3,3-Dimethyl-1H-pyrrolo[3,2-g]quinolin-2(3H)-one derivatives as novel Raf kinase inhibitors

Article abstract of DOI:10.1039/c2md20275a

A series of quinoline derivatives were designed and synthesized as tyrosine kinase inhibitors. Exploration of the structure-activity relationships resulted in compounds that are potent in vitro. In addition, compound 10f was found to be a potent and selec

Full text of DOI:10.1039/c2md20275a

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3,3-Dimethyl-1H-pyrrolo[3,2-g]quinolin-2(3H)-one
derivatives as novel Raf kinase inhibitors
Cite this: DOI: 10.1039/c2md20275a
b
bc
Yanyang Li,ab Xiangfei Shi,b Ning Xie,bc Yanjin Zhao and Shuxin Li
*
Received 13th September 2012
Accepted 13th November 2012
A series of quinoline derivatives were designed and synthesized as tyrosine kinase inhibitors. Exploration of
the structure–activity relationships resulted in compounds that are potent in vitro. In addition, compound
10f was found to be a potent and selective Raf kinase inhibitor.
DOI: 10.1039/c2md20275a
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The relative mortality rate caused by cancer is still very high, but signicant challenges including drug resistance, lack of inhib-
successfully developed target-based therapies have signicantly itor selectivity, lack of inhibitor efficacy and difficulty in drug
changed cancer treatment. The Ras/Raf/MEK/ERK mitogen- target validation for particular disease settings.14 Sorafenib was
activated protein (MAP) kinase signal transduction pathway developed initially as an inhibitor of Raf kinase, but its efficacy
transmits signals from cell-surface receptor tyrosine kinases to in renal and hepatocellular cancer was later attributed to inhi-
the nucleus, which is critical to the survival, growth, and bition of VEGFR2 and PDGFR and potentially other targets.15
proliferation of cells and has been implicated in several human Therefore, we explored structural modications of Sorafenib
cancers.1–4 Raf serine/theronine protein kinases, existing in with the goal of optimizing the activity on C-Raf even further.
three isoforms, A-Raf, B-Raf, and C-Raf (Raf-1), is a crucial
According to the crystal structure of B-Raf in complex with
component of this signal transduction pathway. The three Raf Bay 43-9006, the distal 4-pyridyl ring occupies the ATP adenine
isoforms are all able to interact with Ras and activate the MAP binding pocket of the kinase domain and the lipophilic tri-
kinase pathway. Aberrant activation of the Ras/Raf/MEK/ERK uoromethyl phenyl ring at the opposite end of the molecule
pathway is commonly observed in various cancers.5–7 Thus, inserts into a hydrophobic pocket. Importantly, the urea moiety
therapeutic targeting of individual components of the Ras/Raf/ forms two hydrogen bonds with B-Raf, one with the backbone
MEK/ERK pathway has attracted much attention in the devel- aspartate, and the other with the glutamate side chain.12,16 As
opment of anti-cancer drugs.8
the residues of B-Raf that contact with Bay 43-9006 are
To date, a number of small molecule Raf kinases inhibitors conserved in C-Raf, consistent with much of the structure–
containing diverse scaffolds have received U.S. Food and Drug activity relationship of C-Raf inhibition by Bay 43-9006 and its
Administration approval as cancer treatments in the recent derivatives, the interaction mode can provide signicant infor-
past, which can be divided into ureas, urea bioisosteres, imid- mation for the conduction and validation of ligand-based and
azoles, benzamides, oxindoles and aza-stilbenes.9,10 Among structure-based studies. Systematic analysis of the crystal
them, ureas, especially bis-aryl ureas, have been most exten- structures of protein kinases led us to explore a series of quin-
sively investigated following the success of Sorafenib (tosylate oline derivatives as novel Raf kinase inhibitors with more
salt of Bay 43-9006).11 With the considerable number and variety potent and selective antitumor activities.
