Albumin conjugates of the anticancer drug chlorambucil: Synthesis, characterization, and in vitro efficacy

Article abstract of DOI:10.1002/(SICI)1521-4184(199802)331:2<47::AID-ARDP47>3.0.CO;2-R

In our efforts to improve the selectivity and toxicity profile of antitumor agents, four maleimide derivatives of chlorambucil (1-4) were bound to thiolated human serum albumin which differ in the stability of the chemical link between drug and spacer. 1

Full text of DOI:10.1002/(SICI)1521-4184(199802)331:2<47::AID-ARDP47>3.0.CO;2-R

Full Papers
Albumin Conjugates of the Anticancer Drug Chlorambucil:
Synthesis, Characterization, and In Vitro Efficacy
Felix Kratz*, Ulrich Beyer, Thomas Roth, Mark T. Schütte, Anuschka Unold, Heinz H. Fiebig, and Clemens Unger
Tumor Biology Center, Dept. Med. Oncology, Clin. Res., Breisacher Straße 117, 79106 Freiburg, Germany
Key Words: Chlorambucil; alkylating agents; human serum albumin; protein conjugates
Summary
accumulate in solid tumors due to the enhanced microvascu-
[4,5]
lature of tumor tissue
. This phenomenon has been termed
“
enhanced permeability and retention” in relation to tumor
[
6]
In our efforts to improve the selectivity and toxicity profile of
antitumor agents, four maleimide derivatives of chlorambucil (1–
targeting (“EPR-phenomenon”)
.
Due to our interest in the role which natural plasma proteins
4) were bound to thiolated human serum albumin which differ in
[7,8]
play in the in vivo distribution of anticancer drugs
, we
the stability of the chemical link between drug and spacer. 1 is an
aliphatic maleimide ester derivative of chlorambucil, whereas 2–4
are acetaldehyde, acetophenone, and benzaldehyde carboxylic hy-
drazone derivatives. HPLC stability studies at pH 5.0 with the
related model compounds 5, 7, 8, and 9, in which chlorambucil
was substituted by 4-phenylbutyric acid, demonstrated that the
carboxylic hydrazone derivatives have acid-sensitive properties;
the acid lability of 7 was particular prominent with a half-life of
only a few hours. The alkylating activity of albumin-bound
chlorambucil was determined with the aid of 4-(4-nitrobenzyl)-
pyridine (NBP), demonstrating that on average three equivalents
were protein-bound. Evaluation of the cytotoxicity of free chlor-
ambucil and the respective albumin conjugates in the MCF7
mamma carcinoma and MOLT4 leukemia cell line employing a
propidium iodide fluorescence assay demonstrated that the conju-
gate in which chlorambucil was bound to albumin through an ester
bond was not as active as chlorambucil. In contrast, the conjugates
in which chlorambucil was bound to albumin through carboxylic
hydrazone bonds were as or more active than chlorambucil in both
cell lines. In particular, the conjugate in which chlorambucil was
bound to albumin through an acetaldehyde carboxylic hydrazone
bond exhibited IC50 values which were approximately 4-fold
have developed chlorambucil conjugates of the serum protein
albumin. Besides the fact that albumin has a molecular weight
of 66 500, which is appropriate for the above concept of
passive tumor targeting, this serum protein is suitable as a
potential drug delivery system for the following reasons:
(a) Albumin exhibits a significant uptake in tumor tissue as
[9]
[10]
demonstrated by Matsumura et al.
b) it is a readily available protein in a pure and uniform form
and Sinn et al.
;
(
exhibiting good biological stability; (c) albumin is biodegrad-
able, non-toxic and non-immunogenic.
Chlorambucil was one of the first anticancer agents which
[
11–13]
was used to prepare antibody conjugates
these reports
. However, in
[
11,12]
chlorambucil was either physically ad-
sorbed or chemically linked to the carrier by mere incubation
or by direct coupling using N-hydroxysuccinimide/N,N′-di-
(
MCF7) to 13-fold (MOLT4) 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.
Introduction
®
Chlorambucil (Leukeran ) is a nitrogen mustard which is
used clinically against chronic lymphatic leukemia, lym-
[
1]
phomas, and advanced ovarian and breast carcinomas . 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]
.
A promising approach to circumvent the toxic side effects
of anticancer drugs on normal cells and to improve their
efficacy towards malignant cells is to couple anticancer drugs
to suitable carrier proteins or macromolecules. Expected ad-
vantages 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 adjust-
ment of the chemical properties of the bond between the drug
and the carrier. In addition, polymer or protein conjugates can
3]
Figure 1 Structures of the maleimide derivatives of chlorambucil 1–4.
