Substituent effects on the kinetics of reductively-initiated fragmentation of nitrobenzyl carbamates designed as triggers for bioreductive prodrugs

Article abstract of DOI:10.1039/a904067f

4-Nitrobenzyl carbamates are of interest as triggers for bioreductive drugs, particularly in conjunction with the E. coli B nitroreductase, which efficiently reduces them to the corresponding hydroxylamines. These then fragment to release highly toxic amine-based toxins. While many 4-nitrobenzyl carbamate derivatives have been evaluated as bioreductive drugs, there has been no systematic study of substituent effects on the rate of this fragmentation (which should be as fast as possible following reduction). We therefore prepared a series of 2-, 3- and α-substituted 4-[N-methyl-N-(4-nitrobenzyloxycarbonyl)amino]phenylacetamides as model compounds to study these effects. The majority of the carbamates were prepared by in situ formation of the chloroformate of the appropriate 4-nitrobenzyl alcohol and reaction with methyl 4-(methylamino)phenylacetate, followed by ester hydrolysis and 1,1′-carbonyl-diimidazole (CDI) mediated coupling with N,N-dimethylaminoethylamine. The hydroxylamines were generated by ⁶⁰Co γ-ray irradiation of the nitro compounds in aqueous phosphate-buffered-propan-2-ol. The reactions were analysed by reverse-phase HPLC to determine the maximum half-life (Mt_1/2) of the hydroxylamines generated, and the extent of release of amine from these after 10 half-lives (t_∞). The parent (unsubstituted) hydroxylaminobenzyl carbamate had a Mt_1/2 of 16 min under these conditions, while that of the corresponding α-methyl analogue was 9.5 min. Electron-donating substituents on the benzyl ring also accelerated fragmentation, with the data being fitted to the equation log(Mt_1/2) = 0.57σ + 1.30, where σ represents σ_p for 2-substituents and σ_m for 3-substituents. The acceleration of fragmentation of the hydroxylamines with increasing substituent electron-donation is consistent with the proposed mechanism, and is presumably due to stabilisation of the developing positive charge on the benzylic carbon. The extent of release of amine (t_∞) also increased with increasing substituent electron-donation. These data suggest that the standard 4-nitrobenzyl carbamate trigger for nitroreductase enzyme (NTR) prodrugs can likely be improved on, by increasing the rate of fragmentation by the use of α-methyl and/or electron-donating benzyl substituents. The Royal Society of Chemistry 1999.

Full text of DOI:10.1039/a904067f

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Substituent effects on the kinetics of reductively-initiated
fragmentation of nitrobenzyl carbamates designed as triggers
for bioreductive prodrugs
Michael P. Hay,*a Bridget M. Sykes,b William A. Dennya and Charmian J. O’Connorb
a Auckland Cancer Society Research Centre, Faculty of Medicine and Health Science, and
b Department of Chemistry, The University of Auckland, Private Bag 92019, Auckland,
New Zealand
Received (in Cambridge, UK) 20th May 1999, Accepted 30th July 1999
4-Nitrobenzyl carbamates are of interest as triggers for bioreductive drugs, particularly in conjunction with the
E. coli B nitroreductase, which efficiently reduces them to the corresponding hydroxylamines. These then fragment
to release highly toxic amine-based toxins. While many 4-nitrobenzyl carbamate derivatives have been evaluated as
bioreductive drugs, there has been no systematic study of substituent effects on the rate of this fragmentation
(which should be as fast as possible following reduction). We therefore prepared a series of 2-, 3- and α-substituted
4-[N-methyl-N-(4-nitrobenzyloxycarbonyl)amino]phenylacetamides as model compounds to study these effects. The
majority of the carbamates were prepared by in situ formation of the chloroformate of the appropriate 4-nitrobenzyl
alcohol and reaction with methyl 4-(methylamino)phenylacetate, followed by ester hydrolysis and 1,1Ј-carbonyl-
diimidazole (CDI) mediated coupling with N,N-dimethylaminoethylamine. The hydroxylamines were generated by
60Co γ-ray irradiation of the nitro compounds in aqueous phosphate-buffered-propan-2-ol. The reactions were
analysed by reverse-phase HPLC to determine the maximum half-life (Mt1/2) of the hydroxylamines generated, and
the extent of release of amine from these after 10 half-lives (t∞). The parent (unsubstituted) hydroxylaminobenzyl
carbamate had a Mt1/2 of 16 min under these conditions, while that of the corresponding α-methyl analogue was
9.5 min. Electron-donating substituents on the benzyl ring also accelerated fragmentation, with the data being fitted
to the equation log(Mt1/2) = 0.57σ ϩ 1.30, where σ represents σp for 2-substituents and σm for 3-substituents. The
acceleration of fragmentation of the hydroxylamines with increasing substituent electron-donation is consistent with
the proposed mechanism, and is presumably due to stabilisation of the developing positive charge on the benzylic
carbon. The extent of release of amine (t∞) also increased with increasing substituent electron-donation. These data
suggest that the standard 4-nitrobenzyl carbamate trigger for nitroreductase enzyme (NTR) prodrugs can likely be
improved on, by increasing the rate of fragmentation by the use of α-methyl and/or electron-donating benzyl
substituents.
Introduction
Nitrobenzyl carbamates are of interest as potential bioreduc-
tive drugs, because of their ability to undergo fragmentation
following reduction. In contrast to the reduction of nitrobenzyl
halides1 or quaternary salts,2 which fragment at the radical
anion stage following one-electron reduction,3 nitrobenzyl carb-
amates undergo multi-electron reduction to electron-donating
hydroxylamine or amine species, which then fragment to gener-
Scheme 1 Reduction and fragmentation of nitrobenzyl carbamates.
ate a quinomethane imine and an amine.4 Initial interest
centred around the reduction of compounds such as 1 (R = Me,
Ph) by cellular nitroreductases, with the quinomethane or qui-
nonimine species considered to be the potentially cytotoxic
agent.5 A subsequent study6 considered the nitrobenzyl carb-
amates of 5-fluorouracil 1 (R = 5-FU) as bioreductive agents.
However, the reduction potentials of compounds such as 1 are
too low for efficient reduction by cellular nitroreductases, and
the quinonimines generated were not particularly cytotoxic.
Renewed interest in nitrobenzyl carbamates as bioreductive
drugs was generated by the discovery of an E. coli B nitro-
reductase enzyme (NTR)7 which, in conjunction with NADH
or NADPH, reduces certain aromatic nitro groups to the corre-
sponding hydroxylamines.8 Despite the low reduction poten-
tials of typical 4-nitrobenzyl carbamates (ca. Ϫ490 mV),9 they
undergo reduction by the NTR enzyme (although with widely
varying degrees of activation) to the corresponding unstable
hydroxylamines. The latter, through increased electron release
to the π-system stabilizing the developing positive charge on the
benzylic carbon, readily fragment to release amines (Scheme 1).
The 4-nitrobenzyl carbamate moiety has thus been proposed as
a prodrug “trigger” that may deactivate highly cytotoxic amine
“effectors”.10
The E. coli enzyme has been used to activate such nitrobenzyl
carbamate prodrugs in antibody-11,12 and gene-directed enzyme
prodrug therapy13 (ADEPT and GDEPT, respectively) proto-
cols. Several studies have shown that 4-nitrobenzyl carbamate
derivatives of actinomycin,11 mitomycin,11 enediynes,14 amino-
seco-cyclopropylindoline derivatives,15 and tallimustine deriv-
atives16 are substrates for E. coli NTR. The selectivity of the
NTR enzyme for the 4-nitrobenzyl moiety is well-demonstrated
in the enediyne analogues 2 and 3, where only the 4-nitrobenzyl
isomer 3 was a substrate for the enzyme.14
Factors of prime importance in the design of effective
4-nitrobenzyl carbamate prodrugs for nitroreductase-mediated
prodrug therapy include the efficiency of the trigger unit as an
J. Chem. Soc., Perkin Trans. 1, 1999, 2759–2770
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obtained as reported. 2-Methoxy-4-nitrobenzyl alcohol 8f was
conveniently prepared by methylation of 4-nitrosalicylic acid 9
and diisobutylaluminium hydride (DIBAL-H) reduction of the
resulting ester 10 (Scheme 3).
enzyme substrate and the rate of fragmentation following
enzymic reduction. While kinetic structure–activity relation-
ships (SAR) have been extensively studied for the one-electron
reduction of nitrobenzyl halides3 and quaternary salts,2 the
SAR involved in the reductive fragmentation of 4-nitrobenzyl
carbamates has been neglected, with only the unsubstituted
4-nitrobenzyl derivatives being employed as prodrugs for NTR
activation. The use of substituents on the nitrobenzyl moiety
offers the prospect of modifying the rate of fragmentation of
the reduced prodrug, and also providing a site of attachment
for solubilising functionality.
Scheme 3 Synthesis of alcohol 8f. Reagents: i, CH2N2 Et2O; ii,
DIBAL-H, THF.
In this paper we use a series of model compounds 4a–n to
study the effect of nitrobenzyl substituents on the rate of frag-
mentation of the intermediate hydroxylamines 5a–n (generated
by radiolysis) to release amines 6 or 7 (Scheme 2). Substituents
2-(N-Methyl-N-tert-butyloxycarbonylamino)-4-nitrobenzoic
acid 8h was prepared from 4-nitroanthranilic acid 11 by esterifi-
cation to give 12, reductive methylation of the amine to give 13
and protection of 13 with the tert-butyloxycarbonyl (Boc)
moiety to give the ester 14 (Scheme 4). Alkaline hydrolysis of 14
Scheme 4 Synthesis of alcohol 8h. Reagents: i, (COCl)2, DCM; ii,
MeOH; iii, acetic formic anhydride; iv, BH3ؒDMS, THF; v, di-tert-
butyldicarbonate, DMAP, THF; vi, LiOH, MeOH.
Scheme 2 Reduction and fragmentation of carbamates 4a–n.
at both the 2- and 3-positions were selected to span a wide
range of electronic properties, while also being able to serve
as potential attachments for soluble side chains. The use of
α-substituents was also expected to alter the rate of fragmen-
tation and may be a useful site for the attachment of solubilis-
ing groups.17 The amines 6 and 7, released on fragmentation of
4a–n, were designed as simple models of cyclopropylindoline
and enediyne (e.g. 3) prodrugs, while also being stable, syn-
thetically accessible, water-soluble, and possessing a good UV
signature for HPLC analysis.
gave the acid 15 which was reduced to 8h. Similarly, the isomer
8i was synthesized from the corresponding amine 17, prepared
via a Curtius rearrangement from the 4-nitroisophthalic acid
half ester 16, through intermediates 18–20 (Scheme 5).
The amine 7 was readily available (Scheme 6) from methyl 4-
nitrophenylacetate 21 by catalytic reduction and reductive
amination to 22. Protection of 22 as the Boc derivative 23,
hydrolysis of the ester and coupling of the resulting acid 24
with N,N-dimethylaminoethylamine provided the amide 25.
This was deprotected to give 7, which was conveniently stored
as the hydrochloride salt. The amine 6 was prepared by coup-
ling 4-nitrophenylacetic acid 26 with N,N-dimethylaminoethyl-
amine followed by catalytic reduction of the amide 27 and
conversion to the hydrochloride salt (Scheme 7).
Results and discussion
Synthesis of carbamates 4
Nitrobenzyl alcohols 8a and 8k for the preparation of the
model carbamates were available commercially, and 8d, 8e, 8g,
8l were prepared directly by borane–dimethyl sulfide (BH3ؒ
DMS) reduction of the corresponding acids. 1-(4-Nitrophenyl)-
ethyl alcohol 8c18 and 2,4-dinitrobenzyl alcohol 8j19 were
The majority of the model carbamates 4 were prepared by
in situ formation of the chloroformate of the alcohols 8
and subsequent reaction with the amino ester 22 or methyl
4-aminophenylacetate to give the carbamate esters 28. Basic
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Scheme 8 Preparation of carbamates 4a–i. Method A. Reagents:
i, COCl2, then Cs2CO3, 22 or methyl 4-aminophenyl acetate, DMF;
ii, LiOH, MeOH; iii, CDI, NH2CH2CH2NMe2, DMF.
Scheme 5 Synthesis of alcohol 8i. Reagents: i, SOCl2; ii, NaN3, acet-
one; iii, TMS–ethanol, toluene; iv, TBAF, THF; v, acetic formic
anhydride; vi, BH3ؒDMS, THF; vii, di-tert-butyldicarbonate, DMAP,
THF; viii, LiOH, MeOH.
Scheme 9 Preparation of carbamates 4j–l. Method B. Reagents:
i, COCl2, THF.
Scheme 10 Preparation of carbamates 4m, n. Method C. Reagents:
i, HCl, THF.
Radiolytic reduction of carbamates 4
Radiolytic reduction, rather than enzymic reduction by E.
coli NTR enzyme, was used in order to investigate the sub-
stituent effects on carbamate fragmentation without the com-
plicating influence of the kinetics of enzyme activation. The
radiolysis of water is a useful method to study reductively-
triggered reactions in aqueous media, because powerful, tran-
sient reductants can be generated in controlled quantities (via
the exposure time of a given volume of solution at a known
dose-rate) in solutions buffered over a wide pH range (2–11).
It also provides information on the stoichiometry of reduc-
tive fragmentation, and hence the identity of the activated
species that undergoes fragmentation. 60Co γ-rays produce
approximately equal amounts of reducing aquated electrons
Scheme 6 Synthesis of amine 7. Reagents; i, H2, Pd/C, EtOH; ii, acetic
formic anhydride; iii, BH3ؒDMS, THF; iv, di-tert-butyldicarbonate,
DMAP, THF; v, LiOH, MeOH; vi, CDI, NH2CH2CH2NMe2, DMF;
vii, HCl, MeOH.
(eϪaq) and oxidising hydroxyl radicals (OH ) in water, and the
ؒ
presence of propan-2-ol produces a reducing environment by
converting hydroxyl radicals to reducing 2-hydroxypropan-2-
ؒ
yl radicals, (CH3)2C OH.
