Design, synthesis and biological evaluation of benzyl 2-(1H-imidazole-1-yl) pyrimidine analogues as selective and potent Raf inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.05.030

The synthesis of a novel series of (4-aminobenzyl/benzoyl)-1H-imidazol-1-yl pyrimidin-2-yl derivatives 9, 10, 18, 19 and their in vitro antiproliferative activities against the A375P human melanoma cell line and the U937 human leukemic monocyte lymphoma cell line are described. Potent antiproliferative effects were found from 9l, 9s and 10c; 10c was found to be a highly potent and selective BRAF V600E and CRAF inhibitor (IC₅₀ = 38.3 nM and 8.79 nM).

Full text of DOI:10.1016/j.bmcl.2014.05.030

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of benzyl
2-(1H-imidazole-1-yl) pyrimidine analogues as selective
and potent Raf inhibitors
Minjung Kim a, Junghun Lee a, Kyungjin Jung a, Hyangmi Kim a, Waqar Aman a, Jae-Sang Ryu b,
Jung-Mi Hah a,
⇑
a Department of Pharmacy, College of Pharmacy & Institute of Pharmaceutical Science and Technology, Hanyang University, 55 Hanyangdaehak-ro, Sangnok-gu, Ansan,
Kyeonggi-do 426-791, Republic of Korea
b College of Pharmacy & Graduate School of Pharmaceutical Sciences, Global Top 5 Research Program, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemun-Gu, Seoul
120-750, 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 (4-aminobenzyl/benzoyl)-1H-imidazol-1-yl pyrimidin-2-yl derivatives
9, 10, 18, 19 and their in vitro antiproliferative activities against the A375P human melanoma cell line
and the U937 human leukemic monocyte lymphoma cell line are described. Potent antiproliferative
effects were found from 9l, 9s and 10c; 10c was found to be a highly potent and selective BRAF V600E
and CRAF inhibitor (IC50 = 38.3 nM and 8.79 nM).
Received 11 April 2014
Revised 8 May 2014
Accepted 10 May 2014
Available online xxxx
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
(4-Aminobenzyl)-1H-imidazol-1-yl
pyrimidin-2-yl derivatives
Antiproliferative activity
Melanoma
BRAF V600E
CRAF
Selectivity
Despite the profound progress in cancer research over the years,
it is still the second leading cause of death worldwide. Extensive
studies of the molecular mechanisms of cancer have revealed
many specific molecular targets for its therapy. In fact, targeted
cancer therapy has been very successful, especially in the case of
kinase inhibitors. Protein kinases are involved in many kinds of cell
signaling pathways including cell cycle regulation, apoptosis, and
proliferation; the dysregulation of specific protein kinases has been
implicated in several cancers.
N
RHN
RHN
O
N
O
N
N
H
R'
R'
N
N
NH
RHN
N
N
N
N
O
O
N
N
Previous report
N
R'
H
N
In the case of melanoma, the mitogen-activated protein kinase
(MAPK; RAS-RAF-MEK-ERK)1–4 and the phosphoinositide 3-
kinase-AKT pathways are upregulated;5,6 and alteration of signal-
ing through both pathways plays a major role in melanoma
progression. Since the findings that around 70% of melanomas have
the V600E mutation,7 there have been significant amounts of small
molecule inhibitors developed, which target BRAF V600E. The
recent success of Vemurafenib shows the importance of selective
BRAF V600E inhibition. Additionally, there is a significant portion
Present report
Figure 1. Incorporation of methylene and carbonyl groups into phenyl-1H-imida-
zol-1-yl pyrimidin-2-yl derivatives.15
of melanomas (>33%) that do not contain BRAF V600E mutations,
which will require alternative therapeutic targets.8,9 One gene,
CRAF (or RAF1), which is closely related to BRAF, is involved in
abnormal proliferation in melanoma. Similar to BRAF, CRAF is
involved in activating a MAPK signal transduction system;10 unlike
⇑
Corresponding author. Tel.: +82 31 400 5803; fax: +82 31 400 5958.
E-mail address: jhah@hanyang.ac.kr (J.-M. Hah).
http://dx.doi.org/10.1016/j.bmcl.2014.05.030
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kim, M.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.030

2
M. Kim et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
the development of potent and selective CRAF inhibitors, as well
as BRAF V600E inhibitors, would be ideal for melanoma treatment.
