Pyrazoloadenine Inhibitors of the RET Lung Cancer Oncoprotein Discovered by a Fragment Optimization Approach

Article abstract of DOI:10.1002/cmdc.202100013

A fragment-based drug-discovery approach was used on a pyrazoloadenine fragment library to uncover new molecules that target the RET (REarranged during Transfection) oncoprotein, which is a driver oncoprotein in ～2 % of non-small-cell lung cancers. The fragment library was screened against the RET kinase and LC-2/ad (RET-driven), KM-12 (TRKA-driven matched control) and A549 (cytotoxic control) cells to identify selective scaffolds that could inhibit RET-driven growth. An unsubstituted pyrazoloadenine fragment was found to be active on RET in a biochemical assay, but reduced cell viability in non-RET-driven cell lines (EC₅₀=1 and 3 μM, respectively). To increase selectivity for RET, the pyrazoloadenine was modeled in the RET active site, and two domains were identified that were probed with pyrazoloadenine fragment derivatives to improve RET affinity. Scaffolds at each domain were merged to generate a novel lead compound, 8 p, which exhibited improved activity and selectivity for the RET oncoprotein (A549 EC₅₀=5.92 μM, LC-2/ad EC₅₀=0.016 μM, RET IC₅₀=0.000326 μM).

Full text of DOI:10.1002/cmdc.202100013

Communications
doi.org/10.1002/cmdc.202100013
ChemMedChem
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Pyrazoloadenine Inhibitors of the RET Lung Cancer
Oncoprotein Discovered by a Fragment Optimization
Approach
Debasmita Saha,[a] Katie Rose Ryan,[b] Naga Rajiv Lakkaniga,[a, c] Erica Lane Smith,[a] and
Brendan Frett*[a]
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lung cancer growth.[5] To target RET fusions, small molecule
A fragment-based drug-discovery approach was used on a
kinase inhibitors have been developed to obstruct RET signal-
ling. Vandetanib and cabozantinib, with FDA approval for
thyroid cancer, are in phase II clinical trials for RET-rearranged
NSCLC (NCT 01639508 for cabozantinib, NCT 01823068 for
vandetanib).[6–9]
pyrazoloadenine fragment library to uncover new molecules
that target the RET (REarranged during Transfection) oncopro-
tein, which is a driver oncoprotein in ~2% of non-small-cell
lung cancers. The fragment library was screened against the
RET kinase and LC-2/ad (RET-driven), KM-12 (TRKA-driven
matched control) and A549 (cytotoxic control) cells to identify
selective scaffolds that could inhibit RET-driven growth. An
unsubstituted pyrazoloadenine fragment was found to be
active on RET in a biochemical assay, but reduced cell viability
in non-RET-driven cell lines (EC50 =1 and 3 μM, respectively). To
increase selectivity for RET, the pyrazoloadenine was modeled
in the RET active site, and two domains were identified that
were probed with pyrazoloadenine fragment derivatives to
improve RET affinity. Scaffolds at each domain were merged to
Pyrazoloadenines, with appropriate structural attributes,
inhibit a wide range of protein kinases such as BTK (Bruton’s
tyrosine kinase), CDK1/2 (cyclin-dependent kinase 1/2), Src
kinase, and RIPK1 (receptor-interacting protein kinase).[10–13]
Modification of pyrazoloadenine has generated RET inhibitors
for papillary thyroid cancer (PTC) for example, PP1 and PP2
(Figure S1 in the Supporting Information).[14,15] Perceiving the
plasticity of pyrazoloadenines, we sought to use a pyrazoloade-
nine fragment library to test a fragment-based discovery
approach by screening the library against the RET kinase. With
generate
a novel lead compound, 8p, which exhibited
our prior interest in developing kinase inhibitors,[16–19]
a
improved activity and selectivity for the RET oncoprotein (A549
pyrazoloadenine-based fragment library was screened against
RET in biochemical assays as well as cellular assays employing
LC-2/ad, a RET-CCDC6 fusion-driven NSCLC cell line, and
matched controls to determine oncoprotein specificity (TRK-
driven, KM-12) and cytotoxicity (A549). This approach led to the
discovery of 8p, a highly potent and selective pyrazoloadenine-
based inhibitor of RET.
