Antitumor agents: Development of highly potent glycosidic duocarmycin analogues for selective cancer therapy

Article abstract of DOI:10.1002/anie.200600936

(Chemical Equation Presented) Better and better: The glycosidic prodrug (+)-1, which is based on duocarmycin antibiotics, was synthesized for selective cancer therapy. The drug was developed within the context of "antibody-directed enzyme prodrug therapy"

Full text of DOI:10.1002/anie.200600936

Communications
therefore, should be the targeted removal of malignant cells
without impairment of normal cell populations. The approach
that we have adopted for selective cancer treatment is
“
antibody-directed enzyme prodrug therapy” (ADEPT).
With this method, nontoxic compounds (prodrugs) are
enzymatically transformed into highly cytotoxic agents in
[
1,2]
cancerous tissue in a targeted manner.
In this binary
approach, selectivity is achieved through the use of mono-
clonal antibodies that bind to tumor-associated antigens and
are covalently bound to the required enzyme. Other
approaches for selective cancer therapy depend on the
[
3]
[4]
application of immunoconjugates, angiogenesis inhibitors,
[
5]
[6]
antitumor vaccines, kinase inhibitors, and conjugates
between a neuropeptide and an antitumor agent.
[
7]
Based on our previous work, which dates back to 1985, we
have defined the main criterion for the ADEPT concept to be
that the cytotoxic agent derived from the prodrug should
[
]
achieve an IC *** value of < 10 nm. Furthermore, the
5
0
comparative toxicity ratio between the prodrug and drug,
which we define as the QIC (QIC = IC (prodrug)/
5
0
50
50
[
8]
IC (prodrug+enzyme)), should exceed 1000.
5
0
Herein we describe a new prodrug, (+)-2a, which we have
developed for selective cancer therapy. The compound is
based on the cytotoxic duocarmycin antibiotics, which for
example, include duocarmycin SA (1). Not only does this
Prodrugs
DOI: 10.1002/anie.200600936
Antitumor Agents: Development of Highly
Potent Glycosidic Duocarmycin Analogues for
Selective Cancer Therapy**
Lutz F. Tietze,* Felix Major, and Ingrid Schuberth
compound fulfill the outlined criteria, but owing to its
excellent QIC50 value, its good water solubility, and its easy
Dedicated to Professor Gerhard Erker
on the occasion of his 60th birthday
[
1b,c,9]
synthesis, it exceeds all prodrugs prepared to date by us
[
1,10]
and others.
Duocarmycin SA was isolated from Streptomyces DO-113
[
11]
Effective chemotherapy of malignant tumors is one of the
great challenges of modern medicine. Chemotherapy is
generally connected with severe side effects as the selectivity
of traditional cytostatic agents is mainly based on the
difference in the proliferation rate between malignant and
normal cells. In comparison with cells particularly from the
hematopoietic system, intestinal tract, and hair follicles, this
difference is very small. The aim of modern cancer therapy,
and has an IC50 value of 10 pm in the L1210 tumor cell line.
The spirocyclopropylcyclohexadienone moiety, which is nec-
essary for biological activity, can be formed in situ from the
corresponding seco compound with a free phenolic hydroxy
[
12]
group. In prodrug (+)-2a, the phenolic hydroxy group is
protected as a galactoside so that spirocyclization cannot
occur. The prodrug can, however, be easily converted to give
the required seco compound, (+)-3, which further reacts to
give the cytotoxic agent (+)-4 (Scheme 1). The N,N-dime-
[
13]
[
*] Prof. Dr. L. F. Tietze, Dipl.-Chem. F. Major, Dr. I. Schuberth
Institut für Organische und Biomolekulare Chemie
Georg-August-Universität Göttingen
thylaminoethoxyindole carboxylic acid component
not
only facilitates intercalation in the minor DNA groove, but
also increases water solubility owing to its salification.
