Computer-aided design and synthesis of 3-carbonyl-5-phenyl-1H-pyrazole as highly selective and potent BRAFV600E and CRAF inhibitor

Article abstract of DOI:10.1080/14756366.2019.1599366

BRAF belongs to the upstream portion of the MAPK pathway, which is involved in cell proliferation and survival. When mutations occur in BRAF, downstream MEK and ERK are phosphorylated irrespective of RAS, resulting in melanoma-like cancer. Over the years, small molecules targeting BRAFV600E have been discovered to be very effective melanoma drugs, but they are known to cause the BRAF paradox. Recently, it was shown that this paradox is caused by the heterodimer phenomenon of BRAF/CRAF. Here, we suggest one method by which paradoxical activation can be avoided by selectively inhibiting BRAFV600E and CRAF but not wild-type BRAF. From previous report of N-(3-(3-alkyl-1H-pyrazol-5-yl) phenyl) aryl amide as a selective inhibitor of BRAFV600E and CRAF, we present compounds that offer enhanced selectivity and efficacy with the aid of molecular modelling.

Full text of DOI:10.1080/14756366.2019.1599366

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Computer-aided design and synthesis of 3-
carbonyl-5-phenyl-1H-pyrazole as highly selective
and potent BRAFV600E and CRAF inhibitor
Jinwoong Kim, Byeongha Choi, Daseul Im, Hoyong Jung, Hyungwoo Moon,
Waqar Aman & Jung-Mi Hah
To cite this article: Jinwoong Kim, Byeongha Choi, Daseul Im, Hoyong Jung, Hyungwoo
Moon, Waqar Aman & Jung-Mi Hah (2019) Computer-aided design and synthesis of 3-
carbonyl-5-phenyl-1H-pyrazole as highly selective and potent BRAFV600E and CRAF
inhibitor, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 1314-1320, DOI:
10.1080/14756366.2019.1599366
To link to this article: https://doi.org/10.1080/14756366.2019.1599366
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1314–1320
https://doi.org/10.1080/14756366.2019.1599366
SHORT COMMUNICATION
Computer-aided design and synthesis of 3-carbonyl-5-phenyl-1H-pyrazole as
highly selective and potent BRAFV600E and CRAF inhibitor
Jinwoong Kima, Byeongha Choia, Daseul Ima, Hoyong Junga, Hyungwoo Moona, Waqar Amanb and Jung-Mi Haha
aDepartment of Pharmacy and Institute of Pharmaceutical Science and Technology, College of Pharmacy, Hanyang University, Ansan,
b
Gyunggido, Republic of Korea; Kohat University of Science and Technology, Kohat, Khyber Pukhtunkhwa, Pakistan
ABSTRACT
ARTICLE HISTORY
Received 7 February 2019
Revised 18 March 2019
Accepted 20 March 2019
BRAF belongs to the upstream portion of the MAPK pathway, which is involved in cell proliferation and
survival. When mutations occur in BRAF, downstream MEK and ERK are phosphorylated irrespective of
RAS, resulting in melanoma-like cancer. Over the years, small molecules targeting BRAFV600E have been
discovered to be very effective melanoma drugs, but they are known to cause the BRAF paradox.
Recently, it was shown that this paradox is caused by the heterodimer phenomenon of BRAF/CRAF. Here,
we suggest one method by which paradoxical activation can be avoided by selectively inhibiting
BRAFV600E and CRAF but not wild-type BRAF. From previous report of N-(3-(3-alkyl-1H-pyrazol-5-yl) phe-
nyl) aryl amide as a selective inhibitor of BRAFV600E and CRAF, we present compounds that offer
enhanced selectivity and efficacy with the aid of molecular modelling.
