Development of dual-acting prodrugs for circumventing multidrug resistance

Article abstract of DOI:10.1016/j.bmcl.2008.11.063

We have developed a novel dual-acting maleimide-bearing prodrug that incorporates the anticancer agent doxorubicin and an inhibitor of the P-glycoprotein efflux pump that is over-expressed in multidrug resistant tumor cells. Additionally, the prodrug cont

Full text of DOI:10.1016/j.bmcl.2008.11.063

Bioorganic & Medicinal Chemistry Letters 19 (2009) 995–1000
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of dual-acting prodrugs for circumventing multidrug resistance
*
Khalid Abu Ajaj, Felix Kratz
Tumor Biology Center, Macromolecular Prodrugs, Breisacher Strasse 117, D-79106 Freiburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed a novel dual-acting maleimide-bearing prodrug that incorporates the anticancer
agent doxorubicin and an inhibitor of the P-glycoprotein efflux pump that is over-expressed in multidrug
resistant tumor cells. Additionally, the prodrug contains a 1,6-self-immolative spacer coupled to the
dipeptide Phe-Lys that acts as a substrate for cathepsin B. The prodrug, once bound through its maleimide
moiety to the cysteine-34 group of human serum albumin, was cleaved by cathepsin B and in tumor
homogenates demonstrating a release of the anticancer agent doxorubicin and the inhibitor.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 9 October 2008
Revised 17 November 2008
Accepted 18 November 2008
Available online 24 November 2008
Keywords:
Multidrug resistance
Albumin-binding prodrug
Doxorubicin
Intrinsic or acquired multidrug resistance (MDR) is a major
problem in the treatment of malignant diseases. A number of bio-
chemical mechanisms have been described that are responsible for
the multidrug resistance phenotype that include changes in the cel-
lular target of the respective drug, alterations in enzymatic activa-
tion and detoxification mechanisms, defective apoptotic pathways,
membranechangesaswellaseliminationof thedrugfromthetumor
cell through the action of drug efflux pumps.1 A frequent cause of
drug resistance results from an elevated expression of the cell-mem-
brane transporters that function by increasing the efflux of cytotoxic
drugs from cancer cells and allows them to survive by lowering the
intracellular concentrations of the drugs. The cell-membrane efflux
pumps include P-glycoprotein (P-gp), multiple resistance protein
(MRP) and breast cancer resistance protein (BCRP) which belong to
the ATP-binding cassette (ABC) transporter family.2–4
The best-studied efflux pump is the P-glycoprotein (ABCB1),
energy-dependent, transmembrane efflux pump, which is a 170-
kDa cell-membrane protein encoded by the MDR1 gene. Its bio-
chemistry and pharmacology has been intensely studied.5,6 The
cytotoxic drugs that are most frequently associated with MDR and
which are substrates for the P-glycoprotein are hydrophobic as well
as amphiphilic compounds including taxanes, vinca alkaloids, anth-
racyclines, epipodophyllotoxins, actinomycinD, andmitomycinC.7,8
A great number of studies have evaluated the expression of
MDR1 mRNA or P-gp demonstrating an over-expression in leuke-
mia and solid tumors.9 For example, data from studies comprising
breast cancer patients treated with cytotoxic agents showed a sig-
nificant increase in the proportion of patients with tumors express-
ing P-gp subsequent to therapy.9 In addition, patients with P-gp
expressing tumors were threefold less likely to achieve an objec-
tive response after treatment compared with patients whose
tumors were MDR1/P-gp negative.10 The extent of P-gp expression
correlated with in vitro resistance to doxorubicin and paclitaxel in
functional assays.11
Consequently, there has been a concerted effort over the past 20
years to develop specific P-gp modulators that circumvent MDR.12
MDR modulators of the first generation in combination with stan-
dard chemotherapy regimens demonstrated initial success in clini-
cal trials against retinoblastoma13 and acute myeloid leukemia.14
However, due to the lack of specificity and selectivity of 1.generation
MDR modulators high doses were required resulting in adverse
effects, for example, cardiotoxicity and immunosuppression.5,6,15
Furthermore, the MDR modulators were substrates for metabolic
enzyme systems, for example, the P450 cytochrome system (P450
3A4), resulting in unpredictable modification of the pharmacoki-
netic profile of the co-administered anticancer agents with a con-
comitant dose reduction of the latter.2
MDR modulators of the second generation were analogues of
the 1.generation modulators.16 Although these drugs were better
tolerated, they still altered the pharmacokinetic profile of the co-
administered anticancer drugs and required a dose reduction of
60% or more.17–21
3.generation MDR modulators demonstrated high selectivity for
P-gp with inhibitory activities in the low nanomolar range. Several
clinical studies have been performed and demonstrated that the
pharmacokinetic profile of co-administered drugs was not
altered.2,22–25 Although some objective responses were achieved,
combination therapy with these MDR modulators caused unac-
ceptable toxicity, for example, severe neurotoxicity.26
In summary, although other resistance mechanisms play a role
in solid tumors due to the heterogeneity and genomic instability of
* Corresponding author. Tel.: +49 761 2062930; fax: +49 761 2062905.
