Discovery and synthesis of N2,N4-substitued- cycloalkyl[d]pyrimidine-2,4-diamine analogs: The first examples of small-molecular FGFR-1 activator

Article abstract of DOI:10.1016/j.cclet.2014.06.010

A series of novel, cycloalkyl-modified pazopanib analogs 2 and 3 were designed and synthesized. Their kinase modulatory effects on FGFR-1, VEGFR-2, PDGFR-β, and c-KIT were evaluated by the caliper mobility shift assay. Introduction of cycloalkyl into the pyrimidine linker of pazopanib almost abolished the four kinases inhibitory potency of compounds 2 and 3, but surprisingly, resulted in good activation effects on FGFR-1. Compounds 3d and 3g showed double-digit, nanomolar, selective activation effects on FGFR-1, and could be classified as first-generation small molecular activators of FGFR-1 kinase.

Full text of DOI:10.1016/j.cclet.2014.06.010

Chinese Chemical Letters 25 (2014) 989–994
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Discovery and synthesis of N2,N4-substitued-cycloalkyl[d]pyrimidine-
2,4-diamine analogs: The first examples of small-molecular
FGFR-1 activator
c
a
a
a,
Bao-Li Li a, Fang Xiao b, , Wen-Chao Lu , Yu-Yun Sun , Jin Zhu , Jian Li
*
*
a Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
b Department of Pharmacy, The Second Hospital of Jilin University, Jilin 130041, China
c China Resources Double-Crane Pharmaceutical Co., Ltd., Beijing 100121, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A series of novel, cycloalkyl-modified pazopanib analogs 2 and 3 were designed and synthesized. Their
Received 21 April 2014
Received in revised form 3 June 2014
Accepted 4 June 2014
kinase modulatory effects on FGFR-1, VEGFR-2, PDGFR-b, and c-KIT were evaluated by the caliper
mobility shift assay. Introduction of cycloalkyl into the pyrimidine linker of pazopanib almost abolished
the four kinases inhibitory potency of compounds 2 and 3, but surprisingly, resulted in good activation
effects on FGFR-1. Compounds 3d and 3g showed double-digit, nanomolar, selective activation effects on
FGFR-1, and could be classified as first-generation small molecular activators of FGFR-1 kinase.
ß 2014 Fang Xiao and Jian Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights
reserved.
Available online 18 June 2014
Keywords:
Cycloalkyl[d]pyrimidine derivatives
FGFR-1
Activator
Chemical probe
1. Introduction
Inspired by the structure of pazopanib (1, Fig. 1) [9] as a
representative anti-angiogenesis drug, recently we initiated a
The fibroblast growth factor receptor
1
(FGFR-1) is
a
program aimed at seeking small molecular, multi-target, anti-
angiogenesis agents. In order to obtain novel analogs of pazopanib,
two types of novel cycloalkyl[d]pyrimidine skeletons (2 and 3)
derivedfromthe pyrimidine linkerofpazopanib, weredesigned, and
a totalof 16 analogs (2, 3, Fig. 1) containing thesetwoskeletons were
synthesized, and tested in four pro-angiogenic kinase inhibitory
transmembrane tyrosine kinase receptor (TKR) that belongs to
the immunoglobulin (Ig) superfamily [1]. FGFR-1 was first cloned
and identified by Lee et al., as a member of the FGFR superfamily
[2], which consists of four distinct isoforms (FGFR-1 to FGFR-4)
found among several tissue types and expressed to different
extents under varying conditions [3,4]. The pivotal roles of the
signaling of FGFRs in regulating key cellular processes of cell
growth, proliferation, wound healing, embryogenesis and angio-
genesis are well documented [3,5,6].
Of the 4 known human FGFRs, FGFR-1 is one of the most important
members and has been deemed a strong candidate for drug
development as an anti-tumor target [7,8]. Since the 1990s, numerous
small molecular inhibitors targeting the kinase domain of FGFR-1 have
been reported [6], including pyridopyrimidines, indolinones, quina-
zolines/quinolines, indoles, and imidazopyridines. Despite the signifi-
cant progress, very limited efforts have been devoted toward the
development of FGFR-1 activators, especially small molecular
activators, and none reported to date. In this paper, we report the
identification of N2,N4-substitued-cycloalkyl[d]pyrimidine-2,4-dia-
mines as first-generation small molecular activators of FGFR-1 kinase.
assays (FGFR-1, VEGFR-2, PDGFR-b, and c-KIT). Unexpectedly, our
research identified 11 analogs that showed good kinase-selective
activation effects on FGFR-1 to a certain degree. To the best of our
knowledge, these activators are the first examples of a small-
molecular FGFR-1 activator, which might be used as an effective
chemical probe for the study of FGFR-1 related signaling pathway.
