Design, synthesis, and in vitro evaluation of quinolinyl analogues for α-synuclein aggregation

Article abstract of DOI:10.1016/j.bmcl.2018.02.031

Here we report the synthesis and in vitro evaluation of 25 new quinolinyl analogues for α-synuclein aggregates. Three lead compounds were subsequently labeled with carbon-11 or fluorine-18 to directly assess their potency in a direct radioactive competitive binding assay ng both α-synuclein fibrils and tissue homogenates from Alzheimer's disease (AD) cases. The modest binding affinities of these three radioligands toward α-synuclein were comparable with results from the Thioflavin T fluorescence assay. However, all three ligand also showed modest binding affinity to the AD homogenates and lack selectivity for α-synuclein. The structure–activity relationship data from these 25 analogues will provide useful information for design and synthesis of new compounds for imaging α-synuclein aggregation.

Full text of DOI:10.1016/j.bmcl.2018.02.031

Accepted Manuscript
Design, synthesis, and in vitro evaluation of quinolinyl analogues for α-synu-
clein aggregation
Xuyi Yue, Dhruva D. Dhavale, Junfeng Li, Zonghua Luo, Jialu Liu, Hao Yang,
Robert H. Mach, Paul T. Kotzbauer, Zhude Tu
PII:
S0960-894X(18)30124-0
https://doi.org/10.1016/j.bmcl.2018.02.031
BMCL 25627
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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9 February 2018
14 February 2018
Please cite this article as: Yue, X., Dhavale, D.D., Li, J., Luo, Z., Liu, J., Yang, H., Mach, R.H., Kotzbauer, P.T.,
Tu, Z., Design, synthesis, and in vitro evaluation of quinolinyl analogues for α-synuclein aggregation, Bioorganic
& Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.02.031
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Bioorganic & Medicinal Chemistry Letters
Design, synthesis, and in vitro evaluation of quinolinyl analogues for α-synuclein
aggregation
Xuyi Yue a, Dhruva D. Dhavale b, Junfeng Li a, Zonghua Luo a, Jialu Liu b, Hao Yang a, Robert H. Mach c,
Paul T. Kotzbauer b, Zhude Tu a,*
a Department of Radiology, Washington University School of Medicine, St Louis, MO
b Department of Neurology, Washington University School of Medicine, St Louis, MO
c Department of Radiology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA
ARTICLE INFO
ABSTRACT
Here we report the synthesis and in vitro evaluation of 25 new quinolinyl analogues for α-
synuclein aggregates. Three lead compounds were subsequently labeled with carbon-11 or
fluorine-18 to directly assess their potency in a direct radioactive competitive binding assay ng
both α-synuclein fibrils and tissue homogenates from Alzheimer’s disease (AD) cases. The
modest binding affinities of these three radioligands toward α-synuclein were comparable with
results from the Thioflavin T fluorescence assay. However, all three ligand also showed modest
binding affinity to the AD homogenates and lack selectivity for α-synuclein. The structure-
activity relationship data from these 25 analogues will provide useful information for design and
synthesis of new compounds for imaging α-synuclein aggregation.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
α-Synuclein fibrils
quinolinyl analogue
radiosynthesis
2017 Elsevier Ltd. All rights reserved.
PET radiotracer
Thioflavin T fluorescence assay
Parkinson's disease
Parkinson’s disease (PD) is a chronic and progressive neurodegenerative
disease. The classical motor symptoms of PD include bradykinesia, resting
tremor, rigidity, while non-motor symptoms involve depression, olfactory
malfunction, and cognitive impairment, all of which significantly
compromise the patient’s daily life.1 PD is characterized by the loss of
dopamine neurons within the substantia nigra pars compacta along with the
formation of intracellular fibrillar Lewy bodies (LBs) and Lewy neuritis
inclusions (LNs). Aggregated α-synuclein is the predominant component of
LBs and LNs.1 α-Synuclein is a highly soluble when unfolded presynaptic
protein that consists of 140 amino acids which is ubiquitously expressed in
the brain.2 It plays an important role in recycling synaptic vesicles and in
classes of small molecular probes have been reported: fluorescent probes, and
radiolabeled probes.12 Fluorescent probes, such as Thioflavin T, Thioflavin S,
N-arylaminonaphthalene sulfonate analogues, cyanine dyes, Evans blue,
LDS798, quinoxalin analogues, and Nile red analogues, have not shown
selectivity for Lewy bodies versus Aβ plaques.12 For the radiolabeled probes,
the benzoxazole derivative, [2-[2-(2-dimethylaminothiazol-5-yl-)ethenyl]-6-
[2-[18F]fluoroethoxy]-benzoxazole ([18F]BF227), 11C-Pittsburgh compound-B
([11C]PIB), and chalcone analogs [125I]IDP-4 have been evaluated for imaging
α-synuclein (Figure 1).13 [18F]BF227 failed to bind to Aβ-free Dementia with
Lewy Bodies or age-matched control samples while it bound to AD brain
homogenates.13 [18F]BF227 did not show promise in imaging α-synuclein
aggregation in an accelerated mouse model of synucleinopathy which
develops α-synuclein deposits in brainstem and thalamus,.14 Preclinical
evaluation of PIB as an α-synuclein specific PET tracer has been
inconsistent.15 The higher affinity of PIB for Aβ proteins over α-synuclein
poses another clear concern. The single-photon emission computed
tomography (SPECT) tracer [125I]IDP-4 reportedly has high binding affinity
for α-synuclein fibrils (Kd = 5.4 nM); however its high molecular weight and
high lipophilicity caused low brain uptake in vivo.16
4
regulating the synthesis, storage, and release of neurotransmitters.3, The
accumulation of misfolded α-synuclein protein is widely recognized as the
pathological hallmark of PD and other synucleinopathies including Dementia
with Lewy bodies and Multiple System Atrophy (MSA).5 Furthermore, Braak
staging of brain tissue from PD cases suggests that the appearance of α-
synuclein inclusions precedes dopaminergic loss6 and that α-synuclein
deposition may occur years prior to the appearance of motor symptoms.7, 8 An
additional concern is that treatment of PD with dopaminergic drugs alters the
striatal uptake of dopaminergic imaging biomarkers used for monitoring
disease progression;9 poor correlation has been reported between PET
measures with these tracers and clinical status in therapeutic trials of PD
Recently, we reported several radiolabeled tricyclic ligands [11C]SIL5,
[
125I]SIL23, and [18F]SIL26 which showed reasonable in vitro binding affinity
11
progression.10, Therefore, the strategy of imaging deposited α-synuclein
and selectivity for recombinant α-synuclein fibrils compared to Aβ and tau
fibrils (Figure 1). However, in vitro evaluation of these radioligands in
postmortem PD cases showed only modest affinity for tissue homogenates
from PD cases, this lack of selectivity discouraged further evaluation of these
radiotracers for PET imaging of α-synuclein aggregates in human brain.17-19
We also reported a series of indolinone and indolinone-diene analogues
which bind to α-synuclein fibrils.20 Although the indolinone-diene analogues
had a higher binding affinity for α-synuclein fibrils than the related
indolinone congeners, most indolinone-diene analogues exist as two
inseparable regioisomers, which can make identification of clear structure-
activity relationships challenging. This issue was overcome by introduction of
distinct substituents into the benzene ring of the -ene and -diene moieties (i.e.,
aggregates rather than dopaminergic changes could provide a more accurate
definition of early-stage premotor PD. Identifying
a suitable positron
emission tomography (PET) radioligand that is able to map the α-synuclein
aggregation in the brain at early stage of PD is imperative for development of
disease-modifying therapeutics aimed at slowing or terminating the
progression of PD. The use of PET in assessing α-synuclein aggregation in
the brain has been hampered by the lack of agents having high binding
potency and high selectivity for α-synuclein over other proteins associated
with neurodegenerative disease, particularly, amyloid beta (Aβ) and tau
protein. Investigators have put tremendous effort into developing highly
potent and highly selective α-synuclein PET radiotracers.12 To date, two
1

[18F]WC-58a, Figure 1).20 Thioflavin T indirect competetive binding and
direct homologous radioligand competitive binding assays with the single
isomer clearly indicated favorable binding affinity of these ligands towards α-
synuclein fibrils and good selectivity (> 50-fold) towards Aβ and tau fibrils.
