Synthesis and structure-activity relationships of nitrobenzyl phosphoramide mustards as nitroreductase-activated prodrugs

Article abstract of DOI:10.1016/j.bmcl.2011.05.009

A series of nitrobenzyl phosphoramide mustards and their analogs was designed and synthesized to explore their structure-activity relationships as substrates of nitroreductases from Escherichia coli and trypanosomes and as potential antiproliferative and antiparasitic agents. The position of the nitro group on the phenyl ring was important with the 4-nitrobenzyl phosphoramide mustard (1) offering the best combination of enzyme activity and antiproliferative effect against both mammalian and trypanosomatid cells. A preference was observed for halogen substitutions ortho to benzyl phosphoramide mustard but distinct differences were found in their SAR of substituted 4-nitrobenzyl phosphoramide mustards in E. coli nitroreductase-expressing cells and in trypanosomatids expressing endogenous nitroreductases.

Full text of DOI:10.1016/j.bmcl.2011.05.009

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3986–3991
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships of nitrobenzyl
phosphoramide mustards as nitroreductase-activated prodrugs
Longqin Hu a,b, , Xinghua Wu a, Jiye Han a, Lin Chen a, Simon O. Vass c, Patrick Browne d, Belinda S. Hall e,
⇑
Christopher Bot e, Vithurshaa Gobalakrishnapillai e, Peter F. Searle c, Richard J. Knox d, Shane R. Wilkinson e
a Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
b The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
c University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
d Morvus Technology Ltd., Ty Myddfai, Llanarthne, Carmarthen, SA32 8HZ, UK
e Queen Mary Pre-Clinical Drug Discovery Group, School of Biological & Chemical Sciences, Queen Mary University of London, Mile End Road, London, E1 4NS, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of nitrobenzyl phosphoramide mustards and their analogs was designed and synthesized to
explore their structure–activity relationships as substrates of nitroreductases from Escherichia coli and
trypanosomes and as potential antiproliferative and antiparasitic agents. The position of the nitro group
on the phenyl ring was important with the 4-nitrobenzyl phosphoramide mustard (1) offering the best
combination of enzyme activity and antiproliferative effect against both mammalian and trypanosomatid
cells. A preference was observed for halogen substitutions ortho to benzyl phosphoramide mustard but
distinct differences were found in their SAR of substituted 4-nitrobenzyl phosphoramide mustards in
E. coli nitroreductase-expressing cells and in trypanosomatids expressing endogenous nitroreductases.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 1 April 2011
Revised 28 April 2011
Accepted 2 May 2011
Available online 7 May 2011
Keywords:
Nitroreductase
Prodrug
Phosphoramide mustard
Antiproliferative
Trypanosoma
Leishmania
Targeted prodrug therapy has been extensively investigated to
improve the selectivity of cytotoxic agents toward tumor cells.1–4
By design, the prodrug remains inactive until activated by biochem-
ical mechanisms unique to cancerous cells, resulting in site-specific
release of cytotoxic moieties. Among the mechanisms explored,
enzyme-mediated activationofprodrugs is of great promise.1,5–9 En-
zymes used in the targeted activation can be of endogenous origin,
such as DT-diaphorase, b-glucuronidase, prostate-specific antigen
(PSA) and cytochrome P450 enzymes, or derived from an exogenous
source, such as the bacterial carboxypeptidase and nitroreductase
(NTR). Delivery of prokaryotic enzymes into cancerous cells can be
carried out by antibody-directed enzyme prodrug therapy (ADEPT),
where tumor-specific antibodies are coupled to the ‘exogenous’ pro-
tein by either direct chemical conjugation or by expression as a re-
combinant fusion protein.1,10,11 Alternatively, gene-directed
enzyme prodrug therapy (GDEPT) can be used, where the gene
encoding the activator is targeted to and then expressed in the can-
cerous cell.12–15 In both systems, delivery of the exogenous enzyme/
gene is followed by the administration of a prodrug which then
undergoes activation in the targeted cells to produce the toxin. Both
ADEPT and GDEPT can utilize NTRs, enzymes that catalyze reduction
of an aromatic nitro group to the corresponding hydroxylamine. The
large electronic change resulting from conversion of the electron-
withdrawing nitro to the electron-donating hydroxylamino group
provides an effective ‘switch’ mechanism for the activation of pro-
drugs and the subsequent release of cytotoxic agents.16,17
The NTR enzymes used in cancer prodrug design belong to the
oxygen-insensitive Type I class. These are associated with prokary-
otes and a subset of protozoan parasites, but are absent from most
higher eukaryotes.18 This difference in distribution is the basis of
drug selectivity for many nitroaromatic antimicrobial drugs19 and
is currently being exploited to develop improved anti-trypanoso-
matid treatments and some of our nitrobenzyl phosphoramide
mustard prodrugs have been shown to be activated by nitroreducta-
ses from Trypanosoma brucei.20 Here, we report the synthesis and
structure–activity relationships of a range of nitrobenzyl phospho-
ramide mustards as prodrugs activated by NTRs fromEscherichia coli,
T. brucei,Trypanosoma cruzi, and Leishmania major. This work reveals
that there are subtle differences between the bacterial and parasite
NTRs and identifies compounds with potential for the treatment of
several parasitic diseases and cancer.
