Preparation of 3-substituted-1-isopropyl-1 H-pyrazolo[3,4-d]pyrimidin-4- amines as RET kinase inhibitors

Article abstract of DOI:10.1021/jm3003944

A series of 3-substituted-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines have been designed, synthesized, and evaluated as RET protein kinase inhibitors. On the basis of docking results, a small library of pyrazolopyrimidine compounds with an extended hydrophobic side arm was synthesized. The most promising of the compounds (7a) displayed efficient inhibition in vitro and good selectivity when tested on a panel of kinases. Furthermore, 7a inhibited GDNF-induced RET phosphorylation of ERK1/2 in MCF-7 breast cancer cells at concentrations as low as 100 nM.

Full text of DOI:10.1021/jm3003944

Brief Article
pubs.acs.org/jmc
Preparation of 3-Substituted-1-Isopropyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amines as RET Kinase Inhibitors
Peter Diner,† John P. Alao,† Johan Soderlund,‡ Per Sunnerhagen,† and Morten Grøtli*,†
́
̈
†Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Goteborg, Sweden
̈
‡Department of Medicinal Chemistry, R&I iMED, AstraZeneca R&D, SE-43183 Molndal, Sweden
̈
S
* Supporting Information
ABSTRACT: A series of 3-substituted-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines have been designed, synthesized, and
evaluated as RET protein kinase inhibitors. On the basis of docking results, a small library of pyrazolopyrimidine compounds
with an extended hydrophobic side arm was synthesized. The most promising of the compounds (7a) displayed efficient
inhibition in vitro and good selectivity when tested on a panel of kinases. Furthermore, 7a inhibited GDNF-induced RET
phosphorylation of ERK1/2 in MCF-7 breast cancer cells at concentrations as low as 100 nM.
Small-molecule inhibitors have been used to block the auto-
and substrate phosphorylation of RET in an ATP competitive
way, thereby preventing its oncogenic activity.7 Several classes
of inhibitors have been used as RET inhibitors, including
pyrazole-pyrimidines (e.g., 1), indolocarbazoles, 2-indolinone
derivatives, and a 4-anilinoquinazoline (2).4,8−13
Previously, we have used the pyrazolopyrimidines success-
fully as a scaffold for the inhibition of the PFPK7 kinase of
Plasmodium falciparum;14 therefore, we were also interested in
expanding the use of the pyrazolopyrimidine scaffold to other
kinases. Furthermore, the pyrazolopyrimidine-based compound
1 has previously been used successfully as an inhibitor of several
TKs that are considered important cancer targets.15,16
In this work, we present the design, synthesis, and biological
evaluation of a small library of 3-substituted pyrazolopyrimi-
dines that inhibit the RET kinase in low nanomolar
concentrations (down to 8 nM) in vitro. The most active
compounds have been evaluated in RET kinase-expressing
MCF-7 breast cancer cells and RET-fusion protein-expressing
TPC1 papillary thyroid carcinoma cell lines.
INTRODUCTION
■
Protein kinases constitute 1.7% of the protein products
encoded in the human genome.1 It encodes 518 protein
kinases, of which the majority are serine/threonine kinases and
90 are tyrosine kinases (TKs). The TKs are divided into
nonreceptor and receptor tyrosine kinases, of which the
receptor tyrosine kinases (RTKs) are a major family of
disease-related proteins that are promising therapeutic targets.2
Under normal conditions, RTK activity is closely regulated, but
deregulation of the RTKs can lead to oncogenic conversion,3
which occurs via three main mechanisms: formation of fusion
proteins with altered localization and/or specificity through
retroviral transduction or genomic rearrangements, point
mutations leading to constitutive activation and/or potentially
altered binding of ATP, and overexpression of the normal
protein sequence.
RET (REarranged during Transfection) is a transmembrane
RTK essential for the development of the sympathetic,
parasympathetic, and enteric nervous systems as well as the
kidney.4 Its extracellular part contains four cadherin-like
repeats, a calcium-binding site, and a cysteine-rich domain.
