Significant Differences in Biological Parameters between Prodrugs Cleavable by Carboxypeptidase G2 That Generate 3,5-Difluoro-phenol and -aniline Nitrogen Mustards in Gene-Directed Enzyme Prodrug Therapy Systems

Article abstract of DOI:10.1021/jm030966w

Nine new nitrogen mustard compounds derived from 2,6-difluoro-4-hydroxy- (3a-e) and 2,6-difluoro-4-amino- (4a-d) aniline were synthesized as potential prodrugs. They were designed to be activated to their corresponding 3,5-difluorophenol and -aniline (4)-

Full text of DOI:10.1021/jm030966w

J . Med. Chem. 2004, 47, 2651-2658
2651
Sign ifica n t Differ en ces in Biologica l P a r a m eter s betw een P r od r u gs Clea va ble
by Ca r boxyp ep tid a se G2 Th a t Gen er a te 3,5-Diflu or o-p h en ol a n d -a n ilin e
Nitr ogen Mu sta r d s in Gen e-Dir ected En zym e P r od r u g Th er a p y System s
I. Niculescu-Duvaz, I. Scanlon, D. Niculescu-Duvaz, F. Friedlos, J . Martin, R. Marais, and C. J . Springer*
Cancer Research-UK Centre for Cancer Therapeutics at the Institute of Cancer Research, 15 Cotswold Road,
Sutton, Surrey, SM2 5NG, U.K.
Received J uly 22, 2003
Nine new nitrogen mustard compounds derived from 2,6-difluoro-4-hydroxy- (3a -e) and 2,6-
difluoro-4-amino- (4a -d ) aniline were synthesized as potential prodrugs. They were designed
to be activated to their corresponding 3,5-difluorophenol and -aniline (4)-nitrogen mustards
by the enzyme carboxypeptidase G2 (CPG2) in gene-directed enzyme prodrug therapy (GDEPT)
models. The compounds were tested for cytotoxicity in the MDA MB-361 breast adenocarcinoma.
The cell line was engineered to express stably either CPG2 tethered to the cell surface stCPG2-
(Q)3 or â-galactosidase (â-Gal) as control. The cytotoxicity differentials were calculated between
CPG 2-expressing and -nonexpressing cells and yielded different results for the two series of
prodrugs despite their structural similarities. While the phenol compounds are ineffective as
prodrugs, their aniline counterparts exhibit outstanding activity in the tumor cell lines
expressing CPG2. {3,5-Difluoro-4-[bis(2-chloroethyl)amino]phenyl}carbamoyl-L-glutamic acid
gave a differential of >227 in MDA MB361 cells as compared with 19 exhibited by
4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid, 1a , which has been in clinical
trials.
Sch em e 1
In tr od u ction
In order to increase the selectivity of anticancer
agents, targeting strategies such as antibody-directed
enzyme prodrug therapy (ADEPT)1 and gene-directed
enzyme prodrug therapy (GDEPT)2-6 have been devel-
oped. These strategies do not require highly specific
anticancer drugs, as they both utilize prodrugs, which
release highly cytotoxic drugs at the tumor after activa-
tion by an exogenous enzyme expressed in tumor
cells.7-14 The targeting of the genes for the exogenous
enzyme is achieved by using viral or nonviral delivery
vectors. Several enzyme/prodrug systems have been
investigated, and a number of clinical trials using
suicide gene therapy are ongoing.6
A GDEPT system based on the enzyme carboxypep-
tidase G2 (CPG2, glutamate carboxypeptidase EC
3.4.17.11) from Pseudomonas aeruginosa type RS16 has
been developed.15 CPG2 catalyzes the scission of amid-
ic,16 urethanic, or ureidic17,18 linkages between an
aromatic nucleus and L-glutamic acid. The enzyme has
been expressed both intracellularly19 and extracellu-
larly20 in a series of tumor cell lines. The extracellularly
expressed enzyme obviates the need for the prodrug to
enter the tumor cell for activation. This also improves
the bystander effect.20
Ra tion a le of th e Design . 4-[(2-Mesyloxyethyl)(2-
chloroethyl)amino]benzoyl-L-glutamic acid (CMDA, 1a )
and 4-{[bis(2-iodoethyl)amino]phenyl}oxycarbonyl-L-
glutamic acid (ZD2767P, 1b) have both been used in
ADEPT clinical trials. They are substrates for CPG2 (KM
) 2.0-3.4 µM and kcat ) 29.5-583 s-1).11,21 Cleavage of
CMDA by CPG2 releases 4-[bis(2-mesyloxyethyl)(2-
chloroethyl)amino]benzoic acid as the drug.
Further work indicated that two prodrugs related to
CMDA: 3-fluoro-4-[bis(2-chloroethyl)amino]benzoyl-L-
glutamic acid22 and 3,5-difluoro-4-[bis(2-iodoethyl)ami-
no]benzoyl-L-glutamic acid, 223 were superior to CMDA
in terms of cytotoxic differential, potency, and the
bystander effect in vitro and in vivo in a number of
CPG2/GDEPT systems. The reasons for these improve-
ments are unclear.
To further investigate the 3,5-difluorinated analogues
of ZD2767P, 3a -e and the 3,5 difluorinated analogues
* To whom correspondence should be addressed. Tel: 020 8722 4214.
Fax: 020 8643 6902. E-mail: caroline.springer@icr.ac.uk.
10.1021/jm030966w CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/07/2004

2652 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Niculescu-Duvaz et al.
Sch em e 2a
Reagents and conditions: (a) 3,4-Dihydro-2H-pyran, PPTS; (b) imidazolyl carbonate, DCM followed by NaN3 soln; (c) boiling toluene;
(d) di-tert-butyl-L-glutamate, THF, rt; (e) PPTS, EtOH, 65 °C; (f) HCO2NH4, Pd/C, EtOH; (g) ethylene oxide, AcOH; rt; (h) CF3CO2H, rt;
(i) di-tert-butyl-4-nitrophenyloxycarbonyl-L-glutamate, Amberlyst 27; Ad ) adamantanyl.
of the corresponding {4-[bis(2-chloroethyl)amino] phenyl}-
carbamoyl-L-glutamic acid, 4a -d , both series have been
synthesized (see Scheme 3).
This paper describes the synthesis and the physico-
chemical and biological properties of the new fluorinated
potential prodrugs.
Following purification, the 4-[bis(2-hydroxyethyl)-
amino]phenol, 11, underwent transesterification with
di-tert-butyl 4-nitrophenyloxycarbonyl-L-glutamate in
the presence of the strong base ion exchanger, Am-
berlyst 27, to afford the key intermediate, 13 (Z ) O).
The intermediates 12 and 13 were converted into their
alkylating analogues in the usual way (see Scheme
3).25,26 The final deprotection, with formic acid of the
protected prodrugs 14-22, led to the desired nitrogen
mustard prodrugs, 3a -e and 4a -d .
