4698
E. L. Piatnitski et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4696–4698
Table 3. Enzymatic activities of selected compounds toward VEGFR-
1 and VEGFR-2 kinases9
References and notes
1. (a) Hamilton, W. J.; Boyd, J. D.; Mossman, H. W. Human
Embryology; William & Wilkins: Baltimore, 1962; (b)
Risau, W. Nature 1997, 386, 671.
2. Klagsbrun, M.; DÕAmore, P. Annu. Rev. Physiol. 1991, 53,
217.
3. Folkman, J. J. Natl. Cancer Inst. 1991, 82, 4.
4. Bold, G.; Altmann, K.-H.; Jorg, F.; Lang, M.; Manley, P.
W.; Traxler, P.; Wietfeld, B.; Bruggen, J.; Buchdunger, E.;
Cozens, R.; Ferrari, S.; Pascal, F.; Hofmann, F.; Martiny-
Baron, G.; Mestan, J.; Rosel, J.; Sills, M.; Stover, D.;
Acemoglu, F.; Boss, E.; Emmenegger, R.; Lasser, L.;
Masso, E.; Roth, R.; Schlachter, C.; Vetterli, W.; Wyss,
D.; Wood, J. M. J. Med. Chem. 2000, 43, 2310.
5. Hennequin, L. F.; Stokes, E. S. E.; Thomas, A. P.;
Johnstone, C.; Ple, P. A.; Ogilvie, D. J.; Dukes, M.;
Wedge, S. R.; Kendrew, J.; Curwen, J. O. J. Med. Chem.
2002, 45, 1300.
Compound
% Inhibition of
VEGFR-1 at 10 lMa
% Inhibition of
VEGFR-2 at 10 lMa
4
5
60
73
53
51
86
93
71
74
6
10
a Average of n = 3.
and 25 to 5 and 3). Mono-methyl substitution at the
amide group was tolerated, although the activity seems
to be reduced in some cases (26 and 27), whilst bis-
methylation at the amide group resulted in an inactive
compound (28). This alkylation strategy could also be
extended to include substitution with a group to aid
in solubility (29 and 30). Substitution with bulkier
groups, such as tert-butyl and iso-propyl, resulted in
a complete loss of activity (31 and 32), suggesting a rel-
atively restricted binding area. The amide moieties in
(3) and (4) could be replaced by a methoxycarbonyl
subunit (33 and 34) without significant loss of activity.
However, two sulfonamide replacements of the amide
resulted in no inhibitory activity against VEGFR-2
(35 and 36), although it should be noted that these
are disubstituted analogs. Removal of the amide group,
or its substitution with another small group (Me, OMe,
OEt, Cl) in compounds (37)–(48), completely eliminat-
ed the activity toward VEGFR-2 kinase. Thus, the
presence of a carbonyl-containing group (amide or
methyl ester) proved to be important for activity in this
class of compounds.10
6. See Lu, D.; Jimenez, X.; Zhang, H.; Bohlen, P.; Witte, L.;
Zhu, Z. Int. J. Cancer 2002, 97, 393, for an antibody
approach for VEGFR-2 inhibition.
7. VEGFR tyrosine kinase inhibition is determined by
measuring the phosphorylation level of poly-Glu-Ala-
Tyr-biotin (pGAT-biotin) peptide in a Homogeneous
Time-Resolved Fluorescence (HTRF) assay. Into a black
96-well Costar plate is added 2 ll/well of 25x compound in
100% DMSO (final concentration in the 50 ll kinase
reaction is typically 1 nM to 10 lM). Next, 38 ll of
reaction buffer (25 mM Hepes, pH 7.5, 5 mM MgCl2,
5 mM MnCl2, 2 mM DTT, and 1 mg/ml BSA) containing
0.5 mmol pGAT-biotin and 3–4 ng KDR enzyme is added
to each well. After 5–10 min preincubation, the kinase
reaction is initiated by the addition of 10 ll of 10 lM ATP
to reaction buffer, after which the plate is incubated at
room temperature for 45 min. The reaction is stopped by
addition of 50 ll KF buffer (50 mM Hepes, pH 7.5, 0.5 M
KF, 1 mg/ml BSA) containing 100 mM EDTA and
0.36 lg/ml PY20K (Eu-cryptate labeled anti-phosphoty-
rosine antibody, CIS Bio International). After 30 min,
100 ll of 10 nM SV-XL (modified APC-labeled Streptavi-
din, CIS Bio International) in KF buffer is added, and
after an additional 2 h incubation at room temperature,
the plate is read in a RUBYstar HTRF Reader.
A number of compounds described above were also test-
ed against a small kinase cross-reactivity panel including
VEGFR-1, a related receptor tyrosine kinase. The com-
pounds showed little or no activity against EGFR,
FGF-R1, InsR, c-Met, or IGF-1R at a screening con-
centration of 10 lM.11 However, preliminary data indi-
cated a significant inhibitory activity for both VEGFR-1
and VEGFR-2 kinases (Table 3).
8. Guery, S.; Parrot, I.; Rival, Y.; Wermuth, C. G. Synthesis
2001, 699.
9. These compounds were prepared by an alternative route
that will be detailed in a full report of this work.
10. An alternative arylphthalazine chemotype lacking the
carbonyl moiety was found to be very active against
VEGFR-2. Results will be disclosed in due course.
11. Compounds (3–29) were tested against this small kinase
panel. For example, the percentage inhibition for com-
pound (5) against these kinases at a screening concentra-
tion of 10 lM is as follows: FGF-R1, 36%; c-met, 12%;
EGFR, 14%; IGF-1R, 20%; InsR, 25% (average of n = 3).
A percentage inhibition of <40% at 10 lM is considered to
be inactive in our hands.
In summary, this Letter describes a new class of arylph-
thalazine compounds as inhibitors of the VEGFR
kinase family members.
Acknowledgment
We thank Dr. Marc Labelle for helpful suggestions
during the preparation of the manuscript.