9.5 Hz), 126.3,
124.0, 77.9 (d, J ) 5.7 Hz), 48.8 (d, J ) 4.6 Hz), 42.4, 41.0 (d, J
) 2.3 Hz), 33.4 (d, J ) 3.4 Hz); 31P NMR (121 MHz, CDCl3) δ
14.6 (s); IR (film) 3400, 3120, 2920, 2760, 1590, 1500, 1435, 1330,
1200, 1080, 940, 900, 730 cm-1; MS (FAB+, 3NBA) m/z (relative
intensity) 382.1 (MH+, 3.3), 384.2 (MH+ + 2, 1.7), 386.0 (MH+
+ 4, 3.9); HRMS (FAB+) m/z calcd for C13H19N3O4PCl2 (MH+)
382.0490, found 382.0482; HRMS (FAB+) m/z calcd for C13H21-
N3O4P35Cl37Cl (MH+ + 2) 384.0461, found 384.0462.
3-(tert-Butyldiphenylsilyloxy)-1-(4-nitrophenyl)propan-1-ol (23).
To a cooled solution of 1-(4-nitrophenyl)propane-1,3-diol (630 mg,
2.55 mmol) and imidazole (866 mg, 12.7 mmol) in DMF (15 mL)
at -30 °C was added slowly dropwise tert-butyldiphenylsilyl
chloride (683 µL, 2.63 mmol). The reaction solution was stirred at
-30 to -20 °C for an additional 1.2 h and quenched by the addition
of water (5 mL). The solution was diluted with ethyl acetate (100
mL), washed with saturated NaCl, dried over Na2SO4, and
evaporated in vacuo. The residue was purified by flash column
chromatography (hexanes/acetone, 9:1 to 7:1) to give the desired
product 23 as a yellow oil (1.06 g, 95%): 1H NMR (300 MHz,
CDCl3) δ 8.20 (d, J ) 8.1 Hz, 2H), 7.70-7.30 (m, 2H), 5.20-
5.10 (m, 1H), 4.05 (br s, 1H), 3.90-3.80 (m, 2H), 2.00-1.90 (m,
2H), 1.10 (s, 9H); 13C NMR (50 MHz, CDCl3) δ 152.0, 147.3,
135.6, 132.8, 132.7, 130.1, 128.0, 126.5, 123.7, 73.2, 62.9, 40.5,
27.0, 19.2; IR (film) 3400, 2940, 2920, 1840, 1500, 1410, 1335,
1100, 685 cm-1; MS (FAB, 3NBA) m/z (relative intensity) 436.2
(MH+, 2.2), 418.1 (MH+ - 18, 2.0), 378.1 (MH+ - 56, 1.5);
HRMS (FAB) m/z calcd for C25H30NO4Si (MH+) 436.1944, found
436.1932.
3-(tert-Butyldiphenylsilyloxy)-1-(4-nitrophenyl)propyl Azide
(24). To a solution of 3-(tert-butyldiphenylsilyloxy)-1-(4-nitro-
phenyl)propane-1-ol (5.6 g, 12.8 mmol) and triphenylphosphine
(4.36 g, 16.1 mmol) in THF (80 mL) at 0 °C was added DEAD
(2.89 g, 16.6 mmol) and a solution of hydrazoic acid in benzene
(37.7 mL, 1.2 M). The reaction mixture was stirred at room
temperature for 2 h, quenched by adding saturated NaHCO3 (20
mL), and poured into ethyl acetate (200 mL). The organic phase
was washed with saturated NaHCO3, dried over Na2SO4, and
evaporated in vacuo. The residue was purified by flash column
chromatography (hexanes/acetone, 10:1) to give the desired product
24 as a yellow oil (5.69 g, 97%): 1H NMR (300 MHz, CDCl3) δ
8.14 (d, J ) 8.7 Hz, 2H), 7.66-7.53 (m, 5H), 7.39-7.28 (m, 7H),
4.84 (dd, J ) 6.3, 8.1 Hz, 1H), 3.80-3.70 (m, 1H), 3.56-3.50 (m,
1H), 1.92-1.83 (m, 2H), 1.04 (s, 9H); 13C NMR (50 MHz, CDCl3)
δ 147.8, 147.2, 135.6, 133.4, 130.0, 127.9, 127.8, 124.1, 62.2, 59.9,
39.3, 27.0, 19.3; IR (film) 2890, 2820, 2070, 1500, 1405, 1325,
3.81 (t, J ) 5.25 Hz, 2H), 2.16 (br s, 3H), 1.95-1.89 (m, 2H); 13
NMR (50 MHz, CDCl3) δ 153.4, 126.9, 124.1, 61.6, 55.5, 40.1;
IR (film) 3300, 2900, 1580, 1495, 1330, 1040, 835, 730, 680 cm-1
C
;
MS (FAB, 3NBA) m/z (relative intensity) 197.1 (MH+, 100.0),
180.1 (MH+ - 18, 13.9), 181.1 (MH+ - 17, 9.8); HRMS (FAB)
m/z calcd for C9H13N2O3 (MH+) 197.0926, found 197.0946.
