Derivatization, molecular docking and in vitro acetylcholinesterase inhibitory activity of glycyrrhizin as a selective anti-Alzheimer agent

Article abstract of DOI:10.1080/14786419.2018.1462177

Acetylcholinesterase inhibitors (AChE-Is) increase both level and duration of action of acetylcholine (ACh); thus, alleviate symptoms of Alzheimer’s disease (AD). Glycyrrhizin, is the main active compound in liquorice root. Its aglycone, glycyrrhetinic acid, has shown several beneficial pharmacological activities. This study reports the synthesis and screening of a series of glycyrrhetinic acid analogs as AChE-Is. Fourteen derivatives were prepared, of which five derivatives are recorded as new viz., 3-phenyl-carbamoyl-18β-glycyrrhetinic acid (J9), 3-acetyl-18β-glycyrrhetinic-30-anilinamide (J10), 3-acetyl-18β-glycyrrhetinic-30-ethanolamide (J11), 3-acetyl-18β-glycyrrhetinic-30-n-butylamide (J12) and 18β-glycyrrhetinic acid-30-prenyl ester (J14), in addition to nine known derivatives (J1-J8 & J13). Compounds J12, J11, J0 and J3 showed remarkable AChE-I activity with IC₅₀ values of 3.43, 5.39, 6.27 and 8.68?μM, respectively. These results are in full agreement with the docking study. The active compounds were non-cytotoxic to normal cells (WI-38).

Full text of DOI:10.1080/14786419.2018.1462177

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Derivatization, molecular docking and in vitro
acetylcholinesterase inhibitory activity of
glycyrrhizin as a selective anti-Alzheimer agent
Fatma M. Abdel Bar, Diaaeldin M. Elimam, Amira S. Mira, Fardous F. El-
Senduny & Farid A. Badria
To cite this article: Fatma M. Abdel Bar, Diaaeldin M. Elimam, Amira S. Mira, Fardous
F. El-Senduny & Farid A. Badria (2018): Derivatization, molecular docking and in vitro
acetylcholinesterase inhibitory activity of glycyrrhizin as a selective anti-Alzheimer agent, Natural
Product Research, DOI: 10.1080/14786419.2018.1462177
To link to this article: https://doi.org/10.1080/14786419.2018.1462177
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Natural Product research, 2018
https://doi.org/10.1080/14786419.2018.1462177
Derivatization, molecular docking and in vitro
acetylcholinesterase inhibitory activity of glycyrrhizin as a
selective anti-Alzheimer agent
Fatma M. Abdel Bara,b, Diaaeldin M. Elimamb, Amira S. Mirab, Fardous F. El-Sendunyc
and Farid A. Badriab
aFaculty of Pharmacy, department of Pharmacognosy, Prince sattam Bin abdulaziz university, al-Kharj, saudi
arabia; bFaculty of Pharmacy, department of Pharmacognosy, Mansoura university, Mansoura, egypt; cFaculty
of science, chemistry department, Biochemistry division, Mansoura university, Mansoura, egypt
ABSTRACT
ARTICLE HISTORY
received 8 august 2017
accepted 3 april 2018
Acetylcholinesterase inhibitors (AChE-Is) increase both level and
duration of action of acetylcholine (ACh); thus, alleviate symptoms
of Alzheimer’s disease (AD). Glycyrrhizin, is the main active compound
in liquorice root. Its aglycone, glycyrrhetinic acid, has shown several
beneficial pharmacological activities. This study reports the synthesis
and screening of a series of glycyrrhetinic acid analogs as AChE-Is.
Fourteen derivatives were prepared, of which five derivatives are
recorded as new viz., 3-phenyl-carbamoyl-18β-glycyrrhetinic acid
(J9), 3-acetyl-18β-glycyrrhetinic-30-anilinamide (J10), 3-acetyl-18β-
glycyrrhetinic-30-ethanolamide (J11), 3-acetyl-18β-glycyrrhetinic-30-
n-butylamide (J12) and 18β-glycyrrhetinic acid-30-prenyl ester (J14),
in addition to nine known derivatives (J1-J8 & J13). Compounds J12,
J11, J0 and J3 showed remarkable AChE-I activity with IC50 values
of 3.43, 5.39, 6.27 and 8.68 μM, respectively. These results are in full
agreement with the docking study. The active compounds were non-
cytotoxic to normal cells (WI-38).
