Self-immolative nitrogen mustard prodrugs for suicide gene therapy

Article abstract of DOI:10.1021/jm980425k

Four new potential self-immolative prodrugs derived from phenol and aniline nitrogen mustards, four model compounds derived from their corresponding fluoroethyl analogues and two new self-immolative linkers were designed and synthesized for use in the suicide gene therapy termed GDEPT (gene-directed enzyme prodrug therapy). The self-immolative prodrugs were designed to be activated by the enzyme carboxypeptidase G2 (CPG2) releasing an active drug by a 1,6-elimination mechanism via an unstable intermediate. Thus, N-[(4-{[4-(bis{2- chloroethyl}amino)phenoxycarbonyloxy]methyl}phenyl)carbamoyl]-L-glutamic acid (23), N-[(4-{[4(bis{2- chloroethyl}amino)phenoxycarbonyloxy]methyl}phenoxy)carbonyl]-L-glutamic acid (30), N-[(4-{N-(4-(bis[2- chloroethyl]amino}}pheny)carbamoyloxy]methyl}phenoxy)carbonyl]-L-glutmic acid (37), and N-[(4-{[N-(4-{bis[2- chloroethyl]amino}phenyl)carbamoyloxy]methyl}phenyl)carbamoyl]-L-glutamic acid (40) were synthesized. They are bifunctional alkylating agents in which the activating effects of the phenolic hydroxyl or amino functions are masked through an oxycarbonyl or a carbamoyl bond to a benzylic spacer which is itself]inked to a glutamic acid by an oxycarbonyl or a carbamoyl bond. The corresponding fluoroethyl compounds 25, 32, 42, and 44 were also synthesized. The rationale was to obtain model compounds with greatly reduced alkylating abilities that would be much less reactive with nucleophiles compared to the corresponding chloroethyl derivatives. This enabled studies of these model compounds as substrates for CPG2, without incurring the rapid and complicated decomposition pathways of the chloroethyl derivatives. The prodrugs were desired to be activated to their corresponding phenol and aniline nitrogen mustard drugs by CPG2 for use in GDEPT. The synthesis of the analogous novel parent drugs (21b, 51) is also described. A colorectal cell line was engineered to express CPG2 tethered to the outer cell surface. The phenylenediamine compounds were found to behave as prodrugs, yielding IC₅₀ prodrug/IC₅₀ drug ratios between 20- and 33-fold (for 37 and 40) and differentials of 12-14-fold between CPG2-expressing and control LacZ- expressing clones. The drugs released are up to 70-fold more potent than 4- [(2-chloroethyl)(2-mesyloxyethyl)amino]benzoic acid that results from the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMBA) which has been used previously for GDEPT. These data demonstrate the viability of this strategy and indicate that self-immolative prodrugs can be synthesized to release potent mustard drugs selectively by cells expressing CPG2 tethered to the cell surface in GDEPT.

Full text of DOI:10.1021/jm980425k

J . Med. Chem. 1998, 41, 5297-5309
5297
Self-Im m ola tive Nitr ogen Mu sta r d P r od r u gs for Su icid e Gen e Th er a p y
Dan Niculescu-Duvaz, Ion Niculescu-Duvaz, Frank Friedlos, J anet Martin, Robert Spooner, Lawrence Davies,
Richard Marais, and Caroline J . Springer*
CRC Centre for Cancer Therapeutics, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, U.K.
Received J uly 21, 1998
Four new potential self-immolative prodrugs derived from phenol and aniline nitrogen mustards,
four model compounds derived from their corresponding fluoroethyl analogues and two new
self-immolative linkers were designed and synthesized for use in the suicide gene therapy
termed GDEPT (gene-directed enzyme prodrug therapy). The self-immolative prodrugs were
designed to be activated by the enzyme carboxypeptidase G2 (CPG2) releasing an active drug
by a 1,6-elimination mechanism via an unstable intermediate. Thus, N-[(4-{[4-(bis{2-chloro-
ethyl}amino)phenoxycarbonyloxy]methyl}phenyl)carbamoyl]-L-glutamic acid (23), N-[(4-{[4-
(bis{2-chloroethyl}amino)phenoxycarbonyloxy]methyl}phenoxy)carbonyl]-L-glutamic acid (30),
N-[(4-{[N-(4-{bis[2-chloroethyl]amino}phenyl)carbamoyloxy]methyl}phenoxy)carbonyl]-L-glutam-
ic acid (37), and N-[(4-{[N-(4-{bis[2-chloroethyl]amino}phenyl)carbamoyloxy]methyl}phenyl)-
carbamoyl]-L-glutamic acid (40) were synthesized. They are bifunctional alkylating agents in
which the activating effects of the phenolic hydroxyl or amino functions are masked through
an oxycarbonyl or a carbamoyl bond to a benzylic spacer which is itself linked to a glutamic
acid by an oxycarbonyl or a carbamoyl bond. The corresponding fluoroethyl compounds 25, 32,
42, and 44 were also synthesized. The rationale was to obtain model compounds with greatly
reduced alkylating abilities that would be much less reactive with nucleophiles compared to
the corresponding chloroethyl derivatives. This enabled studies of these model compounds as
substrates for CPG2, without incurring the rapid and complicated decomposition pathways of
the chloroethyl derivatives. The prodrugs were designed to be activated to their corresponding
phenol and aniline nitrogen mustard drugs by CPG2 for use in GDEPT. The synthesis of the
analogous novel parent drugs (21b, 51) is also described. A colorectal cell line was engineered
to express CPG2 tethered to the outer cell surface. The phenylenediamine compounds were
found to behave as prodrugs, yielding IC50 prodrug/IC50 drug ratios between 20- and 33-fold
(for 37 and 40) and differentials of 12-14-fold between CPG2-expressing and control LacZ-
expressing clones. The drugs released are up to 70-fold more potent than 4-[(2-chloroethyl)-
(2-mesyloxyethyl)amino]benzoic acid that results from the prodrug 4-[(2-chloroethyl)(2-
mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA) which has been used previously for
GDEPT. These data demonstrate the viability of this strategy and indicate that self-immolative
prodrugs can be synthesized to release potent mustard drugs selectively by cells expressing
CPG2 tethered to the cell surface in GDEPT.
In tr od u ction
the extrusion process that generates the drug. The site
of activation will usually be separated from the site of
extrusion. The advantage of self-immolation is that it
increases the diversity of prodrugs which can be acti-
vated by a certain enzyme.
The major objective in cancer treatment is to kill
tumor cells selectively without harming normal cells.1
A strategy for achieving selectivity is called suicide gene
therapy. One approach aims to deliver toxin genes to
the cancer cells.2 An alternative approach has been
named gene-directed enzyme prodrug therapy (GDEPT)3
or virally-directed enzyme prodrug therapy (VDEPT).4
These therapies consist of tumor-specific conversion of
prodrugs to active drugs following the delivery of genes
for exogenous enzymes.5
Self-immolative prodrugs have been proposed for
activation by tumor enzymes.6-8 A self-immolative
prodrug can be defined as generating an unstable
intermediate which, following the activation process,
will extrude the active drug in a number of subsequent
steps.6,8 A cascade of events are required. First, the
activation process, which is enzymatic, culminates in
Self-immolative mustard prodrugs for activation by
Escherichia coli nitroreductase in a GDEPT system have
previously been synthesized and evaluated in tumor
cell lines.9 An alternative candidate for GDEPT is car-
boxypeptidase G2 (CPG2) from Pseudomonas sp.10 since
it has no mammalian homologue.11 This enzyme cata-
lyzes the scission of an amidic,11,12 urethanic, or ure-
idic13,14 bond between an aromatic nucleus and L-glutam-
ic acid.
The enzyme CPG2 has recently been mutated and
expressed tethered to the outer cell membrane in the
human breast carcinoma cell line MDA MB 361.15 The
expression of CPG2 tethered to the surface of the cell
overcomes the need for a prodrug to penetrate the tumor
cell membrane. Incubation of the transfected lines with
the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]-
* Corresponding author. Tel: +44 181-643 8901. Fax: +44 181-
643 6902. E-mail: caroline@icr.ac.uk.
10.1021/jm980425k CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/25/1998

5298 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Niculescu-Duvaz et al.
Sch em e 1
benzoyl-L-glutamic acid (CMDA) leads to an increased
sensitivity.15 The prodrugs described herein were de-
signed to release more potent active drugs than that
cleaved from CMDA.
This report describes the first example of self-immo-
lative prodrugs for activation by CPG2. It also describes
the construction of the colorectal cell line LS174T made
to express CPG2 tethered to the cell surface (LS174T-
stCPG2(Q)3). Self-immolative prodrugs depicted as 1 in
Scheme 1 were designed to be activated by CPG2 to
generate the unstable intermediates 1a , further releas-
ing the active drug by a 1,6-elimination mechanism.
obtain compounds with greatly reduced alkylating abili-
ties that would not react with nucleophiles as quickly
as the corresponding chloroethyl derivatives. This en-
abled studies of these compounds as substrates for
CPG2, without incurring the rapid and complicated
decomposition pathways of the chloroethyl derivatives.
A broad panel of potential prodrugs with carbonate,
carbamate, and ureido linkages were considered in order
to find the optimal structural features responsible for
the stability, substrate specificity for CPG2, and efficacy
in cell lines transfected with the stCPG2(Q)3 gene, as
well as for further QSAR and optimization studies.
Ra tion a le
Ch em istr y
For the synthesis of the novel nitrogen mustards
prodrugs of general formula 1 (see Scheme 1), two self-
immolative linkers: (4-hydroxymethylphenyl)carbam-
oyl-L-glutamic acid 12 and (4-hydroxymethylphenyl)-
oxycarbonyl-L-glutamic acid 20 were designed, synthe-
sized, and studied. The ureidic and carbamic linkages
in 12 and 20, respectively, between glutamic acid and
the aromatic nucleus have a dual purpose. They provide
a substrate cleavable by CPG2 and release an amino or
hydroxy group required for the 1,6-elimination.
Aniline and phenol nitrogen mustards were chosen
since the drugs are potent16 and it is possible to reduce
greatly their chemical reactivity through acylation. They
were transformed to the corresponding self-immolative
prodrugs 23, 30, 37, and 40 by coupling with 12 or 20
through an oxycarbonyl or carbamoyl bond. The carbon-
ate and carbamate bonds deactivate the phenol and
aniline mustard, respectively by acylation, also acting
as leaving groups in the 1,6-elimination, thus generating
the drug via an irreversible process. The mechanism of
activation of the prodrugs by CPG2 is presented in
Scheme 1 and the physicochemical and kinetic data are
in the Physicochemical and Kinetic Data section.
The corresponding fluorine mustard analogues 25, 32,
42, and 44 were also synthesized. The rationale was to
Two series of protected linkers (i.e., di-tert-butyl and
diallyl L-glutamyl esters) were made since different
deprotection strategies were needed to obtain the final
prodrugs.
The starting material for 12, 4-nitrobenzyl alcohol,
was protected as tert-butyldiphenylsilyl ether 2a 17,18 and
reduced by hydrogen transfer (Pd/C (10%) and am-
monium formate in EtOH) to the corresponding amine
3a . The amine after conversion to isocyanate 4 (with
triphosgene at 70 °C, in toluene) was coupled with di-
tert-butyl L-glutamate, 5a , in THF, at room temperature
and in basic medium, leading to the protected linker
7a . An alternative route to compound 7a is the direct
coupling of the amine 3a with the di-tert-butyl L-
glutamyl isocyanate 6a under the conditions described
above. Compound 6a was obtained from di-tert-butyl
L-glutamate, 5a , by treatment with triphosgene and
triethylamine at -78 °C in toluene. By exploiting this
route, a one-pot procedure was devised to obtain com-
pound 7a directly, in good yield, starting from di-tert-
butyl L-glutamate and amine 3a . During this conden-
sation the pyroglutamate analogue 10, of compound 7a ,
was formed as byproduct and was further deprotected
with tetra-n-butylammonium fluoride in THF at room

