Identification of two novel RET kinase inhibitors through MCR-based drug discovery: Design, synthesis and evaluation

Article abstract of DOI:10.1016/j.ejmech.2014.09.023

From an MCR fragment library, two novel chemical series have been developed as inhibitors of RET, which is a kinase involved in the pathology of medullary thyroid cancer (MTC). Structure activity relationship studies (SAR) identified two sub-micromolar tractable leads, 6g and 13g. 6g was confirmed to be a Type-II RET inhibitor. 13g and 6g inhibited RET in cells transformed by RET/C634. A RET DFG-out homology model was established and utilized to predict Type-II inhibitor binding modes.

Full text of DOI:10.1016/j.ejmech.2014.09.023

European Journal of Medicinal Chemistry 86 (2014) 714e723
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Identification of two novel RET kinase inhibitors through MCR-based
drug discovery: Design, synthesis and evaluation
Brendan Frett a, Marialuisa Moccia c, Francesca Carlomagno c, Massimo Santoro c,
, b, *
Hong-yu Li a
a College of Pharmacy, Department of Pharmacology and Toxicology, The University of Arizona, Tucson, AZ 85721, USA
b The University of Arizona Cancer Center, 1515 N Campbell Ave, Tucson, AZ 85724, USA
c
ꢀ
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita di Napoli “Federico II”, 80131 Naples, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
From an MCR fragment library, two novel chemical series have been developed as inhibitors of RET,
which is a kinase involved in the pathology of medullary thyroid cancer (MTC). Structure activity rela-
tionship studies (SAR) identified two sub-micromolar tractable leads, 6g and 13g. 6g was confirmed to be
a Type-II RET inhibitor. 13g and 6g inhibited RET in cells transformed by RET/C634. A RET DFG-out
homology model was established and utilized to predict Type-II inhibitor binding modes.
Published by Elsevier Masson SAS.
Received 11 June 2014
Received in revised form
5 September 2014
Accepted 7 September 2014
Available online 8 September 2014
Keywords:
MCR
RET
Medicinal chemistry
Lead discovery
Targeted therapeutics
Kinase
1. Introduction
Research manuscripts have been published on RET inhibition
[10e16] and anticancer drugs vandetanib [17] and cabozantinib
In 1985, the RET (Rearranged during transfection) gene was
identified as a novel oncogene activated by DNA rearrangement.
The isolated oncogene resulted from a recombination event be-
tween two unlinked human DNA segments, which occurs during
the transfection process [1]. It was discovered that point mutations
in RET lead to the development of medullary thyroid cancer (MTC)
[2e9]. The identification of a small molecule inhibitor for the RET
kinase represents an important strategy for the intervention of RET-
driven cancers; however, the target has largely been neglected.
[18] were found to exhibit RET activity. However, both drugs were
developed as vascular endothelial growth factor receptor
2
(VEGFR2) inhibitors and have VEGFR2 as the primary target. To
improve efficacy and toxicity profiles, it is highly desirable to
develop RET selective inhibitors for the treatment of MTC. We
sought out to utilize MCR enabling chemistries to assist in the
discovery of novel RET inhibitors.
Multicomponent reactions (MCRs) encompass a broad synthetic
landscape where multiple inputs react to create a diversified final
product [19,20]. The synthetic ease and facile analoging ability
make MCR enabling chemistries an attractive tool for drug-
discovery to rapidly identify novel, tractable leads. In the drug-
discovery realm, MCR chemistry has been utilized to identify
novel therapeutics for tuberculosis [21], viral infections [22], ma-
laria [23], etc. Therefore, MCR enabling chemistries represent a
highly validated means to generate biologically relevant chemo-
types to treat human disease. Despite the large scope and appli-
cation of kinase directed MCRs [24e27], no group has displayed the
ability to generate RET kinase inhibitors utilizing MCR enabling
chemistries. RET represents an unmet therapeutic target, and
tailoring an MCR for lead development can help facilitate the dis-
covery of novel inhibitor functionality.
Abbreviations: RET, rearranged during transfection; MTC, medullary thyroid
cancer; VEGFR-2, vascular endothelial growth factor receptor 2; MCR, multicom-
ponent reaction; SAR, structure activity relationship; DFG, tyrosine-phenylalanine-
glycine; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DTT, dithio-
threitol; ATP, adenosine triphosphate; DMF, dimethyl formamide; DMSO, dimethyl
sulfoxide; FAM, carboxyfluorescein; DCM, dichloromethane; MeOH, methanol; TLC,
thin layer chromatography; NMR, nuclear magnetic resonance; HPLC, high-per-
formance liquid chromatography; THF, tetrahydrofuran; IPA, isopropyl alcohol;
EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;
HAOt, 1-hydroxy-7-
azabenzotriazole; DIPEA, N,N-diisopropylethylamine; EtOAc, ethyl acetate.
* Corresponding author. College of Pharmacy, Department of Pharmacology and
Toxicology, The University of Arizona, Tucson, AZ 85721, USA.
E-mail address: hongyuli@pharmacy.arizona.edu (H.-y. Li).
http://dx.doi.org/10.1016/j.ejmech.2014.09.023
0223-5234/Published by Elsevier Masson SAS.

B. Frett et al. / European Journal of Medicinal Chemistry 86 (2014) 714e723
715
In order to utilize MCR enabling chemistries to target the RET
kinase we have generated a kinase inhibitor library utilizing a
modified MCR protocol. The MCR is based on the
ꢁ
GroebkeeBlackburneBienayme reaction [28], but glyoxylic acid is
employed in order to create monosubstitution on the 5 membered
ring of imidazopyridine (Scheme 1) [29,30]. The modified MCR
permits the rapid generation of diverse, drug-like scaffolds that
contain kinase inhibitor functionality. By using this chemistry, we
have synthesized more than 100 imidazopyridine analogues. The
screening of these analogues, followed by non-MCR hit optimiza-
tion, produced two novel chemical scaffolds that inhibited the RET
kinase at sub-micromolar concentrations. We report herein the
progress and development of RET kinase inhibitors derived from an
imidazopyridine MCR chemical library.
2. Results and discussion
2.1. MCR hit generation
MCR kinase library screening was completed with a mid-
throughput screening platform utilizing microfluidics technology
established by Caliper® [31]. The assay contains a microfluidics chip
that can separate phosphorylated peptide from unphosphorylated
starting peptide. The starting peptide is FAM tagged, and can be
tracked in each well using the EZ Reader® plate reader. With this
screening technique, an MCR kinase library with 100 kinase frag-
ments was screened and RET active 1 was identified (Fig. 1, A).
Based on the structure of compound 1, the warhead region
(imidazopyridine) is predicted to bind to the RET hinge, while the
phenyl ring system is predicted to engage a lipophilic pocket
adjacent to the RET allosteric pocket. This is an atypical binding
modality [32], permitting the fragment to bind the kinase ‘back-
ward’ in comparison to the typical binding of ATP to the active site.
Computational modeling showed the phenyl ring system was in
close proximity to the allosteric pocket on RET and therefore adding
allosteric functionality to compound 1 was hypothesized to in-
crease potency (Fig. 1, B).
