Design and synthesis of novel photoaffinity reagents for labeling VEGF receptor tyrosine kinases

Article abstract of DOI:10.1016/j.tetlet.2006.02.119

Novel biotin-tagged photoaffinity probes based on a trifunctional tertiary amine scaffold were synthesized and evaluated as vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors. Probes 3a-c inhibit VEGF induced proliferation in HUVE cells, w

Full text of DOI:10.1016/j.tetlet.2006.02.119

Tetrahedron Letters 47 (2006) 2915–2919
Design and synthesis of novel photoaffinity reagents for labeling
VEGF receptor tyrosine kinases
Sun-Young Han, Seo Hyun Choi, Myung Hee Kim, Woo Ghil Lee, Seong Hwan Kim,
Yong Ki Min* and Bum Tae Kim
Bio-Organic Science Division, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea
Received 14 January 2006; revised 15 February 2006; accepted 20 February 2006
Available online 9 March 2006
Abstract—Novel biotin-tagged photoaffinity probes based on a trifunctional tertiary amine scaffold were synthesized and evaluated
as vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors. Probes 3a–c inhibit VEGF induced proliferation in HUVE
cells, with IC50 values of 29.7, 33.3, and 37.7 lM, respectively. Moreover, we identified the interaction of 3b with VEGFR-2 in
photoaffinity labeling experiment using HUVE cells.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Angiogenesis is a process of new blood vessel formation
from preexisting blood vessels and is essential for the
growth of solid tumors and their metastasis.1 Vascular
endothelial growth factor receptors (VEGFRs) stimu-
late endothelial proliferation and play a critical role in
endothelial cell survival/growth and tumor angiogene-
sis.2 Recently, Casanovas et al. have demonstrated that
the inhibition of VEGFR-2 in endothelial cells is impor-
tant to prevent tumor growth.3 Several VEGFR-2 inhib-
itors have been shown to inhibit tumor angiogenesis.4
VEGFR-2 was confirmed by the disappearance (or
reduction) of the corresponding band under the condi-
tion in the presence of two-fold molar excess of non-
labeled mother compound, CB676475 (data not shown).
However, in the case of probe 2, a photolabel tag and
a bioactive ligand have been attached to two functional
groups of biocytin, which has a number of disadvan-
tages to insert a spacer between biotin and a photo-
affinity moiety/ligand and to facilitate interactions
between ligand and proteins in cells. This led to the de-
sign of a trifunctional scaffold for photoaffinity labeling,
which can change the length of the side chain and en-
hance the inhibitory potency. Here, we developed an
efficient synthesis of trifunctional probes that contain a
photoreactive group, a biotin tag, and a bioactive ligand
(see Fig. 1).
Photoaffinity labeling is a powerful method in the chem-
ical proteomic approach of protein functions. This
method is especially useful for the identification of
ligand-binding sites of target proteins and the investiga-
tion of ligand–receptor interactions.5 Biotinylated
photoaffinity probes can be applied to separate labeled
proteins from complex mixtures as well as to study the
interactions of ligand–protein in living cells.6
To introduce the side chains of various lengths between
a photophore and biotin, we prepared the mono-Boc-
protected compounds 4–6 by treatment of diamines with
di-ter-butyl dicarbonate (Boc2O) (Scheme 1).8 Trifunc-
tional tertiary amine derivatives (22–24) were prepared
as illustrated in Scheme 2. Reductive amination of
monoprotected amines (4–6) with perfluorobenzalde-
hyde afforded the pentafluorobenzylamine derivatives
7–9 in good yields.9 The biotinylated precursors 13–15
were synthesized by deprotection of the Boc group with
trifluoroacetic acid (TFA) followed by coupling with
biotin in the presence of 1-ethyl-3-(3-dimethylaminoprop-
yl)carbodiimide hydrochloride (EDC).10 The interme-
diates (16–18) were obtained by the condensation of
Previously, we have described the preparation of a
photoaffinity probe 2 that is a potent VEGFR-2 inhibi-
tor.7 Moreover, we carried out the photocrosslinking
experiment using HUVE cells and identified the interac-
tion of 2 with VEGFR-2. The specific binding of 2 with
Keywords: Biotin-tagged photoaffinity probe; Vascular endothelial
growth factor receptors; Ligand–receptor interactions; Inhibitor; Tri-
functional probe.