2. Experimental
2.1. Chemistry
All purchased starting materials were used without further
purification. Analytical thin-layer chromatography (TLC) was HSGF
254 (150–200
mm thickness; Yantai Huiyou Co., China). Yields
were not optimized. Melting points were measured in capillary
tubes on a SGW X-4 melting point apparatus without correction.
Nuclear magnetic resonance (NMR) spectroscopy was performed
on a Bruker AMX-400 NMR (IS as tetramethylsilane). Chemical
*
Corresponding authors.
E-mail addresses: xiaofang77@sina.com (F. Xiao), jianli@ecust.edu.cn (J. Li).
http://dx.doi.org/10.1016/j.cclet.2014.06.010
1001-8417/ß 2014 Fang Xiao and Jian Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

990
B.-L. Li et al. / Chinese Chemical Letters 25 (2014) 989–994
Fig. 1. Chemical structures of pazopanib (1) and cycloalkyl[d]pyrimidine analogs 2 and 3.
Scheme 1. Synthetic route of the target compounds 2 and 3. Reagents and conditions: (a) urea, MWI, 450 w, 12 min, yield 50%; (b) POCl3, r.t., 2 h, yield 46%; (c) conc. H2SO4,
KNO3, 0 8C, 1.5 h, yield 68%–72%; (d) isoamyl nitrite, AcOH, N2, r.t., 1.5 h, yield 88%–91%; (e) dimethyl carbonate, NaH, NMF, reflux, 5 h, yield 28%–35%; (f) SnCl2, conc. HCl,
DME, r.t., 3 h, yield 65%–76%; (g) NaHCO3, THF/EtOH (v/v = 1/4), reflux, 50 h, yield 48%–60%; (h) R5-Ph-NH2, conc. HCl, EtOH, N2, reflux, 24 h, yield 20%–46%; (i) conc. HCl,
EtOH, N2, reflux, 48 h, yield 23%.
shifts were reported in parts per million (ppm,
d
) downfield from
of 13 or 14 with various anilines was accomplished using acidic
conditions in ethanol to yield the desired cycloalkyl[d]pyrimidines
(2 and 3). Compound 4 was prepared by condensation of 14 with
ethanol using acidic conditions.
5-[[4-[N-(2,3-Dimethyl-2H-indazol-6-yl)-N-methylamino]-
6,7-dihydro-5H-cyclopenta[d]pyrimidin-2-yl]amino]-2-methyl-
benzenesulfonamide (2a): White solid, mp: 256–258 8C, yield: 40%
(last step), purity: 98% (tR 7.3 min, CH3OH/H2O = 80/20, v/v). 1H-
TMS. Proton coupling patterns were described as singlet (s),
doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br).
Low- and high-resolution mass spectra (LRMS and HRMS) were
obtained with electric and electrospray ionization (EI and ESI) on a
Finnigan MAT-95 and LCQ-DECA spectrometer. The purities of
compounds 2 and 3 was determined by Agilent 1100, and checked
with a UV/vis detector setting
The synthetic route of cycloalkyl[d]pyrimidine analogs 2 and 3
is described in Scheme 1. Various -ketoesters (5) and urea were
l = 254 nm.
NMR (400 MHz, DMSO-d6):
d 10.39 (s, 1H), 8.36 (s, 1H), 7.80
b
(d, 1H, J = 8.6 Hz), 7.66 (d, 1H, J = 8.5 Hz), 7.53 (s, 1H), 7.46–7.36
(m, 3H), 7.00 (d, 1H, J = 8.7 Hz), 4.08 (s, 3H), 3.55 (s, 3H), 2.82–2.74
(m, 2H), 2.64 (s, 3H), 2.58 (s, 3H), 1.80–1.60 (m, 4H). HRMS (EI) m/z
calcd. for C24H27N7O2S (M+): 477.1947, found: 477.1948.
5-[[4-[N-(2-Methyl-2H-indazol-6-yl)-N-methylamino]-6,7-
dihydro-5H-cyclopenta[d]pyrimidin-2-yl]amino]-2-methyl-ben-
zenesulfonamide (2b): White solid, mp: 262–264 8C, yield: 46%
(last step), purity: 99% (tR 6.1 min, CH3OH/H2O = 90/10, v/v). 1H-
placed in a domestic microwave oven and irradiated at a power of
450 W for 12 min to afford compounds 6, which were converted to
the corresponding aryl chlorides (7) in the presence of POCl3. The
2-substituted-anilines (8) were nitrated to yield 2-substituted-5-
nitroaniline (9). Cyclization of 9 with isoamyl nitrite in acetic acid
to give 3-R1-6-nitroindazole (10), followed by methylation with
dimethyl carbonate to give 2-methyl-3-R1-6-nitroindazole (11).