Our efforts have been focused on the design of phosphorami-
dates to deliver the cytotoxic phosphoramide mustard alkylating
agent to targeted cells. Our strategy was to introduce an elec-
tron-withdrawing nitro group onto the benzyl phosphoramide
⇑
Corresponding author. Tel.: +1 732 445 5291; fax: +1 732 445 6312.
E-mail addresses: longhu@rutgers.edu, longhu@rci.rutgers.edu (L. Hu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.009

L. Hu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3986–3991
3987
mustard that could then undergo bioreducutive activation by Type
I NTRs (Scheme 1). Upon reduction, the nitro group in compound 1
is converted to the corresponding electron-donating hydroxyla-
mino group as shown in compounds 2 resulting in redistribution
of electrons within the structure. This triggers the cleavage of the
benzylic C–O bond linking the phosphoramide functionality and
thus promotes the release of the cytotoxic phosphoramide mustard
(3), the active metabolite of cyclophosphamide.
We previously reported that 4-nitrobenzyl phosphoramide
mustard (1) was an excellent substrate for nfsB, an E. coli Type I
NTR. This compound exhibited excellent antiproliferative activity
against V79 cells expressing nfsB with a subnanomolar IC50 and a
selectivity ratio of more than 160,000.17,21 Previous SAR studies
using derivatives of 1 demonstrated that the benzylic C–O bond
is crucial for prodrug activation.17,21 Here, we focused on modifica-
tions to the mustard, the linker, and the aromatic ring to gain a bet-
ter understanding of the electronic and steric effects involved in
enzyme substrate activity and subsequent drug release.
substituted 4-nitrobenzoic acids 10 as shown in Scheme 2. Our ear-
lier efforts demonstrated that reduction of methyl 4-nitrobenzoate
with DIBAL at À78 °C in THF afforded less than 40% of the desired
alcohol product with about 50% of the starting material recovered.
By using a modified procedure of sodium borohydride reduction of
activated esters,22 we were able to obtain the desired alcohols 11
in moderate yields. Typically, the N-hydroxybenzotriazoyl (HOBt)
activated esters of 10 were formed in situ followed by reduction
with sodium borohydride in THF. To avoid the problem of benzyl
benzoate ester formation, we added the solutions of OBt activated
esters dropwise to a suspension of sodium borohydride in THF and
we were able to achieve high yield of product formation. The substi-
tuted 4-nitrobenzyl phosphoramide mustards 19a–19l were syn-
thesized via the 3-step one-pot procedure in which the alcohols
were first deprotonated with n-BuLi followed by phosphorylation
with bis(2-chloroethyl)phosphoramidic dichloride and then
aminolysis.17,23
The compounds synthesized were evaluated in a series of
growth inhibition experiments against mammalian cells and try-
panosomatid parasites. In experiments involving mammalian cells,
inhibition data obtained with control lines (V79 and SKOV3) were
compared with values generated using variants expressing E. coli
nfsB (V79NTR+ and SKOV3NTR+). The parasites used in screening
were the extracellular, bloodstream trypomastigote of T. brucei,
the intracellular, amastigote of T. cruzi (the two medically relevant
parasite forms that replicate in the human host) and the L. major
promastigote, the form present in the sandfly gut.