Self-Immolative Nitrogen Mustard Prodrugs
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5299
Sch em e 2a
a
(a) TBDPSCl, imidazole, DMF (THF) or 3,4-dihydropyran, PPTS, CH2Cl2, rt; (b) Pd/C (10%), HCO2NH4, EtOH; (c) (Cl3CO)2CO, NEt3,
toluene, 70 °C; (d) (Cl3CO)2CO, NEt3, toluene, -78 °C; (e) THF, NEt3, rt; (f) Bu4NF, THF, rt or AcOH, THF, H2O; (g) 4-nitrophenyl
chloroformate, CH3CN or THF, NEt3, rt; (h) Pd(Ph3P)4, pyrrolidine, rt.
Sch em e 3a
temperature to 11 (see Scheme 2). A similar deprotec-
tion afforded the linker 8a which was purified by column
chromatography and activated as 4-nitrophenyl carbon-
ate 9a (see Scheme 2).
The diallyl analogue 7c was obtained using a different
protection strategy, since the diallyl ester was unstable
to tetra-n-butylammonium fluoride even at room tem-
perature. The corresponding O-2′-tetrahydropyranyl
ether 7b was prepared by the sequence shown in
Scheme 3. When the protected nitro derivative 2b was
a
(a) HCO2NH4, Pd/C, EtOH.
reduced by hydrogen transfer to the amine 3b, a
mixture of compounds 3b, 13, and 14 resulted that were
separated and purified by chromatography so that a
yield of only 35-40% of the desired amine 3b was
obtained (see Scheme 3).
removed using AcOH in aqueous THF to give the linker
8a , which was purified by column chromatography and
activated as 4-nitrophenyl carbonate 9b (see Scheme 2).
The deprotected 12 was prepared from 8b by the
removal of the allylic groups with Pd(0) and pyrrolidine.
For the synthesis of the second self-immolative linker
20 (see Scheme 4), 4-hydroxybenzaldehyde was pro-
This amine was coupled with the diallyl L-glutamyl
isocyanate 6b, and the tetrahydropyranyl group was

5300 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Niculescu-Duvaz et al.
Sch em e 4a
a
(a) HS(CH2)3SH, BF3‚Et2O, CHCl3, 20 °C; (b) NEt3, toluene or CHCl3, 20 °C; (c) Hg(ClO4)2, THF, 20 °C; (d) NaBH3CN; (e) 4-nitrophenyl
chloroformate, THF, NEt3; (f) Pd(Ph3P)4, pyrrolidine, rt.
tected with 1,3-propanedithiol and boron trifluoride
etherate, at room temperature, to give 1519 in very good
yield. Reacting 15 with di-tert-butyl L-glutamyl isocy-
anate, 6a , in toluene (NEt3) and di-tert-butyl 4-[2′-(1′,3′-
dithianyl)]phenoxycarbonyl-L-glutamate gave 16. The
deprotection of this intermediate with Hg(ClO4)2 in THF
at room temperature led to the corresponding aldehyde
17a . Subsequently, it was found that the aldehydes
17a ,b could be prepared by the direct coupling of the
unprotected 4-hydroxybenzaldehyde with the isocyan-
ates 6a ,b. The reduction of the aldehydes 17a ,b with
sodium cyanoborohydride yielded the desired (4-hy-
droxymethylphenoxy)carbonyl-L-glutamates 18a ,b. Both
linkers 18a ,b were activated as 4-nitrophenyl carbon-
ates 19a ,b. The deprotection to 20 was achieved as
described above for linker 12.
the final prodrugs. The sensitivity of both carbamates
and especially carbonates to basic and acidic media is
widely recognized.23
The compounds fall into two categories: the phenol-
derived and the aniline-derived self-immolative nitrogen
mustards. Different procedures were employed to obtain
the corresponding prodrugs 23, 25, 30, and 32 from
4-bis(2-chloroethyl)- or 4-bis(2-fluoroethyl)aminophenol
(21a ,b) by condensation with the linkers 8a ,b and
18a ,b. Coupling 21a ,b with 9b in the presence of the
strongly basic anion exchanger Amberlyst 27 in aceto-
nitrile resulted in the protected prodrugs 22 (X ) Cl)
and 24 (X ) F). The removal of allylic groups, leading
to the corresponding prodrugs 23 (X ) Cl) and 25 (X )
F), was achieved with Pd(0) and morpholine as allyl
scavanger.
The syntheses of aniline and phenol nitrogen mustard
prodrugs 23, 30, 37, and 40 and the corresponding
fluorine mustard analogues 25, 32, 42, and 44 require
a new approach since the obvious route via the chloro-
formates of linkers 8a ,b and 18a ,b was not available.
The failure of the direct phosgenation of some similar
acylated benzylic derivatives has been reported previ-
ously.20,21 Only one example of successful phosgenation
of a similar 4-substituted benzylic alcohol has been
claimed in a patent.22 Our attempts to obtain chloro-
formates from the linkers using phosgene, diphosgene,
or triphosgene under a variety of experimental condi-
tions were also unsuccessful. The chloroformates are
formed but are unstable above -40 °C21 and difficult to
handle. For this reason they were converted at -78 °C
to the corresponding fluoroformates. Another difficulty
lay with the deprotection procedures required to obtain
The same procedure failed when used with the
activated linker 19b leading unexpectedly to nitrogen
mustard 26 (see Scheme 5). Therefore, a new procedure
was designed taking advantage of the fluorine affinity
for silicon.24 Accordingly, linker 18b was converted into
the corresponding fluoroformate by reaction with phos-
gene, KF‚HF, and 18-crown-6 ether at -78 °C in CH2-
Cl2 and coupled, at room temperature, in a one-pot
procedure with the silylated nitrogen mustards 28a ,b.
However, the coupling with the fluorine nitrogen mus-
tard proceeded with very low yield (5%). Therefore the
derivative 21b was converted with phosgene to the
corresponding chloroformate 27a and coupled with
linker 18b to give compound 31 (X ) F). The protected
nitrogen mustard prodrugs 29 (X ) Cl) and 31 (X ) F)
were deprotected to 30 (X ) Cl) and 32 (X ) F) with
Pd(0) and morpholine (see Scheme 5).

Self-Immolative Nitrogen Mustard Prodrugs
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5301
Sch em e 5a
a
(a) Amberlyst 27, CH3CN; (b) Pd(Ph3)4, morpholine; (c) COCl2, KF‚HF, 18-crown-6, -78 °C; (d) Me3SiCN; (e) COCl2, NEt3, CH2Cl2; (f)
NEt3, THF.
The aniline-derived self-immolative nitrogen mus-
6). A more convenient procedure uses the 4-bis(2-
chloroethyl)- or 4-bis(2-fluoroethyl)aminophenyl isocy-
anates (35a ,b) (prepared from the corresponding aniline
nitrogen mustards 34 (X ) Cl) and 51 (X ) F) with
triphosgene) which were reacted with linkers 8a ,b and
18a in the presence of dibutyltin dilaurate to give the
protected prodrugs 36, 39, 41, and 43, respectively. The
protected prodrugs 36 and 41 were deprotected to 37
and 42 with formic acid. The final deprotection of 38
tards 37, 40, 42, and 44 were obtained by a different
route. In a first attempt the linker 8a was activated by
treatment with 4,6-diphenylthieno[3,4-d][1,3]dioxol-2-
one 5,5-dioxide25,26 to give the corresponding carbonate
33. This intermediate is stable only at low temperature
(-78 °C) and could not be isolated. However, the pro-
tected prodrug 38 was obtained in a one-pot procedure,
using the nitrogen mustard 34, in 10% yield (see Scheme

5302 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Niculescu-Duvaz et al.
Sch em e 6a
a
(a) 4,6-Diphenylthieno[3,4-d][1,3]dioxol-2-one 5,5-dioxide; (b) dibutyltin dilaurate, toluene; (c) HCO2H; (d) Pd(Ph3P)4, pyrrolidine.
with formic acid failed. Therefore, the self-immolative
prodrugs 40 and 44 were obtained by deprotection with
Pd(0) and pyrrolidine of the corresponding diallyl L-
glutamates 39 and 43 (see Scheme 6).
The corresponding active drugs 21 (a , X ) Cl; b, X )
F), 34 (X ) Cl), and 51 (X ) F) were obtained according
to Scheme 7. The starting materials were 4-fluoroni-
trobenzene for the aniline series and 4-amino-O-ben-
zylphenol for the phenol series. The fluorinated nitrogen
mustards were obtained by the nucleophilic displace-
ment of the mesyl group (after mesylation of 46 and 48)
with KF and 18-crown-6 ether, for both series.
25) showed little difference between the bischloro (23)
and bisfluoro (25) congeners, implying that their insta-
bility lies elsewhere in the molecule. By contrast, and
as expected, the bisfluoro compounds 42 and 44 were
much more stable than their bischloro counterparts 37
and 40. However, the relative stability of 30 as com-
pared with 23 is surprising. It proved possible to
determine the KM’s of the compounds 12, 20, 40, 42, and
44. All were similar, in the <5 µM range. Prodrug 37
was shown to be a very poor substrate which required
large amounts of enzyme (data not shown); thus full
kinetic data could not be obtained. The kcat for the
isolated linker moieties 12 and 20 were determined.
P h ysicoch em ica l a n d Kin etic Da ta
Biologica l Eva lu a tion
The chemical half-lives of the candidate prodrugs and
parent drugs were determined by HPLC. The results
are shown in Table 1. The KM with CPG2 was deter-
mined for each of the novel potential prodrugs that had
a t1/2 greater than several minutes and for the depro-
tected linkers. The kinetic data are shown in Table 1.
The aqueous stability of the compounds (pH 7.4; 37
°C) varied considerably. The least stable prodrugs (23,
Cytotoxicity Assa ys. The synthesized prodrugs and
some of the corresponding drugs were tested for cyto-
toxicity on the colon carcinoma cell line LS174T-stCPG2-
(Q)3 made to express the surface-tethered CPG2. The
plasmid pMCEF stCPG2(Q)315 encodes a CPG2 mol-
ecule that is expressed on the outer cell surface. It bears
three asparagine-to-glutamine [(Q)3] mutations that

