Discovery of Selective, Substrate-Competitive, and Passive Membrane Permeable Glycogen Synthase Kinase-3β Inhibitors: Synthesis, Biological Evaluation, and Molecular Modeling of New C-Glycosylflavones

Article abstract of DOI:10.1021/acschemneuro.8b00010

Glycogen synthase kinase-3β (GSK-3β) is a key enzyme responsible for tau hyperphosphorylation and is a viable therapeutic target of Alzheimer's disease (AD). We developed a new class of GSK-3β inhibitors based on the 6-C-glycosylflavone isoorientin (1). The new inhibitors are passive membrane permeable and constitutively attenuate GSK-3β mediated tau hyperphosphorylation and amyloid neurotoxicity in an AD cellular model. Enzymatic assays and kinetic studies demonstrated that compound 30 is a GSK-3β substrate-competitive inhibitor with distinct kinase selectivity, isoform-selectivity and over 310-fold increased potency as compared to 1. Structure-activity relationship analyses and in silico modeling suggest the mechanism of actions by which the hydrophobic, π-cation, and orthogonal multipolar interactions of 30 with the substrate site are critical for the GSK-3β inhibition and selectivity. The results provide new insights into GSK-3β drug discovery. The new inhibitors are valuable chemical probes and drug leads with therapeutic potential to tackle AD and other GSK-3β relevant diseases.

Full text of DOI:10.1021/acschemneuro.8b00010

Subscriber access provided by MT ROYAL COLLEGE
Article
Discovery of Selective, Substrate-Competitive, and Passive Membrane
Permeable Glycogen Synthase Kinase-3# Inhibitors: Synthesis, Biological
Evaluation, and Molecular Modeling of New C-Glycosylflavones
Zhibin Liang, and Qing X. Li
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00010 • Publication Date (Web): 30 Jan 2018
Downloaded from http://pubs.acs.org on January 31, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Chemical Neuroscience is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 63
ACS Chemical Neuroscience
1
2
3
4
5
6
1
7
8
Discovery of Selective, SubstrateꢀCompetitive, and Passive Membrane Permeable
Glycogen Synthase Kinaseꢀ3β Inhibitors: Synthesis, Biological Evaluation, and
Molecular Modeling of New CꢀGlycosylflavones
9
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4
Zhibin Liang and Qing X. Li*
5
6
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa,
Honolulu, Hawaii 96822, United States
7
ACS Paragon Plus Environment
1

ACS Chemical Neuroscience
Page 2 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
9
0
1
ABSTRACT: Glycogen synthase kinaseꢀ3β (GSKꢀ3β) is a key enzyme responsible for tau
hyperphosphorylation and is a viable therapeutic target of Alzheimer’s disease (AD). We
developed a new class of GSKꢀ3β inhibitors based on the 6ꢀCꢀglycosylflavone isoorientin (1).
The new inhibitors are passive membrane permeable and constitutively attenuate GSKꢀ3β
mediated tau hyperphosphorylation and amyloid neurotoxicity in an AD cellular model.
Enzymatic assays and kinetic studies demonstrated that compound 30 is a GSKꢀ3β substrateꢀ
competitive inhibitor with distinct kinase selectivity, isoformꢀselectivity and over 310ꢀfold
increased potency as compared to 1. Structureꢀactivity relationship analyses and in silico
modeling suggest the mechanism of actions by which the hydrophobic, πꢀcation and orthogonal
multipolar interactions of 30 with the substrate site are critical for the GSKꢀ3β inhibition and
selectivity. The results provide new insights into GSKꢀ3β drug discovery. The new inhibitors are
valuable chemical probes and drug leads with therapeutic potential to tackle AD and other GSKꢀ
3β relevant diseases.
1
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
12
1
3
4
1
15
16
17
18
19
20
2
1
2
Keywords: CꢀGlycosylflavone, computerꢀaided drug design, glycogen synthase kinaseꢀ3,
2
substrateꢀcompetitive inhibitor, kinase selectivity, tauopathy, Alzheimer’s disease
2
3
Introduction
24
Alzheimer’s disease (AD) is an irreversible and progressive neurological disorder that causes
1
25
26
27
28
29
memory loss, cognitive decline, and eventually death due to severe brain degeneration. This
devastating and fatal disease, unfortunately, cannot be prevented, cured or even slowed.
Although the exact cause of AD remains unclear, increasing evidence suggests that βꢀamyloid
(Aβ) is plausibly the pathogenic initiator of AD and tau aggregation and distribution in the brain
1ꢀ2
cortices correlate closely with neuronal loss and cognitive decline in AD progression. The tau
ACS Paragon Plus Environment
2

Page 3 of 63
ACS Chemical Neuroscience
1
2
3
3
0
1
protein stabilizes the structural integrity of microtubules in neurons to maintain healthy axonal
transport. In AD, aberrant phosphorylation of tau proteins disrupts their association, leading to
destabilization of microtubules and mislocalization of the abnormal tau to the soma and
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
32
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
3
3
3
3
3
4
5
6
7
8
dendrites. The subsequent aggregation of hyperphosphorylated tau results in formation and
disposition of toxic neurofibrillary tangles (NFTs), triggering synaptic dysfunction, neuronal cell
death and cognitive impairment. This aberrant signaling cascade gives rise to the tauopathy in
4
5ꢀ6
AD. The continued failures of antiꢀAβ approaches in AD clinical studies drive researchers
4, 7
embrace an alternative approach to investigate antiꢀtau strategies for AD treatment.
Glycogen synthase kinaseꢀ3β (GSKꢀ3β) is a key protein kinase in the cascade leading to
aberrant tau hyperphosphorylation. Elevated GSKꢀ3β activity has been implicated in AD and
39
8
4
0
1
other tauopathies. Overactive GSKꢀ3β hyperphosphorylates over 70% of the potential
4
phosphorylation sites on tau proteins in AD brains, which impairs the healthy interactions of tau
9
42
proteins with microtubules. The development of selective GSKꢀ3β inhibitors modulating
1
0ꢀ11
4
4
4
4
4
4
3
4
5
6
7
8
aberrant tau phosphorylation is therefore a promising strategy for AD chemotherapy.
To
date, the USꢀFDA has approved four AD drugs, but they only ameliorate temporary symptoms,
1
0
and none targets GSKꢀ3β.
Traditional GSKꢀ3β inhibitors target the highly conserved ATP site. However, the limited
selectivity of those inhibitors raises safety concerns owing to offꢀtarget effects and, therefore,
1
1
remains a major challenge in GSKꢀ3β based drug development. Despite substantial efforts in
49
developing GSKꢀ3β inhibitors in the past decades, to date only lithium carbonate and tideglusib
10
5
0
1
(a TDZD compound) have been studied in clinical trials for AD. Lithium carbonate shows a
1
1
5
weak inhibition (IC , 2 mM), while tideglusib (IC , 100 nM) is an irreversible and timeꢀ
50
50
1
2ꢀ13
52
dependent inhibitor of GSKꢀ3β.
In recent years, strategies have been employed to search for
ACS Paragon Plus Environment
3

ACS Chemical Neuroscience
Page 4 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
3
4
selective and reversible GSKꢀ3β inhibitors, particularly those that are not ATPꢀsite directed. It is
known that the substrate domain of GSKꢀ3β is less conserved with a unique folding different
5
1
1, 14
55
from other kinases.
Inhibitors targeting this site are thought to be more specific and selective
11, 14
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
5
5
5
6
6
6
7
8
9
0
1
than the ATPꢀcompetitive inhibitors.
Yet few substrateꢀcompetitive inhibitors of GSKꢀ3β
15ꢀ18
have been reported.
New, potent, selective and reversible inhibitors targeting the substrate
site on GSKꢀ3β are potential diseaseꢀmodifying therapies for AD.
We have undertaken a different approach to discover potential substrateꢀcompetitive inhibitors
of GSKꢀ3β from natural sources. Natural products are valuable starting points for drug discovery
as they have been naturally selected and optimized under evolutionary pressure and obtained
1
9
62
privileged structures for protein binding. CꢀGlycosylflavones and their aglycones (Figure 1)
6
3
4
omnipresent in plants are important phytochemicals noted for antiꢀoxidation, antiꢀinflammation,
2
0ꢀ22
6
antiꢀcancer, antiꢀcardiovascular diseases, and cognitive enhancement.
We recently screened
2
3
65
corn silks for GSKꢀ3β inhibitors and isolated a 6ꢀCꢀglycosylflavone, isoorientin (1). Compound
1 alleviates tau hyperphosphorylation and amyloid neurotoxicity through selective and reversible
6
6
6
6
7
7
6
7
8
9
0
1
23
GSKꢀ3β inhibition, by which the mechanism of action is substrate competition rather than the
2
3
common ATP competition. In addition, a recent study showed that 1 and related natural
flavones attenuate Aβ burden and neuroinflammation in an APPswe/PSEN1dE9 mouse model of
24
23
AD. 1 from maize crop is conceivably safe as supported by in vivo subchronic toxicity studies
2
5
of corn silkꢀderived flavones in mice and rats. 1 is a promising medicinal natural product with a
2
3ꢀ25
72
novel mode of action for reducing AD burdens.
However, the lack of druggable potency
7
3
4
(IC , 185 µM), commonly suffered by bioactive natural products, makes it challenging in
5
0
7
pharmaceutical applications. Potency improvement, through structureꢀactivity relationship
(SAR)ꢀbased optimization, pharmacological and pharmacokinetic evaluations, as well as testing
75
ACS Paragon Plus Environment
4

