Design and Synthesis of Novel 2,4-Diamino-5-pyrazol-4-yl Pyrimidine Derivatives as Selective Tyro3 Kinase Inhibitors

Full text of DOI:10.1002/bkcs.11541

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DOI: 10.1002/bkcs.11541
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D. Kim et al.
KOREAN CHEMICAL SOCIETY
Design and Synthesis of Novel 2,4-Diamino-5-pyrazol-4-yl Pyrimidine
Derivatives as Selective Tyro3 Kinase Inhibitors
Dukwoon Kim,† Kyung Won Lee,‡,§ Hyunseok Jung,† Miok Kim,‡,§ Joo-Youn Lee,¶
Yeonkyung Lee,k Jong Yeon Hwang,‡,§,** Youngki Min,‡,§ Chang Hoon Lee,‡,§, and Sung
*
Yun Cho‡,§,
*
†Department of Chemistry, Sungkyunkwan University, Suwon 440-746, South Korea
‡Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience & Biotechnology,
Daejeon 34141, South Korea
§Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 34114,
South Korea. *E-mail: sycho@krict.re.kr
¶Drug Information Platform Center, Korea Research Institute of Chemical Technology, Daejeon 34114,
South Korea
kCollege of Pharmacy, Chungnam National University, Daejeon 34134, South Korea
**Department of Medicinal and Pharmaceutical Chemistry, University of Science and Technology,
Daejeon 305-550, South Korea
Received April 19, 2018, Accepted June 26, 2018
Keywords: TAM, Tyro3 kinase inhibitors, Anticancer agent
The TAM (Tyro3, Axl, Mer) is a small family of kinase
which was initially identified and cloned as a full-length
Tyro3 by Crosier et al.1 The TAM receptor tyrosine kinases
and their cognate ligands GAS6 (growth arrest-specific 6)
and Pros1 (protein S) play an important role in the pro-
cesses of chronic inflammatory2 and autoimmune diseases.3
Knockouts of TAM receptors develop autoimmune dis-
eases, including rheumatoid arthritis and lupus.4 Moreover,
the TAM family of receptor tyrosine kinase and Gas6 are
aberrantly overexpressed in multiple hematological and epi-
thelial malignancies in solid tumor.5 Although, the role of
Gas6/TAM receptor in solid tumors is not fully understood,
Gas6/TAM expression was shown to be higher in tumor tis-
sue, and the correlation to survival was not confirmed for
different tumors.6 Of the TAM receptor, Tyro3 is less stud-
ied and recently identified as Tyro3 expression decreases
survival of malignant melanoma cell.7 Expression level of
Tyro3 has been evaluated in Gas6-mediated Akt-phosphor-
ylation, and Tyro3 was specifically overexpressed in
numerous melanoma cell lines.7 Short-hairpin RNA-
mediated knockdown of Tyro3 led to significant cell death
via apoptotic mechanism regardless of BRAF mutation or
NRAS mutation status in melanoma cell lines, which reflect
the strong possibility for the efficient therapeutics in resis-
tant or refractory melanoma patients.7 Recently, it has been
reported that Tyro3 is being proposed as a drug target for
breast cancer, colorectal cancer, multiple myeloma and lung
carcinoma.8 It has been reported that Tyro3 was emerged
as a potential therapeutic target in breast cancers especially
for estrogen receptor positive/HER2 (human epidermal
growth factor receptor 2)-non-amplified (luminal type) and
estrogen negative/Her2 amplified (HER2 type cell) not
triple negative cells by Tyro3 knockdown experiment.9 Fur-
thermore, Tyro3 was strongly upregulated in the tumor tis-
sue of 42% of patients, and Tyro3 was deeply correlated
with the key marker AFP (alpha-fetoprotein) and ALT (ala-
nine aminotransferase) in hepatocellular carcinoma.10
Thus, the development of inhibitors against TAM recep-
tors, particularly for Tyro3 selective inhibitor,11 has been
considered as a possible methodology to overcome the side
effects5,12 of TAM receptor inhibition and improve resis-
tance from anticancer agents. To date, a number of Tyro3
inhibitors have been identified and being investigated in
biological testing or in the early stage of preclinical trial.
