1,4-Dihydropyrazolo[4,3-d]imidazole phenyl derivatives: A novel type II Raf kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.04.039

The synthesis of a novel series of 1,4-dihydropyrazolo[4,3-d]imidazole phenyl derivatives 1a-b, 2a-v and their antiproliferative activities against A375P and WM3629 human melanoma cell line were described. Most compounds showed competitive antiproliferative activities to sorafenib, the reference standard. Among them, pyrazoloimidazole phenyl urea compounds 2a, 2d, 2g, 2i, 2t exhibited potent activities on WM3629 cell lines (IC₅₀ = 0.56-0.86 μM). Especially, 2t was found to be a potent and selective C-Raf inhibitor, showing a possibility as melanoma therapeutics.

Full text of DOI:10.1016/j.bmcl.2010.04.039

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3805–3808
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1,4-Dihydropyrazolo[4,3-d]imidazole phenyl derivatives: A novel type II Raf
kinase inhibitors
Hana Yu, Yunkyung Jung, Hwan Kim, Junghun Lee, Chang-Hyun Oh, Kyung Ho Yoo, Taebo Sim,
*
Jung-Mi Hah
Life/Health Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a novel series of 1,4-dihydropyrazolo[4,3-d]imidazole phenyl derivatives 1a–b, 2a–v and
their antiproliferative activities against A375P and WM3629 human melanoma cell line were described.
Most compounds showed competitive antiproliferative activities to sorafenib, the reference standard.
Among them, pyrazoloimidazole phenyl urea compounds 2a, 2d, 2g, 2i, 2t exhibited potent activities
Received 20 January 2010
Revised 29 March 2010
Accepted 10 April 2010
Available online 18 April 2010
on WM3629 cell lines (IC50 = 0.56–0.86
lM). Especially, 2t was found to be a potent and selective
C-Raf inhibitor, showing a possibility as melanoma therapeutics.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
1,4-Dihydropyrazolo[4,3-d]imidazole
phenyl derivatives
Antiproliferative activity
Melanoma cell line
Selectivity
The understanding of molecular mechanism for cancer has been
dramatically progressed over the last decade. Since it is known that
cancer is mainly caused by abnormal increased expression or acti-
vation of oncogenes and the corresponding proteins, it is rational
to target a protein that is a key component of the oncogenic path-
way as protein-targeted cancer therapies.
The Ras-Raf-Mek-Erk signal transduction cascade is well-
known protein pathway that regulates cell growth, differentiation
and proliferation.1 Like other oncogenic pathway, overexpression
or mutation of protein members of this pathway is a driving factor
for numerous cancers.2–4
Especially mutations of the Raf protein have been found in
approximately 7% of human cancers5,6 with particulary high fre-
quency in melanoma (50–70%), ovarian (35%), thyroid (30%), and
colorectal (10%) cancers. There have been 40 reported B-Raf muta-
tions, and the most frequent mutation of Raf protein (>85%) is va-
line substitution by glutamic acid at position 600 (V600E), which
shows a 500-fold increase in catalytic activity, providing cancer
cells with both proliferation and survival signals.7 Therefore,
B-Raf V600E is a high-interest therapeutic target for the treatment
of human cancers. However, there is now a growing consensus
being made that C-Raf is also associated significantly with disease
progression and cell proliferation in a subset of melanoma.8–10
A number of inhibitors, designed to target Raf family proteins
(A, B, and C) are known. These include benzylidene oxindoles,11
PLX4720,12 and sorafenib13–16 (Fig. 1: Launched for RCC). The
bi-aryl urea sorafenib is a potent inhibitor of preactivated C-Raf
and B-Raf as well as oncogenically activated B-Raf kinases
(V600E B-Raf: IC50 = 43 nM).
Crystal structure of V600E B-Raf kinase domains in complex
with sorafenib showed that this inhibitor is namely type II kinase
inhibitor, having the activation segment in an inactive conforma-
tion.17 Most of known kinase inhibitors belong to type I inhibitors,
which bind to the ATP binding site through the formation of hydro-
gen bonds to the kinase ‘hinge’ residues and through hydrophobic
interactions in and around the region occupied by the adenine ring
of ATP. However, type II inhibitors typically use the ATP binding
site, but they also exploit unique hydrogen bonding and hydropho-
bic interactions made possible by the DFG residues of the activa-
tion loop being folded away in an inactive conformation.