A typical plot of the change in the composition of an
aqueous solution (50 µM) of carbamate 4 with the extent of
reductive activation by 60Co γ-rays is shown in Fig. 1. Up to 6
stoichiometric equivalents of reducing radicals were added,
and the compositions of the reduced solutions were examined
by HPLC. Fig. 1 shows that 4b is consumed with 4-fold
stoichiometry, generating a transient species assumed to be the
corresponding hydroxylamine 5b. The instability of these com-
pounds precluded formal identification, but reactions of 5b
with Na3[Fe(CN)5NH3] [sodium amminepentacyanoferrate()]
are consistent with a hydroxyamino or nitroso compound.20
There was also proportional release of the corresponding
amine 6 or 7 with up to 4 reducing equivalents, but little further
release if more than 4 reducing equivalents were used. The sub-
sequent production of amine 7 after reduction of 4b to the
hydroxylamine 5b may be attributed to the slow fragmentation
kinetics of 5b (Mt1/2 = 16 min).
Scheme 7 Synthesis of amine 6. Reagents: i, CDI, NH2CH2CH2NMe2,
DMF; ii, H2, Pd/C, EtOH; iii, HCl, MeOH.
hydrolysis of 28 to the acids 29, followed by 1,1Ј-carbonyl-
diimidazole (CDI)-mediated coupling with N,N-dimethyl-
aminoethylamine, gave carbamates 4 (Method A, Scheme 8).
Carbamates 4j, 4k, and 4l were not obtainable by Method A
because of decomposition under the reaction conditions, and
were made instead by direct coupling of the corresponding
alcohols 8j, 8k, and 8l to the amide 7, with purification by semi-
preparative HPLC (Method B, Scheme 9). Finally, carbamates
4m and 4n were made by direct HCl-mediated removal of the
Boc group from 4f and 4g, respectively (Method C, Scheme
10).
J. Chem. Soc., Perkin Trans. 1, 1999, 2759–2770
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Table 1 Fragmentation of 4-nitrobenzyl carbamates
Table 3 Fragmentation of 3-substituted 4-nitrobenzyl carbamates
Compound
R2
R3
Mt1/2/mina
t0b (%)
t∞c (%)
Compound
R1
σm
Mt1/2
t0
t∞
5a
5b
5c
H
H
Me
H
Me
Me
10
16
9.5
52
40
55
54
49
62
5k
5l
NO2
CO2Me
OMe
N(Me)CO2tBu
H
Me
0.71
0.37
0.12
0.07
0.00
Ϫ0.07
Ϫ0.30
Ϫ0.34
65
20
17
15
16
n.d.
14
22
22
44
37
43
40
n.d.
42
49
24
57
58
50
49
n.d.
44
52
5g
5ia
5b
a Maximum half-life, see text for derivation. b % Amine released at
earliest time measurable. c % Amine released after >10 half-lives. Values
were determined in triplicate and were reproducible to within 0.6%.
5eb
5n
NHMe
NHOH
5kc
a σp (NHCO2Me) used as best approximation. b Fragmentation too fast
to measure. c Data collected for 8-electron reduction of 4k. Values were
determined in triplicate and were reproducible to within 0.6%.
Table 2 Fragmentation of 2-substituted 4-nitrobenzyl carbamates
Compound R1
σp
Mt1/2/min
to (%) t∞ (%)
5j
NO2
H
NHOH
N(Me)CO2tBu
Me
OMe
NHMe
0.78 88
0.0 16
Ϫ0.04 16
Ϫ0.15 23
Ϫ0.17 n.d.
Ϫ0.27 12
18
40
68
30
n.d.
48
65
33
49
73
54
n.d.
55
71
occurred with 4-fold stoichiometry, which is consistent with
reduction to the hydroxylamine 5. While all of the initial carb-
amates 4 were consumed after the addition of 4 reducing
equivalents, only about 50% (22–68%) of the expected amines
were produced at the earliest analysis time (assigned t0) (Tables
1–3). When the reduced solutions were maintained under
anaerobic conditions at 20 ЊC for a further 10 half-lives
(assigned t∞), the presence of 5 was undetectable, yet little
further amine was released. The unstable nature of 5 and the
finite time required for its radiolytic generation, prevented
quantification of chromatographic peaks and the direct
measurement of the rate of fragmentation. Rather, quantifi-
cation of the amine 6 or 7 released from 4 after reduction with 4
reducing equivalents was used to estimate the maximum half-
life (Mt1/2) for the fragmentation of hydroxylaminobenzyl-
5b
5ja
5hb
5dc
5f
5m
Ϫ0.84
7.2
a Data collected for 8-electron reduction of 4j. b σp (NHCO2Me) used as
best approximation. c Fragmentation rates not determined. Values were
determined in triplicate and were reproducible to within 0.6%.
carbamates 5. Assuming first order conditions, the half-life (t1/2
)
of species R is calculated from the equation ln([R]0/[R]t) =
t(ln2/t1/2). The ratio [R]0/[R]t was taken as the fraction of
nitrobenzyl carbamate 4 which had not released amine 6 or 7
after 4-fold reduction; i.e., [R]0 = 50 µM (the concentration of
the nitrobenzyl carbamate 4 before radiolytic reduction),
[R]t = 50 µM Ϫ [amine]t, and t = time between beginning 4-fold
reduction and measuring the concentration of 6 or 7 by HPLC
(approximately 12 minutes). This method yields a maximum
value for the half-life of fragmentation. The rate of reduction
and of fragmentation both contribute to the rate of amine
release, but the two sequential processes are combined within
the Mt1/2 value. However, the rate of reduction is constant
(dictated by the dose rate of the γ-rays), and Mt1/2 indicates the
relative rates of amine release via 5 which are presented in
Tables 1–3.
Fig. 1 Representative plot of changes in the composition of solutions
of 4b with changing extent of radiolytic reduction. Plot depicts (Left-
hand y-axis) the peak areas of 4b (᭹) and the major transient reduction
product of 4b, assumed to be the corresponding hydroxyaminobenzyl
carbamate 5 (᭿) and (right-hand y-axis) the % release of amine 7 (᭡).
[4b]0 = 50 µM, pH 7.4 10 mM phosphate buffer, 4% (v/v) isopropyl
alcohol, 20 ЊC.
Structure–activity relationships
The unsubstituted hydroxyaminobenzyl carbamate 5b (gener-
ated from 4b) had a Mt1/2 of 16 minutes (Table 1). This is
relatively long, and under biological conditions may permit
substantial loss of material by side reactions not involving
(activating) amine release. The α-methyl analogue 5c had a
Mt1/2 of 9.5 min. This significant acceleration of fragmentation
is presumably due to stabilisation of developing positive charge
on the benzylic carbon. Compound 5a which releases a primary
amine 6 fragmented about twice as rapidly (Mt1/2 = 10 minutes)
as 5b, indicating the nature of the leaving amine (6 instead of 7)
also has some effect.
The remaining compounds (Tables 2 and 3) all released
amine 7, so that carbamate substituent effects alone could be
discerned. In support of the proposed mechanism, electron-
donating substituents at the 2-position on the benzyl ring also
accelerated fragmentation, presumably by a similar effect. Thus
the electron-donating 2-OMe (5f), 2-NHMe (5m) and 3-NHMe
(5n) analogues were more unstable than the parent com-
pound 5c (Mt1/2 values of 12, 7.2 and 14 min, compared with 16
min). Conversely the strongly electron-withdrawing 2-NO2 and
3-NO2 substituents significantly slowed fragmentation of 5j and
These data were used to estimate the maximum half-life
(Mt1/2) of the hydroxyaminobenzyl carbamates 5. Assum-
ing first order conditions, half-life (t1/2) is calculated from the
equation ln([R]0/[R]t) = t(ln2/t1/2), where [R] is the concentration
of reactant at time t. Approximately 12 minutes were required
to effect 4-electron reduction and assay the samples, thus only
the fraction of the reduced carbamate which had not frag-
mented within 12 minutes could be calculated and substituted
into the above equation. This gave an estimate of the maximum
half-life of 5, and these data are presented in Tables 1–3.
Solutions of the nitrobenzyl carbamates 4 (50 µM, pH 7.4, 10
mM phosphate buffer, 4% (v/v) propan-2-ol, 20 ЊC) were
reduced with 4-fold stoichiometry and assayed for 5 by HPLC
either immediately (ca. 12 minutes after reduction was com-
menced) to give t0, or after ten half-lives to give t∞.
Radiolytic reduction of the 4-nitrobenzyl carbamates 4
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5k (Mt1/2 values of 88 min and 65 min respectively). In the case
of these compounds it is not known unequivocally which nitro
group undergoes reduction.
spectra. Spectra were obtained in deuteriochloroform unless
otherwise specified, and are referenced to Me4Si. Chemical
shifts and coupling constants were recorded in units of ppm
and Hz, respectively. Mass spectra were determined on a VG-
70SE mass spectrometer using an ionizing potential of 70 eV at
a nominal resolution of 1000. High resolution spectra were
obtained at nominal resolutions of 3000, 5000, or 10000 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 sodium sulfate,
unless otherwise noted. Solvents were evaporated under
reduced pressure on a rotary evaporator. Thin-layer chrom-
atography was carried out on aluminium-backed silica gel
plates (Merck 60 F254) with visualisation of components by UV
light (254 nm) or exposure to I2. Column chromatography was
carried out on silica gel, (Merck 230–400 mesh). DCM refers to
dichloromethane; THF refers to tetrahydrofuran dried over
sodium benzophenone ketyl; DMF refers to dry dimethyl
formamide; EtOAc refers to ethyl acetate; ether refers to diethyl
ether; light petroleum refers to petroleum ether, boiling range
40–60 ЊC; MeOH refers to methanol; EtOH refers to ethanol.
All solvents were freshly distilled. Compounds 28a–n, 29a–n,
and 4a–n are named as phenylacetic acid derivatives in order to
provide a consistent numbering scheme throughout.
The 2- and 3-Me derivatives 5d and 5e fragmented, with
apparent 3-fold stoichiometry, too quickly to measure under
our assay system. For the 12 compounds for which data was
available, there was a quantitative relationship between Mt1/2
and substituent electronic properties, measured as σp for
2-substituents and σm for 3-substituents [eqn. (1)] (n is the
sample number, r is the Pearson coefficient, s is the standard
error estimate and F is the F statistic for Goodness of Fit.
log(Mt1/2) = 0.57( 0.10)σ ϩ 1.30( 0.05)
n = 12 r = 0.87 s = 0.15 F = 31.3
(1)
Note that, while the five 2-substituted analogues should
ideally be represented by σo substituent constants, these are not
readily available. Reliable σo values are reported21 for only three
of the five substituents, and these do not differ markedly
(>25%) from the corresponding σp values. A recalculation of
eqn, (1) using these values (not shown) did not change the
conclusions to be drawn from it.
If the correlations are performed separately for the 2- and
3-substituted analogues, the intercept is identical but the σ
coefficient differs (0.67 for the 2- substituted, 0.46 for the
3- substituted). This greater dependence of the 2-substituted
compounds on substituent electronic properties would be con-
sistent with some resonance stabilisation of the putative devel-
oping benzylic cation in the transition state of the carbamate
fragmentation.
The extent of release of amine 7 after 10 half-lives (t∞) was
clearly increased with increasing substituent electron-donation.
Thus the 2-NHMe analogue 5m produced a maximum of 71%
of 7 (t∞) whereas the 2-NO2 derivative 5j produced a maximum
of only 33% of 7. This property also correlated significantly
with σ values, although the relationship was not as strong [eqn.
(2)], and is more difficult to understand.
General preparation of nitrobenzyl alcohols 8d,e,g,l
BH3ؒDMS (20 mmol) was added dropwise to a stirred solution
of nitrobenzoic acid (10 mmol) and trimethyl borate (40 mmol)
in THF (100 cm3) at 20 ЊC. The solution was heated at reflux
temperature for 6 h and cooled to 5 ЊC. The reaction was
quenched carefully with MeOH (2 cm3), the mixture stirred for
5 min, water (2 cm3) was added, and the mixture stirred for 5
min. 5 M HCl (10 cm3) was added and the mixture heated at
50 ЊC for 30 min. The solvent was removed under reduced pres-
sure, the residue partitioned between EtOAc (100 cm3) and
water (100 cm3). The organic fraction was dried, the solvent
removed under reduced pressure, and the residue chromato-
graphed, eluting with 50% EtOAc–light petroleum to give the
nitrobenzyl alcohol 8.
log(t∞) = Ϫ0.21( 0.07)σ ϩ 1.70( 0.03)
n = 12 r = 0.71 s = 0.10 F = 10.3
(2)
2-Methyl-4-nitrobenzyl alcohol 8d. Preparation from 2-
methyl-4-nitrobenzoic acid as described above gave alcohol 8d
(93%) as colourless needles, mp (from EtOAc–light petroleum)
99–100 ЊC (Found: C, 57.7; H, 5.7; N, 8.5. C8H9NO3 requires C,
57.5; H, 5.4; N, 8.4%); νmax/cmϪ1 3310, 1522, and 1343; δH 2.16
(1 H, t, J 5.3, OH), 2.40 (3 H, s, CH3), 4.76 (2 H, d, J 5.3,
CH2O), 7.29 (1 H, d, J 8.3, 6-H), 8.03 (1 H, dd, J 8.3 and 2.4,
5-H), and 8.28 (1 H, d, J 2.4, 3-H); δC 18.8, 62.3, 121.6, 122.3,
130.9, 140.0, 143.4, and 146.5.
The varying degree of release of the amine (as low as 25% in
some cases) is of concern. Prodrugs of low pKA effectors release
to the least extent, with fragmentation of the hydroxylamine
competing with another reaction that forms an unknown
species. The nature of this species is currently under study.
These data suggest that the standard 4-nitrobenzyl carb-
amate trigger for NTR prodrugs may be improved upon.
Electron-donating substituents in the 2-position and α-methyl
substitution of the nitrobenzyl moiety will both favour acceler-
ated fragmentation of the 4-hydroxylamines produced by
enzymic reduction. Substituents such as 2-alkoxy or 2-amino-
alkyl also provide potential linkage points for the attachment of
side chains designed to improve prodrug solubility. Of course,
the effect of such substitutions on the kinetics of enzymic
reduction would have to be determined. Rates of hydroxy-
lamine fragmentation are also likely to be influenced by the
nature of the released amine, with prodrugs of primary amines
fragmenting more rapidly than a comparable secondary amine.
However, there may be less flexibility to alter the amine-based
“effector”.
3-Methyl-4-nitrobenzyl alcohol 8e. Preparation from 3-
methyl-4-nitrobenzoic acid as described above gave alcohol22 8e
(90%) as a white solid, mp (from EtOAc–light petroleum) 60–
61 ЊC (Found: C, 57.2; H, 5.4; N, 8.4. C8H9NO3 requires C,
57.5; H, 5.4; N, 8.4%).