To obtain large amount of aniline prodrugs for in vivo
assessment, a shorter synthetic access route was de-
veloped (Scheme 4) via the nitration of 2,6-difluro-4-
bis(2-hydroxyethyl)aniline, 23. This intermediate was
converted to the corresponding amine, 25, which was
converted to the intermediate, 12 (Z ) NH).
P h ysicoch em ica l a n d Kin etic Da ta . The kinetics
and the chemical half-lives of the prodrugs 3c and 4c
were determined by HPLC (see Experimental Section).
In contrast with {3,5-difluoro-4-[bis(2-chloroethyl)amino]-
phenyl}oxycarbonyloxy-L-glutamic acid, 3c, which is not
a subtrate for CPG2, {3,5-difluoro-4-[bis(2-chloroethyl)-
amino]phenyl}carbamoyl-L-glutamic acid, 4c, proved to
be a good substrate for the enzyme (KM ) 7.7 µM and
kcat ) 29.0 s-1). The chemical half-lives of prodrugs 3a ,
3c, 4a -c are summarized in Table 1.
Cytotoxicity Assa ys. The prodrugs were tested for
cytotoxicity in the line MDA MB 361 breast adenocar-
cinoma. The cell line was engineered to stably express
either CPG2 tethered on the cell surface stCPG2-(Q)3
or â-galactosidase (â-Gal) as the control. For compara-
Resu lts a n d Discu ssion s
Ch em istr y. Nine new potential prodrugs (compounds
3a -e and 4a -d ) derived from 2,6-difluoro-4-amino- and
2,6-difluoro-4-hydroxyaniline nitrogen mustards have
been synthesized.
3,5-Difluoro-4-[bis(2-hydroxyethyl)amino]benzoic acid,
5,23 was used as the starting material for the aniline
series. The compound was protected as 4-bis(2-tetrahy-
dropyranyloxyethyl)aminobenzoic acid, 6, and converted
into the corresponding azide (using diimidazolyl carbon-
ate and NaN3). The intermediate was purified by
preparative HPLC and submitted to the Curtius rear-
rangement (in boiling toluene). The isocyanate formed
was coupled to the di-tert-butyl L-glutamate to obtain
the di-tert-butyl {4-[bis(2-tetrahydropyranyloxyethyl)-
amino]phenyl}carbamoyl-L-glutamate, 8, which after
deprotection gave intermediate 12 (Z ) NH) (see
Scheme 2).
The starting material for the corresponding p-hy-
droxyaniline series was 4-nitro-3,5-difluorophenyloxy-
carbonyl-adamantane, 9.24 After reduction and hydroxy-
ethylation (see Scheme 2), the intermediate 10 was
obtained in satisfactory yield and was deprotected by
exposure to TFA at room temperature.

Prodrugs Cleavable by Carboxypeptidase G2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2653
Sch em e 3a
a
Reagents and conditions: (a) Mes2O, DMAP, DCM, rt; (b) NaI, Me2CO, reflux; (c) LiBr, THF, reflux; (d) NaCl, CH3CN, 60 °C; (e) LiCl,
DMA, rt; (f) TFA; (g) HCO2H.
Sch em e 4a
Ta ble 1. Prodrugs: Half-Lives and Cytotoxicity Differential
between Expressing and Nonexpressing Cell Lines
MDA-MB361
IC50 (µM)
degree of activation
(fold)
half-life
compound
t
1/2 min
â-Gal
3125
2.2
Q3
CMDA
3a
3b
3c
3d
3e
4a
4b
4c
59
6.2
-
45.0
-
165
19
0.9
0.9
2.4
4.0
-
3.6
-
-
19.9
151.9
805
22.5
439.1
15.9
51
0.9
0.3
50.6
19.6
>227
1
-
2.2
0.58
31.0
-
>1000
>2000
>2000
8.8
4d
>2000
In Vivo An titu m or Activity. Compound 4c showed
good antitumor activity in vivo compared to untreated
control tumors grown of the MDA MB 361 engineered
to express stably stCPG2(Q)3 in CD1 nu/ nu mice (see
Experimental Section and Figure 1).
a
Reagents and conditions: (a) AcOH, ethylene oxide; (b) fuming
HNO3, DCM; (c) H2, Pd/C, THF; (d) NEt3, THF.
Discu ssion
tive purposes with benzoic acid mustard prodrugs, the
MDA MB 361 cell line was treated with CMDA (the
clinical ADEPT candidate). The degree of activation,
defined as the ratio of the IC50 of the prodrug in the
â-Gal- and CPG2-expressing cell lines, was calculated
for the new compounds and compared to CMDA (see
Table 1). The phenol prodrugs were inactive against
MDA MB 361 stCPG2(Q)3 cells, compared with 19-fold
activation for CMDA in these cells. The aniline prodrugs
were much more effective than CMDA in CPG2-
expressing cells. {3,5-Difluoro-4[bis(2-chloroethyl)amino]-
phenyl}carbamoyl-L-glutamic acid, 4c, exhibited a very
high differential of >227-fold in this system. The
compound was chosen as a candidate for further devel-
opment.
Previous data have shown that mono-22 and difluoro-
analogues of CMDA23 exhibit improved cytotoxic and
antitumor activity. To compare this series with the
corresponding phenyloxycarbonyl- and phenylcarbam-
oyl-nitrogen mustards the new derivatives 3a -e and
4a -d have now been synthesized. These prodrugs
release nitrogen mustard drugs after cleavage by CPG2.
The drugs are more cytotoxic than the nitrogen mus-
tards derived from 3,5-difluoro-4-aminobenzoic acid.
The use of I and Br instead of Cl as leaving groups in
nitrogen mustards series generally leads to drugs with
shorter half-lives and increased potency (IC50 ) 0.5-
2.7 µM).11,27 As a general observation, carbamoyl-
prodrugs are more effective than their oxycarbonyl-
prodrug counterparts. This occurs despite the improved

2654 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Niculescu-Duvaz et al.
system, but with a resolution set to 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 Bruker AC250 spectrometer (250 MHz) at 30 °C (303 K)
unless otherwise stated. IR spectra (film) were recorded on a
Perkin-Elmer 1720X FT-IR spectrometer. Elemental analyses
were determined by Butterworth Laboratories Ltd. (Tedding-
ton, Middlesex, UK) and are within 0.4% of theory except when
stated. The chemical stability of the prodrugs and their
propensity to behave as substrate for CPG2 were determined
by HPLC. Preparative HPLC purifications were performed on
an Axial Chromatospac Prep 10 (J obin-Yvon) using Merck
Kieselgel 60 (0.015-0.040) (Art. 15,111).
