New mustard prodrugs for antibody-directed enzyme prodrug therapy: Alternatives to the amide link

Article abstract of DOI:10.1021/jm950671l

Antibody-directed enzyme prodrug therapy (ADEPT) is a two-step approach for the treatment of cancer which seeks to generate a potent cytotoxic agent selectively at a tumor site. In this work described the cytotoxic agent is generated by the action of an enzyme CPG2 on a relatively nontoxic prodrug. The prodrug 1 currently on clinical trial is a benzamide and is cleaved by CPG2 to a benzoic acid mustard drug 1a. We have synthesized a series of new prodrugs 3-8 where the benzamide link has been replaced by, for example, carbamate or ureido. Some of these alternative links have been shown to be good substrates for CPG2 and therefore new candidates for ADEPT. The active drugs 3a and 4a derived from the best of these prodrugs are potent cytotoxic agents (1-2 μM) some 100 times more than 1a. The prodrugs 3 and 4 are some 100-200-fold less cytotoxic, in a proliferating cell assay, than their corresponding active drugs 3a and 4a.

Full text of DOI:10.1021/jm950671l

1100
J . Med. Chem. 1996, 39, 1100-1105
New Mu sta r d P r od r u gs for An tibod y-Dir ected En zym e P r od r u g Th er a p y:
Alter n a tives to th e Am id e Lin k
Robert I. Dowell,*,† Caroline J . Springer,‡ David H. Davies,† Elizabeth M. Hadley,† Philip J . Burke,§
F. Thomas Boyle,† Roger G. Melton, Thomas A. Connors,‡ David C. Blakey,† and Anthony B. Mauger‡
Cancer Research Department, Zeneca Pharmaceutical, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K.,
CRC Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, U.K., CRC Labs, Charing Cross Hospital,
London, U.K., and PHLS, Porton Down, U.K.
Received September 11, 1995X
Antibody-directed enzyme prodrug therapy (ADEPT) is a two-step approach for the treatment
of cancer which seeks to generate a potent cytotoxic agent selectively at a tumor site. In this
work described the cytotoxic agent is generated by the action of an enzyme CPG2 on a relatively
nontoxic prodrug. The prodrug 1 currently on clinical trial is a benzamide and is cleaved by
CPG2 to a benzoic acid mustard drug 1a . We have synthesized a series of new prodrugs 3-8
where the benzamide link has been replaced by, for example, carbamate or ureido. Some of
these alternative links have been shown to be good substrates for CPG2 and therefore new
candidates for ADEPT. The active drugs 3a and 4a derived from the best of these prodrugs
are potent cytotoxic agents (1-2 µM) some 100 times more than 1a . The prodrugs 3 and 4 are
some 100-200-fold less cytotoxic, in a proliferating cell assay, than their corresponding active
drugs 3a and 4a .
Ta ble 1. Structure, Biological Data, Enzyme Kinetics, and
Chemical Reactivity of Prodrugs
In tr od u ction
Antibody-directed enzyme prodrug therapy1,2 (ADEPT)
is a two-step approach for the treatment of cancer which
seeks to generate a potent cytotoxic agent selectively
at the tumor site or its metastases. In the first step, a
tumor selective antibody chemically linked to an enzyme
(conjugate) is administered and allowed to localize at
the tumor site. After a suitable time, to allow clearance
of the antibody-enzyme conjugate from other tissues,
this is followed, in the second step, by the administra-
tion of a relatively nontoxic prodrug. Since the conju-
gate will have cleared from the blood and other tissues
the prodrug will be enzymatically converted to active
drug only at the tumor site and the usual nonspecific
toxicity associated with cytotoxic agents will be mini-
mized. Potentially therefore one of the major advan-
tages of ADEPT is the ability to deliver, to the tumor,
a highly potent cytotoxic drug, e.g. an alkylating agent,
in concentrations that cannot be achieved by conven-
tional chemotherapy. Clinical studies to assess the
potential of ADEPT in patients with advanced colorectal
cancer are ongoing.3-5 To maximize the benefit of
ADEPT therapy it is beneficial to (a) have high tumor
to normal tissue ratio of the conjugate, (b) ensure the
prodrug to drug conversion is maximal at tumor site,
and (c) generate a potent drug that will act locally and
not diffuse away to normal tissue. Nitrogen mustard
drugs are good candidates for ADEPT since their
cytotoxicity is dose related and have the advantage that
the active drug is non cell cycle specific so that total
cell kill in a solid tumor will be maximized.