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
0365-6233/98/0202/0047 $17.50 +.50/0

48
Kratz and co-workers
cyclohexylcarbodiimide (DCC) [13]. These direct methods up by the cell through endocytosis [6,15]. During internaliza-
have the disadvantage that during preparation polymeric tion the pH is reduced from 7.4 to 5.0, and this pH change can
products are likely to be formed and the resulting conjugates be exploited through acid-cleavage of a predetermined break-
are chemically not well defined with respect to the chemical ing point so that the drug can be released inside the tumor cell.
As a first step in anticancer drug development, unbound
chlorambucil as well as the albumin conjugates, which we
obtained with the above mentioned derivatives, were sub-
sequently evaluated for their inhibitory efficacy in two tumor
cell lines. In this paper we report on the synthesis and char-
acterization of the outlined albumin conjugates, their antipro-
liferative efficacy and the relationship between acid-
sensitivity, serum stability, and cytotoxicity.
link between drug and carrier protein.
For these reasons we recently synthesized maleimide de-
rivatives of chlorambucil which differed in the stability of the
chemical link (ester and carboxylic hydrazone bond) between
the drug and the spacer molecule (see Figure 1) [14]. The
introduced maleimide group is able to bind selectively to
sulfhydryl groups of carrier proteins. The rationale for vary-
ing the stability of the chemical link was to assess the signifi-
cance of the pH-dependent stability of the link between drug
and carrier for in vitro and in vivo activity. Albumin is taken
Results
Scheme 1
Synthesis of the model com-
pounds 5, 7, 8, and 9 and their
stability at pH 5.0. In order to
assess the significance of the
acid-sensitivity 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
in 1–4. Unfortunately, due to the
rapid hydrolysis of the chlorine
atoms of chlorambucil (detection
Scheme 2
of a number of peaks within a few
hours with the aid of HPLC [
16]
)
aqueous stability studies of 1–4
with respect to the chemical link
between the maleimide spacer
and chlorambucil could not be
carried out. We therefore synthe-
sized the model compounds 5, 7,
8
, and 9 in which chlorambucil
was substituted by 4-phenyl-
butyric acid.
The aliphatic ester of 4-phenyl-
butyric acid was synthesized by
reacting 4-phenylbutyric acid
with an excess of 2-hydroxy-
ethylmaleimide in CH Cl and
2
2
addition of N-cyclohexyl-N′-(2-
morpholinoethyl)-carbodiimide
metho-p-toluenesulfonate and
catalytic amounts of dimethyl-
aminopyridine (Scheme 1). The
carboxylic hydrazone deriva-
tives 7–9 were obtained by reac-
tion of 4-phenylbutyric acid
hydrazide (trifluoroacetate) [pre-
pared by reacting phenylbutyric
acid chloride with tert.-butylcar-
bazate and subsequent cleavage
with CF COOH] with 2-male-
3
imidoacetaldehyde, 3-maleim-
idoacetophenone, or 3-maleim-
idobenzaldehyde (Scheme 2).
Arch. Pharm. Pharm. Med. Chem. 331, 47–53 (1998)

Albumin Conjugates of Chlorambucil
49
, 7, 8, and 9 were characterized through H NMR and 13C
1
5
NMR spectroscopy as well as elemental analysis. Charac-
teristic peaks of the introduced maleimide groups are singlets
in the range from 6.8 to 7.2 ppm for the proton signals of the
1
double bond in the H NMR spectra and at 134–135 ppm and
1
69–170 ppm for the carbon atoms of the double bonds and
13
carbonyl groups in the C NMR spectra (recorded in
CDCl ). In the H NMR and C NMR spectra of 7–9 only
1
13
3
one set of signals is observed showing the presence of only
one isomer. From a steric point of view the E-stereoisomer is
favored, and we therefore tentatively suggest that this stereo-
isomer is present which is in accordance with studies on
simple hydrazones [
17]
.
The stability of 5, 7, 8, and 9 was studied at pH 5.0 on a
reverse-phase C-18-HPLC column. Stability studies were
carried out by first reacting 5, 7, 8, and 9 quantitatively in a
Michael addition with one equivalent of 2-mercaptoethanol
to produce the addition product as a single peak (5, 7, 8, and
9
were not used directly for HPLC-studies due to the slow
hydrolysis of the maleimide group at pH 5.0–7.0). The de-
crease of this peak area for 5, 7, 8, and 9 in the chromatograms
was used to calculate the respective half-lives. Whereas the
hydrolysis of 5 was slow (< 20% decomposition after 96 h),
the half-lives of the carboxylic hydrazones at pH 5.0 were t1/2
Figure 2. Chromatogram of A-2 at pH = 7.4. Chromatograms were per-
formed on a size-exclusion column and recorded at 280 nm. Retention times:
6.35 min (dimeric albumin conjugate), 7.20 min (monomeric albumin con-
jugate).