of ureas identied as inhibitors of C-Raf kinase, Sorafenib,
According to the research on nonclassical bioisosteric theory
initially and mainly targeting C-Raf, has nally emerged as a and structure–activity relationships (SAR) of Sorafenib, we
successful representative of ureas, approved by the FDA for the explored structural modication of Sorafenib on the basis of
treatment of advanced renal cell carcinoma (RCC) in 2005 and retaining the urea functional moiety with the goals of improving
unresectable hepatocellular carcinoma (HCC) in 2007.12,13 the antitumor activities and optimizing the activity on Raf
However, despite some motivating factors, Sorafenib faces kinase even further. Principle changes focused on the hinge
binding moiety – the linear methyl amide side group of the
pyridyl moiety was changed to quinoline derivatives to enhance
the interaction with Raf. In addition, a variety of hydrophobic
aChinese PLA Postgraduate Medical School, Beijing 100853, China. E-mail: yysymc@
163.com; Fax: +86 10 6821 4653; Tel: +86 10 6693 2289
aromatic rings were introduced into the end of the inhibitors
bInstitute of Radiation and Irradiation Medicine, Academy of Military Medical Science,
for the study of structure–activity relationships (SAR). Subse-
Beijing 100850, China. E-mail: lisx28@163.com; Fax: +86 10 6821 4653; Tel: +86 10
quently, we investigated hydrogen, uoro and chloro groups on
the central phenyl ring with a view to increasing the potency of
inhibitors (Fig. 1).
6693 2289
cDepartment of Medicinal Chemistry, Jiangxi University of Traditional Chinese
Medicine, Nanchang 330006, China. E-mail: xiening575@163.com; Fax: +86 10
6821 4653; Tel: +86 10 6693 2289
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Fig. 1 The structure and the target compounds 10a–l.
In this paper, based on Sorafenib as the lead compound, we
designed and synthesized a series of quinoline derivatives and
tested their antitumor activities on the in vitro growth of human
cancer cell lines HepG2, A549 and KCC-853 using the MTT
assay.17 Most of the compounds exhibited excellent anti-
proliferative activities against all three tested cell lines. Further,
their inhibitory activities against Raf kinase were investigated,
and the selective prole of the best compound 10f was assessed
against a panel of 20 protein kinases.
The general synthesis method for the target compounds
10a–l was devised and is shown in Scheme 1. The ethyl ester 2
can be prepared by treating 1 with thionyl chloride in ethanol.
Synthesis of compound 3 can be achieved by treating 2 with
nitric acid and sulphuric acid. Cyclization to intermediate 4 can
be performed by reuxing intermediate 3 in a mixed solvent of
ethanol and H2O with iron powder (reduced) and ammonium
chloride. Compound 5 can be synthesized by reuxing inter-
mediate 4 with 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-
4,6-dione in isopropanol. Preparation of compound 6 was ach-
ieved by heating intermediate 5 in biphenyl and diphenyl ether.
Scheme 1 Reagent and conditions: (a) SOCl2, ethanol, reflux, 88.0%; (b) nitric
acid, sulphuric acid, rt, 54.5%; (c) Fe, NH4Cl, EtOH, H2O, reflux, 64.1%; (d) iso-
propanol, 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione, reflux,
78.2%; (e) biphenyl, diphenyl ether, 240 ꢀC, 48.2%; (f) POCl3, PCl5, reflux, 83.2%;
(g) diphenyl ether, 160 ꢀC, 47.1%; (h) Fe, NH4Cl, EtOH, H2O, reflux, 80.2%; (i)
N,N-dimethylaniline, pyridine, phenyl chloroformate, 77.2%; (j) 5-nitropyridin-2-
ol, diphenyl ether, 160 ꢀC, 42.3%; (k) Fe, NH4Cl, EtOH, H2O, reflux, 78.5%; (l)
N,N-dimethylaniline, pyridine, phenyl chloroformate, 80.3%.