*
Corresponding author. Tel.: +82 42 860 7029; fax: +82 42 861
0307; e-mail: ykmin@krict.re.kr
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.02.119

2916
S.-Y. Han et al. / Tetrahedron Letters 47 (2006) 2915–2919
Cl
F
NH
O
O
N
N
1 (CB676475)
F
F
F
O
O
F
N3
F
F
N3
F
HN
HN
NH
NH
O
H
N
H
N
N
O
F
NH
NH
S
O
S
n
O
O
2
NH
O
O
O
3a (n=0)
3b (n=1)
3c (n=2)
O
O
O
O
Cl
Cl
N
N
N
N
H
N
N
H
F
F
Figure 1. Structures of CB676475 (1) and biotin-tagged photoaffinity probes.
H
N
n=0, 4
n=1, 5
n=2, 6
a
H2N
O
H2N
NH2
O
O
n
n
O
Scheme 1. Reagents and conditions: (a) (i) n = 0, (Boc)2O, CHCl3, 0 ꢁC to rt, 24 h, 98%; (ii) n = 1, NaOH, MeOH/THF, rt, overnight, 97%; (iii)
n = 2, (Boc)2O, dioxane, rt, 5 h, 97%.
F
F
F
O
H
N
F
F
F
F
F
F
O
a
b
d
N
H
N
H
O
H2N
O
n
+
O
n
F
O
c
n=0, 7, 64%
n=1, 8, 49%
n=2, 9, 77%
CHO
F
O
F
F
O
HN
F
F
O
F
F
NH
N
N
n
H
N
H
H
S
NH2
F
F
O
n
F
F
n=0, 13, 56% in two steps
n=1, 14, 67% in two steps
n=2, 15, 72% in two steps
n=0, 10
n=1, 11
n=2, 12
O
O
F
O
O
HN
HN
e
F
O
F
F
O
NH
NH
N
N
H
N
N
H
n
n
O
S
O
S
N3
F
F
F
O
F
O
n=0, 16, 63%
n=1, 17, 83%
n=2, 18, 85%
n=0, 19, 82%
n=1, 20, 87%
n=2, 21, 88%
f
O
F
F
O
HN
F
O
NH
N
N
H
n
O
S
N3
F
OH
n=0, 22, 77%
n=1, 23, 92%
n=2, 24, 97%
Scheme 2. Reagents and conditions: (a) (i) MgSO4, NEt3, MeOH, rt, 1.5 h, (ii) NaBH4, 0 ꢁC, 2 h; (b) TFA, CH2Cl2, rt, 1 h; (c) EDC, MeOH/CH3CN
(1/3), rt, 5 h; (d) methyl bromoacetate, DIEA, DMF, 5 ꢁC to rt, 12 h; (e) NaN3, Bu4NN3 (cat.), DMF, 80 ꢁC, 12 h; (f) 1 N NaOH, MeOH, rt, 3 h.

S.-Y. Han et al. / Tetrahedron Letters 47 (2006) 2915–2919
2917
Table 1. Inhibition of HUVEC proliferation by novel biotin-tagged
photoaffinity probes
O
F
F
O
HN
F
O
NH
N
N
H
IC50 (lM)a
n
Compound
O
S
N3
F
n=0, 22
n=1, 23
n=2, 24
1
1.7 0.3
41.7 4.7
29.7 1.5
33.3 4.2
37.7 2.5
OH
2
3a
3b
3c
a
O
F
O
HN
a IC50 values are expressed as the average of at least three
determinations.