Fig. 1. A) A novel RET hit (1, RET IC50 ~2.0 mM) from an MCR-based library. The scaffold
is predicted to bind the kinase in an atypical, backward binding mode to that of ATP. B)
Computational binding of 1 in the RET kinase. Compound 1 is predicted to bind the
hinge of RET by hydrogen bonding to A807. The phenyl ring system of 1 accesses a
lipophilic pocket close to the RET allosteric pocket. The RET hinge is shown in red, the
DFG loop is shown in blue, and the c-Helix is shown in purple. Computational ex-
periments were completed with AutoDock Vina [39]. (For interpretation of the refer-
ences to colour in this figure legend, the reader is referred to the web version of this
article.)
2.2. RET inhibitor optimization
A synthetic protocol was developed to grow MCR active 1 into
the RET allosteric pocket (Scheme 2). The solvent exposed tert-butyl
moiety was replaced with a hydrogen to decrease lipophilicity at
the solvent region. Using Fischer esterification, starting acid 2 was
esterified to compound 3. Employing developed Suzuki coupling
conditions, compound 4 was generated from a coupling reaction
between an amino-pyridine boronic ester and compound 3. Com-
pound 4 was cyclized to compound 5 using chloroacetaldehyde.
After, compound 5 was saponified with base to produce 6 and
subsequently coupled to various anilines using EDC to generate
compounds 6ae6g.
position increase potency by engaging a conserved hydrophobic
region. Compound 6f is >6 times more potent than 6b because the
eCF3 moiety from compound 6f is more lipophilic and bulky than
the eCH3 moiety on 6c. Interestingly, the SAR trend does not hold
true for compound 6e, which contains a hydrogen atom at the meta
position. Likely, the hydrophobicity of the unsubstituted aniline on
6e creates high-binding affinity at the hydrophobic region. From
MCR active 1, a series of RET inhibitors were developed, which
uncovered compound 6g, a RET lead inhibitor with sub-micromolar
activity.
6g was strategically designed to engage the conserved hydro-
phobic region of RET (Fig. 2). Taking into account the increase in
activity achieved with 6f, it was hypothesized that exchanging the
eCF3 moiety with a tert-butyl group would have a favorable impact
on activity (Fig. 2). Because of computational modeling results, 3-
amino-5-tert-butylisoxazole was used as the amine input to
generate compound 6g, which displayed ~75 times increase in ac-
tivity when compared to 6f. It is hypothesized that the increase in
activity is a direct result from better engagement in lipophilic
contacts at the allosteric pocket (Fig. 2).
As predicted, growing MCR active 1 into the allosteric region of
RET drastically increased inhibitor potency as seen with compound
6g (RET IC50 ¼ 0.21 0.04
m
M) (Table 1). Other compounds, 6e (RET
M), also dis-
IC50 ¼ 18.8 4.6
m
M) and 6f (RET IC50 ¼ 15.7 2.9
m
played an increase in activity. The structure activity relationship
(SAR) trend on RET is that bulky, lipophilic moieties at the meta
Compound 6g was subjected to an incubation assay to deter-
mine the kinetics of RET inhibition. In general, compounds that
access the kinase allosteric pocket bind the DFG-out kinase
conformation and incubation is typically required to achieve
ꢁ
Scheme 1. Three component GroebkeeBlackburneBienayme reaction with glyoxylic
acid.

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B. Frett et al. / European Journal of Medicinal Chemistry 86 (2014) 714e723
Scheme 2. Synthesis of Compounds (6ae6g). i) EtOH, H2SO4, 100 ꢀC, 12 h. ii) Pd2(dba)3, P(Cy)3, Na2CO3, 4:1 DMF/H2O, MWI 130 ꢀC, 30 min. iii) Chloroacetaldehyde, Na2CO3, EtOH,
100 ꢀC, 48 h. iv) LiOH, 1:1 THF/H2O, 100 ꢀC, 5 h. v) EDC, HOAt, DIPEA, DMF, rt.
maximal inhibition [33]. With an increase in incubation time, 6g
achieved greater RET inhibition (Fig. 3). A linear line was fit to the
IC50 vs incubation time and the IC50 value for no incubation time
was shown to bind RET in the DFG-out fold (Fig. 3), there is not a
large difference in biochemical vs cellular IC50s again suggesting
the compound is not directly competitive with ATP. 6g represents a
strong lead candidate that can be further developed into a RET
advanced lead.
(Y ¼ 0) was determined to be 0.44 0.03
mM. Therefore, through
incubation, RET IC50 increases greater than 2 times and supports
the hypothesis that 6g binds the DFG-out fold of RET. This suggests
6g is a Type-II kinase inhibitor that is not directly competitive with
the binding of ATP.
2.4. Homology model development
Compounds based on the structure of 6g have very ridged, liner
geometries and work was completed to identify a more flexible
scaffold to define additional SARs on RET. An ether linker was hy-
pothesized to be tolerated from a computational modeling study,
and therefore a synthetic protocol was developed (Scheme 3).
Using Fischer esterification, compound 7 was generated from
7a. Compound 8 was synthetized employing a strong base to couple
7 with 3,6-dichloropyridazine. Buchwald conditions were utilized
to convert compound 8 into compound 10, an amino-pyridazine.
Cyclization of compound 10 with chloroacetaldehyde generated
intermediate 11. Compound 11 was hydrolyzed with base to
generate compound 12, which was subsequently coupled to various
anilines using EDC to generate compounds 13aeg.
In order to better understand activity for compounds 6g and 13g
a novel RET DFG-out homology model was generated utilizing
Swiss-Model [34e36]. Crystal structure coordinates of a VEGFR-2
DFG-out structure was used as a template [37], and the RET
amino acid sequence [38] was employed to build a RET DFG-out
crystal structure. The resulting RET DFG-out model clearly dis-
plays the correct shift in the DFG-loop, which opens up the allo-
steric pocket of the kinase for inhibitors to access. There are two
interchangeable folds of the RET kinase: DFG-in, the kinase is active
and the allosteric pocket is closed; DFG-out, the kinase is not active
and the allosteric pocket is open [33]. The developed DFG-out
model served as a tool to evaluate binding modes of compounds
6g and 13g.
Through scaffold hopping from a novel MCR-based hit 1, com-
pound 13g (RET IC50 ¼ 0.75 0.03
mM) was identified, generating
an additional RET lead inhibitor. The SAR on the scaffold based on
compound 13g displayed a more direct trend than SAR from com-
pound 6g (Table 1). On average, compounds generated from
Scheme 3 displayed higher potency on RET, and bulkier groups at
the meta position of the allosteric region produced compounds
with higher potency. Similar to the case of compound 6g, com-
pounds 13aeg engage a hydrophobic region in the allosteric
pocket, which increases compound potency. Interestingly, despite
having on average weaker RET inhibitors, Scheme 2 produced the
most potent compound, 6g. Also, both 6g and 13g contain a t-butyl
isoxazole structural moiety in the allosteric pocket and are at least
10-times more potent than any other compound with different
substituents at the same region.