3,5-Diflu or o-4-[bis(2-h ydr oxyeth yl)am in o]ben zoic Acid
(5). A solution of 3,4,5-trifluorobenzonitrile (5.0 g, 0.032 mmol)
and diethanolamine (9.0 mL, 0.94 mmol) in DMA (40 mL) was
heated a 100 °C in the CEM Discover series microwave for 1
h at 30 W power. After solvent removal, the residue was
redissolved in AcOEt (100 mL), extracted with water (2 × 100
mL), dried, and evaporated under vacuum. After purification
by preparative HPLC (DCM:EtOH, 95:5), 4-[bis(2-hydroxy-
ethyl)amino]benzonitrile was obtained (4.15 g, 53%) as a white
solid (48 h reaction time and 35% yield reported by standard
heating).23 The hydrolysis of this compound by reflux in NaOH/
ethanol/water solution for 3 h led to the corresponding 3,5-
difluorobenzoic acid 5 (mp 147-149 °C).23
3,5-Diflu or o-4-[bis(2-tetr a h yd r op yr a n yloxyeth yl)a m i-
n o]b en zoic Acid (6). To a stirred solution of 4-[bis(2-
hydroxyethyl)amino]-3,5-difluorobenzoic acid (1.83 g, 7.0 mmol)
and 3,4-dihydro-2H-pyran (1.60 mL, 1.47 g, 17.50 mmol) in
DCM (100 mL) was added pyridinium-p-toluenesulfonate (1
g, 4.0 mmol), and the reaction mixture was refluxed for 3 h.
The mixture was stirred for an additional 18 h at room
temperature, washed with water (2 × 100 mL), dried (MgSO4),
and evaporated under vacuum. The product resulted as an oil
(2.86 g, 95.2%) and was purified by HPLC using cyclohexane:
AcOEt 1:1 as eluent. A solid resulted: mp 70-2 °C. νmax/cm-1
(film): 3081 (OH), 2944, 2872 (CH2), 1721 (CO, acid); 1H NMR,
F igu r e 1. Tumor growth in vivo in CD1 nu/nu mice, following
treatment with prodrug 4c versus control group. All tumors
100% stCPG(Q)3-expressing MDA MB 361 cells. Controls
injected with 5% DMSO in water. Points shown are mean
tumor volumes over the whole group.
kinetics (kcat and KM) of the oxycarbonyl linker itself.11,26
The differential obtained in tumor cells expressing
stCPG2(Q)3 appears to be dependent on optimal chemi-
cal reactivity of the prodrugs and their corresponding
drugs, despite the relatively low kcat for the new pro-
drugs. This is in good agreement with previous observa-
tions of the in vitro and in vivo behavior of the benzoic
acid mustard prodrugs in CPG2-based GDEPT sys-
tems.23
A suprising difference in activity is demonstrated
between phenols (3a -e) and aniline (4a -d ) compounds.
As shown in Table 1, the phenol prodrugs are not
substrates for CPG2. The reason for this behavior is
unclear since the similar nonfluorinated prodrug ZD
2767P is a reasonable substrate for the enzyme and the
corresponding difluorinated nitrogen mustard 2 is an
excellent substrate for CPG2.23 A difference in the
positioning of the difluoro-prodrugs in the active site of
CPG2 may explain these results.
δ
H 1.28-1.55 (m, 12H, H3′+H4′+H5′), 3.32-3.45 (m, 8H, N(CH2-
CH2OTHP)2), 3.57-3.71 (m, 4H, H6′), 4.49 (s, 2H, H2′), 7.45
(d, 2H, Harom2+6, J H-F ) 10.36 Hz); MS (FAB), m/z: 452 (M+
23, 100); acc mass: (C21H29F2NO6Na) calcd 452.1861, found
452.1850. Anal. (C21H29F2NO6) C, H, N.
+
3,5-Diflu or o-4-[bis(2-tetr a h yd r op yr a n yloxyeth yl)a m i-
n o]ben zoyl Azid e (7). To a solution containing the acid 6
(11.8 g, 27.5 mmol) in 200 mL of dry THF was added the
imidazolyl carbonate (4.70 g, 29.0 mmol) under stirring and
nitrogen at room temperature. After 1 h a solution of 5 M NaN3
(12.5 g of NaN3 in 30 mL of H2O) was added, and the reaction
mixture stirred for an additional 3 h. The reaction mixture
was poured into 1.0 L of H2O and extracted with ether (2 × 1
L), dried, and evaporated to an oily residue. This was redis-
solved in dry benzene (2 × 100 mL) and evaporated each time
under vacuum. The final purification was carried out by HPLC
Con clu sion s
Nine new potential prodrugs belonging to the 3,5-
difluorophenol- and 3,5-difluoroaniline-nitrogen mus-
tard series are synthesized and studied. Compounds
3a -e are shown not to act as substrates for CPG2,
whereas 4a -d are excellent prodrugs for CPG2, show-
ing activity in tumor cell lines engineered to express
stCPG2(Q)3. The {3,5-difluoro-4[bis(2-chloroethyl)amino]-
phenyl}carbamoyl-L-glutamic acid, 4c, is the most ef-
fective prodrug on MDA MB361 cell line expressing
stCPG2(Q)3 in vitro, and further studies in vivo show
that it is significant at reducing the rate of tumor
growth compared to control (see Figure 1).
(cyclohexane:AcOEt, 2:1) to give an oil (11.05 g, 88.4%). νmax
/
cm-1 (film); 2943, 2871 (CH2), 2146 (CO, azide); H NMR, δH
1.33-1.55 (m, 12H, 4×H3′+4×H4′+4×H5′), 3.32-3.44 (m, 8H,
N(CH2CH2OTHP)2), 3.60-3.70 (m, 4H, 4×H6′), 4.47 (s, 2H,
2×H2′), 7.45 (d, 2H, Harom2+6, J H-F ) 10.36 Hz); MS (FAB),
m/z: 455 (M+ + 1, 55), 412 (M+ - N3, 45); acc mass:
(C21H29F2N4O5)calcd455.2106,found455.2120.Anal.(C21H28F2N4O5)
C, H.