F
O
NH
N
N
H
n
O
N
S
N3
F
O
n=0, 25
n=1, 26
n=2, 27
F
O
O
carbodiimide (DCC). The quinazoline derivative (28)
was prepared from 3,4-dimethoxybenzoic acid, as de-
scribed previously.7 Finally, the target biotin-tagged
photoprobes 3a–c were obtained by the condensation
of the NHS esters (25–27) with 28 in moderate yield.13
Cl
+
HN
N
O
O
F
O
In order to confirm whether a series of quinazoline
derivatives (3a–c) effectively inhibit HUVEC prolifera-
tion, we determined their inhibitory activity against
VEGF-stimulated HUVEC proliferation using Cell
Counting Kit-8 (CCK-8) assay (Dojindo laboratories)
(Table 1).14 The probe 2 had an IC50 of 41.7 lM, which
was 25-fold less potent than CB676475 (IC50 = 1.7 lM).
All probes 3a–c were found to be more potent inhibitors
compared to compound 2, with IC50 values of 29.7, 33.3,
and 37.7 lM, respectively. Activity was dependent on
the chain length of the linker between biotin and the
photoaffinity moiety/ligand. The decrease of side chain
length resulted in more potent inhibitors, which could
be explained by the decrease of unfavorable steric inter-
actions of the bulky biotinylated photoactive moiety
with the target enzymes. The result suggested that novel
probes based on a trifunctional amine scaffold are very
useful to probe the structural requirements for optimal
biological activity.
H2N
N
28
b
O
F
O
HN
F
N3
O
NH
N
N
n
H
O
S
F
F
HN
O
O
N
O
F
H
N
N
Cl
n= 0, 3a, 51% in two steps
n= 1, 3b, 34% in two steps
n= 2, 3c, 53% in two steps
The UV absorption maxima of probes 3a–c in MeOH
showed at 250 and 330 nm. The decrease in absorption
at 250 nm was observed after 254 nm UV irradiation,
suggesting the decomposition of the azido group (data
not shown). In photoaffinity labeling experiment using
HUVE cells, VEGFR-2 was detected in all lines (1) pro-
teins only, (2) UV-irradiated proteins, (3) proteins mixed
with 3b, and (4) after proteins mixed with 3b, UV-irradi-
ated—at ca. 220-kDa using VEGFR-2 specific antibody
(Fig. 2).15,16 Proteins in the absence or presence of 3b
were irradiated at 254 nm twice for 2 min. When used
both streptavidin and biotin antibodies, which can de-
tect 3b, the immunoreactivities were detected only in
the fourth line at similar molecular weight position with
that of VEGFR-2. This indicated the interaction of 3b
Scheme 3. Reagents and conditions: (a) DCC, NHS, DMF, rt,
overnight; (b) DMF, rt, overnight.
secondary amines (13–15) with methyl bromoacetate.11
Treatment of intermediates (16–18) with NaN3 in addi-
tion of catalytic amount of Bu4NN3 in DMF at 80 ꢁC
afforded the desired azides (19–20), which were con-
19
firmed by F NMR.11,12 Finally, intermediates (22–
24) were obtained by hydrolysis of methyl esters (19–
21) under basic conditions. The synthesis of novel tri-
functional probes (3a–c) is summarized in Scheme 3.
The N-hydroxysuccinimide (NHS) esters (25–27) were
synthesized by the condensation of acid derivatives
(22–24) with N-hydroxysuccinimide using dicyclohexyl-
3b
UV-crosslinking
250-kDa
Used antibody
Streptavidin-HRP
VEGFR-2-HRP
Biotin-HRP
Figure 2. Western blot analysis showing the interaction of 3b with VEGFR-2.