3-Methoxy-4-nitrobenzyl alcohol 8g. Preparation from 3-
methoxy-4-nitrobenzoic acid as described above gave alcohol 8g
(83%) as cream coloured needles, mp (from EtOAc–light pet-
roleum) 96.5–97.0 ЊC (lit.23 mp (from benzene–light petroleum)
96 ЊC); νmax/cmϪ1 3260, 1618, 1512, and 1283; δH 2.23 (1 H, br s,
OH), 3.97 (3 H, s, OCH3), 4.77 (2 H, d, J 4.3, CH2O), 6.96 (1 H,
dd, J 8.2 and 1.3, 6-H), 7.15 (1 H, d, J 1.3, 2-H), and 7.84 (1 H,
d, J 8.2, 5-H); δC 56.5, 64.0, 111.1, 117.7, 126.0, 138.3, 148.3,
and 153.4.
Experimental
Analyses were carried out in the Microchemical Laboratory,
University of Otago, Dunedin, NZ. Melting points were deter-
mined on an Electrothermal 2300 Melting Point Apparatus. IR
spectra were recorded on a Midac FT-IR as KBr discs, unless
otherwise stated. NMR spectra were obtained on a Bruker
3-Methoxycarbonyl-4-nitrobenzyl alcohol 8l. Preparation
from 3-methoxycarbonyl-4-nitrobenzoic acid as described
above gave alcohol 8l (89%) as a tan solid, mp (from EtOAc–
light petroleum) 56–58 ЊC (Found: C, 51.4; H, 4.4; N, 6.7.
AM-400 spectrometer at 400 MHz for 1H and 100 MHz for 13
C
J. Chem. Soc., Perkin Trans. 1, 1999, 2759–2770
2763

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C9H9NO5 requires C, 51.2; H, 4.3; N, 6.6%); νmax/cmϪ1 3287,
1724, 1532, and 1360; δH 2.30 (1 H, br s, OH), 3.92 (3 H, s,
OCH3), 4.82 (2 H, s, CH2O), 7.60 (1 H, dd, J 8.4, and 1.4, 6-H),
7.68–7.70 (1 H, m, 2-H), and 7.91 (1 H, d, J 8.4, 5-H); δC 53.3,
63.4, 124.2, 127.3, 128.0, 129.0, 146.8, 147.0, and 166.2.
(150 cm3) and sat. aq. NaHCO3 (150 cm3). The aqueous phase
was extracted with EtOAc (2 × 50 cm3), the combined organic
fraction dried and the solvent removed under reduced pressure.
The residue was chromatographed, eluting with a gradient (20–
50%) of EtOAc–light petroleum to give (i) methyl 2-methyl-
amino-4-nitrobenzoate 13 (0.63 g, 27%) as orange needles, mp
(from EtOAc–light petroleum) 120–121 ЊC (Found C, 51.2; H,
4.65; N, 13.2. C9H10N2O4 requires C, 51.4; H, 4.8; N, 13.3%);
νmax/cmϪ1 3376, 1689, 1547, 1345, and 1238; δH 2.98 (3 H, d,
J 5.1, NCH3), 3.90 (3 H, s, OCH3), 7.34 (1 H, dd, J 8.7 and 2.2,
5-H), 7.46 (1 H, d, J 2.2, 3-H), 7.88 (1 H, br s, NH), and 8.02
(1 H, d, J 8.7, 6-H); δC 29.7, 52.1, 105.4, 108.2, 114.3, 132.9,
151.9, 152.3, and 167.8; (ii) starting material (0.25 g, 11%)
spectroscopically identical to an authentic sample; and (iii) 2-
methylamino-4-nitrobenzyl alcohol 8m (1.0 g, 49%) as orange
prisms, mp (from EtOAc–light petroleum) 135–137 ЊC (Found:
C, 52.5; H, 5.5; N, 15.4. C8H10N2O3 requires C, 52.7; H, 5.5; N,
15.4%); νmax/cmϪ1 3146, 1622, 1533, and 1367; δH 1.75 (1 H, br s,
NH), 2.94 (3 H, s, NCH3), 4.74 (2 H, s, CH2O), 5.14 (1 H, br s,
OH), 7.15 (1 H, d, J 8.1, 6-H), 7.40 (1 H, d, J 2.1, 3-H), and 7.48
(1 H, dd, J 8.1 and 2.1, 5-H); δC 30.2, 64.0, 104.0, 111.0, 128.8,
130.0, 149.3, and 149.4.
2-Methoxy-4-nitrobenzyl alcohol 8f. An etheral solution of
diazomethane (CAUTION) was added to a solution of 4-
nitrosalicylic acid 9 (1.0 g, 5.46 mmol) in ether (50 cm3) until a
yellow colour persisted and the solution stood at 20 ЊC for 4 h.
The reaction was quenched with glacial acetic acid (2 cm3),
poured into sat. aq. NaHCO3 solution and extracted with ether
(2 × 50 cm3). The combined organic fractions were dried and
the solvent removed under reduced pressure to give ester 10
(1.11 g, 96%) as white needles, mp (from ether) 89–90 ЊC (lit.24
mp (from MeOH) 86–88 ЊC) (Found: C, 51.5; H, 4.3; N, 6.7.
C9H9NO5 requires C, 51.2; H, 4.3; N, 6.6%).
A solution of 10 (0.9 g, 4.26 mmol) in THF (20 cm3) was
added dropwise to a stirred solution of DIBAL-H (1 M solution
in toluene, 13.4 cm3, 13.4 mmol) in THF (20 cm3) at 2 ЊC and
the solution stirred at 2 ЊC for 15 min. The solvent was removed
under reduced pressure and the residue partitioned between
EtOAc (100 cm3) and water (100 cm3). The aqueous fraction
was extracted with EtOAc (2 × 50 cm3) and the combined
organic fraction dried and the solvent removed under reduced
pressure. The residue was chromatographed, eluting with 50%
EtOAc–light petroleum, to give alcohol 8f (0.74 g, 93%) as
cream needles, mp (EtOAc–light petroleum) 103–104 ЊC
(Found: C, 52.4; H, 4.8; N, 7.4. C8H9NO4 requires C, 52.5; H,
4.95; N, 7.65%); νmax/cmϪ1 3310, 1523, 1250, and 1036; δH 2.27
(1 H, br s, OH), 3.96 (3 H, s, OCH3), 4.76 (2 H, d, J 5.5, CH2O),
7.52 (1 H, d, J 8.3, 6-H), 7.71 (1 H, d, J 2.1, 3-H), and 7.86 (1 H,
dd, J 8.3 and 2.1, 5-H); δC 55.9, 60.7, 105.0, 116.0, 127.9, 136.6,
148.3, and 157.1.
A solution of 13 (1.51 g, 7.18 mmol), di-tert-butyl dicarbo-
nate (3.14 g, 14.37 mmol) and dimethylaminopyridine (DMAP)
(80 mg, 0.72 mmol) in THF (100 cm3) was heated at reflux
temperature for 24 h. More di-tert-butyl dicarbonate (1.0 g,
4.58 mmol) was added and the solution heated at reflux
temperature for a further 24 h. The solvent was removed under
reduced pressure and the residue chromatographed, eluting
with 10% EtOAc–light petroleum to give methyl 2-[N-methyl-
N-(tert-butyloxycarbonyl)amino]-4-nitrobenzoate 14 (2.21 g,
97%) as a yellow oil, νmax(thin film)/cmϪ1 1734, 1713, 1531,
1350, and 1157; δH (2 rotamers) 1.31 and 1.52 (9 H, 2s,
C(CH3)3), 3.29 and 3.33 (3 H, 2s, NCH3), 3.91 and 3.93 (3 H, 2s,
OCH3), 7.95–8.03 (1 H, m, 6-H), and 8.08–8.18 (2 H, m, 5-H
and 3-H); δC 28.0 and 28.2 (3), 37.4 and 37.8, 52.8, 81.2 and 81.5,
121.0, 123.2, 126.1, 131.8, 144.5, 150.3, 153.4, and 165.3; m/z
310.1162 (Mϩ. C14H18N2O6 requires 310.1165); m/z 310 (Mϩ,
2%), 237 (10), 210 (40), and 57 (100).
2-[N-Methyl-N-(tert-butyloxycarbonyl)amino]-4-nitrobenzyl
alcohol 8h and 2-methylamino-4-nitrobenzyl alcohol 8m
Oxalyl chloride (4.3 cm3, 49.4 mmol) was added dropwise to
a mixture of 4-nitroanthranilic acid 11 (6.0 g, 32.9 mmol)
and DMF (2 drops) in DCM (100 cm3) at 2 ЊC and the mix-
ture stirred at 20 ЊC for 16 h. The solvent was removed under
reduced pressure and the residue dissolved in ice cold MeOH
(100 cm3) and the solution stirred for 16 h. The solvent was
removed under reduced pressure and the residue partitioned
between EtOAc (200 cm3) and sat. aq. NaHCO3 solution (200
cm3). The organic fraction was washed with 0.1 M NaOH
(100 cm3), water (100 cm3), brine (50 cm3), dried and the
solvent removed under reduced pressure. The residue was
chromatographed, eluting with 20% EtOAc–light petroleum,
to give methyl 4-nitroanthranilate 12 (4.48 g, 69%) as orange
A solution of 14 (2.21 g, 7.12 mmol) in MeOH (50 cm3) and
1 M LiOH solution (36 cm3, 36 mmol) was stirred at 50 ЊC for
30 min. The solution was cooled to 2 ЊC, diluted with water
(50 cm3), extracted with ether (50 cm3), and acidified to pH 4
with 1 M HCl. The mixture was extracted with EtOAc (3 × 50
cm3), the combined extracts dried and the solvent removed
under reduced pressure to give 2-[N-methyl-N-(tert-butyloxy-
carbonyl)amino]-4-nitrobenzoic acid 15 (2.1 g, 99%) as a tan
hygroscopic foam (Found: C, 52.5; H, 5.9; N, 9.1. C13H16N2O6
requires C, 52.7; H, 5.4; N, 9.5%); νmax(thin film)/cmϪ1 3457,
1710, 1533, 1350, and 1157; δH [(CD3)2SO] (2 rotamers) 1.32
and 1.40 (9 H, 2s, C(CH3)3), 3.20 (3 H, s, NCH3), 7.98 (1 H, d,
J 8.5, 6-H), 8.17 (1 H, dd, J 8.5 and 2.2, 5-H), 8.20 (1 H, br s,
3-H), and 13.53 (1 H, br s, CO2H); δC 27.4 and 27.3 (3), 37.6 and
37.1, 82.4 and 80.1, 121.1, 122.6, 124.9, 131.8, 143.5, 149.7 and
149.4, 152.6, and 166.3.
BH3ؒDMS reduction of 15 as described above gave alcohol
8h (41%) as a pale orange oil, νmax(thin film)/cmϪ1 3447, 1705,
1682, 1528, 1350, and 1157 cmϪ1; δH (2 rotamers) 1.27 and 1.35
(9 H, 2s, C(CH3)3), 3.23 (3 H, s, NCH3), 4.60–4.70 (2 H, m,
CH2O), 7.74 (1 H, d, J 8.4, 6-H), 8.03 (1 H, br s, 3-H), and 8.22
(1 H, dd, J 8.4 and 2.2, 5-H); δC 28.2 (3), 38.0, 60.6, 82.0, 122.1,
122.4, 128.4, 130.9, 145.6, 147.8, and 155.7; m/z 282.1214 (Mϩ.
C13H18N2O5 requires 282.1216); m/z 282 (Mϩ, 1%), 209 (10), 182
(20), and 57 (100).
needles, mp (from EtOAc–light petroleum) 154–156 ЊC; νmax
/
cmϪ1 3493, 3381, 1701, 1587, 1518, 1348, and 1252; δH 3.94
(3 H, s, OCH3), 6.05 (2 H, br s, NH2), 7.40 (1 H, dd, J 8.8 and
2.3, 5-H), 7.50 (1 H, d, J 2.3, 3-H), and 8.00 (1 H, d, J 8.8,
6-H); δC 52.2, 110.6, 111.1, 114.9, 132.8, 150.7, 151.3, and 167.3.
Formic acid (1.35 cm3, 35.9 mmol) was added dropwise to
acetic anhydride (2.75 cm3, 29.2 mmol) at 2 ЊC and then the
mixture was heated at 50 ЊC for 30 min. The mixture was
cooled to 2 ЊC and THF (20 cm3) added. A solution of 12
(2.2 g, 11.2 mmol) in THF (20 ml) was added dropwise and the
stirred solution allowed to warm to 20 ЊC over 1 h. The solvent
was removed under reduced pressure and the residue dissolved
in THF (100 cm3). The solution was cooled to 2 ЊC and
BH3ؒDMS (2.8 cm3, 28 mmol) added and the solution stirred at
2 ЊC for 3 h. The reaction was quenched carefully with MeOH
(2 cm3), stirred for 5 min, water (2 cm3) added and the mixture
stirred for 5 min. 1 M HCl (10 cm3) was added and the mixture
heated at 50 ЊC for 30 min. The solvent was removed under
reduced pressure and the residue partitioned between EtOAc
3-[N-Methyl-N-(tert-butyloxycarbonyl)amino]-4-nitrobenzyl
alcohol 8l
A mixture of 5-methoxycarbonyl-2-nitrobenzoic acid 16 (2.07
g, 9.19 mmol) (derived from 4-nitroisophthalic acid by Fischer
2764
J. Chem. Soc., Perkin Trans. 1, 1999, 2759–2770

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esterification) and DMF (2 drops) in SOCl2 (100 cm3) was
heated at reflux temperature for 3 h. The mixture was evapor-
ated to dryness under reduced pressure, dissolved in acetone
(50 cm3) and a solution of NaN3 (0.90 g, 13.8 mmol) in water
(5 cm3) added in one portion. The mixture was stirred at 20 ЊC
for 15 min, partitioned between benzene (150 cm3) and water
(150 cm3) and the aqueous fraction extracted with benzene (50
cm3). The combined organic extracts were dried and the solvent
removed under reduced pressure. The residue was dissolved in
xylene (100 cm3) and 2-(trimethylsilyl)ethanol (1.60 cm3, 11.0
mmol) added. The solution was heated at reflux temperature for
16 h, the solvent removed under reduced pressure and the resi-
due dissolved in THF (100 cm3). A solution of tetrabutyl-
ammonium fluoride (TBAF) (1 M solution in THF, 14 cm3, 14
mmol) in THF was added and the solution stirred at 20 ЊC for
30 min. The solvent was removed under reduced pressure and
the residue chromatographed, eluting with a gradient (20–50%)
of EtOAc–light petroleum, to give methyl 3-amino-4-nitro-
benzoate 17 (1.60 g, 89%) as orange needles, mp (from EtOAc–
light petroleum) 193–194.5 ЊC; νmax/cmϪ1 3493, 3370, 1720,
1633, 1580, 1320, and 1246; δH 3.90 (3 H, s, OCH3), 7.15–7.18
(3 H, m, 6-H and NH2), 7.71 (1 H, d, J 1.7, 2-H), 8.08 (1 H, d,
J 8.9, 5-H); δC 51.7, 114.6, 120.5, 125.3, 132.2, 134.9, 144.7, and
164.7; which was used without further characterisation.
