Novel Furan-2-yl-1 H-pyrazoles Possess Inhibitory Activity against α-Synuclein Aggregation

Article abstract of DOI:10.1021/acschemneuro.0c00252

A series of novel furan-2-yl-1H-pyrazoles and their chemical precursors were synthesized and evaluated for their effectiveness at disrupting α-synuclein (α-syn) aggregation in vitro. The compounds were found to inhibit α-syn aggregation with efficacy comp

Full text of DOI:10.1021/acschemneuro.0c00252

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Article
Novel furan-2-yl-1H-pyrazoles possess inhibitory
activity against #-synuclein aggregation
Philip Ryan, Mingming Xu, Kousar Jahan, Andrew K. Davey, Prasad V. Bharatam, Shailendra
Anoopkumar-Dukie, Michael Kassiou, George D Mellick, and Santosh RUDRAWAR
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.0c00252 • Publication Date (Web): 18 Jun 2020
Downloaded from pubs.acs.org on June 29, 2020
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ACS CHEMICAL NEUROSCIENCE MANUSCRIPT:
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Novel furan-2-yl-1H-pyrazoles possess inhibitory activity
against α-synuclein aggregation
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Philip Ryan1,2,3, Mingming Xu4, Kousar Jahan5, Andrew K. Davey1,2,3, Prasad V. Bharatam5,
Shailendra Anoopkumar-Dukie1,2, Michael Kassiou6, George D. Mellick4†, Santosh
Rudrawar1,2,3,†
1School of Pharmacy and Pharmacology, Griffith University, Gold Coast, QLD 4222, Australia.
2Quality Use of Medicines Network, Griffith University, Gold Coast, QLD 4222, Australia.
3Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD 4222, Australia
4Griffith Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia.
5National Institute of Pharmaceutical Education and Research (NIPER) SAS Nagar, 160062, India
6School of Chemistry, The University of Sydney, NSW 2006, Australia.
†Corresponding authors:
1) Dr Santosh Rudrawar
2) Prof George Mellick
E-mail address: s.rudrawar@griffith.edu.au
E-mail address: g.mellick@griffith.edu.au
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Abstract: A series of novel furan-2-yl-1H-pyrazoles and their chemical precursors were
synthesised and evaluated for their effectiveness at disrupting α-synuclein (a-syn) aggregation
in vitro. The compounds were found to inhibit a-syn aggregation with efficacy comparable to
the promising drug candidate anle138b. The results of this study indicate that compounds 8b,
8l and 9f may qualify as secondary leads for the structure-activity relationship studies aimed
to identify the suitable compounds for improving the modulatory activity targeted at α-syn
self-assembly related to Parkinson’s disease.
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Keywords: a-Synuclein aggregation; Parkinson’s disease; neurodegenerative diseases;
anle138b; Molecular modelling studies; aminopyrazole.
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INTRODUCTION
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Neurodegenerative diseases (NDs) are causing significant increase in the societal burden, with
their growing prevalence of cases in aged population. Though symptoms can be treated for
some NDs, there are no therapies that address the underlying progressive, disease-related
processes. Various novel strategies have been pursued towards the diagnosis and treatment of
NDs such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). A number of
proteopathic NDs share common defining characteristics, specifically the misfolding and self-
assembly of the culprit proteins at the molecular level leading to the accretion and propagation
of toxic soluble species and the appearance of insoluble fibrillar structures.1 Ultimately these
prion-like processes lead to the neuronal cell death and the onset of symptoms.
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Regarding AD and PD, mounting evidence indicates that the pathogenesis is associated with
the arrival of soluble, oligomeric protein complexes, rather than the consequential fibrillar
deposits.2 Therefore, modern therapeutic approaches aim to arrest oligomer formation, and
related processes, rather than targeting the larger aggregate structures.3 Recently, the
diphenylpyrazole anle138b (1, Figure 1) was identified as a potent modulator of protein
oligomerisation.4 It displays potent antiprion activity with high CNS bioavailability and low
cytotoxicity, even able to prolong survival in prion-infected mice.4 The compound 1 is also
able to inhibit the formation and accumulation of α-synuclein (α-syn) oligomers in the brain,
reduce PD-associated motor deficits and prolong the survival of various animal models of PD.4-
5 In addition, anle138b effectively bind to the aggregated tau and inhibit tau aggregation. It also
possesses a tendency to ameliorate AD symptoms, and increases survival time and improve
cognition in treated transgenic PS19 mice.6 When administered orally, anle138b ameliorates
Aβ-induced deficits in synaptic plasticity and memory formation in the APPPS1ΔE9 mouse
model for amyloid pathology.7 Finally, oral administration of anle138b in Multiple System
Atrophy (MSA) model mice reverses impaired motor function; reduces levels of α-syn
oligomers and glial cytoplasmic inclusions; and results in the preservation of dopaminergic
neurons and reduction of microglial activation in the substantia nigra.5 The compound 1 is
undergoing pre-clinical development for use as a PD therapeutic agent, and has been
recommended for use in clinical trials for MSA and AD.6, 8
Reports suggest that compound 1 modulates the oligomer formation by targeting structure-
dependant epitopes. This may partly explain its broad spectrum of activity against
aggregopathies. Well-defined structure-activity relationships (SARs) have provided insight on
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the key structural features that determine its inhibitory potency.4, 9 The pyrazole ring, of all
heterocycles investigated, is most closely associated with the strong antiprion activity.
Molecular dynamics (MD) simulations suggest that the ring is effective at establishing
stabilising, transient, intermolecular hydrogen bonding interactions with the peptide backbone,
burying itself into the oligomeric structures.8 This binding mode is then strengthened by
hydrophobic contacts formed between the phenyl rings and the proteins’ aromatic and aliphatic
side chains. Simulations suggest that the compound binds larger, ordered β-sheet filaments at
alternative sites however, burying into hydrophobic pockets at the expense of these hydrogen
bonding interactions.
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Since the first reports on compound 1, a number of research groups have produced powerful
H-bonding ligands effective at modulating protein aggregation. The heterocycles 2,6-
diaminopyridines and 3-substituted indoles, such as 2 and 3, respectively (Figure 1), have been
identified as key binding motifs, when appropriately functionalised, and effective at inhibiting
the Aβ1-42 aggregation in vitro.10-11 Both 2 and 3 boast favourable BBB permeability in male
Swiss mice, though 2 exhibited poor metabolic stability, possibly accounting for its rapid
clearance. Mach and co-workers were able to tune the binding affinity of their novel scaffold
(4) for binding α-syn fibrils by altering the distance between hydrogen bonding groups. Their
SAR data suggested that hydrogen bonding may play a more important role in binding α-syn
than Aβ or tau fibrils.12
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10-12
Figure 1. Structures of recently reported
inhibitors of amyloid formation and the generalised structure of the
furan-2-yl-1H-pyrazoles investigated in this work.
Pyrazoles effectively form bidentate hydrogen bond contacts to amide hydrogens and carbonyl
oxygens of the peptide backbones (Figure 2).13-14 It was speculated that the binding may
improve as the number of H-bonding interactions increases accordingly. Aminopyrazole and
related structures have been identified as potent ligands bearing high affinity for the top face
of extending peptide strands.15 We were interested to probe how an increase in the number of
potential hydrogen bond participating groups might affect the inhibitory activity of the
analogues of anle138b. Consequently, it was decided to investigate a series of furan-2-yl-1H-
pyrazoles bearing similar substitutions to the anle138b compound in the hope of improving
biological activity. It was hypothesized that the furan oxygen may act as a hydrogen bond
acceptor, without majorly altering the overall topological surface areas or lipophilicities of the
resultant molecular structures. The furan ring was also chosen due to its relative structural
simplicity, and its introduction would not increase the hydrogen bond donor count in the
molecular structure. The hydrogen bond donor count represents a key CNS differentiating
parameter.16 A recent analysis, from Eli Lilly, found the median HBD count for currently
marketed CNS active drugs is 1, while the median HBA count is 4.16
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Figure 2. Binding scheme of a peptide backbone with i) methylaminopyrazole, ii) anle138b (1) and iii) a furan-
2-yl-1H-pyrazole investigated here highlighting the differences in hydrogen-bond donor and acceptor properties.
For the current study, novel furan-2-yl-1H-pyrazoles and related chemical precursors were
chosen for the activity against α-syn aggregation. The ThT fluorescence assays were employed
to characterise the inhibitory activity and the MS-binding assay was used to probe the nature
of the binding interaction with the protein.17 The results of inhibition studies led to the
identification of a number of highly active pyrazole and pyrazoline derivatives more potent
than the compound 1 in vitro. Molecular modelling studies were also conducted to examine the
compounds’ structural features and to evaluate binding energies. The human tau protein is a
key protein involved in various proteopathic NDs. As with α-syn, tau exhibits a tendency to
aggregate to form oligomers, which further leads to the generation of insoluble mass in the
brain through aggregation. Although the exact mechanism of aggregation remains unknown it
has been hypothesized that hydrogen bond formation in between peptides could play a major
role. Furthermore, in vitro studies revealed that tau undergoes polymerization with the aid of
an inducer conceivably α-syn via phosphorylation as reported by Gaisson et al.18 Hence, the
molecular modelling analysis has been carried out using tau protein.
RESULTS AND DISCUSSION
Chemistry
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Previously, anle138b has been generated via the 1,3-diketo-intermediate.4 Considering rapid
access to library of novel compounds, the synthesis of desired compounds was envisaged here
via chalcone intermediates. Chalcones have displayed the inhibitory activity against α-syn
aggregation,19 as well as high-affinity binding for the protein, and therefore, the synthetic
precursors of the pyrazoles were evaluated also. Synthesis involved an initial Suzuki-Miyaura
cross-coupling reaction of 5-bromo-2-furaldehyde 5 with various appropriately substituted
phenylboronic acids before condensation with substituted acetophenone and subsequent
generation of either the pyrazoline or pyrazole ring under microwave conditions (Scheme 1).
