Structural modifications of 2,3-indolobetulinic acid: Design and synthesis of highly potent α-glucosidase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.102957

A series of nineteen nitrogen-containing lupane triterpenoids was obtained by modification of C2, C3, C20 and C28 positions of betulonic acid and their α-glucosidase inhibiting activity was investigated. Being a leader compound from our previous study, 2,3-indolo-betulinic acid was used as the main template for different modifications at C-(28)-carboxyl group to obtain cyano-, methylcyanoethoxy-, propargyloxy- and carboxamide derivatives. 20-Oxo- and 29-hydroxy-20-oxo-30-nor-analogues of 2,3-indolo-betulinic acid were synthesized by ozonolysis of betulonic acid followed by Fischer indolization reaction. To compare the influence of the fused indole or the seven-membered A-ring on the inhibitory activity, lupane A-azepanones with different substituents at C28 were synthesized. The structure-activity relationships revealed that the enzyme inhibition activity dramatically increased (up to 4730 times)when the carboxylic group of 2,3-indolo-betulinic acid was converted to the corresponding amide. Thus, the IC₅₀ values for glycine amide and L-phenylalanine amides were 0.04 and 0.05 μM, respectively. This study also revealed that 2,3-indolo-platanic acid is 4.5 times more active than the parent triterpenoid with IC₅₀ of 0.4 μM. Molecular modeling suggested that improved potency is due to additional polar interactions formed between C28 side chain and a sub-pocket of the α-glucosidase allosteric site.

Full text of DOI:10.1016/j.bioorg.2019.102957

Accepted Manuscript
Structural modifications of 2,3-indolobetulinic acid: design and synthesis of
highly potent α-glucosidase inhibitors
Elmira F. Khusnutdinova, Anastasiya V. Petrova, Ha Nguyen Thi Thu, Anh Le
Thi Tu, Tra Nguyen Thanh, Cham Ba Thi, Denis A. Babkov, Oxana B. Kazakova
PII:
S0045-2068(19)30150-6
https://doi.org/10.1016/j.bioorg.2019.102957
102957
DOI:
Article Number:
Reference:
YBIOO 102957
Bioorganic Chemistry
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Revised Date:
Accepted Date:
22 February 2019
17 April 2019
28 April 2019
Please cite this article as: E.F. Khusnutdinova, A.V. Petrova, H. Nguyen Thi Thu, A. Le Thi Tu, T. Nguyen Thanh,
C. Ba Thi, D.A. Babkov, O.B. Kazakova, Structural modifications of 2,3-indolobetulinic acid: design and synthesis
of highly potent α-glucosidase inhibitors, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.102957
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Structural modifications of 2,3-indolobetulinic acid: design and synthesis of highly
potent α-glucosidase inhibitors
Elmira F. Khusnutdinovaa,*, Anastasiya V. Petrovaa,b, Ha Nguyen Thi Thuc, Anh Le Thi Tuc, Tra
Nguyen Thanhc, Cham Ba Thic, Denis A. Babkovd and Oxana B. Kazakovaa
aUfa Institute of Chemistry UFRS RAS, 71 pr. Oktyabrya, Ufa 450054, Russian Federation
bBashkir State University, 32 Validy Str., Ufa 450076, Russian Federation
c Institute of Chemistry, Vietnamese Academy of Science and Technology, 18 Hoang Quoc Viet Str., Cau
Giay Dist., Hanoi, Vietnam
d Scientific Center for Innovative Drugs, Volgograd State Medical University, Novorossiyskaya st. 39,
Volgograd 400087, Russian Federation
*corresponding author
e-mail: elmaH@inbox.ru
Fax: +7 347 2356066
Key words: natural products, lupanes, triterpenoids, betulinic acid, indole, synthesis, type II diabetes, α-
glucosidase inhibitor, acarbose
2

Abstract
A series of nineteen nitrogen-containing lupane triterpenoids was obtained by
modification of C2, C3, C20 and C28 positions of betulonic acid and their α-glucosidase
inhibiting activity was investigated. Being a leader compound from our previous study, 2,3-
indolo-betulinic acid was used as the main template for different modifications at C-(28)-
carboxyl group to obtain cyano-, methylcyanoethoxy-, propargyloxy- and carboxamide
derivatives. 20-Oxo- and 29-hydroxy-20-oxo-30-nor-analogues of 2,3-indolo-betulinic acid were
synthesized by ozonolysis of betulonic acid followed by Fischer indolization reaction. To
compare the influence of the fused indole or the seven-membered A-ring on the inhibitory
activity, lupane A-azepanones with different substituents at C28 were synthesized. The structure-
activity relationships revealed that the enzyme inhibition activity dramatically increased (up to
4730 times) when the carboxylic group of 2,3-indolo-betulinic acid was сonverted to the
corresponding amide. Thus, the IC50 values for glycine amide and L-phenylalanine amides were
0.04 and 0.05 μM, respectively. This study also revealed that 2,3-indolo-platanic acid is 4.5
times more active than the parent triterpenoid with IC50 of 0.4 μM. Molecular modeling
suggested that improved potency is due to additional polar interactions formed between C28 side
chain and a sub-pocket of the α-glucosidase allosteric site.
1. Introduction
Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia. Type 2
diabetes mellitus (T2DM) accounts for 90% of cases of diabetes and is characterized by
individuals with postprandial hyperglycemia associated with low production of insulin,
resistance to insulin, or both [1]. One strategy used to control T2DM is the use of inhibitors of
digestive enzymes such as α-glucosidase that is present in the intestine which catalyzes the
digestion of complex carbohydrates, converting them into easily absorbable monosaccharides
[2]. In this regard, α-glucosidase inhibitors play a valuable role to prevent or delay the digestion
or absorption of carbohydrates and suppress postprandial hyperglycemia, making such inhibitors
useful in the management of type 2 diabetes. Thus, the design and development of new α-
glucosidase inhibitors is a noteworthy goal for pharmacologists [3,4]. Consumption of different
natural compounds is known to result in anti-diabetic effects, offering exciting possibilities for
the future development of successful therapies [5].
Triterpenoids are widely distributed in both plant and animal kingdoms and possess
interesting bioactivity [6, 7], such as anticancer [8], antiviral [9], anti-inflammatory [10],
antibacterial [11], antiparasitic [12], as well as antidiabetic [13–18]. Thus, an increasing number
of publications were focused on pentacyclic triterpenoids, particularly oleanolic, ursolic and
3

