Nitroaryl Phosphoramides as Novel Prodrugs for E. coli Nitroreductase Activation in Enzyme Prodrug Therapy

Article abstract of DOI:10.1021/jm034133h

Cyclic and acyclic nitroaryl phosphoramide mustard analogues were activated by E. coli nitroreductase, an enzyme explored in GDEPT. The more active acyclic 4-nitrobenzyl phosphoramide mustard (7) showed 167 500x selective cytotoxicity toward nitroreductase-expressing V79 cells with an IC₅₀ as low as 0.4 nM. This is about 100x more active and 27x more selective than CB1954 (1). The superior activity was attributed to its better substrate activity (k_cat/K_m 19x better than 1) and/or excellent cytotoxicity of phosphoramide mustard released.

Full text of DOI:10.1021/jm034133h

4818
J . Med. Chem. 2003, 46, 4818-4821
Letters
for the targeted delivery of toxic alkylating species to
hypoxic tumor cells.9,10 For activation by E. coli nitrore-
ductase, four classes of prodrugs have been described:
dinitroaziridinylbenzamides (e.g., 1, CB1954), dini-
trobenzamide mustards (e.g., 2, SN 23862,), 4-nitroben-
zylcarbamates (3), and nitroindolines (e.g., 4).8,11,12 Of
the four classes, the first two are considered the most
promising when used in conjunction with NTR.13 Com-
pound 1, currently under Phase I clinical trial in
conjunction with the virally delivered NTR enzyme,14
has high selectivity (>1000-fold) in cell lines transfected
with NTR and has potent and long-lasting inhibition of
nitroreductase-transfected tumors in mice. The related
mustard 2 has similar selectivity and good bystander
effects in animal models. Here, we would like to report
a class of nitroaromatics that are excellent substrates
of NTR and that release a known highly cytotoxic
phosphoramide mustard or like-reactive species upon
NTR-reduction.
Nitr oa r yl P h osp h or a m id es a s Novel
P r od r u gs for E. coli Nitr or ed u cta se
Activa tion in En zym e P r od r u g Th er a p y
Longqin Hu,*,† Chengzhi Yu,† Yongying J iang,†
J iye Han,† Zhuorong Li,† Patrick Browne,‡
Paul R. Race,§ Richard J . Knox,‡
Peter F. Searle,§ and Eva I. Hyde§
Department of Pharmaceutical Chemistry,
Ernest Mario School of Pharmacy, Rutgers, The State
University of New J ersey, 160 Frelinghuysen Road,
Piscataway, New J ersey 08854; Enact Pharma PLC,
Porton Down Science Park, Salisbury, Wiltshire,
SP4 0J Q, UK; and University of Birmingham,
Edgbaston, Birmingham B15 2TT, UK
Received J une 27, 2003
Abstr a ct: Cyclic and acyclic nitroaryl phosphoramide mus-
tard analogues were activated by E. coli nitroreductase, an
enzyme explored in GDEPT. The more active acyclic 4-ni-
trobenzyl phosphoramide mustard (7) showed 167 500× selec-
tive cytotoxicity toward nitroreductase-expressing V79 cells
with an IC50 as low as 0.4 nM. This is about 100× more active
and 27× more selective than CB1954 (1). The superior activity
was attributed to its better substrate activity (kcat/Km 19×
better than 1) and/or excellent cytotoxicity of phosphoramide
mustard released.
Ch em istr y. The design of our nitroaromatics is based
on the activation mechanism of the clinically useful
anticancer drug cyclophosphamide. Cyclophosphamide
is an anticancer prodrug, which has to be activated by
the cytochrome P-450 enzyme in the liver.15-17 Hepatic
cytochrome P-450 oxidation converts cyclophosphamide
to 4-hydroxyphosphamide, which ultimately forms the
cytotoxic alkylating species, phosphoramide mustard,
following ring opening and â-elimination. Our efforts
have been focused on designing phosphoramide ana-
logues incorporating site-specific activation mechanisms
such as nitro reduction in order to move the site of
activation from liver into the tumor tissues.
