Computer aided drug discovery of highly ligand efficient, low molecular weight imidazopyridine analogs as FLT3 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.02.052

The FLT3 kinase represents an attractive target to effectively treat AML. Unfortunately, no FLT3 targeted therapeutic is currently approved. In line with our continued interests in treating kinase related disease for anti-FLT3 mutant activity, we utilized pioneering synthetic methodology in combination with computer aided drug discovery and identified low molecular weight, highly ligand efficient, FLT3 kinase inhibitors. Compounds were analyzed for biochemical inhibition, their ability to selectively inhibit cell proliferation, for FLT3 mutant activity, and preliminary aqueous solubility. Validated hits were discovered that can serve as starting platforms for lead candidates.

Full text of DOI:10.1016/j.ejmech.2015.02.052

Accepted Manuscript
Computer Aided Drug Discovery of Highly Ligand Efficient, Low Molecular Weight
Imidazopyridine Analogues as FLT3 Inhibitors
Brendan Frett, Nick McConnell, Catherine C. Smith, Yuanxiang Wang, Neil P. Shah,
Hong-yu Li
PII:
S0223-5234(15)00148-8
10.1016/j.ejmech.2015.02.052
EJMECH 7734
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 January 2015
Revised Date: 19 February 2015
Accepted Date: 27 February 2015
Please cite this article as: B. Frett, N. McConnell, C.C. Smith, Y. Wang, N.P. Shah, H.-y. Li, Computer
Aided Drug Discovery of Highly Ligand Efficient, Low Molecular Weight Imidazopyridine Analogues as
FLT3 Inhibitors, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.02.052.
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ACCEPTED MANUSCRIPT
For submission as a Short communication
Computer Aided Drug Discovery of Highly Ligand
Efficient, Low Molecular Weight Imidazopyridine
Analogues as FLT3 Inhibitors
1
,†
1,†
3
1
3,4
Brendan Frett , Nick McConnell , Catherine C. Smith , Yuanxiang Wang , Neil P. Shah , and
1
,2,*
Hong-yu Li
1
College of Pharmacy, Department of Pharmacology and Toxicology, The University of Arizona,
Tucson, AZ 85721, USA, 520-626-0083, hongyuli@pharmacy.arizona.edu
2
The University of Arizona Cancer Center, 1515 N Campbell Ave, Tucson, AZ 85724, USA
3
Division of Hematology/Oncology, University of California, San Francisco, CA
4
Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA
†
Authors completed equally to manuscript

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Abstract: The FLT3 kinase represent an attractive target to effectively treat AML.
Unfortunately, no FLT3 targeted therapeutic is currently approved. In line with our continued
interests in treating kinase related disease, we utilized pioneering synthetic methodology in
combination with computer aided drug discovery and identified low molecular weight, highly
ligand efficient, FLT3 kinase inhibitors. Compounds were analyzed for biochemical inhibition,
their ability to selectively inhibit cell proliferation, for FLT3 mutant activity, and preliminary
aqueous solubility. Validated hits were discovered that can serve as starting platforms for lead
candidates.
Keywords: FLT3, kinase inhibitor, AML, ligand efficiency, computer aided drug discovery
Abbreviations: FLT3, Fms-like tyrosine kinase 3; acute myeloid leukemia, AML; ITD, internal
tandem duplication; juxtamembrane, JM; RAS, rat sarcoma; RAF, rapidly accelerated
fibrosarcoma; JAK, janus kinase; STAT, signal transducer and activator of transcription; PI3K,
phosphoinositide 3-kinase; AKT, protein kinase B; TK, tyrosine kinase; FDA, food and drug
administration; PK, pharmacokinetic; LE, ligand efficiency; CADD, computer aided drug
discovery; MW, molecular weight; MCR, multi-component reaction; MWI, microwave
irradiation; IC50, half maximal inhibitory concentration; GI50, half of maximal inhibition of cell
proliferation; STR, short tandem repeat; DCM, dichloromethane; MeOH, methanol; NMR,
nuclear magnetic resonance; DMSO, dimethyl sulfoxide; MHz, megahertz; LC/MS, liquid
chromatography mass spectrometry; HPLC, high performance liquid chromatography; FT-ICR,
fourier transfer ion-cyclotron resonance; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; ATP, adenosine triphosphate;
FAM, carboxyfluorescein; DMF, dimethylformamide; pTSA, p-toluenesulfonic acid; adsorption,
distribution, metabolism, and excretion, ADME

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1. Introduction
FLT3 (Fms-like tyrosine kinase 3) is a member of the class III family of cytokine receptor
tyrosine kinases.[1] FLT3 signaling produces pro-survival and growth signals in cells and is
important for the normal development of hematopoietic stem cells and progenitor cells.[2]
Pathological activating mutations in the FLT3 kinase represent the most common genetic
alteration in patients with acute myeloid leukemia (AML), occurring in approximately one third
of all cases, and are associated with a poor prognosis.[3] The most common FLT3 gain of
function alterations are FLT3-ITD (internal tandem duplication) mutations, which typically occur
in the juxtamembrane (JM) domain-coding sequence of FLT3.[4] The ITD event produces
constitutive ligand-independent activation of FLT3;[5] therefore, the catalytic regulation of FLT3
is compromised leading to autophosphorylation and continuous activation of the RAS/RAF,
JAK/STAT, and PI3K/AKT pathways (Figure 1).[6] FLT3-ITD mutations are present in about 20-
25% of all AML patients.[7] An additional 5-10% of AML patients have activating point
mutations in the FLT3 kinase domain.
The growing body of evidence that suggests FLT3 activity is essential for a large
population of AML has sparked enormous therapeutic interests. A number of tyrosine kinase
(TK) inhibitors have been tested as FLT3 inhibitors.[8-25] TK inhibitors such as cabozantinib,[8]
sorafenib,[9-12] sunitinib,[12-14] ponatinib,[15-17] crenolanib,[18-21] and quizartinib[22-23]
have all displayed FLT3 activity, and crenolanib[24] and quizartinib[25] are currently undergoing
FDA trials. Issues with current FLT3 inhibitors stem from problems with poor pharmacokinetic
and toxicity profiles and also vulnerability to drug-resistant mutations.[24-26] Crenolanib has
displayed significant drawbacks in a phase II solid tumor trial because of issues with hepatic

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toxicity and a poor pharmacokinetic (PK) profile, requiring the drug to be administered three
times daily;[24], leading to modest clinical activity to date. Quizartinib cannot be dosed at a
high enough concentration to inhibit secondary kinase domain mutations because of dose-
limiting myelosuppression.[26] Although numerous kinase inhibitors have displayed FLT3
activity, issues with toxicity, poor drug properties, and vulnerability to drug-resistant mutation
have caused unfavorable therapeutic profiles with no agent achieving FDA approval.
In our continued effort to develop novel kinase inhibitors for human disease,[27-28] we
identified novel FLT3 validated hits with low molecular weights and high ligand efficiency (LE)
[
29] values utilizing an innovative synthetic methodology.[30-31] While the compounds are not
as active as known FLT3 inhibitors, low molecular weights (MW), high LE values, and rapid
synthesis permit the hit compounds to serve as exceedingly flexible starting points to develop
FLT3 lead candidates. Further, the inhibitors are based upon the imidazopyridine core, which is
a unique heterocycle for FLT3 inhibition. Herein we discuss the identification of novel
imidazopyridine FTL3 inhibitors using innovative synthetic mythology in combination with
computer aided drug discovery (CADD).

