Identification of 2-subsituted benzothiazole derivatives as triple-functional agents with potential for AD therapy

Article abstract of DOI:10.1039/c5ra25788c

A novel series of 2-subsituted benzothiazole derivatives as MTDLs were designed and synthesized for AD therapy using pharmacophore-combine strategy. The benzothiazole moiety from ThT and the HPO moiety from deferiprone were connected with vinyl linker to

Full text of DOI:10.1039/c5ra25788c

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Identification of 2-subsituted benzothiazole
derivatives as triple-functional agents with
Cite this: RSC Adv., 2016, 6, 17318
potential for AD therapy†
Liu Jiang,a Minkui Zhang,a Li Tang,a Qinjie Weng,a Yanhong Shen,b Yongzhou Hua
a
*
and Rong Sheng
A novel series of 2-subsituted benzothiazole derivatives as MTDLs were designed and synthesized for AD
therapy using pharmacophore-combine strategy. The benzothiazole moiety from ThT and the HPO
moiety from deferiprone were connected with vinyl linker to achieve target compounds. The biological
evaluation results revealed that the majority of them demonstrated desirable triple functions by
interfering with Ab aggregation, oxidative stress and metal dyshomeostasis simultaneously. The two most
attractive compounds 9c and 9i exhibited excellent self-Ab1–42 aggregation inhibitory activity, efficient
ABTSc+ scavenging activity, potent biometals chelating properties, as well as disaggregation activity
against previous formed Ab1–42 fibrils. In addition to these advantages, both of them displayed no
cytotoxicity to human glioma U251 cells up to 50 mM, thereby meriting further investigation.
Received 3rd December 2015
Accepted 14th January 2016
DOI: 10.1039/c5ra25788c
www.rsc.org/advances
Evidences show that the rising Ab monomer level in the
brain (resulting from unbalanced Ab production and clearance)
1 Introduction
promotes the formation of dimers and larger oligomers, and
then oligomers aggregate progressively to form protobrils,
brils and plaques of brils, the major histopathological hall-
mark of AD.19–21 Aggregated Ab species are neurotoxic, so
compounds capable of inhibiting Ab aggregation may exert
potential therapeutic effect on AD.22–24 Benzothiazole derivative
thioavin T (ThT) is the most widely used Ab aggregation
indicator in vitro, which displays uorescence enhancement
and a characteristic red shi when binding to Ab aggregates.25,26
Recently, its analogue utemetamol 18F has been approved by
FDA as the PET tracer for imaging b-amyloid (Ab) deposition in
AD patients.27 In addition, natural stilbene derivative resveratrol
also exhibits extraordinary binding affinity to amyloid aggre-
gates, and its analogue orbetaben 18F launched in clinic for Ab
deposits imaging.28–30 Due to their high affinity to Ab, benzo-
thiazole and stilbene scaffold have been frequently incorpo-
rated into different molecules for attaining Ab aggregation
inhibitory activity.31,32
Meanwhile, remarkably high concentration of biometals
(Cu, ꢀ0.4 mM. Zn, ꢀ1.0 mM. Fe, ꢀ0.9 mM) was found to co-
localize with the amyloid deposits in AD-affected brains.
Research reveals that these biometals can rapidly facilitate Ab
aggregation through binding to three histidines (H6, H13 and
H14) of Ab peptide. Besides, the binding of iron and copper to
Ab species generates reactive oxygen species (ROS), which may
result in oxidative damage to biological molecules.11–18 Owing to
these, blocking metals-induced Ab aggregation and reducing
oxidative stress have been regarded as a promising approach for
AD therapy. During the pursuit of MTDLs, deferiprone for iron
Alzheimer's disease (AD), which causes progressive memory
loss and other irreversible cognitive impairments, is the most
common form of dementia that afflicts more than 24 million
people worldwide.1 Despite the tremendous efforts devoted to
this serious neurodegenerative disease since its discovery more
than 100 years ago, the exact etiology of AD still remains
elusive.2 So far, no therapeutic agents have been available other
than several drugs approved for the symptomatic treatment of
AD, including three cholinesterase (AChE) inhibitors (donepe-
zil, rivastigmine, and galantamine) and one N-methyl-D-aspar-
tate (NMDA) receptor antagonist (memantine).3,4
Numerous evidences indicate that diverse factors are
responsible for the initiation and progression of this complex
disease, including amyloid-b (Ab) aggregation, s-protein hyper-
phosphorylation, oxidative stress, metal ion dyshomeostasis,
decreased levels of acetylcholine, neuroinammation in
central nervous system (CNS), and so on.5–18 Reasoning this,
recently more efforts have been focused on the development
of multi-target-directed-ligands (MTDLs), which interfere
with two or more causes of AD simultaneously and may
achieve better therapeutic efficacy through complementary
mechanism.5,19
aCollege of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
E-mail: shengr@zju.edu.cn; Fax: +86-571-8820-8458; Tel: +86-571-8820-8458
bCollege of Chemistry and Environmental Science, Anyang Institute of Technology,
Anyang 455000, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra25788c
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bioisostere 3-hydroxy-4-pyranone from maltol (a wildly used
food avoring agent)39 to ameliorate the toxic prole of
compounds. To investigate the structural activity relationship
(SAR) of these newly designed compounds and nd optimal
compounds for further development, the substituent in the
benzothiazole ring was modied. The benzoxazole and N-
methyl-benzoimidazole were introduced into the molecule as
the bioisostere of benzothiazole. In these newly designed
compounds, the benzothiazole or similar moieties and stilbene-
similar scaffold may demonstrate Ab aggregation inhibitory
activity, and the 3-hydroxy-4-pyridinone (3-hydroxy-4-pyranone)
moiety may exert metal chelating and anti-oxidant activity. In
addition, all the structural elements of compounds were
selected to adhere to the values of Lipinski's rules (Table S1†).
To conrm the rationality of our design strategy, the
molecular docking study of 2-substituted-benzothiazole deriv-
ative 9a and HL12 with Ab1–42 (PDB ID: 1IYT) were performed
using SYBYL-X 1.3 soware package (Tripos, Inc.).40
Fig. 1 Structures of lead compounds.
overload in thalassemia major has attracted much attention as
an oral metal chelator, in which 3-hydroxy-4-pyridinone (HPO)
plays a vital role in chelating activity on copper, zinc and iron
ions as well as potential radical scavenging activity. Orvig group
has reported several series of HPO derivatives as MTDLs with
potential AD therapeutic effect.32–36 Among them, HL5 and HL12,
incorporating both benzothiazole and HPO moieties, exhibit
metal chelating activity, along with Ab aggregation inhibitory
potency.32,33 In addition, HL12 also demonstrates potent anti-
oxidant activity similar to Trolox. However, the relatively high
cytotoxicity hinders its further development (Fig. 1).32
As shown in Fig. 3, although both HL12 and compound 9a
are located nearby the N-terminus (residues 1–17) of Ab1–42
,
they interacted with the Ab1–42 residues in different manners.
The 3-hydroxy group of HL12 formed hydrogen bond with
ꢀ
Lys16 at a distance of 2.84 A, and the benzothiazole moiety
interacted with His13 through another hydrogen bond
ꢀ
(2.99 A), which was similar to the docking result of HL12 with
2 Rational design of novel MTDLs
32
Ab1–40
.
For compound 9a, the oxygen of pyranone moiety
ꢀ
As a continuation of our previous work on 3-substituted indole
and 1-phenyl-3-hydroxy-4-pyridinone derivatives as multifunc-
tional agents for AD therapy,37,38 herein, we report a series of 2-
subsituted benzothiazole derivatives (9a–o) as the novel multi-
functional agents capable of interfering with Ab aggregation,
metal dyshomeostasis and oxidative stress simultaneously
(Fig. 2).
To develop a novel MTDLs capable of targeting the above
three AD pathological factors, we undertook a rational drug
design using pharmacophore-combination strategy. The ben-
zothiazole moiety from ThT (Ab aggregation inhibition phar-
macophore) was connected with 3-hydroxy-4-pyridinone from
deferiprone (metal chelating and radical scavenging pharma-
cophore) using a vinyl linker. The newly designed 2-benzo-
thiazole derivatives share the similar scaffold to resveratrol,
which may be benecial to improve the binding affinity with Ab
as well as to enhance the antioxidant activity through conju-
gation effect. In addition, the HPO moiety was replaced by its
formed hydrogen bond (2.42 A) with Leu17 of Ab1–42. In
addition, the benzothiazole moiety was embedded in a groove
formed by Lys16 and Phe20 and interacted with the amino
ꢀ
group of Lys16 at a distance of 2.96 A. On the basis of
the molecular modeling results, we considered that the
compound 9a could bind monomeric Ab so that it was able to
inhibit self-induced Ab aggregation by disturbing the
formation of b-sheets.
