Doxorubicin prodrug on the basis of tert-butyl cephalosporanate sulfones

Article abstract of DOI:10.1016/j.bmcl.2003.11.071

Doxorubicin-cephalosporin prodrug adapted to the development of elastases for the liberation of parent drug was synthesized on the basis of cephalosporanate sulfone esters.

Full text of DOI:10.1016/j.bmcl.2003.11.071

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1007–1010
Doxorubicin prodrug on the basis of tert-butyl cephalosporanate
sulfones
Grigory Veinberg,* Irina Shestakova, Maxim Vorona, Iveta Kanepe, Ilona Domrachova
and Edmunds Lukevics
Latvian Institute of Organic Synthesis, 21 Aizkraukles Street, Riga, LV 1006, Latvia
Received 9 July 2003; revised 10 November 2003; accepted 25 November 2003
Abstract—Doxorubicin–cephalosporin prodrug adapted to the development of elastases for the liberation of parent drug was
synthesized on the basis of cephalosporanate sulfone esters.
# 2003 Elsevier Ltd. All rights reserved.
The design of doxorubicin containing prodrugs in gen-
eral is focused on the creation of specific carriers selec-
tively liberating cytotoxic agent in organism sites
affected by cancer and preventing healthy ones from
unnecessary toxic exposure. There are various approa-
ches to solve this problem one of which presumes the
covalent attachment of doxorubicin amino group to 3-
carbonyloxymethyl moiety of cephalosporanic acids.
The employment of monoclonal antibody (MoAb)-b-
lactamase conjugate system to split cephalosporin
molecule according to a specific mechanism confines the
spread of expelling drug by the region chosen for
antitumor treatment.1ꢀ3
Presented investigation was targeted at the exploration
of the alternative type of doxorubicin–cephalosporin
prodrug, which structural design was adapted to the
involvement of elastases usually generated during
metastasizing process (Scheme 1).4
Such a concept predetermined the selection of cephalo-
sporanate sulfone esters acting as irreversible or
Scheme 1. The concept of elastase mediated splitting of doxorubicin-cephalosporin prodrug.
* Corresponding author. Tel.: +371-755-5946; fax: +371-755-0338; e-mail: veinberg@osi.lv
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.071

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G.Veinberg et al./ Bioorg.Med.Chem.Lett.14 (2004) 1007–1010
reversible elastase inhibitors for the role of the drug
carrier.5 Target prodrugs 6a,b containing an urethane
bridge between doxorubicin and cephalosporin parts
were obtained by three steps conversion of tert-butyl
deacetoxycephalosporanate sulfones (2a,b) into their
prodrugs at small 1.5 mg/kg/day dosages failed.8 The
increase in a dosage up to 5 mg/kg/day positively chan-
ged the effectiveness of the treatment, which resulted in
30% and 22% tumor growth inhibition by prodrugs 6a
and 6b reaching 80% and 60% of doxorubicin activity
respectively (Table 2).
3-(4-nitrophenoxy)carbonyloxymethyl
5a,b capable to condense with 1, employing already
derivatives
developed synthetic pathways (Scheme 2).1,6,7
In the case of a more slowly growing melanoma B16
transplanted in male C57BL/6 mice the inhibition of
tumor growth was achieved even at small dosage
of prodrug 6a (Table 2, Nos 1–3). Its enlargement up to
2 and 5 mg/kg/day increased the effectiveness of both
prodrugs 6a and 6b, especially on the eleventh and
fourteenth day (Table 2, Nos 4–6). However their final
tumor inhibiting effect was 2 and 4 times weaker than in
the case of doxorubicin. Specially carried out experiments
showed that tumor growth in a control group treated
with physiologic solution (PS) containing 2% DMSO
was comparatively 10–15% faster than in the control
group treated only with PS. The agarose additions to both
solutions did not influence this process. It enabled to
presume and then to prove experimentally (Table 2 Nos 7,
8) that the curing properties of 6a could be more impress-
ive if its effectiveness was estimated against the control
group treated only with PS without DMSO additive.
According to the HPLC data 6a and 6b did not contain
even the traces of the parent drug. They were also stable
at pH 7.5 in phosphate buffer at least for 24 h. Their
treatment with slightly basic water solutions provided
hydrolytic splitting of b-lactam ring and liberation of
doxorubicin proved by TLC and HPLC.
Cytotoxic activity of doxorubicin, starting deacetoxy-
cephalosporanate sulfones 2a,b and prodrugs 6a,b in
vitro was tested on standard monolayer tumor cell lines:
MG-22A (mouse hepatoma), HT-1080 (human fibro-
sarcoma), B16 (mouse melanoma) and Neuro 2A
(mouse neuroblastoma) (Table 1).8 Obtained IC50 data
evidenced that prodrugs 6a,b being less cytotoxic than
doxorubicin (1) were much more potent in this respect
than starting cephalosporanates 2a,b.
The in vivo treatment of rapidly growing Sarcoma
S-180 tumor transplanted in male JRC mice with
A significant difference in the activity demonstrated by
6a and 6b during their in vivo testing against melanoma
Scheme 2. The synthesis of doxorubicin–cephalosporin prodrugs.
Table 1. Cytotoxic properties of doxorubicin, prodrugs and starting deacetoxycephalo-sporanate sulfones in vitro
Compd
Concentrations providing 50% cell killing effect IC50 (mg/mL)
MG-22A B16
HT-1080
MTTb
Neuro 2A
CVa
CV
MTT
CV
MTT
CV
MTT
1
0.17
6
8
0.02
0.4
0.31
6
7
0.08
0.5
<0.001
6
3.4
0.03
0.07
0.73
2
3.0
0.04
0.07
0.001
3
>100
<0.001
2
>100
0.004
2
>100
<0.001
2
>100
2a
2b
6a
6b
0.03
0.09
0.04
0.5
0.001
0.04
0.008
0.04
a CV, crystal violet coloration.
b MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide coloration.