The intracellular part contains a typical TK domain. The RET
protein is activated by ligands of the glial-derived neurotrophic
factor (GDNF) family, such as GDNF, neurturin, artemin,
persephin, and a coreceptor, GFR-α. The GDNF ligand/
receptor complex brings together two RET proteins, thereby
triggering autophosphorylation and intracellular signaling.5
Deregulation and dysfunction of RET can lead to several
different thyroid cancers, including multiple endocrine neo-
plasia 2 syndromes (MEN2A and MEN2B), familial medullary
thyroid carcinoma (FMTC), and papillary thyroid carcinoma
(PTC).4 Point mutations in the RET gene are the cause of the
three cancer syndromes MEN2A, MEN2B, and FMTC, while
PTC is related to chromosomal inversions or translocations
affecting the intracellular kinase domain.
RESULTS AND DISCUSSION
■
The RET kinase has been characterized by X-ray crystallog-
raphy, with several small molecule inhibitors. Among them, 2
(Figure 1, PDB 2IVU)10 and 1 (Figure 1, PDB 2IVV)10 have
been shown to inhibit the RET kinase in nanomolar
concentrations and 2 is currently undergoing phase II clinical
trials. Both inhibitors are ATP competitive and bind into the
ATP binding site with small adjustments compared to AMP.10
In the X-ray structures, 1 forms two hydrogen bonds to the
kinase (N to NH of Ala807 and NH2 to CO of Glu805),
whereas 2 only forms one hydrogen bond (N to NH of
Ala807).10 Furthermore, the tolyl group of 1 and the
bromofluorophenyl group of 2 both occupy a hydrophobic
pocket in the back of the ATP binding site.10 We were
interested in developing inhibitors, based on the pyrazolopyr-
Recent studies have demonstrated that RET is overexpressed
in a subset of estrogen-receptor (ER)-positive breast cancers
and that crosstalk between ER and RET is important in
responses to endocrine therapy.5,6
Received: February 2, 2012
Published: May 7, 2012
© 2012 American Chemical Society
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Table 1. Synthesis of Compounds 7a−7p, 8a, 8b, and 9
compd
R2
yield (%)
7a
7b
7c
7d
7e
7f
phenyl
81
79
68
63
88
88
90
78
71
50
70
68
77
56
97
51
78
86
81
Figure 1. Initial docking of 7a (yellow, A and B) into the crystal
structure of the RET kinase (2IVV and 2IVU) containing the original
ligands 1 (blue, A) and 2 (blue, B), respectively. Compound 7a has the
same binding mode as 1 and interacts with the same hydrophobic
pocket in the RET kinase that is seen for both 1 (blue, A) and 2 (blue,
B).
4-fluorophenyl
4-pyridinyl
3-pyridinyl
2-pyridinyl
4-aminophenyl
3-aminophenyl
2-aminophenyl
4-trifluoromethyl-phenyl
1-methyl-1H-imidazol-5-yl
1-napthyl
7g
7h
7i
imidine scaffold, with a hydrophobic arm that utilizes the
hydrophobic back pocket. Our design strategy was to extend
the pyrazolopyrimidine with an alkynyl linker in the 3-position
of the pyrazole ring leading to compound 7a (Figure 1).
Compound 7a was docked into the X-ray structures of RET
7j
7k
7l
6-methoxy-2-napthyl
2,5-dimethylphenyl
prop-1-en-2-yl
t-butyl
7m
7n
7o
7p
8a
8b
9
(2IVV and 2IVU) using Schrodinger’s Glide. The docking
̈
suggests a similar binding mode of 7a and 1 in both crystal
structures, although compound 7a is slightly shifted out of the
pocket (Figure 1A). Moreover, the extended hydrophobic arm
of compound 7a fits deeper into the hydrophobic back pocket
than the inhibitors 1 and 2 (Figure 1A,B). This suggests that
compound 7a could potentially inhibit the RET kinase;
therefore, we were interested in synthesizing a small library
of compounds with an extended linker between the pyrazolo-
pyrimidine scaffold and the carbocyclic part of the inhibitor.