However, the high log P value of the most potent compound limited the in
vivo application of [18F]WC-58a.20 More recently, researchers used the tau
compounds 1 – 16 in high yields.26 The final compounds include diverse
substituents with a fluoroethoxy group in the 6-position, or a methoxy group
in the 3–8-position of the quinolinyl fragment. Methoxy and halogen
substituents were also introduced to the six-membered aromatic ring (pyridyl
fragment); these substituents facilitate convenient carbon-11 or fluorine-18
radiolabeling.
PET
ligand
2-((1E,3E)-4-(6-([11C]methylamino)pyridin-3-yl)buta-1,3-
dienyl)benzo[d]thiazol-6-ol ([11C]PBB3) (Figure 1) to assess α-synuclein
pathology by in vitro fluorescence and autoradiographic studies; although α-
synuclein pathology in Lewy body disease was not detected by [11C]PBB3
PET, the tracer was able to image MSA cases having high densities of glial
cytoplasmic inclusions.21
Figure 1. Reported radiotracers for α-synuclein.
Here we report our continuing efforts on the development of PET tracers
17-20
for imaging α-synuclein aggregation.12,
New analogues containing a
quinolinyl moiety and six-membered aromatic rings linked by either a double
bond or an oxadiazole bridge were synthesized and their in vitro binding
properties were characterized (Figure 2). The rationales behind the design of
these new compounds include: 1) LDS798 contains multiple double bounds
and is able to label Lewy bodies and penetrate the blood brain barrier,
therefore we introduced a quinolinyl group that has a double bond side
chain;12 2) N-arylaminonaphthalene sulfonate analogue 2,6-ANS shows
binding to α-synuclein fibrils, therefore, we introduced heteroatoms on both
aromatic rings of 2,6-ANS and modified the secondary amine bridge of our
new analogues; 3) an oxadiazole fragment was used to replace the bridged
double bond in order to: optimize the lipophilicity of the target compounds,
restrict the flexibility of the molecular structure, and overcome the potential
isomerization of the double bound. This strategy was based on the
observation that oxadiazole moieties have been employed for radiotracers that
target Aβ in Alzheimer’s disease, neurotransmitter α7 nicotinic acetylcholine
receptors in neuropsychiatric diseases, and type 2 cannabinoid receptor for
neuroinflammation.22-25 As described in detail below, the in vitro binding
affinity of the 25 newly synthesized quinolinyl analogues was measured using
a Thioflavin T fluorescence assay (Table 1); this screening identified six
compounds with binding affinity <60 nM towards recombinant α-synuclein
fibrils. Subsequently, we carbon-11 or fluorine-18 radiolabeled three
compounds to perform direct homologous radioactive competitive binding
assays, results showed modest binding potency for α-synuclein fibrils; further
characterization using AD tissue homogenates also revealed modest binding
with AD tissue (Figure 3).
Next, we synthesized additional analogues in which the bridged double
bond in compounds 1 – 16 was replaced with an oxadiazole moiety. Lower
lipophilicity could be expected by this substitution which has been used
successfully for other CNS radiotracers.22-25 Reaction of commercially
available 6-methoxyquinoline-2-carbonitrile 17 with hydroxylamine
hydrochloride afforded carboximidamide 18 in near quantitative yield, which
was used for next condensation reaction without further purification.
Removal of the methyl group from compound 17 gave phenol 19, which was
alkylated to produce the 6-position modified compounds 20 – 22. Compounds
20 – 22 were converted to corresponding carboximidamides 23 – 25 using
similar strategy as used to produce 18. Condensation of compounds 18, and
23 – 25 with nicotinic acid or isonicotinic acid afforded compounds 26 – 30.
By similar strategy, the four imidazole pyridine analogues 31 – 34 were
obtained in moderate yield (Scheme 2).
Figure 2. Rationale of newly designed α-synuclein compounds.
The binding affinities of the quinolinyl compounds for recombinant α-
synuclein fibrils were determined using a Thioflavin T assay as previously
reported (Table 1).17 Ten of the 25 compounds produced measurable
displacement of Thioflavin T in the competitive binding assay, However the
maximum displacement of ThioT binding was only ~ 50%, indicating that the
The synthesis of quinolinyl analogues with a double bond bridge is
outlined in Scheme 1. Condensation of 2-methylqinoline derivatives with
aldehyde in the presence of p-toluenesulfonamide at 130 °C afforded
2

binding interaction of quinolinyl compounds with α-synuclein fibrils did not
completely overlap with that of Thioflavin T. Fifteen of the compounds did
not produce sufficient displacement of Thioflavin T (at concentrations up to
10 μM) to enable measurement of Ki values. For phenyl-containing
compounds 1–4, no binding was observed for compounds 1 and 4, while the
6-OMe derivative, compound 2, showed a Ki value of 192 nM and the 7-OMe
derivative 3 had a Ki value > 500 nM (Table 1, entries 1-4). Among the
pyridyl containing compounds 5 – 10, methoxy substitution on the 5- or 6-
position of the quinolinyl ring resulted in modest affinity in compounds 7 and
8 with respective Ki values of 56 nM, 52 nM , while substitution on the 3-, 4-,
7-, or 8-position resulted in no measurable binding potency (Table 1, entries
5 – 10). Among the 6-position substituted structures, compounds 11–16 had
additional substituents on the pyridyl ring. Compounds 12, 13, and 14 showed
high binding affinity with Ki values of 54, 52, and 18 nM respectively;
compound 16 displayed moderate binding and compounds 11 and 15 had no
binding (Table 1, entries 11–16). For oxadiazole containing compounds 26–
34, one fragment was the 6-position modified quinolinyl moiety while the
other included a pyridyl or (pyridyl)imidazole fragment. Compound 28
displayed high binding affinity with a Ki value of 15 nM, but the other
compounds had very weak potency (Table 1, entries 17-25). Six of the 25
new compounds displayed modest potency with Ki values < 60 nM;
compounds 14 and 28 were slightly more potent with Ki values of 18 and 15
nM respectively.
measures direct competition between the radioligand and the unlabeled
compound. Such assays can provide greater accuracy and sensitivity than the
Thioflavin T method. The syntheses of precursors for radiolabeling are
illustrated in Scheme 3. Reaction of commercially available 2-methoxy-6-
hydroxyquinoline with bromomethyl methyl ether afforded compound 35,
which was subjected to condensation with 2-methoxyisonicotinaldehyde to
provide compound 36. Acidolysis of the MOM protecting group in compound
36 gave the radiolabeling precursor 37. The oxadiazole containing
radiolabeling precursor 38 was obtained from compound 29 using the same
procedure used to synthesize compound 37.
Table 1. Binding affinities (Ki) of new compounds determined by Thioflavin
T indirect competitive binding assay.
The radiosyntheses of [11C]13, [18F]14, and [18F]28 are shown in Scheme
4. The one-step procedure for the radiosynthesis of [11C]13 progressed
smoothly with [11C]methyl iodide in the presences of aqueous sodium
hydroxide solution to afford [11C]13 in good yield and high specific activity
(50 ± 10% yield, specific activity >148 GBq/µmol, decay corrected to end of
bombardment, EOB). A two-step procedure was used for radiosynthesis of
Thioflavin T assay
Entry
Compound
Log P b
Ki (nM) a
1
1
NB
4.61
4.61
4.6
[18F]14 and [18F]28. The radioactive intermediate 2-[18F]fluoroethyl tosylate
28, 29
was synthesized following routine methods,27
followed by alkylation of
2
2
192 ± 1.0
680 ± 139
NB
the phenol in precursor compound 37or 38 to respectively afford [18F]14 or
[18F]28. We have found this two-step procedure to be much reliable and
reproducible than one-step methods; an additional benefit is that phenol
3
3
4
4
4.61
3.27
3.27
3.27
3.27
3.27
3.27
3.89
4.17
3.86
4.05
3.83
4.10
3.42
4.01
4.20
4.13
4.67
3.62
4.28
3.81
3.74
precursors are stable during storage. [18F]14 was produced in 21 ± 7%
yield, specific activity >27 GBq/µmol (decay corrected to end of
synthesis, EOS); [18F]28 was produced in 30 ± 4% yield, specific
activity >37 GBq/µmol (decay corrected to EOS).