≈
4h for7, t1/2 ≈ 20 h for 8, and t1/2 ≈ 65 h for 9. Thishydrolytic
classification can be expected from the character of the
concentration of the conjugates was determined using the
BCA-protein-assay from Pierce (USA). In this way the num-
ber of equivalents of chlorambucil bound to albumin was
calculated. The ratio of chlorambucil/albumin in the conju-
gates was 2.7–3.0 which corresponded well with the number
of introduced HS-groups (see above).
When free chlorambucil was reacted with thiolated albumin
and the protein separated by gel filtration, the collected
sample contained > 2.5 HS-groups/protein and less than 10%
of chlorambucil with respect to the amount of protein present,
demonstrating that chlorambucil does not couple to thiolated
proteins under these experimental conditions.
chemical link involved [
18]
.
1)
Preparation of albumin conjugates (A-1, A-2, A-3, A-4) .
The albumin conjugates were prepared by reacting 1–4 with
thiolated albumin. The sulfhydryl group adds to the double
bond of the maleimide group in a fast and selective reaction
forming a stable thioether bond. During thiolation using
iminothiolane the formation of disulfide bonds was prevented
by addition of 0.001 M EDTA and degassing all buffers with
argon. Under these conditions the number of introduced
HS-groups was highly reproducible, an average number of
2.8–3.0 HS-groups being introduced. For preparing the con-
jugates the respective maleimide derivative was added to the
thiolated protein sample, and A-1, A-2, A-3, and A-4 were
Stability in 10% serum. In order to determine the loss of
alkylating activity of chlorambucil and our albumin conju-
gates A-1–A-4 in 10% serum, we incubated chlorambucil and
®
isolated by gel chromatography (Sephadex G-25). In order
to rule out the presence of unbound drug as well as polymeric
by-products which may be formed in the coupling step, the
purity of the conjugates was determined with an analytical
HPLC size exclusion column (Bio-Sil SEC 250). We have
previously shown that this column is suitable for separating
and detecting serum proteins in the presence of low-molecu-
lar weight compounds [ . As an example a typical chroma-
togram of A-2 recorded at λ = 280 nm is depicted in Fig. 2,
showing a main peak at 7.2 min corresponding to that of
monomeric albumin; 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 albumin also shows this
peak with a peak area of approximately 3–5%).
7]
The amount of chlorambucil bound to albumin was deter-
mined with the aid of the NBP-assay [ which was modified
accordingly (see Experimental Section) whereas the protein
19]
Figure 3. Time-dependent stability of the alkylating activity of the chloram-
bucil-albumin conjugates in comparison to free chlorambucil incubated with
10% human blood serum at 37 °C. Averages obtained in two independent
experiments are presented (determinations were carried out in triplicate).
Standard deviations were below ± 5% and are not shown; the value at t = 0 h
was set as 100%.
—
——
1
)
The abbreviations used are: HSA, human serum albumin; A-1, A-2, A-3,
A-4 refer to the respective chlorambucil albumin conjugates prepared with
the maleimide derivatives 1–4.
Arch. Pharm. Pharm. Med. Chem. 331, 47–53 (1998)

50
Kratz and co-workers
Table 2. Comparison of acute toxicity of chlorambucil and the chlorambucil-
albumin conjugate A-2 in NMRI mice.
the conjugates with 10% human serum at T = 37 °C and
determined the loss of alkylating activity with the aid of the
NBP-assay over a period of four days. The results of these
incubation studies are shown in Fig. 3. An initial rapid de-
crease of alkylating activity for free chlorambucil is observed
——————————————————————————————
Compound Dose, ip,
day 1, 2,
Number
of mice
Mortality
(days of
death)
Average increase
in body weight after
16 days
and 3
(
approximately 50% of activity remaining after 5 h). This
decrease in alkylating activity is significantly smaller for the
albumin conjugates A-1–A-4 (approximately 50% of activity
remaining after 25 to 35 h), there being little difference
between the individual albumin conjugates. Stability studies
carried out in 10% fetal calf serum (FCS) did not differ
significantly to those performed in 10% human serum (data
not shown).
——————————————————————————————
Chlor-
20 mg/kg
3
2 (2, 3)
9%
ambucil
A-2
20 mg/kg
3
0
22%
——————————————————————————————
Discussion
Cell culture experiments. The newly synthesized albumin
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. Respective IC50 val-
ues are summarized in Table 1. Albumin 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).
