Nitroarylmethylcarbamate prodrugs of doxorubicin for use with nitroreductase gene-directed enzyme prodrug therapy

Article abstract of DOI:10.1016/j.bmc.2005.03.055

A series of nitrobenzyl- and nitroimidazolylmethyl carbamate prodrugs of doxorubicin were prepared and evaluated for their potential use in nitroreductase (NTR) mediated gene-directed enzyme prodrug therapy (GDEPT). The carbamate prodrugs and doxorubicin

Full text of DOI:10.1016/j.bmc.2005.03.055

Bioorganic & Medicinal Chemistry 13 (2005) 4043–4055
Nitroarylmethylcarbamate prodrugs of doxorubicin for use with
nitroreductase gene-directed enzyme prodrug therapy
Michael P. Hay,* William R. Wilson and William A. Denny
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland,
Private Bag 92019, Auckland, New Zealand
Received 27 February 2005; revised 30 March 2005; accepted 30 March 2005
Available online 25 April 2005
Abstract—A series of nitrobenzyl- and nitroimidazolylmethyl carbamate prodrugs of doxorubicin were prepared and evaluated for
their potential use in nitroreductase (NTR) mediated gene-directed enzyme prodrug therapy (GDEPT). The carbamate prodrugs
and doxorubicin were tested in a cell line panel comprising parental and NTR transfected human (SKOV3/SKOV3–NTRneo
,
WiDr/WiDr–NTRneo), Chinese hamster (V79/V79–NTRpuro) and murine (EMT6/EMT6–NTRpuro) cell line pairs, and were com-
pared with the established NTR substrates CB 1954 (an aziridinyl dinitrobenzamide) and the analogous dibromomustard SN
29427. The low solubility of the prodrugs (from 3 to 39 lM) precluded the determination of IC50 values against the parent cell lines
in some instances. All of the prodrugs were unstable in culture medium with 5% added fetal calf serum over a 24 h period, although
release of doxorubicin was not observed. The prodrugs were 20- to >336-fold less toxic than doxorubicin in the human cells lines
SKOV3 and WiDr, with overall less deactivation seen in the V79 cell line (11- to >286-fold) and EMT6 cell line (1.8- to >178-fold).
Prodrugs with the nitrobenzyl unit directly conjugated to doxorubicin showed modest selectivity for NTR across the cell line panel
(1- to 5.9-fold) but this was increased to between >10- and >370-fold with the interpolation of an 4-aminobenzyl spacer unit between
the bioreductive unit and doxorubicin. A 2-nitroimidazolylmethyl carbamate provided deactivation of doxorubicin (8- to 124-fold)
but showed only modest selectivity for NTR (2- to 14-fold) across the panel. The interpolation of a 4-aminobenzyl spacer gave
slightly lower deactivation (3- to 64-fold) and similar selectivity for NTR (>1.2- to >12-fold) for 2- and 5-nitroimidazolylmethyl
prodrugs. The activity of two nitrobenzyl prodrugs containing an aminobenzyl spacer, providing excellent selectivity for NTR+ve
cells in culture, was evaluated against EMT6 tumours comprising ca. 10% NTR+ve cells, but neither showed statistically significant
levels of killing even of NTR+ve cells. This lack of activity in tumours, despite potent and selective activity in culture, indicates that
pharmacokinetic optimization is needed to achieve in vivo efficacy against solid tumours with this new class of NTR prodrugs.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
tard 2 (SN 29427) shows increased in vivo activity and a
larger bystander effect compared to CB 1954 in preclin-
ical models.22 A second class of prodrugs under investi-
gation is based on the 4-nitrobenzylcarbamate moiety
(3), which undergoes reduction to the hydroxylamine
and subsequent fragmentation to release a cytotoxic
amine.23–25 Masking the amine function as a carbamate
provides significant deactivation of the cytotoxin by
either electronic effects, where electronic release from
the amine is required for activation of the cytotoxin
[rp(NH ) = ꢀ0.66, rp(OCONH) = ꢀ0.17], or by steric effects
limitin2g binding to the site of action. A variety of cyto-
toxic agents bearing a critical amine group have been
examined as their 4-nitrobenzylcarbamate prodrugs for
NTR including aniline mustard,26 mitomycin,26 enediy-
nes,27,28 seco-cyclopropylindoline derivatives,29 pyrrolo-
benzodiazepines,30 anthracyclines26 and tallimustine
analogues.31 Related nitrobenzyl phosphorodiamidate32
The development of gene-directed enzyme prodrug ther-
apy (GDEPT)1–5 using an oxygen-insensitive nitrore-
ductase from Escherichia coli B (the nfsB gene product
NTR) has been the subject of considerable study6 since
the first description of NTR.7–10 Two main classes of
nitroaromatic substrate have been identified as prodrugs
activated by NTR: 2,4-dinitrobenzamides and 4-nitro-
benzylcarbamates. The first-identified NTR prodrug,
the 2,4-dinitrobenzamide, CB 1954 (1)7,11–13 has ad-
vanced to clinical trial14–16 and structure–activity rela-
tionships (SAR) have been determined for related
aziridines17 and nitrogen mustards.18–21 The bromomus-
Keywords: Prodrug; Doxorubicin; Nitroreductase.
*
Corresponding author. Tel.: +64 9 3737599x86598; fax: +64 9
3737502; e-mail: m.hay@auckland.ac.nz
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.03.055

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M. P. Hay et al. / Bioorg. Med. Chem. 13 (2005) 4043–4055
and nitrobenzyl phosphoroamide33 prodrugs of alkylat-
ing agents have also been reported.
2. Results and discussion
2.1. Chemistry
Recently we extended the 4-nitrobenzylcarbamate class
of prodrugs to include 2-alkoxy-4-nitrobenzylcarba-
mates (e.g., 4), where the addition of the 2-alkoxy sub-
stituents improved solubility and provided faster
fragmentation kinetics.24 Several prodrugs of 5-amino-
benz[e]indoline alkylating agents were good substrates
for NTR in human cell lines in vitro and were more
selectively cytotoxic to NTR+ve cells than the unsubsti-
tuted 4-nitrobenzyl carbamate.34 Additionally, we iden-
tified a range of nitroheterocyclic methyl carbamates of
a 5-aminobenz[e]indoline alkylating agent (e.g., 5),
which were good substrates for NTR in human cell lines
in vitro and were as selectively cytotoxic to NTR+ve
cells as the 2-alkoxy-4-nitrobenzylcarbamate pro-
drugs.35,36
4-Nitrobenzyl alcohol 15 and 2-methoxy-4-nitrobenzyl
alcohol 1624 were activated as the 4-nitrophenyl carbon-
ates 1826 and 19 (Scheme 1). Displacement of the car-
bonates 18 and 19 with 6 gave 7 and 8, respectively, in
excellent yield. Similarly, reaction of the THP-protected
propyloxy ether 17, prepared by alkylation of methyl
4-nitrosalicylate with the protected iodopropanol and
subsequent DIBAL-H reduction of the ester, with 4-
nitrophenylchloroformate gave carbonate 20. Deprotec-
tion under acidic conditions gave 21, which reacted with
6 to give carbamate 9. Similarly, 2-nitroimidazole-5-
methanol (22) was activated as the carbonate 2349 and
reacted with 6 to give a directly linked 2-nitroimidazole
carbamate 10.
In an effort to consolidate the initial promise shown by 4
and 5 as prodrugs for NTR we sought to conjugate the
bioreductive elements identified in our previous stud-
ies24,34–36 with doxorubicin (6) via its 30-amino group.
Doxorubicin (6) has been a popular choice37–42 as a
cytotoxic effector in numerous prodrug strategies be-
cause of the requirement of a free 30-amino group for
activity, high potency, broad-spectrum activity and
established clinical efficacy. The 4-nitrobenzylcarbamate
linked to the 30-amine of doxorubicin (7) has been pre-
pared previously and although 7 was a substrate for
NTR, the authors concluded doxorubicin was not re-
leased upon reduction.26 There is evidence43–48 that the
rate of activation of some anthracycline prodrugs is en-
hanced by inclusion of a 4-aminobenzyloxycarbonyl
ꢀspacerꢁ, which undergoes spontaneous fragmentation
via a 1,6-elimination process.23
An iterative process was used to obtain carbamates with
a 4-aminobenzyl spacer between the nitroaryl trigger
and 6. Reaction of 4-nitrobenzylchloroformate 24 with
4-aminobenzyl TBDMS ether 25 in the presence of
HOBT50 gave carbamate 26, which was deprotected to
give 27 (Scheme 2). Activation of alcohol 27 with 4-
nitrophenylchloroformate gave carbonate 30, which re-
acted with 6 to give carbamate 11. Similarly, reaction
of carbonate 19 with 25 gave carbamate 28, which was
deprotected to give 29. Activation of alcohol 29 as the
4-nitrophenyl carbonate 30 facilitated reaction with 6
to give carbamate 12. Similar reaction sequences with
2-nitroimidazolyl-5-methyl carbonate (23) and 5-nitro-
imidazolyl-2-methyl carbonate (35)49 produced the
corresponding nitroimidazole-spacer-doxorubicin pro-
drugs 13 (Scheme 3) and 14 (Scheme 4), respectively.
2.2. Solubility and stability
We report here the synthesis of, and in vitro and in vivo
studies on directly linked 4-nitroarylcarbamates 7–11
and 4-nitroarylcarbamates containing a ꢀself-immola-
tiveꢁ linker 12–14 as prodrugs of doxorubicin (6) for
an NTR-mediated GDEPT approach.
The purity of the compounds 7–14 was shown by HPLC
analysis to be 98% or greater. The amount of effector 6
detected was generally less than 0.1%, although 0.3%
was detected in samples of 11 and 14 and 0.5% in 10.

M. P. Hay et al. / Bioorg. Med. Chem. 13 (2005) 4043–4055
4045
Scheme 1. Reagents: (i) NO2PhOCOCl, DIEA, THF; (ii) HCl, MeOH; (iii) DOX, Et3N, DMF.
˚
Scheme 2. Reagents: (i) (25), Et3N, HOBT, 4 A sieves, THF; (ii) HCl, MeOH; (iii) 4-NO2PhOCOCl, DIEA, THF; (iv) DOX, Et3N, DMF.
˚
Scheme 3. Reagents: (i) (25), Et3N, HOBT, 4 A sieves, THF; (ii) HCl, MeOH; (iii) 4-NO2PhOCOCl, DIEA, THF; (iv) DOX, Et3N, DMF.

