Multisubstituted quinoxalines and pyrido[2,3-d]pyrimidines: Synthesis and SAR study as tyrosine kinase c-Met inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.08.075

Two series of new analogues were designed by replacing the quinoline scaffold of our earlier lead 2 (zgwatinib) with quinoxaline and pyrido[2,3-d]pyrimidine frameworks. Moderate c-Met inhibitory activity was observed in the quinoxaline series. Among the p

Full text of DOI:10.1016/j.bmcl.2012.08.075

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6368–6372
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Multisubstituted quinoxalines and pyrido[2,3-d]pyrimidines: Synthesis and SAR
study as tyrosine kinase c-Met inhibitors
Kui Wu a, , Jing Ai b, , Qiufeng Liu c, , TianTian Chen c, Ailing Zhao d, Xia Peng b, Yuanxiang Wang d,
c,
b,
d,
Yinchun Ji b, Qizheng Yao a, , Yechun Xu , Meiyu Geng , Ao Zhang
⇑
⇑
⇑
⇑
a Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
b Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
c Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
d Synthetic Organic & Medicinal Chemistry Laboratory, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2012
Revised 7 August 2012
Accepted 20 August 2012
Available online 27 August 2012
Two series of new analogues were designed by replacing the quinoline scaffold of our earlier lead 2 (zgw-
atinib) with quinoxaline and pyrido[2,3-d]pyrimidine frameworks. Moderate c-Met inhibitory activity
was observed in the quinoxaline series. Among the pyrido[2,3-d]pyrimidine series, compounds 13a–c
possessing an O-linkage were inactive, whilst the N-linked analogues 15a–c retained c-Met inhibitory
potency. Highest activity was observed in the 3-nitrobenzyl analog 15b that showed an IC50 value of
6.5 nM. Further structural modifications based on this compound were undergoing.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
c-Met kinase
3,5-Diamino-7-trifluoroquinoline
hERG
Antitumor activity
Structure–activity relationship (SAR)
Similar to most of the receptor tyrosine kinases (RTK), the hepa-
tocyte growth factor (HGF) c-Met is a regulator of many critical
cellular processes including embryological development, cell
growth, differentiation, neovascularization and tissue regenera-
tion.1,2 Aberrantly high expression of HGF/c-Met has been impli-
We recently reported20,21 a series of highly potent c-Met inhib-
itors structurally featured by multisubstituted quinolines. One of
these compounds, 3-(4-methylpiperazin-1-yl)-N-(3-nitrobenzyl)-
7-(trifluoromethyl)quinolin-5-amine (2)20, also named zgwatinib
( Fig. 1), displayed high c-Met potency both at enzymatic
(0.93 nM) and cellular (230 nM) levels, thereafter was selected
for earlier preclinical investigations. Meanwhile, a series of triazol-
o[4,3-b]pyridazine analogues were designed21 by merging the
3-piperazinyl-7-trifluoromethylquinoline core of 220 and the tria-
zolopyridazine core of 3,15 another potent c-Met inhibitor reported
by Amgen, into one molecule to improve the memberane perme-
ability and then eradicate the discrepancy between the enzymatic
and cellur potency of 2 (Fig. 2). Unfortunately, these new triazol-
o[4,3-b]pyridazin-3-ylmethanamines 421 showed much reduced
inhibitory effects to c-Met enzyme.
As a continuation of our study toward the identification of po-
tent c-Met inhibitors, we decided to take advantage of the
widely-used quinoxazoline skeleton as a privileged scaffold of
RTK inhibitors and modifiy it with the three key substutients of
compound 4 or lead 2, thereby designing a new series of quinoxa-
line analogues I (Fig. 2). Meanwhile, replacement of the quinoline
core 2 with a pyrido[2,3-d]pyrimidine artwork and concurrent
incorporation of the two side chains of 1 led to another series of
new analogues II (Fig. 2). Herein, in this report we disclose the
synthesis and c-Met inhibition study of these two series of new
analogues.
cated in
a
variety of solid tumors.3–9 Therefore, c-Met has
emerged as an attractive molecular target and inhibition of HGF/
c-Met signaling pathway has shown great therapeutic benefit as
novel cancer therapy.10–15 Among the large number of c-Met-tar-
geting small molecules reported recently,16–18 crizotinib (1, PF-02
341066, Fig. 1) has been the only one approved by FDA as a first-
in-class c-Met inhibitor antitumor drug. However, this drug is
indeed a dual inhibitor with equal potency at both c-Met and
ALK (anaplastic lymphoma kinase) kinases.19 Therefore, a direct
correlation between the clinical anti-cancer benefit and the
c-Met potency is dampened by the polypharmacy profile, and
highly selective and potent c-Met inhibitors are emergently
needed as probes to validate clinical efficacy of the c-Met targeting
strategy.
