Design, synthesis and antitumor activity of 4-aminoquinazoline derivatives targeting VEGFR-2 tyrosine kinase

Article abstract of DOI:10.1016/j.bmcl.2011.11.061

We report herein the design and synthesis of novel 4-aminoquinazoline derivatives based on the inhibitors of VEGFR-2 tyrosine kinases. The VEGFR-2 inhibitory activities of these newly synthesized compounds were also evaluated and compared with that of ZD6

Full text of DOI:10.1016/j.bmcl.2011.11.061

Bioorganic & Medicinal Chemistry Letters 22 (2012) 110–114
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and antitumor activity of 4-aminoquinazoline derivatives
targeting VEGFR-2 tyrosine kinase
Bing Yu a,b, Li-da Tang b,c, Yi-liang Li c, Shu-hui Song d, Xiao-liang Ji d, Mu-sen Lin d, Chun-Fu Wu a,
⇑
a Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China
b Research Center of New Drug Evaluation, Tianjin Institute of Pharmaceutical Research, Tianjin 300193, People’s Republic of China
c Tianjin Key Laboratory of Molecular Drug & Drug Discovery, Tianjin Institute of Pharmaceutical Research, Tianjin 300193, People’s Republic of China
d Tianjin University of Traditional Chinese Medicine, Tianjin, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein the design and synthesis of novel 4-aminoquinazoline derivatives based on the inhib-
itors of VEGFR-2 tyrosine kinases. The VEGFR-2 inhibitory activities of these newly synthesized com-
pounds were also evaluated and compared with that of ZD6474. We found that most of target
compounds had good inhibitory potency. In particular, compounds 1h, 1n and 1o were found to be 6,
2 and 2-fold more potent than the positive control ZD6474. The leading compound 1h also showed an
in vivo activity against HepG2 human tumor xenograft model in BALB/c-nu mice.
Received 3 August 2011
Revised 15 November 2011
Accepted 16 November 2011
Available online 23 November 2011
Keywords:
4-Aminoquinazolines
Synthesis
Ó 2011 Elsevier Ltd. All rights reserved.
VEGFR-2
Angiogenesis of tumor mass is the process of forming new blood
vessels from existing ones of host, and is strictly controlled by the
balance of a number of angiogenic positive factors and negative
ones.1 The break of the balance is thought to drive angiogenesis,
which is involved in the expansion and metastasis of solid tumor.2
The blockade of the tyrosine kinase VEGFR-2 signaling pathway
may disrupt the angiogenesis process of solid tumor so that the
developing tumor cells required blood flow grow slowly, even stop
growth because of lack of nutrient and growth factors supported by
freshly forming vessels.3 Antiangiogenic therapy appears to have its
optimum efficacy if given daily or intermittently over a long time
period.4 It is directed mainly at proliferating capillary endothelial
cells and does not cause bone marrow suppression, gastroinintesti-
nal symptoms or hair loss.5 Antiangiogenic therapy has also been
proposed as a potential strategy to avoid drug resistance as inhibitor
of angiogenesis targets endothelial cells of blood capillaries, the
progenitors of new blood vessels, which show relative genetic sta-
bility based on their normal complement of chromosome.6 Angio-
genesis inhibitors increase uptake of chemotherapeutic drugs in a
tumor.7 After the completion of chemotherapy, radiation or surgery,
Figure 1. Examples of clinical VEGFR-2 kinase inhibitors.
⇑
Corresponding author. Tel./fax: +86 24 23843567.