Table 1 illustrates the structure and activities of the first series
of nitroarylmethyl phosphoramidates and analogs. When the posi-
tional effect of the nitro group on the benzene ring was investi-
gated, bacterial enzyme and mammalian antiproliferative
activities were highest when using 1, where the nitro substituent
is in the para-position. Lower activity levels were observed when
the nitro group was moved to the ortho- (12) or meta-positions
(13): the 2-nitro analog (12) was the worst substrate, presumably
due to the steric effect of the ortho substitution. When enzyme
activities were compared between the positional isomers, only a
2 to 5-fold difference was detected whereas a 225- to 325-fold dif-
ference was observed in their antiproliferative properties against
mammalian cells expressing E. coli nfsB. This suggests that intra-
cellular phosphoramide mustard release occurs more readily from
1 than from the other analogues, thus enhancing its antiprolifera-
tive effect. Analysis of effects against trypanosomatid pathogens
revealed that both 1 and 13 were reduced by the T. brucei NTR
(TbNTR), but only 1 displayed any antiparasitic activity in the
concentration range tested and this was restricted to T. brucei
bloodstream forms.
A series of phosphoramide mustard-based analogs of compound
1 was first designed and synthesized. These included 2- and
3-nitrobenzyl (12, 13),
a-methylated 4-nitrobenzyl (14A, 14B),
and 5-nitropyrid-2-yl methyl (15) phosphoramide mustards,
4-nitrobenzyl carbamate (16) and sulfamidite (17) mustards plus
the 4-nitrobenzyl phosphoramide bromomustards (18). The aryl-
methyl phosphoramide mustards 12–15 and 18 were synthesized
starting from the corresponding arylmethyl alcohol in a 3-step
one-pot procedure as published previously.17 The carbamate and
sulfamidite analogs 16 and 17 were synthesized by reacting
4-nitrobenzyl alcohol with phosgene or thionyl chloride followed
by quenching with nitrogen mustard.
A second series of substituted 4-nitrobenzyl phosphoramide
mustards 19a–19l was designed to introduce various functional
groups like electron-donating or electron-withdrawing groups to
the aromatic benzene ring in order to modulate the electron density
of the aromatic ring system to affect the release of cytotoxic phos-
phoramide mustard 3 via 1,6-elimination and to allow the 4-
hydroxylamino arylmethyl phosphoramide mustard intermediate
2 to permeate across cell membranes and release the phosphora-
mide mustard 3 inside neighboring cells, thus, increasing bystander
effect. The substituted 4-nitrobenzyl phosphoramide mustards
19a–19l were synthesized starting from the corresponding
Cl
Cl
O
P
NH2
O
P
NH2
NTR
O
N
O
N
R
R
HOHN
O2N
Cl
Cl
2
1
R = H
19 R = EDG or EWG
Nu
Introduction of a methyl group at the benzylic carbon of 1
resulted in two diastereomers (14A, 14B) that were separable on
silica gel. This modification did not significantly affect the com-
pound’s biological properties in the mammalian system: the two
diastereomers had similar enzymatic activities against the bacte-
rial enzyme as compared to 1 and gave IC50 values approximately
2- to 3-fold higher when screened against V79NTR+ and SKOVNTR+
Nu
R
DNA
R
HOHN
HOHN
O
4
5
Nu
Cl
Cl
Nu
O
P
NH2
O
DNA
O
P
N
O
N
O P N
NH2
NH2
a
Cl
Cl
R = 2-OCH3
1.HOBt/DCC
2.NaBH4
THF, r.t.
COOH
R
b
c
d
e
f
R = 3-OCH3
R = 3-CH3
7
6
3
OH
R
R = 2-CF3
O2N
O2N
R
R = 3-CONH2
R = 2-CONH2
R = 3-COOCH3
R = 2-CH2CONH2
R = 2-F
Nu
Nu
Nu
10
11
O
P
NH2
O
Cl
g
h
i
O
N
O P N
O
P
NH2
cross-linking DNA strands
leading to cytotoxicity
1. BuLi
NH2
2. Cl2PON(CH2CH2Cl)2
O
N
j
R = 3-F
R = 2,6-diF
R = 2-Cl
3. NH3
8
9
k
l
Nu
O2N
Cl
THF, -78 °C
19a-l
Scheme 1. Proposed mechanism of activation of 4-nitrobenzyl phosphoramide
mustards.