E-mail address: felix@tumorbio.uni-freiburg.de (F. Kratz).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.063

996
K. Abu Ajaj, F. Kratz / Bioorg. Med. Chem. Lett. 19 (2009) 995–1000
Phe-Lys-PABC-P-gp-inhibitor
O
P-gp-inhibitor
drug
O
N
N
Phe-Lys-PABC-doxorubicin
O
O
Figure 1. General design of albumin-binding dual-acting prodrugs.
the tumor cell population, it is feasible that P-gp is responsible for
multidrug resistance in a defined subpopulation of cancer patients
and thus represents a molecular target for circumventing MDR.
inhibitor of P-gp are transported simultaneously on a molecular
level to resistant cancer cells that express P-gp, thus preventing
the efflux of the anticancer drug with a concomitant increase in
tumor cell death.
Therapeutic strategies that combine
a MDR modulator with
anticancer agents have the following inherent disadvantages27
:
For the design of the prodrug (see Fig. 1), we selected to
introduce the dipeptide Phe-Lys that is cleaved by cathepsin B, a
cysteine protease that is over-expressed in solid tumors. Recently,
we described the synthesis of maleimide bifunctional linkers that
can be applied in the synthesis of dual-acting prodrugs with enzy-
matically cleavable linkers.28 The linkers contain two arms with
the 1,6-self-immolative para-aminobenzyloxycarbonyl (PABC)
spacer and the dipeptide Phe-Lys. In addition, they contain a malei-
mide group for conjugation with a suitable polymer or protein car-
rier. In our previous work, we have investigated a passive targeting
approach in which maleimide-bearing prodrugs bind selectively to
the cysteine-34 position of circulating albumin after intravenous
administration. This macromolecular prodrug concept is based on
two features: (i) rapid and selective binding of a maleimide func-
tionalized prodrug to the cysteine-34 position of endogenous albu-
min after intravenous administration, and (ii) release of the
albumin-bound drugs predominantly at the tumor site due to the
incorporation of an acid-sensitive or an enzymatically cleavable
bond between the drug and the carrier.29–31
(a) MDR modulators often alter the pharmacokinetic profile of
the co-administered anticancer drugs resulting in a considerable
dose reduction, (b) systemic toxicity of the MDR modulators, (c)
sufficient concentrations of the MDR modulator and the anticancer
drug in resistant tumor cell are not achieved simultaneously since
the therapy schedule for two pharmacokinetically unrelated drugs
remains unpredictable and needs to be assessed empirically, (d) a
lack of tumor targeting for the MDR modulators as well as antican-
cer agents.
The goal of this work was to develop dual-acting prodrugs
that avoid these disadvantages and prove more efficacious in
the potential treatment of cancer patients with intrinsic or
acquired resistance caused by an over-expression of the P-glyco-
protein. Such prodrugs consist of an anticancer agent, a P-glyco-
protein inhibitor and a thiol-binding moiety that allows coupling
to a suitable carrier (Fig. 1). Dual-acting prodrugs for circum-
venting P-gp mediated MDR have not been described to date
and could have the advantage that an anticancer agent and an
O
H2N
b
H
N
H2N
HN
H
N
H
N
H
N
a
O
O
O
N
N
N
N
N
N
1
2
ONT-093
H
a: replacement of the isopropyl amino groups in ONT-093 with a primary amino group (to bind to a peptide)
and a tertiary amine (to prevent compitition reactions)
b: replacement of the primary aromatic amino group in 1 with a primary aliphatic group
Figure 2. Structure of ONT-093 and its chemical modifications.
N
OEt
N
N
H
O
O
O
O
O
N
a
5
O
N
H
b
BocHN
H2N
BocHN
c
3
N
Reagents and conditions:
N
a: Boc2O, DCM, tert-butanol, 60 ºC, 87%
b: NH4OAc, AcOH, 140 ºC, 70%
c: TFA/DCM (50%), RT, 80%
O
N
H
H2N
1
Scheme 1. Synthesis of ONT-093 derivative 1.