G.Veinberg et al./ Bioorg.Med.Chem.Lett.14 (2004) 1007–1010
1009
Table 2. Antitumor effect of doxorubicin and prodrugs in vivo
Table 3. Physiological changes in cardiomyocytes after the treatment
of male JRC mice with doxorubicin and prodrugs
Nos Compda
Dosageb
(mg/kg/day)
Tumor growth inhibition
GI on fixed date (%)
Compd
Dosagea
(mg/kg/day)
Physiologic changes in
cardiomyocytesa
S-180
B 16
Healthy miceb
Mice with transplanted
Sarcoma S-180b
7
7
9
28
11
70
75
13
65
86
11
25
86
16
60
26
1
2
3
4
5
6
7
8
1
1.5
1.5
1.5
5.0
5.0
5.0
2.0
2.0d
6a
6b
1
6a
6b
1
No effect
No effect
35
72
ꢀ10c
CB
MTT
CB
MTT
ꢀ49c
1
1.5
1.5
1.5
5.0
5.0
5.0
83ꢁ6
92ꢁ4
87ꢁ2
60ꢁ6
90ꢁ6
40ꢁ1
85ꢁ6
54ꢁ6
39ꢁ4
87ꢁ4
90ꢁ6
106ꢁ16
92ꢁ4
114ꢁ10
225ꢁ12
270ꢁ12
130ꢁ10
230ꢁ21
360ꢁ12
37
30
22
75
39
16
70
6a
6b
1
6a
6b
81ꢁ6
70ꢁ5
6a
56
75ꢁ4 102ꢁ7
90ꢁ4
a Doxorubicin was administered in physiological solution (PS,
0.9%NaCl, pH 6.2); prodrugs 6a,b in PS containing 2% DMSO and
0.3% agarose additive.
b Administration schedule: 1, 2, 3, 4, 7 days.
c Negative data indicate the activation of tumor growth.
d The treatment of control group with physiological solution.
a Administration schedule: 1, 2, 3, 4, 7 days.
b Cells isolated on the 9th day of experiment.
same tendencies in the changes of its intensity (Table 3).
Such an increase in redox activity regardless to the che-
mical nature of administered compounds could be
explained as a specific response of cardiomyocytes to
pathologic alterations caused by tumor transplantation.
B16 evidences about the more pronounced involvement
of doxorubicin in the curing process in the case of 6a.
That is why at the present stage of comparative biolo-
gical studies we assume that compound 6a to more
extent than 6b meets the requirements of the prodrug
according to the mechanism proposed.
Obtained data evidence that the application of tert-
butyl 7a-chlorocephalosporanate sulfone as a prodrug
carrier provided 6a with antitumor efficacy comparable
with the parent drug both in vitro and in vivo. Its con-
siderable lower cardiotoxicity correlates with the antici-
pated difference in the concentrations of prodrug
splitting elastases in metastasizing and healthy organs.
As a result their relatively smaller amount in animals
heart prevented cardiomyocytes from unnecessary
exposure to toxic doxorubicin.
It is well known that one of the most serious toxic con-
sequences of doxorubicin treatment is connected with its
irreversible cardiotoxic action. That is why there were
carried out the comparative studies of physiologic con-
dition of cardiomyocytes after injections of doxorubicin
and prodrugs in healthy mice and in mice with trans-
planted tumor. In both series of experiments cardio-
myocytes were isolated from the treated animals on the
9th day,10 and changes in the amount of their intracel-
lular peptides and mitochondrial redox activity against
control cells taken from the untreated animals were
analysed using Coomassie Brilliant Blue (CB) and MTT
coloration, respectively.
References and notes
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According to the CB test there was not observed a sub-
stantial difference in the amount of peptides in cardio-
myocytes after the treatment of healthy mice with
doxorubicin (1) and prodrugs 6a,b at the same dosages.
Contrary to it the MTT test revealed 60% decrease in
mitochondrial redox activity after the long term expo-
sure of cardiomyocytes to 1 contrasting with its 15%
and 46% reduction in the case of 6a and 6b (Table 3).
Such a specificity of the MTT test correlates with the
known ability of doxorubicin to inhibit the oxidation of
mitochondrial palmitate serving as a major source of
energy in cardiomyocytes .12 At higher dosages a similar
decrease and even stimulation of redox activity in car-
diomyocytes was observed after doxorubicin and 6b
administration.
4. Liotta, L. A.; Roo, C. N. Tumor Invasation and Methastasis.
New Concepts in Neoplasma as Applied to Diagnostic
Pathology; Williams and Wilkins: Baltimore, 1986.
5. (a) Doherty, J. B.; Ashe, B. M.; Barker, P. L.; Blacklock,
T. J.; Butcher, J. W.; Chandler, G. O.; Dahlgren, M. E.;
Davies, P.; Dorn, C. P., Jr.; Finke, P. E.; Firestone, R. A.;
Hagmann, W. K.; Halgren, T.; Knight, W. B.; Maycock,
A. L.; Navia, M. A.; O’Grady, L.; Pisano, J. M.; Shah,
S. K.; Thompson, K. R.; Weston, H.; Zimmerman, M. J.
Med.Chem. 1990, 33, 2513. (b) Shah, S. K.; Brause, K. A.;
Chandler, G. O.; Finke, P. E.; Ashe, B. M.; Weston, H.;
Knight, W.; Maycock, A. L.; Doherty, J. B. J.Med.
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6. Veinberg, G.; Vorona, M.; Grigan, N.; Kanepe, I.;
Shestakova, I.; Strakovs, A.; Lukevics, E. Chem.Heterocycl.
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7. Doxorubicin hydrochloride (69.5 mg, 0.12 mM) water
solution (10 mL) was neutralized with 5% Na2CO3 and
antibiotic was extracted with dichloromethane (50 mL).
Organic phase was evaporated and the residue was redis-
solved in THF (4 mL). Obtained solution was added to
Similar trends were detected in cardiomyocytes isolated
from mice with transplanted Sarcoma S-180 and treated
with doxorubicin and prodrugs 6a,b. According to the
MTT test they were characterized with abnormally high
level of redox activity exceeding 2–3 times the respective
one observed in healthy mice, however, preserving the