Page 5 of 63
ACS Chemical Neuroscience
1
2
3
7
7
6
7
administration routes in vivo are therefore necessary to develop analogues of 1 with therapeutic
potential.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
78
In our recent paper, preliminary examination of the predicted 3D structure of the GSKꢀ3βꢀ1
complex revealed that the substrate pocket of GSKꢀ3β favors specific interactions with both the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
79
80
81
82
83
84
23
Cꢀglycone and flavone moieties in 1. In particular, a cleft comprised of Phe67, Val87, Leu88
2
6
and Phe93 on GSKꢀ3β is critical for substrate recognition. This concave cleft could
accommodate a hydrophobic moiety favoring ligand binding, which is in the vicinity of the
primary hydroxyl group on Cꢀglycone of 1. This raises our hypothesis that introduction of a
hydrophobic functional group at this primary alcohol enhances affinity and selectivity of 1 to
GSKꢀ3β. In addition, methylation of the phenolic hydroxyls can improve bioavailability,
85
27
8
6
7
metabolic stability and potency of flavones.
8
In the present study, a new series of 1 analogues containing a 6ꢀCꢀglycosylflavone scaffold
were semiꢀsynthesized to target the substrate site on GSKꢀ3β. Potency, selectivity, passive
membrane permeability, antiꢀtau hyperphosphorylation, and antiꢀAβ neurotoxicity of the new
inhibitors were evaluated with molecular and cellular studies, SAR analysis and molecular
modeling.
88
89
90
91
92
9
3
4
Results
9
Chemistry
95
Design and Synthesis. A series of new analogues of 1 were designed and synthesized (Table
1). The semiꢀsynthesis of 6ꢀCꢀglycosylflavones from 1 was carried out according to the route
outlined in Scheme 1. The four phenolic hydroxyls on the flavone core of 1 were first selectively
9
6
7
9
ACS Paragon Plus Environment
5

ACS Chemical Neuroscience
Page 6 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
8
9
methylated by TMSCHN in a methanolic toluene solution according to a previously described
2
2
8
9
method. The resulting tetraꢀmethylated product (5) then underwent an oxoammonium saltꢀ
2
9
+
−
100
mediated oxidation. We optimized an oxidation protocol using a [TEMPO] [BF ] salt in a
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
1
1
1
01
02
03
04
05
06
pyridine base solution at room temperature, which chemoselectively transformed the primary
alcohol to a carboxylic acid without affecting the secondary hydroxyls on the Cꢀglycone. This
method successfully afforded the desired product (6) in over 95% yield which was confirmed by
ESIꢀTOFꢀMS and NMR analyses. The carboxylic acid derivative 6 was then subjected to methyl
esterification to afford 7. It is known that an ester bond is susceptible to metabolic hydrolysis.
Our chemical design was focused on amide analogues with better physiochemical stability. A
small library of hydrophobic amides (8ꢀ31) was generated by coupling 6 with a series of organic
amines via HCTU catalysis. Aliphatic, alicyclic, aromatic and fluorinated amines were selected
for the solutionꢀphase amidation (Table 1). The resulting hydrophobic amides were evaluated on
their SAR, cytotoxicity, antiꢀAD activity, and passive membrane permeability. All final products
were characterized by NMR and HRMS, and were over 95% purity determined by HPLCꢀUV at
210, 254, and 340 nm.
107
1
08
09
1
110
1
1
1
11
12
13
ACS Paragon Plus Environment
6

Page 7 of 63
ACS Chemical Neuroscience
1
2
3
1
14
Table 1. Chemical Structures of Natural and SemiꢀSynthetic CꢀGlycosylflavones.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
15
cmpd
no.
Functional group
X2
R1
H
X1
R2
H
1
5
6
7
8
9
H
2
2
O
O
CH
CH
CH
CH
CH
H
H
3
3
3
3
3
O
O
O
O
O
H
O
CH
3
NH
NH
1
0
CH
3
3
O
O
NH
NH
11
CH
1
2
CH
CH
CH
3
3
3
O
O
O
NH
NH
NH
13
14
15
16
CH
O
O
NH
NH
3
3
CH
ACS Paragon Plus Environment
7

ACS Chemical Neuroscience
Page 8 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
7
CH
3
O
NH
1
8
9
CH
3
3
O
O
NH
NH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
CH
2
0
1
CH
3
O
NH
2
CH
CH
3
O
O
NH
NH
22
23
3
CH
CH
O
O
NH
NH
3
2
4
5
3
2
CH
CH
3
O
O
NH
NH
26
3
2
7
CH
CH
CH
3
3
3
O
O
O
NH
NH
NH
28
2
9
0
3
CH
3
3
O
O
NH
NH
(S)
CF3
3
1
CH
ACS Paragon Plus Environment
8

Page 9 of 63
ACS Chemical Neuroscience
1
2
3
1
16
17
Biological Activities and Relevance
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
StructureꢀActivity Relationship and LigandꢀLipophilic Efficiency of CꢀGlycosylflavones
to GSKꢀ3β. The semiꢀsynthetic 6ꢀCꢀglycosylflavones (5ꢀ31) were assayed on GSKꢀ3β inhibition
in comparison to four natural flavones with structural similarities (two 6ꢀCꢀglycosylflavones
isoorientin 1 and isovitexin 2, one 8ꢀCꢀglycosylflavone orientin 3, and a flavone aglycone
luteolin 4). Among the four flavones (1ꢀ4, Figure 1), 4 showed the highest potency against GSKꢀ
3β (IC , 3.1 ꢁM in Table 2), but it is nonspecific and promiscuous as noted in the literature and
118
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
119
120
121
122
123
124
5
0
23, 30
our previous studies.
1 and 2 with Cꢀglycosides at 6ꢀposition showed a moderate potency
against GSKꢀ3β with an IC value of 185 and 194 ꢁM, respectively (Figure 1 and Table 2). In
50
125
contrast, 3 with an 8ꢀCꢀglycoside was inactive (IC , > 5 mM). The results demonstrated that the
50
1
26
27
presence and position of Cꢀglycone on the flavone core are critical for GSKꢀ3β inhibition, which
2
3
1
agrees with our previous observation.
128
The tetraꢀmethylated alcohol (5) and tetraꢀmethylated carboxylic acid (6) slightly decreased
the potency (IC , 237 and 239 ꢁM, respectively) in comparison with 1, indicating a trivial
129
130
131
132
133
134
5
0
contribution of the phenolic hydroxyl groups to GSKꢀ3β inhibition. However, a methyl ester (7)
(IC , 135 ꢁM) increased the potency by 1.37ꢀfold as compared to 1 (IC , 185 ꢁM), suggesting
50
50
hydrophobic groups at the primary hydroxyl position are preferred for GSKꢀ3β inhibition.
Remarkably, transforming the primary alcohol to corresponding hydrophobic amides (8ꢀ31)
(Table 2) significantly increased the potency against GSKꢀ3β as most analogues displayed IC50
values less than 50 ꢁM and ten of them (9, 13, 16, 17, 20, 21, 27, 29, 30 and 31) were less than
20 ꢁM. As shown in Figure 2A, the aliphatic (e.g., 9 and 13) and alicyclic amides (e.g., 16 and
17) exhibited a higher affinity to GSKꢀ3β than the aromatic amides (e.g., 20ꢀ23). Small (14 and
15) or large (18 and 19) alicyclic rings showed a less affinity than the cyclopentyl (16) or
135
1
36
37
1
138
ACS Paragon Plus Environment
9