Reductive formylation of 17 (2.34 g, 11.9 mmol) as described
above for 12 gave methyl 3-methylamino-4-nitrobenzoate 18
(1.07 g, 43%) as an orange powder, mp (from EtOAc–light
petroleum) 129–130 ЊC; νmax/cmϪ1 3376, 1730, 1576, 1319, and
1226; δH 3.08 (3 H, d, J 5.1, NCH3), 3.97 (3 H, s, OCH3), 7.23
(1 H, dd, J 8.9 and 1.7, 6-H), 7.54 (1 H, d, J 1.7, 2-H), 8.00
(1 H, br s, NH), and 8.21 (1 H, d, J 8.9, 5-H); δC 29.9, 52.7,
115.1, 115.3, 127.0, 133.8, 136.5, 145.7, and 165.8; m/z 210.0633
(Mϩ, C9H10N2O4 requires 210.0641); m/z 210 (Mϩ, 100%),
179 (20), and 161 (80); which was used without further
characterisation.
A solution of 18 (1.20 g, 5.7 mmol), di-tert-butyl dicarbonate
(2.49 g, 11.4 mmol) and DMAP (64 mg, 0.6 mmol) in THF
(100 cm3) was heated at reflux temperature for 96 h. The solvent
was removed under reduced pressure and the residue par-
titioned between EtOAc (150 cm3) and water (150 cm3). The
organic fraction was dried, the solvent removed under reduced
pressure and the residue chromatographed, eluting with 5%
EtOAc–light petroleum, to give methyl 3-[N-methyl-N-(tert-
butyloxycarbonyl)amino]-4-nitrobenzoate 19 (1.77 g, 99%) as
a yellow oil; νmax(thin film)/cmϪ1 1730, 1717, 1533, 1352, and
1157; δH 1.27 (9 H, s, C(CH3)3), 3.33 (3 H, s, NCH3), 3.99
(3 H, s, OCH3), and 7.92–8.02 (3 H, m, 2-H, 5-H, and 6-H);
δC 27.8 (3), 37.6, 52.9, 82.0, 124.8, 128.0, 130.0, 134.7, 137.5,
148.9, 152.7, and 164.7; m/z 310.1161 (Mϩ. C14H18N2O6
requires 310.1165); m/z 310 (Mϩ, 1%), 251 (2), 237 (5),
210 (10), and 57 (100); which was used without further
characterisation.
C, 55.3; H, 6.4; N, 9.9%); νmax/cmϪ1 3510, 1707, 1512, 1358, and
1157; δH (2 rotamers) 1.29 and 1.49 (9 H, 2s, C(CH3)3), 3.27 and
3.28 (3 H, 2s, NCH3), 4.74 and 4.78 (2 H, 2s, CH2O), 7.34–7.36
(m, 2 H, 2-H and 5-H), and 7.88–7.93 (1 H, m, 6-H); δC 28.2 and
27.7 (3), 37.4 and 37.3, 63.5, 81.6, 124.7, 125.3, 126.9, 137.4,
145.0, 147.8, and 154.9 and 153.3.
Preparation of N-(NЈ,NЈ-dimethylaminoethyl) 4-(methylamino)-
phenylacetamide 7 (Scheme 6)
A mixture of methyl 4-nitrophenylacetate 21 (6.44 g, 33.0
mmol) and Pd/C (100 mg) in EtOH (100 cm3) was stirred under
H2 (60 psi) for 1 h. The mixture was filtered through Celite
washed with EtOH (2 × 20 cm3) and the solvent removed under
reduced pressure. The residue was dissolved in THF (50 cm3)
and added to a solution of acetic formic anhydride (prepared
by adding formic acid (4.0 cm3, 106 mmol) dropwise to acetic
anhydride (8.1 cm3, 86.0 mmol) as described above) in THF (50
cm3) at Ϫ10 ЊC. The solution was stirred at Ϫ10 ЊC for 30 min,
allowed to warm to 20 ЊC, the solvent removed under reduced
pressure and the residue partitioned between EtOAc (150 cm3)
and sat. aq. NaHCO3 (150 cm3). The aqueous fraction was
extracted with EtOAc (100 cm3) and the combined organic frac-
tion dried and the solvent removed under reduced pressure. The
residue was dissolved in THF (100 cm3) and BH3ؒDMS (8.25
cm3, 82.5 mmol) added slowly. The solution was stirred at 20 ЊC
for 1 h, MeOH (10 cm3) carefully added, the mixture stirred for
15 min. 1 M HCl (10 cm3) was added and the mixture stirred at
40 ЊC for 30 min. The solvent was removed under reduced pres-
sure and the residue partitioned between EtOAc (200 cm3) and
sat. aq. NaHCO3 solution (100 cm3). The aqueous fraction was
extracted with EtOAc (100 cm3), the combined organic fraction
dried and the solvent removed under reduced pressure. The
residue was chromatographed, eluting with a gradient (20–40%)
EtOAc–light petroleum, to give methyl 4-(methylamino)phenyl-
acetate 22 (4.48 g, 76%) as a colourless oil which was used
directly.
A solution of 22 (4.48 g, 25.0 mmol), di-tert-butyl dicarbon-
ate (8.18 g, 37.5 mmol) and DMAP (0.28 g, 2.5 mmol) in
THF (100 cm3) was heated at reflux temperature for 16 h. The
solution was cooled to 20 ЊC and the solvent removed under
reduced pressure. The residue was partitioned between EtOAc
(100 cm3) and sat. aq. NaHCO3 solution (100 cm3) and the
aqueous fraction extracted with EtOAc (2 × 50 cm3). The com-
bined organic fraction was washed with water (2 × 50 cm3),
brine (50 cm3), dried, and the solvent removed under reduced
pressure. The residue was chromatographed, eluting with a
gradient (10–30%) of EtOAc–light petroleum to give methyl
4-[N-methyl-N-(tert-butyloxycarbonyl)amino]phenylacetate 23
(4.30 g, 62%) as a pale yellow gum (Found: C, 64.6; H, 7.6; N,
5.2. C15H21NO4 requires C, 64.5; H, 7.6; N, 5.0%); νmax(thin
film)/cmϪ1 1738, 1699, 1366, and 1153; δH 1.45 (9 H, s,
C(CH3)3), 3.24 (3 H, s, NCH3), 3.66 (2 H, s, CH2O), 3.69 (3 H, s,
OCH3), and 7.17–7.23 (4 H, m, 2-H, 3-H, 5-H, and 6-H); δC
28.3 (3), 37.2, 40.6, 52.0, 80.3, 125.5 (2), 129.4 (2), 130.9, 142.8,
154.7, and 171.9.
1  LiOH (25 cm3, 25 mmol) was added to a stirred solution
of 19 (1.75 g, 5.64 mmol) in MeOH (50 cm3) and the mixture
stirred at 50 ЊC for 30 min. The mixture was cooled to 5 ЊC,
washed with ether (50 cm3) and the pH adjusted to 4.0 with 1 M
HCl. The suspension was extracted with EtOAc (3 × 50 cm3),
the combined organic fraction dried, and the solvent removed
under reduced pressure to give 3-[N-methyl-N-(tert-butyloxy-
carbonyl)amino]-4-nitrobenzoic acid 20 (1.48 g, 89%) as a pale
yellow powder, mp (from EtOAc–light petroleum) 176–178 ЊC
(Found: C, 52.95; H, 5.6; N, 9.5. C13H16N2O6 requires C, 52.7;
H, 5.4; N, 9.5%); νmax/cmϪ1 3150, 1720, 1694, 1532, 1372, and
1155; δH 1.23 (9 H, s, C(CH3)3), 3.28 (3 H, s, NCH3), 7.98 (1 H,
dd, J 8.5 and 1.7, 6-H), 8.03 (1 H, d, J 1.6, 2-H), 8.10 (1 H, br d,
J 8.5, 5-H), and 13.86 (1 H, br s, CO2H); δC 27.2 (3), 36.9, 80.8,
125.2, 128.1, 129.3, 135.8, 136.4, 148.1, 151.9, and 165.3.
BH3ؒDMS reduction of 20 as described above gave alcohol 8i
(92%) as yellow prisms, mp (from EtOAc–light petroleum) 106–
107 ЊC (Found: C, 55.1; H, 6.6; N, 10.25. C13H18N2O5 requires
A solution of 23 (4.00 g, 14.3 mmol) in MeOH (50 cm3) and
1 M LiOH (73 cm3, 73 mmol) was stirred at 50 ЊC for 30 min.
The solution was cooled to 2 ЊC, washed with ether (50 cm3),
and the pH adjusted to 4.0 with 5 M HCl. The suspension was
stirred at 2 ЊC for 30 min and filtered to give 4-[N-methyl-N-
(tert-butyloxycarbonyl)amino]phenylacetic acid 24 (3.67 g,
97%) as a white powder, mp (from MeOH–water) 114–116 ЊC
(Found: C, 63.3; H, 7.1; N, 5.2. C14H19NO4 requires C, 63.4; H,
7.2; N, 5.3%); νmax/cmϪ1 3101, 1732, 1659, 1377, and 1155;
δH 1.45 (9 H, s, C(CH3)3), 3.24 (3 H, s, NCH3), 3.61 (2 H, s,
CH2O), 7.18–7.24 (4 H, m, 2-H, 3-H, 5-H, and 6-H), and 10.55
(1 H, br s, CO2H); δC 28.3 (3), 37.3, 40.4, 80.5, 125.6 (2), 129.5
(2), 130.3, 142.9, 154.8, and 177.0.
A solution of 24 (2.09 g, 7.88 mmol) and CDI (1.92 g, 15.8
J. Chem. Soc., Perkin Trans. 1, 1999, 2759–2770
2765

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mmol) in DMF (20 cm3) was stirred at 50 ЊC for 10 min. NЈ,NЈ-
Dimethylaminoethylamine (1.73 cm3, 15.8 mmol) was added
and the solution stirred at 20 ЊC for 2 h. The solution was
poured into water (250 cm3) and extracted with EtOAc (3 × 100
cm3), the combined organic fractions washed with brine (50
cm3), dried and the solvent removed under reduced pressure.
The residue was chromatographed, eluting with 0.5% Et3N–
10% MeOH–EtOAc to give N-(NЈ,NЈ-dimethylaminoethyl)
4-[N-methyl-N-(tert-butyloxycarbonyl)amino]phenylacetamide
25 (2.33 g, 88%), as a colourless oil, νmax/cmϪ1 3324, 1701, 1655,
1512, 1365, and 1153; δH 1.45 (9 H, s, C(CH3)3), 2.23 (6 H, s,
N(CH3)2), 2.44 (2 H, t, J 6.0, CH2N), 3.24 (3 H, s, NCH3), 3.31–
3.36 (2 H, m, CH2N), 3.49 (1 H, br s, CONH), 3.56 (2 H, s,
CH2CO), and 7.18–7.25 (4 H, m, 2-H, 3-H, 5-H, and 6-H); δC
28.3 (3), 36.7, 37.3, 43.0, 44.8 (2), 57.7, 80.3, 125.7 (2), 129.5 (2),
132.1, 142.7, 154.7, and 171.1; m/z 335.2200 (Mϩ. C18H29N3O3
requires 335.2209); m/z 335 (100%, Mϩ), 278 (5), 262 (50), 235
(30), and 219 (90).
A solution of 25 (1.0 g, 2.98 mmol) in HCl-saturated MeOH
(50 cm3) was stirred at 20 ЊC for 1 h. The solvent was removed
under reduced pressure to give acetamide 7 as the dihydro-
chloride salt (0.87 g, 95%) as a hygroscopic foam, νmax(thin
film)/cmϪ1 3435, 3230, 2672, and 1667; δH 2.75 (6 H, d, J 5.3,
N(CH3)2), 2.89 (3 H, s, NCH3), 3.12–3.17 (2 H, m, CH2N),
3.41–3.47 (2 H, m, CH2N), 3.62 (2 H, s, CH2CO), 7.43 (2 H, br
d, J 8.6, 3-H and 5-H), 7.49 (2 H, br d, J 8.6, 2-H and 6-H), 8.66
(1 H, t, J 5.6, CONH), 10.64 (1 H, br s, NHCl), and 11.35 (1 H,
br s, NHCl); δC 34.3, 36.1, 41.9, 42.5 (2), 55.8, 121.8 (2), 130.5
(2), 136.4, 136.5, and 170.3; m/z 235.1684 (Mϩ. C13H21N3O
requires 235.1685); m/z 235 (Mϩ, 20%), 120 (20), and 58 (100).
Preparation of methyl esters 28a–i. General method
Alcohol 8 was converted to the chloroformate by adding a solu-
tion of phosgene (CAUTION) in benzene (10 mmol) dropwise
to a stirred solution of 8 (2 mmol) in THF (50 cm3) at 0 ЊC
under N2. The solution was stirred at 20 ЊC for 16 h and the
solvent removed under reduced pressure. The residue was dis-
solved in DMF (20 cm3), Cs2CO3 (2.5 mmol) and a solution of
amine 6 or 7 (2.2 mmol) in DMF (5 cm3) added. The mixture
was stirred at 20 ЊC for 16 h, poured into water (100 cm3), and
extracted with EtOAc (3 × 50 cm3). The combined organic
extract was washed with 1 M HCl (2 × 50 cm3), water (50 cm3),
brine (40 cm3), dried, and the solvent removed under reduced
pressure. The residue was chromatographed, eluting with a
gradient (20–50%) of EtOAc–light petroleum to give the ester
28. Yields and characterisations of individual compounds are
given below.