Among the nitroaryl phosphoramides designed and
synthesized were compounds 5, 6, and 7, each with a
strategically placed nitro group on the benzene ring in
the para position to the benzylic carbon. Compound 5
is a cyclophosphamide analogue with the cyclophospha-
mide ring fused with a benzene ring and a nitro group
placed in the para position to the benzylic carbon.
Compound 6 is a 4-nitrophenyl-substituted cyclophos-
phamide analogue, and 7 is an acyclic nitrobenzyl phos-
phoramide mustard. The nitro group is a strong electron-
withdrawing group (Hammett σp electronic parameter
) 0.78) and is converted to an electron-donating hy-
droxylamino group (σp ) -0.34) upon NTR-reduction.
This large difference in electronic effect (∆σp ) 1.12)
is exploited to effect the formation of the highly cyto-
toxic phosphoramide mustard or like-reactive species
as shown in Scheme 1. After reduction by NTR, the
resulting hydroxylamines 8, 10, and 12 will relay their
In tr od u ction . Prodrug design is an important strat-
egy that has been proven to work for many drugs in
improving their undesirable physicochemical and bio-
logical properties.1-3 Recently, prodrug strategies have
also been used in targeted drug delivery including
antibody-directed enzyme prodrug therapy (ADEPT)
and gene-directed enzyme prodrug therapy (GDEPT).
In these approaches, an enzyme is delivered site-
specifically by chemical conjugation or genetic fusion to
a tumor specific antibody or by an enzyme gene delivery
system into tumor cells. This is then followed by the
administration of a prodrug, which is selectively acti-
vated by the delivered enzyme at the tumor cells. A
number of these systems are in development and have
been reviewed.4 Among the enzymes under evaluation
is the nfsB gene product of Escherichia coli, an oxygen-
insensitive flavin mononucleotide-containing nitrore-
ductase (NTR). This flavoprotein is capable of reducing
certain aromatic nitro groups to the corresponding
hydroxylamines in the presence of the cofactor NADH
or NADPH.5-7
Reduction of aromatic nitro groups to hydroxylamines
or amines represents a very large electronic change and
can provide an efficient “switch” that can be exploited
to generate potent cytotoxins.8 This approach was used
* To whom correspondence should be addressed. Phone: 732-445-
5291. Fax: 732-445-6312. E-mail: LongHu@rci.rutgers.edu.
† Rutgers, The State University of New J ersey.
‡ Enact Pharma PLC, Salisbury, Wiltshire, UK.
§ University of Birmingham, Edgbaston, Birmingham, UK.
10.1021/jm034133h CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/10/2003

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4819
Sch em e 1. Proposed Mechanism of Activation of Nitroaryl Phosphoramides by Bioreduction
Sch em e 2. Synthesis of 7-Nitrobenzocyclophosphamide
Sch em e 3. Synthesis of
5a
6-(4-Nitrophenyl)cyclophosphamide 6 Isomersa
a
Reagents and conditions: (i) Ac2O, pyridine, 6 h, 84%; (ii) NBS/
CCl4, hυ, 20 h, 56%; (iii) CaCO3, dioxane-H2O, ∆, 3 h, 42%; (iv) 6
N
HCl, rt, 16 h, 100%; (v) bis(2-chloroethyl)phosphoramidic
dichloride, Et3N, EtOAc, 48 h, 23%.