2.5. RET computational modeling
Compounds developed from the MCR-hit 1 were created to ac-
cess the allosteric pocket of RET, which is only available in the DFG-
out fold and a DFG-out homology model was developed and uti-
lized to computationally model 6g and 13g (Figs. 5 and 6). Com-
pound 6g is predicted to bind to the hinge of RET with
imidazopyridine, making a hydrogen bond with A807 and piepi
stacking with Y806. The amide of compound 6g sits on a ‘bridge’
held up by two hydrogen bonds with E775 and D892 (from the DFG
motif), which allows the inhibitor to enter the allosteric pocket.
This is a typical binding modality that is also observed with ima-
tinib (PDB# 3K5V). Within the allosteric pocket, there is a hydro-
phobic region containing conserved valine, leucine, and isoleucine
residues that the tert-butyl moiety engages (Fig. 5). Compound 13g
is predicted to bind RET in a similar mode to compound 6g, where
the imidazopyridine ring system makes a hydrogen bond with
A807 and piepi stacks with Y806 at the RET hinge. At the ‘bridge’
region, the aryl-ring system from engages L758 through an ion-
induced dipole interaction. Also at the ‘bridge’ region, E775 and
D892 (from the DFG motif) hydrogen bond to 13g. This permits
access into the allosteric pocket where the tert-butyl moiety can
engage the hydrophobic region (Fig. 6). Because of similar biding-
modes observed with compounds 6g and 13g, both compounds
display somewhat similar potency. However, compound 6g was
found ~3 times more potent than compound 13g in the RET
biochemical assay. In cell-based assays, 6g was found ~10 times
more potent than 13g.
2.3. Cell-based studies
To further evaluate RET inhibition of 13g and 6g, the compounds
were progressed into cell-based assays. The assay determined the
amount of RET target inhibition by monitoring phosphorylation
status of Y905 and Y1062 on the RET kinase domain (Fig. 4). Y1062
is responsible for activating PI3K/AKT and RAS/ERK pathways and is
important to inhibit to block RET oncogene signaling. 13g was
found active on RET in RAT1 cells transformed with a RET/C634R
oncogene at an IC50 between 2.5 and 10
corresponds well to the determined biochemical IC50 of
0.75 0.03 M. 6g was found active on RET in RAT1 cells trans-
formed with a RET/C634R oncogene at an IC50 between 0.25 and
0.50 M (Fig. 4, B). Like 13g, the cell activity of 6g corresponds well
to the determined biochemical IC50 of 0.21 0.04 M. Because 6g
mM (Fig. 4, A). This value
m
m
m

B. Frett et al. / European Journal of Medicinal Chemistry 86 (2014) 714e723
717
Table 1
drug-discovery campaigns. A RET DFG-out homology model was
generated and utilized to model both lead inhibitors. The tert-butyl
moiety was found essential for efficient potency, and engages a
hydrophobic region in the allosteric pocket of RET. An incubation
based binding study was completed that identified 6g binds RET
slowly, typical of inhibitors that bind the DFG-out form of the ki-
nase. Further, 13g and 6g inhibited RET in RAT1 cells transformed
by RET/C634R. Both compounds are being developed into advanced
leads through optimization of potency and activity, and the work
will be published in due course.
RET SAR for compounds 6aeg and 13aeg.a
<!–Col Count:2– > Compound
RET IC50 (mM)
6a
6b
6c
6d
6e
6f
6g
>100
>100
>100
67.8 1.1
18.8 4.6
15.7 2.9
0.21 0.04
22.3 7.7
20.4 5.6
19.3 2.8
17.2 3.1
14.3 1.9
9.1 2.6
13a
13b
13c
13d
13e
13f
13g
Vandetanib
4. Experimental Section
4.1. General experimental
0.75 0.03
0.102 0.008
All solvents were reagent grade or HPLC grade and all starting
materials were obtained from commercial sources and used
without further purification. Purity of final compounds was
assessed using a Shimadzu ultra-high throughput LC/MS system
(SIL-20A, LC-20AD, LC-MS 2020, Phenomenex® Onyx Monolithic C-
18 Column) at variable wavelengths of 254 nM and 214 nM (Shi-
madzu PDA Detector, SPD-MN20A) and was >95%, unless otherwise
noted. The HPLC mobile phase consisted of a watereacetonitrile
gradient buffered with 0.1% formic acid. 1H NMR spectra were
recorded at 400 MHz and 13C spectra were recorded at 100 MHz,
both completed on a Varian 400 MHz instrument (Model#
4001S41ASP). Compound activity and kinetics were determined
with the EZ Reader II plate reader (PerkinElmer®, Walthman, USA).
Vandetanib was obtained from LC Laboratories® (Woburn, USA,
Lot# BTB-105). All compounds were purified using silicagel
(0.035e0.070 mm, 60 Å) flash chromatography, unless otherwise
noted. Microwave assisted reactions were completed in sealed
vessels using a Biotage Initiator microwave synthesizer.
a
IC50 values represent the concentration required to inhibit enzyme ac-
tivity by 50%. The screening was completed at 9
duplicate.
mM ATP concentration in
4.2. Synthesis and characterization
4.2.1. 5-Phenylpyridin-2-amine
5-Chloropyridin-2-amine (500 mg, 3.89 mmol), phenyl boronic
acid (711 mg, 5.83 mmol), and Na2CO3 (11.67 mmol) were added to
a mixture of 4:1 DMF/Water (10 mL). The resulting solution was
stirred and degassed with argon for 5 min. P(Cy)3 (49.0 mg,
0.175 mmol) and Pd2(dba)3 (53.4 mg, 0.58 mmol) were then added
Incubation Kinetics of 6g
0.5
0.4
R2
0 86
= .
Fig. 2. A) Computational modeling of 6f in the RET kinase. The eCF3 moiety engages
hydrophobic regions at the back of the allosteric pocket. B) Computation modeling of
6g in the RET kinase. The tert-butyl group is more bulky than the eCF3 moiety and is
predicted to better engage hydrophobic regions at the back of the RET allosteric pocket.
The RET hinge is shown in red, the DFG loop is shown in blue, and the c-Helix is shown
in purple. Computational experiments were completed with AutoDock Vina [39]. (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
0.3
0.2
0.1
0.0
3. Conclusion
MCR fragment-based drug discovery has been utilized to un-
cover two novel kinase inhibitors (6g and 13g) from active frag-
ment 1, and established ‘proof-of-concept’ for MCR-directed RET hit
to lead generation. The binding geometries of both compounds are
unique for the RET kinase and present as valid starting points for
0
20
40
60
Incubation Time (min)
Fig. 3. Incubation assay to determine binding kinetics of compound 6g. IC50 without
incubation: 0.44 0.03 M, IC50 after 3 min incubation: 0.42 0.05 M, IC50 after
20 min incubation: 0.38 0.03 M, IC50 after 60 min incubation: 0.21 0.06 M.
m
m
m
m
m

718
B. Frett et al. / European Journal of Medicinal Chemistry 86 (2014) 714e723
Scheme 3. Synthesis of Compounds (13aeg). i) EtOH, H2SO4, 100 ꢀC, 12 h. ii) NaH, DMF, 80 oC, 48 h. iii) Diphenylmethanimine, xantphos, Pd2(dba)3, K3PO4, dioxane, reflux, 12 h. iv)
MeOH, 12 M HCl, 0 ꢀC-RT, 12 h. v) Chloroacetaldehyde, Na2CO3, EtOH, 100 ꢀC, 48 h. vi) LiOH, 1:1 THF/H2O, 100 ꢀC, 5 h. vii) EDC, HOAt, DIPEA, DMF, RT.