1
Exp er im en ta l Section
All starting materials, reagents, and anhydrous solvents
(i.e., THF packed under N2) were purchased from Aldrich,
unless otherwise stated. Kieselgel 60 (0.043-0.060) was used
in gravity columns (Art 9385 and 15111, Merck). TLC was
performed on precoated sheets of Kieselgel 60 F254 (Art 5735,
Merck). Melting points were determinated on a Kofler hot-
stage (Reichert Thermovar) melting point apparatus and are
uncorrected. Low resolution EI and FAB mass spectra were
performed on a VG-2AB-SE double focusing magnetic sector
mass spectrometer (Fisons Instruments, Warrington, Manches-
Di-ter t-b u t yl {3,5-Diflu or o-4-[b is(2-t et r a h yd r op yr a n -
yloxyeth yl)am in o]ph en yl}car bam oyl-L-glu tam ate (8). The
azide 7 (2.80 g, 6.53 mmol) was heated in 150 mL of dry
toluene and submitted to condensation without further puri-
fication. The reaction completion was monitored by IR (ν )
2146 cm-1). The solvent was evaporated under vacuum and
the residue dissolved in 10 mL of THF. The clear solution
obtained from di-tert-butyl-L-glutamate‚HCl (1.70 g, 6.5 mmol)
and NEt3 (0.97 mL, 7.0 mmol) in 50 mL of THF, after filtration,
was added dropwise (15 min) at room temperature with
stirring to the solution containing the previously prepared
ter, UK), operating at
resolution accurate mass spectra were determined on the same
a resolution of 1000 amu. High-

Prodrugs Cleavable by Carboxypeptidase G2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2655
isocyanate. The reaction was finished in 30 min (disappearance
of the ν ) 2268 cm-1). The solvent was evaporated, the residue
dissolved in AcOEt (50 mL), washed with H2O (2 × 50 mL),
and evaporated under vacuum. A thick oil was obtained which
after HPLC (cyclohexane:AcOEt 2.5:1) gave (4.22 g, 94.3%) of
8 (oil). νmax/cm-1 (film); 2942, 2872 (CH2), 1731 (CO, ester); 1H
NMR, δH 1.38-1.63 (m, 30H, t-Bu: 18H; 2×H3′+2×H4′+-
2×H5′: 12H), 1.71-1.96 (2 × m, 2H, CH2CHNH), 2.18-2.33
(m, 2H, CH2CO2tBu), 3.20-3.30 (m, 8H, N(CH2CH2OTHP)2),
3.57-3.68 (m, 4H, H6′), 4.10-4.16 (m, 1H, CH-G), 4.49 (s, 2H,
Di-ter t-bu tyl {3,5-Diflu or o-4-[bis(2-h ydr oxyeth yl)am in o]-
p h en yl}oxyca r bon yl-L-glu ta m a te (12). To a stirred solution
containing the phenol 11 (1.50 g, 5.07 mmol) and di-tert-butyl
4-nitrophenyloxycarbonyl-L-glutamate (2.26 g, 5.3 mmol) in
THF (60 mL) was added 5.0 g Amberlyst 27 (OH-) at once.
The reaction mixture was kept at room temperature for 3.5 h.
The resin was filtered, the solvent evaporated under vacuum,
and the residue redissoved in AcOEt (20 mL). The solution
was washed (2 × 25 mL of H2O), dried, and evaporated again,
giving an oil. After purification by column chromatography
1
H2′), 6.57 (d, 1H, NH-G, J ) 8.02 Hz), 7.04 (d, 2H, Harom2+6
,
(AcOEt), a solid resulted (1.28 g, 48.8%), mp 109-110 °C. H
J H-F ) 11.23 Hz), 8.79 (s, 1H, NHPh); MS (FAB), m/z: 708
(M+ + 23, 26), 685 (M, 25); acc mass: (C34H53F2N3O9) calcd
685.3750, found 685.3733. Anal. (C34H53F2N3O9) C, H, N.
NMR, δH 1.40 (s, 9H, t-Bu), 1.42 (s, 9H, t-Bu), 1.93-2.08 (m,
2H, CH2CHNH), 2.33 (t, 2H, CH2CO2, J ) 7.99 Hz), 3.13 (t,
4H, NCH2, J ) 6.37 Hz), 3.41 (q, 4H, CH2OH, J ) 6.12 Hz),
3.93-3.99 (m, 1H, CHNH), 4.44 (t, 2H, OH, J ) 5.19 Hz), 6.87
(d, 2H, Harom2+6, J H-F ) 9.80 Hz), 8.24 (d, 1H, NH-G, J ) 7.80
Hz); MS (FAB), m/z: 540 (M+ + 23, 98), 518 (M+ + 1,100); acc
mass: (C24H38F2N3O7) calcd 518.2678, found 518.2656. Anal.
(C24H38F2N3O7) C, H, F, N.
Di-ter t-bu tyl {3,5-Diflu or o-4-[N,N-bis(2-h yd r oxyeth yl)-
a m in o]p h en yl}ca r ba m oyl-L-glu ta m a te (13). Pyridinium-
p-toluene sulfonate (0.17 g, 0.67 mmol) was added to a stirred
solution of 8 (2.30 g, 3.35 mmol) in 100 mL of EtOH. The
reaction mixture was heated for 3 h at 65 °C, the solvent
evaporated, and the residue dissolved in AcOEt (100 mL). The
solution was washed with H2O (2 × 100 mL), dried (MgSO4),
and evaporated under vacuum giving an oil (1.53 g, 88.6%)
Di-ter t-b u t yl {3,5-Diflu or o-4-[b is(2-m esyloxyet h yl)-
a m in o]p h en yl}oxyca r bon yl-L-glu ta m a te (14). To a solu-
tion of compound 12 (0.330 g, 0.64 mmol) in 30 mL of DCM
were added mesyl anhydride (0.334 g, 1.92 mmol), DMAP
(0.033 g), and triethylamine (0.313 mL, 2.25 mmol) under
stirring at room temperature. The reaction mixture was stirred
for 2 h, and then the solution washed with citric acid 10% (30
mL), dried, and evaporated under vacuum. After HPLC
purification (cyclohexane:AcOEt, 1:1), a clear oil was obtained
1
which crystallizes on standing mp 109-110 °C. H NMR, δH
1.18 (s, 18H, t-Bu), 1.65-1.99 (2×m, 2H, CH2NH-G), 2.23-
2.34 (m, 2H, CH2CO2H), 3.07 (t, 4H, NCH2, J ) 6.25 Hz), 3.37
(q, 4H, CH2OH), 4.39 (t, 2H, OH, J ) 5.37 Hz), 6.58 (d, 1H,
NH-G, J ) 8.04 Hz), 7.05 (d, 2H, Harom2+6, J m ) 4.38 Hz, J H-F
) 15.62 Hz), 8.81 (s, 1H, NHPh); MS (FAB), m/z: 540 (M+
+
23, 100), 518 (M+ + 1, 40); acc mass: (C24H38F2N3O7) calcd
1
(0.290 g, 67.2%). H NMR, δH 1.40 (s, 9H, t-Bu), 1.42 (s, 9H,
518.2678, found 518.2664. Anal. (C24H38F2N3O7) C, H, F, N.
t-Bu), 1.72-2.05 (m, 2H, CH2CHNH), 2.34 (t, 2H, CH2CO2, J
) 8.24 Hz), 3.10 (s, 6H, CH3SO2), 3.45 (t, 4H, NCH2, J ) 5.35
Hz), 3.90-4.05 (m, 1H, CHNH), 4.20 (t, 4H, CH2OSO2CH3, J
) 6.12 Hz), 6.94 (d, 2H, Harom2+6, J H-F ) 9.52 Hz), 8.26 (d, 1H,
NH-G, J ) 7.89 Hz); MS (FAB), m/z: 697 (M+ + 23, 18), 675
(M+ + 1, 22); acc mass: (C26H41F2N2O12S2) calcd 675.2042,
found 675.2069. Anal. (C26H40F2N2O12S2) C, H, F, N, S.
4-Nitr o-3,5-diflu or oph en yloxycar bon yladam an tan e (9).
