S. J. Gregson et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1845±1847
1847
Table 1. In vitro cytotoxicity (human ovarian) and thermal denaturation data (calf thymus DNA) for the novel C2-exo unsaturated PBDs 19±22
and parent compounds 3 and 4
Cytotoxicity (mM)a
Induced ÁTm (ꢀC)b,d after incubation at 37 ꢀC for:
SKOV-3
A2780
A2780cisR
RFc
CH1d
CH1cisR
RFc
0 h
4 h
18 h
19
20
21e
22e
3
0.39
0.35
0.135
0.081
1.70
0.46
0.15
0.36
0.20
0.14
0.066
0.155
0.48
2.4
6.9
1.9
1.9
2.4
2.8
0.066
0.017
0.035
0.031
0.082
0.145
0.084
0.082
0.056
0.031
0.11
1.3
4.8
1.6
1.0
1.3
1.0
1.46
1.01
0.84
0.92
0.24
0.20
1.95
1.22
1.47
1.65
0.43
0.40
2.38
1.33
1.86
2.34
0.57
0.40
0.029
0.071
0.034
0.064
0.17
4
0.145
aDose required to inhibit cell growth by 50% compared with PBD-free controls after incubation for 96 h at 37 ꢀC.
bFor a 5:1 molar ratio of duplex-form CT-DNA (100 mM) and ligand (20 mM) in aqueous buer (10 mM NaH2PO4/Na2HPO4+1 mM Na2EDTA,
pH 7.00Æ0.01). All values are Æ<0.08±0.13 ꢀC.
cRF=resistance factor (IC50 cisplatin-resistant/parent).
dFor comparison: IC50 value for tomaymycin (2) in CH1 is 0.00013 mM, and ÁTm values are 0.97, 2.38 and 2.56 ꢀC after incubation at 37 ꢀC for 0, 4
and 18 h, respectively.
eEvaluated as a mixture of E/Z geometric isomers.
for 20; Â3.4 for 22) and SKOV-3 lines (Â5.0 for 22).
Furthermore, 22 was 7.2-fold more cytotoxic in the cis-
platin-resistant A2780cisR line than 4. It should be noted
that tomaymycin, which diers in structure from 21
only in having a C8-OH rather than a C8-OCH3 sub-
stituent, raises Tm signi®cantly over the three time
points and is 270-fold more cytotoxic in CH1. This
establishes the importance of a free hydroxyl group at
the C8-position in maximising both DNA stabilisation
and cytotoxicity.
2. Thurston, D. E. In Molecular Aspects of Anticancer Drug±
DNA Interactions; Neidle, S., Waring. M. J., Eds.; Macmillan
Press: UK, 1993; Vol. 1, p. 54.
3. Puvvada, M. S.; Hartley, J. A.; Jenkins, T. C.; Thurston, D.
E. Nucleic Acids Res. 1993, 21, 3671.
4. Thurston, D. E.; Bose, D. S.; Howard, P. W.; Jenkins, T.
C.; Leoni, A.; Baraldi, P. G.; Guiotto, A.; Cacciari, B.; Kel-
land, L. R.; Foloppe, M.-P.; Rault, S. J. Med. Chem. 1999, 42,
1951.
5. Gregson, S. J.; Howard, P. W.; Barcella, S.; Nakamya, A.;
Jenkins, T. C.; Kelland, L. R.; Thurston, D. E. Bioorg. Med.
Chem. Lett. 2000, 10, 1849.
6. Althuis, T. H.; Hess, H. J. J. Med. Chem. 1977, 20, 146.
7. 6-Nitroveratic acid is commercially available from Sigma-
Aldrich.
In conclusion, we have developed a versatile synthetic
route to C2-exo unsaturated PBD analogues which is
illustrated by the syntheses of 19±22. The versatile
ketone intermediates 15a,b can be eciently synthesised
on a large scale (i.e., >20 g), and should be applicable to
the synthesis of a wide range of C2-exo and C2-endo5
derivatives. Cytotoxicity and thermal denaturation data
for 19±22 con®rm that C2-unsaturation enhances both
cytotoxic potency and DNA-binding anity.
8. Fukuyama, T.; Liu, G.; Linton, S. D.; Lin, S.-C.; Nishino,
H. Tetrahedron Lett. 1993, 34, 2577.
1
9. Based on H and 13C NMR assignments, see: Langley, D.
R.; Thurston, D. E. J. Org. Chem. 1987, 52, 91 and Mori, M.;
Uozumi, Y.; Ishikura, M.; Ban, Y. Tetrahedron 1986, 42, 3793.
10. Barkley, M. D.; Cheatham, S.; Thurston, D. E.; Hurley,
L. H. Biochemistry 1986, 25, 3021.
11. Kelland, L. R.; Abel, G.; McKeage, M. J.; Jones, M.;
Goddard, P. M.; Vallenti, M.; Murrer, B.; Harrap, K. R.
Cancer Res. 1993, 53, 2581.
Acknowledgements
12. Data for 19 [a]D22=+583.14ꢀ (c=1.42, CHCl3); H NMR
1
(270 MHz, CDCl3) d 7.69 (d, 1H, J=4.39 Hz), 7.51 (s, 1H),
6.82 (s, 1H), 5.21±5.17 (m, 2H), 4.44±4.23 (s, 2H), 3.96±3.81
(m, 7H), 3.17±3.08 (m, 1H), 2.95 (d, 1H, J=14.29 Hz); 13C
NMR (67.8 MHz, CDCl3) d 164.7, 162.6, 151.5, 147.6, 141.6,
140.8, 119.8, 111.2, 109.4, 109.4, 56.2, 56.1, 53.8, 51.4, 35.5;
MS (EI), m/z (relative intensity) 272 ([M]Á+, 100), 257 (19), 243
(7), 230 (6), 212 (3), 191 (16), 164 (19), 136 (22), 93 (6), 82 (7),
80 (3), 53 (3); IR (neat) 3312 (br), 3083, 2936, 2843, 1624,
1603, 1505, 1434, 1380, 1264, 1217, 1180, 1130, 1096, 1069,
1007, 895, 837, 666, 594, 542 cm 1; exact mass calcd for
C15H16N2O3 m/z 272.1161, obsd m/z 272.1154.
The Cancer Research Campaign (UK) is thanked for pro-
viding ®nancial support for this work (SP1938/0301 to
DET/TCJ, and SP1938/0401 to DET). Additional support
was provided by Yorkshire Cancer Research (to TCJ).
References and Notes
1. Thurston, D. E. British Journal of Cancer 1999, 80 (Sup-
plement 1), 65.