Self-Immolative Nitrogen Mustard Prodrugs
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5303
Sch em e 7a
Expression of stCPG2(Q)3 in the LS174T results in an
increased sensitivity to the treatment with CMDA
compared with the control LS174T-LacZ-expressing
cells. The IC50 of the assayed compounds against the
stCPG2(Q)3-expressing cell clone, are presented in
Table 1.
The phenol compounds (25 and 30) do not act as
prodrugs, since they are as toxic as the corresponding
drug in stCPG2(Q)3-expressing and LacZ-expressing
cells. By contrast, the phenylenediamine compounds are
successful prodrugs. Good IC50 prodrug/IC50 drug ratios
of 20-fold (for 37) and 33-fold (for 40) and differentials
of 12-14-fold between CPG2-expressing and control
LacZ-expressing clones are achieved (see Figure 1A,B).
The data that support these figures derive from the IC50
of the prodrug 37 in the control LS174T-LacZ line of
11.5 µM and the IC50 of the drug 34 in the test line of
0.34 µM resulting in a IC50 differential (11.5/0.34) of 33-
fold. The result for the IC50 of the prodrug 40 in the
control LS174T-LacZ line was 7 µM and the IC50 of the
drug of 0.34 µM in the test line resulting in a IC50
differential (7/0.34) of 20-fold. The drugs released are
between 7- and 70-fold more potent than the 4-[(2-
chloroethyl)(2-mesyloxyethyl)amino]benzoic acid (52)
released by CMDA (see Figure 1C).
These data demonstrate the viability of this strategy
and indicate that self-immolative prodrugs can be
synthesized to release potent mustard drugs selectively
by CPG2 tethered to the cell surface.
a
Su m m a r y
(a) Mesyl chloride, Py, 70 °C; (b) mesyl anhydride, then KF,
18-crown-6, MeCN; (c) TFA, pentamethylbenzene; (d) SOCl2,
CH2Cl2; (e) mesyl anhydride, then F-, CHCl3; (f) H2, Pd/C.
Four new potential self-immolative prodrugs derived
from phenol and aniline nitrogen mustards, four model
compounds derived from their corresponding fluorinated
analogues, and two new self-immolative linkers, all
substrates for CPG2, were designed and synthesized.
The phenylenediamine compounds were found to
behave as prodrugs, yielding IC50 prodrug/IC50 drug
ratios between 20- and 33-fold (for 37 and 40) and
differentials of 12-14-fold between CPG2-expressing
and control LacZ-expressing clones. The drugs released
are up to 70-fold more potent than 4-[(2-chloroethyl)-
(2-mesyloxyethyl)amino]benzoic acid that results from
the prodrug CMDA which has been used previously for
GDEPT.
Ta ble 1. Kinetic and Cytotoxicity (LS174T-stCPG2(Q)3) Data
for Phenol and Aniline Self-Immolative Prodrugs and Their
Bisfluoro Counterparts
linker (L),
prodrug (P),
t1/2,
prodrug/
drug
b
kcat
,
compd or drug (D)a t1/2, min
s-1
IC50, µM
NA
NA
0.48
264.1
0.42
128.0
12
20
L1
L2
P
P
P
P
P
P
P
602
+++c
NA
NA
0.3
NA
13.7
NA
8.1
7.6
NA
0.4
NA
NA
NA
NA
NA
17
stable +++d
23 (Cl)
25 (F)
30 (Cl)
32 (F)
37 (Cl)
40 (Cl)
42 (F)
44 (F)
21a (Cl)
34 (Cl)
21b (F)
51 (F)
52 (Cl)
CMDA
2.9
2.4
143
stable
51
+
+
+
+
+
0.62 ( 0.21
0.46 ( 0.10
256.7
48
++
stable ++
P
528
10.4
6.3
++
NA
NA
183.5
Exp er im en ta l Section
D
D
D
D
D
P
3.78 ( 1.83
0.34 ( 0.04
322.9
121.3
27.78 ( 1.27
24.74 ( 3.04
All starting materials, reagents, and anhydrous solvents
were purchased from Aldrich, unless otherwise stated. The di-
tert-butyl L-glutamate was bought from Novabiochem and
diallyl L-glutamate from Fluka. 4-Bis(2-hydroxyethyl)amino-
nitrobenzene was obtained using the known procedure.27
Kieselgel 60 (0.043-0.060) was used in gravity columns
(Art. 9385, Merck). TLC was performed on precoated sheets
of Kieselgel 60 F254 (Art. 5735, Merck). Preparative HPLC
was performed on an Axxial Chromatospac Prep 10 (J obin-
Yvon), using Merck Kieselgel 60 (0.015-0.040) (Art. 15,111).
stable NA
1314
41
NA
NA
+++
984
a
This column describes the status of the compounds, being a
prodrug (P), a drug (D), or a linker (L). For +++, kcat > 50 s-1
;
b
for ++, kcat ) 50-10 s-1; for +, kcat < 10 s-1; for all, KM < 5 µM.
c kcat ) 65.4 s-1 and kcat/KM ) 21.1 s-1 µM. kcat ) 140.0 s-1 and
d
k
cat/KM ) 83.3 s-1 µM; NA, not applicable.
Reverse-phase HPLC was performed on
a ThermoQuest
system using a 5-mm C18300A column J upiter Phenomenex.
Melting points were determined on a Kofler hot-stage (Reichert
Thermovar) melting point apparatus and are uncorrected.
Low-resolution EI and FAB spectra were performed on a VG-
2AB-SE double focusing magnetic sector mass spectrometer
(Fisons Instruments, Warrington, U.K.), operating at a resolu-
tion of 1000. High-resolution accurate mass spectra were
determined on the same system, but with a resolution set to
were otherwise inappropriately glycosylated. Two sub-
lines were engineered and cloned. The control was
transfected with the LacZ gene (LacZ), while the test
line stCPG2(Q)3 expresses CPG2 tethered on the outer
cell surface.15 The LS174T-stCPG2(Q)3 cells were treated
with the known prodrug CMDA for comparison with the
synthesized prodrugs and the corresponding drugs.

5304 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Niculescu-Duvaz et al.
4-Nitr oben zyl 2′-Tetr a h yd r op yr a n yl Eth er (2b). To a
stirred solution of 4-nitrobenzyl alcohol (1.53 g, 10.0 mmol) in
CH2Cl2 (50 mL) were added 3,4-dihydropyran (1.36 mL, 1.26
g, 15.0 mmol) and pyridinium p-toluenesulfonate (0.25 g). After
1.5 h no starting material could be detected. The reaction
mixture was evaporated to 4-5 mL, AcOEt (25 mL) added,
and the solution washed with H2O (3 × 50 mL) and brine, dried
(MgSO4), and evaporated under vacuum. The product 2b (2.46
g, 99%) resulted as an oil: 1H NMR δH 1.51-1.63 (m, 4H, 2H4′
+ 2H5′), 1.66-1.74 (m, 2H, 2H3′), 3.50 (s, 1H, H6′), 3.78 (m,
1H, H6′), 4.62 (d, 1H, CH2-Ph, J ) 3.8 Hz), 4.72 (m, 1H, H2′),
4.81 (d, 1H, CH2-Ph), 7.62 (d, 2H, Harom, J ) 8.5 Hz), 8.22 (d,
2H, Harom); MS m/z 260 (M+ + 23, 15), 236 (M+ - 1, 45), 136
(4-NO2C6H5CH2+, 100).
4-Am in oben zyl ter t-Bu tyld ip h en ylsilyl Eth er (3a ). To
a stirred solution of 2a (5.00 g, 12.80 mmol) in EtOH (100 mL)
were added 10% Pd/C (1.50 g) and ammonium formate (4.60
g) in one portion at 20 °C. After 1.5 h no starting material
could be detected. The catalyst was removed by filtration, the
filtrate concentrated under vacuum, and the residue parti-
tioned between EtOAc and H2O. The organic layer was dried
(MgSO4) and concentrated to give 3a (4.24 g, 91%) as an oil:
ν
max/cm-1 (film) 3433, 3378 (NH2), 2931, 2857 (CH2); 1H NMR
δH 1.00 (s, 9H, t-Bu), 4.57 (s, 2H, PhCH2), 4.98 (s broad, 2H,
NH2), 6.52 (d, 2H, Harom3+5, J ) 8.25 Hz), 6.96 (d, 2H, Harom2+6),
7.42-7.46 (m, 5H, Harom), 7.62-7.65 (m, 5H, Harom); MS m/z
361 (M+, 8), 304 (M+ - t-Bu, 100), 199 (Ph2SiOH+, 100). Anal.
(C23H27NOSi) C, H; N: calcd, 3.87; found, 4.33.
4-Am in oben zyl O-2′-Tetr a h yd r op yr a n yl Eth er (3b).
The same procedure as for 3a , starting from 2b (8.53 g, 35.9
mmol), led to a three-component reaction mixture (6.40 g) after
workup, which was separated by column chromatography
(cyclohexane:AcOEt, 1:1). The desired compound 3b eluted
first as an oil (2.59 g, 38%): νmax/cm-1 (film) 3447, 3360 (NH2),
2943, 2870 (CH2); 1H NMR δH 1.42-1.53 (m, 4H, 2H4′ + 2H5′),
1.55-1.72 (m, 2H, 2H3′), 3.45 (s, 1H, H6′), 3.79 (m, 1H, H6′),
4.22 (d, 1H, PhCH2, J ) 11.1 Hz), 4.37 (d, 1H, PhCH2), 4.60
(d, 1H, H2′), 5.00 (s, 2H, NH2), 6.52 (d, 2H, Harom, J ) 8.23
Hz), 6.97 (d, 2H, Harom); MS (EI) m/z 207 (M+, 100), 106 (H2-
NC6H4CH2+, 100). Anal. (C12H17NO2) C, H, N.
F igu r e 1. Sensitivity of LS174T cells expressing either control
LacZ or stCPG2(Q)3 to challenge with prodrugs or drugs: (2)
cells expressing stCPG2(Q)3 in the presence of the prodrug;
(b) cells expressing stCPG2(Q)3 in the presence of the drug;
(0) cells expressing LacZ in the presence of the prodrug.
Survival is determined by sulforhodamine assay, and the
results are expressed as the proportion of cells surviving
relative to similar untreated cells: A, incubation with prodrug
37 or drug 34 (mean of six separate experiments); B, incuba-
tion with prodrug 40 or drug 34 (mean of seven separate
experiments); C, incubation with prodrug CMDA or drug 52
(mean of three separate experiments).
The fraction eluting second, as an oil (1.15 g, 18%), was the
4-N-(5′-h yd r oxyp en tyl)tolu id in e, 13: νmax/cm-1 (film) 3353
1
(NH-, OH, broad), 2934, 2861 (CH2); H NMR δH* 1.39-1.55
(m, 6H, CH2b+c+d), 2.13 (s, 3H, PhCH3), 2.93 (q, 2H, CH2a, J )
6.60 Hz), 3.39 (q, 2H, CH2e, J ) 6.10 Hz), 4.31 (t, 1H, OH or
NH, J ) 5.19 Hz), 5.19 (t, 1H, NH or OH), 6.45 (d, 2H, Harom
,
J ) 8.15 Hz), 6.86 (d, 2H, Harom); MS (EI) m/z 193 (M+, 18),
120 (CH3C6H4NHCH2+, 100) (*for numbering see formula 13).
The third fraction, eluting as an oil (0.88 g, 9%), was N-(5′-
h yd r oxyp en t yl)a m in ob en zyl 2′-O-t et r a h yd r op yr a n yl
eth er , 14: νmax/cm-1 (film) 3367 (NH, OH, broad), 2937, 2863
8000-10000. Masses are measured by peak matching the
unknown with a mass of known composition. Reported spectra
are by FAB unless otherwise stated. NMR spectra were
determined in Me2SO-d6 on a 250-MHz spectrometer Brucker
AC250 at 30 °C (303 K) unless otherwise stated. IR spectra
(film) were recorded on a Perkin-Elmer 1720X FT-IR spec-
trometer. Elemental analyses were determined by Butterworth
Laboratories Ltd. (Teddington, Middlesex, U.K.).
1
(CH2); H NMR δH* 1.36-1.59 (m, 12H, 2Hb + 2Hc + 2Hd
+
2H3′ + 2H4′ + 2H5′), 2.97 (q, 2H, 2Ha, J ) 5.91 Hz), 3.40 (q,
2H, 2He), 3.50 (m, 1H, H6′), 3.80 (m, 1H, H6′), 4.24 (d, 1H,
PhCH2, J ) 11.10 Hz), 4.32 (t, 1H, OH or NH, J ) 5.23 Hz),
4.48 (d, 1H, PhCH2), 4.60 (d, 1H, H2′), 5.50 (t, 1H, NH or OH),
6.51 (d, 2H, Harom, J ) 8.81 Hz), 7.02 (d, 2H, Harom); MS (EI)
m/z 293 (M+, 52), 192 (C5H10NHC6H4CH2+, 100) (*for number-
ing see formula 13).
4-Nitr oben zyl ter t-Bu tyld ip h en ylsilyl Eth er (2a ). To a
stirred solution of 4-nitrobenzyl alcohol (1.00 g, 6.50 mmol)
and imidazole (0.97 g, 14.10 mmol) in DMF (10.0 mL) was
added tert-butyldiphenylchlorosilane (1.98 g, 1.87 mL, 7.20
mmol) over 10 min under nitrogen, at room temperature. The
reaction mixture was stirred for an additional 5 h, diluted with
75 mL of Et2O, washed with water (5 × 15 mL), dried (MgSO4),
and evaporated under vacuum. An oil, which crystallized on
standing, was obtained. Recrystallization from aqueous 70%
EtOH afforded 2.36 g (93%) of a white solid: mp 80-81 °C;
ter t-Bu tyld ip h en ylsilyl 4-Oxym eth ylp h en yl Isocya n -
a te (4). To a stirred solution of 3a (0.63 g, 1.70 mmol) and
triethylamine (0.25 mL, 0.18 g, 1.80 mmol) in toluene (10 mL)
heated at 70 °C was added triphosgene (0.18 g, 0.61 mmol) in
one portion. The reaction was monitored by IR (νNCO 2275
cm-1). After 5 h the reaction mixture was filtered and the
filtrate evaporated under vacuum, giving 4 (0.65 g, 99%) as
an oil which was used without further purification: νmax/cm-1
(film) 2931, 2857 (CH2), 2275 (NCO); 1H NMR δH 1.03 (s, 9H,
t-Bu), 4.76 (s, 2H, PhCH2), 7.23 (d, 2H, Harom3+5, J ) 8.38 Hz),
7.35 (d, 2H, Harom2+6), 7.37-7.48 (m, 5H, Harom), 7.62-7.71 (m,
5H, Harom); MS (EI) m/z 330 (M+ - t-Bu, 52), 286 (M+ - t-Bu-
NCO, 48), 199 (Ph2SiOH+, 100).
ν
max/cm-1 (film) 2931, 2857 (CH2), 1521, 1345 (NO2); 1H NMR
δH 1.06 (s, 9H, t-Bu), 4.92 (s, 2H, PhCH2), 7.42-7.46 (m, 5H,
H
arom), 7.63-7.65 (m, 7H, Ph + Harom2+6), 8.23 (d, 2H, Harom3+5
,
-
J ) 8.23 Hz); MS (EI) m/z 334 (M+ - t-Bu, 100), 288 (M+
t-Bu - NO2, 10), 256 (M+ - t-Bu - Ph, 20), 199 (Ph2SiOH+,
Di-ter t-bu tyl N-[(4-{[ter t-Bu tyldiph en ylsilyl]oxym eth yl}-
p h en yl)ca r b a m oyl]-L-glu t a m a t e (7a ) a n d ter t-Bu t yl 1-
100). Anal. (C23H25NO3Si) C, H, N.