Methyl 4-[N-(4-nitrobenzyloxycarbonyl)amino]phenylacetate
28a. Ester 28a was obtained (42%) as a pale yellow solid, mp
(from EtOAc–light petroleum) 115.5–116 ЊC (Found: C, 59.2;
H, 4.55; N, 8.0. C17H16N2O6 requires C, 59.3; H, 4.7; N, 8.1%);
νmax/cmϪ1 3333, 1732, 1716, 1605, 1541, and 1324; δH 3.59 (2 H,
s, CH2CO), 3.69 (3 H, s, OCH3), 5.29 (2 H, s, CH2O), 6.82 (1 H,
br s, NHCO2), 7.23 (2 H, br d, J 8.1, 3-H and 5-H), 7.34 (2 H, br
d, J 8.1, 2-H and 6-H), 7.54 (2 H, br d, J 8.7, 2Ј-H and 6Ј-H),
8.22 (2 H, ddd, J 8.7, 2.4, and 2.0, 3Ј-H and 5Ј-H); δC 40.4, 52.0,
65.4, 118.9 (2), 123.8 (2), 128.3 (2), 129.4, 130.0 (2), 136.4,
143.4, 147.7, 152.8, and 172.0.
Methyl
4-[N-methyl-N-(4-nitrobenzyloxycarbonyl)amino]-
Preparation of NϪ(NЈ,NЈ-dimethylaminoethyl) 4-aminophenyl-
acetamide 6 (Scheme 7)
phenylacetate 28b. Ester 28b was obtained (76%) as a cream
solid, mp (from EtOAc) 73–74 ЊC (Found: C, 60.1; H, 5.1; N,
7.5. C18H18N2O6 requires C, 60.3; H, 5.1; N, 7.8%); νmax/cmϪ1
1740, 1723, 1607, 1516, 1342, and 1157; δH 3.33 (3 H, s, NCH3),
3.64 (2 H, s, CH2CO), 3.71 (3 H, s, OCH3), 5.23 (2 H, s, CH2O),
7.21 (2 H, br d, J 8.1, 3-H and 5-H), 7.27 (2 H, d, J 8.4, 2-H and
6-H), 7.41 (2 H, br s, 2Ј-H and 6Ј-H), and 8.18 (2 H, d, J 8.4,
3Ј-H and 5Ј-H); δC 37.9, 40.5, 52.1, 65.8, 123.7 (2), 126.0 (2),
127.4 (2), 129.9 (2), 132.3, 141.8, 143.9, 147.5, 154.9, and 171.8.
A mixture of 4-nitrophenylacetic acid 26 (1.0 g, 5.52 mmol) and
CDI (1.34 g, 8.28 mmol) in DMF (10 cm3) was stirred at 50 ЊC
for 10 min. The solution was cooled to 20 ЊC, NЈ,NЈ-dimethyl-
aminoethylamine (1.21 cm3, 11 mmol) was added dropwise and
the solution stirred for 2 h. The solution was poured into water
(150 cm3) and extracted with EtOAc (3 × 75 cm3). The com-
bined organic extracts were washed with water (2 × 50 cm3),
brine (50 cm3), dried, and the solvent removed under reduced
pressure. The residue was chromatographed, eluting with a
gradient (0–30%) of MeOH–EtOAc, to give N-(NЈ,NЈ-dimethyl-
aminoethyl) 4-nitrophenylacetamide 27 (0.70 g, 58%) as a brown
solid, mp (from EtOAc) 89–90 ЊC (Found: C, 57.2; H, 6.6; N,
Methyl 4-{N-methyl-N-[1-(4-nitrophenyl)ethyloxycarbonyl]-
amino}phenylacetate 28c. Ester 28c was obtained (61%) as a
pale yellow gum, νmax(thin film)/cmϪ1 1736, 1707, 1518, 1346,
and 1155; δH 1.52 (3 H, d, J 6.6, CH3), 3.31 (3 H, s, NCH3), 3.65
(2 H, s, CH2CO), 3.72 (3 H, s, OCH3), 5.88 (1 H, q, J 6.6, OCH),
7.19 (2 H, d, J 8.3, 3-H and 5-H), 7.29 (2 H, d, J 8.3, 2-H and
6-H), 7.38 (2 H, br s, 2Ј-H and 6Ј-H), 8.17 (2 H, d, J 8.6, 3Ј-H
and 5Ј-H); δC ((CD3)2SO) 22.7, 37.7, 40.6, 52.1, 72.7, 123.8 (2),
126.0 (3), 126.5 (2), 129.8 (2), 141.9, 147.3, 149.6, 154.5, and
171.8; m/z (DEI) 372.1325 (Mϩ. C19H20N2O6 requires
372.1321); m/z (DEI) 372 (Mϩ, 50%), 328 (20), 313 (60), 223
(45), and 178 (100).
16.6. C12H17N3O3 requires C, 57.35; H, 6.8; N, 16.7%); νmax
/
cmϪ1 3304, 3061, 1665, 1540, 1508, and 1350; δH 2.20 (6 H, s,
N(CH3)2), 2.41 (2 H, t, J 5.9, CH2N), 3.30–3.35 (2 H, m,
CH2N), 3.63 (2 H, s, CH2CO), 6.28 (1 H, br s, CONH), 7.47
(2 H, dd, J 8.8 and 2.4, 2-H and 6-H), and 8.18 (2 H, dd, J 8.8
and 2.4, 3-H and 5-H); δC 36.9, 43.1, 45.0 (2), 57.5, 123.7 (2),
130.1 (2), 142.7, 147.0, and 169.1; m/z (CI) 252.1354 (MHϩ.
C12H18N3O3 requires 252.1348); m/z (CI) 252 (MHϩ, 100%), 222
(95), and 151 (10).
A solution of 27 (0.68 g, 3.10 mmol) in EtOAc (30 cm3) was
stirred with Pd/C (50 mg) under H2 (60 psi) for 1 h. The mixture
was filtered through Celite, washed with EtOAc (50 cm3) and
the solvent removed under reduced pressure. The residue was
dissolved in EtOAc (30 cm3) and HCl-saturated EtOAc (30 cm3)
added. The solvent was removed under reduced pressure to give
acetamide 6 (0.84 g, 92%) as a green foam, mp (from EtOAc)
70 ЊC (decomp.) (Found: C, 48.8; H, 7.2; N, 14.3; Cl, 24.1.
Methyl 4-[N-methyl-N-(2-methyl-4-nitrobenzyloxycarbonyl)-
amino]phenylacetate 28d. Ester 28d was obtained (53%) as a
pale yellow oil, νmax/cmϪ1 1734, 1707, 1518, 1346, and 1153;
δH 2.38 (3 H, s, CH3), 3.33 (3 H, s, NCH3), 3.66 (2 H, s,
CH2CO), 3.71 (3 H, s, OCH3), 5.19 (2 H, s, CH2O), 7.23–7.34
(5 H, m, 2-H, 3-H, 5-H, 6-H, and 6-H), and 8.19–8.21 (2 H, m,
3-H and 5-H); δC 19.0, 37.9, 40.6, 52.0, 64.2, 121.5, 122.6, 126.1
(2), 130.3 (2), 130.9, 131.0, 136.5, 141.7, 142.3, 146.4, 154.9, and
171.8; m/z (DEI) 372.1317 (Mϩ. C19H20N2O6 requires
372.1321); m/z (DEI) 372 (Mϩ, 60%), 312 (15), 269 (20), 178
(80), and 150 (100).
C12H21Cl2N3O requires C, 49.0; H, 7.2; N, 14.3; Cl, 24.1%); νmax
/
cmϪ1 3447, 3316, 2866, 1655, and 1530; δH 2.75 (6 H, s,
N(CH3)2), 3.12 (2 H, t, J 5.9, CH2N), 3.40–3.44 (2 H, m,
CH2N), 3.66 (2 H, s, CH2CO), 7.30 (2 H, br d, J 8.3, 2-H and
6-H), 7.38 (2 H, br d, J 8.3, 3-H and 5-H), 8.58 (1 H, br s,
CONH), 10.37 (1 H, br s, NHCl), and 10.54 (2 H, br s, NH2);
δC 33.9, 41.5, 42.1 (2), 55.5, 122.8 (2), 130.3 (2), 130.4, 135.7,
and 170.3.
Methyl 4-[N-methyl-N-(3-methyl-4-nitrobenzyloxycarbonyl)-
amino]phenylacetate 28e. Ester 28e was obtained (52%) as a
pale yellow oil, νmax/cmϪ1 1736, 1707, 1518, 1343, and 1153;
δH 2.56 (3 H, s, CH3), 3.32 (3 H, s, NCH3), 3.64 (2 H, s, CH2CO),
2766
J. Chem. Soc., Perkin Trans. 1, 1999, 2759–2770

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3.71 (3 H, s, OCH3), 5.16 (2 H, s, CH2O), 7.09–7.34 (6 H, m,
2-H, 3-H, 5-H, 6-H, 2Ј-H, and 6Ј-H), and 7.94 (1 H, d, J 8.8,
5Ј-H); δC 20.5, 37.9, 40.5, 52.1, 65.8, 125.0, 125.5 (2), 126.0,
129.9 (2), 130.4, 131.4, 133.9, 141.8, 142.1, 148.4, 155.0, and
171.8; m/z (DEI) 372.1317 (Mϩ. C19H20N2O6 requires
372.1321); m/z (DEI) 372 (Mϩ, 100%), 328 (15), 313 (20) and
269 (100).
solvent removed under reduced pressure. The solid was
recrystallised from EtOAc to give acid 29. Yields and character-
isations of individual compounds are given below.
4-[N-(4-Nitrobenzyloxycarbonyl)amino]phenylacetic acid 29a.
Acid 29a was obtained (80%) as a tan powder, mp (from
MeOH–water) 182–183 ЊC (Found: C, 58.4; H, 4.3; N, 8.4.
C16H14N2O6 requires C, 58.2; H, 4.3; N, 8.5%); νmax/cmϪ1 3393,
1736, 1690, 1610, 1541, 1344, and 1223; δH [(CD3)2SO] 3.49
(2 H, s, CH2CO), 5.30 (2 H, s, CH2O), 7.17 (2 H, d, J 8.4, 3-H,
5-H), 7.40 (2 H, d, J 8.4, 2-H, 6-H), 7.69 (2 H, d, J 8.6, 2Ј-H,
6Ј-H), 8.26 (2 H, d, J 8.7, 3Ј-H, 5Ј-H), 9.87 (1 H, s, NHCO2),
and 12.26 (1 H, br s, CO2H); δC [(CD3)2SO] 39.9, 64.4, 118.1 (2),
123.5 (2), 128.4 (2), 129.1, 129.6 (2), 137.3, 144.6, 147.0, 153.0,
and 172.7.
Methyl
4-[N-methyl-N-(2-methoxy-4-nitrobenzyloxy-
carbonyl)amino]phenylacetate 28f. Ester 28f was obtained (66%)
as a pale yellow oil, νmax/cmϪ1 1736, 1707, 1518, 1348, 1250, and
1155; δH 3.34 (3 H, s, NCH3), 3.64 (2 H, s, CH2CO), 3.71 (3 H, s,
OCH3), 3.95 (3 H, s, OCH3), 5.23 (2 H, s, CH2O), 7.22–7.31
(4 H, m, 2-H, 3-H, 5-H, and 6-H), 7.53 (1 H, d, J 8.3, 6Ј-H),
7.71 (1 H, d, J 2.1, 3Ј-H), and 7.88 (1 H, dd, J 8.3 and 2.1,
5Ј-H); δC 37.8, 40.6, 52.1, 55.9, 62.1, 105.0, 115.7, 126.0 (2),
127.8, 129.9 (2), 132.9, 136.6, 141.9, 148.3, 155.0, 157.1, and
171.8; m/z (DEI) 388.1264 (Mϩ. C19H20N2O7 requires
388.1271); m/z (DEI) 388 (Mϩ, 20%), 344 (35), 285 (20), 178
(30), and 166 (100).
4-[N-Methyl-N-(4-nitrobenzyloxycarbonyl)amino]phenyl-
acetic acid 29b. Acid 29b was obtained (89%) as a white solid,
mp (from EtOAc) 125–126 ЊC (Found: C, 59.2; H, 4.6; N, 8.1.
C17H16N2O6 requires C, 59.3; H, 4.7; N, 8.1%); νmax/cmϪ1 3443,
3109, 1703, 1692, 1610, 1541, 1346, and 1167; δH [(CD3)2SO]
3.26 (3 H, s, NCH3), 3.58 (2 H, s, CH2CO), 5.24 (2 H, s, CH2O),
7.26–7.30 (4 H, m, 2-H, 3-H, 5-H, 6-H), 7.56 (2 H, br d, J 8.7,
2Ј-H, 6Ј-H), 8.21 (2 H, d, J 8.7, 3Ј-H, 5Ј-H), and 12.36 (1 H,
br s, CO2H); δC [(CD3)2SO] 37.5, 40.0, 65.3, 123.5 (2), 125.6
(2), 127.9 (2), 129.8 (2), 132.9, 141.3, 144.6, 146.9, 154.3, and
172.6.
Methyl
4-[N-methyl-N-(3-methoxy-4-nitrobenzyloxy-
carbonyl)amino]phenylacetate 28g. Ester 28g was obtained
(67%) as a white powder, mp (from EtOAc–light petroleum) 80–
83 ЊC (Found: C, 58.9; H, 5.2; N, 7.3. C19H20N2O7 requires C,
58.8; H, 5.2; N, 7.2%); νmax/cmϪ1 1736, 1707, 1611, 1518, 1344,
and 1157; δH 3.33 (3 H, s, NCH3), 3.63 (2 H, s, CH2CO), 3.71
(3 H, s, OCH3), 3.86 (3 H, br s, OCH3), 5.17 (2 H, s, CH2O),
6.89–6.93 (2 H, m, 2-H and 6-H), 7.21–7.23 (2 H, m, 2Ј-H and
6Ј-H), 7.30 (1 H, br d, J 8.4, 3-H and 5-H), and 7.81 (1 H, d,
J 8.3, 5-H); δC 37.9, 40.4, 52.1, 56.4, 65.9, 111.9, 118.5, 125.8,
126.1 (2), 129.9 (2), 132.4, 138.7, 141.8, 143.7, 153.1, 154.9, and
171.7.
