Design and synthesis of N-(4-aminopyridin-2-yl)amides as B-RafV600E inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.04.076

B-Raf^V600E was an effective target for the treatment of human cancers. Based on a pan-Raf inhibitor TAK-632, a series of N-(4-aminopyridin-2-yl)amide derivatives were designed as novel B-Raf^V600E inhibitors. Detailed structure-activi

Full text of DOI:10.1016/j.bmcl.2016.04.076

Bioorganic & Medicinal Chemistry Letters 26 (2016) 2760–2763
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of N-(4-aminopyridin-2-yl)amides as B-RafV600E
inhibitors
a,y
a,y
a
b,c,y
b
b,c
b
b
Xiaokai Li , Jiayi Shen , Li Tan
, Zhang Zhang , Donglin Gao , Jinfeng Luo , Huimin Cheng ,
a
b,
⇑
d,b,
⇑
Xiaoping Zhou , Jie Ma , Ke Ding , Xiaoyun Lu
a
College of Pharmacy, Jilin University, Changchun 130021, China
State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, #190 Kaiyuan Avenue, Guangzhou 510530, China
University of Chinese Academy of Sciences, #19 Yuquan Road, Beijing 100049, China
b
c
d
School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China
a r t i c l e i n f o
a b s t r a c t
B-RafV600E was an effective target for the treatment of human cancers. Based on a pan-Raf inhibitor TAK-
6
Detailed structure–activity studies of the compounds revealed that most of the compounds displayed
Article history:
Received 2 March 2016
Revised 7 April 2016
Accepted 25 April 2016
Available online 26 April 2016
32, a series of N-(4-aminopyridin-2-yl)amide derivatives were designed as novel B-RafV600E inhibitors.
V600E
WT
potent enzymatic activity against B-Raf
, and good selectivity over B-Raf . One of the most promis-
V600E
ing compound 4l exhibited potent inhibitory activity with an IC50 value of 38 nM for B-raf
, and dis-
M,
respectively. It also displayed good selectivity on both enzymatic and cellular assays over B-Raf . These
played antiproliferative activities against colo205 and HT29 cells with IC50 values of 0.136 and 0.094
l
Keywords:
WT
V600E
B-Raf
inhibitor
V600E
inhibitors may serve as lead compounds for further developing novel B-Raf
drugs.
inhibitors as anticancer
N-(4-Aminopyridin-2-yl)amides
Structural modification
Selective
Ó 2016 Elsevier Ltd. All rights reserved.
The Ras-Raf-Mek-Erk signaling pathway plays a critical role in
regulating cell growth, proliferation and differentiation.1 Among
Cl
N
NH
2
the serine/threonine kinases Raf (A-Raf, B-Raf, and C-Raf) family,
the B-Raf aberrant activation or constitutively activating mutation
N
S
F
O
S
O
F
O
F
2,3
H
N
N
H
has been identified in various cancers. The single point mutation
S
O
F
N
6
00
600
N
F
Val
? Glu
accounts for more than 90% of the B-Raf oncogenic
O
V600E
N
H
mutants. Thus, B-Raf
treatment of human cancers harboring this mutation.
became as an effective target for the
4
Vemurafenib (1)
Dabrafinib (2)
Many classes of B-Raf small molecule inhibitors have been iden-
5
tified. Two selective inhibitors vemurafenib (1) and dabrafenib (2)
Figure 1. The chemical structures of FDA-approved B-Raf inhibitors vemurafenib
have been approved by US FDA (Fig. 1),6 and shown significant
,7
and dabrafinib.
efficacy against metastatic and unresectable melanoma bearing
V600E
8,9
B-Raf
mutation in clinical. However, intrinsic and acquired
inhibitors to overcome this clinical resistance have been published
by our group.
As part of our continuous efforts to identify new molecules that
resistance in clinic limits the therapeutic benefit of current used
1
3,14
1
0,11
drugs.
