Novel oxazolxanthone derivatives as a new type of α-glucosidase inhibitor: synthesis, activities, inhibitory modes and synergetic effect

Article abstract of DOI:10.1016/j.bmc.2018.05.008

Xanthone derivatives have shown good α-glucosidase inhibitory activity and have drawn increased attention as potential anti-diabetic compounds. In this study, a series of novel oxazolxanthones were designed, synthesized, and investigated as α-glucosidase inhibitors. Inhibition assays indicated that compounds 4–21 bearing oxazole rings exhibited up to 30-fold greater inhibitory activity compared to their corresponding parent compound 1b. Among them, compounds 5–21 (IC₅₀ = 6.3 ± 0.4–38.5 ± 4.6 μM) were more active than 1-deoxynojirimycin (IC₅₀ = 60.2 ± 6.2 μM), a well-known α-glucosidase inhibitor. In addition, the kinetics of enzyme inhibition measured by using Lineweaver–Burk analysis shows that compound 4 is a competitive inhibitor, while compounds 15, 16 and 20 are non-competitive inhibitors. Molecular docking studies showed that compound 4 bound to the active site pocket of the enzyme while compounds 15, 16, and 20 did not. More interestingly, docking simulations reveal that some of the oxazolxanthone derivatives bind to different sites in the enzyme. This prediction was further confirmed by the synergetic inhibition experiment, and the combination of representative compounds 16 and 20 at the optimal ratio of 4:6 led to an IC₅₀ value of 1.9 ± 0.7 μM, better than the IC₅₀ value of 7.1 ± 0.9 μM for compound 16 and 8.6 ± 0.9 μM for compound 20.

Full text of DOI:10.1016/j.bmc.2018.05.008

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel oxazolxanthone derivatives as a new type of
a-glucosidase
inhibitor: synthesis, activities, inhibitory modes and synergetic effect
⇑
Sen-Miao Ding, Tian Lan, Gao-Jie Ye, Jia-Jun Huang, You Hu, Yi-Ran Zhu, Bo Wang
School of Chemistry, Sun Yat-sen University, 135 Xingang West Road, Guangzhou 510275, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Xanthone derivatives have shown good
a-glucosidase inhibitory activity and have drawn increased
Received 20 March 2018
Revised 3 May 2018
Accepted 5 May 2018
Available online xxxx
attention as potential anti-diabetic compounds. In this study, a series of novel oxazolxanthones were
designed, synthesized, and investigated as -glucosidase inhibitors. Inhibition assays indicated that com-
pounds 4–21 bearing oxazole rings exhibited up to 30-fold greater inhibitory activity compared to their
corresponding parent compound 1b. Among them, compounds 5–21 (IC50 = 6.3 0.4–38.5 4.6 M)
were more active than 1-deoxynojirimycin (IC50 = 60.2 6.2 M), a well-known -glucosidase inhibitor.
In addition, the kinetics of enzyme inhibition measured by using Lineweaver–Burk analysis shows that
compound 4 is a competitive inhibitor, while compounds 15, 16 and 20 are non-competitive inhibitors.
Molecular docking studies showed that compound 4 bound to the active site pocket of the enzyme while
compounds 15, 16, and 20 did not. More interestingly, docking simulations reveal that some of the oxa-
zolxanthone derivatives bind to different sites in the enzyme. This prediction was further confirmed by
the synergetic inhibition experiment, and the combination of representative compounds 16 and 20 at the
a
l
l
a
Keywords:
Oxazolxanthones
a-Glucosidase inhibitors
Docking
Synergetic effect
Non-competitive and competitive
optimal ratio of 4:6 led to an IC50 value of 1.9 0.7
pound 16 and 8.6 0.9 M for compound 20.
l
M, better than the IC50 value of 7.1 0.9
lM for com-
l
Ó 2018 Elsevier Ltd. All rights reserved.
1. Introduction
extended p-conjugated systems (e.g., 1c), exhibit greater inhibitory
effects than compound 1a. Our investigations suggested that the 3-
hydroxyl group of 1b may not be required for the inhibitory activ-
ity because the 3-arylacyloxyxanthone derivative 1d shows greater
inhibitory effects than 1b.19 This finding also implies that the addi-
tion of aromatic moieties by esterification at the 3-OH of 1b is an
effective strategy to increase the inhibition activity. However, as
oral therapies for diabetes, ester-bearing compounds may have
considerable limitations because of their susceptibility to hydroly-
sis by intestinal esterases.
In response to these issues, we have been keenly interested in
investigating modifications of the 3-OH that may bring about
improved stability and higher bioactivity. Previously, our quantita-
tive structure-activity relationship (QSAR) study on xanthone
derivatives found that the number of H-bonds formed, the number
of aromatic rings and the chemical softness value are positively
correlated with the inhibitory effect.16 This finding suggests that
a
-Glucosidase (EC 3.2.1.20) is an enzyme which catalyzes the
hydrolysis of glycosidic bonds in complex carbohydrates, playing
a key role in the digestion of dietary carbohydrates and in the pro-
cessing of glycoproteins and glycolipids. Inhibitors of
dase can delay carbohydrate digestion and prolong the overall
a-glucosi-
carbohydrate digestion time, thereby helping to reduce elevated
blood sugar levels after a meal.1–4 Amongst the
a-glucosidase inhi-
bitors, acarbose, miglitol, and voglibose have being used as first-
line treatments for type 2 diabetes in the clinical setting.5–7 More-
over, a-glucosidase inhibitors have become important therapies in
viral infections, including HIV and influenza, and also have thera-
peutic potential in metastatic cancer, obesity, and lysosomal stor-
age diseases.8–11
The hetero-tricyclic structure of xanthone (1a, Chart 1) has been
found in a wide variety of natural products bearing various bioac-
tivities.12,13 In previous studies, we have shown that xanthone
adding heteroaromatic rings can bring additional
p-systems and
derivatives could act as
found that the derivatives bearing hydroxyl groups (e.g., 1b) or
a
-glucosidase inhibitors.14–18 We have
heteroatoms as H-bond donors/acceptors, and which improve the
chemical softness, might be a good way to enhance both the stabil-
ity and the bioactivity. Among the various heteroaromatic rings,
oxazole rings have particularly drawn our attention, thanks to their
unique properties as key building blocks for compounds having
various bioactivity, including anti-inflammatory,20 antibacterial,21
⇑
Corresponding author.
E-mail address: ceswb@mail.sysu.edu.cn (B. Wang).
https://doi.org/10.1016/j.bmc.2018.05.008
0968-0896/Ó 2018 Elsevier Ltd. All rights reserved.