Self-Immolative Nitrogen Mustard Prodrugs
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5305
[N -(4-{[t er t -b u t y ld ip h e n y ls ily l]o x y m e t h y l}p h e n y l)-
ca r ba m oyl]-5-oxop yr r olid in e-2-ca r boxyla te (10). Meth od
A: To a solution containing 6.03 g (13.10 mmol) of isocyanate
4 and 3.9 mL (2.64 g, 26.2 mmol) of triethylamine in 60 mL of
THF was added at room temperature, during 30 min and
under stirring, a solution of di-tert-butyl L-glutamate hydro-
chloride (3.90 g, 13.10 mmol) in 20 mL of THF. After 3 h the
reaction was complete. The precipitate was filtered and the
solvent removed under vacuum, leaving an oil. The oil was
redissolved in AcOEt (25 mL), washed with H2O (25 mL),
aqueous HCl (2%) (25 mL), aqueous Na2CO3 (2%) (25 mL), and
brine (2 × 25 mL), dried, and evaporated again giving 7.53 g
of an oil. The product was purified by column chromatography
(AcOEt:cyclohexane, 2:1). The first eluting compound was 7a
(5.31 g, 63%) as a solid: mp 89-90 °C (hexane); νmax/cm-1 (film)
3359 (NH), 2932, 2857 (CH2), 1729 (CdO, ester), 1670 (CdO,
urea), 1154 (C-O); 1H NMR δH 1.03 (s, 9H, t-Bu), 1.40 (s, 9H,
t-Bu-G), 1.43 (s, 9H, t-Bu-G), 1.68-2.00 (2m, 2H, CH2(NH)-
CH), 2.18-2.32 (m, 2H, CH2CO2), 4.08-4.12 (m, 1H, CH(NH)-
CH2), 4.68 (s, 2H, PhCH2), 6.38 (d, 1H, NH-G, J ) 8.12 Hz),
Di-ter t-bu tyl N-[(4-{Hydr oxym eth yl}ph en yl)car bam oyl]-
L-glu ta m a te (8a ). Compound 7a (5.15 g, 8.0 mmol) was
dissolved in THF (100 mL) and tetrabutylammonium fluoride
solution in THF (1 M, 20.0 mL, 2.5 equiv) was added, in one
portion, under stirring at room temperature. The reaction was
finished in 3 h. The reaction mixture was evaporated under
vacuum. The residue was dissolved in AcOEt (50 mL), washed
with H2O (3 × 100 mL), dried (MgSO4), and evaporated again.
A yellow oil (5.08 g) resulted which was purified by column
chromatography (AcOEt:cyclohexane, 3:1) yielding an oil (1.88
g, 58%) which crystallized on standing: mp 103-4 °C (aqueous
MeOH, 60%); νmax/cm-1 (film) 3370 (broad, NH+OH), 2967
(CH3), 2930, 2857 (CH2), 1716 (CdO, ester), 1678 (CdO,
amide), 1153 (C-O). Anal. (C21H32N2O6) C, H, N. 1H NMR and
low-resolution mass spectra are in Supporting Information.
The fraction eluting second was ter t-b u t yl 1-[N-(4-
{h yd r oxym eth yl}p h en yl)ca r ba m oyl]-5-oxop yr r olid in e-2-
ca r boxyla te (11) (0.356 g, 13%): white solid, mp 157-9 °C;
ν
max/cm-1 (film) 3354 (NH2), 2977, 2932 (CH2), 1719 (CdO,
ester), 1676 (CdO, amide); 1H NMR δH 1.41 (s, 9H, t-Bu),
1.90-2.08 (2m, 2H, CH2(NH)CH), 2.37 (t, 2H, CH2CO, J ) 7.67
Hz), 4.23 (t, 1H, CH(N)CH2), 4.52 (d, 2H, CH2, J ) 5.64 Hz),
5.23 (t, 1H, CH2OH), 7.28 (d, 2H, Harom3+5, J ) 8.36 Hz), 7.39
(d, 2H, Harom2+6), 8.44 (s, 1H, PhNH); MS (EI) m/z 334 (M+,
16), 278 (M+ - t-Bu, 82). Anal. (C17H22N2O5) C, H; N: calcd,
8.38; found, 7.92.
7.19 (d, 2H, Harom3+5, J ) 8.41 Hz), 7.32-7.47 (m, 7H, Harom
+
Harom2+6), 7.62-7.70 (m, 5H, Harom), 8.54 (s, 1H, NH-Ph); MS
(EI) m/z 590 (M+ - t-Bu + 1, 2), 534 (M+ - 2t-Bu, 5), 478 (M
- 3t-Bu, 100), 199 (Ph2SiOH+, 100). Anal. (C37H50N2O6Si) C,
H, N.
The compound eluting second was 10, an oil (0.39 g, 5%),
Diprop-2-enylN-[(4-{Hydroxymethyl}phenyl)carbamoyl]-
L-glu ta m a te (8b). The intermediate 7b (0.610 g, 1.3 mmol)
was hydrolyzed at 45 °C in a mixture of AcOH:THF:H2O (24.5
mL, 4:2:1) for 3.5 h. The reaction mixture was diluted with
H2O (50 mL) and extracted with ether (2 × 25 mL) and then
with AcOEt (2 × 30 mL). The pooled organic layers were
washed with H2O (2 × 30 mL), dried (MgSO4), and evaporated
to dryness (with addition of toluene, 2 × 30 mL). An oil
resulted which was purified by preparative HPLC (cyclohexa-
ne:AcOEt, 1:2), leading to 8b (0.221 g, 45%): νmax/cm-1 (film)
3354 (NH2, OH, broad), 1737 (CdO, ester), 1659 (CdO, amide).
Anal. (C19H24N2O6) C, H, N. 1H NMR and low-resolution mass
spectra are in Supporting Information.
which was deprotected without further characterization.
Meth od B (on e-p ot syn th esis of com p ou n d 7a ): To a
solution of di-tert-butyl L-glutamate hydrochloride (4.14 g, 14.0
mmol) and triphosgene (1.39 g, 4.67 mmol) in toluene, cooled
at -78 °C, was added triethylamine (3.90 mL, 2.83 g, 28 mmol)
in toluene (10 mL) dropwise over 30 min. The reaction ws
allowed to reach room temperature and finished in 50 min
(monitored by IR, νNCO 2253 cm-1). To this mixture was added
a solution containing 4-aminobenzyl tert-butyldiphenylsilyl
ether (3a ) (5.00 g, 13.8 mmol) and triethylamine (1.95 mL, 14.0
mmol) in 30 mL of toluene over 5-10 min. The reaction was
monitored by IR (disappearance of the νNCO 2253 cm-1 peak)
and was finished in 14-20 h. The reaction mixture was
filtered, washed with H2O (200 mL), aqueous HCl (1%) (200
mL), aqueous Na2CO3 (1%) (200 mL), and H2O (2 × 200 mL),
dried (MgSO4), and evaporated under vacuum to give an oil
(9.90 g). The product was deprotected without further purifica-
tion.
Di-ter t-bu tyl
N-[(4-{[4-Nitr op h en oxyca r bon yloxy]-
m eth yl}p h en yl)ca r ba m oyl]-L-glu ta m a te (9a ). To a stirred
solution of 8a (0.200 g, 0.49 mmol) in dry THF (10 mL) were
added 4-nitrophenyl chloroformate (0.11 g, 0.5 mmol) and
triethylamine (0.1 mL, 0.6 mmol) at room temperature. The
reaction was complete after 1 h. The formed precipitate was
filtered and the solution concentrated under vacuum. AcOEt
(10 mL) was added; the solution was washed with brine (2 ×
10 mL), dried (MgSO4), and evaporated again, giving an oil
which was purified by column chromatography (0.160 g, 57%)
and repurified by preparative HPLC (0.140 g, 50%): mp 55-6
°C; νmax/cm-1 (film) 3349 (NH2), 2979, 2932 (CH2), 1767 (Cd
O, carbonate), 1716 (CdO, ester), 1652 (CdO, amide), 1527,
1349 (NO2). Mass (C28H36N3O10) calcd, 574.2401; found,
574.2420. Anal. (C28H35N3O10) C, H, N. 1H NMR and low-
resolution mass spectra are in Supporting Information.
Dip r op -2-en yl N-[(4-{[4-Nit r op h en oxyca r b on yloxy]-
m eth yl}ph en yl)car bam oyl]-L-glu tam ate (9b). Starting from
8b (0.190 g, 0.50 mmol), 9b, obtained by the same procedure
as 9a , was purified by preparative HPLC to a solid (0.132 g,
48.6%): mp 106-7 °C; νmax/cm-1 (film) 3356 (NH2), 2933 (CH2),
1766 (CdO, carbonate), 1738 (CdO, ester), 1660 (CdO, amide),
1525, 1346 (NO2). Mass (C26H27N3O10Na) calcd, 564.1594;
found, 564.1590. Anal. (C26H27N3O10) H, N; C: calcd, 57.67;
found, 58.09. 1H NMR and low-resolution mass spectra are in
Supporting Information.
Dip r op -2-en yl
N-[(4-{[2′-O-t et r a h yd r op yr a n yl]oxy-
m eth yl}ph en yl)car bam oyl]-L-glu tam ate (7b) was obtained,
starting from diallyl L-glutamate p-toluenesulfonate (1.08 g,
3.1 mmol) and 3b (0.52 g, 2.5 mmol) by the one-pot procedure.
An oil resulted which was separated by preparative HPLC
using cyclohexane:AcOEt (1.5:1) as eluent (0.75 g, 65%): νmax
/
1
cm-1 (film) 3362 (NH2), 2944, 2871 (CH2); H NMR δH 1.47-
1.50 (m, 4H, 2H4′ + 2H5′), 1.60-1.88 (m, 3H, 2H3′ + CH2(NH)-
CH), 1.88-2.05 (m, 1H, -CH2CH(NH)-), 2.37-2.45 (m, 2H,
CH2CO2), 3.45 (s, 1H, H6′), 3.80 (m, 1H, H6′), 4.20-4.31 (m,
1H, CH(NH)CH2), 4.35 (d, 1H, PhCH2, J ) 11.55 Hz), 4.53-
4.63 (m, 5H, PhCH2 + CH2O, allyl), 5.18-5.33 (m, 4H, CH2d
allyl), 5.80-6.00 (m, 2H, CHd allyl), 6.60 (d, 1H, NH-G, J )
8.03 Hz,), 7.20 (d, 2H, Harom, J ) 8.47 Hz), 7.35 (d, 2H, Harom),
8.47 (s, 1H, NH-Ph); MS (EI) m/z 461 (M+ + 1, 15). Mass
(C24H32N2O7Na) calcd, 483.2107; found, 483.2120.
Dip r op -2-en yl
N-[(4-{[ter t-b u t yld ip h en ylsilyl]oxy-
m eth yl}p h en yl)ca r ba m oyl]-L-glu ta m a te (7c) was synthe-
sized by the same method starting from 5.0 g (13.8 mmol) of
amine 3a . An oil resulted which was purified by column
chromatography (cyclohexane:AcOEt, 2:1) leading to 7c (6.47
g, 47%): νmax/cm-1 (film) 3382 (NH2), 1739 (CdO, ester), 1651
[(4-Hydr oxym eth ylph en yl)car bam oyl]-L-glu tam ic Acid
(12). In the stirred solution of 8b (0.200 g, 0.53 mmol) in CH2-
Cl2 (15 mL) were added Pd(PPh3)4 (40 mg) and pyrrolidine (1
mL, 11.8 mmol). On addition of pyrrolidine, a precipitate
started to form immediately. After 40 min of stirring, the
solvent was removed and the precipitate washed with AcOEt
and CH2Cl2. The residue was dissolved in methanol (10 mL)
and passed through a weakly acid resin IRC50 ion-exchange
column. After eluting with methanol (50 mL), the eluate was
evaporated under vacuum to yield 12 (0.135 g, 86%) as a solid;
1
(CdO, amide); H NMR δH 1.03 (s, 9H, t-Bu), 1.85-2.11 (2m,
2H, CH2(NH)CH), 2.46-2.52 (m, 2H, CH2CO2), 4.32-4.35 (m,
1H, CH(NH)CH2), 4.56 (dd, 2H, CH2d allyl, J ) 4.62 Hz), 4.62
(dd, 2H, CH2d allyl), 4.69 (s, 2H, PhCH2), 5.17-5.38 (m, 4H,
CH2O allyl), 5.80-6.00 (m, 2H, CHd allyl), 6.60 (d, 1H, NH-
G, J ) 8.03 Hz), 7.20 (d, 2H, Harom3+5, J ) 8.49 Hz), 7.34-7.48
(m, 7H, Harom + Harom2+6), 7.63-7.72 (m, 5H, Harom), 8.57 (s,
1H, NH-Ph).

