α-Glucosidase Inhibitors Based on Oleanolic Acid for the Treatment of Immunometabolic Disorders

Abstract

Using oleanolic acid as a starting compound, a series of new oleanane-type triterpenic derivatives were synthesized via O-acylation (with nicotinic, isonicotinic, and methoxycinnamic acid acyl chlorides), N-amidation (with cyclic- or polyamines), the Mannich reaction (with secondary cyclic amines), and Claisen–Schmidt condensation (with aromatic aldehydes), and their potencies as treatments for immunometabolic disorders were investigated. The compounds were evaluated against α-glucosidase and PTP1B enzymes and LPS-stimulated murine macrophages. It was found that the target compounds are highly effective α-glucosidase inhibitors but lack activity against PTP1B. A leading compound, N-methylpiperazine methylated 2,3-indolo-oleanolic propargyl amide 15, is also a micromolar inhibitor of NO synthesis in LPS-stimulated macrophages and suppresses oxidative bursts in neutrophils with similar efficiency. These results, in addition to its ability to stimulate glucose uptake in rat fibroblasts and improve maltose tolerance in rats, allow us to consider compound 15 a promising prototype drug for the treatment of immunometabolic defects in type 2 diabetes.

Full text

Citation: Petrova, A.V.; Babkov, D.A.;
Khusnutdinova, E.F.; Baikova, I.P.;
Kazakova, O.B.; Sokolova, E.V.;
Spasov, A.A. α-Glucosidase
Inhibitors Based on Oleanolic Acid
for the Treatment of
Immunometabolic Disorders. Appl.
Sci. 2023,13, 9269. https://doi.org/
10.3390/app13169269
Academic Editor: Emanuel Vamanu
Received: 3 July 2023
Revised: 29 July 2023
Accepted: 3 August 2023
Published: 15 August 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
applied
sciences
Article
α-Glucosidase Inhibitors Based on Oleanolic Acid for the
Treatment of Immunometabolic Disorders
Anastasiya V. Petrova 1, Denis A. Babkov 2 ,* , Elmira F. Khusnutdinova 1, Irina P. Baikova 1,
Oxana B. Kazakova 1, * , Elena V. Sokolova 2and Alexander A. Spasov 2
1Ufa Institute of Chemistry, Ufa Federal Research Centre, Russian Academy of Science, 71 Pr.,
Oktyabrya Ufa 450054, Russia; ana.orgchem@gmail.com (A.V.P.); elmah@inbox.ru (E.F.K.);
poljr@anrb.ru (I.P.B.)
2Scientific Center for Innovative Drugs, Volgograd State Medical University, 39, Novorossiyskaya St.,
Volgograd 400087, Russia; sokolova210795@gmail.com (E.V.S.); aspasov@mail.ru (A.A.S.)
*Correspondence: denis.a.babkov@gmail.com (D.A.B.); obf@anrb.ru (O.B.K.)
Abstract:
Using oleanolic acid as a starting compound, a series of new oleanane-type triterpenic
derivatives were synthesized via O-acylation (with nicotinic, isonicotinic, and methoxycinnamic acid
acyl chlorides), N-amidation (with cyclic- or polyamines), the Mannich reaction (with secondary
cyclic amines), and Claisen–Schmidt condensation (with aromatic aldehydes), and their potencies
as treatments for immunometabolic disorders were investigated. The compounds were evaluated
against
α
-glucosidase and PTP1B enzymes and LPS-stimulated murine macrophages. It was found
that the target compounds are highly effective
α
-glucosidase inhibitors but lack activity against
PTP1B. A leading compound, N-methylpiperazine methylated 2,3-indolo-oleanolic propargyl amide
15
, is also a micromolar inhibitor of NO synthesis in LPS-stimulated macrophages and suppresses
oxidative bursts in neutrophils with similar efficiency. These results, in addition to its ability to
stimulate glucose uptake in rat fibroblasts and improve maltose tolerance in rats, allow us to consider
compound
15
a promising prototype drug for the treatment of immunometabolic defects in type
2 diabetes.
Keywords:
triterpenoids; oleanolic acid; indole; Mannich reaction; type 2 diabetes;
α
-glucosidase
inhibiting activity; PTP1B
1. Introduction
Type 2 diabetes, marked by persistent hyperglycemia, is tied to obesity, insulin re-
sistance, and systemic inflammation (metaflammation) that damages beta cells in the
pancreas [
1
]. The disease contributes significantly to the prevalence of non-communicable
diseases globally. Managing it often involves inhibiting
α
-glucosidase, an enzyme that
helps convert complex carbohydrates into glucose. Existing pharmaceutical inhibitors,
including acarbose and miglitol, help lower post-meal glucose levels but have drawbacks.
They can be contraindicated for patients with gastrointestinal disorders and may not
effectively control blood sugar levels. Thus, the pursuit of new compounds to act as
α
-
glucosidase inhibitors for diseases like type 2 diabetes is critical [
2
]. They could potentially
enhance glycemic control, decrease long-term complications, and improve overall disease
management [3].
Currently, a large number of discoveries in the field of inhibitors are devoted to the syn-
thesis of N-heterocyclic compounds. Benzimidazoles, thiadiazoles, thiazole, and triazoles
are recommended as good inhibitors of
α
-glucosidase, acetylcholinesterase, and butyryl-
cholinesterase [
4
–
7
]. Among the various classes of compounds obtained from natural
resources, triterpenoids also show potent antidiabetic activity, including
α
-glucosidase-
inhibiting effects [
8
–
11
]. For example, oleanolic acid (OA) derivatives, such as oxime
esters, showed inhibitory activities against
α
-glucosidase and
α
-amylase [
12
], a series of
Appl. Sci. 2023,13, 9269. https://doi.org/10.3390/app13169269 https://www.mdpi.com/journal/applsci

Appl. Sci. 2023,13, 9269 2 of 16
benzylidene derivatives exhibited excellent to moderate inhibitory effects, and inhibition
kinetics results showed that the leading analogs were reversible and mixed-type inhibitors
of
α
-glucosidase and
α
-amylase [
13
,
14
]. OA indole derivatives with various electron-
withdrawing groups possessed anti-
α
-glucosidase activities and demonstrated the capacity
to form ligand-enzyme complexes with
α
-glucosidase enzymes [
15
]. However, in the field
of metabolic disorders, researchers have not achieved such progress as with CDDO (an
OA derivative), a potent anticancer agent which was evaluated in phase I clinical trials
for the treatment of advanced solid tumors and lymphoma via the activation of Nrf2 in
peripheral blood mononuclear cells and the inhibition of NF-
κ
B and cyclin D1 in tumor
biopsies [
16
]. All this proves that oleanolic acid is a promising scaffold for obtaining new
pharmacological agents, and the search for new inhibitors based on it remains an urgent
and priority task.
Keeping in mind the importance of these types of compounds, in this study, oleanolic
acid derivatives containing nicotinic, isonicotinic, methoxycinnamic, succinic, polyamines,
amides, arylidene, propargylaminoalkyl, and azepano fragments were synthesized, and
their potencies as antidiabetic agents were investigated. It was found that the major-
ity of the compounds exhibit significant
α
-glucosidase-inhibiting properties. The N-
methylpiperazine methylated 2,3-indolo-oleanolic propargyl amide with the most potential,
obtained via a one-pot Cu (I)-catalyzed Mannich reaction, was the most active compound
acting as a noncompetitive inhibitor, with a Ki (the dissociation constant of the enzyme-
inhibitor complex) value estimated at 3.01
µ
M. The leading compound demonstrated that
it can prevent inflammatory responses and the generation of ROS in immune cells and act
synergistically with acarbose to ameliorate hyperglycemia. This exploration can provide
alternative therapies while fostering a better understanding of immunometabolic disorders,
thereby paving the path for targeted and potent treatments.
2. Materials and Methods
2.1. Chemistry
2.1.1. General
The spectra were recorded at the Center for the Collective Use ‘Chemistry’ at the Ufa
Institute of Chemistry of the UFRC RAS and at the RCCU “Agidel” of the UFRC RAS.
1
H
and
13
C NMR spectra were recorded using a “Bruker Avance-III” (Bruker, Billerica, MA,
USA, 500 and 125.5 MHz, respectively,
δ
, ppm, Hz) in CDCl
3
, internal standard tetramethyl-
silane. Mass spectra were obtained using an LCMS-2010 EV liquid chromatograph–mass
spectrometer (Shimadzu, Kyoto, Japan). Melting points were detected on a microtable
«Rapido PHMK05» (Nagema, Dresden, Germany). Optical rotations were measured via a
Perkin-Elmer 241 MC polarimeter (PerkinElmer, Waltham, MA, USA) with a tube length of
1 dm. Thin-layer chromatography analyses were performed on Sorbfil plates (Sorbpolimer,
Krasnodar, Russia), using the solvent system chloroform/ethyl acetate in a ratio of 40:1.
Substances were detected via a 10% of sulfuric acid solution with subsequent heating at
100–120
◦
C for 2–3 min. All chemicals were of reagent grade (Sigma-Aldrich). Compounds
6, 7
[
17
],
8
[
18
],
9
[
19
],
10
[
20
],
11–13
,
15
and
16
[
21
];
18
[
22
],
19
[
23
],
20
,
24,
and
27
[
24
];
21
,
22
, and
25;
and
28
[
25
],
29,
and
30
[
22
] were prepared as described previously. All spectral
data are provided in the Supplementary Materials file.
2.1.2. General Procedure for the Synthesis of Compounds 4and 5
To a solution of compound
2
(1 mmol; 0.44 g) in dry pyridine (15 mL), 3-(3-methoxyphenyl)
acryloyl chloride (2 mmol; 0.39 g) or nicotinoyl chloride (2 mmol; 0.28 g) and DMAP (cat.) was
added. The reaction mixture was refluxed for 4 h, poured into H
2
O/H
+
, filtered, washed with
water until neutral, and dried. The residue was purified via Al
2
O
3
column chromatography
(eluent petroleum ether-ethyl acetate 20:1→5:1).