K. Abu Ajaj, F. Kratz / Bioorg. Med. Chem. Lett. 19 (2009) 995–1000
997
Albumin demonstrates preferential tumor uptake in various
tumor animal models32 due to the enhanced permeability and
retention of macromolecules for solid tumors.33 In situ binding of
maleimide-based prodrugs to the cysteine-34 position of circulat-
ing albumin is meanwhile a preclinically and clinically validated
technology of increasing the therapeutic index of anticancer
agents. Besides an acid-sensitive prodrug of doxorubicin, the (6-
maleimidocaproyl)hydrazone derivative of doxorubicin [INNO-
206, previously DOXO-EMCH] that has been studied clinically34, a
series of albumin-binding prodrugs with enzymatically cleavable
linkers have been developed in the past 5 years.29,33–39
In our first example of the synthesis of such dual-acting
prodrugs, we focused on its potential use for the treatment of resis-
tant breast cancer. An anticancer agent of choice for such an
OEt
OH
N
N
H
O
O
O
O
O
O
O
H
5
N
NO
a
N
O
O
O
b
O
NH
O
O
O
O
Fmoc
H
N
H2N
N
N
H
H
N
H
O
3
4
10
Mtt
HN
N
NO
HN
Mtt
O
O
N
O
O
O
O
N
H
R
d
7
N
H
N
N
O
OH
OH
O
O
OH
H
H
O
NH
NH
c
6
2
R = Fmoc
R = H
OH
OH
O
O
Fmoc
O
O
CH
H
N
N
N
O
O
Mtt
HN
N
O
O
NH
O
O
N
O
O
H
N
H
O
OH
O
N
H
N
H
O
N
O
O
N
H
H
11
N
H
O
NH
HN
8
9
R = Fmoc
R = H
Mtt
NH
R
N
e
N
Mtt
HN
O
O
O
N
H
N
O
O
N
H
H
N
H
NH
O
O
OH
OH
O
OH
NH
OH
O
O
O
O
O
CH
H
N
N
O
O
O
O
NH
O
N
O
O
H
N
H
OH
N
H
O
12
Reagents and conditions:
HN
a: (1) Fmoc-β-alanine, oxalic acid chloride, DCM, RT
(2) Et3N, DCM, RT, 79%
Mtt
f
N
b: (1) NH4OAc, AcOH, 140 ˚C, 70%
(2) piperidine/DMF (20%), RT, 94%
c: (1) Et3N, DMF, RT, 86%
N
O
TFA*H N
O
O
N
H
(2) piperidine/DMF (20%), RT, 72%
d: doxorubicin-HCl, Et3N, DMF, RT, 75%
e: HATU, HOBt, DIPEA, DMF, RT, 70%
f: TFA/DCM (0.5%), RT, 40%
N
O
O
N
H
H
N
H
NH
NH
O
O
OH
OH
O
O
OH
OH
O
O
O
O
CH
H
N
N
O
O
O
O
NH
O
N
O
O
H
N
H
OH
N
H
O
13
NH *TFA
Scheme 2. Synthesis of the prodrug 13.