E-mail address: chunfuw@gmail.com (C.-F. Wu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.061

B. Yu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 110–114
111
preclinical trials, sorafenib is shown to have broad spectrum
antitumor activity in mouse models and is found to prevent the
growth of tumors but not to reduce tumor size.9 Sorafenib is ap-
proved by FDA for patients with metastatic renal cell carcinoma
and hepatocellular carcinoma.10 Although most of the VEGF recep-
tor inhibitors currently on the market or under development are
generally characterized by prominent anti-tumor growth effect,
they also have respective shortage. For example, the water-solubil-
ity of Sorafenib and ABT-869 is not satisfactory,11 while ZD6474
(Fig. 1) has good physico–chemical property but prolonging QT
interval.12
According to the structural analysis, both Sorafenib and
ABT-869 contain aromatic urea fragment. The crystal structure of
the complex between sorafenib and VEGFR-2 reveals that the urea
portion occupies the back hydrophobic pocket with additional
hydrogen-bonding interactions. When sorafenib is bound to VEG-
FR-2, it forms hydrogen bonds with the backbone-NH of Cys919,
the side chain carboxylate of Glu885, and backbone-NH of
Asp1046 (Fig. 2). The terminal phenyl moiety occupies the hydro-
phobic pocket created by rearrangement of the protein. Analysis of
the X-ray structure also suggests sufficient space is available for
the introduction of various substituents around the terminal ben-
zene ring.
Figure 2. Predicted binding mode of sorafenib in the X-ray structure of VEGFR-2
(PDB code 2RL5). Sorafenib (yellow carbon) was docked into the X-ray structure of
VFGFR-2 using Glide method of Schrodinger software. Key hydrogen bond contacts
are shown in dotted line.
The introduction of carbamoylpyridyl increases the lipophilicity
of the compounds (logP = 5–6). This increased lipophilicity is
expected to decrease its water solubility of sorafenib. ZD6474
shows good water solubility, we reasoned that the basicity of
the N-methylpiperidine (pKa = 8.1) improves the solubility of
anilinoquinazolines. Base on the fragment drug design, we describe
here the chemistry, SAR, and biological testing for these series. The
quinazoline core was modified at position 4 of the aryl with differ-
ent bulky substituents such as amide, carbamate or urea. These
moieties appeared to be appropriate for modification in order to
improve solubility as well as to enhance inhibitory potency against
VEGFR-2. Based on the above hypothesis, we designed and synthe-
sized the following compounds (I) (Fig. 3).
Figure 3. Structure of compounds 1a–o.
antiangiogenic therapy may be continued for years because of its
low toxicity, to prolong dormancy of microscopic metastases or to
stabilize residual disease.8
Recently, a number of VEGF receptor inhibitors have been
reported. Sorafenib, marketed by Bayer and Onyx pharmaceuticals,
has been shown to be a potent inhibitor of VEGF receptors in vitro. In
A general synthesis of aromatic urea and amide derivatives is
shown in Schemes 1 and 2. Arylamines 4a–i were treated with
triphosgene at the presence of triethylamine to form isocyanates
5a–i, which reacted with aniline derivatives followed by hydrolysis
Scheme 1. Synthesis of aromatic urea compounds 7a–i. Reagents and conditions: (a) triphosgene, CH2Cl2, rt, triethylamine, 60–75%; (b) CH2Cl2, rt, 58–83%; (c) pd/C,
methonal, 80–86%.
Scheme 2. Synthetic of aromatic amide compounds 11a–c. Reagents and conditions: (a) SOCl2, 60 °C, 86–93%; (b) CH2Cl2, rt, K2CO3, 90–95%; (c) Pd/C, methonal, 92–96%.

112
B. Yu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 110–114
Table 1
under basic conditions to provide the ureas 6a–i. Using palladium-
charcoal as the catalyst, reduction of 6a–i performed smoothly in
methanol to give 7a–i. On the other hand, carboxylic acid 8 reacted
with thionyl chloride to give acyl chloride 9, which reacted with
aniline derivatives to form amides 10a–c. The nitro group of
10a–c was reduced to amino group by Pd/C-catalyzed hydrogena-
tion to give 11a–c.