Km
kcat
t1/2
(min)
kcat
Km
/
IC50
(µM)
compd
X
Y
link
(µM) (s-1
)
1
2
3
4
5
6
7
8
Cl MeSO3 CO
3.4 583
3.4 700
1.0
46
660
31
171.5 >500
206
49
2.5
1.0 >500
0.08 100
Cl Cl
Cl Cl
Cl Cl
Cl Cl
Cl Cl
Cl Cl
Cl Cl
CO
OCO
>500
200
200
49
NHCO 3.0
CH2CO 16
7.5 16.9
15
4
33
32.2
SCO
OCS
50
NSa
CH2CS NSa
a
NS, not a substrate.
the amide bond of the benzoylglutamate of prodrug 1 is
cleaved to generate the benzoic acid mustard drug 1a
and glutamic acid (Figure 1). The benzoic acid mustard
drug of type 1a is, however, a weak cytotoxic drug (IC50
) 80 µM). This necessitates high doses of prodrug in
therapy studies, and furthermore the benzoic acid based
drug 1a has a long chemical half-life (t1/2) which
increases the risk of drug generated at the tumor
escaping to the periphery and enhancing toxicity.6
These considerations lead us to search for alternative
systems which would be cleaved by CPG2 to release
more potent cytotoxic drugs with shorter chemical half-
lives. We initially chose our targets to be release of the
phenol and aniline mustards 3a and 4a . Other groups
in the ADEPT area have also chosen to release these
compounds as the active drug moiety.7
The ADEPT system currently under clinical investi-
gation4,8,15 is based on a conjugate of the bacterial
carboxypeptididase enzyme (CPG2) and the F(ab′)2
fragment of murine antibody A5B7. With this system,
This paper describes our investigations into modifica-
tions to the amide moiety of 1 and 2 and the design of
novel prodrugs (3-8 ) (Table 1). Some of these prodrugs
are transformed by the action of CPG2 into potent
cytotoxic agents which are thus attractive for a CPG2-
based ADEPT system (Figure 2).
† Macclesfield.
‡ Sutton.
§ London.
Porton Down.
X Abstract published in Advance ACS Abstracts, February 1, 1996.
0022-2623/96/1839-1100$12.00/0 © 1996 American Chemical Society

New Mustard Prodrugs for ADEPT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1101
F igu r e 1.
chloride gas in ether to give 4 as its hydrochloride salt
(Scheme 1). The phenylacetic acid drug 5a was made
from 4-nitrophenylacetic acid tert-butyl ester10 12 by
first reducing the nitro group using 30% palladium on
carbon/hydrogen and then reacting the resulting aniline
13 with ethylene oxide in 1:1 acetic acid/water to give
the diol 14. This diol was converted to the dichloro
mustard using methanesulfonyl chloride in pyridine to
give 15. On treatment with trifluoroacetic acid the
phenylacetic acid drug 5a was generated (Scheme 2).
The corresponding prodrug 5 was prepared as shown
in Scheme 2. A DCCI-mediated coupling of 4-nitrophen-
ylacetic acid 16 with di-tert-butyl glutamate acid gave
a good yield of 17 as an oil. This was reduced with
hydrogen and 30% palladium on carbon to give the
amine 18. Treatment with ethylene oxide gave the diol
19 which was converted to the dichloro compound 20
with methanesulfonyl chloride in pyridine. Treatment
of 20 with trifluoroacetic acid gave 5 as its trifluoroac-
etate salt which failed to crystallize. Compound 20
reacted with Lawesson’s reagent to give the thioamide
21 which after deprotection with trifluoroacetic acid
gave 8 (Scheme 2). Compound 7 was prepared by the
route shown in Scheme 3 from the phenol drug 3a by
treatment with pentafluorophenyl chlorothioformate in
chloroform in the presence of triethylamine to give the
thiocarbonate 22 which was reacted with di-tert-butyl
F igu r e 2.
Ch em istr y
The prodrugs 1 and 2 are known compounds.8,16 The
prodrug 3 was made as shown in Scheme 1 from the
known phenol mustard 3a 9 by reaction with either
phosgene or 4-nitrophenyl chloroformate to give the
intermediate chloroformate 3b or 4-nitrophenyl carbon-
ate 3c, respectively, which was then reacted with
dibenzyl glutamate to give 9. This dibenzyl ester was
hydrogenated in the presence of 30% palladium on
carbon to give 3. The prodrug 4 was made from the
amino mustard 4a 9 by treatment with triphosgene in
chloroform to give the intermediate isocyanate 10 which
was not isolated but reacted with di-tert-butyl glutamate
to give 11 as an oil. This oil was treated with hydrogen
Sch em e 1a
a
(i) 4-Nitrophenyl chloroformate; (ii) NaHCO3/phosgene; (iii) Z ) dibenzyl glutamate, Z ) S, Z ) NH, di-tert-butyl glutamate; (iv) Z )
O, H2, 30% Pd/C; Z ) S-formic acid; Z ) NH, HCl, ether; (v) triphosgene, CHCl3.

1102 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Dowell et al.
Sch em e 2a
a
(i) Di-tert-butyl glutamate, DCCI, DMF; (ii) H2, 30% Pd/C; (iii) ethylene oxide; (iv) MeSO2Cl, pyridine; (v) CF3CO2H, CH2Cl2; (vi)
Lawesson’s reagent, CH2Cl2.
