Discovery and initial SAR of pyrimidin-4-yl-1H-imidazole derivatives with antiproliferative activity against melanoma cell lines

Article abstract of DOI:10.1016/j.bmcl.2010.01.064

The synthesis of a novel series of pyrimidin-4-yl-1H-imidazol-2-yl derivatives 7, 8, 9 and their antiproliferative activities against A375P human melanoma cell line and WM3629 cell line were described. Most compounds showed superior antiproliferative activities compared to Sorafenib, the well-known RAF inhibitor. Among them, 7a exhibited potent activities on both cell lines (IC₅₀ = 0.62 and 4.49 μM, respectively) and turned out to be a selective and potent CRAF inhibitor.

Full text of DOI:10.1016/j.bmcl.2010.01.064

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1573–1577
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and initial SAR of pyrimidin-4-yl-1H-imidazole derivatives with
antiproliferative activity against melanoma cell lines
Junghun Lee, Hwan Kim, Hana Yu, Jae Yoon Chung, 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 pyrimidin-4-yl-1H-imidazol-2-yl derivatives 7, 8, 9 and their antipro-
liferative activities against A375P human melanoma cell line and WM3629 cell line were described. Most
compounds showed superior antiproliferative activities compared to Sorafenib, the well-known RAF
inhibitor. Among them, 7a exhibited potent activities on both cell lines (IC50 = 0.62 and 4.49 lM, respec-
tively) and turned out to be a selective and potent CRAF inhibitor.
Received 8 December 2009
Revised 12 January 2010
Accepted 13 January 2010
Available online 21 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Pyrimidin-4-yl-1H-imidazol-2-yl
derivatives
Antiproliferative activity
Melanoma cell line
BRAF
CRAF
Selectivity
Skin cancer is the most common form of cancer in the United
States.1 Although melanoma is the least common type of skin can-
cer; it is the most serious form of the disease. Melanoma occurs in
melanocytes, a type of cell in the skin that produces the pigment
that gives skin its natural color. Melanoma begins when melano-
cytes become malignant, which can occur on any skin surface.
Once melanoma cells migrates to other organs, it is notoriously
refractory to current treatment regimens, which induce responses
in only 10–20% of patients.2,3
In the vast majority of melanomas, the mitogen-activated pro-
tein kinase (MAPK; RAS-RAF-MEK-ERK) and the phosphoinositide
3-kinase-AKT pathways are up-regulated,4,5 and alteration of sig-
naling through both pathways plays a major role in melanoma
progression.
Previous generations of RAF inhibitors include pyrazole com-
pounds from Pfizer,12 SB-590885,13 PLX4720,14 sorafenib15
(Fig. 1: Launched for RCC), and PLX4032 (Phase II for melanoma
and colorectal cancer). The bi-aryl urea sorafenib is a potent inhib-
itor of preactivated CRAF and BRAF as well as oncogenically acti-
vated BRAF kinases (V600E BRAF: IC50 = 43 nM).
Type I inhibitor
F
NOH
O
O
S
O
OH
Cl
N
N
F
H
R
X
N
N
N
H
N
N
N
Especially, the discovery of activating V600E BRAF mutations in
approximately 50% of melanomas has raised the expectations for
targeted therapy.6,7 As a result, the pharmacological targeting of
BRAF/MAPK signaling in melanoma is now being intensively stud-
ied in both the clinical and preclinical settings.8
Pfizer
CN
PLX-4720
N
H
SB-590885
N
O
Type I I inhibitor
HN
However, there is now a growing consensus being made that
there are significant ratio of melanomas (>33%) that do not contain
BRAF V600E mutations that may involve alternative therapeutic
targets9, and one of which is closely related CRAF (or Raf-1).10,11
Cl
CF3
O
F
N
O
N
F
N
H
N
H
N
H
O
N
N
PLX-5568
NH
Sorafenib
N
* Corresponding author. Tel.: +82 2 958 6438; fax: +82 2 958 5189.
E-mail address: jhah@kist.re.kr (J.-M. Hah).
Figure 1. Structure of known V600E RAF inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.064