2-[Bis(2-chloroethyl)amino]-4-(4-nitrophenyl)-2H-1,3,2-oxaza-
phosphorinane 2-Oxide (4). To a solution of 3-(4-nitrophenyl)-
3-amino-1-propanol (65 mg, 0.33 mmol) in anhydrous ethyl acetate
(40 mL) at 0 °C was added a solution of bis(2-chloroethyl)-
phosphoramidic dichloride (103 mg, 0.4 mmol) and triethylamine
(111 µL, 0.8 mmol) in ethyl acetate (10 mL). The reaction solution
was stirred at room temperature for 48 h and filtered to remove
the precipitate. The filtrate was washed with saturated NaCl, dried
over Na2SO4, and evaporated in vacuo. The residue was purified
by flash column chromatography (hexanes/ethyl acetate, 3:1 for
cis-(()-4, followed by chloroform/methanol, 30:1 for trans-(()-
4) to give the desired product as two diastereoisomers.
cis-(()-4: yellow solid (17 mg, 14%); mp (CHCl3-MeOH)
118-120 °C; 1H NMR (300 MHz, CDCl3) δ 8.16 (d, J ) 9.0 Hz,
2H), 7.71 (d, J ) 9.0 Hz, 2H), 4.74 (t, J ) 7.2 Hz, 1H), 1H),
4.31-4.16 (m, 2H), 3.64-3.33 (m, 8H), 3.09 (d, J ) 3.6 Hz, 1H),
2.26-2.21 (m, 1H), 2.04-1.96 (m, 1H); 13C NMR (50 MHz,
CDCl3) δ 150.5, 147.7, 127.6, 124.2, 64.9 (d, J ) 6.8 Hz), 56.7,
48.7 (d, J ) 4.6 Hz), 42.3, 33.2 (d, J ) 8.4 Hz); 31P NMR (121
MHz, CDCl3) δ 9.6 (s); IR (film) 3350, 3180, 2900, 1700, 1585,
1500, 1325, 1210, 1110, 1090, 970, 930, 840, 720, 680 cm-1; MS
(FAB, 3NBA) m/z (relative intensity) 382.0 (MH+, 56.7), 384.0
(MH+ + 2, 38.8), 386.0 (MH+ + 4, 3.9); HRMS (FAB) m/z calcd
for C13H19N3O4PCl2 (MH+) 382.0490, found 382.0491; HRMS
(FAB) m/z calcd for C13H19N3O4P35Cl37Cl (MH+ + 2) 384.0461,
found 384.0467.
trans-(()-4: yellow solid (21.3 mg, 17%); mp (CHCl3-MeOH)
1
139.5-141 °C; H NMR (300 MHz, CDCl3) δ 8.24 (d, J ) 8.1
Hz, 2H), 7.54 (d, J ) 8.1 Hz, 2H), 4.81 (dd, J ) 4.8, 9.9 Hz, 1H),
4.65-4.56 (m, 1H), 3.70-3.48 (m, 8H), 2.85 (br s, 1H), 2.00-
1.92 (m 2H); 13C NMR (50 MHz, CDCl3) δ 150.0 (d, J ) 12.2

4342 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Jiang et al.
Hz), 149.9, 120.1, 124.4, 66.2 (d, J ) 5.7 Hz), 56.7, 48.6 (d, J )
5.0 Hz), 42.4, 34.7 (d, J ) 3.0 Hz); 31P NMR (121 MHz, CDCl3)
δ 14.2 (s); IR (KBr) 3447, 3112, 2995, 2876, 1521, 1449, 1349,
1222, 1193, 1109, 914, 874, 750 cm-1; MS (FAB, 3NBA) m/z
(relative intensity) 382.0 (MH+, 65.9), 384.0 (MH+ + 2, 42.9),
386.0 (MH+ + 4, 6.1); HRMS (FAB) m/z calcd for C13H19N3O4-
PCl2 (MH+) 382.0490, found 382.0464; HRMS (FAB) m/z calcd
for C13H19N3O4PCl2 (MH+) 382.0490, found 382.0464; HRMS
(FAB) m/z calcd for C13H19N3O4P35Cl37Cl (MH+ + 2) 384.0461,
found 384.0440.
SKOV NTR cells were generated by infecting SKOV3 cells with
the retrovirus rv.RD18.LNC-nr38 and selecting clones of transduced
cells with 1 mg/mL G418. NTR expression was confirmed by
Western blots. For prodrug sensitivity assays, SKOV NTR or
parental SKOV3 cells were plated in 96-well plates at 20 000 cells
per well and allowed to adhere for 24 h. The medium was
exchanged for fresh medium containing a range of prodrug
concentrations. After 18 h of exposure to prodrug, the medium was
removed and replaced with fresh medium lacking prodrug, and cell
survival was determined by MTT assay after an additional 2 days.