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S.-M. Ding et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Chart 1. Structures of xanthone derivatives 1a–d.
antifungal22 antiproliferative,23 anti-tuberculosis,24 muscle
relaxant,25 HIV inhibitory26 and hypoglycemic activity.27
With these considerations in mind, we designed and synthe-
sized a series of oxazole-containing xanthone derivatives 4–18
(Scheme 1) with the aim to clarify the role of the oxazole ring in
acetone with Pd/C as catalyst to produce compound 3. Compounds
4–16 were obtained in 33–59% yields by heating the appropriate
acid with compound 3 in Eaton’s reagent (7.5% W/W P2O5).
Demethylation of compound 16 in CH2Cl2 with BBr3 gave com-
pound 17. Hydrogenation of compound 8 in acetone with Pd/C as
catalyst produced compound 18. Compounds 19–21 were prepared
in 88–92% yields from the methylation of compounds 6, 11 and 16,
respectively, with CH3I in the presence of K2CO3 under reflux.32,33
Compounds 1b and 2–21 were characterized by HRMS (EI or ESI)
and NMR (1H and 13C). HMQC and HMBC were also performed to
determine the chemical structure of compound 2.
the inhibition of
a-glucosidase. We also synthesized compounds
19–21 in order to clarify the necessity of the 5-OH group in com-
pounds 4–18 and to gain further insight into the structure–activity
relationship of oxazolxanthone derivatives as a-glucosidase inhibi-
tors. We conducted a series of inhibition assays and kinetic assays
to characterize the inhibitory activities of these compounds, and
we also investigated the potential for synergistic inhibition. Addi-
tionally, we carried out molecular docking studies to gain insight
2.2. Determination of the structure of compound 2
into possible modes of binding with a-glucosidase.
Under the nitration condition, both hydrogen atoms (H-2 and
H-4) of compound 1b are able to be substituted. Therefore it was
necessary to determine the structure of the intermediate product
(compound 2) by 2D-NMR (HMQC and HMBC). In the 1H NMR
spectrum of compound 1b (Fig. S1), two aromatic protons H-2
and H-4 were observed at 6.41 and 6.22 ppm, respectively, as dou-
blets due to their long-distance coupling of 2.1 Hz. However, in the
range of 6–7 ppm, only one singlet was observed at 6.37 ppm for
compound 2 (Fig. S5). This indicated that the nitro group was
installed on the xanthone skeleton at either the C-2 or C-4 posi-
tions. The exact position was determined using HMBC and HMQC.
2. Results and discussion
2.1. Synthesis of xanthone derivatives 1b and 2-21
The synthetic route of xanthone derivatives 1b and 2–21 is
shown in Scheme 1. Compound 1b was synthesized from the con-
densation of hydroxybenzoic acid with phloroglucinol in the pres-
ence of anhydrous zinc chloride and phosphoryl chloride.28,29
Compound 2 was obtained from the nitration of compound 1b with
70% HNO3 in acetic acid,30,31 and then was hydrogenated in
Scheme 1. Synthesis of compounds 1b, 2–21.
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S.-M. Ding et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
As can be seen from HMBC (Fig. S7), a sharp singlet was observed
at 13.07 ppm that was assignable to the chelated 1-OH group. This
1-OH showed strong correlations with the carbon signals at 163.8,
101.7 and 98.4 ppm. On the other hand, HMQC (Fig. S8) indicated
that hydrogen signals at 6.37 ppm showed strong correlations with
the carbon signals at 98.4 ppm. These results indicated that the H-
2 was preserved and H-4 was substituted by a nitro group.
and 3, as well as 1-deoxynojirimycin as a positive control for com-
parison are shown in Table 1. Some structure–activity relation-
ships may be derived from examination of the IC50values of
compounds 4–21.
The first observation is that the weak inhibitory activity of xan-
thones can be amplified by inserting one oxazole ring. As can be
seen, the inhibitory activity of compound 4, which has one conju-
gated oxazole ring, was increased by about 3-fold compared to
compound 1b, 4.5-fold compared to compound 2, and 2-fold com-
pared to compound 3.
2.3. a-Glucosidase inhibitory activity
More significantly, compound 5, which has one cyclohexyl
group attached to the oxazole ring, showed higher activity, and
compounds 6 and 7, with one more benzene ring attached to the
oxazole ring, were much more active than compound 4 and even
The inhibitory activities of compounds 1b and 2–21 in yeast a-
glucosidase were evaluated by method described previously.14–19
The IC50 values obtained, along with those of compounds 1b, 2,
more active than 1-deoxynojirimycin, a well-known a-glucosidase
Table 1
inhibitor. These results suggest that the addition of either a cyclo-
hexyl or a benzene ring may enhance the interaction of these com-
In vitro a-glucosidase inhibitory activity of compounds 1b, 2–21.
pounds with the enzyme through the hydrophobic or
p-stacking
effect. Compounds 8 and 9, which bear electron-withdrawing sub-
stituents such as the nitryl or trifluoromethyl groups on the linked
benzene ring, showed decreased inhibitory activity compared to
compounds 6 and 7, while compounds 16, 17 and 18, which have
electron donating groups such as the methoxy, hydroxyl and
amino groups, showed significantly greater inhibitory activity.
Furthermore, the introduction of various halogen groups at the
linked benzene ring (compounds 10–15) increased the inhibitory
activity. And the increase in inhibitory activity was not affected
by the number of halogens or their positions of substitution. Com-
pound 15 showed the highest inhibitory activity, with an IC50 value
of 6.3 0.4 mM, a nearly 30-fold increase compared to compound
1b.
In order to further clarify the role of the 5-OH group in com-
pounds 4–21, compounds 6, 11 and 16 were methylated to pro-
duce compounds 19–21, respectively. Comparison of the
inhibitory activity of compounds 6, 11 and 16 with 19–21 showed
that the corresponding 5-OH or 5-OMe derivatives possess similar
IC50, suggesting that the H-bond donor effect might be the most
possible interaction between these groups and the enzyme. An
interesting observation is that compounds 19–21 showed better
solubility than the corresponding compounds having a 5-OH, for
which solubility may be negatively impacted by an intramolecular
H-bond. This observation provides important insights for the fur-
Compound
R1
R2
H
IC
(lM)
50
1b
2
3
4
5
186.5 12.4
286.4 10.5
126.6 9.3
64.4 5.0
OH
OH
38.5 4.6
6
OH
OH
OH
OH
OH
OH
OH
OH
18.3 0.7
19.1 1.5
24.5 5.1
26.9 3.8
11.0 1.1
8.8 0.6
6.7 1.2
8.4 0.6
7
8
9
10
11
12
13
ther design of new
oxazolxanthone.
a-glucosidase inhibitors using the skeleton of
2.4. Inhibition kinetics of a–glucosidase
In order to gain further insight into how these xanthone deriva-
tives interact with yeast -glucosidase, the mode of inhibition for
14
15
OH
OH
8.9 0.5
6.3 0.4
a
compounds 4, 15, 16 and 20 as representative examples were
determined from Lineweaver-Burk plots using the methods
described previously.34,35 The double reciprocal plots are shown
in Fig. 1. As can be seen, the double reciprocal plot of compound
4 showed straight lines with the same Vmax, which indicates that
compound 4 is a competitive inhibitor of
a-glucosidase, while
16
OH
7.1 0.9
7.3 0.9
8.3 1.8
19.6 0.6
8.6 0.9
9.9 0.2
60.3 6.2
the double reciprocal plots of compounds 15, 16 and 20 showed
straight lines with the same Michaelis constant (Km), suggesting
that compounds 15, 16 and 20 are non-competitive inhibitors of
17
OH
18
OH
a
-glucosidase. The inhibition constants (Ki) were 6.8
pound 15 (6 M), 5.3 M for compound 16 (5 M) and 7.4
for compound 20 (7 M), respectively.
lM for com-
l
l
l
lM
19
OCH3
OCH3
OCH3
l
20
2.5. Molecular docking studies
21
To gain further insights into the binding mode of these oxazolx-
anthone derivatives, molecular docking studies were performed.