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M. P. Hay et al. / Bioorg. Med. Chem. 13 (2005) 4043–4055
˚
Scheme 4. Reagents: (i) (25), Et3N, HOBT, 4 A sieves, THF; (ii) HCl, MeOH; (iii) 4-NO2PhOCOCl, DIEA, THF; (iv) DOX, Et3N, DMF.
The solubility of compounds (1, 2, 7–14) in a-MEM cul-
ture medium with 5% added fetal calf serum (FCS) was
determined by HPLC analysis of the supernatant of a
saturated solution (Table 1). The prodrugs 7–14 had sol-
ubilities ranging from 3 to 39 lM: considerably less sol-
uble than CB 1954 (1) and similar to the bromomustard
2. The addition of a 2-OMe group provided an apprecia-
ble increase in solubility over the unsubstituted nitro-
benzyl analogue 7 in the directly linked prodrug 8, but
this effect was not seen when a 4-aminobenzyl linker unit
was present, that is, 11 and 12. The prodrugs 7–14 were
unstable in a-MEM + 5% FCS, with 20–87% prodrug
remaining after 24 h whereas both 1 and 2 were stable
under these conditions.
12 were >286- to >336-fold less toxic than 6 in the hu-
man and hamster cell lines (SKOV, WiDr and V79)
and 57- to >178-fold less toxic than 6 in the murine cell
line (EMT6). The 2- and 5-nitroimidazole spacer-linked
prodrugs 13 and 14 were more toxic than their nitroben-
zyl counterparts 11 and 12; with 13 and 14 being 34- to
64-fold less toxic than 6 in SKOV, WiDr and V79 cells,
and 3- to 4-fold less toxic than 6 in EMT6 cells. Directly
linked prodrugs 7–10 displayed modest selectivity for
NTR in human cell lines (1.8- to 14-fold) and lower
selectivity in the rodent lines with only the 2-nitroimi-
dazole 10 being selective across the panel (2.6- to 14-
fold). Similarly, the nitroimidazole prodrugs 13 and 14
containing a spacer unit displayed only modest selectiv-
ity. However, the nitrobenzyl prodrugs containing a
spacer unit, 11 and 12, showed good to excellent selec-
tivity (>16- to >370-fold) although solubility prevented
determination of an upper limit. The selectivity for
NTR observed for 11 and 12 was comparable to 1 and
2 in the human cell line pairs and only ca. 2.5- to 6-fold
lower in the EMT6 cell line pair.
2.3. In vitro cytotoxicity
The effectors and prodrugs were evaluated for cytotoxic-
ity in four pairs of cell lines, each comprising a transfec-
tant stably expressing NTR and its non-NTR-expressing
counterpart. The parental lines were SKOV3 (human
ovarian carcinoma), WiDr (human colon carcinoma),
V79puro (Chinese hamster fibroblast) and EMT6 (mouse
mammary carcinoma). Cytotoxicity was measured as
IC50 values following an 18 h drug exposure in the
NTRꢀve lines, and these values are reported in Table
1, together with the ratios of the IC50 values between
the NTRꢀve and NTR+ve lines which are a measure
of selectivity for NTR-expressing cells. The compounds
were compared with the established NTR prodrugs CB
1954 (1) and the related dibromomustard 2 in order to
evaluate their potential as GDEPT candidates.22 Doxo-
rubicin (6) is a potent cytotoxin with IC50 values ranging
from 30 to 56 nM against the cell line panel (Table 1)
and showed no selectivity for NTR+ve cells. The di-
rectly-linked prodrugs 7–10 were ca. 20- to 124-fold less
toxic than 6 to the human NTR-ve cell lines (SKOV and
WiDr) and similarly ca. 11- to 88-fold less toxic in the
hamster NTRꢀve line (V79), but were only 1.4- to 8-
fold less toxic against the mouse cell line (EMT6). The
nitrobenzyl prodrugs containing a spacer unit, 11 and
12, were considerably less toxic overall, to the extent
that cytotoxicity in NTRꢀve cell lines could not be
quantified at the solubility limit. Compounds 11 and
2.4. In vivo evaluation
Prodrugs 11 and 12 were selected for in vivo evaluation
because of their selectivity for NTR across the cell line
panel. The maximum tolerated dose of 11 given by intra-
peritoneal (i.p.) injection in DMSO to C3H mice was
>1000 lmol kgꢀ1, whereas the MTD of 12 given i.p. in
DMSO to Swiss nude mice was >750 lmol kgꢀ1. The
MTD of 1 was 240 lmol kgꢀ1 given i.p. in DMSO to
C3H mice whereas 2 was considerably less toxic
(>1330 lmol kgꢀ1).34 The activity of 11 and 12 were
evaluated against NTR-expressing EMT6 tumours com-
prising mixtures of NTRꢀve and NTR+ve cells. In this
model nude mice are inoculated with 2:1 mixtures of
EMT6–NTRpuro and EMT6 (NTRꢀve) cells, respec-
tively, providing ca. 10% NTR+ve cells at the time of tu-
mour treatment.34,51 This tumour model represents the
likely situation in a GDEPT protocol where low rates
of transfection of tumour tissue are expected. Com-
pound 11 provided no killing of either NTR+ve or
ꢀve tumour cells and although a small decrease in tu-
mour cells was observed after treatment with 12 these

M. P. Hay et al. / Bioorg. Med. Chem. 13 (2005) 4043–4055
4047
Figure 1. Comparison of in vivo activity and bystander effects for CB
1954 (1) bromomustard 2, doxorubicin prodrugs 11 and 12. Prodrugs
given at 200, 1330, 1000 and 750 lmol kgꢀ1 i.p. For experiments with
1, 2 and 12, control tumours at excision comprised 10.3% EMT6–
NTRpuro, and for experiments with 11 control tumours at excision
comprised 9.5% EMT6–NTRpuro, as assessed by the proportion of
puromycin-resistant cells. Filled bars, EMT6–NTRpuro; shaded bars,
EMT6. *EMT6–NTRpuro < 10ꢀ4 clonogens/g.
effects were not significant (Fig. 1). In contrast, we have
previously demonstrated34 that dinitrobenzamides 1 and
2 given at 200 and 1330 lmol kgꢀ1, respectively, show
significant killing of activator (EMT6–NTRpuro) cells
and target (EMT6) cells, indicating the operation of a
bystander effect.
2.5. Discussion
Directly linked compounds showed modest deactivation
of doxorubicin and relatively low selectivity for NTR. In
the case of 2-nitroimidazole 13 the addition of a spacer
did not provide any significant increase in either the
deactivation of the cytotoxin or in selectivity for NTR
compared to 10. However, in the two nitrobenzyl ana-
logues, 11 and 12, the addition of a linker unit provided
a substantial deactivation of 6 and also an increase in
selectivity for NTR+ve cells, in accord with the previ-
ously noted requirement for a spacer for optimal pro-
drugs of doxorubicin.43–48 The instability of the
prodrugs in culture medium is a potential problem for
this class of compounds even though release of effector
6 was not detected.
The high MTD values for 11 and 12, >1000 and
>750 lM kgꢀ1, respectively, suggest the prodrug is effec-
tively deactivating doxorubicin, which has an MTD of
23.7 lmol kgꢀ1 also in C3H mice.52 However, it has
not been established that the compounds distribute from
the intraperitoneal injection site. The lack of in vivo
activity against both activator and target cells suggests
either poor plasma pharmacokinetics leading to low
amounts of prodrug reaching the tumour or poor

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M. P. Hay et al. / Bioorg. Med. Chem. 13 (2005) 4043–4055
tumour penetration resulting in little activation of the
prodrug in the activator (NTR+ve) cell population.
The high molecular weight of 12 (ca. 900 Da), the pres-
ence of seven hydrogen bond donors and 20 hydrogen
bond acceptors, and a calculated log D7.4 of 5.6 (ACD
log D Calculator v7.0: Advanced Chemistry Develop-
ment Inc, Toronto, Canada) altogether present an unfa-
vorable profile for drug distribution53 and studies of the
penetration of anthracyclines through a three-dimen-
sional cell culture models have shown that doxorubicin
itself displays limited drug penetration.54,55
an ionizing potential of 70 eV at a nominal resolution of
1000. High-resolution spectra were obtained at nominal
resolutions of 3000, 5000 or 10,000 as appropriate. All
spectra were obtained as electron impact (EI) using
PFK as the reference unless otherwise stated. Solutions
in organic solvents were dried with anhydrous Na2SO4.
Solvents were evaporated under reduced pressure on a
rotary evaporator. Thin-layer chromatography was car-
ried out on aluminium-backed silica gel plates (Merck
60F254) with visualization of components by UV light
(254 nm) or exposure to I2. Column chromatography
was carried out on silica gel, (Merck 230–400 mesh).
All compounds designated for biological evaluation
were analyzed by reverse phase HPLC using an Agilent
1100 liquid chromatograph, an Altima C18 (5 lM)
stainless steel column (150 mm · 3.2 mm i.d.) and an
Agilent 1100 diode array detector. Chromatograms were
run using various gradients of aqueous (0.045 M ammo-
nium formate and formic acid at pH 3.5) and organic
(80% MeCN/MilliQ water) phases. DCM refers to
dichloromethane; DIEA refers to diisopropylethyl-
amine; DMF refers to dry dimethylformamide; EtOAc
refers to ethyl acetate; HOBT refers to 1-hydroxybenzo-
triazole; MeOH refers to methanol; pet. ether refers to
petroleum ether, boiling range 40–60 ꢁC; THF refers
to tetrahydrofuran dried over sodium benzophenone ke-
tyl. All solvents were freshly distilled. Doxorubicin
hydrochloride (6) in a lactate buffer was dissolved in
water, neutralized with dilute aqueous NH3 and ex-
tracted with CHCl3, dried and the solvent evaporated
immediately prior to use.
3. Conclusions
These studies of nitrobenzyl- and nitroimidazolylmethyl
carbamates of doxorubicin identified two nitrobenzyl
carbamates, 11 and 12, linked to doxorubicin via an
aminobenzyl carbamate spacer, as potential prodrugs
for NTR. These compounds were considerably less po-
tent than doxorubicin across the cell line panel and
showed marked selective cytotoxicity to NTR+ve cells
in vitro, but did not show any significant in vivo activity
against tumours comprising NTR+ve and ꢀve cells in
mice. As a clinical agent, doxorubicin is an appealing
drug candidate for a GDEPT approach from a regula-
tory point of view. However, the lack of in vivo activity
of the prodrugs may reflect the physicochemical proper-
ties of these compounds that are probably outside the
acceptable range for drug-like properties. This points
to the importance of early ꢀdrugabilityꢁ screening in drug
discovery.53 This work brings to a conclusion a series of
exploratory studies24,25,27,28,34–36 searching for prodrugs,
based on the 4-nitrobenzylcarbamate motif, for NTR-
mediated GDEPT. In the course of these studies we
have identified 2-alkoxy-4-nitrobenzyl, 2-nitro-5-methyl-
imidazolyl and 2-methyl-5-nitroimidazolyl carbamates
as potential prodrugs. These carbamates provided con-
siderable deactivation of cytotoxins and displayed good
in vitro selectivity for activation by NTR in vitro, but
were not active against NTR-expressing cells in tu-
mours. Further development of these triggers is contin-
gent on the optimization of pharmacokinetic and
pharmacodynamic parameters of such prodrugs to en-
sure in vivo activity.
4.1.1. Preparation of methanols. {4-Nitro-2-[3-(tetrahy-
dro-2H-pyran-2-yloxy)propoxy]phenyl}methanol (17). A
mixture of methyl 4-nitrosalicylate (2.3 g, 11.7 mmol)
and K2CO3 (2.42 g, 17.5 mmol) in DMF (25 mL) was
stirred at 20 ꢁC for 20 min. A solution of 3-iodopropyl
tetrahydro-2H-pyran-2-yl ether (4.7 g, 17.5 mmol) in
DMF (5 mL) was added and the mixture stirred at
100 ꢁC for 2 h. The mixture was poured into water, ex-
tracted with EtOAc (3 · 100 mL), the combined organic
extracts washed with water (2 · 50 mL) and brine
(50 mL), dried and the solvent evaporated. The residue
was purified by chromatography, eluting with 20%
EtOAc/pet. ether to give methyl 4-nitro-2-[3-(tetrahy-
dro-2H-pyran-2-yloxy)propoxy]benzoate (3.66 g, 92%)
1
as a colourless oil, H NMR d 7.89 (d, J = 8.5 Hz, 1H,
4. Experimental
4.1. General
H-6), 7.80–7.84 (m, 2H, H-3, H-5), 4.60–4.62 (m, 1H,
OCHO), 4.27 (t, J = 6.2 Hz, 2H, CH2O), 3.95–4.00 (m,
1H, CH2O), 3.94 (s, 3H, OCH3), 3.79–3.86 (m, 1H,
CH2O), 3.59–3.66 (m, 1H, CH2O), 3.47–3.52 (m, 1H,
CH2O), 2.13–2.17 (m, 2H, CH2), 1.78–1.84 (m, 1H,
CH2), 1.68–1.75 (m, 1H, CH2), 1.47–1.62 (m, 4H,
2 · CH2); 13C NMR d 164.5 (CO2), 158.6 (C-2), 150.7
(C-4), 132.0 (C-6), 126.2 (C-1), 114.8 (C-5), 107.9 (C-
3), 99.0 (OCO), 66.5 (CH2O), 63.4 (CH2O), 62.4
(CH2O), 52.5 (OCH3), 30.6 (CH2), 29.3 (CH2), 25.4
(CH2), 19.6 (CH2); MS m/z 339 (M+, 2), 322 (12), 239
(20), 222 (40), 85 (100); HRMS calcd for C16H21NO7
(M+) m/z 339.1318. Found 339.1317.
Analyses were carried out in the Microchemical Labora-
tory, University of Otago, Dunedin, NZ. Melting points
were determined on an Electrothermal 2300 Melting
Point Apparatus. NMR spectra were obtained on a Bru-
1
ker Avance 400 spectrometer at 400 MHz for H and
100 MHz for 13C spectra. Spectra were obtained in
CDCl3 unless otherwise specified and are referenced to
Me4Si. Chemical shifts and coupling constants were re-
corded in units of ppm and hertz, respectively. Assign-
ments were determined using APT, COSY, HSQC and
HMBC two-dimensional experiments. Mass spectra
were determined on a VG-70SE mass spectrometer using
DIBALH (1 M in DCM, 34 mL, 34 mmol) was added
dropwise to a solution of benzoate (3.46 g, 10.2 mmol)

