Novel cytotoxic oxopyridoindolizines: Isopropyl-7,8,9-trichloro-6,7,8,9- tetrahydro-5-oxopyrido[2,3-a]-indolizine-10-carboxylates (OPIC)

Article abstract of DOI:10.3390/70800628

A series of eight new alkyl-7,8,9-trichloro-6,7,8,9-tetrahydro-5- oxopyrido[2,3-a]-indolizine-10-carboxylates (OPIC), analogues of camptothecin (CPT), were prepared in a one-pot reaction of 2,2′-bipyridine-3,3′- dicarboxylic acid (BPA) with a mixture of thionyl chloride/chlorine, followed by addition of the appropriate alcohol. This led to a mixture of OPIC compounds 3a-d, 4a-d and 3,3′-dialkoxycarbonyl-2,2′-bipyridines (BPE, 2a-d). The isopropyl OPIC 3c and its corresponding diastereoisomer 4c showed marked activity against three cancer cell lines compared to other analogs. These same diastereoisomers also displayed high cytotoxic activity against five leukemia cell lines, thus the presence of an isopropyl substituent on the carboxylic ester, as opposed to other alkyl substituents, appears to play a key role in the cytotoxic potency of this new class of compounds.

Full text of DOI:10.3390/70800628

Molecules 2002, 7, 628-640
molecules
ISSN 1420-3049
http://www.mdpi.org
Novel Cytotoxic Oxopyridoindolizines: iso-Propyl-7,8,9-tri-
chloro-6,7,8,9-tetrahydro-5-oxopyrido[2,3-a]-indolizine-10-
carboxylates (OPIC)
Franco Zunino,1 Ann T. Kotchevar,2 Michael Waring,3 Maria Daoudi,4 Najib Ben Larbi,4
Mostafa Mimouni,5 Najat Sam,5 Assou Zahidi5 and Taibi Ben-Hadda,4,5,*
1
Oncologia Sperimentale B, Instituto Nationale per lo Studio e la Cura dei Tumori, Via Venezian,
20133 Milano, Italy.
2
Department of Chemistry and Biochemistry, 1 University Plaza, Long Island University, Brooklyn,
NY 11201, USA.
3
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD,
UK.
4
Department of Chemistry, Sciences Faculty of D'Har Elmehraz, Fès 30000, Morocco.
5
Department of Chemistry, Sciences Faculty of Oujda, Oujda 60000, Morocco.
* Author to whom correspondence should be addressed; E-mail: benhadda@sciences.univ-oujda.ac.ma
Tel: +212 61 75 31 35 Fax : + 212 56 50 06 03.
Received: 16 May 2002; in revised form: 5 August 2002 / Accepted: 5August 2002 / Published:31 August
2002
Abstract: A series of eight new alkyl-7,8,9-trichloro-6,7,8,9-tetrahydro-5-oxopyrido[2,3-a]-
indolizine-10-carboxylates (OPIC), analogues of camptothecin (CPT), were prepared in a
one-pot reaction of 2,2'-bipyridine-3,3'-dicarboxylic acid (BPA) with a mixture of thionyl
chloride/chlorine, followed by addition of the appropriate alcohol. This led to a mixture of
OPIC compounds 3a-d, 4a-d and 3,3'-dialkoxycarbonyl-2,2'-bipyridines (BPE, 2a-d). The
isopropyl OPIC 3c and its corresponding diastereoisomer 4c showed marked activity against
three cancer cell lines compared to other analogs. These same diastereoisomers also
displayed high cytotoxic activity against five leukemia cell lines, thus the presence of an
isopropyl substituent on the carboxylic ester, as opposed to other alkyl substituents, appears
to play a key role in the cytotoxic potency of this new class of compounds.

629
Molecules 2002, 7
Keywords: Oxopyridopyndolizine carboxylate (OPIC), Cycloacylation, Camptothecin,
Cancer, Chlorination, N-ligands.
_________________________________________________________________________________
Introduction
The natural alkaloid camptothecin (CPT, Scheme 1), extracted from Camptotheca acuminata by
Wall and Wani [1], has demonstrated efficacy in the treatment of several cancers. However, interest in
its application as an anti-tumour agent has declined due to its toxic secondary effects. The cellular
target of the alkaloid has been shown to be topoisomerase I [2], an enzyme essential for religation of
DNA during a number of critical cellular processes, including replication, transcription and repair [3].
Camptothecin, topoisomerase I and DNA form a so-called "cleavable complex" [4] that results in
topoisomerase I-mediated DNA breaks by preventing DNA religation. These results have prompted the
synthesis of a variety of derivatives and analogues of camptothecin. Topotecan (Hycamtin) and
irinotecan (Camptosar) are two of the leading examples [5-7]. Much effort, including that leading to
the two drugs above cited, has been devoted towards increasing the water solubility of the
camptothecin analogues in order to obtain compounds with an improved pharmacological profile and
enhanced efficacy against human tumors [8-10].
Scheme 1.
O
4
O
7
3
5
4a
9
Cl
N
2
N
7
10b
6
10a
N
1
O
8
N
Cl
10
9
O
O
Cl
H3C
OH
O
OPIC
R
CPT
1
From structure-activity relationship studies [11] it appears that the E-ring lactone and the natural
20S-configuration are essential for anti-tumour activity. While the activity of compounds with
substitutions in rings C and D is critically dependent on the size and type of the substituents [12], most
structural modifications have concerned rings A and B where wide possibilities of variation exist,
especially at positions 7, 9, 10 and 11. Recently the structure of topoisomerase I covalent and non-
covalent complexes with a 22-base pair DNA duplex has been solved by X-ray analysis. Based on the
crystal structure and structure-activity relationships, a mode of binding for camptothecin has been
proposed [13]. In this and in the analogous compound [14], there is plenty of room for substitution at
position 7 without steric interference.
A number of camptothecin derivatives have been developed and tested against various cancer cell
lines in the past few years and have indicated the importance of lipophilic groups at position 7 of