Compound 9 was prepared starting from 4-nitro-3,5-difluo-
rophenol (10 g, 57.1 mmol).24 After purification by column
chromatography, a solid was obtained (14.90 g, 73.9%). Re-
crystallized from MeOH aq 95%, mp 74-75 °C. νmax/cm-1 (film);
2916, 2856 (CH2), 1766 (CO, carbonate) 1544, 1347 (NO2); 1H
NMR, δH 1.64 (s, 6H, CH2, adam), 2.12 (s, 6H, CH2, adam), 2.20 (s,
Di-ter t-bu tyl {3,5-d iflu or o-4-[bis(2-m esyloxyeth yl)a m -
in o]p h en yl}ca r ba m oyl-L-glu ta m a te (15) was prepared us-
ing the same procedure as for compound 14. Starting from
intermediate 13 (1.28 g, 2.47 mmol) and after purification by
HPLC (cyclohexane:AcOEt, 1:1), a solid resulted (1.00 g,
3H, CH, adam), 7.60 (dd, 2H, Harom2+6, J H-F ) 11.23 Hz, J m
)
2.01 Hz); MS (FAB), m/z: 376 (M+ + 23, 17), 354 (M+ + 1,
52), 309 (M+ - CO2 + 1,100); acc mass: (C17H17F2NO5Na) calcd
376.0972, found 376.0950. Anal. (C17H17F2NO5) C, H, N.
{3,5-Diflu or o-4-[b is(2-h yd r oxyet h yl)a m in o]p h en yl}-
oxyca r bon yla d a m a n ta n e (10). 9 (14.34 g, 40.6 mmol) was
submitted to reduction by hydrogen transfer using 2 g of Pd/C
10% and finely ground ammonium formate (18.9 g, 300 mmol)
in 200 mL of EtOH under vigorous stirring. The amine
obtained, after a conventional workup, as a red oil (13.8 g)
was submitted without purification to hydroxyethylation. The
compound was dissolved in 300 mL of AcOH, ethylene oxide
(17.6 mL, 400 mM) was added at room temperature, and the
reaction mixture was stirred for 48 h. The solvent was
evaporated under vacuum and the oily residue washed with
NaHCO3 sol. 5% (3 × 100 mL) and water, dried (MgSO4), and
evaporated again. An oil resulted, which was purified by HPLC
(AcOEt), and then a solid resulted: 6.91 g (41.3% with respect
to 9). Recrystallized from n-hexane:AcOEt, 20:7, mp 78-9 °C.
1H NMR, δH 1.63 (s, 6H, CH2, adam), 2.10 (s, 6H, CH2, adam), 2.18
(s, 3H, CH, adam), 3.14 (t, 4H, NCH2, J ) 6.27 Hz), 3.41 (q, 4H,
1
59.8%), mp 61-63 °C. H NMR, δH 1.39 (s, 9H, t-Bu), 1.42 (s,
9H, t-Bu), 1.72-2.08 (m, 2H, CH2CHNH), 2.23-2.32 (m, 2H,
CH2CO2), 3.11 (s, 6H, CH3SO2), 3.35-3.41 (m, 4H, NCH2), 4.16
(t, 4H, CH2OSO2CH3, J ) 5.44 Hz), 6.61 (d, 1H, NH-G, J )
8.19 Hz), 7.10 (dd, 2H, Harom2+6, J H-F ) 14.97 Hz, J m ) 3.77
Hz), 8.88 (s, 1H, NHPh); MS (FAB), m/z: 696 (M+ + 23, 5),
674 (M+ + 1, 14); acc mass: (C26H42F2N3O11S2) calcd 673.2151,
found 673.2176. Anal. (C26H41F2N3O11S2) C, H, F, N.
Di-ter t-bu tyl {3,5-Diflu or o-4-[bis(2-iod oeth yl)a m in o]-
p h en yl}oxyca r bon yl-L-glu ta m a te (16). To a solution of
compound 14 (0.200 g, 0.30 mmol) in 30 mL of acetone was
added sodium iodide (0.674 g, 10 equiv) under stirring, and
the solution was refluxed for 5 h. The solvent was evaporated,
the residue redissolved in AcOEt (25 mL), washed (2 × 25 mL
H2O), dried and evaporated again. An oil resulted which was
purified by HPLC (cyclohexane:AcOEt, 3:1) leading to a solid
1
(0.146 g, 75.6%) mp 46-8 °C. H NMR, δH 1.40 (s, 9H, t-Bu),
CH2OH), 4.42 (t, 2H, OH, J ) 5.30 Hz), 7.02 (d, 2H, Harom2+6
,
-
J H-F ) 9.86 Hz); MS (FAB), m/z: 412 (M+ + 1, 8), 336 (M+
1.42 (s, 9H, t-Bu), 1.72-2.07 (m, 2H, CH2CHNH), 2.33 (t, 2H,
CH2CO2, J ) 7.96 Hz), 3.22 (t, 4H, NCH2), 3.44 (t, 4H, CH2I,
CO2 + 1, 16); acc mass: (C21H28F2NO5) calcd 412.1936, found
412.1940. Anal. (C21H28F2NO5) C, H, F, N.
J ) 7.16 Hz), 3.92-4.06 (m, 1H, CHNH), 6.94 (d, 2H, Harom2+6
,
J H-F ) 9.69 Hz), 8.26 (d, 1H, NH-G, J ) 7.77 Hz); MS (FAB),
m/z: 739 (M+ + 1, 18); acc mass: (C24H35F2I2N2O6) calcd
739.0553, found 739.0500. Anal. (C24H34F2I2N2O6) C, H, F, N.
4-[Bis(2-h yd r oxyeth yl)a m in o]-3,5-d iflu or op h en ol (11).
Compound 10 (2.0 g, 4.9 mmol) was deprotected with TFA (150
mL) by stirring for 2 h at room temperature. The solvent was
evaporated under vacuum, and then the residue was redis-
solved in 20 mL of ACOEt and evaporated again (five times).
A clear oil (3.07 g) was obtained which was submitted to HPLC
(AcOEt). After purification, an oil was obtained (0.86 g, 75.5%)
1H NMR, δH 3.02 (t, 4H, NCH2, J ) 5.58 Hz), 3.35 (t, 4H, CH2-
OH), 4.39 (s, broad, 2H, OH), 6.39 (d, 2H, Harom2+6, J H-F ) 9.86
Di-ter t-bu tyl {3,5-Diflu or o-4-[bis(2-iod oeth yl)a m in o]-
p h en yl}ca r ba m oyl-L-glu ta m a te (17). The same procedure
was used to obtain 17 (0.186 g, 67.6%) starting from the
intermediate 15 (0.250 g, 0.37 mmol). After HPLC purification,
a thick oil resulted. 1H NMR, δH 1.40 (s, 9H, t-Bu), 1.42 (s,
9H, t-Bu), 1.69-1.99 (m, 2H, CH2CHNH), 2.24-2.30 (m, 2H,
CH2CO2), 3.17 (t, 4H, NCH2), 3.38 (t, 4H, CH2Br, J ) 7.21 Hz),
4.10-4.17 (m, 1H, CHNH), 6.60 (d, 1H, NH-G, J ) 7.97 Hz),
7.10 (d, 2H, Harom2+6, J H-F ) 11.37 Hz), 8.87 (s, 1H, NHPh);
Hz), 10.05 (s, broad, 1H, OHphenol); MS (FAB), m/z: 256 (M+
+
23, 100), 234 (M+ + 1, 72); acc mass: (C10H14F2NO3) calcd
234.0942, found 234.0936. Anal. (C10H13F2NO3) C, H, N.

