Design, synthesis, and biological evaluation of furosemide analogs as therapeutics for the proteopathy and immunopathy of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2021.113565

β-Amyloid (Aβ) triggered proteopathic and immunopathic processes are a postulated cause of Alzheimer's disease (AD). Monomeric Aβ is derived from amyloid precursor protein, whereupon it aggregates into various assemblies, including oligomers and fibrils, which disrupt neuronal membrane integrity and induce cellular damage. Aβ is directly neurotoxic/synaptotoxic, but may also induce neuroinflammation through the concomitant activation of microglia. Previously, we have shown that furosemide is a known anthranilate-based drug with the capacity to downregulate the proinflammatory microglial M1 phenotype and upregulate the anti-inflammatory M2 phenotype. To further explore the pharmacologic effects of furosemide, this study reports a series of furosemide analogs that target both Aβ aggregation and neuroinflammation, thereby addressing the combined proteopathic-immunopathic pathogenesis of AD. Forty compounds were synthesized and evaluated. Compounds 3c, 3g, and 20 inhibited Aβ oligomerization; 33 and 34 inhibited Aβ fibrillization. 3g and 34 inhibited the production of TNF-α, IL-6, and nitric oxide, downregulated the expression of COX-2 and iNOS, and promoted microglial phagocytotic activity, suggesting dual activity against Aβ aggregation and neuroinflammation. Our data demonstrate the potential therapeutic utility of the furosemide-like anthranilate platform in the development of drug-like molecules targeting both the proteopathy and immunopathy of AD.

Full text of DOI:10.1016/j.ejmech.2021.113565

European Journal of Medicinal Chemistry 222 (2021) 113565
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis, and biological evaluation of furosemide analogs as
therapeutics for the proteopathy and immunopathy of Alzheimer's
disease
,
Zhiyu Wang a b, Yanfei Wang a, Jagadeesh Prasad Pasangulapati a, Kurt R. Stover a,
, b, c, d, *
Xiaojing Liu a, Stephanie (Wohnig) Schier a, Donald F. Weaver a
a Krembil Research Institute, University Health Network, Toronto, Canada
b Faculty of Pharmacy, University of Toronto, Ontario, Canada
c Faculty of Medicine, University of Toronto, Ontario, Canada
d Department of Chemistry, University of Toronto, Ontario, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
b
-Amyloid (A
heimer's disease (AD). Monomeric A
gates into various assemblies, including oligomers and fibrils, which disrupt neuronal membrane
integrity and induce cellular damage. A is directly neurotoxic/synaptotoxic, but may also induce neu-
b
) triggered proteopathic and immunopathic processes are a postulated cause of Alz-
Received 26 February 2021
Received in revised form
18 April 2021
Accepted 12 May 2021
Available online 2 June 2021
b
is derived from amyloid precursor protein, whereupon it aggre-
b
roinflammation through the concomitant activation of microglia. Previously, we have shown that furo-
semide is a known anthranilate-based drug with the capacity to downregulate the proinflammatory
microglial M1 phenotype and upregulate the anti-inflammatory M2 phenotype. To further explore the
pharmacologic effects of furosemide, this study reports a series of furosemide analogs that target both A
aggregation and neuroinflammation, thereby addressing the combined proteopathic-immunopathic
pathogenesis of AD. Forty compounds were synthesized and evaluated. Compounds 3c, 3g, and 20
Keywords:
Furosemide
Alzheimer's disease
b
b
-Amyloid
inhibited A
TNF- , IL-6, and nitric oxide, downregulated the expression of COX-2 and iNOS, and promoted microglial
phagocytotic activity, suggesting dual activity against A aggregation and neuroinflammation. Our data
b oligomerization; 33 and 34 inhibited Ab fibrillization. 3g and 34 inhibited the production of
Protein misfolding
Neuroinflammation
3-Hydroxyanthranilate
a
b
demonstrate the potential therapeutic utility of the furosemide-like anthranilate platform in the
development of drug-like molecules targeting both the proteopathy and immunopathy of AD.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
A
b
impairs cellular function through various mechanisms. It
disrupts cell membrane integrity, destabilizing homeostasis [4]. It
also engages 7-nicotinic acetylcholine receptors inducing the
production of astrocytic glutamate [5]. This action activates extra-
synaptic neuronal N-methyl- -aspartate (NMDA) receptors, fol-
Alzheimer's disease (AD) is the most common type of dementia.
a
b
-Amyloid (A
pathology [1]. A
by -secretase and
monomer, whereupon it aggregates into various assemblies
including oligomers and fibrils [2]. A oligomers are soluble and
b
) deposition is regarded as a central event in AD
is cleaved from amyloid precursor protein (APP)
-secretase sequentially, and secreted as a
b
D
b
g
lowed rapidly by a decrease in mEPSC (miniature excitatory
postsynaptic current) frequency with accompanying generation of
nitric oxide (NO), hyperphosphorylation of tau, and activation of
b
diffusible; amyloid fibrils are larger and insoluble [2]. The fibrils
further aggregate into amyloid plaques which deposit extracellu-
larly within the central nervous system (CNS) [3].
caspase-3 [5]. In addition, soluble Ab oligomers can be internalized
by endocytic vesicles and interact with various organelles, inducing
endoplasmic reticulum (ER) stress, mitochondria damage, and
endosomal and lysosomal membrane leakage [6].
In addition to its direct neuronal toxicity, Ab also induces neu-
roinflammation through the activation of immune cells. Normally,
neuroinflammation is a complex set of immune responses initiated
by infections, traumatic injury, or toxic substances [7]. As the
* Corresponding author. Krembil Discovery Tower, 60 Leonard Ave. 4KD-477,
Toronto, ON, M5T 0S8, Canada.
E-mail address: Donald.Weaver@uhnresearch.ca (D.F. Weaver).
https://doi.org/10.1016/j.ejmech.2021.113565
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
Abbreviation
HBA
HBD
IL-
iNOS
LPS
hydrogen bond acceptor
hydrogen bond donor
interleukin-
inducible NO synthase
lipopolysaccharides
3-HAA
3-hydroxylanthranilic acid
-amyloid
A
b
b
AD
Alzheimer's disease
APP
BBB
amyloid precursor protein
blood brain barrier
mEPSC
NIS
miniature excitatory postsynaptic currents
N-iodosuccinimide;
CD
CDI
CNS
cluster of differentiation
1,10-carbonyldiimidazole
central nervous system
cyclooxygenase-2
Dulbecco's modified eagle medium
dess martin periodinane
dimethyl sulfoxide;
Dulbecco's phosphate-buffered saline;
Escherichia coli
enzyme-linked immunosorbent assay
endoplasmic reticulum
fetal bovine serum
NMDA
NO
NSAIDs
PK
ROS
SAR
SD
SIeV
SR
N-methyl-D-aspartate
nitric oxide;
nonsteroidal anti-inflammatory drugs
pharmacokinetic
reactive oxygen species
structure-activity relationship;
standard deviation
series IeV
scavenger receptor
trifluoroacetic acid
tetramethylbenzidine;
tumor necrosis factor-alpha
COX-2
DMEM
DMP
DMSO
DPBS
E. coli
ELISA
ER
TFA
TMB
TNF-a
FBS
FITC
fluorescein isothiocyanate
brain's resident immune cells, microglia respond to such stimuli
and are activated to mediate neuroinflammation [8]. In AD,
microglia surround plaques and phagocytotically degrade the
endogenous tryptophan metabolite, was a bifunctional inhibitor
against A aggregation and neuroinflammation [22,23]. Seeking
b
known drugs with an anthranilate backbone in common with 3-
HAA, we selected furosemide (4-chloro-N-furfuryl-5 sulfamoylan-
thranilic acid, shown in Fig. 1 (B)) as a potential platform for initial
study. Furosemide is a well-described loop diuretic used to treat
edema [24]. As a structural analog of 3-HAA, furosemide provides a
drug-like platform suitable for chemical modification to achieve an
misfolded-A
b [9]. The soluble peptides are transported into the
lysosomes via macroautophagy, chaperone-mediated autophagy,
and endocytosis [9]. By the secretion of growth factors and che-
mokines, they also promote the proliferation and recruitment of
neural precursor cells to the site of injury [10]. However, accumu-
lated A
b
peptide chronically activates microglia to proinflammatory
agent targeting Ab oligomerization and fibrillization; in addition,
and less phagocytic phenotypes, which secrete reactive oxygen
species (ROS) and proinflammatory cytokines, including tumor
we have previously shown that furosemide downregulates the
microglial “M1” proinflammatory phenotype and upregulate the
“M2” anti-inflammatory phenotype [25]. To further explore the
potentially beneficial pharmacologic effects of furosemide, herein
necrosis factor-alpha (TNF-
Accordingly, AD may be conceptualized as a combined proteo-
pathic and immunopathic disease. Drug design targeting A
mediated AD progression is challenging. A aggregation is a
complicated process e the A monomers rapidly form oligomers
that subsequently aggregate into fibrils, causing a complexity of A
a), interleukins (IL)-1b, and IL-6 [11].
b
-
we report that furosemide analogs inhibit both Ab aggregation and
b
lipopolysaccharide (LPS)-induced proinflammatory immune re-
sponses, thereby affording a drug-like platform with novel com-
bined anti-proteopathic/anti-immunopathic dual activities.
b
b
aggregates, which structurally change over time [12]. A number of
studies have suggested that small soluble oligomers are the pri-
mary toxic species in the CNS [13,14]; however, oligomers and fi-
brils also both activate microglia through different receptors and
2. Results
2.1. Rational design and synthesis of furosemide analogs
induce neuroinflammation [15,16]. Inhibition of A
been proposed as a possible therapeutic approach to AD. Tan et al.
demonstrated that diaportheone A analogs inhibited A aggrega-
b aggregation has
Furosemide was selected as a known drug with an anthranilate
core structure analogous to 3-HAA a metabolite of tryptophan and
an endogenous brain compound demonstrated to inhibit both A
aggregation and the release of proinflammatory cytokines. We
synthesized and evaluated proof-of-concept analogs of furosemide
exploring the capacity of the furosemide-like anthranilate platform
to be modified into a brain penetrant agent capable of poly-
pharmacological targeting of both the proteopathic (Ab aggrega-
tion) and immunopathic (proinflammatory cytokine release)
mechanisms of AD.
Furosemide has three “faces” (designated as Regions I-III in Fig.1
(B)) appended to a central aromatic core. Region I consists of a furan
ring. Such aromatic ring systems are central to p-electron mediated
intermolecular interactions [26,27], and, as exemplified by poly-
phenolic natural products [28], probably are crucial to small
molecule interactions with Ab. Region II consists of a sulfonamide
group. Sulfonamides are a common feature within many drugs
(including antibiotics, anti-inflammatories and allergy medica-
tions) and are able to interact with a diversity of targets including
b
tion and associated neuronal cell death [17]. Gao et al. designed and
synthesized ranitidine analogs which showed potent activity
b
against A
noids promoted disaggregation of A
b
aggregation [18]. Windsor et al. revealed that biflavo-
fibrils due to their ability to
b
disrupt fibril structure, suggesting another class of compounds as
putative therapeutic agents [19]. Other studies have devised small
molecules to target neuroinflammation as potential AD treatments.
Lee et al. identified a compound designated CA140 that inhibited
microglial activation, demonstrating a beneficial role in alleviating
neuroinflammation [20]. Yi et al. reported that compound SAS-
0132 exhibited neuroprotective activity and reduced cognitive
deficits through inhibition of neuroinflammation in experimental
models of AD [21].
This study aims to identify drug candidates that may concomi-
tantly inhibit
inflammation.
hydroxyanthranilic acid (3-HAA, shown in Fig.
A
b
oligomerization/fibrillization and neuro-
Previous work demonstrated that 3-
(A)), an
1
2

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
Fig. 1. (A) 3-HAA is a metabolite of tryptophan. (B) Structure of furosemide. (C) Representative structures of different series (SI-SV) modification. SI (3a-3q): substitution of sul-
fonamide hydrogen with different functional groups. SI-A (3r): replacement of furan with tetrahydrofuran; SII (11ae11g): modification of sulfonamides to amides; SIII (12, 16e20,
24, 26): modification of carboxylic acid to different bioisosteres. SIV (27e29): sulfonamide removal and different halogen substituents at X; SV (32e38): carboxylic acid replaced by
2,2,2-trifluoro-1-hydroxy ethyl substituent; furan or thiazole ring attached at R4 position.
zinc metalloenzyme carbonic anhydrase, dopamine receptors and
ion channels [29]. Region III consists of a carboxylate, another
common moiety within many drugs, but one which imparts sig-
nificant polarity thereby strongly impacting pharmacokinetics and
drug distribution across barriers such as the blood-brain barrier. To
explore the structure-activity contributions of these three molec-
ular faces, five analog series (Series IeV; SI-SV) were prepared, as
(18e20), tetrazole (24), and oxadiazolone (26). Scheme 5 depicts
the synthetic route for compounds 16e20 from the 3c single pre-
cursor molecule. To synthesize 16, 3c was reduced with BH3-THF
complex followed by oxidation with DMP to afford aldehyde in-
termediate 13. After protection by Boc anhydride (14), tri-
fluoromethylation was then conducted, and Boc was deprotected
under acidic condition to generate 16. Compounds 17e20 were
obtained from coupling reactions between 3c with varying bases in
the presence of reagent 1,10-carbonyldiimidazole (CDI). In addition,
we devised and optimized an integrated synthetic route to prepare
compounds 24 and 26 employing intermediate precursor 23. As
outlined in Scheme 6, intermediate 2c from Scheme 1 was reacted
with CDI and ammonium hydroxide to yield intermediate amide 21.
This primary amide was then converted to a nitrile (22) by cyanuric
chloride. Subsequent aromatic substitution introduced furfuryl-
amine to obtain precursor molecule 23, which was then submitted
to reaction with sodium azide to afford final compound 24. In
parallel, an oxadiazolone derivative was prepared by intermolec-
ular addition of hydroxylamine to nitrile and subsequent reaction
with CDI to generate compound 26.
In the SIV analogs (Table 4), the sulfonamide was removed, and
different halogens were explored on the central phenyl ring. As
shown in Scheme 7, compounds 27e29 were obtained through
cross-coupling reactions. In SV (Table 5), the carboxylic acid was
replaced with a 2,2,2-trifluoro-1-hydroxy ethyl substituent, and
furan or thiazole rings were attached to the R4 position. Com-
pounds 32e38, as depicted in Scheme 8, were synthesized from
commercially available halide-substituted 2-nitrobenzoaldehyde
as a building block with subsequent trifluoromethylation to
afford intermediates 30a-30d, which were reduced by either H2 or
iron to obtain anilines 31a-31d. Reductive amination with varying
aldehydes generated final compounds 32e38.
described in Fig. 1 (C). Series I, II, III investigated activity against A
oligomerization; Series IV and V investigated activity against A
b
b
fibrillization. All series were evaluated for anti-inflammatory
properties.
Series I substitutions (Table 1) involved replacement of a sul-
fonamide hydrogen by different functional groups. As shown in
Scheme 1, compounds 3a-3q of SI were obtained starting from
chlorosulfonation
of
commercially available
4-chloro-2-
fluorobenzoic acid and subsequent reaction with varying amines
to afford intermediates 2a-2q. Aromatic substitutions were then
performed with furfurylamine to afford 3a-3q. (Amine 5 used in the
synthesis of 3e was obtained from initial trifluoromethylation of 4-
cyanobenzaldehyde followed by reduction with LiAlH4 (Scheme 2)).
The SI-A substitution was the replacement of the furan ring with a
tetrahydrofuran (Scheme 3).
Series II modifications (Table 2) focused on the replacement of
sulfonamides with amides, as represented by compounds 11a-11g.
As described in Scheme 4, iodination of 4-chloro-2-fluorobenzoic
acid was initially performed with N-iodosuccinimide (NIS) to
afford intermediate 6, after which the carboxylic acid was then
protected by an ester. Upon reaction with a Grignard reagent, car-
bon dioxide (CO2) was introduced to obtain intermediate 8. The
carboxylic acid was then converted to varying amides (9a-9g)
through the reactions with thionyl chloride and amines. Following
substitutions with furfurylamine and ester deprotection, final
products 11a-11g were obtained.
AD arises from parallel, interconnected proteopathic and
immunopathic disease mechanisms. Two fundamental proteo-
Series III substitutions (Table 3) replaced the carboxylate moiety
with
a
variety of bioisosteric replacements, including 2,2,2-
pathic disease mechanisms include A
b oligomerization and Ab
trifluoro-1-hydroxy ethyl substituent (16), ester (17), amides
fibrillization; two fundamental immunopathic disease mechanisms
3