ACS Chemical Neuroscience
Page 10 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
39
40
cyclohexyl (17) analogues, plausibly due to the size of the hydrophobic concave cleft in the
substrate site on GSKꢀ3β. A branched isopropyl group (9, 30, and 31) showed a higher affinity
than a linear propyl group (8, 28, and 29). Monoꢀfluorination of phenyl (24), benzyl (25), ethyl
(26) or propyl (28) groups did not improve affinity as compared to nonꢀfluorinated counterparts
1
141
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
142
143
144
145
146
147
(e.g., 8, 20, and 21). It is interesting that a trifluoromethyl (CF ) group has a significant effect on
3
potency. Compounds 27, 29, 30 and 31 containing a CF moiety consistently improved binding
3
affinity to GSKꢀ3β in comparison with no fluorine or monoꢀfluorinated counterparts (e.g., 8, 9,
26 and 28) (Figure 2A). In particular, 30 (IC , 0.59 ꢁM) with a (S)ꢀCF group increased the
5
0
3
potency against GSKꢀ3β by 310ꢀfold in comparison with 1, and is about 4ꢀfold more potent than
its epimer 31 with a (R)ꢀCF group (IC , 2.3 ꢁM). All new analogues were not panꢀassay
148
3
50
23, 30ꢀ31
1
49
50
interference compounds to GSKꢀ3β as determined with a detergentꢀbased assay.
1
Ligandꢀlipophilic efficiency (LiPE) is a parameter commonly used in drug design to assess the
quality of compound candidates. Lipophilicity is the most important druglike physiochemical
property that highly correlates to absorption, distribution, metabolism, excretion and toxicity
151
152
153
154
155
156
157
32
(ADMET) profiles and ultimately to the pharmacological response for oral drugs. High potency
(large pIC ) is a desirable feature in drug candidates, as it reduces the risk of offꢀtarget and
50
nonspecific pharmacology. Correlation between lipophilicity (CLogP) and potency (pIC )
5
0
33
provides a valuable parameter to estimate druglikeness. Many of the semiꢀsynthetic analogues
clustered in an upperꢀright range of the LiPEs between 2 and 4, indicating that both lipophilicity
(CLogP) and GSKꢀ3β inhibitory potency (pIC50) have been increased relative to 1 (Figure 2B).
Particularly, 30 has the highest LiPE value (> 4) among the analogues, suggesting a unique
158
1
59
60
1
contribution of the CF moiety of 30 to improving potency and lipophilicity.
3
161
ACS Paragon Plus Environment
10

Page 11 of 63
ACS Chemical Neuroscience
1
2
3
1
62
63
Table 2. Comparison of Natural and SemiꢀSynthetic CꢀGlycosylflavones on GSKꢀ3β Inhibition
and CLogP
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
b
b
cmpd
GSKꢀ3β inhibition CLogP
cmpd
no.
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
GSKꢀ3β inhibition CLogP
a
a
no.
1
IC (ꢁM)
IC (ꢁM)
5
0
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
184.9 ± 1.4
194.1 ± 1.0
5153 ± 31
3.1 ± 1.3
0.21
0.80
0.21
2.31
1.28
0.80
1.21
1.57
1.35
2.10
1.97
2.63
3.16
1.10
1.43
1.99
9.0 ± 1.3
2.55
3.16
3.17
2.21
2.34
1.54
1.45
2.61
2.48
0.77
1.31
1.00
1.29
1.62
1.62
2
26.0 ± 1.2
33.5 ± 0.8
19.7 ± 1.1
15.8 ± 1.2
35.6 ± 1.3
35.1 ± 1.0
56.2 ± 1.2
21.7 ± 1.2
34.8 ± 1.0
19.3 ± 0.8
49.5 ± 1.2
17.2 ± 0.9
0.59 ± 0.04
2.3 ± 0.5
3
4
5
239.2 ± 1.2
237.3 ± 1.4
135.0 ± 1.3
29.2 ± 1.1
5.4 ± 0.1
6
7
8
9
1
0
33.3 ± 1.1
31.1 ± 1.2
28.0 ± 1.1
9.4 ± 0.9
11
1
2
13
1
1
1
4
5
6
25.8 ± 1.1
61.9 ± 1.2
13.1 ± 1.1
a
b
1
1
64
65
IC values were the mean of quadruplicate of each of two independent experiments. CLogP
50
values were calculated by a fragmentꢀbased method. cmpd, compound.
34
1
66
67
1
Evaluations of 1, 9, 17, 21, 30 on AntiꢀTau Hyperphosphorylation Mediated by GSKꢀ3β.
Compounds 9, 17, 21 and 30 were selected for further evaluation on their effect against GSKꢀ3β
mediated tau hyperphosphorylation relative to 1, as they are the most potent GSKꢀ3β inhibitors
within each of the aliphatic, alicyclic, aromatic and fluorinated amide analogues, respectively.
We recently established an in vitro GSKꢀ3β assay using a wholeꢀcell lysate of human SHꢀSY5Y
168
1
69
70
1
171
ACS Paragon Plus Environment
11

ACS Chemical Neuroscience
Page 12 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
23
1
72
73
neuroblastomas and demonstrated that the pS396 site on tau protein is GSKꢀ3β specific. Direct
1
GSKꢀ3β inhibition by isoorientin 1 led to its consequent effect against GSKꢀ3β mediated tau
2
3
174
hyperphosphorylation on the pS396 site in an ex vivo protein matrix of the SHꢀSY5Y lysate. In
analogy, an aliquot of lysate was fortified with GSKꢀ3β (wt/wt 0.25%), and incubated with 9, 17,
21, or 30 at different concentrations (2, 10, and 50 ꢁM) for 2 h followed by an enzyme linked
immunosorbent assay (ELISA) with an antiꢀtau pS396 antibody. TDZDꢀ8 (10 ꢁM) and 1 (100
ꢁM) were used as reference controls. Quantitative ELISA measurements substantiated that
introducing exogenous GSKꢀ3β significantly increased phosphorylation by approximately 2.5ꢀ
fold (p < 0.0001) at the site pS396 on tau proteins as compared to their basal levels (lysate
fortified with heatꢀinactivated GSKꢀ3β) (Figure 3). In contrast, treatment of 9, 17, 21, or 30
effectively attenuated tau hyperphosphorylation in a doseꢀdependent manner (p < 0.0001). All
four analogues showed vast improvements of antiꢀtau hyperphosphorylation in comparison to the
natural product 1. Compound 30 at 10 ꢁM exerted an antiꢀtau effect comparable to TDZDꢀ8 (a
known potent GSKꢀ3β inhibitor, IC = 2 ꢁM) at the same concentration (p < 0.05). These results
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
175
176
177
178
179
180
181
1
82
83
1
184
185
186
187
188
189
190
5
0
demonstrated that the new 6ꢀCꢀglycosylflavone analogues indeed alleviate tau
hyperphosphorylation, of which 30 is the most promising candidate suitable for further in vivo
pharmacological assessments.
GSKꢀ3β Kinetic Studies on the Inhibition Mode of 30. To determine the GSKꢀ3β inhibitory
mechanism, the most potent analogue 30 (Figure 4A) was assayed to competitively replace ATP
or the GSKꢀ3β substrate GS2 (a peptide derived from human muscle glycogen synthase). Under
a constant concentration of the substrate GS2 (17 ꢁM), ATP concentrations varied from 2 to 50
ꢁM and 30 concentrations varied from 0 to 5 ꢁM. The Lineweaver‒Burk plots show a
191
1
92
93
1
194
ACS Paragon Plus Environment
12

Page 13 of 63
ACS Chemical Neuroscience
1
2
3
1
95
96
convergence of intersecting lines on the xꢀaxis, indicating an unaltered Michaelis‒Menten
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
constant (K ) but a reduced GSKꢀ3β activity (increased 1/Vmax) when the concentration of 30
m
197
increased (Figure 4B(a)). This suggested no competition between ATP and 30. In the second set
of experiments under a constant concentration of ATP (10 ꢁM), substrate GS2 concentrations
varied from 8 to 66 ꢁM and 30 concentrations varied from 0 to 5 ꢁM. The Lineweaver‒Burk
plots show that all lines intersected at the same point on the yꢀaxis, suggesting an unchanged
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
198
199
200
201
202
203
1/Vmax but an increase of K when the concentration of 30 increased (Figure 4B(b)). The data
m
demonstrated that 30 competed with the substrate GS2. The enzyme inhibitory behaviors of 30
2
3
are similar to that of the parent compound 1 and therefore confirm that the new analogues are
204
indeed reversible and substrateꢀcompetitive inhibitors of GSKꢀ3β.
2
05
06
2
Kinase Selectivity Profile of 30. It is prudent to evaluate kinase selectivity in the early phases
of drug discovery. Compound 30 was screened against a panel of 41 human protein kinases for
207
9
208
209
210
211
212
213
selectivity relevant to AD and other CNS disorders. 30 at 5 ꢁM showed an overall good
selectivity as it effectively inhibited GSKꢀ3β by decreasing 92.3% kinase activity (p < 0.0001) in
comparison to the control (100% kinase activity), whereas it showed only marginal or weak
inhibition against 40 out of 41 kinases in the test panel (Figure 4C). Notably, between the two
GSKꢀ3 isoforms (GSKꢀ3α and GSKꢀ3β), 30 at 5 ꢁM displayed a 12.3ꢀfold selectivity to GSKꢀ3β
(92.3% inhibition) versus GSKꢀ3α (7.5% inhibition). The exceptional selectivity of 30 to GSKꢀ
3β could minimize risk of offꢀtarget effects.
214
2
15
16
2
Evaluation of 30 on Cytotoxicity and AntiꢀAmyloid Neurotoxicity. To investigate whether
semiꢀsynthetic 6ꢀCꢀglycosylflavone analogues exert neuroprotection against Aβꢀinduced
217
ACS Paragon Plus Environment
13