(Figure 1) Among the compounds, LDC1267 (Max-Planck-
Gesellschaft, Munich, Germany) displayed strong potency
against Axl, Tyro3, and Mer kinases. UNC569 (University
of North Carolina, Chapel Hill, NC, USA) was observed in
some degree of selectivity against Tyro3 over Axl, and Mer
kinase. 1 and 2 (Pfizer, New York, NY, USA) were identi-
fied and developed as highly selective pyrimidine deriva-
tives against Tyro3.11
In this report, we synthesized a series of novel N-cyclo-
hexylpyrimidin-4-amine derivatives that were substituted
with aniline moieties and evaluated structural-activity rela-
tionship (SAR) studies for TAM kinase inhibitory activity
by me-too approach focused on the structures 2 (Figure 1).
Some of the compounds displayed excellent selectivity
toward Tyro3 in vitro enzyme assay. The general synthetic
methods adopted in the preparation of N-cyclohexyl-5-pyra-
zol-4-yl-phenylpyrimidin-2,4-diamine derivatives are out-
lined in Scheme 1.11
Starting from a 5-bromo-2,4-dichloropyrimidine, 4-cyclo-
hexylamino-5-bromo-2-chloropyrimidine (3) was obtained by
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assay against TAM receptors (Tyro3, Axl, Mer kinases)” in
Appendix S1 (Supporting information)] toward Tyro3, Axl,
and Mer kinases, respectively. The results were summarized in
Table 1. Bromo compound (5a) displayed low degree of
inhibitory activity in Axl. Introduction of 3,5-
dichlorophenylamino at 2 position of pyrimidine, compound
5b, micromolar range of inhibitory activity toward Tyro3 was
observed. Interestingly, introduction of 4-methylphenylamino
both at 2 and 4 positions, compound (6a), resulted in highly
potent inhibitory activity toward Axl kinase. When we intro-
duced methylpyrazole group at 5 position of pyrimidines
instead of phenyl group, no inhibitory activity toward Tyro3
was observed with compound (4a), which has chlorine at
2 position of pyrimidine. We decided to introduce aromatic
groups at 2 position, such as 5-methylisoxazol-3-amine, that
resulted in low degree of inhibitory activity (6b). In contrast,
introduction of 5-(tert-butyl)isoxazol-3-amine at 2 position of
pyrimidine (6c), slight increase of inhibitory activity against
Axl and Mer was observed with no inhibitory activity toward
Tyro3. Introduction of benzylamine group (6d) at 2 position
resulted in increase of Tyro3 and Axl inhibitory activity with
no activity in Mer kinase. On the contrary, introduction of
polar substituent 2-(4-pyridyl)ethylamine group (6f ) displayed
results with the inhibitory activity in Axl and Tyro3 with no
enzymatic activity in Mer kinase inhibition. In case of
piperazin-1-yl(thiophen-2-yl)methanone (6g) at the same posi-
tion of pyrimidine, selectivity and inhibitory activity were
almost equivalent to that of compound 6d. More potent inhibi-
tory activity against Mer tyrosine kinase was observed with tri-
fluoromethylbenzylamine group (6e) at 3 position of phenyl
substituent with moderate activity in Tyro3 and Axl kinases.
We next decided to introduce phenylamine instead of hetero-
cyclic aromatic amine group. In this result, inhibitory activity
and selectivity toward Tyro3 were dramatically increased with
high degree of inhibitory activity against Tyro3 enzyme. Intro-
duction of phenylamine (6h) secured selectivity toward Tyro3
with micromolar inhibitory activity except Axl and Mer
enzymes. Enhancement of inhibitory activity was observed in
the introduction of 3-chlorophenylamine (6i) with no inhibi-
tory activity in Axl and low micromolar range of Mer kinase.
