Synthesis and in vitro efficacy of transferrin conjugates of the anticancer drug chlorambucil

Article abstract of DOI:10.1021/jm9704661

One strategy for improving the selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing soluble macromolecules or carrier proteins. Thus, five maleimide derivatives of chlorambucil were bound to thiolated human seru

Full text of DOI:10.1021/jm9704661

J . Med. Chem. 1998, 41, 2701-2708
2701
Syn th esis a n d in Vitr o Effica cy of Tr a n sfer r in Con ju ga tes of th e An tica n cer
Dr u g Ch lor a m bu cil
Ulrich Beyer,‡ Thomas Roth,‡ Peter Schumacher,‡ Gerhard Maier,‡ Anuschka Unold,‡ August W. Frahm,§
Heinz H. Fiebig,‡ Clemens Unger,‡ and Felix Kratz*,‡
Department of Medical Oncology, Clinical Research, Tumor Biology Center, Breisacher Strasse 117, 79106 Freiburg, Germany,
and Chair of Pharmaceutical Chemistry, Faculty of Chemistry and Pharmacy, University of Freiburg,
Hermann-Herder-Strasse 9, 79104 Freiburg, Germany
Received J uly 16, 1997
One strategy for improving the selectivity and toxicity profile of antitumor agents is to design
drug carrier systems employing soluble macromolecules or carrier proteins. Thus, five
maleimide derivatives of chlorambucil were bound to thiolated human serum transferrin which
differ in the stability of the chemical link between drug and spacer. The maleimide ester
derivatives 1 and 2 were prepared by reacting 2-hydroxyethylmaleimide or 3-maleimidophenol
with the carboxyl group of chlorambucil, and the carboxylic hydrazone derivatives 5-7 were
obtained through reaction of 2-maleimidoacetaldehyde, 3-maleimidoacetophenone, or 3-male-
imidobenzaldehyde with the carboxylic acid hydrazide derivative of chlorambucil. The
alkylating activity of transferrin-bound chlorambucil was determined with the aid of 4-(4-
nitrobenzyl)pyridine (NBP) demonstrating that on average 3 equivalents were protein-bound.
Evaluation of the cytotoxicity of free chlorambucil and the respective transferrin conjugates in
the MCF7 mammary carcinoma and MOLT4 leukemia cell line employing a propidium iodide
fluorescence assay demonstrated that the conjugates in which chlorambucil was bound to
transferrin through non-acid-sensitive linkers, i.e., an ester or benzaldehyde carboxylic
hydrazone bond, were not, on the whole, as active as chlorambucil. In contrast, the two
conjugates in which chlorambucil was bound to transferrin through acid-sensitive carboxylic
hydrazone bonds were as active as or more active than chlorambucil in both cell lines.
Especially, the conjugate in which chlorambucil was bound to transferrin through an
acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 3-18-
fold lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this
conjugate can be administered at higher doses in comparison to unbound chlorambucil. The
structure-activity relationships of the transferrin conjugates are discussed with respect to
their pH-dependent acid sensitivity, their serum stability, and their cytotoxicity.
In tr od u ction
tumor-associated antigens) since the transferrin recep-
tor is essential for cell growth.7 Furthermore, trans-
ferrin has been used as a delivery system for toxins and
DNA8 and is a stable, commercially available protein,
which has been intensively studied and characterized.6
Chlorambucil (Leukeran) is a nitrogen mustard which
is used clinically against chronic lymphatic leukemia,
lymphomas, and advanced ovarian and breast carcino-
mas.1 The clinical application of this anticancer drug,
which exhibits its cytotoxicity due to its alkylating
properties, is, however, limited by its toxic side effects
such as nausea, myelotoxicity, and neurotoxicity.2
One approach to overcome the toxicity of anticancer
drugs to normal tissue is to attach cytotoxic drugs to
suitable carrier proteins which accumulate in tumor
tissue. Due to our interest in the role which human
plasma proteins play in the in vivo distribution of
anticancer drugs,3,4 we have developed chlorambucil
conjugates of the serum protein transferrin, the iron-
(III) transport protein. Transferrin exhibits a signifi-
cant uptake in tumor tissue due to high amounts of
specific transferrin receptors (150 000-1 000 000/cell)
on the cell surface of tumor cells.5,6 Antigenic hetero-
geneity or modulation is not present (in comparison to
Chlorambucil was one of the first anticancer agents
which was used to prepare antibody conjugates.9-11
However, in these reports9,10 chlorambucil was either
physically adsorbed or chemically linked to the carrier
by mere incubation or by direct coupling using N-
hydroxysuccinimide/N,N′-dicyclohexylcarbodiimide
(DCC).11 These direct methods have the disadvantage
that during preparation polymeric products are likely
to be formed and the resulting conjugates are chemically
not well-defined with respect to the chemical link
between drug and carrier protein.
We therefore wanted to improve the coupling tech-
nique in order to prepare better defined protein conju-
gates of chlorambucil in which the stability of the bond
between carrier protein and chlorambucil could also be
varied. An effective method of preparing protein con-
jugates is to introduce a maleimide group into the drug,
which is then able to bind selectively to sulfhydryl
groups of carrier proteins through its carbon double
* To whom correspondence should be addressed. Tel: 0049-761-
2062176. Fax: 0049-761-2061899. E-mail: felix@tumorbio.uni-
freiburg.de.
‡ Tumor Biology Center.
§ University of Freiburg.
S0022-2623(97)00466-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/25/1998

2702 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Beyer et al.
Sch em e 1
(prepared by reacting the acid chloride of chlorambucil
with tert-butyl carbazate and subsequent cleavage with
CF3COOH) with 2-maleimidoacetaldehyde, 3-maleimi-
doacetophenone, or 3-maleimidobenzaldehyde (see
Scheme 2).
Characteristic peaks of the introduced maleimide
groups are singlets in the range from 6.8 to 7.2 ppm for
1
the proton signals of the double bond in the H NMR
spectra and at 134-135 and 169-170 ppm for the
carbon atoms of the double bonds and carbonyl groups
in the 13C NMR spectra. Furthermore, the proton signal
of the carboxyl group (12.1 ppm in chlorambucil) is not
1
present in the spectra of 1, 2, and 5-7. In the H NMR
and 13C NMR spectra of 5-7 (recorded in CDCl3) only
one set of signals is observed showing the presence of
only one steresoisomer. From a steric point of view the
E-isomer is favored, and we therefore tentatively sug-
gest that this stereoisomer is present which is in
accordance with studies on simple hydrazones.15
The molecular ion peak of 1, 2, and 5-7 was observed
in the mass spectra (EI/CI, FAB) indicating that the
chlorine atoms were not lost or substituted during
synthesis which is also confirmed by elemental analysis
(see the Experimental Section). In addition, we deter-
mined the alkylating activity of our derivatives with the
aid of a nitrobenzylpyridine (NBP)-based assay accord-
ing to Epstein et al.16 This reagent produces a violet-
colored complex (ꢀ565 ) 15 100 M-1 cm-1) with alkylating
agents under basic conditions. Compounds 1, 2, and
5-7 exhibited 97-100% alkylating activity when com-
pared to pure chlorambucil, which was set as the
standard.
bond.12 Recently, we developed a number of maleimide
compounds for this purpose.13
Chlorambucil is a suitable agent for chemical modi-
fication due to the presence of only one carboxyl group
in the molecule, and thus we synthesized five maleimide
derivatives of chlorambucil which differed in the stabil-
ity of the chemical link (aliphatic and aromatic ester or
carboxylic hydrazone bond) between the drug and the
spacer group. The rationale for varying the stability of
the chemical link was to assess the significance of the
pH-dependent stability of the link between drug and
carrier for in vitro and in vivo activity. Transferrin is
taken up by the cell through receptor-mediated endocy-
tosis. During internalization the pH is reduced from
7.4 to 5.5-5.0, and this pH change can be exploited
through acid cleavage of a predetermined breaking point
so that the drug can be released inside the tumor cell.14
In this paper we report on the synthesis and charac-
terization of chlorambucil-transferrin conjugates, their
antiproliferative efficacy, and the relationship between
pH-dependent stability, serum stability, and cytotoxic-
ity.