4-{N-Methyl-N-[1-(4-nitrophenyl)ethyloxycarbonyl]amino}-
phenylacetic acid 29c. Acid 29c was obtained (75%) as a white
powder, mp (from MeOH) 117–118.5 ЊC (Found: C, 60.3; H,
5.0; N, 7.7. C18H18N2O6 requires C, 60.3; H, 5.1; N, 7.8%); νmax
/
cmϪ1 3414, 2980, 1705, 1607, 1516, and 1344; δH [(CD3)2SO]
1.45 (3 H, d, J 6.3, CH3), 3.15 (3 H, s, NCH3), 3.59 (2 H, s,
CH2CO), 5.85 (1 H, q, J 6.3, OCH), 7.28 (4 H, br s, 2-H, 3-H,
5-H, 6-H), 7.53–7.57 (2 H, m, 2Ј-H, 6Ј-H), 8.20 (2 H, d, J 8.7,
3Ј-H, 5Ј-H), and 12.37 (1 H, br s, CO2H); δC [(CD3)2SO] 22.3,
37.3, 40.0, 72.1, 123.6 (2), 125.4 (2), 126.6 (2), 129.7 (2), 132.7,
141.3, 146.7, 149.8, 153.7, and 172.6.
Methyl
4-(N-methyl-N-{2-[N-methyl-N-(tert-butyloxy-
carbonyl)amino]-4-nitrobenzyloxycarbonyl}amino)phenyl-
acetate 28h. Ester 28h was obtained (94%) as a pale yellow oil,
νmax(thin film)/cmϪ1 1736, 1707, 1527, 1348, and 1153; δH (two
rotamers) 1.33 and 1.51 (9 H, 2s, C(CH3)3), 3.18 (3 H, s, NCH3),
3.32 (3 H, s, NCH3), 3.64 (2 H, s, CH2CO), 3.71 (3 H, s, OCH3),
5.16 (2 H, s, CH2O), 7.21 (2 H, br d, J 8.3, 2-H and 6-H), 7.30
(2 H, br d, J 8.3, 3-H and 5-H), 7.40 (1 H, br s, 6Ј-H), 8.00 (1 H,
br s, 3Ј-H), and 8.09 (1 H, br d, J 8.0, 5Ј-H); δC 28.2 (3), 37.0,
38.0, 40.5, 52.1, 62.6, 81.3, 122.0, 122.3, 126.1 (2), 128.4, 130.0
(2), 132.6, 141.8, 142.1, 147.8, 153.8, 154.9, and 171.7; 2Ј-C not
observed; m/z (DEI) 487.1969 (Mϩ. C24H29N3O8 requires
487.1956); m/z (DEI) 487 (Mϩ, 5%), 431 (21), 209 (90), and 57
(100).
4-[N-Methyl-N-(2-methyl-4-nitrobenzyloxycarbonyl)amino]-
phenylacetic acid 29d. Acid 29d was obtained (80%) as a pale
yellow powder, mp (from EtOAc) 107–111 ЊC (Found: C, 60.4;
H, 5.0; N, 7.8. C18H18N2O6 requires C, 60.3; H, 5.0; N,
7.8%); νmax/cmϪ1 3086, 2944, 1720, 1703, 1528, 1344, and 1157;
δH [(CD3)2SO] 2.36 (3 H, s, CH3), 3.24 (3 H, s, NCH3), 3.58 (2 H,
s, CH2CO), 5.20 (2 H, s, CH2O), 7.28 (4 H, br s, 2-H, 3-H, 5-H,
6-H), 7.47 (1 H, d, J 8.3, 6Ј-H), 8.06–8.09 (2 H, m, 3Ј-H and
5Ј-H), and 12.40 (1 H, br s, CO2H); δC [(CD3)2SO] 18.5, 37.5,
40.0, 64.0, 122.2, 122.6 (2), 125.6 (2), 129.8 (2), 136.7, 131.3,
141.3, 144.6, 145.6, 154.2, and 172.6.
Methyl
4-(N-methyl-N-{3-[N-methyl-N-(tert-butyloxy-
carbonyl)amino]-4-nitrobenzyloxycarbonyl}amino)phenyl-
acetate 28i. Ester 28i was obtained (89%) as a pale yellow oil,
νmax(thin film)/cmϪ1 1736, 1711, 1528, 1354, and 1155; δH 1.27
and 1.47 (9 H, 2s, C(CH3)3), 2.93 (3 H, s, NCH3), 3.25 (3 H, s,
NCH3), 3.64 (2 H, s, CH2CO), 3.71 (3 H, s, OCH3), 5.20 (2 H, s,
CH2O), 7.20–7.30 (6 H, m, 2-H, 3-H, 5-H, 6-H, 2Ј-H, and
6Ј-H), and 7.80 (1 H, br d, J 8.5, 5Ј-H); δC 28.2 (3), 37.3, 37.9,
40.5, 52.1, 65.4, 81.7, 125.0, 125.3, 126.0 (2), 127.0, 129.9 (2),
132.4, 137.5, 141.7, 143.1, 145.3, 153.0, 154.8, and 171.7; m/z
(DEI) 487.1959 (Mϩ. C24H29N3O8 requires 487.1956); m/z
(DEI) 487 (Mϩ, 10%), 399 (20), 387 (40), 178 (50), 165 (70), and
57 (100).
4-[N-Methyl-N-(3-methyl-4-nitrobenzyloxycarbonyl)amino]-
phenylacetic acid 29e. Acid 29e was obtained (59%) as a cream
powder, mp (from EtOAc) 118–119 ЊC (Found: C, 60.5; H, 5.1;
N, 7.8. C18H18N2O6 requires C, 60.3; H, 5.0; N, 7.8%); νmax/cmϪ1
3034, 1707, 1613, 1516, 1339, and 1169; δH [(CD3)2SO] 2.51
(3 H, s, CH3), 3.25 (3 H, s, NCH3), 3.58 (2 H, s, CH2CO), 5.16
(2 H, s, CH2O), 7.26–7.34 (4 H, m, 2-H, 3-H, 5-H, and 6-H),
7.34–7.37 (2 H, m, 2Ј-H, 6Ј-H), 7.97 (1 H, d, J 8.2, 5Ј-H), and
12.36 (1 H, br s, CO2H); δC [(CD3)2SO] 19.6, 37.5, 40.0, 65.2,
124.6 (2), 125.5, 129.8 (2), 130.0, 130.9, 132.9, 141.3, 142.5 (2),
148.0, 154.2, and 172.5.
Hydrolysis of esters 28a–i. General method
4-[N-Methyl-N-(2-methoxy-4-nitrobenzyloxycarbonyl)-
amino]phenylacetic acid 29f. Acid 29f was obtained (85%) as a
pale yellow powder, mp (from MeOH–water) 173.5–175.5 ЊC
(Found: C, 57.8; H, 5.0; N, 7.4. C18H18N2O7 requires C, 57.8; H,
4.85; N, 7.5%); νmax/cmϪ1 3094, 2945, 1721, 1699, 1518, 1345,
and 1155; δH [(CD3)2SO] 3.26 (3 H, s, NCH3), 3.58 (2 H, s,
A solution of ester 28 (2 mmol) in MeOH (40 cm3) and 1 M
NaOH solution (10 mmol) was stirred at 20 ЊC for 1 h. The
solution was washed with ether (20 cm3), the pH of the aqueous
fraction adjusted to 2 with 5 M HCl, and extracted with EtOAc
(3 × 30 cm3). The combined organic extract was dried and the
J. Chem. Soc., Perkin Trans. 1, 1999, 2759–2770
2767

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CH2CO), 3.95 (3 H, s, OCH3), 5.15 (2 H, s, CH2O), 7.26–7.31
(4 H, m, 2-H, 3-H, 5-H, and 6-H), 7.39–7.41 (1 H, m, 6Ј-H),
7.77 (1 H, d, J 2.0, 3Ј-H), 7.83 (1 H, dd, J 8.3 and 2.0, 5Ј-H),
and 12.36 (1 H, br s, CO2H); δC [(CD3)2SO] 37.5, 40.0, 56.2,
61.5, 105.3, 115.5, 125.5 (2), 127.9, 129.8 (2), 132.5, 132.9,
141.3, 148.0, 154.2, 156.6, and 172.6.
νmax/cmϪ1 3385, 3283, 1738, 1649, 1607, 1539, 1346, and 1125;
δH [(CD3)2SO] 2.75 (6 H, d, J 5.0, N(CH3)2), 3.34 (2 H, s,
CH2CO), 3.39–3.43 (4 H, m, 2 × CH2N), 3.47 (3 H, br
s, CH3OH), 5.30 (2 H, s, CH2O), 7.19 (2 H, d, J 8.4, 3-H, 5-H),
7.39 (2 H, d, J 8.4, 2-H, 6-H), 7.68 (2 H, d, J 8.6, 2Ј-H, 6Ј-H),
8.27 (2 H, d, J 8.7, 3Ј-H, 5Ј-H), 8.46 (1 H, t, J 5.5, CONH), 9.88
(1 H, s, NHCO2), and 10.55 (1 H, s, NHCl); δC [(CD3)2SO] 33.9,
41.6, 42.1 (2), 49.0, 55.6, 64.4, 118.5 (2), 123.6 (2), 128.4 (2),
129.4 (2), 130.1, 137.2, 144.6, 147.0, 153.0, and 170.8.
4-[N-Methyl-N-(3-methoxy-4-nitrobenzyloxycarbonyl)-
amino]phenylacetic acid 29g. Acid 29g was obtained (83%) as a
cream powder, mp (from MeOH–water) 158–160 ЊC (Found: C,
57.85; H, 4.85; N, 7.3. C18H8N2O7 requires C, 57.8; H, 4.85; N,
7.5%); νmax/cmϪ1 3119, 3055, 1701, 1614, 1516, 1340, and 1165;
δH [(CD3)2SO] 3.26 (3 H, s, NCH3), 3.58 (2 H, s, CH2CO), 3.86
(3 H, s, OCH3), 5.17 (2 H, s, CH2O), 7.01 (1 H, br d, J 8.3,
6Ј-H), 7.23 (1 H, br s, 2Ј-H), 7.27–7.33 (4 H, m, 2-H, 3-H, 5-H,
and 6-H), 7.86 (1 H, d, J 8.3, 5Ј-H), and 12.37 (1 H, br s,
CO2H); δC [(CD3)2SO] 37.5, 40.0, 56.5, 65.3, 112.1, 118.4, 125.2
(2), 125.7, 129.8 (2), 133.0, 138.2, 141.3, 144.0, 152.0, 154.2, and
172.6.
N-[2-(N,N-Dimethylamino)ethyl]
4-[N-methyl-N-(4-nitro-
benzyloxycarbonyl)amino]phenylacetamide hydrochloride 4b.
Acetamide hydrochloride 4b was obtained (58%) as a pale yellow
gum (Found: C, 55.65; H, 6.3; N, 12.1; Cl, 8.1. C21H27ClN4O5
requires C, 55.9; H, 6.0; N, 12.4; Cl, 7.9%); νmax/cmϪ1 3289,
1701, 1651, 1610, 1541, 1346, and 1167; δH [(CD3)2SO] 2.75
(6 H, d, J 3.9, N(CH3)2), 3.12–3.15 (2 H, m, CH2N), 3.25 (3 H, s,
NCH3), 3.40–3.44 (2 H, m, CH2N), 3.48 (2 H, s, CH2CO), 5.24
(2 H, s, CH2O), 7.26–7.31 (4 H, m, 2-H, 3-H, 5-H, 6-H), 7.56
(2 H, br d, J 8.7, 2Ј-H, 6Ј-H), 8.22 (2 H, d, J 8.7, 3Ј-H, 5Ј-H),
8.56 (1 H, t, J 5.5, CONH), and 10.49 (1 H, br s, NHCl);
δC [(CD3)2SO] 33.9, 37.6, 41.6, 42.1 (2), 55.6, 65.3, 123.5 (2),
125.5 (2), 127.9 (2), 129.6 (2), 133.9, 141.2, 144.6, 146.9, 154.3,
and 170.6.
4-(N-Methyl-N-{2-[N-methyl-N-(tert-butyloxycarbonyl)-
amino]-4-nitrobenzyloxycarbonyl}amino)phenylacetic acid 29h.
Acid 29h was obtained (90%) as a colourless oil, νmax(thin film)/
cmϪ1 3202, 1710, 1528, 1348, and 1153; δH [(CD3)2SO] 1.28 and
1.46 (9 H, 2s, C(CH3)3), 3.11 (3 H, s, NCH3), 3.26 (3 H, s,
NCH3), 3.58 (2 H, s, CH2CO), 5.10 (2 H, s, CH2O), 7.26–7.31
(4 H, m, 2-H, 3-H, 5-H, and 6-H), 7.53–7.56 (1 H, m, 6Ј-H),
8.14–8.17 (2 H, m, 3Ј-H and 5Ј-H), and 12.60 (1 H, br s,
CO2H); δC [(CD3)2SO] 27.6 (3), 36.6, 37.6, 40.0, 59.7, 80.2,
122.0, 122.2, 125.6, 128.5 (2), 128.6, 129.8 (2), 133.0, 141.2,
142.1, 147.3, 153.0, 154.2, and 172.5; m/z (DEI) 473.1795. (Mϩ.
C23H27N3O8 requires 473.1798); m/z (DEI) 473 (Mϩ, 5%), 417
(2), 373 (5), 209 (90), 165 (90), and 57 (100).
N-[2-(N,N-Dimethylamino)ethyl] 4-{N-methyl-N-[1-(4-nitro-
phenyl)ethyloxycarbonyl]amino}phenylacetamide hydrochloride
4c. Acetamide hydrochloride 4c was obtained (85%) as a yellow
gum, νmax(thin film)/cmϪ1 3407, 1701, 1658, 1516, 1346, and
1159; δH [(CD3)2SO] 1.45 (3 H, d, J 6.6, CH3), 2.81 (6 H, d, J 4.0,
N(CH3)2), 3.14 (2 H, s, CH2CO), 3.25 (3 H, s, NCH3), 3.36–3.41
(4 H, m, 2 × CH2N), 5.84 (1 H, q, J 6.6, OCH), 7.25–7.32 (4 H,
m, 2-H, 3-H, 5-H, and 6-H), 7.54–7.58 (2 H, m, 2Ј-H, 6Ј-H),
8.22 (2 H, d, J 8.7, 3Ј-H, 5Ј-H), 8.58 (1 H, t, J 5.5, CONH), and
10.56 (1 H, br s, HCl); δC [(CD3)2SO] 22.3, 33.9, 37.4, 41.6, 42.1
(2), 55.5, 72.1, 123.6 (2), 125.4 (2), 126.7 (2), 129.5 (2), 133.7,
141.2, 146.7, 149.8, 153.7, and 170.6; m/z (DEI) 428.2055 (Mϩ.
C22H28N4O5 requires 428.2060); m/z (DEI) 428 (Mϩ, 60%), 409
(80), 385 (10), 235 (70), and 164 (100).