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2. Results and Discussion
2.1 Virtual Library Generation
Recently, a new synthetic methodology has been developed that permits the rapid
generation of imidazopyridine analogues that appear to have kinase-inhibitor functionality
Scheme 1).[30] A virtual library was constructed around the synthesis using a select
(
compound (Table 1; 1) along with additional compounds (Table 1; 2 and 3) and were utilized to
help identify potential kinase inhibitors (Table 1). In total, the virtual library contained about
5
0 structures that could be synthesized utilizing this novel, one-pot synthesis. The overarching
goal was to exploit rapid synthetic diversity with help from computer aided drug discovery
CADD) to expeditiously evaluate the usefulness of imidazopyridine-based kinase inhibitors.
(
2.2 Computer Aided Drug Discovery (CADD)
Using AutoDock Vina,[32] the virtual library was screened against multiple kinase active
sites. AutoDock Vina provides docking scores in terms of ΔG values and a ΔG value of less than
8.0 kcal/mol was defined as a hit. Computational hit compounds were investigated further for
-
plausible binding modes and novelty of structure in Discovery Studio 3.5. In general, the
imidazopyridine library was found to have highly negative docking scores with good ligand
efficiency (LE) values for the FLT3 tyrosine kinase (PDB# 1RJB) (Table 1).[33] Binding analysis
was investigated with 3 because of an increase in computational affinity. Unlike 1 or 2, which
are mono-substituted, 3 contains di-imidazopyridine substitution. Modeling results indicated
substitution off the 3 position interacts with Lys614 (cation-Pi interaction) and substitution at
the 7 position interacts with Val624 (sigma-Pi interaction) in the FLT3 active site (Figure 2).

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Because of the modeling results, the synthesis of a FLT3-directed compound library was
completed utilizing the imidazopyridine core. Importantly, generated inhibitors have low
molecular weights, high LEs, and are validated hits for further FLT3 inhibitor development.
2.3 FLT3 Inhibitor Library Synthesis
Library generation was accomplished using a rapid 2 or 3 step synthesis that was
assisted with microwave irradiation (MWI) (Scheme 2). Although the literature reports
aminopyridine cyclization and C-C bond formation in one step,[30] the synthetic approach was
modified for analoguing purposes and to increase overall yield/purity of the final products. The
most innovative aspect to the synthesis is the use of a ligand-free, C-H activating reaction to
create substitution at the 5 position of imidazopyridine. Traditionally, imidazopyridine is
activated at the 5 position through halogenation,[34-41] requiring additional steps to create a
C-C bond. We took advantage of an innovate ligand-free, C-H activating reaction to lower the
amount of synthetic steps as well as library iteration time.[30-31]
To construct the imidazopyridine core, 2-aminopyridine or 4-chloro-2-aminopyridine
was cyclized with 2-chloroacetaldehyde in 1-butanol under reflux conditions. Imidazopyridine,
4, was subjected to a ligand-free, C-H activating reaction to generate analogues 6-9. To create
di-substituted imidazopyridines, 7-chloroimidazo[1,2-a]pyridine, 5, was reacted with various
boronic acids for substitution at the 7 position creating intermediates 10-13. After, the
analogues were subjected to a ligand-free, C-H activating reaction for substitution at the 5
position yielding compounds 14-29.

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2.4 FLT3 Inhibitor Biochemical Evaluation
After synthesis, compounds were screened against FLT3 at a single point concentration
of 20.0 µM (Table 2). Results from single point activity correlate very well with results from
computational modeling, as di-substituted imidazopyridines displayed the greatest amount of
inhibition. It was found that electron withdrawing functionality at the para position of R1
increased activity. Also, at the R position, 5-membered ring systems with a heteroatom were
2
preferred.
Compounds that displayed strong single point inhibition (> 90%) were subjected to IC50
calculation (Table 2). At the R position, 4-fluorobenzene produced compounds with the best
1
inhibition as seen with 17, 21, 25, and 29, which obtained IC values of 0.015, 0.041, 0.016, and
5
0
0.078 µM, respectively. This series is predicted to engage Lys614 through a cation-Pi
interaction (Figure 2), and the electron withdrawing properties of fluorine likely increase this
interaction. At the R position, thiophene as well as phenyl obtained greatest inhibition as seen
2
2
with 17 and 25, which achieved IC50 values of 0.015 and 0.016 µM, respectively. The R position
is predicted to contact Val624 through a sigma-pi interaction and thiophene and phenyl appear
preferred at this region.
To assess binding energy per atom, ligand efficiency (LE) values were calculated (Table
2
). Inhibitors displayed LE values greater than 0.34, which suggests all compounds are
extremely energy efficient binders. The FLT3 LE value for crenolanib and quizartinib is 0.38 and
.31, respectively. Even though both crenolanib and quizartinib achieve strong inhibition of
0
FLT3, LE has not been optimized. For instance, compound 9, which displayed a moderate FLT3
IC50 of 0.480 µM achieved a strikingly high LE value of 0.55. Compound 9 represents an

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extremely energy efficient binder and crystallography studies are underway to help decipher
important binding interactions. The most potent analogues, 17 and 25, displayed LE values of
0.52 and 0.49, respectively. Both compounds 17 and 25 are strategic progressions from
compound 9 and illustrate a durable conservation in LE with the imidazopyridine scaffold.
Although 17 and 25 achieved low nano-molar inhibition on FLT3, the compounds are still
relatively small and can be further improved for enhanced potency.
2.5 FLT3-Driven Cell-Based Studies
Compounds 17, 21, and 25 were progressed to cell based studies using FLT3-driven cell
lines (Figure 3 and Table 4). In BaF3 cells transformed with a FLT3 oncogene, all compounds
were quite active displaying GI50 values between 0.1 and 1.0 µM (Table 4). In patient-derived
Molm-14 and MV4-11 AML cell lines, which harbor a FLT3 ITD mutation, 17, 21, and 25
displayed similar potency (Table 4). However, in BaF3 cells transformed with a FLT3-ITD
oncogene containing a secondary quizartinib-resistant activation loop (D835V) or gatekeeper
(F691L) point mutation, compounds 17 and 21 were ineffective. While 25 was still able to
maintain activity for both activation loop and gatekeeper mutants with an IC50 of ~1.0 µM,
similar potency was observed in native BaF3 cells. Therefore, it is unclear if compound 25 is
capable of inhibiting these FLT3 mutations or if the compound possesses undesirable cytotoxic
properties. Further studies are underway to resolve this ambiguity. Nevertheless, 17, 21, and
25 are cell-membrane permeable and are able to block growth of FLT3-driven cells in vivo.
Because of high LE values and low molecular weights, the compounds represent validated hits
that can be further optimized into lead candidates.