R2
O
Cl
O
H
N
O
R3
N
N
H
N
H
N
H
CF3
N
N
N
N
H
O
R1
Imidazolopyrazole
Sorafenib
R1 = H, Bn, PMB
R
R
2 = H, Me
3 = -NHAr, Ar, HetAr
* Corresponding author. Tel.: +82 2 958 6438; fax: +82 2 958 5189.
E-mail address: jhah@kist.re.kr (J.-M. Hah).
Figure 1. Structures of sorafenib and imidazolopyrazole derivatives.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.039

3806
H. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3805–3808
R2
N
H
N
H
N
H
N
R4
N
i, ii
NO2
O
N
N
N
N
N
R1
R1
2b,g,h and j
6a ; R1 = Bn
6b ; R1 = PMB
Scheme 2. Reagents and conditions: (i) KOtBu, MeI THF, rt; (ii) R4NCO, THF, rt.
mation, (ii) introducing methyl group in nitrogen of imidazole ring
or/and in the middle phenyl moiety looking for a proper conforma-
tion, and (iii) introducing the various direction of connectivity (m-,
p-) and changing the spacer to the aromatic tail part to understand
the relative spatial arrangements for the two aromatic rings.
The general synthesis of 1,4-dihydropyrazolo[4,3-d]imidazole
derivatives is shown in Scheme 1. The core pyrazole moiety with
two nitrogen functionalities was made from protected hydrazine
and 3-methoxyacrylonitrile in one pot process based on the litera-
ture.18 Then, the nitroso group on pyrazole was reduced to give
diamine19 for subsequent imidazole cyclization. The diaminopy-
razole 5 was reacted with corresponding nitro benzoic acid in
POCl3 to provide 1,4-dihydropyrazolo[4,3-d]imidazole skeleton
6.20 The resulting nitro group in 6 was reduced to amino group
and coupled with various aromatic acids under EDCI/HOBt condi-
tions or directly aromatic isocyanate to give amide (1a–b) or urea
(2a–v) analogues. To investigate an optimal conformation of mid-
dle phenyl ring, we synthesized several compounds with addi-
tional methyl group on imidazole nitrogen. N-methylated
analogues at R3 position (2b, 2g, 2h, 2j) were synthesized as shown
in Scheme 2. The methylation was performed just after the second
cyclization and the rest of reaction was followed to yield the de-
sired analogues.
Figure 2. Docking structure of designed 1,4-dihydropyrazolo [4,3-d] imidazole
(bold, orange) overlayed with sorafenib (thin, cyan) in V600E B-Raf.
The ATP binding and subsequent kinase reaction is prevented in
this inactive kinase conformation, and the binding feature of
sorafenib to B-Raf is interesting enough to prompt us to design a
novel scaffold for Raf inhibitor. Since the amino acids surrounding
this secondary hydrophobic pocket are less conserved relative to
those in the ATP binding pocket, it has been proposed that it
may be easier to achieve kinase selectivity with type II inhibitors.
Type II inhibitors typically have potent cellular activity, pre-
sumably because they recognize (or induce) the DFG-out confor-
mation, which has a lower affinity for ATP than for the active
kinase. Here we wish to report the initial SAR studies of a novel
scaffold, 1,4-dihydropyrazolo[4,3-d]imidazole derivatives as type
II inhibitor for Raf.
To design a new chemical entity (NCE), we need to complement
the size, shape, and electronic properties of molecular targets.
Designing a new type II inhibitor for V600E B-Raf, the hinge binder
was still considered to be the primary pharmacophore, and the
imidazolopyrazole bicyclic ring has attracted considerable atten-
tion of ours as a new hinge binder in the terms of novelty, and
chemical modification accessibility (Fig. 2).