Resu lts a n d Discu ssion
p H-Dep en d en t Sta bility of 8-12. To assess the
significance of pH-dependent stability of the chemical
link between drug and protein for subsequent in vitro
studies, we wanted to determine the stability of the
ester and hydrazone links realized in 1, 2, and 5-7.
Unfortunately, due to the rapid hydrolysis (detection of
a number of peaks within a few hours with the aid of
HPLC17) of the chlorine atoms of chlorambucil, aqueous
stability studies of 1, 2, and 5-7 with respect to the
chemical link between the maleimide spacer and
chlorambucil could not be carried out. We therefore
synthesized the model compounds 8-12 in which
chlorambucil was substituted by 4-phenylbutyric acid
and the maleimide spacer by ethanol, phenol, phen-
ylacetaldehyde, acetophenone, and benzaldehyde, re-
spectively18 (see Chart 1). 8 and 9 were synthesized
according to the literature19 and 10-12 in analogy to
that of 5-7 (synthesis and characterization data are
available as Supporting Information). The pH-depend-
ent stability of 8-12 was studied at pH values of 5.0
and 7.4 on a reverse-phase C18 HPLC column. The
decrease of the peak areas of 8-12 in the chromato-
grams was used to calculate the respective half-lives.
Whereas the half-lives of 8 and 9 were not reached after
4 days at either pH 5.0 or 7.4 (<10-20% decomposition
after 96 h), the decomposition of the carboxylic hydra-
zones at pH 5.0 was fast for 10 and 11 [t1/2 ≈ 5 h (10);
t1/2 ≈ 11 h (11)] but slow for 12 [t1/2 ≈ 90 h]. This
hydrolytic classification can be expected from the char-
acter of the chemical link involved.20
Ch em istr y
Synthesis of Maleimide Derivatives of Chlorambucil.
The route of preparing the maleimide ester derivatives
of chlorambucil (1 and 2) is depicted in Scheme 1. The
aliphatic and aromatic esters of chlorambucil were
synthesized by reacting chlorambucil with an excess of
2-hydroxyethylmaleimide or 3-maleimidophenol in CH2-
Cl2 and addition of DCC or N-cyclohexyl-N′-(2-morpholi-
noethyl)carbodiimide metho-p-toluenesulfonate and cata-
lytic amounts of (dimethylamino)pyridine.
The carboxylic hydrazone derivatives 5-7 were ob-
tained by reaction of the acid hydrazide of chlorambucil

Transferrin Conjugates of Chlorambucil
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2703
Sch em e 2
All hydrazones showed good stability at pH 7.4 (<10%
decomposition after 96 h). In a further set of experi-
ments we analyzed the stability of 8-12 in cell-
conditioned culture medium over a period of 6 days.
Samples were incubated at 37 °C, ultrafiltered (cutoff
MW 10 000), and analyzed through HPLC (see the
Experimental Section) every 24 h. In all cases the
decrease in the original peak area was less than 20%
demonstrating that the model compounds are suf-
ficiently stable under these experimental conditions.
P r ep a r a tion of Tr a n sfer r in Con ju ga tes (T-1, T-2,
T-5, T-6, T-7). (Abbreviations: Tf, human serum trans-
ferrin; T-1, T-2, T-5, T-6, T-7, refer to the respective
chlorambucil-transferrin conjugates prepared with the
maleimide derivatives 1, 2, 5, 6, and 7.) The transferrin
conjugates were prepared by reacting 1, 2, and 5-7 with
thiolated transferrin. The HS group adds to the double
bond of the maleimide group in a fast and selective
reaction forming a stable thioether bond. During thi-
olation using iminothiolane the formation of disulfide
bonds was prevented by addition of 0.001 M EDTA and
degassing all buffers with argon. Under these condi-
tions the number of introduced HS groups was highly
reproducible, with an average number of 3.0-3.2 HS
groups being introduced. For preparing the conjugates
the respective maleimide derivative was added to the
thiolated protein sample and T-1, T-2, T-5, T-6, or T-7
purified by gel chromatography (Sephadex G-25). To
rule out the presence of unbound drug as well as
polymeric byproducts which may be formed in the
coupling step, the purity of the conjugates was deter-
mined with an analytical HPLC size exclusion column
(Bio-Sil SEC 250). Typical chromatograms recorded at
λ ) 280 nm show a main peak at 7.2 min corresponding
to that of monomeric transferrin; a small peak at 6.35
min results from a dimeric product with a peak area of
less than 10% in our conjugates (commercially available
transferrin also shows this peak with a peak area of
approximately 2-3%). In addition, the transferrin
conjugates and native transferrin were analyzed by
agarose electrophoresis as well as SDS-PAGE showing
a dominant band at the position of monomeric trans-
ferrin (MW 80 000)sdata not shown.
The amount of chlorambucil bound to transferrin was
determined with the aid of the NBP assay which was
modified accordingly (see the Experimental Section),
whereas the protein concentration of the conjugates was
determined using the BCA-protein assay from Pierce.

2704 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Ch a r t 1. Structures of Model Compounds 8-12
Beyer et al.
Ta ble 2. IC50 Values (µM)a for the Chlorambucil-Transferrin
Conjugates in Comparison to Free Chlorambucil in the MCF7
Mammary Carcinoma and MOLT4 Leukemia Cell Lines
compound
MCF7
MOLT4
chlorambucil
24.6 ( 1.7
-
6.3 ( 0.8
7.8 ( 0.7
15.3 ( 1.3
0.35 ( 0.08
3.3 ( 0.3
7.5 ( 0.9
T-1
T-2
T-5
T-6
T-7
-
6.8 ( 0.4
23.2 ( 1.5
35.5 ( 3.4
a
IC50 values (50% inhibitory concentration) represent the mean
( standard deviation (n ) 3). Chlorambucil hydrazide (4) showed
comparable activity to T-5 in the MOLT4 cell line and was as active
as chlorambucil in the MCF7 cell line as demonstrated in
subsequent cell culture experiments. Concentrations refer to the
equialkylating activity of the respective compounds.
difference between the individual transferrin conjugates
(see Table 1).