Scheme 2. Synthesis of substituted 4-nitrobenzyl phosphoramide mustards.

Table 1
Nitroreductase activation of nitroarylmethyl phosphoramidates and analogs in V79 cells and trypanosomal parasites
Compd
nfsB Activitya(nmol NADH
oxidized/min/mg)
V79 cells, 72 h exposure
SKOV3 cells, 18 h exposure
TbNTR activitya (nmol
NADH oxidized/min/mg)
Trypanocidal activity
(IC50 M)
, l
IC50
M), NTRÀ
IC50
Ratio
IC50
943
(l
M), NTRÀ IC50
(l
M) NTR+ Ratioc
(NTRÀ/NTR+)
T. cruzi T. brucei L. major
b
b
c
b
b
(l
(l
M) NTR+ (NTRÀ/NTR+
Cl
Cl
O
P
O
N
N
1
1283
256
683
1405
916
ND
59
0.0008
0.18
73,750
>556
0.099
9525
>190
17
246
19
3.4
>10
>10
>10
>10
>10
ND
>30
>30
>30
>30
>30
8.3
NH2
O2N
NO2
Cl
O
P
O
12
>100
>100
57
>1000
947
5.23
55
>10
>10
>10
>10
1.6
NH2
Cl
N
Cl
O
P
O2N
O
13
14A
14B
15
0.26
>385
369
34
NH2
Cl
Cl
O
O
O
P
N
0.002
0.002
0.009
28,500
32,500
8,333
>1000
>1000
ND
0.28
0.49
ND
>3500
>2000
ND
NH2
O2N
O2N
Cl
Cl
O
P
N
65
52
NH2
Cl
Cl
O
N
O
O
P
N
75
ND
NH2
O2N
O2N
Cl
O
Cl
N
N
NDd
45
1.6
28
>300
16
>18
1122
>10
>10
16.23
16
Cl
Cl
O
S
Cl
O
O
ND
ND
62
6
2.9
21
>300
1102
>300
0.09
ND
ND
ND
ND
ND
ND
ND
ND
ND
17
18
O2N
O2N
Br
O
P
N
0.0001
60,000
12244
NH2
Br
a
Nitroreductase substrate activity expressed as the initial velocity (nmol of NADH oxidized/min/mg) in the presence of either E. coli nfsB or T. brucei nitroreductase (TbNTR) by following the change in absorption at 340 nm.
V79 cells were transfected with vector only while a bicistronic construct coding for the E. coli nfsB and a puromycin selective marker was introduced to form the V79NTR+ line. IC50 values are the concentration required to reduce
b
the cell number to 50% of control after the cells were exposed to the drug. The standard errors of all assays were within 10% of the mean between replicates at a given concentration and 10–20% for the fitted IC50 values.
c
Ratio of IC50 values (NTRÀ/NTR+) as an indication of activation by E. coli nitroreductase.
Was not determined.