Appl. Sci. 2023,13, 9269 3 of 16
3,28-Di-O-[3-(3-methoxyphenyl)acrylate]-olean-12(13)-en 4
Beige solid; mp: 114
◦
C; yield 78%; [
α
]
D20
+17
◦
(c0.1, CHCl
3
).
1
H NMR (500 MHz,
CDCl
3
)
δ
7.64 (m, 2H, H3
00
, H8
00
), 7.62 (m, 2H, H3
0
, H8
0
), 7.48 (d, J= 8.7 Hz, 2H, H9
0
, H9
00
),
6.92 (d, J= 1.5 Hz, 2H, H5
0
, H5
00
), 6.86 (d, J= 1.7 Hz, 2H, H7
0
, H7
00
), 6.33 (d, J= 2.7 Hz,
1H, H2
00
), 6.29 (d, J= 2.5 Hz, 1H, H2
0
), 5.23 (s, 1H, H12), 4.64 (dd, J= 9.2 Hz, 1H, H3), 4.17
(d, J= 11.0 Hz, 1H, H28), 3.83 (2s, 6H), 3.80 (m, 1H, H28), 2.16–1.00 (m, 23H, CH, CH
2
),
1.18, 0.99, 0.98, 0.94, 0.91, 0.90, 0.88 (7s, 21H, 7CH
3
).
13C NMR (125.5 MHz, CDCl3
)
δ
167.50 (C1
0
),
167.20 (C1
00
), 161.32 (C6
00
), 161.27 (C6
0
), 144.14, 143.96, 143.67 (C13), 131.97, 129.71, 129.68,
127.30, 127.25, 122.86 (C12), 116.35, 115.87, 114.30, 113.47, 113.40, 80.74 (C3), 70.68 (C28),
55.37 (OCH
3
), 55.32 (C5), 47.55 (C18), 46.27 (C19), 42.60, 41.69, 39.83, 38.34, 37.98, 36.86
(C10), 36.08, 34.05, 33.18, 32.50, 31.55, 30.95 (C20), 28.10, 25.99, 25.65, 23.71, 23.60, 22.37,
18.25, 16.89, 16.82, 16.74, 15.60. MS (APCI) m/z 763.6 [M+H]+(calcd for C50 H66O6, 763.07).
3,28-Di-O-[isonicotinate]-olean-12(13)-en 5
Beige solid; mp: 123
◦
C; yield 72%; [
α
]
D20
+5
◦
(c0.1, CHCl
3
).
1
H NMR (500 MHz,
CDCl
3
)
δ
8.80–8.73 (m, 4H, H4
00
, H6
00
, H4
0
, H6
0
), 7.85–7.80 (m, 4H, H3
00
, H7
00
, H3
0
, H7
0
), 5.25
(s, 1H, H12), 4.79–4.72 (m, 1H, H3), 4.33 (d, J= 11.0 Hz, 1H, H28), 3.97 (d, J= 11.1 Hz, 1H,
H28), 1.02–2.18 (m, 23H, CH, CH
2
), 1.19, 1.01, 0.99, 0.93, 0.91 (7s, 21H, 7CH
3
).
13
C NMR
(125.5 MHz, CDCl
3
)
δ
165.06 (C1
00
), 164.75 (C1
0
), 150.67 (C6
00
, C4
00
), 150.56 (C6
0
), 150.51
(C4
0
), 143.37 (C13), 138.14 (C2
0
), 137.76 (C2
0
), 123.07 (C12), 122.86 (C3
00
, C7
00
, C3
0
, C7
0
), 82.72
(C3), 71.98 (C28), 55.29 (C5), 47.51 (C18), 46.15 (C19), 42.59, 41.69, 40.13, 39.83, 38.25, 38.10,
37.45, 36.85 (C10), 36.24, 34.15, 33.94, 33.13, 32.43, 31.62, 30.91 (C20), 28.20, 26.20, 26.02,
25.62, 23.57, 23.52, 22.45, 21.50, 18.20, 16.94, 16.73, 15.57. MS (APCI) m/z 653.5 [M+ H]
+
(calcd for C42H56 N2O4, 652.92).
2.1.3. Synthesis of Compound 17
N-Methylpiperazine (1.6 mmol; 0.16 mL), paraformaldehyde (0.3 g), NaOAc (5 mmol;
0.41 g), and CuI (0.05 mmol; 9.5 mg) were added to a solution of compound
11
(1 mmol;
0.56 g) in dry dioxane (10 mL). The reaction mixture was stirred under argon for 10 h at
60
◦
C. After the reaction, the mixture was diluted with water and extracted with CHCl
3
(3
×
20 mL). The combined organic layer was washed with water (3
×
50 mL), dried over
CaCl2and evaporated under reduced pressure. The residue was purified by SiO2column
chromatography (eluent CHCl3–MeOH 100:0→90:10), obtaining compound 17.
[3,2b]Indolo-N-(4-(piperazin-1-yl)but-2-yn-1-yl)-olean-12(13)-en-28-amide 17
Yellow solid; mp: 139
°C
; yield 81%; [
α
]
D20 −
21
◦
(c0.1, CHCl
3
).
1
H NMR (500 MHz,
CDCl
3
)
δ
7.45–7.00 (m, 4H), 6.12 (br. s, NH), 5.50 (s, 1H, H12), 3.90 and 4.10 (d, 2H,
J= 17.2 Hz
, NHCH
2
), 3.30 (d, J= 9.1 Hz, NCH
2
), 2.80
−
2.50 (m, 8H, 4CH
2
), 2.30
−
1.30
(m, 23H, CH and CH
2
), 1.32, 1.30, 1.22, 1.20, 0.90, 0.89, 0.81 (7s, 21H, 7CH
3
).
13
C NMR
(
125.5 MHz
, CDCl
3
)
δ
178.03 (CONH), 144.62 (C13), 140.90, 136.16, 128.10, 123.30 (C12),
120.92, 118.81, 117.85, 110.47, 106.50, 81.10 (C
≡
C), 79.11 (C
≡
C), 53.07 (2C), 51.32 (2C), 46.91,
46.66, 46.39, 46.24, 42.20, 40.94, 39.50, 38.01, 37.45, 36.82, 34.11, 34.01, 33.01, 32.26, 31.78,
30.97, 30.76, 29.63, 27.32, 25.60, 23.93, 23.62, 23.31, 19.29, 16.63, 15.67. MS (APCI) m/z 663.5
[M+ H]+(calcd for C44H62 N4O, 663.01).
2.1.4. General Procedure for the Synthesis of Compounds 14 and 26
To a solution of compound 3 (1 mmol; 0.53 g), oleanonic acid (
1 mmol
; 0.45 g), or
21
(
1 mmol
; 0.54 g) in dry CH
2
Cl
2
(15 mL), (COCl)
2
(0.08 mL, 1 mmol), and Et
3
N (2 drops)
were added. The reaction mixture was stirred at room temperature for 2 h, then CH
2
Cl
2
was evaporated to give an intermediate acyl chloride. This residue was dissolved in
CH
2
Cl
2
(5 mL) and a CH
2
Cl
2
solution of piperazine (1.3 mmol; 0.11 g), 2-aminopyridine
(1.3 mmol; 0.12 g) or ammonia solution (1.3 mmol; 0.02 mL), and Et
3
N (1 mmol; 0.14 mL)
was added in each reaction. The reaction mixture was refluxed for 2 h, the organic layer
was diluted with cold water (20 mL) and separated. The aqueous layer was extracted

Appl. Sci. 2023,13, 9269 4 of 16
with CH
2
Cl
2
(
2×15 mL
), the combined extracts were washed with 5% HCl (3
×
15 mL),
H
2
O (
2×15 mL
), dried over CaCl
2
, and the solvent was evaporated in a water jet vacuum.
Purification of the crude product was performed using column chromatography on Al
2
O
3
by elution with a mixture of petroleum ether–chloroform (1:0 →1:1).
[3,2b]Indolo-N-piperazin-olean-12(13)-en-28-amide 14
Yellow solid; mp: 117
◦
C; yield 85%; [
α
]
D20
+4
◦
(c0.1, CHCl
3
).
1
H NMR (500 MHz,
CDCl
3
)
δ
7.45–7.00 (m, 4H), 5.38 (s, 1H, H12), 3.15–2.70 (m, 8H, 4CH
2
), 3.10
−
1.20 (m, 22H,
CH and CH
2
), 1.30, 1.21, 1.19, 0.95, 0.93, 0.91, 0.81 (7s, 21H, 7CH
3
).
13
C NMR (125.5 MHz,
CDCl
3
)
δ
175.34 (C28), 144.17 (C13), 140.91 (C3), 136.12 (C6
0
), 128.22 (C1
0
), 122.09 (C12),
120.89 (C4
0
), 118.82 (C3
0
), 117.98 (C2
0
), 110.36 (C5
0
), 106.81 (C2), 53.27 (2C), 51.63 (2C),
47.59, 46.47, 46.32, 44.60, 43.70, 42.05, 39.26, 38.15, 36.73, 34.01, 33.04, 32.36, 31.01, 30.41,
29.99, 27.99, 25.82, 24.03, 23.44, 23.32, 22.83, 19.31, 16.74, 16.68, 15.59. MS (APCI) m/z 596.5
[M+ H]+(calcd for C40H57 N3O, 595.92).
2-[(E)-pyridine-4-ylmethylene]-3-oxo-olean-12(13)-en-28-carboxamide 26
White solid; mp: 166
◦
C; yield 72%; [
α
]
D20
+49
◦
(c0.1, CHCl
3
).
1
H NMR (500 MHz,
CDCl
3
)
δ
8.71–8.65 (d, J= 6 Hz, 2H), 7.40–7.30 (d, J= 6.2 Hz, 2H), 6.95 (s, 1H), 5.85 (br.s,
NH), 5.40 (d, J= 3.4 Hz, 1H, H12), 2.95–1.25 (m, 22H, CH, CH
2
), 1.24, 1.18, 1.15, 0.90, 0.95,
0.88, 0.87 (7s, 21H, 7CH
3
).
13
C NMR (125.5 MHz, CDCl
3
)
δ
207.09 (C3), 181.01 (C28), 147.92,
145.54, 145.04 (C13), 142.22, 139.08, 133.21, 126.21, 124.69, 122.19 (C12), 53.00, 46.62, 46.49,
45.42, 44.03, 42.69, 42.37, 39.36, 39.13, 36.35, 34.07, 32.97, 32.51, 31.56, 30.73, 29.50, 27.28,
25.72, 25.49, 23.50, 22.68, 20.24, 19.05, 16.59, 15.39. MS (APCI) m/z 543.5 [M+ H]
+
(calcd for
C36H50 N2O2, 542.81).
2.1.5. Synthesis of Compound 23
4-Pyridinecarboxyaldehyde (1.3 mmol; 0.12 mL) and 40% KOH in ethanol (2.5 mL)
were added to a solution of compound
19
(1 mmol; 0.53 g) in ethanol (5 mL) under stirring
and cooling (from
−
5 to 10
◦
C). The mixture was stirred for 24 h at room temperature, pH
was adjusted to neutral values with 5% HCl solution, and the mixture was poured into
cold water (50 mL). The residue was filtered, washed with water and dried, then purified
by column chromatography on Al2O3using petroleum ether-CHCl3 (1:1 to 1:3) as eluent.
N-(pyridin-2-yl)-2-[(E)-pyridine-3-ylmethylene]-3-oxo-olean-12(13)-en-28 carboxamide 23
Beige solid; mp: 154–156
◦
C; yield 71%; [
α
]
D20
+23
◦
(c0.1, CHCl
3
).
1
H NMR (500 MHz,
CDCl
3
)
δ
8.63 (d, J= 4.8 Hz, 2H, H4
00
–H6
00
), 8.49 (d, J= 4.8 Hz, 2H, H3
00
- H7
00
), 8.30 (s, 1H,
NH), 8.20–8.25 (m, 2H, H3
0
–H6
0
), 7.65 (m, 1H, H4
0
), 7.25 (s, 1H, H1
00
), 6.99 (m, 1H, H5
0
),
5.58 (s, J= 3.21 Hz, 1H, H12), 2.93 (d, J= 15.5 Hz, 1H, H1), 2.82 (d, J= 10.4 Hz, 1H, H18),
2.28 (d, J= 15.5 Hz, 1H, H1), 1.00–2.30 (m, 18H, CH, CH
2
), 1.23, 1.12, 0.93, 0.92, 0.82, 0.91,
0.69 (7s, 21H, 7CH
3
).
13
C NMR (125.5 MHz, CDCl
3
)
δ
207.41 (C3), 176.75 (C28), 151.43 (C1
0
),
149.82 (C4
00
, C6
00
), 148.69 (C2), 147.49 (C3
0
), 144.15 (C13), 143.50 (C2
00
), 138.48 (C5
0
), 137.96
(C1
00
), 124.11 (C3
00
, C7
00
), 122.89 (C12), 119.61 (C4
0
), 113.97 (C6
0
), 52.98 (C5), 47.40, 46.53,
45.38, 45.31, 43.96, 42.20 (C4), 42.10, 39.27, 36.31, 34.09, 32.99, 32.52, 31.48, 30.74, 29.48, 27.39,
25.73, 24.03, 23.78, 23.57, 22.02, 20.19, 16.09, 15.36. MS (APCI) m/z 620.5 [M+H]
+
(calcd for
C41H53 N3O2, 619.89).
2.2. Biological Evaluation
Biological Assays
The experimental procedures for
α
-Glucosidase assay, PTP1B inhibition assay, cellular
assay, animal assay, and statistical analysis are included in Supplementary Materials Data:
S2 Biological evaluation.