The substitution on the phenyl rings strongly affects the bioavailability,4 therefore
acetophenones and phenylboronic acids bearing the 1,3-benzodioxole or bulky halogens in the
meta-position (compound 1) were considered for conferring attractive physicochemical
properties. The cross-coupling reactions of the furaldehydes forming 6a-6e were typically
high-yielding, however the reaction with 3-chlorophenylboronic acid afforded very poor yields
of the desired product (6b). All attempts were made to improve the product yield by varying
reaction conditions20 (such as increasing temperature or molar equivalents of reactants of
reactants and/or reagents) but failed. Interestingly, (E)-3-(5-(3-chlorophenyl)furan-2-
yl)acrylaldehyde (SI, Figure S1), the product of ethanol’s condensation with the 2-formyl
derivatives was isolated as major product and the (E)-isomer was formed exclusively. To the
best of our knowledge, this type of one-pot reaction has not been used previously to form this
class of arylated furanoids.
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Chalcones can be readily synthesized by the base-catalysed Claisen-Schmidt condensation
of an aryl aldehyde and acetophenone derivative in a protic polar solvent like ethanol or
methanol. The traditional chalcone synthesis involves the use of inorganic bases (NaOH, KOH,
LiOH or Ba(OH)2).21 Other protocols involve the use of organic bases also, such as
piperidine.22 Employing aldol condensation to form chalcones (7a-7j) was initially attempted
following by established protocols, specifically using NaOH in EtOH at room temperature
overnight.23-24 The diarylated chalcone synthesis worked well (data not included). However,
synthesis using heteroaryl aldehydes (eg. 2-furaldehyde) frequently resulted in poor reaction
yields due to the formation of complex mixtures of side-products which were challenging to
isolate. When conducted under microwave-assisted heating conditions however, the reactions
afforded the desired product quantitatively. Additionally, the reaction time was dramatically
improved, being reduced from upward of 5 h to only 30 min. With chalcones in hand, pyrazoles
(9a-9j) were generated using hydrazine hydrate with sulfur as an oxidant under microwave-
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assisted heating conditions.25 Though the reaction yields were not exceptional, they produced
an acceptable amount of desired compounds for the biological evaluation. The poor yields
suggested that sulfur may not have been an effective oxidizing agent, as the reactions yielding
pyrazolines (8b-8j) were characterized by significantly increased yields. The synthesis of 5-
bromofuran-2-yl pyrazoles 9k and 9l were also attempted but isolation could not be carried out
due to decomposition into the unsubstituted furfural derivatives, which were confirmed by
NMR and MS.
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Scheme 1. Synthesis of chalcone, pyrazoline and pyrazole analogues 7a-9j (A) and 7k-9l (B). Reagents and
conditions: (a) ArB(OH)2, Pd(PPh3)4, PhMe, EtOH, K2CO3(aq), reflux, 16 h; 40-98%; (b) acetophenone (1 eq.),
EtOH, piperidine, MW, 70 °C, 30 min, 80-97%; (c), NH2NH2, EtOH, MW, 70 °C, 1 h, 59-87%; (d); NH2NH2,
EtOH, S, MW, 150 °C, 2 h, 13-42%, (e) acetophenone (1 eq.), EtOH, NaOH, rt, 16 h, 81-97%.
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Biology
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The ability of the compounds to inhibit α-syn aggregation was studied by incubating α-syn
(80 µM), expressed in E.coli and purified using anion exchange, with or without the compounds
(400 µM) under constant agitation for 48 hours before being analysed by ThT fluorescence. It
was decided to see the effect of the compounds at the end-point of the experiment, rather than
any effects on the rate of aggregation, as ThT has been found previously to alter aggregation
kinetics.26 Two positive controls were examined, synthetically produced anle138b and
commercially available EGCG, with six replicates per sample being used.
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Treatment with chalcones 7a-7l reduced fluorescence by values ranging from 58% (7c) to
80% (7e) (Figure 3). It was noted that the substitution of ring A with 3-Br often yielded
compounds more effective at reducing fluorescence than those substituted with the 1,3-
benzodioxole. It was also noted that substitution of ring B with 4-methoxy or 1,3-benzodioxole
groups yielded more effective compounds as compared to those bearing larger groups such as
3-Cl or 4-phenyl. Chalcones 7k and 7l, generated via aldol condensation with unmodified 5-
bromo-2-furaldehyde, were also evaluated. Fluorescence values were decreased five-fold
following treatment with 7k, compared to 1,3-benzodioxole-substituted 7l. Of all the chalcones
evaluated, only 7e and 7k displayed effects comparable to anle138b.
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A
B
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Figure 3. Aggregation of α-syn was measured by ThT fluorescence assay. Chalcones’ (A), pyrazolines’ (B), and
pyrazoles’ (C) effects on fluorescence were examined using a 1:5 α-syn to compound molar ratio (80:400 µM).
The data are expressed as mean of six independent experiments. Data are expressed as percentage (mean ±
standard deviation) of untreated negative control (α-syn only) and compared to positive control treated with
epigallocatechin gallate (EGCG) and anle138b. Colours correlate with ring substitution patterns common between
the compound panels.
Pyrazolines 8b-8l reduced fluorescence by values in the range of 76% (8c) to 94% (8b)
(Figure 3B). In the case of the pyrazolines, more often than not substitution with 1,3-
benzodioxole on ring A was associated with a larger decrease in fluorescence than 3-Br
substitution. Interestingly, these results contrast with the trends observed for the chalcones.
Again, smaller substitutions on ring B tended to reduce fluorescence more significantly.
Pyrazolines 8k and 8l, arising from the reaction of hydrazine with chalcones 7k and 7l
respectively, were also tested. The 1,3-benzodioxole-substituted 8l decreased fluorescence by
four-fold relative to 8k. Under the assay conditions here, the majority of pyrazolines were as
effective as anle138b at reducing levels of fluorescence. Notably three of the pyrazolines, 8b
(p<0.01), 8d (p<0.05), and 8l (p<0.05), decreased fluorescence more significantly than the
anle138b compound.
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The pyrazoles 9a-9j displayed inhibition of α-syn aggregation in the range of 36% (9a) to 91%
(9f) (Figure 3C). Among the pyrazoles examined, the most effective inhibitor was 9f, the
compound bearing 1,3-benzodioxole substitution on ring A and 4-methoxy substitution on ring
B. It was observed that 1,3-benzodioxole substitutions on ring A typically resulted in stronger
reducers of fluorescence than the 3-Br substitution, as observed in the case of the pyrazolines.
Again, smaller substitutions on ring B yielded more effective compounds also. Attempts to
generate pyrazoles brominated at the C-5 position on the furan ring were unsuccessful as the
desired products were subject to rapid degradation. Under the assay conditions here, a majority
of the pyrazoles were less effective than the anle138b compound at reducing levels of ThT
fluorescence with the exception of compounds 9b and 9f which displayed similar potencies.
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Of all the compounds examined, the most significant decreases in ThT fluorescence resulted
from co-incubation of α-syn with pyrazolines 7k, 8b, 8d and 8l and with pyrazole 9f. The most
interesting compounds 8b, 8l and 9f were chosen and tested at two different molar ratios to
examine whether the decreases in fluorescence may be dose-dependent. Interestingly, although
the pyrazole 9f at a 1:5 molar ratio decreased fluorescence by an amount comparable to
anle138b, at stoichiometric concentration it was less effective, seemingly even promoting
fluorescence. Pyrazolines 8b and 8l seemed to reduce fluorescence at both concentrations
tested. Interestingly, 8b decreased fluorescence to a similar degree under both concentrations
tested.
60000
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Control 1:1
1:5
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1:5
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9f
8b
8l
Figure 4. Inhibition of aggregation measured by ThT fluorescence assay at 1:5 and 1:1 molar ratio α-
syn/compound. The data are expressed as mean of 3 independent experiments. Data are expressed as percentage
(mean ± standard deviation) of untreated negative control (α-syn only) and compared to positive control treated
with anle138b. Data was analysed using one-way ANOVA with Dunnett’s post hoc comparison (*p<0.05 vs
control; ***p<0.001 vs control).
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The inhibition of fibril formation was validated using dot blot analysis, a non-denaturing
method for examining protein epitopes. Dot blot analysis has been employed previously to
study α-syn fibril formation and inhibitor-bound α-syn complexes structural conformations.27-
28 Equal volumes of each sample (2 µL) were spotted on nitrocellulose membrane before being
stained with one of either the non-conformation dependent anti-α-syn antibody (1:2,000) or the
conformation dependent anti-α-syn filament antibody (1:8,000) prior to further staining with a
detection antibody.29-30 Development of the blots exhibited notable signal intensity of fibrillary
and total α-syn, which was analysed for quantification (Figure 5). All compounds were found
to decrease concentration of fibrillar α-syn at both molar equivalents tested (1:1, 1:5, α-
syn/compound) relative to the untreated control. Dose-dependent effects were observed for
anle138b and compounds 8b and 8l, with compound 8b decreasing the concentration of fibrillar
α-syn detected most effectively. Interestingly, dose-dependent effects on fibril concentration
were not displayed for compound 9f, which had worked previously to increase fluorescence at
1:1 molar ratio in the ThT assay. Rather compound 9f reduced the concentration of fibrillar α-
syn detected by ~80 % consistently in this case.
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**
**
**
**
**
**
0
Control 1:1
1:5
1:1
1:5
1:1
1:5
1:1
1:5
anle138b
9f
8b
8l
Figure 5. Dot blot analysis of α-syn fibril formation relative to an untreated control. Presence of α-syn in each
sample was confirmed via staining with anti-α-synuclein antibody. In samples treated with conformation
dependent anti-α-synuclein filament antibody, the total α-syn dot blot quantification was used to normalize the
fibrillary α-syn staining. Data are expressed as percentage (mean ± standard deviation) of untreated negative
control (α-syn only, taken as 100, n = 3) and compared to positive control treated with anle138b (1). Data was
analysed using one-way ANOVA with Dunnett’s post hoc comparison (** p<0.01 vs control).
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It is interesting to note that in most of the cases the pyrazoline derivatives were found
to be more effective at reducing ThT fluorescence than their structural analogues (pyrazole
derivatives) possessing equivalent substitution patterns. To rationalize the observed trend in
the biological results, molecular docking analysis has been carried out. The binding affinity
has been estimated in terms of Glide scores,31-32 Induced Fit Docking33-34 (IFD) scores (data
listed in Table S1). The binding energies associated with docked (1st ranked) poses have been
estimated using Prime MMGBSA tool.35 Figure 6 shows two views of binding pocket chosen
to perform molecular docking. The identified binding cavity has a C2 symmetric elliptical cone
shape with hydrophobic component in the centre of the cavity (yellow color, Figure 6: A and
B).
