Discovery of a highly specific and potent pan-RAF inhibitor

Article abstract of DOI:10.1002/bkcs.10917

We describe the structure-based design and synthesis of N-(2,6-difluorophenyl)-3-(9H-purin-6-yl)pyridine-2-amine derivatives as a selective pan-RAF kinase inhibitor. The synthesized compounds showed highly potent and specific inhibition of the BRAF^V600E mutant cell line. Among them, N-(3-((3-(9H-purin-6-yl) pyridine-2-yl)amino)-2,4-difluorophenyl)furan-3-sulfonamide (4b) exhibited the most potent inhibitory activities against protein kinase enzymes BRAF^V600E, BRAF^WT, and CRAF (IC₅₀ of 2, 2, and 1 nM, respectively) and a mutant cell line bearing a BRAF^V600E mutation, A375P (GI₅₀ of 7 nM).

Full text of DOI:10.1002/bkcs.10917

Article
DOI: 10.1002/bkcs.10917
BULLETIN OF THE
S. P. Hong et al.
KOREAN CHEMICAL SOCIETY
Discovery of a Highly Specific and Potent Pan-RAF Inhibitor
†,
Sung Pyo Hong,† Younho Lee,‡ Nam Song Choi,§ Ky-Youb Nam,¶, and Soon Kil Ahn
*
*
†Institute for New Drug Development, Division of Life Sciences, Incheon National University, Incheon
22012, Korea. *E-mail: skahn@inu.ac.kr
‡College of Pharmacy and Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon
21983, Korea
§College of Interdisciplinary & Creative Studies, Konyang University, Chungcheongnam-do, 32992,
Korea
¶Pharos I&BT Co., Ltd., Gyeonggi-do, 14059, Korea. *E-mail: kyn@pharosibt.com
Received July 9, 2016, Accepted August 1, 2016
We describe the structure-based design and synthesis of N-(2,6-difluorophenyl)-3-(9H-purin-6-yl)pyridine-
2-amine derivatives as a selective pan-RAF kinase inhibitor. The synthesized compounds showed highly
potent and specific inhibition of the BRAFV600E mutant cell line. Among them, N-(3-((3-(9H-purin-6-yl)
pyridine-2-yl)amino)-2,4-difluorophenyl)furan-3-sulfonamide (4b) exhibited the most potent inhibitory
activities against protein kinase enzymes BRAFV600E, BRAFWT, and CRAF (IC50 of 2, 2, and 1 nM,
respectively) and a mutant cell line bearing a BRAFV600E mutation, A375P (GI50 of 7 nM).
Keywords: RAF, Pan-RAF inhibitor, Structure-based drug design, Melanoma, Anticancer
Introduction
agents to BRAFV600E kinases as melanoma target drugs.
Another BRAFV600E kinase drug (dabrafenib) has been
approved, and some inhibitors have entered clinical trials.6
However, melanoma rapidly develops resistance to selec-
tive BRAF inhibitors after a median duration of response of
6–7 months. The main reason for resistance is that drug-
bound BRAF activates CRAF and causes the activation of
the MAPK pathway.3 Therefore, the development of a more
effective pan-RAF inhibitor is needed to reduce resistance.
Herein, we report the design and synthesis of an N-(2,6-
difluorophenyl)-3-(9H-purin-6-yl)pyridine-2-amine deriva-
tive as a selective pan-RAF kinase inhibitor.
RAFs are serine/threonine protein kinases. They comprise
three evolutionarily conserved isoforms: ARAF, BRAF,
and CRAF. RAF kinases are core members of the RAS-
RAF-MEK-ERK (MAPK) signal transduction cascade. The
upstream RAS protein is activated by extracellular stimuli,
and then it recruits RAF, which is phosphorylated by adap-
tor proteins of RAS. The phosphorylated RAF activates
MEK which signals forwards to its downstream transcrip-
tion effectors that have cellular proliferation and survival
activities.1 The dimerization of RAF is a main RAS-
regulated event in RAF activation.2 BRAF could stimulate
the catalytic activity of CRAF independently of the intrinsic
kinase activity of BRAF.3
Experimental
All derivatives were synthesized as shown in Scheme 1.