EC50 =5.92 μM,
0.000326 μM).
LC-2/ad
EC50 =0.016 μM,
RET
IC50 =
Introduction
The RET (REarranged during Transfection) proto-oncogene[1]
encodes a transmembrane receptor tyrosine kinase, which is
responsible for activating numerous signalling cascades.[2]
Initially, RET was identified as a driver oncogene in thyroid
cancers.[3] RET mutations were subsequently identified in other
cancers[4] and were found to drive ~2% of non-small-cell lung
cancers (NSCLC). Several RET gene fusions have been identified
in lung adenocarcinomas and inhibition of these fusions impair
Chemistry
In Scheme S1, pyrazoloadenine 1 was iodinated by using N-
°
iodosuccinimide in DMF heated to 85 C for 18 hours to form 2.
Iodinated intermediate 2 was then N-alkylated at the N1-
pyrazole using different alkyl iodides or bromides in the
°
presence of K2CO3 in DMF at 80 C for 12 hours. Fragments 3b–
d
were synthesized by reacting
2
with the respective
[a] Dr. D. Saha, Dr. N. R. Lakkaniga, E. L. Smith, Dr. B. Frett
Department of Pharmaceutical Sciences
College of Pharmacy
hydrochloride salt of the alkyl chloride using NaH in dry DMF
°
under inert atmosphere and heated to 80 C for 24–48 hours.
University of Arkansas for Medical Sciences
Little Rock, AR 72205 (USA)
E-mail: BAFrett@uams.edu
Reacting intermediate
2
with 1-bromo-4-(methylsulfonyl)
benzene under copper catalysis produced compound 3d.
C-3-substituted 1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-
amines 4a–d were obtained by reacting 3a with the corre-
sponding boronic acid using Pd(dppf)Cl2.DCM complex and
K3PO4 in dioxane/H2O (4:1) under microwave irradiation for
[b] Dr. K. R. Ryan
Department of Biochemistry and Molecular Biology
College of Medicine
University of Arkansas for Medical Sciences
Little Rock, AR 72205 (USA)
[c] Dr. N. R. Lakkaniga
°
1 hour at 100 C (Scheme S2).
SmartBio Labs
Suzuki coupling of 3a–k with ethyl 2-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)acetate provided fragments 6a–
e. The ester was subjected to saponification by LiOH in THF/H2O
126, Rajamannar Salai, K. K. nagar, Tamilnadu, Chennai-600078 India.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100013
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(1:1) under microwave irradiation to generate intermediates
7a–c. The acid component was subsequently coupled to
various heterocyclic amines to afford the pyrazolo[3,4-d]
pyrimidin-3-yl)phenyl) acetamide derivatives (Scheme S3). De-
rivative 8s was synthesized according to the synthetic route
depicted in Scheme S4. Reaction of 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl) aniline with triphosgene in presence of
substitution with terminal alcohol 3g, isopentane 3i, pyran 3k,
and 3-methoxypropane 3f improved RET potency and selectiv-
ity against TRKA. Of these, 3-methoxypropane (3f; RET IC50 =
1.9�2.81 μM) was found to be the most potent in reducing cell
viability with 50-fold selectivity over TRKA.
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3
4
5
6
7
8
9
To probe interactions in the kinase back pocket of RET
(Domain I), C-3 pyrazoloadenines fragment derivatives were
generated (Table S2). All substitutions off the C-3 carbon were
found selective for RET over TRKA (4a–d). Of these, phenyl 4a
(RET IC50 =6.82�2.22 μM) and 1-methyl-1H-pyrazole 4d (RET
IC50 =1.044�0.27 μM) were most potent. Analyzing 4a and 4d
in the RET active site revealed that the phenyl ring of 4a
accesses the RET back pocket through a π-π interaction with
Phe893 of the DFG-out motif, while the pyrazole of 4d did not;
hence 4a was considered for additional modification to better
exploit Domain I (Figure S3). To further develop the pyrazoloa-
denine SAR, derivatives were synthesized to expand into the
back pocket of RET. 6a and 6e were found to be most active
against RET with TRKA selectivity, albeit without cell activity.