ACS Chemical Neuroscience
Page 14 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
18
19
neurotoxicity, 30 was assayed in an AD cellular model where Aβ oligomers were administrated
4
2
2
3, 35
2
in human SHꢀSY5Y neuroblastomas.
Compound 30 displayed a good tolerability profile
2
3
220
similar to 1 as no observable cytotoxicity up to a dose of 1000 ꢁM (Figure 5A). On the other
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
221
222
223
224
225
226
hand, treatment of 10 ꢁM Aβ oligomers decreased cell viability to 40% compared with the
4
2
controls (Figure 5B). However, such Aβ neurotoxicity was greatly relieved, as pretreatment of
4
2
SHꢀSY5Y cells with 30 at concentrations from 1.25 to 20 ꢁM for 1 h followed by coꢀincubation
with 10 ꢁM Aβ for 72 h recovered cell viability from 40% to 100% in a doseꢀdependent
42
manner. The neuroprotective potency of 30 (EC , 8.7 ꢁM) (Figure 5C) was a 5.4ꢀfold increase
5
0
2
3
in comparison to its parent compound 1 (EC , 47 ꢁM).
5
0
227
Morphological observations also illustrated that pretreatment by 30 at 10 ꢁM effectively
2
28
29
protected the SHꢀSY5Y cells from Aβ intoxication, as the neuronal cells were healthy and well
4
2
2
differentiated with extended axons and dendrites (Figure 5D). Although a similar neuroprotective
230
activity was observed to 1, 30 exhibited approximately 20ꢀfold improvement of effectiveness in
2
3
231
232
233
234
235
236
comparison to its parent compound (effective dose: 30 in 10 ꢁM versus 1 in 200 ꢁM). In a
good agreement with our previous findings, 30 and the other 6ꢀCꢀglycosylflavone analogues
exerted antiꢀAβ neurotoxicity through protecting neurite outgrowth and neuronal differentiation,
whose functions are constitutively regulated by GSKꢀ3β and tau protein within axonal
7
microtubules.
237
Evaluations of 1, 4, 9, 17, 21, 30 on Passive Membrane Permeability. Oral administration is
noninvasive and is preferred for chronical diseases such as AD and other CNS disorders. It is
advantageous to discover passive membrane permeable AD drugs targeting GSKꢀ3β upon oral
administration. The parallel artificial membrane permeability assay (PAMPA) is used to assess
2
38
39
2
240
ACS Paragon Plus Environment
14

Page 15 of 63
ACS Chemical Neuroscience
1
2
3
2
41
42
passive and transcellular permeability, which is well correlated with in vivo oral absorption rates
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
6
2
for drugs that cross the gastrointestinal barrier. In the PAMPA model, the amount of target
compounds diffused from a donor compartment to an acceptor compartment through a triꢀlayer
phospholipid preꢀcoated membrane was measured to assess oralꢀabsorption potential of drug
candidates. In the present study, 1, 4, 9, 17, 21 and 30 were evaluated with the PAMPA assay.
Theophylline and atenolol known for their low permeability were used as negative controls.
Desipramine known for its high permeability was used as a positive control. The measured Pe
243
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
244
245
246
247
248
249
36
values were compared with the literature data.
The new analogues 9, 17, 21 and 30 demonstrated a significant increase of permeability upon 5
h of incubation in comparison with their parent compound 1 (Table 3). Methylation of phenolic
hydroxyls and transformation of the primary hydroxyl to hydrophobic amides on the Cꢀglycone
of 1 increased the PAMPA permeability (P ) ranging from 2.6 to 4.4ꢀfold relative to 1. Such P
250
2
51
52
2
e
e
253
changes agreed well with the CLogP changes (Table 3). The best PAMPA permeability of 30 is
254
255
256
257
258
259
probably attributed to the CF group. Multiple hydroxyl groups in Cꢀglycone and flavone
3
37
moieties make 1 poorly permeable, which is consistent with the literature reports. For
comparison, luteolin (4), an aglycone flavone, had a medium PAMPA permeability similar to 9,
17, and 21, indicating the liability of the polar Cꢀglycone of 1 in respect to passive diffusion.
Regardless, it is indispensable to assess pharmacokinetics of the new GSKꢀ3β inhibitors for their
in vivo bioavailability which involves the complexity of active and facilitated transports in
addition to passive diffusion.
260
61
2
ACS Paragon Plus Environment
15

ACS Chemical Neuroscience
Page 16 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
62
Table 3. PAMPA Permeability and CLogP Values of Compounds Tested
cmpd
no.
PAMPA Permeability
CLogP
ꢀ6
a
b
P (× 10 cm/s)
R (%)
Permeability c
e
classification
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Isoorientin (1)
Luteolin (4)
9
0.51 ± 0.01
1.42 ± 0.07
1.55 ± 0.06
1.77 ± 0.07
1.35 ± 0.08
2.23 ± 0.21
0.04 ± 0.01
0.29 ± 0.09
13.43 ± 0.91
48
43
46
20
30
30
n/a
40
96
low
0.21
2.31
1.35
2.55
2.34
1.62
ꢀ0.03
ꢀ0.11
4.47
medium
high
17
21
30
high
medium
high
Theophylline
low
d
Atenolol
low
d
Desipramine
high
a
b
2
2
265
266
63
64
P values were the mean of PAMPA measurements (n = 3ꢀ4) with ± SD. Percent recovery
e
c
(R%) measures mass retention of compounds trapped inside the PAMPA membrane. PAMPA
ꢀ
6
ꢀ6
ꢀ
permeability classification: high (P > 1.5 × 10 cm/s), medium (1.0 × 10 cm/s ≤ P ≤ 1.5 × 10
e
e
6
ꢀ6
d
36
cm/s), low (P < 1.0 × 10 cm/s). The measured P values are comparable to the data in ref.
e
e
267
2
68
69
Computational Modeling
2
Key Molecular Interactions between New CꢀGlycosylflavones and the Substrate Site of
GSKꢀ3β. To elucidate the molecular mechanisms by which the new 6ꢀCꢀglycosylflavones
increase the binding affinity and selectivity to GSKꢀ3β relative to 1, compounds 5ꢀ31 were
docked into GSKꢀ3β enzyme. The AutoDock Vina program was used in the present study
270
2
2
2
2
2
71
72
73
74
75
3
8ꢀ40
because it has shown good accuracies for binding pose prediction and scoring function.
This
docking tool has been widely used in drug design and applied in many cases of GSKꢀ3β inhibitor
17, 41
discovery.
ACS Paragon Plus Environment
16

