Synthesis of tetracyclic oxindoles and evaluation of their α-glucosidase inhibitory and glucose consumption-promoting activity

Article abstract of DOI:10.1016/j.bmcl.2020.127264

A series of tetracyclic oxindole derivatives was synthesized by asymmetric 1, 3-dipole reaction in 2–4 steps in 57–86% overall yields. These compounds were evaluated for α-glucosidase inhibitory and glucose consumption-promoting activity in vitro. Compound 4l competitively and reversibly inhibited α-glucosidase (IC₅₀ = 3.64 μM) with activity 14-fold higher than that of acarbose. Docking analysis substantiated these findings. In addition, compound 4l exhibited significant glucose consumption promoting activity at 1 μM.

Full text of DOI:10.1016/j.bmcl.2020.127264

Bioorganic&MedicinalChemistryLetters30(2020)127264
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of tetracyclic oxindoles and evaluation of their α-glucosidase
inhibitory and glucose consumption-promoting activity
Lei Haoa,1, Yujiao Maa,1, Lianbo Zhaoa, Yinan Zhanga, Xinying Zhanga, Ying Mab,
⁎
⁎
Robert H. Dodda,c, Hua Suna, , Peng Yua,
a China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science
& Technology, Tianjin 300457, China
b School of Pharmacy, Tianjin Medical University, Tianjin 300070, China
c Centre de recherche de Gif-sur-Yvette, Institut de Chimie des Substances Naturelles, UPR 2301, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of tetracyclic oxindole derivatives was synthesized by asymmetric 1, 3-dipole reaction in 2–4 steps in
57–86% overall yields. These compounds were evaluated for α-glucosidase inhibitory and glucose consumption-
promoting activity in vitro. Compound 4l competitively and reversibly inhibited α-glucosidase (IC50 = 3.64 μM)
with activity 14-fold higher than that of acarbose. Docking analysis substantiated these findings. In addition,
compound 4l exhibited significant glucose consumption promoting activity at 1 μM.
Tetracyclic oxindole
α-Glucosidase inhibitor
Inhibitory mechanism
Docking analysis
Glucose consumption
α-Glucosidase is a typical exohydrolase that reacts with the 1,4-α-
glucosidic linkage of the nonreducing terminal of substrates, releasing
α-glucose. Under high substrate concentrations, it also catalyzes
transglucosylation, synthesizing oligosaccharides.1 The functions of α-
glucosidase in mammals include intestinal digestion of dietary carbo-
hydrates, glycogen degradation, and glycoprotein folding and matura-
tion.2 Disorder of α-glucosidase function in humans is correlated to
many diseases including diabetes,3 pompe disease,4 azoospermia,5 viral
infection,6 etc. Therefore, α-glucosidase can be considered as an im-
portant target for drug development (Fig 1).
evaluation for the treatment of malaria and human cancer, and its de-
rivatives containing the thiazolidine ring (3).14,15 The introduction of a
spiral ring structure can improve pharmacokinetic properties, stabilize
the configuration of compounds and enhance the overall stability of
compounds.16–18 Mono-carbonyl curcumin analogues has also been
reported to have a significant beneficial effect on diabetes.19 In this
study, a series of spiropyrrolidine oxindoles containing a thiazolidine
ring was diastereoselectively synthesized from isatin, chalcone and 2-
phenylthiazolidine-4-carboxylic acid via 1,3-dipolar cycloaddition and
the resulting compounds were evaluated for their α-glucosidase in-
hibitory and cell-based glucose consumption-promoting activities.
As shown in Scheme 1 1-(4-methoxybenzyl)-5-bromoisatin 5 was
synthesized from 5-bromoisatin and p-methoxybenzyl bromide
(PMBBr) in DMF in the presence of K2CO3 in excellent yield. Compound
5 was then converted to 1-PMB-5-trans-(2-methoxycarbonylethenyl)
isatins 6 by microwave-assisted Heck coupling reaction with methyl
acrylate in 88–90% yields. Finally, equimolar amounts of mono-car-
bonyl curcumin analogues 7, prepared by aldol condensation of sub-
stituted aldehydes and acetone, were reacted with substituted isatins
and L-thiazolidine-4-carboxylic acid to give target compounds 4a-x by
1,3-dipolar reaction in one-pot fashion.8 1H and 13C NMR spectra along
with DEPT, 1H-1H COSY, HSQC, HMBC, and NOESY experiments were
Oxindoles are a class of privileged scaffolds that are highly pre-
valent in natural and synthetic compounds of medicinal interest.7–9
Tetracyclic oxindole alkaloids are naturally present in plants and mi-
crobial metabolites, and exhibit many biological activities. Our pre-
vious study generated, via a tandem Suzuki coupling-Michael addition
reaction, a new class of tetracyclic oxindole alkaloids (1) which ex-
hibited potent α-glucosidase inhibitory activity.10,11 A structure–-
activity relationships study showed that structural modifications at C3,
C4 and C5 of oxindoles had effects on α-glucosidase inhibitory activity
and other anti-diabetic activities. The unique structural features of
spirooxindoles, especially the spiro rings fused at the C3 position of the
oxindole scaffold, together with diverse biological activities, have made
them promising structures for new drug discovery.12,13 Two re-
presentative examples are MI-888 (2), which is currently in preclinical
used to elucidate the structures of the new products. Analysis of the 1
H
NMR spectrum of compound 4b and observation of coupling constant
⁎ Corresponding authors.
E-mail addresses: sunhua@tust.edu.cn (H. Sun), yupeng@tust.edu.cn (P. Yu).
1 The authors have equal contribution.
https://doi.org/10.1016/j.bmcl.2020.127264
Received 28 January 2020; Received in revised form 22 April 2020; Accepted 12 May 2020
Availableonline15May2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