998
K. Abu Ajaj, F. Kratz / Bioorg. Med. Chem. Lett. 19 (2009) 995–1000
approach is doxorubicin since an anthracycline-based regimen is
routinely used to treat patients with breast cancer. In addition,
its chemical structure allows the design of prodrugs with enzymat-
ically cleavable bonds through its amino group.40 Furthermore, we
selected the third generation MDR modulator, ONT-093 (Fig. 2) as
an inhibitor of P-gp because its structure–activity relationships
have been intensely studied and it possesses suitable functional
groups for chemical modification.41 ONT-093 reversed resistance
to doxorubicin and paclitaxel in six different cell lines expressing
P-gp in the low nanomolar range with an average EC50 value of
32 nM. Although ONT-093 shows high specificity for P-gp, it is
not a P-gp transport substrate.42
400
300
200
100
0
HSA-13
366 nm
Doxorubicin reference
reference 2
4 h
0 h
0
10
20
30
40
50
time (min)
The synthesis of ONT-093 derivatives is based on the formation
of an imidazole ring. 2,4,5-Trisubstituted imidazoles can be simply
obtained in high yield from 1,2-diketone and aldehyde in the pres-
ence of ammonium acetate, either under microwave irradiation43
or by heating at high temperatures.41,44 Since the modulator
ONT-093 has no suitable functional groups for direct derivatiza-
tion, a structural modification is required. Zhang et al. could show
that the replacement of the isopropyl amino groups in ONT-093
with dimethyl amino groups or other substituents has only a very
weak effect on the activity of the substance.41 Aided with theses
results, we first modified this inhibitor and replaced one isopropyl
amino group with a primary amino group, and replaced the other
isopropyl amino group with a tertiary amine to prevent any com-
petition reaction thus obtaining 1 (Fig. 2). According to the litera-
ture41, the imidazole ring in 1 (Fig. 2) should be obtained from
the coupling reaction (Scheme 1) of 3 and 5 in the presence of
ammonium acetate in acetic acid at 140 °C. Unfortunately, the
reaction gave in our hands only by-products that may be attributed
to the side reactions of the free amino group in reactant 3. We
could overcome this problem and obtained the desired product 1
by protecting the free aromatic amino group in 3 followed by cou-
pling of this Boc-protected 1,2-diketone with the aldehyde 5 in the
presence of ammonium acetate and acetic acid by heating at 140 °C
and subsequent treatment with trifluoroacetic acid (TFA).45
A non-expected very low reactivity of the aromatic amino group
in 1 and 3 was observed, when reacting these with amino acids
which did not form an amide bond in the presence of various stan-
dard amide-coupling reagents or under reaction conditions that
were applied for aromatic amines.46 The chemical behavior of the
aromatic amino group led us to investigate the introduction of an
aliphatic amino group. Thus, the dione 3 was reacted with Fmoc-
ß-alanine chloride47 to give 4 (Scheme 2). The desired imidazole
400
300
200
100
0
495 nm
HSA-13
Doxorubicin reference
4 h
0 h
0
10
20
30
40
50
time (min)
Figure 4. (A and B) Chromatograms of incubation studies of HSA-13 (385
activated cathepsin B (10 L; 0.095 U) at 37 °C and monitored at 366 nm (A) and
495 nm (B). Doxorubicin and 2 are shown as references.
lM) with
l
product 6 was obtained from the reaction of 1,2-dione 4 and alde-
hyde 5 in the presence of ammonium acetate and acetic acid at
140 °C. Standard Fmoc-cleavage using piperidine in DMF (20%) fur-
nished 2 as the derivative of the P-gp-inhibitor ONT-093.48
In an orientating in vivo study, we first wanted to assess the
efficacy of the ONT-093 derivative 2 in combination with doxoru-
bicin in the MT-3/ADR Adriablastin resistant mamma carcinoma
xenograft model that is based on a cell line that expresses P-glyco-
protein.49 Female nude mice with subcutaneously growing MT-3/
ADR-xenografts were randomly distributed to the experimental
groups (three/group) and injected with (a) saline (on days 13 and
20), (b) doxorubicin (6 mg/kg, i.v, on days 13 and 20), or (c) com-
pound 2 (5 mg/kg, administered i.p. as a solution in 70% PEG
400/30% D-glucose buffer pH 5, on days 12, 13, 19, and 20) and
doxorubicin (6 mg/kg, i.v, on days 13 and 20 after the administra-
tion of 2).
16
Whereas doxorubicin (2 ꢀ 6 mg/kg) was inactive, a combina-
tion of doxorubicin (2 ꢀ 6 mg/kg) and 2 (4 ꢀ 5 mg/kg) exhibited
superior antitumor activity, and mice treated with this combina-
tion showed a clear antitumor response (Fig. 3). However, the com-
bination was accompanied with considerable body weight loss
(ꢁ23%). These preliminary in vivo results support our development
of a more active and less toxic dual-acting prodrug that contains
both doxorubicin and 2 for circumventing MDR.
Solvent
14
Doxorubicin (2 x 6 mg/kg)
12
Doxorubicin (2 x 6 mg/kg) +
compound 2 (4x 5 mg/kg)
10
8
6
4
2
0
For developing such a prodrug we used the recently developed
building block 7 and the bifunctional maleimide spacer 10 that
allow the derivatization with two different pharmaceutically active
compounds independently bound by enzymatically cleavable link-
ers26—see Scheme 2. The introduction of the anticancer agent
doxorubicin in one arm of the linker 10 was performed through
the reaction of its amino group with the carbamate group of 10
to give 11.50 The building unit for the P-gp inhibitor 9 was obtained
from the reaction between 2 and the activated carbonate 7 with
10
15
20
25
30
35
day
Figure 3. Curves depicting tumor growth inhibition of subcutaneously growing
MT-3/ADR-xenografts under therapy with doxorubicin and combination of
a
compound 2 and doxorubicin. Female NMRI-nu mice (three/group) were injected
with 107 MT-3/ADR cells on day 0. Compound 2 (5 mg/kg) was administered i.p. as a
solution in PEG 400/D-glucose buffer pH 5 (7:3) on days 12, 13, 19, and 20. The
treatment with doxorubicin (6 mg/kg, i.v.) was performed on days 13 and 20
without and after the administration of compound 2.
