Synthesis of a novel biotin-tagged photoaffinity probe for VEGF receptor tyrosine kinases

Article abstract of DOI:10.1016/j.bmcl.2005.09.036

A novel biotin-tagged photoaffinity probe was synthesized and evaluated as a vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitor. The probe (2) is a potent VEGFR-2 inhibitor with an IC₅₀ value of 7.1 μM, and inhibi

Full text of DOI:10.1016/j.bmcl.2005.09.036

Bioorganic & Medicinal Chemistry Letters 16 (2006) 129–133
Synthesis of a novel biotin-tagged photoaffinity probe for
VEGF receptor tyrosine kinases
Sun-Young Han, Seok-Soon Park, Woo Ghil Lee, Yong Ki Min* and Bum Tae Kim
Bio-Organic Science Division, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea
Received 8 June 2005; revised 1 September 2005; accepted 13 September 2005
Available online 10 October 2005
Abstract—A novel biotin-tagged photoaffinity probe was synthesized and evaluated as a vascular endothelial growth factor receptor-2
(VEGFR-2) tyrosine kinase inhibitor. The probe (2) is a potent VEGFR-2 inhibitor with an IC50 value of 7.1 lM, and inhibits VEGF-
induced proliferation in human umbilical vein endothelial cells (HUVEC), with an IC50 value of 40.3 lM. This probe will be a useful
reagent for investigating ligand–protein interactions.
Ó 2005 Elsevier Ltd. All rights reserved.
Photoaffinity labeling is a useful method for the identifi-
cation of ligand-binding sites of target proteins and the
investigation of ligand–receptor interactions. Photoac-
tive moieties, such as an azido or a diazirine group, have
been used as precursors for the highly reactive inter-
mediate nitrene or carbene, which is generated upon
photolysis. Perfluorophenyl azides are the most widely
applied photolabeling reagents and belong to a new
class of photolabeling reagents with improved C–H
insertion efficiency compared with their nonfluorinated
analogues.1,2 Biotin-labeling is a powerful technique
for the radioisotope-free detection of photolabeled pro-
teins based on the strong interaction of biotin with
either avidin or streptavidin. Therefore, biotinylated
photoaffinity probes can be applied to separate photola-
beled proteins from complex mixtures as well as to study
the interactions of ligand–protein in living cells using ad-
vanced imaging techniques.3 Recently, much attention
has been devoted to the application of this method to
the investigation of small molecule–target protein
interactions.4
this reason, VEGFRs are attractive therapeutic targets
for the development of novel agents to treat diseases
such as cancer.6,7 Several inhibitors of VEGFRs have
Cl
F
NH
O
O
N
N
CB676475 (1)
Cl
F
NH
F
O
O
F
N3
F
N
O
H
O
N
N
N
H
O
F
HN
O
Vascular endothelial growth factor receptors (VEG-
FRs) are located on the surfaces of vascular endothelial
cells and their activity is crucial for the induction of
tumor angiogenesis, which is the process of new blood
vessel formation from preexisting blood vessels.5 For
2
NH
S
O
N
Keywords: Biotin-tagged photoaffinity probe; Vascular endothelial
growth factor receptors; Inhibitor; Ligand–protein interactions.
H
*
Figure 1. Structures of CB676475 (1) and a biotinylated photoactive
quinazoline analogue (2).
Corresponding author. Tel.: +82 42 860 7029; fax: +82 42 861
0307; e-mail: ykmin@krict.re.kr
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.036