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2.6 Preliminary Pre-formulation Studies
The imidazopyridine scaffold is known to have low water solubility. Therefore,
preliminary salt formation with organic and inorganic acids was investigated to increase water
solubility of compound 17 (Table 3). It is hypothesized that the imidazopyridine of 17 has a pKa
around 6.0, based on the experimentally determined pKa of Ambien® (zolpidem) (pK =
a
6
.16).[42] Inorganic and organic acids with pK s less than 6.0 were stoichiometrically mixed to
a
create the conjugate acid of compound 17 (Table 3). It was found that oxalic acid as well as p-
toluenesulfonic acid drastically increased aqueous solubility. Oxalic acid was found to be the
best co-salt, increasing aqueous solubility from < 0.08 mg/mL to 0.5 mg/mL. Despite the poor
solubility of imidazopyridine compounds, formation of highly-soluble salts can increase water
solubility and improve adsorption, distribution, metabolism, and excretion (ADME) properties.

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3. Conclusion
New synthetic methodology that generates imidazopyridine analogues was utilized for
kinase library synthesis.[30-31] Computational screening identified the imidazopyridine
scaffold was active on the FLT3 kinase. Subsequently, a low molecular weight FLT3-directed
library was constructed and screened. Resulting FLT3 inhibitors were found to be highly ligand
efficient (LE) and possess LE values greater than clinical FLT3 inhibitors. Select compounds
were progressed into cell based assays and were found to block FLT3-driven proliferation at
sub-micromolar IC50 values, and aqueous solubility was increased by forming highly water
soluble salts. The developed analogues represent validated FLT3 hits and can be utilized to
identify potent, lead candidates.

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4. Experimental Section
4.1. General Experimental
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
(Shimadzu PDA Detector, SPD-MN20A) and was >95%, unless otherwise noted. The HPLC
1
H
mobile phase consisted of a water-acetonitrile gradient buffered with 0.1% formic acid.
1
3
NMR spectra were recorded at 400 MHz and C spectra were recorded at 100 MHz, both
completed on a Varian 400 MHz instrument (Model# 4001S41ASP). High-resolution mass
spectrometry was completed using a Bruker 9.4 T Apex-Qh hybrid Fourier transfer ion-cyclotron
resonance (FT-ICR) mass spectrometer. Compound activity was determined with the EZ Reader
II plate reader (PerkinElmer®, Walthman, USA). All compounds were purified using silicagel
(0.035-0.070 mm, 60 Å) flash chromatography, unless otherwise noted. Microwave assisted
reactions were completed in sealed vessels using a Biotage Initiator microwave synthesizer.
4.2 Synthesis and characterization
4.2.1 Imidazo[1,2-a]pyridine (4)
2-aminopyridine (15 g, 0.159 M) was dissolved in 1-butanol (64 mL) in a 250 mL round
bottom flask affixed with a magnetic stir bar. 2-chloroacetaldehyde 50% solution in water (24.3
o
mL, 0.191 M) was added hitherto and the reaction was heated to 130 C for 12 hours. The
reaction solvent was condensed in vacuo and the crude product was adsorbed onto silica. The

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product was purified via flash chromatography utilizing a DCM/MeOH gradient and isolated as a
1
light, yellow oil (17 g, 90%). H NMR (400 MHz, Chloroform-d) δ 8.13 (d, J = 6.7 Hz, 1H), 7.63
+
(
m, 2H), 7.58 (s, 1H), 7.19 – 7.12 (m, 1H), 6.78 (t, J = 6.7 Hz, 1H). ESIMS m/z [M+H] 119.
4.2.2 7-chloroimidazo[1,2-a]pyridine (5)
7-chloroimidazo[1,2-a]pyridine (5) was synthesized according to the procedure outlined
1
for ‘4.2.1 Imidazo[1,2-a]pyridine (4)’ and isolated as an off-white solid (79%). H NMR (400
MHz, DMSO-d ) δ 9.04 (d, J = 7.2 Hz, 1H), 8.46 (s, 1H), 8.21 (s, 1H), 8.12 (s, 1H), 7.59 (dd, J = 7.2,
6
+
2
.0 Hz, 1H). ESIMS m/z [M+H] 153.
4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile (6)
Imidazo[1,2-a]pyridine (4) (100 mg, 0.846 mmol) was dissolved in DMF (4 mL) in a
microwave vial affixed with a magnetic stir bar. 5-bromo-2-fluorobenzonitrile (339 mg, 1.693
mmol) was added, followed by potassium acetate (166 mg, 1.693 mmol) and palladium(II)
o
acetate (19 mg, 0.085 mmol) and the vial was sealed. The reaction was heated to 160 C by
microwave irradiation for 1 hour. The reaction mixture was diluted with ethyl acetate and
washed three times with saturated sodium bicarbonate and three times with deionized water.
The organic layer was dried over anhydrous sodium sulfate and the solvent was removed in
vacuo. The crude product was absorbed onto silica. The product was purified via flash
chromatography utilizing a DCM/MeOH gradient and isolated as a brown solid (132 mg, 66%).
1
H NMR (400 MHz, DMSO-d ) δ 8.77 (d, J = 6.9 Hz, 1H), 8.06 (s, 1H), 8.04 (d, J = 7.7 Hz, 1H), 7.94
6
–
7.90 (m, 1H), 7.79 – 7.75 (m, 1H), 7.74 – 7.71 (m, 1H), 7.44 – 7.38 (m, 1H), 7.07 (t, J = 6.9 Hz,