We used a convergent route for preparing 1,3-disubstituted
pyrazolopyrimidines (Scheme 1 and Table1).17 This route
ferrocenyl
−(CH2)2-Ph
−(CH2)2-4-F-Ph
phenyl
assisted heating at 200 °C for 5 min. The reaction yielded 6 in
nearly quantitative yield and high regioselectivity.18
Compound 6 was then reacted with several aromatic
acetylenes (5 equiv) in the presence of Pd(PPh3)4 (2 mol
%), copper iodide (20 mol %), and Amberlite IRA-67 (5 equiv)
in THF, using microwave assisted heating (120 °C, 15 min), in
order to generate compounds with the general structure 7a−p.
This protocol is a further development of our previous
methodology,18 which now allows for short reaction times
(within minutes) and easy workup (use of ion-exchange resin).
The reactions proceeded in moderate to excellent yields (51−
97%) (Table 1).
Scheme 1. Synthesis of 1,3-Disubstituted
Pyrazolopyrimidines
To include compounds with an aliphatic linker, the triple
bonds of 7a and 7b were hydrogenated using Pd/C in ethanol
at 40 °C for 48 h to yield compounds 8a and 8b in good yields
(78% and 86%, respectively). To include an alkenyl moiety as a
linker, 6 was also reacted with trans-2-phenylvinylboronic acid
(1.5 equiv) in the presence of Pd(PPh3)4 (2 mol %) and
K2CO3 (3 equiv) in dioxane:water (v/v 3:1) at 150 °C (MW)
for 30 min to generate 9 in 81% yield.
The effect of molecules 7a−p, 8a, 8b, and 9 on the activity of
RET was tested in a cell-free inhibition assay at 300 nM for
compounds 7a−p, 8a, 8b, and 9 (Figure 2A). The assay
measured the efficacy of compounds 7a−p, 8a, 8b, and 9 to
decrease the substrate phosphorylation. The most efficient
inhibitor has a phenyl group as R2. Introducing larger groups,
involves the synthesis of a common intermediate, 4-amino-3-
iodo-1H-pyrazolo[3,4-d]pyrimidine (5) that allowed rapid
derivatization of the heterocyclic core scaffold in two steps
(Scheme 1).
Compound 5 was reacted with isopropyl chloride in the
presence of potassium carbonate in DMF and with microwave-
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The efficacy was then measured radiometrically, in the presence
of 7a (10 μM); the remaining activity is a measurement of the
inhibition for each kinase. The inhibition profile of the panel of
53 kinases is a measurement of the selectivity of the compound.
The high inhibitor concentration (10 μM) was chosen in order
to ensure a reliable readout of a potential inhibition even
though this concentration is much higher than the observed
IC50 value for 7a (8 nM). To evaluate the inhibition on the
panel of 53 kinases, we classified the inhibition as strong (0−
25% remaining activity), intermediate (26%−75% remaining
activity), and no inhibition (76%−100% remaining activity).
Out of the 53 kinases, 7a shows “no inhibition” for 29 kinases,
“intermediate inhibition” for 15 kinases, and “strong inhibition”
for 9 kinases (Figure 2S, SI). A closer analysis of the selectivity
screen shows that only a small part of the serine/threonine
kinases (7%, 3 out of 41 kinases, including c-Raf, MLK1,
SAPK2) was inhibited strongly by 7a, while a larger part of the
tyrosine kinases (>50%, 7 out of 13, including Abl, EphA5,
EphB4, Fyn, KDR, Lyn, MLK1) showed strong inhibition.