KEYWORDS
Melanoma; BRAFV600E;
BRAFWT; CRAF; selectivity
1. Introduction
mutant tumors. However, resistance to these drugs often occurs
within 1 year, and the disease then recurs. In addition, secondary
malignant tumors arising from RAS mutations, such as squamous
cell carcinoma and keratocyte sarcoma, may occur during treat-
ment. BRAF inhibitors can bind to wild-type RAF and induce
dimerization to phosphorylate downstream proteins regardless of
RAS activation. This phenomenon is referred to as the BRAF para-
dox.8,9 The mechanism underlying this phenomenon is dimeriza-
tion of RAF, which has emerged as a clinically important issue as
vemurafenib and dabrafenib do not effectively inhibit homodimers
or heterodimers of BRAF or CRAF. Another BRAF paradox mechan-
ism is that first-generation BRAF inhibitors bind not only to wild-
type BRAF, but also to one of the BRAF dimers, turning the aC-
helix into an outward facing position. These inhibitors competi-
tively bind to ATP sites in the DFG IN/aC-helix OUT conformation
of BRAF as type I1/2 inhibitors. As a result, the aC-helix of another
protomer is transformed into an OUT structure, preventing other
inhibitors from binding. Downstream signaling is activated by
unbound protomer. To prevent occurrence of the paradox, block-
ing mutations in RAS together with CRAF, one of the protein kin-
ases of other RAF families, is effective, as is blocking BRAF
mutations. Therefore, it is important to maintain CRAF as well as
BRAFV600E.10
Malignant melanoma is the deadliest of all skin cancers.
Historically, melanoma was very rare, but its prevalence has
increased rapidly over the last 50 years compared to other cancers.
Sunburn and congenital naevi are included among the risk factors
of melanoma and may also be influenced by family history.1 At
present, it is most effective to surgically remove lesion sites in
patients with early melanoma after diagnosis, and lymph node
dissection around the lesion is often considered because radiation
or melanoma cells can be transferred to the surrounding lymph
nodes. Immunotherapy with drugs such as nivolumab or ipilimu-
mab or targeted chemotherapy for BRAF gene mutations is used
to treat the surrounding lymph nodes in patients with meta-
static melanoma.2
The RAS/RAF/MEK/ERK signaling pathway regulates cell growth,
proliferation, and survival. RAF families act as critical modulators
in this signaling pathway and RAS-induced dimerisation of RAF
phosphorylates MEK1, and MEK2 activates the downstream cas-
cade, subsequently phosphorylating ERK1 and ERK2.3 This signal-
ing pathway is the most frequently mutated in human cancers,
with about 50% of melanoma patients. BRAF is found in 10–70%
of patients with thyroid cancer, less than 10% of patients with
colorectal cancer, and in 3–5% of patients with non-small cell
lung cancer (NSCLC).4 V600E is the most abundant mutation in
BRAF; therefore, BRAF V600E is the most popular target of melan-
oma treatment.5
We have suggested a BRAF inhibitor that selectively inhibits
BRAFV600E and CRAF but not BRAFWT for use as a BRAF paradox
breaker and previously described 3-carboxamido-2H-indazole-6-
arylamide (1), a selective CRAF inhibitor with an IC50 of 38.6 nM
(Figure 2). However, the IC50 was only 7.82 lM for BRAFV600E and
9.45 lM for wild-type BRAF.11 As a follow-up study aimed at iden-
tifying selective binding inhibitors of BRAF V600E and CRAF, N-
(3–(3-alkyl-1H-pyrazol-5-yl)phenyl)-aryl amide (2) were developed
The drugs vemurafenib (Zelboraf)6 and dabrafenib (Tafinlar)7
were developed and approved by the FDA as inhibitors targeting
BRAFV600E for use in therapies for advanced melanoma treatment
(Figure 1). These drugs are associated with significant response
and enhanced survival rates in melanoma patients with BRAFV600 with good selectivity for BRAFV600E and CRAF.12
CONTACT Jung-Mi Hah
jhah@hanyang.ac.kr
Department of Pharmacy and Institute of Pharmaceutical Science and Technology, College of Pharmacy, Hanyang
University, 55 Hanyangdaehak-ro, Sangrok-gu, Ansan, Gyunggido, Republic of Korea
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1315
H2N
N
H
H
N
N
N
N
N
H
N
O
N
O
H
N
O
F
O
F
F
H
N
O
O
S
N
S
S
F
S
N
N
O
Cl
O
F
F
F
Vemurafenib
Dabrafenib
PLX7904
Figure 1. Chemical structures of BRAF paradox inducer (Vemurafenib, Dabrafenib) and breakers (PLX7904 and TAK-632).
Figure 2. Selective BRAFV600E and CRAF inhibitors reported and docking mode of N-(3–(3-alkyl-1H-pyrazol-5-yl) phenyl)-aryl amide (2) on BRAFV600E (4G9R).