⇑
Corresponding authors. Tel.: +86 21 50801267; fax: +86 21 50807188 (Y.X.);
tel./fax: +86 21 50806072 (M.G.); tel./fax: +86 21 50806035 (A.Z.).
E-mail addresses: qz_yao@yahoo.com.cn (Q. Yao), ycxu@mail.shcnc.ac.cn (Y. Xu),
mygeng@mail.shcnc.ac.cn (M. Geng), aozhang@mail.shcnc.ac.cn (A. Zhang).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.075

K. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6368–6372
O2N
6369
NH
Cl
O
NH
N
N
N
F
N
Cl
F3C
N
2 (zgwatinib)
H2N
N
In vitro: c-Met enzyme: IC50 = 0.93 nM
1
(Crizotinib, PF-2341066)
BAF3-TRP-Met/-IL3: IC50 = 230 nM
Figure 1. Marketed c-Met inhibitor 1 and our earlier reported compound 2.
NO2
N
N
N
N
R
Ar
N
N
N
N
NH
N
NH
N
NH
N
N
N
F3C
N
F3C
N
4
R'
O
2
(our lead compound)
3
(Ar=3,4,5-trifluorophenyl)
(c-Met Enzyme: IC50 > 30 nM)
(c-Met Enzyme: IC50 = 0.93 nM
(c-Met Enzyme: Ki = 5 nM
Cell: Ki = 230 nM)
Cell: Ki = 2 nM)
(Wang, Y.: OBC-2011-9-5930)
(Wang Y: JMC-2011-54-2127)
(Amgen: JMC-2008-2793)
our current design
R
N
NH
R
N
N
N
Y
R'
NH
X
N
N
Y
X
N
N
N
N
F3C
F3C
N
N
I,
quinoxaline series
II,
pyrido[2,3-d]pyrimidine series
Figure 2. Our new compounds design.
Br
The synthesis of compound series I was started from 3-chloro-5-
bromo-7-trifluoromethylquinoxaline (5)22 which was prepared by
following a literature procedure. As described in Scheme 1, treat-
ment of 5 with variant amines provided 3-amino-quinoxalines
6a–f in 88–95% yields. Pd2(dba)3-catalyzed C–N coupling20 of 6a
with (6-substituted-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methan-
amines 7a–j afforded 8a–j in 64–82% yields. Meanwhile, coupling
of 4-fluorophenyl substituted [1,2,4]triazolo[4,3-b]pyridazin-3-
ylmethanamine 7g with bromides 6b–f yielded N-substituted
piperazines 9a–e in 65–80% yields.
Br
N
N
Cl
R
N
N
THF, reflux
amine
F3C
F3C
N
5
6a-f
(88-95%)
N
N
O
N
N Ac
O
a
,
R=:
b
c
,
,
;
;
e
,
d
N
O
,
f
N
O
,
N
N
;
;
N
N
R'
N
N
Table 1
N
6a
c-Met activity of compounds 8a–ja
N
NH
N
N
Pd2(dba)3, BINAP
Cs2CO3, 1,4-dioxane,
reflux
a
R0
IC50 (nM)
R'
N
Compd
N
N
N
NH2
7a-j
8a
8b
8c
8d
8e
8f
8g
8h
8i
H
MeO
2-Thienyl
3-Thienyl
2-Furyl
Ph
2500 67.0
2700 317
659 129
441 52.5
409 183
1980 328
626 97.2
385 84.7
257 40.3
924 179
0.93 0.18
330
F3C
8a-j
(64-82%)
N
N
N
N
F
4-F-Ph
3-F-Ph
3-Cl-Ph
1-Me-pyrazol-4-yl
—
NH
Pd2(dba)3, BINAP
8j
7g
(R'=4-F-Ph)
6b-f
N
N
R
+
220
421
Cs2CO3, 1,4-dioxane,
reflux
R@R0@H
F3C
a
In vitro kinase assays were performed with the indicated purified recombinant
9a-e
(65-80%)
c-Met kinase domains, IC50s were calculated by Logit method from the results of at
least three independent tests with six concentrations each.
Scheme 1. Synthesis of compounds 8a–j and 9a–e (Series I).