Sch em e 3a
that the cell-killing ability of an aromatic mustard is
related to its chemical reactivity. Hydroxy and amino
groups in the para position of an aromatic nitrogen
mustard are reported12 to increase the chemical reactiv-
ity and thus the cytotoxicity. In the search for a CPG2-
based ADEPT system which would deliver a more
potent drug, we examined alternative linkages to the
amide bond between the aromatic nitrogen mustard and
the glutamate moiety. To deliver such a phenol or
amino drug required the corresponding prodrug to
embody a carbamate 3 or urea 4 moiety. These com-
pounds were synthesized and found to be surprisingly
good substrates for CPG2 with Km and kcat values as
shown in Table 1. From this data it can be seen that
on replacement of the benzamide link by a carbamate
or urea the Km remains almost the same but there has
been a significant drop in the kcat value. These results
show that the compounds 3 and 4 have a similar binding
affinity as 1 and 2 for CPG2 but the enzyme cleaves
the carbamate and urea link less efficiently than the
amide bond (kcat/Km for 3 and 4 are 49 and 2.5,
respectively, whereas 2 is 206). When compounds 3 and
4 were hydrolyzed by the enzyme CPG2 to the phenol
and aniline mustard drugs 3a and 4a , respectively, the
scissile bond is assumed (by analogy with other car-
boxypeptidases13) to be the carbonyl nitrogen bond thus
yielding an unstable carbonic or carbamic acid which
will lose carbon dioxide to give the phenol or aniline
drug. The plasma stability of these carbamate and urea
links has been examined by incubation of 3 and 4 in
mouse plasma at 37 °C for 3 h. During this period no
breakdown to the active drugs 3a and 4a could be
detected. Although the aniline and phenol drugs 4a and
a
(i) Pentafluorophenyl chlorothioformate; Et3N; CHCl3; (ii) di-
tert-butyl glutamate; Et3N; (iii) formic acid.
glutamate in chloroform to give 23. Treatment of 23
with formic acid at room temperature gave 7. Prodrug
6 was prepared from the known11 thiophenol mustard
6a by reaction with phosgene in pyridine to give the
intermediate thiochloroformate 24 which was reacted
immediately with di-tert-butylglutamic acid to give 25
as an oil. This oil on treatment with formic acid
followed by evaporation gave 6 (Scheme 1).
Resu lts a n d Discu ssion
The weak growth inhibition (IC50’s in Table 1) proper-
ties of the dichloro-2a or monomesylbenzoic acid mus-
tard drugs 1a is likely to be due to both the deactivating
effect of the 4-carboxylic acid on the mustard reactivity
and poor cell penetration due to the carboxyl function
which is ionized at physiological pH. In an attempt to
improve both of these factors we turned our attention
to more potent alternative mustard drugs. It is known12

New Mustard Prodrugs for ADEPT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1103
Ta ble 2. Reactivity, Biological Data, and Chemical Reactivity
and Cytotoxicity Ratios of Drugs and Prodrugs
well) microtitre plates for 1 h. The cells were then washed
and incubated for a further 3 days at 37 °C. TCA was then
added and the amount of cellular protein adhering to the plates
was assessed by addition of SRB dye. Cytotoxic potency of
the compounds is expressed as the concentration required to
inhibit cell growth by 50% (IC50).14
ratio of
chemical
reactivity
(min)
ratio of
chemical
compd reactivity to prodrugs
cytoxicity
of drugs
IC50
(µM)
compd
CP G2 En zym e Assa y. The Km and Vmax of the prodrugs
for CPG2 were determined based on the CPG2 assay method
for methotrexate.15 The absorbances of prodrug and corre-
sponding drug were scanned from 200 to 350 nm using a
spectrophotometer (Perkin-Elmer Lambda 2), and the wave-
length was selected where the absorbance difference (due to
release of glutamate from the prodrug) between prodrug and
drug was maximal. The Km and Vmax were then determined
by measuring the initial rate of conversion of prodrug at this
wavelength using a range of prodrug concentrations (1-100
µM) and CPG2 enzyme concentrations (0.05-1 Unit). kcat was
calculated from the Vmax by dividing by the amount of CPG2
in the reaction mixture.
Ch em ica l Hyd r olysis. The rates of hydrolysis, i.e. loss of
the first chloro or mesyl group, of the prodrugs 1-8 and their
corresponding drugs 1a -6a were measured by HPLC analysis
in phosphate buffer (pH 7.4) at 37 °C. Reaction products were
separated on a 25 cm S5ODS1 column using acetonitrile/water/
0.1% TFA as eluant. The t1/2 was calculated using a kinetic
program developed within Zeneca Pharmaceuticals Plc.
