Synthesis of new cytotoxic E-ring modified camptothecins

Article abstract of DOI:10.1016/j.tetlet.2010.09.130

In an effort to decrease the toxicity and improve the stability of the E-ring of camptothecin, new analogues with an 'inverted' lactone ring were designed and synthesized. The compounds retained a good cytotoxic activity on human non-small lung cancer cel

Full text of DOI:10.1016/j.tetlet.2010.09.130

Tetrahedron Letters 51 (2010) 6489–6492
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of new cytotoxic E-ring modified camptothecins
Salvatore Cananzi a, Sabrina Dallavalle a, , Alberto Bargiotti a, Lucio Merlini a, Roberto Artali b,
⇑
Giovanni Luca Beretta c
a Dipartimento di Scienze Molecolari Agroalimentari, Università di Milano, Via Celoria 2, 20133 Milano, Italy
b Dipartimento di Scienze Farmaceutiche ‘P. Pratesi’, Università di Milano, Via Mangiagalli 25, I-20133 Milano, Italy
c Unità di Chemioterapia e Farmacologia Antitumorale Preclinica, Dipartimento di Oncologia Sperimentale e Laboratori, Fondazione IRCCS Istituto Nazionale dei Tumori,
Via Venezian 1, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2010
Revised 22 September 2010
Accepted 27 September 2010
Available online 8 October 2010
In an effort to decrease the toxicity and improve the stability of the E-ring of camptothecin, new
analogues with an ‘inverted’ lactone ring were designed and synthesized. The compounds retained a good
cytotoxic activity on human non-small lung cancer cells H-460.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Camptothecin
Synthesis
Antitumour activity
Topoisomerase I
Molecular docking
Camptothecin (1, CPT) is a naturally occurring pentacyclic alka-
loid first isolated in 1966 from the Chinese tree Camptotheca
acuminata by Wall et al.1 CPT and its derivatives selectively inhibit
the nuclear enzyme Topoisomerase I (TopoI),2 thus representing a
very promising class of anticancer agents. The family of CPT deriv-
atives has been enlarged rapidly during the last decades.3 Among
CPT analogues, Topotecan4 and Irinotecan5 have been approved
as chemotherapeutic drugs in clinical treatment of human cancer
and several other derivatives are in different phases of clinical
trials.6
Topo I inhibition and in vivo potency. Three-dimensional structure
analyses of a ternary complex formed between TopoI, DNA and
Topotecan by X-ray crystallography showed that the drug interacts
both with the DNA base pairs flanking the cleavage sites and three
key aminoacid residues of the enzyme (Asn722, Arg364 and Asp533).7
The E-ring of CPT is essential to the interaction with the TopoI–
DNA cleavable complex.
Unfortunately, under physiological conditions, the presence of
the a-hydroxylactone group results in an equilibrium that favours
the inactive but toxic open carboxylate over the active ring-closed
One of the major limitations of CPT and analogues is the severe
toxicity, stemming in part from the instability of the a-hydroxylac-
tone E-ring. This ring plays a key role in supporting both efficient
lactone form.8 Moreover, the CPT carboxylate binds tightly to ser-
um albumin, which limits the fraction of drug in the active lactone
form.9
In early research, a number of E-ring modified CPT analogues
were prepared. These studies showed that any change in the
E-ring, such as deletion of the hydroxy group at C20 position, ring
⇑
Corresponding author. Tel.: +39 2 50316818; fax: +39 2 50316801.
E-mail address: sabrina.dallavalle@unimi.it (S. Dallavalle).
contraction to
a five-membered lactone, replacement of the
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.09.130

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S. Cananzi et al. / Tetrahedron Letters 51 (2010) 6489–6492
O
O
O
N
N
N
N
N
N
O
3
4
O
2
O
OH
OH
OH
Scheme 1. Examples of E-ring modified CPT analogues.