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The mechanism of aggregation of oligomers involves interpeptide main chain
interaction due to the formation of hydrogen bonds. Anle138b effectively inhibits the
interpeptide main chain interaction, by occupying the C2 symmetric elliptical cavity (especially
the hydrophobic pocket of the macromolecule, the dimer of β-sheet). The important amino
acids responsible for this binding are ILE4, VAL5, PHE10 and TYR6 amino acid residues
(from each monomer). The two nitrogen atoms of the pyrazole ring face away from the centre
of the cavity and form hydrogen bonds with the back bone atoms of the peptide chains (the
dimer of β-sheet near to the proximity of ILE4 of C and D chain). This interaction is responsible
for the inhibition of the interpeptide hydrogen bond formation and thus for the inhibition of the
aggregation. The hydrophobic residues of cavity i.e. TYR6 of D chain & PHE10 of C and D
chains of dimer of β-sheet comes in contact with phenyl rings of molecules via π-π stacking
into the hydrophobic pocket in the docked pose. The lead compound anle138b exhibits very
strong binding affinity -- the Glide score -6.84, IFD score -54.30 and the Prime MMGBSA
binding energy value -949.20 kcal/mol (Table S1). This strong binding affinity is originating
from the specific interaction involving two hydrogen bonds between pyrazole moiety of
compound anle138b with central aliphatic ILE4 of C & D chain and hydrophobic interactions
between phenyl ring of compound with the Phe10 of C and D chains of the β-sheet.
Compound 8b exhibits better binding affinity in comparison to the lead compound because of
the additional hydrogen bond interaction in the pyrazoline moiety (i.e. TYR D: 6 in interpeptide
backbone; Figure 7: A and B). Molecular docking analysis was performed using both the R
and S isomers of the considered compounds. However, the S isomer was considered in further
studies because it showed better binding affinity on a comparative scale. Further, 11
compounds (7c, 7h, 8c, 8d, 8e, 8f, 9c, 9d, 9f, 9g and 9h) can be considered as competitors to
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the lead compound. Two compounds 8h and 8l are worth considering for further modification
because these are in the border line. This conclusion is based on the cut off values Glide score
> -6.0, IFD score > -40 and Prime MMGBSA > -800 kcal/mol. The pyrazoline core is
considerably more capable at engaging with the C2 symmetric cavity, due to the scaffolds
increased flexibility, which may explain the ability of the pyrazolines to reduce ThT
fluorescence more significantly than the other compounds.
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A
B
Figure 6. Identification of the hydrophobic active site of the β-sheets of hTau protein in which the Anle138b and
synthesized compounds bind. (A) Front view and (B) side view of hydrophobic binding site. Yellow color
component in centre represents hydrophobic active site in between two β-sheet.
A
B
7 (A)
7 (B)
7 (C)
7 (D)
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Figure 7. Interactions between 8b and the active site residues of dimer of β-sheet where the interpeptide hydrogen
bonds are formed in between the ILE and TYR residues of backbone peptide chain with the pyrazoline nitrogens
therefore, inhibit the interpeptide hydrogen bond formation. (7A) Front view, (7B) side view. 9f compound
representative of synthesized pyrazole class which interacts with active hydrophobic side via the formation of
hydrogen bond with ILE D: 4 showed in Figure 7(C). Interaction of Anle138b with the binding site involves
formation of the two hydrogen bonds and showed hydrophobic interaction with PHE 10 (7 D).
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The ADME profile of the synthesized compounds was estimated using SWISS ADME profile
online server. The results are listed in Table S2 (ESI). Almost all the synthesized compounds
exhibited good bioavailability properties, satisfy the Lipinski’s rule of five. Most of them are
also CNS permeable as per the computational estimation.
The chemical properties of these species were evaluated using Chem3D Ultra 15.1 (using
ChemPropStd module and the molecular topology property module; Table S3). Clearly, the
chemical properties of 8b are larger in terms of topological diameter, accessible surface area,
polar surface area and the shape attribute in comparison to that of the lead compound anle138b.
In summary, novel pyrazoles analogues were identified which are capable of significantly
inhibiting the aggregation of α-syn characteristically seen in PD pathology. A number of the
compounds were found to possess inhibitory activity in the range of the preclinical drug
anle138b. Compound 9f was identified as a lead pyrazole while compounds 8b and 8l were
identified as lead pyrazolines species towards the inhibition of α-syn aggregation. Molecular
modelling studies employing molecular docking and including induce fit effect provided clues
regarding the inhibitory effect exhibited by the new leads suggested above. In comparison with
anle138b, one or more of these novel compounds may serve as secondary leads for further SAR
studies and development of anle138b analogues possessing desirable physicochemical
properties.
METHODS
General Methods. Proton nuclear magnetic resonance (1H NMR) spectra were recorded
using a Bruker AVANCE DPX 400 spectrometer at a frequency of 400.2 MHz. Carbon nuclear
magnetic resonance (13C NMR) spectra were recorded on a Bruker Avance DPX 400
spectrometer at a frequency of 100 MHz. The spectra are reported as parts per million (ppm)
downfield shift using the solvent peak as internal reference. The data are reported as chemical
shift (δ), multiplicity, relative integral, coupling constant (J, Hz) and assignment where
possible. For pyrazole-containing compounds 9a-9j, one drop of trifluoroacetic acid was added
to the NMR solvent to stabilise tautomerism and simplify interpretation. Low resolution mass
spectra were recorded on a Finnigan LCQ Deca ion trap spectrometer (ESI). High resolution
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mass spectra were recorded on a Bruker 7T Fourier Transform Ion Cyclotron Resonance Mass
Spectrometer (FTICR). Analytical thin layer chromatography (TLC) was performed using
commercially prepared silica plates (Merck Kieselgel 60 0.25 mm F254). Flash column
chromatography was performed using 230-400 mesh Kieselgel 60 silica eluting with analytical
grade solvents. Reagents, catalysts and solvents were purchased from Sigma-Aldrich, AK
Scientific and Chem-Supply and were used as received unless otherwise noted.
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General Procedure for Preparations of Compounds 6a-6e. To a stirred suspension of 5-
bromo-2-furaldehyde (0.50 g, 2.9 mmol) in toluene/ethanol/aq. potassium carbonate (2:1:2
v/v/v 25 mL) was added phenylboronic acid (1.1 equiv., 3.1 mmol). The reaction vessel was
then charged with [tetrakis(triphenylphosphine)]palladium(0) complex (0.01 equiv., 0.032 g,
0.029 mmol) before heating to reflux. The mixture was left stirring for 16 h before being
allowed to cool to room temperature and diluted with ethyl acetate (30 mL), washed with water
(10 mL), brine and then dried over Na2SO4. The solution was then filtered and concentrated
under reduced pressure before being purified by flash chromatography (EtOAc/hexane 1:1) to
give aryl substituted furaldehydes.
5-phenylfuran-2-carbaldehyde (6a): Yellow oil, 93% yield, 1H NMR (500 MHz CDCl3):
δ 9.65 (s, 1H, CHO), 7.84-7.81 (m, 2H, ArH), 7.47-7.38 (m, 3H, ArH), 7.32 (d, J = 3.6 Hz, 1H,
furan CH), 6.84 (d, J = 3.6 Hz, 1H, furan CH); 13C NMR (126 MHz CDCl3): δ 177.3, 159.5,
152.1, 129.7, 129.0 (3 x C), 125.3 (2 x C), 123.6 (broad), 107.7.
1
5-(3-chlorophenyl)furan-2-carbaldehyde (6b): Orange flake, 40% yield, H NMR (400
MHz CDCl3): δ 9.67 (s, 1H, CHO), 7.80 (dd, J = 1.4, 0.6 Hz, 1H, ArH), 7.69 (ddd, J = 1.8, 2.4,
6.4 Hz, 1H, ArH), 7.39-7.36 (m, 2H, 2 x ArH), 7.32 (d, J = 3.8 Hz, 1H, furan CH), 6.86 (d, J
= 3.7 Hz, 1H, furan CH). 13C NMR (101 MHz CDCl3): δ 177.4, 157.7, 152.3, 135.1, 130.6,
130.3, 129.6, 125.3, 123.3, 100.5.
1
5-(4-methoxyphenyl)furan-2-carbaldehyde (6c): Red flake, 98% yield, H NMR (400
MHz CDCl3): δ 9.60 (s, 1H, CHO), 7.76 (d, J = 8.9 Hz, 2H, 2 x ArH), 7.30 (d, J = 3.7 Hz, 1H,
furan CH), 6.97 (d, J = 8.9 Hz, 2H, 2 x ArH), 6.71 (d, J = 3.7 Hz, 1H, furan CH), 3.86 (s, 3H,
CH3); 13C NMR (101 MHz CDCl3): δ 176.9, 160.9, 159.8, 151.6, 127.0 (2 x C), 121.8, 114.8,
114.4 (2 x C), 106.3, 55.4.
5-(benzo[d][1,3]dioxol-5-yl)furan-2-carbaldehyde (6d): Yellow-orange flake, 92% yield.
1H NMR (400 MHz CDCl3): δ 9.60 (s, 1H, CHO), 7.36 (dd, J = 8.2, 1.7 Hz, ArH), 7.90 (d, J =
3.8 Hz, 1H, furan CH), 7.27 (d, J = 1.7 Hz, 1H, ArH), 6.87 (d, J = 8.2 Hz, 1H, ArH), 6.69 (d,
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J = 3.7 Hz, 1H, furan CH), 6.02 (s, 2H, CH2); 13C NMR (101 MHz CDCl3): δ 176.9, 159.4,
5
6
7
151.4, 149.1, 148.3, 123.9, 123.2, 120.1, 108.9, 106.7, 105.6, 101.6.
1
5-([1,1'-biphenyl]-4-yl)furan-2-carbaldehyde (6e): Orange solid, 62% yield, H NMR
8
9
(400 MHz CDCl3): δ 9.67 (s, 1H, CHO), 7.91 (d, J = 8.6 Hz, 2H, ArH), 7.69 (d, J = 8.6 Hz,
2H, ArH), 7.64 (d, J = 7.2 Hz, 2H, ArH), 7.47 (t, J = 7.2 Hz, 2H, ArH), 7.39 (t, J = 7.2 Hz, 1H,
ArH), 7.34 (d, J = 3.7 Hz, 1H, furan CH), 6.88 (d, J = 3.7 Hz, 1H, furan CH); 13C NMR (101
MHz CDCl3): δ 177.2, 159.3, 152.1, 142.4, 140.1, 128.9 (2 x C), 127.9 (2 x C), 127.6 (2 x C),
127.0 (2 x C), 125.8 (2 x C), 123.6 (broad), 107.8.