KEYWORDS
Glycyrrhizin; glycyrrhetinic
acid; ache-I; docking; wound
healing; biocompatibility
assay
CONTACT Farid a. Badria
faridbadria@gmail.com
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1462177.
© 2018 Informa uK limited, trading as taylor & Francis Group

2
F. M. ABDEL BAR ET AL.
1. Introduction
Acetylcholinesterase (AChE) is responsible for the cessation of impulse transmission by rapid
hydrolysis of ACh in almost all cholinergic pathways throughout the nervous system. Thus,
it is a therapeutic target for improving the quality of life in AD patients. AChE-Is prevent
enzymatic hydrolysis of ACh, thus increasing its level and duration of action. However, many
of the side effects of the AChE-Is such as tacrine, physostigmine and velnacrine are attributed
to the peripheral cholinergic effects including nausea, vomiting and diarrhea hence the
search for new safe and effective anticholinesterases (McGleenon et al.1999). Pentacyclic
triterpenes, have been reported by several researchers as AChE-Is (Gurovic et al. 2010;
Schwarz et al. 2014). Glycyrrhizin is a pentacyclic triterpene glycoside present in liquorice
root (Glycyrrhiza glabra) (Sharma and Agrawal 2013). Although, glycyrrhizin acts as AchE
activator, derivatives of its aglycone, glycyrrhetinic acid, has been shown to exhibit significant
AChE inhibition. These derivatives have been altered at positions C-3 and C-30 by several
reactions including coupling with amino acids, esterification or amidation (Schwarz et al.
2014). Thus, glycyrrhetinic acid was selected in the current study as a promising scaffold for
the development of selective AchE-Is suggesting new modifications at A-ring, C-3 and C-30.
2. Results and discussion
2.1. Chemistry
Following glycyrrhizin (J0) isolation, glycyrrhetinic acid J1 was produced via acid hydrolysis
and used as a starting material for the preparation of five new derivatives J9-J12 & J14
(Figure 1), in addition to eight known derivatives viz., 18β-glycyrrhetinic acid J1, 18β-gly-
cyrrhetinic acid-30-methyl ester J2, 3-methoxy-18β-glycyrrhetinic acid J3, 3-meth-
oxy-18β-glycyrrhetinic acid-30-methyl ester J4, 3-oxo-18β-glycyrrhetinic acid J5,
apo-allo-18β-glycyrrhetinic-30-methyl ester J6, 3-acetyl-18β-glycyrrhetinic acid J7, 3-acetyl-
18β-glycyrrhetinic-30-methyl ester J8 and 18β-glycyrrhetinic acid-30-allyl ester J13 (Beseda
et al. 2010; Graebin et al. 2010). All 1H- and 13C-NMR data are available online in supplemental
file.
Compound J9 was prepared from J1 by carbamoylation with phenyl-isocyanate (El Sayed
et al. 2008). 1H-NMR spectrum of compound J9 displayed a downfield-shifted signal at δΗ
4.43 (1H, dd, J = 8.0, 4.0 Hz) of a substituted hydroxyl group (H-3). Extra protons of a
mono-substituted phenyl group were detected. Also, a proton signal at δΗ 6.46 (1H, s) was
assigned to the amide proton (NH). Additionally, the shifted carbon signal at δC 81.8 was
assigned to a substituted hydroxymethine carbon (C-3). Extra signals at δC 153.6 of an amide
carbon (CO), and at δC 138.2 (C-1`), δC 123.2 (C-3`, 5`), δC 120.6 (C-4`) and at δC 118.7 (C-2`,
6`) confirmed the presence of an aromatic substituent. Furthermore, EI+ MS spectrum showed
m/z at 590 [M]+ and 545 [M-CO2]+ confirming the molecular formula C37H51NO5. On the basis
of these spectral evidences, the structure of compound J9 was determined to be 3β-phe-
nylcarbamoyl-18β-glycyrrhetinic acid.