Fig. 3 Docking studies of 9a and HL12 with Ab1–42 (PDB code 1IYT). (A
and B) Binding mode of 9a (colored yellow) with Ab1–42 monomer. The
hydrogen bonds between 9a and residues Leu16 and Lys17 are indi-
cated by red lines. (C and D) Binding mode of HL12 (colored green) with
Fig. 2 Rational design of 2-subsituted benzothiazole derivatives as Ab1–42 monomer. The hydrogen bonds between HL12 and residues
MTDLs.
Leu17, Lys16 are indicated by red lines.
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3 Results and discussion
Synthesis
The synthetic route of 2-substituted-benzothiazole derivatives
9a–o is outlined in Scheme 1. Protection of phenol moiety of
compound 10a and 10b with para-methoxy benzyl chloride
(PMB-Cl) gave 11a and 11b, which were oxidized to benzalde-
hyde derivatives 12a and 12b using MnO2, respectively.
Condensation of 12a, b with 2-methyl-benzothaizole (or 2-
methyl-benzoxazole, or N-methyl-2-methyl-benzoimidazole) in
HAc–Ac2O under microwave condition afforded compounds
13a–h, 13j and 13l, followed by deprotection of –OAc with K2CO3
to furnish target compounds 9a–9h, 9j and 9l. Demethylation of
compounds 9g, 9j and 9l with BBr3 in CH2Cl2 gave 6-hydroxy
derivatives 9i, 9k and 9m, respectively. For the 3-hydroxy-4-
pyridinone derivatives, condensation of 11a with methylamine
gained compound 14, which was oxidized to aldehyde 15 with
MnO2. Condensation of compounds 16a, b with triethyl phos-
phite gave compounds 17a, b, which experienced aldol
condensation with aldehyde 15 to give compound 18a, b.
Finally, deprotection of 18a, b with CF3COOH provided target
compounds 9n and 9o.
Scheme
1 Synthetic route to compounds 9a–9o. Reagents and
conditions. (a) PMB-Cl, K2CO3, DMF, 80 ꢁC. (b) MnO2, CHCl3, reflux. (c)
2-Methyl-benzothaizole, or 2-methyl-benzoxazole, or N-methyl-2-
methyl-benzoimidazole, HAc, Ac2O, microwave. (d) K2CO3, MeOH, r.t.
(e) BBr3, CH2Cl2. (f) CH3NH2, H2O, reflux. (g) P(OEt)3, reflux. (h) NaH,
dry THF, r.t. (i) CF3COOH, CH2Cl2.
Biological evaluation
Inhibition of self-induced Ab1–42 aggregation. The inhibitory
ability of compounds 9a–9o against self-induced Ab1–42
Table 1 Biological activities of compounds 9a–9o
Inhibition of self-induced Ab1–42
aggregation
Compd
X
Y
R2
R1
(%)a
IC50b (mM)
TEAC valuesb
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
HL5
HL12
Curcumin
Trolox
S
S
O
O
NCH3
NCH3
S
S
S
O
O
NCH3
NCH3
S
O
—
O
O
O
O
O
O
O
O
O
O
O
O
H
H
H
H
H
H
OCH3
Br
OH
OCH3
OH
OCH3
OH
H
H
—
—
—
H
CH3
H
CH3
H
CH3
H
H
H
H
H
H
H
H
H
—
—
—
—
43.82 ꢂ 5.54
49.36 ꢂ 2.01
64.95 ꢂ 5.84
59.20 ꢂ 2.48
62.35 ꢂ 3.42
71.41 ꢂ 1.50
67.40 ꢂ 2.30
46.82 ꢂ 3.42
78.26 ꢂ 3.93
65.12 ꢂ 2.53
80.42 ꢂ 5.36
80.27 ꢂ 3.82
73.65 ꢂ 5.25
45.59 ꢂ 4.48
65.73 ꢂ 4.34
64.75 ꢂ 4.86
67.78 ꢂ 3.48
56.81 ꢂ 0.46
n.t.c
n.t.c
4.03 ꢂ 0.02
1.45 ꢂ 0.09
4.29 ꢂ 0.03
1.34 ꢂ 0.25
2.31 ꢂ 0.21
1.55 ꢂ 0.06
2.10 ꢂ 0.021
1.31 ꢂ 0.81
6.03 ꢂ 0.12
2.69 ꢂ 0.32
4.98 ꢂ 0.56
1.38 ꢂ 0.47
3.53 ꢂ 0.23
1.64 ꢂ 0.17
2.79 ꢂ 0.09
0.64 ꢂ 0.43
1.10 ꢂ 0.06
1.56 ꢂ 0.26
1.00 ꢂ 0.24
n.t.c
7.05 ꢂ 0.42
12.45 ꢂ 1.17
10.36 ꢂ 0.67
9.23 ꢂ 1.05
6.32 ꢂ 1.78
n.t.c
5.06 ꢂ 1.02
8.25 ꢂ 0.41
5.38 ꢂ 0.57
5.97 ꢂ 0.73
6.90 ꢂ 0.45
n.t.c
8.56 ꢂ 0.82
9.78 ꢂ 1.23
8.75 ꢂ 1.09
8.48 ꢂ 1.14
n.t.c
O
NCH3
NCH3
—
—
—
—
—
—
—
—
a
The thioavin-T uorescence method was used. The values are expressed as the mean of three independent measurements. All values were
b
c
obtained at a compound concentration of 20 mM. The mean of the three independent experiments. n.t. means not tested.
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aggregation was measured by thioavin T (ThT) uorescence
assay, and curcumin was used as control. Table 1 showed that
all the compounds demonstrated moderate to excellent Ab1–42
self-aggregation inhibitory activity, and seven of them were
more potent (IC50 values ranging from 5.06 mM to 8.25 mM) than
The choice of drug candidates. Based on the above biological
evaluation results, compounds 9c and 9i exhibited the most
desirable triple functions (IC50 values of 7.05, 5.06 mM for Ab
self-aggregation inhibition, TEAC values of 4.29, 6.03 for ABTSc+
scavenging activity and potent chelating activities with copper,
iron and zinc ions). Therefore, the two compounds were chosen
as drug candidates for further biological evaluation.
curcumin (IC50 ¼ 8.48 mM) and two positive controls HL5 (IC50
¼
9.78 mM), HL12 (IC50 ¼ 8.75 mM).
The primary SAR revealed that the modication of benzo-
thiazole ring affected the activity obviously, for instance, the
benzoxazole and N-Me-benzoimidazole rings were more bene-
cial for Ab1–42 aggregation inhibition than benzothiazole (such as
9c, 9e vs. 9a; 9d, 9f vs. 9b). Introduction of methyl at the C-2
position of pyranone impacted slightly on activity, such as 9a
vs. 9b; 9e vs. 9f. The obviously increased activities of compounds
9g, 9j, 9i, 9k (IC50 ¼ 6.32, 8.25, 5.06, 5.38 mM, respectively)
compared with 9a (43.82% inhibition in 20 mM) revealed that
introduction of methoxy or hydroxy moiety into the 6-position of
benzothiazole was benecial for Ab1–42 aggregation inhibition.
Trolox-equivalent antioxidant capacity assay. The antioxi-
dant activities of compounds 9a–9o were evaluated with Trolox-
equivalent antioxidant capacity (TEAC) assay, which is based on
the generation and detection of a blue-colored cation (ABTSc+,
2,20-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) radical
cation) using Trolox as a control.37,38 The TEAC values were
expressed as Trolox equivalents calculated from the ratio of the
slopes of the concentration–response curves, the antioxidant vs.
Trolox (TEAC value ¼ 1.00).