630
Molecules 2002, 7
camptothecin for potent cytotoxic activity. The low solubility in water of these compounds does not
amount to a grave disadvantage due to the possibility of successful administration per os of
camptothecin derivatives [15,16].
We report the synthesis and in vitro anti-tumour activity of a new series of OPIC compounds
(Scheme 1) that are readily separated into their pure diastereoisomers (3a-d and 4a-d). These new
OPIC compounds, deriving from a pyridyl-oxo cyclisation, can be obtained through a simpler and
more economical synthetic method than CPT. The starting material, 2,2'-bipyridine-3,3'-dicarboxylic
acid (1), was obtained in good yield from commercial 1,10-phenanthroline by oxidation using the
KMnO4 procedure described by Ben-Hadda et al [17]. The reaction of 1 with thionyl chloride under an
inert atmosphere provides an unstable chloro-addition intermediate, the 3,3'-dichlorocarbonyl-2,2'-
bipyridine, which is converted into the 3,3’-dialkoxycorbonyl-2,2’-bipyridine (BPE) upon addition of
the desired alcohol. The same method for the synthesis of BPE could be slightly modified and usefully
exploited to prepare the new family of OPIC compounds.
Results and Discussion
Chemistry
Scheme 2 depicts the general reaction. In our first attempt, oxopyridoindolizine-carboxylates (OPIC
3, 4) were prepared from BPA (1) and freshly distilled thionyl chloride. After gentle reflux for 5-24 h,
followed by subsequent addition of the alcohol, the title compounds were isolated in very poor yield (2-
5%). The majority of products (62-93%) were the 3,3'-dialkoxycarbonyl-2,2'-bipyridines (BPE).
Scheme 2.
R
O
N
HO
N
O
O
O
Cl
Cl
1. SOCl2/ Cl2
2. ROH
N
O
N
N
O
+
Cl
N
O
O
O
OH
R
R
BPA : 1
BPE : 2a-d
OPI : 3a-d, 4a-d
[R = Me (a), R = Et (b), R = i-Pr (c), R = i-Bu (d)].
2a: (R = Me) 52%
2b: (R = Et) 81%
2c: (R =i-Pr) 73%
2d: (R = i-Bu) 78%
3a: (R = Me) 7%
3b: (R = Et) 4%
3c: (R =i-Pr) 6%
4a: (R = Me) 31%
4b: (R = Et) 12%
4c: (R =i-Pr) 12%
3d: (R = i-Bu) 3% 4d: (R = i-Bu) 16%

631
Molecules 2002, 7
The use of technical grade thionyl chloride (which contains small amounts of chlorine), under the
same conditions as above led to higher, but still modest, yields of the OPIC compounds: 3-7% for 3a-d
and 12-31% for 4a-d. The thionyl chloride contained a small amount of gaseous chlorine, most likely
produced in situ by thermo and/or photodecomposition of the reagent. This observation led us to
believe that the deliberate addition of chlorine would greatly improve the yield of OPIC. We found
that in addition to improving the yield of OPIC, the addition of gaseous chlorine to the reaction
decreased the reaction time from 5h to 2h. The optimisation and mechanism of the formation of OPIC
has been reported recently in more detail [20].
Evaluation of in vitro Anti-tumour Activity
A series of substituted OPIC (3 and 4) and BPE (2) differing in the substituents on the carboxylate
groups were selected by the National Cancer Institute for evaluation of their in vitro anticancer activity
(Table 1).
Table 1. In vitro cytotoxic activity of selected OPIC derivatives against three cell lines.
Growth Percentages20
Compound
R
Activity
(Lung)
NCI-H460
97
(Breast)
MCF7
89
(CNS)
SF-268
90
2b
2c
2d
3c
4b
4c
4d
Et
Inactive
Inactive
Inactive
Active
i-Pr
i-Bu
i-Pr
Et
105
90
114
74
81
82
96
20
72
99
67
96
Inactive
Active
i-Pr
i-Bu
105
19
62
100
62
100
Inactive
From the data in Table 1 it appears that only the oxopyridoindolizines 3c and 4c show any marked
cytotoxic activity compared to the rest of series (3 and 4). In this case, the degree of lipophilicity of the
carboxylate substituent does not correlate positively with the cytotoxic activity. A direct influence of
the lipophilic group from the pyridyl nucleus appears to be more important for activity. Compounds 3c
and 4c were further tested for their in vitro activity against 60 cell lines (Tables 2-4).