N-[[4-[N,N-Bis(2-ch lor oeth yl)am in o]ph en oxy]car bon yl]-
L-glu ta m ic Acid (3). A solution of dibenzyl N-[[4-[N,N-bis-
(2-chloroethyl)amino]phenoxy]carbonyl]-L-glutamate (6 g, 10.2
mmol) in ethyl acetate (100 mL) was hydrogenated over 30%
palladium on carbon (0.6 g) for 2 h. When the theoretical
amount of hydrogen had been taken up, the catalyst was
removed by filtration and the filtrate evaporated to dryness.
The residue was taken up into hot ether (25 mL) and hexane
added until cloudy. On cooling 3 was obtained as a white
1a
2a
3a
4a
5a
6a
17
274
4.5
2.8
19
80
>100
1.0
1.8
19
100
1:1a
2:2a
3:3a
4:4a
5:5a
6:6a
2.7
2.4
6.9
6.7
1.7
6.4
>6.25
>5
200
111
>26
1
5.0
3a are just 3.8-6 times more chemically reactive than
monomesyl benzoic 1a they are some 40-80 times more
potent in a cell-killing test (Table 2). The results in
Table 1 show the negative consequences of changing the
linkage from carbamate or urea to phenylacetamide 5
or thiocarbamate 6. The isomeric thiocarbamate 7 and
the thiophenlyacetamide 8 were found not to be sub-
strates for CPG2 under the conditions of the test. The
phenylacetic acid prodrug 5 was not considered as a
linkage for further development because of its enzyme
kinetics and the low reactivity and cytotoxicity of the
derived drug 5a . The three sulfur-containing analogues
6, 7, and 8 all have poor enzyme kinetics against CPG2
and are therefore not candidates for ADEPT.
The phenol and aniline mustard drugs 3a and 4a
show a marked increase in chemical reactivity over the
benzoic acid drug 2a and a substantial increase in
cytotoxicity over the corresponding drugs 1a and 2a
(Table 1). The prodrugs 3 and 4 are 50-100 times less
cytotoxic than their corresponding drugs.
crystalline solid: 3.4 g, (79%); mp 87-89 °C. Anal. (C16H29
Cl2N2O6) C, H, N.
-
The title compound was also prepared using the above
method but obtained in a different polymorphic form, not
soluble in ether, with a melting point of 128-130 °C.
Diben zyl N-[[4-[N,N-Bis(2-ch lor oeth yl)am in o]ph en oxy]-
Interestingly the ratio of the chemical reactivities of
the prodrug to drug which can be considered as a
“deactivation factor” varies for the two series. In the
series where there is a heteroatom between the amide
bond and the aromatic ring (as in compounds 3, 4, and
6) this deactivation factor is 2-3-fold larger than in the
series where the amide is directly attached (1 and 2) or
when there is a methylene insertion (5, Table 2).
ca r bon yl]-L-glu ta m a te (9). Ch lor ofor m a te Meth od .
A
suspension of 4-[N,N-bis(2-chloroethyl)amino]phenol hydro-
chloride 3a (0.5 g, 2 mmol) in chloroform (10 mL) was shaken
twice with 50% saturated sodium bicarbonate solution. The
chloroform phase was dried and was added to a mixture of
1.9 M phosgene in toluene (6 mL) and quinoline (0.25 mL, 2
mmol) over 5 min at 15-20 °C. The yellow solution was
allowed to stir at ambient temperature for 1 h, washed twice
with water, dried, and evaporated to an oil. To a solution of
this oil in chloroform (10 mL) was added, in one portion,
L-glutamic acid dibenzyl ester tosylate (1 g, 2 mmol) followed
by triethylamine (0.28 mL, 2 mmol). The mixture was stirred
at ambient temperature for 1 h, washed with water (2 × 5
mL), and evaporated to dryness. The residue was chromato-
graphed, eluting with 2.5% ethyl acetate in dichloromethane
to give 9 as a white solid: 0.62 g (77%); mp 85-7 °C; NMR
(CDCl3) δ 7.35 (s, 5H), 7.30 (s, 5H), 6.98 (d, J ) 13.6 Hz, 2H),
6.62 (d, J ) 13.6 Hz, 2H), 5.68 (1, 1H), 5.19 (s, 2H), 5.11 (s,
2H), 4.5 (m, 1H), 3.65 (m, 8H), 2.47 (m, 2H), 2.04 (m, 2H). Anal.
(C30H32Cl2N2O6‚H2O) C, H, N.
Con clu sion
We have shown that a limited range of new prodrugs
with improved properties for ADEPT can be prepared.
The amide link in 1 can be replaced by a carbamate or
a urea moiety as in compounds 3 and 4, and these
compounds retain recognition and turnover by CPG2.
Other links between the aniline mustard and the
glutamate portion were either unacceptably poor sub-
strates for CPG2 (5 and 6) or were not substrates at all
(7 and 8). The active drugs generated from the prodrugs
3 and 4 were markedly (∼100-fold) more potent than
the benzoic acid drug 1a .