subsequent cleavage of the Fmoc group with diethylamine.51
was then introduced in the prodrug by reacting its free amino
9

K. Abu Ajaj, F. Kratz / Bioorg. Med. Chem. Lett. 19 (2009) 995–1000
999
References and notes
400
300
200
100
0
doxorubicin
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HSA-13
intristic signal from
tumor homogenate
compound 2
22h - 495 nm
0 h - 495 nm
22 h - 366 nm
0 h- 366 nm
-10
0
10
20
30
40
50
time (min)
Figure 5. Chromatograms of incubation studies of HSA-13 (385
homogenate from doxorubicin resistant MT-3/ADR tumors at 366 and 495 nm.
lM) with a
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12. Finally, cleavage of both Mtt protecting groups in 12 with
0.5% TFA in DCM furnished the prodrug 13. Purification by HPLC
and subsequent lyophilization yielded a red powder.52 The prodrug
13 was characterized by analytical HPLC and mass spectra. It
showed good water solubility of ꢂ6.5 mg/mL in glucose buffer
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(5% D-(+)-glucose, pH 5.8) when diluted with 10% PEG 400.
In a next step, we wanted to assess the albumin-binding prop-
erties of the prodrug 13 bound to human serum albumin (HSA). In
analogy to our previous work on the preparation of drug albumin
conjugates27,28, the albumin conjugate of 13 was prepared by
reacting the maleimide moiety of 13 to the cysteine-34 group of
HSA and purifying the albumin-bound prodrug HSA-13 with
size-exclusion chromatography.53 The cleavage properties of
HSA-13 were evaluated using enzymatically active cathepsin B at
pH 5.0 and in tumor homogenates.53 Reverse phase HPLC showed
that the incubation with cathepsin B (Fig. 4) as well as with
homogenates of doxorubicin resistant MT-3/ADR tumors (Fig. 5)
resulted in an efficient cleavage over a period of 22 h (k = 366 nm
for the inhibitor 2 and k = 495 nm for doxorubicin). The cleavage
products were identified by HPLC to be the free anticancer agent
doxorubicin (eluting at 16.7 min) and the inhibitor 2 (eluting at
18.5 min).
In conclusion, we have demonstrated for the first time the syn-
thesis of a dual-acting prodrug which consists of an anticancer
agent and an inhibitor of P-gp independently bound by enzymati-
cally cleavable linkers to a targeting protein-binding moiety. The
prodrug in its albumin-bound form was cleaved specifically by
cathepsin B and in tumor homogenates releasing the free drugs.
These results form a good basis for evaluating the activity of the
new prodrug in a P-gp expressing animal model. Preliminary tox-
icity studies with 13 in nude mice have shown that a dose of
24 mg/kg doxorubicin equivalents can be safely administered
which is at least a threefold increase over the standard dose of
doxorubicin (6–8 mg/kg) that is used in nude mice models.54
30. Kratz, F.; Beyer, U. Drug Deliv. 1998, 5, 281.
31. Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. J. Control. Release 2000, 65,
271.
32. Kratz, F. Expert Opin. Invest. Drugs 2007, 16, 855.
33. Graeser, R.; Chung, D. E.; Esser, N.; Moor, S.; Schachtele, C.; Unger, C.; Kratz, F.
Int. J. Cancer 2008, 122, 1145.
34. Kratz, F.; Mansour, A.; Soltau, J.; Warnecke, A.; Fichtner, I.; Unger, C.; Drevs, J.
Arch. Pharm. (Weinheim) 2005, 338, 462.
35. Ryppa, C.; Mann-Steinberg, H.; Fichtner, I.; Weber, H.; Satchi-Fainaro, R.;
Biniossek, M. L.; Kratz, F. Bioconjug. Chem. 2008, 19, 1414.
36. Chung, D. E.; Kratz, F. Bioorg. Med. Chem. Lett. 2006, 16, 5157.
37. Schmid, B.; Chung, D. E.; Warnecke, A.; Fichtner, I.; Kratz, F. Bioconjug. Chem.
2007, 18, 702.