Reduction of 11 with tin (II) chloride gave 2-methyl-3-R1-6-
NMR (400 MHz, DMSO-d6):
d 9.35 (s, 1H), 8.65 (d, 1H, J = 2.1 Hz),
aminoindazole (12), and condensation with
7
using basic
8.37 (s, 1H), 7.73 (d, 2H, J = 8.7 Hz), 7.37 (s, 1H), 7.20 (m, 3H), 6.97
(dd, 1H, J = 8.8, 1.8 Hz), 4.18 (s, 3H), 3.52 (s, 3H), 2.58 (t, 2H,
J = 7.5 Hz), 1.79–1.74 (m, 2H), 1.66–1.57 (m, 2H); HRMS (EI) m/z
calcd. for C23H25N7O2S (M+): 463.179, found: 463.1788.
conditions in THF and ethanol yielded key intermediate (13),
followed by second methylation with dimethyl carbonate to
give another key intermediate (14). Finally, a second condensation

B.-L. Li et al. / Chinese Chemical Letters 25 (2014) 989–994
991
5-[[4-[(2,3-Dimethyl-2H-indazol-6-yl)amino]-6,7-dihydro-5H-
5-[[4-[N-(2,3-Dimethyl-2H-indazol-6-yl)-N-methylamino]-
cyclopenta[d]pyrimidin-2-yl]amino]-2-methyl-benzenesulfona-
mide (2c): Off-white solid, mp: 298–301 8C, yield: 24% (last step),
purity: 99% (tR 6.2 min, CH3OH/H2O = 90/10, v/v). 1H NMR
5,6,7,8-tetrahydroquinazolin-2-yl]amino]-2-methyl-benzenesul-
fonamide (3a): White solid, mp: 316–320 8C, yield: 27% (last step),
purity: 97% (tR 11.1 min, CH3OH/H2O = 80/20, v/v). 1H NMR
(400 MHz, DMSO-d6):
d
10.40 (s, 1H), 9.94 (s, 1H), 7.86 (s, 1H),
(400 MHz, DMSO-d6): d 10.30 (s, 1H), 8.38 (s, 1H), 7.80 (d, 1H,
7.81 (s, 1H), 7.6 (d, 1H, J = 6.2 Hz), 7.68 (d, 1H, J = 8.6 Hz), 7.59 (s,
2H), 7.21 (d, 1H, J = 8.5 Hz), 7.16 (d, 1H, J = 6.3 Hz), 4.05 (s, 3H), 3.55
(s, 3H), 2.95 (s, 2H), 2.91–2.78 (m, 2H), 2.61 (s, 3H), 2.56 (s, 3H),
2.15 (s, 2H); HRMS (EI) m/z calcd. for C23H25N7O2S (M+): 463.179,
found: 463.1789.
J = 8.8 Hz), 7.65 (d, 1H, J = 8.2 Hz), 7.46–7.38 (m, 4H), 7.03 (dd, 1H,
J = 8.7, 1.6 Hz), 4.07 (s, 3H), 3.54 (s, 3H), 2.70–2.64 (m, 2H), 2.64 (s,
3H), 2.58 (s, 3H), 1.53 (s, 4H), 1.33 (s, 2H); HRMS (EI) m/z calcd. for
C
25H29N7O2S (M+): 491.2103, found: 491.2102.
5-[[4-[(2,3-Dimethyl-2H-indazol-6-yl)amino]-5,6,7,8-tetrahy-
3-[[4-[N-(2,3-Dimethyl-2H-indazol-6-yl)-N-methylamino]-
6,7-dihydro-5H-cyclopenta[d]pyrimidin-2-yl]amino]-benzenesul-
fonamide (2d): White solid, mp: 240–243 8C, yield: 37% (last step),
purity: 99% (tR 6.8 min, CH3OH/H2O = 80/20, v/v). 1H NMR
droquinazolin-2-yl]amino]-2-methyl-benzenesulfonamide (3b):
White solid, mp: 290–293 8C, yield: 20% (last step), purity: 98%
(tR 11.6 min, CH3OH/H2O = 80/20, v/v). 1H NMR (400 MHz, DMSO-
d6):
d 10.41 (s, 1H), 10.04 (s, 1H), 8.42 (s, 1H), 7.81 (d, 1H,
(400 MHz, CDCl3):
d
8.25 (s, 1H), 7.72 (brs, 1H), 7.56 (d, 1H,
J = 8.7 Hz), 7.65 (d, 1H, J = 8.5 Hz), 7.58 (s, 1H), 7.45–7.38 (m, 3H),
7.02 (s, 1H), 4.10 (s, 3H), 2.86–2.75 (m, 2H), 2.65 (s, 3H), 2.58 (s,
3H), 1.59–1.49 (m, 4H), 1.45–1.36 (m, 2H); HRMS (EI) m/z calcd. for
J = 8.6 Hz), 7.42–7.34 (m, 2H), 7.20–7.07 (m, 2H), 6.91 (d, 1H,
J = 8.6 Hz), 4.11 (s, 3H), 3.59 (s, 3H), 2.75–2.66 (m, 2H), 2.64 (s, 3H),
1.91–1.84 (m, 2H), 1.76–1.64 (m, 2H); HRMS (EI) m/z calcd. for
C23H25N7O2S (M+): 463.179, found: 463.1791.