Page 17 of 63
ACS Chemical Neuroscience
1
2
3
42
43
2
76
77
Two Xꢀray crystallographic structures of GSKꢀ3β (PDB codes 1PYX and 1H8F ) were
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
chosen to take into account for potential induced fit effects upon ligand binding. The GSKꢀ3β in
2
+
278
1PYX contains two Mg ions and a ligand ANP, a nonꢀhydrolyzed ATP derivative, at the ATP
2
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
279
280
281
282
283
284
site. Upon binding of Mg ions and an ATPꢀmimic ligand, it is postulated that GSKꢀ3β
undergoes subtle conformational changes in both the ATP and substrate sites for an optimal
4
4ꢀ45
kinase reaction, a phenomenon known as induced fit effects.
The GSKꢀ3β structure in 1PYX
hence adopts a conformation that resembles the native enzyme ready for substrate recognition.
Docking 5ꢀ31 into the substrate site of GSKꢀ3β using 1PYX would give more reliable binding
poses. On the other hand, the GSKꢀ3β in 1H8F is by far the only available Xꢀray crystallographic
structure containing the ligand HEPES in the substrate site. HEPES in 1H8F may cause induced
fit changes of GSKꢀ3β conformation, particularly for the substrate site. Given the new 6ꢀCꢀ
glycosylflavones are substrate competitive, we used 1PYX and 1H8F in molecular docking. The
docking method was validated by reꢀdocking and crossꢀdocking experiments using both ATPꢀ
competitive and substrateꢀcompetitive inhibitors (Supporting Information S1).
285
2
86
87
2
288
289
290
291
292
293
294
The synthetic 6ꢀCꢀglycosylflavones (5ꢀ31) and their parent 1 were thus docked into 1PYX and
2
1H8F. Docking scores of 1PYX show a better linear fit to the experimental pIC50 values (R =
2
0.7844) than that of 1H8F (R = 0.6999), suggesting more accurate predictions of binding poses
using 1PYX than 1H8F (Supporting Information S2). The 1PYX docking dataset was therefore
used in the remainder of the study to analyze the molecular interactions responsible for the
improved potency and selectivity to GSKꢀ3β.
295
2
96
97
The docking results of 1PYX showed that the Cꢀglycone moiety of 5ꢀ31 forms hydrogen bonds
with GSKꢀ3β residues Gln89, Asn95, Arg96 and Glu97 within the substrate pocket, which is
similar to 1 (Supporting Information S3). Moreover, the newly introduced hydrophobic groups
2
298
ACS Paragon Plus Environment
17

ACS Chemical Neuroscience
Page 18 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
99
00
indeed exhibit a favorable ligand pose to the concave surface comprised of Phe67, Val87, Leu88
3
and Phe93 on GSKꢀ3β. The most potent compounds 30 and 31 (IC , 0.59 and 2.3 ꢁM,
5
0
301
respectively) contain a CF moiety, which may form orthogonal multipolar interactions for
3
4
6ꢀ47
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
302
303
304
305
306
307
protein binding.
To visualize such molecular interactions, we conducted flexibleꢀresidue
40, 48
docking
to refine binding poses of 9, 30 and 31 in 1PYX. To prepare GSKꢀ3β structure, side
chains of Ser66, Phe67, Leu88, Gln89, Asp90, Phe93, Asn95 and Lys183 within the substrate
site were treated flexible. The docking results of 30 suggested formation of orthogonal
multipolar interactions. The (S)ꢀCF in 30 shows a favorable geometry within typical F···C
3
distances to Leu88 (backbone amide carbonyl carbon, 3.3 Å), Gln89 (backbone amide carbonyl
carbon, 3.6 Å) and Asp90 (side chain carbonyl carbon, 3.3 and 3.8 Å) (Figure 6C). It also forms
a polar interaction with the backbone NH of Asp90 (F···H distance, 2.6 Å). In contrast, the (R)ꢀ
308
3
09
10
3
CF group of 31 is absent from these interactions probably owing to the orientation and steric
3
311
hindrance by switching CH and CF positions at the stereocenter (Figures 6Bꢀ6C). Therefore,
3
3
3
3
3
3
3
3
12
13
14
15
16
17
only hydrophobic interactions with Phe67, Val87 and Leu88 of GSKꢀ3β exist in 31 (improper
configuration of CF for orthogonal multipolar interactions) as well as 9 (lack of CF ) (Figures
3
3
6Aꢀ6B).
Interestingly, the docking results showed that the catechol Bꢀring of the flavone core in 9, 30
49ꢀ50
and 31 appears to have a πꢀcation interaction with Lys183.
The distance between the catechol
+
Bꢀring center and ε‒NH cation of Lys183 is 4.1 Å at an angle of 71.5° (Figure 6C and
3
+
318
Supporting Information S4). In conjunction, the side chain ε‒NH of Lys183 forms weak
3
3
19
20
hydrogen bonds with two methoxy oxygen atoms of catechol Bꢀring (distances of 3.1 and 3.2 Å).
The orthogonal multipolar and πꢀcation interactions potentially enhance the binding affinity to
GSKꢀ3β.
3
321
ACS Paragon Plus Environment
18

Page 19 of 63
ACS Chemical Neuroscience
1
2
3
3
22
23
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
Potential Causes for the IsoformꢀSelectivity of 30 between GSKꢀ3α and GSKꢀ3β. Isoformꢀ
selectivity of 30 to GSKꢀ3β over GSKꢀ3α (Figure 4C) was assessed with comparative molecular
modeling. Yet no Xꢀray crystallographic structures of GSKꢀ3α available in PDB, a homology
324
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
325
326
327
328
329
330
51
model of GSKꢀ3α was built with the SWISSꢀMODEL server (Supporting Information S5). A
full human GSKꢀ3α amino acid sequence (UniProt code: P49840) was searched against protein
databases in BLAST and HHblits and overall 4470 templates were found. A GSKꢀ3β template
(PDB code 1PYX) resulted in a top ranking with an overall sequence identity of 82.97%, thereby
selected for homology modeling of GSKꢀ3α. A sequence alignment between the two GSKꢀ3
isoforms indicated that GSKꢀ3α has extra amino acid portions flanking the Nꢀ and Cꢀtermini of
GSKꢀ3β (Supporting Information S6). Within the matched sequence portion, most amino acid
differences occur in the Nꢀ and Cꢀterminal regions. In contrast, both the ATP and substrate
domains of GSKꢀ3α/β isoforms are conserved as high as 92.37% identical, in which only few
amino acid residues are different in these regions. The superposition and comparison of GSKꢀ3β
structure (PDB code 1PYX) with the GSKꢀ3α homology model (Figure 7A) implied that those
subtle differences of amino acid residues in the kinase catalytic domain (both ATP and substrate
pockets) may affect substrate recognition as well as ligand binding. Intriguingly, docking
experiment using the GSKꢀ3α homology model showed that 30 resides a similar location in the
substrate site in comparison to GSKꢀ3β docking data (Figures 7Bꢀ7C). However, the resulting
binding pose of 30 in GSKꢀ3α favors neither a πꢀcation interaction of catechol Bꢀring nor the
331
3
32
33
3
334
335
336
337
338
339
340
341
3
42
43
orthogonal multipolar interactions of the (S)ꢀCF group. It is conceivable that the isoformꢀ
3
3
selectivity of 30 to GSKꢀ3β might be in part due to the lack of these critical molecular
interactions in GSKꢀ3α. Instead, the (S)ꢀtrifluoroisopropyl group of 30 simply exerts
344
ACS Paragon Plus Environment
19

ACS Chemical Neuroscience
Page 20 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
45
46
hydrophobic affinity with the homologous residues Phe130, Val150, and Leu151 in the substrate
site of GSKꢀ3α (Figure 7C).
3
347
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
48
Discussion
349
350
351
352
353
GSKꢀ3β plays a central role in signaling pathways of AD. Accumulating evidence has
demonstrated that GSKꢀ3β is activated abnormally by Aβ and in turn hyperphosphorylates tau
1
, 7ꢀ8
proteins in neurons. This eventually triggers the tauopathic cascade in AD progression.
Development of GSKꢀ3β inhibitors as a diseaseꢀmodifying therapy for AD, therefore, is highly
attractive. In recent years, discovery of selective inhibitors targeting the substrate site of GSKꢀ3β
1
1
354
has emerged as a rational and feasible strategy in AD drug design. Unlike the ATPꢀsite directed
GSKꢀ3β inhibitors that frequently bind to many offꢀtarget kinases, inhibitors targeting the
substrate site on GSKꢀ3β plausibly overcome this limitation. GSKꢀ3β phosphorylates its specific
substrates for biological processes such as glucose homeostasis, immune response, neurogenesis,
cell proliferation and apoptosis, and circadian rhythm, which has a plethora of normal functions
3
55
56
3
357
358
359
360
361
362
363
52
for human health. However, GSKꢀ3β is aberrantly overactive in AD. Subtle modulation of
GSKꢀ3β activity at a normal level rather than complete shutꢀoff to prevent disruption of its
8
normal cellular functions will be highly appreciated in AD therapy. Targeting the substrate site
to intervene the GSKꢀ3βꢀsubstrate recognition would be a feasible means to find selective
14
inhibitors. Until now, substrateꢀcompetitive inhibitors of GSKꢀ3β are limited in the structural
1
5
17
364
classes of marine alkaloid manzamines, labdane diterpenoid andrographolide, peptidomimetic
18
16
3
65
66
inhibitors, and synthetic compound ITDZ series. These inhibitors were in the low micromolar
to subꢀmicromolar range of potency, but exhibited a good selectivity to GSKꢀ3β under in vitro or
in vivo evaluations. Such findings substantiate the rationale of developing substrateꢀcompetitive
3
367
ACS Paragon Plus Environment
20