2918
S.-Y. Han et al. / Tetrahedron Letters 47 (2006) 2915–2919
Org. Lett. 2005, 7, 1699–1702; (g) Fujii, T.; Manabe, Y.;
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Bioorg. Med. Chem. Lett. 2006, 16, 129–133.
with VEGFR-2. These results demonstrated that the
probes 3a–c could be powerful photoaffinity reagents
to label VEGFRs involved in various aspects of tumor
angiogenesis.
8. (a) Braun, M.; Camps, X.; Vostrowsky, O.; Hirsch, A.;
Endreß, E.; Bayerl, T. M.; Birkert, O.; Gauglitz, G. Eur. J.
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Nowick, J. S.; Famulok, M. Bioorg. Med. Chem. 2003, 11,
235–249.
In conclusion, we have synthesized novel biotin-tagged
photoaffinity probes 3a–c and estimated the importance
of the side chain length between biotin and a photo-
active moiety/ligand. Moreover, we showed that the
probes can be used in photoaffinity crosslinking to label
VEGFR-2 in HUVE cells. Novel trifunctional reagents
for photoaffinity labeling should play a useful rule to
identify target proteins and to investigate ligand–protein
interactions in living cells. The biochemical application
of these probes is currently under investigation.
11. Redman, J. E.; Ghadiri, M. R. Org. Lett. 2002, 25, 4467–
4469.
Acknowledgements
12. Compound 19: 19F NMR (CFCl3): d À142.91 (m, 2F),
À151.87 (m, 2F). Compound 20: 19F NMR (CFCl3): d
À143.09 (m, 2F), À152.27 (m, 2F). Compound 21: 19F
NMR (CFCl3): d À143.05 (m, 2F), À152.37 (m, 2F).
This research was supported by Chemical Genomics
R&D Project of the Ministry of Science and Technology
(MOST) of Korea.
1
13. Compound 3a: H NMR (300 MHz, CDCl3): d 1.30–1.70
(m, 6H), 2.06 (t, 2H, J = 7.5 Hz), 2.57 (m, 2H), 2.66 (d,
1H, J = 12.6 Hz), 2.85 (m, 1H), 3.05 (m, 1H), 3.12 (s, 2H),
3.24 (m, 2H), 3.48 (m, 2H), 3.66 (m, 2H), 3.79 (s. 2H), 3.97
(m, 5H), 4.22 (m, 1H), 4.30 (m, 2H), 4.44 (m, 1H), 5.55 (s,
1H), 6.25 (s, 1H), 6.83 (m, 1H), 7.17–7.27 (m, 3H), 7.34 (s,
1H), 7.45 (m, 1H), 8.13 (t, 1H, J = 8.7 Hz), 8.21 (br s, 1H),
8.58 (s, 1H); 19F NMR (CFCl3): d À122.63 (s, 1F),
À142.40 (m, 2F), À151.84 (m, 2F); HRMS-FAB (m/z)
[M+H]+ calcd for C40H44ClF5N11O6S, 936.2805, found,
936.2805. Compound 3b: 1H NMR (300 MHz, MeOH-d4):
d 1.30–1.45 (m, 2H), 1.50–1.78 (m, 4H), 2.19 (t, 2H,
J = 7.3 Hz), 2.60–2.75 (m, 3H), 2.88 (m, 1H), 3.10–3.20
(m, 1H), 3.20 (s, 2H), 3.28–3.35 (m, 2H), 3.40–3.55 (m,
6H), 3.68 (m, 2H), 3.80 (s. 2H), 3.86–4.0 (m, 2H), 3.97 (s,
3H), 4.28 (m, 3H), 4.46 (m, 1H), 7.11 (s, 1H), 7.27–7.36
(m, 2H), 7.65 (m, 2H), 8.34 (s, 1H); 19F NMR (CFCl3): d
À123.47 (s, 1F), À142.80 (m, 2F), À151.92 (m, 2F);
HRMS-FAB (m/z) [M+H]+ calcd for C42H48ClF5N11O7S,
980.3068, found, 980.3069. Compound 3c: 1H NMR
(300 MHz, MeOH-d4): d 1.25–1.45 (m, 2H), 1.48–1.78
(m, 4H), 2.15 (t, 2H, J = 7.4 Hz), 2.60–2.73 (m, 3H), 2.88
(m, 1H), 3.05–3.20 (m, 1H), 3.20 (s, 2H), 3.27–3.35 (m,
2H), 3.40–3.60 (m, 10H), 3.67 (m, 2H), 3.78 (s, 2H), 3.84–
4.0 (m, 2H), 3.96 (s, 3H), 4.15–4.30 (m, 3H), 4.45 (m, 1H),
7.08 (m, 1H), 7.26–7.36 (m, 2H), 7.60–7.66 (m, 2H), 8.33
(s, 1H); 19F NMR (CFCl3): d À117.18 (s, 1F), À142.01 (m,
2F), À152.26 (m, 2F); HRMS -FAB (m/z) [M+Na]+ calcd
for C44H51ClF5N11O8SNa, 1046.3149, found, 1046.3146.
