Chemoenzymatic synthesis of new derivatives of glycyrrhetinic acid with antiviral activity. Molecular docking study

Article abstract of DOI:10.1016/j.bioorg.2018.03.018

We present an efficient approach to the synthesis of a series of glycyrrhetinic acid derivatives. Six derivatives, five of them new compounds, were obtained through chemoenzymatic reactions in very good to excellent yield. In order to find the optimal reaction conditions, the influence of various parameters such as enzyme source, nucleophile:substrate ratio, enzyme:substrate ratio, solvent and temperature was studied. The excellent results obtained by lipase catalysis made the procedure very efficient considering their advantages such as mild reaction conditions and low environmental impact. Moreover, in order to explain the reactivity of glycyrrhetinic acid and the acetylated derivative to different nucleophiles in the enzymatic reactions, molecular docking studies were carried out. In addition, one of the synthesized compounds exhibited remarkable antiviral activity against TK + and TK- strains of Herpes simplex virus type 1 (HSV-1), sensitive and resistant to acyclovir (ACV) treatment.

Full text of DOI:10.1016/j.bioorg.2018.03.018

Accepted Manuscript
Chemoenzymatic synthesis of new derivatives of glycyrrhetinic acid with anti-
viral activity. Molecular docking study
M. Antonela Zígolo, Maximiliano Salinas, Laura Alché, Alicia Baldessari,
Guadalupe García Liñares
PII:
S0045-2068(18)30089-0
DOI:
https://doi.org/10.1016/j.bioorg.2018.03.018
YBIOO 2308
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
22 January 2018
9 March 2018
18 March 2018
Please cite this article as: M. Antonela Zígolo, M. Salinas, L. Alché, A. Baldessari, G.G. Liñares, Chemoenzymatic
synthesis of new derivatives of glycyrrhetinic acid with antiviral activity. Molecular docking study, Bioorganic
Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.03.018
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Chemoenzymatic synthesis of new derivatives of glycyrrhetinic acid
with antiviral activity. Molecular docking study
M. Antonela Zígolo,a Maximiliano Salinas,b Laura Alché,b Alicia Baldessaria and
Guadalupe García Liñaresa*
aLaboratorio de Biocatálisis. Departamento de Química Orgánica y UMYMFOR, Facultad de Ciencias Exactas y Naturales,Universidad
de Buenos Aires, Ciudad Universitaria,Pabellón 2, piso 3, C1428EGA Buenos Aires, Argentina.
b
Laboratorio de Virología, Departamento de Química Biológica e IQUIBICEN; Facultad de Ciencias Exactas y Naturales, Universidad
de Buenos Aires. Ciudad Universitaria, Pabellón 2, piso 4, C1428EGA, Buenos Aires, Argentina.
Abstract
We present an efficient approach to the synthesis of a series of glycyrrhetinic acid
derivatives. Six derivatives, five of them new compounds, were obtained through
chemoenzymatic reactions in very good to excellent yield. In order to find the optimal
reaction conditions, the influence of various parameters such as enzyme source,
nucleophile:substrate ratio, enzyme:substrate ratio, solvent and temperature was studied.
The excellent results obtained by lipase catalysis made the procedure very efficient
considering their advantages such as mild reaction conditions and low environmental
impact. Moreover, in order to explain the reactivity of glycyrrhetinic acid and the
acetylated derivative to different nucleophiles in the enzymatic reactions, molecular
docking studies were carried out. In addition, one of the synthesized compounds exhibited
remarkable antiviral activity against TK+ and TK- strains of Herpes simplex virus type 1
(HSV-1), sensitive and resistant to acyclovir (ACV) treatment.
Keywords: Glycyrrhetinic acid; lipase-catalyzed; molecular modeling; antivirals
Corresponding author. Tel.: 5411-4576-3300 ext. 262; fax: 5411-4576-3385; e-mail: alib@qo.fcen.uba.ar