Compound 7 was obtained by chlorination of 6. Compound 8 comparison with Sorafenib. The best compound 10f (with IC50
can be synthesized by reacting 7 with the 4-nitrophenol deriv- values ¼ 6.356, 0.916 and 1.102 mM) exhibited superior anti-
atives in diphenyl ether. Preparation of compound 9 was
proliferative activities to Sorafenib (with IC50 values ¼ 12.669,
accomplished by reuxing 8 with powdered iron and ammo-
5.231 and 6.779 mM) in Hep G2, A549 and KCC-853. Moreover,
nium chloride in water and alcohol. Synthesis of compounds
most of the prepared compounds exhibited superior activities
10a–l was carried out using the arylamine derivatives and against A549 and KCC-853 than Hep G2. This provides valuable
phenyl chloroformate in N,N-dimethylaniline and pyridine.
information, demonstrating the selective behaviour of the
To conrm that the cellular antiproliferative activity is due to target compounds to some extent. We hypothesized that the
cellular inhibition of C-Raf kinase activity, all compounds were enhanced potency could arise from a series of contributing
tested in both a cellular C-Raf assay and cell proliferation factors, including size, electronic properties, and hydrogen-
assays. The antiproliferative activities of compounds 10a–l
against HepG2, A549 and KCC-853 together with that of Sor-
afenib as reference standard and the IC50 (mM) values
bond forming capabilities.
Diverse electron withdrawing groups (e.g. uoro, tri-
uoromethyl) and electron donating groups (e.g. methyl) were
(concentration required to achieve 50% inhibition of the introduced into the phenyl ring that connects with the urea
tumour growth) of the tested compounds on each cell line were group at the end of the inhibitors for investigation of structure–
presented in Table 1. As shown in Table 1, most of compounds activity relationships (SARs). Compounds bearing a uoro
10a–l showed better or similar antiproliferative activities group at the para position of phenyl ring exhibited reasonable
against cancer cell lines Hep G2, A549 and KCC-853 in antitumor activities against the three cell lines (10a, 10b, 10c).
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Table 1 Antiproliferative activity of quinoline derivatives 10a–l
Table 2 C-Raf kinase inhibitory activities of the target compounds 10a-l
Compound
Inhibition (nM)
Compound
Inhibition (nM)
10a
10b
10c
10d
10e
10f
37.3
25.2
22.1
14.7
15.9
8.7
10h
10i
10j
10k
10l
Sorafenib
30.3
28.1
58.5
27.2
18.7
28.5
10g
52.1
IC50 (mM)
Compound
10a
R1
H
F
Ar
HepG2
12.042
15.464
A549
5.463
7.712
KCC-853
10.587
5.642
Table 3 Inhibition percentages of compound 10f at a single dose concentration
of 10 mM over 20 protein kinases
Kinase
Inhibition (%)
Kinase
Inhibition (%)
10b
ABL1
AKT1
c-Kit
c-MET
C-Raf
CDK2
FLT3
FMS
26.2
16.5
18.7
1.5
94.6
7.8
15.3
46.9
0.6
IKKb
JAK2
PDGFRa
PTK2
PKA
PLK1
KDR
SYK
4.7
0.2
0.3
3.6
1.3
4.9
1.4
15.0
22.3
34.1
10c
10d
10e
10f
Cl
H
F
7.353
9.561
8.090
6.356
4.385
3.196
2.155
0.916
6.493
4.227
2.849
1.102
FYN
FGFR1
TIE2
VEGFR2
20.2
Cl
antiproliferative activities. In particular, addition of a uoro
group to the central phenyl ring improved antitumor activities
compared to a hydrogen substituent, whilst a chloro group
further improved potency. Unfortunately, the chloro group led
to a slight reduction in the potency of compound 10k. In this
case, changing the substituent on the end of the phenyl ring
and the central phenyl ring gave improved potency.