M. P. Hay et al. / Bioorg. Med. Chem. 13 (2005) 4043–4055
4049
in THF (100 mL) at 5 ꢁC and the solution stirred at 5 ꢁC
for 1 h. The solution was poured into a solution of so-
dium potassium tartrate (1 M, 100 mL, 0.1 mol) and
stirred for 30 min. The mixture was extracted with
EtOAc (3 · 100 mL), the combined organic fraction
washed with water (100 mL) and brine (50 mL), dried
and the solvent evaporated. The residue was purified
by chromatography, eluting with 50% EtOAc/pet. ether,
to give 17 (3.11 g, 98%) as a pale yellow solid, mp
(EtOAc/pet. ether) 64–65.5 ꢁC; 1H NMR d 7.84 (dd, J =
8.2, 2.1 Hz, 1H, H-5), 7.72 (d, J = 2.1 Hz, 1H, H-3), 7.50
(d, J = 8.2 Hz, 1H, H-6), 4.74 (dd, J = 14.8, 4.2 Hz, 2H,
CH2O), 4.58–4.61 (m, 1H, OCHO), 4.24 (t, J = 6.1 Hz,
2H, CH2O), 3.96 (dt, J = 10.0, 5.8 Hz, 1H, CH2O),
3.80–3.86 (m, 1H, CH2O), 3.62 (dt, 10.0, 5.8 Hz, 1H,
CH2O), 3.46–3.51 (m, 1H, CH2), 2.30 (br s, 1H, OH),
2.08–2.11 (m, 2H, CH2), 1.79–1.85 (m, 1H, CH2),
1.69–1.77 (m, 1H, CH2), 1.48–1.62 (m, 4H, 2 · CH2);
13C NMR d 156.5 (C-2), 148.2 (C-4), 136.8 (C-1),
128.1 (C-6), 115.9 (C-5), 105.8 (C-3), 99.3 (OCO), 65.9
(CH2O), 63.9 (CH2O), 62.8 (CH2O), 60.8 (CH2O), 30.6
(CH2), 29.3 (CH2), 25.3 (CH2), 19.7 (CH2); MS (CI)
m/z 312 (MH+, 0.5), 294 (1), 245 (15), 227 (30), 85
(100); HRMS (CI) calcd for C15H22NO6 (MH+) m/z
312.1447. Found 312.1438. Anal. Calcd for
C15H21NO6: C, 57.9; H, 6.8; N, 4.5. Found: C, 58.1;
H, 6.7; N, 4.5.
(2 · 100 mL), the combined organic fraction washed
with water (50 mL), dried and the solvent evaporated.
The residue was purified by chromatography, eluting
with a gradient (20–50%) of EtOAc/pet. ether to give
27 (442 mg, 75%) as a colourless solid, mp (EtOAc/
pet. ether) 137–140 ꢁC; 1H NMR [(CD3)2SO]: d 9.83
(s, 1H, OCONH), 8.26 (ddd, J = 8.7, 2.4, 1.9 Hz, 2H,
H-30, H-50), 7.69 (d, J = 8.7 Hz, 2H, H-20, H-60), 7.42
(d, J = 8.5 Hz, 2H, H-2, H-6), 7.22 (d, J = 8.5 Hz, 2H,
H-3, H-5), 5.30 (s, 2H, CH2O), 5.07 (t, J = 5.7 Hz, 1H,
OH), 4.42 (d, J = 5.7 Hz, 2H, CH2O); 13C NMR
[(CD3)2SO]: d 153.0 (OCONH), 147.0 (C-40), 144.5 (C-
10), 137.4 (C-1), 136.7 (C-4), 129.3 (C-3, C-5), 127.0
(C-20, C-60), 123.5 (C-30, C-50), 117.9 (C-2, C-6), 64.3
(CH2O), 62.5 (CH2O). Anal. Calcd for C15H14N2O5:
C, 59.6; H, 4.7; N, 9.3. Found: C, 59.7; H, 4.8; N, 9.3.
4.1.4. 2-Methoxy-4-nitrobenzyl 4-({[tert-butyl(dimethyl)-
silyl]oxy}methyl)phenylcarbamate (28). Et3N (0.40 mL,
2.8 mmol) was added to a stirred suspension of carbon-
ate 19 (0.90 g, 2.6 mmol), 4-({[tert-butyl(dimethyl)sil-
yl]oxy}methyl)aniline (25) (0.64 g, 2.7 mmol), HOBT
˚
(0.35 g, 2.6 mmol) and 4 A molecular sieves (900 mg)
in THF (80 mL) and the mixture stirred at 20 ꢁC for
16 h. The solvent was evaporated and the residue parti-
tioned between EtOAc (100 mL) and water (100 mL).
The organic fraction was washed with 1 M HCl
(2 · 40 mL), water (100 mL) and brine (50 mL), dried
and the solvent evaporated. The residue was purified
by chromatography, eluting with 20% EtOAc/pet. ether
to give 28 (0.89 g, 77%) as a white solid, mp (EtOAc/pet.
ether) 120–122 ꢁC; 1H NMR d 7.84 (dd, J = 8.3, 2.1 Hz,
1H, H-50), 7.72 (d, J = 2.1 Hz, 1H, H-30), 7.51 (d,
J = 8.3 Hz, 1H, H-60), 7.35 (d, J = 8.3 Hz, 2H, H-2,
H-6), 7.26 (d, J = 8.3 Hz, 2H, H-3, H-5), 6.76 (br s,
1H, OCONH), 5.30 (s, 2H, CH2O), 4.69 (s, 2H, CH2O-
Si), 3.93 (s, 3H, OCH3), 0.92 [s, 9H, SiC(CH3)3], 0.09
4.1.2. 4-Nitrobenzyl 4-({[tert-butyl(dimethyl)silyl]oxy}-
methyl)phenylcarbamate (26). A solution of 4-nitroben-
zyl chloroformate (24) (1.0 g, 4.6 mmol) in THF
(10 mL) was added dropwise to a stirred solution
of 4-({[tert-butyl(dimethyl)silyl]oxy}methyl)aniline (25)
(0.91 g, 4.2 mmol) and Et3N (0.76 mL, 5.5 mmol) in
THF (20 mL) at 0 ꢁC and the solution was stirred at
20 ꢁC for 16 h. The solvent was evaporated and the res-
idue dissolved in EtOAc (200 mL), washed consecutively
with 1 M HCl (100 mL), water (2 · 100 mL), aqueous
Na2CO3 (100 mL), water (100 mL) and brine (50 mL),
dried and the solvent evaporated. The residue was puri-
fied by chromatography, eluting with 10% EtOAc/pet.
ether to give 26 (1.10 g, 63%) as a white solid, mp
13
[s, 6H, Si(CH3)2]; C NMR d 157.3 (C-20), 153.0
(OCONH), 148.7 (C-40), 137.0 (C-4), 136.4 (C-1),
132.1 (C-10), 128.7 (C-60), 126.9 (C-3, C-5), 118.6
(C-2, C-6), 115.7 (C-50), 105.2 (C-30), 64.6 (CH2O),
61.4 (CH2O), 56.0 (OCH3), 26.9 [SiC(CH3)3], 18.4
[SiC(CH3)3], ꢀ5.2 [Si(CH3)2]. Anal. Calcd for
C22H30N2O6Si: C, 59.2; H, 6.8; N, 6.4. Found: C, 59.0;
H, 6.9; N, 6.3.
1
(EtOAc/pet. ether) 107–109 ꢁC; H NMR d 8.22 (ddd,
J = 8.7, 2.3, 1.8 Hz, 2H, H-30, H-50), 7.55 (d,
J = 8.7 Hz, 2H, H-20, H-60), 7.34 (br d, J = 8.1 Hz, 2H,
H-2, H-6), 7.26 (d, J = 8.6 Hz, 2H, H-3, H-5), 6.73 (br
s, 1H, OCONH), 5.29 (s, 2H, CH2O), 4.70 (s, 2H,
CH2OSi), 0.93 [s, 9H, SiC(CH3)3], 0.09 [s, 6H, Si(CH3)2];
13C NMR d 152.9 (OCONH), 147.7 (C-40), 143.5 (C-10),
137.1 (C-1), 136.1 (C-4), 128.3 (C-20, C-60), 127.1 (C-3,
C-5), 123.8 (C-30, C-50), 118.7 (C-2, C-6), 65.4 (CH2O),
64.5 (CH2O), 25.9 [SiC(CH3)3], 18.4 [SiC(CH3)3], ꢀ5.2
[Si(CH3)2]; MS m/z 416 (M+, 0.2), 401 (1), 359 (50), 241
(40), 206 (55), 162 (55), 132 (100); HRMS calcd for
C21H28N2O5Si (M+) m/z 416.1768. Found 416.1760.
Anal. Calcd for C21H28N2O5Si: C, 60.6; H, 6.8; N, 6.7.
Found: C, 60.7; H, 6.7; N, 6.8.
4.1.5. 2-Methoxy-4-nitrobenzyl 4-(hydroxymethyl)phen-
ylcarbamate (29). HCl (1 M, 4 mL, 4 mmol) was added
to a stirred solution of silylether 28 (0.89 g, 0.2 mmol)
in MeOH (10 mL) and stirred at 20 ꢁC for 1 h. The solu-
tion was poured into brine (50 mL) and extracted with
EtOAc (3 · 50 mL). The combined organic fraction
was washed with water (50 mL), dried and the solvent
evaporated. The residue was purified by chromatogra-
phy, eluting with a gradient (20–50%) of EtOAc/pet.
ether to give 29 (628 mg, 95%) as a white solid, mp
1
(EtOAc/pet. ether) 164–165 ꢁC; H NMR [(CD3)2SO]:
d 9.83 (br s, 1H, OCONH), 7.90 (dd, J = 8.3, 2.1 Hz,
1H, H-50), 7.80 (d, J = 2.1 Hz, 1H, H-30), 7.63 (d,
J = 8.3 Hz, 1H, H-60), 7.41 (d, J = 8.4 Hz, 2H, H-2, H-
6), 7.22 (d, J = 8.4 Hz, 2H, H-3, H-5), 5.21 (s, 2H,
CH2O), 5.07 (t, J = 5.6 Hz, 1H, OH), 4.41 (t, J =
5.6 Hz, 2H, CH2O), 3.97 (s, 3H, OCH3); 13C NMR
4.1.3. 4-Nitrobenzyl 4-(hydroxymethyl)phenylcarbamate
(27). HCl (1 M, 3.9 mL, 3.9 mmol) was added to a stir-
red solution of silyl ether 26 (0.81 g, 1.9 mmol) and the
solution stirred at 20 ꢁC for 15 min. The solution was
poured into brine (50 mL), extracted with EtOAc