5
5
NB
6
6
NB
7
7
56 ± 40
52 ± 38
NB
8
8
9
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
10
11
12
13
14
15
16
26
27
28
29
30
31
32
33
34
NB
NB
54 ± 2
52 ± 21
18 ±1
NB
167 ± 28
NB
630
15 ± 3
NB
Using the direct radioligand competitive binding assay, binding affinities
(Kd values) towards recombinant α-synuclein fibrils of compounds [11C]13,
[18F]14, and [18F]28 were determined to be 21, 79, and 18 nM respectively.
These modest affinities are comparable with the results from the Thioflavin T
fluorescence binding assay, which showed similarly modest Ki values of 52,
18, and 15 nM respectively, towards recombinant α-synuclein fibrils. The
binding affinities of these three radiotracers towards tissue homogenates from
AD cases were also determined in the direct radioligand competitive binding
assay; the Kd values of [11C]13, [18F]14,and [18F]28 were determined to be 13,
55, and 25 nM respectively (Figure 3), suggesting that these three
radioligands lack selectivity for α-synuclein aggregates over AD.
NB
NB
NB
NB
25
NB
a
Ki values (mean ± SD nM) were determined in at least three
experiments, NB; no binding was measurable in the Thioflavin T assay; b
Calculated by ChemBioDraw Ultra 16.0.
Among the six compounds with Ki values <60 nM for recombinant α-
synuclein fibrils in the Thioflavin T fluorescence assay, compounds 13, 14,
and 28 were radiolabeled with carbon-11 or fluorine-18 to further evaluate
their performance in
a homologous competitive binding assay which
3

National Institute of Mental Health (NIMH): NS061025, NS075527,
NS103988, NS058714. Mass spectrometry was provided by the Washington
University Mass Spectrometry Resource, a National Institute of Health
Research Resource (grant P41RR0954). The authors gratefully thank William
H. Margenau and Robert Dennett for their excellent assistance in isotope
production.
References and Notes
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12. Kotzbauer PT, Tu ZD, Mach RH. Clin Transl Imaging. 2017;5(1): 3.
13. Fodero-Tavoletti MT, Mulligan RS, Okamura N, et al. Eur J Pharmacol.
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2014;11(10): 955.
15. Maetzler W, Reimold M, Liepelt I, et al. NeuroImage. 2008;39(3): 1027.
16. Ono M, Cheng Y, Kimura H, et al. PloS one. 2013;8(9): e74104.
17. Yu L, Cui J, Padakanti PK, et al. Bioorg. Med. Chem. 2012;20(15): 4625.
18. Bagchi DP, Yu L, Perlmutter JS, et al. PloS one. 2013;8(2): e55031.
19. Zhang X, Jin H, Padakanti PK, et al. Appl. Sci. 2014;4(1): 66.
20. Chu WH, Zhou D, Gaba V, et al. J. Med. Chem. 2015;58(15): 6002.
21. Koga S, Ono M, Sahara N, Higuchi M, Dickson DW. Mov Disord. 2017,
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2013;21(9): 2635.
Figure 3. Kd values for [11C]13, [18F]14, and [18F]28 determined
by the direct radioligand competitive binding assay.
Significant progress has been made in recent years in the development of
PET probes for imaging Aβ plaques in Alzheimer’s disease; [18F]Florbetapir,
[18F]Florbetaben, [18F]Flutemetamol, have all been approved by FDA to
estimate Aβ neuritic plaque density in adult AD patients with cognitive
impairment.12 However, the development of PET radiotracers for selective
imaging of α-synuclein aggregation in PD has been more challenging. This
may result from the comparatively lower density of aggregated α-synuclein in
the brain compared to Aβ or tau proteins.30 Furthermore, α-synuclein
aggregates are often accompanied by other pathological Aβ and tau protein
aggregates, suggesting that selective detection will be more complicated. As a
general rule, a binding potential (BP = Bmax/Kd) >2 is required for clinical
translation of a radioligand.31 This implies that identification of highly potent
ligands with a Kd value at subnanomolar range will be pivotal for imaging α-
synuclein aggregation. In our current work, we designed and synthesized 25
new compounds. Among them, screening with a ThioflavinT assay identified
six compounds with modest binding affinities for recombinant α-synuclein
fibrils. Three of these leads were subsequently radiolabeled with carbon-11 or
fluorine-18. A direct radioligand binding assay also showed all three
compounds had modest affinity for recombinant α-synuclein fibrils, which
was comparable to the results of the Thioflavin T assay. However, when
tissue homogenates from AD cases were used in the direct homologous
binding assay, these three radioligands lacked selectivity. Further structural
optimization is required to identify new ligands with higher potency and
selectivity for α-synuclein aggregation.
25. Ahamed M, van Veghel D, Ullmer C, Van Laere K, Verbruggen A,
Bormans GM. Front Neurosci-Switz. 2016;10.
26. Yan YZ, Xu K, Fang Y, Wang ZY. J. Org. Chem. 2011;76(16): 6849.
27. Yue X, Bognar C, Zhang X, et al. Appl Radiat Isot. 2016;107: 40.
28. Yue X, Jin H, Liu H, et al. Org. Biomol. Chem. 2017;15(24): 5197.
29. Tu Z, Zhang X, Jin H, et al. Bioorg. Med. Chem. 2015;23(15): 4699.
30. Shah M, Seibyl J, Cartier A, Bhatt R, Catafau AM. J. Nucl. Med.
2014;55(9): 1397.
31. Brust P, van den Hoff J, Steinbach J. Neurosci Bull. 2014;30(5): 777-
811.
32. Experimetal details.
a) General: All reagents and chemicals were purchased from commercial
suppliers and used without further purification unless otherwise stated. 1H
NMR and 13C NMR spectra were recorded at 400 MHz on a Varian
spectrometer with CDCl3, CD3COCD3, CD3SOCD3 as solvent and
tetramethylsilane (TMS) as the internal standard. All chemical shift values
are reported in parts per million (ppm) (δ). Under these conditions, the
chemical shifts (in ppm) of residual solvents are observed at 7.26 (CDCl3),
2.05 (CD3COCD3), 2.50 (CD3SOCD3) for 1H NMR. The following
abbreviations were used to describe peak patterns wherever appropriate: br =
broad, d = doublet, t = triplet, m = multiplet. HRMS analyses were conducted
in Washington University Resource for Biomedical and Bio-organic Mass
Spectrometry. Preparative chromatography was performed on Chemglass
chromatography column using 230 – 400 mesh silica gel purchase from
Silicycle. Analytical TLC was carried out on Merck 60 F254 silica gel glass
plates, and visualization was aided by UV.
Experimental details for the intermediates and target
compounds, radiolabeling procedures, and binding assay methods
are provided in the Notes.32
In conclusion, we designed and synthesized 25 new quinolinyl analogues
for α-synuclein. Thioflavin T fluorescence assay revealed six compounds had
binding affinities <60 nM towards recombinant α-synuclein fibrils. Three of
these compounds were further radiolabeled with carbon-11 or fluorine-18. A
direct radioligand homologous binding assay demonstrated affinities for
recombinant α-synuclein fibrils comparable to those determined by
Thioflavin
T assay. However, the lack of selectivity of these three
radioligands toward α-synuclein fibrils over AD tissues in the direct binding
assay discouraged further in vivo evaluation. Our structure-activity
relationship data may provide useful information for future design and
synthesis of new PET tracers for imaging α-synuclein aggregation in patients
with PD and other diseases with α-synucleinopathies.
General procedure for the synthesis of vinyl quinoline derivatives. A
solution of commercially available quinoline derivatives (1 equiv.), p-
toluenesulfonamide (1 equiv.) and aldehyde (1 equiv.) in toluene (1 M) was
refluxed at 120 °C for 12 h in a reaction tube under N2. After the mixture was
cooled to room temperature, the solvent was removed under reduced pressure.