4-(N-Methyl-N-{3-[N-methyl-N-(tert-butoxycarbonyl)amino]-
-4-nitrobenzyloxycarbonyl}amino)phenylacetic acid 29i. Acid 29i
was obtained (91%) as a white foam (Found: C, 57.6; H, 6.0; N,
8.45. C23H27N3O8.1/2MeOH requires C, 57.7; H, 6.0; N, 8.6%);
νmax/cmϪ1 3360, 1711, 1528, 1356, and 1157; δH [(CD3)2SO] 1.22
and 1.42 (9 H, 2s, C(CH3)3), 3.18 (3 H, s, NCH3), 3.10 (1.5 H, br
s, residual CH3OH), 3.26 (3 H, s, NCH3), 3.58 (2 H, s, CH2CO),
4.00 (0.5 H, m, residual CH3OH), 5.19 (2 H, s, CH2O), 7.26–
7.31 (4 H, m, 2-H, 3-H, 5-H, 6-H), 7.37 (1 H, br d, J 8.3, 6Ј-H),
7.45 (1 H, br s, 2Ј-H), 7.95 (1 H, d, J 8.3, 5Ј-H), and 12.35 (1 H,
br s, CO2H); δC [(CD3)2SO] 27.8 (3), 37.0, 37.5, 39.9, 48.6, 65.0,
80.6, 124.9, 125.3, 125.6 (2), 126.7, 129.8 (2), 132.9, 136.3,
141.3, 143.7, 144.6, 152.1, 154.2, and 172.5.
N-[2-(N,N-Dimethylamino)ethyl] 4-[N-methyl-N-(2-methyl-4-
nitrobenzyloxycarbonyl)amino]phenylacetamide hydrochloride
4d. Acetamide hydrochloride 4d was obtained (63%) as a brown
hygroscopic foam, νmax(thin film)/cmϪ1 3396, 1705, 1672, 1516,
1346, and 1155; δH [(CD3)2SO] 2.75 (6 H, d, J 4.8, N(CH3)2),
2.50 (3 H, s, CH3), 3.11–3.15 (2 H, m, CH2N), 3.24 (3 H, s,
NCH3), 3.40–3.43 (2 H, m, CH2N), 3.47 (2 H, s, CH2CO), 5.20
(2 H, s, CH2O), 7.22–7.31 (4 H, m, 2-H, 3-H, 5-H, 6-H), 7.49
(1 H, d, J 8.3, 6-H), 8.02–8.06 (1 H, m, 3Ј-H), 8.08 (1 H, dd,
J 8.3 and 2.2, 5Ј-H), 8.52 (1 H, t, J 5.5, CONH), and 10.41
(1 H, s, NHCl); δC [(CD3)2SO] 18.5, 33.9, 37.5, 41.6, 42.1 (2),
55.7, 63.9, 122.1, 122.6, 125.5 (2), 129.6 (2), 131.3, 134.0, 136.7,
141.3, 144.6, 145.6, 154.2, and 170.6; m/z (DEI) 428.2049 (Mϩ.
C22H28N4O5 requires 428.2060); m/z (DEI) 428 (Mϩ, 35%), 409
(60), 384 (60), 358 (80), and 327 (100).
Formation of carbamates 4a–i: example of general preparation
by method A (Scheme 8)
A solution of acid 29 (1 mmol) and CDI (1.5 mmol) in DMF
(10 cm3) was heated at 50 ЊC for 10 min. NЈ,NЈ-Dimethyl-
aminoethylamine (2 mmol) was added and the solution stirred
at 20 ЊC for 2 h. The solution was poured into ice–water (100
cm3) and extracted with EtOAc (3 × 50 cm3). The combined
organic fraction was washed with 1 M NaOH solution (10 cm3),
water (3 × 30 cm3), brine (30 cm3), dried, and the solvent
removed under reduced pressure. The residue was chromato-
graphed on neutral alumina, eluting with EtOAc to give the
amide 4. Amide 4 was dissolved in MeOH (10 cm3) and HCl-
saturated MeOH (10 cm3) added and then the solvent was
removed under reduced pressure to give 4 as the HCl salt.
Yields and characterisations of individual compounds are given
below.
N-[2-(N,N-Dimethylamino)ethyl] 4-[N-methyl-N-(3-methyl-4-
nitrobenzyloxycarbonyl)amino]phenylacetamide hydrochloride
4e. Acetamide hydrochloride 4e was obtained (85%) as a tan
hygroscopic foam, νmax(thin film)/cmϪ1 3426, 1705, 1674, 1520,
1345, 1265, and 1161; δH [(CD3)2SO] 2.51 (3 H, s, CH3), 2.75
(6 H, d, J 4.8, N(CH3)2), 3.10–3.15 (2 H, m, CH2N), 3.24 (3 H, s,
NCH3), 3.40–3.45 (2 H, m, CH2N), 3.47 (2 H, s, CH2CO), 5.16
(2 H, s, CH2O), 7.27–7.33 (4 H, m, 2-H, 3-H, 5-H, 6-H), 7.40–
7.45 (2 H, m, 2Ј-H, 6Ј-H), 7.97 (1 H, d, J 8.3, 5Ј-H), 8.56 (1 H,
t, J 5.5, CONH), and 10.50 (1 H, s, NHCl); δC [(CD3)2SO] 19.6,
33.9, 37.5, 41.6, 42.1 (2), 55.6, 65.2, 119.1, 124.7 (2), 125.6 (2),
129.6 (2), 130.9, 132.9, 141.2, 142.5, 148.0, 154.3, and 170.6;
m/z (DEI) 428.2067 (Mϩ. C22H28N4O5 requires 428.2060); m/z
(DEI) 428 (Mϩ, 30%), 409 (100), and 384 (60).
N-[2-(N,N-Dimethylamino)ethyl]
4-[N-(4-nitrobenzyloxy-
carbonyl)amino]phenylacetamide hydrochloride 4a. Acetamide
hydrochloride 4a was obtained (66%) as a tan powder, mp (from
MeOH–EtOAc) 161 ЊC (decomp.) (Found: C, 53.5; H, 5.7; N,
11.9. C20H25ClN4O5.MeOH requires C, 53.8; H, 6.2; N, 12.0%);
2768
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N-[2-(N,N-Dimethylamino)ethyl] 4-[N-methyl-N-(2-methoxy-
4-nitrobenzyloxycarbonyl)amino]phenylacetamide hydrochloride
4f. Acetamide hydrochloride 4f was obtained (76%) as a brown
oil, νmax(thin film)/cmϪ1 3418, 1707, 1658, 1518, 1348, and 1155;
δH [(CD3)2SO] 2.48 (6 H, d, J 4.5, N(CH3)2), 3.14 (2 H, dt, J 6.0
and 5.5, CH2N), 3.25 (3 H, s, NCH3), 3.38–3.48 (4 H, m, CH2N
and CH2CO), 3.97 (3 H, s, OCH3), 5.15 (2 H, s, CH2O), 7.26–
7.32 (4 H, m, 2-H, 3-H, 5-H, 6-H), 7.41–7.43 (1 H, m, 6Ј-H),
7.76 (1 H, d, J 1.8, 3Ј-H), 7.84 (1 H, dd, J 8.3 and 1.8, 5Ј-H),
8.64 (1 H, t, J 5.4, CONH), and 10.76 (1 H, s, NHCl);
δC [(CD3)2SO] 33.9, 37.5, 41.6, 42.1 (2), 55.5, 56.2, 61.5, 105.4,
115.1, 125.5 (2), 128.0, 129.6 (2), 132.5, 134.0, 141.2, 148.0,
154.2, 156.7, and 170.5; m/z (DEI) 444.2013 (Mϩ. C22H28N4O6
requires 444.2009); m/z (DEI) 444 (Mϩ, 40%), 425 (100), 300
(15), 185 (40), and 235 (60).
20 ЊC for 16 h. The solution was poured into sat. aq. NaHCO3
solution (100 cm3), and extracted with EtOAc (3 × 50 cm3). The
combined organic extract was dried and the solvent removed
under reduced pressure. The residue was chromatographed on
neutral alumina, eluting with a gradient (0–10%) MeOH–
EtOAc, to give the carbamate 4. Final purification was achieved
by semi-preparative reverse phase HPLC using a Philips
PU4100 liquid chromatograph, a Phenomenex Bondclone 10
C18 column (300 × 21.2 mm id) monitoring at 254 nm. Chrom-
atograms were run in 50% acetonitrile–ammonium formate
at 10 cm3 minϪ1. Yields and characterisations of individual
compounds are given below.
N-[2-(N,N-Dimethylamino)ethyl] 4-[N-methyl-N-(2,4-dinitro-
benzyloxycarbonyl)amino]phenylacetamide hydrochloride 4j.
Acetamide hydrochloride 4j was obtained (5%) as a red–brown
gum, νmax(thin film)/cmϪ1 3321, 1711, 1657, 1535, 1346, and
1154; δH [(CD3)2SO] 2.55 (6 H, d, J 5.0, N(CH3)2), 2.95 (2 H, t,
J 6.5, CH2N), 3.12–3.16 (2 H, m, CH2N), 3.26 (3 H, s, NCH3),
3.36 (2 H, s, CH2CO), 5.53 (2 H, s, CH2O), 7.26–7.30 (4 H,
m, 2-H, 3-H, 5-H, and 6-H), 7.50–7.54 (1 H, m, 6Ј-H), 8.56
(1 H, dd, J 8.6 and 2.2, 5Ј-H), 8.64 (1 H, t, J 5.4, CONH),
8.78 (1 H, d, J 2.2, 3Ј-H), and 10.76 (1 H, s, NHCl);
δC [(CD3)2SO] 36.7, 37.6, 40.0, 45.0 (2), 58.1, 63.1, 120.3,
125.7 (2), 128.0, 129.5 (2), 129.8, 134.8, 139.3, 140.9, 146.7,
146.8, 153.9, and 169.8; m/z (DCI, NH3) 460.1798 (MHϩ.
C21H26N5O7 requires 460.1832); m/z (DCI, NH3) 460 (MHϩ,
2%), 293 (3), and 236 (100).
N-[2-(N,N-Dimethylamino)ethyl] 4-[N-methyl-N-(3-methoxy-
4-nitrobenzyloxycarbonyl)amino]phenylacetamide hydrochloride
4g. Acetamide hydrochloride 4g was obtained (95%) as a brown
gum (Found: C, 59.2; H, 6.2; N, 12.6. C22H28N4O6 requires C,
59.4; H, 6.35; N, 12.6%); νmax/cmϪ1 3464, 3299, 1707, 1643,
1612, 1518, 1332, and 1161; δH [(CD3)2SO] 2.75 (6 H, d, J 4.8,
N(CH3)2), 3.11–3.14 (2 H, m, CH2N), 3.25 (3 H, s, NCH3), 3.42
(2 H, dt, J 6.3 and 5.9, CH2N), 3.47 (2 H, s, CH2CO), 3.87 (3 H,
s, OCH3), 5.17 (2 H, s, CH2O), 7.00–7.04 (1 H, m, 6Ј-H), 7.20
(1 H, br s, 2Ј-H), 7.26–7.33 (4 H, m, 2-H, 3-H, 5-H, 6-H), 7.87
(1 H, d, J 8.3, 5Ј-H), 8.56 (1 H, t, J 5.5, CONH), and 10.53
(1 H, s, NHCl); δC [(CD3)2SO] 33.9, 37.6, 41.6, 42.1 (2), 56.5,
59.7, 65.3, 112.2, 118.4, 125.2 (2), 125.6, 129.6 (2), 134.0, 138.2,
141.2, 144.0, 152.2, 154.2, and 170.6.
N-[2-(N,N-Dimethylamino)ethyl] 4-[N-methyl-N-(3,4-dinitro-
benzyloxycarbonyl)amino]phenylacetamide hydrochloride 4k.
Acetamide hydrochloride 4k was obtained (26%) as a red–brown
gum, νmax(thin film)/cmϪ1 3342, 1707, 1653, 1543, 1350, and
1155; δH [(CD3)2SO] 2.32 (2 H, t, J 6.7, CH2N), 2.76 (6 H, d,
J 5.0, N(CH3)2), 3.15 (2 H, dt, J 6.6 and 5.7, CH2N), 3.30–
3.38 (5 H, m, NCH3 and CH2CO), 5.27 (2 H, s, CH2O), 7.25–
7.30 (4 H, m, 2-H, 3-H, 5-H, and 6-H), 7.83–7.87 (1 H, m,
6Ј-H), 8.03 (1 H, t, J 5.4, CONH), 8.11 (1 H, br s, 2Ј-H),
8.22 (1 H, d, J 8.3, 5Ј-H), and 10.50 (1 H, s, NHCl);
δC [(CD3)2SO] 36.6, 37.7, 41.7, 44.9 (2), 59.8, 64.5, 123.6,
125.6 (2), 125.9, 129.5 (2), 132.3, 134.5, 141.0, 142.1, 144.7
(2), 154.1, and 170.0; m/z (DCI, NH3) 460.1811. (MHϩ.
C21H26N5O7 requires 460.1832); m/z (DCI, NH3) 460 (MHϩ,
5%), 430 (2), 412 (3), and 236 (100).
N-[2-(N,N-Dimethylamino)ethyl]
4-(N-methyl-N-{2-[N-
methyl-N-(tert-butyloxycarbonyl)amino]-4-nitrobenzyloxycarb-
onyl}amino)phenylacetamide 4h. Acetamide 4h was obtained
(80%) as a pale yellow oil, νmax(thin film)/cmϪ1 3389, 1709, 1670,
1527, 1348, and 1158; δH [(CD3)2SO] 1.45 and 1.27 (9 H, 2s,
C(CH3)3), 2.17 (6 H, d, J 4.5, N(CH3)2), 2.26 (2 H, t, J 6.7,
CH2N), 3.10–3.15 (5 H, m, NCH3 and CH2N), 3.48 (2 H, s,
CH2CO), 3.23 (3 H, s, NCH3), 5.08 (2 H, s, CH2O), 7.24–7.30
(4 H, m, 2-H, 3-H, 5-H, and 6-H), 7.54 (1 H, br s, 3Ј-H), 8.00
(1 H, t, J 5.5, CONH), 8.15–8.19 (2 H, m, 5Ј-H and 6Ј-H);
δC [(CD3)2SO] 27.6 (3), 35.7, 36.4, 36.6, 37.6, 41.7, 45.1 (2), 58.1,
80.2, 122.0, 122.2, 125.5, 127.5, 128.3 (2), 129.3 (2), 134.6,
141.2, 142.1, 147.3, 153.0, 154.2, and 169.7; m/z (DEI) 543.2686
(Mϩ. C27H37N5O7 requires 543.2693); m/z (DCI, NH3) 544
(MHϩ, 100%), 488 (10), 250 (10), and 236 (90).