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
Table 1
Compound structures of Series I and activity against Ab oligomerization and fibrillization.
SI
Compound No.
R1
Inhibition of A
b
oligomerization
Inhibition of Ab fibrillization at
concentration 100
mM
IC50
(
m
M)
R-squared
Inhibition (%)
SD
3a furosemide
3b
NH2
Inactivea
Inactive
N/A
N/A
8.56
29.18
5.37
8.89
3c
53.01 3.37
Inactive
0.886
N/A
Pro-
N/A
fibrillizationb
3d
0.44
11.81
3e
68.5 18.75
0.927
Pro-fibrillization
N/A
3f
41.16 12.78
21.92 7.36
0.87
Pro-fibrillization
Pro-fibrillization
N/A
N/A
3g
0.905
3h
3i
41.15 20.41
Inactive
0.883
N/A
17.63
10.61
N/A
Pro-fibrillization
3j
137.39 11.88
113.13 22.83
Inactive
0.958
0.88
21.29
11.76
N/A
3k
3l
Pro-fibrillization
27.83
N/A
25.62
4.34
3m
47.81 14.35
0.838
39.24
3n
3o
Inactive
N/A
Pro-fibrillization
N/A
167.53 28.97
0.907
17.42
5.31
3p
3q
115.52 13.82
Inactive
0.887
N/A
8
11.30
5.44
19.08
4

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
Table 1 (continued)
SI
Compound No.
R1
Inhibition of A
b
oligomerization
Inhibition of Ab fibrillization at
concentration 100
mM
IC50
(
m
M)
R-squared
Inhibition (%)
SD
SI-A
Compound No.
Inhibition of A
b
oligomerization
Inhibition of A
100
b
fibrillization at concentration
m
M
IC50
136.39 30.66
(
m
M)
R-squared
0.933
Inhibition (%)
17.49
SD
12.03
3r
a
“Inactive” indicates that inhibition is not detected above 200
m
M.
b
“Pro-fibrillization” indicates that the compound facilitates A
b fibrillization.
Scheme 1. Synthesis of SI compounds: 3a-3q. Reagents and conditions: (a) ClSO3H, 0e140 ꢀC, 16 h; (b) R1, Et3N, THF, 0 ꢀC, 16 h; (c) furfurylamine, Et3N, ethylene glycol, 130 ꢀC, 16 h.
Scheme 3. Synthesis of SI-A compound 3r. Reagents and conditions: (a) tetrahy-
Scheme 2. Synthesis of amine 5 for compound 3e. Reagents and conditions: (a)
drofurfurylamine, Et3N, ethylene glycol, 130 ꢀC, 16 h.
TMSCF3, TBAF, THF, -20 - 0 ꢀC, 3 h; (b) LiAlH4, THF, 0 ꢀC, 2 h.
assay (method is described in 5.2.1). Furosemide itself was inac-
include proinflammatory cytokine release and altered phagocy-
tive. Modification of furosemide's primary sulfonamide to a sec-
ondary methyl sulfonamide as in 3c reduced the IC50 to 53.01 mM.
tosis. Using furosemide analogs from the five series to address the
three pharmacophoric faces of furosemide, these four disease
mechanisms (two proteopathic, two immunopathic) were studied.
Additional studies also considered pharmacokinetic and blood-
brain barrier penetrance of anthranilate analogs.
To investigate whether the hydrophobic interaction provided by
the methyl group contributed to this activity, we further synthe-
sized 3j and 3m by introducing ethyl and hexyl groups, respec-
tively. These analogs did not significantly improve activity. We next
explored the incorporation of aromatic systems through the addi-
tion of phenyl or benzyl functionalities, as shown in 3d, 3e, 3g, 3h,
3i, 3l, 3n, and 3o; the most potent compound in SI was 3g with an
2.2. Furosemide analogs inhibit Ab oligomerization
Fig. 2 summarizes the activity of selected furosemide analogs
against A oligomerization examined via an anti-oligomerization
IC50 of 21.92 mM. To quantify the significance of these structural
b
5

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
Table 2
Compound structures of Series II and activity against Ab oligomerization and fibrillization.
SII
Compound No.
R2
Inhibition of A
b
oligomerization
Inhibition of Ab fibrillization at
concentration 100
mM
IC50
(m
M)
R-squared
Inhibition (%)
SD
11a
11b
NH2
Inactivea
Inactive
N/A
N/A
32.37
19.07
8.18
11.59
11c
11d
11e
11f
11g
Inactive
Inactive
Inactive
Inactive
Inactive
N/A
N/A
N/A
N/A
N/A
24.64
18.35
34.05
11.03
29.43
5.12
6.97
7.86
10.98
12.29
a
“Inactive” indicates that inhibition is not detected above 200 mM.
Scheme 4. Synthesis of SII compounds: 11a-11g. Reagents and conditions: (a) NIS, H2SO4, 0 ꢀC, 4 h; (b) SOCl2, MeOH, 70 ꢀC, 2 h; (c) i-PrMgBr, THF, ꢁ40 ꢀC, 2 h; (d) CO2, -40 ꢀC, 16 h;
(e) SOCl2, 60 ꢀC, 2 h; (f) R2; (g) furfurylamine, Et3N, DMF, 80 ꢀC, 16 h; (h) NaOH, THF, r.t., 2 h.
modifications, we statistically analyzed activity differences be-
tween each molecule using a one-way ANOVA with Tukey's mul-
tiple comparisons. Since the activity difference between 3c and 3g
was not statistically significant as shown in Fig. 2, we maintained
the methyl substitution at the sulfonamide moiety.
on anti-Ab oligomerization activity, we replaced the sulfonamide
with amides in SII. No significant improvement in activity was
identified from this modification, as shown in Table 2.
To investigate if the planarity of furan contributes to compound
activity, we modified the furan ring to
a tetrahydrofuran.
To further elucidate the role of the sulfonamide functional group
Comparing the activities of 3c and 3r in Table 1, the IC50 increased
6

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
Table 3
Compound structures of Series III and activity against Ab oligomerization and fibrillization.
SIII
Compound No.
R3
Inhibition of A
b
oligomerization
Inhibition of Ab fibrillization at
concentration 100
Inhibition (%)
15.03
mM
IC50
(
m
M)
R-squared
0.971
SD
12
16
17
117.49 3.95
10.30
Inactivea
N/A
e
e
55.95 26.58
0.834
27.14
5.50
18
19
20
82.41 28.54
0.843
0.958
0.947
Pro-fibrillizationb
Pro-fibrillization
25.17
N/A
N/A
4.98
102.59 22.05
6.56 2.47
24
26
70.48 15.7
Inactive
0.927
N/A
14.06
20.22
5.68
10.85
a
“Inactive” indicates that inhibition is not detected above 200
m
M.
b
“Pro-fibrillization” indicates that the compound facilitates A
b fibrillization.
from 53
may assist ligand-protein interaction (possibly through interactions
such as stacking or cation- interactions).
m
M to 136
m
M, indicating that furan's sp2 planar system
2.4. Furosemide analogs inhibit lipopolysaccharide (LPS)-induced
inflammation
p-
p
p
We then investigated the contribution of the carboxylate moiety
through various carboxylate bioisosteres, including esters, amides
and tetrazoles, shown in SIII. The most potent compound was 20
To investigate anti-inflammatory activity, furosemide analogs
were evaluated using an LPS-stimulated SIM-A9 cell in vitro assay.
SIM-A9, a microglial cell line derived from murine cerebral cortices,
exhibits key characteristics of cultured primary microglia and ex-
presses microglia-specific proteins [30]. Upon stimulation with LPS,
SIM-A9 cells polarize to the “M1” proinflammatory phenotype [30].
This assay investigated whether furosemide analogs inhibited LPS-
induced proinflammatory responses by SIM-A9 cells.
with a 6.56 mM IC50.
2.3. Furosemide analogs inhibit Ab fibrillization
In addition to our investigation of anti-A
tivity, we synthesized additional furosemide analogs to evaluate
activity against A fibrillization examined by ThT assay (experi-
b oligomerization ac-
Multiple furosemide analogs active against Ab aggregation were
screened using the SIM-A9 assay. Prior to these studies, a cyto-
toxicity assay was performed using the commercially available Cell
Counting Kit-8 (CCK-8) to determine the safe concentration for
compound application on SIM-A9 cells. We selected a cut-off at a
2.7 mM concentration to ensure cell viability >90% after 24 h in-
cubation with each compound (results are shown in Fig. S1). Anti-
b
mental method is described in 5.2.2). We completely removed the
sulfonamide moiety in SIV, observing that 28 showed 20% inhibi-
tion of fibrillization at 100 mM concentration. We next modified the
carboxylate to 2,2,2-trifluoro-1-hydroxy ethyl substituent, a weakly
acidic, nonplanar bioisostere of carboxylic acid. As shown in Fig. 3,
this modification significantly increased the inhibitory activity as
evidenced by compounds 28 and 33. In addition, we also noted that
halogen substitution affects activity (Br > Cl > F) as shown by 32,
33, and 34. The most potent compound in this series was 34,
inflammation activity results are summarized in Fig. 4. 3g and 18
significantly inhibited the production of LPS-induced TNF-
and downregulated COX-2 and iNOS expression, demonstrating
dual activities against A oligomerization and inflammation. 33 and
34 significantly inhibited the production of LPS-induced NO, TNF-
IL-6 and downregulated the expression of COX-2 and iNOS,
exhibiting dual activities against A fibrillization and inflammation.
a/IL-6
b
a
,
demonstrating 81.32% inhibition at 100
fibrillization. The spectrum of structure-activity relationships is
shown in Fig. 3.
mM concentration against
A
b
b
7

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
Scheme 5. Synthesis of compounds 16e20. Reagents and conditions: (a) BH3-THF, THF, 60 ꢀC, 16 h; (b) DMP, DMF, 0 ꢀC to r.t., 6 h; (c) Boc2O, Et3N, DMAP, CH2Cl2, r.t., 6 h; (d) TMSCF3,
TBAF, THF, 0 ꢀC to r.t., 6 h; (e) 1 M HCl, MeOH; (f) CDI, THF, 40 ꢀC, 3 h; (g) t-BuOK, MeOH, r.t., 2 h; (h) CDI, THF, 40 ꢀC, 3 h; (i) appropriate amine, r.t., 2 h.
Microglia are phagocytic brain macrophages typically inhibited
by proinflammatory cytokines. To investigate if furosemide analogs
facilitate microglial phagocytosis, we measured phagocytic activity
using FITC-conjugated E. coli bioparticles after a 24 h LPS exposure.
As shown in Fig. 5 (A), 3c, 3g, 19, 20, 37 significantly promoted the
phagocytic activity of SIM-A9 cells compared to the DMSO control
group, as characterized by increased intracellular fluorescein iso-
thiocyanate (FITC) signal. Administration of furosemide analogs
consistently promoted phagocytic activity compared to DMSO in
the LPS treatment group.
In furosemide metabolism, a major metabolite is the glucur-
onidate conjugate of the carboxylic acid [33]. This pathway facili-
tates renal clearance and allows furosemide to exert diuretic effects
at the level of the renal tubule. The glucuronidation process also
targets sulfonamide substituents contributing to furosemide's
inability to traverse the BBB. To avoid glucuronidation and increase
the potential for BBB permeability, we substituted one hydrogen on
the primary sulfonamide with different functional groups in SI, and
further converted the carboxylic acid to primary (18) or tertiary
amides (20). We selected three active compounds shown in Table 6
for further pharmacokinetic (PK) study. Their BBB scores were
calculated, and select PK studies were conducted. The 4.48 BBB
Score for 20 indicates an improved probability for traversing the
BBB. The PK study further confirmed that 20 provided a moderate
plasma exposure and an increased brain/plasma ratio 0.22
compared with furosemide. Although this value is not optimal for a
CNS-active drug, it does demonstrate the capacity of the anthra-
nilate platform to be modified for improved PK and BBB-
penetrance properties.
2.5. Furosemide analogs exhibit varying pharmacokinetic
properties
The anti-proteopathic and anti-immunopathic activities
demonstrate the pharmacodynamic utility of the anthranilate
platform within the context of AD therapeutic development;
however, particularly with CNS diseases, pharmacokinetic (PK)
considerations are also paramount. On average, 70% of an oral
furosemide dose is absorbed, and 97.6% is bound to plasma albumin
[24]. However, the blood-brain barrier (BBB) is relatively imper-
meable to negatively charged carboxylates [31], explaining furo-
semide's inability to traverse the BBB. We therefore also sought to
investigate the BBB permeability of furosemide analogs. To pursue
this objective, we used an algorithm of BBB-permeability predic-
tion, the “BBB Score” [32].
3. Discussion
Multiple factors, including cellular stress, dysregulated meta-
bolism and impaired immune responses contribute, independently
and interdependently, to AD disease progression [34]. Corre-
spondingly, Ab plays multiple roles in AD. First, non-toxic Ab
8

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
Scheme 6. Synthesis of compounds 24 and 26. Reagents and conditions: (a) CDI, THF, 40 ꢀC, 3 h; (b) NH4OH, r.t., 2 h; (c) (NCCl)3, DMF, 0 ꢀC, 3 h; (d) furfurylamine, Et3N, DMF, 50 ꢀC,
16 h; (e) NaN3, Et3NHCl, toluene, 100 ꢀC, 48 h; (f) [NH2OH]Cl, NaHCO3, EtOH, reflux, 16 h; (g) CDI, 1,4-dioxane, reflux, 16 h.
Table 4
Compound structures of Series IV and activity against A
b oligomerization and fibrillization.
SIV
Compound No.
X
Inhibition of A
b
oligomerization
Inhibition of Ab fibrillization at
concentration 100
mM
IC50
(
mM)
R-squared
Inhibition (%)
SD
27
28
29
F
Cl
Br
129.54 25.82
Inactivea
154.42 33.79
0.958
N/A
0.846
12.39
20.18
3.10
12.05
7.22
6.13
a
“Inactive” indicates that inhibition is not detected above 200 mM.
monomers aggregate into toxic oligomers, fibrils, and eventually
plaques [35]. The toxic A aggregates impair cellular integrity and
neuronal function [4]. Inhibitors of A
suggested as potential therapeutic agents with which to ameliorate
b aggregation have been
b
9