betulinic acids due to their antidiabetic properties [19,20]. Oleanolic acid is the hypoglycemic
component of many traditional Chinese medicines [21]. The dammarane type triterpenoid 20(S)-
protopanaxadiol demonstrated a regulatory function in glucose. Betulinic acid shows antidiabetic
activity due to inhibition of human PTP1B activity with an IC50 of 3.5 μM [22]. Oleanolic and
ursolic acids completely inhibited the activity of α-glucosidase in a non-competitive manner and
combination of these compounds displayed a significant synergistic inhibition on α-glucosidase
[23]. The combination of corosolic and oleanolic acids with acarbose showed synergistic
inhibition against α-amylase. The combination of the triterpenic acids with acarbose mainly
exhibited additive inhibition against α-glucosidase. These results provide valuable implications
for the triterpenic acids (ursolic, corosolic, and oleanolic) alone or in combination with acarbose
as a therapeutic agent for the treatment of diabetes mellitus [24].
Semi-synthetic strategies based on triterpenoids are also promising in the discovery of
new antidiabetic agents. Thus, methyl ester analog of betulinic acid with morpholine fragment
exhibited an almost 1.4-fold increase in α-glucosidase inhibitory activity compared to betulinic
acid [25]. Among the N-allylated/N-alkylated niacin and α-amyrin or lupeol hybrids tested for
their α-glucosidase inhibiting activity, N-prenylated α-amyrine-niacin hybrid showed the highest
inhibitory action with IC50 of 5 µM [26]. Among the different ursolic acid derivatives condensed
with a suitable aldehyde at the C2 position, the analog with p-trifluoromethyl benzene moiety
presented a great improvement of α-glucosidase inhibition activity with IC50 of 0.71 μM [27]. 3-
Oxo-derivatives of β-boswellic acid retained good α-glucosidase inhibition compared to 3-
hydroxy acids, indicating that the carboxylic group plays a key role in SAR [28]. It was shown
that in vivo oral hypoglycemic testing, both acute and sub-acute studies demonstrated that the
C2-modified oleanolic acid methyl ester derivatives containing pyridine- or morpholine fragment
had high potency and long duration of action compared to the reference standards pioglitazone,
acarbose and oleanolic acid [29].
Some ring A-fused triterpenoids such as 2,3-isoxazolocyclomusalenone and methyl 2,3-
indolobetulinate inhibited α-glucosidase with IC50 of 20.4 μM and 48.5 μM being 20- and 8-fold
more active than acarbose, respectively [17, 18, 30]. According to our previous studies, the
oxidized 28-oxo-indoloallobetulone derivatives possess antiviral activity [31], and 3-indolo-
betulinic acid was discovered as a new promising template for the synthesis of α-glucosidase
inhibitors [32]. In this work, a series of nitrogen-containing lupane triterpenoids, mainly of 2,3-
indolo-betulinic acid derivatives, was designed, synthesized and the activity of these new and
some previously described lupanes against α-glucosidase was evaluated in vitro.
4

2. Results and discussion
2.1. Design
Previously we have found that 2,3-indolo-betulinic acid 1 inhibited α-glucosidase with
IC50 of 1.8 µM being 221-fold more active than acarbose [32]. In order to get structural insight
and direct further optimization efforts, a molecular modeling study was performed.
Compound 1 was docked into the allosteric site of S. cerevisiae α-glucosidase. Since the
crystal structure of the enzyme is unavailable to date, we utilized the homology model built as
described earlier [33]. It was found that 1 occupies a cleft formed between the loop region and β-
sheet close to the active site (Fig. 1). Indole core interacts with Lys147 side chain via π-alkyl and
π-cation interaction, and with Pro148 via H-bond with a nitrogen atom. Rings A, D, and E form
multiple hydrophobic interactions with lipophilic side chains of Ile149, Pro150, Phe172, Ile415,
and Lys418. Additionally, the carboxylic group of 1 forms H-bond with Ser179 backbone and
Arg175 side chain. At the same time, the carboxylic group protrudes into the unexploited pocket
formed by Ser161, Thr164, and Arg175. Introduction of additional hydrogen bond donors or
acceptors could possibly facilitate inhibitor binding due to additional polar interactions. This
observation prompted us to obtain a series of analogs comprising functionalized C28 carboxyl in
order to explore the possibility of improving the potency of compound 1.
5

Fig. 1. CPK model of compound 1 docked into the allosteric site of S. cerevisiae α-glucosidase; catalytic Asp214
and Glu276 are shown with yellow carbons (A). Proposed interactions of compound 1 with amino acids of the
allosteric site (B).
6

2.2. Chemistry
The lead compound from our previous studies 2,3-indolo-betulinic acid 1 was modified at
C20 and C28 positions. The synthesis of different derivatives at C28 is shown in Scheme 1. It is
well known that structural modification of triterpenoids with the introduction of amine or amide
fragment improve biological properties, and result also in a better bioavailability [6, 34-36]. The
reaction of 1 with ammonia, amino acids (glycine, L-alanine or L-phenylalanine) or imidazole
via the intermediate acyl chloride gave the corresponding amides 2-6 with yields of 71-89%.
Alkynyl derivatives 7 and 8, were synthesized by a similar way with propargylamine or
propargyl alcohol with yields of 81 and 85%. The structure of compounds was confirmed by
NMR spectroscopy. For compounds 2-6 and 8, the signals of amide function C(O)NH were
detected at δ 172-179 ppm (13C NMR) and at δ 5.51-6.12 ppm (1H NMR). Also, for compounds
7 and 8 the signals of the acetylene fragment at δ 71.6-80.0 ppm (13C NMR) and δ 2.21-2.23
ppm (1H NMR) were characteristic.
Many triterpene nitriles are of interest as potential agents. For example, 2-cyano-3,12-
dioxoolean-1,9(11)-dien-28-oic acid (CDDO) bearing a cyanoenone moiety in ring A exhibits
400 times greater anti-inflammatory activity than oleanolic acid [37, 38]. 2,20-Dinitrile-
glycyrrhetinic acid displayed 99% inhibition rate at a dose of 50 mM and exhibited stronger
inhibitory activities against the LPS-induced NO production in RAW 264.7 macrophages
relative to the parent compound [39]. To investigate the effect of the nitrile group on α-
glucosidase inhibitory activity amide 2 was converted to compound 9 by trifluoroacetic
anhydride in CH2Cl2.
Another way to introduce a nitrile fragment to the side chain is by cyanoethylation. The
synthesis of bis- and tri-cyanoethyltriterpenoids [40], selective cytotoxicity of 28-cyanoethoxy-
indolo- [41] and aminopropoxy-triterpenoids were described previously [42]. At first, the
reduction of 2,3-indolobetulinic acid 1 by lithium aluminum hydride afforded 2,3-indolobetulin
10, which gave С28-cyanoethoxy-derivative 11 after treatment with acrylonitrile. In NMR
spectra of 10, signals of the methylene group С28Н2 as doublets at δ 3.28–3.88 (1Н NMR) and δ
68–78 ppm (13C NMR) were observed. In the 13C NMR spectra of 9 and 11, the typical signals of
1
the nitrile group at δ 118–119 ppm were observed. In the H NMR spectrum of 11, signals of
methylene protons of cyanoetoxy fragment at δ 2.59 and 3.68 ppm were characteristic.
7

Scheme 1. Synthesis of C28-derivatives of 2,3-indolo-betulinic acid. Reagents and conditions: (a) (i)
(COCl)2, Et3N, CH2Cl2, 20 °C, 2 h, (ii) ammonia, imidazole, corresponding amino acid (OMe·HCl), propargylamine
or propargyl alcohol, Et3N, CH2Cl2, ∆, 3 h, (iii) 4 N NaOH, CH3OH-THF (1 : 1), 20 °C, 6 h (for synthesis of
compounds 3-5); (b) TFAA, CH2Cl2, ∆, 2 h; (c) LiAlH4, THF, ∆, 3 h; (d) H2C=CHCN, 30% KОН, BnEt3NCl, 1,4-
dioxane, 20 °C, 2 h.
Recently, it was shown that platanic acid derivatives are potent inhibitors of
butyrylcholinesterase and anticancer agents [43-45]. At the same time, the effect of the 20-oxo-
group in lupane core on the inhibition of α-glucosidase was not described. For the synthesis of
platanic acid derivatives bearing an indole fragment, оxidation of betulonic acid with ozone in
CH2Cl2 was carried out. As a result of this reaction, we obtained a mixture of 3-oxo-platanic acid
12 and 29-hydroxy-20-oxo-30-nor-lupane acid 13, also known as messagenic acid [46], which
were isolated by column chromatography with yields of 75% and 18%, respectively (Scheme 2).
8