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General Procedure for Preparations of Compounds 7a-7j. To a stirred suspension of 5-
aryl-2-furaldehyde (0.2 g) in ethanol (2.0 mL) was added substituted acetophenone (1.1 equiv.)
and piperidine (0.03 mL, 0.03 mmol). The reaction vessel was purged with argon and then
heated with microwave irradiation (300 W of max. power) to 70 °C. The pressure limit was set
to 190 psi. Once the desired temperature was reached over 5 minutes, heating was continued
for a further 20 min to maintain temperature. The mixture was then allowed to cool to room
temperature before being concentrated under reduced pressure. The mixture was then
resuspended in ethanol/water (1:1 v/v), isolated using filtration and recrystallised from ethanol
to give highly pure chalcones.
(E)-1-(3-bromophenyl)-3-(5-phenylfuran-2-yl)prop-2-en-1-one (7a): Yellow solid, 93%
yield, 1H NMR (400 MHz CDCl3): δ 8.18 (t, J = 1.8 Hz, 1H, ArH), 7.97 (dt, J = 1.2, 7.8 Hz,
1H, ArH), 7.80-7.79 (m, 2H, ArH), (ddd, J = 0.9, 1.8, 7.8 Hz, 1H, ArH), 7.63 (d, J = 15.2 Hz,
1H, CH), 7.47-7.34 (m, 4H, ArH), 7.42 (d, J = 15.2 Hz, 1H, CH), 6.85 (d, J = 3.6 Hz, 1H, furan
CH), 6.80 (d, J = 3.6 Hz, 1H, furan CH); 13C NMR (101 MHz CDCl3): δ 188.4, 156.8, 151.0,
140.2, 135.5, 131.4, 131.2, 130.2, 129.7, 128.9, 128.8, 126.9, 124.6, 123.0, 119.6, 117.9, 108.4.
(E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(5-phenylfuran-2-yl)prop-2-en-1-one (7b): Orange
solid, 88% yield, 1H NMR (400 MHz CDCl3): δ 7.79-7.77 (m, 2H, ArH), 7.70 (dd, J = 8.2, 1.7
Hz, 1H, ArH), 7.60 (d, J = 15.2 Hz, 1H, CH), 7.57 (d, J = 1.7 Hz, 1H, ArH), 7.47 (d, J = 15.2
Hz, 1H, CH), 7.46-7.42 (m, 2H, ArH), 7.36-7.33 (m, 1H, ArH), 6.92 (d, J = 8.2 Hz, 1H, ArH),
6.80 (d, J = 3.6 Hz, 1H, furan CH), 6.78 (d, J = 3.6 Hz, 1H, furan CH), 6.07 (s, 2H, CH2); 13C
NMR (101 MHz CDCl3): δ 187.6, 156.3, 151.6, 151.2, 148.3, 133.2, 130.1, 129.9, 128.9 (2 x
C), 128.6, 124.6, 124.5 (2 x C), 118.6, 118.4, 108.4, 108.2, 108.0, 101.9; HRMS (ESI)
+
319.0959 ([M + H]+), calcd. for C20H15O4 319.0965.
(E)-1-(3-bromophenyl)-3-(5-(3-chlorophenyl)furan-2-yl)prop-2-en-1-one (7c): Yellow
1
solid. 80% yield, H NMR (400 MHz CDCl3): δ 8.18 (t, J = 1.7 Hz, 1H, ArH), 7.98 (dt, J =
7.8, 1.3 Hz, 1H, ArH), 7.76 (t, J = 1.7 Hz, 1H, ArH), 7.72 (ddd, J = 0.9, 1.9, 7.9 Hz, 1H, ArH),
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7.66 (dt, J = 7.7, 1.4 Hz, 1H, ArH), 7.62 (d, J = 15.2 Hz, 1H, CH), 7.44 (d, J = 15.2 Hz, 1H,
CH), 7.41 (t, J = 7.9 Hz, 1H, ArH), 7.38 (t, J = 7.86 Hz, 1H, ArH), 7.32 (ddd, J = 1.2, 1.9, 8.0
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Hz, 1H, ArH), 6.85 (d, J = 3.6 Hz, 1H, furan CH), 6.82 (d, J = 3.6 Hz, 1H, furan CH); C
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NMR (101 MHz CDCl3): δ 188.3 155.1, 151.4, 140.0, 135.6, 135.0, 131.4 (2 x C), 131.0, 130.2
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(2 x C), 128.6, 127.0, 124.5, 123.0, 122.6, 119.3, 118.6, 109.3.
(E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(5-(3-chlorophenyl)furan-2-yl)prop-2-en-1-one
(7d): Fluorescent yellow solid, 86% yield, 1H NMR (400 MHz CDCl3): δ 7.75 (t, J = 1.7 Hz,
1H, ArH), 7.70 (dd, J = 8.2, 1.7 Hz, 1H, ArH), 7.64 (dt, J = 7.7, 1.3 Hz, 1H, ArH), 7.58 (d, J =
15.2 Hz, 1H, CH), 7.56 (d, J = 1.7 Hz, 1H, ArH), 7.47 (d, J = 15.2 Hz, 1H, CH), 7.37 (t, J =
7.8 Hz, 1H, ArH), 7.30 (ddd, J = 1.3, 1.7, 8.0 Hz, 1H, ArH), 6.93 (d, J = 8.2 Hz, 1H, ArH),
6.80 (d, J = 3.8 Hz, 1H, furan CH), 6.79 (d, J = 3.8 Hz, 1H, furan CH), 6.07 (s, 2H, CH2); 13C
NMR (101 MHz CDCl3): δ 187.6, 154.6, 151.7 (2 x C), 148.3, 135.0, 133.0, 131.5, 130.1,
129.9, 128.4, 124.7, 124.4, 122.5, 119.0, 118.4, 109.1, 108.4, 108.0, 101.9; HRMS (ESI)
+
353.0558 ([M + H]+), calcd. for C20H14ClO4 353.0575.
(E)-1-(3-bromophenyl)-3-(5-(4-methoxyphenyl)furan-2-yl)prop-2-en-1-one
(7e):
1
Yellow solid, 95% yield, H NMR (400 MHz CDCl3): δ 8.17 (t, J = 1.7 Hz, 1H, ArH), 7.97
(dt, J = 1.3, 6.6 Hz, 1H, ArH), 7.73 (d, J = 8.9 Hz, 2H, 2 x ArH), 7.70 (ddd, J = 0.9, 1.9, 7.9
Hz, 1H, ArH), 7.61 (d, J = 15.1 Hz, 1H, CH), 7.39 (t, J = 7.9 Hz, 1H, ArH), 7.39 (d, J = 15.1
Hz, 1H, CH), 6.98 (d, J = 8.9 Hz, 2H, 2 x ArH), 6.83 (d, J = 3.6 Hz, 1H, furan CH), 6.67 (d, J
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= 3.6 Hz, 1H, furan CH), 3.87 (s, 3H, CH3); C NMR (101 MHz CDCl3): δ 188.4, 160.2,
157.1, 150.4, 140.3, 135.4, 131.4, 131.3, 130.2, 126.9, 126.2 (2 x C), 122.9, 122.7, 120.0,
117.2, 114.4 (2 x C), 107.0, 55.4.
(E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(5-(4-methoxyphenyl)furan-2-yl)prop-2-en-1-one
(7f): Orange solid, 93% yield, 1H NMR (400 MHz CDCl3): δ 7.72 (d, J = 8.9 Hz, 2H, 2 x ArH);
7.69 (dd, J = 1.8, 8.1 Hz, 1H, ArH), 7.58 (d, J = 15.3 Hz, 1H, CH), 7.56 (d, J = 2.0 Hz, 1H,
ArH), 7.43 (d, J = 15.3 Hz, 1H, CH), 6.97 (d, J = 8.9 Hz, 2H, 2 x ArH), 6.91 (d, J = 8.0 Hz,
1H, ArH), 6.78 (d, J = 3.6 Hz, 1H, furan CH), 6.65 (d, J = 3.6 Hz, 1H, furan CH), 6.07 (s, 2H,
13
CH2), 3.87 (s, 3H, CH3); C NMR (101 MHz CDCl3): δ 187.7, 160.0, 156.6, 151.6, 150.7,
148.3, 133.3, 130.2, 126.1, 124.5, 122.9, 119.0, 117.7, 114.3, 108.4, 107.9, 106.8, 101.8, 55.4;
+
HRMS (ESI) 349.1070 ([M + H]+), calcd. for C21H17O5 349.1071.
(E)-3-(5-(benzo[d][1,3]dioxol-5-yl)furan-2-yl)-1-(3-bromophenyl)prop-2-en-1-one
(7g): Yellow solid, 90% yield, 1H NMR (400 MHz CDCl3): δ 8.16 (t, J = 1.7 Hz, 1H, ArH),
7.96 (d, J = 7.8, 1H, ArH), 7.70 (ddd, J = 0.9, 1.9, 8.0 Hz, 1H, ArH), 7.60 (d, J = 15.2 Hz, 1H,
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CH), 7.39 (t, J = 7.5 Hz, 1H, ArH), 7.37 (d, J = 15.2 Hz, 1H, CH), 7.32 (dd, J = 8.1, 1.7 Hz,
1H, ArH), 7.24 (d, J = 1.6 Hz, 1H, ArH), 6.89 (d, J = 8.2 Hz, 1H, ArH), 6.82 (d, J = 3.6 Hz,
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1H, furan CH), 6.65 (d, J = 3.6 Hz, 1H, furan CH), 6.03 (s, 2H, CH2); C NMR (101 MHz
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CDCl3): δ 188.3, 156.7, 150.5, 148.3, 148.2, 140.3, 135.5, 131.4, 131.1, 130.2, 126.9, 124.1,
123.0, 119.9, 119.0, 117.4, 108.9, 107.4, 105.0, 101.5 (2 x C).