IC50 values were not reached for the albumin conjugate A-1
in both cell lines (concentration range 0.1–40 µM). In con-
trast, the conjugates A-3 and A-4 were as active as unbound
chlorambucil (IC50 values in the range of 2.5–4µM (MOLT4)
and 14–17 µM (MCF7) respectively). The most active com-
pound in both cell lines is A-2, in which chlorambucil was
bound to albumin through an acetaldehyde carboxylic hydra-
zone bond (IC50 values ≈ 0.3 µM (MOLT4), and ≈ 4 µM
Among naturally occurring proteins, albumin is a potential
carrier due to a suitable molecular size for passive tumor
targeting and due to its biocompatibility. We have therefore
coupled the anticancer drug chlorambucil to this serum pro-
tein. Preferably, these albumin conjugates should be prep-
arable in sufficient purity, they should exhibit stability at
physiological conditions (pH ≈ 7.4), and they should retain
the alkylating activity of the drug.
When preparing our conjugates, the maleimide derivatives
of chlorambucil 1–4 were coupled to thiolated albumin, and
the resulting conjugates were isolated with a purity of > 90%.
Our cell culture experiments demonstrate that the conjugate
in which chlorambucil was bound to albumin through an
acetaldehyde carboxylic hydrazone bond (A-2) revealed IC50
values which were significantly lower than those of chloram-
bucil. The aldehyde carboxylic hydrazone bond in the model
compound 7 shows marked acid-sensitivity with a half-life of
(MCF7) respectively).
Table 1. IC50 values (µM)a for the chlorambucil-albumin conjugates in
comparison to free chlorambucil in the MCF7 mamma carcinoma and
MOLT4 leukemia cell line.
2)
approximately 4 h . The conjugates A-3 and A-4, in which
acid-sensitivity is not that pronounced (half-lives of the
model compounds 8 and 9 in the range of 20–70 h at pH 5.0)
exhibit antiproliferative activity which is comparable to that
of chlorambucil. In contrast, the albumin conjugates prepared
with 1, which contains an ester bond, is not as active as
chlorambucil, IC50 values not being reached in the tested
concentration range.
—
—————————————————————————————
Compound
MCF7
MOLT4
——————————————————————————————
Chlorambucil
14.3 ± 1.1
–
3.9 ± 0.3
–
A-1
A-2
A-3
A-4
These results taken together indicate that antiproliferative
in vitro activity is correlated with the chemical link between
chlorambucil and albumin. The importance of the acid-sen-
sitivity of the linker between anthracyclines and the carrier
protein for in vitro and in vivo anticancer activity has been
3.7 ± 0.3
17.7 ± 1.3
15.8 ± 1.2
0.3 ± 0.02
2.9 ± 0.2
2.7 ± 0.3
[
20]
[21,22]
noted by Greenfield et al.
and our group
. Endocy-
——————————————————————————————
a
tosis is presumably responsible for cellular uptake with sub-
sequent release of chlorambucil in acidic endosomes and/or
IC50 values (50% inhibitory concentration) represent the mean ± standard
deviation from three independent experiments. Concentrations refer to the
equialkylating activity of the respective compounds.
———
2
)
We confirmed the acid-sensitivity of the albumin conjugates through the
following experiment: A-1 – A-4 were incubated at pH 7.4 and pH 5 and
their time-dependent stability was determined by HPLC (Biosil SEC 250
column) over a period of six 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 A-2 and A-4 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
A-2 > A-3 > A-4. 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 A-2 which is the conjugate with the highest in
vitro activity. The conjugate A-1 showed only a small peak at 10.5–11.0 at
both pH-values.
Preliminary toxicity studies. Subsequently, chlorambucil
and A-2 were subjected to acute toxicity studies (dose:
0 mg/kg per day with respect to the amount of chlorambucil
present; administration: ip (intraperitoneal), three consecu-
tive days). Whereas two out of three mice in the group treated
with free chlorambucil died within two or three days after the
first injection, no mortality was observed in the conjugate
group even after 16 days (see Table 2). An increase in body
weight was observed in this group, which is an indication of
very low systemic toxicity.
2
Arch. Pharm. Pharm. Med. Chem. 331, 47–53 (1998)

Albumin Conjugates of Chlorambucil
51
LKB 2150 pump (flow: 1.5 mL/min) and 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 dissolved in 0.15 M
NaCl, 0.01 M NaHCO3, 0.004 M NaH2PO4, pH 7.4, and a 50 µL sample was
injected.
lysosomes, which is well documented for macromolecules
[6,15]
and serum albumin
.
When comparing the serum stability of chlorambucil and
the albumin conjugates with respect to their alkylating activ-
ity, we found an increased stability of the conjugates over a
period of fourdays(Fig. 3). However, there was no significant
difference between the conjugates themselves indicating that
the superior antiproliferative activity of A-2, A-3, and A-4
over A-1 observed in vitro cannot, on the whole, be ascribed
to the prolonged alkylating activity of chlorambucil albumin
conjugates, but rather to their acid-sensitive properties.