Therefore, we extended the selectivity screen to include a larger
number of receptor and nonreceptor TKs. At 10 μM,
compound 7a shows “strong inhibition” for 38 kinases,
“intermediate inhibition” for 23 kinases, and “no inhibition”
for 16 kinases, including the RET kinase. This means that at
high concentrations (10 μM), 7a inhibits about half of the
tested TKs strongly, which has previously been seen for
inhibitors such as 1 and 1NM-1.19 However, the IC50 value for
7a is much lower (8 nM) compared to the concentration (10
μM) that is used in the inhibitory selectivity study. Therefore,
we conducted a second selectivity screen at a concentration
(0.4 μM) that corresponds to a concentration 50 times higher
than the observed IC50 value for compound 7a (see SI) At this
concentration (0.4 μM), the selectivity screen shows that only
six kinases, including RET (EphA1, FGFR1, Flt4, Lck, RET,
and Yes), show “strong inhibition”, 26 kinases show
“intermediate inhibition”, and 44 kinases show “no inhibition.”
GDNF has been shown to induce RET-dependent phosphor-
ylation of the extracellular signal-regulated protein kinases 1/2
(ERK1/2) in serum-starved MCF-7 breast cancer cells.20 MCF-
7 cells were grown for 24 h in low-serum medium (1% fetal
bovine serum; FBS) and then exposed to increasing
concentrations of 7a for 30 min prior to the addition of 10
ng/mL GDNF. The cells were then incubated for a further 45
min and processed for Western blotting. Addition of GDNF
resulted in a rapid increase in ERK1/2 phosphorylation (Figure
3A).
Figure 2. In vitro RET kinase activity assays. (A) Efficacy of 7a−p, 8a,
8b, and 9, using a compound concentration of 300 nM. (B) IC50
values for 7a, 7b, 7g, 7h, 7m, and 9. For experimental details, see
http://www.millipore.com/techpublications/tech1/pf3036.
such as a naphthyl group in 7k and 7l, leads to lower inhibition.
The same effect is also seen in compounds with smaller
substituents (7j, imidazole; 7n, 3-methylbut-3-en; 7o, t-butyl).
On the other hand, substituents in the phenyl ring, such as a
fluorine atom in the 4-position (7b), an amino group in the 3-
or 2-position (7g and 7h), and methyl groups in the 2- and 5-
positions (7m) also showed efficient inhibition. Replacing the
triple bond in the ethynyl linker with a fully saturated linker (8a
and 8b) resulted in a less efficient inhibitory effect. However,
using a double-bond linker, as seen in 9, provides good RET
kinase inhibition. The result shows that six of the compounds,
7a, 7b, 7g, 7h, 7m, and 9, reduce the phosphorylation
considerably (less than 20% remaining activity).
The IC50 values for the most promising compounds, 7a, 7b,
7g, 7h, 7m, and 9, were determined using a commercial
radiometric RET assay (Figure 2B) by measuring the substrate
phosphorylation at different inhibitor concentrations (0.3−
3000 nM). All six compounds showed a strong inhibition of the
RET activity, with IC50 values ranging from 8 to 195 nM
(Figure 2). The results show that the lipophilic substituents
(phenyl, 4-fluoro-substituted, and 2,5-dimethyl-substituted
phenyl) provide the strongest inhibition, which indicates the
hydrophobic nature of the back pocket.
To be an efficient inhibitor, in addition to strong and fast
inhibition of the kinase, the inhibitor should also bind,
selectively, only to the RET kinase. The selectivity of
compound 7a was evaluated toward a panel of 53 serine/
threonine/tyrosine kinases (40 Ser/Thr kinases, 12 Tyr kinases,
and 1 Ser/Thr/Tyr kinase) that were selected in order to
reflect the human kinome (Figure 1S, Supporting Information
(SI)). The panel was selected to cover each of the major kinase
subfamilies, and it was further based on sequence similarity of
the catalytic domain of the kinase as well as experimentally
determined inhibition profiles with common kinase inhibitors.