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1
3
1
1
2
H). C NMR (101 MHz, Chloroform-d) δ 163.56 (d, J = 260.1 Hz), 135.99, 134.49 (d, J = 23.2 Hz)
25.87, 123.55, 123.20, 123.06 (d, J = 3.4 Hz), 118.66, 118.25, 117.35, 115.37, 114.36 (d, J =
+
0.8 Hz), 113.92. ESIMS m/z [M+H] 238
4.2.4 2-(4-(imidazo[1,2-a]pyridin-3-yl)phenyl)acetonitrile (7)
2-(4-(imidazo[1,2-a]pyridin-3-yl)phenyl)acetonitrile (7) was synthesized according to the
procedure outlined for ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile (6)’ and
1
isolated as a brown solid (143 mg, 72.4%) H NMR (400 MHz, Chloroform-d) δ 8.31 (d, J = 7.0
Hz, 1H), 7.71 (s, 1H), 7.68 (d, J = 9.1 Hz, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.22
1
3
(
dd, J = 9.1, 7.0 Hz, 1H), 6.84 (t, J = 6.8 Hz, 1H), 3.84 (s, 2H). C NMR (100 MHz, Chloroform-d) δ
146.27, 132.72, 129.80, 129.21, 128.87, 128.56, 124.78, 124.53, 123.19, 118.28, 117.57, 112.83,
+
2
3.46. ESIMS m/z [M+H] 234
4.2.5 3-(p-tolyl)imidazo[1,2-a]pyridine (8)
3-(p-tolyl)imidazo[1,2-a]pyridine (8) was synthesized according to the procedure
outlined for ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile (6)’ and isolated as a
1
brown solid (120 mg, 68%). H NMR (400 MHz, Chloroform-d) δ 8.29 (d, J = 7.0 Hz, 1H), 7.66 (s,
1H), 7.64 (s, 1H), 7.43 (d, J = 7.9 Hz, 2H), 7.30 (d, J = 7.9 Hz, 2H), 7.17 - 7.13 (m, 1H), 6.78 - 6.74
1
3
(
m, 1H), 2.42 (s, 3H). C NMR (100 MHz, Chloroform-d) δ 145.91, 138.08, 132.18, 129.86,
+
1
28.94, 127.90, 126.24, 123.98, 123.31, 118.09, 112.35, 21.29. ESIMS m/z [M+H] 209

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4.2.6 3-(4-fluorophenyl)imidazo[1,2-a]pyridine (9)
3-(4-fluorophenyl)imidazo[1,2-a]pyridine (9) was synthesized according to the
procedure outlined for ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile (6)’ and
1
isolated as a brown solid (132 mg, 73%). H NMR (400 MHz, Chloroform-d) δ 8.25 – 8.19 (m,
1
3
1
H), 7.69 – 7.62 (m, 2H), 7.51 - 7.47 (m, 2H), 7.22 - 7.15 (m, 3H), 6.81 - 6.78 (m, 1H). C NMR
(100 MHz, Chloroform-d) δ 162.41 (d, J = 248.4 Hz), 145.94, 132.38, 129.88 (d, J = 8.2 Hz),
+
125.26 (d, J = 3.4 Hz), 124.58, 123.02, 118.12, 116.25 (d, J = 21.6 Hz), 112.59. ESIMS m/z [M+H]
213
4.2.7 7-(thiophen-2-yl)imidazo[1,2-a]pyridine (10)
7
-chloroimidazo[1,2-a]pyridine (5) (300 mg, 1.97 mmol) was dissolved in 8:1 DMF:H O
2
(
13.1 mL) in a microwave vial affixed with a magnetic stir bar. Thiophen-2-ylboronic acid was
added followed by sodium carbonate (834 mg, 7.86 mmol). The reaction mixture was degassed
with argon. Pd (dba) (36 mg, 0.039 mmol) and P(Cy) (33 mg, 0.118 mmol) was added to the
2
3
3
reaction mixture and the vial was sealed. The reaction was heated by microwave irradiation to
o
1
30 C for 1 hour. After, the reaction mixture was diluted with ethyl acetate and washed three
times with saturated sodium bicarbonate and three times with deionized water. The organic
layer was dried over anhydrous sodium sulfate and the solvent was removed in vacuo. The
crude product was absorbed onto silica. The product was purified via flash chromatography
1
utilizing a DCM/MeOH gradient and isolated as a brown solid (223 mg, 59%). H NMR (400 MHz,
Chloroform-d) δ 8.07 (dd, J = 7.0, 2.7 Hz, 1H), 7.81 (s, 1H), 7.63 (s, 1H), 7.54 (s, 1H), 7.38 (s, 1H),
1
3
7
.35 – 7.30 (m, 1H), 7.14 – 7.07 (m, 1H), 7.03 (d, J = 5.4 Hz, 1H). C NMR (100 MHz, Chloroform-

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d) δ 145.57, 142.02, 134.43, 130.87, 128.29, 125.88, 125.59, 124.20, 112.82, 112.33, 111.40.
+
ESIMS m/z [M+H] 201
4.2.8 7-(furan-2-yl)imidazo[1,2-a]pyridine (11)
7-(furan-2-yl)imidazo[1,2-a]pyridine (11) was synthesized according to the procedure
outlined in ‘4.2.7 7-(thiophen-2-yl)imidazo[1,2-a]pyridine (10)’ and was isolated as a brown
1
solid (189 mg, 52%). H NMR (400 MHz, Chloroform-d) δ 8.12 – 8.06 (m, 1H), 7.88 (s, 1H), 7.64
(s, 1H), 7.55 (s, 1H), 7.52 (s, 1H), 7.08 (d, J = 7.1 Hz, 1H), 6.74 (d, J = 3.2 Hz, 1H), 6.52 (dd, J = 3.2,
1
3
1
.8 Hz, 1H). C NMR (100 MHz, Chloroform-d) δ 151.85, 145.55, 142.97, 134.39, 127.02,
+
1
25.63, 112.38, 111.97, 110.86, 109.25, 106.85. ESIMS m/z [M+H] 185
4.2.9 7-phenylimidazo[1,2-a]pyridine (12)
7-phenylimidazo[1,2-a]pyridine (12) was synthesized according to the procedure
outlined in ‘4.2.7 7-(thiophen-2-yl)imidazo[1,2-a]pyridine (10)’ and was isolated as a brown
1
solid (266 mg, 70%). H NMR (400 MHz, Chloroform-d) δ 8.12 - 8.07 (m, 2H), 7.96 (s, 1H), 7.72 –
7
7
1
.67 (m, 1H), 7.62-7.59 (m, 2H), 7.53 – 7.49 (m, 1H), 7.48-7.43 (m, 1H), 7.41 – 7.35 (m, 3H),
1
3
.07-7.03 (m, 1H). C NMR (100 MHz, Chloroform-d) δ 145.06, 133.59, 133.08, 129.09, 128.42,
+
27.46, 126.79, 125.75, 114.21, 112.75, 112.00. ESIMS m/z [M+H] 195
4.2.10 7-(4-methoxyphenyl)imidazo[1,2-a]pyridine (13)
7-(4-methoxyphenyl)imidazo[1,2-a]pyridine (13) was synthesized according to the
procedure outlined in ‘4.2.7 7-(thiophen-2-yl)imidazo[1,2-a]pyridine (10)’ and was isolated as a