Preliminary in vivo studies comparing the toxicity of
Synthesis of 5, 7, 8, and 9
4-Phenylbutanoyl-2-hydroxyethylmaleimide (5). 4-Phenylbutyric acid
(1.0 g, 6.08 mmol), 2-hydroxyethylmaleimide (1.72 g, 12.16 mmol), and a
catalytic amount of 4-dimethylaminopyridine (DMAP; 20 mg, 0.16 mmol)
were dissolved in 100 mL anhydrous CH2Cl2 at room temperature. To this
solution N-cyclohexyl-N′-(2-morpholinoethyl)-carbodiimide metho-p-tolu-
enesulfonate (2.97 g, 6.68 mmol), dissolved in 100 mL anhydrous CH2Cl2,
was added dropwise within 1 h and the solution stirred for further 8 h at room
temperature. The turbid solution was filtered and evaporated in vacuo. The
residue was dissolved in 50 mL ethyl acetate, filtered, and chromatographed
on a silica gel column (ethyl acetate/hexane 1:2) to yield 1.33 g (76%) pale
“
equialkylating” high doses of chlorambucil and A-2 in mice
have shown that the albumin conjugate was better tolerated
and no mortality was observed.
In conclusion, our results show that an acid-sensitive link
between chlorambucil and albumin is an effective way of
retaining or improving the alkylating activity of chloram-
bucil. In the light of these results and preliminary studies of
acute toxicity, we will evaluate the antitumor efficacy of
active albumin conjugates of chlorambucil in animal tumor
models.
1
yellow syrup; Rf 0.30 (ethyl acetate/hexane 1:2).– H NMR (CDCl3): λ =
1.92 (tt, 2H, J = 6.7/6.5 Hz, 6-H), 2.29 (t, 2H, J = 6.5 Hz, 7-H), 2.63 (t, 2H,
J = 6.7 Hz, 5-H), 3.79 (t, 2H, J = 6.5 Hz, 3′-H), 4.24 (t, 2H, J = 6.5, 4′-H),
13
6
2
1
.70 (s, 1H, 1′-H), 7.14–7.32 (m, 5H, aromatic H).– C NMR (CDCl3): δ =
6.21 (C-6), 33.38 (C-7), 35.08 (C-5), 36.99 (C-3′), 61.31 (C-4′), 125.99,
28.39, 128.49, 141.37 (aromatic C), 134.24 (C-1′), 170.40 (C-2′), 173.21
(C-8) ppm.– Anal. (C16H17NO4).
4
-Phenylbutanoyl tert.-butoxycarbonyl hydrazide (6). 4-Phenylbutyric
acid (5.0 g, 30.4 mmol) was dissolved in 200 mL anhydrous CH2Cl2. Oxalyl
chloride (3.97mL, 44.2 mmol) was added to this solution 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 was dissolved in 100 mL anhydrous CH2Cl2
and tert.-butylcarbazate (4.25 g, 32.2 mmol), dissolved in 100 mL anhydrous
CH2Cl2, added dropwise while stirring at room temperature. The mixture
was stirred for 36 h at room temperature, then filtered, and the filtrate
evaporated in vacuo. After chromatography over a silica gel column (ethyl
acetate/hexane 1:2) the product was crystallized from ethyl acetate/hexane
in a yield of 6.95 g (82%) as a light brown solid; mp 94 °C.– Rf value: 0.15
Acknowledgements
This work has been supported by the Dr. Mildred Scheel Stiftung der
Deutschen Krebshilfe, FRG and the Deutsche Forschungsgemeinschaft. We
thank Burroughs Wellcome, FRG for providing chlorambucil. The assistance
of Heike Vongerichten in carrying out animal experiments is gratefully
acknowledged.
Experimental
1
1
Chemicals, Materials, and Spectroscopy. Melting points: Büchi 530.– H
(ethyl acetate/hexane 1:2).– H NMR (CDCl3): δ = 2.05 (tt, 2H, J = 6.6/6.4
1
3
NMR and C NMR: Bruker 400 MHz AM 400, Varian 300 (internal
standard: TMS); elemental analysis:Perkin-Elmer Elemental Analyzer 240.–
Analytical HPLC: reverse phase HPLC-column (Spherisorb-C18 ODS 2–
Hz, 6-H), 2.23 (t, 2H, J = 6.4 Hz, 7-H), 2.68 (t, 2H, J = 6.6 Hz, 5-H), 6.63
(s, 1H, NH), 7.15–7.36 (m, 5H, aromatic H), 7.61 (s, 1H, NH).– Anal.
(C15H22N2O3).