Their dehydrated conjugated ene-lactone analogues were only
modestly active in the TopoI cleavage test.13 Other successful ap-
proaches to the synthesis of stabile E-ring CPT derivatives were re-
cently reported. Five-membered lactone-free CPT analogues (3),
appeared to be potent inhibitors of TopoI and showed interesting
cytotoxic activity.16 Compound 417and the natural pyrroloquinazo-
linoquinoline alkaloid luotonin A,18 with an aromatic system as E-
ring, were also found to have significant in vitro cytotoxic activity
(Scheme 1).
These results raise the possibility that novel E-ring modified
analogues of CPT might retain or even possess enhanced antitu-
mour activity.
The present study was undertaken to explore a new structural
change at the E-ring, while keeping a lactone moiety in this portion
of the molecule. Thus, we designed derivatives with an ‘inverted’
lactone ring. (compounds 5 and 6, Scheme 2). In these compounds
the lactone ring is expected to be more stable, because the car-
R
O
N
N
5 R = H
6 R = OMe
O
O
Scheme 2. E-ring modified CPT analogues with an ‘inverted’ lactone ring.
lactone by a lactam, a thiolactone or an imide,10 reduction of the
lactone to lactol,11 synthesis of six-member ring12 or five-member
ring ethers10,13 gave essentially inactive compounds. These results
led to the conclusion that the intact six-membered ring was indis-
pensable for antitumor activity.
However, further investigations demonstrated that other E-ring
modified CPT analogues showed comparable or even better antitu-
mor activity than the parent compound. The first successful
approach was to expand the
additional methylene, thereby generating seven-membered
a-hydroxylactone ring by an
bonyl group, lacking the activating
a-hydroxy substituent typical
a
-hydroxylactone E-ring analogues, which are referred to as
of camptothecins, should be a weaker electrophile than the CPT
homocamptothecins (2),14 expectedly less prone to ring opening.
HomoCPTs were reported to exert potent inhibition of TopoI and
elevated levels of cytotoxicity, while showing enhanced stability
and decreased protein binding in human plasma.15
a
-hydroxylactone moiety (compare the HomoCPTs,14 see above).
Molecular modelling studies were performed to explore the
binding mode for the planned compounds to the covalent TopoI–
DNA complex. This study was based on the X-ray crystal structure
Figure 1. Schematic representation of the proposed binding mode for Topotecan (left) and compound 5 (right) in the TopoI–DNA complex. The side chains of aminoacids
relevant to the discussion are labeled and rendered in stick. Hydrogen bonds are shown as green dotted lines.
Figure 2. (left) Representation of the proposed top-score binding mode for compound 6 in the TopoI–DNA complex. The side chains of aminoacids relevant to the discussion
are labeled and rendered in stick, and hydrogen bonds are shown as green dotted lines. (right) Superimposition of the top-score binding conformations for compounds 5
(green) and 6 (red) inside the TopoI–DNA complex. Ligands are shown in stick, TopoI–DNA complex is rendered by using its solvent accessible surface (SAS) representation.

S. Cananzi et al. / Tetrahedron Letters 51 (2010) 6489–6492
6491
of a ternary complex between a human TopoI construct covalently
HO
O
attached to a DNA duplex with bound Topotecan.7 For comparison
purposes, the same docking protocol used to study the complex
containing Topotecan was applied in the present work.
N
a, b
N
O
14
The intercalation binding site was created by conformational
changes of the phosphodiester bond between the +1 (upstream)
and À1 (downstream) base pairs of the uncleaved strand, which
effectively ‘open’ the DNA duplex, with the evidence of only one di-
rect hydrogen bond between the Asp533 residue of the enzyme and
Topotecan in both the carboxylate and lactone models. The com-
parative analysis of the best poses obtained for compound 5 re-
vealed that the molecule formed base-stacking interactions with
OH
O
O
MeO
N
c
N
O
15
OH
O
OH
both the À1 and +1 base pairs, with a strong
p–p stacking between
MeO
the C-ring and guanosine at position +1 of the cleaved strand (+1G).