5306 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Niculescu-Duvaz et al.
mp 73-6 °C. Mass (C13H17N2O6) calcd, 297.1087; found,
297.1070. Anal. (C13H16N2O6) C, H, N. 1H NMR and low-
resolution mass spectra are in Supporting Information.
Na) calcd, 432.1998; found, 432.1990. Anal. (C21H31NO7) C, H,
N. 1H NMR and low-resolution mass spectra are in Supporting
Information.
Dip r op -2-en yl N-[(4-{H yd r oxym et h yl}p h en oxy)ca r -
bon yl]-L-glu ta m a te (18b). Compound 18b was obtained by
a similar reduction procedure as a clear oil (0.233 g, 78%).
Mass (C19H23NO7Na) calcd, 400.1372; found, 400.1376. Anal.
(C28H35N3O10) H, N; C: calcd, 60.47; found, 60.01. 1H NMR
and low-resolution mass spectra are in Supporting Informa-
tion.
Di-ter t-b u t yl N-[(4-{1,3-Dit h ia n -2-yl}p h en yloxy)ca r -
bon yl]-L-glu ta m a te (16). Compound 16 was prepared by the
one-pot procedure (as described above for compound 7a ,
method B) from di-tert-butyl L-glutamate hydrochloride (1.50
g, 5.0 mmol) and 4-[2′-(1′,3′-dithianyl)]phenol, 15 (1.10 g, 5.0
mmol). The reaction was left for 12 h. The solid formed was
filtered; the solution was washed with aqueous NaOH (1%)
(75 mL) and H2O (2 × 75 mL), dried (MgSO4), and evaporated
under vacuum to yield a solid (2.30 g, 95.8%). Recrystallization
from aqueous 50% EtOH afforded 16 (1.81 g, 72.7%): mp
137-8 °C; νmax/cm-1 (film) 3347 (NH2), 2977, 2945 (CH2), 1728
(CdO, ester); 1H NMR δH 1.41 (s, 9H, t-Bu), 1.42 (s, 9H, t-Bu),
1.62-1.90 (m, 2H, 2H5′), 1.90-2.32 (2m, 2H, CH2(NH)CH),
2.30-2.40 (m, 2H, CH2CO2), 2.86-2.93 (m, 2H, 2H4′or6′), 3.09
(t, 2H, 2H6′or4′, J ) 12.3 Hz), 5.41 (s, 1H, H2′), 7.08 (d, 2H,
Di-ter t-bu tyl
N-[(4-{[4-Nitr op h en oxyca r bon yloxy]-
m eth yl}p h en oxy)ca r bon yl]-L-glu ta m a te (19a ). Activation
of linker 18a (1.0 g, 2.7 mmol) was achieved by the same
procedure as for 9a . After purification, an oil resulted (0.64 g,
41%): νmax/cm-1 (film) 3367 (NH2), 2980, 2933 (CH2), 1767 (Cd
O, carbonate), 1728 (CdO, ester). Anal. (C28H34N2O11) C, H,
N. 1H NMR and low-resolution mass spectra are in Supporting
Information.
Starting from 18b (0.4 g, 1.06 mmol), 19b was obtained by
the same method as an oil (0.448 g, 75.5%). Mass (C26H26N2O11-
Na) calcd, 565.1434; found, 565.1430. Anal. (C26H26N2O11) C,
H, N. 1H NMR and low-resolution mass spectra are in
Supporting Information.
[(4-H yd r oxym et h ylp h en yl)oxyca r b on yl]-L-glu t a m ic
Acid (20). Starting from 18b (150 mg, 0.4 mmol) in CH2Cl2
(12 mL) and using Pd(PPh3)4 (25 mg) and pyrrolidine (152 mL,
1.8 mmol), compound 20 was obtained by the procedure
described above for compound 12. However, the compound
eluting from the IRC50 ion-exchange column, after evaporation
under vacuum, led only to an impure 20 (103 mg). This product
was purified by reverse-phase (C18) preparative HPLC using
as mobile phase a gradient of 0.1% aqueous TFA/acetonitrile.
After lyophilization 20 (36 mg, 30%) was obtained as a white
powder: mp 135-7 °C. Mass (C13H15NO7Na) calcd, 320.0746;
found, 320.0730. Anal. (C13H15NO7) C, H, N. 1H NMR and low-
resolution mass spectra are in Supporting Information.
H
arom3+5, J ) 8.50 Hz), 7.43 (d, 2H, Harom2+6), 8.12 (d, 1H, NH-
G, J ) 7.81 Hz); MS (EI) m/z 497 (M+, 2), 451 (M+ - t-Bu, 2),
359 (M+ - 2t-Bu, 20). Anal. (C24H25NO6S2) C, N; H: calcd, 7.09;
found, 6.68.
Di-ter t-bu tyl N-[(4-For m ylph en yloxy)car bon yl]-L-glu ta-
m a te (17a ). P r oced u r e A: To a stirred solution of dithiane
16 (0.50 g, 1.0 mmol) in CHCl3 (10 mL) and THF (5 mL) was
added dropwise a solution of Hg(ClO4)2‚3H2O (0.91 g, 2.0 mmol)
in THF (5 mL). The reaction was completed in 5 min. The
precipitate was filtered and the solution washed with H2O (2
× 25 mL), aqueous Na2CO3 (25 mL), and H2O (2 × 25 mL),
dried (MgSO4), and evaporated under vacuum. The compound
17a (0.33 g, 81%) was obtained as a clear oil.
P r oced u r e B: To a solution of di-tert-butyl L-glutamyl
isocyanate 6a (prepared from di-tert-butyl L-glutamate hydro-
chloride (0.200 g, 0.67 mmol), triphosgene (0.067 g, 0.22 mmol),
and triethylamine (0.190 mL, 1.35 mmol) according to the
procedure described above) in CHCl3 (15 mL) were added at
room temperature 4-hydroxybenzaldehyde (0.082 g, 0.67 mmol)
and triethylamine (0.190 mL, 1.35 mmol) in CHCl3 (10 mL)
over 10 min. The reaction was heated at reflux for 2 h, when
the νNCO peak disappeared. The solvent was removed under
vacuum and the compound purified by preparative HPLC
(cyclohexane:AcOEt, 6:1). A clear oil resulted (0.197 g, 72%):
Dip r op -2-en yl
N-[(4-{[4-(Bis{2-ch lor oeth yl}a m in o)-
ph en oxy car bon yloxy]m eth yl}ph en yl)car bam oyl]-L-glu ta-
m a te (22). The solution containing the activated linker 9b
(0.200 g, 0.37 mmol), 4-bis(2-chloroethyl)aminophenol (21a (X
) Cl)), (0.140 g, 0.60 mmol) dissolved in CH3CN (10 mL), and
a strongly basic resin-Amberlyst 27 HO- (0.13 g) was stirred
for 20 h. The resin was filtered off, and a further 0.080 g of
fresh resin was added. After 30 min of stirring, the second
portion of resin was filtered off and the solvent evaporated.
The product was purified by column chromatography (cyclo-
hexane:AcOEt, 2:1) to afford 28 (82 mg, 32%) as a solid: mp
134-5 °C. Mass (C30H35N3O8Cl2Na) calcd, 658.1699; found,
658.1690. Anal. (C30H35N3O8Cl2) C, H, N. 1H NMR and low-
resolution mass spectra are in Supporting Information.
ν
max/cm-1 (film) 3347 (NH2), 2979, 2934 (CH2), 1731 (CdO,
1
ester), 1712 (CdO, aldehyde); H NMR δH 1.41 (s, 9H, t-Bu),
1.43 (s, 9H, t-Bu), 1.80-2.02 (2m, 2H, CH2(NH)CH), 2.32-
2.39 (m, 2H, CH2CO2), 3.99-4.04 (m, 1H, -CH(NH)CH2-), 7.34
(d, 2H, Harom3+5, J ) 8.39 Hz), 7.96 (d, 2H, Harom2+6), 8.29 (d,
1H, NH-G, J ) 7.72 Hz), 9.98 (s, 1H, CHO). Anal. (C21H29
NO7) C, H, N.
-
Dip r op -2-en yl
N-[(4-for m ylp h en yloxy)ca r bon yl]-L-
Dip r op -2-en yl
N-[(4-{[4-(Bis{2-flu or oeth yl}a m in o)-
glu ta m a te (17b) was obtained as a clear oil (2.90 g, 76%)
using procedure B: νmax/cm-1 (film) 3347 (NH2), 2979, 2934
(CH2), 1731 (CdO, ester), 1712 (CdO, aldehyde); 1H NMR δH
1.93-2.16 (2m, 2H, CH2(NH)CH), 2.53 (t, 2H, CH2CO2), 4.19-
4.26 (m, 1H, CH(NH)CH2), 4.57 (d, 2H, CH2-allyl, J ) 5.35
Hz), 4.64 (d, 2H, CH2-allyl), 5.18-5.37 (m, 4H, CH2dallyl, J
p h e n o x y c a r b o n y lo x y ]m e t h y l}p h e n y l)c a r b a m o y l]-L -
glu ta m a te (24). 24 (120 mg, 40%) was obtained as a solid,
mp 129-31 °C, by a similar procedure as 22. Mass (C30H36N3-
O8F2) calcd, 604.2470; found, 604.2450. Anal. (C30H35N3O8F2)
C, H, N. 1H NMR and low-resolution mass spectra are in
Supporting Information.
) 5.35 Hz), 5.86-5.94 (m, 2H, CHdallyl), 7.35 (d, 2H, Harom3+5
,
J ) 8.50 Hz), 7.77 (d, 2H, Harom2+6), 8.49 (d, 1H, NH-G, J )
7.77 Hz), 9.98 (s, 1H, CHO); MS m/z 398 (M+ + 23, 16), 376
(M+ + 1, 14). Anal. (C19H21NO7) C, H; N: calcd, 3.73; found,
4.16.
N -[(4-{[4-(B is {2-c h lo r o e t h y l}a m in o )p h e n o x y c a r -
bon yloxy]m eth yl}ph en yl)car bam oyl]-L-glu tam ic Acid (23).
To a solution of 22 (0.200 g, 0.32 mmol) and Pd(PPh3)4 (25
mg, 0.022 mmol) in CH2Cl2 (9 mL) was added morpholine (107
mL, 1.23 mmol), and the mixture stirred for 2 h under argon
at room temperature. The reaction mixture was diluted with
AcOEt (a precipitate formed upon addition of AcOEt). The
solvent was evaporated, and the insoluble residue was washed
with AcOEt. It was dissolved in methanol (10 mL) and passed
through a weakly acid resin, IRC50 ion-exchange column. After
eluting with methanol (50 mL), the eluate was evaporated
under vacuum to yield 23 (0.145 g, 81%) as a solid: mp 73-6
°C. Mass (C24H27N3O8Cl2Na) calcd, 578.1073; found, 578.1070.
Anal. (C24H27N3O8Cl2) C, H, N. 1H NMR and low-resolution
mass spectra are in Supporting Information.
Di-ter t-bu tyl N-[(4-{Hydr oxym eth yl}ph en oxy)car bon yl]-
L-glu ta m a te (18a ). Compound 17a (0.150 g, 0.36 mmol)
dissolved in a mixture of H2O:AcOH:CH3OH (5.2 mL, 1:1:1)
was reduced with sodium cyanoborohydride (0.027 g, 0.43
mmol) at room temperature. The reaction was complete after
1 h. AcOEt (20 mL) was added, the reaction mixture evapo-
rated under vacuum, the residue dissolved in AcOEt (20 mL),
and the solution washed with H2O (20 mL) and brine (2 × 20
mL), dried (MgSO4), and evaporated to dryness. Compound
18a (0.127 g, 86%) was obtained as an oil. The final purifica-
tion was achieved by preparative HPLC (cyclohexane:AcOEt,
2:1): νmax/cm-1 (film) 3352 (NH2, OH, broad), 2979, 2945 (CH2),
1728 (CdO, ester), 1712 (CdO, aldehyde). Mass (C21H31NO7-
N-[(4-{[4-(Bis{2-flu or oeth yl}a m in o)p h en oxyca r bon yl-
oxy]m eth yl}p h en yl)ca r ba m oyl]-L-glu ta m ic a cid (25) was