For instance, a number of colorectal cancer patients
V600E
who were detected to harbor B-Raf
mutation also display
V600E
inherent resistance against vemurafenib.12 As acquired resistance
could target B-Raf
mutant, a series of N-(4-aminopyridin-2-yl)
V600E
V600E
amide derivatives were designed as novel B-Raf
inhibitors by
increasingly observed in the clinic, novel selective B-Raf
inhi-
structural modifications based on a pan-Raf inhibitor TAK-6321
under the ‘atomic economy’ principle (Fig. 2). N-(4-Aminopy-
ridin-2-yl)amide scaffold was designed to bind to the hinge region
instead of 1,3-benzothiazole amide of TAK-632, and substituted
phenylamide targeted the hydrophobic back pocket of B-Raf. Some
5
bitors are still desirable to not only suppress resistance but also
display reduced side effects. Drug discovery efforts directed toward
new B-Raf inhibitors alone or in combination with EGFR kinase
⇑
Corresponding authors. Tel.: +86 20 32015276; fax: +86 20 32015299.
E-mail addresses: ding_ke@gibh.ac.cn (K. Ding), lu_xiaoyun@gibh.ac.cn (X. Lu).
These authors contribute equally to the work.
N-(4-aminopyridin-2-yl)amide derivatives were found as potent
V600E
inhibitors of B-Raf
. In this Letter, we report the synthesis,
y
http://dx.doi.org/10.1016/j.bmcl.2016.04.076
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
0

X. Li et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2760–2763
2761
F
O
N
O
N
S
Structural modifications
H
N
O
R1
N
H
N
H
N
H
R2
O
N
H
CF
3
O
CN
3
(TAK-632)
4a-4t
Figure 2. Design of N-(4-aminopyridin-2-yl)amide derivatives as B- RafV600E inhibitors.
Cl
N
Cl
N
HN
NO
2
O
H
2
N
NO
2
O
4
O
R1
+
Cl
a
R1
N
H
b
H
2
N
R1
N
H
N
5
6
7
8
R2
O
HN
N
NH
2
HO
HN
N
N
H
R2
O
10
O
O
c
R1
d
N
H
R1
N
H
4a-4t
9
Scheme 1. Reagents and conditions: (a) Et
3
N, DCM, overnight, rt, 60–90%; (b) Pd(OAc)
2
, Binap, Cs
2
CO
3
, dioxane, 90 °C, overnight, 55–75%; (c) H
2
, Pd/C, MeOH, rt, overnight,
6
0–85%; (d) HATU, DIEA, DMF, rt, overnight, 70–90%.
structure–activity relationships (SAR) and antiproliferative activity
Further exploration suggested that introduction of various lipophi-
lic groups at the meta-position instead of CF can exerted great
V600E
studies
of
N-(4-aminopyridin-2-yl)amide-based
B-Raf
3
inhibitors.
effect on kinase inhibition. Compounds 4g and 4h with relatively
small lipophilic substituents displayed less potent or comparable
potency to 4a, While compounds with increasing size of lipophilic
substituents such as ethyl, isopropyl, t-butyl, cyclopropyl,
2-methyl-2-propanenitrile, cyclopropanecarbonitrile (4h–4m),
exhibited 1.8- to 10.8-folds improvement on kinase inhibitory
activities. Additionally, we investigated whether cyclopropyl is
the optimal group in the hinge-binding moiety N-(4-aminopy-
ridin-2-yl)amide. It was found that with the increasing size of
cycloalkyl (4r–4t), the corresponding compounds’ potency
decreased gradually. The same trend was observed in alkyl ana-
logues, with compound 4n harboring a methyl group being the
The synthesis of target compounds 4a–4t is outlined in
Scheme 1. Briefly, the amide 7 was afforded by amide condensation
of 4-chloropyridin-2-amine (5) with freshly prepared acyl chloride
6. A Buchwald–Hartwig coupling reaction with amide 7 and
3
-nitroaniline (4) provided substituted 4-aminopyridin 8. Reduc-
tion of the intermediate 8 with hydrogen in the presence of Pd/C
gave the key intermediate 9, which was coupled with variable sub-
stituted benzoic acid 10 to get the desired compounds 4a–4t by
using 1-[bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]
pyridinium 3-oxidhexa fluorophosphate (HATU) and diisopropy-
lethylamine (DIEA) in anhydrous DMF at room temperature. All
desired compounds were given satisfactory analytical and spectro-
scopic data in accordance with their depicted structure.
most potent with IC50 value of 0.05
l
M, in contrast to compound
M.
