1198
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Seidelmann, D.; Menrad, A.; Haberey, M.; Thierauch, K. -H. US Patent
6,878,720 B2, 2005.
17. Chang, Y. S.; Cortes, C.; Polony, B. Proc. Am. Assoc. Cancer Res. (AACR) 2005, 46,
Abstr 2030.
18. Kaufman, S.; Starnes, C.; Coxon, A. Proc. Am. Assoc. Cancer Res. (AACR) 2006, 47,
Abstr 3792.
19. Kiselyov, A. S.; Piatnitski, L. E.; Samet, A. V.; Kisly, V. P.; Semenov, V. V. Bioorg.
Med. Chem. Lett. 2007, 17, 1369. and references cited therein.
20. Kiselyov, A. S.; Balakin, K.; Tkachenko, S. E. Exp. Opin. Invest. Drugs 2007, 16,
83.
VEGFR-2 activity similar to that for the VEGFR-1 (Table 1, for
example, 6, 12, 13, 17). This outcome could be of benefit in the
clinical setting as both receptors are reported to mediate VEGF sig-
naling in the angiogenesis.1–5,20 In our hands, bulky substituents in
the meta-position of Ar1 favored VEGFR-2 suggesting potential for
the development of an inhibitor selective against VEGFR-1 (Table 1,
for example, 3, 9, 11, 22). Further screening of 3–32 against a num-
ber of other receptor (IGF1R, InR, FGFR1, Flt3, EGFR, ErbB2, c-Met,
Ron) and cytosolic (PKA, GSK3b, bcr-Abl, bcr-AblT315, Cdk1/2, Src,
Auroras A and B, Plk1) kinases in HTRF format indicated no signif-
icant cross reactivity (PI < 30%, triplicate measurements) at a con-
**
21. Analytical data for selected compounds—3: 4-(Pyridin-4-ylmethylamino)-N-(3-
(trifluoromethyl)phenyl)isothiazole-3-carboxamide; mp 217–219 °C, 1H NMR
(400 MHz, DMSO-d6) d, ppm: 4.46 (d, J = 7.2 Hz, 2H), 6.38 (br s, exch. D2O,
1H, NH), 7.26 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 7.4 Hz, 1H), 7.45 (d, J = 7.6 Hz, 2H),
7.58 (m, 1H), 8.04 (s, 1H), 8.52 (d, J = 5.6 Hz, 2H), 8.79 (s, 1H), 10.38 (br s, exch.
D2O, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d, ppm: 45.7, 104.9, 117.7, 120.3,
123.0, 124.1, 124.3, 129.4, 130.9, 135.6, 147.5, 148.1, 150.2, 157.4, 163.7; ESI
MS (M+1): 379, (MÀ1): 377; HRMS, exact mass calcd. for C17H13F3N4OS:
378.0762, found: 378.0758. Elemental analysis: calcd for C17H13F3N4OS: C,
53.96; H, 3.46; N, 14.81; found: C, 53.73; H, 3.62, N, 14.58.11: N-(3-tert-
butylphenyl)-4-(pyridin-4-ylmethylamino)isothiazole-3-carboxamide; mp 223–
235 °C; 1H NMR (400 MHz, DMSO-d6) d, ppm: 1.25 (s, 9H, tBu), 4.36 (d,
J = 7.2 Hz, 2H), 6.48 (br s, exch. D2O, 1H, NH), 7.07 (d, J = 7.6 Hz, 1H), 7.16–7.18
(m, 2H), 7.42 (d, J = 7.6 Hz, 2H), 7.69 (s, 1H), 8.55 (d, J = 5.6 Hz, 2H), 8.73 (s, 1H),
10.33 (br s, exch. D2O, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d, ppm: 31.2,
40.9, 45.5, 116.9, 118.6, 120.3, 123.2, 124.7, 128.3, 135.7, 147.5, 148.0, 149.6,
149.9, 157.2, 162.1; ESI MS (M+1): 367, (MÀ1): 365; HRMS, exact mass calcd
for C20H22N4OS: 366.1514, found: 366.1512. Elemental analysis: calcd for
C20H22N4OS: C, 65.55; H, 6.05; N, 15.29; found: C, 65.41; H, 5.87, N, 15.13.