Appl. Sci. 2023,13, 9269 5 of 16
3. Results and Discussion
3.1. Chemistry
Our previous studies confirm that compounds containing such groups as isonico-
tinic, methoxycinnamic, succinic, polyamine, arylidene, amide, propargylaminoalkyl, and
azepane display cytotoxic and antimicrobial activity [
25
,
26
]. Also, we have shown that
among the different types of triterpenoids [
27
–
29
], 2,3-indolo-betulinic acid glycine and
L-phenylalanine amides were discovered as lead compounds with IC
50
values of 0.04
and 0.05
µ
M, being 3784- and 4730-fold more active than acarbose [
30
]. Triterpene type
indoles were highlighted as excellent pharmacophore platforms for the development of
new anti-diabetes drugs [
31
]. We have shown that lupane-type C2-furfurylidene, m-iodo-
benzylidene, and 3-pyridinylidene derivatives exhibited significantly better activity (up to
700 times higher) than acarbose [
32
], as well as the chalcone derivatives of platanic acid
(20-oxo-lupanes) with furfurylidene and trifluoromethylbenzylidene fragments [33].
Thus, to study the inhibitory activity of
α
-glucosidase, a series of oleanolic acid
derivatives with these pharmacophore groups was obtained.
Triterpenic platforms such as oleanolic acid 1, erythrodiol (ED) 2, 2,3-indolo- and C2-
benzylidene derivatives of 3-oxo-OA were successfully used for the synthesis of the desired
compounds
4–18
and
19–28
(Schemes 1–3). Scheme 1outlines the synthesis of compounds
4–10
by O-acylation and N-amidation of hydroxy and carboxy groups at C3 and C28
positions of OA
1
and ED
2
. The reaction of nicotinic, isonicotinic, and methoxycinnamic
acid acyl chlorides or succinic anhydride with
1
or
2
led to mono- and bis-esters
4–8
.
Amides
9
and
10
were obtained from 3-acetoxyoleanolic acid by the reaction of
1
with
spermidine or diethyltriamine by the acyl chloride method in the presence of Et3N.
Appl. Sci. 2023, 13, x FOR PEER REVIEW 5 of 17
among the different types of triterpenoids [27–29], 2,3-indolo-betulinic acid glycine and
L-phenylalanine amides were discovered as lead compounds with IC50 values of 0.04 and
0.05 µM, being 3784- and 4730-fold more active than acarbose [30]. Triterpene type indoles
were highlighted as excellent pharmacophore platforms for the development of new anti-
diabetes drugs [31]. We have shown that lupane-type C2-furfurylidene, m-iodo-benzyli-
dene, and 3-pyridinylidene derivatives exhibited significantly beer activity (up to 700
times higher) than acarbose [32], as well as the chalcone derivatives of platanic acid (20-
oxo-lupanes) with furfurylidene and trifluoromethylbenzylidene fragments [33].
Thus, to study the inhibitory activity of α-glucosidase, a series of oleanolic acid de-
rivatives with these pharmacophore groups was obtained.
Triterpenic platforms such as oleanolic acid 1, erythrodiol (ED) 2, 2,3-indolo- and C2-
benzylidene derivatives of 3-oxo-OA were successfully used for the synthesis of the de-
sired compounds 4–18 and 19–28 (Schemes 1–3). Scheme 1 outlines the synthesis of com-
pounds 4–10 by O-acylation and N-amidation of hydroxy and carboxy groups at C3 and
C28 positions of OA 1 and ED 2. The reaction of nicotinic, isonicotinic, and methox-
ycinnamic acid acyl chlorides or succinic anhydride with 1 or 2 led to mono- and bis-esters
4–8. Amides 9 and 10 were obtained from 3-acetoxyoleanolic acid by the reaction of 1 with
spermidine or diethyltriamine by the acyl chloride method in the presence of Et3N.
Scheme 1. Synthesis of OA and ED derivatives 4–10. Reagents and conditions: (i) LiAlH4, THF, re-
flux; (ii) 3-(3-methoxyphenyl)acryloyl or nicotinoyl chlorides, DMAP, Py, reflux; (iii) succinic anhy-
dride, Py, reflux; (iv) 1. Ac2O, Py, reflux; 2. (COCl)2, CH2Cl2, 25 °C; 3. spermidine or diethyltriamine,
Et3N, CH2Cl2, 25 °C.
Compounds 12–18 were prepared from 2,3-indolo-oleanolic acid 3 [15] (Scheme 2).
First, the coupling of 3 with secondary amines by the acyl chloride method led to amides
12–14. The Mannich reaction of alkynylamide 11 with amines in the presence of a catalytic
amount of copper iodide and sodium acetate produced propargylaminoalkyl derivatives
15–17.
Scheme 1.
Synthesis of OA and ED derivatives
4–10
. Reagents and conditions: (i) LiAlH
4
, THF, reflux;
(ii) 3-(3-methoxyphenyl)acryloyl or nicotinoyl chlorides, DMAP, Py, reflux; (iii) succinic anhydride,
Py, reflux; (iv) 1. Ac
2
O, Py, reflux; 2. (COCl)
2
, CH
2
Cl
2
, 25
◦
C; 3. spermidine or diethyltriamine, Et
3
N,
CH2Cl2, 25 ◦C.

Appl. Sci. 2023,13, 9269 6 of 16
Appl. Sci. 2023, 13, x FOR PEER REVIEW 6 of 17
Scheme 2. Synthesis of 2,3-indolo-oleanolic acid derivatives 12–18. Reagents and conditions: (i) 1.
(COCl)2, CH2Cl2, 25 °C; 2. corresponding amine, Et3N, CH2Cl2; 25 °C; (ii) corresponding amine, PFA,
NaOAc, CuI, 1,4-dioxane, 60 °C; (iii) LiAlH4, THF, reflux.
Scheme 3 describes the Claisen–Schmidt condensation of 3-oxo-OA amide 19 and 20
derivatives with 2- or 4-pyridinecarboxaldehyde or furfural to afford C2-substituted com-
pounds 23–28. Additionally, two other A-ring modified azepano- 29 and seco- 30 deriva-
tives were synthesized [22].
Scheme 2.
Synthesis of 2,3-indolo-oleanolic acid derivatives
12–18
. Reagents and conditions:
(i) 1. (COCl)2
, CH
2
Cl
2
, 25
◦
C; 2. corresponding amine, Et
3
N, CH
2
Cl
2
; 25
◦
C; (ii) corresponding
amine, PFA, NaOAc, CuI, 1,4-dioxane, 60 ◦C; (iii) LiAlH4, THF, reflux.
Appl. Sci. 2023, 13, x FOR PEER REVIEW 6 of 17
Scheme 2. Synthesis of 2,3-indolo-oleanolic acid derivatives 12–18. Reagents and conditions: (i) 1.
(COCl)2, CH2Cl2, 25 °C; 2. corresponding amine, Et3N, CH2Cl2; 25 °C; (ii) corresponding amine, PFA,
NaOAc, CuI, 1,4-dioxane, 60 °C; (iii) LiAlH4, THF, reflux.
Scheme 3 describes the Claisen–Schmidt condensation of 3-oxo-OA amide 19 and 20
derivatives with 2- or 4-pyridinecarboxaldehyde or furfural to afford C2-substituted com-
pounds 23–28. Additionally, two other A-ring modified azepano- 29 and seco- 30 deriva-
tives were synthesized [22].
Scheme 3.
Synthesis of A-ring modified OA derivatives
19–30
. Reagents and conditions:
(i) 1. (COCl)2
, CH
2
Cl
2
, 25
◦
C; 2. Corresponding amine, Et
3
N, CH
2
Cl
2
, 25
◦
C; (ii) corresponding
aldehyde, 40% KOH in EtOH, EtOH; (iii) 1. NH
2
OH
·
HCl, NaOAc, EtOH, reflux; 2. SOCl
2
, 1,4-
dioxane; 3. LiAlH4THF, reflux.