4050
M. P. Hay et al. / Bioorg. Med. Chem. 13 (2005) 4043–4055
[(CD3)2SO]: d 157.0 (C-20), 153.0 (OCONH), 148.2 (C-
40), 137.4 (C-4), 136.7 (C-1), 132.3 (C-10), 128.8 (C-60),
127.0 (C-3, C-5), 117.9 (C-2, C-6), 115.5 (C-50), 105.4
(C-30), 62.5 (CH2O), 60.4 (CH2O), 56.0 (OCH3). Anal.
Calcd for C16H16N2O6: C, 57.8; H, 4.9; N, 8.4. Found:
C, 58.0; H, 4.7; N, 8.5.
˚
mmol), HOBT (0.97 g, 7.2 mmol) and 4 A molecular
sieves (2.5 g) in THF (100 mL) and the mixture stirred
at 20 ꢁC for 48 h. The solvent was evaporated and the
residue partitioned between EtOAc (100 mL) and water
(100 mL). The organic fraction was washed with 1 M
HCl (2 · 40 mL), water (100 mL) and brine (50 mL),
dried and the solvent evaporated. The residue was puri-
fied by chromatography, eluting with 50% EtOAc/pet.
ether to give 36 (2.57 g, 85%) as a white solid, mp
(EtOAc/pet. ether) 145–146 ꢁC; 1H NMR d 7.99 (s,
1H, H-40), 7.32 (br d, J = 8.1 Hz, 2H, H-3, H-5), 7.27
(d, J = 8.1 Hz, 2H, H-2, H-6), 6.96 (br s, 1H, OCONH),
5.30 (s, 2H, CH2O), 4.69 (s, 2H, CH2O), 4.05 (s, 3H,
NCH3), 0.93 [s, 9H, SiC(CH3)3], 0.09 [s, 6H, Si(CH3)2];
4.1.6. (1-Methyl-2-nitro-1H-imidazol-5-yl)methyl 4-({[tert-
butyl(dimethyl)silyl]oxy}methyl)phenylcarbamate
(32).
Et3N (0.26 mL, 1.9 mmol) was added to a stirred sus-
pension of (1-methyl-2-nitro-1H-imidazol-5-yl)methyl
4-nitrophenyl carbonate (23)49 (0.50 g, 1.6 mmol), 4-
({[tert-butyl(dimethyl)silyl]oxy}methyl)aniline (25) (0.40
˚
g, 1.7 mmol), HOBT (0.21 g, 1.6 mmol) and 4 A molecu-
13
lar sieves (500 mg) in THF (80 mL) and the mixture stir-
red at 20 ꢁC for 16 h. The solvent was evaporated and
the residue partitioned between EtOAc (100 mL) and
water (100 mL). The organic fraction was washed with
1 M HCl (2 · 40 mL), water (100 mL) and brine
(50 mL), dried and the solvent evaporated. The residue
was purified by chromatography, eluting with 40%
EtOAc/pet. ether to give 32 (0.43 mg, 66%) as a white
C NMR d 152.3 (OCONH), 147.0 (C-50), 139.6 (C-
20), 137.4 (C-1), 135.8 (C-40), 129.6 (C-4), 126.9 (C-2,
C-6), 118.8 (C-3, C-5), 64.5 (CH2O), 58.0 (CH2O),
33.7 (NCH3), 25.9 [SiC(CH3)3], 18.4 [SiC(CH3)3], ꢀ5.3
[Si(CH3)2]. Anal. Calcd for C19H28N4O5Si: C, 54.3; H,
6.7; N, 13.3. Found: C, 54.5; H, 7.0; N, 13.5.
4.1.9. (1-Methyl-5-nitro-1H-imidazol-2-yl)methyl 4-
(hydroxymethyl)phenylcarbamate (37). HCl (1 M,
16 mL, 16 mmol) was added to a stirred solution of sil-
ylether 36 (1.36 g, 3.2 mmol) in MeOH (50 mL) and stir-
red at 20 ꢁC for 1 h. The solution was poured into brine
(50 mL) and extracted with EtOAc (3 · 50 mL). The
combined organic fraction was washed with water
(50 mL), dried and the solvent evaporated. The residue
was recrystallized to give 37 (0.86 g, 47%) as a white so-
lid, mp (EtOAc/pet. ether) 181–183 ꢁC; 1H NMR
[(CD3)2SO]: d 9.85 (br s, 1H, OCONH), 8.09 (s, 1H,
H-40), 7.40 (d, J = 8.5 Hz, 2H, H-3, H-5), 7.22 (d,
J = 8.5 Hz, 2H, H-2, H-6), 5.29 (s, 2H, CH2O), 4.42 (s,
2H, CH2O), 3.96 (s, 3H, NCH3), 3.79 (br s, 1H, OH);
13C NMR [(CD3)2SO]: d 152.5 (OCONH), 147.8 (C-
50), 139.3 (C-20), 137.4 (C-1), 136.8 (C-4), 131.7 (C-2,
C-6), 127.0 (C-3, C-5), 118.0 (C-4), 62.4 (CH2O), 57.5
(CH2O), 33.4 (NCH3). Anal. Calcd for C13H14N4O5:
C, 51.0; H, 4.6; N, 18.3. Found: C, 51.0; H, 4.6; N, 18.4.
1
solid, mp (EtOAc/pet. ether) 131–132 ꢁC; H NMR d
7.33 (br d, J = 8.8 Hz, 2H, H-3, H-5), 7.27 (d,
J = 8.8 Hz, 2H, H-2, H-6), 7.23 (s, 1H, H-40), 6.83 (br
s, 1H, OCONH), 5.22 (s, 2H, CH2O), 4.69 (s, 2H,
CH2O), 4.05 (s, 3H, NCH3), 0.93 [s, 9H, SiC(CH3)3],
0.09 [s, 6H, Si(CH3)2]; 13C NMR d 152.3 (OCONH),
146.1 (C-20), 137.4 (C-1), 135.8 (C-4), 132.5 (C-50),
129.6 (C-40), 126.9 (C-2, C-6), 118.8 (C-3, C-5), 64.5
(CH2O), 55.4 (CH2O), 34.3 (NCH3), 25.9 [SiC(CH3)3],
18.4 [SiC(CH3)3], ꢀ5.3 [Si(CH3)2]. Anal. Calcd for
C19H28N4O5Si: C, 54.3; H, 6.7; N, 13.3. Found: C,
54.5; H, 6.6; N, 13.4.
4.1.7. (1-Methyl-2-nitro-1H-imidazol-5-yl)methyl 4-(hydr-
oxymethyl)phenylcarbamate (33). HCl (1 M, 2 mL, 2
mmol) was added to a stirred solution of silylether 32
(0.39 g, 0.9 mmol) in MeOH (10 mL) and stirred at
20 ꢁC for 1 h. The solution was poured into brine
(50 mL) and extracted with EtOAc (3 · 50 mL). The
combined organic fraction was washed with water
(50 mL), dried and the solvent evaporated. The residue
was purified by chromatography, eluting with a gradient
(50–100%) of EtOAc/pet. ether to give 33 (247 mg, 87%)
as a pale yellow solid, mp (EtOAc) 180–181 ꢁC; 1H
NMR [(CD3)2SO]: d 9.77 (br s, 1H, OCONH), 7.40 (d,
J = 8.5 Hz, 2H, H-3, H-5), 7.31 (s, 1H, H-40), 7.22 (d,
J = 8.5 Hz, 2H, H-2, H-6), 5.27 (s, 2H, CH2O), 5.08 (t,
J = 5.6 Hz, 1H, OH), 4.42 (d, J = 5.6 Hz, 2H, CH2O),
3.97 (s, 3H, NCH3); 13C NMR [(CD3)2SO]: d 152.6
(OCONH), 146.0 (C-20), 137.2 (C-1), 136.8 (C-4),
133.3 (C-50), 128.7 (C-40), 127.0 (C-2, C-6), 118.0 (C-3,
C-5), 62.4 (CH2O), 55.0 (CH2O), 34.2 (NCH3). Anal.
Calcd for C13H14N4O5: C, 51.0; H, 4.6; N, 18.3. Found:
C, 51.0; H, 4.5; N, 18.2.
4.1.10. Preparation of carbonates. 2-Methoxy-4-nitro-
benzyl 4-nitrophenyl carbonate (19). A solution of 4-
nitrophenyl chloroformate (1.00 g, 5.0 mmol) in THF
(10 mL) was added dropwise to a stirred solution of 2-
methoxy-4-nitrobenzyl alcohol (16)24 (617 mg, 3.3
mmol) and DIEA (0.75 mL, 4.3 mmol) in THF
(40 mL) at 20 ꢁC and the solution stirred for 16 h. The
solution was evaporated and the residue partitioned be-
tween EtOAc (100 mL) and water (100 mL). The organ-
ic fraction was washed with water (3 · 50 mL) and brine
(50 mL), dried and the solvent evaporated. The residue
purified by chromatography, eluting with a gradient
(20–50%) EtOAc/pet. ether to give 19 (928 mg, 80%)
as pale yellow solid, mp (EtOAc/pet. ether) 105–
1
106 ꢁC; H NMR d 8.28 (ddd, J = 9.2, 3.1, 2.1 Hz, 2H,
H-3, H-5), 7.89 (dd, J = 8.3, 2.1 Hz, 1H, H-50), 7.77
(d, J = 2.1 Hz, 1H, H-30), 7.58 (d, J = 8.3 Hz, 1H, H-
60), 7.40 (ddd, J = 8.3, 3.1, 2.1 Hz, 2H, H-2, H-6), 5.41
(s, 2H, CH2O), 4.00 (s, 3H, OCH3); 13C NMR d 157.6
(C-20), 155.4 (OCO2), 152.3 (C-1), 149.2 (C-40), 145.5
(C-4), 129.8 (C-10), 129.3 (C-60), 125.3 (C-2, C-6),
121.7 (C-3, C-5), 115.8 (C-50), 105.5 (C-30), 65.3
4.1.8. 1-Methyl-5-nitro-1H-imidazol-2-yl 4-({[tert-butyl-
(dimethyl)silyl]oxy}methyl)phenylcarbamate (36). Et3N
(1.10 mL, 7.9 mmol) was added to a stirred suspension
of (1-methyl-5-nitro-1H-imidazol-2-yl)methyl 4-nitro-
phenyl carbonate (35)49 (2.31 g, 7.2 mmol), 4-({[tert-
butyl(dimethyl)silyl]oxy}methyl)aniline (25) (1.79 g, 7.9