Stability Test in Aqueous Buffer. A 1 mg sample of the
substrate was dissolved in 1 mL of 50 mM sodium phosphate buffer
(pH 7.4) containing 10% DMSO and incubated at 37 °C. Aliquots
were withdrawn at different time intervals for 72 h and subjected
to reversed-phase HPLC analysis.
E. coli Nitroreductase Assay. The substrate (0.2 mM) was
incubated with 1 mM NADH at 37 °C in 10 mM sodium phosphate
buffer (pH 7.0) in a total volume of 250 µL. The reaction was
initiated by the addition of 1.8 µg of E. coli nitroreductase. For
compound (()-7, 2 mM cysteine was added to protect the
nitroreductase enzyme from inactivation by the phosphoramide
mustard released upon activation (the addition of 2 mM cysteine
had little effect on the half-life of 5-aziridinyl-2,4-dinitrobenzamide
under the same conditions). Aliquots were withdrawn and analyzed
by reversed-phase HPLC analysis. The half-life of reduction by E.
coli nitroreductase was calculated on the basis of the disappearance
of the substrate assuming pseudo-first-order kinetics.
Acknowledgment. We gratefully acknowledge the financial
support of Grant SNJ-CCR 700-009 from the State of New
Jersey Commission on Cancer Research and Grant RSG-03-
004-01-CDD from American Cancer Society (to L.H.), the U.K.
Medical Research Council for Grant G9806623/44940 (to
P.F.S.), and Research Studentship G78/8255 (to S.O.V.). We
also thank Dr. Susan M. Ludeman and Dr. O. Michael Colvin
from Duke University for providing a sample of 4-hydroperoxy-
cylophosphamide and Dr. Eva I. Hyde from University of
Birmingham, U.K., for providing the E. coli nitroreductase
enzyme and for helpful discussions.
Supporting Information Available: NMR, MS, and IR data
of target compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
Cell Culture and Antiproliferative Assays in Vitro. V79
Chinese hamster lung fibroblasts were grown in monolayer culture
in DMEM containing 10% fetal calf serum and 4 mM glutamine.
Cells were maintained in a humidified atmosphere at 37 °C with
5% CO2 and subcultured twice weekly by trypsinization. A
bicistronic eukaryotic expression vector containing the coding
regions for E coli nitroreductase together with puromycin acetyl
transferase (conferring puromycin resistance) driven from a single
CMV promoter was constructed by cloning into the XhoI site of
the vector pIRES-P (EMBL:Z75185; 1) using conventional tech-
niques. Insert orientation and identity were confirmed by diagnostic
restriction digests and dideoxy sequencing using a Sequenase II
kit (Amersham Pharmacia Biotech, St. Albans, Herts, U.K.). The
V79 cells were transfected with the bicistronic vector, and the
positive clones were selected in growth medium containing 10 µg/
mL puromycin and maintained under selective pressure. Cells
expressing E. coli nitroreductase in exponential phase of growth
were trypsinized, seeded in 96-well plates at a density of 1000 cells/
well, and permitted to recover for 24 h. V79 cells transfected with
vector only were used as the controls. Serial dilutions of the drug
solution were performed in situ, and cells were then incubated with
drug for 3 days at 37 °C. The plates were fixed and stained with
SRB before reading with optical absorption at 570 nm; results were
expressed as a percentage of control growth. IC50 values are the
concentration required to reduce cell number to 50% of control
and were obtained by interpolation. For the short drug exposure
experiments, cells were exposed to each test drug for 1 h and culture
media was then replaced with drugfree, fresh medium for continued
incubation. Cell viability assay was performed 3 days after drug
addition. For the control experiment using 4-hydroperoxycyclo-
phosphamide, sealed microtiter plates were used to prevent the
volatile metabolite from contaminating neighboring wells.39
SKOV3 human ovarian carcinoma cells grown in HEPES-
buffered DMEM with 10% FCS were infected with an E1, E3-
deleted replication-defective adenovirus vector vPS1233 expressing
the wild type E. coli nitroreductase from the CMV promoter, using
infection ratios of 10 or 100 plaque forming units (pfu) per cell.
Cells were plated in 96-well plates (at 15 000 or 20 000 cells/well
in different experiments) and incubated for 2 days to allow for
nitroreductase expression. The used medium was exchanged with
fresh medium containing a range of prodrug concentrations up to
1000 µM. After 18 h of incubation, the medium was replaced with
fresh medium (without prodrugs). An MTT assay was performed
3 days after adding prodrug to assess cell viability.
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