1. Introduction
The pentacyclic triterpenoid glycyrrhetinic acid (GA) is the major bio-active constituent
isolated from the roots of Glycyrrhiza glabra, which is a sweet-tasting material and has
greater sweetening power than sugar, making it a widely used as additive in the food
industry [1-3]. GA has been used as a lead compound to search more potent derivatives
with different pharmacological properties. Glycyrrhetinic acid and some derivatives have
been shown to exhibit anti-inflammatory [4, 5] and anti-viral [6, 7] activities. In addition, it
has been reported derivatives of GA as inhibitors of cholinesterases [8] and 11-HSD1 and
11-HSD2 [9-11]. Despite these reports, few potential applications of GA derivatives for
the development of new pharmacological agents have been investigated.
The application of enzymes as biocatalysts in the synthesis and transformation of different
substrates with engaging properties has widely expanded in the last years [12-14]. Enzymes
catalyze diverse type of reactions in a highly regio, chemo, and enantioselective way using
a wide ranging of compounds. Therefore, biocatalysts constitutes a good alternative to
obtaining of compounds through a Green Chemistry approach carrying out chemical
transformations more easly, particularly in substrates with several functional groups [15-
19]. In our laboratory we have studied the application of lipases in diverse reactions like
esterifications, transesterifications and aminolysis of multiple substrates, obtaining a wide
diversity of new compounds, which show interesting biological activities [20, 21].
Recently, we have reported the synthesis of numerous compounds with potential
applications as antiparasitic [22, 23], antitumoral [24] and antiviral agents [25].
Continuing with our project on enzymatic transformations, we report here the results from
the application of lipases in the preparation of a series of glycyrrhetinic acid derivatives.
In addition, all synthesized compounds were biologically evaluated as potential antiviral
agents against TK+ and TK- strains of Herpes simplex virus type 1 (HSV-1), sensitive and
resistant to acyclovir (ACV) treatment, respectively.
HSV-1 is a member of the Herpesviridae family and infects a high proportion of human
population, causing a range of diseases from mild uncomplicated mucocutaneous to more
serious infections, such as keratitis and encephalitis. HSV-1 is the causal agent of cold sore
and encephalitis [26]. The current gold standard treatment for HSV-1 infections is ACV, a

guanosine analogue antiviral drug. With the emergence of Herpesvirus strains resistant to
nucleoside analogues, including ACV, there is an urgent need for new and more effective
treatments for HSV infections [27]. In this work, we evaluated the antiviral capacity of five
glycyrrhetinic acid (GA) derivatives against HSV-1.
2. Results and Discussions
Many glycyrrhetinic acid derivatives were chemically synthesized and displayed a wide
spectrum of applications in different areas. Some of them, with modifications in C-3, C-11
and C-29 positions, were used in the treatment of metabolic diseases [28]. Other derivatives
with modifications at C-3 and C-30, showed a selective inhibition of the enzyme BChE [8].
Some chemically prepared alkanolamides, amides and esters derived from glycyrrhetinic
acid had a high anti-inflammatory activity [11].
In this section we are going to describe the results obtained by applying lipases as catalysts
for obtaining derivatives of glycyrrhetinic acid (GA, 1).
Our initial experiments to obtain the ester derivatives were performed using 1 as substrate
and ethanol as nucleophile. We studied the esterification catalyzed by lipases from several
sources: from Candida antarctica B (CAL B), from Candida rugosa (CRL), Lipozyme
from Rhizomucor miehei (LIP), Rhizopus oryzae lipase (ROL), Lipozyme from
Thermomyces lanuginosus (TLL) and from Carica papaya (CPL), which is the remaining
solid fraction of papaya latex after wash off of proteases, in a variety of solvents of
different polarity (acetonitrile, acetone, diisopropyl ether, dioxane, hexane and toluene).
Unfortunately, none of the enzymes tested catalyzed the esterification reaction of 1 with
ethanol.
Then, in order to obtain amides derivatives, we tried the reaction of 1 with n-butylamine as
model nucleophile. Several different conditions (diverse lipases and solvents) were applied
but the same unsatisfactory results were observed.
Additionally, taking into account the previous successful results in the enzymatic
esterification reaction of acetylated bile acids [21, 22], we examined the possibility of
acetylate the OH group of 1 using ethyl acetate as acylating agents and different lipases as
biocatalysts at several conditions but no reaction was observed. The reaction with activated