Then the residue was purified by silica gel column chromatography to afford
the vinyl quinoline derivatives.
Acknowledgements
General procedure for carboximidamide formation. To a solution of
quinoline-2-carbonitrile derivatives (0.4 M, 1 equiv.) in EtOH/H2O (2/1, v/v)
was added hydroxylamine hydrochloride (1 equiv.), sodium bicarbonate (2
equiv.) successively. The mixture was stirred at 100 °C overnight. Upon
This project was funded by Michael J. Fox Foundation grant supporting a
Consortium of Developing α-Synuclein Imaging Agents. United States – the
National Institutes of Health (NIH)/ National Institute of Neurological
Disorders and Stroke (NINDS), National Institute on Aging (NIA) and
4

cooling to the room temperature, water was added and the reaction mixture
was extracted with EtOAc. The combined organic phase was washed with
water, saturated aqueous sodium chloride, and dried over anhydrous Na2SO4.
The organic phase was then concentrated to afford the carboximidamide
product, which was used without further purification for the next
condensation step.
(E)-6-Methoxy-2-(2-(pyridin-4-yl)vinyl)quinolone (8). Light yellow solid.
MP 132 – 134 ºC. 1H NMR (400 MHz, CDCl3) δ 8.60 (d, J = 6.0 Hz, 2H),
8.03 (d, J = 8.6 Hz, 1H), 7.97 (d, J = 9.2 Hz, 1H), 7.60 (d, J = 8.5 Hz, 1H),
7.52 (d, J = 1.2 Hz, 2H), 7.44 (d, J = 6.0 Hz, 2H), 7.37 (dd, J = 9.2, 2.8 Hz,
1H), 7.05 (d, J = 2.7 Hz, 1H), 3.92 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
158.03, 152.32, 150.28, 144.30, 143.98, 135.26, 133.27, 130.82, 130.24,
128.68, 122.73, 121.22, 119.93, 105.10, 55.55. HRMS (ESI) calcd. for
C17H15N2O [M + H]+ 263.1179, found: 263.1108.
General procedure for oxadiazole formation. To a solution of acid (1
equiv.) in anhydrous DMF (0.18 M) was added EDCI.HCl (1 equiv.),and
HOBt (1 equiv.) successively in a sealed tube. The mixture was stirred at rt
for 30 min. Carboximidamide (1 equiv.) was added in one portion and the
reaction was placed in a pre-heated 130 °C oil bath and continued to stir
overnight. Water was added to quench the reaction and the mixture was
extracted with EtOAc. The combined organic phase was washed with
saturated aqueous sodium bicarbonate solution, water, saturated aqueous
sodium chloride, and dried over anhydrous Na2SO4. The organic phase was
concentrated in vacuum and the residue was subjected to silica gel
chromatography to give the target oxadiazole compound.
(E)-7-Methoxy-2-(2-(pyridin-4-yl)vinyl)quinolone (9). Light yellow solid.
MP 104 – 106 ºC. 1H NMR (400 MHz, CDCl3) δ 8.55 (d, J = 5.7 Hz, 2H),
8.00 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.9 Hz, 1H), 7.54 (d, J = 16.3 Hz, 1H),
7.47 – 7.41 (m, 2H), 7.39 (d, J = 5.8 Hz, 2H), 7.34 (d, J = 2.1 Hz, 1H), 7.11
(dd, J = 8.9, 2.3 Hz, 1H), 3.90 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 161.11,
154.77, 150.31, 149.95, 143.83, 136.25, 133.24, 131.14, 128.50, 122.93,
121.29, 120.02, 117.67, 107.12, 55.53. HRMS (ESI) calcd. for C17H15N2O [M
+ H]+ 263.1179, found: 263.1108.
(E)-8-Methoxy-2-(2-(pyridin-4-yl)vinyl)quinolone (10). Brown solid. MP
74 – 76 ºC. 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 5.8 Hz, 2H), 8.05 (d, J
= 8.6 Hz, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.60 (d, J = 16.4 Hz, 1H), 7.44 (d, J =
16.5 Hz, 1H), 7.41 – 7.34 (m, 3H), 7.30 (d, J = 8.1 Hz, 1H), 6.99 (d, J = 7.6
Hz, 1H), 4.03 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 155.24, 153.80, 150.28,
143.82, 140.03, 136.53, 133.85, 131.02, 128.66, 127.00, 121.26, 119.46,
119.41, 108.12, 56.12. HRMS (ESI) calcd. for C17H15N2O [M + H]+ 263.1179,
found: 263.1109.
General procedure for alkylation of 6-hydroxyquinoline. To a solution of
6-hydroxyquinoline-2-carbonitrile (1 equiv.) in anhydrous THF (0.08 M) was
added Cs2CO3 (1.8 equiv.), 2-fluoroethyl tosylate (1.8 equiv.). The reaction
was stirred at 70 °C overnight in a sealed tube. The mixture was concentrated
and the residue was subjected to silica gel chromatography to afford the
alkylation product.
(E)-5-Methoxy-2-styrylquinoline (1). Yellow solid. MP 83 – 85 ºC. 1H
NMR (400 MHz, CDCl3) δ 8.00 (d, J = 8.4 Hz, 1H), 7.70 – 7.55 (m, 4H),
7.49 (d, J = 8.4 Hz, 1H), 7.43 – 7.33 (m, 4H), 7.30 (t, J = 7.3 Hz, 1H), 7.13
(dd, J = 8.9, 2.3 Hz, 1H), 3.94 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 160.93,
156.08, 149.92, 136.55, 135.96, 134.06, 129.02, 128.75, 128.52, 128.45,
127.20, 122.55, 119.29, 117.26, 107.13, 55.50. HRMS (ESI) calcd. for
C18H16NO [M + H]+ 262.1226, found: 262.1152.
(E)-2-(2-(2-Fluoropyridin-4-yl)vinyl)-6-methoxyquinoline (11). Yellow
1
solid. MP 141 – 143 ºC. H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 5.2 Hz,
1H), 8.02 (d, J = 8.5 Hz, 1H), 7.96 (d, J = 9.2 Hz, 1H), 7.55 (d, J = 8.5 Hz,
1H), 7.49 (d, J = 4.5 Hz, 2H), 7.39 – 7.27 (m, 2H), 7.06 – 6.98 (m, 2H), 3.90
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 164.54 (d, J = 238.4 Hz), 158.17,
151.75, 149.72 (d, J = 8.1 Hz), 147.90 (d, J = 15.2 Hz), 144.27, 135.34,
134.44, 130.83, 128.91 (d, J = 4.0 Hz), 128.83, 122.88, 120.10, 118.94 (d =
4.0 Hz), 106.75 (d, J = 38.4 Hz), 105.05, 55.55. HRMS (ESI) calcd. for
C17H14FN2O [M + H]+ 281.1085, found: 281.1008.
(E)-6-Methoxy-2-styrylquinoline (2). Light yellow solid. MP 137 – 139 ºC.
1H NMR (400 MHz, CDCl3) δ 7.93 (dd, J = 17.7, 8.9 Hz, 2H), 7.61 – 7.47
(m, 4H), 7.37 – 7.27 (m, 4H), 7.24 (t, J = 7.3 Hz, 1H), 6.99 (d, J = 2.7 Hz,
1H), 3.86 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 157.61, 153.68, 144.25,
136.67, 135.05, 133.17, 130.62, 129.03, 128.73, 128.35, 128.26, 127.09,
122.28, 119.53, 105.22, 55.52. HRMS (ESI) calcd. for C18H16NO [M + H]+
262.1226, found: 262.1155.