M. P. Hay et al. / Bioorg. Med. Chem. 13 (2005) 4043–4055
4051
(CH2O), 56.2 (OCH3). Anal. Calcd for C15H12N2O8: C,
51.7; H, 3.5; N, 8.1. Found: C, 51.8; H, 3.3; N, 7.8.
123.5 (C-300, C-500), 122.5 (C-3, C-5), 118.1 (C-30, C-50),
70.2 (CH2O), 64.5 (CH2O). Anal. Calcd for
C22H17N3O9: C, 56.5; H, 3.7; N, 9.0. Found: C, 56.7;
H, 3.7; N, 8.8.
4.1.11. 4-Nitrophenyl 4-nitro-2-[3-(tetrahydro-2H-pyran-
2-yloxy)propoxy]benzyl carbonate (20). Similarly, 17
(0.44 g, 1.4 mmol) gave (i) starting material 17
(176 mg, 40%); and (ii) 20 (380 mg, 56%) as a pale yel-
4.1.14. 4-({[(2-Methoxy-4-nitrobenzyl)oxy]carbonyl}ami-
no)benzyl 4-nitrophenyl carbonate (31). Similarly, reac-
tion of 29 (282 mg, 0.9 mmol) gave 31 (238 mg, 56%)
1
low oil, H NMR d 8.30 (ddd, J = 9.2, 3.1, 2.1 Hz, 2H,
1
H-30, H-50), 7.87 (dd, J = 8.4, 2.1 Hz, 1H, H-5), 7.79
(d, J = 2.1 Hz, 1H, H-3), 7.57 (d, J = 8.4 Hz, 1H, H-6),
7.41 (ddd, J = 9.2, 3.1, 2.1 Hz, 2H, H-20, H-60), 5.42 (s,
2H, CH2O), 4.58–4.61 (m, 1H, OCHO), 4.28 (t,
J = 6.3 Hz, 2H, CH2O), 3.96 (dt, J = 10.0, 6.0 Hz, 1H,
CH2O), 3.78–3.83 (m, 1H, CH2O), 3.59 (dt, J = 10.0,
6.0 Hz, 1H, CH2O), 3.45–3.52 (m, 1H, CH2O), 2.13–
2.18 (m, 2H, CH2O), 1.79–1.86 (m, 1H, CH2), 1.67–
1.76 (m, 1H, CH2), 1.48–1.60 (m, 4H, 2 · CH2); 13C
NMR d 157.0 (C-10), 155.4 (C-2), 153.4 (OCONH),
149.2 (C-4), 145.5 (C-40), 129.9 (C-1), 129.2 (C-6),
125.3 (C-30, C-50), 121.7 (C-20, C-60), 115.6 (C-5),
106.3 (C-6), 99.1 (OCO), 66.1 (CH2O), 65.3 (CH2O),
63.5 (CH2O), 60.4 (CH2O), 30.6 (CH2), 29.4 (CH2),
25.4 (CH2), 19.7 (CH2); MS m/z 476 (M+, 2), 459 (5),
392 (2), 210(30), 85 (100); HRMS calcd for
C22H24N2O10 (M+) m/z 476.1431. Found 476.1425.
as a white powder, mp (EtOAc/DCM) 144–146 ꢁC; H
NMR [(CD3)2SO]: d 10.01 (s, 1H, OCONH), 8.31
(ddd, J = 9.1, 3.4, 2.2 Hz, 2H, H-3, H-5), 7.91 (dd,
J = 8.3, 2.2 Hz, 1H, H-500), 7.81 (d, J = 2.2 Hz, 1H, H-
300), 7.64 (d, J = 8.3 Hz, 1H, H-600), 7.56 (ddd, J = 9.1,
3.4, 2.2 Hz, 2H, H-2, H-6), 7.53 (br d, J = 8.6 Hz, 2H,
H-30, H-50), 7.41 (br d, J = 8.6 Hz, 2H, H-20, H-60),
5.24 (s, 4H, 2 · CH2O), 3.98 (s, 3H, OCH3); 13C NMR
[(CD3)2SO]: d 157.0 (C-200), 155.2 (OCO2), 153.0
(OCONH), 151.9 (C-1), 148.2 (C-400), 145.1 (C-4),
139.4 (C-1), 132.2 (C-10), 129.6 (C-20, C-60), 128.9
(C-600), 128.5 (C-40), 125.3 (C-2, C-6), 122.6 (C-3, C-5),
118.0 (C-30, C-50), 115.5 (C-500), 105.5 (C-300), 70.2
(CH2O), 60.5 (CH2O), 56.2 (OCH3); MS (FAB+) m/z
498 (MH+, 0.5); HRMS (FAB+) calcd for
C23H20N3O10 (MH+) m/z 498.1149. Found 498.1151.
Anal. Calcd for C23H19N3O10: C, 55.5; H, 3.9; N, 8.5.
Found: C, 55.3; H, 3.7; N, 8.5.
4.1.12. 2-(3-Hydroxypropoxy)-4-nitrobenzyl 4-nitro-
phenyl carbonate (21). A solution of ether 20 (207 mg,
0.5 mmol) in THF (20 mL) and 1 M HCl (5 mL) was
stirred at 20 ꢁC for 16 h. The solvent was evaporated
and the residue partitioned between EtOAc (50 mL)
and water (50 mL). The organic fraction was dried, the
solvent evaporated and the residue purified by chroma-
tography, eluting with 50% EtOAc/pet. ether, to give 21
(125 mg, 68%) as a white solid, mp (EtOAc/pet. ether)
4.1.15. 4-({[(1-Methyl-2-nitro-1H-imidazol-5-yl)meth-
oxy]carbonyl}amino)benzyl 4-nitrophenyl carbonate (34).
Similarly, reaction of 33 (219 mg, 0.7 mmol) gave 34
1
(62 mg, 18%) as a white solid, H NMR [(CD3)2SO]: d
9.96 (s, 1H, OCONH), 8.31 (ddd J = 9.2, 3.3, 2.2 Hz,
2H, H-3, H-5), 7.56 (ddd, J = 9.2, 3.3, 2.2 Hz, 2H, H-
2, H-6), 7.51 (d, J = 8.6 Hz, 2H, H-30, H-50), 7.40 (d,
J = 8.6 Hz, 2H, H-20, H-60), 7.31 (s, 1H, H-400), 5.33 (s,
2H, CH2O), 5.24 (s, 2H, CH2O), 3.98 (s, 3H, NCH3);
1
116–117 ꢁC; H NMR d 8.29 (ddd, J = 9.1, 3.2, 2.1 Hz,
2H, H-3, H-5), 7.88 (dd, J = 8.3, 2.1 Hz, 1H, H-50),
7.80 (d, J = 2.1 Hz, 1H, H-30), 7.58 (d, J = 8.3 Hz, 1H,
H-60), 7.40 (ddd, J = 9.1, 3.2, 2.1 Hz, 2H, H-2, H-6),
5.41 (s, 2H, CH2O), 4.30 (t, J = 6.0 Hz, 2H, CH2O),
3.90 (dt, J = 5.4, 4.6 Hz, 2H, CH2O), 2.10–2.15 (m,
2H, CH2), 1.65 (br s, 1H, OH); 13C NMR d 157.0 (C-
20), 155.3 (C-1), 152.3 (OCONH), 149.3 (C-40), 145.5
(C-4), 129.8 (C-10), 129.6 (C-60), 125.4 (C-2, C-6),
121.7 (C-3, C-5), 115.8 (C-50), 106.4 (C-30), 66.2
(CH2O), 65.3 (CH2O), 59.5 (CH2O), 31.7 (CH2). Anal.
Calcd for C17H16N2O9: C, 52.0; H, 4.1; N, 7.2. Found:
C, 52.3; H, 4.1; N, 6.9.
13C NMR [(CD3)2SO]:
d
155.2 (OCO2), 152.6
(OCONH), 151.9 (C-1), 145.1 (C-200), 139.2 (C-10),
133.2 (C-40), 129.6 (C-3, C-5), 128.8 (C-400), 128.7 (C-
500), 125.6 (C-20, C-60), 122.2 (C-2, C-6), 118.8 (C-20,
C-60), 70.2 (CH2O), 55.2 (CH2O), 34.2 (NCH3); MS
(FAB+) m/z 472 (MH+, 1), 443 (0.5); HRMS (FAB+)
calcd for C20H18N5O9 (MH+) m/z 472.1105. Found
472.1106.
4.1.16. 4-({[(1-Methyl-5-nitro-1H-imidazol-2-yl)meth-
oxy]carbonyl}amino)benzyl 4-nitrophenyl carbonate (38).
Similarly, reaction of 37 (0.73 g, 2.4 mmol) gave 38
1
(0.71 g, 63%) as a white solid, H NMR [(CD3)2SO]: d
4.1.13. 4-({[(4-Nitrobenzyl)oxy]carbonyl}amino)benzyl 4-
nitrophenyl carbonate (30). Similarly, reaction of 27
(320 mg, 1.1 mmol) gave 30 (81 mg, 16%) as a white
powder, mp (EtOAc/pet. ether) 165–166.5 ꢁC; 1H
NMR [(CD3)2SO]: d 10.42 (s, 1H, OCONH), 8.32
(ddd, J = 9.0, 3.2, 2.3 Hz, 2H, H-3, H-5), 8.28 (br d,
J = 8.8 Hz, 2H, H-300, H-500), 7.70 (br d, J = 8.8 Hz,
2H, H-200, H-600), 7.58 (ddd, J = 9.0, 3.2, 2.3 Hz, 2H,
H-2, H-6), 7.54 (br d, J = 8.5 Hz, 2H, H-30, H-50), 7.41
(br d, J = 8.5 Hz, 2H, H-20, H-60), 5.32 (s, 2H, CH2O),
5.24 (s, 2H, CH2O); 13C NMR [(CD3)2SO]: d 155.2
(OCO2), 153.0 (OCONH), 151.9 (C-1), 147.0 (C-400),
145.1 (C-4), 144.4 (C-100), 139.3 (C-40), 129.5 (C-20, C-
60), 128.6 (C-10), 128.4 (C-2, C-6), 125.3 (C-200, C-600),
10.02 (s, 1H, OCONH), 8.31 (ddd J = 9.1, 3.3, 2.1 Hz,
2H, H-3, H-5), 8.08 (s, 1H, H-400), 7.57 (ddd, J = 9.1,
3.3, 2.1 Hz, 2H, H-2, H-6), 7.51 (d, J = 8.5 Hz, 2H, H-
30, H-50), 7.40 (d, J = 8.5 Hz, 2H, H-20, H-60), 5.32 (s,
2H, CH2O), 4.70 (s, 2H, CH2O), 3.97 (s, 3H, NCH3);
MS (FAB+) m/z 472 (MH+, 1.5); HRMS (FAB+) calcd
for C20H18N5O9 (MH+) m/z 472.1105. Found 472.1108.
4.1.17. Preparation of carbamates (7–14). 4-Nitrobenzyl
doxorubicincarbamate (7). A solution of 4-nitrobenzyl
4-nitrophenyl carbonate (18) (32 mg, 103 lmol), pre-
pared26 from 4-nitrobenzyl alcohol (15) and 4-nitro-
phenyl chloroformate, in DMF (2 mL) was added to a
solution of doxorubicin (6) (46 mg, 86 lmol) and Et3N