The interactions between compound 5 and TopoI–DNA complex
are given in Figure 1 (together with the experimental Topotecan
conformation), where the interactions listed are hydrogen bonds.
For compound 5, the hydrogen bond between the Asp533 resi-
due and Topotecan is replaced by two strong hydrogen bond inter-
actions between the lactone ring oxygen atom and both the NH1
and NH2 hydrogen atoms of Arg364 (close to Asp533), equal to
2.85 Å and 3.00 Å, respectively. Thus, compound 5 appears to make
use of distinct sets of interactions to stabilize the binding mode at
the intercalation site in TopoI–DNA complex. The promising bind-
ing provides structural support to synthesis of 5 and analogues.
Among them, compound 6, bearing a methoxy group in position
10, showed a calculated free energy (À9.12 kcal/mol) close to the
value calculated for compound 5 (À9.81 kcal/mol).
N
N
O
H
16
O
O
MeO
O
N
N
6
O
O
Scheme 4. Synthesis of compound 6 from 10-hydroxycamptothecin. Reagents and
conditions: (a) CH2N2, MeOH/dioxane 1:1, rt, 2 h, 100%; (b) NaBH4, MeOH, rt, 2 h,
90%; (c) silica-gel supported NaIO4, DCM, rt, 24 h, 60%.
As shown in Figure 2, the ligand is placed in the TopoI–DNA
complex in almost the same orientation as Topotecan and com-
pound 5, but the molecule is shifted towards the entrance of the
while treatment with sodium borohydride in DCM/methanol gave
compound 9 in good yield. The diol was disilylated using TBDMSCl
in DCM and selective primary desilylation was successfully
achieved using catalytic PTSA in methanol.19 To install the carbox-
ylic acid moiety in the right position for the subsequent lactoniza-
tion, one-carbon homologation of the side chain at position 8 of
compound 11 was required. Thus, compound 11 was converted
into the corresponding tosylate, that was immediately treated with
sodium cyanide to furnish compound 13. Finally, deprotection of
the hydroxyl group and simultaneous hydrolysis and cyclization
were achieved using HCl in ethanol (Scheme 3).
binding site. Consequently, the strong
p–p stacking interaction is
now between the B-ring and +1G. Due to the new position of the
ligand, compound 6 is connected to the TopoI–DNA complex
through two new hydrogen bonds between the heterocyclic nitro-
gen of B-ring and NH1Arg364 (2.93 Å) and N2 of guanosine +1
(2.78 Å).
The synthesis of compound 5 was performed using natural cam-
ptothecin as a starting material. CPT was readily reduced with so-
dium borohydride to the corresponding lactol 7, whose 1,2-diol
moiety was oxidatively cleaved with periodic acid to give com-
pound 8.12 Attempts to catalytically reduce the keto group failed,
The synthesis of the corresponding 10-methoxy derivative was
achieved following the same pathway described for compound 13,
O
a
O
O
O
b
c
N
N
N
N
N
N
O
O
O
H
7
8
1
O
O
OH
O
OH
O
O
OH
N
d
e
N
N
N
N
N
9
OH
N
OTBS
OH
10
11
TBSO
TBSO
HO
O
O
O
N
N
g
h
f
N
N
N
5
CN
OTs
12
13
O
O
TBSO
TBSO
Scheme 3. Synthesis of compound 5 from natural camptothecin. Reagents and conditions: (a) NaBH4, MeOH, rt, overnight, 90%; (b) NaIO4, AcOH, rt, overnight, 78%; (c) NaBH4,
DCM/MeOH 8:2, rt, 40 min, 87%; (d) TBDMSCl, imidazole, DMF, rt, 24 h, 70%; (e) PTSA, DCM/MeOH 1:1, 0 °C then rt, 1 h, 68%; (f) TsCl, DMAP, DCM, 0 °C, 24 h; (g) NaCN, DMSO,
70 °C, 2 h, 54%; (h) HCl, EtOH, 90 °C, 1.5 h, 29%.