VEGFR-2 kinase and cell inhibitory activity compounds 1a–o
The synthesis of 4-substituted anilinoquinazolines derivatives
compound 1a–o was conducted as outlined in Scheme 3. Treatment
of 7-fluoroquinazolone 12 with N-methyl-4-piperidinemethanol at
the presence of NaH gave the corresponding C-7-piperidinemethan-
oxyanilinoquinazolines derivatives 13. 4-Chloroquinoline 14 was
prepared by the chlorination of 13 with thionyl chloride, which
was coupled with 7a–I or 11a–c to provide 1a–o.13
Compound
X
R1
H
R2 R3
VEGFR-2
IC50 (nM)
HUVEC-v
IC50 (nM)
1a
CH2
H
3-CF3-4-Cl-
87
258
These compounds were tested for VEGFR-2 kinase activity using
homogeneous time resolved fluorescence (HTRF) method.14 The
catalytic activity of kinases was measured by phosphorylated bio-
tin-peptide conjugate using streptavidin linked-APC and euro-
pium-labeled anti-phosphotyrosine antibody. Subsequently, a cell
proliferation assay was performed to find the potent VEGFR-2
kinase inhibitors among these compounds for their ability to inhibit
VEGF-stimulated proliferation of human umbilical vein endothelial
cells (HUVEC).15 The results are shown in Table 1. Overall, consider-
able relationships between their structures and inhibitory activities
were observed. The urea compounds (X = NH) exhibited higher
activity than the corresponding amides (X = CH2) as seen from 1g–
i versus 1a–c, which demonstrated the importance of hydrogen
bond between carbamido group and GLU885. Based on these find-
ings, we further surveyed the effect of substitution at the nitrogen
atom of carbamido group. Substitutions on the carbamido group
in this study included ethyl, cyclopropyl, cyclohexyl and some kind
of aryl group. As shown in Table 1, the activity imparted to the
synthesized compounds by the R3 was in the order of 3-F-Ph >
3-CF3-4-Cl-Ph ꢀ 3-F-4-F-Ph > 3-Br-4-Me-Ph ꢀ 2-F-4-Br-Ph > cyclo-
propyl > ethyl > cyclohexyl against VEGFR-2 and HUVEC-v cell.
Because of the results that the 3-F-Ph group seemed to be optimal,
we selected 1h as the primer to carry out further research and fo-
cused on the halogenation on benzene ring of the side chain. There
was a decrease in activity when a fluorine or chlorine atom was in-
ducted at 2-position of the benzene ring as seen from 1l, 1m versus
1h. On the other hand, 1h and 1n showed similar potency against
VEGFR-2 while the latter compound was much less active against
HUVEC-v cell. These results indicated that the C-6 methoxy group
Ph
1b
1c
CH2
CH2
H
H
H
H
3-F-Ph
3-F-4-F-Ph
65
73
237
308
1d
1e
NH
NH
H
H
H
H
105
98
587
324
1f
NH
NH
H
H
H
H
237
23
—
1g
3-CF3-4-Cl-
Ph
3-F-Ph
3-F-4-F-Ph
4-CH3-3-Br-
PH
2-F-4-Br-PH
3-F-Ph
92
1h
1i
1j
NH
NH
NH
H
H
H
H
H
H
5.5
25
45
52
89
196
1k
1l
1m
1n
1o
NH
NH
NH
NH
NH
—
H
H
H
F
50
15
17
16
17
35
233
67
132
179
185
187
Cl 3-F-Ph
OCH3
OCH3
—
H
F
—
3-F-Ph
3-F-Ph
—
ZD6474
of the quinazolinone ring played a negative role in drug entering
the cell. This deduction was further supported by the fact that 1l
was two-fold more potent than 1o against VRGFR-2, whereas it
was almost opposite as to HUVEC-v cell. The reason for this is not
clear, but the cellular permeability might influence the antiprolifer-
ative activity.
Table 2 shows the selective inhibitory potencies of compound
1h against a panel of kinases. The compound 1h is potent,
Scheme 3. Synthesis of compounds 1a–o. Reagents and conditions: NaH, DMF, 80 °C, 65–75%; (b) SOCl2, 60 °C, 75–83%; (c) isopropanol, 80 °C, 80–84%.

B. Yu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 110–114
113
Table 2
by the Nature Science Foundation of Tianjin, China (Grant ID No.
09JCZDJC21700).
Kinase selectivity for compound 1h
Kinase test
IC50 (nM)
References and notes
VEGFR-2
VEGFR-1
VEGFR-3
5.5
1920
9.6
1355
1. Carmeliet, P.; Jain, R. K. Nature 2000, 407, 249.
2. (a) Folkman, J. Semin. Oncol. 2001, 28, 536; (b) Ferrara, N.; Gerber, H. P.;
LeCouter, J. Nat. Med. 2003, 9, 669.