(E)-2-(2-(2-Chloropyridin-4-yl)vinyl)-6-methoxyquinoline (12). Yellow
1
solid. MP 156 – 158 ºC. H NMR (400 MHz, CDCl3) δ 8.36 (d, J = 5.2 Hz,
1H), 8.05 (d, J = 8.5 Hz, 1H), 7.97 (d, J = 9.2 Hz, 1H), 7.57 (d, J = 8.5 Hz,
1H), 7.51 – 7.46 (m, 3H), 7.39 – 7.35 (m, 2H), 7.05 (d, J = 2.7 Hz, 1H), 3.93
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 158.19, 152.26, 151.74, 149.97,
147.27, 144.34, 135.33, 134.55, 130.91, 128.84, 128.71, 122.90, 121.69,
120.16, 119.85, 105.05, 55.57. HRMS (ESI) calcd. for C17H14ClN2O [M +
H]+ 297.0789, found: 297.0709.
(E)-7-methoxy-2-styrylquinoline (3). Light yellow solid. MP 89 – 91 ºC. 1H
NMR (400 MHz, CDCl3) δ 7.94 (d, J = 8.4 Hz,1H), 7.66 – 7.49 (m, 4H), 7.43
(d, J = 8.4 Hz,1H), 7.37 – 7.28 (m,4H), 7.24 (dd, J = 13.5, 6.2 Hz, 1H), 7.06
(dd, J = 8.9, 2.3 Hz, 1H), 3.88 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 159.91,
155.07, 148.91, 135.54, 134.95, 133.04, 128.00, 127.73, 127.50, 127.43,
126.18, 121.53, 118.28, 116.24, 106.11, 54.48. HRMS (ESI) calcd. for
C18H16NO [M + H]+ 262.1226, found: 262.1154.
(E)-6-Methoxy-2-(2-(2-methoxypyridin-4-yl)vinyl)quinolone (13). White
1
solid. MP 125 – 127 ºC. H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 5.4 Hz,
1H), 8.02 (d, J = 8.6 Hz, 1H), 7.96 (d, J = 9.2 Hz, 1H), 7.58 (d, J = 8.5 Hz,
1H), 7.47 (d, J = 2.0 Hz, 2H), 7.36 (dd, J = 9.2, 2.7 Hz, 1H), 7.09 (d, J = 5.3
Hz, 1H), 7.04 (d, J = 2.6 Hz, 1H), 6.86 (s, 1H), 3.95 (s, 3H), 3.91 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 164.97, 157.97, 152.46, 147.10, 146.76, 144.29,
135.21, 133.03, 130.81, 130.38, 128.63, 122.64, 119.92, 114.32, 108.79,
105.10, 55.54, 53.49. HRMS (ESI) calcd. for C18H17N2O2 [M + H]+ 293.1285,
found: 293.1207.
(E)-8-Methoxy-2-styrylquinoline (4). Light yellow solid. MP 96 – 97 ºC. 1H
NMR (400 MHz, CDCl3) δ 7.93 (d, J = 8.6 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H),
7.51 (d, J = 7.5 Hz, 2H), 7.45 (d, J = 7.7 Hz, 2H), 7.27 - 7.24 (m, 3H), 7.19
(dd, J = 13.4, 7.3 Hz, 2H), 6.91 (d, J = 7.5 Hz, 1H), 3.98 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 155.14, 155.10, 140.01, 136.56, 136.21, 133.92, 129.69,
128.75, 128.47, 128.33, 127.20, 126.32, 119.41, 119.13, 107.92, 56.07.
HRMS (ESI) calcd. for C18H16NO [M + H]+ 262.1226, found: 262.1154.
(E)-6-(2-Fluoroethoxy)-2-(2-(2-methoxypyridin-4-yl)vinyl)quinolone (14).
White solid. MP 114 – 116 ºC. 1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 5.4
Hz, 1H), 7.92 (dd, J = 8.8, 4.4 Hz, 2H), 7.50 (d, J = 8.6 Hz, 1H), 7.40 (d, J =
4.2 Hz, 2H), 7.34 (dd, J = 9.3, 2.7 Hz, 1H), 7.05 – 6.99 (m, 1H), 6.97 (d, J =
2.7 Hz, 1H), 6.79 (s, 1H), 4.83 – 4.65 (m, 2H), 4.30 – 4.16 (m, 2H), 3.89 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 164.96, 156.71, 152.75, 147.11, 146.68,
144.43, 135.26, 132.91, 131.02, 130.58, 128.45, 122.67, 120.01, 114.31,
108.81, 106.12, 81.70 (d, J = 171.7 Hz), 67.32 (d, J = 20.2 Hz), 53.49. HRMS
(ESI) calcd. for C19H18FN2O2 [M + H]+ 325.1347, found: 325.1262.
(E)-3-Methoxy-2-(2-(pyridin-4-yl)vinyl)quinolone (5). Light yellow solid.
MP 152 – 154 ºC. 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 5.5 Hz, 2H),
7.97 (d, J = 8.4 Hz, 1H), 7.85 (s, 2H), 7.63 (d, J = 8.0 Hz, 1H), 7.49 (t, J = 7.6
Hz, 1H), 7.45 – 7.36 (m, 3H), 7.33 (s, 1H), 3.93 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 151.59, 150.22, 147.28, 144.33, 143.02, 132.17, 129.10, 129.04,
127.10, 126.94, 126.44, 126.28, 121.56, 112.32, 55.50. HRMS (ESI) calcd.
for C17H15N2O [M + H]+ 263.1179, found: 263.1109.
(E)-4-Methoxy-2-(2-(pyridin-4-yl)vinyl)quinolone (6). Light yellow solid.
MP 139 – 141 ºC. 1H NMR (400 MHz, CDCl3) δ 8.60 (d, J = 5.9 Hz, 2H),
8.13 (d, J = 8.3 Hz, 1H), 8.00 (d, J = 8.5 Hz, 1H), 7.73 – 7.63 (m, 1H), 7.55
(d, J = 16.2 Hz, 1H), 7.45 (dd, J = 15.6, 8.1 Hz, 4H), 6.92 (s, 1H), 4.06 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 162.65, 155.71, 150.31, 149.03, 143.75,
133.73, 131.09, 130.24, 128.81, 125.80, 121.72, 121.29, 120.87, 98.33, 55.67.
HRMS (ESI) calcd. for C17H15N2O [M + H]+ 263.1179, found: 263.1127.
(E)-2-(2-(3-Chloropyridin-4-yl)vinyl)-6-methoxyquinoline (15). Light
yellow solid. MP 145 – 146 ºC. H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H),
1
8.46 (d, J = 5.2 Hz, 1H), 8.05 (d, J = 8.6 Hz, 1H), 7.98 (d, J = 9.2 Hz, 1H),
7.86 (d, J = 16.4 Hz, 1H), 7.69 (d, J = 8.6 Hz, 1H), 7.61 (d, J = 5.2 Hz, 1H),
7.53 (d, J = 16.4 Hz, 1H), 7.37 (dd, J = 9.2, 2.7 Hz, 1H), 7.06 (d, J = 2.6 Hz,
1H), 3.93 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 158.21, 152.16, 150.24,
147.77, 144.30, 141.88, 135.53, 135.29, 130.97, 130.83, 128.81, 126.03,
122.79, 120.11, 119.64, 105.09, 55.57. HRMS (ESI) calcd. for C17H14ClN2O
[M + H]+ 297.0789, found: 297.0710.
(E)-5-Methoxy-2-(2-(pyridin-4-yl)vinyl)quinolone (7). Light yellow solid.
1
MP 154 – 156 ºC. H NMR (400 MHz, CDCl3) δ 8.63 – 8.50 (m, 2H), 7.97
(d, J = 8.4 Hz, 1H), 7.59 (d, J = 8.9 Hz, 1H), 7.51 (d, J = 16.3 Hz, 1H), 7.46 –
7.28 (m, 5H), 7.11 (dd, J = 8.9, 2.1 Hz, 1H), 3.89 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 161.06, 154.71, 150.29, 149.89, 143.76, 136.23, 133.18,
131.07, 128.50, 122.89, 121.27, 119.99, 117.64, 107.05, 55.51. C17H15N2O
[M + H]+ 263.1179, found: 263.1173.
