Synthesis of novel arylaminoquinazolinylurea derivatives and their antiproliferative activities against bladder cancer cell line

Article abstract of DOI:10.1016/j.bmcl.2016.08.076

A novel series of arylurea and arylamide derivatives 1a–z, 2a–d having aminoquinazoline scaffold was designed and synthesized. Their in vitro antiproliferative activities against RT112 bladder cancer cell line and inhibitory activities against FGFR3 kinase were tested. Most compounds showed good antiproliferative activities against RT112 bladder cancer cell line, and arylurea compounds 1a–z were more potent than arylamide compounds 2a–d. Among them, eight compounds 1a, 1d–g, 1l, 1y, and 1z showed potent activities with GI₅₀values below submicromolar range. Especially, arylurea compounds 1d and 1g possessing 2,3-dimethyl and 3,4-dimethyl moieties exhibited superior or similar antiproliferative activity (GI₅₀?=?8.8?nM and 30.2?nM, respectively) to AZD4547 (GI₅₀?=?29.2?nM) as a reference standard.

Full text of DOI:10.1016/j.bmcl.2016.08.076

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel arylaminoquinazolinylurea derivatives and their
antiproliferative activities against bladder cancer cell line
Jung Hun Kim a,c, Yeonui Kwak , Chiman Song , Eun Joo Roh , Chang-Hyun Oh , So Ha Lee , Taebo Sim ,
a
a
a
b
a
a
c
a,
⇑
Jung Hoon Choi , Kyung Ho Yoo
a
Chemical Kinomics Research Center, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
Center for Biomaterials, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
Department of Chemistry, Hanyang University, Seoul 133-791, Republic of Korea
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of arylurea and arylamide derivatives 1a–z, 2a–d having aminoquinazoline scaffold was
designed and synthesized. Their in vitro antiproliferative activities against RT112 bladder cancer cell line
and inhibitory activities against FGFR3 kinase were tested. Most compounds showed good antiprolifera-
tive activities against RT112 bladder cancer cell line, and arylurea compounds 1a–z were more potent
than arylamide compounds 2a–d. Among them, eight compounds 1a, 1d–g, 1l, 1y, and 1z showed potent
activities with GI50 values below submicromolar range. Especially, arylurea compounds 1d and 1g pos-
sessing 2,3-dimethyl and 3,4-dimethyl moieties exhibited superior or similar antiproliferative activity
Received 23 June 2016
Revised 18 August 2016
Accepted 24 August 2016
Available online xxxx
Keywords:
Arylaminoquinazolinylureas
Arylaminoquinazolinylamides
Antiproliferative activity
Bladder cancer cell line
Enzymatic activity
FGFR3
(GI50 = 8.8 nM and 30.2 nM, respectively) to AZD4547 (GI50 = 29.2 nM) as a reference standard.
Ó 2016 Elsevier Ltd. All rights reserved.
Bladder cancer represents the fifth most common neoplasms in
developed countries and a major cause of cancer-related morbidity
and death. Incidence and mortality rates have remained relatively
constant over the past four decades, with an estimated 72,570 new
cases and 15,210 deaths predicted for 2013 in the United States
as bladder cancer also exhibit deregulation and mutation of FGFR3,
but whether this contributes to tumorigenesis in vivo has
remained unclear. FGFR3 has long been considered a particularly
promising target for novel therapeutic approaches in bladder
9
cancer. Recently, a number of small molecule as receptor tyrosine
1
alone. About 70% of cases are non-muscle invasive and have a high
kinase inhibitors have been developed to target FGFRs and their
1
0–14
15
propensity to recur, giving bladder cancer the highest recurrence
rate of any cancer.2 The other 30% of cases are muscle invasive
tumors, for which the 5-year survival is approximately 50%. Their
conventional treatment involves surgical resection and intravesical
chemo- or immunotherapy.3
associated FGFs.
AZD4547 (AstraZeneca, Phase II/III) is a
novel and selective inhibitor of FGFR1, FGFR2, FGFR3 tyrosine
1
6
kinases, and PD173074 (Pfizer) serves as an important antitumor
inhibitor because of their good selectivity towards FGFR1 and
FGFR3 (Fig. 1).