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
receptors also activates the secretion of neurotoxic molecular me-
diators, including ROS and proinflammatory cytokines [15,16].
These immunological mechanisms demonstrate that Ab-induced
microglia activation is likewise a chronic, progressive process
occurring along the entire chronological and structural pathway of
Ab oligomerization and fibrillization. Thus, AD pathogenesis may
be conceptualized as having concomitant, parallel protein aggre-
gation (proteopathic) and immunotoxic (immunopathic) pathways
with multiple points of neurotoxicity chronologically along each
pathway and multiple points of inter-pathway crosstalk stimula-
tion mechanistically along each pathway (analogous to a ladder
with multiple rungs).
This study aims to identify a potential drug-like molecular
platform capable of inhibiting not only Ab aggregation at different
stages (oligomerization, fibrillization), but also downregulating the
associated neuroinflammation. Our previous work identified 3-
HAA, an endogenous tryptophan metabolite, as being able to
Scheme 7. Synthesis of compounds 27e29. Reagents and conditions: (a) furfuryl-
amine, CuI, K2CO3, DMSO, 40 ꢀC, 12 h.
neurotoxicity [36,37]. However, aggregation of Ab into oligomers,
inhibit not only A
b aggregation (purportedly by interacting with
fibrils e both of which are neurotoxic e is a chronic, progressive
process, requiring the design of inhibitors targeting both oligo-
merization and fibrillization. Secondly and concomitantly, micro-
A
b
0s HHQK domain) but also the release of proinflammatory cyto-
kines [22,23]. Rather than designing analogs of 3-HAA we sought to
expedite the search by identifying known drugs containing an
anthranilate moiety similar to 3-HAA. This search identified furo-
semide as a candidate compound.
glia, as brain macrophages, engulf both oligomeric and fibrillar A
b
through different scavenger receptors. For instance, the scavenger
receptor (SR)-A mediates microglial internalization of oligomeric
Since we are endeavoring to optimize a compound against
multiple receptors e for which no ligand-receptor crystal struc-
tures nor structural biological data are available e we approached
this task via a systematic fragment optimization of logical structural
molecular subunits within furosemide. Previous mechanistic
A
b
, whereupon A
subsequently degraded by cysteine proteases [38]. However,
microglia exploit different SRs when confronting neurotoxic A
fibrils. SR-BI and CD36 are SRs for fibrillar A , mediating microglial
endocytosis of these larger aggregates [39e42]. In addition to
b oligomers are trafficked toward lysosomes and
b
b
studies have demonstrated that Ab aggregation is temperature and
triggering phagocytosis, binding of Ab to SR-A, SR-BI, and CD36
Table 5
Compound structures of Series V and activity against A
b oligomerization and fibrillization.
SV
Compound No.
X
R4
Inhibition of A
b
oligomerization
Inhibition of Ab fibrillization at
concentration 100
Inhibition (%)
5.14
mM
IC50
(
m
M)
R-squared
SD
32
33
34
35
36
37
F
153.05 48.01
0.840
6.13
Cl
Inactivea
N/A
60.72
81.32
36.80
27.88
33.03
6.49
8.86
16.28
4.28
6.04
5.06
5.03
Br
Inactive
N/A
OMe
Cl
Inactive
N/A
Inactive
N/A
Br
Inactive
N/A
38
OMe
129.69 28.37
0.865
a
“Inactive” indicates that inhibition is not detected above 200 mM.
10

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
Scheme 8. Synthesis of 32e38. Reagents and conditions: (a) TMSCF3, TBAF, THF, 0 ꢀC to r.t., 3 h; (b) H2, Pd/C, MeOH, r.t. 4 h; (c) Fe, NH4Cl, EtOH, reflux; (d) HCOR4, NaBH(OAc)3,
AcOH, dichloroethane, 60 ꢀC, 16 h; (e) NaBH4, EtOH, 60 ꢀC, 1 h.
Fig. 2. Anti-A
Further optimization in SIII improved IC50 to 6.56
show standard deviation ( SD), n ¼ 3. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.
b
oligomerization activity of selected compounds evaluated with oligomerization assay. (A) Series I modification brought the activity to IC50 21.92
mM in compound 3g.
m
M in compound 20. (B), (C), (D) show IC50 curves of compounds 3c, 3g, and 20, respectively, against A oligomerization. Error bars
b
11

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
synthesis of the 2,2,2-trifluoro-1-hydroxyethyl substituted
congener by TMSCF3 and appropriate aldehyde was not enantio-
selective, resulting in these analogs being racemic mixtures. As an
initial stage in the exploration of furosemide as a druggable plat-
form for AD, we employed these analogs as racemic mixtures. In
future studies, we shall separate racemic mixtures by esterification
using Mosher's acid, based on previous literature [50].
A
b
aggregation is a complicated process. Following an initial
rate-limiting nucleation stage, aggregation may proceed by two
different pathways [51]. In primary nucleation, A aggregation oc-
b
curs via monomer oligomerization with subsequent fibrillization.
Alternatively, aggregation can arise from heterogenous contacts
between monomers and fibrils, via secondary nucleation, suggesting
that A
These two pathways have been experimentally confirmed; certain
compounds can selectively inhibit A oligomerization, others
inhibit fibrillization [52]. Since A monomers can divert from the
oligomerization pathway and initiate secondary nucleation, exclu-
sively inhibiting oligomerization may in principle redirect mono-
mers to fibrillization. Our experimental results displayed in Table 1,
show that analogs 3c and 3f inhibited oligomerization but pro-
b oligomerization is not obligate for fibril formation [51].
b
b
Fig. 3. Anti-Ab fibrillization activity of selected compounds tested in ThT assay. The
initial modification of sulfonamide removal in SIV increased the inhibitory activity to
20.18% in 28. The modification of carboxylic acid to 2,2,2-trifluoro-1-hydroxy ethyl
substituent in SV improved inhibition to 60.72% in 33. The final substitution of Cl to Br
optimized the activity to 81.32% in 34. Error bars show SD, n ¼ 3. **, p < 0.01; ****,
p < 0.0001.
moted fibrillization. This suggests A
b oligomerization and fibrilli-
zation may be two competing pathways, and thus inhibition of both
may be important to the amelioration of A
progression.
b-associated disease
concentration-dependent, suggesting that monomeric Ab first as-
sociates through hydrophobic interactions to form soluble oligo-
mers [43,44]. In SI, we blocked one HBD on the sulfonamide moiety
to increase hydrophobicity, aiming to create analogs active against
oligomerization. We started with a simple methyl group replace-
ment. The most fundamental modification during SAR studies is
replacing a hydrogen atom with a methyl group; the importance of
methyl groups in modulating biological activity is well documented
[45]. This replacement afforded 3c and brought the activity from
To investigate activity against neuroinflammation, we evaluated
our compounds on LPS-stimulated SIM-A9 microglial cells. SIM-A9
is a novel cell line from primary glial culture of postnatal murine
cerebral cortices [30]. Upon LPS stimulation, they polarize to a
proinflammatory phenotype via the TLR4 receptor, the same re-
ceptor responsible for A
compounds had already demonstrated inhibitory activity against
aggregation, LPS rather than A oligomers was used to trigger
inflammatory activation, thereby avoiding potential interactions
between A and the furosemide analogs. As shown in Fig. 4, LPS
induced secretion of TNF- , IL-6, NO, and the expression of COX-2
b-induced activation [30,53]. Since our
A
b
b
inactive to a weakly active 53.01
mM IC50 (Although a previous
study suggested that furosemide has antiaggregant properties [46],
we were unable to reproduce this observation). We also blocked
one HBD with aromatic rings, as indicated in compounds 3o, 3d,
and 3i. However, these analogs did not increase activity. We further
blocked two HBDs and synthesized compound 3k with dimethyl
substitution, which reduced activity significantly compared to 3c.
We next explored combining methyl substitution (3c) with benzyl
substitution in compounds 3g and 3h and identified compound 3g
b
a
and iNOS by SIM-A9 cells. The role of these proinflammatory bio-
markers in AD pathology has been demonstrated in multiple
studies, manifesting as impaired synaptic function, induced
apoptosis and augmented neuroinflammation, all correlating with
AD progression [54e57]. The broad inflammation spectrum
induced by LPS provides an opportunity for anti-inflammatory ac-
tivity screening. Treatment with compounds 3g, 33, and 34
significantly inhibited the LPS-stimulated inflammatory response,
as shown in Fig. 4. These results demonstrated that furosemide
with IC50 ¼ 21.92
from a hydrophobic interaction involving the methyl group and
aromatic interactions in the potential binding pocket. In addition,
mM. This improved activity may possibly arise
p
in accord with literature precedent and based on their physical and
spatial similarity, we replaced the sulfonamide with a carboxamide
in SII [47e49]. However, no improvement in activity was identified.
Although the carboxylate functionality is a pharmacophoric
structural moiety in many classes of therapeutic agent, its acidity,
polarity and ability to establish strong electrostatic interactions and
hydrogen bonds, interferes with other drug properties such as
pharmacokinetics. Accordingly, in SIII we replaced the carboxylic
acid with bioisosteres, exhibiting similar volume, shape, physico-
chemical properties and biological effects. This approach afforded
analogs inhibit both Ab aggregation (oligomerization/fibrillization)
and LPS-induced inflammation.
As part of the innate immune response, microglia phagocytose
debris, helping to maintain homeostasis. However, as a component
of AD disease progression, inflammatory cytokines chronically
modulate microglia to a less phagocytic phenotype [11]. To inves-
tigate if our compounds restore or promote microglial phagocytosis
during neuroinflammation, we measured the phagocytic activity of
SIM-A9 cells using FITC-conjugated E. coli bioparticles after 24 h LPS
stimulation, with and without compound treatment. These bio-
particles consist of a series of fluorescently labeled, heat- or
chemically killed bacteria and yeast in a variety of sizes, shapes, and
natural antigenicities. Our results demonstrated that LPS slightly
impaired phagocytosis activity after 24 h incubation. Upon treat-
ment with furosemide analogs (e.g. compounds 3c and 3g),
phagocytic activity significantly increased, suggesting the contri-
compound 20 with IC50 ¼ 6.56
To improve the structural similarity to 3-HAA whilst designing
an inhibitor of A fibrillization, we eliminated the entire sulfon-
amide group; this modification slightly improved anti-A fibrilli-
mM.
b
b
zation activity. We then replaced the carboxylic acid with a 2,2,2-
trifluoro-1-hydroxy ethyl substituent, in which the electronic
properties of the CF3 component afford an acidic OH and in which
the sp3 hybridization enables improved ligand rotational flexibility.
This modification yielded substantive improvements in activity as
exemplified by compounds 28 and 33 shown in Fig. 3. However,
bution to Ab internalization during AD progression.
Finally, we examined the PK properties of selected compounds.
The BBB protects the brain from external insult. Designing mole-
cules to traverse the BBB is a challenging hurdle in CNS drug
12

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
Fig. 4. Anti-inflammatory activity of selected compounds tested in SIM-A9 microglial cells. SIM-A9 cells were stimulated by 5 ng/mL LPS in the presence of furosemide analogs at
2.7 M. Following 24 h incubation, the cell supernatants were harvested to analyze the production of (A) NO using a Griess assay, (B) TNF- and (C) IL-6 using ELISA assays; the cell
m
a
lysates were harvested to analyze the expression of (D) COX-2 and (E) iNOS using Western blot assays. All results were collected from three independent experiments. Error bars
show SD. n ¼ 6 in NO and cytokine measurements, n ¼ 3 in Western blot analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****p < 0.0001.
discovery. Multiple algorithms have been developed to predict BBB
penetration. These models integrate various physicochemical de-
scriptors in devising a prediction algorithm. We designed our
molecules by endeavoring to incorporate the desirable ranges for
these descriptors (e.g. cLogP <5.0; cLogD <4; molecular weight
<500; 40 < polar surface area <120; HBD <3; pKa <10) in accord
with literature precedent [32,58]. To facilitate this multivariable
process, we employed the “BBB Score” as a validated algorithm with
which to predict BBB penetration [32]. BBB score ranges from 0 to 6,
and a score >4 is compatible with a CNS penetrant drug. In our
study, we selected four representative molecules (3a [furosemide],
3g, 18 and 20) to explore BBB score as a function of structural
modification. As summarized in Table 6, the BBB Score improved
from 3.42 (3g), to 3.93 (18) to 4.48 (20). However, the brain/plasma
ratio of 20 as deduced in our PK experiments was not optimal. We
speculate that this may be explained by drug metabolism. A pre-
dicted metabolic pathway for 20 is depicted in Scheme 9 [33,59].
The oxidation of the furan ring affords an epoxide, which either
interacts with a nucleophile such as glutathione (GSH) forming a
conjugate, or undergoes a ring-opening step due to its ring strain.
The so-formed Michael receptor would subsequently interact with
N-acetyl cysteine and N-acetyl lysine and form a conjugate. The
oxidation may also facilitate N-dealkylation and form an aromatic
amine that further undergoes acetylation. In addition, glucur-
onidation occurs in the primary amides upon the oxidation on
dimethyl amino group. These diverse metabolic possibilities
complicate interpretation of our PK studies.
As exemplified by furosemide, the anthranilate platform is drug-
like and readily amenable to analog generation. Collectively, our
results indicate the capacity of the furosemide-like anthranilate
platform to exhibit multi-receptor targeting against various stages
of the parallel yet interdependent proteopathic and immunopathic
disease pathways of AD. In terms of proteopathic processes, an-
thranilates target the two fundamental aggregation processes,
namely oligomerization and fibrillization. In terms of immuno-
pathic processes, anthranilates target the two fundamental
microglial processes, namely proinflammatory cytokine release and
phagocytosis. Finally, the anthranilate platform can be additionally
derivatized to improve PK and BBB-penetrance properties.
13

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
Fig. 5. Phagocytotic function assay using FITC conjugated E. coli bioparticles. Cells were plated in a 96-well cell culture plate. After 24 h compounds and LPS treatment, cells were
given E. coli bioparticles at final concentration 0.125 mg/mL. Following 1hr incubation at 37 ꢀC, the extracellular FITC signal was quenched by 0.4% trypan blue. The intracellular FITC
signal (A) was measured in a plate-reader at Ex 494/Em 518. Pictures (B) were taken in 30 min after quenched by trypan blue. Error bars show SD, n ¼ 3. *, p < 0.05; **, p < 0.01;
***, p < 0.001; ****, p < 0.0001 in control group; #, p < 0.05; ##, p < 0.01 in LPS group. Scale bars: 400 mm.
4. Conclusion
aggregation is a chronic and progressive process in which mono-
mers aggregate into various assemblies, including oligomers and
fibrils, which activate microglia, eventually causing immunotoxic
A
b
self-aggregates into toxic species that impair neuronal
function and chronically induce neuroinflammation.
A
b
neuroinflammation. This complex interaction between
Ab
14