4052
M. P. Hay et al. / Bioorg. Med. Chem. 13 (2005) 4043–4055
(15 lL, 104 lmol) in DMF (5 mL) at 20 ꢁC and the solu-
tion stirred for 16 h. The solvent was evaporated and the
residue purified by chromatography, eluting with a gra-
dient (0–5%) of MeOH/DCM, to give 7 (55 mg, 88%) as
a red solid, mp (DCM) 153–158 ꢁC; 1H NMR d 13.95 (s,
1H, 6-OH), 13.19 (s, 1H, 11-OH), 8.15 (d, J = 8.5 Hz,
2H, H-300), 8.01 (dd, J = 7.7, 0.7 Hz, 1H, H-1), 7.78
(dd, J = 8.0, 7.7 Hz, 1H, H-2), 7.45 (d, J = 8.5 Hz, 2H,
H-200), 7.39 (dd, J = 8.0, 0.7 Hz, 1H, H-3), 5.50 (d,
J = 3.5 Hz, 1H, H-10), 5.30 (d, J = 8.7 Hz, 1H,
OCONH), 5.28 (br s, 1H, H-7), 5.14 (d, J = 13.3 Hz,
1H, CH2O), 5.09 (d, J = 13.3 Hz, 1H, CH2O), 4.75 (s,
2H, H-14), 4.51 (s, 1H, 9-OH), 4.12–4.17 (m, 1H,
H-50), 4.08 (s, 3H, 4-OCH3), 3.83–3.09 (m, 1H, H-30),
3.67 (br s, 1H, H-40), 3.24 (d, J = 18.7 Hz, 1H, H-10),
3.04 (br s, 1H, 14-OH), 2.95 (d, J = 18.7 Hz, 1H,
H-10), 2.34 (d, J = 15.0 Hz, 1H, H-8), 2.17 (dd,
J = 15.0, 4.0 Hz, 1H, H-8), 2.13 (br d, J = 7.2 Hz, 1H,
40-OH), 1.89 (dd, J = 13.3, 4.9 Hz, 1H, H-20), 1.79 (dt,
J = 13.3, 3.8 Hz, 1H, H-20), 1.29 (d, J = 6.6 Hz, 3H, 60-
CH3); 13C NMR d 213.7 (C-13), 187.1 (C-5), 186.6
(C-12), 161.0 (C-4), 156.1 (C-6), 155.5 (C-11), 155.0
(OCONH), 147.5 (C-400), 143.8 (C-100), 135.8 (C-2),
135.4 (C-12a), 133.5 (C-6a), 13.4 (C-10a), 128.1 (C-200,
C-600), 123.7 (C-300, C-500), 120.7 (C-4a), 120.0 (C-1),
118.5 (C-3), 111.6 (C-5a), 111.4 (C-11a), 100.7 (C-10),
76.6 (C-9), 69.8 (C-7), 69.5 (C-40), 67.2 (C-50), 65.4
(C-14), 65.2 (CH2O), 56.6 (4-OCH3), 47.1 (C-30), 35.6
(C-8), 33.9 (C-10), 30.2 (C-20), 16.8 (60-CH3); MS
(FAB+) m/z 723 (MH+, 0.2); HRMS (FAB+) calcd for
C35H35N2O15 (MH+) m/z 723.2037. Found 723.2039.
Anal. Calcd for C35H34N2O15: C, 58.2; H, 4.7; N, 3.9.
Found: C, 57.8; H, 4.9; N, 3.8.
(MH+) m/z 753.2143. Found 753.2100. Anal. Calcd for
C36H36N2O16: C, 57.4; H, 4.8; N, 3.7. Found: C, 57.2;
H, 5.1; N, 3.9.
4.1.19. 2-(3-Hydroxypropoxy)-4-nitrobenzyl doxorubi-
cincarbamate (9). Similarly, reaction of 21 (41 mg,
104 lmol) and 6 (46 mg, 86 lmol) gave 9 (69 mg, 84%)
as a red solid, mp (DCM) 154–160 ꢁC; 1H NMR
[(CD3)2SO]: d 14.00 (s, 1H, 6-OH), 13.24 (s, 1H, 11-
OH), 7.85–7.89 (m, 2H, H-1, H-3), 7.80 (dd, J = 8.3,
1.8 Hz, 1H, H-500), 7.71 (d, J = 1.8 Hz, 1H, H-300), 7.62
(dd, J = 6.6, 2.8 Hz, 1H, H-2), 7.50 (d, J = 8.3 Hz, 1H,
H-600), 7.07 (d, J = 8.0 Hz, 1H, OCONH), 5.42 (s, 1H,
9-OH), 5.14 (br s, 1H, H-10), 5.05 (d, J = 18.4 Hz, 1H,
CH2O), 4.99 (d, J = 18.4 Hz, 1H, CH2O), 4.92 (br s,
1H, H-7), 4.85 (t, J = 6.0 Hz, 1H, 14-OH), 4.74 (d,
J = 5.8 Hz, 1H, 40-OH), 4.58 (d, J = 6.0 Hz, 2H, H-
14), 4.55 (t, J = 5.3 Hz, 1H, H-50), 4.14–4.20 (m, 2H,
CH2O), 3.97 (s, 3H, 4-OCH3), 3.69–3.76 (m, 1H, H-
30), 3.54 (dt, J = 6.0, 5.7 Hz, 2H, CH2O), 3.48 (br s,
1H, H-40), 3.30 (br s, 1H, OH), 2.98 (d, J = 18.2 Hz,
1H, H-10), 2.90 (d, J = 18.2 Hz, 1H, H-10), 2.22 (br d,
J = 14.4 Hz, 1H, H-8), 2.09 (dd, J = 14.4, 5.5 Hz, 1H,
H-8), 1.88–1.92 (m, 1H, H-20), 1.82–1.87 (m, 2H,
CH2), 1.50 (dd, J = 12.4, 3.7 Hz, 1H, H-20), 1.13 (d,
J = 6.4 Hz, 3H, H-60); 13C NMR [(CD3)2SO]: d 213.7
(C-13), 186.4 (C-5), 186.3 (C-12), 160.7 (C-4), 156.0
(C-200), 155.9 (C-6), 154.9 (C-11), 154.4 (OCONH),
147.8 (C-400), 136.1 (C-2), 135.4 (C-12a), 134.5 (C-6a),
134.0 (C-10a), 133.2 (C-100), 127.8 (C-600), 119.9 (C-4a),
119.6 (C-1), 118.9 (C-3), 115.3 (C-500), 110.6 (C-5a),
110.5 (C-11a), 105.8 (C-300), 100.2 (C-10), 74.8 (C-9),
69.8 (C-7), 67.9 (C-40), 66.6 (C-50), 65.6 (CH2O), 63.6
(C-14), 59.8 (CH2O), 57.0 (CH2O), 56.5 (4-OCH3),
47.2 (C-30), 36.5 (C-8), 32.0 (C-10), 31.7 (CH2), 29.7
(C-20), 16.9 (C-60); MS (FAB+) m/z 797 (MH+, 0.3);
HRMS (FAB+) calcd for C38H40N2O17 (MH+) m/z
797.2405. Found 797.2953. Anal. Calcd for
4.1.18. 2-Methoxy-4-nitrobenzyl doxorubicincarbamate
(8). Similarly, reaction of 19 (23 mg, 66 lmol) and 6
(30 mg, 55 lmol) gave 8 (37 mg, 88%) as a red solid,
1
mp (DCM) 159–161 ꢁC; H NMR d 13.97 (s, 1H, 6-
1
OH), 13.22 (s, 1H, 11-OH), 8.02 (dd, J = 8.0, 1.0 Hz,
1H, H-1), 7.77–7.81 (m, 2H, H-2, H-500), 7.66 (br s,
1H, H-300), 7.41 (d, J = 8.0 Hz, 1H, H-600), 7.39 (dd,
J = 8.0, 1.0 Hz, 1H, H-3), 5.52 (br d, J = 3.3 Hz, 1H,
H-10), 5.29 (br s, 1H, H-7), 5.25 (d, J = 8.7 Hz, 1H,
OCONH), 5.13 (2 d, J = 14.0 Hz, 2H, CH2O), 4.75 (s,
2H, H-14), 4.51 (s, 1H, 9-OH), 4.13–4.17 (m, 1H, H-
50), 4.08 (s, 3H, 4-OCH3), 3.90 (s, 3H, 200-OCH3),
3.84–3.88 (m, 1H, H-30), 3.69 (s, 1H, H-40), 3.24 (dd,
J = 18.9, 1.3 Hz, 1H, H-10), 3.03 (s, 1H, 14-OH), 3.01
(d, J = 18.9 Hz, 1H, H-10), 2.34 (br d, J = 14.7 Hz,
1H, H-8), 2.18 (dd, J = 14.7, 4.0 Hz, 1H, H-8), 2.02
(br s, 1H, 40-OH), 1.90 (dd, J = 13.2, 4.7 Hz, 1H, H-
20), 1.79 (dd, J = 13.2, 3.3 Hz, 1H, H-20), 1.30 (d,
J = 6.5 Hz, 3H, H-60); 13C NMR d 213.7 (C-13), 187.1
(C-5), 186.7 (C-12), 161.0 (C-4), 157.0 (C-200), 156.1
(C-6), 155.6 (C-11), 155.2 (OCONH), 148.5 (C-400),
135.8 (C-2), 135.5 (C-12a), 133.5 (C-6a), 133.4 (C-10a),
132.5 (C-100), 128.4 (C-600), 120.8 (C-4a), 119.9 (C-1),
118.5 (C-3), 115.7 (C-500), 111.6 (C-5a), 111.4 (C-11a),
105.1 (C-300), 100.7 (C-10), 76.6 (C-9), 69.8 (C-7), 69.6
(C-40), 67.2 (C-50), 65.5 (C-14), 61.1 (CH2O), 56.7 (4-
OCH3), 56.0 (20-OCH3), 47.1 (C-30), 35.6 (C-8), 34.0
(C-10), 30.2 (C-2), 16.8 (C-60); MS (FAB+) m/z 753
(MH+, 0.3); HRMS (FAB+) calcd for C36H37N2O16
C38H40N2O17 ꢁ H2O: C, 56.6; H, 5.1; N, 3.5. Found: C,
2
56.7; H, 5.1; N, 3.5.
4.1.20. (1-Methyl-2-nitro-1H-imidazol-5-yl)methyl doxo-
rubicincarbamate (10). Similarly, reaction of 2349
(33 mg, 104 lmol) and 6 (46 mg, 86 lmol) gave 10
(44 mg, 70%) as a red solid, mp (DCM) 162–166 ꢁC;
1H NMR [(CD3)2SO]: d 13.96 (s, 1H, 6-OH), 13.21 (s,
1H, 11-OH), 7.82–7.87 (m, 2H, H-1, H-3), 7.58 (dd,
J = 7.5, 2.1 Hz, 1H, H-2), 7.18 (s, 1H, H-400), 7.02 (d,
J = 7.9 Hz, 1H, OCONH), 5.42 (s, 1H, 9-OH), 5.21 (d,
J = 2.6 Hz, 1H, H-10), 5.07 (s, 2H, CH2O), 4.86–4.91
(m, 2H, H-7, 14-OH), 4.73 (d, J = 5.9 Hz, 1H, 4-OH),
4.58 (d, J = 5.9 Hz, 2H, H-14), 4.13–4.17 (m, 1H, H-
50), 3.96 (s, 3H, 4-OCH3), 3.88 (s, 3H, NCH3), 3.66–
3.74 (m, 1H, H-30), 3.41–3.46 (m, 1H, H-40), 2.97 (d,
J = 18.3 Hz, 1H, H-10), 2.87 (d, J = 18.3 Hz, 1H, H-
10), 2.21 (d, J = 14.0 Hz, 1H, H-8), 2.17 (dd, J = 14.0,
5.4 Hz, 1H, H-8), 1.84 (dt, J = 12.8, 3.5 Hz, 1H, H-20),
1.57 (dd, J = 12.8, 3.8 Hz, 1H, H-20), 1.12 (d,
J = 6.4 Hz, 3H, H-60); 13C NMR [(CD3)2SO]: d 213.7
(C-13), 186.3 (C-5), 186.2 (C-12), 160.7 (C-4), 156.0
(C-6), 154.6 (C-11), 154.4 (OCONH), 145.8 (C-200),
136.1 (C-2), 135.4 (C-12a), 134.5 (C-6a), 134.0 (C-10a),
133.8 (C-500), 128.3 (C-400), 119.8 (C-4a), 119.6 (C-1),