Compound J10 was obtained by reacting the acyl chloride of J7 with aniline (Helferich
and Schaefer 1929). In a similar fashion to J9, 1H-NMR spectrum of compound J10 displayed
extra protons of mono-substituted phenyl group. Also, a proton signal at δΗ 4.45 (1H, s) was
assigned to an amide proton (NH). APT experiment displayed quaternary carbon signals at
δC 175.4 assigned to an upfield-shifted carboxylic group at C-30. The carbon signals at δC

NATURAL PRODUCT RESEARCH
3
O
O
29
29
18
30
C
30
C
OR2
OCH3
19
20
17
21
22
28
19
20
17
21
22
28
H
H
12
26
12
26
18
O
11
O
25
11
13
14
25
13
14
9
1
1
9
16
2
3
16
2
3
10
8
15
10
8
15
H
27
H
27
4
7
R1O
5
7
5
24
6
4
6
23
J6
24
23
R1
R2
R2
R1
CH3COO-
CH3
J0
J1
J8
Di-glucuronic acid
H
H
N
3
`
2`
J9
H
H
3`
4`
H
1`
6`
O
5`
2``
3`
6`
1``
2`
4`
5`
CH3
CH3COO-
J2
H
J10
1`
HN
HN
1``
2``
2`
1`
CH3
CH3
CH3COO-
J3
J4
H
J11
J12
OH
1``
2``
4`
HN
CH3
CH3COO-
1`
3`
2`
2`
1`
J5
J7
H
H
J13
J14
H
3`
O
4`
3`
CH3COO-
H
1`
2`
5`
Figure 1. structures of prepared glycyrretinic acid derivatives.
138.2 (C-1`), δC 128.9 (C-3`, C-5`), δC 124.3 (C-4`) and δC 120.2 (C-2`, C-6`) were assigned to
the aromatic substituent at C-30. Also, EI+ MS spectrum shoed m/z at 587 [M]+, 527 [M-H-
acetate]+ and 466 [M-H-benzamido]+ confirming the molecular formula C38H53NO4. Thus, the
structure
of
new
compound
J10
was
determined
to
be
3β-acetyl-18β-glycyrrhetinic-30-anilinamide.
Compound J11 was obtained by reacting the acyl chloride of J7 with 2-amino-ethanol
(Helferich and Schaefer 1929). 1H-NMR spectrum of compound J11 showed extra protons
at δΗ 6.08 (1H, s) of an amide proton (NH) and at δΗ 3.37 (2H, brs, H-1`) & δΗ 3.66 (2H, brs,
H-2`) of ethanolamine moiety. Extra signals at δC 43.9 and 62.6 were assigned respectively
to C-1` and C-2`, of the ethanolamine substituent. Furthermore, EI+ MS spectrum showed
m/z at 555 [M]+, 495 [M-H-acetate]+ and 466 [M-H-ethanolamido]+ confirming the molecular
formula C34H53NO5. Thus, the structure of compound J11 was determined to be
3β-acetyl-18β-glycyrrhetinic-30-ethanolamide.
Compound J12 was obtained by reacting the acyl chloride of J7 with n-butyl-amine
(Helferich and Schaefer 1929).1H-NMR spectrum of compound J12 was very similar to J11
except for the presence of proton signals at δΗ 3.20 (2H, brs, H-1`), δΗ 2.75 (2H, brs, H-2`), δΗ
1.40 (2H, m, H-3`) and at δΗ 0.87 (3H, brs, H-4`) of n-butylamide moiety. The signals at δC 43.3
(C-1`), δC 39.2 (C-2`), δC 20.1 (C-3`), and at δC 13.4 (C-4`) further indicated n-butylamine sub-
stituent. The upfield-shifted carbonyl carbon at δC 175.5 (C-30) indicated n-butylamide sub-
stitution at this position. Additionally, EI+ MS spectrum showed m/z at 567 [M]+, 508
[M-acetate]+ and 466 [M-H-butylamido]+ confirming the molecular formula C36H57NO4. On

4
F. M. ABDEL BAR ET AL.
Table 1. ache-I activity of glycyrrhetinic acid derivatives, their docking scores within the active site of
ache and antioxidant activity (100 μg/ml) represented as % free radical scavenging using aBts·+ assay.