Appl. Sci. 2023,13, 9269 7 of 16
Compounds
12–18
were prepared from 2,3-indolo-oleanolic acid
3
[
15
] (Scheme 2). First,
the coupling of
3
with secondary amines by the acyl chloride method led to amides
12–14
.
The Mannich reaction of alkynylamide
11
with amines in the presence of a catalytic amount
of copper iodide and sodium acetate produced propargylaminoalkyl derivatives 15–17.
Scheme 3describes the Claisen–Schmidt condensation of 3-oxo-OA amide
19
and
20
derivatives with 2- or 4-pyridinecarboxaldehyde or furfural to afford C2-substituted
compounds
23–28
. Additionally, two other A-ring modified azepano-
29
and seco-
30
derivatives were synthesized [22].
3.2. Biological Evaluation
3.2.1. Enzymatic Screening
Based on our previous results [
30
], the synthesized compounds were evaluated as
α
-glucosidase inhibitors. Screening in 100
µ
M concentration was performed, followed by
IC
50
evaluation for active compounds. As Table 1shows, the majority of oleanolic acid
derivatives proved to be effective
α
-glucosidase inhibitors, significantly exceeding the
activity of acarbose.
Table 1. Biological activity of compounds 4–30.
Cmpd Glucosidase IC50 ±SD, µM % NO (10 µM) % MTT Viability
(10 µM)
MTT
CC50 (µM)
NO
IC50 (µM)
42.4 ±0.47 insoluble insoluble
54.56 ±1.1 insoluble insoluble
6752.6 ±536.5 insoluble insoluble
7>600 66.83 43.79
86.5 ±4.4 −51.7 4.74
94.5 ±0.9 105.42 116.07
10 >600 −30.35 1.88
11 139.1 ±73.82 34.22 103.18 >100 >100
12 4.71 ±1.17 98.19 90.61
13 170.2 ±36.04 89.13 100.77
14 38.3 ±6.9 51.97 16.56
15 3.01 ±0.53 26.11 18.24 4.66 4.83
16 13.91 ±3.08 insoluble insoluble
17 223.5 ±44.19 insoluble insoluble
18 12.37 ±3.34 33.37 72.59 >100
19 14.2 ±6.71 insoluble insoluble
20 18.88 ±7.35 59.67 140.65 39.11
21 5.56 ±1.88 75.26 94.15
22 inactive 42.56 111.77 >100
23 6.6 ±1.12 −4.33 6.17
24 inactive 7.69 194.19 >100 7.89
25 79.35 ±14.04 54.7 98.83 38.17
26 38.84 ±6.63 insoluble insoluble
27 >600 −6.13 124.59 35.2 10.71
28 inactive 77.79 82.68
29 inactive −133.47 22.47
30 20.5 ±1−66.87 2.38
Acarbose 436 — — — —
Dexamethasone
— 2.01 98.77 >100 0.003
The structure–activity relationship of compounds
4
–
30
was analyzed according to the
experimental data in Table 1. One can see that the enzyme inhibition was increased when
the 3,28-dihydroxy-groups of erythrodiol were esterified with m-methoxy-cinnamic acid
(compound
4
) and isonicotinic acid (compound
5
). Compounds
4
and
5
exhibited much bet-
ter potent inhibitory activity with IC
50
values of 2.40
±
0.47
µ
M,
4.56 ±1.1 µM
, respectively,
which were about 96- to 182-fold higher than that of acarbose (IC
50 436 µM
). Compound
6
with 3,28-bisnicotinic moieties was not active (IC
50
752.6
µ
M). The replacement of C28

Appl. Sci. 2023,13, 9269 8 of 16
to a free carboxy group in the case of compound
7
was not successful (
IC50 >600 µM
). OA
succinate
8
is more active than
7
with IC
50
value 6.50
±
0.47
µ
M being 98-fold more effi-
cient than the acarbose. 3
β
-Acetoxy-OA spermidine amide
9
exhibited significantly better
activity (IC
50
4.50
±
0.9
µ
M) than another long-chain polyamine amide
10
(
IC50 > 600 µM
).
Modification of 2,3-indolo-OA propargyl amide
11
(IC
50
139.1
±
73.82
µ
M) to Mannich
bases with N-methylpiperazine-
15
and morpholine-
16
moieties led to an increase in
activity with IC
50
values of 3.01
±
0.53
µ
M and 13.91
±
3.08
µ
M, respectively, that were
about 31- to 145-fold higher than that of acarbose. At the same time, compound
17
with
piperazine fragment showed only a weak activity. When the heterocycles, such as N-
methylpiperazine and morpholine, were conjugated directly with an oleanane core via
amide function at C28 (compounds
12
and
13
), the inhibitory activity against
α
-glucosidase
decreased. Meanwhile, piperazine amide
14
exhibited higher activity (IC
50
38.3
±
6.9
µ
M)
than the propargyl containing analogue
17
. The derivatives of erythrodiol with indole
18
and seco-amine
30
fragments showed potent activity with IC
50
values of 12.37
±
3.34
µ
M
and 20.5 ±1µM, respectively, whereas compound 29 with A-azepano-cycle was inactive.
Among the series of C2-derivatives
21
-
28
obtained using Claisen–Schmidt condensa-
tion, 4-pyridinylideno-OA
21
demonstrated an activity with an IC
50
value of
5.56 ±1.88 µM
,
being 78-fold more active than acarbose. The enzyme inhibition activit
y
decreased when
the carboxyl group of the compound
21
was amidated using pyridine-2-amine up to
23
(IC
50
6.6
±
1.12
µ
M), N-methyl-piperazine up to
25
(IC
50
79.35
±
14.04
µ
M), or in the case
of carboxamide derivative
26
(IC
50
38.84
±
6.63
µ
M). The introduction of the furfurylidene
fragment (compound
22)
, as well as the modification of the carboxyl group to N-ethyl-
27
or N-methyl-piperazine amide
28
, led to a negative effect on inhibitory activity. Pyridine
19
or N-ethyl-piperazine
20
amides exhibited high activity with an IC
50
of
14.2 ±6.71 µM
and
18.88
±
7.35
µ
M. Modification of
20
by introducing 2-pyridinylidene-
24
or furfurylidene
27
fragments led to the loss of activity, while the presence of isomeric 4-pyridinylideno
fragments increased the activity of 23 (IC50 6.6 ±1.12 µM) compared to 19 almost twice.
Several studies identified triterpenoid inhibitors of PTP1B, a valuable antidiabetic
target [
34
–
36
]. We have also screened compounds against this enzyme, but no activity was
observed up to 100
µ
M (). This may be attributed to the lack of a 3-hydroxy group, which
appears to be a requisite for PTP1B inhibition [37].
3.2.2. Influence on LPS-Stimulated Macrophages
Considering the pivotal role of immunometabolic disorders in type 2 diabetes, target
compounds were also profiled using a phenotypic screening approach [
38
]. Primary
peritoneal murine macrophages were stimulated with LPS to induce a pro-inflammatory
state. Target compounds were tested for the suppression of LPS-induced nitric oxide
(NO) synthesis at a screening concentration of 10
µ
M. According to the MTT test, seven
compounds (compounds
11, 18, 20, 22, 24, 25,
and
27
) inhibit the formation of NO by
LPS-activated peritoneal macrophages in mice. Of these, compound
24
with an IC
50
value
of 7.8
µ
M was the most active, but this compound has no activity against a-glucosidase.
Compound
20
is also non-cytotoxic, suppresses the synthesis of NO with an IC
50
39.11
µ
M,
and inhibits a-glucosidase with an IC50 of 18.88 µM.
Compound
15
, the most potent glucosidase inhibitor, also actively suppressed NO
synthesis (IC
50
4.8
µ
M), but according to the MTT test, it also reduced cell metabolic activity.
Final conclusions about cytotoxicity can be made after the involvement of additional
research methods. The MTT test can both overestimate and underestimate the actual
cell viability. In particular, such artifacts have been described for triterpene acids [
39
–
41
].
In general, 2,3-indolo-oleanolic acid derivatives appear to be more active than A-ring
modified oleanolic acid derivatives. The anti-inflammatory activity was improved upon
the introduction of the N-alkyl-piperazine moiety at C28 carboxyl, as exemplified by
compounds 15 and 24.
We observed a lack of correlation between
α
-glucosidase and NO inhibition. Similar
results were reported for betulinic acid derivatives [
42
]. Compound
15
appeared to be