Diben zyl N-[[4-[N,N-Bis(2-ch lor oeth yl)am in o]ph en oxy]-
ca r bon yl]-L-glu ta m a te (9). 4-Nitr op h en yl Ch lor ofor m a te
Meth od . 4-Nitrophenyl chloroformate (3.3 g,16 mmol) was
added slowly to a solution of 4-[N,N-bis(2-chloroethyl)amino]-
phenol hydrochloride 3a (4.4 g, 16 mmol) and triethylamine
(4.6 mL, 32 mmol) in chloroform (50 mL). The solution was
allowed to stand at ambient temperature for 4 h and then
evaporated to an oil. The oil was chromatographed, eluting
with hexane/ethyl acetate (3:1) to give the intermediate
carbonate 3c as an oil: 5.5 g (86%), NMR (DMSO-d6) δ 8.36
(d, J ) 9.15 Hz, 2H), 7.68 (d, J ) 9.19 Hz, 2H), 7.21 (d, J )
9.15 Hz, 2H), 6.81 (d, J ) 9.19 Hz, 2H), 3.73 (s, 8H). This oil
(5.5 g, 14 mmol) was dissolved in chloroform (40 mL) and
triethylamine (4.0 mL, 28 mmol) and L-glutamic acid dibenzyl
ester tosylate (13.75 g, 28 mmol) were added. The reaction
Exp er im en ta l Section
Melting points were determined on a Buchi melting point
apparatus and are uncorrected. 1H NMR spectra were re-
corded on a Bruker AC250 or Bruker AM200 instrument with
TMS as an internal standard. Mass spectrum were deter-
mined on a VG-ZAB instrument. All organic extracts were
dried over MgSO4, and evaporations were carried out under
reduced pressure. All microanalyses were within 0.4% of
theory except where stated. All chromatography was done
with Merck 9385 silica gell using the “flash chromatography”
technique.
Cytotoxicity Assa y. The colorectal tumor cell line, LoVo,
was incubated with prodrug, or drug in 96-well (2500 cells/

1104 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Dowell et al.
mixture was heated at 60 °C for 4 h and evaporated to dryness
and the residue chromatographed using 3% ethyl acetate/
chloroform as eluant to give 9, 6.2 g (77%).
mixture was stirred under an atmosphere of hydrogen until
the theoretical amount of hydrogen had been taken up. The
catalyst was removed by filtration through Celite, and the
filtrate was evaporated to an oil 13 (8.5 g). This oil 13 was
dissolved in 1:1 acetic acid/water (140 mL), and a slow stream
of ethylene oxide was passed in until 12 g had been absorbed.
The mixture was allowed to stand at ambient temperature for
48 h, and then evaporated to dryness. The residue was
dissolved in ethyl acetate (150 mL), washed with water and
saturated sodium bicarbonate solution, dried, and evaporated
to a solid, 14 (2.9 g): NMR (DMSO-d6) δ 7.1 (d, J ) 8.3 Hz,
2H), 6.6 (d, J ) 8.3 Hz, 2H), 3.4-3.7 (m, 8H), 3.35 (s, 2H), 1.4
(s, 9H). This solid was dissolved in pyridine (75 mL) and,
under an argon atmosphere, was added dropwise methane-
sulfonyl chloride (4.85 mL). The temperature was kept at 20-
25 °C during the addition, and the mixture was then heated
to 80 °C for 1 h. After cooling the mixture was diluted with
ethyl acetate (500 mL), washed with 10% citric acid solution
(×2) and water, dried, and evaporated to an oil. This oil was
chromatographed, eluting with 9:1 hexane/ethyl acetate gave
15 as an oil: 1.65 g; NMR (DMSO-d6) δ 7.18 (d, J ) 8.2 Hz,
2H), 6.68 (d, J )8.2 Hz, 2H), 3.71 (s, 8H), 3.42 (s, 2H), 1.39 (s,
9H). This oil (1 g) was dissolved in dichloromethane (45 mL)
and trifluoroacetic acid (4.5 mL) added. The mixture was
stirred at ambient temperature for 2 h and then evaporated
to an oil 5a which crystallized: 0.39 g (48%); mp 102-4 °C.
Anal. (C12H15Cl2NO2) C, H, N.
N-[-4-[N,N-Bis(2-ch lor oeth yl)am in o]ph en yl]car bam oyl]-
L-glu ta m ic Acid Hyd r och lor id e (4). A saturated solution
of hydrogen chloride in ether (120 mL) was added to a solution
of 11 (4.4 g, 8.5 mmol) in ethyl acetate (20 mL). After 1 h at
ambient temperature the mixture was evaporated to a solid.