d

L. Hu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3986–3991
3989
cells. In contrast, the two diastereomers were not efficiently re-
duced by the parasite enzyme and displayed no antiparasitic activ-
ity at concentrations tested. Replacing the phenyl ring in 1 with a
pyridinyl ring (15) generated a compound with significant antipro-
liferative activity against the V79NTR+ cells, although the IC50 value
is 10-fold higher than that for 1. This compound also had growth
inhibitory activity against bloodstream form T. brucei and L. major
promastigotes, in these cases better than parental structure: the
Directhalogen substitution on thephenyl ring exhibitedinterest-
ing SAR in terms of antiproliferative and antiparasitic activity. Fluo-
rine substitution at the ortho- (19i) or meta- (19j) positions on the
phenyl ring had different effects on the activation and release of
cytotoxic phosphoramide mustard. When screened against TbNTR,
both compounds generated a significantly higher enzymatic activity
than 1. However, in the case of 19j, this increased turnover when
compared to 1, was not reflected in an increased trypanocidal capac-
ity, although an improvement in leishmanicidal activity was de-
tected. In contrast, the increased enzymatic activity observed with
19i did correlate with increased parasite toxicity in all the pathogens
tested. Similarly, V79NTR+ and SKOV3NTR+ cells were more suscepti-
ble to 19i, generating IC50 values slightly lower than both 1 and
19j. The fact that 19j was a better substrate of NTR but had lower
antiparasitic effect than 19i suggests that the difference in antipro-
liferative and trypanocidal activity is the result of the structural
effects of the fluorine substitutions on the second step of activation
and eventual release of cytotoxic phosphoramide mustard rather
than the first step of nitro reduction. This preference of 2-F substitu-
tion could be due to the balanced electronic inductive and resonance
effect of fluorine: although electron-withdrawing fluorine
decreased the electron density of hydroxylamino intermediate 2,
and thus might impact the 1,6-elimination process, fluorine also
facilitated the cleavage of benzylic C–O bond through its electron-
donating resonance effect. Introduction of two fluorines, one each
side of the benzylic carbon (19k) maintained the subnanomolar
antiproliferative activity towards V79NTR+ cells. However, 19k was
also 4-fold more cytotoxic towards V79NTRÀ and SKOV3NTRÀ cells.
Substitution of a chlorine at the ortho-position (19l) produced a
reduction in antiproliferative activity against V79NTR+ and
SKOV3NTR+. This combined with increased toxicity toward the
appropriate control lines resulted in lower selectivity for 19l.
However, this compound was reduced at a similar rate as 19i and
was a highly effective antiparasitic agent. Against bloodstream form
T. brucei, 19l was 425-fold more effective at inhibiting pathogen
growth than 1 with an IC50 value of 8 nM and gave IC50 values
pyridinylmethyl-containing compound 15 gave an IC50 of 1.6
against T. brucei and 8.3 M against L. major while the benzyl-con-
taining compound 1 yielded values of 3.4 M against T. brucei and
no activity (>30 M) toward L. major. Replacement of the phos-
lM
l
l
l
phoramidate with carbamate (16) or sulfamidate (17) was not ex-
pected to affect enzyme substrate activity, but was found to have
reduced antiproliferative activity against mammalian NTR express-
ing cells by 2 to 3 orders of magnitude, indicating the phosphoram-
idate functionality was critical to prodrug activation and the
resulting cytotoxicity. Similarly, the 4-nitrobenzyl carbamate
analogue 16 was efficiently reduced by the parasite Type I NTR
but this did not translate to a significant anti-trypanosomatid
activity: 16 did not affect trypanosomal growth at concentrations
up to 10
lM, while a low leishmanicidal activity was observed
(16.2 M). As 16 was metabolized by the TbNTR in vitro, inefficient
l
uptake by the parasite and transport into the mitochondrion, the
organelle where this enzyme is located, may account for the poor
anti-trypanosomatid activity. Alternatively, cleavage of the ben-
zylic C–O bond following nitro reduction and subsequent elimina-
tion of the nitrogen mustard may be inefficient for this compound.
Replacement of chlorine with bromine in the mustard portion (18)
resulted in an improvement in cytotoxicity but not selectivity
against V79NTR+ cells. When screened against SKOV3 cells, com-
pound 18 were shown to be similar in cytotoxicity and selectivity
against SKOV3NTR+ cells.