To test the potent antiproliferative activities, the inhibitory
activities of these compounds against C-Raf kinase were further
evaluated in vitro at a xed concentration of 1 mM by the FRET
assay,18 Sorafenib was also used as the positive control drug.
The results summarized in Table 2 show that most of the tested
compounds exhibited a reasonable inhibitory potency against
C-Raf kinase. Moreover, compound 10f (with IC50 ¼ 8.7 nM)
demonstrated more potent inhibitory activities against C-Raf
than the positive control drug Sorafenib (with IC50 values ¼ 28.5
nM). Introduction of the 3,3-dimethyl-1H-pyrrolo[3,2-g]quino-
lin-2(3H)-one moiety led to a signicant improvement in C-Raf
potency and measurable cellular inhibition.
10g
10h
10i
H
F
20.340
10.272
11.197
10.537
4.069
3.852
10.265
3.746
4.391
Cl
10j
H
F
31.687
12.005
6.473
5.026
10.073
4.179
10k
10l
Cl
8.464
5.759
5.231
5.864
6.779
The kinase selective prole of these prepared compounds
was assessed over 20 protein kinases at a single dose concen-
tration of 10 mM, using compound 10f as a representative. The
dose response data are shown in Table 3. Within this table,
Sorafenib
12.669
Systematic evaluation of additional substituent groups at the which includes a range of protein kinases targets of therapeutic
meta position indicted that triuoromethyl led to higher anti- relevance, compound 10f was found to demonstrate excellent
tumor activities than other substituents, suggesting the selectivity over the majority of the targets. Based on the known
importance of the substituent size at the aromatic ring (10d, pathway biology these particular off-target activities are not
10e, 10f). However, introduction of electron donating groups or expected to contribute to inhibition of C-Raf mediated
replacing with an isoxazolyl ring led to
a decrease in signaling.
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In conclusion, the quinoline derivatives presented here are 11 T. B. Lowinger, B. Riedl, J. Dumas and R. A. Smish, Curr.
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17 Cell assay: Hep G2, A549 and KCC-853 were cultured in
RPMI-1640 medium, supplemented with 10% fetal calf
serum (FCS), and maintained in a 5% CO2, 95% humidity
atmosphere at 37 ꢀC. In 96-well plates were seeded 5.0 ꢁ
103 cells per well and incubated for 24 h. The cells were
Acknowledgements
This work was nancially supported by the National Natural
Science Foundation of China (Grant no.30973016).
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then incubated for another 72
h
with various
concentrations of compounds 10a–l. Subsequently, 20 mL
of fresh MTT solution (5 mg mlꢂ1) was added to each well
ꢀ
and incubated with cells at 37 Cfor an additional 4 h. The
supernatant was carefully removed from each well and 100
mL of DMSO was added to each well to dissolve the
formazan crystals which were formed by the cellular
reduction of MTT. Aer mixing with a mechanical plate
mixer, the absorbance of each well was measured by a
plate reader at a test wavelength of 570 nm. IC50 data were
calculated by linear regression analysis as described above.
18 FRET assay: Active C-Raf and inactive Mek were diluted
together with kinase dilution buffer (25 mM Tris, pH 7.5,
0.02 mM EGTA, 0.66 mg mLꢂ1 myelin basic protein, 1 mM
DTT, 0.1 mg mLꢂ1 BSA) to 4 and 20 mg mLꢂ1, respectively,
and 20 mL of this enzyme-substrate mixture was added to
each well of a 96-well plate. Test compound (5 mL) of
desired concentration was added to the mixture and the
kinase reaction was initiated by adding 25 mL of 10 mM g-
[33P] ATP (specic activity approx. 500 cpm pmolꢂ1) for
incubation at 30 ꢀC for 20 min. The reaction was spotted
onto a phosphocellulose mat, washed with 1% phosphoric
acid solution, and the radioactivity counted in the
presence of scintillation uid in a scintillation counter.
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