Introduction of 3,5-dichlorophenylamino group at 2-position
of pyrimidine (6j) displayed excellent selectivity over Axl
and Mer kinases retaining strong inhibitory potency against
Tyro3 kinase. Substitution of 3-chloro-4-methoxyphenylamino
group (6k), 2-methylphenylamino group (6l), and 2-
isopropylphenylamino group (6m) were detrimental to the
inhibitory activity toward all of TAM kinases. Moderate inhib-
itory activity against Tyro3 and Mer kinases was observed in
compound (6n) of 3-methylphenylamino group with no activ-
ity in Axl kinase. Introduction of 3-furyl (6o) and (6p) instead
of pyrazol of compound was turned out to be little beneficial
to inhibition of TAM kinases. In order to assess the binding
mode of 6j on Tyro3 kinase, we performed flexible docking
study using Schrödinger Suite 2018–1 (“Molecular modelling
study for 6j” in Appendix S1). Pyrimidin-2-amine scaffold of
6j made hydrogen bonding with Met596 carbonyl and amide
Figure 1. TAM inhibitors.1
Scheme 1. Synthesis of N4-cyclohexyl-5-aryl-N2-arylpyrimidine-2,-
4-diamine. Reagents and conditions: (a) cyclohexylamine, DIPEA,
DMF, rt., 18 h, 65%; (b) R1B(OH)2, Pd(dppf )2Cl2, K2CO3, DMF,
80 ꢀC, 98%; (c) R2NH2, Et3N, CH2Cl2, rt., 18 h, 39%.
simple substitution of cyclohexylamine in the presence of dii-
sopropylamine in DMF. Palladium catalyzed cross coupling of
3 with the appropriate arylboronic acid yielded 4-cyclohexyla-
mino-5-aryl-2-chloropyrimidines (4).11 Substitution of suitable
arylamine to compound (4) afforded the final 2-arylamin-
4-cyclohexylamino-5-arylpyrimidine derivatives 6. Our initial
structural-activity relationship studies in 2,4-diaminopyrimi-
dine-5-aryl series demonstrated that highly selective Tyro3
compound can be derived from the modification of aryls at
4-position and aryl amine functional group at the 2-position.
In an effort to secure excellent selectivity toward Tyro3 other
than Axl or Mer kinases, we explored and optimized in vitro
enzyme assay by structural modification of parent 2,4-diamine
substituted pyrimidines. To understand the SAR of
2,4-disubstituted pyrimidines, all the compounds were tested
in vitro enzyme assay [“Experimental procedure for enzyme
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Table 1. Results of TAM (Tyro3, Axl, and Mer) inhibition.
Entry
R1
R2
Cl
Tyro3 IC50 (μM)
Axl IC50 (μM)
Mer IC50 (μM)
4a
>10
8.5
>10
5a
5b
6a
6b
Br
Br
p-tolyl
a
p-tolylamino
3,5-dichlorophenylamino
p-tolylamino
>10
0.42
>10
>10
2.8
9.1
0.0015
4.1
>10
1.1
6.2
0.77
6c
a
a
>10
1.1
2.1
0.5
0.4
6d
>10
6e
6f
a
a
3-trifluoromethylbenzylamino
0.61
1.1
0.52
>10
0.085
0.52
6g
a
1.3
4.7
>10
6h
6i
6j
6k
6l
6m
6n
6o
a
a
a
a
a
a
a
phenylamino
1.4
0.39
0.028
>10
>10
>10
>10
>10
>10
>10
6.3
>10
>10
>10
>10
2.4
12
>10
1.2
>10
0.11
>10
3-chlorophenylamino
3,5-dichlorophenylamino
3-Cl-4-methoxyphenylamino
2-methylphenylamino
2-isopropylphenylamino
3-methylphenylamino
4-methylphenylamino
0.16
>10
6p
b
3,5-dichlorophenylamino
0.62
>10
>10
NH in the hinge region. Also pyrimidine ring formed hydro-
phobic interactions with Leu514, Ala538, Phe595, and
Met652. The 1-methylpyrazole moiety of 6j was positioned to
the inside hydrophobic pocket including Val522, Ala571,
Leu593, and Ala662, as well as 3,5-dichlorophenyl group was
oriented toward solvent exposed area by forming hydrophobic
interactions with Leu514. In addition, the orientation of cyclo-
hexylamine sidechain was similar to that of 3-aminopropyl
substituent of inhibitor bound to Tyro3 kinase. As shown in
Figure S1 (Appendix S1), the proposed binding model of 6j
has similar binding mode to that of X-ray co-crystal structure
in complexed with 2,4-diaminopyrimidine-5-carboxamide
analog.
identification of analog 6j as a potent Tyro3 kinase inhibi-
tor with excellent selectivity over other TAM family kinase,
such as Axl and Mer. Besides, compound 6a displayed
excellent inhibitory activity against Axl tyrosine kinase and
selectivity over Tyro3 and Mer kinase. We found that it is
possible to identify selective either Tyro 3 or Axl kinase by
simple structural modification of 2,4,5-trisubstituted pyrimi-
dines. Further studies on the development of potent and
selective Tyro3 inhibitors and biological studies are being
planned.