In the second series, various substitutions were introduced on
the phenyl ring to optimize their NTR substrate activity and their
antiproliferative and antiparasitic activity. As shown in Table 2,
the introduction of an electron donating –OCH3 ortho to the phos-
phoramide mustard substituent on the phenyl ring (19a) generated
a substrate that showed preferential reduction by E. coli nfsB (NTR
activity against 19a was 5-fold higher than against 1), and exhib-
ited similar subnanomolar growth inhibitory effect towards
V79NTR+ cells. Additionally, both compounds displayed comparable
toxicity to controls resulting in similar selectivity ratios. In con-
trast, the T. brucei NTR enzyme reduced both compounds at equiv-
alent rates with 19a showing a slight improvement in antiparasitic
activity against T. brucei and L. major. When –OCH3 (19b) or –CH3
(19c) substituents were introduced meta to phosphoramide mus-
tard substituent, a slight reduction (3.8-fold) in antiproliferative
activity against V79NTR+ cells was observed while sensitivity of
the control cells remained unchanged. Neither of these compounds
displayed any antiparasitic activity and both were considered
very poor substrates for TbNTR. Introduction of electron-with-
drawing –CONH2 (19e) and –COOCH3 (19g) at the meta-position
resulted in a 6- to 8-fold reduction in E. coli nfsB enzymatic activ-
ity, translating to a lower antiproliferative activity towards V79NTR+
and SKOV3NTR+ cells. A similar effect was seen when –CONH2 (19f)
and –CH2CONH2 (19h) substituents were added to the ortho-posi-
tion. Introduction of an electron withdrawing CF3 at the ortho-po-
sition (19d) caused a 5-fold decrease in antiproliferative activity
towards V79NTR+ cells while having no appreciable difference in
its effect on control cells. When screened against SKOV3 cells, a
reduction in sensitivity was observed in both E. coli nfsB expressing
and non-expressing lines, resulting in 19d displaying lower selec-
tivity than 1. Interestingly, all trypanosomatid lines were more
sensitive to 19d than to 1 and this correlated with an increased
rate of reduction by the parasite NTR.
between 3 and 10 lM for T. cruzi and L. major. The antiparasitic activ-
ity was increased further by incorporating two fluorine substituents,
one on each side of the benzylic carbon (19k). Out of all the mustards
analyzed, 19k was the most effective substrate for TbNTR and
displayed the highest potency against all parasite forms: 19k was
485-fold more effective at inhibiting pathogen growth than 1, with
an IC50 value of 7 nM against bloodstream form T. brucei and about
1
lM against T. cruzi and L. major.
For gene-directed prodrug therapy (GDEPT) of cancer, it is unli-
kely that all tumor cells will be transfected by the vector and thus
not all tumor cells will express the prodrug-converting enzyme.
Consequently, some tumor cells would not be directly exposed to
the active agent released from its corresponding prodrug form
upon enzymatic activation. Therefore, the ideal active agent is able
to diffuse into the intercellular fluid and kill neighboring tumor
cells through the bystander effect, which is crucial to the success
of GDEPT as the effect of the drug is amplified. The bystander effect
(BE50) of compounds (1, 19d, 19i, 19j, 19k and 19l) were measured
in SKOV3 cell lines by quantitating the percentage of NTR+ cells in
a mixed population of NTR+ and NTRÀ cells to produce an IC50
midway on a log scale (geometric mean) between those in either
NTR+ or NTRÀ cell type alone; BE50 is a parameter used to compare
the bystander effect of different compounds similar to the TE50 re-
ported previously.15,21 As shown in Table 2, the results showed that
the introduction of one fluorine at either ortho- or meta-positions
and a CF3 or chlorine at the ortho-position improved the bystander
effect by ꢀ2-fold while the introduction of two fluorines at the
ortho positions decreased the bystander effect as compared to
the unsubstituted compound 1. Therefore, out of all the derivatives
synthesized, the 2-fluoro-4-nitrobenzyl phosphoramide mustard

Table 2