4q with t-butyl with IC50 value of 1.03
l
Kinase inhibitory activities of the designed compounds against
Compounds 4i–4n stood out in the above structure activity
studies for they exhibited comparable potency to vemurafenib
and TAK-632. We further determined the selectivity of these
V600E
WT
B-Raf
FRET-based Z -Lyte assay.
32 were used as positive controls to validate the screening condi-
tions. As shown in Table 1, vemurafenib and TAK-632 both
and B-Raf were evaluated by using a well established
0
13,14
FDA-approved drug 1 as well as TAK-
V600E
WT
6
B-Raf
inhibitors over B-Raf . The results are summarized in
WT
Table 2. It was shown that compounds 4i–4n inhibited B-Raf
V600E
potently suppressed the B-Raf
5 nM, respectively, which were similar to the reported data. ,15
Our designed initial lead 4a with a trifluoromethyl group at the
meta position on phenylamide was discovered to exhibit potent
with IC50 values of 59 nM and
with IC50 values of 0.148, 0.240, 0.453, 0.476, 2.031 and
6
1
1.200
lM, respectively, which are 11.4, 12.0, 12.9, 12.5, 25.7 and
V600E
24-folds greater than IC50 values on the according B-Raf
.
While the ratios for vemurafenib and TAK-632 are only 2.0 and
V600E
V600E
B-Raf
kinase inhibitory activity with an IC50 of 0.141
l
M, indi-
5.1-folds, which indicate the good selectivity of these B-Raf
WT
cating this novel scaffold might be a good starting point for devel-
inhibitors over B-Raf . To our knowledge, 4m is one of the best
V600E
V600E
WT
opment of potent inhibitors to target B-Raf
(Table 1).
selective B-Raf
inhibitors over B-Raf
.
Therefore, extensive structure activity studies have been con-
ducted around this new scaffold. Initial investigation revealed that
the position of trifluoromethyl on phenylamide is essential for
maintaining kinase inhibitory activity, since ortho- and para-triflu-
The antiproliferative activities of 4i–4n were examined against
V600E
Colo205 and HT29 cells harboring B-Raf
mutation. The results
were summarized in Table 2. The compounds displayed compara-
V600E
ble or slightly weak antiproliferative effects against B-Raf
oromethyl (4b and 4c) totally abolished their suppressing effect on
mutated cancer cells to that of vemurafenib and TAK-632.
Compounds 4k and 4l potently inhibited the growth of Colo205
and HT29 cells with nanomolar magnitude, which were equally
potent to vemurafenib. Furthermore, the compounds (4i–4n) dis-
played less potent activity against HCT116 harboring B-Raf than
Colo205 and HT29 cells, which are consistent with the kinase
V600E
B-Raf
3
kinase (IC50 >10 lM). Next, we replaced CF with other
electronegative groups on meta-position, the results were summa-
rized in Table 1. It showed that compounds 4d–4f featured with Cl,
WT
NO
2
, CN suffered a significant decrease in biochemical potential to
M.
different extent with IC50 values of 0.785, 1.512 and 3.53
l

2
762
X. Li et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2760–2763
Table 1
V600E
Inhibitory activities of compounds 4a–4t against B-Raf
O
N
O
R1
N
H
N
H
N
H
R2
Compds
R1
R2
B-RafV600E (IC50
, lM)
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
m-CF
3
0.141
>10
o-CF
p-CF
m-Cl
3
Figure 3. Compounds 4k and 4l dose-dependently inhibited the autophosphory-
V600E
lation of B-Raf
in Colo205 cells.