ABTS·+ scavenging activity (%)
Compound
IC50 Activity for AChE-I (μM)
Binding Energy (kcal/mol)
J0
6.27
24.36
> 25
−175.945
−162.354
−146.776
−165.164
−150.589
−142.197
−137.151
−144.3
−159.903
−164.017
−155.731
−171.392
−176.107
−163.465
−150.551
−168.341
8.37
3.14
0
0
0
0
1.04
0
0
0
2.61
10.47
4.71
5.75
4.18
–
J1
J2
J3
8.68
J4
13.46
17.47
> 25
17.23
15.01
17.98
16.09
5.39
3.43
> 25
20.76
1.6 1.02
J5
J6
J7
J8
J9
J10
J11
J12
J13
J14
Galanthamine*
dMso**
*Galantamine was used as a positive control in acetylcholinesterase assay.; **dMso was used as a negative control in wound
healing assay.
the basis of these spectral evidences, the structure of compound J12 was determined to be
3β-acetyl-18β-glycyrrhetinic-30-butylamide.
Compound J14 was prepared from J1 by reaction with prenyl-bromide. 1H-NMR spectrum
of compound J14 displayed signals a signal at δΗ 3.14 (1H, dd, J = 8.0, 4.0 Hz) of unsubstituted
hydroxymethine carbon (H-3). Extra protons were detected at δΗ 5.27 (1H, brs) of an olefinic
proton (H-2`), δΗ 4.45 (2H, m) of an allylic proton (H-1`), δΗ 1.69 (3H, s, H-4`) and δΗ 1.65 (3H,
s, H-5`) of a prenyl group. APT experiment displayed an upfield-shifted signal at δC 176.3
(C-30) was assigned to an esterified carboxylic group at C-30, and a signal at δC 78.8 assigned
to a free hydroxymethine carbon (C-3). Extra signals at δC 18.0 (C-4`), δC 23.3 (C-5`), δC 61.2
(C-1`), δC 128.1 (C-2`), and δC 139.0 (C-3`) were assigned to prenyloxy substituent. Chemical
method was used to confirm the reaction of the prenyl group at C-30 rather than the hydroxyl
group at C-3. This was accomplished by acetylation of compound J14 which produced an
acetyl product indicating a free OH group at C-3 in J14. Furthermore, EI+ MS spectrum
showed m/z at 539 [M + H]+ and 470 [M + H-prenyl]+ confirming the molecular formula
C35H54O4. Therefore, the structure of new compound J14 was determined to be 3β-hy-
droxy-18β-glycyrrhetinic prenyl ester.
2.2. Acetylcholinesterase inhibition activity
AChE inhibition was assessed using Ellman’s method (Thompson et al. 2012), the results are
shown in Table 1. Compounds J12, J11, J0 and J3 showed remarkable AChE inhibition with
IC50 values of 3.43, 5.39, 6.27 and 8.68 μM, respectively, compared to galantamine as a positive
control (IC50 of 1.6 μM). It was revealed that although blocking of the hydroxyl group at C-3
either by sugar (as in J0), CH3 (as in J3) or acetyl (as in J11 & J12) was a common character,
the presence of a hydrogen bond donor group at C-30 in these structures was also crucial
for the inhibitory activity.

NATURAL PRODUCT RESEARCH
5
Figure 2. the binding mode of the most active compounds J0, J3, J11 and J12 with ache (code: 1cFJ)
using Molegro Virtual docker software.
2.3. Docking
To gain insights about the binding interaction of the active compounds with the active site
of AChE, molecular docking experiments were performed (binding energy is shown in Table
1). It is well established that the hydrolysis reaction of ACh is mediated by two sites: the
anionic site that withdraws ACh molecules to the second site; the esteratic site; at which
hydrolysis occurs (Sussman et al. 1991). Another important component of the active center
in AChE is the oxyanion hole which is formed by peptidic NH groups from Gly-118, Gly-119,
and Ala-201 and has an important role in stabilization of high-energy intermediates and the
transition state through hydrogen bonding (Zhang et al. 2002). Figure 2 shows the binding
residues of the most active compounds; J12, J11, J0 and J3. Compound; J12 could bind
through hydrophobic binding to the most important residues of AChE such as the catalytic
triad; Ser-200, Glu-327, His-440, four of the anionic site residues; Asp-72, Tyr-121, Tyr-354
and Trp-279 (Shafferman et al. 1992; Harel et al. 1993) and two residues of the oxyanion hole
residues; Gly-118 and Gly-119.