Biologica l Da ta . The newly synthesized transferrin
conjugates and unbound chlorambucil were evaluated
for inhibitory effects in a mammary carcinoma (MCF7)
and an acute lymphoblastic leukemia (MOLT4) cell line
employing a propidium iodide fluorescence assay. Both
cell lines were transferrin receptor-positive as shown
by flow cytometric analysis with an anti-human CD71
antibody directed against the transferrin receptor (FACS
data are available as Supporting Information). Respec-
tive IC50 values are summarized in Table 2 (cell culture
data is included as Supporting Information). Transfer-
rin and the employed buffer (0.0025 M sodium borate,
0.15 M NaCl, pH 7.2) had no influence on cell growth
in both cell lines (data not shown). All of the tested
compounds showed dose-dependent activity in both cell
lines (concentration range 0.15-40 µM). The transfer-
rin conjugates T-1, T-2, and T-7, which do not contain
an acid-sensitive bond according to our stability studies
using model compounds, are the least active compounds
in both cell lines, IC50 values being reached at high
concentrations (>7 µM) or not at all in the tested
concentration range. This result suggests that the low
antiproliferative effect of these conjugates could be due
either to a slow release of chlorambucil under cell
culture conditions or to interactions with the plasma
membrane, which has been suggested as an additional
target for the activity of alkylating agents besides their
binding to cellular DNA.23
Ta ble 1. Time-Dependent Stability of the Alkylating Activity
of the Chlorambucil-Transferrin Conjugates in Comparison to
Free Chlorambucil Incubated with 10% Human Blood Serum at
37 °C
alkylating activity
(% remaining after hours of incubation)a
compound
t1/2 (h)
24 h
48 h
96 h
chlorambucil
4.5
3.1
4.3
4.0
4.5
5.9
12.2
20.9
22.3
25.0
22.9
24.1
6.6
13.8
15.2
19.5
13.7
14.5
T-1
T-2
T-5
T-6
T-7
11.0
12.1
15.2
10.9
13.4
a
Starting concentrations in the incubated samples were c ∼
330 µM in each case with respect to alkylating activity; this value
at t ) 0 h was set as 100%. Averages obtained in two independent
experiments are presented (determinations were carried out in
triplicate). Standard deviations were below (5% and are not
shown.
In contrast, the acid-sensitive conjugates T-5 and T-6
exhibit antiproliferative activity which is comparable to
or exceeds that of chlorambucil. Above all, T-5 exhibited
IC50 values which were approximately 3-fold lower
(MCF7) or 18-fold lower (MOLT4) than those of chloram-
bucil. Chlorambucil hydrazide (4), which is the hy-
drolysis product after cleavage of the carboxylic hydra-
zone bond, showed comparable activity to T-5 in the
MOLT4 cell line and was as active as chlorambucil in
the MCF7 cell line as demonstrated in subsequent cell
culture experiments. Among the transferrin conjugates
prepared with the carboxylic hydrazone derivatives 5-7,
cytotoxicity is enhanced with increasing acid sensitivity
of the hydrazone link according to pH-dependent stabil-
ity studies carried out with the related model com-
pounds 10-12.24 Furthermore, stability studies of 8-12
in cell-conditioned medium show that the respective
ester and carboxylic hydrazone bonds are not prone to
rapid hydrolysis. These results taken together indicate
In this way the number of equivalents of chlorambucil
bound to transferrin was calculated.21 The ratio of
chlorambucil/transferrin in the conjugates was 2.8-3.1
which corresponded well with the number of introduced
HS groups (see above).
Sta bility in 10% Ser u m . To determine the loss of
alkylating activity of chlorambucil and our transferrin
conjugates in serum, we incubated chlorambucil and the
conjugates with 10% human serum22 at T ) 37 °C and
determined the loss of alkylating activity with the aid
of the NBP assay over a period of 4 days. The results
of these incubation studies are shown in Table 1. There
is an initial rapid decrease of alkylating activity in all
of the compounds (approximately 50% of activity re-
maining after 4-6 h). After this time a gradual loss of
alkylating activity is observed which is larger for
chlorambucil than for the conjugates, there being little

Transferrin Conjugates of Chlorambucil
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2705
that antiproliferative in vitro activity is correlated with
the chemical link between chlorambucil and transferrin.
The importance of the acid sensitivity of the linker
between drug (e.g., anthracyclines) and the carrier
protein for in vitro and in vivo anticancer activity has
been noted by Greenfield et al.25 and our group.26-28
the antitumor efficacy of transferrin conjugates of
chlorambucil in animal tumor models.
Exp er im en ta l Section
Ch em icals, Mater ials, an d Spectr oscopy. Melting points,
Bu¨chi 530; 1H NMR and 13C NMR, Bruker 400 MHz AM 400,
Varian 300 (internal standard, TMS); EI-MS, FAB, Finnigan-
MAT 312; elemental analysis, Perkin-Elmer elemental ana-
lyzer 240; preparative low-pressure chromatography, LiChro-
PrepDiol column (LOBAR, 310-25, 40-63 µm) from Merck
AG with a LKB 2248 pump (flow, 3 mL/min), a LKB 2211
fraction collector, and a Lambda 1000 UV/visible spectropho-
tometer detector from Bischoff (254 or 280 nm); analytical
HPLC, reverse-phase HPLC column (Spherisorb ODS 2-5 µm;
MedChrom, Heidelberg, FRG); silica gel chromatography, silica
gel 60 (0.063-0.100 mm) from Merck AG; TLC, silica-coated
plates 60 F254 from Merck; HPTLC, diol glass-coated plates
F254 (0.2 mm) from Merck AG. Chlorambucil (Mr 304.20) was
a gift from Borroughs Wellcome, FRG. Organic solvents:
HPLC grade (Merck) or analytical grade (gift from BASF AG).
Other organic or inorganic compounds: Merck AG, FRG.
Maleimide spacer molecules were prepared previously.13 Ma-
terials for the preparation of conjugates: human serum trans-
ferrin (Tf) (98%, crystalline, iron-saturated, Mr 80 000) was a
gift from Boehringer Mannheim, FRG. Iminothiolane‚HCl,
5,5′-dithiobis(2-nitrobenzoic acid), and propidium iodide were
purchased from Aldrich-Sigma-Chemie, FRG. The buffers
used were vacuum-filtered through a 0.2-µm membrane (Sar-
torius, FRG) and thoroughly degassed with argon prior to use.
Cell culture media, supplements (L-glutamine, antibiotics,
trypsin versene/EDTA), and fetal calf serum (FCS) were
purchased from Bio Whittaker (Serva, Heidelberg, FRG). All
culture flasks were obtained from Greiner Labortechnik
(Frickenhausen, FRG).
When comparing the serum stability of chlorambucil
and the transferrin conjugates with respect to their
alkylating activity, we found an increased stability of
the conjugates over a period of 4 days. However, there
was no significant difference between the conjugates
themselves indicating that the superior antiproliferative
activity of T-5 and T-6 over T-1, T-2, and T-7 observed
in vitro cannot, on the whole, be ascribed to the
prolonged alkylating activity of chlorambucil-transfer-
rin conjugates, but rather to their acid-sensitive proper-
ties.
Admittedly, our results do not rule out the possibility
that chlorambucil hydrazide might be released extra-
cellularly by the acid-sensitive conjugates T-5 and T-6,
although as we have mentioned above, only small
amounts are liberated at pH 7.4, and the model spacers
demonstrate a good stability in cell-conditioned medium.
The significance of the transferrin receptor for uptake
of the chlorambucil-transferrin conjugates is an im-
portant issue for the outlined approach of tumor cell
targeting, and we are currently investigating the mecha-
nistic aspects of conjugate cell transport (e.g., through
inhibition experiments with antibodies directed against
the transferrin receptor and influence of ionophores on
the inhibitory effects of the conjugates, respectively).