The pyrazoloadenine SAR was further developed by modify-
ing substituents in the back pocket (Domain I) while simulta-
neously altering groups at the solvent front (6a–e, 7a–c;
Domain II; Table S3). Among these 6a, its acidic derivative 7a,
and the N-pyran-substituted derivative 6e were most active.
Extension of the aryl substituent at the C-3 position, via a
carboxylic acid handle, facilitated the exploration of the RET
allosteric pocket. A small library of pyrazoloadenine acetamides
were synthesized using different aromatic and heterocyclic
amines to investigate this pocket (Tables 1 and S4).
°
triethylamine in THF at 60 C for 6 hours provided the
isocyanate 10. Subsequent addition of 5-(tert-butyl)isoxazol-3-
amine to intermediate 10 provided 1-(5-(tert-butyl)isoxazol-3-
yl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)urea
11 which was subjected to Suzuki coupling to furnish the
derivative 8s.
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Results and Discussion
Inhibitor design by a fragment-based optimization approach
The pyrazoloadenine warhead (fragment 1) was subjected to a
biochemical screen (RET and TRKA) and an oncogene-driven
cellular screen (LC-2ad and KM-12). TRKA was employed as an
oncoprotein control because of its similarity to the RET kinase
domain;[20] if compounds exhibit selectivity against TRKA, this
suggests selectivity in the greater kinome. The biochemical
screen indicated selectivity of fragment 1 for RET (RET IC50 =
9.20 μM) over TRK (TRKA IC50 =57.07 μM). However, the frag-
ment was found to reduce cell viability in both oncogene-
driven cell lines (EC50 =1.47 μM and 1.73 μM, respectively for
LC-2ad and KM-12) along with A549 (EC50 =3.02 μM; cytotoxic
control). Despite exhibiting biochemical relevance, fragment 1
was found to be too cytotoxic by nonselectively reducing cell
viability, suggesting the fragment did not possess oncoprotein
selectivity. To remove the cytotoxicity profile, fragment 1 was
visualized in the RET active site to identify ligand-protein
interactions to improve affinity (Figure S2).
Fragment 1 was modeled in a RET DFG-out homology
model since molecules that interact with the DFG-out fold of
RET possess improved selectivity.[21] By analyzing ligand-protein
interactions, it was found that fragment 1 formed two hydrogen
bonds with hinge residues Ala807 and Glu805 and π-π
interactions with Phe893 at the DFG-motif (Figure S2). The
modeling study suggested the possibility for fragment expan-
sion at the solvent front (Domain II) and the allosteric pocket
(Domain I; Figure S2). Using modeling insight, fragment 1
underwent expansion at the R1 position. To probe interactions
at the solvent front (Domain II), several pyrazoloadenines
fragment derivatives were generated (Table S1). N-Methyl
substitution slightly improved potency against RET (RET IC50 =
3.3 μM; LC-2/ad EC50 =1 μM) but was still cytotoxic (A549 EC50 =
1 μM). Introducing polar groups, such as morpholino ethyl 3b
and N,N-dimethylethyl 3c, improved RET activity and lowered
cytotoxicity but cell activity diminished likely from permeability
issues. Substituting with aromatic groups such as 2-methyl
pyridine 3e and 4-(methylsulfonyl)benzene 3d decreased
potency, whereas propanenitrile 3j and methoxyethane 3h
drastically reduced activity (Table S1). On the other hand, N-
In general, when R1 was methyl pyridine, modification with
aryl and heteroaryl amines yielded compounds with weak
activity (8i, j). However, when R3 was 3-cyclopropyl-N-methyl
pyrazol-5-amine (8m), 3-(tert-butyl)-N-methyl pyrazol-5-amine
(8l), or 3-(trifluoromethyl)aniline (8k) compounds exhibited
improved cellular activities consistent with enzymatic activities.
Solubilizing groups, such as morpholino ethyl (8n), exhibited
decent RET inhibition but weak cellular activity, whereas N,N-
dimethylethyl (8o) had diminished RET activity and very poor
cellular activity.