Commercially available 2,6-difluorobenzoic acid 7 was
converted to a 3-nitro substituent 8. A boc-protected amine
9 was synthesized in three steps from the carboxylic acid
8 through Curtius rearrangement. The sulfonyl amide deri-
vatives 11 were synthesized by coupling with aliphatic or
arylsulfonyl chloride after the nitro reduction of compound
9. Following the Boc-deprotection, compound 12 was
coupled with a tetrahydropyran (THP)-protected purine
compound 13, which was made following the method
described by Amgen group.7 The final compounds were
obtained after THP-deprotection (Scheme 1).8
The aberrantly activated MAPK pathway results in carcin-
ogenesis. Pathway mutations can be found in approximately
30% of all cancers. The BRAF mutation causes 8% of the
MAPK pathway mutations. BRAF mutants are found in vari-
ous cancers, such as melanoma (40–70%), papillary thyroid
(36–53%), ovarian serous cancer (~30%), and colorectal can-
cer (5–22%). The most frequently detected mutations (>90%)
in BRAF are the V600E type which leads to constitutive
kinase activity greater than the BRAF wild type.4
The first clinically approved BRAFV600E kinase inhibitor,
vemurafenib (PLX4032) was developed as a melanoma tar-
get drug. The inhibition of BRAF activity using this selec-
tive BRAF target drug can result in the diminished
proliferation and survival of BRAFV600E melanoma.5 After
the development of the drug for the treatment of melanoma,
there have been numerous efforts to develop therapeutic
Results and Discussion
Several BRAF inhibitors have an N-(2,4-difluorophenyl)
propane-1-sulfonamide
1
functional group including
Bull. Korean Chem. Soc. 2016
© 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Wiley Online Library
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Article
BULLETIN OF THE
ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
O
F
HO
F
7
F
(
a)
O
F
F
8
F
H
H
N
(b)
(c)
N
NH2
NO2
NO2
Boc
HO
Boc
F
F
9
10
O
N
N
F
F
H
N
O
H
N
H
N
O
(d)
(e)
(g)
R1
O
1
N
H2N
F
R
N
S
+
S
Boc
O
F
F
N
11
12
13
O
H
N
N
N
N
(f)
N
N
N
N
N
F
F
H
H
N
O
H
N
H
N
O
N
R1
O
R1
O
S
S
N
F
F
14
15
Scheme 1. Reagents and conditions: (a) KNO3, H2SO4, r.t., 0.5 h; (b) (1) oxalyl chloride, DMF, 20ꢀC; (2) NaN3, DCM, 20ꢀC, 0.5 h;
(3) t-BuOH, DCM, reflux, 3 h; (c) H2, Pd/C, MeOH, r.t., overnight; (d) sulfonyl chloride derivatives, pyridine, DCM, r.t., 2 h; (e) 4N HCl
in 1,4-dioxane, EA, r.t., 2 h; (f ) LiHMDS, THF, 0ꢀC, 1 h; (g) HCl, water, reflux, 1 h.
PLX4032.5,9–13 We superimposed all of the released BRAF
X-ray crystal structures with the PLX4032 complex struc-
ture (3OG7.pdb) for the structure-based design of
BRAFV600E kinase inhibitors. The difluorophenyl ring and
isopropyl groups occupy the hydrophobic pockets adjacent
to the Thr529 and Leu505 residues, respectively. Those
binding interactions create a BRAF αC-helix-out conforma-
tion in which the highly conserved salt bridge between
Lys483 and Glu501 is broken. Wang et al. classified the
characteristic inhibitors as type IIB kinase inhibitors. Addi-
tionally, the authors explained that type IIB inhibitors had
minimal preclinical toxicity by lacking significant MAPK
activation or inhibition in BRAFWT cells.14 A research
group from Amgen determined that the hinge binding
group 2 was a BRAF kinase inhibitor. Their reported com-
pounds had selective BRAF kinase inhibitory activity and
good bioavailability. However, they did not introduce an
proliferative effects (GI50: 130 nM) using an MTT assay.
The activity of compound 3a was comparable to that of the
reference compound, PLX4032 (GI50: 90 nM).
We tried to obtain more potent chimeric preclinical can-
didates that contained the aforementioned hinge binder and
type IIB characteristics of a hydrophobic binding group.
Recently, Array BioPharma attempted an approach that was
similar to our study. They changed the hinge binding group
that retained the N-(2,4-difluorophenyl) propane-1-
sulfonamide moiety. The compound exhibited desirable
physicochemical properties and potent efficacy in a
BRAFV600E mutant mouse xenograft model.13
Based on this information, we expected that our chimeric
compounds would have good selectivity and activity to the
enzyme. Therefore, we conducted an MTT cell proliferation
assay against BRAF mutant (A375P) and wild-type (SK-
MEL-2) cells to optimize the inhibitors more rapidly and
effectively.16 We first attempted to optimize the activity by
changing the n-propyl group to other aliphatic functional
groups (Table 1). An isopropyl derivative 3b was shown to
have comparable activity with n-propyl. However, other
N-(2,4-difluorophenyl)propane-1-sulfonamide
functional
group. Furthermore, the compounds showed type IIA char-
acteristics that were confirmed by an X-ray crystal complex
structure (3IDP.pdb).7
We designed a novel chimeric compound for a BRAF
inhibitor that had a previously known hinge binder 2 and a
compound 1 group based on structure analysis as mentioned
earlier (Figure 1). The initial chimeric compound 3a bound
well in the ATP binding pocket of BRAFV600E with type IIB
characteristics based on a docking study (Figure 2).15 The
synthesis and cell-based in vitro assay of the compound 3a
was carried out using a BRAFV600E mutant melanoma cell
line (A375P). The compound 3a had significant anti-
Figure 1. Design of a novel chimeric compound 3a.