Here, we describe 3-carbonyl-5-phenyl-1H-pyrazole derivatives compared to its high selectivity. A new inhibitor design was
attempted by introducing an extra group capable of hydrogen
bonding to hinge residues beside pyrazole. We assessed 3-car-
bonyl-5-phenyl-1H-pyrazole (3) as a new scaffold and performed
docking studies.
that strengthen activity. They were designed by extending the
solvent exposure part in the ribose part of the scaffold through in
silico modeling. 3-carbonyl-5-phenyl-1H-pyrazole has a binding
mode that binds hydrogen with Cys 532 of the hinge and Thr529,
which is a gatekeeper commonly found in BRAFV600E and CRAF.
Glide docking of each compound was attempted to predict select-
ivity for BRAFV600E and CRAF over BRAFWT, thereby increasing
potency while maintaining selectivity.
To design better inhibitors in terms of activity and selectivity,
we conducted docking studies to identify the best compound in a
series of 2, N-(3–(3-isopropyl-1H-pyrazol-5-yl)phenyl)-3-(4-methylpi-
perazin-1-yl)-5-(trifluoromethyl)benzamide. We then extended
compound 2 to a 3-carbonyl-5-phenyl-1H-pyrazole (3) compound
and tried to dock it to BRAFV600E (PDB Code: 4G9R)13 and CRAF
(PDB Code: 3OMV)14 with Maestro (Figure 3). In general, the dock-
ing scores of series 3 were better than those of series 2 on a scale
of 1–2 (10 to 100 times lower in energy). Compounds 2 and 3
were docked to BRAFV600E through induced fit docking and
showed multiple interactions like those shown in Figure 3. Most
importantly, 3-carbonyl-5-phenyl-1H-pyrazole (3) has three hydro-
gen bonds near the hinge site, with the proton of the pyrazole
group hydrogen bonded to Trp531 and the amine of pyrazole to
Thr529. The carbonyl group substituted at the pyrazole ring was
also hydrogen bonded to Cys532 at the hinge. Moreover, Phe595
of the DFG motif and the pyrazole and middle phenyl rings of the
compound have a p–p interaction. The carbonyl of amide is
hydrogen bonded to Lys601 of the activation loop. Likewise, com-
2. Results and discussion
Recently, we described N-(3–(3-alkyl-1H-pyrazol-5-yl)phenyl)-aryl
amide (2) as a selective RAF inhibitor.12 This derivative showed
very selective binding affinity to BRAFV600E and CRAF and was 20
and 126 times more potent than either, respectively, in BRAFWT.
Structural features of these analogues include a bulky 1,3,5-substi-
tuted benzyl group in the type 2 hydrophobic pocket region and
a common ring bond to pyrazole, the hinge binder, with a direct
C-C bond. However, we found that the compound N-(3–(3-alkyl-
1H-pyrazol-5-yl)phenyl)-aryl amide did not effectively hydrogen
bond with the Cys531 of the hinge portion during glide docking
in the Maestro programme. We conclude that this is the same
problem seen in previous series, resulting in low potency pound 3 maintained the interactions of compound 2.

1316
J. KIM ET AL.
Figure 3. Binding mode of 2 and 3 to BRAFV600E (PDB: 4G9R).
Figure 4. Overlapped binding mode between BRAFV600E (PDB: 4G9R, yellow) and CRAF (PDB: 3OMV, green), and overlapped binding mode between BRAFV600E
(PDB: 4G9R, yellow) and modified CRAF (PDB: 3OMV, green).
The PDB for currently available CRAF is 3OMV, which is com- one hydrogen bond with Glu500 in the a-helix of BRAFWT, and
plexed with GDC0879 Reference. However, GDC0879 is a type 1 hinge hydrogen bonds were not formed, resulting in a much
inhibitor for CRAF and binds the DFG motif of CRAF in the state lower docking score than for BRAFV600E (ꢀ9.813 to 1UWH,
of IN. Therefore, to explain the binding mode of our Type II inhibi- ꢀ15.128 to 4G9R). Most importantly, the difference in docking
tors, we needed to modify 3OMV using the Induced Fit module in scores of BRAFV600E and BRAFWT was much greater for the new
Maestro. First, we superimposed the binding mode of 2 to carbonyl series 3 than for series 2 (docking scores of series 3 on
BRAFV600E (PDB: 4G9R) and to CRAF (PDB: 3OMV). Then, the posi- BRAFWT compared to BRAFV600E and CRAF were higher by mag-
tions of several residues around DFG in CRAF were shifted, nitudes of 102–104).