2.4. ABTS·+ Antioxidant assay (Re et al. 1999)
Upon screening of glycyrrhizin and its derivatives, nearly all compounds showed non-sig-
nificant free radical scavenging activity indicating that this mechanism may not be involved
in the Anti-AD of these compounds (Table 1).

6
F. M. ABDEL BAR ET AL.
Table 2. cell viability and wound healing assays of compounds J0, J3, J11 and J12.
Biocompatibility assays
The rate of the wound healing (%)
Compound code
Cell viability (%)
0 h
4 h
24 h
48 h
32
52.6 1.7
59.13 1.32
34.4 0.41
59.2 2.94
–
J0
99
98
99
96
100
22.8
0.0
0.0
0.0
0.0
0.0
–
11.5 0.3
10.8 0.8
21.3 0.49
4.8 0.82
9.5 1.79
–
19.9 0.09
34 0.7
38.57 0.7
12.4 1.86
31.4 2.57
–
2
J3
J11
J12
dMso
cisplatin
*Biocompatibility assay was done at a safe dose of the compounds at 10 μM to test the effect of the compounds on the
viability and healing activity of the normal cells.
Figure 3. effect of J12 on the wound healing of the normal cells (WI-38). the cells were able to migrate
to decrease the space between each other (black arrow) and the wound size was decreased after 48 h
of incubation.
2.5. Evaluation of Selectivity and Safety of compounds of J0, J3, J11 and J12; cell
viability and wound healing assays, (Abdel Bar 2016)
In order to show the selectivity of the active compounds to AChE and to show that they have
no cytotoxic effect on normal cells, cell viability and wound healing assays were performed.
Over than 95% cell viability was obtained at 12.3 μM for the potential AChE-inhibitors; J0,
J3, J11 and J12 (Table 2). So, the biocompatibility was confirmed by running wound healing
assay at 10 μM of each compound. The cells that were treated with J0 or J3 were able to heal
the wound in a rate closer to the untreated cells. While J11 induced the cell migration to
close the wound and around 91% of wound was healed (See supplemental file). On the other
hand, J12-treated cells healed in a slower rate but it did not effect on the growth of the cells
(black arrows in Figure 3). These data demonstrated that the active compounds are not
cytotoxic to normal cells and J11 has healing activity.

NATURAL PRODUCT RESEARCH
7
3. Experimental
For general experimental procedures, preparation of 18-β-glycyrrhetinic acid derivatives,
acetylcholinesterase inhibitory assay, molecular docking, antioxidant [ABTS·+] assay, evalu-
ation of selectivity and safety for the active compounds see supplemental file.
3.1. Plant material
Liquorice roots were collected in August 2014 from the farm of Faculty of Pharmacy,
Mansoura University, Mansoura, Egypt. After botanical identification by department of
Pharmacognosy, voucher specimens (014-Mansoura-1) were kept at the department and
the roots were cleaned and shade-dried then ground using a tooth miller.
Compound J9, 3-phenyl-carbamoyl-18β-glycyrrhetinic acid: yellowish white powder. Rf 0.81
(Pet. ether-Benzene-EtOAc-Acetic acid, 2:4:1.4:0.1) and H2SO4 spray reagent. EI+ MS (m/z):
590 [M]+ and 545 [M-CO2]+ for the molecular formula C37H51NO5. IRνmax (neat): 3344 cm−1