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Table 1. Anti-proliferative activity of alkyl sulfonamide of N-
(2,6-difluorophenyl)-3-(9H-purin-6-yl)pyridine-2-amine
derivatives against A375P (BRAFV600E) and SK-MEL-2
(BRAFWT) cells.
Table 2. Anti-proliferative activity of five-membered heterocyclic
sulfonamide derivatives of N-(2,6-difluorophenyl)-3-(9H-purin-6-
yl) pyridine-2-amine against A375P (BRAFV600E) and SK-MEL-2
(BRAFWT) cells.
Cell viability GI50 (μM)
Cell viability GI50 (μM)
A375P(B-
SK-MEL-2
A375P(B-
SK-MEL-2
Compound
R
RAFV600E
0.13
)
(BRAFWT
>10
)
Compound
R
RAFV600E
0.04
)
(BRAFWT
>10
)
3a
4a
3b
0.123
>10
4b
4c
0.007
4.09
>10
>10
3c
0.66
0.25
>10
>10
3d
4d
>15
>10
3e
3f
0.59
3.24
>10
>10
4e
4f
0.03
0.02
>10
>10
4g
2.25
>10
alkyl derivatives 3c-3f showed slightly decreased A375P
cell anti-proliferation activity.
Next, we evaluated the effect of the five-membered het-
erocyclic groups of the compounds on their inhibitory
effects (Table 2).
4h
4i
0.22
3.82
>10
>10
A couple of furan 4a and 4b and thiophene analogs 4e
and 4f exhibited more potent cell inhibitory activity than an
n-propyl derivative 3a. In particular, N-(3-((3-(9H-purin-6-
yl)pyridine-2-yl)amino)-2,4-difluorophenyl)furan-3-
sulfonamide 4b exhibited 1–9 nanomolar half-growth
inhibitory activity against A375P cells. Substituted furan 4c
and 4d, thiophene 4g and 4h, thiazole 4i, imidazole 4j, and
pyrazole 4k derivatives demonstrated 1000-fold less inhibi-
tory activity than compound 4b.
4j
7.84
1.37
>10
>10
4k
We also prepared several benzene derivatives (Table 3).
Most of the phenyl substituted compounds and pyridinyl
derivatives 5b were equally potent to n-propyl compound
3a. Finally, we prepared several bicyclic sulfonamide deri-
vatives (Table 4). All of the bicyclic sulfonamide analogs
showed decreased inhibitory activity compared to n-propyl
compound 3a.
IIB characteristics of the synthesized compounds. The com-
pound did not completely abolish MEK-ERK phosphoryla-
tion signaling in SK-MEL-2 cells, unlike in the A375P cells
which was also observed in other BRAF inhibitors.17
Figure 3 represents the docking model of the most active
compound 4b. As previously demonstrated, the purine ring
interacted with the amide backbone of C532 which was a
hinge amino acid.7 The purine and pyridine rings had π–π
stacking interactions with W531 and F583 side chains. An
The anti-proliferative effect against SK-MEL-2 (BRAFWT
)
was examined to confirm the cell line selectivity. All of the
test compounds displayed poor activity against the BRAF
wild-type cell line. This result was anticipated from the type
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Table 4. Anti-proliferative activity of bicyclic sulfonamide
Table 3. Anti-proliferative activity of substituted benzene
sulfonamide derivatives of N-(2,6-difluorophenyl)-3-(9H-purin-6-
yl) pyridine-2-amine against A375P (BRAFV600E) and SK-MEL-2
(BRAFWT) cells.
derivatives of N-(2,6-difluorophenyl)-3-(9H-purin-6-yl)pyridine-2-
amine against A375P (BRAFV600E) and SK-MEL-2 (BRAFWT
cells.