3. (a) Peruzzi, B.; Bottaro, D. P. Clin. Cancer Res. 2006, 12, 3657; (b) Folkman, J.
Semin. Oncol. 2002, 29, 15; (c) Ferrara, N. Oncologist 2004, 9, 2.
4. (a) Kerbel, R. S. BioEssays 1991, 13, 31; (b) Crawford, J.; Ozer, H.; Stoller, R.;
Johnson, D.; Lyman, G.; Tabbara, I.; Kris, M.; Grous, J.; Picozzi, V.; Rausch, G.;
Smith, R.; Gradishar, W.; Yahanda, A.; Vincent, M.; Stewart, M.; Glaspy, J. N.
Engl. J. Med. 1991, 325, 164.
PDGFR
a
PDGFRb
FGFR1
Tie-2
EGFR
HER2
AKT
CDK1
Aurora A
2788
>10,000
>10,000
>10,000
>10,000
8710
10,000
2680
5. Wassberg, E.; Christofferson, R. Eur. J. Cancer 1997, 33, 2020.
6. (a) Eikesdal, H. P.; Kalluri, R. Semi. Cancer Biol. 2009, 19, 310; (b) Azam, F.;
Mehta, S.; Harris, A. L. Eur. J. Cancer 2010, 46, 1323.
7. (a) Ma, J.; Waxman, D. J. Clin. Cancer Res. 2009, 15, 578; (b) Doloff, J. C.; Khan,
N.; Ma, J.; Demidenko, E.; Swartz, H. M.; Jounaidi, Y. Curr. Cancer Drug Targets
2009, 6, 777.
8. (a) Griffioen, A. W.; Barendsz-Janson, A. F.; Kevin, H. M.; Hillen, H. F. J. Lab. Clin.
Med. 1998, 132, 363; (b) Kaban, K.; Herbst, R. S. Hemato. Oncol. Clin. North Am.
2002, 16, 1125.
9. (a) Galle, P. R. J. Hepatol. 2008, 49, 871; (b) Adnane, L.; Trail, P. A.; Taylor, I.;
Wilhelm, S. M. Methods Enzymol. 2006, 407, 597; (c) Wilhelm, S. M.; Carter, C.;
Tang, L.; Wilkie, D.; McNabola, A.; Rong, H.; Chen, C.; Zhang, X.; Vincent, P.;
McHugh, M.; Cao, Y.; Shujath, J.; Gawlak, S.; Eveleigh, D.; Rowley, B.; Liu, L.;
Adnane, L.; Lynch, M.; Auclair, D.; Taylor, I.; Gedrich, R.; Voznesensky, A.; Riedl,
B.; Post, L. E.; Bollag, G.; Trail, P. A. Cancer Res. 2004, 64, 7099.
10. (a) Escudier, B.; Eisen, T.; Stadler, W. M.; Szczylik, C.; Oudard, S.; Siebels, M.;
Negrier, S.; Chevreau, C.; Solska, E.; Desai, A. A.; Rolland, F.; Demkow, T.;
Hutson, T. E.; Gore, M.; Freeman, S.; Schwartz, B.; Shan, M.; Simantov, R.;
Bukowski, R. M. N. Engl. J. Med. 2007, 356, 125; (b) Ryan, C. W.; Goldman, B. H.;
Lara, P. N.; Mack, P. C.; Beer, T. M.; Tangen, C. M.; Lemmon, D.; Pan, C. X.;
Drabkin, H. A.; Crawford, E. D. J. Clin. Oncol. 2007, 25, 3296; (c) Abou-Alfa, G. K.;
Schwartz, L.; Ricci, S.; Amadori, D.; Santoro, A.; Figer, A.; De Greve, J.; Douillard,
J. Y.; Lathia, C.; Schwartz, B.; Taylor, I.; Moscovici, M.; Saltz, L. B. J. Clin. Oncol.
2006, 24, 4293; (d) Llovet, J. M.; Ricci, S.; Mazzaferro, V.; Hilgard, P.; Gane, E.;
Blanc, J. F.; De Oliveira, A. C.; Santoro, A.; Raoul, J. L.; Forner, A.; Schwartz, M.;
Porta, C.; Zeuzem, S.; Bolondi, L.; Greten, T. F.; Galle, P. R.; Seitz, J. F.; Borbath, I.;
Häussinger, D.; Giannaris, T.; Shan, M.; Moscovici, M.; Voliotis, D.; Bruix, J. N.