(NH), 2937 & 2869 cm−1 (CH), 1805, 1730 & 1659 cm−1 (C=O), 1601, 1525, 1444 cm−1 (C=C),
1
690 & 754 cm−1 (aromatic C-H bending). H-NMR (400 MHz, CDCl3, J in Hz): 4.43, dd, (12.0,
4.0, H3); 2.21, s (H9); 5.50 (s, H12); 2.61, dd (8.0, 4.0, H18); 0.89, s (Me23); 0.80, s (Me24); 1.10,
s (Me25); 1.07, s (Me26); 1.19, (s, Me27); 0.83, s (Me28); 1.31, s (Me29); 7.19, dd (8.0, 4.0, H2`/6`);
7.28, d (8.0, H3`/5`); 6.96, d (8.0, H4`); 6.46, s (NH). 13C-NMR (100 MHz, CDCl3): 37.4 (C1); 23.9
(C2); 81.8 (C3); 38.3 (C4); 55.2 (C5); 17.4 (C6); 32.8 (C7); 39.0 (C8); 61.8 (C9); 37.1 (C10); 199.5
(C11); 128.6 (C12); 168.3 (C13); 45.4 (C14); 26.5 (C15); 31.0 (C16); 32.0 (C17); 48.3 (C18); 43.3
(C19); 45.4 (C20); 31.9 (C21); 41.1 (C22); 27.5 (C23); 16.4 (C24); 16.8 (C25); 18.8 (C26); 23.4
(C27); 28.4 (C28); 28.1 (C29); 172.2 (C30); 138.2 (C1`); 123.2 (C2`/6`); 128.8 (C3`/5`); 118.7 (C4`);
153.6 (C1``). Compound J10, 3-acetyl-18β-glycyrrhetinic-30-anilinamide: yellowish white pow-
der. Rf 0.79 (Pet. ether-Benzene-EtOAc-Acetic acid, 2:4:1.4:0.1) and H2SO4 spray reagent. EI+
MS (m/z): 587 [M]+, 527 [M-H-acetate]+ and 466 [M-H-benzamido]+ for the molecular formula
C38H53NO4. IRνmax (neat): 1740, 1700 & 1678 cm−1 (C=O), 1540 & 1520 cm−1 (C=C), 670 cm−1
1
(aromatic C-H bending). H-NMR (400 MHz, CDCl3, J in Hz): 4.04, brd (8.0, H3); 2.22, s (H9);
5.53, s (H12); 2.62, brd (12.0, H18); 1.08, s (Me23); 0.80, s (Me24); 1.18, s (Me25); 1.16, s (Me26);
1.27, s (Me27); 0.80, s (Me28); 1.29, s (Me29); 7.24, brs (H 2`/6`); 7.44, d (8.0, H3`/5`); 7.03, brs
(H4`); 4.45, s (NH); 1.97, s (Me2``). 13C-NMR (100 MHz, CDCl3): 37.7 (C1); 23.6 (C2); 80.7 (C3);
38.1 (C4); 55.2 (C5); 17.5 (C6); 36.3 (C7); 38.9 (C8); 60.7 (C9); 37.0 (C10); 199.3 (C11); 124.3 (C12);
165.2 (C13); 45.4 (C14); 26.7 (C15); 32.3 (C16); 35.5 (C17); 38.9 (C18); 43.3 (C19); 44.9 (C20);
33.8 (C21); 41.1 (C22); 28.1 (C23); 16.0 (C24); 16.5 (C25); 16.7 (C26); 20.8 (C27); 18.6 (C28); 21.5
(C29); 175.4 (C30); 138.2 (C1`); 120.2 (C2`/6`); 129.0 (C3`/5`); 124.2 (C4`); 170.8 (C1``); 21.1
(C2``). Compound J11, 3-acetyl-18β-glycyrrhetinic-30-ethanolamide: yellowish white powder.
Rf 0.10 (Pet. ether-Benzene-EtOAc-Acetic acid, 2:4:1.4:0.1) and H2SO4 spray reagent. EI+ MS
(m/z): 555 [M]+, 495 [M-H-acetate]+ and 466 [M-H-ethanolamido]+ for the molecular formula
C34H53NO5. IRνmax (neat): 3421 cm−1 (OH), 1735 & 1652 cm−1 (C=O), 1540 & 1458 cm−1 (C=C).
1H-NMR (400 MHz, CDCl3, J in Hz): 4.45, dd (12.0, 4.0, H3); 2.21, s (H9); 5.50, s (H12); 2.64, dd
(8.0, 4.0, H18); 0.81, s (Me23); 0.81, s (Me24); 1.07, s (Me25); 1.15, s (Me26); 1.20, s (Me27); 0.81,
s (Me28); 1.27, s (Me29); 3.37, brs (H1`); 3.66, brs (H2`); 6.08, s (NH); 1.97, s (Me2``). 13C-NMR
(100 MHz, CDCl3): 37.7 (C1); 23.6 (C2); 80.7 (C3); 38.0 (C4); 55.2 (C5); 17.5 (C6); 33.8 (C7); 38.9
(C8); 60.7 (C9); 36.8 (C10); 199.4 (C11); 124.1 (C12); 165.5 (C13); 45.4 (C14); 26.7 (C15); 31.5
(C16); 32.8 (C17); 48.2 (C18); 43.7 (C19); 45.0 (C20); 31.9 (C21); 42.6 (C22); 28.1 (C23); 16.0