and the reaction was heated under microwave irradiation for
30 min at 130 ꢀC. The crude reaction was adsorbed onto silica and
was consumed. The solvent from the reaction was evaporated
and the reaction was adsorbed onto silica and purified with flash
chromatography using a gradient from 100% DCM to 40% 4:1 DCM/
MeOH to yield compound 3 as yellow crystals (1.153 g, 99%). 1H
purified using flash chromatography utilizing
gradient to yield compound 5-phenylpyridin-2-amine (345 mg,
52.1%). 1H NMR (400 MHz, DMSO-d6)
a DCM/MeOH
d
8.23 (dd, J ¼ 2.5, 0.6 Hz, 1H),
NMR (400 MHz, DMSO-d6)
d
8.21 (d, J ¼ 2.6 Hz,1H), 7.66 (dd, J ¼ 8.6,
7.77 (dd, J ¼ 8.0, 1.5 Hz, 1H), 7.69 (dd, J ¼ 8.6, 2.6 Hz, 2H), 7.53 (d,
J ¼ 7.2 Hz, 2H), 7.38 (d, J ¼ 7.2 Hz, 2H), 6.53 (d, J ¼ 9.3 Hz,1H), 6.10 (s,
2H). ESIMS m/z [MþH]þ 171.
2.6 Hz, 1H), 7.49 (d, J ¼ 8.3 Hz, 2H), 7.26 (d, J ¼ 8.3 Hz, 2H), 6.49 (d,
J ¼ 8.6 Hz, 1H), 6.02 (s, 2H), 4.06 (q, J ¼ 7.1 Hz, 2H), 3.64 (s, 2H), 1.17
(t, J ¼ 7.1 Hz, 3H). ESIMS m/z [MþH]þ 257.
4.2.2. N-(tert-butyl)-6-phenylimidazo[1,2-a]pyridin-3-amine (1)
Glyoxylic acid 50% solution in water (0.097 mL, 0.881 mmol), 5-
phenylpyridin-2-amine (100 mg, 0.588 mmol), and acetic acid
4.2.5. Ethyl 2-(4-(imidazo[1,2-a]pyridin-6-yl)phenyl)acetate (4)
Compound 3 (1.165 g, 4.55 mmol) was added into a reaction
vessel equipped with a stir bar. Ethanol (40 mL) and Na2CO3
(1.205 g, 11.36 mmol) were then added to the reaction vessel and
the reaction was heated to 100 ꢀC in an oil bath. After 5 min,
chloroacetaldehyde 50% solution in water (6.39 mL, 45.5 mmol)
was added dropwise and the reaction was heated to 100 ꢀC for 48 h.
TLC confirmed that all of starting material was consumed, the
solvent was condensed and the reaction was adsorbed onto silica.
The reaction was purified with flash chromatography using a
gradient from 100% DCM to 40% 4:1 DCM/MeOH to yield compound
(5.08 mL, 0.088 mmol) were added to MeOH (1 mL) under stirring.
After 15 min, 2-isocyano-2-methylpropane (0.070 mL, 0.617 mmol)
was added to the reaction and was stirred overnight. After 12 h, the
reaction was adsorbed onto silica and purified using flash chro-
matography using a DCM/MeOH gradient to yield compound 1
(90 mg, 57.7%). 1H NMR (400 MHz, DMSO-d6)
d
8.52 (dd, J ¼ 1.8,
1.0 Hz, 1H), 7.99 (s, 1H), 7.75 (dd, J ¼ 8.0, 1.5 Hz, 1H), 7.69 (dd, J ¼ 8.4,
1.2 Hz, 2H), 7.49 (t, J ¼ 8.0 Hz, 3H), 7.42 (dd, J ¼ 9.3, 1.9 Hz, 1H), 7.15
(s, 1H), 4.61 (s, 1H), 1.17 (s, 9H). ESIMS m/z [MþH]þ 266. HPLC Purity
>95% at 254 nM.
4 (1.210 g, 95%). 1H NMR (400 MHz, CDCl3)
d 8.30 (s, 1H), 7.69 (d,
J ¼ 9.3 Hz,1H), 7.66 (d, J ¼ 1.2 Hz,1H), 7.63 (s, 1H), 7.52 (d, J ¼ 8.2 Hz,
2H), 7.43 (dd, J ¼ 9.3, 1.8 Hz, 1H), 7.40 (d, J ¼ 8.2 Hz, 2H), 4.18 (q,
J ¼ 7.2 Hz, 2H), 3.67 (s, 2H), 1.29 (d, J ¼ 7.2 Hz, 3H). ESIMS m/z
[MþH]þ 281.
4.2.3. Ethyl 2-(4-bromophenyl)acetate (2)
2-(4-bromophenyl)acetic acid (15 g, 69.8 mmol) was added to
ethanol (125 mL). Then, sulfuric acid (0.186 mL, 3.49 mmol) was
added to the reaction and the reaction was heated to 80 ꢀC for 12 h.
The reaction was confirmed complete based on TLC. Solid NaHCO3
was added to the reaction and then the ethanol was evaporated.
The product was extracted with ether and washed 2ꢁ with water
and 1ꢁ with brine. The organic layer was collected, dried with
MgSO4, and condensed to generate compound 2 as a clear oil
4.2.6. 2-(4-(Imidazo[1,2-a]pyridin-6-yl)phenyl)acetic acid (5)
Compound 4 (1.2 g, 4.28 mmol) was added to 1:1 THF/Water
(40 mL) in a pressure reaction vessel. LiOH (1.798 g, 21.4 mmol) was
then added and the reaction was heated to 100 ꢀC for 5 h. TLC
confirmed complete consumption of compound 4. All organic sol-
vent was evaporated and the water solution was extracted with 5ꢁ
DCM and all extracts were discarded. Then, the reaction was acid-
ified with 3 M HCl to pH ~4. The acidified aqueous solution was
extracted 10ꢁ with 4:1 DCM/IPA. All extracts were combined, dried,
and condensed to yield compound 5 (455 mg, 42.1%). 1H NMR
(16.113 g, 95%). 1H NMR (400 MHz, CDCl3)
d
7.45 (d, J ¼ 8.4 Hz, 2H),
7.16 (d, J ¼ 8.4 Hz, 2H), 4.15 (q, J ¼ 7.1 Hz, 2H), 3.56 (s, 2H), 1.25 (t,
J ¼ 7.1 Hz, 3H). ESIMS m/z [MþH]þ 243.