Page 21 of 63
ACS Chemical Neuroscience
1
2
3
3
3
68
69
inhibitors of GSKꢀ3β for AD by which the agents can attain an utmost therapeutic advantage ‒ a
high selectivity to avoid potential side effects.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
370
Herein, we reported a new structural class of substrateꢀcompetitive inhibitors of GSKꢀ3β,
inspired by isoorientin (1), containing a 6ꢀCꢀglycosylflavone scaffold. It is a valuable addition to
the known substrateꢀcompetitive inhibitors and offers new lead compounds for AD. The
inhibitors are new chemical probes allowing to elucidate the underling mechanisms of GSKꢀ3β
inhibition and selectivity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
371
372
373
374
375
376
The SAR analysis, in vitro enzymatic kinetics, and in silico docking studies indicate that both
the presence and position of Cꢀglycone on the flavone core are essential features binding to the
substrate site on GSKꢀ3β. A flavone core (4) alone is promiscuous and tends to sneak in the ATP
site and thus lose the required selectivity (Supporting Information S1). The presence of a Cꢀ
glycone at 8ꢀposition (3, IC , > 5 mM) rather than 6ꢀposition (1, IC , 185 ꢁM) results in an
377
3
78
79
3
5
0
50
380
unfavorable binding pose to the substrate site plausibly due to the loss of key hydrogen bonds
with Gln89, Asp90, Asn95, and Arg96 (Supporting Information S3). Such unique SAR features
inspired us to semiꢀsynthesize a series of novel analogues of 1 that selectively inhibit GSKꢀ3β at
the substrate site. Data suggest that the new lipophilic amide analogues (8ꢀ31) increase the
potency against GSKꢀ3β for 3‒310 fold (Table 2) and passive membrane permeability for 2.6‒
4.4 fold relative to 1 (Table 3). Systematic modifications of the carbonꢀchain length and ring size
381
382
383
384
385
386
and bioisosteric replacement in the R ꢀgroup on the Cꢀglycone of 1 (Table 1) confer a dramatic
2
387
GSKꢀ3β potency improvement. The structural modifications contribute hydrophobic affinities
with Phe67, Val87 and Leu88 in the substrate site of GSKꢀ3β (Supporting Information S3),
which is concurrently supported by the LiPE analysis (LiPE highlights potency changes to the
net of lipophilicity). Compounds 9, 30, and 31 containing an isopropyl moiety have IC values
3
88
89
3
390
5
0
ACS Paragon Plus Environment
21

ACS Chemical Neuroscience
Page 22 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
91
92
of 5.4, 0.59, and 2.3 ꢁM, respectively, which are most potent among the analogues. It suggests
that they adopt a suitable carbonꢀchain length and topological size within the hydrophobic cleft
of the substrate site. LiPE analysis also indicates that 9, 30, and 31 are quality ligands (LiPE
values ≥ 4, Figure 2B). In addition, the new analogues alleviate tau hyperphosphorylation and
Aβ neurotoxicity through GSKꢀ3β inhibition in the human SHꢀSY5Y neuronal model of AD
(Figures 3 and 5).
3
393
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
94
95
3
396
3
97
98
With respect to the GSKꢀ3β selectivity of 30, πꢀcation interaction and orthogonal multipolar
interactions appear to play a critical role. In protein structures, the aromatic side chain of Phe or
3
4
9
399
Tyr usually involves a favorable πꢀcation interaction with the cationic side chain of Lys or Arg.
4
4
4
00
01
02
In analogy, if a small aromatic ligand binds to a protein, potential πꢀcation interactions would be
an important contribution to the binding affinity. Such phenomena have been exemplified in our
5
0
53ꢀ54
previous study as well as other investigations.
Flavonoids seem to be the case ‒ interactions
4
1, 55
403
between the aromatic A/Bꢀrings with the cationic Lys or Arg residues.
In the docking
4
04
05
experiments, we found that the catechol Bꢀring of the flavone core in 30 forms a πꢀcation
interaction with Lys183 of GSKꢀ3β (distance, 4.1 Å; angle, 71.5°). To a certain extent, this might
stabilize the binding conformation and orientation within the substrate site.
4
406
4
07
08
It is known that a fluorine atom has distinct chemical properties contributing to the molecular
4
6
4
recognition. Fluorinated compounds commonly form orthogonal multipolar interactions with
409
the backbone and side chain carbonyl carbons (Asp/Glu/Asn/Gln) or the guanidinium carbon
4
6ꢀ47
410
411
412
(Arg) of amino acid residues in protein structures.
Introduction of a CF group is a common
3
tactic in drug design. It is estimated that substitution of CH for CF may improve 5ꢀ to 10ꢀfold
3
3
4
7, 56
ligand binding affinity.
In the present study, 27 (IC , 19.3 ꢁM) and 29 (IC , 17.2 ꢁM)
50 50
413
containing a 2,2,2ꢀtrifluoroethyl and 3,3,3ꢀtrifluoropropyl, respectively, show an improved
ACS Paragon Plus Environment
22

Page 23 of 63
ACS Chemical Neuroscience
1
2
3
4
14
15
potency relative to their analogue 8 (IC , 29.2 ꢁM) without a CF group. In addition, 30 and 31
5
0
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
containing a 1,1,1ꢀtrifluoroisopropyl are more potent than their analogue 9 without a CF group.
3
416
Docking experiments suggest that the isopropyl (S)ꢀCF group of 30 interacts with the backbone
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
417
418
419
420
421
422
and side chain carbonyls of Leu88, Gln89, and Asp90 (Figure 6C) within typical interaction
47
distances (F···C, 3.0‒3.7 Å). Such orthogonal multipolar interactions might in part give rise to
an increased potency of 30 (IC , 0.59 ꢁM) against GSKꢀ3β approximately 9ꢀfold and 4ꢀfold
50
compared to 9 (IC , 5.4 ꢁM) and 31 (IC , 2.3 ꢁM), respectively. Conversely, 31 with a (R)ꢀCF
3
5
0
50
group may be lacking these critical molecular interactions (Figures 6Bꢀ6C) and thus has weaker
binding affinity than 30. The differential and stereospecific binding of the CF group between 30
3
423
and 31 plausibly explains the high inhibitory selectivity to GSKꢀ3β (Figures 2 and 4).
Interestingly, 30 exhibits isoformꢀselectivity to GSKꢀ3β over GSKꢀ3α, which deserves
discussion. In humans, GSKꢀ3α (51 kDa) and GSKꢀ3β (47 kDa) are derived from different genes
4
24
25
4
5
2
426
and have distinct functions. Both isoforms share an overall sequence identity of 83%,
5
7
427
428
429
430
431
432
especially at the ATP catalytic domain with 93% identity. However, they differ substantially in
the Nꢀ and Cꢀterminal lobes that are thought to cause protein conformational dynamics and
52, 57
differential cellular localization.
Subtle amino acid differences in the substrate site between
two isoforms (Supporting Information S6) putatively affect substrate recognition and preference
58ꢀ59
as noted in the literature.
Wang et al. reported that GSKꢀ3β phosphorylates a substrate
5
8
(phosphatase inhibitorꢀ2) 10ꢀtime faster than GSKꢀ3α, indicating the differential substrate
433
recognition of GSKꢀ3 isoforms. Soutar et al. demonstrated that the specific sites T231, T235, and
59
4
34
35
S396 on tau proteins are solely phosphorylated by GSKꢀ3β but not GSKꢀ3α, giving an evidence
4
of substrate specificity between two isoforms. Those studies pose a possibility to discover
436
isoformꢀselective inhibitors via targeting the substrate site of GSKꢀ3β. Compound 30 attains
ACS Paragon Plus Environment
23

