Synthesis and structure–activity relationship study of pyrazolo[3,4-d]pyrimidines as tyrosine kinase RET inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.03.088

Three series of pyrazolo[3,4-d]pyrimidine derivatives were synthesized and evaluated as RET kinase inhibitors. Compounds 23a and 23c were identified to show significant activity both in the biochemical and the BaF3/CCDC6-RET cell assays. Compound 23c was found to significantly inhibit RET phosphorylation and down-stream signaling in BaF3/CCDC6-RET cells, confirming its potent cellular RET-targeting profile. Different from other RET inhibitors with equal potency against KDR that associated with severe toxicity, 23c did not show significant KDR-inhibition even at the concentration of 1?μM. These results demonstrated that 23c is a potent and selective RET inhibitor.

Full text of DOI:10.1016/j.bmcl.2017.03.088

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationship study of pyrazolo[3,4-d]
pyrimidines as tyrosine kinase RET inhibitors
Chengyan Wang a,d, Hongchun Liu c,d, Zilan Song b, Yinchun Ji c, Li Xing b, Xia Peng c, Xisheng Wang a,
,
⇑
Jing Ai c, , Meiyu Geng c, Ao Zhang b,
⇑
⇑
a Nano Science and Technology Institute, University of Science and Technology of China, Suzhou 215123, China
b CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China
c Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three series of pyrazolo[3,4-d]pyrimidine derivatives were synthesized and evaluated as RET kinase inhi-
bitors. Compounds 23a and 23c were identified to show significant activity both in the biochemical and
the BaF3/CCDC6-RET cell assays. Compound 23c was found to significantly inhibit RET phosphorylation
and down-stream signaling in BaF3/CCDC6-RET cells, confirming its potent cellular RET-targeting profile.
Different from other RET inhibitors with equal potency against KDR that associated with severe toxicity,
Received 11 February 2017
Revised 27 March 2017
Accepted 29 March 2017
Available online xxxx
23c did not show significant KDR-inhibition even at the concentration of 1
strated that 23c is a potent and selective RET inhibitor.
lM. These results demon-
Keywords:
Tyrosine kinase RET
Pyrazolo[3,4-d]pyrimidine
Anticancer
Ó 2017 Elsevier Ltd. All rights reserved.
Cell proliferation
Structure-activity relationship
The transmembrane receptor tyrosine kinase RET (REarranged
during Transfection) is mainly expressed in both central and
peripheral nervous systems.1,2 Collective studies have shown that
activated RET oncogenes via mutations, amplifications or fusions
are involved in the pathogenesis of many human cancers.3 For
example, gain of function mutation in RET is relevant to multiple
endocrine neoplasia (MEN 2A and 2B), familial medullary thyroid
carcinoma (FMTC), and papillary thyroid carcinoma (PTC).4–7
Recently, several chimeric fusion partners of RET (KIF5B-RET,
CCDC6-RET, TRIM33-RET NCOA4-RET) have been identified in 1–
2% of non-small cell lung cancers (NSCLC) that led to abnormal
activation of RET transcription and subsequent tumorigenesis.8
Therefore, RET has been recognized as an emergent molecular tar-
get for cancer treatment.9,10 Small molecule RET-targeting inhibi-
tors cabozantinib (XL-184)11 and vandetanib12 have been granted
FDA’s approval recently for the treatment of MTC. In addition,
ponatinib from Ariad is another RET inhibitor that recently under-
going phase II clinical trials to treat RET-rearranged NSCLC.14,15
Notably, all these RET inhibitors are nonselective with high
potency against several kinases other than RET, especially
VEGFR-2 that correlates to many off-target effects such as rash,
diarrhea, hypertension and others.13,16–18
Therefore, new inhibitors selectively targeting RET are highly
needed both to validate the sole contribution of RET-targeting to
the clinical antitumor efficacy and to minimize the off-target side
effects. Recently, pyrazolo[3,4-d]pyrimidines have been reported
to show selective inhibitory activity against RET, and the represen-
tative compounds 1 (7a in Ref. 20) and 2 (6i in Ref. 21) both
showed single-digit nanomolar potency against RET and much
improved selectivity against VEGFR-2. Unfortunately, these com-
pounds failed to show similar high potency in the cellular
assay.20,21 In this regard, we conducted a structure-activity rela-
tionship study based on the pyrazolo[3,4-d]pyrimidin skeleton,
by ring-closing to form a tricyclic center, insertion of an amido
moiety to fit the hydrophobic pocket and attachment of a func-
tional group to form additional interactions (Fig. 1). Herein, we
report our design and pharmacological investigation on these
new derivatives (3–5, Fig. 1).
The synthesis of compounds 10–12 and 18 was outlined in
Scheme 1. Commercially available iodide 6 was used as the starting
material to synthesize intermediate 7 by following literature pro-
cedures.20 N-Alkylation with 4-bromobut-1-ene in the presence
of potassium carbonate yielded 8. Bromination of 8 with NBS at
⇑
Corresponding authors.