Self-Immolative Nitrogen Mustard Prodrugs
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5307
obtained as a white solid (68 mg, 72%), mp 104-7 °C, by a
procedure similar to 23. Mass (C24H27N3O8F2Na) calcd, 546.1664;
found, 546.1680. 1H NMR and low-resolution mass spectra are
in Supporting Information.
605.2330. Anal. (C30H34N2O9F2): H, N; C: calcd, 59.60; found,
60.02.
Meth od B: Fluoroformate 27 (starting from 0.67 mmol of
18b) was prepared as described for 29. The suspension
containing 27 was cooled at -78 °C, a solution of 28b (0.12 g,
0.44 mmol) in CH2Cl2 (4 mL) added dropwise over 15 min, and
the reaction mixture stirred for a further 30 min. The suspen-
sion was filtered, the filtrate evaporated to dryness, and the
residue purified by preparative HPLC to afford 31 (0.018 g,
7%). 1H NMR and low-resolution mass spectra are in Sup-
porting Information.
N -[(4-{[4-(B i s {2-flu o r o e t h y l}a m i n o )p h e n o x y c a r -
bon yloxy]m eth yl}ph en oxy)car bon yl]-L-glu tam ic Acid (32).
32 (0.04 g, 42%) was obtained as an oil from 31 by the same
method as for 23. Mass (C24H26N2O9F2Na) calcd, 547.1504,
found, 547.1526. 1H NMR and low-resolution mass spectra are
in Supporting Information.
4-[Bis(2-flu or oeth yl)am in o]ph en yl Ch lor ofor m ate (27a).
Over a solution of 21b (0.2 g, 1.0 mmol) in CH2Cl2 (8 mL)
phosgene (20% in toluene, 3.5 mL, 7 mmol) was added. After
5 min of stirring, a solution of NEt3 (0.14 mL, 1 mmol) in CH2-
Cl2 (8 mL) was added dropwise over 15 min. The solvent was
evaporated, the residue taken up in AcOEt, the precipitate
filtered off, and the filtrate evaporate to dryness to afford 27a
(0.184 g, 74.5%) as an oil: IR νmax/cm-1 (film) 1780 (OCOCl),
1515, 1117; 1H NMR (CDCl3) δH 3.73 (2t, 4H, NCH2, J ) 5.20
Hz, J H-F ) 23.63 Hz), 4.61 (2t, 4H, CH2F, J ) 5.22 Hz, J H-F
) 47.15 Hz), 6.68 (d, 2H, Harom2+6, J ) 9.26 Hz), 7.08 (d, 2H,
H
arom3+5).
4-Bis(2-ch lor oeth yl)a m in op h en yl Tr im eth ylsilyl Eth er
(28a ). Phenol nitrogen mustard 21a (X ) Cl) (60 mg, 0.26
mmol) and trimethylsilyl cyanide (40 mL, 0.3 mmol) were
stirred at room temperature without solvent for 30 min.
Boiling hexane (10 mL) was added to the reaction mixture and
the remaining solid filtered off. After evaporation, 28a (70 mg,
88%) as an oil resulted: 1H NMR δH 0.20 (s, 9H, Si(CH3)3),
3.55-3.70 (m, 8H, N(CH2CH2Cl)2), 6.65 (d, 2H, J ) 9.30 Hz,
Di-ter t-bu tyl N-[(4-{[N-(4-{Bis[2-ch lor oeth yl]a m in o}-
p h e n yl)ca r b a m oyloxy]m e t h yl}p h e n oxy)ca r b on yl]-L -
glu ta m a te (36). To a CH2Cl2 solution (3 mL) of 34 (0.147 g,
0.63 mmol) and triethylamine (180 mL, 1.25 mmol) was added
triphosgene (63 mg, 0.21 mmol) in one portion. After 15 min
stirring, the solvent was evaporated, the residue taken up in
THF, and the insoluble salt filtered off. The THF was
evaporated, and isocyanate 35a (X ) Cl) was obtained as an
oil which was used immediately in the next reaction.
To the toluene solution (5 mL) of 18b (60 mg, 0.15 mmol)
was added isocyanate 35a (X ) Cl) (0.60 mmol) in toluene (5
mL) and dibutyltin dilaurate (10 mL). The reaction mixture
was stirred for 12 h at room temperature, filtered, and
evaporated to dryness. Purification was achieved by column
chromatography (cyclohexane:AcOEt, 3:1), when product 36
(90 mg, 90%) was obtained as a solid: mp 44-7 °C; νmax/cm-1
(film) 1728 (CdO, ester, carbamate). Mass (C32H43N3O8Cl2)
calcd, 667.2427; found, 667.2420. Anal. (C32H43N3O8Cl2) C, H,
N, Cl. 1H NMR and low-resolution mass spectra are in
Supporting Information.
Di-ter t-b u t yl N-[(4-{[N-(4-{b is[2-flu or oet h yl]a m in o}-
p h e n yl)ca r b a m oyloxy]m e t h yl}p h e n oxy)ca r b on yl]-L -
glu ta m a te (41) was obtained in a similar manner from 18a
and isocyanate 35b (X ) F) as a solid (0.205 g, 69%): mp 39-
41 °C. Isocyanate 35b (X ) F) was prepared from 51 by the
method previously described for 35a . Mass (C32H43N3O8F2)
calcd, 635.3018; found, 635.3040. Anal. (C32H43N3O8F2) H, N;
C: calcd, 60.46; found, 60.03. 1H NMR and low-resolution mass
spectra are in Supporting Information.
N -[(4-{[N -(4-{Bis[2-ch lor oe t h yl]a m in o}p h e n yl)ca r -
ba m oyloxy]m eth yl}p h en oxy)ca r bon yl]-L-glu ta m ic Acid
(37). Di-tert-butyl ester 36 (0.250 g, 0.37 mmol) was dissolved
in formic acid (10 mL) and stirred under argon at room
temperature for 48 h. The formic acid was evaporated under
vacuum (oil pump) and the residue reevaporated again five
times with CH2Cl2 and toluene to yield 37 as a solid (0.185 g,
89%): mp 99-102 °C; νmax/cm-1 (film) 1713 (CdO, acid). Mass
(C24H27N3O8Cl2Na) calcd, 578.1073; found, 578.1070. Anal.
(C24H27N3O8Cl2) C, H, Cl; N: calcd, 7.57; found, 6.01. 1H NMR
and low-resolution mass spectra are in Supporting Informa-
tion.
H
arom2+6), 6.73 (d, 2H, Harom3+5); MS m/z 305 (M+, 100), 270
(M+ - Cl, 35). Mass (C13H21NOCl2Si) calcd, 305.0769; found,
305.0765. Anal. (C13H21NOCl2Si) C, H, N, Cl.
4-Bis(2-flu or oeth yl)a m in op h en yl Tr im eth ylsilyl Eth er
(28b). Meth od A: 28b (0.65 g, 80%) was synthesized according
to the same procedure as 21b but using excess trimethylsilyl
cyanide and longer time periods: 1H NMR δH 0.19 (s, 9H, Si-
(CH3)3), 3.61 (2t, 4H, NCH2, J ) 5.20 Hz, J H-F ) 24.44 Hz),
4.54 (2t, 4H, CH2F, J ) 5.17 Hz, J H-F ) 47.55 Hz), 6.68 (s,
4H, Harom); MS m/z 273 (M+, 100), 254 (M+ - F, 23). Mass
(C13H21NOF2Si) calcd, 273.1360; found, 273.1345. Anal. (C13H21
NOF2Si) C, H, N.
-
Meth od B: Over a solution of 21b (0.25 g, 1.25 mmol) in
CH2Cl2 (3 mL) trimethylsilyl N,N-dimethylcarbamate (0.475
mL, 2.74 mmol) was added under argon. After 1.5 h, the
solvent was evaporated, the residue taken in hexane, and the
precipitate filtered off. The hexane was evaporated to dryness
to afford 28b (0.24 g, 70%) as an oil.
Dipr op-2-en yl N-[(4-{[4-(Bis{2-ch lor oeth yl}am in o)ph en -
oxyca r bon yloxy]m eth yl}p h en oxy)ca r bon yl]-L-glu ta m a te
(29). Over a solution of 18b (0.375 g, 1.0 mmol) in CH2Cl2 (15
mL) cooled to -78 °C, phosgene (20% in toluene, 0.75 mL, 1.5
mmol), 18-crown-6 ether (0.255 g, 1.0 mmol), and KF‚HF (0.3
g, 3.8 mmol) were added. After stirring for 12 h and allowing
the mixture to warm to room temperature, the fluoroformate
27 was formed (νOCOF 1827 cm-1). The suspension was cooled
to 0 °C, and a solution of 28a (0.41 g, 1.35 mmol) in CH2Cl2 (5
mL) was added in one portion. After 1 h stirring, the suspen-
sion was filtered and the filtrate evaporated to dryness. The
residue was purified by preparative HPLC (cyclohexane:
AcOEt, 3:1) to afford 29 (0.31 g, 49%) as an oil. Mass
(C30H35N2O9Cl2) calcd, 637.1710; found, 637.1726. Anal.
(C30H34N2O9Cl2) C, H, N, Cl. 1H NMR and low-resolution mass
spectra are in Supporting Information.
N -[(4-{[4-(B is {2-c h lo r o e t h y l}a m in o )p h e n o x y c a r -
bon yloxy]m eth yl}ph en oxy)car bon yl]-L-glu tam ic Acid (30).
30 was obtained from 29 by the same method as 23 as a solid
(0.105 g, 78.5%): mp 66-9 °C. Mass (C24H26N2O9Cl2Na) calcd,
579.0913; found, 579.0903. Anal. (C30H35N3O8Cl2) C, H; N:
calcd, 5.04; found, 4.61. 1H NMR and low-resolution mass
spectra are in Supporting Information.
N -[(4-{[N -(4-{B i s [2-flu o r o e t h y l]a m i n o }p h e n y l)-
car bam oyloxy]m eth yl}ph en oxy)car bon yl]-L-glu tam ic acid
(42) was obtained identically from 41 as a solid (60 mg, 82%):
mp 124-7 °C. Mass (C24H27N3O8F2) calcd, 523.1766; found:
523.1780. Anal. (C24H27N3O8F2) C, H, N. 1H NMR and low-
resolution mass spectra are in Supporting Information.
Dip r op -2-en yl N-[(4-{[N-(4-{Bis[2-ch lor oeth yl]a m in o}-
p h e n yl)ca r b a m oyloxy]m e t h yl}p h e n yl)ca r b a m oyl]-L -
glu ta m a te (39). The same procedure as for 36 was used
starting from isocyanate 35a (X ) Cl) (0.54 mmol) in toluene
(5 mL), linker 8b (0.100 g, 0.27 mmol) dissolved in CH2Cl2 (5
mL), and dibutyltin dilaurate (10 mL). 39 was obtained as a
solid. Further recrystallization from hexane:AcOEt (9:2) af-
forded 39 (0.110 mg, 64%) as a pure compound: mp 148-9
°C; νmax/cm-1 (film) 1731 (CdO, ester), 1691 (CdO, carbamate),
1642 (CdO, urea). Mass (C30H36N4O7Cl2Na) calcd, 657.1859;
Dip r op -2-en yl
N-[(4-{[4-(Bis{2-flu or oeth yl}a m in o)-
p h e n o x y c a r b o n y lo x y ]m e t h y l}p h e n o x y )c a r b o n y l]-L -
glu t a m a t e (31). Met h od A: A solution of 18b (0.4 g, 1.08
mmol) in THF (10 mL) and 27a (prepared from 2.0 mmol of
21b) was dissolved in THF (10 mL) and NEt3 (0.25 mL, 1.8
mmol) added. The reaction mixture was stirred for 1.5 h and
the solvent evaporated to dryness. The residue was purified
by preparative HPLC (CH2Cl2:AcOEt, 39:1) to afford 31 (0.21
g, 35%) as an oil. Mass (C30H35N2O9F2) calcd, 605.2311; found,