Fibroblast growth factor receptors (FGFRs) constitute a major
class of receptor tyrosine kinases (RTKs) for a large family of
fibroblast growth factors (FGFs) and contain an extracellular
ligand-binding domain and an intracellular tyrosine kinase
Quinazoline is one of the most widespread scaffolds among nat-
ural and synthetic bioactive compounds. Several compounds pos-
sessing quinazoline scaffold have been recently reported as
1
7–21
potential antiproliferative agents.
In this work, a novel series
4
domain. Overexpression of each FGFR has been identified and
of arylurea and arylamide derivatives 1a–z, 2a–d having an amino-
quinazoline scaffold as a hinge region binding motif, based on the
structural features of AZD4547 and PD173074, was designed and
synthesized (Fig. 1). Their in vitro antiproliferative activities were
linked to a variety of human cancers, including breast and prostate
cancer, as well as multiple myeloma.5 FGFR3 is a member of a
structurally related family of tyrosine kinase receptors (FGFR1–4)
that orchestrate a diverse variety of cellular activities, including
–7
2
2,23
tested over RT112 bladder cancer cell line
(FGFR3 high-
8
proliferation, differentiation, and survival. Epithelial cancers such
expressing bladder cancer). And their inhibitory activities against
FGFR3 kinase are reported.
Arylaminoquinazolinylureas 1a–z were synthesized by the
pathways illustrated in Scheme 1. Ring closure of 2-amino-3-
⇑
Corresponding author. Tel.: +82 2 958 5152; fax: +82 2 958 5189.
E-mail address: khyoo@kist.re.kr (K.H. Yoo).
http://dx.doi.org/10.1016/j.bmcl.2016.08.076
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Kim, J. H.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.08.076

2
J. H. Kim et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
O
HN
N
NH
O
O
N
N
NH
N
O
HN
N
N
N
N
H
O
O
N
O
H
AZD4547
PD173074
O
N
N
N
H
N
H
H
N
N
R2
O
N
H
R1
O
N
H
O
O
1a-z
2a-d
Figure 1. Structures of AZD4547, PD173074, and the lead compounds.
O
NH
2
N
NH
N
N
HO C
2
NO
2
a
NO2
b
NO
c
N
N
2
O
Cl
NO
2
O
N
H
3
4
5
6
O
O
d
e
N
N
N
N
H
N
H
N
NH
2
R1
O
N
H
O
N
H
O
7
1a-z
Scheme 1. Reagents and conditions: (a) formamidine acetate, 2-methoxyethanol, 160 °C, 24 h, 51%; (b) DIPEA, POCl
dimethoxyaniline, Na CO , i-PrOH, 95 °C, 15 h, 79%; (d) Pd/C, H
2 3 2
2
3
, chlorobenzene, 10–90 °C, 5 h, 60%; (c) 3,5-
, EtOH, rt, 24 h, 87%; (e) R NCO, THF, rt, 3 h, 1a: 62%, 1b: 22%, 1c: 45%, 1d: 32%, 1e: 79%, 1f: 24%, 1g: 19%, 1h:
6%, 1i: 33%, 1j: 47%, 1k: 32%, 1l: 39%, 1m: 42%, 1n: 32%, 1o: 29%, 1p: 42%, 1q: 33%, 1r: 52%, 1s: 43%, 1t: 49%, 1u: 41%, 1v: 45%, 1w: 67%, 1x: 22%, 1y: 52%, 1z: 46%.
1
nitrobenzoic acid (3) as a starting material with formamidine acet-
ate in 2-methoxyethanol gave 8-nitroquinazolin-4(1H)-one (4),
The in vitro antiproliferative activity of the newly synthesized
compounds against RT112 bladder cancer cell line was tested.