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
Table 6
dry. Intermediate 1 was obtained as a yellow solid (1.44 g, 90%).
Intermediate 1 was then dissolved in anhydrous THF (0.3 M) and
cooled to 0 ꢀC. Triethylamine (3 eq.) was added to the reaction
mixture. The appropriate amine (1.2 eq.) was dissolved by THF and
added to reaction slowly. The reaction was allowed to warm to
room temperature and monitored by TLC. Upon completion of the
reaction, the mixture was diluted with EtOAc/H2O and layers
separated. The pH of the aqueous phase was adjusted to 3 and
extracted with EtOAc. The organic layers were washed with brine,
dried over Na2SO4 and concentrated under reduced pressure. Col-
umn chromatography gave the desired compounds 2a-2q
(20e88%). Intermediates 2a-2q (1 eq.) were subsequently dissolved
in ethylene glycol (0.3 M) and to this, furfurylamine (3 eq.) and
triethylamine (2 eq.) were added. The reaction mixture was heated
to 130 ꢀC for 16 h. The mixture was diluted with EtOAc/H2O and
layers were separated. The pH of the aqueous phase was adjusted to
3 and extracted with EtOAc. The organic layers were washed with
brine, dried over Na2SO4 and concentrated under reduced pressure.
Column chromatography gave the compounds 3a-3q (33e82%).
4-Chloro-2-((furan-2-ylmethyl)amino)-5-sulfamoylbenzoic acid
(3a, furosemide): compound 3a was obtained as a white solid,
126 mg, 76% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
PK study of selected furosemide analogs.
BBB Score Short PK results, n ¼ 3
Plasma (ng/mL) Brain (ng/
mg)
Brain/Plasma
Ratio
0.5 h
2.5 h
0.5 h 2.5 h 0.5 h
2.5 h
3a, furosemide 3.78
1728.4 1630.7 8.7
7.8
0.1
3.5
0.005
0.005
3g
18
20
3.42
3.93
4.48
1026.1 873.1
0.8
5.2
0.0008 0.00006
38.2
29.3
83.2
0.14
0.12
0.22
301.6
61.1 18.5 0.20
aggregation and neuroinflammation has impeded therapeutics
development. We identified 3-HAA as an endogenous brain mole-
cule targeting protein misfolding and neuroinflammation, and
furosemide as structurally similar known anthranilate-based drug.
Our novel furosemide analogs inhibit neurotoxic Ab aggregation,
both oligomerization and fibrillization. These furosemide analogs
also concurrently inhibit proinflammatory cytokines in LPS-
stimulated SIM-A9 microglial cells. This study identifies the
furosemide-like anthranilate platform as a druggable entity capable
of multi-receptor targeting of the combined proteopathic-
immunopathic pathogenesis of AD.
d
(ppm) 13.36 (s, 1H), 8.62 (t, J ¼ 5.9 Hz, 1H), 8.38 (s, 1H), 7.62 (d,
J ¼ 1.8 Hz, 1H), 7.34 (s, 2H), 7.06 (s, 1H), 6.46e6.26 (m, 2H), 4.58 (d,
J ¼ 5.8 Hz, 2H); 13C NMR (75 MHz, acetone-d6)
d (ppm) 170.2, 154.7,
5. Experimental section
153.0, 144.3, 138.7, 135.6, 128.6, 115.2, 112.1, 109.7, 109.3, 41.2. HRMS
(ESI, m/z) Calculated for C12H10ClN2O5S [M ꢁ H]-: 328.9999, found:
328.9998. Purity by HPLC: 100%, tR ¼ 9.81 min.
5.1. Chemistry
4-Chloro-5-(N-cyclopropylsulfamoyl)-2-((furan-2-ylmethyl)
amino)benzoic acid (3b): compound 3b was obtained as a white
solid, 270 mg, 59% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
All reagents and solvents were purchased from Sigma-Aldrich®,
Combi-blocks®, and Fisher Scientific®.
d
(ppm) 13.46 (s, 1H), 8.72 (t, J ¼ 5.4 Hz, 1H), 8.40 (s, 1H), 7.92 (d,
5.1.1. General characterization methods
J ¼ 2.3 Hz, 1H), 7.63 (s, 1H), 7.08 (s, 1H), 6.51e6.28 (m, 2H), 4.59 (d,
1H/13C NMR spectra were recorded on a Bruker Ultrashield™
(300/75 MHz) spectrometers. Data are presented as follows:
J ¼ 5.7 Hz, 2H), 2.13 (m, 1H), 0.54e0.18 (m, 4H). 13C NMR (75 MHz,
acetone-d6)
d (ppm) 170.1, 155.1, 153.0, 144.3, 139.0, 138.0, 124.7,
chemical shift (in ppm on a
d
scale relative to dTMS ¼ 0), multiplicity
115.3, 112.1, 110.0, 109.4, 41.2, 25.5, 6.8. HRMS (ESI, m/z) Calculated
for C15H14ClN2O5S [M ꢁ H]-: 369.0312, found: 369.0312. Purity by
HPLC: 99%, tR ¼ 10.67 min.
(s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, qn ¼ quintet,
sep ¼ septet, m ¼ multiplet, br ¼ broad), coupling constant (J/Hz)
and integration.
4-chloro-2-((furan-2-ylmethyl)
amino)-5-(N-methylsulfamoyl)
Mass spectra were recorded using electron spray ionization (ESI)
with a Xevo TQ-XS Tandem Triple Quadrupole Mass Spectrometer.
The parent ion ([MþH]þ, [M ꢁ H]-, or [MþNa]þ) is quoted. Purity
was determined using either a Waters Aquity UPLC® (2.1 ꢂ 50 mm,
benzoic acid (3c): compound 3c was obtained as a white solid,
126 mg, 57% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d
(ppm) 13.44 (s, 1H), 8.73 (s, 1H), 8.34 (s, 1H), 7.63 (d, J ¼ 1.8 Hz,
1H), 7.35 (q, J ¼ 4.9 Hz, 1H), 7.08 (s, 1H), 6.46e6.34 (m, 2H), 4.59 (d,
J ¼ 5.8 Hz, 2H), 2.40 (d, J ¼ 4.9 Hz, 3H). 13C NMR (75 MHz, acetone-
1.7
Chrom® SB C18 (4.6 ꢂ 250 nm, 5.0
2487 Dual Absorbance Detector. For both instruments elution
mm particle size) or a Waters 1525 EF Binary HPLC pump/Silia
mm particle size) with a Waters
d6) d (ppm) 170.0, 155.0, 152.9, 144.3, 138.8, 137.7, 123.6, 115.3, 112.1,
l
109.8, 109.3, 41.1, 30.0; HRMS (ESI, m/z) Calculated for
C
13H12ClN2O5S [M ꢁ H]-: 343.0155, found: 343.0159. Purity by
with a water/0.1% formic acid and acetonitrile gradient was used
with a flow rate of 0.60 mL/min (5 and 15 min run times respec-
tively). UV detection was carried out at a wavelength of 254 nm.
Thin layer chromatography (TLC) was carried out on Merch
Kieselgel 60 F254 plates with visualization by ultraviolet light
(254 nm) and potassium permanganate or ninhydrin dips. Flash
chromatography was carried out on RediSep columns using an Isco
CombiFlash SG100c.
HPLC: 100%, tR ¼ 10.24 min.
5-(N-benzylsulfamoyl)-4-chloro-2-((furan-2-ylmethyl)amino)
benzoic acid (3d): compound 3d was obtained as a white solid,
390 mg, 57% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d
(ppm) 8.81e8.60 (m, 1H), 8.29 (s, 1H), 8.12 (t, J ¼ 6.3 Hz, 1H), 7.64
(dd, J ¼ 1.9, 0.9 Hz, 1H), 7.31e7.10 (m, 5H), 6.98 (s, 1H), 6.44 (dd,
J ¼ 3.2, 1.9 Hz, 1H), 6.40e6.30 (m, 1H), 4.56 (d, J ¼ 5.8 Hz, 2H), 4.01
(d, J ¼ 6.3 Hz, 2H). 13C NMR (75 MHz, acetone-d6)
d (ppm) 169.8,
All NMR spectra and intermediate characterizations are listed in
Supplementary Material.
154.6,152.7,144.0,138.8,138.5,137.1,129.6,129.2,128.5,125.1,114.9,
111.9, 109.6, 109.1, 48.1, 40.9. HRMS (ESI, m/z) Calculated for
5.1.2. General synthesis procedures of compounds 3a-3q
C
19H16ClN2O5S [M ꢁ H]-: 419.0468, found: 419.0469. Purity by
Chlorosulfonic acid (4.09 g, 35.09 mmol, 6 eq.) was cooled to
HPLC: 96%, tR ¼ 11.25 min.
0
ꢀC. To this, 4-chloro-2-fluorobenzoic acid (1.02 g, 5.85 mmol, 1
4-Chloro-2-((furan-2-ylmethyl)amino)-5-(N-(4-(2,2,2-trifluoro-
1-hydroxyethyl)benzyl) sulfamoyl)benzoic acid (3e): compound 3e
was obtained as a white solid, 362 mg, 31% yield over 2 steps. 1H
eq.) was added, portion-wise. The reaction was then heated to
140 ꢀC and stirred for 5 h. The reaction was monitored by TLC. After
TLC indicated consumption of starting material, the reaction was
cooled to ꢁ10 ꢀC and ice was added to the mixture. A white solid
precipitated, was washed with water and placed in desiccator to
NMR (300 MHz, DMSO‑d6)
d
(ppm) 13.39 (s, 1H), 8.68 (t, J ¼ 5.8 Hz,
1H), 8.34 (s, 1H), 8.16 (t, J ¼ 6.2 Hz, 1H), 7.64 (s, 1H), 7.36 (d,
J ¼ 8.0 Hz, 2H), 7.26 (d, J ¼ 8.2 Hz, 2H), 7.03 (s, 1H), 6.79 (d,
15

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
16

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
J ¼ 5.6 Hz, 1H), 6.48e6.31 (m, 2H), 5.08 (p, J ¼ 7.3 Hz, 1H), 4.57 (d,
J ¼ 5.7 Hz, 2H), 4.02 (d, J ¼ 6.1 Hz, 2H). 13C NMR (75 MHz, acetone-
HRMS (ESI, m/z) Calculated for C14H14ClN2O5S [M ꢁ H]-: 357.0312,
found: 357.0311. Purity by HPLC: 97%, tR ¼ 11.06 min.
d6)
d
(ppm) 170.4, 168.6, 153.5, 151.5, 142.8, 138.7, 137.4, 135.9, 134.9,
4-Chloro-2-((furan-2-ylmethyl) amino)-5-(N-(4-methoxybenzyl)
sulfamoyl) benzoic acid (3l): compound 3l was obtained as a white
solid, 167 mg, 21% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
127.9, 127.7, 125.3 (q, J ¼ 281.9 Hz), 123.8, 113.8, 110.7, 108.4, 107.9,
71.5 (q, J ¼ 31.2 Hz), 46.5, 39.7. HRMS (ESI, m/z) Calculated for
C
21H17ClF3N2O6S [M ꢁ H]-: 517.0448, found: 517.0451. Purity by
d
(ppm) 13.35 (s, 1H), 8.64 (t, J ¼ 5.9 Hz, 1H), 8.23 (s, 1H), 8.02 (t,
HPLC: 100%, tR ¼ 10.92 min.
J ¼ 6.2 Hz, 1H), 7.63 (s, 1H), 7.08 (d, J ¼ 8.4 Hz, 2H), 6.97 (s, 1H), 6.73
(d, J ¼ 8.4 Hz, 2H), 6.46e6.32 (m, 2H), 4.56 (d, J ¼ 5.8 Hz, 2H), 3.94
(d, J ¼ 6.2 Hz, 2H), 3.67 (s, 3H). 13C NMR (75 MHz, acetone-d6)
4-Chloro-2((furan-2-ylmethyl)
amino)-5-(morpholinosulfony)
benzoic acid (3f): compound 3f was obtained as a white solid,
123 mg, 50% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d
(ppm) 169.9, 160.6, 154.7, 152.9, 144.2, 138.7, 137.2, 130.8, 130.7,
d
(ppm) 13.53 (s, 1H), 8.78 (t, J ¼ 5.8 Hz, 1H), 8.33 (s, 1H), 7.67e7.57
115.0, 112.0, 109.5, 109.2, 56.2, 47.8, 41.1. HRMS (ESI, m/z) Calculated
for C20H18ClN2O6S [M ꢁ H]-: 449.0574, found: 449.0575. Purity by
HPLC: 86%, tR ¼ 11.15 min.
(m, 1H), 7.13 (s, 1H), 6.49e6.33 (m, 2H), 4.60 (d, J ¼ 5.8 Hz, 2H),
3.67e3.49 (m, 4H), 3.13e2.97 (m, 4H). 13C NMR (75 MHz, CDCl3)
d
(ppm) 170.8, 153.8, 150.3, 143.2, 139.6, 138.1, 121.5, 114.7, 111.0,
4-Chloro-2-((furan-2-ylmethyl)
amino)-5-(N-hexylsulfamoyl)
108.4, 107.5, 66.7, 46.0, 40.6. HRMS (ESI, m/z) Calculated for
C
benzoic acid (3 m): compound 3m was obtained as a white solid,
16H16ClN2O6S [M ꢁ H]-: 399.0417, found: 399.0420. Purity by
561 mg, 74% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
HPLC: 97%, tR ¼ 10.85 min.
d (ppm) 13.42 (s, 1H), 8.71 (s, 1H), 8.34 (s, 1H), 7.62 (s, 1H), 7.53 (t,
5-(N-Benzyl-N-methylsulfamoyl)-4-chloro-2-((furan-2-ylmethyl)
amino) benzoic acid (3g): compound 3g was obtained as a white
solid, 269 mg, 34% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
J ¼ 5.6 Hz,1H), 7.06 (s,1H), 6.41 (d, J ¼ 1.7 Hz,1H), 6.36 (d, J ¼ 2.9 Hz,
1H), 4.57 (d, J ¼ 5.5 Hz, 2H), 2.74 (q, J ¼ 6.5 Hz, 2H), 1.40e1.26 (m,
2H), 1.19e1.03 (m, 6H), 0.79 (t, J ¼ 6.6 Hz, 3H). 13C NMR (75 MHz,
d
(ppm) 8.82 (d, J ¼ 5.1 Hz, 1H), 8.41 (s, 1H), 7.64 (s, 1H), 7.40e7.24
(m, 5H), 7.14 (s, 1H), 6.48e6.36 (m, 2H), 4.61 (d, J ¼ 5.8 Hz, 2H), 4.32
(s, 2H), 2.61 (s, 3H). 13C NMR (75 MHz, acetone-d6)
(ppm) 169.7,
acetone-d6) d (ppm) 169.9, 154.8, 152.9, 144.2, 138.8, 137.2, 125.2,
115.2, 112.0, 109.7, 109.2, 44.3, 41.0, 32.8, 31.3, 27.6, 23.9, 15.0. HRMS
(ESI, m/z) Calculated for C18H22ClN2O5S [M ꢁ H]-: 413.0938, found:
413.0939. Purity by HPLC: 99%, tR ¼ 12.15 min.
d
154.9, 152.8, 144.2, 139.1, 138.4, 130.1, 129.7, 129.1, 123.7, 115.6, 112.0,
109.7, 109.3, 55.0, 41.1, 40.9. HRMS (ESI, m/z) Calculated for
4-Chloro-2-((furan-2-ylmethyl)
amino)-5-(N-(4-methylbenzyl)
C
20H18ClN2O5S [M ꢁ H]-: 433.0625, found: 433.0625. Purity by
sulfamoyl) benzoic acid (3n): compound 3n was obtained as a white
HPLC: 99%, tR ¼ 12.24 min.
solid, 259 mg, 33% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
4-Chloro-2-((furan-2-ylmethyl) amino)-5-(N-(1-phenylethyl) sul-
famoyl) benzoic acid (3h): compound 3h was obtained as a white
solid, 188 mg, 24% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d
(ppm) 9.84 (s, 1H), 8.32 (s, 1H), 7.87 (t, J ¼ 6.0 Hz, 1H), 7.62 (s, 1H),
7.14e6.97 (m, 4H), 6.82 (s, 1H), 6.42 (dd, J ¼ 3.1, 1.9 Hz, 1H), 6.34 (d,
J ¼ 3.0 Hz, 1H), 4.46 (d, J ¼ 4.9 Hz, 2H), 3.92 (d, J ¼ 6.1 Hz, 2H), 2.22
d
(ppm) 13.30 (s, 1H), 8.60 (t, J ¼ 5.7 Hz, 1H), 8.20 (s, 1H), 8.13 (d,
(s, 3H). 13C NMR (75 MHz, acetone-d6)
d (ppm) 154.8, 153.3, 144.1,
J ¼ 8.5 Hz, 1H), 7.65 (d, J ¼ 1.0 Hz, 1H), 7.20e7.01 (m, 5H), 6.88 (s,
1H), 6.49e6.29 (m, 2H), 4.53 (d, J ¼ 5.8 Hz, 2H), 4.28 (dt, J ¼ 14.3,
7.0 Hz, 1H), 1.29 (d, J ¼ 7.0 Hz, 3H). 13C NMR (75 MHz, acetone-d6)
138.3, 137.9, 137.5, 136.0, 130.4, 129.5, 125.0, 114.7, 112.1, 109.1, 48.1,
41.0, 21.8. HRMS (ESI, m/z) Calculated for C20H18ClN2O5S [M ꢁ H]-:
433.0625, found: 433.0623. Purity by HPLC: 100%, tR ¼ 11.52 min.
d
(ppm) 170.2, 155.1, 153.3, 144.7, 144.5, 139.1, 137.6, 128.7, 128.1,
4-Chloro-2-((furan-2-ylmethyl)
amino)-5-(N-phenylsulfamoyl)
126.1, 115.4, 115.3, 112.4, 109.7, 109.5, 55.7, 41.4, 24.8. HRMS (ESI, m/
z) Calculated for C20H18ClN2O5S [M ꢁ H]-: 433.0625, found:
433.0624. Purity by HPLC: 99%, tR ¼ 11.38 min.
benzoic acid (3o): compound 3o was obtained as a white solid,
600 mg, 81% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d
(ppm) 13.49 (s, 1H), 10.35 (s, 1H), 8.70 (t, J ¼ 5.8 Hz, 1H), 8.40 (s,
4-Chloro-2-((furan-2-ylmethyl) amino)-5-(N-phenethylsulfamoyl)
benzoic acid (3i): compound 3i was obtained as a white solid,
612 mg, 77% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
1H), 7.60 (dd, J ¼ 1.9, 0.9 Hz, 1H), 7.26e7.17 (m, 2H), 7.10e6.92 (m,
4H), 6.43e6.30 (m, 2H), 4.54 (d, J ¼ 5.7 Hz, 2H). 13C NMR (75 MHz,
acetone-d6)
d (ppm) 169.7, 155.2, 152.7, 144.2, 139.2, 138.8, 138.2,
d
(ppm) 13.41 (s, 1H), 8.68 (t, J ¼ 6.0 Hz, 1H), 8.33 (s, 1H), 7.73e7.52
130.7, 125.7, 123.5, 121.3, 115.3, 112.0, 109.6, 109.3, 41.1. HRMS (ESI,
m/z) Calculated for C18H14ClN2O5S [M ꢁ H]-: 405.0312, found:
405.0312. Purity by HPLC: 100%, tR ¼ 11.10 min.
(m, 2H), 7.26e6.96 (m, 6H), 6.49e6.27 (m, 2H), 4.58 (d, J ¼ 5.9 Hz,
2H), 3.00 (q, J ¼ 7.2 Hz, 2H), 2.68 (q, J ¼ 7.7 Hz, 2H). 13C NMR
(75 MHz, acetone-d6)
d
(ppm) 170.1, 156.1, 153.1, 144.4, 140.5, 139.0,
4-Chloro-5-(N-cyclohexylsulfamoyl)-2-((furan-2-ylmethyl)
amino) benzoic acid (3p): compound 3p was obtained as a white
solid, 409 mg, 54% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
137.4, 130.4, 130.1, 128.0, 125.1, 115.5, 112.2, 109.9, 109.4, 46.2, 41.3,
37.5. HRMS (ESI, m/z) Calculated for C20H18ClN2O5S [M ꢁ H]-:
433.0625, found: 433.0625. Purity by HPLC: 98%, tR ¼ 11.60 min.
d
(ppm) 13.45 (s, 1H), 8.70 (s, 1H), 8.37 (s, 1H), 7.67e7.50 (m, 2H),
7.06 (s, 1H), 6.48e6.32 (m, 2H), 4.57 (d, J ¼ 5.5 Hz, 2H), 1.73e0.81
(m, 11H). 13C NMR (75 MHz, acetone-d6)
(ppm) 169.9, 154.7, 152.9,
4-Chloro-5-(N-ethylsulfamoyl)-2-((furan-2-ylmethyl)
amino)
benzoic acid (3j): compound 3j was obtained a white solid, 127 mg,
d
19% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d
(ppm) 13.44
144.2, 138.8, 136.9, 126.3, 115.2, 112.0, 109.7, 109.2, 54.1, 41.0, 35.1,
26.7, 26.3. HRMS (ESI, m/z) Calculated for C18H20ClN2O5S [M ꢁ H]-:
411.0783, found: 411.0783. Purity by HPLC: 98%, tR ¼ 11.75 min.
4-Chloro-2-((furan-2-ylmethyl) amino)-5-(piperidin-1-ylsulfonyl)
benzoic acid (3q): compound 3q was obtained as a white solid,
466 mg, 64% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
(s, 1H), 8.70 (t, J ¼ 5.7 Hz, 1H), 8.35 (s, 1H), 7.63 (dd, J ¼ 1.9, 0.9 Hz,
1H), 7.52 (t, J ¼ 5.7 Hz, 1H), 7.07 (s, 1H), 6.46e6.32 (m, 2H), 4.58 (d,
J ¼ 5.7 Hz, 2H), 2.80 (p, J ¼ 7.2 Hz, 2H), 0.96 (t, J ¼ 7.2 Hz, 3H). 13C
NMR (75 MHz, acetone-d6) d (ppm) 169.8, 154.9, 152.8, 144.2, 138.8,
137.2, 125.1, 115.2, 112.0, 109.6, 109.2, 41.1, 39.4, 16.0. HRMS (ESI, m/
z) Calculated for C14H14ClN2O5S [M ꢁ H]-: 357.0312, found:
357.0314. Purity by HPLC: 100%, tR ¼ 10.61 min.
d
(ppm) 13.49 (s, 1H), 8.73 (s, 1H), 8.33 (s, 1H), 7.63 (s, 1H), 7.10 (s,
1H), 6.49e6.32 (m, 2H), 4.59 (d, J ¼ 5.7 Hz, 2H), 3.06 (s, 4H),
1.66e1.30 (m, 6H). 13C NMR (75 MHz, acetone-d6)
(ppm) 169.8,
4-Chloro-5-(N,
N-dimethylsulfamoyl)-2-((furan-2-ylmethyl)
d
amino) benzoic acid (3k): compound 3k was obtained as a white
154.9, 152.9, 144.3, 139.4, 138.2, 123.2, 115.6, 112.1, 109.7, 109.3, 47.8,
41.8, 27.0, 25.1. HRMS (ESI, m/z) Calculated for C17H18ClN2O5S
[M ꢁ H]-: 397.0625, found: 397.0623. Purity by HPLC: 100%,
tR ¼ 11.97 min.
solid, 238 mg, 36% yield over 2 steps. 1H NMR (300 MHz, DMSO‑d6)
d
(ppm) 13.50 (s, 1H), 8.76 (s, 1H), 8.33 (s, 1H), 7.63 (dd, J ¼ 1.9,
0.9 Hz, 1H), 7.11 (s, 1H), 6.47e6.35 (m, 2H), 4.60 (d, J ¼ 5.8 Hz, 2H),
2.72 (s, 6H). 13C NMR (75 MHz, acetone-d6)
(ppm) 169.9, 155.0,
153.0, 144.4, 139.4, 138.6, 122.7, 115.8, 112.2, 109.9, 109.4, 41.2, 38.4.
d
17

