Z. Cai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1354–1358
1357
% T.G.I.
respectively. Moreover, the ring constrained analog 35
1500
1250
1000
750
500
250
0
was almost 8-fold more potent than conformation flexi-
ble analog 34 although the terminal amino groups in
both compounds were two carbon atoms away from
the N- or O-oxadiazole core. Compound 35 also dis-
played good cellular potency in HUVEC proliferation
assay.
Vehicle
30 mg/kg
90 mg/kg
0%
66%
The preference of amino group being three carbons or
more away from the N- or O-oxadiazole core with re-
spect to activity was observed as in compounds 27–32
(with acyclic amino side chains), and 36–37 (with het-
erocyclic amine side chains), regardless of conforma-
tional nature of the amino group. All analogs
demonstrated potent inhibitory activity in VEGFR-2
kinase assay as well as in cell proliferation assay except
compounds 28 and 29, which were moderate VEGFR-2
inhibitors in the cellular assay. Compounds 27
(branched alkyl amine side chain), 32 and 33 possessed
strong inhibition with CYP3A4, while compounds with
straight and long terminal side chains as 28–31 tended
to be weak CYP3A4 inhibitors. The O-tethered oxadi-
azole analogs 36 and 37 exhibited moderate activity
against CYP3A4.
14
19
24
29
Days post-implant
Figure 2. Antitumor activity of 37 versus L2987 human non-small cell
lung carcinoma xenografts implanted in athymic mice. Arrows indicate
dosing.
doses. Compound 37 was not efficacious at a low dose
of 30 mg/kg.
In summary, a series of N-cyclopropylamides was iden-
tified as a replacement for methylhydroxamate-substi-
tuted pyrrolotriazine VEGFR-2 kinase inhibitors. SAR
studies of substituents on 1,3,4-oxadiazole provided a
series of compounds with potent enzymatic and
VEGF-stimulated HUVEC cellular inhibitory activity
against VEGFR-2. Among them, compound 37 also
demonstrated in vivo antitumor activity versus L2987
human lung carcinoma xenografts inplanted in athymic
mice.
Compounds that exhibited potent inhibition of HU-
VEC proliferation and a favorable CYP inhibition pro-
file14 were screened for in vitro metabolic stability
studies using human (HLM) and mouse (MLM) liver
microsomes (Table 3). Compounds 30, 31, and 37 dem-
onstrated low metabolic rates in both HLM and
MLM. Therefore, they were further evaluated in mouse
oral exposure studies. After administration of a 50 mg/
kg dose, 30 demonstrated moderate exposure levels of
drug in plasma, and 31provided extremely low oral
adsorption, whereas 37 achieved high systemic expo-
sure of drug with an AUC(0–4 h) of 53.2 lM h. On
the basis of superior oral exposure profile of 37 in
mouse, it was selected for evaluation in an in vivo effi-
cacy study.
References and notes
1. (a) Folkman, J.; Shing, Y. J. Biol. Chem. 1992, 267, 10931;
(b) Folkman, J. J. Semin. Oncol. 2001, 28, 536.
2. (a) Carmeliet, P.; Jain, R. K. Nature 2000, 407, 249; (b)
Carmeliet, P. Nat. Med. 2003, 9, 653.
3. Griffioen, A. W.; Molema, G. Pharmacol. Rev. 2000, 52,
237.
4. Folkman, J.; Klagsbrun, M. Science 1987, 235, 442.
5. Thomas, K. A. J. Biol. Chem. 1996, 271, 603.
6. Gimbrone, M. Drug Discov. Today 1997, 2, 50.
7. Folkman, J. J. Natl. Cancer Inst. 1990, 83, 4.
8. (a) Klebl, B. M.; Muller, G. Expert Opin. Ther. Targets
2005, 9, 975; (b) Kumar, C. C.; Madison, V. Expert Opin.
Emerg. Drugs 2001, 6, 303.
9. (a) Cohen, M. H.; Gootenberg, J.; Keegan, P.; Pazdur, R.
Oncologist 2007, 12, 356; (b) Sandler, A.; Gray, R.; Perry,
M. C.; Brahmer, J.; Schiller, J. H.; Dowlati, A.; Lilen-
baum, R.; Johnson, D. H. N. Eng. J. Med. 2006, 355,
2542; (c) Cardones, A. R.; Banez, L. L. Current Pharm.
Design 2006, 12, 387.
The in vivo anti-tumor activity of 37 was determined in
a L2987 human lung carcinoma xenograft model in
athymic mice. Following once daily oral administration
of 37 at two doses for 14 days (in Fig. 2), tumor growth
inhibition (%TGI) of 66% was achieved at high dose of
90 mg/kg. No adverse events were observed even at all
10. Sepp-Lorenzino, L.; Thomas, K. Expert Opin. Investig.
Drugs 2002, 11, 1447.
Table 3. Metabolic stability and mouse exposure data for 30, 31, and
37
11. Borzilleri, R. M.; Zheng, X.; Qian, L.; Ellis, C.; Cai,
Z.-W.; Wautlet, B. S.; Mortillo, S.; Jayaseelan, R.;
Kukral, D.; Fura, A.; Kamath, A.; Vyas, V.; Tokarski,
J. S.; Barrish, J. C.; Hunt, L. J.; Fargnoli, J.; Bhide, R.
J. Med. Chem. 2005, 12, 3991.
12. Borzilleri, R. M.; Bhide, R.; Barrish, J. C.; D’Arienzo, C.
J.; Derbin, G. M.; Fargnoli, J.; Hunt, L. J.; Jayaseelan, R.;
Kamath, A.; Kukral, D.; Marathe, P.; Mortillo, S.; Qian,
L.; Tokarski, J. S.; Wautlet, B. S.; Zheng, X.; Lombardo,
L. J. J. Med. Chem. 2006, 49, 3776.
Compound
HLM/ MLMa
nmol/min/mg
protein
Mouse oral exposureb,c
Cmax
(lM)
Tmax
(h)
AUC (0–4 h)
lM h
30
31
37
0.098/ 0.020
0.012/ 0.000
0.023/ 0.007
8.0
0.1
1.0
0.5
4.0
21.7
0.2
15.0
53.2
a Compounds at 3 lM concentration incubated in 10 mg of protein
(HLM or MLM) for 10 min.
b Dosed at 50 mg/kg, all values are means of three mice.
c Vehicle: PEG400:water (1:1).
13. Representative characterization data for compound 37:
N-cyclopropyl-2,4-difluoro-5-(5-isopropyl-6-(5-(piperidin-