1-deoxynojirimycin
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S.-M. Ding et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 1. Double-reciprocal plots of the inhibition kinetics of yeast a–glucosidase by compounds 4 (a), 15 (b), 16 (c) and 20 (d).
From the structure-activity relationships discussed above, we were
particularly interested in the effect of the oxazole ring and the
additional phenyl groups. Therefore, compounds 15, 16, 18, 20
and 21, which are among the most efficient inhibitors, were chosen
for the docking simulations. Compounds 4, 6 and the parent com-
pound 1b were also studied for comparison.
phenyl groups of some of these compounds and ionized residues
such as lysine.
As shown in Fig. 3, molecular docking analysis suggests that
compound 4 has a possible binding position close to the active site
of a-glucosidase (Asp214, Glu276 and Asp349), while the other six
docked compounds all bind to allosteric sites in the enzyme, which
is consistent with the results from the enzyme kinetic assay
demonstrating that compound 4 is a competitive inhibitor and
the other compounds are noncompetitive inhibitors. Another inter-
esting result we observed, also shown in Fig. 3, is that compounds
20 and 21 with a methoxy group on the 5-position bind to a differ-
ent position on the enzyme compared to those having an hydroxy
group on the 5-position, i.e. compounds 6, 15, 16 and 18. Taken
together, this suggests the presence of multiple binding modes
Since the X-ray crystal structure of a-glucosidase from Saccha-
romyces cerevisiae is currently unavailable, the homology modeling
provided by SWISS-MODEL Repository, with the model quality
estimation performed,36 was used as the receptor. From the dock-
ing studies, the binding affinity of oxazolxanthone 4 was predicted
to be À33.07 kJÁmolÀ1, greater than the parent compound 1b
(À32.65 kJÁmolÀ1). Also, when a phenyl group was added to the
oxazole ring, as in compounds 6 (À39.76 kJÁmolÀ1), 15 (À42.70
kJÁmolÀ1), 16 (À41.02 kJÁmolÀ1), 18 (À40.60 kJÁmolÀ1), 20
(À38.51 kJÁmolÀ1) and 21 (À40.18 kJÁmolÀ1), the binding affinity
was further increased. This suggests that introducing an oxazole
ring into the core structure of xanthone could lead to stronger
for these inhibitors of
a-glucosidase.
2.6. Synergetic inhibition
binding with
a-glucosidase, and an additional phenyl group
We previously reported that the inhibitors 1, 3, 7-trihydroxyx-
anthone and 1, 3-dihydroxybenzoxanthone interacted with the
enzyme in multiple binding configurations, thus having a syner-
getic effect.18 The observation that compounds 20 and 21 may
have different binding modes from compounds 6, 15, 16, and 18
suggested that they may also exhibit such synergetic inhibitory
effect when used in combination. Therefore, we decided to perform
synergetic inhibition assays.
Compounds 16 and 20, possessing greater inhibitory activity
and minimal structural differences, were selected for investigation
of synergetic inhibition. We investigated their inhibitory effect
individually and in combination in varying molar ratios as
attached to the oxazole ring could further improve the interaction
with the enzyme, which is consistent with the observations in the
enzyme inhibitory essays.
Molecular docking analysis suggests that several types of inter-
actions are formed between the inhibitor and the enzyme upon
binding, as seen in Fig. 2, the heteroatoms in the oxazole ring
can form hydrogen bonds with specific residues in the protein.
Additionally, both the core structure of the oxazolxanthones and
the additional phenyl group can participate in the formation of
p-
p
interactions. Moreover,
p-cation interactions may also exist
as a noncovalent intermolecular force between the additional
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S.-M. Ding et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
Fig. 2. Predicted interactions between a-glucosidase and compounds 21 (a), 20 (b) and 18 (c).
Fig. 3. Binding positions of compound 4 (a) and compounds 6, 15, 16, 18, 20 and 21 (b).
previously described.18,34,35,37 As shown in Fig. 4, compounds 16 (2
M) and 20 (2 M) inhibited the enzyme by 21.7% and 20.3%,
respectively. However, when applied together in a 1:1 M ratio (2
M total), the enzyme was inhibited by 41.9%, a 2-fold increase.
This result suggests that the combination of compounds 16 and
20 indeed produces synergetic inhibition.
Further assays were performed to determine the optimal ratio
of compound 16 to compound 20. A series of mixtures of com-
pounds 16 and 20 with fixed total concentrations (2 lM) but vary-
ing molar ratios were prepared, and inhibition assays were carried
out. As shown in Fig. 4, the optimal molar ratio of compound 16 to
compound 20 was found to be 4:6.
l
l
l
We then measured the IC50 value of the two inhibitors together
at this optimal ratio and obtained an IC50 value of 1.9 0.3
which is 3.5- and 4-fold better than those of compound 16 (IC50
= 7.1 0.9 M) and compound 20 (IC50 = 8.6 0.9 M) alone,
lM,
l
l
respectively. These observations not only confirm the synergetic
inhibition of the two compounds but also suggest that they inter-
act with the enzyme via different modes of binding, as observed in
the molecular docking studies.