This solid was triturated with ether to obtain 4 as a gray
solid: 3.5 g (93%); mp 148-150 °C. Anal. (C16H21Cl2N3O5‚
HCl‚Et2O‚0.5EtOAc) C, H, N.
Di-ter t-bu tyl Bis[[4-[(2-ch lor oeth yl)a m in o]p h en yl]ca r -
ba m oyl]-L-glu ta m a te (11). To a solution of triphosgene (200
mg, 0.67 mmol) in chloroform (10 mL) at 0-5 °C was added
4a (339 mg, 2 mmol) followed by triethylamine (0.83 mL, 6
mmol). After 15 min at room temperature a solution of di-
tert-butyl L-glutamate (0.31 g, 2.4 mmol) in chloroform (5 mL)
was added. The mixture was allowed to stand at ambient
temperature for 18 h, washed with water, dried, and evapo-
rated to dryness. The residue was chromatographed and
elution with hexane/ethyl acetate (3:1) gave 11 as an oil: 0.44
g (32%), NMR (DMSO-d6) δ 7.2 (d, J ) 9.9 Hz, 2H), 6.65 (d, J
) 9.9 Hz, 2H), 4.1 (m, 1H), 3.66 (s, 8H), 1.7-2.2 (m, 4H), 1.38
(s, 9H), 1.42 (s, 9H).
N-[[4-[N,N-Bis(2-ch lor oeth yl)a m in o]p h en yl]a cetyl]-L-
glu ta m ic Acid (5). 1-Hydroxybenzotriazole (4.05 g, 30 mmol)
was added to a solution of 4-nitrophenylacetic acid (16) (5.4 g,
30 mmol) in dimethylformamide (75 mL). Di-tert-butyl L-
glutamate (7.77 g, 30 mmol) and then dicyclohexylcarbodiimide
(6.2 g, 30 mmol) were added to the mixture. The mixture was
then stirred for 18 h at ambient temperature. The mixture
was filtered, and the filtrate was washed with saturated
sodium bicarbonate solution, water, 0.5 M hydrochloric acid,
dried, and evaporated to dryness. The residue was chromato-
graphed and elution with hexane/ethyl acetate (2:1) gave 17
as an oil: 9.7 g (77%); NMR (DMSO-d6) δ 8.2 (d, J ) 9.9 Hz,
2H), 7.45 (d, J ) 9.9 Hz, 2H), 6.45 (d, 1H), 4.45 (m, 1H), 3.15
(s, 2H), 2.1 (m, 4H), 1.4 (d, 9H), 1.35 (s, 9H).
A solution of 17 (9.7 g) in ethyl acetate (200 mL) was
hydrogenated over 30% Pd/C (900 mg). The mixture was then
filtered through Celite, and the filtrate was evaporated to yield
18 as yellow oil (8.2 g, 90%) which was used without further
purification: NMR (DMSO-d6) δ 8.23 (d, J ) 12.0 Hz, 1H),
6.92 (d, J ) 12.3 Hz, 2H), 6.50 (d, J ) 12.3 Hz, 2H), 4.86 (s,
2H), 4.15 (m, 1H), 3.24 (s, 2H), 2.22 (m, 2H), 1.8 (m, 2H), 1.4
(d, 18H). Ethylene oxide (7.1 g) was bubbled into a solution
of 18 (8.2 g) in glacial acetic acid (40 mL) and water (40 mL).
The mixture was then stirred at ambient temperature for 24
h. The solution was evaporated to dryness, redissolved in
ether, washed with water, dried, and evaporated to yield 19
as an oil (6.3 g) which was used without further purification:
NMR (DMSO-d6) δ 8.16 (d, J ) 12.3 Hz, 1H), 7.02 (d, J ) 10.3
Hz, 2H), 6.58 (d, J ) 10.3 Hz, 2H), 4.1 (m, 1H), 3.4 (m, 8H),
3.25 (s, 2H), 2.25 (m, 2H), 1.8 (m, 2H), 1.4 (s, 18H). Meth-
anesulfonyl chloride (2.91 mL) was added dropwise to a
solution of 19 (2.88 g) in pyridine (45 mL) under an argon
atmosphere, maintaining the temperature below 25 °C. The
solution was stirred at 80 °C for 1 h, cooled, poured into 10%
citric acid (500 mL), and extracted with ether. The ether
extract was washed with water, dried, and evaporated to a
brown oil. This oil was chromatographed eluting with hexane/
ethyl acetate (2:1) to give 20 as an oil: 1.3 g (42%); NMR
(DMSO-d6) δ 8.19 (d, J ) 8.3 Hz, 1H), 7.10 (d, J ) 9.9 Hz,
2H), 6.66 (d, J ) 9.9 Hz, 2H), 4.10 (m, 1H), 3.69 (s, 8H), 3.32
(s, 2H), 2.21 (m, 2H), 1.9 (m, 2H), 1.37 (d, 18H). This oil was
dissolved in dichloromethane (1.5 mL), and trifluoroacetic acid
(1.5 mL) was added. The solution was stirred at ambient
temperature for 2 h. The solution was then evaporated to give
5 as an oil (0.8 g): NMR (DMSO-d6) δ 8.22 (d, J ) 11.6 Hz,
1H), 7.10 (d, J ) 11.6 Hz, 2H), 6.66 (d, J ) 11.6 Hz, 2H), 4.19
(m, 1H), 3.69 (s, 8H), 3.32 (s, 2H), 2.24 (m, 2H), 1.9 (m)2H.