130
S.-Y. Han et al. / Bioorg. Med. Chem. Lett. 16 (2006) 129–133
Cl
Cl
Cl
i)
ii)
HN
HN
N
HN
N
O
F
O
O
F
O
O
F
N
N
N
HO
MsO
HO
N
3
4
5
Cl
Cl
iii)
iv)
HN
N
HN
N
O
F
O
O
F
N
N
N3
H2N
O
7
6
Scheme 1. Synthesis of compound 7. Reagents and conditions: (i) 2-bromoethanol, K2CO3, THF/DMF, reflux, 2 h, 88%; (ii) MsCl, NEt3,
THF/DMF, 0 °C, 1 h, 83%; (iii) NaN3, EtOH/H2O, 80 °C, 3 h, 78%; (iv) H2, Pd(OH)2/C, MeOH, 89%.
been shown to inhibit tumor angiogenesis and the
growth of tumors in animal models.8 Recently, 4-anili-
noquinazoline derivatives that selectively inhibit VEG-
FRs (KDR and Flt) have been reported by Hennequin
et al.6 To design probes that are bioactive in the same
range as its parent compound, we analyzed the struc-
ture–activity relationships reported in the various
synthetic derivatives. Structure–activity studies of 4-
anilinoquinazoline inhibitors indicated that the addi-
tion of various substituents at the C-7 position of the
quinazoline ring can retain good inhibitory activity.6,7
In order to synthesize small molecule probes that can
be applied for the study of VEGFRs, we investigated
the introduction of a photoactive moiety and a biotin
group at the C-7 position of the quinazoline ring,
and synthesized chemical probes with perfluorophenyl
azide and/or biotin moiety.
(KDR and Flt) tyrosine kinases. IC50 values for KDR
and Flt are 0.1 and 2 lM, respectively (Fig. 1).6 Based
on the structure of CB676475 (1), a novel biotinylated
photoaffinity probe (2) was synthesized and evaluated.
As shown in Scheme 1, the key intermediate 4-(40-chlo-
ro-20-fluorophenylamino)-6-methoxyquinazolin-7-ol (3)
was prepared from 3,4-dimethoxybenzoic acid, as de-
scribed previously.7,9 The side chain at the C-7 position
of quinazoline ring of compound 4 was introduced by
the reaction of 7-hydroxyanilinoquinazoline 3 with 2-
bromoethanol. The azide 6 was synthesized by the mesy-
lation of alcohol 4 and the replacement of the mesyl
group using NaN3. 7-(20-Aminoethoxy)quinazoline
derivative 7 was obtained by the reduction of the azido
group. The amine 12 was prepared as illustrated in
Scheme 2. The intermediate 9 was synthesized by the
O-alkylation of 3 with 2-acetoxyethyl-20-tosyloxyethyl
ether followed by the deprotection of the O-acetyl
group.10 Compound 9 was treated with methansulfonyl
chloride to give O-mesylated quinazoline 10. Finally,
4-[(40-Chloro-20-fluoro)phenylamino]-6,7-dimethoxyqui-
nazoline (CB676475, 1) is a potent inhibitor of VEGFR
Cl
Cl
Cl
i)
ii)
iii)
HN
HN
N
HN
N
O
F
O
O
F
O
O
F
N
O
N
N
O
O
HO
N
3
O
HO
9
8
Cl
Cl
Cl
iv)
v)
HN
HN
HN
N
O
O
F
O
O
F
O
O
F
N
N
N
O
O
O
N3
N
H2N
N
MsO
12
10
11
Scheme 2. Synthesis of compound 12. Reagents and conditions: (i) CH3CO2(CH2CH2O)2Ts, K2CO3, DMF, 95 °C, 81%; (ii) 30% NH4OH, MeOH,
rt, 12 h, 65%; (iii) MsCl, NEt3, THF/DMF, 0 °C, 1 h, 99%; (iv) NaN3, EtOH/H2O, 80 °C, 3 h, 67%; (v) H2, Pd(OH)2/C, MeOH, 95%.

S.-Y. Han et al. / Bioorg. Med. Chem. Lett. 16 (2006) 129–133
131
Cl
O
Cl
O
HN
NH
HN
N
HN
S
i)
HN
N
NH
O
O
F
+
O
O
F
N
O
H
N
N
S
H2N
O
n
n
NO2
O
n=1-2
13a, n=1
13b, n=2
Cl
Cl
O
F
F
F
F
F
HN
N
ii)
HN
O N
O
N3
F
O
F
+
N3
O
O
F
N
H
N
N
O
H2N
F
O
n
N
n
F
O
15a, n=1
15b, n=2
n=1-2
14
Scheme 3. Synthesis of compounds 13 and 15. Reagents and conditions: (i) NEt3, THF/CH2Cl2, rt, 10 h, 94%; (ii) DMF, rt, overnight, 98%.
compound 12 was obtained from 10 in high yield, under
the same conditions as those used to prepare the amine
7. As shown in Scheme 3, the biotinylated quinazoline
derivatives (13a,b) were prepared by the condensation
of amines 7 or 12 with (+)-biotin 4-nitrophenyl ester.
The 4-azidotetrafluorobenzamide derivatives (15a,b)
were obtained by the condensation of amines 7 or 12
with N-succinimidyl 4-azidotetrafluorobenzoate 14.1
The synthesis of the novel biotinylated photoreactive
derivative 2 is summarized in Scheme 4. The intermedi-
ate 18 was synthesized by the condensation of commer-
cially available biocytin 16 with 14 followed by the
succinimidyl activation of acid 17 with N-hydroxy-
succinimide.11,12 Finally, the target biotin-tagged
photoprobe 2 was obtained by the condensation of the
NHS ester 18 with 12 in excellent yield.
In order to confirm whether a series of quinazoline
derivatives (2, 13a,b, 15a, and b) effectively inhibit VEG-
FR-2, we measured their inhibitory activities against
VEGFR-2 tyrosine kinase.6,13a All compounds were
found to be potent VEGFR-2 inhibitors as shown in
N3
F
O
NH
O
F
F
F
F
F
N3
F
HN
S
HN
S
NH
i)
O
+
H
N
H
NH2
OH
N
NH
OH
F
O
O
O
N
O
O
O
O
O
17
O
16
F
O
F
N3
F
HN
NH
H
F
N
NH
NH
F
O
S
F
N3
HN
S
O
NH
O
O
F
H
N
ii)
iii)
NH
F
O
2
O
O
O
N
O
O
O
O
18
Cl
N
N
N
H
F
Scheme 4. Synthesis of compound 2. Reagents and conditions: (i) NaOH, DMF/H2O (2/1), rt, overnight, 89%; (ii) N-hydroxysuccinimide, DCC,
DMF, rt, 4 h, 77%; (iii) 12, DMF, rt, overnight, 98%.