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(E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(5-(benzo[d][1,3]dioxol-5-yl)furan-2-yl)prop-2-en-
1-one (7h): Yellow solid, 92% yield, 1H NMR (400 MHz CDCl3): δ 7.69 (dd, J = 8.2, 1.7 Hz,
1H, ArH), 7.57 (d, J = 15.2 Hz, 1H, CH), 7.56 (d, J = 1.7 Hz, 1H, ArH), 7.42 (d, J = 15.2 Hz,
1H, CH), 7.30 (dd, J = 8.1, 1.7 Hz, 1H, ArH), 7.24 (d, J = 1.6 Hz, 1H, ArH), 6.91 (d, J = 8.2
Hz, 1H, ArH), 6.88 (d, J = 8.1 Hz, 1H, ArH), 6.77 (d, J = 3.5 Hz, 1H, furan CH), 6.63 (d, J =
3.6 Hz, 1H, furan CH), 6.07 (s, 2H, CH2), 6.02 (s, 2H, CH2); 13C NMR (101 MHz CDCl3): δ
187.6, 156.2, 151.6, 150.8, 148.3, 148.2, 148.1, 133.2, 130.0, 124.6, 124.3, 118.9 (2 x C),
118.0, 108.8, 108.4, 107.9, 107.2, 105.0, 101.8, 101.4; HRMS (ESI) 363.0855 ([M + H]+),
+
calcd. for C21H15O6 363.0863.
(E)-3-(5-([1,1'-biphenyl]-4-yl)furan-2-yl)-1-(3-bromophenyl)prop-2-en-1-one
(7i):
1
Yellow solid, 91% yield, H NMR (400 MHz CDCl3): δ 8.19 (t, J = 1.8 Hz, 1H, ArH), 7.97
(dt, J = 1.7, 7.9 Hz, 1H, ArH), 7.87 (d, J = 8.5 Hz, 2H, ArH), 7.71 (ddd, J = 1.0, 2.0, 7.9 Hz,
1H, ArH), 7.69 (d, J = 8.5 Hz, 2H, ArH), 7.65 (d, J = 7.6 Hz, 2H, ArH), 7.64 (d, J = 15.1 Hz,
1H, CH), 7.47 (t, J = 7.6 Hz, 2H, ArH), 7.45 (d, J = 15.1 Hz, 1H, CH), 7.40 (t, J = 7.8 Hz, 1H,
ArH), 7.38 (t, J = 7.6 Hz, 1H, ArH), 6.87 (d, J = 3.4, 1H, furan CH), 6.84 (d, J = 3.4, 1H, furan
13
CH); C NMR (101 MHz CDCl3): δ 188.4, 156.6, 151.1, 141.5, 140.3, 140.2, 135.5, 131.4,
131.2, 130.2, 128.9 (2 x C), 128.6, 127.7, 127.6 (2 x C), 127.0 (2 x C), 126.9, 125.0 (2 x C),
123.0, 119.7, 117.9, 108.6.
(E)-3-(5-([1,1'-biphenyl]-4-yl)furan-2-yl)-1-(benzo[d][1,3]dioxol-5-yl)prop-2-en-1-one
(7j): Yellow solid, 91% yield, 1H NMR (400 MHz CDCl3): δ 7.85 (d, J = 8.3 Hz, 2H, ArH),
7.71 (dd, J = 1.7, 8.1 Hz, 1H, ArH), 7.68 (d, J = 8.3 Hz, 2H, ArH), 7.67-7.59 (m, 3H, ArH and
CH) 7.57 (d, J = 1.6 Hz, 1H, ArH), 7.49 (d, J = 15.1 Hz, 1H, CH), 7.47 (t, J = 7.5 Hz, 2H,
ArH), 6.92 (d, J = 8.0 Hz, 1H, ArH), 6.82 (s, 2H, 2 x furan CH), 6.08 (s, 2H, CH2); 13C NMR
(101 MHz CDCl3): δ 187.6, 156.1, 151.6, 151.4, 148.3, 141.3, 140.3, 133.2, 130.1, 128.9 (2 x
C), 128.8, 127.7, 127.5 (2 x C), 127.4, 127.4, 127.0 (2 x C), 124.9 (2 x C), 124.6, 118.8, 118.4,
108.4 (2 x C), 101.9.
(E)-3-(5-bromofuran-2-yl)-1-(3-bromophenyl)prop-2-en-1-one (7k): To a stirred
suspension of 5-aryl-2-furaldehyde (0.20 g, 1.1 mmol) in ethanol (5 mL) was added substituted
acetophenone (1 equiv. 1.1 mmol) and aqueous sodium hydroxide (30% v/v, 5 mL) at room
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temperature. The mixture was left stirring for 16 h before being diluted with water (10 mL).
The resulting suspension was isolated by filtration, washed with aqueous ethanol (1:1 v/v) and
dried to give the title compound (0.33 g, 0.93 mmol, 81%) as an off-white solid. 1H NMR (400
MHz CDCl3): δ 8.15 (t, J = 1.7 Hz, 1H, ArH), 7.95 (d, J = 7.8 Hz, 1H, ArH), 7.70 (dd, J = 0.9,
1.7, 8.0 Hz, 1H, ArH), 7.49 (d, J = 15.2 Hz, 1H, CH), 7.38 (t, J = 7.8 Hz, 1H, ArH), 7.36 (d, J
= 15.2 Hz, 1H, CH), 6.68 (d, J = 3.4 Hz, 1H, furan CH), 6.46 (d, J = 3.4 Hz, 1H, furan CH);
13C NMR (101 MHz CDCl3): δ 188.0, 153.4, 139.8, 135.7, 131.5, 130.2, 130.0, 127.0, 126.3,
123.0, 118.8 (2 x C), 114.8.
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(E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(5-bromofuran-2-yl)prop-2-en-1-one (7l): Yellow
solid, 97% yield. 1H NMR (400 MHz CDCl3): δ 7.67 (dd, J = 1.7, 8.2 Hz, 1H, ArH), 7.53 (d,
J = 1.7 Hz, 1H, ArH), 7.46 (d, J = 15.2 Hz, 1H, CH), 7.39 (d, J = 15.2 Hz, 1H, CH), 6.90 (d, J
= 8.2 Hz, 1H, ArH), 6.63 (d, J = 3.4 Hz, 1H, furan CH), 6.44 (d, J = 3.4 Hz, 1H, furan CH),
13
6.06 (s, 2H, CH2); C NMR (101 MHz CDCl3): δ 187.4, 153.7, 151.8, 148.4, 132.8, 128.9,
125.6, 124.8, 119.4, 117.9, 114.5, 108.4, 108.0, 101.9.
General Procedure for Preparations of Compounds 8b-8l. To a suspension of chalcone
(0.10 g) in ethanol (1.5 mL) was added hydrazine hydrate (0.028 mL, 60 % in H2O). The
reaction vessel was purged with argon and then heated with microwave irradiation (300 W of
max. power) to 70 °C. The pressure limit was set at 190 psi. Once the desired temperature was
reached over 5 minutes, heating was continued for a further hour to maintain temperature. The
mixture was then allowed to cool to room temperature before being concentrated under reduced
pressure before being recrystallised from ethanol to give pure pyrazolines.
3-(benzo[d][1,3]dioxol-5-yl)-5-(5-phenylfuran-2-yl)-4,5-dihydro-1H-pyrazole
(8b):
Off-white solid, 80% yield, 1H NMR (400 MHz CDCl3): δ 7.63-7.61 (m, 2H, ArH), 7.36 (t, J
= 7.6 Hz, 2H, 2 x ArH), 7.32 (d, J = 1.5 Hz, 1H, ArH), 7.26-7.22 (m, 1H, ArH), 7.08 (dd, J =
1.7, 8.2 Hz, 1H, ArH), 6.82 (d, J = 8.0 Hz, 1H, ArH), 6.57 (d, J = 3.4, 1H, furan CH), 6.32 (d,
J = 3.4 Hz, 1H, furan CH), 5.99 (s, 2H, CH2), 5.01 (dd, J = 7.7, 10.2 Hz, 1H, CH), 3.37 (dd, J
= 10.2, 16.2 Hz, 1H, CHH), 3.27 (dd, J = 7.8, 16.2 Hz, 1H, CHH); 13C NMR (101 MHz CDCl3):
δ 154.1, 153.8, 152.4, 148.6, 148.1, 130.5, 128.7 (2 x C), 127.5, 127.0, 123.7 (2 x C), 120.8,
108.2, 108.1, 106.1, 105.6, 101.3, 57.3, 38.0; HRMS (ESI) 333.1228 ([M + H]+), calcd. for
+
C20H17N2O3 333.1234.
3-(3-bromophenyl)-5-(5-(3-chlorophenyl)furan-2-yl)-4,5-dihydro-1H-pyrazole (8c):
White powder, 70% yield, 1H NMR (400 MHz CDCl3): δ 7.85 (t, J = 1.8 Hz, 1H, Ar), 7.62 (dt,
J = 1.3, 7.8 Hz, 1H, ArH), 7.60 (t, J = 1.7 Hz, 1H, ArH), 7.50-7.48 (m, 2H, 2 x ArH), 7.29 (t,
J = 8.0 Hz, 1H, ArH), 7.27 (t, J = 7.9 Hz, 1H, ArH), 7.22 (ddd, J = 1.2, 1.9, 8.0 Hz, 1H, ArH),
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6.60 (d, J = 3.4 Hz, 1H, furan CH), 6.35 (d, J = 3.4 Hz, 1H, ArH), 5.07 (t, J = 9.2 Hz, 1H, CH),
3.41 (dd, J = 10.4, 16.2 Hz, 1H, CHH), 3.30 (dd, J = 7.9, 16.2 Hz, 1H, CHH); 13C NMR (101
MHz CDCl3): δ 154.2, 152.5, 151.3, 134.7, 134.5, 132.1, 132.0, 130.1, 130.0, 129.1, 127.4,
124.7, 123.7, 122.8, 121.8, 108.6, 106.8, 57.4, 37.6; HRMS (ESI) 401.0051 ([M + H]+), calcd.
for C19H15BrClN2O+ 401.0051.