electrons to the para-position and facilitate the cleavage
of the benzylic C-O bond, producing the anionic cyto-
toxic species phosphoramide mustard (14) or like-
reactive species (9 and 11). Structurally, the phosphor-
amide portion in 9 and 11 closely resembles phosphor-
amide mustard (14), the reactive alkylating agent pro-
duced following the metabolic activation of cyclophos-
phamide in the liver, and could also be the ultimate cy-
totoxic alkylating agent. In addition, compounds 9, 11,
and 13 possess additional electrophilic centers that
might form adducts with functionally important mac-
romolecules, providing additional mechanisms for cy-
totoxicity. The mechanism of activation proposed here
is similar to that proposed for the hypoxic reductive
activation of 4-nitrobenzyl N,N,N′,N′-tetrakis(2-chloro-
ethyl)phosporodiamidate9 and the reductive activation
of 4-nitrobenzyl carbamates.12,18
a
Reagents and conditions: (i) CH2dCHMgBr, THF, -78 °C to
-60 °C, 4 h, 95%; (ii) MOMCl/DIEA/CH2Cl2, 0 °C to rt, 24 h, 95%;
(iii) B2H6/THF, 0 °C, 5 h, then 3 N NaOH, 30% H2O2, 30 min, 78%;
(iv) MsCl/ Et3N, 0 °C, 15 min then NaN3, 15-crown-5 (cat.), DMF,
91%; (v) PPh3, THF-H2O, 72%; (vi) CBB followed by HOAc,
CH2Cl2, -20 °C, 76%; (vii) bis(2-chloroethyl)phosphoramidic dichlo-
ride, Et3N, EtOAc, 48 h, cis-6, 34%, trans-6, 33%.
Sch em e 4. Synthesis of 4-Nitrobenzyl Phosphoramide
Mustard 7a
a
Reagents and conditions: (i) BuLi,,-78 °C; (ii) bis(2-chloro-
ethyl)phosphoramidic dichloride, THF, -78 °C, 2 h; (iii) NH3, -78
Compounds 5, 6, and 7 were synthesized by the
reaction sequences shown in Schemes 2-4. Bromination
of acetylated 2-methyl-5-nitroaniline (15) followed by
hydrolysis and triethylamine-mediated cyclization with
bis(2-chloroethyl)phosphoramidic dichloride gave com-
pound 5 (Scheme 2).19,20 For the synthesis of compound
6, a Grignard reaction with vinylmagnesium bromide
followed by protection and hydroboration-oxidation was
used to convert 4-nitrobenzaldehyde (18) to monopro-
tected 1,3-diol 20. Subsequent activation, SN2 displace-
ment, and reduction afforded 1,3-amino alcohol 21.
Cyclization of 21 with bis(2-chloroethyl)phosphoramidic
dichloride gave our desired product 6 as a pair of cis
and trans diastereomers, which were separated on flash
silica gel column chromatography (Scheme 3). Here, cis
and trans refer to the relative orientation of the 6-(4-
nitrophenyl) and the 2-oxo substituents on the 1,3,2-
oxazaphosphorinane ring. Compound 7 was synthesized
°C, 30 min, 53% for the three steps.
according to a modified literature procedure (Scheme
4).10 All new compounds were fully characterized by IR,
1H, 13C, and 31P NMR and high-resolution mass spec-
trometry.
Resu lts a n d Discu ssion . Two types of cells were
used to evaluate the antiproliferative activities of our
compounds in combination with E. coli nitroreductase:
the V79 Chinese hamster fibroblast cells and the
SKOV3 human ovarian carcinoma cells. The Chinese
hamster V79 cells were transfected with a bicistronic
vector encoding for the NTR protein and puromycin
resistance protein as the selective marker. The corre-
sponding V79 cells, transfected with vector only, were
used as controls. The SKOV3 human ovarian carcinoma
cells were infected with a replication-defective adenovi-

4820 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Letters
Ta ble 1. >IC50 Values (µM) and IC50 Ratio (NTR-/NTR+) for Compounds 5, 6, and 7 in Comparison to Compound 1a
V79 cells, 72 h drug exposure
IC50,b NTR- IC50,b NTR+ ratioc
V79 cells, 1 h drug exposure
IC50,bNTR- IC50,b NTR+ ratioc
SKOV3 cells, 18 h drug exposure
compound
IC50,bNTR- IC50,bNTR+
ratioc
d
d
d
5
>100
832
608
67
3.0
>33
28 690
22 519
167 500
-
-
-
550
680
55
4.7
5
10
145
cis-6
trans-6
7
0.029
0.027
0.0004
>100
>100
>100
0.34
0.17
0.01
>294
>588
>10 000
>1000
>1000
>1000
>1000
625
>200
>833
>161e
>213
12e
1.2
6.2e
4.7
1
254
0.036
7056
>100
0.31
>323
52e
a
Cells were exposed to each test compound for a given amount of time, and a standard cell viability assay was performed 72 h after
the addition of test compound. Initially, the maximum concentration used was 100 µM in the case of V79 cells and 1000 µM in the case
b
of SKOV3 cells. When necessary, assays were repeated at higher drug concentrations to obtain accurate IC50 values. IC50 values are the
concentration required to reduce cell number to 50% of control after the cells were exposed to the drug for the indicated time. c Ratio of
d
IC50 values (NTR-/NTR+) as an indication of activation by E. coli nitroreductase. Not determined. e 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. Cell lines: V79 (Chinese hamster fibroblast) and SKOV3 (human ovarian carcinoma).