2
4
25
which was then chlorinated with phosphorus oxychloride in the
presence of N,N-diisopropylethylamine to give 4-chloro-8-nitro-
quinazoline (5). Nucleophilic substitution of the chloro group of
The ability of arylaminoquinazolinylureas 1a–z and arylamino-
quinazolinylamides 2a–d to inhibit the growth of RT112 cell line
is summarized in Tables 1 and 2. AZD4547 was selected as a refer-
ence standard in this experiment because of its good selectivity
towards FGFR1, FGFR2, FGFR3 tyrosine kinases. Most compounds
showed good antiproliferative activities against RT112 bladder
cancer cell line. In general, arylaminoquinazolinylureas 1a–z with
a terminal urea moiety as a hydrophobic tail region were more
potent than arylaminoquinazolinylamides 2a–d having a terminal
amide moiety. This may be attributed to that the longer spacer,
urea moiety, may geometrically permit appropriate fitting of the
molecule at the receptor site. Or the terminal NH group of the urea
moiety may form additional hydrogen bond at the binding pocket.
Any or both of these effects would enable optimal drug–receptor
interaction, and hence higher antiproliferative activity. As shown
in Table 1, thirteen compounds showed good potency with GI50
5
with 3,5-dimethoxyaniline was carried out using sodium carbon-
ate in 2-propanol to produce N-(3,5-dimethoxyphenyl)-8-nitro-
quinazolin-4-amine (6). Reduction of the nitro group of 6 using
palladium over carbon in hydrogen atmosphere gave the com-
pound 7 in good yield. Treatment of 7 with the appropriate aryl
isocyanate derivatives afforded the corresponding title compounds
1
a–z, respectively.
Synthesis of arylaminoquinazolinylamides 2a–d was carried
out by the sequence of reactions shown in Scheme 2. Amide cou-
pling of the amino group of compound 7 with the appropriate car-
boxylic acid derivatives in the presence of HOBt, EDCI, and
triethylamine led to the desired title compounds 2a–d,
respectively.
values in 1-digit micromolar range (GI50 = 1.08–6.23 lM) and eight
compounds 1a, 1d–g, 1l, 1y, and 1z exhibited potent activities with
GI50 values below submicromolar range (GI50 = 8.8–471 nM)
against RT112 bladder cancer cell line. The effect of substituents
onto aryl nucleus on potency was also investigated. Compounds
with electron-donating group were generally found to be more
potent than compounds having electron-withdrawing group,
except for compounds 1y,z having nitro moiety. Among them,
dimethylphenylurea compounds 1d–g showed higher potency
compared with compounds bearing the different terminal
O
O
a
N
N
N
N
H
N
NH
2
R2
O
N
H
O
N
H
O
7
2a-d
H, HOBt, EDCI, Et N, DMF, 0–80 °C,
2
Scheme 2. Reagents and conditions: (a) R CO
8 h, 2a: 24%, 2b: 48%, 2c: 29%, 2d: 43%.
2
3
1
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J. H. Kim et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 1
The in vitro antiproliferative activity of arylaminoquinazolinylureas 1a–z against RT112 bladder cancer cell line
O
N
N
H
N
H
N
O
N
H
R1
O
1
a-z
Compd. no.
R1
RT112 (GI50
,
lM)
Compd. no.
R1
RT112 (GI50
2.75
, lM)
O
O
O
O
1
a
0.366
1o
O
1
1
b
c
14.0
1.29
1p
1q
5.91
10.1
1
1
d26
0.0088
0.356
1r
1s
1.74
2.49
Cl
Cl
e
Cl
Cl
Cl
Cl
1
1
1
1
f
0.0632
0.0302
4.89
1t
3.06
6.23
2.46
3.92
Cl
Cl
g26
h
i
1u
1v
1w
Cl
Cl
F
Cl
Cl
F
Cl
F
2.63
CF
3
F
Cl
F
CF
Cl
3
1
1
j
>20.0
2.97
1x
1y
1.08
CF3
CF
3
CF
3
k
0.471
F
CF3
1
l
0.467
1z
O
0.317
CF3
O
1
m
n
>20.0
>20.0
AZD4547
0.0292
0.0150
NO
2
NO
2
1
PD17307427
CF
3
substituents. Especially, arylurea compounds 1d and 1g possessing
,4-dimethyl and 3,4-dimethyl moieties exhibited superior or
similar antiproliferative activity (GI50 = 8.8 nM and 30.2 nM,
respectively) to AZD4547 (GI50 = 29.2 nM) as a reference standard.