acylating agents such as vinyl or isopropenyl acetate or other acylating agents like ethyl
caproate or diethyl succinate conducted to the same unsuccessful results.
From these results and considering the work previously reported [22], we carried out the
chemical acetylation of 1 obtaining 3-acetyl glycyrrhetinic acid (2) in excellent yield
(98%). Then, we studied the esterification (with ethanol) and amidation (with butylamine)
reactions of 2 using several lipases in various solvents at different temperatures, but again
no reaction was observed. In summary, under all reaction conditions, neither 1 nor 2 proved
to be good substrates for nucleophilic agents such as ethanol or n-butylamine.
Lastly, we decided to study the reaction of 1 and 2 with alkanolamines as nucleophiles. We
carried out the lipase catalyzed reaction with ethanolamine (3a) applying the optimal
conditions previously determined for other similar substrates: CAL B as biocatalyst (E/S:
10), hexane as solvent, temperature: 55 °C, a Nu/S: 5. In this case, we observed that while 1
did not reacted, 2 led to the corresponding ethanolamide (4a) (Scheme 1). Therefore,
considering this result, in order to optimize the reaction conditions, we performed different
experiments using 2 as substrate and varying the lipase, solvent, enzyme:substrate ratio
(E/S), nucleophile:substrate ratio (Nu/S) and temperature.
Scheme 1. Chemoenzymatic synthesis of glycyrrhetinic acid derivatives
2.1. Amidation of 3-acetyl glycyrrhetinic acid (2)
Six commercial lipases from different sources were evaluated for enzymatic amidation with
ethanolamine: from the yeasts Candida rugosa (CRL), Candida antarctica B (CAL B) and
Candida antarctica lipase B (CAL B immo plus); from the fungus Rhizomucor miehei
(LIP), Rhizopus oryzae lipase (ROL) and Thermomyces lanuginosus (TLL).
Several solvents were also assayed: hexane, diisopropyl ether, acetonitrile and acetone. As
it was mentioned, the reactions were carried out at 55 °C using E/S: 10 and Nu/S: 5. It was
observed that enzyme activity was variable, giving CAL B the most satisfactory results in

acetonitrile; CAL B was also active in DIPE and hexane, but to a lesser extent (Figure 1).
LIP in DIPE and hexane has also showed activity but with a lower performance than CAL
B showing conversion 53% at 96 h and 46% at 72 h, respectively, whereas with CRL,
ROL, TLIM the enzyme activity were very low. In case of CAL B immo plus, no product
was observed under the conditions tested. In the absence of biocatalyst no product was
detected.
Figure 1. Screening of biocatalyst and solvent for reaction of 2 with ethanolamine at 55 °C
using E/S: 10 and Nu/S: 5.
It is important to note the chemoselective behavior of CAL B since the ethanolamide was
the only obtained product; the isomeric aminoester was not detected. Under chemical
conditions, alkanolamines are susceptible for acylation both at the amino and hydroxy

group. These results are in coincidence with studies which report the same chemoselective
behavior of lipase, solely affording the amide [20, 24, 29-31].
Once determined the appropriate lipase and solvent, the influence of the enzyme:substrate
ratio on this reaction was evaluated at 48 h, using a Nu/S ratio of 5, acetonitrile as solvent
at 55 °C and variable amounts of CAL B. From the results, it can be concluded that a ratio
E/S of 10 is the most acceptable (Figure 2).
100
80
60
40
20
0
1
2
5
10
20
Enzyme/Substrate ratio
Figure 2. Influence of E/S ratio on reaction of 2 with ethanolamine. Reaction conditions:
Enzyme: CAL B; Solvent: Acetonitrile; Temperature: 55 °C; Nu/S: 5; time: 48 h.
100
80
60
40
20
0
1
2
5
10
15
Nucleophile/Substrate ratio
Figure 3. Influence of Nu/S ratio on reaction of 2 with ethanolamine. Reaction conditions:
Enzyme: CAL B; Solvent: Acetonitrile; Temperature: 55 °C; E/S: 10; time: 48 h.

Similarly, the influence of the Nu/S ratio over amidation yield was evaluated at 48 h in
acetonitrile using CAL B (E/S = 10) at 55 °C and variable amounts of nucleophile. As it
was observed, a ratio Nu/S of 5 is enough to afford the best conversion (Figure 3).
Finally, we performed the reaction at different temperatures setting the other reaction
parameters to their optimal determined values (CAL B, acetonitrile, E/S: 10 and Nu/S: 5).
Results showed higher yield with the increase in the temperature. Therefore we selected 55
°C as the best reaction temperature.
Considering the previously mentioned experiments, the determined conditions were applied
for enzymatic reactions of 2 with different alkanolamines (3a-d) obtaining only the
products of amidation (4a-d) in very good yields (Table 1).
Table 1. Enzymatic synthesis of 3-acetylglycyrrhetinic acid derivatives
Nucleophile
H2NCH2CH2OH
H2N(CH2)4OH
Producta
Yield (%)b
83
76
68
73
86
4a
4b
4c
4d
5
H2NCH(CH3)CH2OH
H2NCH2CH(OH)CH3
HO(CH2)4OH
a Reaction conditions: Enzyme: CAL B; Solvent: AcCN; Temperature: 55 °C;
E/S: 10; Nu/S: 5; t: 48 h. b Isolated yield after column chromatography.
As it was exhibited, the acetylated glycyrrhetinic acid (2) lacked reactivity to both alcohols
and amines, whereas amidation reaction was observed in the case of the reaction with
alkanolamines. In view of these experimental results, new questions arose regarding the
chemical structure of the nucleophile used. Therefore, it was of interest to study the
reaction of 2 with diols and diamines, using 1,4-butanediol and 1,4-diaminobutane as
model nucleophiles. The reaction conditions employed were those optimized for the
amidation reaction of 2 with ethanolamine: CAL B as biocatalyst, temperature: 55 ° C, E/S:
10, Nu/S: 5 and acetonitrile as solvent.