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1
brown solid (241 mg, 55%). H NMR (400 MHz, Chloroform-d) δ 8.12 (d, J = 7.1 Hz, 1H), 7.81 (s,
1
7
1
H), 7.65 (d, J = 1.1 Hz, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.54 (s, 1H), 7.04 (dd, J = 7.1, 1.8 Hz, 1H),
1
3
.00 (d, J = 8.8 Hz, 2H), 3.86 (s, 6H), 3.81 (s, 3H). C NMR (100 MHz, Chloroform-d) δ 133.63,
+
30.90, 127.84, 125.56, 114.46, 113.36, 112.26, 111.83, 55.36. ESIMS m/z [M+H] 225
4.2.11 2-fluoro-5-(7-(thiophen-2-yl)imidazo[1,2-a]pyridin-3-yl)benzonitrile (14)
2
-fluoro-5-(7-(thiophen-2-yl)imidazo[1,2-a]pyridin-3-yl)benzonitrile (14) was synthesized
according to the procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile
(6)’ using 7-(thiophen-2-yl)imidazo[1,2-a]pyridine (10) and was isolated as a brown solid (10.7
1
mg, 17%) H NMR (400 MHz, Chloroform-d) δ 8.37 (d, J = 7.2 Hz, 1H), 7.91 (s, 1H), 7.85 (s, 1H),
1
3
7
.78 (t, J = 7.3 Hz, 1H), 7.54 – 7.39 (m, 4H), 7.23 (d, J = 7.0 Hz, 1H), 7.18 – 7.13 (m, 1H). C NMR
(100 MHz, Chloroform-d) δ 152.86, 140.03, 139.38, 136.57, 135.61, 134.42, 132.69, 132.29,
128.54, 126.71, 124.89, 123.13, 122.96, 122.87 (d, J = 3.3 Hz), 114.18 (d, J = 20.9 Hz), 113.30,
+
1
12.75. ESIMS m/z [M+H] 320
4.2.12 2-(4-(7-(thiophen-2-yl)imidazo[1,2-a]pyridin-3-yl)phenyl)acetonitrile (15)
2-(4-(7-(thiophen-2-yl)imidazo[1,2-a]pyridin-3-yl)phenyl)acetonitrile was synthesized
according to the procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile
(6)’ using 7-(thiophen-2-yl)imidazo[1,2-a]pyridine (10) and was isolated as a brown solid (18 mg,
1
2
3
7
9%). H NMR (400 MHz, Chloroform-d) δ 8.28 (dd, J = 7.1, 3.2 Hz, 1H), 7.89 (s, 1H), 7.72 (d, J =
.8 Hz, 1H), 7.62 – 7.56 (m, 2H), 7.52-7.49 (m, 2H), 7.43 (t, J = 3.7 Hz, 1H), 7.38 – 7.34 (m, 1H),
1
3
.14 - 7.11 (m, 2H), 3.85 (s, 2H). C NMR (100 MHz, Chloroform-d) δ 141.76, 133.53, 131.08,

ACCEPTED MANUSCRIPT
129.87, 128.94, 128.59, 128.47, 128.38, 126.32, 126.12, 124.40, 123.15, 117.49, 113.10, 111.86,
+
1
09.99, 23.50. ESIMS m/z [M+H] 316
4.2.13 7-(thiophen-2-yl)-3-(p-tolyl)imidazo[1,2-a]pyridine (16)
7-(thiophen-2-yl)-3-(p-tolyl)imidazo[1,2-a]pyridine (16) was synthesized according to the
procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile (6)’ using 7-
1
(thiophen-2-yl)imidazo[1,2-a]pyridine (10) and was isolated as a brown solid (33 mg, 56%).
H
NMR (400 MHz, Chloroform-d) δ 8.27 (dd, J = 7.3, 1.0 Hz, 1H), 7.86 (dd, J = 1.8, 1.0 Hz, 1H), 7.67
(s, 1H), 7.47 – 7.43 (m, 2H), 7.40 (dd, J = 3.7, 1.2 Hz, 1H), 7.34 – 7.31 (m, 3H), 7.11 (dd, J = 5.1,
1
3
3
1
1
.6 Hz, 1H), 7.06 (dd, J = 7.3, 1.8 Hz, 1H), 2.43 (s, 3H). C NMR (100 MHz, Chloroform-d) δ
46.15, 142.07, 138.23, 133.14, 130.48, 129.96, 129.64, 128.31, 127.85, 125.83, 125.61, 124.13,
+
23.30, 113.11, 111.40, 21.34. ESIMS m/z [M+H] 291
4.2.14 3-(4-fluorophenyl)-7-(thiophen-2-yl)imidazo[1,2-a]pyridine (17)
3
-(4-fluorophenyl)-7-(thiophen-2-yl)imidazo[1,2-a]pyridine (17) was synthesized
according to the procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile
(6)’ using 7-(thiophen-2-yl)imidazo[1,2-a]pyridine (10) and was isolated as a brown solid (25 mg,
1
5
1
7
1
2%). H NMR (400 MHz, Chloroform-d) δ 8.23 – 8.18 (m, 1H), 7.86 (s, 1H), 7.66 (d, J = 1.9 Hz,
H), 7.56 – 7.50 (m, 2H), 7.43 – 7.39 (m, 1H), 7.36 – 7.33 (m, 1H), 7.25 - 7.20 (m, 2H), 7.13 –
1
3
.10 (m, 1H), 7.10 - 7.07 (m, 1H). C NMR (100 MHz, Chloroform-d) δ 162.57 (d, J = 248.8 Hz),
46.21, 141.87, 133.28, 130.81, 129.90 (d, J = 8.2 Hz), 128.35, 127.39, 126.00, 125.13 (d, J = 3.2