Inhibition of self-induced Ab aggregation monitored by
TEM. To further conrm the ability of compounds 9c and 9i
against Ab1–42 aggregation, the inhibitory activities of them
were monitored by transmission electron microscopy (TEM)
using curcumin as a positive control. Aer incubated for 24 h
at 37 ꢁC, compared to Ab1–42 without incubation (Fig. 4a), Ab1–
alone aggregated into well-dened Ab brils (Fig. 4b). On
42
the contrary, few Ab brils were observed in the presence of
compounds 9c and 9i (Fig. 4d and e) under identical condi-
tions, which were similar to that of curcumin (Fig. 4c).
Therefore, the results of TEM and ThT assay proved that
compounds 9c and 9i could effectively inhibit self-induced
Ab1–42 bril formation.
Disaggregation of self-induced Ab aggregation. The ability of
compounds 9c and 9i to disaggregate self-induced Ab1–42
aggregation brils was investigated by thioavin T (ThT) uo-
rescence assay and transmission electron microscopy (TEM)
using curcumin as positive control. Ab1–42 brils were generated
ꢁ
by incubating fresh Ab1–42 for 24 h at 37 C (Fig. 5b), then cur-
cumin, compounds 9c or 9i was added to the sample and
ꢁ
As shown in Table 1, all the compounds displayed excellent
ABTSc+ scavenging activities with TEAC values ranging from
1.31 to 6.03, and the majority of them were superior to that of
curcumin, HL5 and HL12 (TEAC values of 0.64, 1.10, 1.56
respectively). In particular, four 3-hydroxy-4-pyranone deriva-
tives 9a, 9c, 9i, 9k displayed very potent radical scavenging
activities with TEAC values of 4.03, 4.29, 6.03 and 4.98 respec-
tively. The primary SAR indicated that modication of benzo-
thiazole ring signicantly affected the activity with benzoxazole
and N-Me-benzoimidazole, when the compounds containing
benzoxazole moieties exerted similar antioxidant activity, while
N-Me benzoimidazole derivatives displayed compromised
activity (9a, 9c vs. 9e). As expected, in comparison with unsub-
stituted compounds (9a, 9e), the introduction of hydroxy moiety
into R2 position of benzothiazole ring resulted in a remarkable
enhancement in activity (9i, 9m), while the bromo and methoxy
moieties were not favorable for activity (9h, 9j). The methyl
moiety in R1 pyranone ring also decreased the antioxidant
capacities obviously (9a vs. 9b, 9c vs. 9d), indicating the detri-
mental steric effect on antioxidant activity.
incubated for another 24 h at 37 C with constant agitation.
The ThT binding assay revealed that both compound 9c and
9i could effectively disaggregate previously formed self-induced
Ab1–42 brils (49.3% and 47.9%, respectively), which were
a slightly better than curcumin (53.6%) (Fig. S2†).
The TEM assay results in Fig. 5c–e also showed that
compounds 9c and 9i were capable of disassembling the Ab1–42
Metal chelating property. The metal chelating activities of
compounds 9a–o were measured by UV-vis spectroscopy using
deferiprone and maltol as positive control. As expected, all the
compounds displayed good metal chelating activities similar to
deferiprone and maltol. As shown in Fig. S1 (ESI†), signicant
red shi was observed in UV-vis spectra when Cu2+, Zn2+, Fe2+,
Fe3+ was mixed with the compounds 9a–o, deferiprone and
maltol, indicating the formation of metal ion complex. These
results conrmed that the 3-hydroxy-4-pyridinone or 3-hydroxy-
4-pyranone moiety was essential for metal chelating activity.
Fig. 4 (Top) Scheme of inhibition experiments on self-induced Ab
aggregation. TEM images. (a) Ab1–42, 0 h. (b) Ab1–42 alone. (c) Ab1–42
curcumin. (d) Ab1–42 + 9c. (e) Ab1–42 + 9i.
+
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UV spectroscopy of 9c with CuCl2 complex at different concen-
trations were evaluated and the absorbance changes at 341 nm
were plotted. As illustrated in Fig. 6c, the two straight lines
intersected at a mole fraction of 0.686, which implied a 2 : 1 9c–
Cu(II) complex. Similarly, the UV spectroscopy of 9i with CuCl2
complex was shown in Fig. 6d with the absorbance changes at
370 nm being plotted. The Job plot revealed a break at 0.651,
indicating a 2 : 1 stoichiometry for the 9i–Cu(II) complex.
Inhibition of Cu2+-induced Ab aggregation. To investigate
the ability of 2-subsituted benzothiazole derivatives to inhibit
Cu(II)-induced Ab1–42 aggregation, the ThT uorescence and
TEM experiments were carried out. The ThT uorescence assay
revealed that the Cu2+ led to a reduced ThT uorescence in
comparison with Ab self (83.7%, Fig. S3†), probably due to the
emission quenched by the paramagnetic Cu2+ ions-induced
formation of nonbrillar Ab aggregates.41,42 The addition of 9c
and 9i resulted in a dramatic decrease of ThT uorescence
(27.0% and 24.1% respectively), indicating their excellent
activities to inhibit Cu2+ induced Ab1–42 aggregation in
comparison with CQ (46.3%).
Fig. 5 (Top) Scheme of disaggregation experiments on self-induced
Ab1–42 aggregation. TEM images. (a) Ab1–42, 0 h. (b) Ab1–42 alone. (c)
Ab1–42 + curcumin. (d) Ab1–42 + 9c. (e) Ab1–42 + 9i.
The TEM showed that a signicant amount of brillogenesis
brils from self-mediated aggregation with similar activity to (Fig. 7c) was observed in the presence of Cu(II) than for Ab1–42
that of curcumin.
alone (Fig. 7b), and fewer Ab brils were observed when 9c
Metal-chelating characteristics of 9c, 9i. The ability of 9c and (Fig. 7e) or 9i (Fig. 7f) was added to the samples, which were
9i to chelate biometals was studied by UV-vis spectroscopy similar to that of CQ added the sample (Fig. 7d). The above
observation suggested that compounds 9c and 9i were potent
copper chelators with ability to inhibit copper-induced Ab
aggregations.
(Fig. 6). Aer ZnCl2, FeSO4, CuCl2 or FeCl3 was added to
a solution of 9c, an obvious drop of absorbance at 341 nm was
observed (Fig. 6a) indicating the formation of 9c–Zn2+, 9c–Fe2+,
9c–Cu2+, 9c–Fe3+ complex. Similarly, Fig. 5b revealed that
Disaggregation of Cu-induced Ab1–42 aggregation. The
compound 9i was potent chelator for biometals Cu2+, Fe3+, Zn2+ capability of compounds 9c and 9i to disaggregate copper-
and Fe2+.
induced Ab aggregation brils was investigated by Th-T uo-
The stoichiometry of the 9c and 9i with copper complex was rescence assay and TEM as well. Ab1–42 brils were generated by
determined using Job's method. A series of solutions were incubating fresh Ab1–42 with 1.0 equiv. of Cu2+ for 24 h at 37 ꢁC
with constant agitation (Fig. 8b). Compound 9c, 9i or clioquinol
prepared, in which the total concentration of compound 9c or 9i
and CuCl2 remained constant while their proportions varied. The
Fig. 6 (a) UV-vis spectra of compounds 9c with biometals. (b) UV-vis
spectra of compounds 9i with biometals. [9c], [9i] ¼ 50 mM, [Cu2+],
[Zn2+], [Fe2+], [Fe3+] ¼ 25 mM. (c) Determination of the stoichiometry of
complex 9c–Cu(II) by Job's method. (d) Determination of the stoi-
chiometry of complex 9i–Cu(II) by Job's method.
Fig. 7 (Top) Scheme of inhibition experiment on copper induced
Ab1–42 aggregation. TEM images. (a) Ab1–42, 0 h. (b) Ab1–42 alone, 24 h.
(c) Ab1–42 + Cu, 24 h. (d) Ab1–42 + Cu + CQ. (e) Ab1–42 + Cu + 9c. (f)
Ab1–42 + Cu + 9i.
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4 Conclusions
A novel series of 2-substituted-benzothiazole were designed as
potential MTDLs for AD therapy through combination of metal
chelating moiety 3-hydroxy-4-pyranone (or 3-hydroxy-4-
pyridinone) and benzothiazole moiety from ThT with vinyl
linker. Most of these compounds exhibited more potent Ab1–42
aggregation inhibitory activity and more efficient ABTSc+ scav-
enging capability than that of curcumin and two positive
controls HL5 and HL12, which conrmed the rationality of our
drug design strategy. The further in vitro biological evaluation of
two promising compounds 9c and 9i revealed that they were
novel MTDLs with triple functions. Moreover, the cytotoxic
evaluation also demonstrated that 9c and 9i were not toxic to
human glioma U251 cells up to 50 mM, which were much better
than HL5 and HL12. All the above results indicated that these
new hybrids were interesting MTDLs with potential to be
developed as therapeutic agents against AD.