Engl. J. Med. 2008, 359, 378.
11. (a) Wong, C. I.; Koh, T. S.; Soo, R.; Hartono, S.; Thng, C. H.; McKeegan, E.; Yong,
W. P.; Chen, C. S.; Lee, S. C.; Wong, J.; Lim, R.; Sukri, N.; Lim, S. E.; Ong, A. B.;
Steinberg, J.; Gupta, N.; Pradhan, R.; Humerickhouse, R.; Goh, B. C. J. Clin. Oncol.
2009, 27, 4718; (b) Jain, L.; Woo, S.; Gardner, E. R.; Dahut, W. L.; Kohn, E. C.;
Kummar, S.; Mould, D. R.; Giaccone, G.; Yarchoan, R.; Venitz, J.; Figg, W. D. Br. J.
Clin. Pharmacol. 2011, 72, 294.
Figure 4. Effect of compound 1h vehicle on tumor growth in xenograft model.
HepG2 human heparcarcinoma cells (1 Â 107 cells) were implanted sc in the flank
of BALB/c-nu mice. Five days after implantation following once daily oral
administration of compound 1h or vehicle. Data points represent mean SE (n = 8).
12. (a) Ederhy, S.; Cohen, A.; Dufaitre, G.; Izzedine, H.; Massard, C.; Meuleman, C.;
Besse, B.; Berthelot, E.; Boccara, F.; Soria, J. C. Target Oncol. 2009, 4, 89; (b)
ODAC Briefing Document Drug Substance Vandetanib (ZD6474) Date 25
October 2010.
low-nanomolar inhibition of VEGFR-2, VEGFR-3. Other kinases
such as VEGFR-1, PDGFRa, PDGFRb, FGFR1, Tie-2, EGFR, HER2,
AKT, CDK1and AuroraA are only weakly inhibited.
13. Melting points were determined in open capillary tubes on a Büchi reference B-
530 digital melting point apparatus and are uncorrected. NMR spectra were
determined on a Bruker AV-400 (400 MHz) spectrometer in DMSO-d6 or in
CDCl3 at ambient temperature. Low resolution mass spectral (MS) data were
determined on an Agilent 1100 Series LC–MS with UV detection at 254 nm and
a low resonance electrospray mode (ESI). Chemical shifts are reported in ppm
from the solvent resonance (DMSO-d6, 2.49 ppm).
Potential lead candidate compound 1h was evaluated in vivo in
the HepG2 cells, a human hepatomacellar carcinoma cell line, as a
xenograft model in BALB/c-nu mice. Following once daily oral
administration of 1h at three doses for 15 days, tumor growth inhi-
bition (%TGI) of 25.6%, 61.4%, 83.6% was achieved at three doses of
20, 50, 100 mg/kg (Fig 4). No adverse events were observed. Oral
administration 1h was found to be well tolerated in the experi-
ment, and the treated animals exhibited no clinically observable
sign of toxicity or weight loss at the highest test dose of 100 mg/
kg. It elicited robust and dose-proportional tumor responses over
the dose range of 20–100 mg/kg in this tumor model.
In summary, we successfully developed a series of 4-anilinoqui-
nazolines derivatives which showed potent, selective inhibition of
the VEGFR-2 kinases. In the quinazoline series, compounds 1h, 1n,
and 1o displayed the most potent cytotoxic activity with IC50 equal
to 5.5, 16, and 17 nmol, respectively. The lead compound, 1h,
exhibited a good kinase selectivity, antiproliferative potency, oral
exposure, and efficacy in tumor xenograft models. The further
development of SAR was underway to found the more potent lead
antitumor drug.