M. P. Hay et al. / Bioorg. Med. Chem. 13 (2005) 4043–4055
4053
118.9 (C-3), 110.6 (C-5a), 110.5 (C-11a), 100.3 (C-10),
74.9 (C-9), 69.8 (C-7), 67.8 (C-40), 66.6 (C-50), 63.7 (C-
14), 56.5 (4-OCH3), 54.7 (CH2O), 47.3 (C-30), 38.4 (C-
8), 34.1 (NCH3), 32.0 (C-10), 29.7 (C-20), 17.0 (C-60);
MS (FAB+) m/z 727 (MH+, 0.2); HRMS (FAB+)
calcd for C33H35N4O15 (MH+) m/z 727.2099. Found
H-10), 2.20 (br d, J = 14.1 Hz, 1H, H-8), 2.12 (dd,
J = 14.1 Hz, 1H, H-8), 1.85 (dt, J = 12.8, 3.7 Hz, 1H,
H-20), 1.47 (dd, J = 12.8, 4.1 Hz, 1H, H-20), 1.13 (d,
J = 6.5 Hz, 3H, H-60); 13C NMR [(CD3)2SO]: d 213.7
(C-13), 186.4 (C-5), 186.3 (C-12), 160.7 (C-4), 157.0
(C-2000), 156.0 (C-6), 155.2 (C-11), 154.4 (OCONH),
152.9 (OCONH), 148.2 (C-4000), 138.4 (C-400), 136.1 (C-
2), 135.4 (C-12a), 134.6 (C-6a), 134.0 (C-10a), 132.2
(C-100), 131.0 (C-1000), 128.9 (C-2000), 128.6 (C-200, C-600),
119.9 (C-4a), 119.6 (C-1), 118.9 (C-3), 117.9 (C-300, C-
500), 115.4 (C-5000), 110.7 (C-5a), 110.6 (C-11a), 105.4
(C-3000), 100.2 (C-10), 74.9 (C-9), 69.8 (C-7), 67.9 (C-40),
66.6 (C-50), 64.8 (C-14), 63.6 (CH2O), 60.4 (CH2O),
56.5 (OCH3), 56.2 (OCH3), 47.0 (C-30), 36.5 (C-8),
32.0 (C-10), 29.7 (C-20), 16.9 (C-60); MS (FAB+) m/z
902 (MH+, 0.2). Anal. Calcd for C44H43N3O18ÆH2O: C,
57.5; H, 4.9; N, 4.6. Found: C, 57.4; H, 5.1; N, 5.6.
1
727.2075. Anal. Calcd for C33H34N4O15 ꢁ H2O:
2
C, 53.9; H, 4.8; N, 7.6. Found: C, 53.7; H, 4.8; N, 7.3.
4.1.21.
4-({[(4-Nitrobenzyl)oxy]carbonyl}amino)benzyl
doxorubicincarbamate (11). Similarly, reaction of 30
(48 mg, 104 lmol) and 6 (46 mg, 86 lmol) gave 11
(61 mg, 81%) as a red solid, mp (DCM) 160–166 ꢁC;
1H NMR [(CD3)2SO]: d 13.98 (s, 1H, 6-OH), 13.22 (s,
1H, 11-OH), 9.88 (br s, 1H, OCONH), 8.24 (d,
J = 8.7 Hz, 2H, H-3000, H-5000), 7.82–7.86 (m, 2H, H-1,
H-3), 7.66 (d, J = 8.7 Hz, 2H, H-2000, H-6000), 7.58 (dd,
J = 6.6, 2.9 Hz, 1H, H-2), 7.40 (d, J = 8.4 Hz, 2H, H-
300, H-500), 7.22 (d, J = 8.4 Hz, 2H, H-200, H-600), 6.81
(d, J = 7.9 Hz, 1H, OCONH), 5.42 (s, 1H, 9-OH), 5.27
(s, 2H, CH2O), 5.21 (d, J = 2.5 Hz, 1H, H-10), 4.89–
4.92 (m, 1H, H-7), 4.87 (s, 2H, CH2O), 4.85 (br s, 1H,
14-OH), 4.70 (d, J = 5.7 Hz, 1H, 40-OH), 4.59 (d,
J = 6.0 Hz, 2H, H-14), 4.13–4.17 (m, 1H, H-50), 3.95
(s, 3H, 4-OCH3), 3.68–3.78 (m, 1H, H-30), 3.45 (br s,
1H, H-40), 2.98 (d, J = 18.2 Hz, 1H, H-10), 2.90 (d,
J = 18.2 Hz, 1H, H-10), 2.21 (br d, J = 12.8 Hz, 1H,
H-8), 2.08–2.12 (m, 1H, H-8), 1.84 (dt, J = 12.8,
3.5 Hz, 1H, H-20), 1.47 (dd, J = 12.8, 4.5 Hz, 1H, H-
20), 1.14 (d, J = 6.4 Hz, 3H, 60-H); 13C NMR
[(CD3)2SO]: d 213.7 (C-13), 186.4 (C-5), 186.2 (C-12),
160.7 (C-4), 156.0 (C-6), 155.2 (C-11), 154.5 (OCONH),
153.0 (OCONH), 147.0 (C-4000), 144.5 (C-1000). 138.4 (C-
400), 136.1 (C-2),00135.5 (C-12a), 134.5 (C-6a), 131.1 (C-
10a), 128.6 (C-2 , C-600), 128.4 (C-2000, C-6000), 128.2 (C-
100), 123.5 (C-3000, C-5000), 119.8 (C-4a), 119.6 (C-1),
118.7 (C-3), 118.0 (C-300, C-500), 110.7 (C-5a), 110.5 (C-
11a), 100.2 (C-10), 74.9 (C-9), 69.8 (C-7), 67.9 (C-40),
66.6 (C-50), 64.9 (CH2O), 64.4 (CH2O), 63.6 (C-14),
56.5 (4-OCH3), 47.0 (C-30), 36.5 (C-8), 32.0 (C-10),
29.8 (C-20), 16.9 (C-60); MS (FAB+) m/z 872 (MH+,
0.25); HRMS (FAB+) calcd for C43H42N3O17 (MH+)
m/z 872.2514. Found 872.2499. Anal. Calcd for
4.1.23.
4-({[(1-Methyl-2-nitro-1H-imidazol-5-yl)meth-
oxy]carbonyl}amino)benzyl doxorubicincarbamate (13).
Similarly, reaction of 34 (81 mg, 172 lmol) and 6
(45 mg, 86 lmol) gave 13 (57 mg, 75%) as a red solid,
1
mp (DCM) 160–162 ꢁC; H NMR [(CD3)2SO]: d 13.99
(s, 1H, 6-OH), 13.24 (s, 1H, 11-OH), 9.82 (s, 1H,
OCONH), 7.84–7.89 (m, 2H, H-1, H-2), 7.60–7.63 (m,
1H, H-3), 7.40 (d, J = 8.3 Hz, 2H, H-300, H-500), 7.29 (s,
1H, H-4000), 7.23 (d, J = 8.3 Hz, 2H, H-200, H-600), 6.81
(d, J = 8.0 Hz, 1H, OCONH), 5.43 (s, 1H, H-7), 5.25
(s, 2H, CH2O), 5.21 (d, J = 2.9 Hz, 1H, H-10), 4.89–
4.91 (m, 1H, 9-OH), 4.87 (s, 2H, CH2O), 4.84 (dd,
J = 6.3, 5.8 Hz, 1H, 14-OH), 4.69 (d, J = 5.7 Hz, 1H,
4-OH), 4.58 (d, J = 6.0 Hz, 2H, H-14), 4.13–4.17 (m,
1H, H-50), 3.97 (s, 3H, OCH3), 3.95 (s, 3H, NCH3),
3.68–3.75 (m, 1H, H-30), 3.43–3.46 (m, 1H, H-40), 2.99
(d, J = 18.3 Hz, 1H, H-10), 2.91 (d, J = 18.3 Hz, 1H,
H-10), 2.21 (br d, J = 14.1 Hz, 1H, H-8), 2.10 (dd,
J = 14.1 Hz, 1H, H-8), 1.84 (dt, J = 12.9, 3.6 Hz, 1H,
H-20), 1.47 (dd, J = 12.9, 3.8 Hz, 1H, H-20), 1.13 (d,
J = 6.5 Hz, 3H, H-60); 13C NMR [(CD3)2SO]: d 213.7
(C-13), 186.4 (C-5), 186.3 (C-12), 160.7 (C-4), 156.0
(C-6), 155.2 (C-11), 154.4 (OCONH), 152.6 (OCONH),
146.0 (C-2000), 138.2 (C-400), 136.1 (C-2), 135.4 (C-12a),
134.6 (C-6a), 134.0 (C-10a), 13.2 (C-5000), 131.2 (C-100),
128.7 (C-4000), 128.6 (C-200, C-600), 119.9 (C-4a), 119.6
(C-1), 118.9 (C-3), 118.0 (C-300, C-500), 110.7 (C-5a),
110.5 (C-11a), 100.2 (C-10), 74.9 (C-9), 69.8 (C-7), 67.9
(C-40), 66.6 (C-50), 64.8 (C-14), 63.6 (CH2O), 56.5
(OCH3), 55.1 (CH2O), 47.0 (C-30), 36.5 (C-8), 34.1
(NCH3), 32.0 (C-10), 29.8 (C-20), 16.9 (C-60); MS
(FAB+) m/z 876 (MH+, 0.2). Anal. Calcd for
C41H41N5O17ÆH2O: C, 55.1; H, 4.9; N, 7.8. Found: C,
54.7; H, 4.9; N, 7.5.
1
C43H41N3O17 ꢁ H2O: C, 58.6; H, 4.8; N, 4.8. Found: C,
2
58.6; H, 5.1; N, 4.6.
4.1.22. 4-({[(2-Methoxy-4-nitrobenzyl)oxy]carbonyl}ami-
no)benzyl doxorubicincarbamate (12). Similarly, reaction
of 31 (52 mg, 103 lmol) and 6 (45 mg, 86 lmol) gave 12
(61 mg, 80%) as a red solid, mp (DCM) 128–131 ꢁC; 1H
NMR [(CD3)2SO]: d 14.01 (s, 1H, 6-OH), 13.25 (s, 1H,
11-OH), 9.88 (s, 1H, OCONH), 7.87–7.90 (m, 3H, H-
1, H-2, H-5000), 7.79 (d, J = 2.2 Hz, 1H, H-3000), 7.59–
7.63 (m, 2H, H-3, H-6000), 7.41 (d, J = 8.3 Hz, 2H, H-
300, H-500), 7.22 (d, J = 8.3 Hz, 2H, H-200, H-600), 6.81
(d, J = 8.0 Hz, 1H, OCONH), 5.44 (s, 1H, H-7), 5.21
(d, J = 3.0 Hz, 1H, H-10), 5.19 (s, 2H, CH2O), 4.91–
4.94 (m, 1H, 9-OH), 4.87 (s, 2H, CH2O), 4.83 (dd,
J = 6.3, 5.9 Hz, 1H, 14-OH), 4.69 (d, J = 5.7 Hz, 1H,
4-OH), 4.58 (d, J = 6.0 Hz, 2H, H-14), 4.12–4.18 (m,
1H, H-50), 3.97 (s, 3H, OCH3), 3.95 (s, 3H, OCH3),
3.68–3.75 (m, 1H, H-30), 3.43–3.47 (m, 1H, H-40), 2.99
(d, J = 18.4 Hz, 1H, H-10), 2.92 (d, J = 18.4 Hz, 1H,
4.1.24.
4-({[(1-Methyl-5-nitro-1H-imidazol-2-yl)meth-
oxy]carbonyl}amino)benzyl doxorubicincarbamate (14).
Similarly, reaction of 38 (61 mg, 129 lmol) and 6
(45 mg, 86 lmol) gave 14 (50 mg, 66%) as a red solid,
1
mp (DCM) 170–173 ꢁC; H NMR [(CD3)2SO]: d 14.00
(s, 1H, 6-OH), 13.24 (s, 1H, 11-OH), 9.91 (s, 1H,
OCONH), 8.07 (s, 1H, H-4000), 7.86–7.90 (m, 2H, H-1,
H-2), 7.60–7.63 (m, 1H, H-3), 7.40 (d, J = 8.4 Hz, 2H,
H-300, H-500), 7.23 (d, J = 8.4 Hz, 2H, H-200, H-600), 6.81
(d, J = 8.0 Hz, 1H, OCONH), 5.43 (s, 1H, H-7), 5.27

4054
M. P. Hay et al. / Bioorg. Med. Chem. 13 (2005) 4043–4055
(s, 2H, CH2O), 5.21 (d, J = 2.9 Hz, 1H, H-10), 4.92–4.95
(m, 1H, 9-OH), 4.87 (s, 2H, CH2O), 4.84 (dd, J = 6.3,
5.9 Hz, 1H, 14-OH), 4.69 (d, J = 5.7 Hz, 1H, 4-OH),
4.58 (d, J = 6.0 Hz, 2H, H-14), 4.12–4.18 (m, 1H, H-
50), 3.97 (s, 3H, OCH3), 3.95 (s, 3H, NCH3), 3.68–3.75
(m, 1H, H-30), 3.43–3.46 (m, 1H, H-40), 2.98 (d,
J = 18.3 Hz, 1H, H-10), 2.91 (d, J = 18.3 Hz, 1H, H-
10), 2.20 (br d, J = 14.1 Hz, 1H, H-8), 2.11 (dd,
J = 14.1 Hz, 1H, H-8), 1.84 (dt, J = 12.9, 3.7 Hz, 1H,
H-20), 1.47 (dd, J = 12.9, 4.0 Hz, 1H, H-20), 1.12 (d,
J = 6.5 Hz, 3H, H-60); 13C NMR [(CD3)2SO]: d 213.7
(C-13), 186.4 (C-5), 186.3 (C-12), 160.7 (C-4), 156.0
(C-6), 155.2 (C-11), 154.4 (OCONH), 152.4 (OCONH),
147.8 (C-5000), 139.3 (C-2000), 138.2 (C-400), 136.1 (C-2),
135.4 (C-12a), 134.6 (C-6a), 134.0 (C-10a), 131.7 (C-
4000), 131.2 (C-100), 128.6 (C-200, C-600), 119.9 (C-4a),
119.6 (C-1), 118.9 (C-3), 118.0 (C-300, C-500), 110.7 (C-
5a), 110.5 (C-11a), 100.2 (C-10), 74.9 (C-9), 69.8 (C-7),
67.9 (C-40), 66.6 (C-50), 64.8 (C-14), 63.6 (CH2O), 57.6
(CH2O), 56.5 (OCH3), 47.0 (C-30), 36.5 (C-8), 33.4
(NCH3), 32.0 (C-10), 29.7 (C-20), 16.9 (C-60); MS
(FAB+) m/z 876 (MH+, 0.6%); HRMS (FAB+) calcd
for C41H42N5O17 (MH+) m/z 876.2576. Found
876.2573. Anal. Calcd for C41H41N5O17ÆH2O: C, 55.1;
H, 4.9; N, 7.8. Found: C, 55.2; H, 4.9; N, 7.9.
dose increments, and were observed daily for 60 days.
Any animals losing >15% body weight or becoming
moribund during the study were terminated.
4.2.4. In vivo excision assay. Activity against NTR-
expressing and parental (NTRꢀve) tumour cells was as-
sessed by treating mice with tumours containing mix-
tures of EMT6–NTRpuro cells and EMT6 cells. CD-1
nude mice were inoculated subcutaneously with 3 · 106
cells using a 2:1 mixture of EMT6–NTRpuro and
EMT6 cells. When the tumours reached a mean diame-
ter (length · width) of 9 1 mm the animals were ran-
domized to treatment groups (5 animals/group). Mice
were treated i.p. with single doses of prodrugs, at the
MTD as determined in C3H mice and tumours were re-
moved 18 h later to determine cell killing by clonogenic
assay as reported elsewhere.51 Briefly, tumours were dis-
sected, weighed and dissociated in a pronase/collage-
nase/DNAase cocktail. Cell numbers were determined
with a particle counter (Coulter Electronics) and up to
105 cells were plated in medium containing 3 lM puro-
mycin or nonselective medium to quantify survival of
EMT6–NTRpuro and total tumour cells respectively.
Plates were incubated for 8 days, and colonies of >50
cells counted. The plating efficiency of EMT6 cells was
estimated from the difference between plating efficiency
in puromycin and nonselective medium, and the number
of clonogens of both types was calculated per gram of
tumour tissue for control and treated tumours. Statisti-
cal significance of drug effects was determined by ANO-
VA using Dunnettꢁs test to compare groups.
4.2. Biological testing
4.2.1. Cell lines. Four pairs of cell lines, each comprising
a tumour cell line and corresponding transfectant stably
expressing NTR, were grown as monolayers in aMEM
containing 5% fetal bovine serum. V79–NTRpuro, also
known as T79-A3, is a Chinese hamster fibroblast that
expresses NTR from an CMV promoter; the corre-
sponding NTRꢀve line here referred to as V79puro has
been transfected with the empty shuttle vector and is
also known as T78-1.20 SKOV–NTRneo and WiDr–
NTRneo, also known as SC3.2 and WC14, respectively,
are human ovarian and colon carcinoma lines derived
from SKOV3 and WiDr, and also express NTR from
a CMV promoter.56 EMT6–NTRpuro, also known as
EN2A, is a murine breast carcinoma line derived from
EMT6 and expresses NTR from a bicistronic cassette
with an EF-1a promoter.51 Selection for NTR expres-
sion was maintained during passage, but not during
experiments, using 15 lM puromycin (V79–NTRpuro),
Acknowledgements
The authors thank Dr. Frederik Pruijn for helpful dis-
cussions and suggestions; Dr. Maruta Boyd, Dianne
Ferry, Alison Hogg, Li Fong Leong and Susan Pullen
for technical assistance; Frank Friedlos for providing
the V79puro and V79–NTRpuro cell lines; and Dr. Martin
Ford, GlaxoSmithKline, Stevenage, UK, for providing
the SKOV–NTRneo and WiDr–NTRneo cell lines. The
Marsden Fund of New Zealand (M.P.H.), the Health
Research Council of New Zealand (W.R.W.) and the
Auckland Division of the Cancer Society of New Zea-
land (W.A.D.) supported this work.
5 lM puromycin (EMT6–NTRpuro
G418 (WiDr–NTRneo; SKOV–NTRneo).
) or 300 lg/mL
References and notes
4.2.2. Growth inhibition assays. Growth inhibitory
potencies were determined under aerobic conditions
using log-phase cultures in 96-well plates, as described
previously.57,58 Cultures were initiated 24 h before an
18 h drug exposure, with cell densities determined 4–
5 days later by staining with sulforhodamine B. IC50 val-
ues were calculated as the drug concentration providing
50% inhibition of growth relative to controls on the
same plate.
1. Huber, B. E.; Richards, C. A.; Austin, E. A. Adv. Drug
Delivery Rev. 1995, 17, 279–292.
2. Martin, L.-A.; Lemoine, N. R. Cancer Metastasis Rev.
1996, 15, 301–316.
3. Niculaescu-Duvaz, I.; Spooner, R.; Marais, R.; Springer,
C. J. Bioconjugate Chem. 1998, 9, 4–22.
4. Springer, C. J.; Niculescu-Duvaz, I. J. Clin. Invest. 2000,
105, 1161–1167.
5. Xu, G.; McLeod, H. L. Clin. Cancer Res. 2001, 7, 3314–
3324.
4.2.3. Mouse toxicity. Compounds were formulated in
DMSO immediately before use. Groups of six male
C3H mice (ca. 25 g) were treated i.p. with single doses
of compounds at 1 lL/g body weight, using 101/8-fold
6. Denny, W. A. Curr. Pharm. Des. 2002, 15, 1349–1361.
7. Anlezark, G. M.; Melton, R. G.; Sherwood, R. F.; Coles,
B.; Friedlos, F.; Knox, R. J. Biochem. Pharmacol. 1992,
44, 2289–2296.