ACCEPTED MANUSCRIPT
+
Hz), 124.29, 123.03, 116.42 (d, J = 21.8 Hz), 113.12, 111.69. ESIMS m/z [M+H] 295. Predicted:
295.06997; Found: 295.06975
4.2.15 2-fluoro-5-(7-(furan-2-yl)imidazo[1,2-a]pyridin-3-yl)benzonitrile (18)
2-fluoro-5-(7-(furan-2-yl)imidazo[1,2-a]pyridin-3-yl)benzonitrile (18) was synthesized
according to the procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile
(6)’ using 7-(furan-2-yl)imidazo[1,2-a]pyridine (11) and was isolated as a brown solid (12 mg,
1
2
5%). H NMR (400 MHz, Chloroform-d) δ 8.34 (d, J = 7.4 Hz, 1H), 7.96 – 7.92 (m, 1H), 7.77 (s,
1H), 7.65 - 7.60 (m, 1H), 7.58 – 7.52 (m, 1H), 7.28 - 7.19 (m, 1H), 7.06 – 6.98 (m, 1H), 6.96 – 6.90
1
3
(
m, 1H), 6.80 (d, J = 3.5 Hz, 1H), 6.55 - 6.54 (m, 1H). C NMR (100 MHz, Chloroform-d) δ 143.60
(d, J = 17.5 Hz), 136.08, 134.50, 134.03, 123.81, 123.59, 120.76, 119.45, 117.19 (d, J = 4.8 Hz),
+
1
15.19 (d, J = 15.1 Hz), 112.21, 111.81, 111.18, 110.78, 108.46, 107.78. ESIMS m/z [M+H] 304
4.2.16 2-(4-(7-(furan-2-yl)imidazo[1,2-a]pyridin-3-yl)phenyl)acetonitrile (19)
2-(4-(7-(furan-2-yl)imidazo[1,2-a]pyridin-3-yl)phenyl)acetonitrile (19) was synthesized
according to the procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile
(6)’ using 7-(furan-2-yl)imidazo[1,2-a]pyridine (11) and was isolated as a brown solid (22 mg,
1
4
6%). H NMR (400 MHz, Chloroform-d) δ 8.29 – 8.24 (m, 1H), 7.92 (s, 1H), 7.70 (s, 1H), 7.58 (d,
J = 8.1 Hz, 2H), 7.54 – 7.52 (m, 1H), 7.49 (d, J = 8.1 Hz, 2H), 7.12 (dd, J = 7.3, 1.6 Hz, 1H), 6.77 (d,
1
3
J = 3.4 Hz, 1H), 6.52 (dd, J = 3.4, 1.8 Hz, 1H), 3.84 (s, 2H). C NMR (100 MHz, Chloroform-d) δ
151.68, 146.46, 143.20, 133.51, 129.83, 128.99 (d, J = 14.4 Hz), 128.48, 127.24, 124.52, 123.19,
+
1
17.52, 112.09, 111.18, 109.78, 107.23, 23.50. ESIMS m/z [M+H] 300

ACCEPTED MANUSCRIPT
4.2.17 7-(furan-2-yl)-3-(p-tolyl)imidazo[1,2-a]pyridine (20)
7-(furan-2-yl)-3-(p-tolyl)imidazo[1,2-a]pyridine (20) was synthesized according to the
procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile (6)’ using 7-
1
(
furan-2-yl)imidazo[1,2-a]pyridine (11) and was isolated as a brown solid (26 mg, 70%) H NMR
400 MHz, Chloroform-d) δ 8.28 – 8.24 (m, 1H), 7.91 (s, 1H), 7.67 (s, 1H), 7.53 – 7.50 (m, 1H),
(
7.44 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.09 – 7.05 (m, 1H), 6.74 (d, J = 3.4 Hz, 1H), 6.51
1
3
(
dd, J = 3.4, 1.8 Hz, 1H), 2.43 (s, 3H). C NMR (100 MHz, Chloroform-d) δ 151.91, 146.08,
143.02, 138.25, 132.96, 129.94, 129.48, 127.90, 126.80, 126.11, 123.34, 112.02, 111.16, 109.39,
+
1
06.90, 21.33. ESIMS m/z [M+H] 275
4.2.18 3-(4-fluorophenyl)-7-(furan-2-yl)imidazo[1,2-a]pyridine (21)
3-(4-fluorophenyl)-7-(furan-2-yl)imidazo[1,2-a]pyridine (21) was synthesized according
to the procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile (6)’ using
1
7
-(furan-2-yl)imidazo[1,2-a]pyridine (11) and was isolated as a brown solid (29 mg, 69%). H
NMR (400 MHz, Chloroform-d) δ 8.21 - 8.19 (m, 1H), 7.91 (s, 1H), 7.66 (s, 1H), 7.56 – 7.49 (m,
3
1
1
1
H), 7.27 - 7.20 (m, 2H), 7.13 – 7.07 (m, 1H), 6.75 (d, J = 3.4 Hz, 1H), 6.52 (dd, J = 3.4, 1.7 Hz,
1
3
H). C NMR (100 MHz, Chloroform-d) δ 162.55 (d, J = 248.8 Hz), 151.76, 146.19, 143.12,
33.24, 129.91 (d, J = 8.2 Hz), 127.02, 125.16 (d, J = 3.1 Hz), 123.07, 116.40 (d, J = 21.7 Hz),
+
15.79, 112.05, 111.18, 109.62, 107.07. ESIMS m/z [M+H] 279
4.2.19 2-fluoro-5-(7-phenylimidazo[1,2-a]pyridin-3-yl)benzonitrile (22)

ACCEPTED MANUSCRIPT
2-fluoro-5-(7-phenylimidazo[1,2-a]pyridin-3-yl)benzonitrile (22) was synthesized
according to the procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile
6)’ using 7-phenylimidazo[1,2-a]pyridine (12) and was isolated as a brown solid (47 mg, 59%)
(
1
H NMR (400 MHz, Chloroform-d) δ 8.45 (d, J = 7.2 Hz, 1H), 7.92 (s, 1H), 7.87 (s, 1H), 7.81 – 7.75
1
3
(
m, 1H), 7.68 (d, J = 7.9 Hz, 2H), 7.56 – 7.43 (m, 5H), 7.29 – 7.24 (m, 1H). C NMR (101 MHz,
Chloroform-d) δ 163.60 (d, J = 260.2 Hz), 139.21, 137.76, 136.20 (d, J = 8.7 Hz), 135.02, 134.45,
134.08, 129.25, 128.85, 128.19 (d, J = 17.4 Hz), 123.20, 122.99 (d, J = 3.3 Hz), 119.93, 117.29 (d,
+
J = 8.4 Hz), 115.00, 114.29 (d, J = 20.9 Hz), 113.92. ESIMS m/z [M+H] 314
4
.2.20 2-(4-(7-phenylimidazo[1,2-a]pyridin-3-yl)phenyl)acetonitrile (23)
-(4-(7-phenylimidazo[1,2-a]pyridin-3-yl)phenyl)acetonitrile (23) was synthesized
according to the procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile
6)’ using 7-phenylimidazo[1,2-a]pyridine (12) and was isolated as a brown solid (42 mg, 53%).
2
(
1
H NMR (400 MHz, Chloroform-d) δ 8.37 (dd, J = 7.2, 0.9 Hz, 1H), 7.90 (dd, J = 1.8, 0.9 Hz, 1H),
7
7
1
1
.75 (s, 1H), 7.70 – 7.66 (m, 2H), 7.64 – 7.60 (m, 2H), 7.53 – 7.47 (m, 4H), 7.44 – 7.41 (m, 1H),
1
3
.15 (dd, J = 7.2, 1.8 Hz, 1H), 3.85 (s, 2H). C NMR (100 MHz, Chloroform-d) δ 146.84, 138.42,
37.56, 133.52, 129.78, 129.29, 129.24, 129.12, 128.93, 128.49, 128.35, 127.94, 126.73, 123.15,
+
14.98, 112.76, 23.50. ESIMS m/z [M+H] 310
4.2.21 7-phenyl-3-(p-tolyl)imidazo[1,2-a]pyridine (24)
7-phenyl-3-(p-tolyl)imidazo[1,2-a]pyridine (24) was synthesized according to the
procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile (6)’ using 7-