The known protein kinase inhibitors can be divided into type I
and type II, depending on the conformational state of the kinase
with the inhibitors bound. Type I inhibitors bind to the ATP-bind-
ing site through the formation of hydrogen bonds at the hinge
region; type II inhibitors bind to the ATP-binding site, but also
occupy the unique secondary hydrophobic pocket in the inactive
conformation of the kinase domain. In this respect, the approach
toward type II inhibitors could be stronger for the selective kinase
inhibitor.
In this respect, the approach toward type II inhibitors could be
stronger for the selective kinase inhibitor (see Fig. 1).
In our previous studies,15 we synthesized a novel series of
pyrimidine-4-yl-1H-imidazole-2-yl derivatives that showed out-
standing antiproliferative activities against human melanoma cell
lines. Further assessment of the inhibitory activity showed that
the most potent compound on A375P was a very potent BRAF
V600E inhibitor. This same compound also had good potency on
wild-type BRAF, therefore the selectivity was only three-fold. For
that reason, we compared the active site of wild-type BRAF and
V600E BRAF through molecular docking studies with known struc-
tures, and found that addition of methylene/carbonyl moieties to
the imidazole derivatives could boost potency and selectivity. As
shown in the Figure 2, the methylene group between pyrimidine
Figure 2. Docking structures of the designed amino-1H-pyrazole amide scaffold
(bold, green) in BRAF V600E.16
BRAF, CRAF controls lower proteins, such as MST2(STK3) and ASK1
(MAP3K5), independent of the MAPK signal transduction system.11
In addition, CRAF affects mitochondria directly, thereby control-
ling the inhibition of apoptosis, which is achieved via phosphoryla-
tion of BAD through direct coupling with BCL2.12 CRAF seems to be
related to the mutants of NRAS.13 A recent study, which shows that
BRAF can directly phosphorylate CRAF,14 emphasizes the value of
CRAF as a target protein for melanoma therapeutics. Therefore,
NC
EtO
HN
NO2
MeO
MeO
HN
NO2
NO2
NO2
NH
NH
N
i
ii
iii
3
4
1
2
N
N
N
S
MeO2S
HN
HO
N
N
N
N
N
NO2
N
NO2
NO2
N
N
N
iv
v
vi
6
7
5
N
HN
O
HO
N
N
N
N
N
9a - t
R1
H
N
HN
HO
N
N
N
8
NH2
vii
viii
HN
O
HO
N
N
10a - d
R2
N
H
N
N
H
Scheme 1. Reagents and reaction conditions: (i) HCl(g), EtOH, MC, À10 °C, 3 h; (ii) 2,2-dimethoxyethanamine, EtOH 40 °C, 4 h; (iii) 6N HCl in H2O–MeOH, 90 °C, 1 h; (iv) 4-
chloro-2-(methylthio)pyrimidine, Pd(OAc)2, BINAP, K3PO4, toluene, 130 °C; (v) 70% m-CPBA, CH2Cl2, rt, 2 h; (iv) (S)-1-aminopropan-2-ol, THF, 60 °C, 6 h; (vii) Raney nickel, H2,
MeOH; (viii) R1CO2H, EDCI, TEA, HOBt, DMF or R2NCO, THF, rt.
O
O
Cl
NO2
N
N
O
NO2
iv
N
NO2
H
N
O
Cl
N
S
S
N
N
MeO2S
N
N
N
HN
NO2
N
N
i, ii
iii
N
14
15
13
11
O
O
N
N
H
R1'
18a-18k
19a-19d
N
H
N
N
N
O
N
OH
NH2
N
H
N
N
N
v, vi
vii
OH
O
O
N
R2'
N
16
N
H
H
N
N
N
N
H
OH
Scheme 2. Reagent and reaction conditions: (i) Pyridine, TEA, 0 °C?rt; (ii) 10 N NaOH, 1 h; (iii) 4-chloro-2-(methylthio) pyrimidine, NaH, DMF 100 °C, 3 h; (iv) 70% mCPBA,
MC, rt, 2 h; (v) (S)-1-aminopropan-2-ol, THF, 60 °C, 6 h; (vi) Fe, HCl, EtOHÁH2O, 1 h; (vii) R1CO2H, HATU, TEA, DMF or R2NCO, THF.
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M. Kim et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 1
Antiproliferative activities of aminobenzyl, aminobenzoyl-1H-imidazol-1-yl pyrimidine analogues on cancer cell lines.