Scheme 2. Synthesis of 2,3-indoloplatanic acid derivatives. Reagents and conditions: (a) O3, CH2Cl2, −70 ºC; (b)
PhNHNH2, AcOH, ∆, 2 h; (c) (i) (COCl)2, Et3N, CH2Cl2, 20 °C, 2 h, (ii) imidazole, Et3N, CH2Cl2, ∆, 3 h.
We propose the following mechanism for the formation of compounds 12 and 13
formation (Fig. 2). First, 1,3-cycloaddition of ozone to С20(29)-double bond of betulonic acid
led to primary ozonide А, followed by rearrangement to secondary ozonide B and formation of
3-oxo-platanic acid 12. An alternative route based on the 1,2-cycloaddition of ozone to the
double bond with formation of epoxide C, which rearranges to D with the following oxidation to
compound 13 is also possible.
20-Oxo-indoles 14 and 16 were synthesized from 12 and 13 by Fischer reaction. The
interaction of 2,3-indolo-platanic acid with imidazole via the intermediate acyl chloride gave
amide 15.
Fig. 2. Plausible way of formation of compounds 12 and 13 during oxidation of betulonic acid with ozone
In order to compare the potency of triterpenic indoles with other types of derivatives, a
series of A-azepanones and C3,C28-substituted lupanes was also synthesized (Scheme 3).
9

A-azapanone-17-cyano-lup-20(29)-ene 17 was synthesized through reaction of betulonic
aldehyde with hydroxylamine hydrochloride to give 3,28-bisoxime with following Beckmann
rearrangement which affected both A-ring and C28-position. Synthesis of azepanone betulinic
acid 20 included two-step modification of betulonic acid to C3-oxime followed by Beckmann
rearrangement. Amide 21 was obtained by interaction of compound 20 with imidazole via the
intermediate acyl chloride with a yield of 65%. The formation of A ring lactam was confirmed
1
by the signal at δ ~176.0 ppm in 13C NMR, that pointed the presence of C=O group. The H
NMR spectrum of the lactams 17, 20 and 21 showed singlet at δ ~6 ppm typical of NH group.
Finally, compounds 18 and 19 were synthesized according to previously described
methods [8, 47].
Scheme 3. Synthesis of azepanone and 3,28-modified lupane derivatives. Reagents and conditions: (a) (i) PCC,
CHCl3, 20 °C, 2 h; (b) (i) NH2OH·HCl, NaOAc, EtOH, ∆, 5 h, (ii) SOCl2, 1,4-dioxane, 0 °C, 30 min. (c) Jones
reagent, acetone, 0 °C, 2 h; (d) (i) (COCl)2, Et3N, CH2Cl2, 20 °C, 2 h, (ii) imidazole, Et3N, CH2Cl2, ∆, 3 h.
2.3. α-Glucosidase inhibition and structure-activity relationships
All synthesized compounds 1‒21 were screened for in vitro α-glucosidase enzyme
inhibition (Table 1). The 2,3-indolo-betulinic acid amides 2‒6 and 8 demonstrated significant
inhibitory properties with IC50 values ranged from 0.04 μM to 60.74 μM. Carboxamide 2 (IC50
1.74 μM) was found to be 109-times more active than acarbose (IC50 189.20 μM). However, the
inhibition activity of the propargylamide 8 (IC50 60.74 μM), as well as imidazolo-amide 6 (IC50
18.54 μM), was lower than that of 2, albeit still greater than acarbose. However, conjugation of
the carboxylic group of compound 1 by amino acid residue led to a dramatic activity increase.
Compounds 3 (IC50 0.04 μM) and 5 (IC50 0.05 μM) containing glycine and L-phenylalanine
residues exhibited an almost 3784- and 4730-fold higher inhibitory activity compared to
acarbose and 20-30-fold compared to the previously described activity of 2,3-indolobetulinic
acid 1 (IC50 1.8 μM) [32]. Also, the inhibitory activity of L-alanine amide 4 (IC50 0.12 μM) was
less than of compounds 3 and 5.
10

Table 1. Inhibition of -glucosidase by triterpenoids 1-21
Compound
R1
R
IC50, µM
1.80
pIC50
5.74
5.76
7.40
6.92
7.30
4.73
3.74
1
2
3
4
5
6
7
-COOH
-CONH2
1.74
-CONHCH2COOH
CONHCH(CH3)COOH
-CONHCH(Bn)COOH
-COImd
0.04
0.12
0.05
18.54
183.24
-COOCH2C CH
-CONHCH2C CH
8
60.74
76.00
30.12
18.67
0.40
4.22
4.12
4.52
4.73
6.40
9
-CN
-CH2OH
10
11
14
-CHOCH2CH2CN
-COOH
15
-COImd
53.89
4.27
16
-COOH
57.38
4.24
% Inhibition at 100
µM, m±SEM
24.90±4.59
Compound
R
IC50, µM
pIC50
12
13
H
˃ 256
˃ 256
<3.59
<3.59
-OH
14.74±5.76
Compound
17
R
IC50, µM
pIC50
<3.59
-CN
˃ 256
11

20
21
-COOH
-COImd
34.68
6.19
4.46
5.21
Compound
18
R1
R
IC50, µM
pIC50
45.35
4.34
19
˃ 256
<3.59
3.72
Acarbose
189.20
The results are in accordance with our previous study as well [18], indicating that the
propargyl ester of 2,3-indolobetulinic acid 7 with IC50 of 183.24 μM showed almost no activity
comparing with acarbose and less than methyl ester analog. On the other hand, propargylamide 8
(IC50 60.74 μM) exhibited three-fold higher activity than ester 7, indicating that the increased
activity of compound 8 is indeed due to the presence of carboxamide moiety, which can provide
an extra H-bond.
Reduction of the carboxyl group of compound 1 to 2,3-indolo-betulin 10 (IC50 30.12 μM)
led to a decreased inhibition activity. On the other hand, the cyanoethylation of the hydroxy
group of 10 to compound 11 improved IC50 value up to 18.67 μM. Comparison of 1 and 9 (IC50
76.00 μM) showed that the replacement of the carboxyl group by a nitrile group has weakened
the activity against α-glucosidase.
Modification of the isopropenyl fragment of 1 had a positive effect. Thus, 2,3-indolo-
platanic acid 14 showed high activity with IC50 0.40 μM, being 473-fold more active than
acarbose, while 29-hydroxy analog 16 was far less active. Imidazole amide 15 showed also
moderate activity with IC50 53.89 μM in comparison with compounds 1 and 6. At the same time,
analogs of compound 14, acids 12 and 13, are virtually inactive, hence indicating the importance
of indole core presence.
Imidazole amide 21 with A-lactam cycle showed promising activity with IC50 value 6.19
μM which is 30-times more active than acarbose and is 3-times more active than the indole-
bearing analog 6. The replacement of imidazole amide fragment with nitrile in compound 17
resulted in a complete loss of activity. Modification of the 3-hydroxy group of lupanes with the
introduction of cinnamoyloxy- 18 or cyanoethoxy- 19 fragments did not lead to positive results.
12