C
24H27N7O2S (M+): 477.1947, found: 477.1945.
3-[[4-[N-(2,3-Dimethyl-2H-indazol-6-yl)-N-methylamino]-
3-[[4-[(2,3-Dimethyl-2H-indazol-6-yl)amino]-6,7-dihydro-5H-
cyclopenta[d]pyrimidin-2-yl]amino]-benzenesulfonamide (2e):
White solid, mp: 270–273 8C, yield: 20% (last step), purity: 100%
(tR 6.8 min, CH3OH/H2O = 80/20, v/v). 1H NMR (400 MHz, DMSO-
5,6,7,8-tetrahydroquinazolin-2-yl]amino]-benzenesulfonamide
(3c): Pale yellow solid, mp: 263–266 8C, yield: 36% (last step),
purity: 100% (tR 6.6 min, CH3OH/H2O = 90/10, v/v). 1H NMR
(400 MHz, DMSO-d6):
d 9.55 (s, 1H), 8.67 (s, 1H), 7.78 (d, 1H,
d6):
d
9.49 (drs, 1H), 8.06 (m, 1H), 7.57 (d, 1H, J = 9.0 Hz), 7.41 (d,
J = 7.8 Hz), 7.65 (d, 1H, J = 8.8 Hz), 7.41 (t, 1H, J = 7.9 Hz), 7.32 (d,
1H, J = 7.8 Hz), 7.23 (s, 2H), 7.01 (s, 1H), 6.85 (d, 1H, J = 8.8 Hz), 4.01
(s, 3H), 3.46 (s, 3H), 2.62 (t, 2H, J = 6.0 Hz), 2.59 (s, 3H), 1.68 (t, 2H,
J = 5.8 Hz), 1.61–1.54 (m, 2H), 1.42–1.33 (m, 2H); HRMS (EI) m/z
calcd. for C24H27N7O2S (M+): 477.1947 (M+), found: 477.1945.
N2-(3,4-Dimethoxyphenyl)-N4-(2,3-dimethyl-2H-indazol-6-
yl)-N4-methyl-5,6,7,8-tetrahydroquinazoline-2,4-diamine (3d):
White solid, mp: 193–196 8C, yield: 40% (last step), purity: 100%
(tR 12.4 min, CH3OH/H2O = 80/20, v/v). 1H NMR (400 MHz, DMSO-
1H, J = 8.7 Hz), 7.39 (s, 2H), 7.34 (s, 1H), 7.26 (dd, 1H, J = 8.6,
2.5 Hz), 7.22 (dd, 1H, J = 9.1, 2.5 Hz), 4.00 (s, 3H), 2.8–2.77 (m, 4H),
2.58 (s, 3H), 2.09–2.01 (m, 2H); HRMS (EI) m/z calcd. for
C
22H23N7O2S (M+): 449.1634, found: 449.1632.
3-[[4-[N-(2,3-Dimethyl-2H-indazol-6-yl)-N-methylamino]-
6,7-dihydro-5H-cyclopenta[d]pyrimidin-2-yl]amino]-4-methoxy-
benzenesulfonamide (2f): White solid, mp: 187–189 8C, yield: 23%
(last step), purity: 100% (tR 12.4 min, CH3OH/H2O = 80/20, v/v). 1H
NMR (400 MHz, DMSO-d6):
d
8.68 (s, 1H), 8.46 (s, 1H), 8.03 (s, 1H),
d6): d 8.96 (s, 1H), 7.68 (s, 1H), 7.64 (d, 1H, J = 8.8 Hz), 7.20 (d, 1H,
7.69 (s, 1H), 7.56 (d, 1H, J = 8.8 Hz), 7.46 (d, 1H, J = 8.7 Hz), 7.43 (s,
2H), 7.22–7.14 (m, 2H), 3.97 (s, 3H), 3.93 (s, 3H), 2.82–2.73 (m, 4H),
2.56 (s, 3H), 2.08–2.00 (m, 2H); HRMS (EI) m/z calcd. for
J = 8.6 Hz), 6.99 (s, 1H), 6.88–6.80 (m, 2H), 4.01 (s, 3H), 3.72 (s, 3H),
3.70 (s, 3H), 3.43 (s, 3H), 2.64–2.55 (m, 5H), 1.69 (s, 2H), 1.57 (s,
2H), 1.38 (s, 2H); HRMS (EI) m/z calcd. for C26H30N6O2 (M+):
458.243, found: 458.2426.