N
O
O
N
HN
O
9a ~ 9t
N
H
HO
R1'
N
18a-18k
19a-19d
10a ~ 10d
N
N
H
N
N
N
H
R1
N
N
OH
Compd Substitution R1/R2
A375P (GI50
,
U937 (GI50
,
Compd Substitution R1/R2
A375P (GI50
,
U937 (GI50
,
l
M)
lM)
l
M)
lM)
Cl
N
N
9a
3-Amide
12.3
>30
10a
3-Urea
19.2
1.73
Cl
Cl
CF3
9b
9c
3-Amide
3-Amide
NA
NA
16.8
>30
10b
10c
3-Urea
3-Urea
26.2
1.82
15.3
2.73
CF3
Cl
N
N
N
N
9d
3-Amide
7.45
>30
10d
3-Urea
>30
>30
CF3
F
CF3
N
CF3
9e
9f
3-Amide
3-Amide
6.39
10.2
25.8
>30
18a
18b
3-Amide
3-Amide
>30
3.78
2.55
N
O
9.39
OMe
Cl
N
CF3
9g
9h
3-Amide
3-Amide
NA
23.8
>30
18c
18d
3-Amide
3-Amide
NA
4.9
NA
N
O
N
N
>30
27.8
N
H
CF3
9i
9j
9k
9l
3-Amide
3-Amide
3-Amide
3-Amide
>30
8.92
11.2
0.6
18.5
NA
18e
18f
3-Amide
3-Amide
4-Amide
4-Amide
>30
>30
>30
>30
>30
>30
>30
>30
N
Cl
Cl
H
N
10.0
1.68
18g
18h
Cl
Cl
CF3
N
9m
9n
3-Amide
3-Amide
NA
NA
NA
NA
18i
18j
4-Amide
4-Amide
7.07
5.92
NA
NA
O
N
N
N
O
CF3
CN
CF3
S
9o
9p
3-Amide
3-Amide
NA
NA
10.0
23.6
18k
19a
4-Amide
3-Urea
>30
>30
>30
Cl
Cl
O
14.7
Cl
OH
9q
3-Amide
3.97
3.24
19b
3-Urea
>30
>30
Cl
Cl
Cl
(continued on next page)
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4
M. Kim et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 1 (continued)
Compd Substitution R1/R2
A375P (GI50
,
U937 (GI50
M)
,
Compd Substitution R1/R2
A375P (GI50
,
U937 (GI50
M)
,
lM)
l
l
M)
l
CF3
Cl
CF3
O
N
9r
9s
3-Amide
3-Amide
NA
3.3
NA
19c
19d
4-Urea
4-Urea
>30
>30
7.45
11.6
CF3
Cl
Cl
6.83
Cl
CF3
9t
3-Amide
6.6
9.7
Sorafenib
3.4
2.74
N
O
⁄
Maximum potency showed less than 60% of total growth.18
and imidazole in the two newly designed analogues increased the
flexibility, compared to the previous series of compounds, helping
the tail moiety to approach the secondary hydrophobic pocket.
Among the new derivatives, we were able to find a new potent
and selective BRAF V600E kinase and CRAF kinase inhibitor.
The general syntheses of the imidazoyl methylene derivatives
(9a–t, 10a–d) are shown in Scheme 1. The synthesis of the nitro-
benzyl imidazole moiety (4) was started from 2-(4-nitrophenyl)
acetonitrile (1). Under dry hydrogen chloride gas, ethanol was
reacted to yield ethyl 2-(4-nitrophenyl)acetimidate (2), which
was then substituted with 2,2-dimethoxyethanamine to form 3,
and strong acidic cyclization was accomplished to give the nitro-
benzyl imidazole (4). 4-Chloro-2-(methylthio) pyrimidine was
then introduced onto 4 in a modified Buchwald condition and
the methylthio group was oxidized, using mCPBA, to methyl sulf-
oxide (6) for further substitution with (S)-1-aminopropan-2-ol
(7). The nitro group on the benzyl ring was then reduced to give
corresponding amines (8) and was coupled with diverse aromatic
acids under EDCI/HOBt conditions to obtain amide (9a–9t), and
directly with aromatic isocyanate to obtain urea (10a–10d)
derivatives.
The imidazole carbonyl derivatives were synthesized according
to the sequence of reactions shown in Scheme 2. Imidazole (11)
was directly coupled with 3 or 4-nitro benzoyl chloride and then
rearranged in a basic condition.17 Introduction of pyrimidyl moie-
ties and the rest of the syntheses are completed in a similar way as
in Scheme 1.