Fig. 8 (Top) Scheme of disaggregation experiments on copper-
induced Ab1–42 aggregation. TEM images. (a) Ab1–42, 0 h. (b) Ab1–42
+
Cu. (c) Ab1–42 + Cu + CQ. (d) Ab1–42 + Cu + 9c. (e) Ab1–42 + Cu + 9i.
5 Experimental section
General methods and materials
was then added to the sample and incubated for another 24 h
at 37 C.
ꢁ
All reagents and solvents used were reagent grade and
As shown in Fig. S3,† the ThT binding assay demonstrated purchased from commercial resources. Ab1–42 was purchased
that both compound 9c and 9i was able to resolve Ab brils from Chinese Peptide Company. Melting points were deter-
mined with a B-540 Buchi melting-point apparatus. 1H NMR
(30.9% and 26.8% disaggregation respectively).
In comparison with the well-dened Ab brils in Fig. 8b, and 13C NMR were recorded on a Bru¨ker Advance DMX 500 MHz
noticeably fewer Ab1–42 brils were observed when CQ or 9c or 9i or DMX 400 MHz spectrometer. Coupling constants (J) were
was added to the samples (Fig. 8c–e), indicating 9c and 9i could expressed in hertz (Hz) and chemical shis (d) of NMR
efficiently disassemble copper-induced Ab1–42 brils.
were reported in parts per million (ppm) units relative to an
Cytotoxicity. To investigate the safety prole of these ben- internal control (TMS). Low resolution mass spectras (ESI-MS)
zothiazole derivatives, the cytotoxicity of compounds 9c and 9i were gathered on a Finnigan LCQ DecaXP ion trap mass spec-
against human glioma U251 cells was evaluated by SRB (sulfo- trometer. UV-visible spectras were recorded on a U-3010 spec-
rhodamine B) assay, and HL5 and HL12 was used reference
trophotometer. TECAN F200 reader was used for radical
compounds. As illustrated in Fig. 9, compounds 9c and 9i did scavenging assay, ThT uorescence assay. JEM1200EX trans-
not show obvious cytotoxicity at concentrations 50 mM aer 24 h mission electron microscope was used for Ab1–42 aggregation
incubation, while for the controls HL5 and HL12, they demon- and disaggregation assay. All targeted compounds were puried
strated obvious toxicity to U251 cells at concentrations 10 mM. to $94% purity as determined by Agilent 1260 series HPLC
These results indicated that compounds 9c and 9i possessed systems with a Eclipse XDB-C18 (4.6 ꢃ 250 mm, 5 mm); 20.0 mL
more favorable safety prole than HL5 and HL12 and were injection volume; ow rate of 1.0 mL minꢄ1; (mobile phase: (A)
suitable for further development for the treatment of AD.
H2O, (B) MeCN) with a UV detector set at 254 nm.
General procedure for the synthesis of compounds 11a and
11b
To a mixture of compound 10a (42.2 mmol) and K2CO3 (12.2 g,
88.7 mmol) in dry DMF (15 mL) was added PMB-Cl (7.1 mL, 52.8
mmol) in drop wise. The mixture was stirred for 2 h at 80 ꢁC and
quenched with 10 mL H2O. Aer extraction with EtOAc (3 ꢃ 20
mL), the combined organic layer was washed with water, satu-
rated saline and dried over Na2SO4. The solvent was removed in
vacuo and the residue was recrystallized with ethyl acetate to
afford compound 11a. 2-(Hydroxymethyl)-5-((4-methoxybenzyl)
oxy)-4H-pyran-4-one (11a). White solid, yield 84.1%. 1H NMR
(500 MHz, DMSO)/d (ppm): 8.14 (s, 1H, H-6), 7.34 (d, 2H, J ¼ 8.0
Hz, Ph-H-20, H-60), 6.95 (d, 2H, J ¼ 7.5 Hz, Ph-H-30, H-50), 6.30 (s,
1H, H-3), 5.67 (t, 1H, J ¼ 5.5 Hz, OH), 4.85 (s, 2H, Ph-CH2), 4.29
Fig. 9 Effects of 9c, 9i, HL5 and HL12 on U251 cell viability. Data
represent mean ꢂ SD.
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(d, 2H, J ¼ 5.5 Hz, CH2OH), 3.75 (s, 3H, OCH3), ESI-MS (m/z): d (ppm): 8.56 (s, 1H, H-6), 7.83 (d, 1H, J ¼ 7.5 Hz, H-40), 7.78 (d,
263.2 [M + H]+.
1H, J ¼ 8.0 Hz, H-70), 7.66 (d, 1H, J ¼ 16.0 Hz, CH]CHa), 7.51–
6-(Hydroxymethyl)-3-((4-methoxybenzyl)oxy)-2-methyl-4H- 7.49 (m, 1H, H-50), 7.46–7.43 (m, 1H, H-60), 7.39 (d, 1H, J ¼ 16.0
pyran-4-one (11b). White solid, yield 64.0%. 1H NMR (400 MHz, Hz, CHb]CH), 6.98 (s, 1H, H-3), 2.28 (s, 3H, COCH3). ESI-MS
CDCl3)/d (ppm): 7.31 (d, 2H, J ¼ 8.4 Hz, Ph-H-20, H-60), 6.87 (m/z): 298.2 [M + H]+.
(d, 2H, J ¼ 8.8 Hz, Ph-H-30, H-50), 6.45 (s, 1H, H-5), 5.07 (s, 2H,
(E)-6-(2-(Benzo[d]oxazol-2-yl)vinyl)-2-methyl-4-oxo-4H-pyran-
Ph-CH2), 4.44 (s, 2H, CH2OH), 3.80 (s, 3H, OCH3), 2.05 (s, 3H, 3-yl acetate (13d). Yellow solid, yield 52.2%. 1H NMR (400 MHz,
CH3). ESI-MS (m/z): 277.3 [M + H]+.
CDCl3)/d (ppm): 7.78 (d, 1H, J ¼ 8.8 Hz, H-40), 7.57 (d, 1H, J ¼ 8.4
Hz, H-70), 7.44–7.37 (m, 2H, H-50, H-60), 7.32 (s, 2H, CH]CH),
6.50 (s, 1H, H-3), 2.37 (s, 3H, CH3), 2.36 (s, 3H, COCH3). ESI-MS
(m/z): 312.2 [M + H]+.
General procedure for the synthesis of compounds 12a and 12b
A mixture of 11a (2.62 g, 10.0 mmol) and manganese(IV) oxide
(13.04 g, 150.0 mmol) suspended in CHCl3 (65 mL) was reux
overnight. Aer cooling to r.t., the reaction mixture was ltered
through Celite and the ltrate was concentrated to dryness. The
residue was puried by silica gel chromatography eluting with
PE/EtOAc to afford 12a as a white solid. Yield 45.2%. 5-((4-
Methoxybenzyl)oxy)-4-oxo-4H-pyran-2-carbaldehyde (12a). 1H
NMR (400 MHz, CDCl3)/d (ppm): 9.64 (s, 1H, CHO), 7.65 (s, 1H,
H-6), 7.32 (d, 2H, J ¼ 8.8 Hz, Ph-H-2, H-60), 6.98 (s, 1H, H-3), 6.91
(d, 2H, J ¼ 8.8 Hz, Ph-H-30, H-50), 5.07 (s, 2H, Ph-CH2), 3.81 (s,
3H, OCH3). ESI-MS (m/z): 261.1 [M + H]+.
5-((4-Methoxybenzyl)oxy)-6-methyl-4-oxo-4H-pyran-2-carbal-
dehyde (12b). Yellow solid, yield 66.3%. 1H NMR (500 MHz,
CDCl3)/d (ppm): 9.55 (s, 1H, CHO), 7.25 (d, 2H, J ¼ 8.5 Hz, H-20,
H-60), 6.89 (s, 1H, H-5), 6.89 (d, 2H, J ¼ 8.5 Hz, H-30, H-50), 5.09 (s,
2H, Ph-CH2), 3.74 (s, 3H, OCH3), 2.12 (s, 3H, CH3). ESI-MS (m/z):
275.3 [M + H]+.