30: 4-(quinolin-4-ylmethylamino)-N-(3-(trifluoromethyl)phenyl)isothiazole-3-
carboxamide; mp 250–252 °C; 1H NMR (400 MHz, DMSO-d6) d, ppm: 4.29 (d,
J = 7.2 Hz, 2H), 6.51 (br s, exch. D2O, 1H, NH), 7.01 (dd, J1 = 8.0 Hz, J2 = 4.8 Hz,
1H), 7.39 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 7.6 Hz, 1H), 7.59–7.62 (m, 2H), 7.74
(m, 1H), 8.02 (d, J = 6.8 Hz, 1H), 8.13 (s, 1H), 8.19 (d, J = 7.2 Hz, 1H), 8.59 (d,
J = 4.8 Hz, 1H), 8.75 (s, 1H), 10.33 (br s, exch. D2O, 1H, NH); 13C NMR
(100 MHz, DMSO-d6) d, ppm: 43.5, 117.2, 120.6, 121.8, 123.5, 124.2, 124.6,
124.7, 126.7, 127.4, 129.0, 129.2, 129.5, 130.9, 136.5, 142.0, 146.5, 147.3,
150.3, 156.8, 163.1; ESI MS (M+1): 429, (MÀ1): 427; HRMS, exact mass calcd
for C21H15F3N4OS: 428.0919, found: 428.0916. Elemental analysis: calcd for
C21H15F3N4OS: C, 58.87; H, 3.53; N, 13.08; found: C, 58.69; H, 3.68, N, 12.91.
22. VEGFR tyrosine kinase inhibition is determined by measuring the
phosphorylation level of poly-Glu-Ala-Tyr-biotin (pGAT-biotin) peptide in a
Homogenous Time-Resolved Fluorescence (HTRF) assay. Into a black 96-well
centration of 10 lM.
Active in vitro inhibitors of VEGFR-2 were further characterized
in a cell-based phosphorylation ELISA assay (Table 1).23 In general,
good in vitro- to cell-based activity correlation has been found for
these compounds. In our hands, the best compounds displayed 41–
390 nM activity in inhibiting cell-based phosphorylation of VEGFR-
2. Passive diffusion potential for compounds3, 9, 11, 14, 22, 29, 30
across Caco-2 cell monolayer was predictive of cell permeability. In
general, good correlation between cell-based activity and Pm val-
ues was observed for all active compounds (Table 2). This fact indi-
cates that a number of derivatives, including 3, 9, 11 could be
further developed for in vivo studies.
Further, competition assays were conducted for the selected
molecules with varying concentration (0–100 lM) of ATP. Specifi-
cally, five different concentrations of 32P ATP were incubated with
VEGFR-2 along with varying concentrations (absence, IC50, IC90) of
the inhibitors 3, 11 and 14 for 45 min at RT. A double reciprocal
graph of the degree of phosphorylation (1/cpm) against ATP-con-
centration (1/[ATP]) was plotted. The data were analyzed by a
non-linear least-squares program to determine kinetic parameters
using GraphPad software. Determined Ki values for the three se-
lected compounds are listed in Table 3.
Costar plate is added
concentration in the 50
2
l
l
l/well of 25Â compound in 100% DMSO (final
In summary, we have described a series of isothiazoles as po-
tent inhibitors of both VEGFR-2 and VEGFR-1 receptors. Enzymatic
and cellular activities of representative molecules are comparable
to the clinical candidates VatalanibTM and AMG-706. This outcome
could be of benefit in the clinical setting as both receptors are re-
ported to mediate VEGF signaling in the angiogenesis.
l kinase reaction is typically 1 nM to 10 M). Next,
l
38 ll of reaction buffer (25 mM Hepes pH 7.5, 5 mM MgCl2, 5 mM MnCl2,
2 mM DTT, 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
after which the plate is incubated at room temperature for 45 min. The
reaction is stopped by addition of 50 l of KF buffer, (50 mM Hepes, pH 7.5,
0.5 M KF, 1 mg/ml BSA) containing 100 mM EDTA and 0.36 g/ml PY20K (Eu-
ll of 10 lM ATP in reaction buffer,
l
l
cryptate labeled anti-phosphotyrosine antibody, CIS bio international) is added
and after an additional 2 h incubation at room temperature, the plate is read in
a RUBYstar HTRF Reader.
References and notes
1. Bailar, J. C., III; Gornick, H. L. N. Engl. J. Med. 1997, 336, 1569.
2. Boehm, T.; Folkman, J.; Browder, T. Nature 1997, 390, 404.
3. Risau, W. Nature 1997, 386, 671.
4. Klagsbrun, M.; Moses, M. A. Chem. Biol. 1999, 6, R217.
5. Hanahan, D.; Folkman, J. Cell 1996, 86, 353.
6. Olsson, A.-K.; Dimberg, A.; Kreuger, J.; Claesson-Welsh, L. Nat. Rev. Mol. Cell Biol.
2006, 5, 359.