rus vector vPS1233 expressing NTR,21 and the unin-
fected SKOV3 cells were used as controls.
The IC50 values and their ratios (NTR-/NTR+) of
compounds 5, 6, and 7 are provided in Table 1 in
comparison with that of compound 1. In calculating the
ratio of IC50, the value of 100 or 1000 µM was used for
those compounds with an undetermined IC50 >100 or
>1000 µM so the ratio was an underestimate. None of
our test compounds were very cytotoxic in the absence
of NTR, and they were not activated by endogenous
mammalian enzymes, at least not those found in V79
and SKOV3 cells. In all cases, the NTR+ cells were more
cytotoxically affected by the test compounds than the
F igu r e 1. Enzyme kinetics: 7 (b) as a substrate of E. coli
control cells and all gave ratios >1, indicating activation
nitroreductase in comparison with compound 1 (O).
by NTR. For the 72 h drug exposure, the benzene ring-
fused cyclophosphamide 5 was >33× more cytotoxic in
100 pfu/cell of adenovirus expressing NTR, compound
7 showed an IC50 that is about 4× lower than that of
the nitroreductase-expressing Chinese hamster V79
cells than in the control V79 cells that were transfected
with a vector only, while compound 1 showed 7000×
selectivity toward nitroreductase-expressing V79 cells
under the same conditions. The IC50 values of the 6-(4-
nitrophenyl)-substituted cyclophosphamide isomers (cis-6
and trans-6) were about the same as that of compound
1 in nitroreductase-expressing V79 cells while both were
3-4× less toxic to V79 cells not expressing NTR. Thus,
the advantage of cis-6 and trans-6 over compound 1 is
their 22 000-28 000× selective cytotoxicity in targeting
nitroreductase-expressing V79 cells. This selectivity is
3-4× better than compound 1. The acyclic 4-nitrobenzyl
phosphoramide mustard 7 turned out to be the most
active compound with IC50 as low as 0.4 nM and a
selectivity ratio of 167 500 against nitroreductase-
expressing V79 cells upon 72 h exposure. This shows
that 7 is 100× more active and 27× more selective than
compound 1 in targeting nitroreductase-expressing V79
cells.
More importantly, compound 7 was quickly activated
and had long lasting cytotoxic effects in E. coli nitrore-
ductase-expressing Chinese hamster V79 cells. When
the nitroreductase-expressing V79 cells were briefly
exposed to each test compound for 1 h, 7 showed the
best activity with an IC50 as low as 10 nM, while cis-6,
trans-6, and compound 1 had IC50’s around 300 nM. 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
compound 1 and compound 6 isomers. When the level
of NTR expression was made more limiting by infection
with just 10 pfu/cell of the adenovirus, although both
IC50’s were higher, the differential increased such that
the IC50 for compound 7 was about 9× lower than that
for compound 1. The higher IC50’s of all prodrugs in the
SKOV3 cells could be due to the possibly lower levels
of NTR expressed in the virally transfected SKOV3 as
compared to the stably transfected V79 cells. Another
contributory factor could be a greater DNA repair
capability, or resistance to DNA damage-induced apo-
ptosis, in the human ovarian tumor cells.