M. P. Hay et al. / Bioorg. Med. Chem. 13 (2005) 4043–4055
4055
8. Knox, R. J.; Friedlos, F.; Sherwood, R. F.; Melton, R. G.;
Anlezark, G. M. Biochem. Pharmacol. 1992, 44, 2297–
2301.
9. Michael, N. P.; Brehm, J. K.; Anlezark, G. M.; Minton,
N. P. FEMS Microbiol. Lett. 1994, 124, 195–202.
10. Skelly, J. V.; Collins, P. J.; Knox, R. J.; Anlezark, G. M.;
Melton, R. G. J. Mol. Biol. 1994, 238, 852–853.
11. Grove, J. I.; Searle, P. F.; Weedon, S. J.; Green, N. K.;
McNeish, I. A.; Kerr, D. J. Anti-Cancer Drug Des. 1999,
14, 461–472.
12. Djeha, A. H.; Hulme, A.; Dexter, M. T.; Mountain, A.;
Young, L. S.; Searle, P. F.; Kerr, D. J.; Wrighton, C. J.
Cancer Gene Ther. 2000, 7, 721–731.
13. Djeha, A. H.; Thomson, T. A.; Leung, H.; Searle, P. F.;
Young, L. S.; Kerr, D. J.; Harris, P. A.; Mountain, A.;
Wrighton, C. J. Mol. Ther. 2001, 3, 233–240.
31. Lee, M.; Simpson, J. E., Jr.; Woo, S.; Kaenzig, C.;
Anlezark, G. M.; Eno-Amooquaye, E.; Burke, P. J.
Bioorg. Med. Chem. Lett. 1997, 7, 1065–1070.
32. Mulcahy, R. T.; Gipp, J. J.; Schmidt, J. P.; Joswig, C.;
Borch, R. F. J. Med. Chem. 1994, 37, 1610–1615.
33. 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, 4818–4821.
34. Hay, M. P.; Atwell, G. J.; Wilson, W. R.; Pullen, S. M.;
Denny, W. A. J. Med. Chem. 2003, 46, 2456–2466.
35. Hay, M. P.; Sykes, B. M.; Denny, W. A.; Wilson, W. R.
Bioorg. Med. Chem. Lett. 1999, 9, 2237–2242.
36. Hay, M. P.; Anderson, R. F.; Ferry, D. M.; Wilson, W.
R.; Denny, W. A. J. Med. Chem. 2003, 46, 5533–5545.
37. Jungheim, L. N.; Shepherd, T. A. Chem. Rev. 1994, 94,
1553–1566.
14. Chung-Faye, G.; Palmer, C.; Anderson, D.; Clark, J.;
Downes, M.; Baddeley, J.; Hussain, S.; Murray, P. I.;
Searle, P.; Seymour, L.; Harris, P. A.; Ferry, D.; Kerr, D.
J. Clin. Cancer Res. 2001, 7, 2662–2668.
38. Hay, M. P.; Denny, W. A. Drugs Future 1996, 21, 917–
931.
39. Springer, C. J.; Niculescu-Duvaz, I. Expert Opin. Invest.
Drugs 1996, 3, 289–308.
15. Palmer, D. H.; Mautner, V.; Mirza, D.; Buckels, J.; Olliff,
D.; Hull, D.; Mountain, A.; Harris, P.; Djeha, H.; Ellis, J.;
Horne, M.; Hill, S.; Wrighton, C.; Searle, P. F.; Kerr, D.
J.; Young, L. S.; James, N. D. Br. J. Cancer 2002,
86(Suppl 1), S30.
40. Denny, W. A. Curr. Pharm. Des. 1996, 2, 281–
294.
41. Papot, S.; Tranoy, I.; Tillequin, F.; Florent, J.-C.; Gesson,
J.-P. Curr. Med. Chem.—Anti-Cancer Agents 2002, 2, 155–
185.
16. Palmer, D. H.; Mautner, V.; Mirza, D.; Oliff, S.; Gerrit-
sen, W.; van der Sijp, Jr.; Hubscher, S.; Reynolds, G.;
Bonney, S.; Rajaratnam, R.; Hull, D.; Horne, M.; Ellis, J.;
Mountain, A.; Hill, S.; Harris, P. A.; Searle, P. F.; Young,
L. S.; James, N. D.; Kerr, D. J. J. Clin. Oncol. 2004, 22,
1546–1552.
17. Helsby, N. A.; Atwell, G. J.; Yang, S.; Palmer, B. D.;
Anderson, R. F.; Pullen, S. M.; Ferry, D. M.; Wilson, W.
R.; Denny, W. A. J. Med. Chem. 2004, 47, 3295–3307.
18. Anlezark, G. M.; Melton, R. G.; Sherwood, R. F.; Wilson,
W. R.; Denny, W. A.; Palmer, B. D.; Friedlos, F.;
Williams, A. Biochem. Pharmacol. 1995, 50, 609–618.
19. Atwell, G. J.; Boyd, M.; Palmer, B. D.; Anderson, R. F.;
Pullen, S. M.; Wilson, W. R.; Denny, W. A. Anti-Cancer
Drug Des. 1996, 11, 553–567.
42. Rooseboom, M.; Commandeur, J. N. M.; Vermeulen, N.
P. E. Pharmacol. Rev. 2004, 56, 53–102.
43. Haisma, H. J.; van Muijen, M.; Pinedo, H. M.; Boven, E.
Cell Biophys. 1994, 24/25, 185–192.
44. Azoulay, M.; Florent, J.-C.; Monneret, C.; Gesson, J. P.;
Jacquesy, J.-C.; Tillequin, F.; Koch, M.; Bosslet, K.;
Czech, J.; Hoffman, D. Anti-Cancer Drug Des. 1995, 10,
441–450.
45. Houba, P. H. J.; Leenders, R. G. G.; Boven, E.; Scheeren,
J. W.; Pinedo, H. M.; Haisma, H. J. Biochem. Pharmacol.
1996, 52, 455–563.
46. Leenders, R. G. G.; Damen, E. W. P.; Bijsterveld, E. J. A.;
Scheeren, H. W.; van der Meulen-Muileman, I. H.; Boven,
E.; Haisma, H. J. Bioorg. Med. Chem. 1999, 7, 1597–1610.
47. Dubowchik, G. M.; Firestone, R. A. Bioorg. Med. Chem.
Lett. 1998, 8, 3341–3346.
48. Dubowchik, G. M.; Mosure, K.; Knipe, J. O.; Firestone,
R. A. Bioorg. Med. Chem. Lett. 1998, 8, 3341–3346.
49. Hay, M. P.; Wilson, W. R.; Denny, W. A. Tetrahedron
2000, 56, 645–657.
20. Friedlos, F.; Denny, W. A.; Palmer, B. P.; Springer, C. J.
J. Med. Chem. 1997, 40, 1270–1275.
21. Helsby, N. A.; Wheeler, S. J.; Pruijn, F. B.; Palmer, B. D.;
Yang, S.; Denny, W. A.; Wilson, W. R. Chem. Res.
Toxicol. 2003, 16, 469–478.
22. Wilson, W. R.; Pullen, S. M.; Hogg, A.; Helsby, N. A.;
Hicks, K. O.; Denny, W. A. Cancer Res. 2002, 62, 1425–
1432.
23. Carl, P. L.; Chakravarty, P. K.; Katzenellenbogen, J. A. J.
Med. Chem. 1981, 24, 479–480.
24. Hay, M. P.; Sykes, B. M.; OꢁConnor, C. J.; Denny, W. A.
J. Chem. Soc., Perkin Trans. 1 1999, 2759–2770.
25. Sykes, B. M.; Hay, M. P.; Bohinc-Herceg, D.; Denny, W.
A.; OꢁConnor, C. J. J. Chem. Soc., Perkin Trans. 1 2000,
1601–1608.
26. Mauger, A. B.; Burke, P. J.; Somani, H. H.; Friedlos, F.;
Knox, R. J. J. Med. Chem. 1994, 37, 3452–3458.
27. Hay, M. P.; Wilson, W. R.; Denny, W. A. Bioorg. Med.
Chem. Lett. 1995, 5, 2829–2834.
28. Hay, M. P.; Wilson, W. R.; Denny, W. A. Bioorg. Med.
Chem. Lett. 1999, 9, 3417–3422.
29. Tercel, M.; Denny, W. A.; Wilson, W. R. Bioorg. Med.
Chem. Lett. 1996, 6, 2741–2744.
30. Sagnou, M. J.; Howard, P. W.; Gregson, S. J.; Eno-
Amooquaye, E.; Burke, P. J.; Thurston, D. E. Bioorg.
Med. Chem. Lett. 2000, 10, 2083–2086.
50. Dubrovchik, G. M.; King, H. D.; Pham-Kaplita, K.
Tetrahedron Lett. 1997, 38, 5261–5264.
51. Wilson, W. R.; Pullen, S. M.; Hogg, A.; Hobbs, S. M.;
Pruijn, F. B.; Hicks, K. O.. In Suicide Gene Therapy:
Methods and Protocols for Cancer; Springer, C. J., Ed.;
Humana: Totowa, NJ, 2003, pp 403–432.
52. Siim, B. G.; Lee, A. E.; Shalal-Zwain, S.; Pruijn, F. B.;
McKeage, M. J.; Wilson, W. R. Cancer Chemother.
Pharmacol. 2003, 51, 43–52.
53. Lipinski, C. A. J. Pharmacol. Toxicol. Method 2000, 44,
235–249.
54. Kyle, A. H.; Huxham, L. A.; Chiam, A. S. J.; Sim, D. H.;
Minchinton, A. I. Cancer Res. 2004, 64, 6304–6309.
55. Durand, R. E. Cancer Chemother. Pharmacol. 1990, 26,
198–204.
56. Friedlos, F.; Court, S.; Ford, M.; Denny, W. A.; Springer,
C. J. Gene Ther. 1998, 5, 105–112.
57. Finlay, G. J.; Baguley, B. C.; Wilson, W. R. Anal.
Biochem. 1984, 139, 272–277.
58. Palmer, B. D.; Wilson, W. R.; Anderson, R. F.; Boyd, M.;
Denny, W. A. J. Med. Chem. 1996, 39, 2518–2528.