5308 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Niculescu-Duvaz et al.
1
found, 657.1850. Anal. (C30H36N4O7Cl2) C, H, N, Cl. H NMR
4-[Bis(2-flu or oeth yl)a m in o]n itr oben zen e (50). 4-[Bis-
(2′-hydroxyethyl)amino]nitrobenzene (1.4 g, 6.2 mmol), tri-
ethylamine (4.3 mL, 31 mmol), and 4-(dimethylamino)pyridine
(0.120 g, 1.0 mmol) were dissolved in CH2Cl2 (40 mL), and
methanesulfonic anhydride (4.3 g, 25 mmol) dissolved in CH2-
Cl2 (40 mL) was added. After 1 h stirring the reaction mixture
was extracted with aqueous citric acid 10% (100 mL) and the
organic layer washed with water (100 mL), dried (MgSO4), and
evaporated to dryness. The residue was redissolved in chlo-
roform (50 mL), Amberlyst 26 F- was added, and the mixture
was stirred at reflux for 72 h. The reaction mixture was filtered
and the filtrate evaporated to dryness. Purification was
achieved by preparative HPLC (cyclohexane:AcOEt, 3:1) when
50 was obtained as a solid. Recrystallization from hexane-
diethyl ether-chloroform, 6:2:1, yielded 50 (0.290 g, 20%) as
yellow crystals: 1H NMR δH 3.87 (2t, 4H, N-CH2, J ) 5.1 Hz,
J H-F ) 25.3 Hz), 4.64 (2t, 4H, CH2F, J H-F ) 47.5 Hz), 6.92 (d,
2H, Harom2+6, J ) 9.48 Hz), 8.03 (d, 2H, Harom3+5); MS m/z 231
(M+ + 1, 100). Mass (C10H12F2N2O2) calcd, 231.0945; found,
231.0955. Anal. (C10H12F2N2O2) C, H, N.
4-[Bis(2-flu or oeth yl)a m in o]a n ilin e (51). 50 (0.260 g, 1.1
mmol) was dissolved in EtOH (12 mL); 10% Pd/C (0.120 g)
and ammonium formate (0.500 g) were added. The suspension
was stirred for 1.5 h at room temperature. The catalyst
was filtered off and the filtrate evaporated to dryness. Puri-
fication was achieved by preparative HPLC (cyclohexane:
AcOEt, 2:1) to yield 51 (0.190 g, 84%) as a dark-yellow oil: 1H
NMR δH 3.47 (2t, 4H, N-CH2, J ) 5.22 Hz, J H-F ) 25.0 Hz),
and low-resolution mass spectra are in Supporting Informa-
tion.
The same procedure was used to obtain d i-ter t-bu tyl N-[(4-
{[N-(4-{bis[2-ch lor oeth yl]a m in o}p h en yl)ca r ba m oyloxy]-
m eth yl}ph en yl)car bam oyl]-L-glu tam ate (38) (0.310 g, 95%),
mp 68-71 °C, from 18a and 35a . Mass (C32H44N4O7Cl2Na)
calcd, 689.2485; found, 689.2480. Anal. (C32H44N4O7Cl2) C, H,
N. 1H NMR and low-resolution mass spectra are in Supporting
Information. Also obtained was d ip r op -2-en yl N-[(4-{[N-(4-
{bis[2-flu or oeth yl]am in o}ph en yl)car bam oyloxy]m eth yl}-
p h en yl)ca r ba m oyl]-L-glu ta m a te (43) (0.190 g, 75%), mp
152-4 °C, from 8b and 35b. Mass (C30H37N4O7F2) calcd,
1
603.2630; found, 603.2650. Anal. (C30H36N4O7F2) C, H, N. H
NMR and low-resolution mass spectra are in Supporting
Information.
N -[(4-{[N -(4-{Bis[2-ch lor oe t h yl]a m in o}p h e n yl)ca r -
ba m oyloxy]m eth yl}p h en yl)ca r ba m oyl]-L-glu ta m ic Acid
(40). Diallyl ester 39 (0.190 g, 0.3 mmol) dissolved in CH2Cl2
(5 mL) was stirred with Pd(PPh3)4 (15 mg) and pyrrolidine (120
mL, 1.45 mmol). The reaction mixture was kept under argon
for 45 min and then diluted with AcOEt. The solvent was
evaporated, and the insoluble residue was washed with AcOEt.
It was dissolved in methanol (10 mL) and passed through an
IRC50 ion-exchange column (weakly acid resin). After eluting
with 50 mL of methanol, the eluate was evaporated under
vacuum to yield 40 (0.100 g, 60%) as a solid: mp 127-9 °C.
Mass (C24H28N4O7Cl2Na) calcd, 577.1233; found, 577.1230.
4.47 (2t, 4H, CH2F, J F-H ) 47.5 Hz), 6.49 (d, 2H, Harom2+6
,
1
Anal. (C24H28N4O7Cl2) H, N; C: calcd, 51.97; found, 51.54. H
NMR and low-resolution mass spectra are in Supporting
Information.
J ) 8.80 Hz), 6.60 (d, 2H, Harom3+5); MS m/z 200 (M+, 100).
Mass (C10H14N2F2) calcd, 200.1115; found, 200.1130. Anal.
(C10H14N2F2) C, H, N.
The same procedure was used to obtain N-[(4-{[N-(4-{bis-
[2-flu or oet h yl]a m in o}p h en yl)ca r b a m oyloxy]m et h yl}-
p h en yl)ca r ba m oyl]-L-glu ta m ic a cid (44) from 43 as a
glassy solid (0.125 g, 80%): mp 104-7 °C. Mass (C24H29N4O7F2)
calcd, 523.2004; found, 523.2020. Anal. (C24H28N4O7F2) C, H,
N. 1H NMR and low-resolution mass spectra are in Supporting
Information.
Ch em ica l Sta bility Deter m in a tion . Compounds were
prepared as 10 mM concentrates in distilled water (12, 20),
DMSO (23, 25, 37, 40, 42, 44), or MeOH (30) and diluted 100-
fold in CPG2 assay buffer (100 mM Tris-HCl, pH 7.3; 260 µM
ZnCl2; 1 mL) to give 100 µM solutions. Aliquots (10 µL) were
injected onto a Partisphere 5-µm C18 column (110 mm × 4.6
mm) and eluted isocratically (1 mL/min) with 10 mM am-
monium acetate (pH 5.0) containing percentages of methanol
chosen to produce a retention time of 3-4 min and monitored
at the optimum wavelength (12, 10%, 246 nm; 20, 10%, 213
nm; 23, 63%, 255 nm; 25, 50%, 250 nm; 30, 65%, 258 nm; 32,
37, 58%, 268 nm; 40, 58%, 253 nm; 42, 45%, 267 nm; 44, 43%,
251 nm). The amount of starting material remaining after
various periods of incubation at 37 °C was determined either
by repeat injections (7.5-min intervals) from a single vial (12,
20, 30, 32, 37, 40, 42, 44) or by delayed injections (1-min
intervals) from a new vial each time (23, 25).
Kin etic Deter m in a tion s. The optimum wavelength for
spectrophotometric kinetic analysis was established from a
differential spectrum comparing each parent compound with
an equimolar mixture of its products (12, 250 nm; 20, 228 nm;
40, 280 nm; 42, 267 nm; 44, 251 nm). CPG2 (50-250 ng) was
added to CPG2 assay buffer (100 mM Tris-HCl, pH 7.3; 260
µM ZnCl2; 1 mL) containing 1-100 µM compound and the rate
of change of absorbance at the chosen wavelength measured.
In the case of the less stable 40, compound was included in
both reference and sample cuvettes to negate the contribution
of purely chemical loss. Kinetic parameters were derived from
Hanes-Woolfe plots.
Biologica l Meth od s: Cytotoxicity Assa ys. For this
study, LS174T cells were constructed to express surface-
tethered mutant CPG2 stably, by transfection of cells with the
plasmid pMCEFstCPG2(Q)315 using lipofectamine, by the
method previously described. The plasmid pMCEFstCPG2(Q)3
encodes for a CPG2 molecule that is expressed on the outer
cell membrane. It bears three asparagine-to-glutamine [(Q)3]
mutations that were otherwise inappropriately glycosylated.
This plasmid encodes a mutated CPG2 molecule fused between
the signal and the transmembrane domains of the c-erbB2
protein tyrosine kinase receptor under the transcriptional
control of the EF-1a promoter and also encodes neomycin
resistance for selection of stably expressing mammalian clones.
4-[Bis(2-flu or oeth yl)am in o]-O-ben zylph en ol (47). 4-[Bis-
(2′-hydroxyethyl)amino]-O-benzylphenol (1.72 g, 6.0 mmol),
triethylamine (4.3 mL, 31.0 mmol), and 4-(dimethylamino)-
pyridine (0.120 g, 1.0 mmol) were dissolved in CH2Cl2 (40 mL)
followed by methanesulfonic anhydride (4.3 g, 25.0 mmol)
dissolved in CH2Cl2 (40 mL). After 1 h stirring the reaction
mixture was extracted with aqueous citric acid 10% (100 mL)
and the organic layer washed with water (100 mL), dried
(MgSO4), and evaporated to dryness. The residue was redis-
solved in acetonitrile (50 mL), KF (3.8 g, 65 mmol) and 18-
crown-6 ether (0.5 g, 1.9 mmol) were added, and the mixture
stirred at 60 °C for 48 h. The mixture was filtered and the
filtrate evaporated to dryness. Purification was achieved by
preparative HPLC (cyclohexane:CH2Cl2, 2:1) when 47 (1.0 g,
57%) was obtained as an oil: 1H NMR δH 3.60 (2t, 4H, N-CH2,
J ) 5.12 Hz, J H-F ) 22.5 Hz), 4.53 (2t, 4H, CH2F, J H-F ) 47.5
Hz), 5.00 (s, 2H, PhCH2-), 6.72 (d, 2H, Harom2+6, J ) 8.68 Hz),
6.87 (d, 2H, Harom3+5), 7.30-7.45 (m, 5H, Harom); MS m/z 291
(M+, 100), 200 (M+ - PhCH2, 100). Mass (C17H19F2NO) calcd,
291.1435; found, 291.1450. Anal. (C17H19F2NO) C, H, N.
4-[Bis(2-flu or oeth yl)a m in o]p h en ol (21b). 47 (1.0 g, 3.4
mmol) and pentamethylbenzene (3.8 g, 27.7 mmol) were
dissolved in trifluoroacetic acid (25 mL) and stirred for 3 days
at room temperature. TFA was then evaporated and the
residue triturated with hexane. The insoluble oil was parti-
tioned between acetonitrile (80 mL) and hexane (80 mL). The
acetonitrile solution was separated, the solvent evaporated,
and the residue taken in AcOEt (80 mL) and washed with
aqueous NaHCO3 (50 mL) and water (50 mL). The organic
layer was dried (MgSO4) and evaporated to dryness to yield a
dark-yellow oil (0.670 g, 98%): 1H NMR δH 3.54 (2t, 4H, N-CH2,
J ) 5.25 Hz, J H-F ) 25.0 Hz), 4.51 (2t, 4H, CH2F, J H-F ) 47.5
Hz), 6.64 (s, 4H, Harom), 8.62 (s, 1H, OH); MS m/z 201 (M+,
100), 182 (M+ - F, 12). Mass (C10H13F2NO) calcd, 201.0965;
found, 201.0975. Anal. (C10H13F2NO) C, H, N.