(400 MHz, DMSO-d6)
d 12.33 (s, 1H), 8.97 (s, 1H), 8.02 (s, 1H),
4.2.4. Ethyl 2-(4-(6-aminopyridin-3-yl)phenyl)acetate (3)
7.73e7.64 (m, 5H), 7.40 (d, J ¼ 7.9 Hz, 2H), 3.65 (s, 1H). ESIMS m/z
Compound 2 (1.270 g, 5.23 mmol), 5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)pyridin-2-amine (1 g, 4.54 mmol), and Na2CO3
(1.445 g, 13.63 mmol) were added to a 20 mL microwave vessel. The
degassed 4:1 DMF/water v/v (10 mL) was then added to the reac-
tion and the reaction was degassed with argon for 5 min. P(Cy)3
(0.057 g, 0.204 mmol) and Pd2(dba)3 (0.062 g, 0.068 mmol) were
then added to the reaction vessel followed by degassing with argon
for an additional 5 min. The reaction vessel was sealed and heated
under microwave irradiation for 30 min at 130 ꢀC. TLC confirmed all
of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine
[MþH]þ 253.
4.2.7. N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(imidazo[1,2-a]pyridin-
6-yl)phenyl)acetamide (6g)
Compound 5 (40 mg, 0.159 mmol), 5-(tert-butyl)isoxazol-3-
amine (33.3 mg, 0.238 mmol), EDC (60.9 mg, 0.37 mmol), HOAt
(21.56 mg, 0.159 mmol), and DIPEA (0.043 mL, 0.238 mmol) were all
added to a reaction vessel. DMF (1 mL) was added and the reaction
was stirred at room temperature overnight. After the reaction was

B. Frett et al. / European Journal of Medicinal Chemistry 86 (2014) 714e723
719
4.2.8. 2-(4-(Imidazo[1,2-a]pyridin-6-yl)phenyl)-N-(3-
(trifluoromethyl)phenyl)acetamide (6f)
Compound 6f was synthesized according to the procedure
outlined for 6g (17.5 mg, 27.9%). 1H NMR (400 MHz, DMSO-d6)
d
10.54 (s, 1H), 8.88 (s, 1H), 8.09 (s, 1H), 7.94 (s, 1H), 7.78 (d,
J ¼ 8.2 Hz, 1H), 7.62 (m, 4H), 7.56e7.48 (m, 2H), 7.44 (d, J ¼ 7.9 Hz,
2H), 7.37 (d, J ¼ 7.7 Hz, 1H), 3.72 (s, 2H). 13C NMR (101 MHz, DMSO-
d6)
d 170.06, 140.35, 130.44, 130.35, 126.91, 125.18, 124.79, 124.43,
123.05, 120.02 (q, J ¼ 3.7 Hz), 117.33, 115.55 (q, J ¼ 3.9 Hz), 114.07,
43.33. ESIMS m/z [MþH]þ 396. HPLC Purity 98%.
4.2.9. 2-(4-(Imidazo[1,2-a]pyridin-6-yl)phenyl)-N-
phenylacetamide (6e)
Compound 6e was synthesized according to the procedure
outlined for 6g (20.4 mg, 39.3%). 1H NMR (400 MHz, DMSO-d6)
d
10.18 (s, 1H), 8.88 (s, 1H), 7.95 (s, 1H), 7.64 (d, J ¼ 8.2 Hz, 2H), 7.59
7.60e7.58 (m, 4H), 7.57e7.52 (m,1H), 7.44 (d, J ¼ 8.2 Hz, 2H), 7.28 (t,
J ¼ 7.9 Hz, 2H), 7.02 (t, J ¼ 7.4 Hz, 1H), 3.68 (s, 2H). 13C NMR
(101 MHz, DMSO-d6) d 169.41, 139.63, 135.97, 135.44, 134.10, 130.29,
129.16, 126.88, 125.23, 124.81, 124.41, 123.67, 119.55, 117.33, 114.07,
43.37. ESIMS m/z [MþH]þ 328. HPLC Purity 100%.
4.2.10. N-(3-chlorophenyl)-2-(4-(imidazo[1,2-a]pyridin-6-yl)
phenyl)acetamide (6d)
Compound 6d was synthesized according to the procedure
outlined for 6g (18.8 mg, 32.8%). 1H NMR (400 MHz, DMSO-d6)
d
10.38 (s, 1H), 8.88 (s, 1H), 7.95 (s, 1H), 7.81 (d, J ¼ 1.7 Hz, 1H), 7.63
(m, 4H), 7.54 (d, J ¼ 9.4 Hz, 1H), 7.44 (m, 3H), 7.31 (t, J ¼ 8.1 Hz, 1H),
7.08 (dd, J ¼ 8.1, 2.1 Hz, 1H), 3.69 (s, 2H). 13C NMR (101 MHz, DMSO-
Fig. 4. Serum-starved RAT1 RET/C634R cells were treated for 2 h with increasing
concentrations of 13g (A), 6g (B) or left untreated (NT); cell lysates (50 g) were
immunoblotted with phospho-Y1062 ( pTyr1062) or -Y905 ( pTyr905) RET antibodies.
Anti-RET ( RET) was used for normalization. 13g displayed an IC50 between 2.5 and
10.0 M and 6g displayed an IC50 between 0.25 and 0.50 M.
m
d6)
d 169.84, 141.05, 135.60, 135.54, 134.11, 133.51, 130.89, 130.32,
a
a
126.90, 125.18, 124.79, 124.44, 123.40, 118.99, 117.91, 117.34, 114.05,
a
43.33. ESIMS m/z [MþH]þ 362. HPLC Purity 96%.
m
m
complete, the organic layer was condensed and the crude reaction
was transferred using DCM to a silica column and purified using
DCM to 30% 4:1 DCM/MeOH. The desired compound 6g was iso-
lated in moderate yield (20.4 mg, 34.4%). 1H NMR (400 MHz, CDCl3)
4.2.11. N-(3-fluorophenyl)-2-(4-(imidazo[1,2-a]pyridin-6-yl)
phenyl)acetamide (6c)
Compound 6c was synthesized according to the procedure
outlined for 6g (15.7 mg, 28.7%). 1H NMR (400 MHz, DMSO-d6)
d
9.83 (s, 1H), 8.29 (s, 1H), 7.70 (m, 2H), 7.64 (s, 1H), 7.53 (d,
J ¼ 7.6 Hz, 2H), 7.45 (d, J ¼ 7.6 Hz, 2H), 7.40 (d, J ¼ 9.4 Hz,1H), 6.76 (s,
1H), 3.84 (s, 2H), 1.35 (s, 9H). 13C NMR (101 MHz, CDCl3)
181.78,
d
10.41 (s, 1H), 8.88 (s, 1H), 7.94 (s, 1H), 7.64 (d, J ¼ 8.2 Hz, 2H), 7.59
(m, 3H), 7.54 (dd, J ¼ 9.4, 1.6 Hz, 1H), 7.43 (d, J ¼ 8.2 Hz, 2H),
7.35e7.27 (m, 2H), 6.88e6.82 (m, 1H), 3.69 (s, 2H). 13C NMR
d
169.13, 157.97, 144.70, 136.53, 133.94, 133.72, 130.09, 127.44, 126.38,
125.10, 123.04, 117.76, 112.77, 93.42, 43.61, 33.05, 28.62. ESIMS m/z
[MþH]þ 375. HPLC Purity 100%.