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J. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1573–1577
Crystal structure of V600E BRAF kinase domains in complex
with sorafenib showed that the inhibitor held the activation seg-
ment in an inactive conformation, preventing ATP binding and sub-
sequent kinase reaction. Although sorafenib failed to show
therapeutic activity in melanoma, the binding feature is interesting
enough to prompt us to design a novel scaffold for RAF inhibitor,
especially, it occupies the unique secondary hydrophobic pocket
in inactive conformation of kinase domain, namely Type II inhibi-
tor.16 However, there are many numbers of Type I inhibitors for
RAF has been also known having a much better potency (see Fig. 2).
Here we could come up with a novel chimera scaffold for RAF
inhibitors, which possibly have a potent hinge binder and take
advantage of secondary hydrophobic pocket as well.16 The struc-
ture of this series comprises 1-pyrimidinyl imidazole part as a
hinge binder, the middle phenyl ring moiety and various aromatic
tail part connected by amide or urea linkage.
We designed the scaffold to aim the middle phenyl ring moiety
to be orthogonal to the imidazole ring for the effective conforma-
tion for hydrophobic pocket. To get more potent inhibitor, we tried
to modify the structures by (a) introducing the various direction of
connectivity in the middle phenyl moiety (m-, p-) and (b) changing
the aromatic tail part to understand the depth and shape of sec-
ondary hydrophobic pocket in inactive conformation.
The general synthesis of pyrimidin-4-yl-1H-imidazol-2-yl
derivatives is shown in Scheme 1. The nitrophenyl imidazole moi-
ety was synthesized from 2,2-dimethoxyethanamine and imidate
in situ, which was formed from 3- or 4-nitrobenzonitle and meth-
oxide under alkaline catalysis in one pot process based on the
literature.17 Then nitrophenyl imidazole was coupled with 4-
iodo-2-(methylthio)pyrimidine in a modified Buchwald condition.
Further, methylthio group was oxidized using mCPBA to methyl
sulfoxide and substituted with (S)-1-aminopropan-2-ol. Without
protection on hydroxy group, nitro group on phenyl ring was re-
duced to give amine and coupled with various aromatic acids un-
der EDCI/HOBt conditions or directly aromatic isocynate to give
amide (7a–7p) or urea (8a–8j) analogues.
Figure 2. (A) Crystal structure of Sorafenib (cyan) and a docking structure of Pfizer
scaffold (yellow) in V600E BRAF (1UWH); (B) Docking structure of a novel chimera
scaffold (red; Glide in Schrödinger package).
N
N
N
N
MeS
OH
MeO2S
NH2
N
MeS
N
H
N
I
NO2
NO2
N
NO2
N
NC
NO2
N
i
ii
iii
iv
N
2
1
N
3
4
N
N
HO HN
N
N
O
N
N
vi
HO HN
HO HN
7a-p
8a-j
N
N
H
R1
N
NO2
N
v
N
NH2
N
N
vii
N
HO HN
O
N
5
6
R2
N
N
N
H
H
N
Scheme 1. Reagents and conditions: (i) 30% NaOMe, MeOH; 2,2-dimethoxyethanamine, AcOH 50 °C; 6 N HCl in H2O–MeOH; (ii) Pd2(dba)3, BINAP, NaOtBu, toluene, 150 °C,
3 h; (iii) m-CPBA, CH2Cl2, rt, 8 h; (iv) THF, 60 °C, 5 h; (v) 10% Pd/C, MeOH, rt, 6 h; (vi) R1CO2H, EDCI, TEA, HOBt, DMF; (vii) R2NCO, THF, rt, 12 h.
N
O
HN
HO
NH
O
N
N
NH
O
N
H
N
NO2
CF3
Cl
i
HN
9a
N
Cl
N
N
O
HN
HO
NH
N
N
O
N
CF3
9b
Scheme 2. Reagents and conditions: (i) 4-nitrobenzoyl chloride, TEA, pyridine 0 °C to rt.

Table 1
Antiproliferative activity of pyrimidin-4-yl-1H-imidazol-2-yl derivatives and pyrimidin-4-yl-1H-imidazole-2-carbonyl derivatives with Sorafenib as reference compound
Structure
No.
R
GI50
WM3629
(lM)
Structure
No.
R
GI50
WM3629
(lM)
A375P
4.49
A375P
79.2
OH
H
CF3
Cl
OH
H
CF3
Cl
N
N
7a22
0.62
7j
>100
N
N
H
N
R1
N
N
O
CF3
N
N
O
O
N
H
R1
N
N
N
7b
9.13
10.4
7k
2.25
35.6
N
N
CF3
CF3
Cl
N
O
7c
3.49
7.37
6.72
28.3
7l
>100
>100
>100
8.05a
N
N
CF3
N
OMe
7d
7m
O
O
CF3
Cl
7e
7f
NA
NA
7n
7o
7p
>100
>100
>100
2.23
139
O
S
CF3
N
>100
NA
164a
NA
Cl
N
N
O
Cl
CF3
N
7g
>100
O
O
CF3
OMe
7h
7i
22.3
13.8
>100
>100
O
S
N
N
(continued on next page)