It is known that the introduction of amino acid fragments to triterpene core improve drug-
like properties, particularly the poor solubility [6]. The solubility data for compounds 1, 3-5 and
14 have been measured. For the 2,3-indolobetulinic acid 1 solubility in distilled water was
determined as 0.257 µg/mL. The amino acid derivatives 3, 4 and 5 showed a better water
solubility, which was 0.450, 0.510 and 0.530 µg/mL, respectively. The solubility of compound 4
with alanine side chain is higher than the solubility of compound 3 with glycine side chain, while
the inhibitory activity is 3 times lower. On the other hand, the solubility of platanic acid
derivative 14 was found to be 0.263 µg/mL, which is comparable to the solubility of compound
1, while the activity of 14 is 4.5-times higher. Thus, inhibitory properties of triterpenoid
derivatives against α-glucosidase are predominantly influenced by the structure of lupane core
and a substituent at C28.
In summary, from a structure-activity-relationship perspective, among all compounds
tested for the inhibition of α-glucosidase 2,3-indolo-betulinic acid derivatives, we conclude that
the presence of carboxamide group (via an amino acid spacer) at the C28 position is important.
Compounds 3 and 5 with a glycine and L-phenylalanine amide fragments exhibited significantly
better activity with sub-micromolar IC50 values than the parent 2,3-indolobetulinic acid (20-times
higher) [32] and standard drug acarbose (3784-4730 times higher). Moreover, the α-glucosidase
inhibition was more than 400-times effective compared with the activity of acarbose when the
C20(29)-double bond of lupane indole was converted into the corresponding ketone (14, IC50 0.4
μM). Imidazole amide of 2,3-indolo-platanic acid 15, in comparison with the same amide of 2,3-
indolo-betulinic acid 6, was three times less active, which demonstrates a high sensitivity of
activity to structural changes in the lupane core. Meanwhile, when the carbocycle А was
replacement by lactam cycle, imidazole amide of A-lactame 21 exhibited three times higher
activity than the indolo-lupane 6. Reduction of carboxylic group of 2,3-betulonic acid 1 to 2,3-
indole-betulin 10 and its following cyanoethylation led to a decrease of activity. Modification of
C3 position by introducing ethylcyano- and p-methoxy-cinnamic acid fragment did not give
positive results.
Chemoinformatic study of molecular characteristics associated with activity against α-
glucosidase revealed that observed pIC50 values are correlated with the number of H-bond
acceptors (Bravais-Pearson coefficient 0.392) and, especially, H-bond donors (Bravais-Pearson
coefficient 0.648) present in the inhibitor molecule (Fig. 3). Therefore, it could be concluded that
polar interactions play a key role in the binding of the studied triterpene derivatives.
Simultaneously, the introduction of polar moieties has a positive impact on drug-like properties,
such as water solubility.
13

Fig. 3. The contribution of H-bond donors and acceptors to the α-glucosidase inhibitory activity of active 2,3-
indolo-betulinic acid derivatives.
2.4. Molecular docking
Hence, biological evaluation showed derivative 3 as a lead compound. The binding mode
of 3 was investigated as described for parent compound 1 in order to elucidate the structural
basis of its enhanced potency in detail. As shown in Fig. 4, extensive interaction network is
formed by the fused indole core, lupane skeleton, and the C28 side chain. Specifically, the indole
aromatic system maintains hydrophobic interactions with Pro150 and H-bond with Pro148 at the
entrance of the pocket. The triterpene core is well fitted to establish multiple hydrophobic alkyl-
alkyl interactions with Lys147, Phe165, and Phe172, which is similar to what was already
observed for the parent compound 1.
In turn, the amide side chain at C28 appears to form a hydrogen bond network, including
bridged H-bonds with Arg123, Arg180, and Tyr173. These additional interactions are likely
responsible for the improved affinity of compound 3 towards α-glucosidase. Isopropyl moiety at
14

C19 is pointing towards the solvent accessible area. This supports the improved activity of
platanic acid derivative 14 since polar acetyl substituent could compensate for enthalpy penalty
associated with displacement of bonded water molecules [48]. Summary of interactions of
compounds 1 and 3 with the allosteric site of α-glucosidase is shown in Fig. 5. Noteworthy,
unfavorable repulsive interaction of C28 hydroxyl of compound 1 is managed to be replaced
with a favorable one realized via the amide carbonyl group of compound 3.
Fig. 4. Proposed binding mode and interaction of compound 3 with the allosteric site of S. cerevisiae α-
glucosidase. Key amino acids are shown. Protein-ligand interactions are indicated with dashed lines.
15

Fig. 5. Schematic 2D diagram of intermolecular interactions of compound 1 (a) and compound 3 (b) with the
allosteric site of S. cerevisiae α-glucosidase. Key amino acids are shown. Interactions are indicated with dashed
lines (purple for hydrophobic contacts, green for H-bonds, orange for charge attraction).
16

3. Conclusions
Modification of previously discovered lead 2,3-indolo-betulinic acid at the C20 and C28
positions led to the identification of derivatives with up to 45-times improved α-glucosidase
inhibiting activity. The conjugates of 1 with glycine and L-phenylalanine via an amide bond at
C28 were found to be the most active with IC50 values of 0.04 and 0.05 μM, being 3784 and
4730-fold more active than acarbose. Most of the indole-fused modified derivatives with C28-
carboxamide, nitrile, hydroxy-, cyanoethoxy-, cinnamoyloxy- and lactame fragments showed
better potency than the standard drug acarbose. Structure-based activity study suggests the key
role of C28 amide side chain in inhibitor binding via the formation of multiple H-bonds with
previously unexploited polar subpocket of the allosteric site.
Acknowledgment
This work was supported by Federal programs (Nº АААА-А17-117011910023-2 and
АААА-А17-117011910027-0). The synthesis of compounds 1-21 was supported by the Russian
Foundation for Basic Research (project no. 18-33-00313 mol_a to Anastasiya V. Petrova). The
study of α-glucosidase inhibitory activity was supported by Vietnam Academy of Science and
Technology (Nr. QTRU01.02/18-19). Denis A. Babkov gratefully acknowledges Council on
grants of the President of the Russian Federation (SP-595.2018.4).
4. Experimental
4.1. Materials and methods
The spectra were recorded at the Center for the Collective Use ‘Chemistry’ of the Ufa
1
13
Institute of Chemistry UFRS RAS. H and C NMR spectra were recorded on a “Bruker AM-
500” (Germany, 500 and 125.5 MHz respectively, δ, ppm, Hz) in CDCl3, internal standard–
tetramethylsilane. Mass spectra were obtained on a liquid chromatograph–mass spectrometer
LCMS-2010 EV (Shimadzu). Melting points were detected on a micro table “Boetius”. Optical
rotations were measured on a polarimeter “Perkin-Elmer 241 MC” (Germany) in a tube length of
1 dm. Elemental analysis was performed on a Euro EA-3000 CHNS analyzer, the main standard
is acetanilide. IR spectra were recorded on a spectrometer IRPrestige-21 Shimadzu (Japan) in a
paste with vaseline oil. Thin-layer chromatography analyses were performed on Sorbfil plates
(Sorbpolimer, Russian Federation), using the solvent system chloroform-ethyl acetate, 40:1.
Substances were detected by a 10% solution of sulfuric acid solution with subsequent heating at
100–120 °C for 2–3 min. The ozone generator used was Ozone–4K (Russian Federation).
Compounds 1 [49], 8 [50], 10 and 11 [51], 18 [47], 19 [8] and 20 [52] were obtained according
to the methods described previously.
17