6370
K. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6368–6372
Table 3
F
c-Met activity of selected compoundsa
a
a
Cl
Cl
Compd
IC50 (nM)
Compd
IC50 (nM)
Cl
N
F
Br
+
13a
13b
13c
>10
>10
>10
l
l
l
M
M
M
15a
15b
15c
441 110
6.5 0.2
>1000
Et3N
O
N
N
Cl
Cl
CH2Cl2
Br
N
F3C
N
OH
10
a
IC50s were calculated by Logit method from the results of at least three inde-
pendent tests with six concentrations each and expressed as mean S.D.
F3C
N
R
O
11 (66%)
N
N
B
O
Pd2(dba)3
F
HN
N
BINAP
PdCl2(dppf).CH2Cl2
dioxane, reflux
Cs2CO3,
Cs2CO3
potent c-Met inhibitory activity. The substituted phenyl deriva-
tives 8g–i had higher potency than the non-substituted phenyl
analog 8f. [1,2,4]Triazolo[4,3-b]pyridazine analogues (8c–j) with
a larger C6-substituent were more potent than non-substituted
congener 8a or analogues with a small C6-substituent (8b, 8f).
Compared to compound 8g, all the analogues 9a–e bearing
diversely substituted piperazine or morpholine fragments showed
moderate, but statistically similar activity. The IC50 values of these
compounds varied in the range of 300–900 nM. In comparison to
compounds 9a and 9c, or 9b and 9e, higher activity was observed
on compounds with less steric effects.
Cl
Cl
F
R
N
O
N
N
Cl
Cl
N
O
N
N
F3C
N
N
13a-c (80-88%)
F3C
N
N
a,
NH
R =:
b
c
, Me; , -CH2CH2OH
;
12
.
Since the quinoxaline series did not give improved activity, we
then turned our attention to the pyrido[2,3-d]pyrimidine core. Ini-
tially, we wanted to incorporate only the (R)-1-(2,6-dichloro-3-
fluorophenyl)ethoxy component of 1 to make compound 12
(Scheme 2). Quite disappointingly, the coupling of N-methylpipera-
zine with bromide 11 (prepared from 6-bromo-4-chloro-2-(trifluo-
romethyl)pyrido[2,3-d]pyrimidine23 in 66% yield) under variant
conditions, including Pd2(dba)3/BINAP, did not occur. To our delight,
Suzuki coupling24 of 11 with 1-substituted pyrazo-4-ylboranes
under PdCl2(dppf)ꢀCH2Cl2/Cs2CO3 went through smoothly. Com-
pounds 13a–c bearing both (R)-1-(2,6-dichloro-3-fluorophenyl)
ethoxy and 1-substituted-1H-pyrazol-4-yl moieties of 1 were
obtained in 80–88% yields.
Scheme 2. Synthesis of compounds 13a–c.
Table 2
c-Met activity of compounds 9a–ea
a
Compd
9a
R–
IC50 (nM)
485 5.5
309 19.3
N
N
O
N
9b
Ac
O
9c
9d
9e
539 15.3
879 95.5
486 48.4
N
O
Unfortunately, all the three pyrido[2,3-d]pyrimidine analogues
13a–c did not show noticeable enzymatic inhibition to c-Met even
at 10 lM concentrations (Table 3). To determine the role of the oxy-
N
N
gen-linkage between the substituted benzyl and the pyrido[2,3-
d]pyrimidine core in 13a, corresponding N-linked analog 15a was
prepared in 70% yield by treating chloride 10 with corresponding
amine followed by Suzuki coupling under PdCl2(dppf)ꢀCH2Cl2/
Cs2CO3 system24 (Scheme 3).
To our delight, moderate activity was retained in the N-linked
compound 15a with an IC50 value of 441 nM, comparable to that
of quinoxaline series in Tables 1 and 2 and our earlier reported
compounds 4, yet still much less potent than our initial lead 2.
Switching the 2,6-dichloro-3-fluorophenyl to 3-nitrophenyl
delivered compound 15b that showed significantly improved
N
O
a
In vitro kinase assays were performed with the indicated purified recombinant
c-Met kinase domains, IC50s were calculated by Logit method from the results of at
least three independent tests with six concentrations each.
The c-Met enzymatic activity of compounds 8a–j was shown in
Table 1. Compared to compounds 421, most of the newly synthetic
compounds 8a–j displayed reduced activity, except compounds
8d, 8e and 8g–i that showing only slightly decreased or equally
H
N
NH
R
O
O
N
N
NH
B
R
N
RNH2
N
N
Br
NH
N
N
10
Et3N
PdCl2(dppf).CH2Cl2
dioxane, reflux
CH2Cl2
F3C
N
N
14a-c
Cs2CO3,
F3C
N
15a-c (78-86%)
CN
F
NO2
R =: a,
c,
b,
Cl
Cl
Scheme 3. Synthesis of compounds 15a–c.

K. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6368–6372
6371
our earlier observation20,25 that the nitro group in lead 2 made key
interactions with c-Met enzyme.
Fortunately, we were able to obtain the co-crystal structures of
lead 2 and compound 15b bound to the c-Met active domain by
following a literature procedure27 (Fig. 3). As shown in Figure 3A,
both compounds bound to the kinase in a similar manner.28 The
compounds formed both hydrophobic and H-bond interactions
with the kinase. The pyridyl N-atom in both the quinoline and pyr-
ido[2,3-d]pyrimidine cores formed a H-bond to Met1160 in the
hinge region of the kinase, the CF3 and 3-nitrobenzylamino groups
occupied the hydrophobic pockets. It is of note that the nitro group
formed one or two H-bonds with Asp1222 accounting for the ob-
served importance of this group to the c-Met potency. The pipera-
zine in
2 or piperidine in 15b positioned towards solvent.
Therefore, further work will focus on more modification in this re-
gion to generate compounds with more c-Met potency and better
druglikeness.
In summary, we have designed two series of new analogues26
bearing quinoxaline and pyrido[2,3-d]pyrimidine frameworks as
the core skeletons to replace the quinoline scaffold in our earlier
lead
2 (zgwatinib). Moderate c-Met inhibitory activity was
observed in the quinoxalines series of 8a–j and 9a–e. Among the
pyrido[2,3-d]pyrimidine series, compounds 13a–c possessing an
O-linkage were inactive, whilst the N-linked analogues 15a–c re-
tained c-Met inhibitory potency. The 3-nitrobenzyl analog 15b
showed the highest activity with an IC50 value of 6.5 nM. Further
structural modifications based on this compound are undergoing.
Acknowledgments
The authors are grateful to Grants from the Chinese National Sci-
ence Foundation for the Distinguished Young Scholars Program
(81125021 for A.Z.) and for the Innovation Research Group (810
21062for M.G.), and Grants from National Science & Technology Ma-
jor Project on Key New Drug Creation and Manufacturing Program
(2012ZX09103-101-035, 2010ZX09401-401, 2012ZX093010 01-
007). Supports from Shanghai Commission of Science and Technol-
ogy (10JC1417100, 10dz1910104), the Hundred Talents Project of
Chinese Academy of Sciences (to Y.X.), and the National Natural Sci-
ence Foundation of China (81102461) and National Program on Key
Basic Research Project of China (2012CB910704) are also highly
appreciated.
Supplementary data
Supplementary data (general procedure and spectral data) asso-
ciated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2012.08.075.
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28. Protein purification and crystallization. Production of the kinase domain (1038–
1346aa) of recombinant human c-Met followed the protocols of Wang27a with
certain modifications. The cDNA fragment was cloned into the vector pET28a
and the protein was co-expressed with catYopH subcloned in pET15b
(164–468AA)2. The expressed c-Met kinase domain was passed through a
Ni-NTA column (Qiagen) and further purified by QHP ion exchange column
(GE) which eluted with 25 mM Tris pH 8.5, 100 mM NaCl, 10% glycerol, 1 mM
DTT. The protein was concentrated to about 10 mg/mL for further
crystallization.Cocrystallization of the c-Met kinase domain with compound
2 was carried out by mixing a solution of the protein-ligand complex with an
equal volume of precipitant solution (100 mM HEPES pH 7.5, 8% isopropanol,
3 mM TCEP, 16% PEG4K). The protein-ligand complex was prepared by adding
the compound to the protein solution to a final concentration of 1 mM of 2.
Cocrystallization utilized the vapour-diffusion method in hanging drops. The
same cocrystallization protocol was also utilized for compound 15b and the
precipitant solution includes 100 mM Tris pH 7.5, 15% glycerol, 12% MPD, 5%
isopropanol, 14% PEG5KMME. Crystals were flash frozen in liquid nitrogen in
the presence of well solution supplemented with 25% glycerol.
25. Wang, Y.; Ai, J.; Yue, J.; Peng, X.; Ji, Y.; Zhao, A.; Gao, X.; Wang, Y.; Chen, Y.; Liu, G.;
Gao, Z.; Geng, M.; Zhang, A. Med. Chem. Commun., accepted for publication.
http://dx.doi.org/10.1039/C2MD20192E.
26. Analytic data for representative compounds. Compound 8i: 1H NMR (300 MHz,
CDCl3) d 8.57 (s, 1H), 8.16 (d, J = 9.6 Hz, 1H), 7.95 (s, 1H), 7.84 (d, J = 9.6 Hz, 1H),