E-mail addresses: xshengwang77@mail.ustc.edu.cn (X. Wang), jai@simm.ac.cn
(J. Ai), aozhang@simm.ac.cn (A. Zhang).
d
These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2017.03.088
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
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C. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 1. Known RET inhibitors 1–2 and our designed new inhibitors 3–5.
À20 °C produced 9. Compounds 10 and 11 were obtained via Heck
reaction from 9i and 9ii. Meanwhile, compound 12 was prepared
from 9ii by following literature procedures.23 In addition, treating
13 with but-3-en-2-ol via Mitsunobu reaction provided precursor
14. Subsequent Sonagashira coupling of 14 with 1-ethyl-4-
ethynylbenzene in the presence of Pd(PPh3)2Cl2/CuI provided 15,
which then underwent amination reaction with ammonium
hydroxide at 120 °C to yield 16.22 Compound 18 was obtained by
bromination of 16 to give 17, followed by hydroboronation with
9-BBN and subsequent Suzuki coupling in the presence of Pd
(dppf)Cl2.23
cyclopropanecarboxamides 21a–c displayed negligible activities
against RET. Benzamide 21d also lost the potency, whereas hetero-
cyclic amides 21e–i exhibited moderate potency with inhibitory
rates around 50% at the concentrations of 10 nM. The five-mem-
bered heterocycle-substituted carboxamides were slightly more
potent than the six-membered congeners (21f,g,i,j vs 21a–e).
5-Cyclopropylisoxazole 21j and furan-isoxazole 21l retained good
RET inhibition. The reduced activity of benzo[d]isoxazole 21k and
5-phenylisoxazoles 21m–o revealed that the hydrophobic pocket
of RET kinase was slightly narrow and only limited substituents
were tolerant.
Preparation of compounds 21a–o was described in Scheme 2.
Reaction of 4-amino-6-chloropyrimidine-5-carbonitrile with iso-
propyl-hydrazine hydrochloride produced 20.24 Acylation of 20
with corresponding acyl chlorides provided 21a–o. As shown in
Scheme 3, following the literature procedure,21 5-substituted isox-
azolines 23a–i were obtained from appropriate terminal alkynes
with the key intermediate 22.
All the prepared new compounds were screened against RET
kinase. As summarized in Table 1, the tricyclic compounds
exhibited moderate to good potency against RET. Compounds 10
and 11 bearing a 6-methylene-7,8-dihydro-6H-pyrimido[5,4-b]
pyrrolizine tricyclic center showed reduced potency compared
to reference 1 (>100 nM vs 56 nM). The 6,7,8,9-tetrahydropyrim-
ido[5,4-b]indolizine 12 also displayed slightly reduced potency
with an IC50 value of 102 nM. However, compound 18
bearing the 7,8-dihydro-6H-pyrimido[5,4-b]pyrrolizine center
showed an IC50 value of 62.5 nM, which is comparable to that of
reference 1.
Compounds 23a–i are a series of analogues of 2 but bearing a
more flexible side chain on the isoxazoline motif. As showed in
Table 3, hydroxymethyl substituted compound 23a turned out as
the most potent with an IC50 value less than 10 nM against RET
kinase. Capping the free hydroxyl (23b) or extending the carbon
chain (23c,d) of 23a led to reduced inhibitory activity. Esteric sub-
stituent (23e), or alkyl substituents bearing a terminal morpholinyl
(23f–h) or pyrrolyl (23i) moieties nearly lost the inhibitory
potency at the tested concentrations.
In view of the high inhibitory potency against RET at the con-
centration of 100 nM, compounds 23a and 23c were further evalu-
ated. As shown in Table 4, high was observed for 23c with an IC50
value of 61 nM, which is >6-fold more potent than 23a. In the
antiproliferative effect assay against the BaF3 cell harboring the
fusion gene CCDC6-RET, the reference 1, though quite potent in
the biochemical assay, did not show significant antiproliferative
effect in our cellular assay, with IC50 values greater than
1000 nM. Our new compounds 23a and 23c showed moderate cel-
lular potency against BaF3/CCDC6-RET cells with an IC50 value of
454 and 433 nM respectively.
Compounds 21a–o represent a series of 1H-pyrazolo[3,4-d]
pyrimidines bearing a C3-amido moiety. As shown in Table 2,
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C. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Scheme 1. Reagent and conditions: (a) Pd(PPh3)4 amberlitelR-67, Cul, THF, 120 °C, 30 min, 65%; (b) 4-bromobut-1-ene, K2CO3, DMF, 30 °C, 6 h, 83%; (c) NBS, À20 °C 1 h, 38–
47%; (d) NaOH, Pd(dppf)Cl2 70–80%, 12 h, ꢀ14%; (e) 1) 9-BBN, THE, 0 °C – rt, 6 h; 2) NaOH, Pd(dppf)Cl2, 70–80 °C, 12 h, ꢀ11% over two steps; (f) PPh3, DIAD, THF 0 °C – rt, 2 h,
94%; (g) Pd(PPh3)2Cl2, Cul, Et3N, N2, THF, 70 °C, 10 h; (h) ammonia hydrate, 1,4-dioxane, 120 °C,18 h, 56–64% over two steps.