Acknowledgment
38. Fiehn, C.; Kratz, F.; Sass, G.; Muller-Ladner, U.; Neumann, E. Ann. Rheum. Dis.
2008, 67, 1188.
39. Warnecke, A.; Fichtner, I.; Sass, G.; Kratz, F. Arch. Pharm. (Weinheim) 2007, 340,
389.
The support of the Fördergesellschaft der Klinik für Tumorbiol-
ogie is gratefully acknowledged.
40. Dubowchik, G. M.; Firestone, R. A.; Padilla, L.; Willner, D.; Hofstead, S. J.;
Mosure, K.; Knipe, J. O.; Lasch, S. J.; Trail, P. A. Bioconjug. Chem. 2002, 13, 855.
41. Zhang, C.; Sarshar, S.; Moran, E. J.; Krane, S.; Rodarte, J. C.; Benbatoul, K. D.;
Dixon, R.; Mjalli, A. M. Bioorg. Med. Chem. Lett. 2000, 10, 2603.
42. Newman, M. J.; Rodarte, J. C.; Benbatoul, K. D.; Romano, S. J.; Zhang, C.; Krane,
S.; Moran, E. J.; Uyeda, R. T.; Dixon, R.; Guns, E. S.; Mayer, L. D. Cancer Res. 2000,
60, 2964.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.11.063. ESI-Mass
spectrum of prodrug 13. This material is available free of charge
via the Internet.
43. Wolkenberg, S. E.; Wisnoski, D. D.; Leister, W. H.; Wang, Y.; Zhao, Z.; Lindsley,
C. W. Org. Lett. 2004, 6, 1453.

1000
K. Abu Ajaj, F. Kratz / Bioorg. Med. Chem. Lett. 19 (2009) 995–1000
44. Sarshar, S.; Zhang, C.; Moran, E. J.; Krane, S.; Rodarte, J. C.; Benbatoul, K. D.;
Dixon, R.; Mjalli, A. M. Bioorg. Med. Chem. Lett. 2000, 10, 2599.
52. Synthesis of the prodrug 13: Compound 11 (1 equiv, 0.84 mmol, 1.30 g), HATU
(1.1 equiv, 0.93 mmol, 035 g) HOBt (1 equiv, 0.84 mmol, 0.10 g), DIPEA
45. Synthesis of ONT-093 derivative 1: Boc2O (1 mL) was added under nitrogen
atmosphere to a solution of 4 (0.19 mmol, 50 mg) in dry DCM (2 mL) and tert-
butanol (2 mL), and the mixture was heated at 60 °C for 20 h (TLC: ethyl
acetate/hexane, 1:1). The solvents were removed under reduced pressure. The
raw product was precipitated with hexane (50 mL) as a red solid (60 mg, 87%).
C21H24N2O4, ESI-MS: calcd [M+H]+ 369.17, found 369.30. The raw product
(1.0 equiv, 0.14 mmol, 50 mg) and aldehyde 5 (1.5 equiv, 0.21 mmol, 40 mg)
were placed in acetic acid (4 mL). Ammonium acetate (10 equiv, 1.4 mmol,
108 mg) was taken up in acetic acid (2 mL) in a separate flask. Both flasks were
heated at 140 °C. As soon as the solids dissolved, the ammonium acetate
solution was added to the other mixture. The resulting solution was heated at
140 °C for 6 h (TLC, ethyl acetate/hexane, 1:1). The solvent was removed under
reduced pressure. The residue was purified with FC eluting with ethyl acetate/
hexane, 1:1 yielding a yellow solid (52 mg, 70%). C33H38N4O3, ESI-MS: calcd
[M+H]+ 539.29, found 539.10. The product (0.09 mmol, 50 mg) was then
dissolved in a mixture of DCM and TFA (1:1; 4 mL) and stirred at room
temperature for 2 h (TLC: CHCl3/MeOH, 10:1). A red solid precipitated upon
adding the solution to diethyl ether (50 mL). 1 was collected as a TFA-salt by
suction filtration, washed with ether and dried to give 33 mg (80%). C28H30N4O,
ESI-MS: calcd [M+H]+ 439.24, found 439.20.
(2 equiv, 1.68 mmol, 260 lL), and 9 (1 equiv, 0.84 mmol, 1.00 g) were
dissolved in dry DMF (10 mL), and the solution was stirred at rt for 3 h. The
product was precipitated with ether (1 L) and was then purified by FC eluting
with CHCl3/MeOH, 15:1 to give 12 as an orange solid (1.60 g, 70%).