A set of pyrazolo pyrimido phenylacetamides (8a–m) were
synthesized to further define the SAR at the RET back pocket
(Tables 1 and S4). Amide modifications with aromatic amines,
such as 3-(fluoromethyl)aniline (8a), exhibited reduced RET
inhibition (RET IC50 =35.5 μM) in spite maintaining cellular
activity (LC-2/ad EC50 =1.68 μM), whereas 3-(trifluoromethyl)
aniline (8c) exhibited RET inhibition (RET IC50 =0.0562 μM)
consistent with cellular activity (LC-2/ad EC50 =0.37 μM). Five-
membered heterocyclic amines, 3-(tert-butyl)pyrazol-5-amine
(8d) and 3-(tert-butyl)-1-isopropyl pyrazol-5-amine (8f), exhib-
ited RET inhibition consistent with cellular activity. While 3-
cyclopropyl pyrazol-5-amine (8e) exhibited selectivity for RET
over TRKA, the analogue had very poor cellular activity, whereas
3-(tert-butyl)-1-cyclohexyl pyrazol-5-amine (8h) exhibited inhib-
ition of both RET and TRKA while maintaining cellular activity.
When 5-(tert-butyl) isoxazol-3-amine (8b) was utilized as the
amine input, the compound exhibited selectivity for RET over
TRKA (RET IC50 =0.00057 μM; TRKA IC50 =0.202 μM). These
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Table 1. . Inhibition values and SAR for various N-functionalized substituted phenylacetamides.[a]
1
2
3
4
5
6
7
Code R1
R3
IC50 [μM]
RET
EC50 [μM]
LC-2/ad
TRKA
KM-12
A549
8
9
8a
8b
8c
8h
methyl
3-fluoroaniline
5-(tert-butyl)- isoxazol-3
N-methyl-3-(trifluoromethyl)aniline
3-(tert-butyl)-1-cyclohexyl-N-methyl-1H-pyra-
zol
35.52�1.41
3.7�0.25
1.68�0.45
2.11�0.08
9.20�3.11
20.35�0.51
56.3�5.91
methyl
methyl
methyl
0.00057�0.00005
0.0562�0.000004
0.109�0.40
0.20�0.74
4.03�0.93
0.295�0.16
0.024�0.02 0.71�0.07
10
11
12
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0.37�0.04
1.56�0.36
2�0.03
10.46�0.21 >20
8k
8l
8m
8n
2-ethylpyridyl
2-ethylpyridyl
2-ethylpyridyl
4-ethylmorpho-
line
1-methoxypropyl
1-methoxypropyl
1-isobutyl
3-(trifluoromethyl) aniline
3-(tert-butyl)-1-methyl-1H-pyrazol
3-cyclopropyl-1-methyl-1H-pyrazol-5-yl
5-(tert-butyl)isoxazol
0.13�0.07
0.069�0.004
2.93�0.5
4.74�1.45
3.95�0.8
0.49�0.18
0.12�0.06
0.38�0.03
3.0�0.04
>20
7.83�0.80
1.12�0.12
2.57�0.64
>20
>20
>20
>20
>20
0.08�0.02
>20
8p
8q
8r
5-(tert-butyl)isoxazol
3-fluoro-aniline
3-fluoro-aniline
0.000326�0.00005 0.198�0.067 0.016
0.3�0.02
4.38�0.30
>20
5.92�1.33
>20
>20
0.25�0.07
3.48�0.42
1.74�0.43
2.48�0.83
2.41�0.16
>20
[a] All data represent means of at least n=3 independent experiments.
results displayed that the phenyl isoxazole acetamide derivative
8b possessed notable potency against RET-driven lung cancer
with a wide therapeutic window (A549 EC50 =20.52 μM).
Our previous observations from Table S1 suggested that N-
substitutions with isopentane, pyran, and 3-methoxypropane
provided improved RET potency and selectivity over TRKA;
hence pyrazolo[3,4-d]pyrimidin-3-yl phenylacetamide deriva-
tives with N-alkyl groups were synthesized and screened. 3-
Fluorophenyl acetamide (8r) with 1-isobutyl as the N-alkyl
group exhibited greater TRKA inhibition over RET albeit with
poor cellular activity.