C
24H27N7O3S (M+): 493.1896, found: 493.1874.
N2-(3,4-Dimethoxyphenyl)-N4-(2,3-dimethyl-2H-indazol-6-
N2-(3-Bromophenyl)-N4-(2,3-dimethyl-2H-indazol-6-yl)-N4-
methyl-5,6,7,8-tetrahydroquinazoline-2,4-diamine (3e): White
solid, mp: 201–206 8C, yield: 33% (last step), purity: 98% (tR
yl)-N4-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-di-
amine (2g): Pink solid, mp: 210–214 8C, yield: 20% (last step),
purity: 100% (tR 12.1 min, CH3OH/H2O = 80/20, v/v). 1H NMR
9.2 min, CH3OH/H2O = 90/10, v/v). 1H NMR (400 MHz, CDCl3):
d
(400 MHz, CDCl3):
d
7.59 (d, 1H, J = 2.4 H), 7.56 (d, 1H, J = 8.8 Hz),
8.23 (s, 1H), 7.51 (d, 1H, J = 8.1 Hz), 7.39 (d, 1H, J = 8.0 Hz), 7.27 (s,
1H), 7.19 (s, 1H), 7.12 (d, 1H, J = 8.9 Hz), 7.07 (d, 1H, J = 7.7 Hz), 6.86
(d, 1H, J = 9.1 Hz), 4.08 (s, 3H), 3.54 (s, 3H), 2.71–2.64 (m, 2H), 2.60
(s, 3H), 1.79–1.72 (m, 2H), 1.68–1.58 (m, 2H), 1.49–1.35 (m, 2H);
HRMS (EI) m/z calcd. for C24H25BrN6 (M+): 476.1324, found:
476.1316.
7.37 (d, 1H, J = 1.1 Hz), 7.02 (dd, 1H, J = 8.6 Hz, 2.4 Hz), 6.93 (1H, dd,
J = 8.8, 1.7 Hz), 6.85 (d, 1H, J = 8.7 Hz), 4.13 (s, 3H), 3.93 (s, 3H), 3.89
(s, 3H), 3.59 (s, 3H), 2.73–2.62 (m, 5H), 1.90 (t, 2H, J = 7.1 Hz), 1.75–
1.66 (dd, 2H, J = 14.6 Hz, 7.5 Hz); HRMS (EI) m/z calcd. for
C
25H28N6O2 (M+): 444.2274, found: 444.2276.
N2-(3-Bromohenyl)-N4-(2,3-dimethyl-2H-indazol-6-yl)-N4-
N2-(3-Fluorophenyl)-N4-(2,3-dimethyl-2H-indazol-6-yl)-N4-
methyl-5,6,7,8-tetrahydroquinazoline-2,4-diamine (3f): White
solid, mp: 169–172 8C, yield: 25% (last step), purity: 99% (tR
methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine
(2h): Pink solid, mp: 215–218 8C, yield: 33% (last step), purity: 97%
(tR 13.1 min, CH3OH/H2O = 80/20, v/v). 1H NMR (400 MHz, CDCl3):
12.2 min, CH3OH/H2O = 80/20, v/v). 1H NMR (400 MHz, CDCl3):
d
d
8.25 (s, 1H), 7.70 (brs, 1H), 7.56 (d, 1H, J = 8.6 Hz), 7.43–7.35 (m,
7.84 (d, 1H, J = 12.2 Hz), 7.55–7.47 (m, 2H), 7.23–7.14 (m, 2H), 6.86
(d, 1H, J = 9.0 Hz), 6.68 (t, 2H, J = 8.0 Hz), 4.09 (s, 3H), 3.55 (s, 3H),
2.71–2.67 (m, 2H), 2.62 (s, 3H), 1.73 (s, 2H), 1.68 (s,2H), 1.44–1.40
(m, 2H); HRMS (EI) m/z calcd. for C24H25FN6 (M+): 416.2125, found:
416.2119.
2H), 7.19–7.07 (m, 2H), 6.91 (d, 1H, J = 8.6 Hz), 4.11 (s, 3H), 3.59 (s,
3H), 2.75–2.66 (m, 2H), 2.64 (s, 3H), 1.90–1.85 (m, 2H), 1.76–1.64
(m, 2H); HRMS (ESI) m/z calcd. for C23H24BrN6 [M+H]+: 463.1246,
found: 463.12403.