Meth od s for th e P r ep a r a tion of Con ju ga tes. FPLC for
preparation of conjugates: P-500 pump, LCC 501 controller
(Pharmacia), and LKB 2151 UV monitor (at λ ) 280 nm);
buffer, standard borate: 0.0025 M sodium borate, 0.15 M
NaCl, pH 7.2. The protein concentration of the thiolated
samples was determined using the ꢀ values for transferrin (ꢀ280
P r elim in a r y Toxicit y St u d ies. Subsequently,
chlorambucil and T-5 were subjected to acute toxicity
studies (dose, 20 mg/kg per day with respect to the
amount of chlorambucil present; administration: ip, 3
consecutive days). Whereas two out of three mice in
the group treated with free chlorambucil died after the
second or third injection, all mice treated with the
conjugate survived. No body weight loss was observed
in this group.
) 92 300 M-1 cm-1)6 and the concentration of HS groups with
29
Ellmann’s reagent (ꢀ412 ) 13 600 M-1 cm-1
)
with a double-
beam UV/vis spectrophotometer U-2000 from Hitachi. The
protein concentration of the conjugates was determined using
the BCA-protein assay from Pierce. The amount of chloram-
bucil bound to transferrin was determined using a modified
4-nitrobenzylpyridine assay based on the assay of Epstein et
al.16 (see below). Purity of the transferrin conjugates was
determined with the aid of HPLC on a Bio-Sil SEC 250 (300
mm × 7.8 mm) from Bio-RAD; mobile phase, 0.15 M NaCl,
0.01 M NaH2PO4, 5% CH3CN, pH 7.0; with a LKB 2150 pump
(flow, 1.5 mL/min), a Bischoff-Lambda 1000 UV/vis monitor
at λ ) 280 nm, an auto sampler (Merck Hitachi AS400), and
an integrator (Merck Hitachi D2500). Conjugates were dis-
solved in 0.15 M NaCl, 0.01 M NaHCO3, 0.004 M NaH2PO4,
pH 7.4, and a 50-µL sample was injected.
In summary, we have shown that maleimide deriva-
tives of chlorambucil can be bound effectively to thi-
olated transferrin by which transferrin conjugates are
obtained in high purity which retain the alkylating
activity of the drug. Transferrin holds promise as a
drug carrier due to the large number of transferrin
receptors on the surface of tumor cells. Expected
advantages of these protein conjugates are preferable
tissue distribution, prolonged half-life of the drug in the
plasma, and controlled drug release from the carrier
protein by adjustment of the chemical properties of the
bond between the drug and the carrier.
Syn t h esis of Ma leim id e Der iva t ives of Ch lor a m -
bu cil: 1, 2, a n d 5-7. O-[4-[4-(Bis(2-ch lor oeth yl)a m in o)-
p h en yl]b u t a n oyl]-2-h yd r oxyet h ylm a leim id e (1). 4-[4-
(Bis(2-chloroethyl)amino)phenyl]butyric acid (chlorambucil; 1.0
g, 3.3 mmol), 2-hydroxyethylmaleimide (1.4 g, 13.2 mmol), and
a catalytic amount of 4-(dimethylamino)pyridine (DMAP; 20
mg, 0.16 mmol) were dissolved in 100 mL of anhydrous
CH2Cl2 at room temperature. DCC (0.747 g, 3.6 mmol),
dissolved in 100 mL of anhydrous CH2Cl2, was added dropwise
to this solution within 1 h, and the solution stirred for a further
8 h at room temperature. The solution was filtered and
evaporated in vacuo. The residue was dissolved in 50 mL of
ethyl acetate, filtered, and chromatographed on a silica gel
column (ethyl acetate/hexane, 1:2) to yield 0.58 g (41%) of a
light-yellow syrup: Rf 0.26 (ethyl acetate/hexane, 1:2); 1H NMR
(CDCl3) δ 1.71 (tt, 2H, J ) 6.7/6.5 Hz, 8-H), 2.21 (t, 2H, J )
6.5 Hz, 9-H), 2.42 (t, 2H, J ) 6.7 Hz, 7-H), 3.63 (t, 2H, J ) 5.5
Hz, 3′-H), 3.66 (t, 4H, J ) 6.0 Hz, 1-H), 3.68 (t, 4H, J ) 6.0
In our effort to prepare new active formulations of
chlorambucil, we have demonstrated that acid-sensitive
chlorambucil-transferrin conjugates (T-5 and T-6) dem-
onstrate antiproliferative activity in two human tumor
cell lines (MCF7 and MOLT4) which is comparable to
or exceeds that of free chlorambucil. Especially, the
conjugate in which chlorambucil was bound to trans-
ferrin through an acetaldehyde carboxylic hydrazone (T-
5) revealed IC50 values which were significantly lower
than those of chlorambucil. In light of these results and
preliminary studies of acute toxicity, we will evaluate

2706 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Beyer et al.
Hz, 2-H), 4.14 (t, 2H, J ) 5.5 Hz, 4′-H), 6.65 (dd, 2H, J ) 9.5/
1.2 Hz, Ph), 7.03 (dd, 2H, J ) 9.5/1.2 Hz, Ph), 7.05 (s, 2H,
1′-H); 13C NMR (CDCl3) δ 26.21 (C-8), 32.71 (C-9), 33.13 (C-
7), 36.42 (C-3′), 41.08 (C-1), 52.17 (C-2), 61.81 (C-4′), 111.91,
129.19, 129.43, 144.44 (Ph), 134.53 (C-1′), 170.70 (C-2′), 172.52
(C-10); MS-EI m/z 426 (M+ - 1), 377, 286, 181. Anal.
(C20H24N2O4Cl2) C, H, N, Cl.
2H, J ) 8.5/1.3 Hz, Ph), 7.12 (t, 1H, J ) 10.2 Hz, 4′-H), 9.94
(s, 1H, NH); 13C NMR (CDCl3) δ 26.31 (C-8), 32.03 (C-9), 34.27
(C-7), 38.52 (C-3′), 40.65 (C-1), 53.72 (C-2), 112.37, 129.72,
131.01, 144.38 (Ph), 134.46 (C-1′), 138.83 (C-4′), 170.07 (C-2′),
176.28 (C-10); MS-FAB m/z 439 (M+), 286, 136. Anal.
(C20H24N4O3Cl2) C, H, N, Cl.