Self-Immolative Nitrogen Mustard Prodrugs
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5309
(11) Springer, C. J .; Antoniw, P.; Bagshawe, K. D.; Searle, F.; Bisset,
G. M. F.; J arman, M. Novel prodrugs which are activated to
cytotoxic alkylating agents by carboxypeptidase G2. J . Med.
Chem. 1990, 33, 677-681.
(12) Springer, C. J .; Niculescu-Duvaz, I.; Pedley, R. B. Novel prodrugs
of alkylating agents derived from 2-fluoro- and 3-fluoro benzoic
acids for antibody-directed enzyme prodrug therapy. J . Med.
Chem. 1994, 37, 2361-2370.
Single colonies were cloned by limiting dilution, and those
expressing surface-tethered CPG2 were identified by their
ability to degrade methotrexate28 and by Western blot using
a rabbit anti-CPG2 polyclonal serum. Control cells expressing
â-galactosidase have already been described.15
The cytotoxicity of the compounds was assayed by a modi-
fication of the published procedure.29 Cells (2 × 106) were
seeded into 6-well plates, producing confluent monolayers in
48 h. Compounds were dissolved in DMSO at 10 mM (30) or
100 mM (25, 42, 44) immediately prior to treatment, diluted
in full medium, and added to the wells. A similar concentration
of drug solution was added after an incubation of 1 h, and the
cells were incubated for an additional 20 h. The cells were
harvested and reseeded in quadruplicate in 96-well plates at
∼2 × 103 cells/well and incubated until the control wells
achieved confluence. The plates were fixed and stained with
sulforhodamine-B, the extinction at 590 nm was read, the
results are expressed as percentage of control growth, and the
IC50 values were evaluated by interpolation.
(13) Springer, C. J .; Dowell, R. L.; Burke, P. J .; Hadley, E.; Davies,
D. H.; Blakey, D. C.; Melton, R. G.; Niculescu-Duvaz, I. Opti-
mization of alkylating prodrugs derived from phenol and aniline
mustards:
a new clinical candidate prodrug (ZD2767) for
ADEPT. J . Med. Chem. 1995, 38, 5051-5065.
(14) Dowell, R.; Springer, C. J .; Davies, D. H.; Hadley, E. M.; Burke,
P. J .; Boyle, F. T.; Melton, R. G.; Connors, T. A.; Blakey, D. C.;
Mauger, A. B. New mustards prodrugs for antibody-directed
enzyme prodrug therapy: alternative for the amide link. J . Med.
Chem. 1996, 39, 1100-1105.
(15) Marais, R.; Spooner, R. A.; Stribbling, S. M.; Light, Y.; Martin,
J .; Springer, C. J . S. A cell surface tethered enzyme improves
efficiency in gene-directed enzyme prodrug therapy. Nature
Biotechnol. 1997, 15, 1373-1377.
Ack n ow led gm en t. The authors would like to thank
the Cancer Research Campaign for financial support.
We are grateful for the support from Professors K. R.
Harrap and P. Garland.
(16) Ross, W. C. J . Biological alkylating agents; Butterworth: London,
1962.
(17) Corey, E. J .; Venkateswarlu, A. Protection of hydroxyl groups
as tert-butyltrimethylsilyl derivatives. J . Am. Chem. Soc. 1972,
94, 6190-6191.
(18) Hanessian, S.; Lavallee, P. The preparation and the utility of
tert-butyldiphenylsilyl ethers. Can. J . Chem. 1975, 53, 2975-
2977.
(19) Kruse, C. G.; Wijsman, A.; van der Gen, A. Synthetic applications
of 2-chloro-1,3-dithiane. 2. Reaction with carbon nucleophiles.
J . Org. Chem. 1979, 44, 1847-1851.
(20) Alexander, R. P.; Beeley, N. R. A.; O’Driscoll, M.; O’Neil, F. P.;
Millican, T. A.; Pratt, A. J .; Willenbrock, F. W. Cephalosporin
nitrogen mustard carbamate prodrugs for “ADEPT”. Tetrahedron
Lett. 1991, 32, 3269-3272.
(21) Dubowchik, G. M.; Dalton King, H.; Pham-Kaplita, K. Efficient
mitomycin C coupling with stable p-nitro-benzyl carbonates
using N-hydroxybenzotriazole as a catalytic additive. Tetrahe-
dron Lett. 1997, 38, 5261-5264.
(22) Perrone, E.; Alpegiani, M.; Bedeschi, A.; Zarini, F.; Della Bruna,
C.; Franceschi, G. Penem derivatives. EP 0 236 880 A2, 1987.
(23) Greene, T. W.; Wuts, P. G. M. Protective groups in organic
chemistry; J ohn Wiley & Sons: New York, 1991.
(24) Olofson, R. A.; Cuomo, J . A regioselective and stereospecific route
to enol carbonates and carbamates: closer look at a “naked
anion”. Tetrahedron Lett. 1980, 21, 819-822.
(25) Hollitzer, O.; Seewald, A.; Steglich, W. 4,6-Diphenylthieno[3,4-
d][1,3]dioxol-2-one 5,5-dioxide, a novel activating agent for
peptide synthesis. Angew. Chem., Int. Ed. Engl. 1976, 5, 444-
445.
(26) Schmidt, H.; Hollitzer, O.; Seewald, A.; Steglich, W. Eine
einfache Synthese von Harnstoffe, Urethanen und Isocyanaten.
Chem. Ber. 1979, 112, 727-733.
(27) Palmer, B. D.; Wilson, W. R.; Pullen, S. M.; Denny, W. A.
Hypoxia-selective antitumor agents. 3. Relationships between
structure and cytotoxicity against cultured tumor cells for
substituted N,N-bis(2-chloroethyl)anilines. J . Med. Chem. 1990,
33, 112-121.
(28) Minton, N. P.; Atkinson, T.; Sherwood, R. F. Molecular cloning
of the Pseudomonas carboxypeptidase G2 gene and its expression
in Escherichia coli and Pseudomonas putida. J . Bacteriol. 1983,
156, 1222-1227.
(29) Marais, R.; Spooner, R. A.; Light, Y.; Martin, J .; Springer, C. J .
Gene-directed enzyme prodrug therapy with a mustard prodrug/
carboxypeptidase G2 combination. Cancer Res. 1996, 56,
4735-4742.
Su p p or tin g In for m a tion Ava ila ble: Spectral data for the
final compounds 12, 20, 23, 25, 30, 32, 37, 40, 42, and 44, the
protected linkers 8a ,b and 18a ,b, the activated linkers 9a ,b
and 19a ,b, the protected prodrugs 22, 29, 36, 38, and 39, and
the corresponding fluorinated analogues 24, 31, 41, and 43
(16 pages). Ordering information is given on any current
masthead page.
Refer en ces
(1) J arman, M. The development of anticancer drugs. Chem. Br.
1989, 25, 51-54.
(2) Zhang, W. W.; Fujiwara, T.; Grimm, E. A.; Roth, J . A. Advances
in cancer gene theraphy. Adv. Pharmacol. (San Diego) 1995, 6,
289-341.
(3) Bridgewater, G.; Springer, C. J .; Knox, R.; Minton, N.; Michael,
P.; Collins, M. Expression of the bacterial nitroreductase enzyme
in mammalian cells renders them selectively sensitive to killing
by the prodrug CB1954. Eur. J . Cancer 1995, 31A, 2362-2370.
(4) Huber, B. A.; Richards, C. A.; Krenitsky, T. A. Retroviral-
mediated gene therapy for the treatment of hepatocellular
carcinoma: An innovative approach for cancer therapy. Proc.
Natl. Acad. Sci. U.S.A. 1991, 88, 8039-8043.
(5) Niculescu-Duvaz, I.; Spooner, R. A.; Marais, R.; Springer, C. J .
S. Gene-directed enzyme prodrug therapy. Bioconjugate Chem.
1998, 9, 4-22.
(6) Carl, P. L.; Chakravarty, P. K.; Katzenellenbogen, J . A. A novel
connector linkage applicable in prodrug design. J . Med. Chem.
1981, 24, 479-480.
(7) Wakselman, M. 1,4 and 1,6 eliminations from hydroxy- and
amino-substituted benzyl systems: chemical and biochemical
applications. N. J . Chim. 1983, 7, 439-447.
(8) Schmidt, F.; Florent, J .-C.; Monneret, C.; Straub, R.; Czech, J .;
Gerken, M.; Bosslet, K. Glucuronide prodrugs of hydroxy
compounds for antibody directed enzyme prodrug therapy
(ADEPT): a phenol nitrogen mustard carbamate. Bioorg. Med.
Chem. Lett. 1997, 7, 1071-1076.
(9) Friedlos, F.; Denny, W. A.; Palmer, B. D.; Springer, C. J . S.
Mustard prodrugs for activation by Escherichia coli nitroreduc-
tase in gene-directed enzyme prodrug therapy. J . Med. Chem.
1997, 40, 1270-1275.
(10) Sherwood, R. F.; Melton, R. G.; Alwan, S. M.; Hughes, P.
Purification and properties of carboxypeptidase G2 Pseudonomas
sp strain RS-16. Eur. J . Biochem. 1985, 24, 447-453.
J M980425K