(E)-6-(2-(1-Methyl-1H-benzo[d]imidazol-2-yl)vinyl)-4-oxo-4H-
pyran-3-yl acetate (13e). Yellow solid, yield 46.6%. 1H NMR (400
MHz, CDCl3)/d (ppm): 7.94 (s, 1H, H-6), 7.80–7.78 (m, 1H, H-40),
7.63 (d, 1H, J ¼ 16.0 Hz, CH]CHa), 7.41–7.31 (m, 4H, CHb]CH,
H-70, H-50, H-60), 6.55 (s, 1H, H-3), 3.92 (s, 1H, NCH3), 2.35 (s, 3H,
COCH3). ESI-MS (m/z): 311.3 [M + H]+.
(E)-2-Methyl-6-(2-(1-methyl-1H-benzo[d]imidazol-2-yl)vinyl)-
1
4-oxo-4H-pyran-3-yl acetate (13f). Yellow solid, yield 32.6%. H
NMR (400 MHz, CDCl3)/d (ppm): 7.79–7.77 (m, 1H, J ¼ 7.2 Hz, H-
70), 7.55 (d, 1H, J ¼ 16.0 Hz, CH]CHa), 7.41–7.33 (m, 4H, CHb]
CH, H-70, H-50, H-60), 6.49 (s, 1H, H-3), 3.92 (s, 1H, NCH3), 2.37 (s,
3H, CH3), 2.36 (s, 3H, COCH3). ESI-MS (m/z): 325.2 [M + H]+.
(E)-6-(2-(6-Methoxybenzo[d]thiazol-2-yl)vinyl)-4-oxo-4H-pyran-
3-yl acetate (13g). Yellow solid, yield 35.3%. 1H NMR (400 MHz,
CDCl3)/d (ppm): 7.95 (d, 1H, J ¼ 8.8 Hz, H-40), 7.93 (s, 1H, H-6),
7.60 (d, 1H, J ¼ 16.0 Hz, CH]CHa), 7.34 (d, 1H, J ¼ 2.4 Hz, H-70),
7.14 (dd, 1H, J1 ¼ 9.2 Hz, J2 ¼ 2.8 Hz, H-50), 7.08 (d, 1H, J ¼ 16.0
Hz, CHb]CH), 6.53 (s, 1H, H-3), 3.91 (s, 3H, OCH3), 2.35 (s, 3H,
COCH3). ESI-MS (m/z): 344.3 [M + H]+.
General procedure for the synthesis of compounds 13a–g, 13j,
13l
(E)-6-(2-(6-Bromobenzo[d]thiazol-2-yl)vinyl)-4-oxo-4H-pyran-
Compound 12a (440.0 mg, 1.69 mmol) and 2-methyl- 3-yl acetate (13h). Yellow solid, yield 45.6%. 1H NMR (500 MHz,
benzothiazole (252.0 mg, 1.69 mmol) were mixed with acetic CDCl3)/d (ppm): 8.05 (d, 1H, J ¼ 1.5 Hz, H-70), 7.94 (s, 1H, H-6),
anhydride (3.52 mL) and acetic acid (1.76 mL). The mixture was 7.91 (d, 1H, J ¼ 8.5 Hz, H-40), 7.64 (dd, 1H, J1 ¼ 9.0 Hz, J2 ¼ 2.0
ꢁ
stirred for 20 min at 145 C under microwave condition. Aer Hz, H-50), 7.59 (d, 1H, J ¼ 16.0 Hz, CH]CHa), 7.18 (d, 1H, J ¼
removal of solvent, the residue was partitioned with EtOAc (20 16.0 Hz, CHb]CH), 6.58 (s, 1H, H-3), 2.35 (s, 3H, COCH3). ESI-
mL) and water (20 mL). The aqueous solution was extracted MS (m/z): 393.2 [M + H]+.
with EtOAc (20 mL ꢃ 3) and the combined organic phase was
(E)-6-(2-(6-Methoxybenzo[d]oxazol-2-yl)vinyl)-4-oxo-4H-pyran-
washed with brine, and dried over Na2SO4. Aer concentration 3-yl acetate (13j). Yellow solid, yield 35.6%. 1H NMR (500 MHz,
to dryness, the residue was puried by silica gel chromatog- CDCl3)/d (ppm): 7.95 (s 1H, H-6), 7.76 (d, 1H, J ¼ 9.0, H-40), 7.29
raphy eluting with PE/EtOAc to afford 13a as a yellow (d, 1H, J ¼ 16.0 Hz, CH]CHa), 7.23 (d, 1H, J ¼ 15.5 Hz, CHb]
solid. Yield 52.4%. (E)-6-(2-(Benzo[d]thiazol-2-yl)vinyl)-4-oxo-4H- CH), 6.77 (dd, 1H, J1 ¼ 9.0 Hz, J2 ¼ 2.5 Hz, H-50), 6.66 (sd, 1H, J ¼
pyran-3-yl acetate (13a). 1H NMR (400 MHz, CDCl3)/d (ppm): 3.0, H-70), 6.55 (s, 1H, H-3), 3.75 (s, 3H, OCH3), 2.36 (s, 3H,
8.08 (d, 1H, J ¼ 8.0 Hz, H-40), 7.94 (s, 1H, H-6), 7.92 (d, 1H, J ¼ 7.6 COCH3). ESI-MS (m/z): 328.4 [M + H]+.
Hz, H-70), 7.63 (d, 1H, J ¼ 16.0 Hz, CH]CHa), 7.56–7.52 (m, 1H,
(E)-6-(2-(6-Methoxy-1-methyl-1H-benzo[d]imidazol-2-yl)vinyl)-
1
H-50), 7.47–7.43 (m, 1H, H-60), 7.19 (d, 1H, J ¼ 16.0 Hz, CHb] 4-oxo-4H-pyran-3-yl acetate (13l). Yellow solid, yield 42.8%. H
CH), 6.56 (s, 1H, H-3), 2.35 (s, 3H, COCH3). ESI-MS (m/z): 314.3 NMR (500 MHz, CDCl3)/d (ppm): 7.93 (s, 1H, H-6), 7.70 (d, 1H,
[M + H]+.
J ¼ 9.0, H-40), 7.65 (d, 1H, J ¼ 15.5 Hz, CH]CHa), 7.36 (d, 1H, J ¼
(E)-6-(2-(Benzo[d]thiazol-2-yl)vinyl)-2-methyl-4-oxo-4H-pyran-3- 15.5 Hz, CHb]CH), 7.01 (dd, 1H, J1 ¼ 8.0 Hz, J2 ¼ 2.0 Hz, H-50),
1
yl acetate (13b). Yellow solid, yield 63.7%. H NMR (400 MHz, 6.80 (d, 1H, J ¼ 2.5 Hz, H-70), 6.51 (s, 1H, H-3), 3.91 (s, 3H,
CDCl3)/d (ppm): 8.07 (d, 1H, J ¼ 8.0 Hz, H-40), 7.86 (d, 1H, J ¼ 7.6 OCH3), 3.90 (s, 3H, NCH3), 2.35 (s, 3H, COCH3).
Hz, H-70), 7.64 (d, 1H, J ¼ 16.0 Hz, CH]CHa), 7.55–7.52 (m, 1H,
H-50), 7.47–7.42 (m, 1H, H-60), 7.14 (d, 1H, J ¼ 16.0 Hz, CHb]
General procedure for the preparation of 9a–9h, 9j, 9l
CH), 6.48 (s, 1H, H-3), 2.36 (s, 3H, CH3), 2.35 (s, 3H, COCH3).
ESI-MS (m/z): 328.2 [M + H]+.
A mixture of 13a (0.46 mmol) and anhydrous K2CO3 (222 mg,
(E)-6-(2-(Benzo[d]oxazol-2-yl)vinyl)-4-oxo-4H-pyran-3-yl acetate 1.60 mmol) in 15 mL MeOH was stirred for 0.5 h at room
1
(13c). Yellow solid, yield 54.1%. H NMR (500 MHz, DMSO-d6)/ temperature. The solvent was removed under reduced pressure
17324 | RSC Adv., 2016, 6, 17318–17327
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and the residue was acidied with diluted HCl. The solution
was extracted with ethyl acetate (20 mL ꢃ 3) and the combined
organic phase was washed with brine, dried over Na2SO4. Aer
concentration to dryness, the residue was puried by silica gel
chromatography to furnish solid products 9a–9h, 9j, 9l.