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S. Cananzi et al. / Tetrahedron Letters 51 (2010) 6489–6492
6. (a) Dallavalle, S.; Merlini, L.; Penco, S.; Zunino, F. Expert Opin. Ther. Pat.
2002, 837–844; (b) Venditto, V. J.; Simanek, E. E. Mol. Pharmacol. 2010,
7, 307–349.
7. Staker, B. L.; Hjerrild, K.; Feese, M. D.; Behnke, C. A.; Burgin, A. B., Jr.; Stewart, L.
Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15387–15392.
starting from natural 10-OH-CPT. In this case the oxidative cleav-
age with sodium periodate appeared troublesome. Several at-
tempts were performed but they all resulted unsuccessful, giving
complex and unseparable mixtures of products. Finally, it was
found that treatment of a DCM solution of compound 15 with sil-
ica-gel supported NaIO4 afforded compound 16 in a satisfactory
yield. The remaining steps to get compound 6 were the same as de-
scribed previously for compound 5 (Scheme 4).
8. Burke, T. G.; Mi, Z. J. Med. Chem. 1993, 36, 2580–2582.
9. Burke, T. G.; Mi, Z. J. Med. Chem. 1994, 37.
10. Hertzberg, R. P.; Caranfa, M. J.; Holden, K. G.; Jakas, D. R.; Gallagher, G.;
Mattern, M. R.; Mong, S.-M.; Bartus, J. O.; Johnson, R. K.; Kingsbury, W. D. J.
Med. Chem. 1989, 32, 715–720.
11. Govindachari, T. R.; Ravindranath, K. R.; Viswanathan, N. J. Chem. Soc., Perkin
Trans. 1 1974, 1215–1217.
12. Du, W.; Curran, D. P.; Bevins, R. L.; Zimmer, S. G.; Zhang, J.; Burke, T. G. Bioorg.
Med. Chem. 2002, 10, 103–110.
13. Tangirala, R. S.; Antony, S.; Agama, K.; Pommier, Y.; Anderson, B. D.; Bevins, R.;
Curran, D. P. Bioorg. Med. Chem. 2006, 14, 6202–6212.
Compounds 5 and 620 were tested for their antiproliferative
activity on human non-small lung cancer cells H-460 (1 h exposure),
using Topotecan as a reference compound (IC50 = 1.38 0.95
Both analogues 5 and 6, even if still in the racemic form, maintained
a good cytotoxic activity, with IC50 less than 10 M (IC50 8.73 and
7.45 M, respectively). The results suggest that the introduction of
lM).
l
14. Lavergne, O.; Lesueur-Ginot, L.; Rodas, F. P.; Bigg, D. C. H. Bioorg. Med. Chem.
Lett. 1997, 7, 2235–2238.
l
15. (a) Bailly, C. Crit. Rev. Oncol. Hematol. 2003, 45, 91–108; (b) Yeh, T. K.; Li, C. M.;
Chen, C. P.; Chuu, J. J.; Huang, C. L.; Wang, H. S.; Shen, C. C.; Lee, T. Y.; Chang, C.
Y.; Chang, C. M.; Chao, Y. S.; Lin, C. T.; Chang, J. Y.; Chen, C. T. Pharmacol. Res.
2010, 61, 108–115; (c) Giannini, G.; Marzi, M.; Cabri, W.; Marastoni, E.;
Battistuzzi, G.; Vesci, L.; Pisano, C.; Beretta, G. L.; De Cesare, M.; Zunino, F.
Bioorg. Med. Chem. Lett. 2008, 18, 2910–2915.
16. (a) Hautefaye, P.; Cimetière, B.; Pierré, A.; Léonce, S.; Hickman, J.; Laine, W.;
Bailly, C.; Lavielle, G. Bioorg. Med. Chem. Lett. 2003, 13, 2731–2735; (b)
Lansiaux, A.; Léonce, S.; Kraus-Berthier, l.; Bal-Mahieu, C.; Mazinghien, R.;
Didier, S.; David-Cordonnier, M. H.; Hautefaye, P.; Lavielle, G.; Bailly, C.;
Hickman, J. A.; Pierré, A. Mol. Pharmacol. 2007, 72, 311–319.
an ‘inverted lactone’ moiety in ring E gives compounds with retained
antitumour activity, making them worthy of further investigation.