On the other hand, the chlorinated compounds 1i–o by the
introduction of mono-, di- or tri-chloro moiety as well as the fluo-
rinated compounds 1p,q displayed poor potency. This may be
rationalized that the electronic effect of substituents at receptor
site may affect the affinity and potency. In arylaminoquinazoliny-
lamide series, arylamide compounds 2a,b,d did not show the
2
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4
J. H. Kim et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 2
Acknowledgments
The in vitro antiproliferative activity of arylaminoquinazolinylamides 2a–d against
RT112 bladder cancer cell line
This work was funded by the KIST Institutional Program (Grant
No. 2E26090) from Korea Institute of Science and Technology, and
by the Creative Fusion Research Program through the Creative
Allied Project funded by the National Research Council of Science
O
N
N
H
N
R2
O
N
H
&
Technology (CAP-12-1-KIST).
O
2a-d
References and notes
Compd. No.
R2
RT112 (GI50
14.2
,
lM)
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N
N
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Kingsley, C.; Dairkee, S.; Meng, Z.; Chew, K.; Pinkel, D.; Jain, A.; Ljung, B. M.;
Esserman, L.; Albertson, D. G.; Waldman, F. M.; Gray, J. W. Cancer Cell 2006, 10,
2
2
b
c
>20.0
4.37
N
529.
N
6. Kwabi-Addo, B.; Ozen, M.; Ittmann, M. Endocr. Relat. Cancer 2004, 11, 709.
7. Chang, H.; Stewart, A. K.; Qi, X. Y.; Li, Z. H.; Yi, Q. L.; Trudel, S. Blood 2005, 106,
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Turner, N.; Grose, R. Nat. Rev. Cancer 2010, 10, 116.
di Martino, E.; Tomlinson, D. C.; Knowles, M. A. Adv. Urol. 2012, 2012 429213.
O
O
2
d
>20.0
10. Gangjee, A.; Zhao, Y.; Raghavan, S.; Ihnat, M. A.; Disch, B. C. Bioorg. Med. Chem.
010, 18, 5261.
2
11. Chen, Z.; Wang, X.; Zhu, W.; Cao, X.; Tong, L.; Li, H.; Xie, H.; Xu, Y.; Tan, S.;
AZD4547
PD173074
0.0402
0.0150
Kuang, D.; Ding, J.; Quian, X. J. Med. Chem. 2011, 54, 3732.
12. Kumar, S. B.; Narasu, L.; Gundla, R.; Dayam, R.; JARPS. Curr. Pharm. Design 2013,
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1
1
1
1
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X.; Liang, G. Cancer Lett. 2014, 344, 82.
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Rooney, C.; Coleman, T.; Baker, D.; Mellor, M. J.; Brooks, A. N.; Klinowska, T.
Cancer Res. 2012, 72, 2045.
meaningful antiproliferative activities against RT112 bladder can-
cer cell line (Table 2). Compound 2c possessing N,N-dimethy-
laminomethyl moiety as a terminal group exhibited GI50 value of
4
.37 lM.
16. Miyake, M.; Ishii, M.; Koyama, N.; Kawashima, K.; Kodama, T.; Anai, S.;
The in vitro inhibitory activity of all the target compounds 1a–z,
Fujimoto, K.; Hirao, Y.; Sugano, K. J. Pharmacol. Exp. Ther. 2010, 332, 795.
2
a–d against FGFR3 kinase was evaluated (Table 3).28 All the tested
17. Harrington, E. A.; Bebbington, D.; Moore, J.; Rasmussen, R. K.; Ajose-Adeogun,
A. O.; Nakayama, T.; Graham, J. A.; Demur, C.; Hercend, T.; Diu-Hercend, A.; Su,
M.; Golec, J. M.; Miller, K. M. Nat. Med. 2004, 10, 262.
compounds showed weak% inhibitory activities (8.00–18.6%)
against FGFR3 kinase. The precise identification of mode of action
is under way.
As a continuation of our ongoing anticancer development
research project, a novel series of arylaminoquinazolinylureas
1
8. Chen, Z.; Venkatesan, A. M.; Dehnhardt, C. M.; Santos, O. D.; Santos, E. D.;
Ayral-Kaloustian, S.; Chen, L.; Geng, Y.; Arndt, K. T.; Lucas, J.; Chaudhary, I.;
Mansour, T. S. Bìoorg. Med. Chem. Lett. 2009, 19, 4980.