632
Molecules 2002, 7
Scheme 3.
O
O
Cl
Cl
Cl
Cl
N
O
N
O
Cl
N
N
O
O
Cl
4c
3c
Table 2. Comparative study of in vitro anti-tumour activity of diastereoisomers 3c and 4c
(Scheme 3) against leukemia and non-small cell lung cancer cell lines.
GI50
Panel
Cell line
3c
4c
HL-60(TB)
K-563
2.58x10-5
3.34x10-5
3.19x10-5
2.98x10-5
4.96x10-5
> 10-4
3.82x10-5
3.06x10-5
2.25x10-5
2.18x10-5
2.65x10-5
> 10-4
Leukemia
MOLT-4
RPMI-8226
SR
A549/ATCC
EKVX
> 10-4
> 10-4
> 10-4
4.63x10-5
> 10-4
4.62x10-5
> 10-4
> 10-4
2.62x10-5
HOP-62
> 10-4
Non-small
cell lung
HOP-92
> 10-4
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
> 10-4
> 10-4
> 10-4
> 10-4
3.57x10-5

633
Molecules 2002, 7
Table 3. Comparative in vitro anti-tumour activity of diastereoisomers 3c and 4c
against CNS, melanoma, ovarian and renal cancer cell lines.
GI50
Panel
Cell line
3c
4c
SF-268
8.21x10-5
> 10-4
> 10-4
> 10-4
> 10-4
> 10-4
> 10-4
> 10-4
> 10-4
> 10-4
> 10-4
> 10-4
> 10-4
4.79x10-5
> 10-4
SF-295
CNS cancer
SF-539
> 10-4
> 10-4
5.68x10-5
6.88x10-5
2.88x10-5
> 10-4
> 10-4
> 10-4
8.30x10-5
3.41x10-5
> 10-4
SNB-19
U251
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Melanoma
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
786-0
9.63x10-5
> 10-4
3.11x10-5
> 10-4
Ovarian cancer
Renal cancer
> 10-4
> 10-4
> 10-4
> 10-4
3.98x10-5
7.39x10-5
> 10-4
3.17x10-5
5.52x10-5
> 10-4
A498
ACHIN
CAKI-1
RXF 393
SN12C
> 10-4
> 10-4
> 10-4
> 10-4
> 10-4
> 10-4
2.12x10-5
> 10-4
2.18x10-5
> 10-4
TK-10
UO-31
> 10-4
> 10-4

634
Molecules 2002, 7
Table 4. Comparative in vitro anti-tumour activity of diastereoisomers 3c and 4c
against prostate, breast and colon cancer cell lines.
GI50
Panel
Cell line
3c
4c
PC-3
> 10-4
5.66x10-5
> 10-4
3.91x10-5
> 10-4
> 10-4
> 10-4
8.10x10-5
5.10x10-5
3.69x10-5
4.39x10-5
> 10-4
5.06x10-5
7.03x10-5
> 10-4
> 10-4
3.46x10-5
Prostate Cancer
DU-145
> 10-4
MCF7
8.44x10-5
> 10-4
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
MDA-N
Breast Cancer
> 10-4
8.15x10-5
> 10-4
4.41x10-5
> 10-4
BT-549
COLO 205
HCC-2998
HCT-116
HCT-15
> 10-4
> 10-4
> 10-4
Colon Cancer
> 10-4
HT29
> 10-4
KM12
> 10-4
SW-620
3.49x10-5
Conclusions
Chlorination of BPA with thionyl chloride in the presence of gaseous chlorine has been
demonstrated to be a powerful and optimal method for pyridyl-oxo cyclisation in the preparation of a
new heterocyclic family; the oxopyridoindolizine carboxylates (OPIC). This work provides for the first
time a simple one-pot synthetic methodology for the preparation of a wide range of OPIC compounds
which are analogues or hybrids of the natural alkaloid Camptothecin (CPT). The cytotoxic activity of
this new family suggests a promising novel approach to the design of prospective compounds for the
treatment of cancer.
As a guide for future work, the data reported here indicate that the OPIC compounds have a definite
potential efficacy that merits development through modification to both the pyridyl and the chlorinated
rings. We note, however, that although the compounds are nominally camptothecin analogues there is
no necessary presumption that they do in fact resemble camptothecin and thereby target topoisomerase
I. It might be possible to address this question by testing their effect(s) on two cell lines, one of which
has normal topoisomerase I and the other has a mutant (camptothecin-resistant) enzyme. If there were a
difference in GI50 value this would indicate that topoisomerase I is a critical target for the drugs. Given