5
µm from MedChrom, Heidelberg, FRG); silica gel chromatography on
silica gel 60 (0.063–0.100 mm) from Merck KG; TLC: silica coated plates
0 F254 from Merck AG; Chlorambucil (Mr 304.20) was a gift from Bur-
4-Phenylbutyric acid hydrazide (trifluoroacetate salt). 4-Phenylbutanoyl
tert.-butoxycarbonyl hydrazide (0.70 g, 2.51 mmol) was dissolved in 10 mL
anhydrous THF. To the stirred solution were added 10 mL trifluoroacetic
acid, and the mixture was stirred for 1 h. Subsequently, the solvent was
removed under high vacuum and the resulting hydrazide (trifluoroacetate
salt) reacted with 2-maleimidoacetaldehyde, 3-maleimidobenzaldehyde or
3-maleimidoacetophenone to obtain the hydrazone derivatives 7–9 as de-
scribed below.
6
roughs Wellcome, FRG. Organic solvents: HPLC grade (Merck KG) or
analytical grade (gift from BASF AG).– Other organic or inorganic com-
pounds: Merck KG, FRG. Maleimide spacer molecules were prepared pre-
viously . Materials for the preparation of conjugates: human serum
albumin (HSA) (98%, crystalline, Mr 66 500), iminothiolane HCl, 5,5′-
dithio-bis-(2-nitrobenzoic acid), 4-(4-nitrobenzyl)pyridine, and propidium
iodide were purchased from Aldrich-Sigma-Chemie, FRG. The buffers used
were vacuum-filtered through a 0.2 µm membrane (Sartorius, FRG) and
thoroughly degassed with argon prior to use. Cell culture media, supplements
[23]
Carboxylic hydrazone derivative 7 of 4-phenylbutyric acid hydrazide and
2
-maleimidoacetaldehyde. 4-Phenylbutyric acid hydrazide (trifluoroacetate
salt; 0.73 g, 2.51 mmol) was dissolved in 30 mL anhydrous THF and
-maleimidoacetaldehyde (0.39 g, 2.75 mmol) added at room temperature.
2
The reaction mixture was stirred for 36 h. The solution was then evaporated
in vacuo and the residue crystallized from ethyl acetate/hexane to yield 0.34 g
(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).
Methods for the preparation of conjugates. 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 sam-
(
45%) of a white solid.– Mp 113 °C.– Rf 0.27 (ethyl acetate/hexane 1:1).–
H NMR (CDCl3): δ = 1.90 (tt, 2H, J = 6.5/6.4 Hz, 6-H), 2.51 (t, 2H, J = 6.4
1
Hz, 7-H), 2.65 (t, 2H, J = 6.5 Hz, 5-H), 4.33 (t, 2H, J = 2.5 Hz, 3′-H), 6.70
(s, 2H, 1′-H), 7.12 (t, 1H, 4′-H), 7.18–7.39 (m, 5H, Ph), 9.94 (s, 1H, NH).–
13
C NMR (CDCl3): δ = 25.99 (C-6), 32.02 (C-7), 35.08 (C-5), 38.44 (C-3′),
–1
–1
ples was determined using the ε values for albumin ε280 = 35 700 M cm
125.88, 128.35, 128.45, 141.66 (aromatic C), 134.38 (C-1′), 138.72 (C-4′),
170.04 (C-2′), 176.08 (C-8) ppm.– Anal. (C16H17N3O3).
[24]
and the concentration of HS-groups with Ellmanns reagent ε412 =
–
1
–1 [25]
13 600 M cm
with a double-beam UV/VIS-spectrophotometer
Carboxylic hydrazone derivative 8 of 4-phenylbutyric acid hydrazide and
3-maleimidoacetophenone. 4-Phenylbutyric acid hydrazide (trifluoroacetate
salt; 0.73 g, 2.51 mmol) was dissolved in 30 mL anhydrous THF and
3-maleimidoacetophenone (0.59 g, 2.75 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:
0.77 g (82%) yellow powder.– Mp 148 °C.– Rf 0.41 (ethyl acetate/hexane
U-2000 from Hitachi. The protein concentration of the conjugates was
determined using the BCA-protein assay from Pierce (USA). The amount of
chlorambucil bound to albumin was determined using a modified
4
-(4-nitrobenzyl)pyridine assay based on the assay of Epstein et al. [19] (see
below). The purity of the albumin 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
1
2:1).– H NMR (CDCl3): δ = 2.08 (tt, 2H, J = 6.7/6.5 Hz, 6-H), 2.27 (s, 3H,
Arch. Pharm. Pharm. Med. Chem. 331, 47–53 (1998)

52
Kratz and co-workers
CH3), 2.73 (t, 2H, J = 6.5 Hz, 7-H), 2.84 (t, 2H, J = 6.7 Hz, 5-H), 6.84 (s,
90 minutes at 80 °C in a water bath and then cooled to room temperature.