Acknowledgments. This study was financially supported
by the Korea Research Institute of Chemical Technology
(SKM-1714 CHL).
In summary, we synthesized
a novel series of
2,4-disubstituted pyrimidine with aromatic amine and
5 position of pyrimidine with aryl group, such as pyrazole
group and assayed TAM kinases, Tyro3, Axl, and Mer
kinases in vitro enzyme assay. This effort resulted in the
Supporting Information. Additional supporting informa-
tion may be found online in the Supporting Information
section at the end of the article.
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8. (a) S. Zhu, H. Wurdak, Y. Wang, A. Galkin, H. Tao, J. Li,
Proc. Natl. Acad. Sci. 2009, 106, 17025. (b) R. C. Eyalongo,
T. Mukohara, Y. Funakoshi, H. Tomioka, Y. Kataoka,
Y. Shimono, N. Chayahara, M. Toyoda, N. Kiyota,
H. Minami, Anticancer Res. 2014, 34, 3337.
References
1. P. S. Crosier, P. M. Lewis, L. R. Hall, M. R. Vitas,
C. M. Morris, D. R. Beier, C. R. Wood, K. E. Croiser,
Growth Factors 1994, 11, 125.
9. J. De Vos, G. Couderc, K. Tarte, M. Jourdan, G. Requirand,
M. C. Delteil, Blood 2001, 98, 771.
2. C. V. Rothlin, S. Ghosh, E. I. Zuniga, M. B. Oldstone,
G. Lemke, Cell 2007, 2007, 131, 1124.
10. Y. Duan, W. Wong, S. C. Chua, H. L. Wee, S. G. Lim,
B. T. Chua, H. K. Ho, Int. J. Oncol. 2016, 48, 358.
3. S. E. Carrera, P. Y. Chan, L. Joannas, A. E. Errasti,
N. Gagliani, Immunity 2013, 39, 160.
11. (a) N. A. Powell, J. T. Kohrt, K. J. Filipski, M. Kaufman,
D. Sheehan, J. E. Edmunds, A. Delaney, Y. Wang,
F. Bourbonais, D. -Y. Lee, F. Schwende, F. Sun,
P. McConnell, C. Catana, H. Chen, J. Ohren, L. A. Perrin,
Bioorg. Med. Chem. Lett. 2012, 22, 190. (b) T. Traoré,
A. Cavagnino, N. Saettel, F. Radvanyi, S. Piguel, I. Bernard-
Pierrot, V. Stoven, M. Legraverend, Eur. J. Med. Chem.
2013, 70, 789. (c) K. L. Billingsley, K. W. Anderson,
S. L. Buchwald, Angew. Chem. Int. Ed. 2006, 45, 3483.
12. A. Donner, SciBX 2014, 7, 1.
4. P. L. Cohen, R. Caricchio, V. Abraham, T. D. Camenisch,
J. C. Jennette, R. A. Roubey, H. S. Earp, G. Matsushima,
E. A. Reap, J. Exp. Med. 2002, 196, 135.
5. D. K. Graham, D. DeRyckere, K. D. Davies, H. S. Earp, Nat.
Rev. Cancer 2014, 14, 769.
6. T. Schmidt, I. Ben-Batalla, A. Schultze, S. Loges, Cell Mol.
Life Sci. 2012, 69, 1391.
7. S. J. Demarest, J. Gardner, M. C. Vendel, E. Ailor, S. Szak,
F. Huang, A. Doern, X. Tan, W. Yang, D. A. Grueneberg,
E. J. Richards, W. O. Endege, E. Harlow, Biochemistry 2013,
52, 3102.
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