N2-(3-Fluorohenyl)-N4-(2,3-dimethyl-2H-indazol-6-yl)-N4-
methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine
(2i): Pink solid, mp: 222–224 8C, yield: 36% (last step), purity: 99%
(tR 7.7 min, CH3OH/H2O = 80/20, v/v). 1H NMR (400 MHz, CDCl3):
N2-(3-Chloro-4-fluorophenyl)-N4-(2,3-dimethyl-2H-indazol-6-
yl)-N4-methyl-5,6,7,8-tetrahydroquinazoline-2,4-diamine (3 g):
White solid, mp: 180–182 8C, yield: 20% (last step), purity: 95%
(tR 24.6 min, CH3OH/H2O = 95/5, v/v). 1H NMR (400 MHz, DMSO-
d
7.84 (d, 1H, J = 12.1 Hz), 7.55 (d, 1H, J = 8.8 Hz), 7.43 (brs, 1H),
d6): d 9.43 (s, 1H), 8.22 (dd, 1H, J = 6.9, 2.6 Hz), 7.70–7.66 (m, 1H),
7.36 (d, 1H, J = 1.7 Hz), 7.24–7.12 (m, 2H), 6.91 (dd, 1H, J = 8.8,
1.8 Hz), 6.71–6.62 (m, 1H), 4.11 (s, 3H), 3.58 (s, 3H), 2.69 (t, 2H,
J = 7.7 Hz), 2.64 (s, 3H), 1.89 (t, 2H, J = 7.2 Hz), 1.71 (m, 2H); HRMS
(ESI) m/z calcd. for C23H24FN6 [M+H]+: 403.2046, found:
403.2041.
7.65 (d, 1H, J = 8.9 Hz), 7.29 (t, 1H, J = 9.1 Hz), 7.01 (dd, 1H, J = 9.2,
1.3 Hz), 6.83 (dd, 1H, J = 9.2, 1.7 Hz), 4.01 (s, 3H), 3.43 (s, 3H), 2.64–
2.59 (m, 5H), 2.59 (s, 3H), 1.72–1.61 (m, 2H), 1.61–1.50 (m, 2H),
1.42–1.33 (m, 2H); HRMS (ESI) m/z calcd. for C24H24ClFN6 [M+H]+:
451.1813, found: 451.1815.

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B.-L. Li et al. / Chinese Chemical Letters 25 (2014) 989–994
Fig. 2. Concentration dependence of FGFR-1 activation effects by 3d (A) and 3g (B), the concentration of ATP was kept constant at 262
compounds 3d and 3g ranged from 0.05 nmol/L to 333 nmol/L and 0.19 nmol/L to 12500 nmol/L, respectively.
mmol/L, while the concentration of
N-Methyl-N-(2,3-dimethyl-2H-indazol-6-yl)-2-ethoxy-5,6,7,8-
tetrahydroquinazolin-4-amine (4): White solid, mp: 263–268 8C,
yield: 23% (last step), purity: 95% (tR 4.1 min, CH3OH/H2O = 95/5,
5.0 software. The accuracy of the in vitro enzyme assays was
confirmed by the IC50 value of the positive inhibitor, staurosporine,
which is in compliance with the reported value.
v/v). 1H NMR (400 MHz, DMSO-d6):
d 7.65 (d, 1H, J = 8.8 Hz), 6.99
(d, 1H, J = 1.2 Hz), 6.82 (dd, 1H, J = 8.8, 1.8 Hz), 4.29 (q, 2H,
J = 7.0 Hz), 4.01 (s, 3H), 3.35 (s, 3H), 2.61–2.56 (m, 5H), 1.64 (t, 2H,
J = 5.9 Hz), 1.58–1.50 (m, 2H), 1.36–1.28 (m, 5 H); HRMS (ESI) m/z
calcd. for C20H26N5O [M+H]+: 352.2137, found: 352.2224.
3. Results and discussion
3.1. In vitro FGFR-1 modulatory activity
The modulatory activities of the target compounds against
FGFR-1 were determined using the caliper mobility shift assay.
Pazopanib was used as the reference drug. Some preliminary
structure–activity relationships (SARs) can be drawn here. As
compared with pazopanib, cyclization of R3 and R4 (Table 1) into an
aliphatic rings, i.e., cyclopentyl (2a–2i) and cyclohexyl (3a–3g)
rings, almost abolished the FGFR-1 inhibitory potency of the
resulting compounds, with the exceptions of 2c, 3a, and 3b.
Surprisingly, eleven analogs showed good activation effects on
FGFR-1. A subtle interplay between cycloalkyl size and two
substitutions (R2 and R5) seemed to be critical for inhibition/
activation selectivity, however, the R1 substitution effect was
limited (2a vs. 2b). When R2 substitution is hydrogen, analogs (2c,
2e, and 3b) showed, exclusively, the inhibition profile. Compared
with cyclopentyl ring, the cyclohexyl ring is optimal and
significantly improves the activation effects (3d vs. 2g, 3c vs.