Appl. Sci. 2023,13, 9269 9 of 16
dual active and the most potent one, hence, it was nominated as a lead for further studies.
There are a considerable number of studies on the role of intracellular glucosidase enzymes
in pancreatic islet metabolism and macrophage immune response [
43
,
44
], also see [
45
],
however the precise mechanistic basis of intracellular endoplasmic reticulum glucosidase
involvement in NO synthesis remains to be elucidated. Also, multiple other protein targets
can be engaged by triterpenoids, and the factor of the various cellular permeabilities of the
studied compounds cannot be ruled out.
3.2.3. Mechanism of α-Glucosidase Inhibition by Compound 15
The mechanism of the inhibition of
α
-1,4-glucosidase of S. cerevisiae by lead com-
pound
15
was elucidated using a kinetic experiment. Results show that
15
behaves as an
uncompetitive (mixed-type) inhibitor. The linear correlation of the reaction slope with
the concentration of the inhibitor suggests the presence of one allosteric binding site. The
inhibition constant Kiwas estimated at 3.011 µM (Figure 1).
Appl. Sci. 2023, 13, x FOR PEER REVIEW 9 of 17
introduction of the N-alkyl-piperazine moiety at C28 carboxyl, as exemplified by com-
pounds 15 and 24.
We observed a lack of correlation between α-glucosidase and NO inhibition. Similar
results were reported for betulinic acid derivatives [42]. Compound 15 appeared to be
dual active and the most potent one, hence, it was nominated as a lead for further studies.
There are a considerable number of studies on the role of intracellular glucosidase en-
zymes in pancreatic islet metabolism and macrophage immune response [43,44], also see
[45], however the precise mechanistic basis of intracellular endoplasmic reticulum gluco-
sidase involvement in NO synthesis remains to be elucidated. Also, multiple other protein
targets can be engaged by triterpenoids, and the factor of the various cellular permeabili-
ties of the studied compounds cannot be ruled out.
3.2.3. Mechanism of α-Glucosidase Inhibition by Compound 15
The mechanism of the inhibition of α-1,4-glucosidase of S. cerevisiae by lead com-
pound 15 was elucidated using a kinetic experiment. Results show that 15 behaves as an
uncompetitive (mixed-type) inhibitor. The linear correlation of the reaction slope with the
concentration of the inhibitor suggests the presence of one allosteric binding site. The in-
hibition constant K
i
was estimated at 3.011 µM (Figure 1).
Figure 1. Hanes–Woolf plot of kinetic experiment on the inhibition of S. cerevisiae α-glucosidase by
compound 15. Analysis conditions: [E] 0.25 µM, [S] 1000.0 µM, K
m
0.1166 µM.
The studied compounds are inactive against rat intestinal maltase (IC
50
for acarbose
is 7 µM), i.e., they will not affect the absorption of carbohydrates from the intestine.
3.2.4. Cytotoxic Evaluation of Compound 15 towards Macrophages
To determine whether MTT-based cytotoxicity data for compound 15 is reliable, ad-
ditional experiments were performed with primary peritoneal macrophages. Neutral red
(NR) is a cationic dye whose uptake is known to reflect ATP-dependent lysosomal func-
tion. The test with neutral red revealed a similar concentration response as was observed
in the NO and MTT assays (Figure 2). MTT and neutral red uptake assays both show via-
bility with CC
50
values around 5 µM.
To confirm these findings, we have also assessed the morphological alterations of
macrophages treated with 15 or dexamethasone, since this is considered the most reliable
method for evaluating cytotoxicity [46]. The final concentration of compound 15 was set
at 10 µM, which resulted in a complete loss of apparent viability according to MTT and
neutral red uptake, and also a complete suppression of NO synthesis. In the LPS-treated
control sample, the cells acquire an elongated fusiform stellate shape, the cells are spread
out and are tightly aached to the culture plate (Figure 3). Surprisingly, both compound
15 and dexamethasone did not change the rounded shape of the cells to stellate under LPS
Figure 1.
Hanes–Woolf plot of kinetic experiment on the inhibition of S. cerevisiae
α
-glucosidase by
compound 15. Analysis conditions: [E] 0.25 µM, [S] 1000.0 µM, Km0.1166 µM.
The studied compounds are inactive against rat intestinal maltase (IC
50
for acarbose is
7µM), i.e., they will not affect the absorption of carbohydrates from the intestine.
3.2.4. Cytotoxic Evaluation of Compound 15 towards Macrophages
To determine whether MTT-based cytotoxicity data for compound
15
is reliable, ad-
ditional experiments were performed with primary peritoneal macrophages. Neutral red
(NR) is a cationic dye whose uptake is known to reflect ATP-dependent lysosomal function.
The test with neutral red revealed a similar concentration response as was observed in the
NO and MTT assays (Figure 2). MTT and neutral red uptake assays both show viability
with CC50 values around 5 µM.
To confirm these findings, we have also assessed the morphological alterations of
macrophages treated with
15
or dexamethasone, since this is considered the most reliable
method for evaluating cytotoxicity [
46
]. The final concentration of compound
15
was set
at 10
µ
M, which resulted in a complete loss of apparent viability according to MTT and
neutral red uptake, and also a complete suppression of NO synthesis. In the LPS-treated
control sample, the cells acquire an elongated fusiform stellate shape, the cells are spread
out and are tightly attached to the culture plate (Figure 3). Surprisingly, both compound
15
and dexamethasone did not change the rounded shape of the cells to stellate under LPS
stimulation, which may reflect a lack of M1-polarization and explain the suppression of
NO synthesis. However, there are cells with nuclei on the periphery, loss of cytoplasm,
and vacuolization of the membrane, this may indicate the presence of the first phase of
apoptosis. It should be noted that similar features are evident in some of the intact and
dexamethasone-treated cells as well. Cell morphology in the presence of 10
µ
M
15
remains

Appl. Sci. 2023,13, 9269 10 of 16
normal and corresponds to the morphological features of intact cells. Final conclusions
about cytotoxicity can be made after the involvement of additional research methods.
Appl. Sci. 2023, 13, x FOR PEER REVIEW 10 of 17
stimulation, which may reflect a lack of M1-polarization and explain the suppression of
NO synthesis. However, there are cells with nuclei on the periphery, loss of cytoplasm,
and vacuolization of the membrane, this may indicate the presence of the first phase of
apoptosis. It should be noted that similar features are evident in some of the intact and
dexamethasone-treated cells as well. Cell morphology in the presence of 10 µM 15 remains
normal and corresponds to the morphological features of intact cells. Final conclusions
about cytotoxicity can be made after the involvement of additional research methods.
Figure 2. Compound 15 inhibit NO synthesis and neutral red (NR) viability of LPS-stimulated per-
itioneal macrophages. Data are shown as mean ± SD (n = 3).
(a) (b)
(c) (d)
Figure 3. The effect of compound 15 and dexamethasone on the morphology of mouse peritoneal
macrophages. Azur-eosin staining according to Romanovsky; magnification 200× ((a)—Vehicle + 2%
DMSO; (b)—LPS + 2% DMSO; (c)—LPS + 10 µM 15; (d)—LPS + 10 µM Dexamethasone).
Figure 2.
Compound
15
inhibit NO synthesis and neutral red (NR) viability of LPS-stimulated
peritioneal macrophages. Data are shown as mean ±SD (n= 3).
Appl. Sci. 2023, 13, x FOR PEER REVIEW 10 of 17
stimulation, which may reflect a lack of M1-polarization and explain the suppression of
NO synthesis. However, there are cells with nuclei on the periphery, loss of cytoplasm,
and vacuolization of the membrane, this may indicate the presence of the first phase of
apoptosis. It should be noted that similar features are evident in some of the intact and
dexamethasone-treated cells as well. Cell morphology in the presence of 10 µM 15 remains
normal and corresponds to the morphological features of intact cells. Final conclusions
about cytotoxicity can be made after the involvement of additional research methods.
Figure 2. Compound 15 inhibit NO synthesis and neutral red (NR) viability of LPS-stimulated per-
itioneal macrophages. Data are shown as mean ± SD (n = 3).
(a) (b)
(c) (d)
Figure 3. The effect of compound 15 and dexamethasone on the morphology of mouse peritoneal
macrophages. Azur-eosin staining according to Romanovsky; magnification 200× ((a)—Vehicle + 2%
DMSO; (b)—LPS + 2% DMSO; (c)—LPS + 10 µM 15; (d)—LPS + 10 µM Dexamethasone).
Figure 3.
The effect of compound
15
and dexamethasone on the morphology of mouse peritoneal
macrophages. Azur-eosin staining according to Romanovsky; magnification 200
×
((
a
)—Vehicle + 2%
DMSO; (b)—LPS + 2% DMSO; (c)—LPS + 10 µM15; (d)—LPS + 10 µM Dexamethasone).
3.2.5. Oxidative Burst in Neutrophils
Encouraged by the anti-inflammatory activity of
15
along with the intact morphology
of macrophages, we tested the influence of compound
15
on the activation of murine
neutrophils as well. There are many triterpenoids known to affect the phenotype, immune,

Appl. Sci. 2023,13, 9269 11 of 16
and oxidative responses of neutrophils, e.g., celastrol [
47
] and glutinone [
48
]. Hence, it
was of interest to evaluate the influence of compound
15
on the generation of reactive
oxygen species in immune cells. Phagocytosis-induced neutrophil oxidative burst is a
convenient model to study this subject. Compound
15
dose-dependently suppressed the
zymosan-induced formation of reactive oxygen species in mouse neutrophils with an IC
50
of 10 µM. Reducing neutrophil oxidative response has implications for both phagocytosis
efficiency and overall oxidative stress in diabetic conditions (Figure 4).
Appl. Sci. 2023, 13, x FOR PEER REVIEW 11 of 17
3.2.5. Oxidative Burst in Neutrophils
Encouraged by the anti-inflammatory activity of 15 along with the intact morphology
of macrophages, we tested the influence of compound 15 on the activation of murine neu-
trophils as well. There are many triterpenoids known to affect the phenotype, immune,
and oxidative responses of neutrophils, e.g., celastrol [47] and glutinone [48]. Hence, it
was of interest to evaluate the influence of compound 15 on the generation of reactive
oxygen species in immune cells. Phagocytosis-induced neutrophil oxidative burst is a con-
venient model to study this subject. Compound 15 dose-dependently suppressed the zy-
mosan-induced formation of reactive oxygen species in mouse neutrophils with an IC
50
of
10 µM. Reducing neutrophil oxidative response has implications for both phagocytosis
efficiency and overall oxidative stress in diabetic conditions (Figure 4).
Figure 4. Compound 15 suppresses the formation of reactive oxygen species during zymosan-in-
duced phagocytosis in mouse neutrophils. CHL—chemiluminescence.
3.2.6. Free Radical Scavenging in Cell-Free System
To discriminate ROS inhibition elicited by lead compounds in cellulo from direct free
radical scavenging, we assessed the antiradical activity of lead compounds in a cell-free
system. Reactive oxygen species were generated with the hemoglobin-hydrogen peroxide
system and detected as luminol-mediated chemiluminescence. Compound 15 scavenges
reactive oxygen species in a cell-free model system, but in concentrations an order of mag-
nitude greater than the effective concentrations in the cellular model. Thus, the IC
50
for
direct antiradical activity is roughly 85.9 µM (see Supplementary Materials), while oxida-
tive burst in neutrophils is inhibited two-fold at 10 µM. This indicates a specific mecha-
nism of action (probably a protein target).
3.2.7. Glucose Uptake in Fibroblasts
To further assess the antidiabetic potential of the lead compound, a cellular model of
glucose uptake was employed. Rat neonatal myocardial fibroblasts were cultivated in
DMEM containing 25 mM glucose to mimic hyperglycemic conditions. Incubation of fi-
broblasts with 20 µM of 15 for 24 h led to a significant increase in glucose consumption
but, at the same time, to a concomitant drop in viability according to the MTT test (Figure
5).
Figure 4.
Compound
15
suppresses the formation of reactive oxygen species during zymosan-induced
phagocytosis in mouse neutrophils. CHL—chemiluminescence.
3.2.6. Free Radical Scavenging in Cell-Free System
To discriminate ROS inhibition elicited by lead compounds in cellulo from direct free
radical scavenging, we assessed the antiradical activity of lead compounds in a cell-free
system. Reactive oxygen species were generated with the hemoglobin-hydrogen peroxide
system and detected as luminol-mediated chemiluminescence. Compound
15
scavenges
reactive oxygen species in a cell-free model system, but in concentrations an order of
magnitude greater than the effective concentrations in the cellular model. Thus, the IC
50
for direct antiradical activity is roughly 85.9
µ
M (see Supplementary Materials), while
oxidative burst in neutrophils is inhibited two-fold at 10
µ
M. This indicates a specific
mechanism of action (probably a protein target).
3.2.7. Glucose Uptake in Fibroblasts
To further assess the antidiabetic potential of the lead compound, a cellular model
of glucose uptake was employed. Rat neonatal myocardial fibroblasts were cultivated
in DMEM containing 25 mM glucose to mimic hyperglycemic conditions. Incubation of
fibroblasts with 20
µ
M of
15
for 24 h led to a significant increase in glucose consumption but,
at the same time, to a concomitant drop in viability according to the MTT test (Figure 5).