ACS Chemical Neuroscience
Page 24 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
37
38
GSKꢀ3β isoformꢀselectivity probably because of both the πꢀcation interaction with Lys183 and
the orthogonal multipolar interactions with Leu88, Gln89, and Asp90 in GSKꢀ3β on the basis of
the comparative docking of GSKꢀ3α/β (Figures 6C and 7). In contrast, 30 is absent from those
key molecular interactions within the substrate site of GSKꢀ3α (Figure 7C). In addition, the
highly nonꢀconserved regions in GSKꢀ3α, such as the Nꢀ and Cꢀterminal domains (Figure 7A),
can modulate enzyme conformations and thereby may affect the binding of 30. Notwithstanding,
the molecular mechanism suggested by computational modeling requires further verification by
conclusive experimental evidence.
4
439
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
4
4
4
4
4
40
41
42
43
44
45
446
Conclusions
4
47
48
In summary, we described a new class of substrateꢀcompetitive inhibitors of GSKꢀ3β focusing
on modifications of the primary hydroxyl group in the Cꢀglycone of isoorientin (1). The results
demonstrated that the 6ꢀCꢀglycone moiety of the new inhibitors defines the specific binding at
the substrate site rather than the ATP site on GSKꢀ3β. The data also help explore topological
requirements in the substrate site of GSKꢀ3β and highlight the critical SAR. Those inhibitors
effectively attenuate tau hyperphosphorylation and amyloid neurotoxicity in molecular and
cellular AD models by which the mechanism is involved in GSKꢀ3β inhibition via a nonꢀATP
competitive but substrate competitive manner. The inhibitors not only significantly increased the
potency, kinase selectivity, and isoformꢀselectivity, but also improved passive membrane
permeability in comparison with the natural product counterparts. Among them, 30 (IC , 0.59
4
449
4
4
4
4
4
4
50
51
52
53
54
55
456
5
0
4
57
58
ꢁM) showed a potency improvement by 310ꢀfold and a promising profile in various biological
assessments, which warrants further exploration. SAR analyses and in silico mechanistic
investigations suggested that the hydrophobic, πꢀcation and orthogonal multipolar interactions of
4
459
ACS Paragon Plus Environment
24

Page 25 of 63
ACS Chemical Neuroscience
1
2
3
4
60
61
30 with the substrate site lead to selective inhibition against GSKꢀ3β, but neither GSKꢀ3α nor a
broad panel of kinases tested. Nevertheless, additional SAR knowledge and physiochemical
property optimization of the GSKꢀ3β inhibitors based on the 6ꢀCꢀglycosylflavone scaffold are
essential to the CNS drug discovery campaign. Pharmacokinetics and in vivo animal studies of
the new GSKꢀ3β inhibitors will help understand drug delivery, target engagement and efficacy,
which would suggest the therapeutic potential of these agents for AD and other GSKꢀ3β relevant
neuropsychiatric and neurodegenerative diseases.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
462
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
463
464
465
466
467
468
Methods
469
Chemicals and Reagents. All solvents and reagents were from commercial sources and were
4
70
71
used without further purification. Natural flavones (isoorientin, orientin, isovitexin, and luteolin),
+
−
4
staurosporine, TDZDꢀ8, theophylline, atenolol, desipramine, TMSCHN , [TEMPO] [BF ] ,
2
4
472
HCTU, organic amines, and protease inhibitor cocktail were from SigmaꢀAldrich (Saint Louis,
MO). βꢀAmyloid fragment peptide 1ꢀ42 (Aβ ) was from AnaSpec (Fremont, CA). Kinase
473
474
475
476
477
4
2
Selectivity Profiling Assay Kit, ADPꢀGlo Kinase Assay Kit, and CellTiter 96 AQueous One
Solution Cell Proliferation MTX Assay Kit were from Promega (Madison, WI). Human Tau
pS396 ELISA Kit and Cell Extraction Buffer were from Invitrogen (Camarillo, CA). Preꢀcoated
PAMPA plate system was from Corning (Tewksbury, MA).
4
78
General Experimental Procedures. Highꢀresolution mass spectrometric data were obtained
479
on a Bruker maXis Impact nanoLCꢀQTOFꢀMS spectrometer in ESI positive mode. Accurate
+
4
80
81
masses of all analytes were obtained from the pseudoꢀmolecule [M+H] and were within 5 ppm
1
13
4
mass error. H, C and 2D NMR data were recorded with a Varian Unity Inova 500 MHz
482
spectrometer. NMR spectra were referenced to the appropriate residual solvent signal (δ 2.50,
H
ACS Paragon Plus Environment
25

ACS Chemical Neuroscience
Page 26 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
83
84
δ 39.5 for DMSOꢀd ) with chemical shifts reported in δ units (ppm). Resonance multiplicities
C
6
4
are denoted s, d, t, q, m, and br for singlet, doublet, triplet, quartet, multiplet, and broad,
respectively.
485
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
486
487
488
489
490
491
All reactions were monitored by LCꢀMS (Bruker nanoLCꢀQTOFꢀMS). Compounds in crude
reaction mixtures were separated by flash column chromatography on HyperSep C18 (40–63
ꢁm), and purified by semiꢀpreparative reverse phase Agilent HPLC with a diode array detector
(Waters XSELECT CSH PhenylꢀHexyl column, 150 × 10 mm, 5 ꢁm, a linear gradient over 30
min from 10 to 50% aqueous acetonitrile containing 0.1% formic acid, flow rate 2.5 mL/min).
The purity of each compound was determined by analytical reverse phase Agilent HPLC with
a diode array detector (Waters XSELECT CSH FluoroꢀPhenyl column, 150 × 4.6 mm, 3.5 ꢁm,
isocratic elution with 35% aqueous acetonitrile containing 0.1% formic acid, detection at 210,
254, and 340 nm, flow rate of 0.8 mL/min). All the tested compounds were over 95% purity by
HPLCꢀUV at 210, 254, and 340 nm.
492
4
93
94
4
495
4
4
4
4
96
97
98
99
Luminescent measurement was performed on an Agilent Cary Eclipse fluorescence
spectrophotometer. Optical absorbance was measured on a Multiskan GO Microplate
spectrophotometer. Microscopic images were observed under a Nikon Diaphot inverted tissue
culture microscope with Optronics MicroFire microscope camera.
5
00
01
General Procedure for the SemiꢀSynthesis of CꢀGlycosylflavone Analogues.
2
8
5
Preparation of Compound 5. The chemoselective methylation proceeded as described. To a
502
stirred solution of 1 (45 mg, 0.1 mmol) in a mixture of toluene (6 mL) and methanol (4 mL) was
5
03
04
added TMSCHN (2 M in hexane, 0.5 mL, 1 mmol). The reaction solution was stirred at room
2
5
temperature for 8 h and the solvent was evaporated. The residue was purified by RPꢀHPLC
(Waters XSELECT CSH FluoroꢀPhenyl column, 150 × 4.6 mm, 3.5 ꢁm, isocratic elution with
505
ACS Paragon Plus Environment
26

Page 27 of 63
ACS Chemical Neuroscience
1
2
3
5
06
07
35% aqueous acetonitrile containing 0.1% formic acid, detection at 210, 254, and 340 nm, flow
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
rate of 0.8 mL/min) to afford 5.
508
2ꢀ(3,4ꢀDimethoxyphenyl)ꢀ5,7ꢀdimethoxyꢀ6ꢀ((2S,3R,4R,5S,6R)ꢀ3,4,5ꢀtrihydroxyꢀ6ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
509
510
511
512
513
514
(hydroxymethyl)tetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ4Hꢀchromenꢀ4ꢀone (5). Light yellow solid (80%
1
yield). H NMR (DMSOꢀd ) δ 7.65 (dd, J = 8.6, 2.2 Hz, 1H), 7.54 (d, J = 2.2 Hz, 1H), 7.12 (d, J
6
= 8.6 Hz, 1H), 7.09 (br s, 1H), 6.72 (br s, 1H), 4.65 (br d, J = 9.7 Hz, 1H), 4.05 (dd, J = 12.7, 9.2
Hz, 1H), 3.89 (s, 3H), 3.86 (s, 3H), 3.81 (s, 3H), 3.77 (s, 3H), 3.72 (dd, J = 12.6, 12.5 Hz, 1H),
13
3.34 – 3.28 (m, 1H), 3.27 – 3.12 (m, 3H). C NMR (DMSOꢀd ) δ 175.5, 163.4, 160.2, 158.9,
6
158.5, 151.8, 149.1, 123.2, 119.7, 111.9, 111.3, 109.4, 107.1, 107.0, 97.0, 82.0, 79.2, 73.1, 71.3,
+
+
515
70.9, 62.6, 62.0, 61.8, 56.4, 55.8. HRESIꢀTOFMS m/z [M+H] 505.1710 (calcd for C H O
,
2
5
29 11
5
16
17
505.1704, ꢀ1.1 ppm error). HPLC purity: 97.1% (210 nm).
5
Preparation of Compound 6. To a stirred solution of 5 (45 mg, 0.09 mmol) in a mixture of
+
−
518
dichloromethane (6 mL) and pyridine (3 mL) was added [TEMPO] [BF ] (oxoammonium salt,
4
519
520
521
522
523
524
60 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 5 h. The reaction
was quenched by adding drops of methanol and then evaporated to dryness. The residue was
reconstituted in 5% MeOH/H O and then eluted on a HyperSep C18 column using the same
2
solvents to remove the redꢀorange nitroxide. Elution was continued with 90% MeOH/H O and
2
the eluate was collected as the crude product, which was further purified by RPꢀHPLC (Waters
XSELECT CSH FluoroꢀPhenyl column, 150 × 4.6 mm, 3.5 ꢁm, isocratic elution with 35%
aqueous acetonitrile containing 0.1% formic acid, detection at 210, 254, and 340 nm, flow rate of
0.8 mL/min) to afford 6.
525
26
5
ACS Paragon Plus Environment
27