Further experiments have demonstrated that 7a inhibits
GDNF-induced phosphorylation of ERK1/2 at concentrations
as low as 100 nM (Figure 3A). To confirm that the decreased
phosphorylation of ERK1/2 is due to selective inhibition of the
RET kinase by 7a, we compared its effect on ERK1/2
phosphorylation in TPC1 papillary thyroid carcinoma cells,
which express a constitutively active RET fusion protein (RET/
PTC1),21 and HCT116 colon cancer cells, which do not
express the receptor kinase. In these experiments, 7a partially
suppressed ERK1/2 phosphorylation in TPC1. However, in
HCT116 cells, the ERK1/2 phosphorylation was not sup-
pressed, which suggests that the inhibition is RET-dependent
(Figure 3B). Phosphorylation induces the translocation of
ERK1/2 to the nucleus.22 We observed a reduction in nuclear
pERK1/2 in TPC1, but not HCT116 cells, exposed to 200 nM
7a for 24 h (Figure 3C). Together, these findings demonstrate
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Figure 4. Induced fit dockings of 7a, 7b, 7g, 7h, 7m, and 9 into the
crystal structure of the RET kinase (2IVV).
Figure 3. In vivo characterization of 7a. (A) MCF-7 cells were grown
for 24 h in low-serum medium and then exposed to the indicated
doses of 7a for 30 min prior to addition of GDNF. The cells were then
incubated for a further 45 min. Total cell lysates were resolved by
SDS-PAGE, and membranes were probed with antibodies directed
against pERK1/2, ERK1/2, and tubulin. (B) HCT116 and TPC1 cells
were exposed to the indicated concentrations of 7a for 24 h. Total cell
lysates were treated as in Figure 4A. (C) TPC1 and HCT116 cells
grown on coverslips were exposed to 200 nM of 7a for 24 h, fixed, and
stained with antibodies directed against pERK1/2. Following staining
with a fluorescein isothiocyanate-conjugated secondary antibody, cells
were examined by immunofluorescence microscopy. Scale bar: 20 μm.
inhibition (8 nM) suggests that the compound binds more
strongly to the hydrophobic pocket than does 1. Compound 7a
should be a useful tool for studying and understanding the
complex molecular mechanisms that underlie resistance to
endocrine therapy.
EXPERIMENTAL SECTION
■
General. See SI for details. All final compounds were confirmed to
be of >95% purity, except 7f (76%), based on HPLC analysis.
General Procedure A for the Synthesis of 7a−p. Compound 6
(1 equiv), aryl acetylene (5 equiv), Pd(PPh3)4 (2 mol %), Amberlite
IR-67 (5 equiv), and CuI (20 mol %) in THF was heated for 15 min at
120 °C in the microwave. After cooling to ambient temperature, the
reaction mixture was filtered through a pad of Celite. The solvent was
removed in vacuo, and the crude product was purified by flash column
chromatography on silica gel.
that 7a is cell-permeable and a potent RET inhibitor that could
be useful for studying RET-dependent signaling.
The effect of 7a on HCT116 and TPC1 proliferation was
also investigated (Figure 2S, SI). 7a inhibited HCT116 and
TPC1 proliferation, with EC50 values of 2.4 and 3.3 μM,
respectively. TPC1 cells however, were considerably more
sensitive to 1 μM 7a than were HCT116 cells. Furthermore, 7a
and sorafenib inhibited TPC1 proliferation to similar degrees
(EC50: 2.4 and 2.6 μM, respectively; Figure 1S, SI). At the
concentrations used (≤10 μM), no overt cytotoxicity was
observed in any of the cell lines tested.
General Procedure B for the Synthesis of 8a and 8b. The
starting material (1 equiv) was dissolved in ethanol (10 mL, 99.9%).
Pd/C (10% w, 10 mg) was added, and the reaction mixture was put
under H2 atmosphere. The solution was carefully heated to 40 °C for
48 h. The reaction mixture was filtered through a pad of Celite, and
the solvent was removed in vacuo. The crude product was purified by
flash column chromatography on silica gel (MeOH:CHCl3 = 1:20).
Compounds 7a, 7b, 7g, 7h, 7m, and 9 were docked into the
crystal structure of the RET-KD kinase (2IVV), using induced
fit dockings (Schrodinger’s Glide) (Figure 4). The docking
̈
results show that compounds (7a, 7b, 7g, 7h, 7m, and 9) utilize
the hydrophobic back pocket, even though compound 7m, with
a more sterically demanding substituent, is positioned further
out in the ATP binding site (Figure 4E).