Appl. Sci. 2023,13, 9269 12 of 16
Appl. Sci. 2023, 13, x FOR PEER REVIEW 12 of 17
Figure 5. The compound 15 at a concentration of 20 µM after 24 h of incubation suppresses the
viability of neonatal myocardial fibroblasts in rats according to the MTT test, but stimulates glucose
uptake (n = 3). Statistical significance according to the one-way ANOVA test with the Dunnet post-
test (**** p < 0.0001, n.s.—not significant).
3.2.8. Oral Maltose Tolerance In Vivo
Finally, the antidiabetic potential of compound 15 was evaluated in intact animals.
Considering the presumed mode of action, oral maltose tolerance in rats was used. The
data obtained are presented as blood glucose levels vs. time and as the areas under the
glucose time curves (Figure 6). Compound 15 at a dose of 5 mg/kg prevents hyperglyce-
mia in intact rats after a maltose load of 2 g/kg. Substance 15 at a dose of 5 mg/kg improved
maltose tolerance in rats in vivo, reducing the area under the curve relative to the control
by 27.3% and by 58.5% when combined with acarbose. Hence, compound 15 is orally ac-
tive and improves maltose tolerance synergistically with the reference α-glucosidase in-
hibitor acarbose.
Figure 5.
The compound
15
at a concentration of 20
µ
M after 24 h of incubation suppresses the
viability of neonatal myocardial fibroblasts in rats according to the MTT test, but stimulates glucose
uptake (n= 3). Statistical significance according to the one-way ANOVA test with the Dunnet post-test
(**** p< 0.0001, n.s.—not significant).
3.2.8. Oral Maltose Tolerance In Vivo
Finally, the antidiabetic potential of compound
15
was evaluated in intact animals.
Considering the presumed mode of action, oral maltose tolerance in rats was used. The data
obtained are presented as blood glucose levels vs. time and as the areas under the glucose
time curves (Figure 6). Compound
15
at a dose of 5 mg/kg prevents hyperglycemia in intact
rats after a maltose load of 2 g/kg. Substance
15
at a dose of 5 mg/kg improved maltose
tolerance in rats
in vivo
, reducing the area under the curve relative to the control by 27.3% and
by 58.5% when combined with acarbose. Hence, compound
15
is orally active and improves
maltose tolerance synergistically with the reference α-glucosidase inhibitor acarbose.
Appl. Sci. 2023, 13, x FOR PEER REVIEW 12 of 17
Figure 5. The compound 15 at a concentration of 20 µM after 24 h of incubation suppresses the
viability of neonatal myocardial fibroblasts in rats according to the MTT test, but stimulates glucose
uptake (n = 3). Statistical significance according to the one-way ANOVA test with the Dunnet post-
test (**** p < 0.0001, n.s.—not significant).
3.2.8. Oral Maltose Tolerance In Vivo
Finally, the antidiabetic potential of compound 15 was evaluated in intact animals.
Considering the presumed mode of action, oral maltose tolerance in rats was used. The
data obtained are presented as blood glucose levels vs. time and as the areas under the
glucose time curves (Figure 6). Compound 15 at a dose of 5 mg/kg prevents hyperglyce-
mia in intact rats after a maltose load of 2 g/kg. Substance 15 at a dose of 5 mg/kg improved
maltose tolerance in rats in vivo, reducing the area under the curve relative to the control
by 27.3% and by 58.5% when combined with acarbose. Hence, compound 15 is orally ac-
tive and improves maltose tolerance synergistically with the reference α-glucosidase in-
hibitor acarbose.
Figure 6.
Antihyperglycemic activity of
15
in rats after maltose challenge. Data shown as mean
±
SD.
Statistical significance vs. vehicle: two-way ANOVA with Tukey post-test: * p< 0.05, ** p< 0.01.

Appl. Sci. 2023,13, 9269 13 of 16
4. Conclusions
Triterpenoids of natural and semi-synthetic origin are structurally diverse compounds
enriched with biological activity. As a development of our previous works, here we report
the design, synthesis, and biological evaluation of novel oleanane-type triterpenic acid
derivatives against yeast
α
-glucosidase. We have found that the majority of compounds
exhibit significant inhibitory properties. Amide derivatives of 2,3-indolo-oleanolic acid
proved to be especially effective, steadily surpassing the activity of the reference drug,
acarbose. N-methylpiperazine methylated 2,3-indolo-oleanolic propargyl amide
15
was the
most active compound acting as a noncompetitive inhibitor, with K
i
estimated at 3.01
µ
M.
We have found that compound
15
also suppresses LPS-induced NO synthesis in the
low micromolar range, representing anti-inflammatory properties. Further cellular studies
revealed additional diabetes-related aspects of compound
15
modes of action. It inhibits the
production of reactive oxygen species during an oxidative burst in neutrophils with an IC
50
of 10
µ
M. In contrast, the direct radical scavenging activity of compound
15
in a cell-free
system is an order of magnitude lower, indicating that a cellular target mediates its action
on neutrophils. Rat fibroblasts treated with 20
µ
M of
15
demonstrate a two-fold increase
in glucose uptake. Hence, lead compound
15
might be able to alleviate oxidative stress,
inflammation, and hyperglycemia—key pathophysiological alterations of type
2 diabetes
(Figure 7).
Appl. Sci. 2023, 13, x FOR PEER REVIEW 14 of 17
Figure 7. Proposed mechanism of antidiabetic activity of compound 15.
Supplementary Materials: The following supporting information can be downloaded at:
www.mdpi.com/xxx/s1, Figure S1–S24: NMR, IR, and mass spectra of compounds 4, 5, 14, 17,
23, and 26, (references cited in Supplementary Materials are [49–58]).
Author Contributions: O.B.K. brought the idea, managed the chemical research and prepared the
manuscript; A.V.P. and E.F.K. conducted the chemical experiments and prepared the manuscript;
I.P.B. conducted the NMR experiments; D.A.B. and E.V.S. conducted the biological experiments and
prepared the manuscript; A.A.S. managed the biological research. All authors have read and agreed
to the published version of the manuscript.
Funding: This work was supported by Federal programs No. 1021062311392-9-1.4.1 and
1022040400061-8-1.4.1;1.4.3 (Russia).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Appari, M.; Channon, K.M.; McNeill, E. Metabolic Regulation of Adipose Tissue Macrophage Function in Obesity and Diabetes.
Antioxid. Redox Signal. 2018, 29, 297–312. https://doi.org/10.1089/ars.2017.7060.
2. Kashtoh, H.; Baek, K.-H. Recent Updates on Phytoconstituent Alpha-Glucosidase Inhibitors: An Approach towards the Treat-
ment of Type Two Diabetes. Plants 2022, 11, 2722. https://doi.org/10.3390/plants11202722.
Figure 7. Proposed mechanism of antidiabetic activity of compound 15.
The antidiabetic potential of
15
was confirmed
in vivo
using an oral maltose tolerance
test. The treatment of rats with 5 mg/kg of
15
prevents hyperglycemia after a maltose load
of 2 g/kg. Of note, we observed a synergy when the compound was co-administered with
5 mg/kg of acarbose. In conclusion, we have identified methylpiperazine methylated 2,3-