Western blotting analysis of 23c was evaluated in BaF3/CCDC6-
RET cells. We found that 23c inhibited the phosphorylation of RET
and the phosphorylation of STAT3 and AKT, main downstream
effectors of RET signaling, in a dose-dependent manner in the
tested cell line (Fig. 2). These results suggested that 23c exhibited
an effective inhibition of RET signaling. In contrast to its high
potency against RET kinase, 23c did not show significant inhibitory
effect against VEGFR-2 kinase even at the concentration of 1 lM.
These results demonstrated that 23c is a potent and selective
RET inhibitor.
which was not observed in the biding mode of 1. Such additional
hydrogen bonding may partially contribute to the higher potency
of 23c than 1.
In summary, we have designed and synthesized three series of
pyrazolo[3,4-d]pyrimidine derivatives and tested their potency
against RET. Compounds 23a and 23c were identified to show sig-
nificant inhibition against both RET kinase and the proliferation of
BaF3/CCDC6-RET cells. Compound 23c significantly inhibited RET
phosphorylation and down-stream signaling in BaF3/CCDC6-RET
cells confirming its RET-selective targeting profile. More importantly,
different from other RET inhibitors with equal potency against KDR
that associated with severe toxicity, 23c did not show significant
The docking study of 23c was described in Fig. 3. In addition to
the essential interactions formed between RET kinase and the isox-
azol-3-yl-pyrazolo[5,4-d]pyrimidine scaffold, an additional hydro-
gen bond was formed between the hydroxyl and Glu775 (1.8 Å),
KDR-inhibition even at the concentration of 1
lM. These results
demonstrated that 23c is a potent and selective RET inhibitor.
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C. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 2
Biochemical assays against RET kinase.a
Compd
Inhibition, %
100 nM
10 nM
21a
21b
21c
21d
21e
21f
21g
21h
21i
21j
21k
21l
21m
21n
21o
4%
À4%
3%
9%
À15%
À16%
4%
11%
44%
57%
56%
48%
51%
57%
1%
54%
13%
16%
3%
53%
67%
63%
61%
51%
60%
25%
57%
44%
30%
13%
a
Inhibition % means inhibition rate against enzyme with the concentration (nM)
of corresponding compound.
Table 3
Biochemical assays against RET kinase.a
Compd
Inhibition, %
Scheme 2. Reagent and conditions: (a) isopropylhydrazine hydrochloride, MeOH,
TEA, 60 °C, 4 h, 75%; (b) 1) RCOOH, oxalylchloride, cat. DMF, DCM, N2, 0 °C, 3 h; 2)
DIPEA, THF, N2, À10 °C, 3 h, 25–65% in two steps.
100 nM
10 nM
23a
23b
23c
23d
23e
23f
23g
23h
23i
43%b
51%
73%
42%
37%
-21%
42%
23%
29%
18%c
22%
45%
34%
5.4%
0.8%
24%
7%
11%
b
and
a
cInhibition rates at the concentration of 250 nM and 15 nM respectively.
Inh% means inhibition rate against enzyme with the concentration (nM) of
corresponding compound.
Table 4
Antiproliferative effects against BaF3/CCDC6-RET cells.a
Compd
RET (IC50, nM)
BaF3/CCDC6-RET cell (IC50, nM)
23a
23c
1
410 195
61.2 35.5
56.5 2.1
454 7.4
433 15.4
>1000
a
Cell proliferation was detected by microculture tetrozolium (MTT) as described
in Experimental Section. IC50 values are given as the mean SD (nM) from two
separate experiments.
Scheme 3. Reagent and conditions: MeOH/H2O (5/1), N2, 0 °C, -rt, 15–24 h, 15–
30%; PIFA = phenyliodine(III)bis(trifluoroacetate).
Table 1
Biochemical assays of tricyclic compounds against RET kinase.a
Compd
Inhibition, %b
IC50, nM
10
11
12
18
1
49.0%@100 nM
23.0%@100 nM
63.6%@100 nM
77.6%@100 nM
75.1%@100 nM
104 39
ND
102 21
62.5 0.7
56.5 2.1
a
The IC50 values are shown as the mean SD (nM) or estimated values from two
separate experiments.
Fig. 2. 23c suppressed RET phosphorylation and down-stream signaling in BaF3/
b
CCDC6-RET Cells.a
Inhibition % means inhibition rate against enzyme concentration (nM).
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C. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
Fig. 3. Docking of 23c with RET catalytic domain (PDB code 2IVV).
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Acknowledgments
This work was supported by grants from Chinese NSF
(81430080, 81373277, 81473243). Supporting from the National
Program on Key Basic Research Project of China (2015CB910603),
the International Cooperative Program (GJHZ1622) and Key Pro-
gram of the Frontier Science (160621) of the Chinese Academy of
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