The antiproliferative activities of the elongated pyrimidinyl
imidazole derivatives (9a–t, 10a–d; 18a–k, 19a–d) against the
A375P human melanoma cell line and the U937 human leukemic
monocyte lymphoma cell line were examined using Sorafenib as
a reference standard.18
All of the pyrimidin-4-yl-1H-imidazole-2-carbonyl derivatives
(18a–19d) showed poor potencies toward both cell lines; the most
potent compound on the A375P cell lines in this series was 18b.
We tried kinase panel screening for compound 10c over 32 dif-
ferent kinases at a single-dose concentration of 10 l
M19 (Table 2,
done in duplicate). It was revealed that the compound was, indeed,
a selective RAF inhibitor with a excellent selectivity profile. This
compound has an inhibitory activity of 99% on BRAF, BRAF V600E
and CRAF at this concentration; the inhibition activity was less
than 30% in most other kinase tested except c-Kit (KIT), and p38a
(MAPK14).
We further investigated the enzymatic activities of 10c and two
amide derivatives, GW5074 and SB202190, on wild-type BRAF,
BRAF V600E, CRAF, and p38a kinase. Indeed, we found that com-
pared to the urea derivatives, 10c was a very selective BRAF
V600E and CRAF inhibitor, and is a possible therapeutic for
melanoma (Table 3).
Table 2
Percentages of enzymatic inhibition exerted by 10c (10
l
M) on 30 selected protein
% inhibition
kinases19
Kinase
ABL1
AKT1
ALK
Aurora A
BRAF
6.9
12.9
4.9
17.6
99.9
96.1
0
BRAF (V599E)
BTK
c-Kit
c-MET
c-Src
CDK1/cyclin B
EGFR
63.8
17.6
2.1
34.8
0
The results are shown in Table 1. Generally, the antiproliferative
activity toward the A375P cell line was stronger than toward the
U937 cell line. The potency range of the benzyl/benzoyl imidazole
derivatives depended on the substitution pattern of the middle
phenyl ring and more importantly, on the tail group (R1/R2), imply-
ing that the activity is more sensitive to the secondary hydropho-
bic pocket.
ERK1
0
FAK/PTK2
FGFR3
FLT3
FMS
GSK3b
IGF1R
1.2
3.5
68.4
33.6
34.5
0
JAK3
0
Out of all of the derivatives, 9l, 9s, and 10c showed competitive
potencies toward both the A375P and the U937 cancer cell lines
(Table 1). Compounds 9l and 10c showed better potencies than
Sorafenib, and compound 9s demonstrated a similar potency as
Sorafenib against A375P, showing a micromolar scale IC50 value.
Except 10c, the arylurea derivatives (10a–10d) did not show good
antiproliferative activities against either of the cell lines. In the
case of this scaffold, the best activity was encountered with
compounds 9l and 10c. This hydrophobic–trifluoromethyl chloro
benzoyl moiety was preferred in both compounds and seemed to
be the most optimal for activity in this series of compounds.
JNK3
LCK
LYN
MEK1
mTOR/FRAP1
P38a/MAPK14
PKA
PLK1
RAF1
0
5.8
8.1
0
0
94.3
0
9.9
98.7
0.42
0.73
0
RON/MST1R
ROS/ROS1
SYK
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M. Kim et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
Table 3
3. Wellbrock, C.; Karasarides, M.; Marais, R. Nat. Rev. Mol. Cell. Biol. 2004, 5,
875.
4. O’Neill, E.; Kolch, W. Br. J. Cancer 2004, 90, 283.
Enzymatic activities of selected compounds
IC50 (nM)
10c
GW5074
SB202190
5. Martelli, A. M.; Evangelisti, C.; Chappell, W.; Abrams, S. L.; Bäsecke, J.; Stivala,
F.; Donia, M.; Fagone, P.; Nicoletti, F.; Libra, M.; Ruvolo, V.; Ruvolo, P.; Kempf, C.
R.; Steelman, L. S.; McCubrey, J. A. Leukemia 2011, 25, 1080.
6. Chappell, W. H.; Steelman, L. S.; Long, J. M.; Kempf, R. C.; Abrams, S. L.; Franklin,
R. A.; Bäsecke, J.; Stivala, F.; Donia, M.; Fagone, P.; Malaponte, G.; Mazzarino, M.
C.; Nicoletti, F.; Libra, M.; Maksimovic-Ivanic, D.; Mijatovic, S.; Montalto, G.;
Cervello, M.; Laidler, P.; Milella, M.; Tafuri, A.; Bonati, A.; Evangelisti, C.; Cocco,
L.; Martelli, A. M.; McCubrey, J. A. Oncotarget 2011, 2, 135.