2656 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Niculescu-Duvaz et al.
MS (FAB), m/z: 760 (M+ + 23, 25), 737 (M+, 100); acc mass:
(C) calcd 737.0634, found 737.0598. Anal. (C24H35Br2F2N3O5)
C, H, N, F,
{3,5-D i flu o r o -4-[b i s (2-i o d o e t h y l)a m i n o ]p h e n y l}-
oxyca r bon yl-L-glu ta m ic Acid (3a ). Compound 16 (0.069 g,
0.09 mmol) was deprotected at room temperature by stirring
24 h, in 5.0 mL of formic acid. After evaporation of the solvent
under vacuum, the residue was redissolved 3 × 5.0 mL of
AcOEt and 3 × 5.0 mL of DCM and evaporated each time. A
Di-ter t-bu tyl {3,5-Diflu or o-4-[bis(2-br om oeth yl)a m in o]-
p h en yl}oxyca r bon yl-L-glu ta m a te (18). To a solution of
compound 14 (0.197 g, 0.30 mmol) in 25 mL of THF was added
lithium bromide (0.260 g, 10 equiv) under stirring, and the
solution was refluxed for 5 h. After a standard workup, an oil
resulted (0.201 g) which was purified by HPLC (cyclohexane:
AcOEt, 3:1) leading to a foam (0.122 g, 63.1%). 1H NMR, δH
1.40 (s, 9H, t-Bu), 1.42 (s, 9H, t-Bu), 1.72-2.04 (m, 2H, CH2-
CHNH), 2.34 (t, 2H, CH2CO2, J ) 8.24 Hz), 3.50 (s, 8H, N(CH2-
CH2Br)2), 3.92-4.06 (m, 1H, CHNH), 6.94 (d, 2H, Harom2+6, J H-F
) 9.61 Hz), 8.26 (d, 1H, NH-G, J ) 7.75 Hz); MS (FAB), m/z:
645 (M+ + 1, 35); acc mass: (C24H35Br2F2I2N2O6) calcd
1
white solid resulted (0.052 g, 92.3%), mp 38-40 °C. H NMR,
δH 1.90-2.10 (2×m, 2H, CH2CHNH), 2.36 (t, 2H, CH2CO2, J
) 7.91 Hz), 3.21 (t, 4H, NCH2, J ) 7.20 Hz), 3.45 (t, 4H, CH2I),
3.98-4.07 (m, 1H, CHNH), 6.95 (d, 2H, Harom2+6, J H-F ) 9.72
Hz), 8.22 (d, 1H, NH-G, J ) 7.95 Hz); MS (FAB), m/z: 649
(M+ + 23, 38), 627 (M+ + 1, 64), 499 (M+ - I + 1, 52); acc
mass: (C16H19I2F2N2O6) calcd 626.9301, found 626.9281. Anal.
(C16H18I2F2N2O6) C, H, N.
Using a similar procedure, the following prodrugs were
prepared:
643.0830, found 643.0801. Anal. (C24H35Br2F2N2O6.0.1C6H12
C, H, N.
)
{3,5-Diflu or o-4-[b is(2-b r om oe t h yl)a m in o]p h e n yl}-
oxyca r bon yl-L-glu ta m ic a cid (3b): except toluene 3 × 5.0
mL was used instead of the AcOEt in order to remove formic
acid (0.041 g, 86.5%), mp 97-8 °C. 1H NMR, δH 1.81-2.07
(2×m, 2H, CH2CHNH), 2.36 (t, 2H, CH2CO2, J ) 7.79 Hz),
3.50 (s, 8H, N(CH2CH2Br)2), 4.00-4.07 (m, 1H, CHNH), 6.95
(d, 2H, Harom2+6, J H-F ) 9.63 Hz), 8.24 (d, 1H, NH-G, J ) 7.95
Hz); MS (FAB), m/z: 555 (M+ + 23, 17), 533 (M+ + 1, 62), 499
(M+ - I + 1, 52); acc mass: (C16H19Br2F2N2O6) calcd 530.9578,
found 530.9569. Anal. (C16H18Br2F2N2O6) C, H, N:calc 5.22,
found 4.78.
Di-ter t-bu tyl {3,5-Diflu or o-4-[bis(2-br om oeth yl)a m in o]-
p h en yl}ca r ba m oyl-L-glu ta m a te (19). Starting from the
intermediate 15 (0.250 g, 0.37 mmol), the same procedure as
for 18 was used to obtain 19 (0.180 g, 75.7%). After HPLC
1
purification, a foam resulted. H NMR, δH 1.39 (s, 9H, t-Bu),
1.41 (s, 9H, t-Bu), 1.71-1.98 (2m, 2H, CH2CHNH), 2.21 (m,
2H, CH2CO2), 3.37 (d, 4H, CH2N, J ) 6.43), 3.56 (t, 4H, CH2-
Br), 4.09-4.15 (m, 1H, CHNH-G), 6.60 (d, 1H, NH-G, J ) 8.03
Hz), 7.08 (dd, 2H, Harom2+6, J H-F ) 11.29 Hz), 8.85 (s, 1H,
NHPh); MS (FAB), m/z: 666 (M+ + 23, 12), 643 (M+ + 1, 56);
acc mass: (C24H35Br2F2N3O5) calcd 641.0912, found 641.0900.
Anal. (C24H35Br2F2N3O5) C, H, F,
{3,5-Diflu or o-4-[b is(2-ch lor oe t h yl)a m in o]p h e n yl}-
oxyca r bon yl-L-glu ta m ic a cid (3c): 0.063 g, 98.0%, glassy
1
solid. H NMR, δH 1.82-2.10 (2×m, 2H, CH2CHNH), 2.36 (t,
Di-ter t-bu tyl {3,5-Diflu or o-4-[bis(2-ch lor oeth yl)a m in o]-
p h en yl}oxyca r bon yl-L-glu ta m a te (20) a n d Di-ter t-bu tyl
{3,5-Diflu or o-4-[(2-ch lor oeth yl)(2-m esyloxyeth yl)a m in o]-
p h en yl}oxyca r bon yl-L-glu ta m a te (22). To a solution of
compound 14 (0.214 g, 0.31 mmol) in 25 mL of acetonitrile was
added sodium chloride (0.175 g, 10 equiv) under stirring, and
the solution was heated at 65 °C (bath) for 48 h. After solvent
evaporation and workup, an oil resulted (0.196 g) which was
separated and purified by HPLC (cyclohexane:AcOEt, 3:1). The
first fraction was compound 20, which was obtained as an oil
2H, CH2CO2, J ) 7.37 Hz), 3.44 (d, 4H, NCH2, J ) 6.28 Hz),
3.61 (t, 2H, CH2Cl), 4.00-4.12 (m, 1H, CHNH), 6.95 (d, 2H,
H
arom2+6, J H-F ) 9.64 Hz), 8.25 (d, 1H, NH-G, J ) 7.85 Hz);
MS (FAB), m/z: 444 (M+ + 1, 62); acc mass: (C16H19Cl2F2N2O6)
calcd 443.0588, found 443.0588. Anal. (C16H18Cl2F2N2O6) C, H,
N: calcd 6.32, found 5.80.