Anal. (C17H22Cl2N2O5‚0.75H2O‚2.5TFA) C, H, N.
N-[4-[N,N-Bis(2-ch lor oeth yl)a m in o]p h en oxy]th ioca r -
bon yl]-L-glu ta m ic Acid (7). To a suspension of 3a (0.8 g. 3
mmol) in chloroform (10 mL) cooled to -10 °C was added
pentafluorophenyl chlorothioformate (0.53 mL, 3.3 mmol)
followed dropwise by triethylamine (0.88 mL, 6.3 mmol). After
stirring for 60 min at -10 °C the mixture was evaporated to
an oil and chromatographed. Elution with hexane/ethyl
acetate (4:1) gave 22 as a colorless oil: 1.04 g (75%); NMR
(DMSO-d6) δ 7.18 (d, J ) 8.8 Hz, 2H), 6.83 (d, J ) 8.8 Hz,
2H), 3.73 (s, 8H). This oil (0.46 g, 1 mmol) was dissolved in
chloroform (10 mL), and L-glutamic acid di-tert butyl ester (0.69
g, 2.8 mmol) was added. Triethylamine (0.4 mL, 2.8 mmol)
was added, and the mixture was heated at 50 °C for 4 h. After
cooling the mixture was evaporated to dryness and the residue
was chromatographed, eluting with 3% ethyl acetate in
chloroform to give 23 as a colorless oil: 168 mg (31%); NMR
(CDCl3) δ 7.36 (d, J ) 8.9 Hz, 2H), 6.98 (d, J ) 8.9 Hz, 2H),
6.66 (d, 2H), 4.82 (m, 1H), 3.65 (m, 8H), 2.24 (m, 4H), 1.50 (d,
18H).. This oil (0.44 g 0.8 mmol) was dissolved in formic acid
(25 mL) and kept at ambient temperature for 18 h. After
evaporation to dryness the residue was chromatographed
eluting with dichloromethane/ethyl acetate/formic acid (6:4:
0.5) gave 7 as an oil: 160 mg (46%). NMR (CDCl3) δ 7.22 (s,
1H), 6.97 (d, J ) 9.0 Hz, 2H), 6.67 (d, J ) 9.0 Hz, 2H), 5.04
(m, 1H), 3.66 (m, 8H), 2.56 (m, 4H). Anal. (C16H20Cl2N2O5-
S‚0.3EtOAc) C, H, N.
N-[4-[N,N-Bis(2-ch lor oeth yl)a m in o]p h en ylsu lfa m yl]-L-
glu ta m ic Acid (6). To a mixture of 6a (0.5 g, 2 mmol) and
pyridine (0.16 mL, 2 mmol) in anhydrous ether (10 mL) was
added over 5 min a solution of phosgene in toluene (1.1 mL of
1.9 M). The mixture was stirred for 30 min at ambient
temperature, and a white precipitate was removed by filtra-
tion. The filtrate was evaporated to dryness to give the crude
thiochloroformate 24 and redissolved in chloroform (10 mL),
and L-di-tert-butyl glutamate (0.49 g, 2 mmol) was added
followed by triethylamine (0.3 mL, 2 mmol). The mixture was
stirred at ambient temperature for 1 h and then evaporated
to dryness. The residue was chromatographed elution with
3% ethyl acetate in chloroform gave 25 as an oil: 380 mg
(36%); NMR (CDCl3) δ 7.42 (d, J ) 6.0 Hz, 2H), 6.7 (d, J ) 6.0
Hz, 2H), 6.01 (d, 1H), 4.44 (m, 1H), 3.72 (m, 8H), 2.46 (m, 2H),
2.26 (m, 2H), 1.47 (s, 18H).
This oil 25 (0.2 g) was dissolved in dichloromethane (2 mL)
at 0 °C, and trifluoroacetic acid (0.5 mL) was added. The
mixture was stirred at 0 °C for 3 days, evaporated to dryness,
and azeotroped twice with ethyl acetate to give 6 as a colorless
oil: 130 mg (46%); NMR (DMSO-d6) δ 8.36 (d, J ) 10.2 Hz,
1H), 7.26 (d, J ) 6.2 Hz, 2H), 6.78 (d, J ) 6.2 Hz, 2H), 4.19
4-[Bis(2-ch lor oeth yl)a m in o]p h en yla cetic Acid (5a ). To
a solution of 4-nitrophenylacetic acid tert-butyl ester10 (12) (10
g, 42 mmol) in ethyl acetate (150 mL) was added 30%
palladium on carbon (50% moist with water) (1.5 g). The

New Mustard Prodrugs for ADEPT
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1105
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Identification of prodrug, active drug and metabolites in an
ADEPT clinical study. Cell Biophys. 1993, 22, 1-8.