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
5.1.3. General synthesis procedures of compound 3r
10g (20e73%) as white solids. Intermediates 10a-10g (1 eq.) were
then dissolved in anhydrous THF (0.3 M). To this, 2 M NaOH was
added until the pH was adjusted to 9. The reaction mixture was
then stirred at room temperature for 2 h before being diluted with
EtOAc. The layers were separated and the pH of aqueous phase was
adjusted to 3. The organic layers were combined and washed with
brine, dried over Na2SO4 and concentrated under reduced pressure.
Flash chromatography give compounds 11a-11g (36e93%) as white
solids.
4-chloro-5-(N-methylsulfamoyl)-2-(tetrahydrofuran-2-yl)
methyl) amino) benzoic acid (3r):Intermediates 2c (110 mg,
0.4 mmol, 1 eq.) were dissolved in ethylene glycol (0.2 mL) and to
this, tetrahydrofurfurylamine (0.12 mL, 1.2 mmol, 3 eq.) and trie-
thylamine (0.12 mL, 0.8 mmol, 2 eq.) were added. The reaction
mixture was heated to 130 ꢀC for 16 h. The mixture was diluted
with EtOAc/H2O and layers were separated. The pH of the aqueous
phase was adjusted to 3 and extracted with EtOAc. The organic
layers were washed with brine, dried over Na2SO4 and concen-
trated under reduced pressure. Column chromatography gave
compound 3r, 142 mg, 99% yield as white solid. 1H NMR (300 MHz,
5-Carbamoyl-4-chloro-2((furan-2-ylmethyl)amino) benzoic acid
(11a): compound 11a was obtained as a white solid, 35 mg, 93%
yield. 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 13.16 (s, 1H), 8.40 (s,
MeOD)
d
(ppm) 8.53 (s, 1H), 6.98 (s, 1H), 4.21e4.09 (m, 1H), 3.93 (dt,
1H), 7.95 (s, 1H), 7.70e7.57 (m, 2H), 7.35 (s, 1H), 6.90 (s, 1H),
J ¼ 8.5, 6.6 Hz, 1H), 3.78 (dt, J ¼ 13.9, 6.3 Hz, 1H), 3.45 (dd, J ¼ 13.4,
6.45e6.30 (m, 2H), 4.52 (d, J ¼ 5.3 Hz, 2H). 13C NMR (75 MHz,
3.9 Hz, 1H), 2.52 (s, 3H), 2.18e1.88 (m, 3H), 1.81e1.64 (m, 1H). 13C
DMSO‑d6) d (ppm) 170.0,168.4,152.7,152.1,143.6,137.5,134.1,123.5,
NMR (75 MHz, DMSO‑d6)
d
(ppm) 169.6, 154.3, 137.2, 136.4, 121.6,
113.3, 111.5, 109.9, 108.5, 40.1. HRMS (ESI, m/z) Calculated for
C
114.5, 109.4, 77.7, 68.5, 47.3, 29.5, 29.4, 26.3. HRMS (ESI, m/z)
Calculated for C13H16ClN2O5S [M ꢁ H]-: 347.0468, found: 347.0466.
Purity by HPLC: 99%, tR ¼ 9.77 min.
13H12ClN2O4 [MþH]þ: 295.0486, found: 295.0477. Purity by
HPLC: 99%, tR ¼ 9.43 min.
4-Chloro-2-((furan-2-ylmethyl)amino)-5-(methylcarbamoyl)ben-
zoic acid (11b): compound 11b was obtained as a white solid,
5.1.4. General synthesis procedures of compound 11a-11g
108 mg, 70% yield. 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 13.17 (s,
4-Chloro-2-fluorobenzoic acid (5 g, 28.6 mmol, 1 eq.) was dis-
solved in concentrated sulfuric acid (50 mL) at 0 ꢀC. To this, N-
iodosuccinimide (7 g, 31.5 mmol, 1.1 eq.) was added, portion-wise.
The reaction mixture was stirred at 0 ꢀC at 4 h. The reaction was
then warmed up to room temperature and poured onto water
(120 mL). 1 N sodium thiosulfate (40 mL) was added. The precipi-
tate was washed with water and dried. The crude residue was
resuspended in hot 1:1 ethanol:water. The yellow precipitate was
discarded. Additional water was added, and the white precipitate
was collected and dried to afford intermediate 6. Intermediate 6
(3.9 g,12.9534 mmol,1 eq.) was then dissolved in MeOH (40 mL). To
this, SOCl2 (5 mL, 5 eq.) was slowly added at 0 ꢀC. The reaction
mixture was refluxed at 70 ꢀC for 2 h before cooling to room
temperature. MeOH was removed in vacuo and the resulting res-
idue was poured onto an ice-water mixture and extracted with
EtOAc. The combined organic extracts were washed with satd
NaHCO3 solution, dried over Na2SO4, and concentrated under
reduced pressure to afford intermediate 7 (3.55 g, 87%) as a white
solid. The product was directly applied to next steps without pu-
rification. Intermediate 7 (3.6 g, 1 eq.) was dissolved in anhydrous
THF (35 mL) under Ar and cooled to ꢁ40 ꢀC. i-PrMgBr (18 mL,
0.75 M,1.2 eq.) was added and stirred at ꢁ40 ꢀC for 2 h. The reaction
system was then evacuated and purged with dry CO2 and stirred for
another 16 h before being diluted with EtOAc/H2O. The pH of the
aqueous phase was adjusted to 3 and extracted with EtOAc. The
organic layers were washed with brine, dried over Na2SO4 and
concentrated under reduced pressure. Column chromatography
(hexanes-EtOAc-AcOH 4:1:0.05) intermediate 8 (2.61 g, 82%) as a
white solid. Intermediate 8 (1 eq.) was dissolved SOCl2 (5 eq.) and
heated to 60 ꢀC for 3 h. The thionyl chloride was then removed in
vacuo and got a yellow solid. The solid was then resuspended in
anhydrous THF (0.3 M) and cooled to 0 ꢀC. To this, the appropriate
amine (1.2 eq.) was added to reaction and stirred at 0 ꢀC for 2 h
before quenched by satd NaHCO3 solution. The layers were then
extracted with EtOAc. The organic extracts were combined and
washed with brine, dried over Na2SO4 and concentrated under
reduced pressure. Flash chromatography gave the desired com-
pounds 9a-9g (81e91%) as white solids. Intermediates 9a-9g (1 eq.)
were dissolved in anhydrous DMF (0.3 M). To this, furfurylamine (3
eq.) and triethylamine (3 eq.) were added and stirred at 80 ꢀC for
16 h before being diluted with EtOAc/H2O. The layers were sepa-
rated, and the organic layers were combined. The organic layer was
washed with brine, dried over Na2SO4 and concentrated under
reduced pressure. Flash chromatography gave intermediates 10a-
1H), 8.40 (s, 1H), 8.15 (d, J ¼ 4.8 Hz, 1H), 7.88 (s, 1H), 7.65e7.54 (m,
1H), 6.91 (s, 1H), 6.41 (dd, J ¼ 3.2 Hz, 1.9 Hz, 1H), 6.34 (d, J ¼ 3.3 Hz,
1H), 4.52 (d, J ¼ 5.1 Hz, 2H), 2.70 (d, J ¼ 4.6 Hz, 3H). 13C NMR
(75 MHz, DMSO‑d6)
d (ppm) 169.9, 167.0, 152.6, 152.0, 143.5, 137.4,
133.8, 123.6, 113.1, 111.4, 109.8, 108.4, 40.0, 27.0. HRMS (ESI, m/z)
Calculated for C14H12ClN2O4 [M ꢁ H]-: 307.0486, found: 307.0482.
Purity by HPLC: 99%, tR ¼ 9.72 min.
4-Chloro-5-(ethylcarbamoyl)-2((furan-2-ylmethyl)amino)benzoic
acid (11c): compound 11c was obtained as a white solid, 40 mg, 36%
yield. 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 13.17 (s, 1H), 8.41 (s,
1H), 8.21 (d, J ¼ 6.0 Hz, 1H), 7.85 (s, 1H), 7.61 (d, J ¼ 1.8 Hz, 1H), 6.90
(s, 1H), 6.41 (dd, J ¼ 3.2, 1.8 Hz, 1H), 6.34 (d, J ¼ 3.3 Hz, 1H), 4.52 (d,
J ¼ 5.1 Hz, 2H), 3.27e3.11 (m, 2H),1.07 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI,
m/z) Calculated for C15H14ClN2O4 [M ꢁ H]-: 321.0642, found:
321.0641. Purity by HPLC: 95%, tR ¼ 10.13 min.
4-Chloro-5-(dimethylcarbamoyl)-2((furan-2-ylmethyl)amino)
benzoic acid (11d): compound 11d was obtained as a white solid,
25 mg, 37% yield. 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 13.17 (s,
1H), 7.67 (s, 1H), 7.62 (d, J ¼ 2.1 Hz, 1H), 6.95 (s, 1H), 6.45e6.33 (m,
2H), 4.50 (s, 2H), 2.95 (s, 3H), 2.79 (s, 3H). HRMS (ESI, m/z) Calcu-
lated for C15H14ClN2O4 [M ꢁ H]-: 321.0642, found: 321.0650. Purity
by HPLC: 99%, tR ¼ 10.17 min.
4-Chloro-2-((furan-2-ylmethyl)amino)-5-(morpholine-4-
carbonyl)benzoic acid (11e): compound 11e was obtained as a white
solid, 80 mg, 62% yield. 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 13.20
(s, 1H), 8.36 (s, 1H), 7.71 (d, J ¼ 1.4 Hz, 1H), 7.62 (d, J ¼ 2.3 Hz, 1H),
6.96 (d, J ¼ 1.4 Hz, 1H), 6.46e6.32 (m, 2H), 4.51 (d, J ¼ 5.1 Hz, 2H),
3.55 (d, J ¼ 27.2 Hz, 7H), 3.18 (s, 2H). 13C NMR (75 MHz, DMSO‑d6)
d
(ppm) 169.8, 166.5, 152.6, 151.8, 143.6, 136.5, 132.6, 122.5, 112.5,
111.5, 110.4, 108.5, 67.1, 66.9, 47.9, 42.7, 40.1. HRMS (ESI, m/z)
Calculated for C17H18ClN2O5 [MþH]þ: 365.0904, found: 365.0911.
Purity by HPLC: 97%, tR ¼ 10.09 min.
4-Chloro-2-((furan-2-ylmethyl)amino)-5-(piperidine-1-carbonyl)
benzoic acid (11f): compound 11f was obtained as a white solid,
15 mg, 44% yield. 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 13.18 (s,
1H), 8.32 (s, 1H), 7.69e7.58 (m, 2H), 6.95 (d, J ¼ 1.3 Hz, 1H),
6.46e6.32 (m, 2H), 4.51 (d, J ¼ 4.9 Hz, 2H), 3.59 (s, 1H), 3.52 (s, 1H),
3.13 (d, J ¼ 5.5 Hz, 2H), 1.55 (m, 7H). 13C NMR (75 MHz, DMSO‑d6)
d
(ppm) 169.8, 166.2, 152.6, 151.6, 143.6, 136.5, 132.1, 123.4, 112.6,
111.5, 110.3, 108.4, 48.4, 42.9, 40.1, 27.0, 26.2, 24.9. HRMS (ESI, m/z)
Calculated for C18H18ClN2O4 [M ꢁ H]-: 361.0955, found: 361.0954.
Purity by HPLC: 96%, tR ¼ 11.20 min.
4-Chloro-2((furan-2-ylmethyl)amino)-5-(phenylcarbamoyl)ben-
zoic acid (11g): compound 11g was obtained as a white solid,
18