The racemic benzyl ether 5, which was easily obtained by
Tammannstrasse 2, 37077 Göttingen (Germany)
Fax: (+49)551-39-9476
E-mail: ltietze@gwdg.de
[
9c,14]
using published methods,
was used to synthesize prodrug
2
and seco compound 3. To synthesize the diastereomerically
[
**] This research was supported by the Deutschen Forschungsge-
meinschaft (SFB 416) and the Fonds der Chemischen Industrie.
F.M. would like to thank the Studienstiftung des deutschen Volkes
for a PhD scholarship.
pure glycosidic prodrugs as well as the enantiomerically pure
[***] IC : drug concentration at which 50% of cell growth is inhibited.
5
0
6
574
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 6574 –6577

Angewandte
Chemie
deprotection, followed by coupling with 7 and final debenzy-
lation to give 3 in yields of 51–65%. Direct glycosylation of 3
with 6 was not successful.
The cytotoxic effects of the newly synthesized compounds
were tested on human tumor cells by using a colony-forming
assay carried out in triplicate (Table 1). This test reflects the
proliferation capacity of single cells and is based on the
[
9d]
HTCFA assay (human tumor colony forming ability test)
.
In experiments using human bronchial carcinoma cell line
A549, a QIC50 of 6600 was achieved with the mixture of
diastereomeric prodrugs 2a/2b, and for the diastereomerical-
ly pure prodrug (+)-(1S,10R)-2a, a QIC50 value of 4800 was
observed. The cytotoxicity of seco compound (+)-(1S,10R)-3
was determined as IC = 0.75 nm. It was not possible to assign
5
0
the QIC50 value of prodrug (À)-(1R,10S)-2b owing to its low
cytotoxicity. Thus, a higher quantity of this prodrug had to be
used, which then led to solubility problems. Interestingly, the
underlying seco compound (À)-(1R,10S)-3, with an IC value
5
0
of 560 nm, showed an almost 1000-fold lower cytotoxicity than
its enantiomer, (+)-(1S,10R)-3. The results of the cytotoxicity
assays are in good agreement with MS data obtained from
alkylation of duplex DNA with (+)-(1S,10R)-2a, (+)-
[
19]
(
1S,10R)-3, and (À)-(1R,10S)-3.
As a further important
finding from the cytotoxicity assays in vitro, it should be
emphasized that after treatment of prodrug (+)-(1S,10R)-2a
with b-d-galactosidase, the observed cytotoxicity was identi-
cal to that determined for seco compound (+)-(1S,10R)-3.
Scheme 1. Enzymatic toxification of the glycosidic prodrug (+)-
a: a) Transformation into the seco compound (+)-3 and
2
b) Winstein cyclization in situ to give the drug (+)-4, which is
an analogue of the antibiotic (+)-duocarmycin SA (1).
toxins, it was necessary to maintain 5 in an enantio-
merically pure form. Owing to the sensitivity of the free
amine of 5, it was not possible to use classical
resolution methods, such as formation of diastereo-
meric salts or derivatives with enantiomerically pure
reagents. However, efficient preparative HPLC sepa-
ration was carried out by using a column with a chiral
[
15]
stationary phase (Chiralpak IA). The absolute con-
figuration of (+)-5 was determined after derivatization
with 3,5-dibromobenzoic acid by using X-ray crystal
[
16]
structure analysis.