Ca r boxylic Hyd r a zon e Der iva tive 6 of 4-[4-(Bis(2-
ch lor oet h yl)a m in o)p h en yl]b u t yr ic Acid Hyd r a zid e (4)
a n d 3-Ma leim id oa cetop h en on e. 4-[4-(Bis(2-chloroethyl)-
amino)phenyl]butyric acid hydrazide (trifluoroacetate salt, 4;
0.71 g, 1.7 mmol) was dissolved in 30 mL of anhydrous THF
and 3-maleimidoacetophenone (0.54 g, 2.5 mmol) added at
room temperature. The reaction mixture was stirred for 36
h. The solution was evaporated in vacuo and the product
purified by crystallization from ethyl acetate: yield 0.54 g
(63%) of a yellow powder; mp 164 °C; Rf 0.27 (ethyl acetate/
O-[4-[4-(Bis(2-ch lor oeth yl)a m in o)p h en yl]bu ta n oyl]-3-
m a leim id op h en ol (2). N-Cyclohexyl-N′-(2-morpholinoethyl)-
carbodiimide metho-p-toluenesulfonate (1.6 g, 3.6 mmol) was
dissolved in 100 mL of CH2Cl2 and added dropwise within 2 h
to a solution of chlorambucil (1.0 g, 3.3 mmol), 3-maleimi-
dophenol (1.9 g, 9.9 mmol), and a catalytic amount of DMAP
(20 mg, 0.16 mmol) in 50 mL of anhydrous CH2Cl2. The
solution was stirred for a further 6 h at room temperature and
then filtered and the filtrate evaporated in vacuo. The residue
was chromatographed on a silica gel column (ethyl acetate/
hexane, 1:1) to yield 0.60 g (38%) of a yellow syrup: Rf 0.29
(ethyl acetate/hexane, 1:1); 1H NMR (DMSO-d6) δ 1.92 (tt, 2H,
J ) 6.7/6.5 Hz, 8-H), 2.58 (t, 2H, J ) 6.5 Hz, 9-H), 2.63 (t, 2H,
J ) 6.7 Hz, 7-H), 3.66 (t, 4H, J ) 6.2 Hz, 1-H), 3.68 (t, 4H, J
) 6.2 Hz, 2-H), 6.68 (dd, 2H, J ) 9.2/1.6 Hz, Ph), 7.09 (dd,
2H, J ) 9.2/1.6 Hz, Ph), 7.21 (s, 2H, 1′-H), 7.14-7.60 (m, 4H,
Ph); 13C NMR (DMSO-d6) δ 26.28 (C-8), 32.81 (C-9), 33.13 (C-
7), 41.09 (C-1), 52.18 (C-2), 111.96, 120.05, 121.16, 123.89,
129.29, 129.46 144.52, 129.32, 132.40, 150.38 (Ph), 134.64 (C-
1′), 169.54 (C-2′), 171.40 (C-10); MS-CI m/z 475 (M+), 457, 377,
304, 190. Anal. (C24H24N2O4Cl2) C, H, N, Cl.
4-[4-(Bis(2-ch lor oet h yl)a m in o)p h en yl]b u t a n oyl ter t-
Bu toxyca r bon yl Hyd r a zid e (3). Chlorambucil (1.0 g, 3.3
mmol) was dissolved in 50 mL of anhydrous CH2Cl2. To this
solution was added coxalyl chloride (431 µL, 4.8 mmol) and
the solution stirred for 15 h at T ) 35 °C. The yellow solution
was evaporated in vacuo. Remaining amounts of oxalyl
chloride were removed under high vacuum. The thus prepared
acid chloride of chlorambucil was dissolved in 20 mL of
anhydrous CH2Cl2, and tert-butyl carbazate (0.46 g, 3.5 mmol),
dissolved in 20 mL of anhydrous CH2Cl2, was added dropwise
while stirring at room temperature. The mixture was stirred
for 36 h at room temperature and then filtered and the filtrate
evaporated in vacuo. After chromatography over a silica gel
column (ethyl acetate/hexane 2:1) the product was obtained
in a yield of 0.70 g (51%) as a yellow oil: Rf value 0.52 (ethyl
acetate/hexane, 2:1); 1H NMR (CDCl3) δ 1.41 (s, 9H, C(CH3)3),
1.72 (tt, 2H, J ) 6.6/6.4 Hz, 8-H), 2.04 (t, 2H, J ) 6.4 Hz, 9-H),
2.46 (t, 2H, J ) 6.6 Hz, 7-H), 3.62 (s, 4H, 1-H), 3.69 (s, 4H,
2-H), 6.64 (dd, 2H, J ) 9.5/1.5 Hz, Ph), 7.03 (dd, 2H, J ) 9.5/
1.5 Hz, Ph), 8.59 (s, 1H, NH), 9.44 (s, 1H, NH). Anal.
(C19H28N3O3Cl2) C, H, N, Cl.
1
hexane, 3:2); H NMR (CDCl3) δ 2.02 (tt, 2H, J ) 7.6/7.3 Hz,
8-H), 2.21 (2s, 3H, CH3), 2.64 (t, 2H, J ) 7.6 Hz, 9-H), 2.81 (t,
2H, J ) 7.3 Hz, 7-H), 3.62 (t, 4H, J ) 5.8 Hz, 1-H), 3.70 (s,
4H, J ) 6.1 Hz, 2-H), 6.67 (d, 2H, J ) 8.8 Hz, Ph), 6.89 (s, 2H,
1′-H), 7.04 (d, 2H, J ) 8.8 Hz, Ph), 7.36-7.77 (m, 4H, Ph),
8.58 (2s, 1H, NH); 13C NMR (CDCl3) δ 12.50 (CH3), 26.51 (C-
8), 32.36 (C-9), 34.30 (C-7), 40.56 (C-1), 53.66 (C-2), 112.22,
123.72, 125.47, 126.65, 129.21, 129.73, 131.16, 131.62, 144.27,
145.22 (Ph), 134.31 (C-1′), 139.15 (C-9′), 169.34 (C-2′), 175.83
(C-10); MS-FAB m/z 515 (M+), 451, 216. Anal. (C26H28N4O3-
Cl2) C, H, N, Cl.
Ca r boxylic Hyd r a zon e Der iva tive 7 of 4-[4-(Bis(2-
ch lor oet h yl)a m in o)p h en yl]b u t yr ic Acid Hyd r a zid e (4)
a n d 3-Ma leim id oben za ld eh yd e. 4-[4-(Bis(2-chloroethyl)-
amino)phenyl]butyric acid hydrazide (trifluoroacetate salt, 4;
0.5 g, 1.2 mmol) was dissolved in 30 mL of anhydrous THF
and 3-maleimidobenzaldehyde (0.29 g, 1.4 mmol) added at
room temperature. The reaction mixture was stirred for 36
h. The solution was then evaporated in vacuo and the product
purified by chromatography over a LOBAR column (ethyl
acetate/hexane, 3:2) to yield 0.15 g (25%) of a pale-yellow
1
solid: mp 62 °C; Rf 0.43 (ethyl acetate/hexane, 2:1); H NMR
(CDCl3) δ 2.04 (tt, 2H, J ) 7.3/7.0 Hz, 8-H), 2.63 (t, 2H, J )
7.3 Hz, 9-H), 2.81 (t, 2H, J ) 7.0 Hz, 7-H), 3.62 (t, 4H, J ) 5.6
Hz, 1-H), 3.70 (s, 4H, J ) 5.6 Hz, 2-H), 6.65 (d, 2H, J ) 8.4
Hz, Ph), 6.92 (s, 2H, 1′-H), 7.11 (d, 2H, J ) 8.4 Hz, Ph), 7.34-
7.68 (m, 4H, Ph), 7.78 (s, 1H, 9′-H), 9.52 (2s, 1H, NH); 13C NMR
(CDCl3) δ 26.49 (C-8), 32.16 (C-9), 34.29 (C-7), 40.54 (C-1),
53.70 (C-2), 112.31, 124.50, 126.35, 127.39, 129.56, 129.76,
131.12, 131.94, 144.26, 145.28 (Ph), 134.35 (C-1′), 134.97 (C-
9′), 169.26 (C-2′), 176.20 (C-10); MS-FAB m/z 501 (M+). Anal.
(C25H26N4O3Cl2) Calcd: C, 59.9; H, 5.19; N, 11.2; Cl, 14.2.
Found: C, 60.2; H, 5.31; N, 10.9; Cl, 13.6.
Syn th esis of th e Tr a n sfer r in Con ju ga tes T-1, T-2, T-5,
T-6, T-7. All reactions were performed at room temperature
unless otherwise stated. Data for one representative experi-
ment is given.