Work is currently in progress to evaluate the effect of substituents
and to study the Topoisomerase I inhibitory activity of these new
camptothecin analogues.
Acknowledgment
17. Grillet, F.; Baumlová, B.; Prévost, G.; Constant, J. F.; Chaumeron, S.; Bigg, D. C.
H.; Greene, A. E.; Kanazawa, A. Bioorg. Med. Chem. Lett. 2008, 18, 2143–2146.
18. (a) Ma, Z.-Z.; Hano, Y.; Nomura, T.; Chen, Y.-J. Heterocycles 1997, 46, 541–546;
(b) Cagir, A.; Jones, S. H.; Gao, R.; Eisenhauer, B. M.; Hecht, S. M. J. Am. Chem.
Soc. 2003, 125, 13628–13629.
The financial support was provided by MIUR (PRIN funds).
References and notes
1. Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A. T.; Sim, G. A. J.
Am. Chem. Soc. 1966, 94, 3888–3890.
19. Shahid, K. A.; Mursheda, J.; Okazaki, M.; Shuto, Y.; Gotob, F.; Kiyooka, S.-J.
Tetrahedron Lett. 2002, 43, 6377–6381.
20. Characterization of compound 5. Mp 320 °C dec 1H NMR 300 MHz (DMSO-d6)
d: 8.70 (s, 1H), 8.10–8.20 (m, 2H), 7.86 (m, 1H), 7.70 (m, 1H), 7.30 (s, 1H), 5.58
(m, 1H), 5.31 (s, 2H), 3.68 (m, 2H), 2.10 (m, 2H), 1.10 (t, J 7.4 Hz, 3H). HRMS
(ESI+) calcd for C20H16N2O3Na [M+Na]+ 355.10531; found 355.10612.
Characterization of compound 6. mp 278 °C dec 1H NMR 300 MHz (CDCl3) d:
8.28 (s, 1H), 8.10 (d, J 8.46 Hz, 1H), 7.50 (dd, J 8.46, 1.47 Hz, 1H), 7.19 (d, J
1.47 Hz, 1H), 7.10 (s, 1H), 5.41 (m, 1H), 5.31 (s, 2H), 4.00 (s, 3H), 3.89 (AB, 1H),
3.62 (AB, 1H), 2.10 (m, 2H), 1.10 (t, J 7.3 Hz, 3H). HRMS (ESI+) calcd for
2. Hsiang, Y.-H.; Hertzberg, R.; Hecht, S. M.; Liu, L. F. J. Biol. Chem. 1985, 260,
14873–14878.
3. (a) Thomas, C. J.; Rahier, N. J.; Hecht, S. M. Bioorg. Med. Chem. 2004, 12, 1585–
1604; (b) Dallavalle, S.; Merlini, L. In Modern Alkaloids; Fattorusso, E.,
Taglialatela, O., Eds.; Wiley-VCH: Weinheim, 2008; pp 503–520.
4. Sawada, S.; Okajima, S.; Aiyama, R.; Nokata, K. I.; Furuta, T.; Yokokura, T.;
Sugino, E.; Yamaguchi, K.; Miyasaka, T. Chem. Pharm. Bull. 1991, 39, 1446–1450.
5. Kingsbury, W. D.; Boehm, J. C.; Jakas, D. R.; Holden, K. G.; Hecht, S. M.;
Gallagher, G.; Caranfa, M. J.; McCabe, F. L.; Faucette, L. F.; Johnson, R. K.;
Hertzberg, R. P. J. Med. Chem. 1991, 34, 98–107.
C
C
21H19N2O4 [M+H]+ 363.13393; found 363.13426; HRMS (ESI+) calcd for
21H18N2O4Na [M+Na]+ 385.15500; found 385.11626.