L. Hao, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127264
structural alterations at the R2 position.
Examination of the activity of derivatives bearing the bulky PMB or
H at the N-1 position showed that the activity is also quite sensitive
towards derivatization at this position. The absence of a PMB group at
N-1 (compounds 4a-g, j) proved generally detrimental to the inhibitory
activity. Furthermore, the introduction of a 3,4-dimethoxyphenyl (4h)
or furan-2-yl group (4i) at the R2 site led to a significant reduction in
activity. It is also evident that a p-substituted phenyl group is favorable
for activity.
The 2-methoxycarbonylethenyl group was found to have an im-
portant influence on biological activity in our previous study.20 How-
ever, in the present derivatives, replacement of the 5-bromo atom of 4j-
n by the 2-methoxycarbonylethenyl group (4o-x) resulted in similar
activities, except for 4q and 4u (4m vs 4q, 4l vs 4u). These results
revealed that the inhibitory activity was not sensitive to the electronic
and stereo changes of R2 position if tetracyclic oxindoles substituted
with both PMB at N-1 and 2-methoxycarbonylethenyl group at C-5.
The contrasting inhibitory activities of compounds 5a and 4l
prompted us to investigate their binding mode toward α-glucosidase,
especially with regard to the potential role of the spiro ring fused at C-3
of the oxindole moiety. As the crystal structure of α-glucosidase from
Saccharomyces cerevisiae is not available, a docking study was con-
ducted using the crystal structure of isomaltase (PDB 3A4A) from S.
cerevisiae that was reported to be a suitably equivalent template.21 Al-
though five H-bonds can be established by 5a with key residues of the
enzyme’s active pocket (Fig. 2B), only formation of one H-bond (blue
line) as well as Pi interactions (orange line) by 4l with the key residues
Ser 240 and Phe 303, respectively, were observed in the docking study
(Fig. 2D). However, the formation of more electrostatic and van der
Waals interactions by 4l allows it to fit very tightly in the active site
(Fig. 2C). The introduction of the spiro ring and the bulky substituents
at the R2 position completely changed the binding conformation and
location of tetracyclic oxindoles, which possessed one of 4-dimethyla-
minophenyl groups in deep of pocket and a PMB group on the edge of
active pocket. Calculated docking scores of 4l (20.2 kcal/mol) were
higher than for 5a (8.3 kcal/mol), consistent with their activity
(Table 1).
Fig. 1. Structures of 1–3 and 4a-x.
for H-18 (J = 16 Hz) suggested that it possesses a trans-double bond.
Besides, the NOESY spectrum of 4b exhibited correlations for 1H signals
of C-14 (δH 4.33, m, δC 75.4, CH) with C-16 (δH 4.44, d, J = 12 Hz, δC
64.4, CH), suggesting that the α, β-unsaturated ketone and thiazolidine
were located on the same plane. By comparing the spectroscopic data
with reported data,8,14,15 the structure of compound 4b was established
as shown in Scheme 1.
The α-glucosidase inhibitory activities of 4a-x were evaluated ac-
cording to the reported experimental methods.10 Acarbose was used as
a reference compound for activity comparison. As can be seen from the
data in Table 1, 5-bromo-1-(4-methoxybenzyl)indoline-2,3-dione (5a)
was first evaluated and compared with the inhibitory activity of tetra-
cyclic oxindole derivative 4k. Interestingly, introduction of the sub-
stituted spiro ring at the C-3 position of oxindole resulted in a 10-fold
improvement in activity. Replacement of the 4-chlorophenyl group of
4k by a 4-dimethylaminophenyl group (4l) led to a similar activity.
Variation of electronic effect for the para substituents on phenyl group
did not significantly alternate the potency. However, the activity was
significantly reduced in the case of the 2,4-dichlorophenyl derivative
4m. The low activity of compound 4m suggested that the stereo effect is
crucial for the desired activity. These results revealed that the α-glu-
cosidase inhibitory activity of the spirooxindoles is indeed sensitive to
To determine the mechanism of α-glucosidase inhibition by the
most potent compound 4l, kinetic analysis was performed using
Lineweaver-Burk plot analysis. The plot of velocity versus substrate
(pNGP) concentration in the presence of different concentrations of 4l
gave a series of straight lines. This analysis (Fig. 3A) showed that Vmax
(the y-intercept) was unaffected by changing the x-intercept of in-
creasing concentrations of 4l. This result indicates that 4l exhibits
competitive inhibition of α-glucosidase, similar to that of acarbose.11
The plots of the velocity versus enzyme concentrations in the presence
Scheme 1. Synthesis of compounds 4–7.
2