ACCEPTED MANUSCRIPT
1
phenylimidazo[1,2-a]pyridine (12) and was isolated as a brown solid (27 mg, 46%). H NMR (400
MHz, Chloroform-d) δ 8.35 (dd, J = 7.2, 0.8 Hz, 1H), 7.91 – 7.89 (m, 1H), 7.70 (s, 1H), 7.69 – 7.65
(m, 2H), 7.50 – 7.45 (m, 4H), 7.42 – 7.39 (m, 1H), 7.33 (d, J = 7.9 Hz, 2H), 7.09 (dd, J = 7.2, 1.8 Hz,
1
3
1
H), 2.44 (s, 3H). C NMR (100 MHz, Chloroform-d) δ 138.60, 138.25, 137.21, 132.70, 129.96,
129.29, 129.08, 128.22, 127.94, 126.73, 126.17, 125.58, 123.34, 114.82, 112.43, 21.35. ESIMS
+
m/z [M+H] 285
4.2.22 3-(4-fluorophenyl)-7-phenylimidazo[1,2-a]pyridine (25)
3-(4-fluorophenyl)-7-phenylimidazo[1,2-a]pyridine (25) was synthesized according to
the procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile (6)’ using 7-
1
phenylimidazo[1,2-a]pyridine (12) and was isolated as a brown solid (28 mg, 48%). H NMR (400
MHz, Chloroform-d) δ 8.28 (d, J = 7.2 Hz, 1H), 7.90 – 7.87 (m, 1H), 7.69 (s, 1H), 7.68 - 7.65 (m,
2
=
1
1
H), 7.57 – 7.52 (m, 2H), 7.51 – 7.46 (m, 2H), 7.42 – 7.37 (m, 1H), 7.26 – 7.20 (m, 2H), 7.11 (dd, J
1
3
7.2, 1.9 Hz, 1H). C NMR (100 MHz, Chloroform-d) δ 162.54 (d, J = 248.6 Hz), 146.54, 138.50,
37.36, 133.16, 129.93 (d, J = 8.2 Hz), 129.28, 129.10, 128.29, 126.72, 125.29 (d, J = 3.5 Hz),
+
23.03, 116.40 (d, J = 21.8 Hz), 114.94, 112.62. ESIMS m/z [M+H] 289
4.2.23 2-fluoro-5-(7-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)benzonitrile (26)
2-fluoro-5-(7-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)benzonitrile
(26)
was
synthesized according to the procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-
yl)benzonitrile (6)’ using 7-(4-methoxyphenyl)imidazo[1,2-a]pyridine (13) and was isolated as a
1
brown solid (38 mg, 62%). H NMR (400 MHz, Chloroform-d) δ 8.42 (d, J = 7.3 Hz, 1H), 7.85 (s,

ACCEPTED MANUSCRIPT
1
–
1
1
1
H), 7.80 – 7.76 (m, 1H), 7.63 - 7.60 (m, 3H), 7.52 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 9.9 Hz, 1H), 7.26
1
3
7.23 (m, 1H), 7.03 (dd, J = 8.8, 3.4 Hz, 2H), 3.88 (s, 3H). C NMR (100 MHz, Chloroform-d) δ
63.60 (d, J = 259.9 Hz), 160.35, 147.89, 139.01, 136.20 (d, J = 8.7 Hz), 134.56 (d, J = 26.6 Hz),
33.24, 129.98, 127.96, 123.13, 122.92 (d, J = 3.4 Hz), 114.68, 114.20 (d, J = 20.8 Hz), 113.77,
+
09.99, 100.07 (d, J = 15.4 Hz), 99.60, 55.44. ESIMS m/z [M+H] 344
4.2.24 2-(4-(7-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)phenyl)acetonitrile (27)
2-(4-(7-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)phenyl)acetonitrile
(27)
was
synthesized according to the procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-
yl)benzonitrile (6)’ using 7-(4-methoxyphenyl)imidazo[1,2-a]pyridine (13) and was isolated as a
1
brown solid (31 mg, 46%). H NMR (400 MHz, Chloroform-d) δ 8.32 (d, J = 7.3 Hz, 1H), 7.82 (s,
1H), 7.71 (s, 1H), 7.63 - 7.59 (m, 4H), 7.49 (d, J = 8.0 Hz, 2H), 7.10 (dd, J = 7.3, 1.6 Hz, 1H), 7.01
1
3
(
d, J = 8.7 Hz, 2H), 3.86 (s, 3H), 3.84 (s, 2H). C NMR (100 MHz, Chloroform-d) δ 159.93, 147.03,
137.21, 133.38, 130.79, 129.66, 129.31, 128.90, 128.41, 127.84, 124.44, 123.06, 117.56, 114.54,
+
1
13.95, 112.56, 55.41, 23.49. ESIMS m/z [M+H] 340
4.2.25 7-(4-methoxyphenyl)-3-(p-tolyl)imidazo[1,2-a]pyridine (28)
7
-(4-methoxyphenyl)-3-(p-tolyl)imidazo[1,2-a]pyridine (28) was synthesized according to
the procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile (6)’ using 7-
4-methoxyphenyl)imidazo[1,2-a]pyridine (13) and was isolated as a brown solid (29 mg, 52%).
(
1
H NMR (400 MHz, Chloroform-d) δ 8.32 (d, J = 7.2 Hz, 1H), 7.81 (s, 1H), 7.67 (s, 1H), 7.63 – 7.58