(101 MHz, DMSO-d6)
d
169.82, 162.56 (d, J ¼ 241.3 Hz), 141.33 (d,
J ¼ 11.1 Hz), 135.63, 135.53, 134.10, 130.82 (d, J ¼ 9.6 Hz), 130.33,
126.90, 125.19, 124.81, 124.43, 117.33, 115.26 (d, J ¼ 2.7 Hz), 114.08,
110.13 (d, J ¼ 21.1 Hz), 106.30 (d, J ¼ 26.3 Hz), 43.33. ESIMS m/z
[MþH]þ 346. HPLC Purity 100%.
Fig. 5. Compound 6g computationally docked in the DFG-out RET homology model. RET: carbon ¼ blue; oxygen ¼ red; nitrogen ¼ purple. Inhibitor: carbon ¼ green; oxygen ¼ red;
nitrogen ¼ purple. Computational experiments were completed with AutoDock Vina [39]. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)

720
B. Frett et al. / European Journal of Medicinal Chemistry 86 (2014) 714e723
Fig. 6. Compound 13g computationally docked in the DFG-out RET homology model. RET: carbon ¼ blue; oxygen ¼ red; nitrogen ¼ purple. Inhibitor: carbon ¼ yellow;
oxygen ¼ red; nitrogen ¼ purple. Computational experiments were completed with AutoDock Vina [39]. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
4.2.12. 2-(4-(Imidazo[1,2-a]pyridin-6-yl)phenyl)-N-(m-tolyl)
acetamide (6b)
on a silica column using a gradient from hexanes to EtOAc (50%).
The desired product 8 was isolated in moderate yield (3.02 g, 36%).
Compound 6b was synthesized according to the procedure
1H NMR (400 MHz, CDCl3)
d
7.49 (d, J ¼ 9.1 Hz, 1H), 7.38 (t,
outlined for 6g (19.2 mg, 35.5%). 1H NMR (400 MHz, DMSO-d6)
J ¼ 7.9 Hz, 1H), 7.21e7.14 (m, 3H), 7.11 (ddd, J ¼ 8.2, 2.4, 0.9 Hz, 1H),
d
10.11 (s, 1H), 8.88 (s, 1H), 7.94 (s, 1H), 7.65e7.59 (m, 4H), 7.54 (dd,
3.70 (s, 3H), 3.65 (s, 2H). ESIMS m/z [MþH]þ 279.
J ¼ 9.4, 1.7 Hz, 1H), 7.45e7.41 (m, 3H), 7.38 (d, J ¼ 7.8 Hz, 1H), 7.15 (t,
J ¼ 7.8 Hz, 1H), 6.83 (d, J ¼ 7.8 Hz, 1H), 3.66 (s, 2H), 2.24 (s, 3H). 13
C
4.2.16. Methyl 2-(3-((6-((diphenylmethylene)amino)pyridazin-3-
yl)oxy)phenyl)acetate (9)
NMR (101 MHz, DMSO-d6)
d 169.34, 139.56, 138.32, 136.02, 135.43,
134.10, 130.26, 129.00, 126.87, 125.22, 124.81, 124.41, 124.37, 120.09,
117.33, 116.74, 114.07, 43.40, 21.62. ESIMS m/z [MþH]þ 342. HPLC
Purity 100%.
Compound 8 (2.768 g, 9.93 mmol), diphenylmethamine (1.980 g,
10.93 mmol), and potassium phosphate (10.53 g, 49.7 mmol) were
added to dioxane (40 mL) and the reaction was degassed with
argon for 10 min. Xantphos (0.287 g, 0.497 mmol) and Pd2(dba)3
(0.454 g, 0.497 mmol) were added to the reaction. The reaction was
then sealed under positive argon pressure and heated under reflux
for 12 h. After the overnight, the reaction was condensed and
adsorbed onto silica. The reaction was purified with a silica column
using flash chromatography with a hexane/EtOAc gradient to yield
4.2.13. N-(3-bromophenyl)-2-(4-(imidazo[1,2-a]pyridin-6-yl)
phenyl)acetamide (6a)
Compound 6a was synthesized according to the procedure
outlined for 6g (17.1 mg, 26.5%). 1H NMR (400 MHz, DMSO-d6)
d
10.43 (s, 1H), 8.94 (s, 1H), 8.01 (s, 2H), 7.69 (m, 4H), 7.60 (d,
J ¼ 8.8 Hz,1H), 7.55 (d, J ¼ 7.9 Hz, 1H), 7.48 (d, J ¼ 8.1 Hz, 2H), 7.31 (t,
compound 9 (1.02 g, 24.5%). 1H NMR (400 MHz, CDCl3)
d 7.82 (d,
J ¼ 7.9 Hz, 1H), 7.27 (d, J ¼ 8.1 Hz, 1H), 3.75 (s, 2H). 13C NMR
J ¼ 7.6 Hz, 2H), 7.52 (t, J ¼ 7.3 Hz,1H), 7.43 (t, J ¼ 7.6 Hz, 2H), 7.31 (m,
Hz, 4H), 7.19 (d, J ¼ 6.5 Hz, 2H), 7.11e7.06 (m, 2H), 7.06e7.02 (m,
1H), 6.96 (d, J ¼ 9.1 Hz, 1H), 6.88 (d, J ¼ 9.1 Hz, 1H), 3.69 (s, 3H), 3.61
(s, 2H). ESIMS m/z [MþH]þ 424.
(101 MHz, DMSO-d6) d 169.82, 141.19, 135.60, 135.53, 134.11, 131.20,
130.32, 126.90, 126.29, 125.18, 124.79, 124.44, 122.00, 121.85, 118.29,
117.34, 114.04, 43.33. ESIMS m/z [MþH]þ 406. HPLC Purity 100%.
4.2.14. Methyl 2-(3-hydroxyphenyl)acetate (7)
2-(3-hydroxyphenyl)acetic acid (15 g, 99 mmol) was added to
methanol (197 mL). Sulfuric acid (3.94 mL, 73.9 mmol) was added
and the reaction was heated reflux for three days. The reaction was
cooled to room temperature and solid Na2CO3 was added until the
pH was neutral. Solvent was evaporated and the reaction was re-
dissolved in ether and washed 3ꢁ with water and 2ꢁ with brine.
The organic layer was collected, dried, and condensed to generate
compound 7 as a dark oil (14.764 g, 90%). 1H NMR (400 MHz, CDCl3)
4.2.17. Methyl 2-(3-((6-aminopyridazin-3-yl)oxy)phenyl)acetate
(10)
Compound 9 (1.02 g, 2.409 mmol) was dissolved in methanol
(100 mL) and was cooled to 0 ꢀC. 12 M HCl (2.007 mL, 24.09 mmol)
was added to the reaction and stirred for 30 min at 0 ꢀC. The re-
action was removed from the ice bath and stirred at room tem-
perature for 12 h. The reaction was purified with flash
chromatography using a silica column and a gradient from DCM to
50% 4:1 DCM/MeOH. Compound 10 was isolated as a sticky-yellow
d
7.17 (t, J ¼ 7.8 Hz,1H), 6.82 (d, J ¼ 7.8 Hz,1H), 6.77 (t, J ¼ 1.8 Hz,1H),
oil (456 mg, 73%). 1H NMR (400 MHz, DMSO-d6)
d
7.30 (t, J ¼ 7.8 Hz,
6.73 (dd, J ¼ 8.1, 2.5 Hz, 1H), 5.56 (s, 1H), 3.70 (s, 3H), 3.58 (s, 2H).