7. Zachary, I. Biochem. Soc. Trans. 2003, 31, 1171.
23. Cell-based assay for VEGFR-2 inhibition: (i) Transfection of 293 cells with DNA
expressing FGFR1/VEGFR-2 chimera:
A chimeric construct containing the
extracellular portion of FGFR1 and the intracellular portion of VEGFR-2 was
transiently transfected into 293 adenovirus-transfected kidney cells. DNA for
transfection was diluted to a 5
medium and incubated at room temperature for 30 min with 40
l
g/ml final concentration in a serum-free
l/mL of
l of the Lipofectamine/
l
Lipofectamine 2000, also in serum-free media. 250
l
DNA mixture was added to 293 cells suspended at 5 Â 105 cells/ml. 200
ll/well
8. Perona, R. Clin. Transl. Oncol. 2006, 8, 77.
of the suspension was added to a 96-well plate and incubated overnight.
9. Jin, T.; Nakatani, H.; Taguchi, T. World J. Gastroenterol. 2006, 12, 703.
10. Itakura, J.; Ishiwata, T.; Shen, B. Int. J. Cancer 2000, 85, 27.
11. Saaristo, A.; Karpanen, T.; Alitalo, K. Oncogene 2000, 19, 6122.
12. Ferrara, N.; Hillan, K. J.; Gerber, H. P.; Novotny, W. Nat. Rev. Drug Discov. 2004,
3, 391.
Within 24 h, media was removed and 100 ll of media with 10% fetal bovine
serum was added to the now adherent cells followed by an additional 24 h
incubation. Test compounds were added to the individual wells (final DMSO
concentration was 0.1%). Cells were lysed by re-suspension in 100 ll Lysis
buffer (150 mM NaCl, 50 mM Hepes pH 7.5, 0.5% Triton X-100, 10 mM NaPPi,
50 mM NaF, 1 mM Na3VO4) and rocked for 1 h at 4 °C.(ii) ELISA for detection of
tyrosine-phosphorylated chimeric receptor: 96-well ELISA plates were coated
13. The anti-angiogenic aptamer MacugenTM (Pegaptanib sodium, Eyetech
Pharmaceuticals, New York, NY and Pfizer) has been approved to treat
neovascular age-related macular degeneration; see: Fine, S. L.; Martin, D. F.;
Kirkpatrick, P. Nat. Rev. Drug Discov. 2005, 4, 187.
using 100
4 °C. FGFR1 was prepared in a buffer made with 16 ml of a 0.2 M Na2CO3 and
34 ml of a 0.2 M NaHCO3 with pH adjusted to 9.6. Concurrent with lysis of the
transfected cells, FGFR1 coated ELISA plates were washed three times with
PBS + 0.1% Tween-20, blocked by addition of 200 l/well of a 3% BSA in PBS for
1 h and washed again. 80 l of lysate were then transferred to the coated and
ll/well of 10 lg/ml of aFGFR1 antibody, and incubated overnight at
a
14. The anti-angiogenic antibody AvastinTM (Bevacizumab, Genentech, San
Francisco, CA) has been approved for the treatment of colorectal cancer, see:
Culy, C. Drugs Today 2005, 41, 23.
15. 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.
16. (a) Manley, P. W.; Furet, P.; Bold, G.; Brüggen, J.; Mestan, J.; Meyer, T.; Schnell,
C.; Wood, J. J. Med. Chem. 2002, 45, 5697; (b) Manley, P. W.; Bold, G.; Fendrich,
G.; Furet, P.; Mestan, J.; Meyer, T.; Meyhack, B.; Strauss, A.; Wood, J. Cell. Mol.
Biol. Lett. 2003, 8, 532; (c) Altmann, K.-H.; Bold, G.; Furet, P.; Manley, P. W.;
Wood, J. M.; Ferrari, S.; Hofmann, F.; Mestan, J.; Huth, A.; Krüger, M.;
a
l
l
blocked wells and incubated for 1 h at 4 °C. The plates were washed three
times with PBS + 0.1% Tween-20. To detect bound phosphorylated chimeric
receptor, 100
Transduction Laboratories)) were added (final concentration 0.5
PBS) and incubated for 1 h. The plates were washed six times with PBS + 0.1%
Tween-20. Enzymatic activity of HRP was detected by adding 50 l/well of
equal amounts of the Kirkegaard & Perry Laboratories (KPL) Substrate A and
l
l/well of anti-phosphotyrosine antibodies (RC20:HRP),
lg/ml in
l
Substrate B. The reaction was stopped by addition of 50
H2SO4, absorbance was measured at 450 nm.
ll/well of a 0.1 N