4642
C. J. Burns et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4639–4642
Wilson, N.; Dilley, R. J.; Wan, S.; Charman, S. A.; Shackleford, D. M.; Fida, R.;
Malcontenti-Wilson, C.; Christophi, C.; Wilks, A. F., submitted for publication.
9. Burns, C. J.; Fantino, E.; Phillips, I. D.; Segal, D.; Su, S.; Harte, M. F.; Dublevic, V.;
Joffe, M.; Malcontenti-Wilson, C.; Christophi, C.; Shnyder, S. D.; Wilks, A. F., in
preparation.
10. Efficacy study of CYT997 in combination with carboplatin and etoposide in
glioblastoma. ClinicalTrials.gov Identifier NCT00650949.
11. Efficacy study of CYT997 in multiple myeloma. ClinicalTrials.gov Identifier
NCT00664378.
of 26 with borane dimethyl sulfide complex utilizing a chiral
pyrrolidine catalyst12 yielded the R alcohol 27. Conversion of the
R alcohol to the S amine 28 proceeded via azide formation and
Staudinger reduction,13 with concomitant stereoinversion. Base
catalysed addition of the weakly nucleophilic amine 28 to 2,4-di-
chloro-5-methylpyrimidine gave 29. Subsequent palladium cata-
lysed Suzuki–Miyaura reaction14 with the appropriate boronate
yielded compound 24 as a pale beige solid.15
In summary, we have discovered CYT997 (24), a potent small
molecule tubulin polymerization inhibitor and vascular disrupting
agent that has completed Phase I trials by both intravenous18 and
oral19 routes and is now in Phase II clinical trials for the treatment
of selected cancers.10,11
12. Moriyasu, M.; Shioiri, T. Synlett 1997, 273–274.
13. Gololobov, Y. G.; Zhmurova, I. N.; Kasukhin, L. F. Tetrahedron 1981, 37, 437–
472.
14. Kotha, S.; Lahiri, K.; Kashinath, D. Tetrahedron 2002, 9633–9695.
15. All compounds were characterized by 1H NMR and/or reverse phase LC–MS and
MS. Compound 24: A mixture of 6-chloro-N-[(1S)-1-pyridin-3-ylbutyl]pyrazin-
2-amine 29 (5.0 g, 18.1 mmol), 1-ethyl-3-[2-methoxy-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolane-2-yl)phenyl]-urea (6.94 g, 21.7 mmol) and aqueous
sodium carbonate (13.6 mL, 2 M, 27.1 mmol) in toluene (120 mL) and n-
propanol (40 mL) was degassed with nitrogen for 15 min. After this time
tetrakis(triphenylphosphine)palladium(0) (2.09 g, 1.81 mmol) was added and
the mixture was heated at reflux for 44 h. The mixture was allowed to cool to
room temperature and then diluted with ethyl acetate (150 mL). The organic
solution was washed with water (3 ꢁ 100 mL), brine (100 mL), dried (Na2SO4)
and filtered through Celite to give a dark brown foam which was purified by flash
chromatography (silica, ethyl acetate/methanol), followed by hot methanol
charcoal filtration and trituration with diethyl ether to give N-[2-methoxy-4-(6-
{[(1S)-1-pyridin-3-ylbutyl]amino}pyrazin-2-yl)phenyl]-N0-ethylurea 24 (4.7 g,
60%) as a pale beige solid. 1H NMR (CDCl3, 300 MHz) d 0.99 (t, J 7.2 Hz, 3H), 1.19 (t,
J 7.2 Hz, 3H), 1.36–1.53 (m, 2H), 1.83–2.05 (m, 2H), 2.11 (d, J 0.9 Hz, 3H), 3.28–
3.37 (m, 2H), 3.92 (s, 3H), 4.63 (t, J 4.8 Hz, 1H), 4.77 (d, J 6.6 Hz, 1H), 5.29–5.36 (m,
1H), 6.83 (br s, 1H), 7.22–7.26 (m, 1H), 7.68 (dt, J 7.8, 1.8 Hz, 1H), 7.72 (d, J 1.8 Hz,
1H), 7.86 (dd, J 8.7, 1.8 Hz, 1H), 8.06 (s, 1H), 8.07 (d, J 8.7 Hz, 1H), 8.47 (dd, J 5.5,
1.8 Hz, 1H), 8.69 (d, J 2.1 Hz, 1H).
References and notes
1. Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A. T. Med. Res. Rev. 1998, 18,
259–296.
2. Carlson, R. O. Expert Opin. Investig. Drugs 2008, 17, 707–722.
3. Tozer, G. M.; Kanthou, C.; Baguley, B. C. Nat. Rev. Cancer 2005, 5, 423–435.
4. Kanthou, C.; Tozer, G. M. Expert Opin. Ther. Targets 2007, 11, 1443–1457.
5. DU145 cells [ATCC HTB-81, human prostate carcinoma] (2 ꢁ 104 cells/mL)
and PC-3 cells [ATCC CRL-1435 human prostate adenocarcinoma]
(2 ꢁ 105 cells/mL) were cultured in microtest plates in DMEM and
Ham’s F12 K medium, respectively, supplemented with 10% FBS and the
test compound for 72 h at 37 °C in 5% CO2. Cell proliferation was
assessed using Alamar BlueTM (Serotec).
6. Tubulin assembly was conducted in MES buffer (100 mM MES, pH 6.4, 1 mM
EGTA, 0.5 mM MgCl2) using microtubules purified from bovine brain. Glycerol
(5%) and GTP (1 mM) were added to the tubulin mixture before the addition of 19
in order to promote polymerization. In the case of the negative control no GTP
16. The S amine 28 was obtained with an ee of 80%.
17. We required multi-kilogram amounts of 24 to support clinical trials. However,
the asymmetric reduction of 26 and conversion to amine 28 via the azide was
not considered viable on large scale. Therefore, we obtained the S amine 28 in
bulk quantities as its racemate and resolved it into its pure enantiomer through
salt formation with mandelic acid.
18. Phase I dose-escalation study for CYT997. ACTR number: ACTRN12605000793617.
19. Phase I accelerated dose-escalation study of CYT997 given as an oral capsule
every two weeks in patients with advanced solid tumours. ACTR number:
was added. The assay was commenced by addition of 10
lL of 19 (3 lM in 1%
DMSO) to 100 g tubulin/reaction) in a
l
L of the tubulin reaction mixture (ꢂ72 l
pre-warmed 96-well plate. Absorbance was read every 60 s at a wavelength of
340 nm for a period of 40 min using a BMG Polarstar plate reader at 37 °C.
7. Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; Garcia-Kendall, D.
Experientia 1989, 45, 209–211.
8. Burns, C. J.; Fantino, E.; Phillips, I. D.; Su, S.; Harte, M. F.; Bukczynska, P. E.;
Dublevic, V.; Frazzetto, M.; Joffe, M.; Kruszelnicki, I.; Wang, B.; Wang, Y.;