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3-(benzo[d][1,3]dioxol-5-yl)-5-(5-(3-chlorophenyl)furan-2-yl)-4,5-dihydro-1H-
pyrazole (8d): White solid, 59 % yield, 1H NMR (400 MHz CDCl3): δ 7.61 (t, J = 1.8 Hz, 1H,
ArH), 7.49 (dt, J = 7.7, 1.3 Hz, 1H, ArH), 7.31 (d, J = 1.6 Hz, 1H, ArH), 7.88 (t, J = 7.9 Hz,
1H, ArH) 7.21 (ddd, J = 1.1, 1.9, 7.9 Hz, 1H, ArH), 7.07 (dd, J = 8.1, 1.7 Hz, 1H, ArH), 6.82
(d, J = 8.1 Hz, 1H, ArH), 6.60 (d, J = 3.4 Hz, 1H, furan CH), 6.33 (d, J = 3.3 Hz, 1H, furan
CH), 6.00 (s, 2H, CH2), 4.99 (dd, J = 10.3, 8.0 Hz, 1H, CH), 3.37 (dd, J = 16.1, 10.3 Hz, 1H,
13
CHH), 3.25 (dd, J = 16.1, 7.9 Hz, 1H, CHH); C NMR (101 MHz CDCl3): δ 154.9, 152.3,
151.9, 148.5, 148.1, 134.7, 132.2, 130.0, 127.3, 127.0, 123.7, 121.7, 120.7, 108.3, 108.1, 106.8,
+
106.1, 101.3, 57.4, 38.1; HRMS (ESI) 367.0827 ([M + H]+), calcd. for C20H16ClN2O3
367.0844.
3-(3-bromophenyl)-5-(5-(4-methoxyphenyl)furan-2-yl)-4,5-dihydro-1H-pyrazole (8e):
Off-white solid, 68% yield, 1H NMR (400 MHz CDCl3): δ 7.85 (s, 1H, ArH), 7.62 (d, J = 8.0
Hz, 1H, ArH), 7.55 (d, J = 8.9 Hz, 2H, 2 x ArH), 7.47 (d, J = 8.0 Hz, 1H, ArH), 7.26 (t, J = 8.0
Hz, 1H, ArH), 6.90 (d, J = 8.9 Hz, 2H, 2 x ArH), 6.43 (d, J = 3.4 Hz, 1H, furan CH), 6.29 (d,
J = 3.4 Hz, 1H, furan CH), 5.04 (dd, J = 7.8, 10.2 Hz, 1H, CH), 3.82 (s, 3H, CH3), 3.66 (dd, J
= 10.2, 16.2 Hz, 1H, CHH), 3.28 (dd, J = 7.7, 16.2 Hz, 1H, CHH); 13C NMR (101 MHz CDCl3):
δ 159.2, 154.0, 153.1, 150.4, 134.7, 131.7, 130.1, 129.0, 125.2 (2 x C), 124.6, 123.6, 122.8,
114.1, 108.2, 104.0, 57.5, 55.3, 37.5; HRMS (ESI) 397.0540 ([M + H]+), calcd. for
+
C20H18BrN2O2 397.0546.
3-(benzo[d][1,3]dioxol-5-yl)-5-(5-(4-methoxyphenyl)furan-2-yl)-4,5-dihydro-1H-
pyrazole (8f): Off-white solid, 62% yield, 1H NMR (400 MHz CDCl3): δ 7.55 (d, J = 8.9 Hz,
2H, 2 x ArH), 7.32 (d, J = 1.7 Hz, 1H, ArH), 7.08 (dd, J = 1.7, 8.4 Hz, 1H, ArH), 6.90 (d, J =
8.9 Hz, 2H, 2 x ArH), 6.82 (d, J = 8.0 H, 1H, ArH), 6.43 (d, J = 3.4 Hz, 1H, furan CH), 6.29
(d, J = 3.4 Hz, 1H, furan CH), 5.99 (s, 2H, CH2), 5.00 (dd, J = 7.6, 10.1 Hz, 1H, CH), 3.82 (s,
3H, CH3), 3.36 (dd, J = 10.2, 16.2 Hz, 1H, CHH), 3.27 (dd, J = 7.6, 16.2 Hz, 1H, CHH); 13C
NMR (101 MHz CDCl3): δ 159.1, 153.8, 153.5, 152.0, 148.5, 148.1, 127.1, 125.2 (2 x C),
123.7, 120.7, 114.1 (2 x C), 108.1, 108.0, 106.1, 104.0, 101.3, 57.3, 55.3, 38.0; HRMS (ESI)
363.1324 ([M + H]+), calcd. for C21H19N2O4 363.1339.
+
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3-(benzo[d][1,3]dioxol-5-yl)-5-(5-(benzo[d][1,3]dioxol-5-yl)furan-2-yl)-4,5-dihydro-
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1H-pyrazole (8h): Yellow solid, 70% yield, H NMR (400 MHz CDCl3): δ 7.31 (d, J = 1.7
Hz, 1H, ArH), 7.13 (dd, J = 1.7, 8.2 Hz, 1H, ArH), 7.08 (d, J = 1.7 Hz, 1H, ArH), 7.07 (dd, J
= 1.7, 8.2 Hz, 1H, ArH), 6.82 (d, J = 8.2 Hz, 1H, ArH), 6.80 (d, J = 8.2 Hz, 1H, ArH), 6.41 (d,
J = 3.3 Hz, 1H, furan CH), 6.29 (d, J = 3.3 Hz, 1H, furan CH), 5.99 (s, 2H, CH2), 5.97 (s, 2H,
CH2), 4.99 (dd, J = 7.8, 10.1 Hz, 1H, CH), 3.35 (dd, J = 10.2, 16.2 Hz, 1H, CHH), 3.26 (dd, J
= 7.9, 16.2 Hz, 1H, CHH); 13C NMR (101 MHz CDCl3): δ 153.7, 153.3, 152.7, 148.6, 148.1,
148.0, 147.1, 126.9, 125.0, 120.8, 117.7, 108.6, 108.3, 108.1, 106.2, 104.5 (2 x C), 101.3,
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+
101.1, 57.2, 38.0; HRMS (ESI) 377.1109 ([M + H]+), calcd. for C21H17N2O5 377.1132.
5-(5-([1,1'-biphenyl]-4-yl)furan-2-yl)-3-(3-bromophenyl)-4,5-dihydro-1H-pyrazole
(8i): Off-white solid, 87% yield, 1H NMR (400 MHz CDCl3): δ 7.86 (t , J = 1.7 Hz, 1H, ArH),
7.69 (d, J = 7.9 Hz, 2H, 2 x ArH), 7.64-7.60 (m, 5H, ArH), 7.48 (ddd, J = 1.2, 1.6, 8.0 Hz, 1H,
ArH), 7.46-7.43 (m, 2H, 2 x ArH), 7.35 (t, J = 7.43 Hz, 1H, ArH), 7.27 (t, J = 7.9 Hz, 1H,
ArH), 6.61 (d, J = 3.4 Hz, 1H, furan CH), 6.35 (d, J = 3.4 Hz, 1H furan CH), 5.08 (dd, J = 7.5,
10.1 Hz, 1H, CH), 3.40 (dd, J = 10.3, 16.2 Hz, 1H, CHH), 3.32 (dd, J = 7.6, 16.2 Hz, 1H,
CHH); 13C NMR (101 MHz CDCl3): δ 153.9, 153.7, 150.8, 140.5, 140.2, 134.6, 131.9, 130.1,
129.4, 129.1, 128.8 (2 x C), 127.4 (3 x C), 126.9 (2 x C), 124.6, 124.1 (2 x C), 122.8, 108.5,
105.8, 57.5, 37.6; HRMS (ESI) 443.0733 ([M + H]+), calcd. for C25H20BrN2O+ 443.0754.
5-(5-([1,1'-biphenyl]-4-yl)furan-2-yl)-3-(benzo[d][1,3]dioxol-5-yl)-4,5-dihydro-1H-
pyrazole (8j): Off-white solid, 78% yield, 1H NMR (400 MHz CDCl3): δ 7.70-7.69 (m, 2H, 2
x ArH), 7.62-7.64 (m, 4H, 4 x ArH), 7.45 (t, J = 7.5 Hz, 2H, 2 x ArH), 7.36 (d, J = 7.5 Hz, 1H,
ArH), 7.33 (d, J = 1.6 Hz, 1H, ArH), 7.09 (dd, J = 1.4, 7.9 Hz, 1H, ArH), 6.83 (d, J = 8.1 Hz,
1H, ArH), 6.61 (d, J = 3.4 Hz, 1H, furan CH), 6.34 (d, J = 3.4 Hz, 1H, furan CH), 6.00 (s, 2H,
CH2), 5.02 (dd, J = 7.8, 10.2 Hz, 1H, CH), 3.38 (dd, J = 10.2, 16.1 Hz, 1H, CHH), 3.29 (dd, J
= 7.7, 16.1 Hz, 1H, CHH); 13C NMR (101 MHz CDCl3): δ 154.3, 153.6, 152.2, 148.5, 148.1,
140.5, 140.1, 129.5, 128.8 (2 x C), 127.4, 127.3 (2 x C), 127.0, 126.9 (2 x C), 124.1 (2 x C),
120.7, 108.3, 108.1, 106.1, 105.8, 101.3, 57.4, 38.0; HRMS (ESI) 409.1539 ([M + H]+), calcd.
+
for C26H21N2O3 409.1547.
5-(5-bromofuran-2-yl)-3-(3-bromophenyl)-4,5-dihydro-1H-pyrazole (8k): Off-white
solid, 70% yield, 1H NMR (400 MHz CDCl3): δ 7.81 (t, J = 1.9 Hz, 1H, ArH), 7.59 (d, J = 7.8
Hz, 1H, ArH), 7.47 (d, J = 8.0 Hz, 1H, ArH), 7.25 (t, J = 7.8 Hz, 1H, ArH), 6.24 (s, 2H, 2 x
furan CH), 4.96 (dd, J = 7.8, 15.8 Hz, 1H, CH), 3.35 (dd, J = 10.4, 16.2 Hz, CHH), 3.20 (dd, J
= 7.6, 16.2 Hz, 1H, CHH); 13C NMR (101 MHz CDCl3): δ 134.8, 133.1, 131.0, 130.7, 129.6,
126.0, 125.2, 123.4, 114.1, 112.8, 100.2, 54.4, 38.1 (total 2 atoms not observed).