19. Cai, X.; Zhai, H. X.; Wang, J.; Forrester, J.; Qu, H.; Yin, L.; Lai, C. J.; Bao, R.; Qian,
C. J. Med. Chem. 2010, 53, 2000.
1
a–z and arylaminoquinazolinylamides 2a–d was designed and
2
0. Barlaam, B.; Acton, D. G.; Ballard, P.; Bradbury, R. H.; Cross, D.; Ducray, R.;
Germain, H.; Hudson, K.; Klinowska, T.; Magnien, F.; Ogilvie, D. J.; Olivier, A.;
Ross, H. S.; Smith, R.; Trigwell, C. B.; Vautier, M.; Wright, L. Bìoorg. Med. Chem.
Lett. 2008, 18, 1799.
1. Beria, I.; Bossi, R. T.; Brasca, M. G.; Caruso, M.; Ceccarelli, W.; Fachin, G.;
Fasolini, M.; Forte, B.; Fiorentini, F.; Pesenti, E.; Pezzetta, D.; Posteri, H.; Scolaro,
A.; Re Depaolini, S.; Valsasina, B. Bìoorg. Med. Chem. Lett. 2011, 21, 2969.
2. Zhao, G.; Li, W. Y.; Chen, D.; Henry, J. R.; Li, H. Y.; Chen, Z.; Zia-Ebrahimi, M.;
Bloem, L.; Zhai, Y.; Huss, K.; Peng, S. B.; McCann, D. J. Mol. Cancer Ther. 2011, 10,
synthesized. Among them, arylurea compounds 1d and 1g possess-
ing 2,4-dimethyl and 3,4-dimethyl moieties exhibited superior or
similar antiproliferative activities against RT112 bladder cancer
cell line to AZD4547 as a reference standard. In our series, the com-
pound 1d can be used as a promising lead for the development of
new potent inhibitors against bladder cancer cell line.
2
2
2
2200.
3. Guagnano, V.; Furet, P.; Spanka, C.; Bordas, V.; Le Douget, M.; Stamm, C.;
Brueggen, J.; Jensen, M. R.; Schnell, C.; Schmid, H.; Wartmann, M.; Berghausen,
J.; Drueckes, P.; Zimmerlin, A.; Bussiere, D.; Murray, J.; Graus Porta, D. J. Med.
Chem. 2011, 54, 7066.
4. Gomtsyan, A.; Bayburt, E. K.; Schmidt, R. G.; Zheng, G. Z.; Perner, R. J.;
Didomenico, S.; Koenig, J. R.; Turner, S.; Jinkerson, T.; Drizin, I.; Hannick, S. M.;
Macri, B. S.; McDonald, H. A.; Honore, P.; Wismer, C. T.; Marsh, K. C.; Wetter, J.;
Stewart, K. D.; Oie, T.; Jarvis, M. F.; Surowy, C. S.; Faltynek, C. R.; Lee, C.-H. J.
Med. Chem. 2005, 48, 744.
Table 3
Inhibition rates of arylaminoquinazolinylureas 1a–z and arylaminoquinazoliny-
lamides 2a–d against FGFR3 at a concentration of 10 lM
2
Compd. no.
FGFR3 (% inhibition)
Compd. No.
FGFR3 (% inhibition)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
13.7
18.1
13.0
17.0
8.90
12.4
10.6
8.10
11.9
12.6
10.1
18.2
9.60
10.1
10.6
11.1
1q
1r
1s
1t
1u
1v
1w
1x
1y
1z
2a
2b
2c
2d
PD173074
13.8
8.00
14.8
11.2
12.0
11.6
17.7
12.0
8.50
8.20
12.5
16.8
12.5
18.6
99.9
25. Cellular antiproliferation assay. RT112 cells were grown in RPMI1640
supplemented with 10% FBS and 1% antibiotics, and plated at a density of
5000 cells/well in 96-well plates. The day after cell seeding, a series of
compounds were added to wells at six dose points of 1:10 serial dilution in
DMSO. After 72 h exposure, cell viability was measured using 3-(4,5-
dimethylthiazol)-2,5-diphenyltetrazolium (MTT) assay (Promega). Values
were normalized to DMSO-treated wells. GI50 values were calculated using
Prism 5.0 software (Graphpad). Each assay was performed in duplicate and
replicated twice.