ACCEPTED MANUSCRIPT
(m, 2H), 7.49 – 7.44 (m, 2H), 7.36 – 7.31 (m, 2H), 7.07 – 7.03 (m, 1H), 7.03 – 6.98 (m, 2H), 3.86
1
3
(
s, 3H), 2.44 (s, 2H). C NMR (100 MHz, Chloroform-d) δ 159.82, 146.64, 138.08, 136.73,
132.78, 131.04, 129.92, 128.97, 127.86, 127.82, 126.35, 123.22, 114.49, 113.88, 112.17, 55.39,
+
2
1.33. ESIMS m/z [M+H] 315
4.2.26 3-(4-fluorophenyl)-7-(4-methoxyphenyl)imidazo[1,2-a]pyridine (29)
3
-(4-fluorophenyl)-7-(4-methoxyphenyl)imidazo[1,2-a]pyridine (29) was synthesized
according to the procedure outlined in ‘4.2.3 2-fluoro-5-(imidazo[1,2-a]pyridin-3-yl)benzonitrile
(6)’ using 7-(4-methoxyphenyl)imidazo[1,2-a]pyridine (13) and was isolated as a brown solid (28
1
mg, 50%). H NMR (400 MHz, Chloroform-d) δ 8.25 (d, J = 7.2 Hz, 1H), 7.83 – 7.80 (m, 1H), 7.66
(s, 1H), 7.61 (d, J = 8.7 Hz, 2H), 7.56 – 7.52 (m, 2H), 7.24 - 7.20 (m, 2H), 7.07 (dd, J = 7.2, 1.8 Hz,
1
3
1
H), 7.01 (d, J = 8.7 Hz, 2H), 3.86 (s, 3H). C NMR (100 MHz, Chloroform-d) δ 162.49 (d, J =
248.4 Hz), 159.90, 146.71, 137.04, 132.99, 130.88, 129.87 (d, J = 8.2 Hz), 129.51, 127.84, 125.38
+
(d, J = 3.4 Hz), 122.94, 116.37 (d, J = 21.7 Hz), 114.53, 113.92, 112.45, 55.40. ESIMS m/z [M+H]
319
4.3. FLT3 Biochemical Inhibition Assay[27-28]
Kinase activity was measured in a microfluidics assay that monitors the separation of a
phosphorylated product from substrate. 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 [PerkinElmer®, Walthman, USA]). In 96-well polypropylene
plates (Greiner, Frickenhausen, Germany) compound stocks (20 mM in DMSO) were diluted

ACCEPTED MANUSCRIPT
into kinase buffer (50 mM HEPES, 0.075% Brij-35, 0.1 % Tween 20, 2 mM DTT, 10 mM MgCl2,
and 0.02% NaN ) in 12-point ½log dilutions (2 mM–6.32 nM). After, 1 μL was transferred into a
3
384-well polypropylene assay plate (Greiner, Frickenhausen, Germany). The FLT3 enzyme
(Invitrogen™, Grand Island, USA) was diluted in kinase buffer to a concentration of 2 nM and 5
µL of the enzyme mixture was transferred to the assay plate. The inhibitors/FLT3 enzyme were
incubated for 60 minutes with minor shaking. A substrate mix was prepared containing ATP
(Ambresco®, Solon, USA) and 5FAM tagged FLT3 peptide (peptide #22, 5’ FAM-EPLYWSFPA,
PerkinElmer®, Walthman, USA) dissolved in kinase buffer, and 5 µL of the substrate mix was
added to the assay plate. Running concentrations were as follows: ATP (190 µM), peptide (1.5
µM), compound 12-point ½log dilutions (0.2 mM–0.632 nM). For positive control, no inhibitor
was added. For negative control, no enzyme was added. For running control, quizartinib was
utilized. The plate was run until 10-20% conversion based on the positive control wells. The
following separation conditions were utilized: upstream voltage -500V; downstream voltage, -
1900V; chip pressure -0.8. 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 IC values were obtained from the half-life value of the curve. IC values
5
0
50
were generated in duplicate and error was calculated from the standard deviation between
values.

ACCEPTED MANUSCRIPT
4.6. Computational Modeling[27-28], [31]
Computational modeling studies were completed using AutoDock Vina,[31] AutoDock
Tools, and Discovery Studio 3.5. Using AutoDock Tools, kinase crystal structures were prepared
as follows: 1) All hydrogens were added as ‘Polar Only’ 2) A grid box for the ATP binding site
was created. Compounds to be computationally modeled were assigned appropriate rotatable
bonds using AutoDock Tools. To computational model the compounds, AutoDock Vina[31] was
employed. AutoDock Vina[31] provides docking scores in terms of ΔG values. After the
modeling study, kinase targets with high affinity ΔG values were visualized and analyzed with
Discovery Studio 3.5.
4.7. Cell Cultures
Stable BaF3 populations expressing activated FLT3 were generated by retroviral spinfection
with the appropriate mutated plasmid followed by selection and growth factor withdrawal as
previously described.[26] The BaF3 cell line was originally obtained from the laboratory of
Charles Sawyers and has not been authenticated. MV4;11 and Molm14 cells were obtained
from the laboratory of Scott Kogan and authenticated by Promega STR analysis in June 2013.
All cell lines were mycoplasma-free. Cells were incubated with compounds for 48 hours and
proliferation was assessed using CellTiter-Glo (Promega; Madison, WI) according to the
manufacturer’s recommendation on a SpectraMax M3 microplate reader using SpectraMax
Software (Molecular Devices; Sunnyvale, CA). All cell viability data shown is reflective of
experiments performed a minimum of three times.

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4.8. Preliminary Pre-Formulation Studies
Compound 17 was dissolved in a 1:1 DCM/MeOH solution at a concentration of 1.0
mg/mL. 1.0 mL of solution was transferred to a 13.0 mL glass tube, followed by a
stoichiometric equivalent of an organic or inorganic acid found in Table 3. The organic solvent
was evaporated and the newly formed salt complex was dried under high vacuum. In
increments of 1.0 mL, 0.2 µM filtered, deionized water was added to the salt of compound 17
and vortexed. The resulting aqueous solution was visually inspected for precipitate using a
laser diode at a wavelength of 600 nM (INFINITER® 100 Red Laser Pointer). Water was added
until no precipitate was present or until the solubility was determined to be below 0.08 mg/mL.

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Acknowledgements
This work was graciously supported by two training grants from The National Institutes
of Health (3T32GM008804-10S1; T32 GM008804), University of Arizona startup funding, The
Caldwell Health Sciences Research Fellowship, and the Leukemia & Lymphoma Society.
Appendix A. Supplementary Data
Supplementary data related to this article can be found at

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