3
>10
0.785
1.512
3.53
m-NO
2
Colo205 cells. The results further support strong target inhibition
of the newly designed compounds.
m-CN
g
h
i
m-CH
3
2
1.21
In summary, a series of N-(4-aminopyridin-2-yl)amide deriva-
V600E
tives are designed and synthesized as new B-raf
inhibitors.
m-CH
CH
3
0.11
The compounds 4i–4n displayed good inhibitory activities against
m-CH(CH
3
)
2
0.013
0.020
0.035
V600E
WT
B-Raf
, and good selectivity over B-Raf , which are better than
j
m-C(CH
)
3 3
the control drugs vemurafenib and TAK-632. Compound 4l was the
most promising compound with the potent inhibitory activity for
k
m-
V600E
B-raf
(IC50 = 38 nM), and good antiproliferative activity
M) and HT29 (IC50 = 0.094 M),
respectively. Therefore, compound 4l might be a promising lead
against colo205 (IC50 = 0.136
l
l
4
4
l
0.038
0.079
m-
CN
CN
V600E
compound for development of novel selective B-raf
overcoming the clinical acquired resistance.
inhibitors
m
m-
m-
m-
m-
m-
m-
m-
4
4
4
4
4
4
4
n
o
p
q
r
CH
3
0.050
0.125
0.420
1.03
Acknowledgments
CH
2
CH
3
We thank National High Technology Research and Development
(863) for Young Scientists program (No. 2015AA020906),
Guangdong Natural Science Funds for Distinguished Young Scholar
3 2
CH(CH )
C(CH
3
)
3
(2015A030306042),
Guangdong
Natural
Science
Funds
(
21302186), the Pearl River S&T Nova Program of Guangzhou
0.182
0.434
0.563
(201506010086), and Guangdong special branch program
Technology Young talents (2014TQ01R341).
s
t
Supplementary data
m-
—
—
Vemurafenib
TAK-632
—
—
0.059
0.015
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.04.
0
76.
Table 2
Kinase and cell inhibitory activities of compounds 4i–4n
References and notes
Compds Kinase inhibition WT:
Cell growth inhibition (IC50, lM)
1
2
.
.
Peyssonnaux, C.; Eychène, A. Biol. Cell. 2001, 93, 53.
(
IC50
,
l
M)
V600E
Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.;
Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.;
Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.;
Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.;
Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.;
Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.;
Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W.; Leung, S. Y.; Yuen, S. T.; Weber,
B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.; Marshall, C. J.;
Wooster, R.; Stratton, M. R.; Futreal, P. A. Nature 2002, 417, 949.
B-RafV600E B-RafWT
Colo205
B-Raf
HT29
HCT116
V600E
) (B-RafV600E) (B-Raf
WT
(
)
4
4
4
4
4
4
i
j
k
l
m
n
0.013
0.020
0.035
0.038
0.079
0.050
0.148
0.240
0.453
0.476
2.031
1.200
0.119
0.077
11.4
12.0
12.9
12.5
25.7
24.0
2.0
0.444
0.787
0.111
0.136
0.725
0.131
0.044
0.025
0.462
0.991
0.092
0.094
0.791
0.236
0.156
0.033
1.055
1.385
0.221
1.091
1.351
0.367
14.58
0.362
3
4
.
.
Maurer, G.; Tarkowski, B.; Baccarini, M. Oncogene 2011, 30, 3477.
Holderfield, M.; Deuker, M. M.; McCormick, F.; McMahon, M. Nat. Rev. Cancer
Vemurafenib 0.059
TAK-632 0.015
2014, 14, 455.
5.1
5
6
.
.
Wang, X.; Kim, J. J. Med. Chem. 2012, 55, 7332.
Bollag, G.; Hirth, P.; Tsai, J.; Zhang, J.; Ibrahim, P. N.; Cho, H.; Spevak, W.; Zhang,
C.; Zhang, Y.; Habets, G.; Burton, E. A.; Wong, B.; Tsang, G.; West, B. L.; Powell,
B.; Shellooe, R.; Marimuthu, A.; Nguyen, H.; Zhang, K. Y.; Artis, D. R.;
Schlessinger, J.; Su, F.; Higgins, B.; Iyer, R.; D’Andrea, K.; Koehler, A.; Stumm,
M.; Lin, P. S.; Lee, R. J.; Grippo, J.; Puzanov, I.; Kim, K. B.; Ribas, A.; McArthur, G.