2d, 3e vs. 2h, and 3f vs. 2i). Halogen (2h–2i and 3e–3f) or multiple-
halogen (3g) substitutions on the phenyl ring (R5) substantially
increase the activation effects of the analogs. Replacement of the
2-aniline substituent with an ethoxy group cannot be tolerated
(4), leading to a complete loss of activity. Remarkably, the
activation effects of analogs 3d and 3g reach double-digit,
nanomolar activities with EC50 values of 68 nmol/L and
99 nmol/L, respectively (Fig. 2). To the best of our knowledge,
these two compounds are the most potent, small molecule FGFR-1
activators reported to date. The molecular mechanism of the
activation effect has been speculated to be allosteric behavior
induced by the FGFR-1 activator. The allosteric binding site and
allosteric mechanism on FGFR-1 is under investigation in our
laboratories.
2.2. Kinase enzyme assays
For the kinase modulatory assay, glutathione-S-transferase
(GST) fusion enzymes were purchased from Millipore (c-KIT) or
Carna (VEGFR-2, PDGFR-b and FGFR-1) and were used without
further purification. Ultrapure ATP and DMSO were purchased
from Sigma. FAM labeled, fluorescent peptide substrates were
synthesized by GL Biochem. Compounds were prepared with
DMSO at a final concentration of 2% in the assay system. Assay
mixtures were prepared with a 5
compounds, followed by 10
enzymes and initiated by adding 10
m
L addition of synthesized
of assay buffer containing
L assay buffer (50 mmol/L
m
L
m
HEPES, 10 mmol/L MgCl2, 0.0015% Brij-35, and 2 mmol/L DTT)
containing ATP and substrate. All kinase reactions were carried
out at 3
and final enzyme concentrations were varied for each target
(10 nmol/L PDGFR- , 4 nmol/L FGFR-1, 1.2 nmol/L VEGFR-2 and
mmol/L peptide substrate and Km of ATP for each kinase
b
6 nmol/L c-KIT). The reaction mixtures were incubated at 28 8C for
1–2 h depending on kinase activity. The reactions were terminat-
ed by the addition of 25
mL of stop buffer (100 mmol/L HEPES pH
7.5, 0.015% Brij-35, 0.2% Caliper coating reagent and 50 mmol/L
EDTA). Reaction products were separated on the Caliper instru-
ment according to the manufacturer’s instructions. Percent
inhibition and percent activation were calculated by using the
following equations.
max ꢀ sample
Percent inhibition ¼
ꢁ 100%
(1)
(2)
max ꢀ min
sample
Percent activation ¼
ꢁ 100%
max
3.2. Kinase selectivity profile
where max represents DMSO control and min represents low
control.
One of the great challenges in kinase ligand discovery is the
design of small-molecule modulators with a selectivity profile. To
further investigate the specificity of these FGFR-1 activators
discovered and, reported herein, we thus tested the modulatory
effects of the eleven FGFR-1 activators against other three kinase
To calculate the half-maximal inhibitory concentration (IC50
)
and the half-maximal activation concentration (EC50), the percent
of remaining kinase activity is plotted against the compound
concentration, and the data were fitted using the GraphPad

B.-L. Li et al. / Chinese Chemical Letters 25 (2014) 989–994
993
Table 1
In vitro FGFR-1 modulatory effects of cycloalkyl[d]pyrimidine analogs.
Compounds
R1
R2
R3
R4
R5
Modulatory effects on FGFR-1
IC50 (nmol/L)
EC50 (nmol/L)
Activation (%)a
Bottom (c, nmol/L)
Top (c, nmol/L)
2a
2b
2c
2d
2e
2f
CH3
H
CH3
CH3
H
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
3-SO2NH2, 4-CH3
3-SO2NH2, 4-CH3
3-SO2NH2, 4-CH3
3-SO2NH2
NAb
3178
2800
NA
106 (0.19)
98 (0.19)
149 (12,500)
156 (12,500)
NA
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
12,192
NA
CH3
H
3159
NA
106 (0.19)
149 (12,500)
3-SO2NH2
>50,000
>50,000
NA
CH3
CH3
CH3
CH3
CH3
H
2-MeO,5-SO2NH2
3,4-di-MeO
3-Br
NA
2g
2h
2i
3478
2099
3722
NA
113 (0.19)
100 (0.19)
90 (0.19)
164 (12,500)
155 (12,500)
148 (12,500)
NA
3-F
NA
3a
3b
3c
3d
3e
3f
3-SO2NH2, 4-CH3
3-SO2NH2, 4-CH3
3-SO2NH2
12,135
39,801
NA
NA
CH3
CH3
CH3
CH3
CH3
1679
68
96 (0.19)
96 (0.05)
86 (0.19)
102 (0.19)
103 (0.19)
145 (12,500)
151 (333)c
3,4-di-MeO
3-Br
NA
NA
890
2058
99
135 (12,500)
171 (12,500)
167 (12,500)
3-F
NA
3g
3-Cl,4-F
NA
4
NA
NA
Pazopanib
CH3
CH3
H
H
3-SO2NH2, 4-CH3
317
NA
a
Percent activation relative to the negative control at the concentration indicated in parenthesis.
b
c
No significant activity observed at 50
mmol/L concentration.