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
153 mg, 40% yield. 1H NMR (300 MHz, DMSO‑d6)
d
(ppm) 13.24 (s,
gave compound 16 (16 mg, 93%) as a white solid. 1H NMR
1H), 10.29 (s, 1H), 8.49 (s,1H), 7.98 (s, 1H), 7.72e7.59 (m, 3H), 7.32 (t,
J ¼ 7.8 Hz, 2H), 7.07 (t, J ¼ 7.2 Hz, 1H), 6.99 (s, 1H), 6.42 (dd, J ¼ 3.2,
1.8 Hz, 1H), 6.37 (s, 1H), 4.56 (d, J ¼ 5.5 Hz, 2H). 13C NMR (75 MHz,
(300 MHz, CDCl3)
d
(ppm) 7.71 (s, 1H), 7.41 (dd, J ¼ 1.9, 0.9 Hz, 1H),
6.78 (s, 1H), 6.39e6.24 (m, 3H), 5.10 (q, J ¼ 7.3 Hz, 1H), 4.72 (t,
J ¼ 5.4 Hz, 1H), 4.39 (d, J ¼ 5.6 Hz, 2H), 3.56 (s, 1H), 2.59 (d,
J ¼ 5.4 Hz, 3H). HRMS (ESI, m/z) Calculated for C14H13ClF3N2O4S
[M ꢁ H]-: 397.0237, found: 397.0238.
DMSO‑d6)
d (ppm) 169.8, 165.3, 152.6, 152.3, 143.6, 140.1, 137.6,
133.9,129.6,124.5,123.8,120.4,113.2,111.5,109.7,108.5, 40.0. HRMS
(ESI, m/z) Calculated for C19H14ClN2O4 [M ꢁ H]-: 369.0642, found:
369.0656. Purity by HPLC: 97%, tR ¼ 9.29 min.
Methyl
4-chloro-2-((furan-2-ylmethyl)amino)-5-(N-methyl-
sulfamoyl)benzoate (17): Intermediate 3c (100 mg, 0.29 mmol,1 eq.)
and CDI (52 mg, 0.319 mmol, 1.1 eq.) were dissolved in THF (2 mL),
under Ar. The mixture was stirred at 40 ꢀC and a yellow homoge-
nous solution formed after 3 h. In a flask, MeOH (0.5 mL) and t-
BuOK (39 mg, 0.348 mmol, 1.2 eq.) were dissolved in THF (0.35 mL).
The MeOH solution was then added to the mixture at room tem-
perature and stirred for a further 2 h. The mixture was diluted with
EtOAc and washed with satd NaHCO3 solution. The organic layer
and aq layer were separated, and aq layer extracted with further
EtOAc. The organic extracts were combined and dried with Na2SO4,
and the solvent was removed under reduced pressure. Flash chro-
matography (hexanes-EtOAc 75:25) gave compound 17 (40 mg,
5.1.5. General synthesis procedures of compounds 12, 16e20, 24,
and 26
2-Chloro-4-((furan-2-ylmethyl)amino)-5-(hydroxymethyl)-N-
methylbenzenesulfonamide (12): Intermediate 3c (100 mg,
0.29 mmol, 1 eq.) was dissolved in anhydrous THF (3 mL). To this, a
solution of BH3-THF (1 M in THF, 0.87 mL, 0.87 mmol, 3 eq.) was
added and stirred overnight at 60 ꢀC. The reaction was monitored
using TLC. The reaction was diluted with EtOAc and washed with
stad NaHCO3 solution. The organic layer was dried over Na2SO4,
filtered, and solvent removed under reduced pressure. Flash chro-
matography (hexanes-EtOAc 70:30) gave compound 12 (77 mg,
40%) as a white solid. 1H NMR (300 MHz, CDCl3)
d (ppm) 8.67 (d,
80%) as a white solid. 1H NMR (300 MHz, CDCl3)
d
(ppm) 7.74 (s,1H),
J ¼ 6.1 Hz, 1H), 8.62 (s, 1H), 7.41 (s, 1H), 6.88 (s, 1H), 6.40e6.26 (m,
7.41 (dd, J ¼ 1.9, 0.9 Hz, 1H), 6.80 (s, 1H), 6.33 (m, 1H), 5.92 (s, 1H),
2H), 4.75 (q, J ¼ 5.2 Hz, 1H), 4.46 (d, J ¼ 5.6 Hz, 2H), 3.9 (s, 3H), 2.62
4.76 (d, J ¼ 5.5 Hz, 1H), 4.71 (s, 2H), 4.41 (d, J ¼ 5.6 Hz, 2H), 2.60 (d,
(d, J ¼ 5.4 Hz, 3H). 13C NMR (75 MHz, acetone-d6)
d (ppm) 168.8,
J ¼ 5.5 Hz, 3H), 1.74 (s, 1H). 13C NMR (75 MHz, acetone-d6)
d
(ppm)
154.6, 152.8, 144.3, 138.8, 137.2, 123.7, 115.4, 112.1, 109.7, 109.3, 53.3,
41.2, 30.0. HRMS (ESI, m/z) Calculated for C14H14ClN2O5S [M ꢁ H]-:
357.0312, found: 357.0313. Purity by HPLC: 100%, tR ¼ 11.47 min.
4-Chloro-2-((furan-2-ylmethyl)amino)-5-(N-methylsulfamoyl)
153.7, 152.3, 143.9, 133.4, 132.7, 125.4, 124.0, 113.4, 112.0, 108.9, 63.5,
41.5, 30.0. HRMS (ESI, m/z) Calculated for
C13H15ClN2O4SNa
[MþNa]þ: 353.0339, found: 353.0341. Purity by HPLC: 100%,
tR ¼ 9.93 min.
benzamide (18): Intermediate 3c (800 mg, 2.3202 mmol, 1 eq.) and
CDI (413.5 mg, 2.5522 mmol, 1.1 eq.) were dissolved in anhydrous
THF (8 mL), under Ar. The mixture was stirred at 40 ꢀC and a yellow
homogenous solution formed after 3 h. The 28% NH4OH solution
(0.5 mL, 2.784 mmol, 1.2 eq.) was then added to the mixture at
room temperature and stirred for a further 2 h. The mixture was
diluted with EtOAc and washed with satd NaHCO3. The organic
layer and aq layer were separated, and aq layer extracted with
further EtOAc. The organic extracts were combined and dried with
Na2SO4, and the solvent was removed under reduced pressure.
Flash chromatography (hexanes-EtOAc 70:30) gave compound 18
(679 mg, 85%) as a white solid. 1H NMR (300 MHz, DMSO‑d6)
2-chloro-4-((furan-2-ylmethyl)amino)-N-methyl-5-(2,2,2-
trifluoro-1-hydroxyethyl)benzene sulfonamide (16): as shown in
Scheme 5, the synthesis route of 16 started from compound 12 as
intermediate. Intermediate 12 (77 mg, 0.233 mmol, 1 eq.) was
dissolved in DMF (2 mL) at 0 ꢀC. To this, Dess-Martin periodinane
(118.5 mg, 0.279 mmol, 1 eq.) was added, portion-wise and the
reaction was stirred for 6 h, warming to room temperature. The
reaction was monitored by TLC and when the starting material was
consumed, the reaction was poured into EtOAc, washed with 1:1
10% Na2S2O3/satd NaHCO3. The organic layer was dried over
Na2SO4, filtered, and solvent removed under reduced pressure.
Flash chromatography (hexanes-EtOAc 70:30) afforded intermedi-
ate 13 (50 mg, 66%) as a white solid. Aldehyde intermediate 13
(28 mg, 0.085 mmol, 1 eq.) was dissolved in anhydrous CH2Cl2
(2 mL) and cooled to 0 ꢀC. To this, triethylamine (13 mg,
0.128 mmol, 1.5 eq.), Boc2O (37 mg, 0.170 mmol, 2 eq.), and DMAP
(10 mg, 0.085 mmol,1 eq.), were added. The reaction was stirred for
3 h, allowing to warm to room temperature. The reaction was
diluted with CH2Cl2 and washed with brine. The organic layer was
dried over Na2SO4, filtered, solvent removed under reduced pres-
sure. Flash chromatography (hexanes-EtOAc 7:3) gave intermediate
14 (37 mg, 99%) as a yellow solid. Intermediate 14 (36.5 mg,
0.085 mmol, 1 eq.) was then dissolved in anhydrous THF (3 mL) and
the mixture cooled to 0 ꢀC. To this, TMSCF3 (0.04 mL, 0.17 mmol, 2
eq.) and TBAF (1 M in THF, 0.04 mL, 0.043 mmol, 0.5 eq.) were
added, dropwise. The mixture was left to stir overnight, allowing to
warm to room temperature. When TLC indicated consumption of
aldehyde 14, water (1 mL) was added to the reaction and stirred for
1 h. The reaction was diluted with EtOAc and washed with brine.
The organic layer was dried over Na2SO4, filtered, solvent removed
under reduced pressure. Flash chromatography (hexanes-EtOAc
85:15) gave intermediate 15 (21 mg, 50%) as a white solid. Inter-
mediate 15 (21 mg, 0.042 mmol) was dissolved in MeOH (2 mL) and
5 M HCl (2 mL) was added. The reaction was stirred at room tem-
perature for 1 h before being diluted with EtOAc/H2O. The organic
layers were combined, washed by brine, dried over Na2SO4, and
concentrated under reduced pressure. Column chromatography
d
(ppm) 8.89 (t, J ¼ 5.8 Hz, 1H), 8.21 (s, 1H), 8.09 (s, 1H), 7.62 (d,
J ¼ 18 Hz, 1H), 7.46 (s, 1H), 7.23 (q, J ¼ 4.9 Hz, 1H), 6.98 (s, 1H),
6.46e6.32 (m, 2H), 4.50 (d, J ¼ 5.8 Hz, 2H), 2.39 (d, J ¼ 4.9 Hz, 3H).
13C NMR (75 MHz, acetone-d6)
d (ppm) 172.0, 154.5, 153.3, 144.2,
137.2,134.5,122.9,115.0,113.8,112.1,109.2, 41.2, 30.0. HRMS (ESI, m/
z) Calculated for C13H13ClN3O4S [M ꢁ H]-: 342.0315, found:
342.0315. Purity by HPLC: 100%, tR ¼ 9.95 min.
4-Chloro-2-((furan-2-ylmethyl)amino)-N-methyl-5-(N-methyl-
sulfamoyl)benzamide (19): Intermediate 3c (200 mg, 0.58 mmol, 1
eq.) and CDI (103.4 mg, 0.638 mmol, 1.1 eq.) were dissolved in THF
(2 mL), under Ar. The mixture was stirred at 40 ꢀC and a yellow
homogenous solution formed after 3 h. Methylamine solution
(0.35 mL, 2 M in THF,1.2 eq.) was then added to the mixture at room
temperature and stirred for a further 2 h. The mixture was diluted
with EtOAc and washed with satd NaHCO3. The organic layer and aq
layer were separated and aq layer extracted with further EtOAc. The
organic extracts were combined and dried over Na2SO4, and the
solvent was removed under reduced pressure. Flash chromatog-
raphy (hexanes-EtOAc 3:1) gave compound 19 (51 mg, 25%) as a
white solid. 1H NMR (300 MHz, CDCl3)
d (ppm) 8.86 (s, 1H), 8.04 (s,
1H), 7.44e7.37 (m, 1H), 6.85 (s, 1H), 6.39e6.25 (m, 3H), 4.77 (q,
J ¼ 5.5 Hz, 1H), 4.41 (d, J ¼ 5.6 Hz, 2H), 2.98 (d, J ¼ 4.8 Hz, 3H), 2.61
(d, J ¼ 5.4 Hz, 3H). 13C NMR (75 MHz, acetone-d6)
d (ppm) 170.0,
154.1, 153.3, 144.2, 136.8, 133.6, 123.0, 114.9, 114.9, 112.1, 109.2, 41.2,
30.0, 27.3. HRMS (ESI, m/z) Calculated for C14H17ClN3O4S [MþH]þ:
358.0628, found: 358.0634. Purity by HPLC: 100%, tR ¼ 10.50 min.
19