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J. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1573–1577
To investigate an optimal location of hydrophobic tail and
urea- or amide-linkage, we attempted extra carbonyl insertion be-
tween imidazole and phenyl ring and synthesized two compounds
with pyrimidin-4-yl-1H-imidazole-2-carbonyl derivatives (9a, 9b),
which is shown in Scheme 2. The imidazole and nitrobenzoyl chlo-
ride was coupled in basic condition to form core structure and the
rest of reactions were completed as the same as in Scheme 1.
We examined the antiproliferative activity of these pyrimidinyl
imidazole derivatives in two melanoma cell lines (human A375P
and WM3629).18,19 Unlike well-known A375P melanoma cell lines,
the WM3629 cell lines were used since it could represent non-
V600E mutated melanoma cell lines (>33%). Instead of V600E
mutation, this cell line depends on low activity mutant BRAF
(D594G) and more importantly, CRAF for the proliferation.9,20 We
chose sorafenib as the reference standard because it has been
extensively studied for melanoma and also for CRAF inhibition.
The antiproliferative activity of pyrimidin-4-yl-1H-imidazol-2-yl
derivatives and pyrimidin-4-yl-1H-imidazole-2-carbonyl derivatives
are shown in Table 1. Generally, they show a variety of potency range
dependingonthesubstitutionpatternofmiddlephenylringandmore
importantly, on the tail group (R) implying that the activity is more
sensitive to the secondary hydrophobic pocket (see Table 2).
When the middle phenyl ring has m- orientation, the amide
compounds with relatively short tail possessed good potency,
especially 7a showed the best potency toward WM3629 cell line
(GI50 = 620 nM) in this series and it was better than the reference
sorafenib. But when the tail has been extended to bicyclic struc-
ture, the smaller aromatic ring was preferred as second ring and
the substitution pattern was favored in 1, 3, 5-position (7c > 7b,
7d). The five-membered aromatic ring-extended bicyclic tail did
not improve the potency at all (7f–7i).
In case of m-oriented urea derivatives (8a–8f), the compound
with less bulky substitutions on the phenyl ring tail showed rela-
tively better potency on both cell lines. It was quite surprising to
see that the small difference in substitution on 3-position of
Table 2
Percentages of enzymatic inhibitions exerted by 7a (10
lM) on selected 30 Protein
Kinases
Kinase
% Inhibition
AKT1
ALK
c-Src
CDK1/cyclinB
CDK2/cyclinE
CDK5/p25
CHK1
DMPK
DNA-PK
EGFR/ERBB1
ERK2/MAPK1/P42MAPK
FAK/PTK2Axl(h)
Flt3(h)
5.1
0.8
60
21
60
2.4
5.9
7.6
9.4
1.7
10
0
82
GSK3b
14
IGF-1R
JAK3
JNK1/MAPK8
LYN/LYN A
MEK1
3.6
0.21
60
21
60
1.5
89
mTOR/FRAP1
P38
a/MAPK14
p70S6K/RPS6KB1
17
PAK4
PKA
PKCa
CRAF
60
6.8
1.1
99
RON/MST1R
ROS/ROS1
SYK
5.0
0.19
13
TRKB/NTRK2
60

J. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1573–1577
1577
benzoic acid tail made a big difference in their cellular activity (8a,
8c vs 8b).
Overall the compounds with middle phenyl ring of p-substitu-
References and notes
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8. Eisen, T.; Ahmad, T.; Flaherty, K. T.; Gore, M.; Kaye, S.; Marais, R. Br. J. Cancer
2006, 95, 581.
9. Smalley, K. S. M.; Xiao, M.; Villaneva, J.; Nguyen, T. K.; Flaherty, K. T.; Letrero,
R.; Van Belle, P.; Elder, D. E.; Wang, Y.; Nathanson, K. L.; Herlyn, M. Oncogene
2009, 28, 85.
10. Jilaveanu, L.; Zito, C. R.; Aziz, S. A.; Conrad, P. J.; Schmitz, J. C.; Sznol, M.; Camp,
R. L.; Rimm, D. L.; Kluger, H. M. Clin. Cancer Res. 2009, 15, 5704.
11. 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.
12. Bennett, M. J. et al. WO2007105058A2.
13. King, A. J.; Patrick, D. R.; Batorsky, R. S.; Ho, M. L.; Do, H. T.; Zhang, S. Y.; Kumar,
R.; Huang, P. S., et al Cancer Res. 2006, 23, 11100.
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15. Wilhelm, S. M.; Adnane, L.; Newell, P.; Villanueva, A.; Llovet, J. M.; Lynch, M.
Mol. Cancer Ther. 2008, 7, 3129.
16. Liu, Y.; Gray, N. S. Nat. Chem. Biol. 2006, 2, 358.
17. Voss, M. E.; Beer, C. M.; Mitchell, S. A.; Blmgren, P. A.; Zhichkin, P. E.
Tetrahedron 2008, 64, 645.
18. 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 (three-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.
tion (7j–7p, 8g–8j) showed mild potency toward both cell lines.
Considering that the ratio of GI50 for A375P/WM3629 could be
used as the rough index for selectivity of BRAF/CRAF, compounds
with middle phenyl ring of m-substitution and sorafenib showed
5–10-fold selective for WM3629 over A375P. However, it seems
that the compounds with middle phenyl ring of p-substitution
show less selectivity or even a reverse trend, especially in case of
8i and 8j.
In case of pyrimidin-4-yl-1H-imidazole-2-carbonyl derivatives
(9), the most potent compound on A375P cell lines in this series
(9a) was found. As we expected in docking experiment (data not
shown), the amide derivative was better than the corresponding
urea (9b).
Generally, the activity toward WM3629 cell line showed higher
than A375P, but there are several compounds (7j, 7m, 7n, 8a, 8c
and data not presented) which show the reverse trend, and that
could be a valuable information for designing a selective each iso-
type of RAF inhibitor.
We further tried kinase panel screening of the compound 7a
over 30 different kinases at
a single dose concentration of
10
l
M21 (done in duplicate) and it was revealed that the com-
pound was indeed a selective CRAF inhibitor with a superb
selectivity profile. While this compound has inhibitory activity
of 99% on CRAF at this concentration, the inhibition activity
was below than 20% in most other kinase tested except p38
and Flt3.
a,
In conclusion,
a series of pyrimidin-4-yl-1H-imidazol-2-yl
derivatives based on the structural features of sorafenib showed
potent antiproliferative activities against both A375P and
WM3629 human melanoma cell lines. Especially, in our series,
7a, 7c, 8j, 9a exhibited competitive activities on A375P cell line
and 7a, 8b showed superior activities on WM3629 cell line
compared to sorafenib. These results suggest that pyrimidin-4-
yl-1H-imidazol-2-yl scaffold possesses a possibility as a thera-
peutic agent for melanoma and all these findings will be com-
bined for further development of potent and selective RAF
inhibitors.
19. WM3629 cell line was supplied from Dr. Merlyn lab at Wistar Institute
(Philadelphia, PA, US) and maintained in Tu2% medium according to the
literature 9. The procedure for GI50 determination (MTT assay) was the same as
in A375P cell line.
20. Montagut, C.; Sharma, S. V.; Shioda, T.; McDermott, U.; Ulman, M.; Ulkus, L. E.;
Dias-Santagata, D.; Stubbs, H.; Lee, D. Y.; Singh, A.; Drew, L.; Haber, D. A.;
Settleman, J. Cancer Res. 2008, 68, 4853.
21. We used Reaction Biology Corp. Kinase HotSpotSM service (www.
reactionbiology.com) for screening of 7a.
Acknowledgments
22. Selected data. Compound 7a: 1H NMR (400 MHz, DMSO-d6) d 10.60 (1H, s),
8.37 (1H, s), 8.30 (1H, d, J = 5.24 Hz), 8.24 (1H, dd, J = 1.94 Hz, J = 1.83 Hz), 7.94
(1H, s), 7.92 (1H, s), 7.84 (1H, d, J = 8.79 Hz), 7.73 (1H, br s), 7.39 (1H, t,
J = 8.02 Hz), 7.30 (1H, br s), 7.21 (1H, s), 7.09 (1H, d, J = 6.72 Hz), 4.50 (1H, br s),
3.77 (1H, m), 3.61 (1H, m), 2.89 (2H, m), 1.26 (3H, d, J = 8.13 Hz); MS m/z 533
(M+H)+. Compound 8b: 1H NMR (400 MHz, CDCl3) d 8.20 (1H, s), 8.16 (1H, s),
7.55–7.51 (2H, m), 7.27 (1H, s), 7.23–7.18 (3H, m), 7.13–7.07 (3H, m), 6.91 (1H,
J = 7.13 Hz), 6.22 (1H, s), 5.61–5.60 (1H, br s), 4.98–4.96 (1H, br s), 3.48–3.47
(1H, m), 3.18–3.00 (2H, m), 1.27 (3H, t, J = 9.24 Hz); MS m/z 499 (M+H)+.
This research was supported by Korea Institute of Science and
Technology (KIST) and Basic Science Research Program through
the National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology (2009-0087992;
J.M.H.). We also thank Dr. Merlyn lab at Wistar Institute for the
kind supply of WM3629 cell line.