Table 1 shows the antiproliferative activitiy21,22 (GI50 values) of
those analogues 1a, 2a–h against A375P21 and WM362922 human
melanoma cell line together with that of sorafenib as a reference
compound. The initial compounds with 3-chloro-5-trifluoromethyl
benzoyl group as a tail (1a, 2a–h) showed competitive antiprolifer-
ative activities against A375P and WM3629 human melanoma cell
line. While an amide linkered compound 1a showed a competitive
activity, urea-linker compounds 2a, 2g exhibited better potency
than reference sorafenib. Especially, the p-directed urea 2g with
extra R3 methyl group possessed the best potency on WM3629 cell
line. However, the free pyrazole or p-methoxybenzyl moiety on
The structure of this series comprises 1,4-dihydropyrazolo[4,3-
d]imidazole part, the middle phenyl ring moiety and various aro-
matic tail part connected by amide or urea linkage. Specifically,
we modified the structures by (i) replacing hydrogen in pyrazole
N-2 position with aromatic rings to get extended structural infor-
R2
H
NH2
NH2
NO
NH2
OMe
N
i
ii
iii
NO2
N
N
N
N
N
N
N
CN
R1
R1
R1
4a ; R1 = Bn
5a ; R1 = Bn
6a ; R1 = Bn
3
4b ; R1 = PMB
5b ; R1 = PMB
6b ; R1 = PMB
R2
R2
R2
H
N
H
N
R4
H
H
N
H
N
H
N
iv
v
N
R4
NH2
O
N
O
N
N
N
N
N
N
N
N
R1
R1
R1
7a ; R1 = Bn
1a ~1c
2a ~ 2v
7b ; R1 = PMB
Scheme 1. Reagents and conditions: (i) R1HNNH2, NaNO2, EtOH, 50 °C; (ii) SnCl2Á2H2O, EtOH, 80 °C; (iii) 2-R2-4-NO2–C6H3–CO2H, POCl3, DMAP reflux; (iv) SnCl2Á2H2O, EtOH,
80 °C; (v) R4CO2H, EDCI, TEA, HOBt, DMF or R4NCO, THF.

H. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3805–3808
3807
Table 1
Antiproliferative activity of pyrazoloimidazole phenyl amide/urea derivatives 1a–c, 2a–v
R2
R2
N
H
R3
N
R4
H
N
H
N
R3
N
N
R4
O
O
N
N
N
N
2a~v
1a-c
N
R1
R1
R1
R2
R3
R4
GI50 (lM)
a
Compd
Orientation
A375P
WM3629
1a24
2a
m-Phenyl
m-Phenyl
Bn
Bn
Me
Me
H
H
2.06
3.79
1.14
0.85
Cl
2b
2c
2d
2e
2f
m-Phenyl
m-Phenyl
p-Phenyl
p-Phenyl
m-Phenyl
p-Phenyl
p-Phenyl
PMB
H
Bn
H
Bn
Me
Me
H
Me
Me
Me
Me
H
H
H
H
Me
Me
Me
>100
>100
6.65
>100
56.3
4.16
51.5
>100
>100
0.75
14.4
5.85
0.56
3.77
CF3
2g
2h
Bn
PMB
1b
2i
2j
2k
2l
m-Phenyl
m-Phenyl
m-Phenyl
p-Phenyl
p-Phenyl
H
Bn
Bn
H
Me
Me
Me
Me
Me
H
H
Me
H
NA
4.29
>100
>100
>100
NA
0.72
>100
NA
>100
O
N
PMB
H
CF3
2m
2n
2o
m-Phenyl
m-Phenyl
p-Phenyl
PMB
Bn
Bn
Me
Me
H
H
H
H
>100
7.73
51.7
>100
1.29
0.98
F
F
F
2p
2q
m-Phenyl
p-Phenyl
PMB
Bn
Me
H
H
H
15.3
>100
1.35
42.8
Cl
Cl
2r
2s
m-Phenyl
p-Phenyl
H
H
Me
Me
H
H
NA
NA
NA
NA
Cl
N
Cl
N
2t24
p-Phenyl
H
Me
H
2.24
0.86
CF3
N
2u
2v
p-Phenyl
p-Phenyl
H
PMB
Me
Me
H
H
NA
NA
NA
NA
S
Sorafenib
5.58
0.65
a
Values are average of two or more experiments; NA: not active.