C159H168N16O26,
ESI-MS: calcd [M+H]+ 2718.23, found 2718.24, and calcd
[M + Na]+ 2740.23, found 2740.26; HPLC analysis: (Nucleosil 100-5-C-18
(Macherey-Nagel); flow rate: 1 mL/min; mobile phase A: 70% H2O, 30%
acetonitrile, 0.1% TFA; mobile phase B: 20% H2O, 80% acetonitrile, 0.1% TFA;
k = 366 nm; retention time 39.12 min) > 94% of peak area. 12 (300 mg) was
dissolved in DCM (6 mL) and then treated with a solution of 1% TFA in DCM
(6 mL), and stirred at rt for 24 h after which no 12 was detectable by HPLC. The
product was then precipitated with DCM (120 mL), washed with ether
(100 mL), centrifuged and then purified by HPLC (25 cm Nucleosil C-18
column (100-7, 250 ꢀ 21 mm) with a pre-column (100-7, 50 ꢀ 21 mm) from
Macherey–Nagel; flow rate: 10 mL/min; mobile phase A: 70% H2O, 30%
acetonitrile, 0.1% TFA; mobile phase B: 20% H2O, 80% acetonitrile, 0.1% TFA)
to yield after lyophilization 13 as a red solid (97 mg, 40%). HPLC analysis
(25 cm Nucleosil C-18 column (100-5, 250 ꢀ 4 mm) from Macherey–Nagel);
flow rate: 1 mL/min; k = 495 and 366 nm; retention time: 29.39 min) >99% of
peak area. C119H136N16O26, ESI-MS: calcd [M+H]2+ 1103.50, found 1103.51, and
calcd [M + 3H]3+ 736.00, found 736.00. (see Supporting information of mass
spectrum of 13).
46. Luyt, L. G.; Katzenellenbogen, J. A. Bioconjug. Chem. 2002, 13, 1140.
47. Oku, A.; Yamaura, Y.; Harada, T. J. Org. Chem. 1986, 51, 3732.
48. Synthesis of ONT-093 derivative 2: Fmoc-b-alanine chloride was prepared
from Fmoc-b-alanine (1 equiv, 27.3 mmol, 4.4 g) and oxalic acid chloride
(0.8 equiv, 21.8 mmol, 1.85 mL) in dry DCM (50 mL). The solution of Fmoc-b-
alanine chloride (21.8 mmol) was added slowly to a mixture of 3 (1 equiv,
16.4 mmol, 4.4 g) and anhydrous triethylamine (1.25 equiv, 20.5 mmol,
2.8 mL) in dry DCM (80 mL). The solution was stirred at rt for 1 h. Excess
amount of the acid chloride was quenched with MeOH (10 mL). The residue
was purified by FC eluting with CHCl3/MeOH, 30:1 to give 4 as a yellow solid
(7.3 g, 79%). C34H31N3O5, ESI-MS: calcd [M+H]+ 562.23, found 562.0. 1,2-
Diketone 4 (1.0 equiv, 9.8 mmol, 5.5 g) and aldehyde 5 (1.5 equiv, 14.7 mmol,
2.75 g) were placed in acetic acid (30 mL). Ammonium acetate (10 equiv,
98 mmol, 7.5 g) was taken up in acetic acid (7.5 mL) in a separate flask. Both
flasks were heated at 140 °C. As soon as the solids dissolved, the ammonium
acetate solution was added to the mixture of 4 and 5. The resulting solution
was heated at 140 °C for 3 h. Removal of the solvent under reduced pressure
and purification of the residue with FC eluting with CHCl3/MeOH, 20:1 gave 6
as a yellow solid (5.7 g, 80%). C46H45N5O4, ESI-MS: calcd [M+H]+ 732.35, found
732.30. A solution of 6 (3 g) in piperidine/DMF (20%, 10 mL) was stirred at rt for
15 min. Product 2 was precipitated with ether (500 mL) as a yellow solid (1.9 g,
92%). C31H35N5O2, ESI-MS: calcd [M+H]+ 510.28, found 510.30.