A
typical procedure utilized is demonstrated for compound 1a as
a
representative example. mixture of 4-chloro-7-piperidinemethoxy-
A
quinazoline 100.0 mg (0.344 mmol), 11a 112.8 mg (0.344 mmol), and
isopropanol (15 mL) was stirred at 85 °C for 2 h. The mixture was
concentrated under reduced pressure, diluted with AcOEt and washed with
water, brine, dried over anhydrous magnesium sulfate, and concentrated under
reduced pressure. The residue was purified by basic silica gel column
chromatography to afford of 1a (186.5 mg, yield 93.0%). Compound 1a:1H
NMR (400 MHz, DMSO-d6): d 1.67–1.76 (m, 2H), 1.95–2.08 (m, 3H), 2.67–2.68
(m, 3H), 2.97–3.00 (m, 2H), 3.17 (s, 1H), 3.38–3.41 (m, 2H), 3.73–3.79 (m, 2H),
4.05–4.07 (m, 2H), 7.41–7.48 (m, 4H), 7.61–7.66 (m, 2H), 7.94–8.00(m, 1H),
8.30–8.34 (m, 1H), 8.77 (s, 1H), 9.05–9.07 (m, 1H), 11.42–11.50 (m, 1H), 11.85
(s, 1H). LC–MS (APCI+): m/z 583 (MH+).
Compound 1b: 1H NMR (400 MHz, DMSO-d6): d 1.32–1.35 (m, 2H), 1.74–1.77
(m, 3H), 1.83–1.88 (m, 2H), 2.15 (s, 3H), 2.76–2.79 (m, 2H), 3.66 (s, 2H),3.98–
4.00 (m, 2H), 4.30–4.33 (m, 1H), 7.13–7.14 (m, 1H), 7.20–7.23 (m, 1H), 7.30–
7.32 (m, 2H), 7.64–7.66 (m, 1H), 7.75–7.77 (m, 2H), 7.83–7.86 (m, 1H), 8.19–
8.20 (m, 1H), 8.42–8.47 (m, 2H), 9.62 (s, 1H), 10.58 (s, 1H). LC–MS (APCI+): m/z
499 (MH+).
Compound 1c: 1H NMR (400 MHz, DMSO-d6): d 1.32–1.39 (m, 2H), 1.69–1.77
(m, 4H), 1.86–1.91 (m, 2H), 2.16 (s, 3H), 2.78–2.81 (m, 2H), 3.62 (s, 2H), 3.98–
4.00 (m, 2H), 7.13–7.23 (m, 2H), 7.30–7.38 (m, 3H), 7.74–7.81 (m, 2H), 8.42–
8.47 (m, 2H), 9.61 (s, 1H), 10.37 (s, 1H). LC–MS (APCI+): m/z517 (MH+).
Compound 1d: 1H NMR (400 MHz, DMSO-d6):d1.15–1.20 (m, 2H), 1.22–1.39
(m, 4H), 1.70–1.76 (m, 4H), 1.85–1.90 (m, 1H), 2.16 (s, 3H), 2.78–2.80 (m, 2H),
3.20 (t,2H), 6.54–6.58 (m, 2H), 7.07 (s, 1H), 7.13–7.22 (m, 2H), 7.30–7.33 (m,
Acknowledgments
The authors thank Dr. Meng, F. C. and Dr. He, H. W. for the
molecular biology and in vivo assays. The work was supported

114
B. Yu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 110–114
2H), 8.24–8.35 (m, 2H), 9.33 (s, 1H). LC–MS (APCI+): m/z 435 (MH+).
(m, 2H), 2.10 (m, 1H), 2.72 (s, 1H), 2.97–3.03 (m, 2H),3.45 (m, 2H), 4.00–4.23
(m, 5H), 4.94 (s, 1H),6.73–6.77 (m, 1H),7.10–7.12 (m, 1H), 7.28–7.60 (m, 7H),
8.30 (s, 1H), 8.76 (s, 1H),9.59 (s, 1H), 9.70 (s, 1H), 11.37 (s, 1H). LC–MS (APCI+):
m/z 531 (MH+).
Compound 1e: 1H NMR (400 MHz, DMSO-d6): d 1.22–1.39 (m, 4H),1.71–1.77
(m, 4H), 1.85–1.90 (m, 1H), 2.16 (s, 3H), 2.78–2.80 (m, 4H), 3.95 (s, 1H), 3.98–
4.00 (m, 2H), 4.94 (s, 1H), 6.54–6.58 (m, 2H), 7.07 (s, 1H), 7.13–7.22 (m, 2H),
7.30–7.33 (m, 2H), 8.24–8.35 (m, 2H), 9.33 (s, 1H). LC–MS (APCI+): m/z 446
(MH+).