)
Cell viability GI50(μM)
A375P(B-
SK-MEL-2
Cell viability GI50(μM)
Compound
R
RAFV600E
0.14
)
(BRAFWT
>10
)
A375P(B-
SK-MEL-2
5a
Compound
R
RAFV600E
8.37
)
(BRAFWT
>10
)
6a
5b
5c
0.07
0.67
>10
>10
6b
6c
0.81
7.19
>10
>10
5d
0.43
>10
5e
5f
3.5
>10
>10
>10
0.66
>100
6d
6e
6f
12.87
0.58
0.68
>10
>10
>10
5g
5h
5i
54.62
0.76
>10
>10
5j
0.16
0.59
>10
>10
5k
6g
6h
2.22
2.93
>10
>10
5l
0.23
1.00
>10
>10
5m
5n
1.05
>10
group with T529 and I527. The other site is near the L505
residue where it interacts with the furan ring. As shown in
our binding model and the released X-ray crystal structure
(3IDP.pdb), the intramolecular hydrogen bonding between
the nitrogen of the purine and the hydrogen of the amine
linker constrains the hinge binder conformation (Figure 3).15
We checked the protein kinase activities and selectivity
of the most potent compound 4b. The kinase inhibitory
activities (IC50) of compound 4b against mutant BRAF,
wild-type BRAF, and CRAF were 2, 2, and 1 nM, respec-
tively (Table 5).18 The cell line growth inhibitory activities
were 7 and >10 000 nM against A375P (BRAFV600E) and
SK-MEL-2 (BRAFWT), respectively. While the ATP-
competitive BRAF inhibitors turn off the MAPK pathway
5o
5p
5q
21.03
0.97
5.91
>10
>10
>10
additional two hydrogen bonding interactions existed
between the sulfonyl oxygen of compound 4b and the hydro-
gen of the F595 and G596 backbone amides. There were two
van der Waals interactions sites. One was the difluorophenyl
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Table 6. Percentages of enzymatic inhibition by compound 4b at
a concentration of 10 μM on 30 selected protein kinases.
Kinase
% Inhibition
ABL1
AKT1
16
1
ALK
2
c-Kit
c-Src
CDK2/cyclin E
CDK5/p25
CHK1
DMPK
EGFR
ERK2/MAPK1
FAK/PTK2
FLT3
GSK3b
IGF1R
JAK2
JNK3
KDR/VEGFR2
MEK1
mTOR/FRAP1
P38a/MAPK14
p70S6K/RPS6KB1
PAK2
47
43
12
12
<0
<0
<0
<0
8
39
36
<0
<0
<0
9
Figure 2. The superimposed model of BRAF inhibitors, PLX4032
(green) based on an X-ray crystal structure (3OG7.pdb) and the
Amgen compound (cyan) and compound 3a from docking using
PyMol. The PLX4032 and Amgen compounds are depicted by
lines with yellow sticks indicating the binding site residues. The
compound 3a is represented by a magenta stick. The dashed line
indicates the hydrogen bonding interactions.
<0
4
4
<0
4
in cells bearing BRAF mutations, they lead to conforma-
tional changes to the wild-type BRAF kinase domain indu-
cing heterodimerization with CRAF causing a paradoxical
activation of MAPK pathway in cells bearing BRAF wild-
type cells with RAS mutations.16,19 Compound 4b also
showed selectivity against various other protein kinases. It
demonstrated weak inhibition against the selected 29 kinases
at a concentration of 10 μM (Table 6).20
PDGFRa
PKA
PKCa
PLK1
RAF1/CRAF
SYK
9
6
2
<0
99
8
TIE2/TEK
TRKB
38
6
Conclusion
In summary, compound 4b, which has type IIB characteris-
tics, was structure-based designed and synthesized. It
exhibited a high degree of kinase selectivity and had more
potent inhibitory activities against pan-RAF kinases and
cell lines bearing a BRAFV600E mutation than vemurafenib
(PLX4032). Preclinical development is ongoing for com-
pound 4b, and the design and synthesis of type IIB pan-
RAF inhibitors are also in progress.
Acknowledgment. This work was supported by the
Incheon National University Research Grant in 2014.
Table 5. The IC50 profiles of compound 4b against three RAF
protein kinases.
RAF kinase IC50(nM)
Figure 3. Compound 4b docking pose is represented by a
magenta stick with the yellow stick binding site residues of
BRAFV600E. The dashed line indicates hydrogen bonding interac-
tions. Intramolecular hydrogen bonding between the purine and
linker amine is also shown.
BRAFWT
BRAFV600E
CRAF
Compound 4b
Staurosporine
2
61
2
80
1
150
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docking studies, the structure of BRAF was taken from PDB
3OG7 (http://www.rcsb.org), in which the structure was
solved in a complex bound to PLX4032. Docking process
was performed using the program AutoDock 4.2.
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1
and spectral data. Selected data for compound 4b: H NMR
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8.21 (d, J = 4 Hz, 1H), 7.89 (s, 1H), 7.20 (d, J = 7 Hz, 2H),
7.10 (t, J = 7 Hz, 1H), 6.74 (s, 1H); 13C NMR (100 MHz,
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