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
4-Chloro-2-((furan-2-ylmethyl) amino)-N, N-dimethyl-5-(N-
methylsulfamoyl) benzamide (20): Intermediate 3c (300 mg,
0.87 mmol, 1 eq.) and CDI (183 mg, 1.13 mmol, 1.2 eq.) were dis-
solved in anhydrous THF (3 mL), under Ar. The mixture was stirred
at 40 ꢀC and a yellow homogenous solution formed after 3 h. The
dimethylamine solution (0.6 mL, 2 M in THF, 1.2 eq.) was then
added to the mixture at room temperature and stirred for a further
2 h. The mixture was diluted with EtOAc and washed with satd
NaHCO3 solution. The organic layer and aq layer were separated
and aq layer extracted with further EtOAc. The organic extracts
were combined and dried with Na2SO4, and the solvent was
removed under reduced pressure. Flash chromatography (hexanes-
EtOAc 3:1) gave compound 20 (237 mg, 73%) as a white solid. 1H
Intermediate 23 (127.2 mg, 0.391 mmol, 1 eq.) was dissolved in
EtOH (2 mL). In a separate flask, the hydroxylamine hydrochloride
(55 mg, 0.782 mmol, 2 eq.) was dissolved in NaHCO3 solution
(100 mg, 1.2 M, 3 eq.). This solution was then added to the reaction
mixture and heated to reflux for 16 h. The reaction mixture was
then diluted with EtOAc/H2O and the layers were separated. The
organic layers were combined, dried over Na2SO4 and concentrated
under reduced pressure. The crude product 25 was used directly in
the next step without further purification. To a stirred solution of
intermediate 25 (64 mg, 0.1882 mmol, 1 eq.) in 1,4-dioxane (1 mL)
was added CDI (45.8 mg, 0.282 mmol, 1.5 eq.) and DBU (31.5 mg,
0.207 mmol, 1.1 eq.). The reaction was heated to reflux for 16 h
before being diluted with EtOAc/H2O. The layers were separated
and the pH of aq layer was adjusted to 5. The aq layer was then
extracted with EtOAc and the organic layers were combined,
washed with brine, dried over Na2SO4 and concentrated under
reduced pressure. Flash chromatography (CH2Cl2eMeOH 95:5)
gave compound 26 (62 mg, 86%) as a white solid. 1H NMR
NMR (300 MHz, CDCl3)
1H), 6.86 (s, 1H), 6.31 (m, 2H), 4.80 (s, 1H), 4.36 (s, 2H), 3.08 (s, 6H),
2.59 (s, 3H). 13C NMR (75 MHz, acetone-d6)
(ppm) 169.6, 153.4,
d
(ppm) 7.84 (s, 1H), 7.39 (dd, J ¼ 1.9, 0.8 Hz,
d
151.5, 144.0, 135.2, 133.4, 123.9, 119.5, 114.5, 112.0, 109.1, 41.3, 30.0.
HRMS (ESI, m/z) Calculated for C15H17ClN3O4S [M ꢁ H]-: 370.0628,
found: 370.0630. Purity by HPLC: 100%, tR ¼ 9.96 min.
(300 MHz, DMSO‑d6
d (ppm) 13.23 (s, 1H), 8.18e8.11 (m, 1H), 7.63
2-Chloro-4-((furan-2-ylmethyl) amino)-N-methyl-5-((1H-tetra-
zol-5-yl) benzenesulfonamide (24): Intermediate 2c (800 mg,
3.01 mmol, 1 eq.) and CDI (633 mg, 3.91 mmol, 1.3 eq.) were dis-
solved in anhydrous THF (9 mL) under Ar. The mixture was stirred
at 40 ꢀC and a yellow homogenous solution formed after 3 h.
Ammonium hydroxide solution (28%, 0.8 mL, 1.3 eq.) was added at
room temperature and stirred for a further 2 h. The mixture was
diluted with EtOAc and washed with satd NaHCO3 solution. The
layers were separated and aq layer was extracted with EtOAc. The
organic extracts were combined and dried with Na2SO4, and the
solvent was removed under reduced pressure. Flash chromatog-
raphy (CH2Cl2eMeOH 95:5) gave intermediate 21 (664 mg, 83%) as
a white solid. Intermediate 21 (50 mg, 0.188 mmol, 1 eq.) was then
dissolved in anhydrous DMF (1 mL) at 0 ꢀC. To this, cyanuric chlo-
ride (52 mg, 0.282 mmol, 1.5 eq.) was added and stirred for 3 h,
warming to room temperature. The reaction mixture was then
diluted with EtOAc/H2O. The organic layers were combined and
washed with brine, dried over Na2SO4 and the solvent was removed
under reduced pressure. Flash chromatography (hexanes-EtOAc
7:3) gave intermediate 22 (44 mg, 94%) as a white solid. Interme-
diate 22 (114 mg, 0.460 mmol, 1 eq.) was subsequently dissolved in
anhydrous DMF (2 mL) at 0 ꢀC. To this, furfurylamine (0.06 mL,
0.690 mmol, 1.5 eq.) and triethylamine (0.1 mL, 0.69 mL 1.5 eq.)
were added. The reaction mixture was heated to 50 ꢀC for 16 h. The
mixture was then diluted with EtOAc/H2O and layers separated. The
pH of the aqueous phase was adjusted to 3 and extracted with
EtOAc. The organic layers were washed with brine, dried over
Na2SO4 and concentrated under reduced pressure. Flash chroma-
tography (hexanes-EtOAc 3:1) gave intermediate 23 (150 mg, 72%)
as a white solid. Intermediate 23 (107 mg, 0.328 mmol, 1 eq.), NaN3
(28 mg, 0.427 mmol, 1.3 eq.) and Et3N$HCl (59 mg, 0.427 mmol, 1.3
eq.) were then dissolved in toluene (2 mL). The reaction was heated
to 100 ꢀC and stirred for 48 h. The reaction was then quenched with
H2O and the layers were separated. The aq layer was extracted with
EtOAc. The combined organic extracts were washed with brine,
dried over Na2SO4 and concentrated under reduced pressure. Flash
chromatography (hexanes-EtOAc-AcOH 3:1:0.02) gave compound
24 (120 mg, 99%) as a white solid. 1H NMR (300 MHz, DMSO‑d6)
(d, J ¼ 1.4 Hz, 2H), 7.44e7.34 (m, 1H), 7.16 (s, 1H), 6.46e6.36 (m, 2H),
4.63 (d, J ¼ 5.6 Hz, 2H), 2.41 (d, J ¼ 4.9 Hz, 3H). 13C NMR (75 MHz,
DMSO‑d6)
d (ppm) 160.2, 158.3, 152.0, 150.2, 143.8, 135.9, 133.2,
123.6, 114.6, 111.5, 108.8, 104.9, 29.31. HRMS (ESI, m/z) Calculated
for C14H12ClN4O5S [M ꢁ H]-: 383.0217, found: 383.0211. Purity by
HPLC: 97%, tR ¼ 10.64 min.
5.1.6. General synthesis procedures of compounds 27-29
The appropriate benzoic acid (1 eq.), furfurylamine (2 eq), CuI
(0.1 eq), K2CO3 (2 eq.) and N,N-dimethylglycein (0.2 eq.) were dis-
solved in DMSO (0.3 M). The reaction was heated to 40 ꢀC for 12 h
before being diluted with EtOAc/1 M HCl solution. The layers were
separated and the pH of aq layer was adjusted to 3. The aq layer was
then extracted with EtOAc and the organic layers were combined,
washed with brine, dried over Na2SO4, and concentrated under
reduced pressure. Column chromatography gave the desired com-
pounds 27e29 (34e71%).
4-Fluoro-2-((furan-2-ylmethyl) amino)benzoic acid (27): com-
pound 27 was obtained as a white solid, 431 mg, 71% yield. 1H NMR
(300 MHz, DMSO‑d6)
d (ppm) 12.79 (s, 1H), 8.34 (s, 1H), 7.84 (dd,
J ¼ 8.8, 7.1 Hz, 1H), 7.66e7.54 (m, 1H), 6.65 (dd, J ¼ 12.7, 2.4 Hz, 1H),
6.45e6.32 (m, 3H), 4.45 (d, J ¼ 5.1 Hz, 2H). 13C NMR (75 MHz,
acetone-d6)
d
(ppm) 170.4, 168.7 (d, J ¼ 248.8 Hz), 154.8 (d,
J ¼ 12.6 Hz), 153.6, 143.9, 136.3 (d, J ¼ 11.8 Hz), 111.9, 108.8, 108.5,
103.7 (d, J ¼ 22.8 Hz), 99.1 (d, J ¼ 26.2 Hz), 41.1. HRMS (ESI, m/z)
Calculated for C12H9FNO3 [M ꢁ H]-: 234.0566, found: 234.0567.
Purity by HPLC: 100%, tR ¼ 11.21 min.
4-Chloro-2-((furan-2-ylmethyl) amino) benzoic acid (28): com-
pound 28 was obtained as a white solid, 151 mg, 34% yield. 1H NMR
(300 MHz, DMSO‑d6)
d (ppm) 12.95 (s, 1H), 8.28 (s, 1H), 7.78 (d,
J ¼ 8.5 Hz, 1H), 7.61 (d, J ¼ 2.1 Hz, 1H), 6.89 (d, J ¼ 2.0 Hz, 1H), 6.61
(dd, J ¼ 8.5, 2.0 Hz, 1H), 6.41 (dd, J ¼ 3.2, 1.9 Hz, 1H), 6.34 (d,
J ¼ 3.1 Hz, 1H), 4.48 (d, J ¼ 4.4 Hz, 2H). 13C NMR (75 MHz, acetone-
d6)
d (ppm) 170.7, 153.8, 153.5, 144.1, 141.9, 135.2, 116.6, 112.7, 112.1,
110.6, 108.9, 41.2. HRMS (ESI, m/z) Calculated for C12H9ClNO3
[M ꢁ H]-: 250.0271, found 250.0271. Purity by HPLC: 100%,
tR ¼ 11.66 min.
4-Bromo-2-((furan-2-ylmethyl) amino) benzoic acid (29): com-
pound 29 was obtained as a white solid, 188 mg, 46% yield. 1H NMR
d
(ppm) 8.59 (s, 1H), 8.47 (s, 1H), 7.64 (s, 1H), 7.41 (d, J ¼ 5.1 Hz, 1H),
7.21 (s, 1H), 6.43 (s, 2H), 4.69 (d, J ¼ 4.8 Hz, 2H), 2.43 (d, J ¼ 4.9 Hz,
3H). 13C NMR (75 MHz, acetone-d6)
(ppm) 153.0, 151.2, 144.3,
(300 MHz, DMSO‑d6)
d (ppm) 12.96 (s, 1H), 8.25 (s, 1H), 7.69 (d,
d
J ¼ 8.5 Hz, 1H), 7.64e7.54 (m, 1H), 7.03 (d, J ¼ 1.6 Hz, 1H), 6.74 (dd,
136.7, 133.9, 124.6, 115.3, 112.1, 109.4, 41.3, 31.6. HRMS (ESI, m/z)
Calculated for C13H12ClN6O3S [M ꢁ H]-: 367.0380, found: 367.0381.
Purity by HPLC: 97%, tR ¼ 10.58 min.
J ¼ 8.5, 1.6 Hz, 1H), 6.45e6.29 (m, 2H), 4.48 (s, 2H). 13C NMR
(75 MHz, acetone-d6) d (ppm) 170.6,153.6, 153.3, 143.9, 135.0, 130.6,
119.4, 115.6, 111.9, 110.8, 108.7, 41.0. HRMS Calculated for
2-Chloro-4-((furan-2-ylmethyl)amino)-N-methyl-5-(5-oxo-2,5-
C
12H9BrNO3 [M ꢁ H]-: 293.9766, found: 293.9766. Purity by HPLC:
dihydro-1,2,4-oxadiazol-3-yl)benzenesulfonamide
(26):
98%, tR ¼ 11.85 min.
20