Compound 1o: 1H NMR (400 MHz, DMSO-d6):d1.60–1.69 (m, 2H),1.97–2.02
(m, 2H), 2.12 (m, 1H), 2.73 (s, 3H), 2.94–3.03 (m, 1H),4.03 (s, 1H), 4.05–4.07 (m,
2H), 6.76–6.78 (m, 1H), 7.11–7.13 (m, 1H), 7.28–7.52 (m, 4H), 7.75–7.7 (m, 4H),
8.14–8.19 (m, 1H), 8.37 (s, 1H),8.84 (s, 1H),8.92 (s, 1H), 9.81 (s, 1H), 11.47 (s,
1H). LC–MS (APCI+): m/z 549 (MH+).
Compound 1f: 1H NMR (400 MHz, DMSO-d6):d1.14–1.91 (m, 15H), 2.16 (s, 3H),
2.72–2.87 (m, 5H), 3.97–3.99 (m, 2H), 6.01–6.03 (m, 1H), 7.10–7.11 (m, 1H),
7.17–7.20 (m, 1H), 7.34–7.36 (m, 2H), 7.59–7.61 (m, 2H), 8.25 (s, 1H), 8.38–
8.42 (m, 2H), 9.51 (s, 1H). LC–MS (APCI+): m/z 488 (MH+).
14. HTRF assays are homogeneous time-resolved assays that generate a signal by
FRET between donor and acceptor molecules. When formatted for kinase
assays, the Eu-cryptate is usually conjugated to a phospho-specific antibody
and is presented upon binding of the antibody to the phosphorylated product,
while the streptavidin-conjugated allophycocyanin binds to the biotin on the
substrate to complete the detection complex. When the two entities get close
proximity and upon excitation, energy transfer occurs and APC emits a specific
long-lived fluorescence at 665 nm. The kinases were purified as the
intracellular domain of human VEGFR-2 fused by GST. The catalytic activity
of the kinase was detected by using a biotinylated synthetic peptide as a
Compound 1g: 1H NMR (400 MHz, DMSO-d6):d1.32–1.38 (m, 2H), 1.74–1.89
(m, 5H), 2.15 (s, 3H), 2.77–2.79 (m, 2H), 7.12 (s, 1H),7.19–7.22 (m, 1H), 7.46–
7.48 (2H), 7.57–7.71 (m, 4H), 8.13–8.14 (m, 1H), 8.40–8.45 (m, 2H), 9.35 (s,
1H), 9.57 (s, 1H), 9.71 (s, 1H). LC–MS (APCI+): m/z 584 (MH+).
Compound 1h: 1H NMR (400 MHz, DMSO-d6):d1.30–1.38 (m, 2H), 1.75–1.77
(m, 3H), 1.86–1.97 (m, 2H), 2.16 (s, 3H), 2.78–2.81 (m, 2H), 3.98–4.00 (m, 2H),
6.74–6.78 (m, 1H), 7.05–7.51 (m, 7H), 7.64–7.71 (m, 2H), 8.40–8.45 (m, 2H),
8.71 (s, 1H), 8.88 (s, 1H), 9.57 (s, 1H). LC–MS (APCI+):m/z 500 (MH+).
Compound 1i: 1H NMR (400 MHz, DMSO-d6):d1.33–1.36 (m, 2H), 1.75–1.77
(m, 3H), 1.84–1.90 (m, 2H), 2.15 (s, 3H),2.77–2.80 (m, 2H),3.98–4.00 (m,
2H),7.10–7.12 (m, 2H),7.19–7.22 (m, 1H),7.29–7.36 (m, 1H),7.42–7.45 (m,
2H),7.63–7.71 (m, 3H),8.40–8.45 (m, 2H),8.70 (s, 1H),8.85 (s, 1H),9.57 (s, 1H).
LC–MS (APCI+): m/z 518 (MH+).
substrate,
biotin-aminohexyl-EEEEYFELVAKKKK-NH2,
for
VEGFR-2.