635
Molecules 2002, 7
the rather low GI50 values of the present compounds, around 200 µM, it would be prudent first to seek
new derivatives having GI50 values around 1 micromolar or less so that the assay can be conducted
more efficiently, though the interest in compounds 3c and 4c because of their particular
stereochemistry remains undiminished. A further consideration relates to the poor solubility of the
chloro derivatives in water; for the cell line assays to work it is important to prepare derivatives that are
more soluble. Substituting the chloro atoms with some other groups such as alkyloxy or amino is
possible and might produce the desired effect.
Acknowledgements
This work was supported by grants from the Ministry of Education of Morocco (PROTARS No.
P1T2/27), Cancer Research U.K., France (AI N° 98/ 160/ SM), and Willaya of Oujda. We wish to
thank Professor Bruce Baguley, University of Auckland School of Medicine, New Zealand, for helpful
discussions. We would also like to thank Advanced Chemistry Development, Inc., Toronto, Canada for
the free on-line nomenclature of organic compounds. We are indebted to TAACF and NCI of USA for
biological tests.
Experimental
General
1
Melting points are determined by using a Buchi 510 apparatus and are uncorrected. H- and
13C-NMR spectra were recorded on a Bruker WP-80 operating at 200.131 MHz and AC 250 at 250.14
MHz or an AM 300 at 300.134 MHz spectrometer. The numbering used in the assignment of the NMR
spectra is shown in Scheme 4. Mass spectra were recorded on a Platform II Micro Mass spectrometer,
and FTIR spectra on a Nicolet 205 instrument.
General procedure for the synthesis of 3,3'-di-alkyloxycarbonyl-2,2'-bipyridines (BPE: 2) and alkyl-
7,8,9-trichloro-6,7,8,9-tetrahydro-5-oxopyrido [2,3-a]indolizine-10-carboxylates (OPIC: 3 and 4).
The 3,3'-dihydroxycarbonyl-2,2'-bipyridine (1) used in this work was prepared from 1,10-
phenanthroline 1 by a procedure described previously [17]. 2,2'-bipyridine-3,3'-dicarboxylic acid (BPA,
600 mg, 2.5 mmol) was added to technical grade thionyl chloride (10 mL) and the mixture was
refluxed for 1 h. The excess thionyl chloride was removed under vacuum to leave a yellow residue.
Toluene (20 mL) and an alcohol (ROH, 1 mL) were added and the solution was heated under reflux for
3 h. Chloroform (40 mL) was added and the organic phase was washed with a cooled solution of
sodium bicarbonate (2.5%). The organic layer was dried over sodium sulfate and the chloroform was