(E)-2-(2-(Benzo[d]thiazol-2-yl)vinyl)-5-hydroxy-4H-pyran-4-one
(E)-5-Hydroxy-2-(2-(6-methoxybenzo[d]thiazol-2-yl)vinyl)-4H-
pyran-4-one (9g). Yellow solid, yield 90.9%. 1H NMR (400 MHz,
DMSO-d6)/d (ppm): 9.31 (s, 1H, OH), 8.10 (s, 1H, H-6), 7.93 (d,
1H, J ¼ 9.2 Hz, H-40), 7.73 (d, 1H, J ¼ 2.0 Hz, H-70), 7.57 (d, 1H, J
¼ 16.0 Hz, CH]CHa), 7.40 (d, 1H, J ¼ 16.0 Hz, CHb]CH), 7.17
(dd, 1H, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz, H-50), 6.78 (s, 1H, H-3), 3.86 (s,
3H, OCH3). 13C NMR (100 MHz, DMSO-d6)/d (ppm): 174.3, 161.9,
159.6, 158.6, 148.3, 146.7, 139.9, 136.9, 127.5, 127.2, 124.2,
117.0, 114.7, 105.2, 56.2. ESI-MS (m/z): 302.4 [M + H]+. HPLC
purity ¼ 98.92%, Rt 15.79 min.
1
(9a). Yellow solid, yield 80.1%. H NMR (500 MHz, DMSO-d6)/
d (ppm): 9.35 (s, 1H, OH), 8.16 (d, 1H, J ¼ 8.0 Hz, H-40), 8.12 (s,
1H, H-6), 8.05 (d, 1H, J ¼ 8.0 Hz, H-70), 7.62–7.47 (m, 4H, H-50, H-
60, CH]CH), 6.81 (s, 1H, H-3). 13C NMR (125 MHz, DMSO-d6)/
d (ppm): 175.1, 165.3, 160.1, 154.6, 147.6, 140.6, 136.0, 129.1,
128.2, 128.1, 127.4, 123.7, 115.9. ESI-MS (m/z): 272.5 [M + H]+.
HPLC purity ¼ 98.42%, Rt 14.84 min.
(E)-2-(2-(6-Bromobenzo[d]thiazol-2-yl)vinyl)-5-hydroxy-4H-
pyran-4-one (9h). Yellow solid, yield 76.2%. 1H NMR (400 MHz,
DMSO-d6)/d (ppm): 9.49 (brs, 1H, OH), 8.49 (d, 1H, J ¼ 2.0 Hz, H-
6), 8.16 (s, 1H, J ¼ 1.6 Hz, H-70), 7.99 (d, 1H, J ¼ 8.8 Hz, H-40),
7.72 (dd, 1H, J1 ¼ 8.4 Hz, J2 ¼ 2.0 Hz, H-50), 7.64 (d, 2H, J ¼ 16.4
Hz, CH]CHa), 7.55 (d, 2H, J ¼ 16.4 Hz, CHb]CH), 6.83 (s, 1H,
H-3). 13C NMR (100 MHz, DMSO-d6)/d (ppm): 176.8, 164.2,
155.4, 152.8, 147.5, 137.2, 132.3, 131.0, 130.5, 128.9, 125.5,
116.0, 113.0, 103.1. ESI-MS (m/z): 351.2 [M + H]+. HPLC purity ¼
95.55%, Rt 24.80 min.
(E)-6-(2-(Benzo[d]thiazol-2-yl)vinyl)-3-hydroxy-2-methyl-4H-
pyran-4-one (9b). Yellow solid, yield 70.1%. 1H NMR (400 MHz,
CDCl3)/d (ppm): 8.08 (d, 1H, J ¼ 7.6 Hz, H-40), 7.92 (d, 1H, J ¼ 8.0
Hz, H-70), 7.66 (d, 1H, J ¼ 15.2 Hz, CH]CHa), 7.55 (t, 1H, J ¼ 7.2
Hz, H-50), 7.47 (t, 1H, J ¼ 7.2 Hz, H-60), 7.18 (d, 1H, J ¼ 15.6 Hz,
CHb]CH), 6.52 (s, 1H, H-3), 2.47 (s, 3H, CH3). 13C NMR (100
MHz, CDCl3)/d (ppm): 170.2, 163.9, 153.5, 152.9, 147.8, 147.1,
140.1, 134.9, 127.8, 126.9, 126.4, 123.6, 121.8, 114.6, 18.3. ESI-
MS (m/z): 286.5 [M + H]+. HPLC purity ¼ 98.83%, Rt 20.81 min.
(E)-2-(2-(Benzo[d]oxazol-2-yl)vinyl)-5-hydroxy-4H-pyran-4-one
(9c). Yellow solid, yield 78.4%. 1H NMR (400 MHz, CD3OD/
CDCl3)/d (ppm): 7.88 (s, 1H, H-6), 7.70 (d, 1H, J ¼ 7.2 Hz, H-40),
7.52 (d, 1H, J ¼ 7.2 Hz, H-70), 7.30–7.24 (m, 4H, H-50, H-60, CH]
CH), 6.53 (s, 1H, H-3). 13C NMR (100 MHz, CD3OD/CDCl3)/
d (ppm): 168.7, 164.5, 163.5, 159.6, 154.3, 145.5, 143.0, 133.2,
130.5, 129.1, 124.5, 124.2, 118.1, 114.6. ESI-M (m/z): 256.5 [M +
H]+. HPLC purity ¼ 98.26%, Rt 15.07 min.
(E)-5-Hydroxy-2-(2-(6-methoxybenzo[d]oxazol-2-yl)vinyl)-4H-
1
pyran-4-one (9j). Yellow solid, yield 86.2%. H NMR (500 MHz,
CD3Cl, CD3OD)/d (ppm): 8.01 (s, 1H, H-6), 7.61 (d, 1H, J ¼ 8.5
Hz, H-40), 7.42 (d, 1H, J ¼ 16.0 Hz, CH]CHa), 7.31 (d, 1H, J ¼
16.0 Hz, CHb]CH), 7.19 (d, 1H, J ¼ 2.5 Hz, H-70), 7.03 (dd, 1H,
J1 ¼ 9.0 Hz, J2 ¼ 2.5 Hz, H-50), 6.67 (s, 1H, H-3), 3.90 (s, 3H,
OCH3). ESI-MS (m/z): 285.2 [M + H]+. HPLC purity ¼ 98.74%, Rt
15.08 min.
(E)-5-Hydroxy-2-(2-(6-methoxy-1-methyl-1H-benzo[d]imida-
zol-2-yl)vinyl)-4H-pyran-4-one (9l). Yellow solid, yield 76.2%. 1H
NMR (500 MHz, CD3OD)/d (ppm): 7.95 (s, 1H, H-6), 7.50 (d, 1H,
J ¼ 16.0 Hz, CH]CHa), 7.44 (d, 1H, J ¼ 8.5 Hz, H-40), 7.28 (d, 1H,
J ¼ 15.5 Hz, CHb]CH), 6.96 (d, 1H, J ¼ 1.5 Hz, H-70), 6.86 (dd,
1H, J1 ¼ 9.0 Hz, J2 ¼ 2.0 Hz, H-50), 6.57 (s, 1H, H-3), 3.85 (s, 3H,
OCH3), 3.80 (s, 3H, NCH3). ESI-MS (m/z): 299.1 [M + H]+. HPLC
purity ¼ 96.88%, Rt 32.05 min.
(E)-6-(2-(Benzo[d]oxazol-2-yl)vinyl)-3-hydroxy-2-methyl-4H-
pyran-4-one (9d). Yellow solid, yield 77.4%. 1H NMR (400 MHz,
CD3OD/CDCl3)/d (ppm): 7.71 (d, 1H, J ¼ 7.2 Hz, H-40), 7.53 (d,
1H, J ¼ 7.2 Hz, H-70), 7.39–7.30 (m, 4H, H-50, H-60, CH]CH), 6.50
(s, 1H, H-3), 2.42 (s, 3H, CH3). 13C NMR (100 MHz, CD3OD/
CDCl3)/d (ppm): 173.3, 169.1, 167.6, 156.7, 153.4, 146.8, 135.3,
130.5, 129.1, 124.2, 123.9, 120.3, 117.4, 114.6, 15.21. ESI-MS (m/
z): 270.4 [M + H]+. HPLC purity ¼ 98.93%, Rt 18.44 min.