Table 2
substitution of fluorine in benzoic acid tail (2n) was tolerated but
the chlorine substitution (2r, 2s) was not, implying the secondary
hydrophobic pocket is sensitive to the conformation of tail group.
Some of pyrazoloimidazole phenyl urea derivatives (2i, 2t) showed
superior antiproliferative activities to sorafenib in both cell lines
Enzymatic activities of selected compounds
Compd
IC50 (nM) on C-Raf
1a
2a
2d
2g
2i
146
76
370
NA
156
839
50
(4.29 lM, 720 nM and 2.68 lM, 860 nM, respectively). Especially,
it was surprising that the free pyrazoloimidazole urea compound
2t showed the best potency toward A375P cell line in these series.
However, the flat bicyclic tail group such as methyl benzothiazole
(2u, 2v) did not seem to be proper for secondary hydrophobic
pocket.
For several compounds with competitive cellular potency, C-Raf
inhibitory activities (IC50) were determined as shown in Table 2.23
Most compounds tested are potent C-Raf inhibitors confirming that
the primary cell line test was valuable. Assuming they are all type II
inhibitors and the contact of hydrophobic tail group is important, it
seems urea-linkered compounds seems more effective (1a < 2a)
2p
2t
pyrazole ring with this tail did not seem to be active for cellular
activity (2c, 2e).
Further, we continued the syntheses of more compounds with
various hydrophobic tail groups, focusing on urea-linkered deriva-
tives. The analogues with m-orientation of middle benzene ring
were preferred in case of urea derivatives (2i >> 2l, 2n > 2o,
2p > 2q) than p-orientation. It was interesting to see that the ortho

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H. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3805–3808
Table 3
References and notes
Percentages of enzymatic inhibitions by compound 2t (10
kinases
l
M) on selected 30 Protein
% Inhibition
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16. Lee, J. T.; McCubrey, J. A. Curr. Opin. Invest. Drugs 2003, 4, 757.
17. Liu, Y.; Gray, N. S. Nat. Chem. Biol. 2006, 2, 358.
Kinase
Abl(h)
ALK(h)
AMPK(r)
Aurora-A(h)
Axl(h)
Bmx(h)
BTK(h)
CDK2/cyclinE(h)
CDK5/p35(h)
CK2(h)
37
61
5
10
6
16
10
33
0
8
0
cKit(h)
C-Raf(h)
cSRC(h)
EphA1(h)
FGFR1(h)
Flt3(h)
Fms(h)
IGF-1R(h)
IKKb(h)
IR(h)
JAK2(h)
KDR(h)
Met(h)
p70S6 K(h)
96
20
30
26
53
42
35
16
0
0
8
0
4
18. Fukuda, Y.; Shikita, S.; Murakami, T.; Oku, M.; Ota, H.; Sone, M.
WO2003062207A1.
PDGFR
Plk1(h)
Syk(h)
Tie2(h)
TrkB(h)
a
(h)
0
8
16
82
62
19. Holschbach, M. H.; Wutz, W.; Olsson, R. A. Tetrahedron Lett. 2003, 44, 41.
20. Barralough, P.; Black, J. W.; Cambridge, D.; Firmin, D.; Gerskowitch, V. P.; Glen,
R. C.; Giles, H.; Gillam, J. M., et al Arch. Pharm. 1992, 325, 225.
21. A375P cells were purchased from American Type Culture Collection (ATCC,
Rockville, MD, US) and maintained in DMEM medium (Welgene, Daegu, Korea)
supplemented with 10% FBS (Welgene) and 1% penicillin/streptomycin
(Welgene) in a humidified atmosphere with 5% CO2 at 37 °C. A375P cells
were taken from culture substrate with 0.05% trypsin–0.02% EDTA and plated
at a density of 5 Â 103 cells/well in 96 well plates and then incubated at 37 °C
for 24 h in a humidified atmosphere with 5% CO2 prior to treatment of various
concentrations (threefold serial dilution, 12 points) of test compounds. The
A357P cell viability was assessed by the conventional 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. MTT assays
were carried out with CellTiter 96Ò (Promega) according to the manufacturer’s
instructions. The absorbance at 590 nm was recorded using EnVision 2103
(Perkin Elmer; Boston, MA, US). The IC50 was calculated using GraphPad Prism
4.0 software.