7. Garnett, M. J.; Marais, R. Cancer Cell 2004, 4, 313.
8. Dumaz, N.; Hayward, R.; Martin, J.; Ogilvie, L.; Hedley, D.; Curtin, J. A.; Bastian,
B. C.; Springer, C.; Marais, R. Cancer Res. 2006, 66, 9483.
9. Kim, M.-H.; Kim, M.; Yu, H.; Kim, H.; Lee, J.; Yoo, K.-H.; Sim, T.; Hah, J.-M. Bioorg.
Med. Chem. 2011, 9, 1915.
10. Kyriakis, J. M. et al Nature 1992, 358, 417.
11. Chen, J. et al Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7783.
12. von Gise, A. et al Mol. Cell Biol. 2001, 21, 2324.
13. Dumaz, N. et al Cancer Res. 2006, 66, 9483.
14. Dhomen, N.; Marais, R. Curr. Opin. Genet. Dev. 2007, 17, 31.
15. Lee, J.; Kim, H.; Yu, H.; Chung, J.; Oh, C.-H.; Yoo, K. H.; Sim, T.; Hah, J.-M. Bioorg.
Med. Chem. Lett. 2010, 20, 1573.
BRAF
186
38.3
571
8.79
10.71
5.4
ND
ND
ND
8.66
ND
BRAF (V599E)
P38a/MAPK14
cRAF1
4.40
Considering that BRAF V600E and CRAF are significantly associ-
ated with disease progression and cell proliferation in a subset of
melanomas,11–13 the finding of a dual Raf-kinase inhibitor, 10c,
was valuable. 10c is especially valuable because its selectivity for
mutant over wild-type BRAF was much better than compounds
previously reported.15
In conclusion, a new series of (S)-1-((4-(2-(4-aminobenzyl)-1H-
imidazol-1-yl) pyrimidin-2-yl) amino) propan-2-ol derivatives
were synthesized, and they showed potent antiproliferative activ-
ities against both the A375P and U937 cell lines. Particularly, com-
pounds 9l, 9s and 10c showed competitive activities on the A375P
cell line and 9l, 9n and 9r exhibited superior activities on the U937
cell lines, compared to the standard compound. The IC50 values of
16. The 3D X-ray protein structure of BRAF V600E as a complex with a ligand was
obtained from the PDB (code: 1uwj) and prepared using Protein Preparation
Wizard of the Schrödinger Maestro program. All water molecules were
removed from the structure and it was selected as a template. The structures
of inhibitors were drawn using Chemdraw, and their 3D conformations were
generated using the Schrödinger LigPrep program with the OPLS 2005 force
field. Molecular docking of compound into the structure of BRAF (PDB code:
1uwj) was carried out using Schrodinger Glide (Version 9.2).
9l, 9s and 10c were 0.6
cell line, respectively. Each of the compounds 9l, 9n and 9r had
1.68 M, 1.29 M and 1.37 M IC50 values against the U937 cell
lM, 3.3 lM, and 1.82 lM against the A375P
l
l
l
17. Malamas, M.S.; Erdal, J.J.; Gunawan, I.S. et al. US Pat. Appl. 2005,
US20050282826 A1 20051222.
line, respectively. Especially in our series, 10c exhibited competi-
tive activities on both cancer cell lines and was found to be a very
potent and selective BRAF V600E/CRAF dual inhibitor having IC50
values of 38.3 nM and 8.79 nM respectively.
18. MTT assays on A375P cells and U937 cell were performed as previously
reported. 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
concentration (3-fold 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.
Acknowledgment
This work was supported by the research fund of Hanyang Uni-
versity (HY-2012-N).
References and notes
1. Avruch, J.; Khokhlatchev, A.; Kyriakis, J. M.; Luo, Z.; Tzivion, G., et al. Recent
Prog. Horm. Res. 2001, 56, 127.
2. Khazak, V.; Astsaturov, I.; Serebriiskii, I. G.; Golemis, E. A. Expert. Opin. Ther.
Targets 2007, 11, 1587.
19. We used Reaction Biology Corp. Kinase HotSpotSM service (www.reactionbiology.
com) for screening of 10c.
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