4-[4-(Bis(2-ch lor oeth yl)a m in o)p h en yl]bu tyr ic Acid Hy-
d r a zid e (4). 4-[4-(Bis(2-chloroethyl)amino)phenyl]butanoyl
tert-butoxycarbonyl hydrazide (0.70 g, 1.7 mmol) was dissolved
in 10 mL of anhydrous THF. To the stirred solution was added
10 mL of trifluoroacetic acid, and the mixture stirred for 1 h.
Subsequently, the solvent was removed under high vacuum
and the resulting hydrazide of chlorambucil (trifluoroacetate
salt) reacted with 2-maleimidoacetaldehyde, 3-maleimidoac-
etophenone, and 3-maleimidobenzaldehye to obtain the hy-
drazone derivatives 5-7 as described below.
(1) Th iola tion of Tr a n sfer r in Usin g Im in oth iola n e: 64
mg of Tf was dissolved in 4.0 mL of degassed buffer (0.1 M
sodium borate, 0.001 M EDTA, 0.15 M NaCl, pH 8.0, c(Tf) ≈
4.0 × 10-4 M), and 66 µL of a 8 × 10-2 M iminothiolane‚HCl
solution (2.75 mg of iminothiolane‚HCl dissolved in 250 µL of
the same buffer) was added. After 60 min thiolated transferrin
was isolated through size exclusion chromatography (Sephadex
G-25F, Pharmacia; column, d ) 2.0 cm, l ) 10 cm; buffer,
standard borate). The average number of introduced HS
groups was 3.1. (A smaller number of thiol groups can be
introduced by reducing the amount of added iminothiolane,
e.g., 40 µL of the iminothiolane solution are added for
introducing two HS groups.) To ensure that Fe(III) had not
been released during thiolation, the Fe(III) concentration was
determined using ꢀ[Tf]465 ) 4650 M-1 cm-1 10 and showed that
samples of thiolated transferrin contained at least 95% iron.
The sample of thiolated transferrin (7.0 mL) was used directly
for the synthesis of the conjugate.
Ca r boxylic Hyd r a zon e Der iva tive 5 of 4-[4-(Bis(2-
ch lor oet h yl)a m in o)p h en yl]b u t yr ic Acid Hyd r a zid e (4)
a n d 2-Ma leim id oa ceta ld eh yd e. 4-[4-(Bis(2-chloroethyl)-
amino)phenyl]butyric acid hydrazide (trifluoroacetate salt, 4;
0.72 g, 1.7 mmol) was dissolved in 30 mL of anhydrous THF
and 2-maleimidoacetaldehyde (0.35 g, 2.5 mmol) added at room
temperature. The reaction mixture was stirred for 36 h. The
solution was then evaporated in vacuo and the product purified
by chromatography over a LOBAR column (ethyl acetate/
hexane, 2:1) to yield 0.30 g (45%) of a pale-yellow solid: mp
1
68 °C; Rf 0.31 (ethyl acetate/hexane, 2:1); H NMR (CDCl3) δ
1.90 (tt, 2H, J ) 6.5/6.4 Hz, 8-H), 2.54 (t, 2H, J ) 6.4 Hz, 9-H),
2.59 (t, 2H, J ) 6.5 Hz, 7-H), 3.63 (t, 4H, J ) 6.0 Hz, 1-H),
3.69 (t, 4H, J ) 6.0 Hz, 2-H), 4.37 (t, 2H, J ) 2.5 Hz, 3′-H),
6.63 (dd, 2H, J ) 8.5/1.3 Hz, Ph), 6.80 (s, 2H, 1′-H), 7.10 (dd,
(2) Rea ction of Ma leim id e Der iva tive 5 w ith Th iola ted
Tr a n sfer r in : 150 µL of a solution of 5 (Mr 439.1) in dimeth-
ylformamide (2.0 mg dissolved in 150 µL of dimethylforma-

Transferrin Conjugates of Chlorambucil
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2707
mide) was added to 7.0 mL of thiolated sample and homog-
enized, and the slightly turbid mixture was kept at room
temperature for 10 min. Concentration of this mixture to a
volume of approximately 2.0 mL was carried out with CEN-
TRIPREP-10-concentrators from Amicon, FRG (10 min at 4
°C and 4500 rpm). The concentrated sample was centrifuged
for 5 min with a Sigma 112 centrifuge, the supernatant loaded
on a Sephadex G-25F column (d ) 1.0 cm, l ) 10 cm; buffer,
standard borate), and the conjugate isolated. The concentra-
tion of bound chlorambucil was 390 ( 20 µM and that of
transferrin 135 ( 6 µM which corresponds to approximately
2.9 equivalents of chlorambucil bound to transferrin.
11.5 min. The UV absorption was detected at a wavelength
of 280 nm (11, 12) or 254 nm (8-10).
For stability studies in cell-conditioned culture medium 0.5
mL of the respective stock solution and 0.5 mL of acetonitrile
were added to 9.0 mL of cell-conditioned culture medium (from
MCF7 cells) and the samples incubated at T ) 37 °C. For
8-10 8.0 mL of 2-propanol had to be added to keep the
compounds in solution during the course of the experiment.
In each case three 400-µL samples were ultrafiltered with
Centrisart C-4 Eppendorfs (from Sartorius AG, FRG; 10 000
rpm, 45 min) at t ) 0, 24, 48, 72, 96, and 120 h, and 20 µL of
the filtrate was analyzed on a reverse-phase HPLC column
(acetonitrile/water, 1/1) and detected as described above.
Biology. Human tumor cells were grown at 37 °C in a
humidified atmosphere (95% air, 5% CO2) in monolayer (MCF7
cells) or suspension (MOLT4 cells) RPMI 1640 culture medium
with phenol red supplemented with 10% heat-inactivated FCS,
300 mg/L glutamine, and 1% antibiotic solution (5.000 µg of
gentamycin/mL). Cells were trypsinized and maintained twice
a week. The concentration of chlorambucil in the stock
solution of the conjugates was 720 µM; chlorambucil was
freshly dissolved in standard borate containing 5% ethanol at
the same concentration.
P r op id iu m Iod id e F lu or escen ce Assa y. The fluores-
cence assay was performed according to the method of Dengler
et al.30 Briefly, cells were harvested from exponential phase
cultures growing in RPMI culture medium by trypsinization,
counted, and plated in 96-well flat-bottomed microtiter plates
(50 µL of cell suspension/well, 1.0 × 105 cells/mL). After a 24-h
recovery in order to allow cells to resume exponential growth,
100 µL of culture medium (6 control wells/plate) or culture
medium containing drug was added to the wells. Each drug
concentration was plated in triplicate. After 6 days of continu-
ous drug exposure nonviable cells were stained by addition of
25 µL of a propidium iodide solution (50 µg/mL). Fluorescence
(FU1) was measured using a Millipore Cytofluor 2350 micro-
plate reader (excitation 530 nm, emission 620 nm). Micro-
plates were then kept at -18 °C for 24 h, which resulted in a
total cell kill. After thawing of the plates and a second
fluorescence measurement (FU2), the amount of viable cells
was calculated by FU2 - FU1. Growth inhibition was ex-
pressed as treated/control × 100 (%T/C).
Deter m in a tion of th e Alk yla tin g Activity of Der iva -
tives 1, 2, a n d 5-7 a n d Resp ective Tr a n sfer r in Con ju -
ga t es w it h 4-(4-Nit r ob en zyl)p yr id in e (NBP )sMod ified
Accor d in g to Ep stein et a l.16 All determinations were
carried out three times and mean values calculated.