(E)-2-(2-(3-Bromopyridin-4-yl)vinyl)-6-methoxyquinoline (16). White
solid. MP 104 – 106 ºC. 1H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 8.49 (d,
J = 5.1 Hz, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.99 (d, J = 9.2 Hz, 1H), 7.82 (d, J
= 16.3 Hz, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.62 (d, J = 5.2 Hz, 1H), 7.51 (d, J =
16.3 Hz, 1H), 7.38 (dd, J = 9.2, 2.7 Hz, 1H), 7.07 (d, J = 2.7 Hz, 1H), 3.94 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 158.22, 152.71, 152.16, 148.31, 144.31,
5

143.67, 135.66, 135.30, 130.98, 128.81, 128.72, 122.79, 122.02, 120.65,
119.59, 105.10, 55.58. HRMS (ESI) calcd. for C17H14BrN2O [M + H]+
341.0284, found: 341.0190.
NMR (101 MHz, CDCl3) δ 175.02, 169.27, 158.97, 144.31, 144.15, 143.53,
136.47, 135.88, 131.95, 130.15, 126.16, 123.29, 120.50, 119.52, 119.12,
114.18, 110.72, 104.88, 55.64. HRMS (ESI) calcd. for C19H14N5O2 [M + H]+
344.1142, found: 344.1052.
6-Hydroxyquinoline-2-carbonitrile (19). To
a
solution of 6-
methoxyquinoline-2-carbonitrile (552 mg, 3 mmol) in CH2Cl2 (20 mL) was
added AlCl3 (1.0 g, 7.5 mmol). The reaction was stirred at 45 °C overnight in
a sealed tube. TLC showed the starting material remained so a second portion
of AlCl3 was added (1.0 g, 7.5 mmol). Water was added to quench the
reaction and the mixture was extracted with EtOAc. The combined organic
phase was washed with saturated bicarbonate solution, saturated sodium
chloride, respectively. The concentrated residue was subjected to silica gel
chromatography (hexane/EtOAc, 3/1, v/v) to afford 6-hydroxyquinoline-2-
carbonitrile (268 mg, 52% yield). 1H NMR (400 MHz, CD3COCD3) δ 9.41 (s,
1H), 8.22 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 9.2 Hz, 1H), 7.68 – 7.62 (m, 1H),
7.44 – 7.38 (m, 1H), 7.20 (d, J = 1.8 Hz, 1H).
5-(Imidazo[1,2-a]pyridin-7-yl)-3-(6-isopropoxyquinolin-2-yl)-1,2,4-
oxadiazole (32). Brown yellow solid. MP 203 – 205 ºC. 1H NMR (400 MHz,
CD3SOCD3) δ 8.84 (d, J = 7.0 Hz, 1H), 8.48 (d, J = 7.4 Hz, 2H), 8.31 – 8.15
(m, 2H), 8.09 (d, J = 8.9 Hz, 1H), 7.90 (s, 1H), 7.65 (d, J = 7.1 Hz, 1H), 7.48
(d, J = 14.6 Hz, 2H), 4.93 – 4.77 (m, 1H), 1.39 (s, 3H), 1.38 (s, 3H). 13C
NMR (101 MHz, CD3SOCD3) δ 175.05, 169.14, 157.06, 143.68, 143.38,
143.34, 136.72, 135.96, 131.48, 130.38, 128.64, 124.61, 120.84, 119.40,
117.73, 116.05, 110.34, 107.76, 70.33, 22.10. HRMS (ESI) calcd. for
C21H18N5O2 [M + H]+ 372.1455, found: 372.1356.
3-(6-(2-Fluoroethoxy)quinolin-2-yl)-5-(imidazo[1,2-a]pyridin-7-yl)-1,2,4-
oxadiazole (33). Brown yellow solid. MP 188 – 190 ºC. 1H NMR (400 MHz,
CDCl3) δ 8.66 – 8.44 (m, 1H), 8.24 – 8.11 (m, 4H), 7.75 (d, J = 41.1 Hz, 1H),
7.67 – 7.62 (m, 1H), 7.44 – 7.37 (m, 1H), 7.08 – 7.03 (m, 2H), 4.89 – 4.66
(m, 2H), 4.40 – 4.23 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 175.05, 169.22,
157.38, 144.45, 144.14, 143.86, 136.45, 135.98, 132.18, 129.97, 126.17,
123.30, 121.20, 120.60, 119.11, 114.20, 110.71, 105.86, 81.63 (d, J = 171.7
Hz), 67.41 (d, J = 21.2 Hz). HRMS (ESI) calcd. for C20H15FN5O2 [M + H]+
376.1204, found: 376.1104.
(Z)-6-(2-Fluoroethoxy)-N'-hydroxyquinoline-2-carboximidamide (20). 1H
NMR (400 MHz, CD3COCD3) δ 8.31 (d, J = 8.5 Hz, 1H), 7.91 (d, J = 9.3 Hz,
1H), 7.72 (d, J = 8.5 Hz, 1H), 7.46 (dd, J = 9.3, 2.8 Hz, 1H), 7.36 (d, J = 2.8
Hz, 1H), 4.81 – 4.67 (m, 2H), 4.41 – 4.32 (m, 2H).
6-(Methoxymethoxy)quinoline-2-carbonitrile (21). 1H NMR (400 MHz,
CD3COCD3) δ 8.31 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 9.2 Hz, 1H), 7.69 (d, J =
8.5 Hz, 1H), 7.46 (dd, J = 9.2, 2.7 Hz, 1H), 7.41 (s, 1H), 5.26 (s, 2H), 3.34 (s,
3H).
5-(Imidazo[1,2-a]pyridin-7-yl)-3-(6-(methoxymethoxy)-quinolin-2-yl)-
1,2,4-oxadiazole (34). Light yellow solid. MP 209 – 210 ºC. 1H NMR (400
MHz, CDCl3) δ 8.58 (s, 1H), 8.30 – 8.06 (m, 5H), 7.80 – 7.65 (m, 3H), 7.50 –
7.39 (m, 1H), 7.37 (s, 1H), 5.28 (s, 2H), 3.48 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 175.06, 169.25, 156.40, 144.57, 144.16, 144.02, 136.49, 136.29,
132.03, 129.98, 126.16, 123.49, 120.46, 119.49, 119.14, 114.19, 110.71,
108.82, 94.47, 56.31. HRMS (ESI) calcd. for C20H16N5O3 [M + H]+ 374.1248,
found: 374.1148.
6-Isopropoxyquinoline-2-carbonitrile (22). 1H NMR (400 MHz,
CD3COCD3) δ 8.41 (d, J = 8.5 Hz, 1H), 7.99 (d, J = 9.2 Hz, 1H), 7.81 (dd, J
= 8.5, 1.2 Hz, 1H), 7.51 – 7.48 (m, 1H), 7.43 (d, J = 2.3 Hz, 1H), 4.98 – 4.76
(m, 1H), 1.39 (d, J = 6.0 Hz, 6H).
3-(6-Methoxyquinolin-2-yl)-5-(pyridin-3-yl)-1,2,4-oxadiazole (26). White
solid. MP 175 – 177 ºC. 1H NMR (400 MHz, CDCl3) δ 9.46 (s, 1H), 8.78 (d,
J = 4.7 Hz, 1H), 8.50 (d, J = 8.0 Hz, 1H), 8.19 – 8.12 (m, 3H), 7.45 (dd, J =
7.9, 4.9 Hz, 1H), 7.37 (dd, J = 9.2, 2.6 Hz, 1H), 7.05 (d, J = 2.6 Hz, 1H), 3.88
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 174.47, 169.17, 158.97, 153.44,
149.31, 144.28, 143.36, 135.86, 135.53, 131.90, 130.14, 123.77, 123.31,
120.47, 120.45, 104.86, 55.61. HRMS (ESI) calcd. for C17H13N4O2 [M + H]+
305.1033, found: 305.0950.
6-(Methoxymethoxy)-2-methylquinoline (35). Light yellow liquid. 1H NMR
(400 MHz, CDCl3) δ 7.85 (dd, J = 8.7, 4.2 Hz, 2H), 7.31 (dd, J = 9.2, 2.7 Hz,
1H), 7.23 (d, J = 2.7 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 5.19 (s, 2H), 3.43 (s,
3H), 2.62 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 156.83, 154.55, 144.10,
135.36, 129.90, 127.18, 122.23, 109.29, 94.53, 56.09, 24.98. HRMS (ESI)
calcd. for C12H14NO2 [M + H]+ 204.1019, found: 204.0962.