(E)-5-Hydroxy-2-(2-(1-methyl-1H-benzo[d]imidazol-2-yl)vinyl)-
4H-pyran-4-one (9e). Yellow solid, yield 90.0%. 1H NMR
(400 MHz, CD3OD/CDCl3)/d (ppm): 8.0 (brs, 1H, OH), 7.85 (s,
1H, H-6), 7.65 (d, 1H, J ¼ 8.8 Hz, H-40), 7.39–7.36 (m, 2H, CH]
CHa, H-70), 7.30–7.24 (m, 3H, H-50, H-60, CHb]CH), 6.57 (s, 1H,
H-3), 3.84 (s, 3H, NCH3). 13C NMR (100 MHz, CD3OD/CDCl3)/
d (ppm): 170.9, 165.8, 159.0, 157.3, 152.5, 146.3, 144.1, 139.8,
130.9, 127.9, 127.5, 123.5, 123.1, 113.7, 33.7. ESI-MS (m/z): 269.5
[M + H]+. HPLC purity ¼ 98.30%, Rt 13.30 min.
(E)-5-Hydroxy-2-(2-(6-hydroxybenzo[d]thiazol-2-yl)vinyl)-4H-
pyran-4-one (9i). To a solution of compound 9g (303 mg, 1.0
mmol) in CH2Cl2 (10 mL) was added 5 mL BBr3/CH2Cl2 solution (1
mol Lꢄ1) in drop wise under ice-water bath. The mixture was
stirred for 30 min and quenched with 5 mL cooled methanol. The
solvent was removed under reduced pressure and the residue was
puried with silica gel chromatography to afford 9i. White solid,
1
61.4% yield. H NMR (400 MHz, DMSO-d6)/d (ppm): 10.0 (s, 1H,
OH), 9.32 (s, 1H, OH), 8.10 (s, 1H, H-6), 7.85 (d, 1H, J ¼ 8.8 Hz, H-
40), 7.54 (d, 1H, J ¼ 16.0 Hz, CH]CHa), 7.41 (s, 1H, H-70), 7.33 (d,
1H, J ¼ 16.0 Hz, CHb]CH), 7.02 (d, 1H, J ¼ 8.0 Hz, H-50), 6.76 (s,
1H, H-3). 13C NMR (100 MHz, DMSO)/d (ppm): 174.3, 160.7, 159.6,
157.0, 157.0, 147.4, 139.9, 136.9, 127.7, 124.3, 117.2, 114.5, 107.1.
ESI-MS (m/z): 288.3 [M + H]+. HPLC purity ¼ 97.15%, Rt 12.69 min.
(E)-5-Hydroxy-2-(2-(6-hydroxybenzo[d]oxazol-2-yl)vinyl)-4H-
pyran-4-one (9k). The same procedure as described for 9i was
(E)-3-Hydroxy-2-methyl-6-(2-(1-methyl-1H-benzo[d]imidazol-
2-yl) vinyl)-4H-pyran-4-one (9f). Yellow solid, yield 65.6%. 1H
NMR (400 MHz, DMSO-d6)/d (ppm): 7.65 (d, 1H, J ¼ 7.6 Hz, H-
70), 7.61 (d, 1H, J ¼ 7.6 Hz, H-40), 7.55 (s, 2H, CH]CH), 7.31–7.23
(m, 2H, H-50, H-60), 6.75 (s, 1H, H-3), 3.96 (s, 3H, NCH3), 2.38
(s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)/d (ppm): 175.8,
159.1, 149.5, 148.9, 146.2, 143.2, 136.7, 126.9, 123.3, 123.0,
120.3, 119.4, 113.4, 111.0, 30.2, 14.6. ESI-MS (m/z): 283.4 [M +
H]+. HPLC purity ¼ 96.18%, Rt 15.23 min.
1
used with 9j to yield 9k as a yellow solid, yield 84.2%. H NMR
(500 MHz, DMSO-d6)/d (ppm): 10.12 (s, 1H, OH), 9.43 (s, 1H,
OH), 8.10 (s, 1H, H-6), 7.59 (d, 1H, J ¼ 16.0 Hz, CH]CHa), 7.46
(d, 1H, J ¼ 16.0 Hz, CHb]CH), 7.05 (s, 1H, H-70), 6.89 (d, 1H,
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J ¼ 8.0 Hz, H-50), 6.82 (s, 1H, H-3). 13C NMR (125 MHz, DMSO- mmol) in small portions under ice-water bath. Aer stirring for
d6)/d (ppm): 175.3, 164.6, 157.1, 155.1, 137.6, 124.2, 120.4, 120.2, additional 10 min at room temperature, compound 15 (194.0
116.1, 108.9, 103.8, 102.4, 100.3, 91.2. ESI-MS (m/z): 272.0 [M + mg, 0.71 mmol) in anhydrous THF (3 mL) was added in drop
H]+. HPLC purity ¼ 98.48%, Rt 11.66 min.
wise. The mixture was stirred overnight at room temperature
(E)-5-Hydroxy-2-(2-(6-hydroxy-1-methyl-1H-benzo[d]imidazol-2- and quenched with water (5 mL), the product precipitated as
yl) vinyl)-4H-pyran-4-one (9m). The same procedure as a yellow solid (172.0 mg, 60.0%). (E)-2-(2-(Benzo[d]thiazol-2-yl)
described for 9i was used with 9l to yield 9m as a yellow solid vinyl)-5-((4-methoxybenzyl)oxy)-1-methylpyridin-4(1H)-one (18a).
1
72.0%. H NMR (500 MHz, CD3OD)/d (ppm): 7.71 (s, 1H, H-6), 1H NMR (500 MHz, CDCl3)/d (ppm): 8.16 (d, 1H, J ¼ 8.0 Hz, H-40),
7.27 (d, 1H, J ¼ 16.0 Hz, CH]CHa), 7.23 (d, 1H, J ¼ 9.0 Hz, H-40), 8.06 (d, 1H, J ¼ 8.0 Hz, H-70), 7.70 (s, 1H, H-6), 7.66 (d, 1H, J ¼
7.12 (d, 1H, J ¼ 16.0 Hz, CHb]CH), 6.78–6.75 (m, 2H, H-50, H- 16.0 Hz, CH]CHa), 7.62 (d, 1H, J ¼ 16.0 Hz, CHb]CH), 7.58–
70), 6.37 (s, 1H, H-3), 3.67 (s, 3H, NCH3). 13C NMR (125 MHz, 7.55 (m, 1H, H-50), 7.51–7.48 (m, 1H, H-60), 7.37 (d, 2H, J ¼ 8.5 Hz,
CD3OD)/d (ppm): 163.9, 163.0, 152.5, 150.3, 145.4, 140.0, 137.6, H-200, H-600), 6.96 (d, 2H, J ¼ 8.5 Hz, H-300, H-500), 6.71 (s, 1H, H-3),
134.8, 133.7, 129.6, 123.3, 119.7, 119.5, 101.9, 95.9, 36.0. ESI-MS 4.96 (s, 2H, Ph-CH2), 3.78 (s, 3H, OCH3), 3.76 (s, 3H, NCH3). ESI-
(m/z): 285.1 [M + H]+. HPLC purity ¼ 99.39%, Rt 6.91 min.
MS (m/z): 405.5 [M + H]+.
2-(Hydroxymethyl)-5-((4-methoxybenzyl)oxy)-1-methylpyridin-
(E)-2-(2-(Benzo[d]oxazol-2-yl)vinyl)-5-((4-methoxybenzyl)oxy)-
4(1H)-one (14). The mixture of compound 11a (3.0 g, 11.5 mmol), 1-methylpyridin-4(1H)-one (18b). Light yellow solid, yield
H2O (138 mL) and 30% aqueous methylamine (36 mL) was 54.0%. 1H NMR (500 MHz, CDCl3)/d (ppm): 7.73 (d, 1H, J ¼ 8.5
reuxed for 4 h. Aer cooling to room temperature, the mixture Hz, H-40), 7.56 (d, 1H, J ¼ 15.0 Hz, CH]CHa), 7.51 (d, 1H, J ¼ 6.5
was extracted with CH2Cl2 (30 mL ꢃ 3). The combined extract was Hz, H-70), 7.38–7.30 (m, 4H, H-50, H-60, H-200, H-600), 7.04 (d, 1H,
washed with saturated saline, dried over anhydrous sodium J ¼ 15.6 Hz, CHb]CH), 6.94 (s, 1H, H-6), 6.86 (d, 2H, J ¼ 10.0 Hz,
sulfate, and concentrated under reduced pressure to afford 14 as H-300, H-500), 6.77 (s, 1H, H-3), 5.13 (s, 2H, Ph-CH2), 3.77 (s, 3H,
a yellow solid, yield 93%. 1H NMR (500 MHz, DMSO-d6)/d (ppm): OCH3), 3.63 (s, 3H, NCH3). ESI-MS (m/z): 389.4 [M + H]+.