rotated, and energy minimized as similarly as possible to the
After docking studies of 3-carbonyl-5-phenyl-1H-pyrazole (3) on
structure of BRAFV600E. We monitored the docking of compound three RAF proteins, we synthesized two kinds of carbonyl group-
2 on CRAF each time we changed the structure. Through this pro- ester and amide-linked pyrazoles as new scaffolds under the
cess, a protein structure of CRAF for type 2 inhibitors was created, assumption that inclusion of a hydrophobic tail would impart
and the binding mode was confirmed, as shown in Figure 4. In selectivity and potency that can be increased by extending the
induced fitted-CRAF and 2, the pyrazole ring has a hydrogen solvent exposure region.
The general synthesis of 3-carbonyl-5-phenyl-1H-pyrazole (3) is
bond with Cys424, and the hinge residue and amide bond were
tied through two hydrogen bonds to the backbone of Asp486 of shown in Scheme 1. The starting material 1-(3-nitrophenyl) ethan-
DFG. In addition, Phe487 and the pyrazole group of the DFG motif 1-one (14) was treated with a solution of sodium methoxide in
exhibit a p–p interaction, and the carboxylic acid of Glu393 of the methanol, and diethyl oxalate was added dropwise to yield 15.16
N-lobe and proton of amide are hydrogen bonded. Finally, imid- Then, hydrazine monohydrate was reacted with 15 and acetic acid
azole and N-lobe Lys375 undergo p–cation interactions, contribu- as a solvent at room temperature to obtain core intermediate pyr-
ting to the stable binding of 2 to CRAF.
azole 16.17 Sequentially, the nitro group of pyrazole 16 was
We also attempted docking studies of compounds 2 and 3 on reduced to aniline 17 through hydrogenation. Then, 17 was
BRAFWT (Figure 5). In binding modes 2 and 3, we observed only reacted with various benzoic acids with different substituents by

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1317
Figure 5. Binding mode of 23b to CRAF (PDB: 3OMV, glide docking) and to BRAFWT (PDB: 1UWH).15
O
O
O
HN
N
O2
OCH3
O
N
O2
N
NO2
O
O
i
ii
15
14
16
CF3
HN
HN
O
N
O
N
17
H
N
NH2
R1
O
O
v
iv
iii
O
18a-d
CF3
HN
NH
N
H
N
R2
R1
vi
O
O
CF3
HN
HO
N
20a-d, 21a-c
N
H
N
R1
O
CF3
O
HN
O
H
N
R2
19a-d
R1
O
vii
O
23a-d, 24
N
N
viii
N
N
H
Boc
21a-c
22a-c
Scheme 1. Synthesis of 3-carbonyl-5-phenyl-1H-pyrazole derivatives. (i) Diethyloxalate, NaOMe, MeOH, rt, 24h; (ii) NH2NH2ꢁH2O, AcOH, rt, 4 h; (iii) H2, Pd/C, MeOH, rt,
30 min, 98%; (iv) benzoic acid, HATU, TEA, THF, rt, 24h; (v) 1 M NaOH, THF, reflux, 2h; (vi) Alkylamine, HATU, DIPEA, THF, rt; (vii) Alkylalcohol, EDCI, HOBt, DMF, rt; (viii)
TFA, DCM, rt, 30 min.
amide coupling to obtain 18a–d.18 After conversion of methyl with several kinds of alkylamines, including HATU and DIPEA. The
ester (18) to its acid (19) using 1 M NaOH solution, the final amide final ester products 23a–d and 24 were synthesized by ester cou-
products 20a–d and 21a–c were synthesised by amide coupling pling with alkyl alcohols, EDCI, HOBt, or DMAP.19

1318
J. KIM ET AL.
Table 1. Enzymatic activities of 3-carbonyl-5-phenyl-1H-pyrazole derivatives.
CF3
HN
X
N
H
N
R2
R1
O
O
IC50 (µM)
Docking score
BRAFV600E BRAFWT
No.