4.2. Chemistry
4.2.1. General procedure for the synthesis of compounds 2-7, 15 and 21
To a solution of compound 1 (1 mmol; 0.53 g), 14 (1 mmol; 0.53 g) or 20 (1 mmol; 0.47
g) in CH2Cl2 (20 mL) (COCl)2 (3 mmol; 0.26 mL) was added and stirred at room temperature
during 2 h. The mixture was concentrated to dryness under reduced pressure and resulting acid
chlorides were dissolved in CH2Cl2 (10 mL), 3 drops of Et3N and following reagents were added:
a) 1.5 mmol of conc. the solution of NH3 (for the synthesis of compound 2);
b) 1.5 mmol of corresponding amino acid (OMe·HCl). After isolation and purification (as
shown below), the deprotection was performed according to [53];
c) 1.5 mmol of propargyl alcohol (for synthesis of compound 7);
d) 1.5 mmol of imidazole (for synthesis of compounds 6, 15 or 21);
After completion of the reactions (TLC control) the organic layers were treated with H2O
(3×50 mL), separated, washed with 5% HCl, H2O (3×50 mL) until neutral pH, dried over
CaCl2, and evaporated under reduced pressure. The residue was purified by column
chromatography on Al2O3 using petroleum ether/CHCl3 (1 : 1) as eluent for 2-7, 15 and
CHCl3/CH3OH (50 : 1) for 21.
4.2.1.1. N-[3,2b]indolo-lup-20(29)-en-28-amide (2). Beige powder; yield (89%); m.p.:
183 °C; [α]20 = +15° (c 0.10, CHCl3); IR (ύ max, cm-1): 1641 (C C), 1656 (CONH), 3398
D
1
(NH); H NMR (300 MHz, CDCl3): δ 0.81, 1.00, 1.05, 1.15, 1.20, 1.65 (s, 18H, 6CH3),
1.62−3.15 (m, 23H, CH and CH2), 4.61 and 4.70 (both s, 2J = 2.0 Hz, 2H, H-29), 5.51 (br. s, 2H,
NH2), 7.02–7.58 (m, 4H, Ar-H), 7.64 (1H, br. s, NH) ppm; 13C NMR (75 MHz, CDCl3): δ 14.82,
15.98, 16.36, 19.09, 19.27, 21.41, 23.16, 25.38, 27.16, 29.15, 29.74, 30.86, 33.47, 34.00, 34.13,
37.26, 37.52, 38.27, 41.01, 42.79, 47.82, 48.76, 49.26, 53.23, 106.99 (C-2), 109.80 (C-arom),
110.34 (C-29) , 117.87 (C-arom), 118.89 (C-arom), 120.94 (C-arom), 128.34 (C-arom), 136.15
(C-arom), 140.89 (C-3), 150.47 (C-20) 179.56 (C-28) ppm; MS: m/z 526. [M + H]+. Anal. calcd
for С36H50N2O: C, 82.08; H, 9.57; N, 5.32. Found: C, 82.09; H, 9.55; N, 5.31.
4.2.1.2. N-glycine-[3,2b]indolo-lup-20(29)-en-28-amide (3). Beige solid; yield (75%);
m.p.: 202 °C; [α]20D = +7° (c 0.10, CHCl3); IR (ύ max, cm-1): 1643 (C C), 1661 (CONH), 3386
1
(NH); H NMR (300 MHz, CDCl3): δ 0.89, 1.05, 1.21, 1.27, 1.58, 1.70 (s, 18H, 6CH3),
2
1.68−3.22 (m, 23H, CH and CH2), 4.05 (m, 2H, H-37), 4.62 and 4.78 (both s, J = 2.0 Hz, 2H,
H-29), 6.10 (br. s, 1H, NH), 7.0−7.48 (m, 4H, Ar-H), 7.76 (1H, br. s, NH) ppm; 13C NMR (75
MHz, CDCl3): δ 14.70, 15.89, 16.36, 19.27, 19.49, 21.50, 22.54, 23.14, 25.80, 29.51, 30.77,
30.85, 33.64, 34.13, 37.28, 37.90, 38.32, 40.84, 41.11, 42.58, 46.71, 49.51, 50.06, 52.28, 53.33,
55.81, 107.05 (C-2), 109.52 (C-arom), 110.31 (C-29), 117.90 (C-arom) , 118.86 (C-arom),
120.90 (C-arom), 128.34 (C-arom), 136.15 (C-arom), 140.90 (C-3), 150.84 (C-20), 174.91 (C-
18