(a ) Ch lor a m bu cil a n d Ch lor a m bu cil Der iva tives 1, 2,
a n d 5-7: 50-µL samples (c ≈ 300 µM) of chlorambucil or the
respective chlorambucil derivative dissolved in anhydrous THF
were filled into 10-mL tubes. To this solution were added 50
µL of acetic acid (5%), 100 µL of buffer (pH 7.4, 0.0025 M
sodium borate, 0.15 M NaCl) 195 µL of distilled water, 50 µL
of ethanol, and 55 µL of a NBP solution (500 mg of NBP
dissolved in 10 µL of ethanol). The sealed tubes were heated
for 90 min at 80 °C in a water bath and then cooled to room
temperature. To the samples was added 500 µL of a triethy-
lamine/acetone solution (1/1, v/v), and the resulting violet color
was determined at 565 nm by spectrophotometry against a
blank (ꢀ565 ) 15 100 M-1 cm-1).
(b) Tr a n sfer r in Con ju ga tes of Ch lor a m bu cil: 50-µL
samples of T-1, T-2, T-5, T-6, or T-7 were filled into tubes and
diluted with buffer to 100 µL. To this solution were added 50
µL of acetic acid (5%), 245 µL of distilled water, 50 µL of
ethanol, and 55 µL of the NBP solution. The samples were
heated for 90 min at 80 °C and analyzed as above.
For the cell culture experiments and serum stability studies
the alkylating activity of T-1, T-2, T-5, T-6 and T-7 was
adjusted to 720 or 1000 µM by concentrating the samples using
CENTRIPREP-10-concentrators.
Mod ified P r oced u r e for Deter m in a tion of th e Alk y-
la tin g Activity of th e Tr a n sfer r in -Ch lor a m bu cil Con -
ju ga tes In cu ba ted w ith 10% Ser u m . A 100-µL portion of
the respective conjugate serum sample (see below) was filled
into 10-mL tubes. To this solution were added 50 µL of acetic
acid (5%), 245 µL of water, 50 µL of ethanol, and 55 µL of the
NBP solution. The sealed tubes were heated for 90 min at 80
°C in a water bath and then cooled to room temperature; 400
µL of the samples was filled into Eppendorf tubes and
centrifuged for 5 min; 250 µL of the supernatant was mixed
with 250 µL of an Et3N/acetone solution (1/1, v/v), and the
resulting violet-colored solution was determined at 565 nm by
spectrophotometry against a blank (sample without conjugate).
F low Cyt om et r ic An a lysis of Tr a n sfer r in R ecep t or
Exp r ession . Adherent growing cells (MCF7) were detached
from culture dishes by trypsinization or employed as cell
suspension (MOLT4). The following steps were performed at
5 °C. Cells were washed twice with PBS and resuspended in
PBS at a density of 1 × 107 cells/mL; 5 µL of anti-human CD71
antibody (biotinylated, monoclonal antibody from mouse; Di-
anova, Hamburg) was added to 100 µL of cell suspension (106
cells), and cells were incubated for 45 min. Cells were then
washed twice with PBS and resuspended in 500 µL of PBS, 2
µL of streptavidin-DTAF [(dichlorortrianzinylamino)fluo-
roscein; Coulter Immunotech, Hamburg] was added, and cells
were incubated for 30 min. Cells were washed twice with PBS
and resuspended in 500 µL of PBS, and FACS analysis was
performed using a FACSort (Becton Dickinson, Heidelberg)
with the Lysis II software. Controls were prepared as de-
scribed above, except that isotype-specific antibody (anti-goat
IgG, biotinylated, monoclonal from mouse; Sigma) was used
instead of anti-CD71 antibody.
Toxicity Stu d ies. Toxicity studies were carried out at the
Chemistry Department of the University Heidelberg, Experi-
mental Division. Using NMRI mice (3 female mice/group) as
animal models, 20 mg/kg chlorambucil (5.0 mg of chlorambucil
dissolved in 10 mL of 0.3 M NaHCO3, 0.15 M NaCl) and T-5
(1650 µM with respect to its alkylating activity) were admin-
istered by ip injection on 3 consecutive days (due to solubility
problems of free chlorambucil, acute toxicity studies could not
be performed with one ip injection alone at higher doses so
that the above-mentioned administration schedule was cho-
sen). Whereas two out of three mice in the group treated with
free chlorambucil died within 1 or 2 days after the second and
third injections, no deaths were recorded in the conjugate
10% Ser u m Sta bility Stu d ies w ith T-1, T-2, T-5, T-6, T-7.
Preparation of the stock solution containing 10% serum: 330
µL of human blood serum was filled into a 10-mL glass tube.
To the serum were added 1970 µL of buffer (pH 7.4, 0.0025 M
sodium borate, 0.15 M NaCl) and 1000 µL of the respective
transferrin-chlorambucil conjugate (c ) 1000 µM). The stock
solution was incubated at 37 °C for the hydrolysis studies. The
alkylating activity of the samples (3 × 100 µL) was determined
at t ) 0, 2, 4, 6, 8, 24, 32, 48, 72, and 96 h.
Hyd r olysis Stu d ies of 8-12. Stock solutions of the
4-phenylbutyric acid derivatives (c ) 5 × 10-3 M for 8, 2 ×
10-3 M for 9-12) in acetonitrile were prepared; 150 µL of the
respective stock solution was added to 1350 µL of a buffer at
pH 5.0 (0.01 M NaOAc, 0.15 M NaCl) or of a buffer at pH 7.4
(0.15 M NaCl, 0.004 M NaH2PO4, 0.025 M NaHCO3). The
solutions were incubated at room temperature; 20-µL samples
of the solutions (pH 5.0 and 7.4) were analyzed at different
times (t ) 0, 2, 4, 8, 24, 32, 48, 72, and 96 h) on a reverse-
phase HPLC column (acetonitrile/water, 1/1): retention times
for 8, 16.9 min; 9, 28.9 min; 10, 10.3 min; 11, 16.0 min; 12,

2708 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Beyer et al.
(16) Epstein, J .; Rosenthal, R. W.; Ess, R. J . Use of γ-(4-Nitrobenzyl)-
pyridine as Analytical Reagent for Ethylenimines and Alkylating
Agents. Anal. Chem. 1955, 27, 1435-1439.
group even after 14 days of administration. No body weight
loss was observed in this group.
(17) Chatterji, D. C.; Yeager, R. L.; Galleli, J . F. Kinetics of Chloram-
bucil Hydrolysis Using High-Pressure Liquid Chromatography.
J . Pharm. Sci. 1982, 71, 50-54.
(18) The maleimide spacer molecules were not used for the synthesis
of the model compounds because the maleimide group is slowly
hydrolyzed at physiological pH making it difficult to evaluate
the stability of the ester and carboxylic hydrazone bond at pH
7.4 in the presence of the maleimide group.
Ack n ow led gm en t. This work has been supported
by the Dr. Mildred Scheel Stiftung der Deutschen
Krebshilfe, FRG, and the Deutsche Forschungsgemein-
schaft. We thank Borroughs Wellcome, FRG, for provid-
ing chlorambucil.
Su p p or tin g In for m a tion Ava ila ble: Syntheses and char-
acterization of 10-12; cell culture data of T-1, T-2, T-5, T-6,
T-7, and chlorambucil in MCF7 and MOLT4 cell lines; and
flow cytometric analysis of transferrin receptor expression in
MCF7 and MOLT4 cell lines (4 pages). Ordering information
is given on any current masthead page.