3. Conclusions
In summary, a series of novel oxazolxanthone derivatives have
been synthesized, and their inhibitory activities against
dase were evaluated. The results indicate that oxazolxanthones
display potent inhibition of -glucosidase. The structure–activity
a-glucosi-
a
relationship analysis suggests that the introduction of an oxazole
ring, as well as additional phenyl groups attached to the oxazole
ring, can enhance inhibitory activity. Addition of electron donating
groups and halogens to the additional phenyl ring can further
enhance the inhibitory activity, while the addition of electron
withdrawing groups reduce the inhibitory activity. Compound 15
shows the highest inhibitory activity, nearly 30-fold more than
compound 1b. Lineweaver–Burk analysis revealed that the repre-
sentative oxazolxanthones 15, 16 and 20 inhibit
a–glucosidase
Fig. 4. Inhibitory activities of compounds 16, 20 and varying combinations of the
two.
via a non-competitive mechanism, while compound 4 inhibits a–
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S.-M. Ding et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
glucosidase via a competitive mechanism. Docking studies showed
that hydrogen bonding interactions with the oxazole ring, stack-
ing interactions with oxazolxanthone, and -cation interactions
involving the additional phenyl group play a crucial role in mediat-
ing the inhibitory effect. In addition, the prediction of multiple
binding modes by molecular docking studies was verified by the
observation of synergetic inhibition involving compounds 16 and
7.61 (d, J = 8.4 Hz, 1H), 7.43 (t, J = 7.4 Hz, 1H), 6.29 (s, 1H); 13C
NMR (101 MHz, DMSO-d6) d 180.58, 156.00, 153.62, 142.99,
135.90, 125.72, 124.61, 124.46, 120.07, 118.23, 116.56, 102.40,
97.83; HRMS (ESI) calcd for C13H8NO4 [MÀH]À: 242.0453 found
242.0456.
p
p
4.4. General procedure for the preparation of compounds 4–16
20 in
a-glucosidase, suggesting that the two inhibitors may bind
simultaneously to the enzyme in different allosteric locations to
generate good synergetic inhibition of enzyme activity.
The derivatives were synthesized by heating compound 3 (1.0
mmol) with suitable acid (1.2 mmol) in Eaton’s reagent (7.5% W/
W P2O5, 24.0 g) and stirring at 100–120 °C for 3 h. At the end of
the reaction period, the residue was poured into an ice-water mix-
ture and neutralized with an excess of NaOH (10%) solution, and
the residue was filtered and dissolved in chloroform. After the
evaporation of solvent under vacuum, the crude product was
obtained and the residue after evaporation was purified by flash
chromatography (chloroform/petroleum ether 10:1) to give com-
pounds 4–16. (33%-59% yield).
4. Experimental and methods
4.1. General information
Melting points were determined using a SGW X-4 digital melt-
ing point apparatus, and the temperatures were not corrected.
High resolution (HR) mass spectra were measured on an LTQ Orbi-
trap Elite or TSQ Quantum XLS mass spectrometer. IR spectra were
measured on a Bruker EQUINOX55 Fourier transformation infrared
spectrometer with KBr pellets. The NMR (1H, 13C, HMQC and
HMBC) were recorded on Bruker AVANCE 400, Varian INOVA
500NB and Bruker Avance III 600 instruments in CDCl3, DMSO-d6
or a solvent mixture of CDCl3/CF3COOD with tetramethylsilane
(TMS) as an internal standard. UV spectra were recorded on a Shi-
madzu UV-3250 scanning spectrophotometer.
4.4.1. 5-hydroxy-2-methyl-6H-xantheno [4, 3-d] oxazol-6-one (4)
Yellow solid (57% yield). Mp 234–236 °C; IR (KBr): 3085, 2927,
1657, 1630, 1612, 1577, 1484, 1451, 1388, 1278, 1222, 1132, 1106,
1090, 976, 896, 787, 764, 749, 647 cmÀ1; 1H NMR (400 MHz, CDCl3)
d 12.99 (s, 1H), 8.34 (dd, J = 8.0, 1.7 Hz, 1H), 7.80 (ddd, J = 8.7, 7.1,
1.7 Hz, 1H), 7.72–7.65 (m, 1H), 7.52–7.40 (m, 1H), 6.91 (s, 1H), 2.69
(s, 3H); 13C NMR (101 MHz, CDCl3) d 181.85, 163.25, 160.27,
156.99, 155.68, 146.84, 135.65, 126.11, 124.76, 121.97, 120.78,
118.08, 106.10, 93.39, 14.40; HRMS (ESI) calcd for C15H10O4N [M
+H]+:268.0593 found 268.0603.
1-Deoxynojirimycin, p-nitrophenyl (PNP)-a-D-glucopyranoside,
and -glucosidase (from Saccharomyces cerevisiae) were purchased
a
from Sigma (St. Louis, MO, USA). Other reagents and solvents were
purchased from commercial sources and used without further
purification. Analytical thin layer chromatography was performed
on pre-coated silica gel (GF-254), and column chromatographic
purifications were performed using 300–400 mesh silica gel. Spots
were visualized under ultraviolet light at 254 and 366 nm.
Compound 1b was prepared as previously reported.17
4.4.2. 2-cyclohexyl-5-hydroxy-6H-xantheno [4, 3-d] oxazol-6-one (5)
Yellow solid (50% yield). Mp 256–258 °C; IR (KBr): 3087, 2921,
2851, 1659, 1610, 1532, 1470, 1449, 1389, 1350, 1285, 1257, 1219,
1189, 1105, 975, 905, 868, 812, 763, 743, 703, 666, 648, 610, 525
cmÀ1 1H NMR (400 MHz, CDCl3) d 13.00 (s, 1H), 8.33 (dd, J = 7.9,
;
1.7 Hz, 1H), 7.79 (ddd, J = 8.7, 7.0, 1.7 Hz, 1H), 7.70 (dd, J = 8.4,
1.1 Hz, 1H), 7.45 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 6.91 (s, 1H), 3.00
(tt, J = 11.6, 3.6 Hz, 1H), 2.05 (ddt, J = 120.8, 13.0, 2.6 Hz, 4H),
1.76 (td, J = 11.7, 3.0 Hz, 4H), 1.44 (qt, J = 14.1, 2.8 Hz, 2H); 13C
NMR (101 MHz, CDCl3) d 181.86, 169.99, 160.21, 156.74, 155.70,
147.05, 135.55, 126.08, 124.68, 121.83, 120.77, 118.18, 106.04,
93.44, 93.38, 38.03, 30.48(2C), 25.68(2C), 25.62; HRMS (ESI) calcd
for C20H16O4N [MÀH]À:334.1084 found 334.1084.