20a
R1
R2
X
BRAFV600E BRAFWT
CRAF
1.5
CRAF
-10.84
16.4
2.80
NH
-12.289
-12.923
-11.749
-10.421
-11.372
-11.851
20b
20c
NH
NH
0.98
0.91
13.1
14.7
0.59
0.58
-10.207
-11.772
20d
22a
22b
22c
NH
NH
NH
NH
2.70
2.09
1.26
1.51
> 20
> 20
12.9
1.15
1.6
-13.847
-13.314
-12.212
-13.149
-10.118
-11.437
-9.538
-9.774
-8.741
-9.909
-10.013
-9.750
0.83
0.58
>15.2
23a
23b
O
O
0.68
0.10
> 20
> 20
0.13
0.02
-13.832
-14.543
-13.669
-12.276
-12.096
-11.625
23c
O
0.33
> 20
0.06
-12.480
-12.426
-11.170
23d
24
O
O
0.68
0.40
> 20
> 20
0.16
0.21
-15.054
-12.659
-13.229
-8.344
-11.435
-13.342
The IC50 values for BRAFV600E and CRAF were measured for the pyrazole group, which is an extended hinge binder and the
each of the 12 compounds synthesised (Table 1). The com- part exposed to solvent, is superior to amide in terms of potency.
pounds were all designed as 1, 3, 5-substituted benzyl group Compounds that were substituted on the solvent exposure part
hydrophobic tails to maintain the selectivity of the previ- with morpholine rather than piperazine showed lower IC50. Of
ous series.
these 12 compounds, 23b showed the highest potency for
For the R1 group of the hydrophobic tail, the IC50 values of the BRAFV600E and CRAF. Compound 23b is a compound in which the
compounds increase in the order 2-methylimidazole < 4-methyli- hydrophobic tail is substituted with 2-methylimidazole, and the
midazole < morpholine ¼ 4-methylpiperazine. The ester linkage of site exposed to the solvent is substituted with morpholine linked

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1319
Table 2. Percentages of enzymatic inhibition exerted by 23b (10 lM) on 35 selected protein kinases
100
90
80
70
60
50
40
30
20
10
0
Table 3. Enzymatic activities of compound 23b.
concentration. The inhibition activity was less than 20% in most
IC50
23b (lM)
Selectivity (fold) other kinases, including BRAFWT.
BRAFWT
BRAFV600E
CRAF
>20
0.10 (>200 fold)
0.02 (1000 fold)
–
Considering that BRAFV600E and CRAF are significantly associ-
ated with disease progression and cell proliferation in a subset of
melanomas and are potential paradox breakers, the finding of a
dual RAF-kinase inhibitor, 23b, was valuable. Our findings are sig-
nificant for medicinal chemistry in two different ways. First,
through in silico modeling, we were able to design highly select-
ive and potent inhibitors for BRAFV600E and CRAF that were dif-
ferentiated from similar BRAFWT. In addition, we confirmed the
enzyme inhibitory activity and selectivity of the designed com-
pounds. Second, we discovered a small molecule capable of more
selective binding to BRAFV600E and CRAF than BRAFWT, suggest-
ing a new direction for development of RAF paradox chemical
inhibitors. Our findings also provide a way to improve the narrow
therapeutic window of previous melanoma drugs, a particular dis-
advantage of pan-RAF inhibitors.
>200
>1000
A37521
SW62021
0.07 lM (GI50
>10 lM (GI50
)
)
via ester. Compound 23 b is seven times more potent for
BRAFV600E and 5 times more potent for CRAF than the previously
N-(3–(3-alkyl-1H-pyrazol-5-yl)phenyl)-aryl amide derivatives.
Next, we used kinase panel screening in duplicate for com-
pound 23b over 35 different kinases at a single-dose concentra-
tion of 10 lM (Table 2). The compound was indeed a selective
BRAFV600E and CRAF inhibitor with an excellent selectivity profile.
This compound has an inhibitory activity of more than 90% on
CRAF and BRAF V600E at a concentration of 10 lM; the inhibition
activity was less than 20% for most other kinases, including wild-
type BRAF. We further determined the IC50 value of 23b on wild-
type BRAF to be over than 20 lM, where we could confirm select-
ivity (Table 3). In order to investigate the effect of 23b in living
cells, the human colon cancer cell line SW260 expressing wild-
type BRAF and the malignant melanoma A375 cell line expressing
BRAFV600E were treated with increasing concentrations of 23b.
The resulting MTT assays showed that in the cells expressing
BRAFV600E (A375 cell line) 23b resulted in inhibition of the prolif-
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
eration (GI50
BRAFWT (SW620).
¼
0.07 lM), and not in the cells expressing
This work was financially supported by the National Research
Foundation of Korea grant [NRF-2017R1A2B4006447; J.-M. Hah].
3. Conclusions
References
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¼
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