removed under reduced pressure. The crude product was purified on a silica gel column (l = 30 cm, θ =
3 cm). Three white solids were successively obtained. Compound 4 was eluted first using a (10:90)

636
Molecules 2002, 7
mixture of petroleum ether/dichloromethane. Derivative 3 eluted next using a 5;95 ratio of
ether/dichloromethane. Finally, the diester 2 was recovered by elution with (40:60) ether/acetone.
Scheme 4. Labelling used for NMR assignments [a (R = Me), b (R = Et), c (R = i-Pr), d (R = i-Bu)]
R
O
O
O
O 1'
N
Cl
Cl
3
4
6
6'
4'
4
4
8
8
N
O
N
O
2
9
9
5
5'
3
3
Cl
Cl
2'
N
10
11
N
1 O
10
11
N
1 O
1 O
3'
2
2
Cl
Cl
O
R
R
R
2a-d (BPE)
3a-d (OPIC)
4a-d (OPIC)
1
3,3'-di-Methoxycarbonyl-2,2'-bipyridine (2a) : Yield 52%; white powder; m.p. 135-136 °C; H-NMR
3
4
(250.14 MHz, CDCl3) δ: 8.74 (dd, 2 H, H6/H6', JH6-H5 = 4.83 Hz, JH6-H4 = 1.62 Hz), 8.37 (dd, 2 H,
3
4
3
H4/H4', JH4-H5 = 7.94 Hz, JH4-H6 = 1.62 Hz), 7.44 (dd, 2 H, H5/H5', JH5-H4 = 7.94 Hz, 3JH5-H6 = 4.83
Hz), 3.66 (s, 6 H, 2 CH3); Analysis, Calcd. (Found) for C14H12N2O4: C 61.76 (60.13), H 4.42 (4.45), N
10.29 (9.82); MS m/z: 273.0 (Calcd for C14H12N2O4 : 272.26). IR (KBr) ν cm-1: 1720 (C=O, s), 1582,
1582 (C=C, m), 1440 (C=N, m), 1307, 1299 (C-O, m).
1
3,3'-di-Ethoxycarbonyl-2,2'-bipyridine (2b): Yield 81%; white powder; m.p. 89-90 °C; H-NMR
3
4
(250.14 MHz, CDCl3) δ ppm: 8.74 (dd, 2 H, H6/H6', JH6-H5 = 4.80 Hz, JH6-H4 = 1.56 Hz), 8.36 (dd, 2
3
4
3
3
H, H4/H4', JH4-H5 = 7.93 Hz, JH4-H6 = 1.56 Hz), 7.42 (dd, 2 H, H5/H5', JH5-H4 = 7.93 Hz, JH5-H6
=
3
3
4.80Hz), 4.08 (qu, 4 H, 2 CH2, JCH2-CH3 = 7.15 Hz), 1.02 (t, 6 H, 2 CH3, JCH3-CH2 = 7.15 Hz);
Analysis, Calcd. (Found) for C16H16N2O4: C 64.01 (63.56.), H 5.33 (5.62), N 9.33 (9.13); MS m/z :
301.10 (Calcd. for C16H16N2O4 : 300.316). IR (KBr) ν cm-1: 1724 (C=O, s), 1578, 1565 (C=C, w),
1423 (C=N, m), 1277 (C-O, w).
3,3'-di-iso-Propyloxycarbonyl-2,2'-bipyridine (2c): Yield 73%; white powder; m.p. 81-82 °C; 1H-NMR
3
4
(250.14 MHz, CDCl3) δ ppm: 8.73 (dd, 2 H, H6/H6', JH6-H5 = 4.80 Hz, JH6-H4 = 1.70 Hz), 8.37 (dd, 2
H, H4/H4', 3JH4-H5 = 7.93 Hz, 4JH4-H6 = 1.70 Hz), 7.42 (dd, 2 H, H5/H5', 3JH5-H4 = 7.93 Hz, 3JH5-H6 = 4.80
Hz), 4.95 (qu, 2 H, CH, 3JCH-CH3 = 6.31 Hz), 0.97 (d, 12 H, 4 CH3, 3JCH3-CH = 6.31 Hz); Analysis Calcd.
(Found) for C18H20N2O4: C 65.85 (65.78.), H 6.10 (6.22), N 8.53 (8.38); MS m/z : 329.10 (Calcd. for
C18H20N2O4 : 328.371); IR (KBr) ν cm-1: 1695 (C=O, s), 1535, 1565 (C=C, w), 1415 (C=N, m), 1260
(C-O, w).