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3-(benzo[d][1,3]dioxol-5-yl)-5-(5-bromofuran-2-yl)-4,5-dihydro-1H-pyrazole (8l): Off-
white solid, 71% yield, 1H NMR (400 MHz CDCl3): δ 7.29 (d, J = 1.6 Hz, 1H, ArH), 7.05 (dd,
J = 1.7, 8.1 Hz, 1H, ArH), 6.81 (d, J = 8.1 Hz, 1H, ArH), 6.25 (d, J = 3.4 Hz, 1H, furan CH),
6.23 (d, J = 3.4 Hz, 1H, furan CH), 5.99 (s, 2H, CH2), 4.93 (dd, J = 7.8, 10.3 Hz, 1H, CH), 3.33
(dd, J = 10.3, 16.3 Hz, 1H, CHH), 3.19 (dd, J = 7.8, 16.3 Hz, 1H, CHH); 13C NMR (101 MHz
CDCl3): δ 156.2, 153.3, 148.9, 148.2, 126.7, 121.8, 121.1, 112.2, 109.3, 108.3, 106.3, 101.5,
57.1, 38.1.
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General Procedure for Preparations of Compounds 9a-9j. To a suspension of chalcone
(0.100 g) and elemental sulfur (0.022 g, 3 eq.) in ethanol (1.00 mL) was added hydrazine
hydrate (0.046 mL, 60 % in H2O). The reaction vessel was purged with argon and then heated
with microwave irradiation (300 W of max. power) to 150 °C. The pressure limit was set at
190 psi. Once the desired temperature was reached over 5 minutes, heating was continued for
a further two hours to maintain temperature. The mixture was then allowed to cool to room
temperature before being concentrated under reduced pressure, redissolved in ethyl acetate and
filtered to remove solid sulfur powder. The filtrate was then concentrated before being purified
by flash chromatography.
3-(3-bromophenyl)-5-(5-phenylfuran-2-yl)-1H-pyrazole (9a): Off-white solid, 22%
yield, 1H NMR (400 MHz DMSO-d6): δ 8.08 (t, J = 1.8 Hz, 1H, ArH), 7.88 (dt, J = 1.3, 7.8
Hz, 1H, ArH), 7.85-7.83 (m, 2H, ArH), 7.54 (ddd, J = 0.9, 1.9, 8.0 Hz, 1H, ArH), 7.48-7.44
(m, 2H, ArH), 7.42 (t, J = 7.8 Hz, 1H, ArH), 7.34-7.30 (m, 1H, ArH), 7.23 (s, 1H, CH), 7.04
(d, J = 3.5 Hz, 1H, furan CH), 6.91 (d, J = 3.5 Hz, 1H, furan CH); 13C NMR (101 MHz DMSO-
d6): δ 158.5, 152.9, 131.5, 131.0, 130.4, 129.4, 128.1, 124.6, 124.0, 122.8, 109.0, 108.2, 100.3
(3 carbon atoms not observed); HRMS (ESI) 365.0282 ([M + H]+), calcd. for C19H14BrN2O+
365.0284.
3-(benzo[d][1,3]dioxol-5-yl)-5-(5-phenylfuran-2-yl)-1H-pyrazole (9b): Off-white solid,
19% yield, 1H NMR (400 MHz DMSO-d6): δ 7.82 (d, J = 7.8 Hz, 2H, ArH), 7.46 (t, J = 7.8
Hz, 2H, ArH), 7.41 (d, J = 1.6 Hz, 1H, ArH), 7.36 (dd, J = 1.6, 8.0 Hz, 1H, ArH), 7.32 (t, J =
7.4 Hz, 1H, ArH), 7.07 (d, J = 3.6 Hz, 1H, furan CH), 7.03 (s, 1H, CH), 7.00 (d, J = 7.9 Hz,
1H, ArH), 6.86 (d, J = 3.6 Hz, 1H, furan CH), 6.07 (s, 2H, CH2); 13C NMR (101 MHz DMSO-
d6): δ 152.7, 148.3, 147.5, 130.5, 129.4 (2 x C), 128.0, 123.9 (2 x C), 119.5, 109.1, 108.6,
+
108.2, 106.2, 101.7, 99.4; HRMS (ESI) 331.1060 ([M + H]+), calcd. for C20H15N2O3 331.1077.
3-(3-bromophenyl)-5-(5-(3-chlorophenyl)furan-2-yl)-1H-pyrazole (9c): Off-white solid,
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15% yield, H NMR (400 MHz DMSO-d6): δ 8.07 (t, J = 1.7 Hz, 1H, ArH), 7.92 (t, J = 1.7
Hz, 1H, ArH), 7.88 (dt, J = 1.2, 7.7 Hz, 1H, ArH), 7.80 (dt, J = 1.3, 7.8 Hz, 1H, ArH), 7.55
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(ddd, J = 1.0, 1.9, 8.0 Hz, 1H, ArH), 7.48 (t, J = 7.9 Hz, 1H, ArH), 7.42 (t, J = 7.9 Hz, 1H,
ArH), 7.37 (ddd, J = 1.0, 2.0, 8.0 Hz, 1H, ArH), 7.27 (s, 1H, CH), 7.22 (d, J = 3.6 Hz, 1H, furan
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CH), 6.94 (d, J = 3.6 Hz, 1H, furan CH); C NMR (101 MHz DMSO-d6): δ 150.9, 133.9,
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131.9, 131.1, 130.9, 130.6, 127.7, 127.3, 124.2, 123.0, 122.3, 122.1, 109.3, 108.7, 100.1 (total
4 carbon atoms not observed); HRMS (ESI) 398.9886 ([M + H]+), calcd. for C19H13BrClN2O+
398.9894.
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3-(benzo[d][1,3]dioxol-5-yl)-5-(5-(3-chlorophenyl)furan-2-yl)-1H-pyrazole (9d): Off-
white solid, 31% yield, 1H NMR (400 MHz DMSO-d6): δ 7.88 (t, J = 1.7 Hz, 1H, ArH), 7.77
(dt, J = 1.2, 8.0 Hz, 1H, ArH), 7.47 (t, J = 7.9 Hz, 1H, ArH), 7.41 (d, J = 1.5 Hz, 1H, ArH),
7.37-7.34 (m, 2H, 2 x ArH), 7.19 (d, J = 3.5 Hz, 1H, furan CH), 7.07 (s, 1H, CH), 7.00 (d, J =
8.2 Hz, 1H, ArH), 6.89 (d, J = 3.5 Hz, 1H, furan CH), 6.06 (s, 2H, CH2); 13C NMR (101 MHz
DMSO-d6): δ 150.6, 147.8, 147.1, 133.9, 132.0, 130.9, 127.2, 122.9, 122.0, 119.1, 109.3,
108.7, 108.3, 105.7, 101.2, 99.2 (total 4 carbon atoms not observed); HRMS (ESI) 365.0685
+
([M + H]+), calcd. for C20H14ClN2O3 365.0688.
3-(3-bromophenyl)-5-(5-(4-methoxyphenyl)furan-2-yl)-1H-pyrazole (9e): Off-white
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solid, 21% yield, H NMR (400 MHz DMSO-d6): δ 8.06 (t, J = 1.7, 1H, ArH), 7.87 (dt, J =
0.9, 7.8 Hz, 1H, ArH), 7.76 (d, J = 8.8 Hz, 2H, 2 x ArH), 7.54 (ddd, J = 0.9, 1.8, 8.0 Hz, 1H,
ArH), 7.42 (t, J = 7.9 Hz, 1H, ArH), 7.18 (s, 1H, CH), 7.03 (d, J = 8.9 Hz, 2H, 2 x ArH), 6.92
(d, J = 3.4 Hz, 1H, furan CH), 6.87 (d, J = 3.4 Hz, 1H, furan CH), 3.80 (s, 3H, CH3); 13C NMR
(101 MHz DMSO-d6): δ 159.0, 158.6, 131.1, 130.6, 127.7, 125.2 (2 x C), 124.2, 122.9, 114.5
(2 x C), 108.6, 106.0, 99.6, 55.3 (total 5 carbon atoms not observed); HRMS (ESI) 395.0373
+
([M + H]+), calcd. for C20H16BrN2O2 395.0390.
3-(benzo[d][1,3]dioxol-5-yl)-5-(5-(4-methoxyphenyl)furan-2-yl)-1H-pyrazole (9f): Off-
white solid, 13% yield, 1H NMR (400 MHz DMSO-d6): δ 7.74 (d, J = 8.9 Hz, 2H, 2 x ArH)
4.41 (d, J = 1.6 Hz, 1H, ArH), 7.36 (dd, J = 1.6, 7.9 Hz, 1H, ArH), 7.03 (d, J = 8.9 Hz, 2H, 2
x ArH), 7.00 (d, J = 7.9 Hz, 1H, ArH), 6.99 (s, 1H, CH), 6.90 (d, J = 3.5 Hz, 1H, furan CH),
6.82 (d, J = 3.5 Hz, 1H, furan CH), 6.06 (s, 2H, CH2), 3.80 (s, 3H, CH3); 13C NMR (101 MHz
DMSO-d6): δ 159.3, 152.9, 148.2, 147.5, 125.5 (2 x C), 123.4, 119.4, 114.8 (2 x C), 109.1,
108.6, 106.4, 106.1, 101.6, 99.2, 55.7 (total 4 carbon atoms not observed); HRMS (ESI)
+
361.1160 ([M + H]+), calcd. for C21H17N2O4 361.1182.
5-(5-(benzo[d][1,3]dioxol-5-yl)furan-2-yl)-3-(3-bromophenyl)-1H-pyrazole (9g): Off-
white solid, 29% yield, 1H NMR (400 MHz DMSO-d6 + TFA): δ 8.05 (s, 1H, ArH), 7.86 (d,
J = 7.5 Hz, 1H, ArH), 7.53 (d, J = 7.8 Hz, 1H, ArH), 7.42-7.39 (m, 2H, 2 x ArH), 7.35 (d, J =
7.5 Hz, 1H, ArH), 7.19 (s, 1H, CH), 7.01 (d, J = 7.8 Hz, 1H, ArH), 6.95 (d, J = 3.1 Hz, 1H,
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furan CH), 6.86 (d, J = 3.1 Hz, 1H, furan CH), 6.07 (s, 2H, CH2); 13C NMR (101 MHz DMSO-
d6): δ 152.5, 148.0, 147.0, 134.1, 131.1, 130.6, 127.7, 124.4, 124.2, 122.4, 117.7, 108.9, 108.7,
106.7, 104.3, 101.4, 99.7; HRMS (ESI) 409.0169 ([M + H]+), calcd. for C20H14BrN2O3
+
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409.0182.