4.2. Synthesis of 1, 3-dihydroxy-4-nitro-9H-xanthen-9-one (2)
Nitric acid (70%, 1.0 mL) in acetic acid (5.0 mL) was slowly
added to a solution of compound 1b (20.0 mmol) in acetic acid
(20.0 mL). The mixture was stirred at 50 °C for 2.0 h and then
poured into ice-cooled water (200 mL). The formed precipitates
were filtered, washed with water. The crude products was purified
by flash chromatography (EtOAc/petroleum ether, 1:3) to yield
compound 2 (3.2 g, 58.6%) as a yellow solid. Mp 226–228 °C; IR
(KBr): 3346, 3277, 2920, 1662, 1611, 1519, 1465, 1392, 1339,
4.4.3. 5-hydroxy-2-phenyl-6H-xantheno [4, 3-d] oxazol-6-one (6)
Yellow solid (52% yield). Mp 260–262 °C; IR (KBr): 3072, 2917,
1658, 1611, 1470, 1447, 1391, 1283, 1155, 1098, 1021, 899, 813,
1285, 1194, 938, 899, 753, 701, 643 cmÀ1 1H NMR (600 MHz,
;
785, 748, 699, 685 cmÀ1 1H NMR (400 MHz, CDCl3) d 13.11 (s,
;
DMSO-d6) d 13.04 (s, 1H), 8.15 (dd, J = 7.9, 1.7 Hz, 1H), 7.89 (ddd,
J = 8.6, 7.1, 1.7 Hz, 1H), 7.61 (dd, J = 8.5, 0.9 Hz, 1H), 7.54 (ddd, J
= 8.0, 7.2, 1.0 Hz, 1H), 6.37 (s, 1H). 13C NMR (151 MHz, DMSO-d6)
d 180.04, 163.78, 158.73, 155.18, 149.91, 136.82, 125.98, 125.82,
122.52, 120.24, 118.35, 101.75, 98.35. HRMS (ESI) calcd for
1H), 8.35 (dd, J = 8.0, 1.6 Hz, 1H), 8.28 (dd, J = 7.4, 2.2 Hz, 1H),
7.82 (ddd, J = 8.6, 7.0, 1.7 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.55
(dt, J = 5.4, 2.4 Hz, 3H), 7.48 (t, J = 7.2 Hz, 1H), 7.02 (s, 1H); 13C
NMR (101 MHz, CDCl3) d 181.87, 162.67, 160.78, 156.81, 155.74,
147.45, 135.68, 131.78, 129.01(2C), 127.58(2C), 126.34, 126.14,
124.84, 123.00, 120.85, 118.24, 106.46, 93.59; HRMS (ESI) calcd
for C20H12O4N [M+H]+:330.0760 found 330.0750.
C
13H6O6N [MÀH]À:272.0200 found 272.0201.
4.3. Synthesis of 1, 3-dihydroxy-4-amino-9H-xanthen-9-one(3)
4.4.4. 5-hydroxy-2-(p-tolyl)-6H-xantheno [4, 3-d] oxazol-6-one (7)
Yellow solid (55% yield). Mp 263–265 °C; IR (KBr): 3022, 2920,
1655, 1639, 1610, 1471, 1442, 1412, 1385, 1269, 1226, 1184, 1156,
1138, 1047, 1018, 920, 809, 788, 762, 722, 681, 635 cmÀ1; 1H NMR
(400 MHz, CDCl3) d 13.02 (s, 1H), 8.37 (dd, J = 7.8, 1.7 Hz, 1H), 8.17
(d, J = 8.2 Hz, 2H), 7.81 (td, J = 7.7, 6.9, 1.7 Hz, 1H), 7.75 (d, J = 8.6
Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H), 7.34 (d, J = 8.0 Hz, 2H), 7.00 (s,
1H), 2.46 (s, 3H); 13C NMR (101 MHz, CDCl3/CF3COOD) d 182.08,
164.26, 160.96, 155.57, 155.32, 146.07, 145.98, 137.03, 130.51
(2C), 128.27(2C), 126.06, 125.79, 120.18, 119.94, 117.96, 117.90,
To a solution of 2 (2.7 g,) in acetone (200 mL) Pd-C (0.3 g) was
added as catalyst and hydrogenation was carried out at 1 bar for
12 h at 40 °C. The resulting suspension was filtered and concen-
trated under reduced pressure to get crude products which were
recrystallized from ethyl acetate to yield pure products 3 (2.2 g,
90.5%). Mp 196–197 °C; IR (KBr): 3081, 2921, 2851, 1659, 1610,
1576, 1532, 1470, 1350, 1284, 1257, 1219, 1142, 999, 975, 868,
812, 763, 743, 703, 666, 610 cmÀ1
;
1H NMR (400 MHz, DMSO-d6)
d 12.13 (s, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 7.9 Hz, 1H),
Please cite this article in press as: Ding S.-M., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.05.008

S.-M. Ding et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
7
106.85, 94.12, 21.80. HRMS (ESI) calcd for C21H12O4N
(dd, J = 8.0, 1.7 Hz, 1H), 8.28 (dd, J = 8.8, 5.2 Hz, 2H), 7.83 (ddd, J
[MÀH]À:342.0771 found 342.0772.
= 8.7, 7.0, 1.7 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.48 (ddd, J = 8.1,
7.0, 1.1 Hz, 1H), 7.23 (d, J = 8.6 Hz, 2H), 7.01 (s, 1H); 13C NMR
(101 MHz, CDCl3/CF3COOD) d 182.79, 168.08, 165.51, 162.02 (d, J
= 259.0 Hz), 156.02, 155.98, 146.75, 137.69, 130.87 (d, J = 9.6 Hz),
119.29(d, J = 2.92 Hz),126.36(2C), 120.35, 118.82, 118.30, 117.27
(d, J = 22.9 Hz,107.22, 94.62; HRMS (ESI) calcd for C20H9O4NF
[MÀH]À:346.0521 found 346.0522.
4.4.5. 5-hydroxy-2-(4-nitrophenyl)-6H-xantheno [4, 3-d] oxazol-6-
one (8)
Yellow solid (33% yield). Mp 272–274 °C; IR (KBr): 3083, 2921,
2851, 1695, 1611, 1577, 1532, 1452, 1389, 1351, 1218, 1188, 1157,
1106, 999, 976, 930, 870, 762, 744, 704, 668, 647 cmÀ1 1H NMR
;
(400 MHz, CDCl3/CF3COOD) d 8.58–8.49 (m, 4H), 8.40 (dd, J = 8.1,
1.6 Hz, 1H), 8.02 (ddd, J = 8.7, 7.2, 1.7 Hz, 1H), 7.75 (dd, J = 8.6,
1.0 Hz, 1H), 7.66 (ddd, J = 8.1, 7.2, 1.1 Hz, H), 7.26 (s, 1H); 13C
4.4.10. 2-(2-fluorophenyl)-5-hydroxy-6H-xantheno [4, 3-d] oxazol-6-
one (13)
NMR (101 MHz, CDCl3/CF3COOD)
d
186.91, 165.74, 165.17,
Yellow solid (56% yield). Mp 264–266 °C; IR (KBr): 3074, 2799,
1657, 1631, 1610, 1577, 1471, 1450, 1391, 1336, 1282, 1260, 1222,
1158, 1143, 1098, 1057, 1025, 975, 901, 864, 814, 787, 764, 748,
160.82, 160.03, 154.14, 151.93, 141.50, 136.27, 134.66, 133.44,
133.11, 130.22, 129.60, 128.96, 125.64, 124.36, 122.28, 111.14,
98.44; HRMS (ESI) calcd for C20H9O6N2 [MÀH]À:373.0466 found
373.0467.