637
Molecules 2002, 7
3,3'-di-iso-Butyloxycarbonyl-2,2'-bipyridine (2d): Yield 78%; white powder; m.p. 88-89 °C; 1H-NMR
3
4
(250.14 MHz, CDCl3) δ ppm: 8.74 (dd, 2 H, H6/H6', JH6-H5 = 4.83 Hz, JH6-H4 = 1.66 Hz), 8.37 (dd, 2
H, H4/H4', 3JH4-H5 = 7.93 Hz, 4JH4-H6 = 1.66 Hz), 7.44 (dd, 2 H, H5/H5', 3JH5-H4 = 7.93 Hz, 3JH5-H6 = 4.83
3
3
Hz), 3.84 (d, 2 H, CH2, JCH-CH2 = 6.6 Hz), 1.68 (m, 2 H, 2 CH), 0.76 (d, 12 H, 4 CH3, JCH3-CH = 6.6
Hz); Analysis, Calcd. (Found) for C20H24N2O4: C 67.42 (67.55), H 6.74 (6.82), N 7.86 (7.87); MS m/z:
357.10 (Calc. for C20H24N2O4 : 356.425); IR (KBr) ν cm-1 : 1713 (C=O, s), 1589/1561 (C=C, w), 1470
(C=N, m), 1289 (C-O, w).
(7R,8R,9S)-Methyl-7,8,9-trichloro-6,7,8,9-tetrahydro-5-oxopyrido[2,3-a]indolizine-10-carboxylate
(3a): Yield 7%; white powder; m.p. 125-126 °C; 1H-NMR (200,131 MHz; CDCl3) δ ppm: 8.86 (dd, 1
3
4
3
4
H, H2, JH2-H3 = 4.9 Hz, JH2-H4 = 1.6 Hz), 8.14 (dd, 1 H, H4, JH4-H3 = 7.8 Hz, JH4-H2 = 1.6 Hz), 7.48
3
3
3
(dd, 1 H, H3, JH3-H4 = 7.8 Hz, JH3-H2 = 4.9 Hz), 6.47 (d, 1 H, H8, JH8-H9 = 3 Hz), 5.26 (d, 1 H, H10,
3JH10-H9 = 9.8 Hz), 4.52 (dd, 1 H, H9, JH9-H8 = 3.05 Hz, JH9-H10 = 9.75 Hz), 3.96 (s, 3 H, CH3); MS
m/z: 347.0 (Calcd. for C13H9N2O3Cl3: 346.60). IR (KBr) ν cm-1: 1744 (C=O, s), 1715 (C=O, s),
1604,1584 (C=C, w), 1437 (C=N, m), 1292 (C-O, w).
3
3
(7R,8R,9S)-Ethyl-7,8,9-trichloro-6,7,8,9-tetrahydro-5-oxopyrido[2,3-a]indolizine-10-carboxylate (3b):
Yield 4%; white powder; m.p. 104-106 °C; 1H-NMR (200.131 MHz, CDCl3) δ ppm: 8.85 (dd, 1 H, H2,
3JH2-H3 = 4.8 Hz, 4JH2-H4 = 1.6 Hz), 8.14 (dd, 1 H, H4, 3JH4-H3 = 7.8 Hz, 4JH4-H2 = 1.6 Hz), 7.48 (dd, 1 H,
H3, 3JH3-H4 = 7.8 Hz, 3JH3-H2 = 4.8 Hz), 6.46 (d, 1 H, H8, 3JH8-H9 = 3.2 Hz), 5.305 (d, 1 H, H10, 3JH10-H9
=
9.8 Hz), 4.53 (dd, 1 H, H9, 3JH9-H8 = 3.2 Hz, 3JH9-H10 = 9.8 Hz), 4.46 (m, 2 H, CH2, 3J = 7.12 Hz), 1.37
3
(t, 3 H, CH3, J = 7.12 Hz); MS m/z : 360.9 (Calcd. for C14H11N2O3Cl3 : 360.63); IR (KBr) ν cm-1 :
1752 (C=O, s), 1718 (C=O, s), 1604, 1583 (C=C, w), 1457 (C=N, m).
(7R,8R,9S)-iso-Propyl-7,8,9-trichloro-6,7,8,9-tetrahydro-5-oxopyrido[2,3-a]indolizine-10-carboxylate
(3c): Yield 6%; white powder; m.p. 99-102 °C; 1H-NMR (200.131 MHz, CDCl3) δ ppm: 8.83 (dd, 1 H,
H2, 3JH2-H3 = 4.8 Hz, 4JH2-H4 = 1.4 Hz), 8.12 (dd, 1 H, H4, 3JH4-H3 = 7.7 Hz, 4JH4-H2 = 1.4 Hz), 7.51 (dd, 1
H, H3, 3JH3-H4 = 7.7 Hz, 3JH3-H2 = 4.8 Hz), 6.47 (d, 1 H, H8, 3JH8-H9 = 3.03 Hz), 5.33 (m, 1 H, H13), 5.26
3
3
(d, 1 H, H10, 3JH10-H9 = 9.7 Hz), 4.52 (dd, 1 H, H9, JH9-H8 = 3.03 Hz, JH9-H10 = 9.76 Hz), 1.39 (d, 3 H,
3
3
CH14, J = 6.2 Hz), 1.34 (d, 3 H, H14, J = 6.2 Hz); MS m/z : 374.90 (Calcd. for C15H13N2O3Cl3 :
374.653); IR (KBr) ν cm-1: 1745 (C=O, s), 1715 (C=O, s), 1603, 1584 (C=C, w), 1392 (C=N, m). The
crystallographic structure of 3c has been recently reported [22].
(7R,8R,9S)-iso-Butyl-7,8,9-trichloro-6,7,8,9-tetrahydro-5-oxopyrido[2,3-a]indolizine-10—carboxylate
(3d): Yield 3%; white powder; m.p. 95-97 °C; 1H-NMR (200.131 MHz, DCCl3) δ ppm: 8.90 (dd, 1 H,
H2, 3JH2-H3 = 4.86 Hz, 4JH2-H4 = 1.6 Hz), 8.18 (dd, 1 H, H4, 3JH4-H3 = 7.8 Hz, 4JH4-H2 = 1.6 Hz), 7.52 (dd,
3
3
3
1 H, H3, JH3-H4 = 7.8 Hz, JH3-H2 = 4.86 Hz), 6.52 (d, 1 H, H8, JH8-H9 = 3.07 Hz), 5.26 (d, 1 H, H10,
3JH10-H9 = 9.75 Hz), 4.56 (dd, 1 H, H9, JH9-H8 = 3.08 Hz; JH9-H10 = 9.75 Hz), 4.21 (m, 2 H, CH2, J =
6.67 Hz), 2.09 (m, 1 H, CH, J = 6.7 Hz), 1.04 (d, 6 H, 2 CH3, J = 6.7 Hz); IR (KBr) λ cm-1: 1750
3
3
3
3
3