(19) Hwa, J . C. H.; Fleming, W. A. Sulfonation of ethyl γ-phenylbu-
tyrate with sulfuric acid. J . Org. Chem. 1957, 22, 1106-1107.
(20) Reeves R. L. In The Chemistry of the Carbonyl Group; Patai, S.,
Ed.; Interscience Publishers: New York, 1966; pp 567-619.
(21) When free chlorambucil was reacted with thiolated transferrin
and the protein separated by gel filtration, the collected sample
contained >2.5 HS groups/protein and less than 10% of chloram-
bucil with respect to the amount of protein present demonstrat-
ing that chlorambucil does not couple to thiolated proteins under
these experimental conditions.
Refer en ces
(1) Armitage, J . O. Leukaran (Chlorambucil). Current Clinical
Guide, 2nd ed.; Borroughs Wellcome Co. Oncology Products
Wellcome Oncology NCM Publ. Inc.: New York, 1993; pp 37-
45.
(2) Dorr, R. T.; Fritz, W. L. Cancer Chemotherapy Handbook;
Elsevier Science: New York, 1982; p 486.
(3) Kratz, F.; Hartmann, M.; Keppler, B. K.; Messori, L. The Binding
Properties of Two Antitumour Ruthenium(III) Complexes to
Apotransferrin. J . Biol. Chem. 1994, 269, 2581-2588.
(4) Kratz, F. Interactions of Antitumor Metal Complexes with
Serum Proteins. Perspectives for Anticancer Drug Development.
In Metal Complexes as Antitumor Agents; Keppler, B. K., Ed.;
VCH: Weinheim, 1993; pp 393-427.
(5) Testa, U.; Pelosi, E.; Peschle, C. The Transferrin Receptor. Crit.
Rev. Oncogen. 1993, 4, 241-276.
(6) Brock, J . H. Transferrins. In Metalloproteins, Part 2: Metal
Proteins with Nonredox Roles; Harrison, P. M., Ed.; VCH:
Weinheim, 1985; pp 183-263.
(7) Cazzola, M.; Bergamaschi, G.; Dezza, L.; Arosio, P. Manipula-
tions of Cellular Iron Metabolism for Modulating Normal and
Malignant Cell Proliferation: Achievements and Prospects.
Blood 1990, 75, 1903-1919.
(8) Wagner, E.; Curiel, D.; Cotten, M. Delivery of Drugs, Proteins
and Genes into Cells using Transferrin as a Ligand for Receptor-
mediated Endocytosis. Adv. Drug Delivery Rev. 1994, 14, 113-
135.
(22) Stability studies carried in 10% FCS did not differ significantly
from those performed in 10% human serum.
(23) Grunicke, H.; Doppler, W.; Hofmann, J .; Lindner, H.; Maly, K.;
Oberhuber, H.; Ringsdorf, H.; Roberts, J . J . Plasma Membrane
as Target of Alkylating Agents. Adv. Enzyme Regul. 1985, 24,
247-261.
(24) We confirmed
a pH-dependent stability of the transferrin
conjugates through the following experiment: T-1, T-2, T-5, T-6,
and T-7 were incubated at pH 7.4 and 5, and their time-
dependent stability was determined by HPLC (Biosil SEC 250
column) over a period of 6 days. Chlorambucil or hydrolysis
products such as chlorambucil hydrazide are seen on this column
at around 10.5-11.0 min (detection at 260 nm). When the acid-
sensitive conjugates T-5-T-7 were incubated at pH 5.0, they
showed a distinct peak at 10.5 min with time which steadily
increased in size during the course of the experiment in the order
T-5 > T-6 > T-7. At pH 7.4, however, only very small peaks were
observed after 6 days of incubation. The appearance of the peak
at 10.5 min at pH 5 was especially pronounced in T-5 which is
the conjugate with the highest in vitro activity. The conjugates
T-1 and T-2 showed only a small peak at 10.5-11.0 min at both
pH-values.
(25) Greenfield, R. S.; Kaneko, T.; Firestone, R. A. (6-Maleimidoca-
proyl)hydrazone of Doxorubicin - a New Derivative for the
Preparation of Immunoconjugates of Doxorubicin. Bioconjugate
Chem. 1993, 4, 521-527.
(26) Kratz, F.; Beyer, U.; Kru¨ger, M.; Schumacher, P.; Zahn, H.; Roth,
T.; Fiebig, H. H.; Unger, C. Synthesis of New Maleimide
Derivatives of Daunorubicin and Biological Activity of Acid
Labile Transferrin Conjugates. Biorg. Med. Chem. Lett. 1997,
7, 617-622.
(27) Fichtner, I.; Beyer, U.; Falken, U.; Meyer, G.; Schumacher, P.;
Unger, C.; Kratz, F. Efficacy of Acid Labile Transferrin and
Albumin Doxorubicin Conjugates in In Vitro and In Vivo Breast
Carcinoma Systems. 2nd International Symposium on Polymer
Therapeuticals, Kumamoto, J apan, 1997.
(28) Kratz, F.; Beyer, U.; Roth, T.; Tarasova, N.; Collery, P.; Lech-
eneault, F.; Cazabat, A.; Schumacher, P.; Unger, C.; Falken, U.
J . Pharm. Sci. 1998, 87, 338-346.
(29) Ellmann, G. L. Tissue Sulfhydryl Groups. Arch. Biochem.
Biophys. 1959, 82, 70-77.
(30) Dengler, W. A.; Schulte, J .; Berger, D. P.; Mertelsmann, R.;
Fiebig, H. H. Development of a Propidium Iodide Fluorescence
Assay for Proliferation and Cytotoxicity Assays. Anti-Cancer
Drugs 1995, 6, 522-532.
(9) Ghose, T.; Norvell, S. T.; Gulcu, A.; Cameron, D.; Bodurtha, A.;
MacDonald, A. Immunochemotherpy of Cancer with Chloram-
bucil Carrying Antibodies. Br. Med. J . 1972, 3, 495-499.
(10) Dullens, H. F.; De Weger, R. A.; Vennegoor, C.; Den Otter, W.
Antitumour Effect of Chlorambucil in
a Murine Melanoma
System. Eur. J . Cancer 1975, 15, 69-75.
(11) Smyth, M. J .; Pietersz, G. A.; Classon, B. J .; McKenzie, I. F. C.
Specific targeting of Chlorambucil to Tumours with the Use of
Monoclonal Antibodies. J . Natl. Cancer Inst. 1986, 76, 503-510.
(12) Ghose, T.; Blair, A. H. The Design of Cytotoxic-agent-antibody
Conjugates. Crit. Rev. Ther. Drug Carrier Syst. 1987, 3, 263-
359.
(13) Beyer, U.; Kru¨ger, M.; Schumacher, P.; Unger, C.; Kratz, F.
Synthesis of New Bifunctional Maleimide Compounds for the
Preparation of Chemoimmunoconjugates. Chem. Monthly 1997,
128, 91-102.
(14) Wellho¨ner, H. H.; Srinivasacher, K.; Neville, D. M.; Erdmann,
G. Uptake and Concentration of Bioactive Macromolecules by
K562 Cells via the Transferrin Cycle Utilizing an Acid-labile
Transferrin Conjugate. J . Biol. Chem. 1991, 266, 4309-4314.
(15) Benassi, R.; Taddei, F. Theoretical Study of Conformational
Changes in Simple Hydrazones. J . Chem. Soc., Perkin Trans. 2
1985, 10, 1629-1632.
J M9704661