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G.Veinberg et al./ Bioorg.Med.Chem.Lett.14 (2004) 1007–1010
7a-chloro-3-(4-nitrophenyloxycarbonyloxymethyl)cephal-
84%. Rf 0.68 dichloromethane-methanol (60:1). IR spectra:
cmꢀ1: 3400, 1790, 1720 cmꢀ1. HPLC analysis: Symmetry
C18 (3.9ꢂ150 mm), v=1 mL/min. Mobil phase: acetoni-
trile (0.1 M): phosphate buffer (pH 2.5) (70:30). Detector:
UV 254 nm. Purity 95%. Retention time: 3.82 min.
1H NMR (CDCl3), d: 2.00–2.18 (2H, m, 8-H2); 3.74–4.10
(4H, m, 10-H2, 9-COCH2O); 4.03 (3H, s, 4-OCH3); 4.44
(1H, br.s CH2OH); 5.24 (1H, m, 7-H); 5.52 (1H, br.s, 9-
H); 7.18 (1H, d, 3H); 7.78 (1H, t, 2H); 8.03 (1H, br.s, 1H);
13.26 (1H, br.s, 11-OH); 14.00 (br.s, 6-OH) naphthacene
fragment. 1.24 (3H, d, 6a-CH3); 2.00–2.18 (2H, m, 3a-H2);
3.63 (1H, br.s, 5a-H); 3.74 (1H, br.s, 4a-H); 4.06 (1H, s,
5a-OH); 4.20 (2H, d, 6a-H); 5.22 (1H, m, 2a-H) pyranosyl
fragment. 1.55 (9H, s, t-C4H9); 2.38 (3H, s, OCOCH3);
2.96, 3.11 (2H, AB-q, J=19, 2b-H2); 4.72, 5.07 (2H, AB-
q, J=14, 3b-CH2OCO); 5.52 (1H, br.s, 6b-H); 6.54 (1H,
br.s, ¼CH–) cephem fragment.
osporanate 1,1-dioxide 5a (63 mg, 0.12 mM) in THF (4
mL). After stirring for 24 h at room temperature target
7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-hydroxyacetyl-1-
methoxy-10-[2,3,6-trideoxy-3-[[4-tert-butoxycarbonyl-7a-
chloro-1,1-dioxo-ceph-3-em-3-yl]methoxycarbonylamino]-
a-l-lyxo-hexopyranosyl]oxy]-5,12-naphtacenedione (6a)
was isolated using column chromatography on Silicagel
Merck (70–230 mesh) and dichloromethane–methanol
(60:1) as eluent from fractions characterized with Rf 0.51
giving 74 mg (68%) of red crystalline powder. IR spectra:
cmꢀ1: 3500, 3400, 1810, 1720, 1620, 1580 cmꢀ1. HPLC
analysis: Symmetry C18 (3.9ꢂ150 mm), v=1 mL/min.
Mobil phase: acetonitrile (0.1 M)–phosphate buffer (pH
2.5) (70:30). Detector: UV 254 nm. Purity 97%. Retention
time: 6.55 min.
1H NMR (CDCl3), d: 2.05–2.18 (2H, m, 8-H2); 3.60–3.95
(4H, m, 10-H2, 9-COCH2O); 4.05 (3H, s, 4-OCH3); 4.45