400 µL of the samples were filled into Eppendorf tubes and centrifuged for
5 minutes. 250 µL of the supernatant were mixed with 250 µL of a Et3N/ace-
tone solution (1/1, v/v), and the resulting violet colored solution was deter-
mined at 565 nm by spectrophotometry against a blank (sample without
conjugate).
1
3
2
(
1
(
H, 1′-H), 7.15–7.72 (m, 9H, aromatic H), 9.75 (s, 1H, NH).– C NMR-data
CDCl3): δ = 12.68 (CH3), 26.33 (C-6), 32.33 (C-7), 35.48 (C-5), 123.77,
25.52, 125.82, 126.64, 128.31, 128.56, 129.22, 131.56, 141.88, 145.53
aromatic C), 134.30 (C-1′), 139.23 (C-9′), 169.38 (C-2′), 176.05 (C-8)
ppm.– Anal. (C22H21N3O3).
Serum stability studies (10% serum). Preparation of the stock solution
containing 10% serum: 330 µL human blood serum were filled into a 10 mL
glass tube. To the serum were added 1970 µL buffer (pH = 7.4; 0.0025 M
sodium borate, 0.15 M NaCl) and 1000 µL of the respective albumin-
chlorambucil conjugate (c = 1000 µM). The stock solution was incubated at
Carboxylic hydrazone derivative 9 of 4-phenylbutyric acid hydrazide and
3
-maleimidobenzaldehyde. 4-Phenylbutyric acid hydrazide (trifluoroacetate
salt; 0.50 g, 1.72 mmol) was dissolved in 50 mL anhydrous THF and
-maleimidobenzaldehyde (0.42 g, 2.06 mmol) added at room temperature.
The reaction mixture was stirred for 24 h. The solution was evaporated in
vacuo and the product purified by repeated crystallization from ethyl ace-
3
3
1
7 °C for the hydrolysis studies. The alkylating activity of the samples (3 ×
00 µL) was determined at t = 0, 2, 4, 6, 24, 32, 48, and 72 h.
tate/hexane.– Yield: 0.45 g (72%) yellow powder.– Mp 108 °C.– Rf 0.24
1
(
ethyl acetate/hexane 2:1).– H NMR (CDCl3): δ = 2.10 (tt, 2H, J = 6.8/6.7
Hydrolysis studies with 5, 7, 8, and 9 at pH 5.0. Stock solutions of 5, 7, 8,
Hz, 6-H), 2.74 (t, 2H, J = 6.8 Hz, 7-H), 2.80 (t, 2H, J = 6.7 Hz, 5-H), 6.89
–2
and 9 (c = 10 M) in acetonitrile were prepared. To 50 µL of the respective
(
s, 2H, 1′-H), 7.12–7.64 (m, 9H, aromatic H), 7.81 (s, 1H, 9′-H), 9.84 (s, 1H,
–2
stock solution were added 50 µL of a mercaptoethanol solution (10 M in
1
3
NH).– C NMR-data (CDCl3): δ = 26.36 (C-6), 32.16 (C-7), 35.44 (C-5),
acetonitrile) and 900 µL of buffer (0.01 M NaOAc, 0.15 M NaCl; pH 5.0).
The solutions were incubated at room temperature for 30 minutes. Then
1
1
(
24.51, 125.81, 126.35, 127.28, 128.30, 128.536, 129.49, 131.82, 135.12,
41.76 (aromatic C), 134.29 (C-1′), 142.16 (C-9′), 169.24 (C-2′), 176.18
C-8) ppm.– Anal. (C21H19N3O3).
Synthesis of the albumin conjugates A-1–A-4. All reactions were per-
formed at room temperature unless otherwise stated. Data for one repre-
sentative experiment is given:
20 µL samples were analyzed at t = 0, 2, 4, 8, 24, 32, 48, 72, and 96 h on a
reverse phase HPLC-column (acetonitrile/water 40/60 for 7–9; acetoni-
trile/water 50/50 for 5); retention times for 5: 4.4 min, 7: 7.1 min, 8: 3.62
min, 9: 8.1 min. The UV-absorption was detected at a wavelength of 212 nm
(
5, 7) or 280 nm (8, 9).
(1) Thiolation of albuminusing iminothiolane –54 mg HSA weredissolved
Biology. Human tumor cells were grown at 37 °C in a humidified atmos-
in 4.0 mL degassed buffer (0.1 M sodium borate, 0.001 M EDTA, 0.15 M
phere (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
–
4
–2
NaCl – pH = 8.0, c (HSA) ≈ 2.0 × 10 M) and 130 µL of a 4 × 10
M
.