638
Molecules 2002, 7
(C=O, s), 1715 (C=O, s), 1582 (C=C, w), 1473 (C=N, m); MS m/z : 388.90 (Calc. for C16H15N2O3Cl3 :
388.68); IR (KBr) ν cm-1 : 1751 (C=O, s), 1713 (C=O, s), 1584 (C=C, w), 1475 (C=N, m).
(7S,8R,9S)-Methyl-7,8,9-trichloro-6,7,8,9-tetrahydro-5-oxopyrido[2,3-a]indolizine-10--carboxylate
(4a): Yield 31%; white powder; m.p. 121-122 °C; 1H-NMR (250,14 MHz; CDCl3) δ ppm: 8.9 (dd, H2,
3
4
3JH2-H3 = 4.9 Hz, 4JH2-H4 = 1.6 Hz), 8.2 (dd, 1 H, H4, JH4-H3 = 7.8 Hz, JH4-H2 = 1.6 Hz), 7.53 (dd, 1 H,
H3, 3JH3-H4 = 7.8 Hz, 3JH3-H2 = 4.9 Hz), 6.49 (dd, 1 H, H8, 3JH8-H9 = 1.33 Hz, 4JH8-H10 = 2.1 Hz), 5.35 (t, 1
H, H9, 3JH9-H8 = 1.33 Hz, 3JH9-H10 = 1.33 Hz), 5.03 (dd, 1 H, H10, 3JH10-H9 = 1.33 Hz, JH8-H10 = 2.1 Hz),
4
4.0 (s, 3 H, CH3); Analysis Calcd. (Found) for C13H9N2O3Cl3: C 44.94 (44.98), H 2.59 (2.63), N 8.06
(7.99); MS m/z: 347.0 (Calcd. for C13H9N2O3Cl3: 346.60); IR (KBr) ν cm-1: 1744 (C=O, s), 1717
(C=O, s), 1601-1581 (C=C, w), 1434 (C=N, m), 1297 (C-O, w).
(7S,8R,9S)-Ethyl-7,8,9-trichloro-6,7,8,9-tetrahydro-5-oxopyrido[2,3-a]indolizine-10--carboxylate
(4b): Yield 12%; white powder; m.p. 102-103 °C; 1H-NMR (300.134 MHz, CDCl3) δ ppm: 8.90 (dd, 1
3
4
H, H2, JH2-H3 = 4.8 Hz, JH2-H4 = 1.6 Hz), 8.21 (dd, 1 H, H4, 3JH4-H3 = 7.8 Hz, 4JH4-H2 = 1.6 Hz), 7.53
3
3
3
4
(dd, , 1 H, H3, JH3-H4 = 7.8 Hz, JH3-H2 = 4.8 Hz), 6.49 (dd, 1 H, H8, JH8-H9 = 1.45 Hz, JH8-H10 = 2.04
Hz), 5.35 (t, 1 H, H9, 3JH9-H8 = 1.42 Hz, 3JH9-H10 = 1.45 Hz), 5.00 (dd, 1 H, H10, 3JH10-H9 = 1.45 Hz, 4JH10-
H8 = 2.04 Hz), 4.48 (m, 2 H, CH2, J = 7.12 Hz), 1.39 (t, 3 H, CH3, J = 7.12 Hz). {1H} C NMR
(MHz, CDCl3) δ ppm: 154.5 (C2), 132.2 (C4), 125.2 (C3), 62.5 (C13), 60.8 (C8), 52.8 (C10), 57.1 (C9),
14.5 (C14); Analysis, Calcd. (Found) for C14H11N2O3Cl3: C 46.97 (47.44), H 3.05 (3.33), N 7.74 (7.35);
MS m/z: 360.9 (Calcd. for C14H11NO3Cl3 : 360.63); IR (KBr) ν cm-1 : 1750 (C=O, s), 1716 (C=O, s),
1609, 1580 (C=C, w), 1457 (C=N, m). The crystallographic structure of 4b has been reported [20].
3
3
13
(7S,8R,9S)-iso-Propyl-7,8,9-trichloro-6,7,8,9-tetrahydro-5-oxopyrido[2,3-a]indolizine-10-carboxylate
(4c): Yield 12%; white powder; m.p. 97-99 °C; 1H-NMR (300.134 MHz, CDCl3) δ ppm: 8.90 (dd, 1 H,
H2, 3JH2-H3 = 4.8 Hz, 4JH2-H4 = 1.43 Hz), 8.21 (dd, 1 H, H4, 3JH4-H3 = 7.8 Hz, 4JH4-H2 = 1.43 Hz), 7.53 (dd,
1 H, H3, 3JH3-H4 = 7.8 Hz, 3JH3-H2 = 4.8 Hz), 6.48 (dd, 1 H, H8, 3JH8-H9 = 1.4 Hz, 4JH8-H10 = 2.1 Hz), 5.34
(t, 1 H, H9, 3JH9-H8 = 1.4 Hz, 3JH9-H10 = 1.4 Hz), 5.35 (m, 1 H, H13), 1.38 (d, 3 H, CH3, 3J = 6.2 Hz), 1.32
3
(d, 3 H, CH3, J = 6.3 Hz); Analysis Calcd. (Found) for C15H13N2O3Cl3: C 47.97 (47.96), H 3.46
(3.52), N 7.46 (7.39); MS m/z : 374.90 (Calcd. for C15H13N2O3Cl3: 374.653); IR (KBr) ν cm-1: 1741
(C=O, s), 1711 (C=O, s), 1600, 1582 (C=C, w), 1396 (C=N, m).
(7S,8R,9S)-iso-Butyl-7,8,9-trichloro-6,7,8,9-tetrahydro-5-oxopyrido[2,3-a]indolizine-10--carboxylate
1
(4d): Yield 16%; white powder; m.p. 96-97°C; H -NMR (200.131 MHz, DCCl3) δ ppm: 8.88 (dd, 1
4
H, H2, 3JH2-H3 = 4.86 Hz, JH2-H4 = 1.6 Hz), 8.21 (dd, 1 H, H4, 3JH4-H3 = 7.8 Hz, 4JH4-H2 = 1.6 Hz), 7.52
3
3
3
4
(dd, 1 H, H3, JH3-H4 = 7.8 Hz, JH3-H2 = 4.86 Hz), 6.50 (dd, 1 H, H8, JH8-H9 = 1.39 Hz, JH8-H10 = 2.04
Hz), 5.36 (t, 1 H, H9, 3JH9-H8 = 1.39 Hz, 3JH9-H10 = 1.39 Hz), 5.03 (dd, 1 H, H10, 3JH10-H9 = 1.39 Hz, 4JH10-
H8 = 2.04 Hz), 4.22 (m, 2 H, CH2, 3J = 6.7 Hz), 2.09 (m, 1 H, H14, 3J = 6.7 Hz), 1.0 (d, 6 H, 2 CH3, 3J =
6.7 Hz); Analysis Calcd. (Found) for C16H15N2O3Cl3: C 49.32 (49.23), H 3.85(3.89), N 7.19 (6.99);