ESIMS m/z [MþH]þ 167.
1H), 7.09 (d, J ¼ 9.3 Hz, 1H), 7.02 (d, J ¼ 7.8 Hz, 1H), 6.96e6.89 (m,
3H), 6.20 (s, 2H), 3.66 (s, 2H), 3.59 (s, 3H). ESIMS m/z [MþH]þ 260.
4.2.15. Methyl 2-(3-((6-chloropyridazin-3-yl)oxy)phenyl)acetate
(8)
Compound 7 (4.07 mL, 30.1 mmol) was added to DMF in a re-
action vessel cooled to 0 ꢀC. NaH 60% dispersion (1.565 g,
39.1 mmol) was added to the reaction and the reaction was stirred
at 0 ꢀC for 45 min. 3,6-Dichloropyridazine (6.01 g, 39.1 mmol) was
added and the reaction was raised to 80 ꢀC using an oil bath and
was heated for 48 h. After the reaction was complete as judged by
TLC, the solvent was evaporated and the reaction was adsorbed
onto silica. The reaction was purified using flash chromatography
4.2.18. Methyl 2-(3-(imidazo[1,2-b]pyridazin-6-yloxy)phenyl)
acetate (11)
Compound 11 was synthesized according to the procedure
outlined for compound 4 (290 mg, 98%). 1H NMR (400 MHz, CDCl3)
d
7.95 (d, J ¼ 9.7 Hz, 1H), 7.71 (s, 1H), 7.64 (s, 1H), 7.40 (t, J ¼ 7.8 Hz,
1H), 7.19 (d, J ¼ 7.8 Hz, 1H), 7.17e7.12 (m, 2H), 6.90 (d, J ¼ 9.7 Hz,
1H), 3.71 (s, 3H), 3.67 (s, 2H). ESIMS m/z [MþH]þ 284. HPLC Purity
70%.

B. Frett et al. / European Journal of Medicinal Chemistry 86 (2014) 714e723
721
4.2.19. 2-(3-(Imidazo[1,2-b]pyridazin-6-yloxy)phenyl)acetic acid
(12)
Compound 12 was synthesized according to the procedure
outlined for 5 (451.4 mg, 95%). 1H NMR (400 MHz, DMSO-d6)
12.41
4.2.25. 2-(3-(Imidazo[1,2-b]pyridazin-6-yloxy)phenyl)-N-(m-tolyl)
acetamide (13b)
Compound 13b was synthesized according to the procedure
outlined for 6g (20.4 mg, 51.1%). 1H NMR (400 MHz, CDCl3)
7.88 (d,
d
d
(s,1H), 8.14 (d, J ¼ 9.6 Hz,1H), 8.04 (s,1H), 7.63 (s,1H), 7.42e7.34 (m,
1H), 7.16e7.15 (m, 3H), 7.08 (d, J ¼ 9.6 Hz,1H), 3.61 (s, 2H). ESIMS m/
z [MþH]þ 270.
J ¼ 9.6 Hz, 1H), 7.82 (s, 1H), 7.61 (d, J ¼ 5.5 Hz, 2H), 7.42 (t, J ¼ 7.8 Hz,
1H), 7.33 (s, 1H), 7.25e7.19 (m, 3H), 7.17e7.13 (m, 2H), 6.90 (d,
J ¼ 7.5 Hz, 1H), 6.85 (d, J ¼ 9.6 Hz, 1H), 3.73 (s, 2H), 2.28 (s, 3H). 13
C
NMR (101 MHz, CDCl3)
d 168.57, 159.60, 153.46, 138.89, 137.67,
4.2.20. N-(5-(tert-butyl)isoxazol-3-yl)-2-(3-(imidazo[1,2-b]
pyridazin-6-yloxy)phenyl)acetamide (13g)
137.40, 136.65, 133.06, 130.45, 128.76, 128.12, 126.56, 125.30, 122.01,
120.53, 120.02, 117.23, 116.96, 111.58, 44.54, 21.43. ESIMS m/z
[MþH]þ 359. HPLC Purity 100%.
Compound 13g was synthesized according to the procedure
outlined for 6g (14.7 mg, 33.7%). 1H NMR (400 MHz, CDCl3)
d 9.70 (s,
1H), 7.93 (d, J ¼ 9.6 Hz, 1H), 7.70 (s, 1H), 7.63 (s, 1H), 7.42 (t,
J ¼ 8.0 Hz), 7.30e7.24 (m, 2H), 7.16 (dd, J ¼ 8.0, 2.2 Hz, 1H), 6.87 (d,
J ¼ 9.6 Hz, 1H), 6.72 (s, 1H), 3.81 (s, 2H), 1.31 (s, 9H). 13C NMR
4.2.26. N-(3-fluorophenyl)-2-(3-(imidazo[1,2-b]pyridazin-6-yloxy)
phenyl)acetamide (13a)
(101 MHz, CDCl3)
d 181.79, 168.69, 159.53, 157.90, 153.43, 135.79,
133.05, 130.26, 128.17, 126.50, 122.05, 120.16, 117.35, 111.47, 93.40,
Compound 13a was synthesized according to the procedure
43.83, 33.02, 28.57. ESIMS m/z [MþH]þ 392. HPLC Purity 100%.
outlined for 6g (21.0 mg, 52.0%). 1H NMR (400 MHz, CDCl3)
d 8.19 (s,
1H), 7.88 (d, J ¼ 9.6 Hz, 1H), 7.66e7.60 (m, 2H), 7.48e7.40 (m, 2H),
4.2.21. 2-(3-(Imidazo[1,2-b]pyridazin-6-yloxy)phenyl)-N-(3-
(trifluoromethyl)phenyl)acetamide (13f)
7.24e7.11 (m, 5H), 6.86 (d, J ¼ 9.6 Hz, 1H), 6.78 (t, J ¼ 8.3 Hz, 1H),
3.74 (s, 2H). 13C NMR (101 MHz, Chloroform-d)
d 168.75, 162.89 (d,
Compound 13f was synthesized according to the procedure
J ¼ 244.9 Hz), 159.59, 153.46, 139.40 (d, J ¼ 10.8 Hz), 137.39, 136.34,
133.04 (d, J ¼ 9.7 Hz), 130.35, 130.00 (d, J ¼ 8.6 Hz), 128.12, 126.52,
121.95, 120.11, 117.30, 114.99 (d, J ¼ 2.8 Hz), 111.26, 111.20, 107.28
(dd, J ¼ 26.7, 7.4 Hz), 44.27. ESIMS m/z [MþH]þ 363. HPLC Purity
100%.