Appl. Sci. 2023,13, 9269 14 of 16
indolo-oleanolic propargyl amide
15
as a promising lead compound for the development
of agents against immunometabolic disorders. Future studies are warranted to assess its
safety and long-term efficacy in animal models of diabetes.
Supplementary Materials:
The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/app13169269/s1, Figure S1–S24: NMR, IR, and mass spectra of compounds
4,5,14,17,23, and 26, (references cited in Supplementary Materials are [49–58]).
Author Contributions:
O.B.K. brought the idea, managed the chemical research and prepared the
manuscript; A.V.P. and E.F.K. conducted the chemical experiments and prepared the manuscript;
I.P.B. conducted the NMR experiments; D.A.B. and E.V.S. conducted the biological experiments and
prepared the manuscript; A.A.S. managed the biological research. All authors have read and agreed
to the published version of the manuscript.
Funding:
This work was supported by Federal programs No. 1021062311392-9-1.4.1 and 1022040400061-8-
1.4.1;1.4.3 (Russia).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Appari, M.; Channon, K.M.; McNeill, E. Metabolic Regulation of Adipose Tissue Macrophage Function in Obesity and Diabetes.
Antioxid. Redox Signal. 2018,29, 297–312. [CrossRef]
2.
Kashtoh, H.; Baek, K.-H. Recent Updates on Phytoconstituent Alpha-Glucosidase Inhibitors: An Approach towards the Treatment
of Type Two Diabetes. Plants 2022,11, 2722. [CrossRef] [PubMed]
3.
Coleman, R.L.; Scott, C.A.B.; Lang, Z.; Bethel, M.A.; Tuomilehto, J.; Holman, R.R. Meta-Analysis of the Impact of Alpha-
Glucosidase Inhibitors on Incident Diabetes and Cardiovascular Outcomes. Cardiovasc. Diabetol.
2019
,18, 135. [CrossRef]
[PubMed]
4.
Khan, S.; Ullah, H.; Taha, M.; Rahim, F.; Sarfraz, M.; Iqbal, R.; Iqbal, N.; Hussain, R.; Ali Shah, S.A.; Ayub, K.; et al. Synthesis, DFT
studies, molecular docking and biological activity evaluation of thiazole-sulfonamide derivatives as potent alzheimer’s inhibitors.
Molecules 2023,28, 559. [CrossRef]
5.
Rahim, F.; Ullah, H.; Hussain, R.; Taha, M.; Khan, S.; Nawaz, M.; Nawaz, F.; Gilani, S.J.; Jumah, M.N.B. Thiadiazole based
triazole/hydrazone derivatives: Synthesis,
in vitro α
-glucosidase inhibitory activity and in silico molecular docking study. J. Mol.
Struct. 2023,1287, 135619. [CrossRef]
6.
Hayat, S.; Ullah, H.; Rahim, F.; Ullah, I.; Taha, M.; Iqbal, N.; Khan, F.; Khan, M.S.; Shah, S.A.A.; Wadood, A.; et al. Synthesis,
biological evaluation and molecular docking study of benzimidazole derivatives as
α
-glucosidase inhibitors and anti-diabetes
candidates. J. Mol. Struct. 2023,1276, 134774. [CrossRef]
7.
Nipun, T.S.; Khatib, A.; Ibrahim, Z.; Ahmed, Q.U.; Redzwan, I.E.; Saiman, M.Z.; Supandi, F.; Primaharinastiti, R.; El-Seedi, H.R.
Characterization of
α
-glucosidase inhibitors from Psychotria malayana jack leaves extract using lc-ms-based multivariate data
analysis and in-silico molecular docking. Molecules 2020,25, 5885. [CrossRef]
8.
Atta-ur-Rahman; Zareen, S.; Choudhary, M.I.; Akhtar, M.N.; Khan, S.N. alpha-Glucosidase inhibitory activity of triterpenoids
from Cichorium intybus.J. Nat. Prod. 2008,71, 903–910. [CrossRef]
9.
Ouyang, J.K.; Dong, L.M.; Xu, Q.L.; Wang, J.; Liu, S.B.; Qian, T.; Yuan, Y.F.; Tan, J.W. Triterpenoids with
α
-glucosidase inhibitory
activity and cytotoxic activity from the leaves of Akebia trifoliata. RSC Adv. 2018,8, 40483–40489. [CrossRef]
10.
Ding, H.; Hu, X.; Xu, X.; Zhang, G.; Gong, D. Inhibitory mechanism of two allosteric inhibitors, oleanolic acid and ursolic acid on
α-glucosidase. Int. J. Biol. Macromol. 2018,107 Pt B, 1844–1855. [CrossRef]
11.
Khan, A.; Khan, I.; Halim, S.A.; Rehman, N.U.; Karim, N.; Ahmad, W.; Khan, M.; Csuk, R.; Al-Harrasi, A. Anti-diabetic potential
of
β
-boswellic acid and 11-keto-
β
-boswellic acid: Mechanistic insights from computational and biochemical approaches. Biomed.
Pharmacother. 2022,147, 112669. [CrossRef] [PubMed]
12.
Deng, X.Y.; Ke, J.J.; Zheng, Y.Y.; Li, D.L.; Zhang, K.; Zheng, X.; Wu, J.Y.; Xiong, Z.; Wu, P.P.; Xu, X.T. Synthesis and bioactivities
evaluation of oleanolic acid oxime ester derivatives as
α
-glucosidase and
α
-amylase inhibitors. J. Enzym. Inhib. Med. Chem.
2022
,
37, 451–461. [CrossRef] [PubMed]
13.
Tang, C.; Zhu, L.; Chen, Y.; Qin, R.; Mei, Z.; Xu, J.; Yang, G. Synthesis and biological evaluation of oleanolic acid derivative–
chalcone conjugates as α-glucosidase inhibitors. RSC Adv. 2014,4, 10862–10874. [CrossRef]

Appl. Sci. 2023,13, 9269 15 of 16
14.
Ke, J.J.; Lin, J.; Zhang, X.; Wu, X.Z.; Zheng, Y.Y.; Hu, C.M.; Kang, Y.; Zhang, K.; Xiong, Z.; Ma, Z.Q. Synthesis of Benzylidene
Analogs of Oleanolic Acid as Potential
α
-Glucosidase and
α
-Amylase Inhibitors. Front. Chem.
2022
,10, 911232. [CrossRef]
[PubMed]
15.
Wu, P.; He, H.; Ma, H.; Tu, B.; Li, J.; Guo, S.; Chen, S.; Cao, N.; Zheng, W.; Tang, X.; et al. Oleanolic acid indole derivatives as novel
α-glucosidase inhibitors: Synthesis, biological evaluation, and mechanistic analysis. Bioorg Chem. 2021,107, 104580. [CrossRef]
16.
Hong, D.S.; Kurzrock, R.; Supko, J.G.; He, X.; Naing, A.; Wheler, J.; Lawrence, D.; Eder, J.P.; Meyer, C.J.; Ferguson, D.A.; et al. A
phase I first-in-human trial of bardoxolone methyl in patients with advanced solid tumors and lymphomas. Clin. Cancer Res.
2012,18, 3396–3406. [CrossRef]
17.
Kazakova, O.B.; Medvedeva, N.I.; Baikova, I.P.; Tolstikov, G.A.; Lopatina, T.V.; Yunusov, M.S.; Zaprutko, L. Synthesis of
triterpenoid acylates: Effective reproduction inhibitors of influenza A (H1N1) and papilloma viruses. Russ. J. Bioorg Chem.
2010
,
36, 771–778. [CrossRef]
18.
Reyes-Zurita, F.J.; Medina-O’Donnell, M.; Ferrer-Martin, R.M.; Rufino-Palomares, E.E.; Martin-Fonseca, S.; Rivas, F.; Martínez,
A.; García-Granados, A.; Pérez-Jiménez, A.; García-Salguero, L.; et al. The oleanolic acid derivative, 3-O-succinyl-28-O-benzyl
oleanolate, induces apoptosis in B16-F10 melanoma cells via the mitochondrial apoptotic pathway. RSC Adv.
2016
,6, 93590–93601.
[CrossRef]
19.
Spivak, A.Y.; Khalitova, R.R.; Nedopekina, D.A.; Gubaidullin, R.R. Antimicrobial properties of amine- and guanidine-
functionalized derivatives of betulinic, ursolic and oleanolic acids: Synthesis and structure/activity evaluation. Steroids
2020
,
154, 108530. [CrossRef]
20.
Youqing, S.; Defa, S.; Jianbin, T.; Meihua, S. Oleanolic Acid Derivative as well as Preparation Method and Application Thereof.
CN102532246B, 18 June 2014.
21.
Khusnutdinova, E.F.; Petrova, A.V.; Kukovinets, O.S.; Kazakova, O.B. Synthesis and Cytotoxicity of 28-N-Propargylaminoalkylated
2,3-Indolotriterpenic acids. Nat. Prod. Commun. 2018,13, 665–668. [CrossRef]
22.
Kazakova, O.; Smirnova, I.; Lopatina, T.; Giniyatullina, G.; Petrova, A.; Khusnutdinova, E.; Csuk, R.; Serbian, I.; Loesche, A.
Synthesis and cholinesterase inhibiting potential of A-ring azepano- and 3-amino-3,4-seco-triterpenoids. Bioorg Chem.
2020
,
101, 104001. [CrossRef] [PubMed]
23.
Kazakova, O.; Rubanik, L.; Smirnova, I.; Poleschuk, N.; Petrova, A.; Kapustina, Y.; Baikova, I.; Tret’yakova, E.; Khusnutdinova, E.
Synthesis and
in vitro
activity of oleanolic acid derivatives against Chlamydia trachomatis and Staphylococcus aureus.Med. Chem.
Res. 2021,30, 1408–1418. [CrossRef]
24.
Kazakova, O.; Mioc, A.; Smirnova, I.; Baikova, I.; Voicu, A.; Vlaia, L.; Maca
s
,
oi, I.; Mioc, M.; Drăghici, G.; Avram, ¸S.; et al. Novel
Synthesized N-Ethyl-Piperazinyl-Amides of C2-Substituted Oleanonic and Ursonic Acids Exhibit Cytotoxic Effects through
Apoptotic Cell Death Regulation. Int. J. Mol. Sci. 2021,22, 10967. [CrossRef] [PubMed]
25.
Khusnutdinova, E.; Petrova, A.; Zileeva, Z.; Kuzmina, U.; Zainullina, L.; Vakhitova, Y.; Babkov, D.; Kazakova, O. Novel A-Ring
Chalcone Derivatives of Oleanolic and Ursolic Amides with Anti-Proliferative Effect Mediated through ROS-Triggered Apoptosis.
Int. J. Mol. Sci. 2021,22, 9796. [CrossRef]
26.
Kazakova, O.B.; Brunel, J.M.; Khusnutdinova, E.F.; Negrel, S.; Giniyatullina, G.V.; Lopatina, T.V.; Petrova, A.V. A-Ring-Modified
Triterpenoids and Their Spermidine–Aldimines with Strong Antibacterial Activity. Molbank 2019,2019, M1078. [CrossRef]
27.
Smirnova, I.E.; Kazakova, O.B.; Viet, D.Q.; Thuc, N.T.; Linh, T.P.; Huong, D.T.T. Synthesis and evaluation of 29-norcycloartane
triterpenoids as α-glucosidase inhibitors. Med. Chem. Res. 2015,24, 2177–2182. [CrossRef]
28.
Khusnutdinova, E.F.; Smirnova, I.E.; Giniyatullina, G.V.; Medvedeva, N.I.; Yamansarov, E.Y.; Kazakov, D.V.; Kazakova, O.B.;
Linh, P.T.; Viet, D.Q.; Huong, D.T. Inhibition of Alpha-Glucosidase by Synthetic Derivatives of Lupane, Oleanane, Ursane and
Dammarane Triterpenoids. Nat. Prod. Commun. 2016,11, 33–35. [CrossRef]
29.
Khusnutdinova, E.F.; Smirnova, I.E.; Kazakova, O.B.; Petrova, A.V.; Ha, N.T.T.; Viet, D.Q. Synthesis and evaluation of 2,3-
indolotriterpenoids as new α-glucosidase inhibitors. Med. Chem. Res. 2017,26, 2737–2742. [CrossRef]
30.
Khusnutdinova, E.F.; Petrova, A.V.; Thu, H.N.T.; Tu, A.L.T.; Thanh, T.N.; Thi, C.B.; Babkov, D.A.; Kazakova, O.B. Structural
modifications of 2,3-indolobetulinic acid: Design and synthesis of highly potent
α
-glucosidase inhibitors. Bioorg Chem.
2019
,
88, 102957. [CrossRef]
31.
Wang, J.; Lu, S.; Sheng, R.; Fan, J.; Wu, W.; Guo, R. Structure-Activity Relationships of Natural and Synthetic Indole-Derived
Scaffolds as α-Glucosidase Inhibitors: A Mini-Review. Mini Rev. Med. Chem. 2020,20, 1791–1818. [CrossRef]
32.
Khusnutdinova, E.F.; Ha, N.T.T.; Giniyatullina, G.V.; Anh, L.T.T.; Poptsov, A.I.; Kazakova, O.B. Synthesis and alpha—Inhibitory
activity of lupane type C2-benzylidene-triterpenoids. Vietnam. J. Chem. 2021,59, 612–619.
33.
Khusnutdinova, E.; Galimova, Z.; Lobov, A.; Baikova, I.; Kazakova, O.; Thu, H.N.T.; Tuyen, N.V.; Gatilov, Y.; Csuk, R.; Serbian,
I.; et al. Synthesis of messagenin and platanic acid chalcone derivatives and their biological potential. Nat. Prod. Res.
2022
,
36, 5189–5198. [CrossRef] [PubMed]
34.
Zhang, Y.N.; Zhang, W.; Hong, D.; Shi, L.; Shen, Q.; Li, J.Y.; Li, J.; Hu, L.H. Oleanolic acid and its derivatives: New inhibitor of
protein tyrosine phosphatase 1B with cellular activities. Bioorg. Med. Chem. 2008,16, 8697–8705. [CrossRef] [PubMed]
35.
Ramírez-Espinosa, J.J.; Rios, M.Y.; Paoli, P.; Flores-Morales, V.; Camici, G.; de la Rosa-Lugo, V.; Hidalgo-Figueroa, S.; Navarrete-
Vázquez, G.; Estrada-Soto, S. Synthesis of oleanolic acid derivatives:
In vitro
,
in vivo
and in silico studies for PTP-1B inhibition.
Eur. J. Med. Chem. 2014,87, 316–327. [CrossRef] [PubMed]