5-(2-Methoxypyridin-4-yl)-3-(6-methoxyquinolin-2-yl)-1,2,4-oxadiazole
(27). White solid. MP 188 – 189 ºC. 1H NMR (400 MHz, CDCl3) δ 8.33 (d, J
= 5.3 Hz, 1H), 8.24 – 8.04 (m, 3H), 7.64 (d, J = 5.2 Hz, 1H), 7.54 (s, 1H),
7.38 (dd, J = 9.3, 2.7 Hz, 1H), 7.06 (d, J = 2.6 Hz, 1H), 3.95 (s, 3H), 3.90 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 174.68, 169.31, 164.88, 159.01, 148.40,
144.31, 143.32, 135.88, 133.30, 131.93, 130.16, 123.34, 120.45, 114.49,
109.81, 104.86, 55.62, 53.98. HRMS (ESI) calcd. for C18H15N4O3 [M + H]+
335.1139, found: 335.1051.
(E)-6-(Methoxymethoxy)-2-(2-(2-methoxypyridin-4-yl)-vinyl)quinolone
(36). Light yellow solid. MP 108 – 110 ºC. 1H NMR (400 MHz, CDCl3) δ
8.12 (d, J = 5.4 Hz, 1H), 7.97 (dd, J = 8.8, 5.5 Hz, 2H), 7.54 (d, J = 8.6 Hz,
1H), 7.46 – 7.36 (m, 3H), 7.29 – 7.27 (m, 1H), 7.05 (d, J = 5.4 Hz, 1H), 6.83
(s, 1H), 5.26 (s, 2H), 3.92 (s, 3H), 3.49 (s, 3H). 13C NMR (101 MHz, CDCl3)
δ 164.94, 155.36, 152.90, 147.08, 146.67, 144.55, 135.53, 132.97, 130.81,
130.61, 128.47, 122.89, 119.84, 114.30, 109.10, 108.80, 94.46, 56.18, 53.47.
HRMS (ESI) calcd. for C19H19N2O3 [M + H]+ 323.1390, found: 323.1309.
3-(6-(2-Fluoroethoxy)quinolin-2-yl)-5-(2-methoxypyridin-4-yl)-1,2,4-
oxadiazole (28). White solid. MP 235 – 237 ºC. 1H NMR (400 MHz, CDCl3)
δ 8.34 (d, J = 5.3 Hz, 1H), 8.25 – 8.11 (m, 3H), 7.65 (d, J = 5.3 Hz, 1H), 7.55
(s, 1H), 7.44 (dd, J = 9.3, 2.7 Hz, 1H), 7.09 (d, J = 2.7 Hz, 1H), 4.88 – 4.68
(m, 2H), 4.37 – 4.24 (m, 2H), 3.96 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
174.75, 169.28, 164.90, 157.78, 148.42, 144.46, 143.67, 136.00, 133.28,
132.18, 130.00, 123.38, 120.56, 114.49, 109.83, 105.84, 81.63 (d, J = 172.7
Hz), 67.41 (d, J = 21.2 Hz), 54.00. HRMS (ESI) calcd. for C19H16FN4O3 [M +
H]+ 367.1201, found: 367.1104.
(E)-2-(2-(2-methoxypyridin-4-yl)vinyl)quinolin-6-ol (37). To a solution of
compound 36 (48 mg, 0.15 mmol) in CH2Cl2 (1 mL) was added CF3COOH (1
mL) at 0 ºC. The mixture was stirred at room temperature for 2 h. Saturated
sodium bicarbonate was added to neutralize the reaction and the mixture was
extracted with EtOAc. The combined organic layer was washed with
saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated
in vacuo, and the residue was subjected to silica gel chromatography
(hexane/EtOAc 1/1) to afford product 37 as a yellow solid (40 mg, 97%
1
yield). MP 244 – 245 ºC. H NMR (400 MHz, CD3SOCD3) δ 10.17 (s, 1H),
8.27 – 8.13 (m, 2H), 7.91 (d, J = 9.1 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.72 –
7.64 (m, 2H), 7.37 (dd, J = 6.8, 4.5 Hz, 2H), 7.19 (d, J = 2.5 Hz, 1H), 7.09 (s,
1H), 3.91 (s, 3H). 13C NMR (101 MHz, CD3SOCD3) δ 164.87, 156.30,
151.92, 147.59, 147.28, 143.25, 135.20, 133.76, 130.85, 130.03, 129.19,
122.87, 120.72, 114.92, 108.80, 108.69, 53.61. HRMS (ESI) calcd. for
C17H15N2O2 [M + H]+ 279.1128, found: 279.1052.
3-(6-(Methoxymethoxy)quinolin-2-yl)-5-(2-methoxypyridin-4-yl)-1,2,4-
oxadiazole (29). White solid. MP 180 – 181 ºC. 1H NMR (400 MHz, CDCl3)
δ 8.33 (d, J = 5.2 Hz, 1H), 8.22 – 8.15 (m, 3H), 7.64 (d, J = 5.2 Hz, 1H), 7.54
(s, 1H), 7.44 (d, J = 9.2 Hz, 1H), 7.35 (s, 1H), 5.27 (s, 2H), 3.96 (s, 3H), 3.48
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 174.72, 169.29, 164.88, 156.43,
148.40, 144.56, 143.81, 136.30, 133.28, 132.01, 129.99, 123.55, 120.41,
114.48, 109.81, 108.79, 94.46, 56.30, 53.98. HRMS (ESI) calcd. for
C19H17N4O4 [M + H]+ 365.1244, found: 365.1405.
2-(5-(2-Methoxypyridin-4-yl)-1,2,4-oxadiazol-3-yl)quinolin-6-ol (38). To a
solution of compound 34 (26 mg, 0.07 mmol) in CH2Cl2 (2 mL) was added
CF3COOH (0.5 mL) at 0 ºC. The mixture was stirred at room temperature for
3 h. Saturated sodium bicarbonate was added to neutralize the reaction and
the mixture was extracted with EtOAc. The combined organic layer was
washed with saturated sodium chloride, dried over anhydrous sodium sulfate,
concentrated in vacuo, and the residue was subjected to silica gel
chromatography (hexane/EtOAc 1/1) to afford product 38 as a yellow solid
(18 mg, 79% yield). MP 217 – 219 ºC. 1H NMR (400 MHz, CD3COCD3) δ
9.17 (s, 1H), 8.37 (d, J = 5.2 Hz, 1H), 8.23 (d, J = 8.6 Hz, 1H), 8.07 (d, J =
8.6 Hz, 1H), 7.97 (d, J = 9.1 Hz, 1H), 7.63 – 7.58 (m, 1H), 7.43 – 7.35 (m,
2H), 7.21 (d, J = 2.4 Hz, 1H), 3.88 (s, 3H). 13C NMR (101 MHz, CD3SOCD3)
δ 172.81, 167.72, 163.16, 155.98, 147.82, 141.58, 140.85, 134.57, 132.06,
130.02, 129.02, 122.26, 119.05, 113.42, 107.64, 107.17, 53.74. HRMS (ESI)
calcd. for C17H13N4O3 [M + H]+ 321.0982, found: 321.0982.
3-(6-Isopropoxyquinolin-2-yl)-5-(2-methoxypyridin-4-yl)-1,2,4-oxadiazole
(30). Light yellow solid. MP 171 – 173 ºC. 1H NMR (400 MHz, CDCl3) δ
8.32 (d, J = 5.2 Hz, 1H), 8.20 – 8.03 (m, 3H), 7.63 (dd, J = 5.2, 0.8 Hz, 1H),
7.53 (s, 1H), 7.34 (dd, J = 9.3, 2.6 Hz, 1H), 7.04 (d, J = 2.5 Hz, 1H), 4.66 (dt,
J = 12.1, 6.0 Hz, 1H), 3.95 (s, 3H), 1.37 (s, 3H), 1.35 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 174.64, 169.33, 164.87, 157.27, 148.38, 144.02, 143.13,
135.74, 133.31, 131.97, 130.21, 124.21, 120.33, 114.48, 109.79, 106.77,
70.30, 53.96, 21.87. HRMS (ESI) calcd. for C20H19N4O3 [M + H]+ 363.1452,
found: 363.1353.