A.; Sosman, J. A.; Chapman, P. B.; Flaherty, K. T.; Xu, X.; Nathanson, K. L.; Nolop,
K. Nature 2010, 467, 596.
Rheault, T. R.; Stellwagen, J. C.; Adjabeng, G. M.; Hornberger, K. R.; Petrov, K. G.;
Waterson, A. G.; Dickerson, S. H.; Mook, R. A., Jr.; Laquerre, S. G.; King, A. J.;
Rossanese, O. W.; Arnone, M. R.; Smitheman, K. N.; Kane-Carson, L. S.; Han, C.;
Moorthy, G. S.; Moss, K. G.; Uehling, D. E. ACS Med. Chem. Lett. 2013, 4, 358.
inhibition results. For example, compound 4l inhibited colo205 and
HT29 cells with IC50 values of 0.136 and 0.094 M, which is almost
l
1
2 times more selective than that of HCT116.
V600E
The B-Raf
kinase inhibitory activity of compounds 4k and
7.
4
l were further validated by using western-blot analysis. As shown
in Figure 3, compounds 4k and 4l both displayed dose-dependent
inhibition against the autophosphorylation of B-Raf
V600E
in

X. Li et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2760–2763
2763
8
.
.
Bollag, G.; Tsai, J.; Zhang, J.; Zhang, C.; Ibrahim, P.; Nolop, K.; Hirth, P. Nat. Rev.
Drug. Disc. 2012, 11, 873.
Ballantyne, A. D.; Garnock-Jones, K. P. Drugs 2013, 73, 1367.
12. Walker, E. M.; Rodriguez, A. I.; Kohn, B.; Ball, R. M.; Pegg, J.; Pocock, J. R.; Nunez,
R.; Peterson, E.; Jakary, S.; Levine, R. A. J. Clin. Oncol. 2010, 28, 634.
13. Cheng, H.; Chang, Y.; Zhang, L.; Luo, J.; Tu, Z.; Lu, X.; Zhang, Q.; Lu, J.; Ren, X.;
Ding, K. J. Med. Chem. 2014, 57, 2692.
14. Li, Y.; Cheng, H.; Zhang, Z.; Zhuang, X.; Luo, J.; Long, H.; Zhou, Y.; Xu, Y.;
Taghipouran, R.; Li, D.; Patterson, A.; Smaill, J.; Tu, Z.; Wu, D.; Ren, X.; Ding, K.
ACS Med. Chem. Lett. 2015, 6, 543.
15. Okaniwa, M.; Hirose, M.; Arita, T.; Yabuki, M.; Nakamura, A.; Takagi, T.;
Kawamoto, T.; Uchiyama, N.; Sumita, A.; Tsutsumi, S.; Tottori, T.; Inui, Y.; Sang,
B. C.; Yano, J.; Aertgeerts, K.; Yoshida, S.; Ishikawa, T. J. Med. Chem. 2013, 56,
6478.
9
1
1
0. Johannessen, C. M.; Boehm, J. S.; Kim, S. Y.; Thomas, S. R.; Wardwell, L.;
Johnson, L. A.; Emery, C. M.; Stransky, N.; Cogdill, A. P.; Barretina, J.; Caponigro,
G.; Hieronymus, H.; Murray, R. R.; Salehi-Ashtiani, K.; Hill, D. E.; Vidal, M.;
Zhao, J. J.; Yang, X.; Alkan, O.; Kim, S.; Harris, J. L.; Wilson, C. J.; Myer, V. E.;
Finan, P. M.; Root, D. E.; Roberts, T. M.; Golub, T.; Flaherty, K. T.; Dummer, R.;
Weber, B. L.; Sellers, W. R.; Schlegel, R.; Wargo, J. A.; Hahn, W. C.; Garraway, L.
A. Nature 2010, 468, 968.
1. Lito, P.; Rosen, N.; Solit, D. B. Nat. Med. 2013, 19, 1401.