For poor solubility, the top concentration of 3d was set as 333 nmol/L.
Table 2
In vitro kinase selectivity profiles of cycloalkyl[d]pyrimidine analogs.
Compounds
Modulatory effects
FGFR-1
VEGFR-2
PDGFR-
b
c-KIT
IC50 (nmol/L)
EC50 (nmol/L)
IC50/EC50 (nmol/L)
IC50/EC50 (nmol/L)
IC50/EC50 (nmol/L)
2a
NAa
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
317
3178
2800
3159
3478
2099
3722
1679
68
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
32
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
141
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
>50,000/NA
27
2b
2d
2g
2h
2i
3c
3d
3e
890
3f
2058
99
3g
Pazopanib
NA
a
No significant activity observed at 50 mmol/L concentration.
receptors, including VEGFR-2, PDGFR-
showed that all FGFR-1 activators had no inhibition or activation
effects on VEGFR-2, PDGFR- , and c-KIT (Table 2). These results
suggested, to a certain extent, that these activators might be
kinase-selective activators of FGFR-1.
b
, and c-KIT. The results
4. Conclusion
b
In summary, inspired by the structure of pazopanib (1), two
types of novel, cycloalkyl[d]pyrimidine skeletons (2–3) derived
from the pyrimidine linker of pazopanib were designed. A total of

994
B.-L. Li et al. / Chinese Chemical Letters 25 (2014) 989–994
References
16 analogs containing these two skeletons were synthesized, and
tested in four pro-angiogenic, kinase inhibitory assays (FGFR-1,
[1] D.E. Johnson, L.T. Williams, Structural and functional diversity in the FGF receptor
multigene family, Adv. Cancer Res. 60 (1993) 1–41.
VEGFR-2, PDGFR-b, and c-KIT). Unexpectedly, 11 analogs were
found to show good kinase-selective activation effects on FGFR-1,
and two analogs (3d and 3g) showed double-digit, nanomolar
selective activation effects on FGFR-1, however, the precise
molecular mechanism of the activation effect still needs further
investigation. To the best of our knowledge, these activators are the
first examples of small-molecular FGFR-1 activators, which might
be used as effective chemical probes for the study of FGFR-1 related
signaling pathways. Further exploration of the functions of these
new activators is under investigation in our laboratories.
[2] P.L. Lee, D.E. Johnson, L.S. Cousens, V.A. Fried, L.T. Williams, Purification and
complementary DNA cloning of a receptor for basic fibroblast growth factor,
Science 245 (1989) 57–59.
[3] V.P. Eswarakumar, I. Lax, J. Schlessinger, Cellular signaling by fibroblast growth
factor receptors, Cytokine Growth Factor Rev. 16 (2005) 139–149.
[4] N. Turner, R. Grose, Fibroblast growth factor signalling: from development to
cancer, Nat. Rev. Cancer 10 (2010) 116–129.
[5] I. Ahmad, T. Iwata, H.Y. Leung, Mechanisms of FGFR-mediated carcinogenesis,
Biochim. Biophys. Acta 1823 (2012) 850–860.
[6] H.K. Ho, A.H.L. Yeo, T.S. Kang, B.T. Chua, Current strategies for inhibiting FGFR
activities in clinical applications: opportunities, challenges and toxicological con-
siderations, Drug Discov. Today 19 (2014) 51–62.
[7] V.D. Acevedo, R.D. Gangula, K.W. Freeman, et al., Inducible FGFR-1 activation leads
to irreversible prostate adenocarcinoma and an epithelial-to-mesenchymal tran-
sition, Cancer Cell 12 (2007) 559–6571.
Acknowledgments
[8] P.U. Magnusson, A. Dimberg, S. Mellberg, A. Lukinius, L. Claesson-Welsh, FGFR-1
regulates angiogenesis through cytokines interleukin-4 and pleiotrophin, Blood
110 (2007) 4214–4222.
[9] P.A. Harris, A. Boloor, M. Cheung, et al., Discovery of 5-[[4-[(2,3-dimethyl-2H-
indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methyl-benzenesulfona-
mide (Pazopanib), a novel and potent vascular endothelial growth factor receptor
inhibitor, J. Med. Chem. 51 (2008) 4632–4640.
Financial support of this research provided by the National
Natural Science Foundation of China (Nos. 21222211, 21372001,
91313303), the Program for New Century Excellent Talents in
University (No. NCET-12-0853), and the Fundamental Research
Funds for the Central Universities are gratefully acknowledged.