than the amide. When the tail group becomes bulkier, then the p-
orientation of middle phenyl ring seems preferred as C-Raf inhibi-
tor (2a < 2t). The best compound 2t has showed an IC50 value of
50 nM, while the IC50 value for the GW5074 was 2.32 nM.
We further tried kinase panel screening of the best compound
2t over 30 different kinases at a single dose concentration of
10 l
M23 (Table 3) and it was revealed that the compound has a de-
cent selectivity profile. While this compound has inhibitory activ-
ity of 96% on C-Raf at this concentration, the inhibition exerted in
most other kinases tested in activity was below 50%.
22. WM3629 cell line was supplied from Dr. Merlyn lab at Wistar Institute
(Philadelphia, PA, US) and maintained in Tu2% medium according to the
literature.10 The procedure for GI50 determination (MTT assay) was the same as
in A375P cell line.
23. We used Millipore KinaseProfilerTM service (www.millipore.com) for
screening of 2t and IC50ProfilerExpress for IC50 measurement. Assay protocol:
In conclusion, a series of novel scaffold, 1,4-dihydropyrazol-
o[4,3-d]imidazole phenyl derivatives based on the structural fea-
tures of sorafenib has been synthesized and showed potent
antiproliferative activities against A375P human and WM3629
melanoma cell line. Furthermore, one of the best compound 2t in
this series has been confirmed as a potent and selective C-Raf ki-
nase inhibitor. These results suggest that the more development
of pyrazoloimidazole phenyl scaffold is a very promising way for
new therapeutics for melanoma, especially non-V600E classes.
In a final reaction volume of 25
25 mM Tris pH 7.5, 0.02 mM EGTA, 0.66 mg/mL myelin basic protein,
10 mM Mg acetate and
33P-ATP] (specific activity approx. 500 cpm/pmol,
concentration as required). The reaction is initiated by the addition of the Mg–
ATP mix. After incubation for 40 min at room temperature, the reaction is
lL, C-Raf (h) (5–10 mU) is incubated with
[
stopped by the addition of
5 lL of a 3% phosphoric acid solution. Ten
microliters of the reaction is then spotted onto a P30 filter mat and washed
three times for 5 min in 75 mM phosphoric acid and once in methanol prior to
drying and scintillation counting.
24. Selected data 1a: 1H NMR (400 MHz, DMSO-d6) d 12.17 (1H, s), 10.61 (1H, s),
8.41 (1H, d), 8.27 (1H, dd, J = 8.41 Hz, 1.92 Hz), 8.03 (1H, d, J = 2.22 Hz), 7.93
(1H, d, J = 8.39 Hz), 7.79 (1H, dd, J = 8.30 Hz, 2.26 Hz), 7.43 (1H, s), 7.35 (1H, d,
J = 8.22 Hz), 7.32–7.24 (5H, m), 5.42 (2H, s), 3.37 (3H, s); MS m/z 481 (M+H)+.
2t: 1H NMR (400 MHz, DMSO-d6) d 12.14 (1H, s), 9.24 (1H, s), 8.24 (1H, s), 8.14
(1H, s), 7.71 (1H, d, J = 0.99 Hz), 7.66 (1H, d, J = 7.11 Hz), 7.60 (1H, d, J = 8.4 Hz),
7.51 (1H, s), 7.41 (1H, d, J = 8.32 Hz), 7.29 (1H, s), 3.3 (3H, s), 2.49 (3H, s); MS m/
z 510 (M+H)+.
Acknowledgments
This research was supported by Korea Institute of Science and
Technology and Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by the Minis-
try of Education, Science and Technology (2009-0087992; J.M.H.).