The galactosides 2 were prepared from 5 after
removal of the benzyl protecting group with palladium
on activated carbon and ammonium formate as the
[
17]
hydrogen source. Glycosylation was carried out with
by using the trichloroacetimidate method developed
6
[
18]
by Schmidt. Subsequently, BF ·OEt was employed
3
2
for N-tert-butoxycarbonyl (N-Boc) deprotection and
the obtained secondary free amines were coupled with
[
13]
the indolecarboxylic acid hydrochloride 7. Finally,
base-catalyzed solvolysis of the acetyl groups resulted
in the desired galactosides 2 in very good final yields of
Scheme 2. Synthesis of prodrugs 2 and seco compounds 3: a) Pd/C/NH HCO , THF,
4
2
4
08C, 15 min; b) 6, BF ·OEt , CH Cl , molecular sieves (4 Š), À108C, 3.5 h, then
3
2
2
2
BF ·OEt , RT, 5 h; c) 7, EDC·HCl, DMF, RT, 20 h; d) NaOMe/MeOH, RT, 2 h; e) 4m HCl/
3
2
3
7–43% in four steps (Scheme 2). The free toxins 3,
EtOAc, RT, 2 h; f) 7, EDC·HCl, DMF, RT, 19 h; g) 4m HCl/EtOAc, RT, 2 h, followed by
which were required as control compounds for the Pd/C/NH HCO , THF, 408C, 2 h. Bn=Benzyl, DMF=N,N-dimethylformamide ,EDC=3-
4
2
in vitro experiments, were obtained from 5 by N-Boc (3-dimethylaminopropyl)-1-ethylcarbodiimide.
Angew. Chem. Int. Ed. 2006, 45, 6574 –6577
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Communications
This confirms the principle of reversible detoxification, the
stability of the prodrugs under test conditions, and the
nondisruptive effects of the enzyme activity.
Table 1: Results of the HTCFA assay for the in vitro analysis of the
cytotoxicity of 2 and 3 against human bronchial carcinoma cells of line
[
a]
A549.
[
b]
Compound
b-d-gal.
IC50 [nm]
QIC50
6600
3
3
2
2
a/2b
a/2b
À
+
À
+
À
+
À
À
À
7.9”10
1.2
3.6”10
0.75
>7.8”10
5.5”10
1.5
(
(
(
(
+)-(1S,10R)-2a
+)-(1S,10R)-2a
À)-(1R,10S)-2b
À)-(1R,10S)-2b
4800
4
2
>140
rac-3
Figure 1. Comparison of the cytotoxicities in vitro of various antitumor
agents against human bronchial carcinoma cells of line A549. (^) (+)-
(
(
+)-(1S,10R)-3
À)-(1R,10S)-3
0.75
5.6”10
2
À1
(1S,10R)-2a in the presence of b-d-galactosidase (4 UmL ):
4
IC50 =0.75 nm; (~) carmustine (8): IC =2.6”10 nm; (*) melphalan
50
[a] Cells were incubated with the corresponding compounds for 24 h at
3
(9): IC50 =3.4”10 nm; (
&
) doxorubicin (10): IC50 =45 nm. R=relative
3
78C; after 12 days incubation, the clone formation rates were
clone-formation rate.
determined in comparison with untreated control cells (b-d-galactosi-
dase: Escherichia coli, 4 UmL ). [b] Presence or absence of b-d-
À1
galactosidase.
Received: March 9, 2006
Published online: September 8, 2006
Keywords: antitumor agents · cytostatic agents · duocarmycin ·
.
glycosides · prodrugs
[
1] Reviews: a) W. A. Denny, Cancer Invest. 2004, 22, 604 – 619;
b) L. F. Tietze, T. Feuerstein, Curr. Pharm. Des. 2003, 9, 2155 –
2
8
175; c) L. F. Tietze, T. Feuerstein, Aust. J. Chem. 2003, 56, 841 –
54; d) W. A. Denny, Eur. J. Med. Chem. 2001, 36, 577 – 595;
e) M. Jung, Mini-Rev. Med. Chem. 2001, 1, 399 – 407; f) G. Xu,
H. L. McLeod, Clin. Cancer Res. 2001, 7, 3314 – 3324; g) K. N.
Syrigos, A. A. Epenetos, Anticancer Res. 1999, 19, 605 – 614;
h) G. M. Dubowchik, M. A. Walker, Pharmacol. Ther. 1999, 83,
67 – 123; i) C. J. Springer, I. Niculescu-Duvaz, Adv. Drug Deliv-
ery Rev. 1997, 26, 151 – 172; j) L. N. Jungheim, T. A. Shepherd,
Chem. Rev. 1994, 94, 1553 – 1566.