14. Wood, J. M.; Bold, G.; Buchdunger, E.; Cozens, R.;
Ferrari, S.; Frei, J.; Hofmann, F.; Mestan, J.; Mett, H.;
O’Reilly, T.; Persohn, E.; Rosel, J.; Schnell, C.; Stover, D.;
Theuer, A.; Towbin, H.; Wenger, F.; Woods-Cook, K.;
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15. Photoaffinity labeling experiment—HUVEC cells were
homogenized in buffer consisting of 10 mM Tris–HCl
(pH 7.5), 150 mM NaCl, 0.05% (v/v) Tween 20, 1 mM
PMSF, and one protease inhibitor cocktail tablet (Roche,
Germany) at 4 ꢁC and centrifuged at 10,000g for 15 min.
The BCA protein assay Kit (Pierce, IL) was used to
determine the concentration of protein in the supernatant.
Proteins (100 lg in 50 ll) were incubated with 3b (100 lM)
at 4 ꢁC for 2 h, and placed on an ice tray under a UV light
source (BIO-LINK with 5 · 8 W tubes, 254 nm, Vilber
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S.-Y. Han et al. / Tetrahedron Letters 47 (2006) 2915–2919
2919
Lourmat, France) twice for 2 min. Compound 3b was first
dissolved in dimethyl sulfoxide (DMSO; Sigma, MO), and
then diluted with homogenate buffer, so that the final
DMSO concentration was 1% in the samples. Irradiated
protein samples (20 lg) were mixed with sample buffer
(100 mM Tris–HCl, 2% sodium dodecyl sulfate, 1%
2-mercaptoethanol, 2% glycerol, 0.01% bromophenol
blue, pH 7.6), incubated at 50 ꢁC for 10 min, and loaded
onto 8% polyacrylamide gels. Electrophoresis was per-
formed using the Mini Protean 3 Cell (Bio-Rad). Proteins
separated on the gels were transferred onto nitrocellulose
membrane (Scheicher & Schnell BioScience, Germany),
and the membrane was incubated in blocking buffer
(10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20,
3% nonfat dry milk). The membrane was incubated for
2 h at room temperature with 1:1000 diluted primary
antibody. Horse radish peroxidase (HRP)-conjugated
antibody against VEGFR-2 and antibodies against strep-
tavidin and biotin were purchased from Santa Cruz
Biotechnology Inc. (CA) and Calbiochem (Merck Bio-
sciences, Korea), respectively. After washing three times
for 15 min with blocking buffer, and developed with
SuperSignal West Femto Maximum Sensitivity Substrate
(Pierce). The immunoreactivity was detected using LAS-
3000 luminescent image analyzer (Fuji Photo Film Co.,
Ltd, Japan).
16. Takahashi, T.; Shibuya, M. Oncogene 1997, 14, 2079–
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