688, 672, 648, 611 cmÀ1 1HNMR (400 MHz, CDCl3) d 13.13 (s,
;
1H), 8.36 (d, J = 7.8 Hz, 1H), 8.26 (t, J = 7.5 Hz, 1H), 7.83 (dd, J =
8.6, 6.8 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.55 (q, J = 7.3 Hz, 1H),
7.48 (t, J = 7.5 Hz, 1H), 7.33 (dd, J = 16.6, 8.8 Hz, 2H), 7.04 (s, 1H);
13C NMR (101 MHz, CDCl3/CF3COOD) d 183.21, 161.52 (d, J =
260.0 Hz), 161.43, 160.60 (d, J = 4.3 Hz), 156.38, 156.24, 147.60,
137.90, 136.35 (d, J = 9.2 Hz), 130.98, 126.70, 126.59, 125.94 (d, J
= 3.7 Hz), 120.73, 120.28, 118.61, 118.02 (d, J = 21.1 Hz), 113.01
(d, J = 10.9 Hz), 107.51, 94.82; HRMS (ESI) calcd for C20H9O4NF
[MÀH]À:346.0521 found 346.0520.
4.4.6. 5-hydroxy-2-(4-(trifluoromethyl) phenyl)-6H-xantheno [4,3-d]-
oxazol-6-one (9)
Yellow solid (49% yield). Mp 269–270 °C; IR (KBr): 3047, 2961,
2922, 2852, 1936, 1636, 1472, 1452, 1613, 1390, 1324, 1287, 1261,
1220, 1175, 1142, 1114, 1064, 1016, 899, 869, 910, 789, 758, 695,
638, 611 cmÀ1; 1H NMR (600 MHz, CDCl3) d 13.14 (s, 1H), 8.39 (d, J
= 8.1 Hz, 2H), 8.35 (dd, J = 7.9, 1.7 Hz, 1H), 7.83 (ddd, J = 8.6, 7.0,
1.7 Hz, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.75 (dd, J = 8.5, 1.0 Hz, 1H),
7.49 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 7.02 (s, 1H); 13C NMR (101
MHz, CDCl3/CF3COOD) d 177.04, 157.00, 156.30, 151.44, 150.74,
142.47, 131.67, 129.27 (q, J = 32.9 Hz), 123.24(2C), 121.56 (q, J =
3.8 Hz), 121.30, 120.65, 118.69 (q, J = 272.4 Hz), 116.57, 115.64,
113.19, 101.85, 88.99; HRMS (EI) calcd for C21H10O4NF3 [M+]: m/
z = 397.0556 found 397.0554.
4.4.11. 2-(3-fluorophenyl)-5-hydroxy-6H-xantheno [4, 3-d] oxazol-6-
one (14)
Yellow solid (44% yield). Mp 268–270 °C; IR (KBr): 3428, 3075,
2961, 2921, 1658, 1632, 1610, 1577, 1472, 1450, 1392, 1334, 1282,
1259, 1221, 1144, 1102, 1024, 975, 865, 812, 764, 744, 687, 647,
609, 526 cmÀ1 1H NMR (600 MHz, CDCl3) d 13.14 (s, 1H), 8.36
;
(dd, J = 7.9, 1.6 Hz, 1H), 8.07 (dt, J = 7.7, 1.2 Hz, 1H), 7.98 (dt, J =
9.4, 2.0 Hz, 1H), 7.84 (ddd, J = 8.6, 7.0, 1.7 Hz, 1H), 7.77 (dd, J =
8.4, 1.1 Hz, 1H), 7.53 (td, J = 8.0, 5.5 Hz, 1H), 7.49 (ddd, J = 8.1,
7.0, 1.1 Hz, 1H), 7.26 (dd, J = 16.6, 2.7 Hz, 1H), 7.03 (s, 1H). 13C
NMR (101 MHz, CDCl3/CF3COOD) d 186.24, 167.21 (d, J = 248.6
Hz), 166.52 (d, J = 3.3 Hz), 165.16, 160.38, 159.85, 151.39, 140.77,
135.50 (d, J = 8.1 Hz), 131.03 (d, J = 8.6 Hz), 130.36, 129.70,
127.82 (d, J = 3.4 Hz), 125.33, 124.68, 124.19 (d, J = 21.3 Hz),
122.23, 119.04 (d, J = 24.5 Hz), 110.91, 98.07; HRMS (ESI) calcd
for C20H9O4NF [MÀH]À:346.0521 found 346.0522.
4.4.7. 2-(4-bromophenyl)-5-hydroxy-6H-xantheno [4, 3-d] oxazol-6-
one (10)
Yellow solid (54% yield). Mp 276–278 °C; IR (KBr): 3031, 2918,
1658, 1633, 1610, 1578, 1470, 1403, 1387, 1333, 1283, 1258, 1218,
1097, 1069, 1041, 1008, 975, 900, 829, 779, 766, 751, 723, 682, 648,
613 cmÀ1 1H NMR (500 MHz, CDCl3) d 13.08 (s, 1H), 8.37 (dd, J =
;
7.9, 1.7 Hz, 1H), 8.15 (d, J = 8.6 Hz 2H), 7.82 (ddd, J = 8.7, 7.0, 1.7 Hz,
1H), 7.75 (d, J = 8.5 Hz, 1H), 7.68 (d, J = 8.6 Hz, 2H), 7.48 (ddd, J =
8.0, 7.0, 1.1 Hz, 1H), 7.01 (s, 1H); 13C NMR (101 MHz, CDCl3/CF3-
COOD) d 182.51, 163.40, 160.65, 155.82, 155.68, 146.66, 137.30,
133.10(2C), 129.38, 129.27(2C), 125.98, 125.95, 122.21, 119.97,
119.25, 117.94, 106.80, 94.23; HRMS (ESI) calcd for C20H9O4NBr
[MÀH]À:405.9720 found 405.9724.
4.4.12. 5-hydroxy-2-(2, 4, 6-trifluorophenyl)-6H-xantheno[4,3-d]-
oxaz-ol-6-one(15)
Yellow solid (57% yield). Mp 275–277 °C; IR (KBr): 3081, 2929,
1749, 1658, 1613, 1579, 1475, 1392, 1358, 1281, 1251, 1226, 1180,
4.4.8. 2-(4-chlorophenyl)-5-hydroxy-6H-xantheno [4, 3-d] oxazol-6-
one (11)
1133, 1042, 974, 924, 825, 809, 761, 747, 678, 625, 637, 615 cmÀ1
;
1H NMR (400 MHz, CDCl3) d 13.14 (s, 1H), 8.37 (dd, J = 8.0, 1.7 Hz,
1H), 7.86–7.80 (m, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.49 (t, J = 7.5 Hz,
1H), 7.04 (s, 1H), 6.91 (t, J = 8.7 Hz, 2H); 13C NMR (101 MHz, CDCl3/
CF3COOD) d 186.51, 170.93 (t, J = 15.5 Hz), 168.36 (t, J = 15.4 Hz),
167.35 (dd, J = 15.4, 7.4 Hz), 165.15, 160.32, 159.88, 159.05,
151.58, 141.10, 130.36, 129.95, 124.74, 124.55, 122.27, 110.98,
106.26 (td, J = 25.8, 4.1 Hz), 98.27; HRMS (ESI) calcd for C20H7O4-
NF3 [MÀH]À:382.0332 found 382.0332.