(1H, br.s CH2OH); 5.20–5.28 (1H, m, 7-H); 5.50 (1H, m,
9-H); 7.35 (1H, d, 3H); 7.63 (1H, t, 2H); 8.12 (1H, br.s,
1H); 13.20 (1H, br.s, 11-OH); 13.96 (br.s, 6-OH) naph-
thacene fragment. 1.24 (3H, d, 6a-CH3); 1.75–1.90 (2H,
m, 3a-H2); 3.62 (1H, br.s, 5a-H); 3.78 (1H, br.s, 4a-H);
4.09 (1H, s, 5a-OH); 4.21 (2H, d, 6a-H); 5.20–5.28 (1H, m,
2a-H) pyranosyl fragment. 1.50 (9H, s, t-C4H9); 2.40 (3H,
s, OCOCH3); 2.86, 3.18 (2H, AB-q, J=19, 2b-H2); 4.62,
5.02 (2H, AB-q, J=14, 3b-CH2OCO); 4.78 (1H, br.s, 6b-H);
5.24 (1H, br.s, 7b-H) cephem fragment.
8. Methodology of in vitro and in vivo antitumor assays is
presented in ref 9.
9. Veinberg, G.; Vorona, M.; Shestakova, I.; Kanepe, I.;
Zharkova, O.; Mezapuke, R.; Turovskis, I.; Kalvinsh, I.;
Lukevics, E. Bioorg.Med.Chem. 2000, 8, 1033.
10. Mouse cardiomyocytes were obtained from hearts of 6
weeks old ICR male mice and isolated according to stan-
dard procedures. Cells were collected by gravity at room
temperature, resuspended in DMEM, 4% FBS, 2 mM
glutamine, 13 mM NaHCO3, 10 mM HEPES, 50 m/mL
penicillin, 50 mg/mLstreptomycine and placed on 96-well
plates. The plates were precoated overnight with myocyte
culture medium. After 4 h of incubation in ‘Hareus’
thermostat at 37ꢃ in a humidified 95% air/ 5% CO2 the
medium was changed while serum was omitted.11
7,8,9,10-Tetrahydro-6,8,11-trihydroxy-8-hydroxyacetyl-1-
methoxy-10-[2,3,6-trideoxy-3-[[4-tert-butoxycarbonyl-7(Z)-
tert-butoxycarbonylmethylene-1,1-dioxo-ceph-3-em-3-
yl]methoxycarbonylamino]-a-l-lyxo-hexopyranosyl]oxy]-
5,12-naphtacenedione (6b) was prepared from doxo-
rubicin base and tert-butyl 3-(4-nitrophenyloxycarbonyl-
oxy)methyl-7(Z)-tert-butoxycarbonylmethylenecephalos-
poranate 1,1-dioxide (5b) in the same manner as 6a. Yield
11. Linssen, M. C. J. G.; Vork, M. M.; de Jong, Y. F.; Glatz,
J. J. C.; Vusse, G. J. Mol.Cell.Biochem. 1990, 98, 19.
12. Sayed-Ahmed, M. M.; Sharawy, S.; Shouman, S. A.;
Osman, A.-M. M. Pharmacol.Research 1999, 39, 1043.