{3,5-D i flu o r o -4-[b i s (2-i o d o e t h y l)a m i n o ]p h e n y l}-
ca r ba m oyl-L-glu ta m ic a cid (4a ): except toluene 3 × 5.0 mL
was used instead of the AcOEt in order to remove formic acid
1
1
(0.112 g, 94.7%), mp 78-80 °C. H NMR, δH 1.75-2.05 (2×m,
(0.102 g, 59.5%). H NMR, δH 1.40 (s, 9H, t-Bu), 1.42 (s, 9H,
2H, CH2CHNH), 2.28 (t, 2H, CH2CO2, J ) 7.98 Hz), 3.17 (t,
4H, NCH2, J ) 7.20 Hz), 3.38 (t, 4H, CH2I), 4.04-4.23 (m, 1H,
t-Bu), 1.79-2.04 (2×m, 2H, CH2CHNH), 2.33 (t, 2H, CH2CO2,
J ) 7.88 Hz), 3.45 (t, 4H, NCH2, J ) 6.28 Hz), 3.61 (t, 4H,
CH2Cl), 3.93-4.02 (m, 1H, CHNH), 6.94 (d, 2H, Harom2+6, J H-F
) 9.64 Hz), 8.27 (d, 1H, NH-G, J ) 7.75 Hz); MS (FAB), m/z:
577 (M+ + 23, 13), 555 (M+ + 1, 45), 519 (M+ - Cl + 1, 22);
acc mass: (C24H35Cl2F2N2O6) calcd 555.1840, found 555.1842.
Anal. (C24H34Br2F2N2O6) C, H, Cl, N.
CHNH), 6.61 (d, 2H, NH-G, J ) 7.93 Hz), 7.10 (d, 2H, Harom2+6
,
J H-F ) 11.35 Hz), 8.91 (s, 1H, NH-G); MS (FAB), m/z: 626
(M+ + 1, 64); acc mass: (C16H20I2F2N3O5) calcd 625.9461, found
625.9447. Anal. (C16H19I2F2N3O5) C, H, N.
{3,5-Diflu or o-4-[b is(2-b r om oxyet h yl)a m in o]p h en yl}-
ca r ba m oyl-L-glu ta m ic a cid (4b): 0.080 g, 93.7%, mp 81-3
The second fraction was compound 22, resulting also as an
oil (0.032 g, 16.8%). In another experiment a yield of 21.7%
was achieved. 1H NMR, δH 1.40 (s, 9H, t-Bu), 1.42 (s, 9H, t-Bu),
1.75-1.98 (2×m, 2H, CH2CHNH), 2.34 (t, 2H, CH2CO2, J )
7.87 Hz), 3,31 (s, 3H, CH3SO2-), 3.41-3.45 (m, 4H, NCH2), 3.61
(t, 2H, CH2Cl, J ) 6.27 Hz), 3.93-4.02 (m, 1H, CHNH), 4.20
(t, 2H, CH2OSO2CH3, J ) 5.30 Hz), 6.94 (d, 2H, Harom2+6, J H-F
) 9.60 Hz), 8.26 (d, 1H, NH-G, J ) 7.75 Hz); MS (FAB), m/z:
637 (M+ + 23, 24), 615 (M+ + 1, 35), 579 (M+ - Cl + 1, 9), 519
(M+ - Mes + 1, 12); acc mass: (C25H38ClF2N2O9S) calcd
615.1955, found 615.1942. Anal. (C25H38ClF2N2O9S) C, H, N,
S.
1
°C. H NMR, δH 1.75-2.08 (2×m, 2H, CH2CHNH), 2.25-2.35
(m, 2H, CH2CO2), 3.44 (s, 8H, N(CH2CH2Br)2), 4.11-4.21 (m,
1H, CHNH), 6.62 (d, 1H, NH-G, J ) 7.95 Hz), 7.10 (d, 2H,
H
arom2+6, J H-F ) 11.29 Hz), 8.1 (s, 1H, NHPh); MS (FAB), m/z:
532 (M+ + 1, 50), 452 (M+ - Br + 1, 12); acc mass: (C16H20
Br2F2N3O5) calcd 529.9738, found 529.9732. Anal. (C16H19
Br2F2N2O6) C, H, N.
-
-
{3,5-Diflu or o-4-[b is(2-ch lor oe t h yl)a m in o]p h e n yl}-
ca r ba m oyl-L-glu ta m ic a cid (4c): 0.079 g, 94.0%, mp 74-6
°C. 1H NMR, δH 1.76-2.05 (2×m, 2H, CH2CHNH), 2.28 (t, 2H,
CH2CO2, J ) 6.11 Hz), 3.38 (d, 4H, NCH2, J ) 6.44 Hz), 3.57
(t, 2H, CH2Cl), 4.14-4.23 (m, 1H, CHNH), 6.62 (d, 1H, NH-G,
J ) 7.94 Hz), 7.10 (d, 2H, Harom2+6, J H-F ) 11.32 Hz), 8.91 (s,
1H, NHPh); MS (FAB), m/z: 464 (M+ + 23, 11), 442 (M+ + 1,
100); acc mass: (C16H20Cl2F2N2O6) calcd 442.0748, found
442.0757. Anal. (C16H19Cl2F2N3O5) C, H, N: calcd 9.50, found
8.91.