7.51 (s, 1H, H-6), 7.33 (d, 2H, J ¼ 9.0 Hz, H-20, H-60), 6.93 (d, 2H, J ¼
8.5 Hz, H-30, H-50), 6.20 (s, 1H, H-3), 5.53 (t, 1H, J ¼ 6.0 Hz, OH),
General procedure for the preparation of 9n and 9o
4.90 (s, 2H, Ph-CH2), 4.36 (d, 2H, J ¼ 5.5 Hz, CH2OH), 3.75 (s, 3H,
OCH3), 3.57 (s, 3H, N-CH3). ESI-MS (m/z): 276.3 [M + H]+.
The mixture of compound 18a (101 mg, 0.25 mmol) with 5 mL
5-((4-Methoxybenzyl)oxy)-1-methyl-4-oxo-1,4-dihydropyridine- CF3COOH/CH2Cl2 (1 : 1) was stirred for 1 h at room tempera-
2-carbaldehyde (15). Compound 15 was prepared according to ture. Aer removal of solvent under reduced pressure, the
the general procedure for synthesis compound 12a and 12b. residue was puried with silica gel chromatography to get 9n as
Yellow solid, yield 44.3%. 1H NMR (500 MHz, DMSO-d6)/d (ppm): white solid, yield 91.2%. (E)-2-(2-(Benzo[d]thiazol-2-yl)-vinyl)-5-
1
9.75 (s, 1H, CHO), 7.71 (s, 1H, H-6), 7.36 (d, 2H, J ¼ 8.5 Hz, H-20, hydroxy-1-methylpyridin-4(1H)-one. H NMR (400 MHz, CDCl3/
H-60), 6.95 (d, 2H, J ¼ 8.5 Hz, H-30, H-50), 6.81 (s, 1H, H-3), 4.98 (s, CD3OD)/d (ppm): 8.17 (s, 1H, H-6), 8.05 (d, 1H, J ¼ 8.0 Hz, H-40),
2H, Ph-CH2), 3.89 (s, 3H, OCH3), 3.76 (s, 3H, N-CH3). ESI-MS (m/ 7.99 (d, 1H, J ¼ 8.0 Hz, H-70), 7.82 (d, 1H, J ¼ 15.6 Hz, CH]CHa),
z): 274.3 [M + H]+.
7.71 (d, 1H, J ¼ 15.6 Hz, CHb]CH), 7.59–7.48 (m, 3H, H-50, H-60,
H-3), 4.20 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3/CD3OD)/
d (ppm): 163.6, 159.9, 151.7, 145.7, 143.6, 134.5, 131.9, 130.3,
127.1, 126.5, 124.3, 122.4, 121.8, 110.7, 43.9. ESI-MS (m/z): 285.4
[M + H]+. HPLC purity ¼ 99.01%, Rt 17.33 min.
General procedure for the preparation of 17a and 17b
The mixture of 2-bromomethyl-benzothiazole (16a) (500 mg, 2.19
mmol) with P(OEt)3 (0.86 mL) was reuxed over night. The excess
P(OEt)3 was removed under reduced pressure and the residue
was puried by silica gel column chromatography to afford 17a.
Yellow oil, yield 98%. Diethyl (benzo[d]thiazol-2-ylmethyl) phos-
phonate (17a). 1H NMR (500 MHz, CDCl3)/d (ppm): 7.92 (d, 1H, J
¼ 8.0 Hz, H-3), 7.77 (d, 1H, J ¼ 8.0 Hz, H-7), 7.39 (t, 1H, J ¼ 8.0 Hz,
H-4), 7.30 (t, 1H, J ¼ 7.5 Hz, H-6), 4.10–4.05 (m, 4H, 2 ꢃ CH2CH3),
3.67 (d, 2H, J ¼ 21.5 Hz, CH2P]O), 1.24 (t, 6H, J ¼ 7.5 Hz, 2 ꢃ
CH2CH3). ESI-MS (m/z): 286.2 [M + H]+.
Diethyl (benzo[d]oxazol-2-ylmethyl)phosphonate (17b). Light
yellow oil, yield 92.0%. 1H NMR (500 MHz, CDCl3)/d (ppm): 7.63–
7.60 (m, 1H, H-3), 7.45–7.43 (m, 1H, H-7), 7.27–7.25 (m, 2H, H-5, H-
6), 4.13–4.09 (m, 4H, 2 ꢃ CH2CH3), 3.52 (d, 2H, J ¼ 21.5 Hz,
CH2P]O), 1.26 (t, 6H, J ¼ 7.5 Hz, 2 ꢃ CH2CH3). ESI-MS (m/z):
269.2 [M + H]+.
(E)-2-(2-(Benzo[d]oxazol-2-yl)vinyl)-5-hydroxy-1-methylpyridin-
1
4(1H)-one (9o). Yellow solid, 74.6% yield. H NMR (400 MHz,
DMSO-d6)/d (ppm): 8.29 (s, 1H, H-6), 7.90 (d, 1H, J ¼ 16.0 Hz,
CH]CHa), 7.85 (d, 1H, J ¼ 7.6 Hz, H-40), 7.81 (d, 1H, J ¼ 8.0 Hz,
H-70), 7.67 (s, 1H, H-3), 7.57 (d, 1H, J ¼ 15.6 Hz, CHb]CH), 7.53–
7.44 (m, 2H, H-50, H-60), 4.18 (s, 3H, CH3). 13C NMR (100 MHz,
DMSO-d6)/d (ppm): 161.0, 160.4, 150.4, 145.8, 143.6, 141.8,
133.2, 128.5, 127.2, 125.8, 123.4, 120.8, 112.0, 111.4, 44.5. ESI-
MS (m/z): 269.5 [M + H]+. HPLC purity ¼ 99.57%, Rt 9.09 min.
HL5 and HL12 was prepared according to the references.32,33
HL5 1H NMR (500 MHz, CD3OD/CDCl3)/d (ppm): 8.45 (d, 1H,
J ¼ 7.0, H-6), 8.17 (t, 2H, J ¼ 7.5, H-40, H-70), 7.74 (t, 1H, J ¼ 8.0, H-
50), 7.69 (t, 1H, J ¼ 7.5, H-60), 7.36 (d, 1H, J ¼ 6.0, H-5), 2.58 (s,
3H, CH3). 13C NMR (125 MHz, CD3OD/CDCl3)/d (ppm): 162.3,
157.3, 149.2, 144.2, 141.8, 138.3, 135.6, 127.9, 127.8, 124.4,
122.4, 111.0, 13.8. ESI-MS (m/z): 245.1 [M + H]+.
HL12 1H NMR (500 MHz, CD3OD/CDCl3)/d (ppm): 7.80 (d, 1H,
General procedure for the preparation of 18a and 18b
To a solution of compound 17a (200.0 mg, 0.71 mmol) in 3 mL J ¼ 8.0, H-6), 7.95 (d, 1H, J ¼ 8.0, H-40), 7.79 (d, 1H, J ¼ 7.0, H-70),
anhydrous THF was added sodium hydride (34.1 mg, 1.42 7.55 (t, 1H, J ¼ 8.0, H-50), 7.52 (t, 1H, J ¼ 8.0, H-60), 6.52 (d, 1H, J
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Acknowledgements
The authors thank the National Natural Science Foundation of
China (No. 21172193), Zhejiang Provincial Natural Foundation of
China (No. R2110297) for nancial support. We also thank Jia-
nyang Pan (Pharmaceutical Informatics Institute, Zhejiang
University) for performing NMR and MS for structure elucidation.
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RSC Adv., 2016, 6, 17318–17327 | 17327