8
F. M. ABDEL BAR ET AL.
(C24); 16.5 (C25); 16.7 (C26); 20.8 (C27); 18.6 (C28); 21.4 (C29); 179.7 (C30); 43.9 (C1`); 62.6
(C2`); 170.8 (C1``); 21.1 (C2``). Compound J12, 3-acetyl-18β-glycyrrhetinic-30-n-butylamide:
yellowish white powder. Rf 0.71 (Pet. ether-Benzene-EtOAc-Acetic acid, 2:4:1.4:0.1) and H2SO4
spray reagent. EI+ MS (m/z): 567 [M]+, 508 [M-acetate]+ and 466 [M-H-butylamido]+ for the
molecular formula C36H57NO4. IRνmax (neat): 3394 cm−1, (NH), 2965, 2930 & 2870 cm−1 (C-H)
1
and 1735 & 1650 cm−1 (C=O), 1532 & 1462 cm−1 (C=C). H-NMR (400 MHz, CDCl3, J in Hz):
4.43, dd (12.0, 4.0, H3); 2.21, s (H9); 5.50, s (H12); 2.64, dd (8.0, 4.0, H18); 0.81, s (Me23); 0.75,
s (Me24); 1.06, s (Me25); 1.06, s (Me26); 1.10, s (Me27); 0.81, s (Me28); 1.30, s (Me29); 3.20, brs
(H1`); 2.75, brs (H2`); 1.40, m (H3`); 0.87, brs (Me4`); 5.49, s (NH); 1.97, s (Me2``). 13C-NMR
(100 MHz, CDCl3): 37.5 (C1); 23.6 (C2); 80.7 (C3); 38.1 (C4); 55.1 (C5); 17.4 (C6); 32.8 (C7); 38.9
(C8); 61.8 (C9); 37.1 (C10); 199.7 (C11); 128.5 (C12); 169.0 (C13); 45.4 (C14); 26.5 (C15); 31.6
(C16); 31.9 (C17); 48.3 (C18); 42.1 (C19); 43.5 (C20); 31.9 (C21); 42.6 (C22); 28.1 (C23); 16.3
(C24); 16.6 (C25); 18.8 (C26); 23.3 (C27); 29.6 (C28); 28.5 (C29); 175.5 (C30); 43.3 (C1`); 39.2
(C2`); 20.1 (C3`); 13.4 (C4`); 170.8 (C1``); 21.1 (C2``). Compound J14, 18β-glycyrrhetinic acid-
30-prenyl ester: yellowish white powder. Rf 0.40 (Pet. ether-EtOAc 4:1) and H2SO4 spray rea-
gent. EI+ MS (m/z): 539 [M + H]+ and 470 [M + H-prenyl]+ for the molecular formula C35H54O4.
1
IRνmax (neat): 3420 cm−1 (OH), 2927 & 2868 cm−1 (C-H), 1727 & 1659 cm−1 (C=O). H-NMR
(400 MHz, CDCl3, J in Hz): 3.14, dd (8.0, 4.0, H3); 2.27, s (H9); 5.56, s (H12); 2.71, brd (12.0, H18);
0.93, s (Me23); 0.73, s (Me24); 1.06, s (Me25); 1.06, s (Me26); 1.06, s (Me27); 0.73, s (Me28); 1.36,
s (Me29); 4.45, m (H1`); 5.27, brs, H2`); 1.69, s (Me4`); 1.65, s (Me5`). 13C-NMR (100 MHz, CDCl3):
37.7 (C1); 26.6 (C2); 78.8 (C3); 39.1 (C4); 55.1 (C5); 17.5 (C6); 32.9 (C7); 39.1 (C8); 61.9 (C9); 37.2
(C10); 200.0 (C11); 128.6 (C12); 169.0 (C13); 45.4 (C14); 27.4 (C15); 31.3 (C16); 31.8 (C17); 48.4
(C18); 43.3 (C19); 44.0 (C20); 31.8 (C21); 41.3 (C22); 28.1 (C23); 15.5 (C24); 16.3 (C25); 18.8
(C26); 25.6 (C27); 28.5 (C28); 28.2 (C29); 176.3 (C30); 61.2 (C1`); 118.8 (C2`); 139.0 (C3`); 18.0
(C4`); 23.3 (C5`).