To demonstrate the positional requirement of nitro
group para to the benzylic oxygen, analogues of 5 and
6 with ring O and NH transposed and an analogue of 7
with the nitro group moved to the meta position were
also synthesized and evaluated. Even though those
analogues of 5, 6, and 7 were NTR substrates, they were
shown to have no significant selective cyctotoxicity
against nitroreductase-expressing V79 and SKOV3 cells
(data not shown). These results indicate that nitrore-
ductase reduction is an important first step but not
sufficient for enhanced cytotoxicity in nitroreductase-
expressing cells. This is consistent with the mechanism
proposed in Scheme 1.
Furthermore, compound 7 was confirmed to be a
much better substrate for NTR than compound 1 in
enzyme assays. As shown in Figure 1, compound 7 has
a Km of 195 ( 14 µM, and a kcat of 14.03 ( 0.35 s-1 while
compound 1 has a Km of 881 ( 42 µM and kcat of 6.60 (
0.11 s-1, which were similar to the published values.7
In SKOV3 human ovarian cancer cells infected with

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4821
(3) Bundgaard, H. Design of Prodrugs; Elsevier: Amsterdam, 1985;
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Since compound 1 is reduced to a 1:1 mixture of
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(4) Melton, R. G.; Knox, R. J . Enzyme-prodrug strategies for cancer
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(5) Bridgewater, J .; Minton, N. P.; Michael, N. P.; Knox, R.;
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1
cytotoxic product,13,22 the productive /2kcat of 3.30 s-1
is used for comparison purposes. This gave a specificity
constant of 71 949 M-1 s-1 (kcat/Km) for compound 7 and
3746 M-1 s-1 (1/2kcat/Km) for compound 1. Thus, com-
pound 7 is 19 times better as a substrate of NTR than
compound 1. This at least partially contributed to the
better activity and selectivity in cell culture assays of
compound 7 in comparison with compound 1.
It should also be noted that compound 7 upon reduc-
tive activation releases phosphoramide mustard, which
is the active metabolite of the clinical drug cylcophos-
phamide.15-17 The excellent activity of compound 7 in
nitroreductase-expressing cells was unexpected consid-
ering 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 un-
der hypoxic conditions.9 This suggests that either this
class of compounds were poor substrates of the human
reductase(s) present or the expression of these reduc-
tase(s) was limited under the hypoxic assay conditions
used.
Con clu sion s. In summary, we have developed a
superior class of nitroaryl phosphoramides as potential
prodrugs for nitroreductase-mediated enzyme-prodrug
therapy. These nitroaryl phosphoramides have low
cytotoxicity before reduction and are converted to phos-
phoramide mustard or like-reactive species upon biore-
duction. The excellent biological activity of these com-
pounds correlates well with their substrate activity for
E. coli nitroreductase and is consistent with the ex-
pected high cytotoxicity of the reactive species released
upon reduction. Work is in progress in our laboratories
to further evaluate the biological activity of these ana-
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S. J . et al. Effect of nitroreduction on the alkylating reactivity
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Ack n ow led gm en t. We gratefully acknowledge the
financial support of grant SNJ -CCR 700-009 from the
State of New J ersey Commission on Cancer Research
and grant RSG-03-004-01-CDD from American Cancer
Society (to L.H.), grant G9806623/44940 from UK Medi-
cal Research Council (to P.F.S.) and a CASE studentship
from BBSRC (to P.R.R.).
Su p p or tin g In for m a tion Ava ila ble: Experimental sec-
tion including spectroscopic data, and enzyme and cell culture
assay conditions. This material is available free of charge via
the Internet at http://pubs.acs.org.
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