[
[
[
2] K. D. Bagshawe, Br. J. Cancer 1987, 56, 531 – 532.
3] A. M. Wu, P. D. Senter, Nat. Biotechnol. 2005, 23, 1137 – 1146.
4] a) T. Arndt, U. Arndt, U. Reuning, H. Kessler in Cancer
Therapy: Molecular Targets in Tumor–Host Interactions (Ed.:
G. F. Weber), Horizon Bioscience, Norfolk, 2005, 93 – 141; b) D.
Ribatti, A. Vacca, F. Merchionne, M. Presta, Mini-Rev. Med.
Chem. 2005, 5, 313 – 317; c) K. A. El Sayed, Mini-Rev. Med.
Chem. 2005, 5, 971 – 993.
Scheme 3. The antitumor agents carmustine (8), melphalan (9), and
doxorubicin (10).
[
5] a) S. Dziadek, D. Kowalczyk, H. Kunz, Angew. Chem. 2005, 117,
7
798 – 7803; Angew. Chem. Int. Ed. 2005, 44, 7624 – 7630; b) S.
Dziadek, A. Hobel, E. Schmitt, H. Kunz, Angew. Chem. 2005,
17, 7803 – 7808; Angew. Chem. Int. Ed. 2005, 44, 7630 – 7635.
6] a) M. E. M. Noble, J. A. Endicott, L. N. Johnson, Science 2004,
03, 1800 – 1805; b) C. Garcia-Echeverria, D. Fabbro, Mini-Rev.
Med. Chem. 2004, 4, 273 – 283.
1
To compare the compounds 2 and 3 that we have
developed with other cytotoxic drugs, HTCFA assays were
performed on the A549 cell line with carmustine (8),
melphalan (9) and doxorubicin (10), which are currently
used as anticancer agents (Scheme 3 and Figure 1). The
results confirm that in the presence of the enzyme b-d-
galactosidase, the new prodrug (+)-(1S,10R)-2a has a higher
biological activity than compounds 8–10. Consequently, the
new compound has considerable benefits for use within the
framework of the ADEPT concept.
[
3
[7] a) M. Langer, F. Kratz, B. Rothen-Rutishauser, H. Wunderli-
Allenspach, A. G. Beck-Sickinger, J. Med. Chem. 2001, 44,
1341 – 1348; b) M. Langer, A. G. Beck-Sickinger, Curr. Med.
Chem.: Anti-Cancer Agents 2001, 1, 71 – 93.
[
[
8] L. F. Tietze, T. Herzig, T. Feuerstein, I. Schuberth, Eur. J. Org.
Chem. 2002, 1634 – 1645.
9] a) L. F. Tietze, T. Feuerstein, A. Fecher, F. Haunert, O. Panknin,
U. Borchers, I. Schuberth, F. Alves, Angew. Chem. 2002, 114,
6
576
www.angewandte.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 6574 –6577

Angewandte
Chemie
785 – 787; Angew. Chem. Int. Ed. 2002, 41, 759 – 761; b) L. F.
Tietze, M. Lieb, T. Herzig, F. Haunert, I. Schuberth, Bioorg.
Med. Chem. 2001, 9, 1929 – 1939; c) L. F. Tietze, T. Herzig, A.
Fecher, F. Haunert, I. Schuberth, ChemBioChem 2001, 2, 758 –
765; d) L. F. Tietze, R. Hannemann, W. Buhr, M. Lögers, P.
Menningen, M. Lieb, D. Starck, T. Grote, A. Döring, I.
Schuberth, Angew. Chem. 1996, 108, 2840 – 2842; Angew.
Chem. Int. Ed. Engl. 1996, 35, 2674 – 2677.
[
10] a) G. Wei, N. A. Loktionova, A. E. Pegg, R. C. Moschel, J. Med.
Chem. 2005, 48, 256 – 261; b) H. Cheng, X. Cao, M. Xian, L.