639
Molecules 2002, 7
MS m/z: 388.90 (Calc. for C16H15N2O3Cl3 : 388.68); IR (KBr) ν cm-1 : 1750 (C=O, s), 1715 (C=O, s),
1582 (C=C, w), 1473 (C=N, m).
References and Notes
1. Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A. T.; Sim, G. A. J. Amer. Chem.
Soc. 1966, 88, 3888.
2. Hsiang, Y. H.; Hertzberg, R.; Hecht, S.; Liu, L. F. J. Bioi. Chem. 1985, 260, 14873.
3. Wang, J. C. Annu. Rev. Pharmacol. Toxicol. 1996, 94, 194.
4. Pommier, Y. Sem. Oncol. 1996, 23, 1.
5. Potmesil, H., Pinedo, H., Eds.; Camptothecins: New Anticancer Agents. CRC: Boca Raton, 1995.
6. Pantaziz, P., Giovannella, B. C.; Eds.; The Camptothecins: From discovery to the patient; Ann. N.
Y.: Acad. Sci. 1996, 803.
7. Lerchen, H. G. Drugs 1999, 2, 896.
8. Robinson, C.; Robinson, K.; Castaner, J. Drugs of Future,1996, 21, 881.
9. Eckhardt, S. G., Baker, S. D., Eckhardt, J. R.;Burke, T. G.; Warner, D. L.; Kuhn, J. G.; Rodriguez,
G.; Fields, S.; Thurman, A.; Smith, L.; Rothenberg, M. L.; White, L.; Wissel, P.; Kunka, R.;
Depee, S.; Littlefield, D.; Burris, H. A.; Von Hoff, D. D.; Rowinsky, E. K. Clinic Cancer Res.
1998, 4, 595.
10. Kumazawa, E.; Jimbo, T.; Ochi, Y; Tohgo, A. Cancer Chemother. Pharmacol. 1998, 42, 210.
11. Kawato, Y.; Terasawa, H., In Progress in Medicinal Chemistry, Ellis, G. P., Luscombe, D. K.
Eds.; Elsevier: London, 1997.
12. Dallava11e, S.; Ferrari, A.; Merlini, L ; Penco, S.; Carenini, N.; De Cesare M.; Perego, P.; Pratesi,
G.; Zunino, F. Bioorg. Med. Chem. Lett. 2001, 1.
13. Redinbo, R. R.; Stewart, L.; Kuhn, P.; Champoux, J. J.; Hol, W. G. J. Science 1998, 279, 1504.
14. Fan, J.; Weinstein, J. N.; Kohn, K. W.; Shi, L. M.; Pommier, Y. J. Med. Chem. 1998, 41, 2216.
15. Pantazis, P. Leuk. Res. 1995, 19, 775.
16. Cai, Q.; Lindsey, J. R.; Zhang, R. Int. J. 0ncol. 1997, 10, 953.
17. Ben-Hadda T.; Sam, N.; Le Bozec, H.; Dixneuf, P. H.; Inorg. Chem. Comm. 1999, 2, 460.
18. Lewis; Wedekind, Proc. Chem., 1909, 25, 59.
19. Pascal, P ; Nouveau Traitй de Chimie Minйrale, Tome XVI, Edis. Masson et Cie: Paris; 1960, pp
153-156.
20. Sam, N.; Elkadiri, S.; Le Bozec, H.; Toupet, L.; Daoudi, M.; Bitit, N.; Ben Hadda, T. and Dixneuf,
P.H. J. Chem. Soc., Perkin Trans. 1, 2002, 1688-1692.
21. In the current protocol, each cell line is inoculated and preincubated on a microtiter plate. Test
agents are then added at a single concentration and the culture incubated for 48 hours. End-point
determinations are made with Alamar blue (Biotechniques 1996, 21, 780). Results for each test
agent are reported as the percent growth of the treated cells compared to the untreated controls.
Compounds which reduce the growth of any one of the cell lines to approximately 32% or less

640
Molecules 2002, 7
(negative numbers indicate cell kill) are passed on for evaluation in the full panel of 60 cell lines
over a 5-log dose range. All compounds were tested at a final concentration of 10-4 M.
22. Le Bozec, H.; Toupet, L.; Daoudi, M.; Ben Larbi, N.; Sam, N.; Mimouni, M. and Ben Hadda, T.
Orgchem/0206001, 2002. Available from http://preprint.chemweb.com/Orgchem/0206001.
Sample Availability: Not available.
© 2002 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.