Yellow solid (53% yield). Mp 271–273 °C; IR (KBr): 3034, 2922,
1659, 1633, 1611, 1578, 1484, 1470, 1451, 1406, 1388, 1333,
1282,1258, 1217, 1161, 1140, 1091, 1042, 1011, 975, 918, 901,
833, 764, 751, 684, 663, 648 cmÀ1 1H NMR (500 MHz, CDCl3) d
;
13.07 (s, 1H), 8.37 (dd, J = 8.0, 1.6 Hz, 1H), 8.22 (d, J = 8.5 Hz, 2H),
7.82 (td, J = 7.6, 6.8, 1.6 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.53 (d, J
= 8.5 Hz, 2H), 7.50–7.46 (m, 1H), 7.01 (s, 1H); 13C NMR (101
MHz, CDCl3/CF3COOD) d 182.34, 163.07, 160.64, 155.94, 155.65,
146.82, 140.23, 137.03, 130.01(2C), 129.19(2C), 126.05, 125.77,
122.40, 120.14, 120.05, 117.96, 106.73, 94.09; HRMS (EI) calcd
for C20H10O4NCl [M+]: m/z = 363.0293 found 363.0281.
4.4.13. 5-hydroxy-2-(4-methoxyphenyl)-6H-xantheno [4, 3-d] oxazol-
6-one (16)
Yellow solid (59% yield). Mp 247–248 °C; IR (KBr): 3004, 2942,
2836, 1659, 1610, 1578, 1504, 1473, 1450, 1421, 1389, 1335, 1282,
1257, 1228, 1214, 1166, 1140, 1095, 1046, 1024, 975, 915, 861,
4.4.9. 2-(4-fluorophenyl)-5-hydroxy-6H-xantheno [4, 3-d] oxazol-6-
one (12)
784, 760, 747, 637, 608 cmÀ1 1H NMR (400 MHz, CDCl3) d 13.07
;
Yellow solid (59% yield). Mp 265–267 °C; IR (KBr): 3423, 3049,
2926, 1657, 1635, 1609, 1578, 1472, 1453, 1416, 1389, 1335, 1228,
1161, 1141, 1099, 1041, 1010, 976, 901, 812, 784, 760, 748, 729,
(s, 1H), 8.33 (dd, J = 7.9, 1.6 Hz, 1H), 8.19 (d, J = 8.8 Hz, 1H), 7.80
(ddd, J = 8.6, 7.0, 1.7 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.46 (t, J =
7.2 Hz, 1H), 7.02 (d, J = 8.8 Hz, 2H), 6.98 (s, 1H), 3.90 (s, 3H); 13C
NMR (101 MHz, CDCl3) d 181.86, 162.80, 162.51, 160.42, 156.73,
640, 611 cmÀ1 1HNMR (400 MHz, CDCl3) d 13.11 (s, 1H), 8.36
;
Please cite this article in press as: Ding S.-M., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.05.008

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S.-M. Ding et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
155.75, 147.07, 135.60, 129.38(2C), 126.10, 124.73, 123.06, 120.82,
118.81, 118.21, 114.43(2C), 106.31, 93.52, 55.50; HRMS (ESI) calcd
for C21H12O5N [MÀH]À:358.0721 found 358.0723.
175.77, 162.69, 159.91, 155.16, 154.64, 149.57, 134.19, 131.69,
128.99(2C), 127.50(2C), 126.89, 126.44, 124.58, 124.52, 123.31,
117.63, 110.18, 89.70, 57.00; HRMS (ESI) calcd for C21H14NO4 [M
+H]+:344.0933 found 344.0923.
4.5. Synthesis of 5-hydroxy-2-(4-hydroxyphenyl)-6H-xantheno [4, 3-
d] oxazol-6- one (17)
4.7.2. 5-methoxy-2-(4-methoxyphenyl)-6H-xantheno [4, 3-d] oxazol-
6-one (20)
Compound 16 was dissolved in anhydrous CH2CI2 (10.0 mL) and
cooled to À8.0 °C under N2, then BBr3 (2.0 mL, 1.0 M in CH2CI2, 2.0
mmol) was added dropwise. The mixture was warmed to room
temperature and stirred for 24 h, H2O was added and the resulting
mixture extracted with CH2CI2, (3 Â 25 mL). The combined CH2CI2
extracts were washed with brine, dried (Na2SO4), and concentrated
under vacuum, The remaining material was purified by column
chromatography (acetone - petroleum ether, 1:2) to produce com-
pound 17. Yellow solid (81% yield). Mp 268–270 °C; IR (KBr): 3326,
3073, 2925, 1657, 1611, 1573, 1509, 1471, 1452, 1393, 1334, 1284,
White solid (92% yield). Mp 233–235 °C; IR (KBr): 3483, 3067,
2949, 1659, 1635, 1609, 1586, 1466, 1421, 1398, 1376, 1333,
1302, 1262, 1225, 1197, 1134, 1160, 1103, 1069, 1044, 109, 921,
894, 826, 800, 749, 723, 685, 644, 612 cmÀ1 1HNMR (400 MHz,
;
CDCl3) d 8.35 (dd, J = 7.7, 1.4 Hz, 1H), 8.22–8.18 (m, 2H), 7.73–
7.66 (m, 2H), 7.39 (ddd, J = 8.1, 6.4, 1.8 Hz, 1H), 7.04 (d, J = 8.9
Hz, 2H), 7.03 (s, 1H), 4.08 (s, 3H), 3.90 (s, 3H). 13C NMR (101
MHz, CDCl3) d 171.06, 158.08, 157.68, 154.77, 150.29, 149.89,
144.48, 129.38, 124.53(2C), 122.10, 119.86, 119.67, 118.51,
114.16, 112.85, 109.66(2C), 105.26, 84.93, 52.21, 50.73; HRMS
(ESI) calcd for C22H16O5N [M+H]+:374.1023 found 374.1014.
1257, 1229, 1141, 1112, 1097, 1042, 976, 900, 830, 762, 747 cmÀ1
;
1H NMR (400 MHz, DMSO-d6) d 12.99 (s, 1H), 10.37 (s, 1H), 8.24
(dd, J = 7.9, 1.7 Hz, 1H), 8.02 (d, J = 8.7 Hz, 2H), 7.96 (ddd, J = 8.6,
7.1, 1.7 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H),
7.20 (s, 1H), 6.96 (d, J = 8.8 Hz, 2H); 13CNMR (101 MHz, DMSO-
d6) d 181.64, 162.64, 161.46, 159.74, 156.45, 155.47, 146.67,
136.75, 129.60(2C), 125.87, 125.50, 122.90, 120.38, 118.48,
116.81, 116.55(2C), 106.01, 93.72; HRMS (ESI) calcd for
4.7.3. 2-(4-chlorophenyl)-5-methoxy-6H-xantheno [4, 3-d] oxazol-6-
one (21)
White solid (88% yield). Mp 248–250 °C; IR (KBr): 3481, 3064,
2941, 1657, 1625, 1607, 1583, 1464, 1426, 1376, 1324, 1191,
1173, 1134, 1222, 1112, 1046, 1025, 1011, 921, 899, 823, 724,
C
20H10O5N [MÀH]À:344.0564 found 344.0566.