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S.-Y. Han et al. / Bioorg. Med. Chem. Lett. 16 (2006) 129–133
Table 1. Inhibitory activity of 4-anilinoquinazoline derivatives
0.3
Compound
IC50 (lM)a
a
1.0
0.5
VEGFR-2
HUVEC
b
c
1
1.5
4.8
1.5
<1
1.0
14.7
1.9
13a
13b
15a
15b
2
d
e
f
250
300
Wavelength (nm)
0.2
4.1
3.2
<1
7.1
40.3
a IC50 values are expressed as the average of at least three
determinations.
0.1
0.0
Table 1. The biotin-tagged photoaffinity probe 2 was a
potent VEGFR-2 inhibitor (IC50 = 7.1 lM) being only
4.7-fold less potent than CB676475 (IC50 = 1.5 lM).
On the basis of their activities against VEGFR-2, qui-
nazoline derivatives were evaluated in the VEGF-stimu-
lated HUVEC proliferation assay (Table 1).13b The
biotinylated compounds 13a,b inhibited VEGF-depen-
dent HUVEC proliferation with IC50 values of 14.7
and 1.9 lM, respectively. The 4-azidotetrafluoroaryl
derivatives 15a,b were found to be more potent inhibi-
tors, with IC50 values of 4.1 and 3.2 lM, respectively.
The extension of side chain at the C-7 position of quinaz-
oline ring resulted in more potent inhibitors 13b and 15b
compared to compounds 13a and 15a. The probe 2 inhib-
ited HUVEC proliferation with an IC50 value of
40.3 lM. The inhibitory ability of probe 2 was 40-fold
less potent than that of CB676475, which could be ex-
plained by unfavorable steric interactions of the bulky
biotinylated photoactive moiety with the target enzymes.
Although the inhibition effect was slightly decreased
compared to that of CB676475, the probe 2 is potentially
useful in selective photolabeling of target proteins.
300
Wavelength (nm)
400
200
Figure 2. UV spectra for the photolysis of 2 (6 lM) in methanol at (a)
0, (b) 5, (c) 10, (d) 20, (e) 40, and (f) 60 s. The inset shows the UV
absorption spectrum of N-succinimidyl 4-azidotetrafluorobenzoate 14
(10 lM) in methanol.
advanced imaging techniques. Further studies of VEG-
FRs using this probe are in progress.
Acknowledgment
This research was supported by Chemical Genomics
R&D Project of the Ministry of Science and Technology
(MOST) of Korea.
Photodecomposition of probe 2 was examined by
monitoring the UV absorbance change in the range
between 200 and 400 nm in quartz cell.14 The absorp-
tion spectra of photolysis are presented in Figure 2.
The absorption maxima of 2 were observed at 250
and 330 nm. The photolysis of 2 in methanol was test-
ed by UV irradiation at 254 or 365 nm at a distance
of 5 cm from a UV lamp (VL-4LC, 4 W). Upon UV
irradiation, the photolysis rate of 2 at 254 nm was
faster than that at 365 nm (data not shown), and
the decrease in absorption at 250 nm after 254 nm
UV irradiation was greater than that at 330 nm, sug-
gesting that the photodecomposition of the azidotetra-
fluorophenyl group resulted in the decrease in
absorption at 250 nm. These results demonstrated that
the probe 2 could be a powerful photoaffinity reagent
to label VEGFRs involved in various aspects of tumor
angiogenesis.
Supplementary data
Supplementary data associated with this article can be
found in the online version at doi:10.1016/
j.bmcl.2005.09.036.
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