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Preparation and characterization of compounds 7−9, H- and
13C NMR spectra, HPLC purity, and effects of our RET
inhibitors in human cancer cells. This material is available free
of charge via the Internet at http://pubs.acs.org.
CONCLUSIONS
Taken together, the data presented here indicate that 7a is,
indeed, a potent RET kinase inhibitor, and the strong cell-free
■
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Indolin-2-one RET Inhibitors. Bioorg. Med. Chem. 2010, 18, 1482−
1496.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +46-31-786 9017. Fax: +46-31-772 3840. E-mail:
grotli@chem.gu.se.
(13) Mologni, L.; Sala, E.; Riva, B.; Cesaro, L.; Cazzaniga, S.;
Redaelli, S.; Marin, O.; Pasquato, N.; Donella-Deana, A.; Gambacorti-
Passerini, C. Expression, Purification, and Inhibition of Human RET
Tyrosine Kinase. Protein Expression Purif. 2005, 41, 177−185.
(14) Klein, M.; Diner, P.; Dorin-Semblat, D.; Doerig, C.; Grøtli, M.
Synthesis of 3-(1,2,3-Triazol-1-yl)- and 3-(1,2,3-Triazol-4-yl)-Substi-
tuted Pyrazolo[3,4-d]Pyrimidin-4-Amines via Click Chemistry:
Potential Inhibitors of the Plasmodium falciparum PfPK7 Protein
Kinase. Org. Biomol. Chem. 2009, 7, 3421−3429.
(15) Tatton, L.; Morley, G. M.; Chopra, R.; Khwaja, A. The Src-
Selective Kinase Inhibitor PP1 also Inhibits Kit and Bcr-Abl Tyrosine
Kinases. J. Biol. Chem. 2003, 278, 4847−4853.
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J.; Ubersax, J.; Blethrow, J.; Morgan, D. O.; Shokat, K. M. Structural
Basis for Selective Inhibition of Src Family Kinases by 1. Chem. Biol.
1999, 6, 671−678.
(17) Wu, T. Y.; Schultz, P. G.; Ding, S. One-Pot Two-Step
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(18) Klein, M.; Morillas, M.; Vendrell, A.; Brive, L.; Gebbia, M.;
Wallace, I. M.; Giaever, G.; Nislow, C.; Posas, F.; Grøtli, M. Design,
Synthesis and Characterization of a Highly Effective Inhibitor for
Analog-Sensitive (as) Kinases. PLoS One 2011, 6, e20789.
(19) Bain, J.; Plater, L.; Elliott, M.; Shpiro, N.; Hastie, C. J.;
McLauchlan, H.; Klevernic, I.; Arthur, J. S.; Alessi, D. R.; Cohen, P.
The Selectivity of Protein Kinase Inhibitors: a Further Update.
Biochem. J. 2007, 408, 297−315.
(20) Boulay, A.; Breuleux, M.; Stephan, C.; Fux, C.; Brisken, C.;
Fiche, M.; Wartmann, M.; Stumm, M.; Lane, H. A.; Hynes, N. E. The
Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the
ERalpha Pathway in Breast Cancer. Cancer Res. 2008, 68, 3743−3751.
(21) Schweppe, R. E.; Klopper, J. P.; Korch, C.; Pugazhenthi, U.;
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a grant from the European
Commission (the CELLCOMPUT project, contract no.
043310) to M.G., from Percy Falk’s foundation to J.P.A., and
from the Swedish Cancer Fund (10-0633) to P.S. This is a
publication from the Chemical Biology Platform at the
University of Gothenburg.
ABBREVIATIONS USED
■
TK, tyrosine kinases; RTK, receptor tyrosine kinases; RET,
rearranged during transfection; GDNF, glial-derived neuro-
trophic factor; MEN2, multiple endocrine neoplasia 2
syndromes; FMTC, familial medullary thyroid carcinoma;
PTC, papillary thyroid carcinoma; DMF, dimethylformamide;
THF, tetrahydrofuran
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