2
6. Selected data. Compound 1d: Mp 177–179 °C; 1H NMR (400 MHz, DMSO-d
6
) d
.73 (s, 1H), 9.66 (s, 1H), 9.06 (s, 1H), 8.72 (s, 1H), 8.65 (d, J = 7.8 Hz, 1H), 7.57
d, J = 8.2 Hz, 1H), 7.56 (t, J = 8.1 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.25 (s, 2H),
9
(
7
3
4
.06 (t, J = 7.8 Hz, 1H), 6.94 (d, J = 7.4 Hz, 1H), 6.31 (s, 1H), 3.78 (s, 6H), 2.26 (s,
Na [M+Na]+
25 5 3
H), 2.18 (s, 3H); HRMS (ESI, positive) calcd for C25H N O
1
66.1957, found 466.1847. Compound 1g: Mp 187–188 °C; H NMR (400 MHz,
Please cite this article in press as: Kim, J. H.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.08.076

J. H. Kim et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
DMSO-d
d, J = 8.3 Hz, 1H), 7.58 (t, J = 8.1 Hz, 1H), 7.32 (s, 1H), 7.27 (s, 3H), 7.06 (d,
J = 8.2 Hz, 1H), 6.32 (s, 1H), 3.79 (s, 6H), 2.21 (s, 3H), 2.17 (s, 3H); HRMS (ESI,
6
) d 9.69 (s, 2H), 9.68 (s, 1H), 9.46 (s, 1H), 8.67 (d, J = 7.8 Hz, 1H), 8.11
LANCE reaction buffer (1 mM EGTA, 10 mM MgCl
2
, 2 mM DTT, 0.01% Tween-
(
20, 50 mM Tris–HCl, and pH 7.5). The FGFR3 kinase domain of 1.0 nM was
incubated with 100 nM ULight-poly GT and various concentrations (10 M,
M, and 0.1 M) of test compounds in the presence or absence of 10 M ATP
l
+
positive) calcd for C25
H
25
N
5
O
3
Na [M+Na] 466.1957, found 466.1854.
1
l
l
l
2
2
7. Lamont, F. R.; Tomlinson, D. C.; Cooper, P. A.; Shnyder, S. D.; Chester, J. D.;
Knowles, M. A. Brit. J. Cancer 2011, 104, 75.
8. In vitro kinase assay. Kinase activities of human FGFR3 kinase domain (a.a.
for 1 h. The EDTA solution and Eu-anti-phospho-Tyr (PT66) antibody
(PerkinElmer, USA) were prepared at 4Â concentrations (40 mM and 8 nM,
respectively) in the 1Â LANCE Detection buffer (PerkinElmer, USA). The kinase
reaction was terminated by adding 10 mM EDTA and further incubated for
5 min. Eu-anti-phospho-Tyr (PT66) antibody was added to the kinase reaction
mixture at a final concentration of 2 nM for the detection of phosphorylation of
substrate and the mixture was incubated for 1 h. The fluorescence signal was
4
36–806; Carna Biosciences, Japan) were measured using LANCE Time-
resolved fluorescence resonance energy transfer (TR-FRET) assays
PerkinElmer, USA) with the FlexStation 3 microplate reader (Molecular
Devices, USA). All kinase assays were performed at a final volume of 10 L in
(
l
white 384-well plates at room temperature and microplates were sealed with
microplate sealing tapes (Corning, USA) during incubation. The FGFR3 kinase
domain, ULight-poly GT (PerkinElmer, USA) used as a substrate, and ATP were
measured using the FlexStation
(excitation wavelength of 320 nm, emission wavelength of 665 nm, a time
3 microplate reader in TR-FRET mode
delay of 50 s between excitation and emission detection, and an integration
l
prepared at 4Â concentrations (4.0 nM, 400 nM, and 40
l
M, respectively) in
time of 100 ls).
Please cite this article in press as: Kim, J. H.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.08.076