ACS Chemical Neuroscience
Page 28 of 63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
27
28
(2S,3S,4R,5R,6S)ꢀ6ꢀ(2ꢀ(3,4ꢀDimethoxyphenyl)ꢀ5,7ꢀdimethoxyꢀ4ꢀoxoꢀ4Hꢀchromenꢀ6ꢀyl)ꢀ
5
3,4,5ꢀtrihydroxytetrahydroꢀ2Hꢀpyranꢀ2ꢀcarboxylic acid (6). Light yellow solid (95% yield).
1
529
H NMR (DMSOꢀd ) δ 7.65 (dd, J = 8.5, 2.0 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.12 (d, J = 8.5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
530
531
532
533
534
Hz, 1H), 7.09 (br s, 1H), 6.76 (d, J = 6.6 Hz, 1H), 4.60 (d, J = 10.1 Hz, 1H), 4.02 (m, 1H), 3.89
13
(s, 3H), 3.85 (s, 3H), 3.82 (s, 3H), 3.75 (s, 3H), 3.30 – 3.16 (m, 3H). C NMR (DMSOꢀd ) δ
6
175.5, 173.0, 162.2, 160.4, 158.9, 158.5, 151.9, 149.2, 123.2, 119.7, 111.9, 111.4, 109.4, 107.1,
+
107.0, 97.1, 79.0, 78.5, 74.1, 72.5, 71.3, 63.5, 62.6, 56.6, 56.1. HRESIꢀTOFMS m/z [M+H]
+
519.1502 (calcd for C H O , 519.1497, ꢀ0.9 ppm error). HPLC purity: 97.8% (210 nm).
2
5
27 12
5
35
Preparation of Compound 7. To a stirred solution of 6 (1 mg, 2 ꢁmol) in a mixture of toluene
536
(1.5 mL) and methanol (1 mL) was added TMSCHN (2 M in hexane, 2 ꢁL, 4 ꢁmol). The
2
5
37
38
reaction solution was stirred at room temperature for 1 h and the solvent was evaporated. The
residue was purified by RPꢀHPLC (Waters XSELECT CSH FluoroꢀPhenyl column, 150 × 4.6
mm, 3.5 ꢁm, isocratic elution with 35% aqueous acetonitrile containing 0.1% formic acid,
detection at 210, 254, and 340 nm, flow rate of 0.8 mL/min) to afford 7.
5
539
5
40
541
542
543
544
545
Methyl (2S,3S,4R,5R,6S)ꢀ6ꢀ(2ꢀ(3,4ꢀdimethoxyphenyl)ꢀ5,7ꢀdimethoxyꢀ4ꢀoxoꢀ4Hꢀchromenꢀ
6ꢀyl)ꢀ3,4,5ꢀtrihydroxytetrahydroꢀ2Hꢀpyranꢀ2ꢀcarboxylate (7). Light yellow solid (87% yield).
1
H NMR (DMSOꢀd ) δ 7.69 (dd, J = 8.5, 2.1 Hz, 1H), 7.56 (d, J = 2.1 Hz, 1H), 7.15 (br s, 1H),
6
7.12 (d, J = 8.5 Hz, 1H), 6.80 (d, J = 5.5 Hz, 1H), 4.57 (d, J = 9.6 Hz, 1H), 4.00 (m, 1H), 3.89 (s,
1
3
3H), 3.85 (s, 3H), 3.80 (s, 3H), 3.73 (s, 3H), 3.63 (s, 3H), 3.29 – 3.13 (m, 3H). C NMR
546
(DMSOꢀd ) δ 175.6, 172.9, 162.1, 160.1, 159.0, 158.4, 151.8, 149.1, 123.2, 119.7, 111.9, 111.4,
6
5
47
48
109.4, 107.1, 107.0, 97.0, 79.0, 78.5, 74.1, 72.5, 71.3, 63.4, 62.6, 56.6, 56.0, 51.8. HRESIꢀ
+
+
5
TOFMS m/z [M+H] 533.1656 (calcd for C H O , 533.1654, ꢀ0.5 ppm error). HPLC purity:
2
6
29 12
549
97.1% (210 nm).
ACS Paragon Plus Environment
28

Page 29 of 63
ACS Chemical Neuroscience
1
2
3
5
50
51
Preparation of Compounds 8ꢀ31. To a stirred solution of 6 (5 mg, 9.7 ꢁmol) in a mixture of
dimethylformamide (1 mL) and DIPEA (0.5 mL) was added HCTU (10 mg, 24 ꢁmol) and then
stirred for 10 min at room temperature. To this solution was added corresponding organic amines
(each 50 ꢁmol) and stirred at room temperature for 5 h.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
552
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
53
554
555
556
557
The reaction was quenched by adding 1 N HCl followed by evaporation of solvents to dryness.
The residue was purified by RPꢀHPLC (Waters XSELECT CSH FluoroꢀPhenyl column, 150 ×
4.6 mm, 3.5 ꢁm, isocratic elution with 35% aqueous acetonitrile containing 0.1% formic acid,
detection at 210, 254, and 340 nm, flow rate of 0.8 mL/min) to afford the final products.
5
58
(2S,3S,4R,5R,6S)ꢀ6ꢀ(2ꢀ(3,4ꢀDimethoxyphenyl)ꢀ5,7ꢀdimethoxyꢀ4ꢀoxoꢀ4Hꢀchromenꢀ6ꢀyl)ꢀ
559
3,4,5ꢀtrihydroxyꢀNꢀpropyltetrahydroꢀ2Hꢀpyranꢀ2ꢀcarboxamide (8). Light yellow solid (90%
1
5
60
61
yield). H NMR (DMSOꢀd ) δ 7.67 (d, J = 8.7 Hz, 1H), 7.53 (d, J = 2.2 Hz, 1H), 7.13 (s, 1H),
6
5
7.12 (d, J = 8.7 Hz, 1H), 6.76 (d, J = 5.9 Hz, 1H), 4.67 (br d, J = 10.1 Hz, 1H), 4.06 (q, J = 10.3
562
Hz, 1H), 3.87 (s, 3H), 3.83 (s, 3H), 3.77 (s, 3H), 3.71 (s, 3H), 3.60 (m, 1H), 3.50 (m, 1H), 3.23
1
3
563
564
565
566
567
(m, 1H), 2.99 (m, 2H), 1.36 (m, 2H), 0.78 (td, J = 7.4, 2.2 Hz, 3H). C NMR (DMSOꢀd ) δ
6
175.7, 168.7, 163.4, 160.5, 160.2, 158.4, 151.6, 149.3, 123.1, 120.4, 112.2, 111.9, 109.6, 107.2,
107.0, 96.8, 80.6, 79.0, 74.4, 71.6, 69.6, 63.9, 63.1, 56.7, 56.1, 40.8, 22.6, 11.5. HRESIꢀTOFMS
+
+
11
m/z [M+H] 560.2135 (calcd for C H NO , 560.2126, ꢀ1.6 ppm error). HPLC purity: 98.6%
2
8
34
(210 nm).
5
68
(2S,3S,4R,5R,6S)ꢀ6ꢀ(2ꢀ(3,4ꢀDimethoxyphenyl)ꢀ5,7ꢀdimethoxyꢀ4ꢀoxoꢀ4Hꢀchromenꢀ6ꢀyl)ꢀ
569
3,4,5ꢀtrihydroxyꢀNꢀisopropyltetrahydroꢀ2Hꢀpyranꢀ2ꢀcarboxamide (9). Light yellow solid
1
5
70
71
(90% yield). H NMR (DMSOꢀd ) δ 7.67 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 2.2 Hz, 1H), 7.14 (br s,
6
5
1H), 7.12 (d, J = 9.7 Hz, 1H), 6.77 (d, J = 5.1 Hz, 1H), 4.67 (d, J = 9.7 Hz, 1H), 4.05 (m, 1H),
ACS Paragon Plus Environment
29