5-(Imidazo[1,2-a]pyridin-7-yl)-3-(6-methoxyquinolin-2-yl)-1,2,4-
1
oxadiazole (31). Light yellow solid. MP 277 – 279 ºC. H NMR (400 MHz,
CDCl3) δ 8.58 (s, 1H), 8.27 – 8.14 (m, 4H), 7.80 (s, 1H), 7.72 – 7.63 (m, 2H),
7.39 (dd, J = 9.2, 2.8 Hz, 1H), 7.08 (d, J = 2.7 Hz, 1H), 3.91 (s, 3H). 13C
6

authenticated by co-injecting with the reference standard solution. The entire
procedure took ~2 h for two-step radiolabeling. [18F]14 was produced in 21 ±
7% yield, specific activity > 27 GBq/µmol (decay corrected to the end of
synthesis), [18F]28 was produced in 30 ± 4% yield, specific activity > 37
GBq/µmol (decay corrected to the end of synthesis).
b) Radiochemistry:
Radiosynthesis of [11C]13. Briefly, [11C]CH3I was produced on-site from
[11C]CO2 using a GE PETtrace MeI Microlab. [11C]CH3I was bubbled for a
period of 3 – 4 min into a solution of precursor 37 (1 – 2 mg) in anhydrous
DMF (0.2 mL) containing 3.0 – 5.0 µL of aqueous sodium hydroxide solution
(5.0 M) at room temperature. When the trapping of radioactivity was
completed, the sealed reaction vessel was heated at 85 ºC for 5 min. After the
heating source was removed, 1.7 mL of the HPLC mobile phase (acetonitrile
in 0.1 M ammonium formate, 50/50, v/v, pH 4.5) was added into to the
reaction vessel. The mixture was loaded onto a reversed phase HPLC system
(Agilent Zorbax, SB-C18, 250 × 9.6 mm, 5 µ, UV at 254 nm, flow rate at 4
mL/min) to purify the mixture. The target fraction with retention time of 20
min for [11C]13 was collected into a vial that contained 50 mL sterile water
for injection. After finishing the collection, the collected fraction was passed
through a C-18 Plus Sep-Pak® cartridge to remove the mobile phase solvent,
and the target tracer was retained on the cartridge. Then the Sep-Pak cartridge
was rinsed using 20 mL of sterile water. Finally, the [11C]13 trapped on the
Sep-Pak was eluted with 0.6 mL of ethanol, following with 5.4 mL 0.9%
sodium chloride solution, passing through a 0.22 µ sterile filter into a sterile
pyrogen-free glass vial. For quality control (QC), an aliquot was assayed by
an analytical HPLC system, UV at 254 nm. [11C]13 was authenticated by co-
injecting with the corresponding reference standard, 13. [11C]13 was
produced in 50 ± 10% yield with >99% radiochemical purity, specific activity
>148 GBq/µmol (decay corrected to EOB). The radiosynthesis typically took
1 hour.
c) In vitro binding assays
Thioflavin T competitive binding fluorescence assay: Indirect affinity
determination (Ki) using Thioflavin T fluorescence assay with recombinant α-
synuclein fibrils.
The binding affinities of new compounds were determined by a Thioflavin T
competitive binding assay. Briefly, the binding assay was conducted in
microplates which contained 3 μM Thioflavin T and 0.5 μM α-synuclein
fibrils in 30 mM Tris–HCl pH 7.4, 0.1% BSA plus the test compound at
concentrations ranging from 3 nM to 10 μM. The mixture was incubated at
room temperature for 1.5 h. Fluorescence was determined in a Biotek plate
reader using a 440/30 excitation filter and a 485/20 emission filter. All data
points were performed in triplicate. Nonspecific fluorescence was measured
in parallel reactions containing ThioT plus each concentration of competitor
but no fibrils, and these measurements were subtracted from the reactions
with fibrils to yield fibril-specific fluorescence. Data were analyzed using
Graphpad Prism software (version 4.0) to obtain EC50 values by fitting the
data to the equation
.
Baseline response is (bottom) and maximum response is (top). Ki values were
calculated from EC50 values using the equation
. We used an affinity constant value of 1850 nM for
Radiosynthesis of [18F]14 and [18F]28 via [18F]fluoroethyl tosylate:
[18F]Fluoride produced in Washington University School of Medicine
Cyclotron Facility was first passed through an ion-exchange resin and was
then eluted with 0.02 M potassium carbonate (K2CO3) solution. 1.0 – 1.5 mg
of the appropriate phenol precursor (37 or 38) was dissolved in Cs2CO3
saturated solution in anhydrous DMSO (200 µL). After vortexing for 1 min,
the phenol precursor solution was added into a reaction tube containing
[18F]fluoroethyl tosylate, which was readily prepared as described in
reference 29. The reaction vessel was capped and briefly swirled, and then
kept at 100 ºC for 15 min. Subsequently, the residue was diluted with 3 mL of
the appropriate HPLC mobile phase (acetonitrile in 0.1 M ammonium
formate, 55/45, v/v, pH 4.5 for [18F]14; acetonitrile in 0.1 M ammonium
formate, 51/49, v/v, pH 4.5 for [18F]28) then loaded onto a semi-preparative
HPLC (Agilent Zorbax, SB-C18, 250 × 9.6 mm, 5 µ) system for purification
at 4.0 mL/min flow rate. The HPLC system contains a 5 mL injection loop, a
UV detector at 254 nm and a radioactivity detector. After HPLC collection of
the desired fraction, (the retention time for [18F]14 was 18 min, for [18F]28
was 22 min), the product fraction was diluted with ~50 mL sterile water. The
product was then trapped on a C-18 Sep-Pak Plus cartridge and washed with
20 mL water. The trapped product was eluted with ethanol (0.6 mL) followed
by 5.4 mL of 0.9% saline. After passing through a 0.22 µ sterile filter into a
sterile pyrogen-free glass vial, the final product was ready for quality control
(QC) analysis and direct binding assays. For quality control, an aliquot of
sample was assayed by an analytical HPLC system. The sample was
Thioflavin-T towards recombinant α-synuclein fibrils.
Direct radioactive competitive binding assay: Affinity determination (Kd)
using direct radioactive competitive binding assay with either recombinant α-
synuclein fibrils or brain tissue homogenates from AD cases.
These direct binding assays used a fixed concentration of either fibrils or AD
tissue and the radioligand ([11C]13, [18F]14,and [18F]28) and varying
concentration ranges of corresponding homologous non-radiolabeled standard
reference compound. In brief, the standard homologous reference compound
was diluted in 30 mM Tris-HCl pH 7.4, 0.1% BSA. Reactions were incubated
at 37 ºC for 1 h before quantifying bound radioligand. Bound and free
radioligand were separated by vacuum filtration through 1.0 µm glass fiber
filters in 96-well filter plates (Millipore), followed by three 200 µL washes
with ice-cold assay buffer. Filters containing the bound ligand were mixed
with 150 µL of Optiphase Supermix scintillation cocktail (PerkinElmer) and
counted immediately. All data points were performed in triplicate. The
dissociation constant (Kd) and the maximal number of binding sites (Bmax
values were determined by fitting the data to the equation. Bound =
(Bmax*[Radioligand])/([Radioligand] [Unlabeled compound] Kd)
)
+
+
+
Bottom by nonlinear regression using Graphpad Prism software (version 4.0),
where (Bottom) is nonspecific binding and [Radioligand], [Unlabeled
compound], and Kd are expressed in nM.
7

Graphical Abstract
Leave this area blank for abstract info.
Design, synthesis, and in vitro evaluation of
quinolinyl analogues for α-synuclein aggregation
Xuyi Yue a, Dhruva D. Dhavale b, Junfeng Li a,
Zonghua Luo a, Jialu Liu b, Hao Yang a,
Robert H. Mach c, Paul T. Kotzbauer b, Zhude Tu a,*
8