Nitroreductase activation of substituted 4-nitrobenzylmethyl phosphoramide mustards in V79 cells, SKOV3 cells, and trypanosomal parasites
Cl
6
3
O
P
NH2
1
5
O
N
R
2
O2N
4
Cl
1, 19a-l
Compd
R
nfsB Activitya (nmol NADH
oxidized/min/mg)
V79 cells, 72 h exposure
SKOV3 cells, 18 h exposure
TbNTR Activitya (nmol
NADH oxidized/min/mg)
Trypanocidal activity
(IC50
M)b
,
l
b
b
b
b
b
IC50
M) NTRÀ
IC50
Ratioc
IC50
M) NTRÀ
IC50
M) NTR+
Ratioc
IC50
(
l
M) 25%
BEd
T. brucei
T. cruzi
L. major
50
(
l
(
l
M) NTR+
(
l
(
l
NTR+ 75% NTRÀ
1
H
1283
6978
2444
894
NDe
211
ND
167
ND
ND
59
41
48
48
0.0008
0.0005
0.003
0.003
0.004
0.03
0.04
0.1
0.05
0.0004
0.003
0.0008
0.002
73,750
82,000
16,000
16,000
12,500
>3,330
>2,500
690
>2,000
122,750
16,000
20,880
13,100
943
847
641
477
172
>1000
>100
435
>100
266
246
257
140
0.099
0.023
0.44
0.45
0.31
6.75
0.8
0.43
1.1
0.08
0.21
0.17
0.12
9,520
3680
1460
1060
570
>150
>125
1000
>90
3,290
1,200
1520
1,120
0.98
ND
ND
ND
1.01
ND
ND
ND
ND
0.37
0.48
1.37
0.54
5.6%
ND
ND
ND
2.8%
ND
ND
ND
ND
3.9%
4.2%
9.2%
3.2%
246
239
0
158
654
ND
ND
ND
ND
832
1116
1454
706
3.4
1.2
>10
>10
0.15
ND
ND
ND
ND
0.27
3.2
>10
>10
>10
>10
7.97
ND
ND
ND
ND
9.54
>10
0.99
9.14
>30
15.6
>30
>30
5.88
ND
ND
ND
ND
4.72
9.05
1.29
3.10
19a
19b
19c
19d
19e
19f
19g
19h
19i
19j
19k
19l
2-OCH3
3-OCH3
3-CH3
2-CF3
50
3-CONH2
2-CONH2
3-COOCH3
2-CH2CONH2
2-F
3-F
2,6-diF
2-Cl
>100
>100
69
>100
49
48
17
26
ND
ND
ND
0.007
0.008
a
Nitroreductase substrate activity expressed as the initial velocity (nmoles of NADH oxidized/min/mg) in the presence of either E. coli nfsB or T. brucei nitroreductase (TbNTR) by following the change in absorption at 340 nm.
V79 cells were transfected with vector only while a bicistronic construct coding for the E. coli nfsB and a puromycin selective marker was introduced to form the V79NTR+ line. For SKOV3, NTR-expressing cells (SKOV3NTR+) were
b
compared with untransfected parental line. IC50 values are the concentration required to reduce the cell number to 50% of control after the cells were exposed to the drug. The standard errors of all assays were within 10% of the
mean between replicates at a given concentration and 10–20% for the fitted IC50 values.
c
Ratio of IC50 values (NTRÀ/NTR+) as an indication of activation by E. coli nitroreductase.
d
BE50 values refer to percentage of NTR+ cells in a mixed population of NTR+ and NTRÀ cells to produce an IC50 midway on a log scale (geometric mean) between those in either NTR+ or NTRÀ cell type alone.
e
Was not determined.

L. Hu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3986–3991
3. Huang, P. S.; Oliffa, A. Curr. Opin. Genet. Dev. 2001, 11, 104.
3991
19i showed the best profile in GDEPT of cancer with excellent
activity, selectivity and improved bystander effect although the
selectivity of 19i was slightly lower than that of 1 in SKOV3 cells.
In summary, 4-nitrobenzyl phosphoramide mustards were iden-
tified as excellent substrates of nitroreductases from E. coli and
T. brucei. In vitro cell culture assays showed that they were highly
cytotoxic against NTR+ mammalian cell lines and trypanosomes
with some having relatively low cytotoxicity against non-type I
NTR expressing cells. The excellent bystander effect and selectivity
suggest that our nitroarylmethyl phosphoramide mustards are
good candidates for use in combination with nitroreductase in
ADEPT or GDEPT of cancer and as potential chemotherapeutic
agents for the treatment of trypanosomal infections.
4. Denny, W. A. Aust. J. Chem. 2004, 57, 821.
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Acknowledgments
17. Hu, L.; Yu, C.; Jiang, Y.; Han, J.; Li, Z.; Browne, P.; Race, P. R.; Knox, R. J.; Searle, P.
F.; Hyde, E. I. J. Med. Chem. 2003, 46, 4818.
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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.) and grants G9806623/44940 from the UK Medical
Research Council and C1007/A6688 from Cancer Research UK (to
P.F.S.).
21. Jiang, Y.; Han, J.; Yu, C.; Vass, S. O.; Searle, P. F.; Browne, P.; Knox, R. J.; Hu, L. J.
Med. Chem. 2006, 49, 4333.
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