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
5.1.7. General synthesis procedures of compounds 32-38
143.8, 137.0, 132.6, 127.1 (q, J ¼ 283.2 Hz), 119.0, 117.5, 112.7, 112.0,
108.6, 71.7 (q, J ¼ 31.8 Hz), 41.9. HRMS (ESI, m/z) Calculated for
The appropriate benzaldehyde (1 eq.) was dissolved in anhy-
drous THF (0.3 M) and cooled to ꢁ20 ꢀC. To this, TMSCF3 (1.2 eq.)
and TBAF (0.2 eq.) were added, slowly. The reaction was allowed to
attain room temperature and stirred for 3 h followed by the addi-
tion of 1 mL of 1 M HCl solution. The mixture was then diluted with
EtOAc/H2O and the layers were separated. The organic layer was
dried over Na2SO4, filtered, solvent removed under reduced pres-
sure. Column chromatography gave the desired intermediates 30a-
30d (53e88%).
C
13H12ClF3NO2 [MþH]þ: 306.0509, found: 306.0504. Purity by
HPLC: 100%, tR ¼ 12.03 min.
1-(4-Bromo-2-((furan-2-ylmethyl) amino) phenyl)-2, 2, 2-
trifluoroethan-1-ol (34): compound 34 was obtained as a white
solid, 84 mg, 50% yield. 1H NMR (300 MHz, CDCl3)
d (ppm) 7.39 (s,
1H), 7.04 (d, J ¼ 8.1 Hz, 1H), 6.96e6.85 (m, 2H), 6.38e6.21 (m, 2H),
5.21e4.96 (m, 2H), 4.32 (d, J ¼ 5.2 Hz, 2H), 2.98 (s, 1H). 13C NMR
(75 MHz, acetone-d6) d (ppm) 154.2, 149.8, 143.7, 132.8, 125.2, 125.1,
Synthesis method of 31a and 31d: Intermediates 30a and 30d (1
eq.) were dissolved in MeOH (0.3 M) and PdeC (1 mmol starting
material with 20e30 mg PbeC) was added. To this, 15 psi H2 was
applied to the reaction mixture with oscillation shaking for 4 h. The
reaction was monitored by TLC. After TLC indicated consumption of
starting material, the reaction mixture was filtered through celite
and quenched by satd ammonium chloride. The layers were sepa-
rated and the aqueous phase was extracted with EtOAc. The com-
bined organic extracts were washed with brine, dried over Na2SO4
and concentrated under reduced pressure to afford intermediate
compounds 31a and 31d (77e86%) as white solids. The crude
products were used directly in the next step without further
purification.
Synthesis method of 31b and 31c: Fe powder (100 eq.), and
NH4Cl powder (50 eq.) were dissolved in EtOH (0.05 M) and placed
in a flask with reflux condenser. The mixture was then heated to
reflux. In a separate flask, intermediates 30b and 30c (1 eq.), was
dissolved in EtOH (0.1 M). The EtOH solution was then added to the
reaction mixture and stirred. The reaction was monitored by TLC.
After TLC indicated consumption of starting material, the reaction
mixture was filtered and quenched by satd ammonium chloride.
The layers were separated and the aq phase was extracted with
EtOAc. The combined organic extracts were washed with brine,
dried over Na2SO4 and concentrated under reduced pressure to
afford intermediate compounds 31b and 31c (49e71%) as white
solids. The crude products were used directly in the next step
without further purification.
Aniline intermediates 31a-31d (1 eq.) were dissolved in anhy-
drous 1,2-dichloroethane (0.3 M), under Ar. Following the addition
of appropriate aldehyde (2 eq.) and AcOH (0.5 eq.), the reaction was
stirred at 60 ꢀC for 1 h. Na(OAc)3BH (1.5 eq.) was then added to the
reaction and stirred for 16 h. The reaction solvent was removed
under reduced pressure and the crude residue was resuspended in
EtOH. NaBH4 (4 eq.) was added and stirred at 60 ꢀC for 1 h. The
reaction was quenched with EtOAc/H2O and the layers were sepa-
rated. The aq phase was extracted with EtOAc. The combined
organic extracts were washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. Column chromatography
gave the compounds 32e38 (29e67%)
120.4, 119.4, 115.5, 111.9, 108.5, 71.6 (q, J ¼ 31.7, 31.2 Hz), 41.8. HRMS
(ESI, m/z) Calculated for C13H10BrF3NO2 [M ꢁ H]-: 347.9847, found:
347.9847. Purity by HPLC: 99%, tR ¼ 12.07 min.
2,
2,
2-trifluoro-1-(2-((furan-2-ylmethyl)
amino)-4-
methoxyphenyl) ethan-1-ol (35): compound 35 was obtained as a
white solid, 32 mg, 29% yield. 1H NMR (300 MHz, DMSO‑d6)
d
(ppm)
7.56 (dd, J ¼ 1.9, 0.9 Hz, 1H), 7.16 (d, J ¼ 8.2 Hz, 1H), 6.72 (d,
J ¼ 5.4 Hz, 1H), 6.37 (dd, J ¼ 3.2, 1.8 Hz, 1H), 6.24 (dd, J ¼ 3.1, 0.9 Hz,
1H), 6.22e6.15 (m, 2H), 6.11 (t, J ¼ 5.8 Hz, 1H), 5.34e5.16 (m, 1H),
4.29 (d, J ¼ 5.8 Hz, 2H), 3.66 (s, 3H). 13C NMR (75 MHz, acetone-d6)
d
(ppm) 163.1, 155.0, 150.0, 143.6, 132.3,127.4 (q, J ¼ 282.9 Hz), 113.1,
112.0, 108.4, 103.0, 99.4, 72.2 (q, J ¼ 31.4 Hz), 56.1, 42.2. HRMS (ESI,
m/z) Calculated for
C
14H15F3NO3 [MþH]þ: 302.1004, found:
302.0989. Purity by HPLC: 100%, tR ¼ 11.27 min.
1-(4-Chloro-2-((thiazol-4-ylmethyl) amino) phenyl)2, 2, 2-
trifluoroethan-1-ol (36): compound 36 was obtained as a white
solid, 65 mg, 65% yield. 1H NMR (300 MHz, CDCl3)
d (ppm) 8.73 (s,
1H), 7.26 (s, 1H), 7.03 (d, J ¼ 8.0 Hz, 1H), 6.68 (d, J ¼ 8.9 Hz, 2H), 5.73
(s, 1H), 5.05 (q, J ¼ 7.4 Hz, 2H), 4.47 (s, 2H). HRMS (ESI, m/z):
Calculated for C12H9ClF3N2OS [M ꢁ H]-: 321.0076, found: 321.0077.
Purity by HPLC: 100%, tR ¼ 11.19 min.
1-(4-Bromo-2-((thiazol-4-ylmethyl) amino) phenyl)-2, 2, 2-
trofluoroethan-1-ol (37): compound 37 was obtained as a white
solid, 41 mg, 50% yield. 1HNMR (300 MHz, DMSO‑d6)
d (ppm) 9.08
(d, J ¼ 1.9 Hz,1H), 7.35 (d, J ¼ 1.9 Hz,1H), 7.21 (d, J ¼ 8.2 Hz,1H), 6.95
(d, J ¼ 5.3 Hz, 1H), 6.81e6.65 (m, 2H), 6.57 (t, J ¼ 5.9 Hz,1H), 5.38 (p,
J ¼ 6.9 Hz, 1H), 4.47 (d, J ¼ 5.8 Hz, 2H). 13C NMR (75 MHz, acetone-
d6) d (ppm) 157.1, 155.3, 150.0, 132.9, 129.0 (m), 125.4, 120.5, 119.5,
116.3, 115.7, 71.82 (q, J ¼ 32.0 Hz), 45.21. HRMS (ESI, m/z): Calcu-
lated for C12H9BrF3N2OS [M ꢁ H]-: 364.9571, found: 364.9569.
Purity by HPLC: 98%, tR ¼ 11.33 min.
2, 2, 2-Trifluoro-1-(4-methoxy-2-(((thiazol-4-ylmethyl) amino)
phenyl) ethan-1-ol (38): compound 38 was obtained as a white
solid, 75 mg, 47% yield. 1H NMR (300 MHz, DMSO‑d6)
d (ppm) 9.06
(d, J ¼ 1.9 Hz,1H), 7.34 (d, J ¼ 1.9 Hz,1H), 7.15 (d, J ¼ 8.5 Hz,1H), 6.72
(d, J ¼ 5.4 Hz, 1H), 6.26 (s, 1H), 6.17 (dd, J ¼ 8.5, 2.4 Hz, 1H), 6.07 (d,
J ¼ 2.4 Hz, 1H), 5.34e5.20 (m, 1H), 4.56e4.29 (m, 1H), 3.61 (s, 3H).
13C NMR (75 MHz, acetone-d6)
d (ppm) 163.4, 158.1, 155.3, 150.3,
132.5, 129.6 (m), 116.2, 113.4, 103.2, 99.7, 72.6 (m), 56.3, 45.7. HRMS
(ESI, m/z) Calculated for C13H14F3N2O2S [MþH]þ: 319.0728, found
319.0729. Purity by HPLC: 97%, tR ¼ 10.30 min.
2, 2, 2-Trifluoro-1-(4-fluoro-2-((furan-2-ylmethyl) amino)phenyl)
ethan-1-ol (32): compound 32 was obtained as a white solid,
103 mg, 67% yield. 1H NMR (300 MHz, CDCl3)
d (ppm) 7.38 (dd,
J ¼ 1.9, 0.8 Hz, 1H), 7.14 (dd, J ¼ 8.3, 6.4 Hz, 1H), 6.53e6.39 (m, 2H),
6.34 (dd, J ¼ 3.2, 1.9 Hz, 1H), 6.25 (dd, J ¼ 3.2, 0.9 Hz, 1H), 5.22 (s,
1H), 5.06 (q, J ¼ 7.4 Hz, 1H), 4.31 (s, 2H), 2.89 (s, 1H). 13C NMR
5.2. Biology
SIM-A9 was purchased from ATCC®, #CRL-3265. Primary anti-
bodies were purchased from Cell Signaling Technology®, tubulin
(#3873); COX-2 (#12282); iNOS (#2982). Secondary antibodies
were purchased from Jackson Immuno Research Laboratories, Inc.,
anti-rabbit IgG (#111-035-045); anti-mouse IgG (#115-035-003).
Mouse cytokine ELISA kits were purchased from ThermoFisher®,
(75 MHz, acetone-d6) d (ppm) 167.5, 164.3, 154.3, 143.7, 132.8, 111.9,
108.5, 104.0, 103.7, 99.9, 99.6, 71.9, 41.9. HRMS (ESI, m/z) Calculated
for C13H12F4NO2 [MþH]þ: 290.0804, found: 290.0797. Purity by
HPLC: 100%, tR ¼ 11.61 min.
1-(4-Chloro-2-((furan-2-ylmethyl) amino) phenyl)-2, 2, 2-
trifluoroethan-1-ol (33): compound 33 was obtained as a white
TNF-
a
(#88-7324-88); IL-6 (88-7064-88).
solid, 55 mg, 58% yield. 1H NMR (300 MHz, CDCl3)
d (ppm) 7.39 (m,
1H), 7.10 (d, J ¼ 8.1 Hz, 1H), 6.83e6.68 (m, 2H), 6.34 (dd, J ¼ 3.3,
5.2.1. b-Amyloid oligomerization assay
This assay was adapted from LeVine [60]. Biotinylated Ab1-42
was purchased from AnaSpec (Freemont, CA, United States). An
1.9 Hz, 1H), 6.25 (d, J ¼ 3.2 Hz, 1H), 5.22e4.92 (m, 2H), 4.32 (s, 2H),
2.95 (s, 1H). 13C NMR (75 MHz, acetone-d6)
d
(ppm) 154.3, 149.8,
21

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
ELISA plate (Costar 9018) was coated with 50
m
L of a stock solution
typhimurium)-containing (5 ng/mL final concentration) medium.
The conditioned media and lysates were harvested for cytokine and
cell marker examination after 24 h incubation.
containing 1
(10 mM, pH 7.50). The plate was sealed and stored at 4 ꢀC overnight
prior to blocking for 2 h at room temperature with 200 L/well of
OFB-T buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.50,
0.1% (v/v) Tween 20). Then, 20 L of Ab1-42 stock solution (0.1 mg/
mg/mL NeutrAvidin in sodium phosphate buffer
m
5.2.4. Nitric oxide measurement by Griess assay
m
Nitric oxide production from the conditioned media of SIM-A9
cell cultures was measured using a Griess assay [62]. Conditioned
medium and sulfanilamide were mixed in a microwell plate to form
a transient diazonium salt. Then, N-naphthyl-ethylenediamine was
added to all wells to form a stable azo compound by incubating for
5e10 min in the dark at room temperature. Absorbance was
measured between 520 nm and 550 nm. The concentration of NO
production was quantified against a standard curve plot.
mL) was treated with HFIP and dried under argon. One hundred
microliters of trifluoroacetic acid (TFA) was added to the tube, and
the sample was dissolved prior to drying under argon. HFIP was
added and dried under argon to remove residual TFA. The bio-
tinylated Ab1-42 was then dissolved in 870
the solution was added to each well of a 96-well polypropylene
plate (Costar 3365) followed by 100 L of test compound diluted in
mL of DMSO, and 2 mL of
m
OFB buffer (various concentrations). The plate was incubated for 1 h
at room temperature without shaking, and then stopped by the
5.2.5. Enzyme-inked immunosorbent assay (ELISA)
addition of 50
m
L of 0.3% (v/v) Tween 20 in MilliQ water. 50
m
L of the
Cytokines were quantified using ELISA kits (Thermofisher)
following the manufacturer's instructions. Briefly, the high-binding
biotinylated Ab1-42/compound solution was added to each well of
the NeutrAvidin-coated plate (after removing blocking solution)
and the plate was sealed and incubated for 2 h with shaking at
150 rpm. The plate was washed three times with TBS-T solution
(20 mM Tris-HCl, 34 mM sodium chloride, pH 7.50, 0.1% (v/v)
plates were coated at 100 mL/well with diluted capture anti-bodies
(1:250) overnight at 4 ꢀC. The coated plates were blocked with the
diluent for 1 h before the performance of the assay. Each sample
was diluted accordingly and added to the plates for 2 h incubation
Tween 20), then 50
mL of Streptavidin-HRP (1:10,000) in OFB-T
at room temperature. Plates were then washed with 250 mL/well
buffer was added and the plate was sealed and incubated for 1 h
with shaking at 150 rpm. The plate was again washed three times
PBS with 0.05% Tween-20 and incubated with detection anti-bodies
(1:250 in assay diluent) for 1 h at room temperature. After another
washing step, 1:250 diluted avidin-HRP (horseradish peroxidase)
was added and incubated for 30 min. 100 mL TMB-substrate was
added after the final washing and the plate was incubated in the
dark until the signal was sufficiently developed. The reaction was
with TBS-T, followed by the addition of 100
dine (TMB)/H2O2 substrate solution to each well. The reaction was
stopped after 10e30 min by the addition of 100 L of 2% (v/v)
mL tetramethylbenzi-
m
aqueous sulfuric acid prior to reading absorbance at 450 nm in a
plate reader.
stopped by 50 mL 2 N sulfuric acid. The absorbance was measured at
450 nm with a correction of 570 nm using a plate reader.
5.2.2.
b-Amyloid ThT aggregation assay
This assay was adapted from LeVine [61]. Ab1-40 (>95%) was
purchased from AnaSpec (Freemont, CA, United States) and stored
at ꢁ80 ꢀC. All other reagents were of highest available purity,
purchased from Sigma-Aldrich (Oakville, ON, Canada), and used
without further purification. All water used in the assays was
micropore filtered and deionized water. Ab1-40 (1 mg) was dis-
solved in hexafluoro-2 propanol (HFIP) and sonicated for 30 min to
disassemble any pre-formed aggregates. HFIP was removed using a
stream of argon gas prior to the dissolution of Ab1-40 in 1.00 mL Tris
base (20 mM, pH 10.0) using vortex and 10 min sonication. The
solution was then further diluted with 4.70 mL of Tris base followed
by adjusting to pH 7.40 using concentrated hydrochloric acid and
then filtered. The pretreated Ab1-40 was diluted with an equal
5.2.6. Western blotting
Cells were washed twice with ice-cold PBS and harvested in
RIPA buffer supplemented with protease inhibitor cocktail. The
whole-cell extracts were then centrifuged at 22,000ꢂg for 20 min
at 4 ꢀC to remove cell debris. Protein concentrations were quanti-
fied using the Micro BCA protein assay kit. The absorbance was
measured at 595 nm using a microplate reader. Equal amounts of
cellular protein were separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
onto polyvinylidene difluoride (PVDF) membranes at 100 V for
90 min. The membranes were blocked for 1 h in Tris-buffered saline
(TBS), pH 7.4, with 0.1% Tween-20 (TBS-T) containing 10% skim
milk. The membrane blot was then incubated overnight at 4 ꢀC with
volume (5.70 mL) of 8
NaCl) and 200 L aliquots of this solution (20
ThT in Tris (20 mM, pH 7.40,150 mM NaCl)) were added to wells of a
black polystyrene 96-well plate. 4 L of test compound solutions at
various concentrations were added to each well. Each sample was
performed in triplicate and MilliQ water alone served as vehicle
control. Plates were sealed and incubated in a microplate reader
(Tecan Genios) at 37 ꢀC with fluorescence measurements recorded
m
M ThT in Tris (20 mM, pH 7.40, 300 mM
primary antibodies against iNOS (1:1000), COX-2 (1:1000), IL-1b
m
mM Ab1-40 and 4
m
M
(1:1000), and tubulin (1:1000) in TBS-T containing 5% skim milk.
The membrane was washed with TBS-T 3 ꢂ 10 min and incubated
with goat anti-rabbit (or anti-mouse) IgG-horseradish peroxidase
(1:5000) for 1 h. After the washing step, the immunoblotting was
visualized by chemiluminescence HRP-substrate.
m
5.2.7. Phagocytosis assay
(
lex ¼ 450 nm, lem ¼ 480 nm) every 15 min after first being shaken
SIM-A9 cells were seeded in 96-well plates designed for fluo-
rescence study. After the same activation method described above,
cells were treated with FITC-conjugated E. coli bioparticles at
0.125 mg/mL final concentration. Followed by 1 h incubation, the
extracellular FITC-particles were quenched by a 0.4% trypan blue/
PBS solution. The intracellular fluorescent signal was measured on
a plate reader at Ex 494/Em 518.
at high intensity for 15 s and then allowed to settle for 10 s before
each reading.
5.2.3. Assay for testing anti-inflammation activity using SIM-A9 cell
line
SIM-A9 (ATCC® CRL-3265™) cells were maintained in Dulbec-
co's modified eagle medium: nutrient mixture F-12 (DMEM-F12)
with 10% fetal bovine serum (FBS), 5% horse-serum and antibiotic-
antimycotic. SIM-A9 cells were seeded in each well of a 12-well
tissue culture plate at a density of 0.25 ꢂ 106 cells/well, one day
prior to the experiment. The culturing medium was replaced with
an LPS (Sigma-Aldrich, L6511, Salmonella enterica serotype
5.2.8. Pharmacokinetic study
All procedures on animals were approved by the University
Health Network Animal Care Committee and adhered to the Ca-
nadian Council on Animal Care guidelines. Compounds were
administered 5 mg/kg PO to male CD-1 mice (n ¼ 3/time point).
22

Z. Wang, Y. Wang, J.P. Pasangulapati et al.
European Journal of Medicinal Chemistry 222 (2021) 113565
Blood was collected via cardiac puncture at 0.5 and 2.5 h time
points followed by transcardial perfusion with PBS, and then the
brains were removed and homogenized with three volumes of
phosphate buffered saline. The samples were protein precipitated
with cold acetonitrile and then centrifuged. The supernatants were
diluted with two volumes of 0.1% formic acid-containing water. The
concentration of plasma and brain were determined by LC-MSMS.
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approved the final draft. S.W. designed trifluoroethanol analogs and
approved the final draft. D.F.W. conceived, designed and supervised
the entire study, reviewed drafts of the paper and approved the
final draft.
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Funding sources
This work was supported by a Tier 1 Canada Research Chair
(Donald F. Weaver). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the
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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgement
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
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