704, 661, 616 cmÀ1 1HNMR (400 MHz, CDCl3) d 8.35 (dd, J = 8.0,
;
1.6 Hz, 1H), 8.20 (d, J = 8.3 Hz, 2H), 7.71 (t, J = 7.6 Hz, 1H), 7.67
(d, J = 8.3 Hz, 1H), 7.52 (d, J = 8.3 Hz, 2H), 7.40 (t, J = 7.4 Hz, 1H),
7.03 (s, 1H), 4.09 (s, 3H). 13CNMR (101 MHz, CDCl3) d 175.69,
161.70, 160.09, 155.14, 154.60, 149.60, 137.97, 134.25, 129.39
(2C), 128.71(2C), 126.91, 124.92, 124.60, 124.52, 123.31, 117.59,
4.6. Synthesis of 2-(4-aminophenyl)-5-hydroxy-6H-xantheno [4, 3-d]
oxazol-6-one (18)
To a solution of 8 (127 mg) in acetone (25.0 mL), Pd-C (20 mg)
was added as catalyst and hydrogenation was carried out at 1
bar for 12 h at 40 °C. The resulting suspension was filtered and
concentrated under reduced pressure to obtain crude products
which were recrystallized from ethyl acetate to yield pure product
18 as a yellow solid (106 mg, 86%). Mp 231–233 °C; IR (KBr): 3478,
3350, 3217, 2924, 1656, 1608, 1576, 1503, 1471, 1453, 1390, 1332,
1283, 1263, 1229, 1176, 1159, 1139, 1097, 1043, 1007, 975, 900,
110.28, 89.65, 57.03; HRMS (ESI) calcd for
C21H13NO4Cl [M
+H]+:378.0539 found 378.0533.
4.8. Biological studies
4.8.1. a-glucosidase inhibitory assays
The inhibitory activities of compounds 1b, 2–16 were measured
;
822, 785, 761, 749, 699, 682, 643 cmÀ1 1H NMR (400 MHz,
by using the methods similar to those described previously.14–19
DMSO-d6) d 12.97 (s, 1H), 8.25 (d, J = 7.9 Hz, 1H), 7.96 (t, J = 7.6
Hz, 1H), 7.86–7.83 (m, 3H), 7.56 (t, J = 7.6 Hz, 1H), 7.17 (s, 1H),
6.69 (d, J = 8.3 Hz, 2H), 6.04 (s, 2H); 13C NMR (101 MHz, DMSO-
d6) d 181.86, 163.70, 159.44, 156.55, 155.67, 153.15, 146.45,
136.86, 129.38(2C), 126.02, 125.56, 123.29, 120.54, 118.60,
114.06(2C), 112.38, 106.07, 93.77; HRMS (ESI) calcd for
Typically, a-glucosidase activity was assayed in 50 mM phosphate
buffer (pH 6.8) containing 5% v/v DMSO, and PNP glycoside was
used as a substrate. The inhibitors were pre-incubated with the
enzyme at 37 °C for 30 min, and then the substrate was added.
The enzymatic reaction was carried out at 37 °C for 60 s and mon-
itored spectrophotometrically by measuring the absorbance at 400
nm. The assay was performed in triplicate with five different inhi-
bitor concentrations around the IC50 values that were roughly esti-
mated in the first round of experiments, and the mean values were
taken.
C
20H11O4N2 [MÀH]À:343.0713 found 343.0723.
4.7. General procedure for the preparation of 19–21
A mixture of compounds 6, 11, 16(1.0 equiv.), K2CO3 (20.0
equiv.) and CH3I (24.0 equiv.) in acetone (5.0 mL) was stirred at
room temperature for 24 h. The reaction mixture was added to
25.0 mL of 2.0 M HCl and extracted with EtOAc (3 Â 25 mL). The
combined organic layers were dried, filtered and evaporated under
vacuum. The crude product was purified by column chromatogra-
phy (acetone-chloroform, 1:100) to produce compounds 19–21
(88–92% yield).
4.8.2. Kinetics of enzyme inhibition
The inhibition types of the selected compounds were deter-
mined from Lineweavere-Burk plots, using the methods similar
to those reported previously.18,34,35 Typically, two different con-
centrations of each compound around the IC50 values were chosen.
With each concentration,
a-glucosidase activity was assayed by
varying the concentration of PNP glycoside. The enzyme reaction
was performed using the conditions described above. The mixtures
of the enzyme and the inhibitor were dissolved in 50 mM phos-
phate buffer (pH 6.8) containing 5% v/v DMSO, pre-incubated at
37 °C for 30 min, and then the substrate was added. The enzyme
reaction was carried out at 37 °C for 60 s, and monitored spec-
trophotometrically by measuring the absorbance at 400 nm. Inhi-
bition types and Ki values of the inhibitors were determined
using double-reciprocal plots.
4.7.1. 5-methoxy-2-phenyl-6H-xantheno [4, 3-d] oxazol-6-one (19)
White solid (91% yield). Mp 272–274 °C; IR (KBr): 3485, 3070,
2949, 1658, 1635, 1609, 1582, 1466, 1425, 1376, 1303, 1193,
1170, 1134, 1225, 1103, 1046, 1028, 1011, 920, 897, 833, 726,
709, 665, 615 cmÀ1 1HNMR (400 MHz, CDCl3) d 8.36 (dd, J = 7.8,
;
1.5 Hz, 1H), 8.28 (dd, J = 7.2, 2.7 Hz, 2H), 7.77–7.65 (m, 2H), 7.56
(d, J = 2.0 Hz, 2H), 7.55 (d, J = 1.9 Hz, 1H), 7.41 (ddd, J = 8.1, 6.3,
1.9 Hz, 1H), 7.06 (s, 1H), 4.10 (s, 3H); 13CNMR (101 MHz, CDCl3) d
Please cite this article in press as: Ding S.-M., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.05.008

S.-M. Ding et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
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a
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We are thankful to Prof. Wen-Hua Chen (Southern Medical
University) and Prof. Zhuofeng Ke (Sun Yat-sen University) for
their helpful discussions and comments. We are thankful to Prof.
Jeff Lawton (Eastern University) for his generously providing us
editorial assistance. This work was supported by the National Nat-
ural Science Foundation of China (Nos. 21272290 and J1103305)
and the Fund for Undergraduate Innovation Program from Sun
Yat-sen University.
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2018.05.008.
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