28), 179.62 (C-38) ppm; MS: m/z 584 [M + H]+. Anal. calcd for С38H52N2O3: C, 78.04; H, 8.96;
N, 4.79. Found: C, 78.05; H, 8.94; N, 4.77.
4.2.1.3. N-L-alanine-[3,2b]indolo-lup-20(29)-en-28-amide (4). White solid; yield (71% );
m.p.: 206 °C; [α]20D = +34° (c 0.10, CHCl3); IR (ύ max, cm-1): 1641 (C C), 1644 (CONH), 3446
1
(NH); H NMR (300 MHz, CDCl3): δ 0.75, 1.02, 1.18, 1.27, 1.43, 1.42, 1.69 (s, 21H, 7CH3),
2
1.70−3.20 (m, 23H, CH and CH2), 4.58 and 4.72 (both s, J = 2.0 Hz, 2H, H-29), 4.55 (m, 1H,
H-37), 6.12 (br. s, 1H, NH), 7.0−7.42 (m, 4H, H-arom), 7.62 (br. s, 1H, NH) ppm; 13C NMR (75
MHz, CDCl3): δ 14.13, 15.87, 16.36, 18.44, 19.51, 21.50, 22.69, 23.14, 25.79, 29.36, 29.70,
30.84, 31.93, 33.65, 34.12, 37.27, 38.31, 40.87, 41.48, 42.52, 46.74, 47.82, 49.49, 50.12, 52.42,
53.31 (CH-37), 55.65, 107. 07 (C-2), 109.43 (C-arom), 110.29 (C-20), 117.91 (C-arom), 118.88
(C-arom), 120.92 (C-arom), 128.33 (C-arom), 136.13 (C-arom), 140.85 (C-3), 150.93 (C-20),
174.00 (C-28), 178.80 (C-39) ppm; MS: m/z 598 [M + H]+. Anal. calcd for С39H54N2O3: C,
78.22; H, 9.09; N, 4.68. Found: C, 78.23; H, 9.07; N, 4.66.
4.2.1.4. N-L-phenylalanine-[3,2b]indolo-lup-20(29)-en-28-amide (5). Beige solid; yield
(75%); m.p.: 134 °C; [α]20 = +13° (c 0.10, CHCl3); IR (ύ max, cm-1): 1642 (C C), 1665
D
1
(CONH), 3453 (NH); H NMR (300 MHz, CDCl3): δ 0.85, 0.90, 0.97, 1.19, 1.28, 1.68 (s, 18H,
2
6CH3), 1.06−3.16 (m, 26H, CH and CH2), 4.60 and 4.73 (both d, J = 2.5, 2H, H-29), 4.85 (m,
1H, H-37), 5.92 (br. s, 1H, NH), 7.04−7.38 (m, 8H, H-arom), 7.84 (br. s, 1H, NH) ppm; 13C
NMR (75 MHz, CDCl3): δ 14.62, 15.89, 16.33, 19.25, 19.44, 21.43, 23.11, 25.72, 29.34, 30.68,
30.81, 33. 54, 33.56, 34.11, 37.25, 37.77, 38.04, 38.05, 38.27, 40.75, 42.46, 46.58, 49.44, 49.86,
52.76, 53.29, 55.77, 106.94 (C-2), 109.47 (C-29), 110.34 (C-arom), 117.86 (C-arom), 118.80 (C-
arom), 120.84 (C-arom), 128.30 (C-arom), 128.72 (C-41), 128.72 (C-43), 128.98 (C-40), 128.98
(C-44), 136.15 (C-arom), 136.37 (C-41), 140.87 (C-3), 150.79 (C-20), 175.99 (C, C-28), 178.57
(C-45) ppm; 15N NMR (СDCl3): δ 110.85 (N); 116.56 (N Ind) ppm; MS: m/z 674 [M + H]+.
Anal. calcd for С45H58N2O3: C, 80.08; H, 8.66; N, 4.15. Found: C, 80.07; H, 8.65; N, 4.14.
4.2.1.5. N-(1H-imidazol-1-yl)-[3,2b]indolo-lup-20(29)-en-28-amide (6). Yellow powder;
yield (74%); m.p.: 171 °C; [α]20 = +3° (c 0.10, CHCl3); IR (ύ max, cm-1): 1641 (C C), 1668
D
1
(CONH), 3430 (NH); H NMR (75 MHz, CDCl3): δ 0.88, 1.02, 1.05, 1.20, 1.30, 1.70 (s, 18H,
2
6CH3), 1.72−3.01 (m, 22H, CH and CH2), 4.68 and 4.82 (both s, J = 2.0 Hz, 2H, H-29),
7.01−7.41 (m, 4H, H-arom), 7.25 (s, 1H, Ar-H Imd) (s, 1H, H-38), 7.59 (s, 1H, H-37), 7.95 (br.
13
s, 1H, NH), 8.32 (s, 1H, H-39) ppm; C NMR (CDCl3, 75 MHz): δ 14.79, 15.76, 16.43, 19.45,
21.57, 23.14, 25.66, 29.77, 29.86, 30.85, 33.16, 33.23, 33.54, 34.16, 36.99, 37.19, 37.35, 38.34,
40.77, 42.21, 45.28, 49.56, 51.50, 53.35, 57.79, 106.85 (C-2), 110.31 (C-29), 110.39 (C-arom),
117.55 (C-arom), 117.87 (CH, C-37), 118.86 (C-arom), 120.92 (C-arom), 128.35 (CH, C-38),
129.34 (C-arom), 136.19 (CH, C-39), 137.28 (C-arom), 140.94 (C-3), 149.78 (C-20), 172.88 (C-
19

28) ppm; MS: m/z 577 [M + H]+. Anal. calcd for С39H51N3O: C, 81.06; H, 8.90; N, 7.27; Found:
C, 81.05; H, 8.93; N, 7.25.
4.2.1.6. Prop-2-yn-1-yl-[3,2b]indolo-lup-20(29)-en-28-oat (7). Yellow solid; yield
(85%); m.p.: 207 °C; [α]20 = +51° (c 0.10, CHCl3); IR (ύ max, cm-1): 1642 (C C), 1710
D
(C O); 2210 (С CH); 1H NMR (300 MHz, CDCl3): δ 0.73, 1.00, 1.02, 1.20, 1.27, 1.68 (s, 18H,
6CH3), 1.32−3.10 (m, 23H, CH and CH2), 2.21 (t, J = 2.4 Hz, 1H, C CH), 4.65−4.75 (m, 2H,
CH2), 4.60 and 4.68 (both s, 2J = 2.0 Hz, 2H, H-29), 6.59−7.40 (m, 4H, H-arom), 7.70 (br. s, 1H,
NH) ppm; 13C NMR (75 MHz, CDCl3): δ 14.79, 15.84, 16.38, 19.28, 19.41, 21.49, 23.16, 25.75,
29.78, 30.54, 30.87, 31.97, 33.63, 34.15, 36.84, 37.32, 38.34, 38.51, 40.93, 42.50, 46.92, 49.48,
49.55, 51.36, 53.34, 56.67, 74.33 (C-39), 78.18 (C-38), 107.07 (C-2), 109.78 (C-arom), 110.33
(C-20), 117.91 (C-arom), 118.91 (C-arom), 120.96 (C-arom), 128.39 (C-arom), 136.18 (C-
arom), 140.89 (C-3), 150.44 (C-20), 175.25 (C-28) ppm; MS: m/z 565 [M + H]+. Anal. calcd for
С39H51NO2: C, 82.78; H, 9.09; N, 2.48. Found: C, 82.77; H, 9.10; N, 2.46.
4.2.1.7. N-(1H-imidazol-1-yl)-[3,2b]indolo-20-oxo-29-norlupan-28-amide (15). Yellow
solid; yield (78%); m.p.: 200 °C; [α]20D = +56° (c 0.10, CHCl3); IR (ύ max, cm-1): 1642 (C C),
1
1663 (CONH), 1733 (C O), 3419 (NH); H NMR (75 MHz, CDCl3): δ 0.87, 1.00, 1.09, 1.22,
1.29, 2.23 (s, 18H, 6CH3), 1.32−3.28 (m, 23H, CH and CH2), 7.03−7.40 (m, 4H, H-arom), 7.19
(s, 1H, H-38), 7.55 (s, 1H, H-37), 7.94 (br. s, 1H, NH), 8.41 (s, 1H, H-39) ppm; 13C NMR
(CDCl3, 75 MHz): δ 14.76, 15.66, 16.39, 19.19, 21.57, 23.13, 27.56, 28.20, 29.89, 30.37, 30.83,
32.60, 33.4, 34.15, 36.19, 36.62, 37.27, 38.34, 40.65, 42.09, 49.23, 49.40, 51.31, 53.29, 57.12,
106.76 (C-2), 110.37 (C-arom), 117.49 (C-37), 117.89 (C-arom), 118.85 (C-arom), 120.93 (C-
arom), 128.28 (C-38), 128.93 (C-arom), 136.18 (C-39), 137.06(C-arom), 140.88 (C-3), 172.80
(C-28), 211.39 (C-20) ppm; MS: m/z 580 [M + H]+. Anal. calcd for С38H49N3O2: C, 78.72; H,
8.52; N, 7.25. Found: C, 78.73; H, 8.51; N, 7.27.
4.2.1.8. N-(1H-imidazol-1-yl)-3-oxo-3-aza-3-homo-lup-20(29)-en-28-amide (21). Beige
solid; yield (65%); m.p.: 201 °C; [α]20D = +10° (c 0.10, CHCl3); IR (ύ max, cm-1): 1680 (С O),
1
1659 (CONH), 3230 (NH); H NMR (300 MHz, CDCl3): δ 0.80, 0.85, 0.94, 1.03, 1.07, 1.62
2
(6s,18H, 6CH3), 1.70−3.00 (m, 25H, CH and CH2), 4.62 and 4.76 (both d, J = 2.0 Hz, 2H, H-
13
29), 5.82 (br. s, 1H, NH), 7.21 (s, 1H, H-32), 7.51 (s, 1H, H-33), 8.29 (s, 1H, H-31) ppm; C
NMR (75 MHz, CDCl3): δ 14.43, 15.75, 18.19, 19.36, 22.05, 22.41, 25.73, 27.14, 29.57, 30.56,
31.88, 33.00, 33.26, 33.53, 36.91, 37.06, 39.21, 40.22, 40.62, 42.18, 45.08, 51.05, 51.26, 53.03,
56.28, 57.62, 110.25 (C-29), 117.38 (C-33), 129.45 (C-32), 138.64 (C-31), 149.54 (C-20),
172.82 (C-28), 176.56 (C-3) ppm; MS: m/z 519 [M + H]+. Anal. calcd for С33H49N3O2: C, 76.26;
H, 9.50; N, 8.08. Found: C, 76.24; H, 9.51; N, 8.09.
20