On the other hand, 3-fluorophenylacetamide 8q with 3-
methoxypropyl as the N-alkyl substituent inhibited RET without
selectivity. Hence, after extensive SAR development from the
initial fragment 1, it was identified that fragment 3f and
compound 8b exhibited greatest RET selectivity with reduced
cytotoxicity. Merging 3f and 8b into a single molecule
generated 8p. The core of 8p was identified from the initial
biochemical screen and interactions at both the solvent and
back pocket of RET were optimized by expanding fragment 1.
Merging 3f and 8b resulted in a twofold improvement in RET
potency and cellular activity (LC-2/ad EC50 =0.01 μM; Figure 1).
Global kinase selectivity of 8p was assessed using a 97-
kinase panel with three RET point mutations at a concentration
of 20 nM. The results indicate that 8p exhibits excellent
selectivity with a 0.10 S35 value defined as the percentage of
the kinases showing >90% inhibition out of the total number
of kinases profiled (Figure S4). Interestingly, 8p was also found
to have high affinity for mutant forms of RET including
RETM918T, RETV804L, and RETV804M.
Figure 1. Scaffold development strategy that led to RET inhibitor 8p.
gen bonds with Ala807 and Glu805 at the hinge region.
Pyrazoloadenine and the phenyl linker π–π stack with Phe893
of the DFG-motif. The 3-methoxypropane substituent orients
towards the solvent front whereas the amide linker and oxygen
of the isoxazole moiety form two hydrogen bonds with the
backbone atoms of Asp892. This interaction permits access into
the allosteric pocket, which is occupied by tert-butyl isoxazole.
To gain insight into the binding of 8p, in-silico docking
studies were performed with the RET kinase DFG [Asp892,
Phe893, Gly894]-out computational model (Figure 2). The bind-
ing of 8p to the RET DFG-out form of the kinase suggests 8p is
a type-2 inhibitor.[21] The pyrazolopyrimidine core of 8p hydro-
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Keywords: fragment-based drug discovery · oncoproteins ·
pyrazoloadenines · RET inhibitor
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Figure 2. Computational modeling of 8p in a RET DFG-out homology model.
Hydrogen bonds are illustrated in blue and π–π stacking interactions in
brown.
Conclusion
This study used a fragment-optimization approach, based on
both biochemical and cell-based screens, along with computa-
tional studies to identify selective, pyazoloadenine-based RET
inhibitors. Modeling uncovered two domains for fragment
expansion to probe interactions within the RET active site. SAR
studies led to the discovery of 8p, which is a selective RET
inhibitor with sub-nanomolar IC50 values against RET (IC50 =
0.00032 μM) and high potency against LC-2/ad cells (EC50 =
0.01 μM). Kinome-wide selectivity profiling revealed that 8p
exhibits excellent selectivity for RET and RET mutants over other
similar kinases. In conclusion, this research illustrates the
usefulness of an fragment-based optimization approach to
rapidly probe an active site to refine a promiscuous molecule
for a specific kinase oncoprotien.
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Acknowledgements
This work was supported by the National Institutes of General
Medical Sciences (P20GM109005), a grant from the American
Thyroid Association, a UAMS College of Pharmacy Seed grant, and
a 2020 UAMS College of Pharmacy Summer Research Fellowship.
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Manuscript received: January 7, 2021
Conflict of Interest
Revised manuscript received: February 1, 2021
Accepted manuscript online: February 8, 2021
Version of record online: ■■■, ■■■■
The authors declare no conflict of interest.
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COMMUNICATIONS
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Refining a promiscuous molecule:
Based on on biochemical and cell-
based screens plus computational
studies, a fragment-optimization
approach was used to identify pyazo-
loadenine-based inhibitors of the RET
oncoprotein. Modeling uncovered
two domains to probe interactions
within the RET active site; then SAR
studies led to compound 8p, which
exhibits excellent selectivity for RET
and RET mutants over other similar
kinases.
Dr. D. Saha, Dr. K. R. Ryan, Dr. N. R.
Lakkaniga, E. L. Smith, Dr. B. Frett*
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Pyrazoloadenine Inhibitors of the
RET Lung Cancer Oncoprotein Dis-
covered by a Fragment Optimiza-
tion Approach
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