2,6-Diflu or o-[bis(2-h yd r oxyeth yl)]a n ilin e (23). 2,6-Di-
fluoroaniline 22 (6.45 g, 50 mmol) was dissolved in AcOH (400
mL), and ethylene oxide (22.5 mL) was added. The reaction
was stirred at room temperature for 3 days. The solvent was
evaporated, first under water vacuum then under pump
vacuum, to afford 2 (12.0 g) as an oil, still containing ethylene
glycol monoacetate as an impurity. This mixture was used
crude, without further purification, in the next step. 1H NMR,
δH, (ppm): 3.16 (t, 4H, N(CH2CH2OH)2, J ) 6.33 Hz), 3.37-
Di-ter t-bu tyl {3,5-Diflu or o-4-[bis(2-ch lor oeth yl)a m in o]-
p h en yl}ca r ba m oyl-L-glu ta m a te (21). A similar procedure
starting from 15 (0.232 g. 0.34 mmol) was used to obtain
compound 21. Lithium chloride (0.085 g, 6 equiv) was added
and the reaction carried out in DMA, by stirring 48 h at room
temperature. An oil resulted which after purification on HPLC
1
(cyclohexane:AcOEt, 3:1) gave (0.130 g, 67.6%). H NMR, δH
1.39 (s, 9H, t-Bu), 1.41 (s, 9H, t-Bu), 1.72-1.98 (2×m, 2H, CH2-
CHNH), 2.23-2.30 (m, 2H, CH2CO2), 3.38 (t, 4H, NCH2, J )
6.43 Hz), 3.57 (t, 4H, CH2Cl), 4.04-4.16 (m, 1H, CHNH), 6.60
(d, 1H, NH-G, J ) 7.96 Hz), 7.09 (d, 2H, Harom2+6, J H-F ) 11.28
Hz), 8.86 (d, 1H, NHPh); MS (FAB), m/z: 576 (M+ + 23, 20),
553 (M+, 96), 518 (M+ - Cl, 33); acc mass: (C24H35Cl2F2N3O5)
calcd 553.1922, found 553.1906. Anal. (C24H35Cl2F2N3O5) C, H,
N.

Prodrugs Cleavable by Carboxypeptidase G2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2657
3.45 (m, 4H, N(CH2CH2OH)2), 4.43 (t, 2H, OH, J ) 5.28 Hz),
6.96-7.13 (m, 3H, Harom3,4,5).
27 g) (Charles River UK). After 4 days, six mice per group were
allocated by stratified distribution of tumor sizes. Compound
4c was prepared at a concentration of 12 mg/mL in dimethyl
sulfoxide/1.26% (w/v) sodium bicarbonate (1:19 by volume).
Mice were dosed intraperitoneally with either 0.2 mL per 20
g bodyweight 5% (v/v) DMSO in water for injection of controls
or compound 4c at 360 mg/kg bodyweight three times over 24
h. Six courses of treatment, where one course is defined as 3
doses over 24 h, were given at weekly intervals. Tumor growth
was assessed by caliper measurement in three dimensions, and
volumes were calculated from the formula length × width ×
depth × 0.5236. Mice were culled when tumor size exceeded 2
cm in one dimension or 1.5 cm in two dimensions or when
tumors became ulcerated.
2,6-Diflu or o-4-n itr o-[bis(2-h yd r oxyeth yl)]a n ilin e (24).
Crude 2,6-difluoro[bis(2-hydroxyethyl)]aniline 2 (12 g) from the
previous step was cooled on an ice-bath, and a solution of
fuming HNO3 (18 mL) in DCM (100 mL) was added in portions,
with stirring. After 15 min, the reaction was neutralized with
solid Na2CO3 added in portions until CO2 evolution stopped.
The solid was filtered off, the filtrate was evaporated, and the
residue was purified by column chromatography (eluent cy-
clohexane: ethyl acetate 1:1 then 1:2) to afford 3 (4.2 g, 32%
1
over two steps) as a yellow solid. H NMR, δH, (ppm): 3.41 (t,
4H, N(CH2CH2OH)2, J ) 5.35 Hz), 3.47-3.55 (m, 4H,
N(CH2CH2OH)2), 4.60 (t, 2H, OH, J ) 4.99 Hz), 7.92 (d, 2H,
H
arom3,5, J H-F ) 10.47 Hz). MS, m/z: 263 (M+ + H, 100), 218
((M+ + H - NO2, 97).
Ack n ow led gm en t. The authors would like to thank
the Cancer Research Campaign (grant numbers SP2330/
0201 and SP2330/0102) and the Institute of Cancer
Research for financial support.
2,6-Diflu or o-4-a m in o-[bis(2-h yd r oxyeth yl)]a n ilin e (25).
4-Nitro-2,6-difluoro-[bis(2-hydroxyethyl)]aniline 24 (3.9 g) was
dissolved in THF (70 mL), palladium on carbon 10% catalyst
(1 g) was added, and the reaction mixture was stirred under
H2 atmosphere for 4 h. The catalyst was filtered off and the
solvent evaporated to afford a brown oil (3.4 g, 98%). 1H NMR,
δH, (ppm): 2.97 (t, 4H, N(CH2CH2OH)2, J ) 6.62 Hz), 3.29-
3.37 (m, 4H, N(CH2CH2OH)2), 4.30 (t, 2H, OH, J ) 5.38 Hz),
5.45 (s, 2H, NH2), 6.13 (d, 2H, Harom3,5, J H-F ) 11.36 Hz). MS,
m/z: 233 (M+ + H, 100).
Aqu eou s Ha lf-Life Deter m in a tion . Compounds were
prepared as 10 mM concentrates in DMSO 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 Synergi Polar RP phenyl phase column (150 ×
4.6 mm, 4 µm, Phenomenex) and eluted isocratically (1 mL/
min) with 0.1% TFA solution containing percentages of
methanol (65-85%) that gave retention times of 3-4 min, and
the elute was monitored at 250-275 nm. The amount of
starting material remaining after various periods of incubation
was determined either by repeat injection from a single vial
(3c, 4c) or by delayed injections from a new vial each time
(all others). The results were expressed as fraction of starting
materials as a function of time, and the half-life was deter-
mined by nonlinear regression to a one-phase exponential
decay, constraining the maximum to 1 and the minimum to 0
(Graphpad Prism, Graphpad Software Inc., San Diego, CA).
En zym e Kin etics. Reactions were set up containing CPG2
assay buffer (1 mL), CPG2 (50 mU) and prodrug (5-50 µM in
steps of 5) from concentrates as above. The vials were
incubated at 37 °C, and the amount of prodrug remaining in
the mixture was determined by HPLC as above at 0, 5, 10,
15, and 20 min poststart. The rate of loss of compound in µM/
min was determined by regression. The rate of chemical-only
loss, calculated from the first derivative of the equation for
exponential decay, was subtracted, and the kinetic parameters
were derived from the nonlinear regression to the Michaelis-
Menten equation (Graphpad Prism).
Cytotoxicity Deter m in a tion . MDA MB 361 (5 × 105) cells
were seeded into six-well tissue culture dishes, yielding
similarly confluent monolayer at 48 h. Prodrugs were dissolved
in DMSO at 100 fold the highest dose (3a -e: 10mM; 4a ,b:
100 mM; 4c,d : 200 mM), immediately prior to treatment and
administered in a two-stage protocol, as described previously.
After exposure to prodrug, the cells were harvested, diluted,
reseeded, and grown until the control wells reached confluence,
and the extent of cell growth was assayed by sulforhodamine-B
dye. The results were expressed as the percentage of control
growth against log dose, and the IC50 was determined by
nonlinear regression to a log dose-effect sigmoid, constraining
the minimum to be positive (GraphPad Prism, GraphPad
Software Inc., San Diego, CA).
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