outlined for 6g (17.2 mg, 37.4%). 1H NMR (400 MHz, CDCl3)
d 8.28 (s,
1H), 7.87 (d, J ¼ 9.6 Hz, 1H), 7.79 (s, 1H), 7.71 (d, J ¼ 7.9 Hz, 1H),
7.63e7.61 (m, 2H), 7.44 (t, J ¼ 7.9 Hz, 1H), 7.39 (t, J ¼ 7.9 Hz, 1H), 7.33
(d, J ¼ 7.6 Hz, 1H), 7.23 (d, J ¼ 7.6 Hz, 1H), 7.21e7.14 (m, 2H), 6.86 (d,
J ¼ 9.6 Hz, 1H), 3.77 (s, 2H). 13C NMR (101 MHz, Chloroform-d)
d
168.91, 159.59, 153.48, 138.38, 137.36, 136.18, 133.07, 131.29 (q,
J ¼ 32.4 Hz), 130.40, 129.50, 128.09, 126.52, 122.87, 121.98, 120.94,
120.20, 117.31, 116.75e116.26 (m), 111.69, 44.23. ESIMS m/z [MþH]þ
413. HPLC Purity 100%.
4.3. RET biochemical inhibition assay
Kinase activity was measured in a microfluidic assay that
monitors the separation of a phosphorylated product from sub-
strate. The assay was run using a 12-sipper chip on a Caliper EZ
Reader II (PerkinElmer®, Walthman, USA) with separation buffer
(100 mM HEPES, 10 mM EDTA, 0.015% Brij-35, 0.1% CR-3 [Perki-
nElmer®, Walthman, USA]). In 96-well polypropylene plates
(Greiner, Frickenhausen, Germany) compound stocks (20 mM in
DMSO) were diluted into kinase buffer (50 mM HEPES, 0.075% Brij-
35, 0.1% Tween 20, 2 mM DTT,10 mM MgCl2, and 0.02% NaN3) in 12-
4.2.22. 2-(3-(Imidazo[1,2-b]pyridazin-6-yloxy)phenyl)-N-
phenylacetamide (13e)
Compound 13e was synthesized according to the procedure
outlined for 6g (14.4 mg, 37.5%). 1H NMR (400 MHz, CDCl3)
d 7.87 (d,
J ¼ 9.6 Hz, 2H), 7.61 (d, J ¼ 6.0 Hz, 2H), 7.47 (d, J ¼ 8.0 Hz, 2H), 7.41
(d, J ¼ 8.0 Hz, 1H), 7.28e7.19 (m, 4H), 7.15 (d, J ¼ 8.0 Hz, 1H), 7.08 (t,
J ¼ 7.4 Hz, 1H), 6.85 (d, J ¼ 9.6 Hz, 1H), 3.74 (s, 2H). 13C NMR
(101 MHz, CDCl3)
d 168.60, 159.59, 153.47, 137.76, 137.40, 136.60,
133.06,130.33,128.95,128.12,126.56,124.49,122.00,120.04,119.89,
point ½log dilutions (2 mMe6.32 nM). After, 1 mL was transferred
into a 384-well polypropylene assay plate (Greiner, Frickenhausen,
Germany). The RET enzyme (Invitrogen™, Grand Island, USA) was
117.24, 111.59, 44.32. ESIMS m/z [MþH]þ 345. HPLC Purity 100%.
4.2.23. N-(3-bromophenyl)-2-(3-(imidazo[1,2-b]pyridazin-6-
yloxy)phenyl)acetamide (13d)
diluted in kinase buffer to a concentration of 2 nM and 5 mL of the
enzyme mixture was transferred to the assay plate. The inhibitors/
RET enzyme were incubated for 60 min with minor shaking. A
substrate mix was prepared containing ATP (Ambresco®, Solon,
USA) and 5FAM tagged RET peptide (peptide #22, 50 FAM-
EPLYWSFPA, PerkinElmer®, Walthman, USA) dissolved in kinase
Compound 13d was synthesized according to the procedure
outlined for 6g (19.4 mg, 41.1%). 1H NMR (400 MHz, CDCl3)
d 8.03 (s,
1H), 7.88 (d, J ¼ 9.6 Hz, 1H), 7.72 (s, 1H), 7.62 (s, 2H), 7.45e7.40 (m,
2H), 7.24e7.05 (m, 4H), 6.86 (d, J ¼ 9.6 Hz, 1H), 3.74 (s, 2H). 13C NMR
(101 MHz, CDCl3)
d
168.71, 159.59, 153.49, 139.07, 137.39, 136.24,
buffer, and 5
Running concentrations were as follows: ATP (9
(1.5 M), compound 12-point ½log dilutions (0.2 mMe0.632 nM).
mL of the substrate mix was added to the assay plate.
133.07, 130.54, 130.24, 128.14, 128.07, 127.41, 126.52, 122.86, 121.98,
120.18, 118.28, 117.28, 111.64, 44.46. ESIMS m/z [MþH]þ 423. HPLC
Purity 100%.
mM), peptide
m
For positive control, no inhibitor was added. For negative control,
no enzyme was added. For running control, vandetanib (LC Labo-
ratories®, Lot# BTB-105) was utilized. The plate was run until
10e20% conversion based on the positive control wells. The
following separation conditions were utilized: upstream
voltage ꢂ500 V; downstream voltage, ꢂ1900 V; chip pressure ꢂ0.8.
Percent inhibition was measured for each well comparing starting
peptide to phosphorylated product peaks relative to the baseline.
Dose response curves, spanning the IC50 dose, were generated in
GraphPad Prisim 6 and fit to an exponential one-phase decay line
and IC50 values were obtained from the half-life value of the curve.
IC50 values were generated in duplicate and error was calculated
from the standard deviation between values.
4.2.24. N-(3-chlorophenyl)-2-(3-(imidazo[1,2-b]pyridazin-6-yloxy)
phenyl)acetamide (13c)
Compound 13c was synthesized according to the procedure
outlined in for 6g (16 mg, 37.9%). 1H NMR (400 MHz, CDCl3)
d 8.00
(s, 1H), 7.88 (d, J ¼ 9.6 Hz, 1H), 7.62 (s, 2H), 7.58 (t, J ¼ 1.8 Hz, 1H),
7.43 (t, J ¼ 7.8 Hz,1H), 7.34 (d, J ¼ 8.0 Hz,1H), 7.24e7.13 (m, 4H), 7.06
(d, J ¼ 8.0 Hz, 2H), 6.86 (d, J ¼ 9.6 Hz, 1H), 3.74 (s, 2H). 13C NMR
(101 MHz, CDCl3)
d 168.70, 159.59, 153.50, 138.93, 137.39, 136.25,
134.58, 133.07, 130.55, 129.95, 128.11, 126.52, 124.48, 121.98, 120.19,
119.89, 117.78, 117.26, 111.63, 44.47. ESIMS m/z [MþH]þ 379. HPLC
Purity 100%.

722
B. Frett et al. / European Journal of Medicinal Chemistry 86 (2014) 714e723
4.4. RET kinetic assay
Appendix A. Supplementary data
The assay was completed identically to the RET Biochemical
Inhibition Assay, but compounds were incubated with RET for 60,
20, and 3 min before addition of the substrate mix. The IC50 verses
incubation time graph was fit to a liner equation (y ¼ mx þ b) in
GraphPad Prisim 6 and extrapolated to determine 0 min incubation.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.09.023.
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Acknowledgments
This work was supported by a training grant from The National
Institutes of Health (3T32GM008804-10S1), University of Arizona
startup funding, ACS-IRG grant 4288802, The Caldwell Health Sci-
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