4.2.2. Synthesis of [3,2b]indolo-lup-20(29)-en-17-nitrile (9)
To a solution of amide 2 (0.53 g; 1 mmol) in anhydrous CH2Cl2 (20 mL) (CF3CO)2O (1
mmol; 0.14 mL) was added and refluxed for 2 h. Then poured into H2O (50 mL) and extracted
with CH2Cl2 (3×50 mL). Organic layers were washed with 5% HCl and H2O (3×50 mL) until
neutral pH, dried over CaCl2, and evaporated under reduced pressure. The crude product was
purified by column chromatography (SiO2; CHCl3/petroleum ether (1 : 1)). Yellow powder; yield
(89%); m.p.: 164 °C; [α]20D = ‒15° (c 0.10, CHCl3); IR (ύ max, cm-1): 1641 (C C), 2251 (C N);
1H NMR (300 MHz, CDCl3): δ 0.90, 1.02, 1.14, 1.15, 1.28, 1.72 (s, 18H, 6CH3), 1.80−2.88 (m,
2
23H, CH and CH2), 4.70 and 4.82 (both s, J = 2.0 Hz, 2H, H-29), 7.05−7.48 (m, 4H, H-arom),
13
8.12 (br. s, 1H, NH) ppm; C NMR (75 MHz, CDCl3): δ 14.94, 15.81, 16.42, 17.45, 19.41,
21.29, 23.16, 25.17, 29.53, 30.88, 31.06, 33.67, 34.15, 35.87, 37.33, 38.32, 41.43, 42.40, 48.63,
49.12, 49.17, 49.35, 53.28, 53.28, 53.46, 106.85 (C-2), 110.41 (C-20), 111.03 (C-arom), 117.86
(C-arom), 118.89 (C-arom), 120.96 (C-arom), 123.56 (C-17), 128.36 (C-arom), 136.17 (C-
arom), 140.89 (C-3), 148.17 (C-29) ppm; MS: m/z 508 [M + H]+. Anal. calcd for С36H48N2: C,
84.98; H, 9.51; N, 5.51. Found: C, 84.97; H, 9.53; N, 5.50.
4.2.3. Oxidation of betulonic acid with ozone
A rapid stream of ozone was passed through a solution of betulonic acid (2 mmol; 0.908
g) in CH2Cl2 (30 mL) at ‒40 ºC until the starting compound disappeared (TLC control). The
solvent was removed under reduced pressure, and the residue was purified by column
chromatography on SiO2 eluting with CHCl3 and CH3OH (from 10:0 to 1:1) giving compounds
12 and 13.
4.2.3.1. 3-Oxo-platanic acid (12). White solid; yield (75%); m.p.: 231 °C; [α]20 = +17°
D
(c 0.01, CHCl3); lit.: m.p. 231 °C; [α]D = +5.4º (c 0.39, CHCl3) [43]; IR (ύ max, cm-1): 1687
(CO-OH), 1705 (C O), 1733 (C O); 1H NMR (300 MHz, CDCl3): δ 0.90, 0.94, 1.00, 1.01, 1.06
(5s, 15H, 5CH3), 2.18 (s, 3H, CH3), 1.02−3.24 (m, 26H, CH and CH2); 13C NMR (75 MHz,
CDCl3): δ 14.60, 15.25, 15.66, 15.98, 19.57, 20.94, 21.34, 26.70, 27.15, 28.22, 29.64, 30.12,
31.35, 33.38, 34.01, 36.65, 37.50, 39.46, 40.44, 42.23, 47.24, 48.97, 49.60, 51.10, 54.64, 56.14,
181.68 (C-28), 212.41 (C-20), 218.52 (C-3). MS: m/z 457 [M + H]+. Anal. calcd for С29H44O4:
C, 76.27; H, 9.71. Found: C, 76.25; H, 9.70.
4.2.3.2. 29-Hydroxy-20-oxo-30-norlupan-28-oic acid (13). Yellow powder; yield (18%);
m.p.: 147 °C; [α]20 = +37° (c 0.01, CHCl3); IR (ύ max, cm-1): 1699 (CO-OH), 1713 (C O),
D
1
1727 (C O), 3450 (OH); H NMR (300 MHz, CDCl3): δ 0.90, 0.95, 1.00, 1.00, 1.06 (5s, 15H,
13
5CH3), 1.02−2.48 (m, 26H, CH and CH2), 3.20 (br. s, 1H, OH), 4.22−4.44 (m, 2H, H-30); C
NMR (75 MHz, CDCl3): δ 14.54, 15.65, 15.94, 17.21, 19.56, 20.97, 21.26, 26.67, 27.29, 28.93,
29.58, 31.24, 33.38, 34.01, 36.77, 36.85, 37.43, 39.46, 40.46, 42.26, 46.48, 47.28, 49.42, 49.56,
21