Fang, T. B. Cai, J. J. Ji, J. B. Tunac, D. Sun, P. G. Wang, J. Med.
Chem. 2005, 48, 645 – 652; c) M. Y. Torgov, S. C. Alley, C. G.
Cerveny, D. Farquhar, P. D. Senter, Bioconjugate Chem. 2005, 16,
717 – 721; d) E. Bouvier, S. Thirot, F. Schmidt, C. Monneret,
Bioorg. Med. Chem. 2004, 12, 969 – 977; e) T. Kline, M. Y.
Torgov, B. A. Mendelsohn, C. G. Cerveny, P. D. Senter, Mol.
Pharm. 2004, 1, 9 – 22; f) E. Bouvier, S. Thirot, F. Schmidt, C.
Monneret, Org. Biomol. Chem. 2003, 1, 3343 – 3352; g) H.
Townes, K. Summerville, B. Purnell, M. Hooker, E. Madsen, S.
Hudson, M. Lee, Med. Chem. Res. 2002, 12, 248 – 253; h) N.
Amishiro, S. Nagamura, C. Murakata, A. Okamoto, E. Kobaya-
shi, M. Asada, K. Gomi, T. Tamaoki, M. Okabe, N. Yamaguchi,
K. Yamaguchi, H. Saito, Bioorg. Med. Chem. 2000, 8, 381 – 391.
11] a) M. Ichimura, T. Ogawa, K. Takahashi, E. Kobayashi, I.
Kawamoto, T. Yasuzawa, I. Takahashi, H. Nakano, J. Antibiot.
[
1990, 43, 1037 – 1038; b) M. Ichimura, T. Ogawa, S. Katsumata,
K. Takahashi, I. Takahashi, H. Nakano, J. Antibiot. 1991, 44,
1045 – 1053; c) D. L. Boger, D. S. Johnson, Angew. Chem. 1996,
108, 1542 – 1580; Angew. Chem. Int. Ed. Engl. 1996, 35, 1438 –
1474.
[
12] a) L. F. Tietze, F. Haunert, T. Feuerstein, T. Herzig, Eur. J. Org.
Chem. 2003, 562 – 566; b) R. Baird, S. Winstein, J. Am. Chem.
Soc. 1963, 85, 567 – 578.
[
[
[
13] J. B. J. Milbank, M. Tercel, G. J. Atwell, W. R. Wilson, A. Hogg,
W. A. Denny, J. Med. Chem. 1999, 42, 649 – 658.
14] For the synthesis of 5, the toxic nBu SnH, which was formerly
3
used, was replaced by tris(trimethylsilyl)silane.
15] Preparative HPLC separation: Chiralpak IA column (250 ꢀ
2
0 mm, particle size: 5 mm), mobile phase: n-heptane/dichloro-
À1
methane (4:1), flow rate: 18 mLmin
, a = 2.05, (+)-5:
9
9.9% ee, (À)-5: 99.9% ee.
[
[
16] L. F. Tietze, F. Major, J. Magull, unpublished results.
17] a) S. Ram, L. D. Spicer, Tetrahedron Lett. 1987, 28, 515 – 516;
b) T. Bieg, W. Szeja, Synthesis 1985, 76 – 77.
[
18] a) R. R. Schmidt, Angew. Chem. 1986, 98, 213 – 236; Angew.
Chem. Int. Ed. Engl. 1986, 25, 212 – 235; b) W. Dullenkopf, J. C.
Castro-Palomino, L. Manzoni, R. R. Schmidt, Carbohydr. Res.
1996, 296, 135 – 147.
[
19] L. F. Tietze, B. Krewer, H. Frauendorf, F. Major, I. Schuberth,
Angew. Chem. 2006, 118, 6720 – 6724; Angew. Chem. Int. Ed.
2006, 45, 6570 – 6574.
Angew. Chem. Int. Ed. 2006, 45, 6574 –6577
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
6577