53. Cleavage studies of HSA-13 with cathepsin
homogenatePreparation of the albumin conjugate HSA-13: 13 (1.6 mg) was
dissolved in PEG 400 (Aldrich, 25 L) and was then diluted with 5% Glucose
solution (225 L, pH 3.5-5, Braun). 13 (600 L, 2600 M) was subsequently
B
and with tumor
l
l
l
l
incubated with HSA (5% octalbin, 5.4 mL) and ethanol (Lichrosolv, 3 mL)
under slight stirring at 37 °C for 1 h. The albumin conjugate HSA-13 was
obtained after subsequent size-exclusion chromatography (Sephacryl S-100,
50 mM sodium acetate buffer, pH 5.0) followed by concentrating the
solution with CENTRIPREPÒ YM-10-concentrators (Amicon, FRG) (4 °C and
4500 rpm) to 2.75 mL. The content of anthracycline in the sample was
determined
7.4) = 10650 Mꢁ1 cmꢁ1
conjugate HSA-13 of 385
thawed prior to use.Cleavage studies with cathepsin B: 500
stock solution (385 M) were mixed with 10 cathepsin
Calbiochem (Bad Soden, FRG), 0.4 g/mL, 23.8 U/mg) and 50 lL
using
the
yielding
M. Samples were kept frozen at ꢁ80 °C and
L of the HSA-13
(from
of buffer
-cysteine (8 mM). The
L) were removed at
e
-value
for
doxorubicin
[495
(pH
]
a
concentration of doxorubicin in the
l
l
l
lL
B
l
(50 mM sodium acetate buffer, pH 5.0) containing
L
mixture was incubated at 37 °C and aliquots (60
l
various time points and analyzed by HPLC.Cleavage studies with tumor
homogenate: 340 L of the HSA-13 stock solution (385 M) were mixed with
homogenates of doxorubicin resistant MT-3/ADR tumors (170 L, 400 mg
tumor/800 L sodium acetate buffer, pH 5) and incubated at 37 °C. Aliquots
(60 L) were removed at various time points and analyzed by HPLC. HPLC
l
l
49. Stein, U.; Walther, W.; Lemm, M.; Naundorf, H.; Fichtner, I. Int. J. Cancer 1997,
72, 885.
50. Synthesis of EMC-Glu-Phe-Lys(Mtt)-PABC-doxorubicin 11: Triethylamine
l
l
l
(1 equiv, 1.31 mmol, 182
l
L) was added to
a
solution of 10 (1 equiv,
conditions were flow rate: 1 mL/min, k = 495 nm and 366 nm; mobile phase
A: 85% H2O, 15% acetonitrile, 0.1% TFA, containing heptane-1-sulfonic acid
(2 g/L); mobile phase B: 20% H2O, 80% acetonitrile, 0.1% TFA, containing
heptane-1-sulfonic acid (2 g/L); gradient: 0–10 min increase to 20% mobile
phase B; 10–35 min increase to 100% mobile phase B; 35–40 min decrease
to 100% mobile phase A; 40–60 min 100% mobile phase A, injection volume:
1.31 mmol, 1.50 g) and doxorubicin-HCl (1 equiv, 1.31 mmol, 0.761 g) in dry
DMF (20 mL), and the solution was stirred at room temperature for 18 h (TLC:
CHCl3/MeOH 7:1). The product was precipitated with ether (1 L) and it was
purified by FC eluting with CHCl3/MeOH 15:1 to give 11 as a red solid (1.52 g,
75%). C85H91N7O21; ESI-MS: calcd [M+H]+1546.63, found 1546.00.
51. Synthesis of Phe-Lys(Mtt)-PABC-inhibitor 9:
2.56 mmol, 1.3 g), (1.03 equiv, 2.63 mmol, 2.75 g) and anhydrous
triethylamine (2 equiv, 5.12 mmol, 718 L) in dry DMF (35 mL) was stirred
A
mixture of
2
(1 equiv,
50 lL; column: symmetry 300 (250 ꢀ 4.6 mm) from Waters.
7
54. Preliminary toxicity studies of 13: Female nude mice were randomly
l
distributed to the experimental groups (two/group) and injected with
at rt for 16 h. The product 8 was precipitated with ether yielding a yellow solid
(3.1 g, 86%). 8 (3 g) was then dissolved in 20% piperidine/DMF (30 mL), and the
solution was stirred at rt for 10 min. Product 9 was finally precipitated with
compound 13 (i.v, administered as a solution in 10% PEG 400/90% D-glucose
buffer pH 5) at the following doses: (a) 33.6 mg/kg = 8 mg/kg doxorubicin
equivalents, (b) 67.1 mg/kg = 16 mg/kg doxorubicin equivalents, or (c)
100.8 mg/kg = 24 mg/kg doxorubicin equivalents. The body weight change
was observed daily for 13 days.
ether (500 mL) as
a yellow solid (1.8 g, 72%). C74H79N9O6, ESI-MS: calcd
[M+H]+1190.52, found 1190.30.