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3-(benzo[d][1,3]dioxol-5-yl)-5-(5-(benzo[d][1,3]dioxol-5-yl)furan-2-yl)-1H-pyrazole
(9h): Off-white solid, 13% yield, 1H NMR (400 MHz DMSO-d6): δ 7.40 (d, J = 1.6 Hz, 2H, 2
x ArH), 7.35 (dd, J = 1.6, 8.0 Hz, 1H, ArH), 7.33 (dd, J = 1.6, 8.0 Hz, 1H, ArH), 7.02 (s, 1H,
CH), 7.01 (d, J = 8.0 Hz, 1H, ArH), 6.99 (d, J = 8.0 Hz, 1H, ArH), 6.94 (d, J = 3.5 Hz, 1H,
furan CH), 6.82 (d, J = 3.5 Hz, 1H, furan CH), 6.07 (s, 2H, CH2), 6.06 (s, 2H, CH2); 13C NMR
(101 MHz DMSO-d6): δ 148.4, 148.2, 147.5, 124.9, 119.4, 117.9, 109.3, 109.1, 108.5, 107.0,
106.1, 104.6, 101.7, 101.6, 99.3 (total 5 carbon atoms not observed); HRMS (ESI) 375.0976
+
([M + H]+), calcd. for C21H15N2O5 375.0976.
5-(5-([1,1'-biphenyl]-4-yl)furan-2-yl)-3-(3-bromophenyl)-1H-pyrazole (9i): Off-white
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solid, 42% yield, H NMR (400 MHz DMSO-d6): δ 13.57 (bs, 1H, NH), 8.09 (s, 1H, ArH),
7.93 (d, J = 8.4 Hz, 2H, 2 x ArH), 7.89 (d, J = 7.8 Hz, 1H, ArH), 7.79-7.73 (m, 4H, 4 x ArH),
7.55 (d, J = 7.8 Hz, 1H, ArH), 7.49 (t, J = 7.5 Hz, 2H, 2 x ArH), 7.47 (t, J = 7.8 Hz, 1H, ArH),
7.38 (t, J = 7.5 Hz, 1H, ArH) 7.25 (s, 1H, CH), 7.15 (d, J = 3.5 Hz, 1H, furan CH), 6.94 (d, J
= 3.5 Hz, 1H, furan CH); 13C NMR (101 MHz DMSO-d6): δ 152.7, 139.9, 139.6, 131.5, 131.0,
129.5 (2 x C), 128.1 (2 x C), 127.6 (2 xC), 127.0 (2 x C), 124.6 (3 x C), 124.5, 122.8, 108.5,
100.4 (total 6 carbon atoms not observed); HRMS (ESI) 441.0592 ([M + H]+), calcd. for
+
C26H19N2O3 441.0597.
5-(5-([1,1'-biphenyl]-4-yl)furan-2-yl)-3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole (9j):
Off-white solid, 29% yield, 1H NMR (400 MHz DMSO-d6): δ 7.91 (d, J = 8.4 Hz, 1H, ArH),
7.78 (m, 4H, 4 x ArH), 7.49 (t, J = 7.8 Hz, 2H, 2 x ArH), 7.42 (d, J = 1.66, 1H, ArH), 7.38 (tt,
J = 1.2, 7.3 Hz, 1H, ArH), 7.37 (dd, J = 1.7, 7.9 Hz, 1H, ArH), 7.14 (d, J = 3.5 Hz, 1H, furan
CH), 7.06 (s, 1H, CH), 7.01 (d, J = 7.9 Hz, 1H, ArH), 6.89 (d, J = 3.5 Hz, 1H, furan CH), 6.07
13
(s, 2H, CH2); C NMR (101 MHz DMSO-d6): δ 152.4, 148.3, 147.5, 139.9, 139.5, 129.6,
129.5 (2 x C), 128.1, 127.6 (2 x C), 127.0 (2 x C), 124.5 (2 x C), 119.5, 109.1, 108.5, 106.2,
101.7, 99.4 (total 5 carbon atoms not observed); HRMS (ESI) 407.1379 ([M + H]+), calcd. for
+
C26H19N2O3 407.1390.
3-(benzo[d][1,3]dioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole (1): Yellow powder, 65%
yield, 1H NMR (400 MHz DMSO-d6): δ 8.01 (t, J = 1.7 Hz, 1H, ArH), 7.82 (dt, J = 1.3, 7.8
Hz, 1H, ArH), 7.51 (ddd, J = 0.9, 1.9, 7.9 Hz, 1H, ArH), 7.40 (t, J = 7.9 Hz, 1H, ArH), 7.38 (d,
J = 1.6 Hz, 1H, ArH), 7.33 (dd, J = 1.7, 8.1 Hz, 1H, ArH), 7.20 (s, 1H, ArH), 7.01 (d, J = 8.1
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Hz, 1H, ArH), 6.06 (s, 2H, CH2); C NMR (101 MHz DMSO-d6): δ 158.6, 158.2, 147.9,
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147.1, 134.5, 131.0, 130.4, 127.5, 124.0, 122.3, 119.0, 116.7, 108.8, 105.6, 101.3, 99.9.
In Vitro Biological Evaluation. Expression and Purification of α-Synuclein. Escherichia
coli BL21 (DE3) cells containing the human α-syn gene inserted into a pET-22b (+) vector
(Novagen, Merck, MA, USA) were induced with 1 mM isopropyl β-D-1-
thiogalactopyranoside. α-syn was first purified following a non-chromatographic protocol
reported by Volles and Lansbury.36 Further purification using anion exchange followed
protocols reported by Ventura and co-workers.37
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In Vitro Inhibition of α-Synuclein Aggregation. A stock solution of ThT (5 mM) in glycine-
NaOH buffer (pH 8.0) was prepared and used with homogenous monomeric α-syn, made using
commonly employed protocols described by Rahimi et al.38 Inhibition of α-syn aggregation
was performed by incubation of a mixture of α-syn monomers (80 µM) suspended in
aggregation buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.05% NaN3) with or without
candidate inhibitor compounds (80 µM and 400 µM) at 37°C with constant shaking (1000 rpm
in a thermomixer) for 48 h. For each treatment group, pre-incubated α-syn samples (60 µL)
were mixed with a ThT solution (240 µL, 50 µM) for 10 min, the solution was dispensed in
triplicate (n=3) into 96-well plates which were analysed using a fluorimeter (ex. 440 nm/em.
500 nm). The fluorescence intensity of samples were compared to an untreated negative control
and a positive control treated with epigallocatechin gallate (EGCG, 80 µM and 400 µM) to
obtain percentage inhibition values.
Dot blot assay: Briefly, samples (2 µL) were immobilized on nitrocellulose membrane
(Amersham Protran, GE Healthcare) and were dried at rt. The membrane was blocked at rt for
1 h using 3% bovine serum albumin (BSA) and then was washed with Tris-buffered saline
(TBST, 0.05% Tween 20, 50 mM Tris–HCl, 150 mM NaCl, pH 7.5). The membranes were
incubated overnight at 4 °C with anti-α-Synuclein antibody (1:2000, BD Biosciences)39
and anti-α-Synuclein filament antibody (1:8,000, Abcam).40 The membranes were washed with
TBST and then incubated with secondary anti-mouse and anti-rabbit IgG (1:20,000, Thermo
Scientific) at rt for 2 h. The blots were incubated further with ECL reagent (Millipore) for 5
min, before being developed.
Molecular modelling: Molecular docking analysis was carried out using Schrodinger software
package41 with (and without) Induce fit docking option in Glide module. Geometry
optimization of initial 3D structures of all the compounds was performed using OPLS3 force
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field.42 The structures of designed compounds were prepared using LigPrep module of Maestro
software. To prepare the macromolecular environment, the protein preparation wizard of
maestro software was employed. The coordinates for dimer of β-sheet were taken from the
hTau homodimer protein identified by PDB Id 4E0M, which consists of four chains A, B, C
and D respectively, in which A, B and C, D were arranged in antiparallel manner via hydrogen
bonds to form aggregates to each other. Water molecules, inorganic phosphate, chain A and
chain B of β-sheet were deleted. The amino acids present in the dimer are GLN, GLU9, ILE4,
LYS13, PHE10, SER1, TYR6 and VAL5 respectively between C and D chains.
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SiteMap43 module in association with Glide software was used to identify the cavity employed
in performing molecular docking. The grid for molecular docking was generated with outer
and inner box of 26 Å and 10 Å respectively using auto option in Induced Fit Docking (IFD)
module of Maestro software. The receptor van der Waals and ligand van der Waals scaling
were kept to 0.50 and for selected 20 poses. Refinement of conformation of the residues during
IFD was done on residues within 5.0 Å. SP precision mode was used for IFD purpose. Prime
MMGBSA option within application tools of Maestro package was used to estimate the binding
free energies between the docked compounds and macromolecular environment which
indicated no conformational interference. The validation of the protocol was performed by re-
docking anle138b. The standard comparison of anle138b and our compounds were drawn by
comparing their G score values and IFD scores.
Statistical Analysis: All data were analyzed by GraphPad Prism (GraphPad Software) using
one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison as the post hoc
test.
ASSOCIATED CONTENT
Supporting Information:
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/
The copy of scanned NMR (1H and 13C) spectra and molecular modelling data.
Corresponding Author
* s.rudrawar@griffith.edu.au
* g.mellick@griffith.edu.au
Author Contributions
S.R. and P.R. conceived and designed the research, P.R. and M.X., performed experimental
work and contributed to data analysis and interpretation. K.J. is involved in the molecular
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modelling work. All authors contributed in writing, reviewing and the revision of the
manuscript.
Funding
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Financial support to S.R. from an Australian Research Council - Discovery Early Career
Research Award (ARC DECRA: DE140101632) is gratefully acknowledged. P.R. received
Griffith University Postgraduate Research Scholarship (GUPRS). This work is supported by
Menzies Health Institute Queensland (MHIQ), Griffith University Collaborative
Interdisciplinary Grants.
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Acknowledgements
P.R. is supported by Griffith University Postgraduate Research Scholarship.
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