Appl. Sci. 2023,13, 9269 16 of 16
36.
Zhang, W.; Hong, D.; Zhou, Y.; Zhang, Y.; Shen, Q.; Li, J.Y.; Hu, L.H.; Li, J. Ursolic acid and its derivative inhibit protein tyrosine
phosphatase 1B, enhancing insulin receptor phosphorylation and stimulating glucose uptake. Biochim. Et Biophys. Acta
2006
,
1760, 1505–1512. [CrossRef]
37.
Zhao, B.T.; Nguyen, D.H.; Le, D.D.; Choi, J.S.; Min, B.S.; Woo, M.H. Protein tyrosine phosphatase 1B inhibitors from natural
sources. Arch. Pharm. Res. 2018,41, 130–161. [CrossRef]
38.
Agrawal, N.K.; Kant, S. Targeting inflammation in diabetes: Newer therapeutic options. World J. Diabetes
2014
,5, 697–710.
[CrossRef]
39.
Liu, W.M.; Dalgleish, A.G. MTT assays can underestimate cell numbers. Cancer Chemother. Pharmacol.
2009
,64, 861–862. [CrossRef]
40.
Stepanenko, A.A.; Dmitrenko, V.V. Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in
over/underestimation of cell viability. Gene 2015,574, 193–203. [CrossRef]
41.
Es-Saady, D.; Simon, A.; Jayat-Vignoles, C.; Chulia, A.J.; Delage, C. MCF-7 cell cycle arrested at G1 through ursolic acid, and
increased reduction of tetrazolium salts. Anticancer. Res. 1996,16, 481–486.
42.
Gundoju, N.; Bokam, R.; Yalavarthi, N.R.; Azad, R.; Ponnapalli, M.G. Betulinic acid derivatives: A new class of
α
-glucosidase
inhibitors and LPS-stimulated nitric oxide production inhibition on mouse macrophage RAW 264.7 cells. Nat. Prod. Res.
2018
,
33, 1462182. [CrossRef] [PubMed]
43.
Mosén, H.; Salehi, A.; Henningsson, R.; Lundquist, I. Nitric oxide inhibits, and carbon monoxide activates, islets acid
α
-glucoside
hydrolase activities in parallel with glucose-stimulated insulin secretion. J. Endocrinol.
2006
,190, 681–693. [CrossRef] [PubMed]
44.
Akesson, B.; Henningsson, R.; Salehi, A.; Lundquist, I. Islet constitutive nitric oxide synthase and glucose regulation of insulin
release in mice. J. Endocrinol. 1999,163, 39–48. [CrossRef] [PubMed]
45.
Tian, G.; Wilcockson, D.; Perry, V.H.; Rudd, P.M.; Dwek, R.A.; Platt, F.M.; Platt, N. Inhibition of
α
-glucosidases I and II increases
the cell surface expression of functional class A macrophage scavenger receptor (SR-A) by extending its half-life. J. Biolog Chem.
2004,279, 39303–39309. [CrossRef] [PubMed]
46.
Borenfreund, E.; Puerner, J.A. Toxicity determined
in vitro
by morphological alterations and neutral red absorption. Toxicol. Lett.
1985,24, 119–124. [CrossRef] [PubMed]
47.
Yu, Y.; Koehn, C.D.; Yue, Y.; Li, S.; Thiele, G.M.; Hearth-Holmes, M.P.; Mikuls, T.R.; O’Dell, J.R.; Klassen, L.W.; Zhang, Z.; et al.
Celastrol Inhibits Inflammatory Stimuli-Induced Neutrophil Extracellular Trap Formation. CMM 2015,15, 401–410. [CrossRef]
48.
Sharma, K.R. Immunomodulatory Studies on Triterpenoids from Scoparia dulcis Linn. Biochem. Pharmacol.
2015
,4, 1000182.
[CrossRef]
49.
Elya, B.; Basah, K.; Mun’im, A.; Yuliastuti, W.; Bangun, A.; Septiana, E.K. Screening of
α
-Glucosidase Inhibitory Activity from
Some Plants of Apocynaceae, Clusiaceae, Euphorbiaceae, and Rubiaceae. J. Biomed. Biotechnol. 2012,2012, 281078. [CrossRef]
50.
Spasov, A.A.; Babkov, D.A.; Prokhorova, T.Y.; Sturova, E.A.; Muleeva, D.R.; Demidov, M.R.; Osipov, D.V.; Osyanin, V.A.;
Klimochkin, Y.N. Synthesis and biological evaluation of 2-acylbenzofuranes as novel
α
-glucosidase inhibitors with hypoglycemic
activity. Chem. Biol. Drug Des. 2017,90, 1184–1189. [CrossRef]
51.
Fujisawa, T.; Ikegami, H.; Inoue, K.; Kawabata, Y.; Ogihara, T. Effect of two
α
-glucosidase inhibitors, voglibose and acarbose, on
postprandial hyperglycemia correlates with subjective abdominal symptoms. Metabolism 2005,54, 387–390. [CrossRef]
52.
Lubben, T.; Clampit, J.; Stashko, M.; Trevillyan, J.; Jirousek, M.R. In Vitro Enzymatic Assays of Protein Tyrosine phosphatase 1B.
In Current Protocols in Pharmacology; Enna, S.J., Ed.; Wiley: Hoboken, NJ, USA, 2001. [CrossRef]
53.
Song, M.; Park, J.E.; Park, S.G.; Lee, D.H.; Choi, H.K.; Park, B.C.; Ryu, S.E.; Kim, J.H.; Cho, S. NSC-87877, inhibitor of SHP-1/2
PTPs, inhibits dual-specificity phosphatase 26 (DUSP26). Biophys. Res. Commun. 2009,381, 491–495. [CrossRef] [PubMed]
54.
Villarreal, F.J.; Kim, G.N.N.; Ungab, D.; Printz, M.P.; Dillmann, W.H. Identification of functional angiotensin II receptors on rat
cardiac fibroblasts. Circulation 1993,88, 2849–2861. [CrossRef] [PubMed]
55.
Dubey, R.K.; Gillespie, D.G.; Mi, Z.; Jackson, E.K. Exogenous and Endogenous Adenosine Inhibits Fetal Calf Serum–Induced
Growth of Rat Cardiac Fibroblasts: Role of A 2B Receptors. Circulation 1997,96, 2656–2666. [CrossRef] [PubMed]
56.
Chen, Y.; Fan, J.; Cao, L.; Han, T.; Zeng, M.; Xu, Y.; Ling, Z.; Yin, Y. Unique mechanistic insights into the beneficial effects of
angiotensin-(1-7) on the prevention of cardiac fibrosis: A metabolomic analysis of primary cardiac fibroblasts. Exp. Cell Res.
2019
,
378, 158–170. [CrossRef] [PubMed]
57.
Kopprasch, S.; Pietzsch, J.; Graessler, J. Validation of different chemilumigenic substrates for detecting extracellular generation of
reactive oxygen species by phagocytes and endothelial cells. Luminescence 2003,18, 268–273. [CrossRef]
58.
Percie du Sert, N.; Hurst, V.; Ahluwalia, A.; Alam, S.; Avey, M.T.; Baker, M.; Browne, W.J.; Clark, A.; Cuthill, I.C.; Dirnagl, U.; et al.
The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLOS Biol. 2020,18, e3000410. [CrossRef]
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