54.70, 56.11, 56.61, 68.07 (C-30), 181.76 (C-28), 213.21 (C-20), 218.41 (C-3). MS: m/z 473 [M
+ H]+. Anal. calcd for С29H44O5: C, 73.69; H, 9.38. Found: C, 73.67; H, 9.37.
4.2.4. Synthesis of compounds 14 and 16
A mixture of compound 12 (1 mmol; 0.46 g) or 13 (1 mmol; 0.47 g) and PhNHNH2 (3.5
mmol; 0.42 mL) in glacial AcOH (20 mL) was refluxed for 15 h. Then poured into water (50
mL) and the precipitate was filtered off and washed. The residue was purified by flash
chromatography (SiO2, petroleum ether/Et2O (1 : 1))
4.2.4.1. [3,2b]Indolo-20-oxo-30-norlupan-28-oic acid (14). Yellow solid; yield (91%);
m.p.: 147 °C; [α]20D = ‒8° (c 0.10, CHCl3); IR (ύ max, cm-1): 1638 (C C), 1700 (CO-OH), 1735
1
(C O); H NMR (300 MHz, CDCl3): δ 0.79, 0.92, 1.00, 1.09, 1.21 (s, 15H, 5CH3), 1.22−2.08
(m, 14H, CH and CH2), 2.21 (s, 3H, CH3), 2.20−3.29 (m, 9H, CH and CH2), 6.98−7.46 (m, 4H,
13
H-arom), 7.68 (br. s, 1H, NH) ppm; C NMR (75 MHz, CDCl3): δ 14.78, 15.78, 16.32, 19.19,
21.40, 23.13, 24.55, 27.39, 28.34, 29.86, 30.20, 30.83, 31.43, 33.42, 34.11, 36.74, 37.76, 38.30,
40.64, 42.30, 49.19, 49.30, 51.23, 53.18, 56.32, 106.88 (C-2), 110.34 (C-arom), 119.96 (C-
arom), 120.95 (C-arom), 124.44 (C-arom), 129.01 (C-arom), 136.13 (C-arom), 140.85 (C-3),
181.86 (C-28), 212.32 (C-20) ppm; MS: m/z 529 [M + H]+. Anal. calcd for С35H47NO3: C,
79.35; H, 8.94; N, 2.64. Found: C, 79.34; H, 8.92; N, 2.65.
4.2.4.2. [3,2b]Indolo-29-hydroxy-20-oxo-30-norlupan-28-oic acid (16). Beige solid;
yield (89 %); m.p.: 151 °C; [α]20 = ‒8° (c 0.10, CHCl3); IR (ύ max, cm-1): 1642 (C C), 1794
D
1
(CO-OH), 1725 (C O), 3450 (OH); H NMR (300 MHz, CDCl3): δ 0.82, 0.89, 0.95, 1.00, 1.06
(5s, 15H, 5CH3), 1.02−2.48 (m, 24H, CH and CH2), 3.20 (br. s, 1H, OH), 4.20−4.35 (m, 2H, H-
30), 7.08−7.39 (m, 4H, H-arom), 7.48 (br. s, 1H, NH) ppm; 13C NMR (75 MHz, CDCl3): δ
14.54, 15.65, 17.21, 19.56, 20.97, 21.26, 26.67, 27.29, 28.93, 29.58, 31.24, 33.38, 34.01, 36.77,
37.43, 39.46, 40.46, 42.26, 46.48, 47.28, 49.42, 54.70, 56.11, 56.61, 68.44 (C-30), 106.88 (C-
arom); 110.34 (C-arom), 117.92 (C-arom), 118.88 (C-arom), 124.44 (C-arom), 128.35 (C-arom),
136.13 (C-arom), 140.85 (C-arom), 181.27 (C-28), 213.13 (C-20); MS: m/z 546 [M + H]+. Anal.
calcd for С35H47NO4: C, 77.03; H, 8.68, N, 2.57. Found: C, 77.05; H, 8.67, N, 2.56.
4.2.5. Synthesis of 3-oxo-3-homo-3-aza-lup-20(29)-en-17-nitrile (17)
To a solution of 3,28-dioximino-lup-20(29)-en (1 mmol; 0.47 g), obtained according [54]
in dried 1,4-dioxane (15 mL), SOCl2 (3 mmol; 0.22 mL) was added drop wise with cooling. The
obtained mixture was stirred at room temperature for 30 min and then poured into H2O (50 mL).
The precipitate was filtered off, washed with water until neutral pH. The product was purified by
column chromatography, using CHCl3 for elution. Yellow solid; yield (69%); m.p.: 195 °C;
[α]20D = +27° (c 0.10, CHCl3); IR (ύ max, cm-1): 1680 (С O), 3230 (NH), 2246 (C N); 1H NMR
(300 MHz, CDCl3): δ 0.82, 1.03, 1.04, 1.21, 1.30, 1.62 (6s, 18H, 6CH3), 1.70−2.68 (m, 25H, CH
22

and CH2), 4.61 and 4.73 (both d, 2J = 2.0 Hz, 2H, H-29), 5.92 (br. s, 1H, NH) ppm; 13C NMR (75
MHz, CDCl3): δ 14.65, 15.88, 18.21, 19.36, 21.78, 22.39, 25.25, 27.13, 28.94, 29.46, 30.93,
31.62, 33.38, 33.70, 35.75, 39.07, 40.29, 40.63, 41.35, 42.41, 48.48, 48.96, 49.06, 50.82, 53.09,
56.70, 111.04 (C-29), 123.40 (C-17), 147.94 (C-20), 176.61 (C-3) ppm; MS: m/z 450 [M + H]+.
Anal. calcd for С30H46N2O: C, 79.95; H, 10.29; N, 6.22. Found: C, 79.93; H, 10.28; N, 6.21.
4.3. Solubility assay
The solubility of compounds 1, 3-5 and 14 was examined simultaneously in distilled
water and different 10 µM sodium phosphate solutions of pH 7.1 to 11.8. Each triterpene
derivatives was dissolved in the aqueous media (100 mg/mL) and heated to 100 °C for one hour
under reflux. The sample was cooled to room temperature. Filtered and analyzed daily, until
saturation according [55].
4.4. Pharmacological studies
4.4.1. -Glucosidase Inhibition Assay Method
The -glucosidase inhibition assay was performed according to the method of [56, 57] with
slight modification. Compounds were dissolved in DMSO at a concentration of 40 mg/ml. A
series of dilutions for each compound were prepared in 100 mM phosphate buffer (pH 6.8) to
final concentrations of 256, 64, 16, 4 and 1 g/mL. In a 96-well plate, a reaction mixture
containing 10 µL of compounds of varying concentrations, 40 µL of 100 mM phosphate buffer
(pH 6.8), 25 µL of α-glucosidase (0.4 U/mL, Sigma G0660) were pre-incubated for 10 min at
37 °C. DMSO was added to the respective control wells to a 0.64% final concentration. Then 25
µL of a 2.5 mM p-nitrophenyl--D-glucopyranoside (pNPG, Sigma N1377) solution was added
to the mixture as a substrate. After further incubation at 37 C for 30 min, the reaction was
stopped by addition of 100 µL of 0.2 M sodium carbonate. The amount of released p-nitrophenol
from pNPG was measured at 410 nm with microplate reader equipped spectrophotometer.
Acarbose was used as a positive control, and all assays were carried out in triplicate. The %
inhibition was obtained using the formula:
% Inhibition = 100% × (ODcontrol – ODsample) / ODcontrol
IC50 value was defined as the concentration of compound exhibiting 50% inhibition of α-
glucosidase activity under the assay conditions.
4.4.2. Chemoinformatic methods
Library preparation, calculation of molecular descriptors and compound properties were
conveniently performed with OSIRIS DataWarrior 4.7.2 [58].
4.4.3 Molecular docking
23

Input structures were prepared with MarvinSketch 18.8.0 (ChemAxon Ltd.) [59]. Ligands
were hydrogenized and conformations with minimal potential energy were obtained using the
MMFF94 force field. Protein and ligand structures followed standard preparation procedure
using AutoDocTools 1.5.6. We used AutoDock Vina 1.1.2 [60] to perform all docking runs. Grid
box with 70×80×80 Å dimensions was centered on the enzyme molecule to include the entire
3D-space around the protein. The top-score binding poses were used in subsequent analysis.
Protein-ligand interactions were analyzed with Discovery Studio Visualizer 17.2.0.16349
(Dassault Systemes Biovia Corp.) [61].
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