(7) (a) Kadow, J .; Kaneko, T.; Senter, P. D.; Vrudhula, V. M.
Prodrugs for beta-lactamases and uses thereof. European patent
0484870 A2, 1991. (b) Wallace, P. M.; Senter, P. D. In vitro and
in vivo activities of monoclonal antibody-alkaline phosphatase
conjugates in combination with phenol mustard phosphate.
Bioconjugate Chem. 1991, 2, 349-352.
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F.; J arman, M. Novel prodrugs which are activated to cytotoxic
alkylating agents by carboxypepdidase G2. J . Med. Chem. 1990,
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(9) Dowell, R. I.; Burke, P. J .; Springer, C. J .; Mauger, A. B. Amino
acid linked nitrogen mustard derivatives and their use as
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tumors. World Patent WO 9402450 A1.
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(13) The Enzymes, 3rd ed.; Boyer, P. D., Ed.; Chapter 1.
(14) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J .;
Vistica, D.; Warren, J . T.; Bokesch, H.; Kenny, S.; Boyd, M. R.
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Purification and properties of carboxypepdidase G2 from
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(16) Springer, C. J .; Bagshaw, K. D.; Sharma, S. K.; Boden, J . A.;
Antoniw, P.; Rogers, G. T.; Burke, P. J .; Sherwood, R. F.; Melton,
R. G. Ablation of human choriocarcinoma xenographs in nude
mice by Antibody Directed Enzyme Prodrug Therapy (ADEPT)
with three novel compounds. Eur. J . Cancer 1991, 27, 1361-66.
(m, 1H), 3.86 (s, 8H), 2.23 (m, 2H), 1.89 (m, 2H). Anal.
(C16H20Cl2N2O5‚3TFA) C, H, N.
N-[[4-[N,N-Bis(2-ch lor oeth yl)am in o]ph en yl]th ioacetyl]-
L-glu ta m ic Acid (8). To a solution of the dichloro-tert-butyl
ester 20 (0.84 g, 1.6 mmol) in dichloromethane (30 mL) was
added Lawesson’s reagent (0.78 g, 1.6 mmol). The mixture
was allowed to stir at ambient temperature for 18 h, evapo-
rated to dryness, and chromatographed. Elution with hexane/
ethyl acetate (4:1) gave 21 as an oil: 0.68 g (80%); NMR
(DMSO-d6) δ 10.2 (d, J ) 9.9 Hz, 1H), 7.2 (d, J ) 8.3 Hz, 2H),
6.7 (d, J ) 8.3 Hz, 2H), 4.7 (m, 1H), 3.85 (s, 2H), 3.7 (s, 8H),
2.0-2.3 (m, 4H), 1.5 (s, 9H), 1.3 (s, 9H). This ester 21 (400
mg, 0.7 mmol) was dissolved in formic acid (30 mL) and
allowed to stand at ambient temperature for 18 h. The
solution was evaporated to an oil 8: 200 mg (60%); NMR
(DMSO-d6) δ 10.2 (d, J ) 10.1 Hz, 1H), 7.1 (d, J ) 7.3 Hz,
2H), 6.6 (d, J ) 7.3 Hz, 2H), 4.9 (m, 1H), 3.85 (m, 1H), 3.7 (s,
8H), 2.0-2.3 (m, 4H). Anal. (C17H22Cl2N2O4S. 0.5EtOAc) C,
H, N.
Ack n ow led gm en t. We thank Mr. S. Nicholson for
providing the chemical reactivity and mouse plasma
stability data.
Refer en ces
(1) Bagshawe, K. D. Antibody directed enzymes revive anti-cancer
prodrug concept. Br. J . Cancer 1987, 56, 531-2.
(2) Bagshawe, K. D.; Springer, C. J .; Searle, F.; Antoniw, P.;
Sharma, S. K.; Melton, R. G.; Sherwood, R. F. A cytotoxic agent
can be generated selectively at cancer sites. Br. J . Cancer 1988,
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Boden, J . A.; Rogers, G. T.; Burke, P. J .; Melton, R. G.; Sherwood,
R. F. Antibody Directed Enzyme Prodrug Therapy (ADEPT)
Clinical Report. Disease Markers 1991, 9, 233-8.
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Antibody Directed Enzyme Prodrug Therapy (ADEPT): A review
of some theoretical, experimental and clinical aspects. Anal.
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(5) Bagshawe, K. D.; Springer, C. J . Tumour Targeting, in press.
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