L. Hao, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127264
Fig. 2. Binding modes of compounds 5a and 4l in the active site of α-glucosidase. (A) and (C) Compounds 5a and 4l dock on the α-glucosidase. (B) and (D)
Interactions of compounds 5a and 4l and the active site of α-glucosidase.
Fig. 3. Inhibitory kinetics of compound 4l on α-
glucosidase. (A) Lineweaver − Burk plots of 4l [(▲)
5, (■) 4, and (●) 0μM]. (B) Relationship of the α-
glucosidase activity and enzyme concentration (4, 6,
and 8 milliunits/mL) at different concentrations of
4l [(●) 0, (■) 4, and (▲) 5 μM].
To further study the anti-diabetic activity of compound 4l, the
glucose consumption in HepG2 cells was evaluated using metformin as
a positive control (Fig. 4). Compound 4l was observed to significantly
promote glucose consumption at 5 and 1 μM. Compound 4l and met-
formin did not demonstrate any cytotoxicity to HepG2 cells at testing
concentrations.
In conclusion, twenty-four spirooxindoles were synthesized and
evaluated for their α-glucosidase inhibitory and glucose consumption-
promoting activity. Compound 4l was identified as a new α-glucosidase
inhibitor with an IC50 value of 3.64 μM acting in a competitive and
reversible inhibitory mode. Molecular docking studies showed that 4l
has a high binding affinity with α-glucosidase due to the presence of
hydrogen bonding, Pi, van der Waals, and electrostatic interactions,
together with a good shape match to the active pocket. In addition,
Fig. 4. Compound 4l promoted glucose consumption in HepG2 cells. Data are
compound 4l showed glucose consumption-promoting activity in
the average of three independent experiments, each performed in duplicate.
HepG2 cells at 1 μM, comparable to metformin at a concentration of
***P < 0.001 vs. Control.
1 mM. In light of these results, compound 4l presents considerable
potential as a lead compound for the treatment of diabetes and other
of different concentrations of compound 4l gave a family of straight
lines (Fig. 3B). The concentrations of 4l and enzyme increased, but the
intercept on the X-axis and Y-axis remained constant, indicating that
compound 4l is a reversible inhibitor. These results provide evidence
that the inhibition of α-glucosidase by 4l is competitive and reversible.
diseases involving α-glucosidase.
Declaration of Competing Interest
The authors declare that they have no known competing financial
3

L. Hao, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127264
Table 1
α-Glucosidase inhibitory activity of compounds 4a-x.
.
Compds
R2
R1
IC50 (μM)a
5a
–
PMB
H
39.92
7.49
16.13
18.85
4.81
9.80
4.47
7.40
> 50
> 50
4.90
3.90
3.64
14.1
4.09
4.73
8.33
4.49
8.44
4.68
4.14
9.47
7.43
4.47
3.81
50.3
4.78
4a
4-chlorophenyl
phenyl
0.10
4b
4c
H
0.52
0.13
4-methylphenyl
4-dimethylaminophenyl
2-chlorophenyl
2-bromophenyl
2,4-dichlorophenyl
3,4-dimethoxyphenyl
furan-2-yl
H
4d
4e
H
0.19
H
0.04
0.03
0.09
4f
H
4g
H
4h
4i
H
H
4j
4-chlorophenyl
4-chlorophenyl
4-dimethylaminophenyl
2,4-dichlorophenyl
2-bromophenyl
4-chlorophenyl
phenyl
H
0.17
0.33
0.42
1.04
0.04
0.38
0.10
0.34
0.47
0.08
0.16
1.17
0.06
0.34
0.07
0.47
4k
4l
PMB
PMB
PMB
PMB
PMB
PMB
PMB
PMB
PMB
PMB
PMB
PMB
PMB
PMB
4m
4n
4o
4p
4q
4r
2,4-dichlorophenyl
4-bromophenyl
2-bromophenyl
(1,1′-biphenyl)-4-yl
4-dimethylaminophenyl
4-diethylaminophenyl
4-fluorophenyl
4-methoxyphenyl
4s
4t
4u
4v
4w
4x
Acarboseb
a
b
Results are the average of three independent experiments, each done in duplicate.
Acarbose was used as a reference compound.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127264.
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