N-[2-(N,N-Dimethylamino)ethyl] 4-[N-methyl-N-(3-methyl-
oxycarbonyl-4-nitrobenzyloxycarbonyl)amino]phenylacetamide
hydrochloride 4l. Acetamide hydrochloride 4l was obtained
(12%) as a tan gum; νmax(thin film)/cmϪ1 3415, 1736, 1707, 1655,
1533, 1346, and 1155; δH [(CD3)2SO] 2.27 (2 H, t, J 6.7, CH2N),
2.66 (6 H, d, J 4.5, N(CH3)2), 3.11–3.16 (2 H, m, CH2N), 3.24 (3
H, s, NCH3), 3.41 (2 H, s, CH2CO), 3.87 (3 H, s, OCH3), 5.23 (2
H, s, CH2O), 7.25–7.30 (4 H, m, 2-H, 3-H, 5-H, and 6-H), 7.69–
7.75 (2 H, m, 2Ј-H and 6Ј-H), 8.03 (1 H, t, J 5.4, CONH), 8.06
(1 H, d, J 8.4, 5Ј-H), 10.35 (1 H, s, NHCl); δC [(CD3)2SO] 36.7,
37.6, 41.6, 45.1 (2), 53.2, 58.1, 64.8, 124.4, 125.5 (2), 126.4,
128.0, 129.3 (2), 130.8, 134.5, 141.0, 143.2, 146.8, 154.2, 164.9,
and 169.8; m/z (DEI) 472.1956 (Mϩ. C23H28N4O7 requires
472.1958); m/z (DEI) 472 (Mϩ, 1%), 402 (2), 357 (3), 71 (20),
and 58 (100).
N-[2-(N,N-Dimethylamino)ethyl]
4-(N-methyl-N-{3-[N-
methyl-N-(tert-butoxycarbonyl)amino]-4-nitrobenzyloxycarb-
onyl}amino)phenylacetamide 4i. Acetamide 4i was obtained
(66%) as a pale yellow oil, νmax(thin film)/cmϪ1 3359, 1709, 1659,
1528, 1345, and 1155; δH [(CD3)2SO] 1.42 and 1.22 (9 H, 2s,
C(CH3)3), 2.13 (6 H, d, J 4.0, N(CH3)2), 2.27 (2 H, t, J 6.6,
CH2N), 3.13 (2 H, dt, J 6.6, 5.8, CH2N), 3.19 (3 H, s, NCH3),
3.26 (3 H, s, NCH3), 3.40 (2 H, s, CH2CO), 5.19 (2 H, s, CH2O),
7.24–7.29 (4 H, m, 2-H, 3-H, 5-H, 6-H), 7.36–7.43 (2 H, m,
2Ј-H, 6Ј-H), and 7.95–8.03 (2 H, m, 5Ј-H and CONH);
δC [(CD3)2SO] 27.7 (3), 36.7, 37.0, 37.6, 41.6, 45.1 (2), 58.1, 64.9,
80.6, 124.9, 125.3, 125.6 (2), 126.7, 129.3 (2), 134.5, 136.3,
141.0, 143.7, 144.6, 152.1, 154.2, and 169.8; m/z (DEI) 543.2687
(Mϩ. C27H37N5O7 requires 543.2693); m/z (CI, NH3) 544 (MHϩ,
80%), 367 (30), 236 (70), and 71 (100).
Formation of carbamates 4m,n: example of general preparation
by method C (Scheme 10)
Formation of carbamates 4j–l: example of general preparation by
method B (Scheme 9)
HCl-saturated MeOH (10 cm3) was added to a stirred solution
of 4h or 4i (0.26 mmol) in MeOH (10 cm3) and the solution
stirred at 20 ЊC for 4 h. The solvent was removed under reduced
pressure and the residue dissolved in water (10 cm3), the pH
adjusted to 10 with aq. Na2CO3 solution, and extracted with
CHCl3 (3 × 25 cm3). The combined organic extracts were
A solution of the chloroformate of the alcohol 8 (2 mmol) in
THF (50 cm3) was prepared in situ using phosgene and the
alcohol 8 as described in Method A above. To this solution
amine 7 in THF (5 cm3) was added and the solution stirred at
J. Chem. Soc., Perkin Trans. 1, 1999, 2759–2770
2769

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washed with brine (30 cm3), dried, and the solvent removed
under reduced pressure. The residue was chromatographed on
neutral alumina, eluting with a gradient (0–10%) MeOH–
EtOAc, to give the carbamate 4. Yields and characterisations of
individual compounds are given below.
Acknowledgements
This work was supported by the US National Cancer
Institute (Contract NO1-CM 47019), the Auckland Division of
the Cancer Society of New Zealand, and a University of
Auckland Post-Doctoral Fellowship (B. M. S).
N-[2-(N,N-Dimethylamino)ethyl] 4-[N-methyl-N-(2-methyl-
amino-4-nitrobenzyloxycarbonyl)amino]phenylacetamide hydro-
chloride 4m. Acetamide hydrochloride 4m was obtained (28%) as
a pale yellow oil, νmax(thin film)/cmϪ1 3340, 1707, 1650, 1576,
1338, and 1145; δH [(CD3)2SO] 2.65 (6 H, d, J 4.9, N(CH3)2),
2.75 (2 H, t, J 6.7, CH2N), 2.87 (3 H, s, NCH3), 3.11–3.16 (2 H,
m, NCH2), 3.23 (3 H, s, NHCH3), 3.39–3.43 (2 H, m, CH2N),
3.47 (2 H, s, CH2CO), 5.21 (2 H, s, CH2O), 5.50 (1 H, s,
NHCH3), 7.24–7.35 (7 H, m, 2-H, 3-H, 5-H, 6-H, 3Ј-H, 5Ј-H,
and 6Ј-H), 8.61 (1 H, t, J 5.5, CONH), and 10.71 (1 H, br s,
NHCl); δC [(CD3)2SO] 33.9, 34.3, 37.5, 41.6, 42.1 (2), 55.5, 63.7,
113.1, 114.0, 125.4 (2), 128.9, 129.5 (2), 129.8, 133.9, 141.7,
146.9, 149.3, 154.2, and 170.5; m/z (DEI) 443.2160 (Mϩ.
C22H29N5O5 requires 443.2169); m/z (DEI) 443 (Mϩ, 15%), 426
(10), 396 (30), and 235 (100).
References
1 D. L. Kirkpatrick, K. E. Johnson and A. C. Sartorelli, J. Med.
Chem., 1986, 29, 2048.
2 M. Tercel, W. R. Wilson, R. F. Anderson and W. A. Denny, J. Med.
Chem., 1996, 39, 1084.
3 (a) P. Neta and D. Behar, J. Am. Chem. Soc., 1980, 102, 4798;
(b) J. P. Pays, S. T. Blumer, S. Barel-Tosh, D. Behar and P. Neta,
J. Am. Chem. Soc., 1983, 105, 320; (c) R. K. Norris, S. D. Barker and
P. Neta, J. Am. Chem. Soc., 1984, 106, 3140.
4 P. L. Carl, P. K. Charkravarty and J. A. Katzenellenbogen, J. Med.
Chem., 1981, 24, 479.
5 B. A. Teicher and A. C. Sartorelli, J. Med. Chem., 1980, 23, 955.
6 T.-S. Lin, L. Wang, I. Antonini, L. A. Crosby, D. A. Shiba,
D. L. Kirkpatrick and A. C. Sartorelli, J. Med. Chem., 1986, 29, 84.
7 N. P. Michael, J. K. Brehm, G. M. Anlezark and N. P. Minton,
FEMS Microbiol. Lett., 1994, 124, 195.
8 (a) G. M. Anlezark, R. G. Melton, R. F. Sherwood, B. Coles,
F. Friedlos and R. J. Knox, Biochem. Pharmacol., 1992, 44, 2289;
(b) R. J. Knox, F. Friedlos, R. F. Sherwood, R. G. Melton and
G. M. Anlezark, Biochem. Pharmacol., 1992, 44, 2297.
9 P. Wardman, Environ. Health Perspect., 1985, 64, 309.
10 (a) M. P. Hay and W. A. Denny, Drugs Future, 1996, 21, 917;
(b) W. A. Denny, Curr. Pharm. Des., 1996, 2, 281.
11 A. B. Mauger, P. J. Burke, H. H. Somani, F. Friedlos and R. J. Knox,
J. Med. Chem., 1994, 37, 3452.
12 G. M. Anlezark, R. G. Melton, R. F. Sherwood, W. R. Wilson,
W. A. Denny, B. D. Palmer, R. J. Knox, F. Friedlos and A. Williams,
Biochem. Pharmacol., 1995, 50, 609.
13 (a) J. A. Bridgewater, C. J. Springer, R. J. Knox, N. P. Minton,
N. P. Michael and M. K. Collins, Eur. J. Cancer, 1995, 31A, 2362;
(b) F. Friedlos, W. A. Denny, B. D. Palmer and C. J. Springer,
J. Med. Chem., 1997, 40, 270.
14 M. P. Hay, W. R. Wilson and W. A. Denny, Bioorg. Med. Chem.
Lett., 1995, 5, 2829.
N-[2-(N,N-Dimethylamino)ethyl] 4-[N-methyl-N-(3-methyl-
amino-4-nitrobenzyloxycarbonyl)amino]phenylacetamide hydro-
chloride 4n. Acetamide hydrochloride 4n was obtained (28%) as
a pale yellow oil, νmax(thin film)/cmϪ1 3387, 1707, 1655, 1626,
1580, 1336, and 1157; δH [(CD3)2SO] 2.78 (6 H, d, J 4.9,
N(CH3)2), 2.88 (3 H, s, NCH3), 3.12–3.16 (2 H, m, NCH2), 3.25
(3 H, br s, NHCH3), 3.40–3.43 (2 H, m, CH2N), 3.47 (2 H, s,
CH2CO), 3.55 (1 H, s, NHCH3), 5.11 (2 H, s, CH2O), 6.55–6.59
(1 H, m, 6-H), 6.77 (1 H, br s, 2-H), 7.28–7.35 (4 H, m, 2-H, 3-
H, 5-H, and 6-H), 8.03 (1 H, d, J 8.8, 5-H), 8.19 (1 H, s, NHCl),
8.51 (1 H, t, J 5.4, CONH), and 10.16 (1 H, br s, NHCl); δC
[(CD3)2SO] 29.6, 34.1, 37.7, 41.6, 42.4 (2), 55.7, 65.5, 111.3,
113.1, 125.8 (2), 126.5, 129.7 (2), 130.0, 134.0, 141.3, 145.8,
146.0, 154.3, and 170.8; m/z (DEI) 443.2165 (Mϩ. C22H29N5O5
requires 443.2169); m/z (DEI) 443 (Mϩ, 25%), 426 (20), 408
(60), 396 (20), and 235 (100).
15 M. Tercel, W. R. Wilson and W. A. Denny, Bioorg. Med. Chem.
Lett., 1996, 6, 2741.
16 M. Lee, J. E. Simpson, Jnr., S. Woo, C. Kaenzig, G. M. Anlezark,
E. Eno-Amooquaye and P. J. Burke, Bioorg. Med. Chem. Lett., 1997,
7, 1065.
17 R. T. Mulcahy, J. J. Gipp, J. P. Schmidt, C. Joswig and R. F. Borch,
J. Med. Chem., 1994, 37, 1610.
18 V. J. Shiner, Jnr., W. E. Buddenbaum, B. L. Murr and G. Lamety,
J. Am. Chem. Soc., 1968, 90, 418.
19 J. M. Brown, C. Christodoulou, A. S. Modak, C. B. Reese and
H. T. Serafinowska, J. Chem. Soc., Perkin Trans. 1, 1989, 1751.
20 E. Boyland and R. Nery, Analyst, 1964, 89, 95.
21 C. Hansch and A. Leo, in Substituent Constants for Correlation
Analysis in Chemistry and Biology, Wiley, New York, 1979.
22 D. S. Dhanoa, S. W. Bagley, R. S. L. Chang, V. J. Liotta, T.-B. Chen,
S. D. Kivlighn, G. J. Zingao, P. K. S. Sieg, A. A. Patchett and
W. J. Greenlee, J. Med. Chem., 1993, 36, 4239.
23 S. B. Hanna, Y. Iskander and A. Salama, J. Chem. Soc., 1961, 221.
24 Y. Imakura, K. Okimoto, T. Konishi, M. Hisazumi, J. Yamazaki,
S. Kobuyashi and S. Yamashita, Chem. Pharm. Bull., 1992, 40,
1691.
Radiolytic reduction of nitrobenzyl carbamates 4a–n
Solutions of the carbamates 4 (50 µM) in 10 mM phosphate
buffer (5 cm3, pH 7.4) and propan-2-ol (4%, v/v) were degassed
by evacuation under reduced pressure, and radiolytically
reduced at 20 ЊC by γ-irradiation, using a 60Co source. The dose
rate of the 60Co source was measured as 0.721 J dmϪ3 sϪ1 using
the NaCl modified Fricke dosimeter,25 and corrected for decay
of 60Co in subsequent irradiations. The time, t, required to form
1 molar equivalent of reducing species in a solution of prodrug
(50 µM, 5 cm3) was calculated from t = n/(Gdv), where n = mole
of prodrug, G = radiation chemical yield (taken as 0.62 µmol
ؒ
J
Ϫ1, G(eϪaq) ϩ G( H) ϩ G(OH)) d = dose rate and v = the volume
of radiolysis solution. Samples were analysed by HPLC, inject-
ing 0.05 cm3 samples through a 0.025 cm3 sample loop, on
an Econosphere C-18 analytical column (reverse phase, 5 µm
particle size, 250 × 4.6 mm id). Samples were eluted using
both gradient and isocratic solvent systems, comprised of mix-
tures of MeOH, pH 6.5, 10 mM phosphate buffer and 10 mM
heptane sulfonic acid, at a flow rate of 0.8 cm3 minϪ1. An
HP1040M series II detector permitted collection of the spectra
of chromatographic peaks. HPLC was used to quantify the
release of amines 6 or 7, using authentic samples to prepare
calibration plots.
25 H. Fricke and E. J. Hart, in Radiation Dosimetry, ed. F. H. Attix and
W. C. Roesch, Academic Press, New York, 1966, vol 2, p. 167.
Paper 9/04067F
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J. Chem. Soc., Perkin Trans. 1, 1999, 2759–2770