Phosphorylated substrate is measured by streptavidin linked-APC and
europium-labeled anti-phosphorylated tyrosine antibody. In briefly the assay
method as follow: the working solution (100 nM TK-substrate, 3
VEGFR-2, 100 nM ATP, 1 mM DTT, 1 mM MnCl2, 5 mM MgCl2 and 20 nM SEB in
10 l reaction volume) incubated at 37 °C for 30 min; the detection solution
(6.25 nM Streptavidin-XL665, 5 l/well TK antibody-cryptate)was added to
lg/ml GST-
Compound 1j: 1H NMR (400 MHz, DMSO-d6):d1.30–1.39 (m, 2H),1.74–1.77 (m,
3H), 1.85–1.90 (m, 1H), 2.16 (s, 3H), 2.27 (s, 3H), 2.77–2.80 (m, 2H), 3.94 (s,
1H), 3.97–3.99 (m, 2H), 7.06–7.66 (m, 8H), 7.81 (s, 1H), 8.38 (s, 1H),8.55
(d,1H),9.38 (s, 1H). LC–MS (APCI+): m/z605 (MH+).
l
l
stop reaction, and placed at room temperature for 30 min for determination
(excitation at 314 nm, emission at 665 nm/emission at 620 nm).
Compound 1k: 1H NMR (400 MHz, DMSO-d6):d1.70–1.73 (m, 2H), 1.85–1.90
(m, 2H), 2.36 (s, 3H), 2.78–2.80 (m, 4H), 3.98–4.00 (m, 1H), 6.54–6.58 (m,
2H),7.13–7.22 (m, 2H), 7.30–7.33 (m, 5H), 8.24–8.35 (m, 2H), 9.53 (s, 1H) 10.33
(s, 1H). LC–MS (APCI+): m/z 579 (MH+).
15. Cell assay: HUVEC were grown in M199 containing 10% FBS and kanamycin
(50 U mlÀ1) in a humidified 5% CO2 incubator at 37 °C. After the cells had
grown to confluence, they were disaggregated in trypsin solution, washed with
M199 containing 10% FBS, centrifuged at 125 g for 5 min, re-suspended, and
then subcultured according to standard protocols. Cells from passages 4–8
were used. HUVEC proliferation in the presence of growth factors was
evaluated using Sulforhodamine B (SRB) assay. Briefly, HUVECs were plated
in 96-well plates (1000 cells/well) and dosed with tested compound +VEGF
(15 ng/ml). The cultures were incubated for 48 h (37 °C; 5% CO2) and then
plused with SRB and reincubated for 15 min. Cells were harvested and assayed
using a ELISA counter to read at 490 nm. IC50 data were interpolated as
described above.
Compound 1l: 1H NMR (400 MHz, DMSO-d6):d1.64–1.71 (m, 2H), 1.97–2.00 (m,
2H), 2.70–2.71 (m, 3H),2.93–3.60 (m, 4H),4.08–4.27 (m, 1H),6.76–6.81 (m,
1H),7.12–8.19 (m, 9H), 8.71–8.99 (m, 2H),9.87–9.94 (m, 1H),10.52 (s, 1H). LC–
MS (APCI+): m/z 518 (MH+).
Compound 1m: 1H NMR (400 MHz, DMSO-d6):d1.45–1.48 (m, 2H), 1.86–1.89
(m, 3H), 2.43 (s, 3H),2.49 (s, 2H),3.08 (s, 2H),4.03–4.04 (m, 2H),6.77–6.81 (m,
1H),7.10–7.12 (m, 1H),7.18 (s, 1H),7.23–7.34 (m, 2H),7.49–7.52 (m, 1H)7.72–
7.74 (m, 1H),8.04–8.06 (m, 1H),8.13–8.14 (m, 1H),8.36 (s, 1H),8.44–8.46 (m,
1H),8.55 (s, 1H),9.63 (s, 1H),9.70 (s, 1H). LC–MS (APCI+): m/z 534 (MH+).
Compound 1n: 1H NMR (400 MHz, DMSO-d6):d1.60–1.69 (m, 2H),1.85–1.99