.
iminothiolane HCl solution (5.5 mg iminothiolane HCl dissolved in 1.0 mL
of the same buffer) were added. After 60 min thiolated albumin is 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 3.0 . The sample of thiolated albumin (7.0–
.5 mL) was used directly for the synthesis of the conjugate.
2) Reaction of maleimide derivative 2 with thiolated albumin – 150 µL of
(
5.000 µg gentamycin/mL). Cells were trypsinized and maintained twice a
week. The concentration of chlorambucil in the stock solution of the conju-
gates was 720 µM; chlorambucil was freshly dissolved in standard borate
containing 5% ethanol at the same concentration.
3)
7
Propidium iodide fluorescence assay: The fluorescence assay was per-
formed according to the method of Dengler et al.[ . Briefly, cells were
harvested from exponential phase cultures growing in RPMI culture medium
by trypsinization, counted, and plated in 96 well flat-bottomed microtitre
26]
(
a solution of 2 (Mr 439.1) in dimethylformamide (2.0 mg dissolved in 150
µL dimethylformamide) were added to 7.0 mL thiolated sample, homoge-
nized and the slightly turbid mixture was kept at room temperature for
5
plates (50 µL cell suspension/well, 1.0 × 10 cells/mL). After a 24 h recovery
1
0 min. Concentration of this mixture to a volume of approximately 2.0 mL
in order to allow cells to resume exponential growth, 100 µL culture medium
(6 control wells per plate) or culture medium containing drug was added to
the wells. Each drug concentration was plated in triplicate. After 6 days of
continuous drug exposure nonviable cells were stained by addition of 25 µL
of a propidium iodide solution (50 µg/mL). Fluorescence (FU1) was meas-
ured using a Millipore Cytofluor 2350 microplate reader (excitation 530 nm,
emission 620 nm). Microplates were then kept at –18 °C for 24 h, which
resulted in a total cell kill. After thawing of the plates and a second fluores-
cence measurement (FU ) the amount of viable cells was calculated by FU
®
was carried out with CENTRIPREP -10-concentrators from Amicon, FRG
10 min at 4 °C and 4500 rpm). The concentrated sample was centrifuged for
min with a Sigma 112 centrifuge, the supernatant loaded on a Sephadex
(
5
G-25F column (d = 1.0 cm, l = 10 cm) and the conjugate isolated (retention
volume: 3.6 – 7.5 mL, buffer: standard borate). The concentration of bound
chlorambucil was 334 ± 15 µM and that of albumin 118 ± 8 µM which
corresponds to approximately 2.8 equivalents of chlorambucil bound to
albumin.
2
2
Determination of the alkylating activity of the albumin conjugates A-1–A-4
with 4-(4-nitrobenzyl)pyridine (NBP) – modified according to Epstein et
–
FU1. Growth inhibition was expressed as treated/control × 100 (%T/C).
Toxicity studies. Toxicity studies were carried out at the Chemistry De-
[19]
al.
All determinations were carried out three times and mean values
partment of the University Heidelberg, Experimental Division. Using NMRI
mice (three female mice per group) as animal models, 20 mg/kg of chloram-
bucil (5.0 mg chlorambucil dissolved in 10 mL 0.3 M NaHCO3, 0.15 M
NaCl) and A-2 (1650 µM with respect to its alkylating activity) were
administered by ip injection on three 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 chosen). Relevant data are summarized in
Table 2.
calculated. 50 µL samples of A-1, A-2, A-3, or A-4 were filled into tubes and
diluted with standard borate to 100 µL. To this solution were added 50 µL
acetic acid (5%), 245 µL distilled water, 50 µL ethanol, and 55 µL of a NBP
solution (500 mg NBP dissolved in 10 mL 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 were added 500 µL of a triethylamine/acetone
solution (1/1, v/v), and the resulting violet color was determined at 565 nm
–
1
–1
by spectrophotometry against a blank (ε565 = 15 100 M cm ).
For the cell culture experiments and serum stability studies the alkylating
activity of the A-1, A-2, A-3, and A-4 was adjusted to 720 or 1000 µM by
concentrating the samples using CENTRIPREP -10-concentrators.
References
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albumin-chlorambucil conjugates incubated with 10% serum. 100 µL of the
respective conjugate serum sample (see below) were filled into 10 mL tubes.
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ethanol, and 55 µL of the NBP solution. The sealed tubes were heated for
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1
Arch. Pharm. Pharm. Med. Chem. 331, 47–53 (1998)

Albumin Conjugates of Chlorambucil
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Received: August 29, 1997 [FP244]
Arch. Pharm. Pharm. Med. Chem. 331, 47–53 (1998)