4. Conclusion
This is a further study on the possible contribution of the commonly known triterpene
‘Glycyrrhizin’ as well as its synthetic derivatives as potential AChE-Is. Synthetic reactions
yielded five new and nine known derivatives. Compounds J12, J11, J0 and J3 showed the
strongest inhibitory activity of AChE. These results are in full agreement with docking study.
The compounds showed non-significant antioxidant power that excluded such pathway in
their AD effect. Furthermore, the most active analogs were investigated for their safety and
selectivity using biocompatibility assays. The results demonstrated that the active com-
pounds were not cytotoxic to normal cells and the cells were able to heal the wound in a
rate closer to the untreated cells.
Disclosure statement
No potential conflict of interest was reported by the authors.
References
Abdel Bar FM. 2016. Dihydropyridinone alkaloid artifacts from Curcuma longa and their anti-migration
activity against HepG2 Cells *. Rec Nat Prod. 10:582–592.

NATURAL PRODUCT RESEARCH
9
Beseda I, Czollner L, Shah PS, Khunt R, Gaware R, Kosma P, Stanetty C, Ruiz-Ruiz MC, Amer H, Mereiter
K, et al. 2010. Synthesis of glycyrrhetinic acid derivatives for the treatment of metabolic diseases.
Bioorg Med Chem. 18:433–454.
El Sayed KA, Laphookhieo S, Yousaf M, Prestridge JA, Shirode AB, Wali VB, Sylvester PW. 2008.
Semisynthetic and Biotransformation Studies of (1 S ,2 E ,4 S ,6 R ,7 E ,11 E )-2,7,11-Cembratriene-
4,6-diol. J Nat Prod. 71:117–122.
Graebin CS, Verli H, Guimarães JA. 2010. Glycyrrhizin and glycyrrhetic acid: scaffolds to promising new
pharmacologically active compounds. J Braz Chem Soc. 21:1595–1615.
Gurovic MS, Castro MJ, Richmond V, Faraoni MB, Maier MS, Murray AP. 2010. Triterpenoids with
acetylcholinesterase inhibition from Chuquiraga erinacea D. Don. subsp. erinacea (Asteraceae).
Planta Med. 76:607–610.
Harel M, Schalk I, Ehret-Sabatier L, Bouet F, Goeldner M, Hirth C, Axelsen PH, Silman I, Sussman JL. 1993.
Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase.
Proc Natl Acad Sci. 90:9031–9035.
Helferich B, Schaefer W. 1929. Organic Synthesis JB Conant ed. 9:32.
McGleenon BM, Dynan KB, Passmore AP. 1999. Acetylcholinesterase inhibitors in Alzheimer’s disease.
Br J Clin Pharmacol. 48:471–480.
Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. 1999. Antioxidant activity applying
an improved ABTS radical cation decolorization assay. Free Radic Biol Med. 26:1231–1237.
Schwarz S, Lucas S, Sommerwerk S, Csuk R. 2014. Amino derivatives of glycyrrhetinic acid as potential
inhibitors of cholinesterases. Bioorg Med Chem. 22:3370–3378.
Shafferman A, Velan B, Ordentlich A, Kronman C, Grosfeld H, Leitner M, Flashner Y, Cohen S, Barak D,
Ariel N. 1992. Substrate inhibition of acetylcholinesterase: residues affecting signal transduction
from the surface to the catalytic center. EMBO J. 11:3561.
Sharma V, Agrawal RC. 2013. Glycyrrhiza glabra: A plant for the future. MJPMS. 2:15–20.
Sussman JL, Harel M, Frolow F, Oefner C, Goldman A, Toker L, Silman I. 1991. Atomic structure of
acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science.
253:872–879.
Thompson PA, Wright DE, Counsell CE, Zajicek J. 2012. Statistical analysis, trial design and duration in
Alzheimer’s disease clinical trials: a review. Int psychogeriatrics. 24:689–697.
ZhangY, Kua J, McCammon JA. 2002. Role of the catalytic triad and oxyanion hole in acetylcholinesterase
catalysis: an ab initio QM/MM study. J Am Chem Soc. 124:10572–10577.