We observed that in the case of reaction with 1,4-butanediol, the corresponding
hydroxyester (5) was obtained in 86% yield at 48 h reaction (Table 1). However with 1,4-
diaminobutane no product was observed. These results are very interesting and suggest that
the presence of an additional hydroxyl group in the nucleophile is essential for the reaction
to occur.
2.2. Molecular modeling
In order to comprehend the lack of reactivity of 1 to the studied reactions, as well as the
reactivity of 2 to specific nucleophiles, we applied computational analysis. Using molecular
modeling we were able to understand the structural and functional relationship between the
active site of the lipase and the ligand, giving an idea of the selective biological behavior of
the enzyme on this family of compounds. In addition, the differences observed for
substrates 1 and 2 and the different reactivity of acetylated glycyrrhetinic acid (2) with
alcohols, amines, alkanolamines, diols and diamines were explained.
Taking into account the interaction distances with the aminoacids of the catalytic site
(Asp187-His224-Ser105), the binding energy and the population of the clusters, we have
selected an optimal interaction conformation between the enzyme and each substrate.
Initially, we carried out docking between CAL B and substrates 1 and 2. In the case of
compound 2, an appropriate interaction conformation was chosen, on which a second
computational study was carried out to evaluate the different nucleophiles used in the
reactions.

Figure 4. Docking of glycyrrhetinic acid (1) with CAL B. Interaction conformation obtained for the
single cluster found at the active site of lipase. Catalytic triad: Serine 105 (green), Histidine 224
(blue), Aspartic acid 187 (red). Oxyanion hollow: Glutamine 106 (brown), Threonine 40 (pink)
Docking evaluations were performed for glycyrrhetinic acid (1) and acetylated
glycyrrhetinic acid (2) generating two hundred conformers for each one using the
Lamarckian genetic algorithm. In the case of 1, a single cluster was obtained, in which the
glycyrrhetinic acid get into the active site through the hydroxyl group, located at 2.92 Å
from the catalytic serine and having a binding energy of -3.05 kcal/mol (Figure 4). For
compound 2, four clusters were found; the binding energies and the corresponding
frequencies are shown in the Figure 5. The interaction conformations of these clusters in
the active site of lipase and its arrangement with respect to catalytic triad are shown in the
Figure 6.

Figure 5. Histogram corresponding to the docking of acetylated glycyrrhetinic acid (2) with CAL
B.
Figure 6. Docking of acetylated glycyrrhetinic acid (2) with CAL B. Conformation interaction
acquired for each cluster at the active site of lipase. Cluster 1 (white); cluster 2 (light blue); cluster 3
(violet); cluster 4 (pink). Catalytic triad: Serine 105 (green), Histidine 224 (blue), Aspartic acid 187
(red). Oxyanion hollow: Glutamine 106 (light blue), Threonine 40 (pink)
According to the results obtained, docking of 1 with CAL B shows that although this
substrate is located at a short distance from the active site, it has higher binding energy than
2. Consequently, the reaction is less favorable. Besides, 1 gets into the active site of the
lipase through the hydroxyl group, while 2 enters through the carboxylic acid group, as it is
observed on the obtained clusters with binding energies between -7.60 and -6.45 kcal/mol
(Figure 5).
Finally we have evaluated the effect of the different nucleophiles in the reactions with 2.
We have chosen cluster 2, the most populated since it was the most similar to the
experimentally acquired conformation. In order to explain the experimental results, a
second docking study was carried out between CAL B-cluster 2 and the five tested type of

nucleophiles: alcohol (ethanol), amine (butylamine), alkanolamine (ethanolamine), diol
(1,4-butanediol) and diamine (1,4-diaminobutane).
Results showed four clusters for ethanol, three clusters for ethanolamine, five clusters for
butylamine and a only cluster for 1,4-butanediol and 1,4-diaminobutane. In all cases the
most populous cluster corresponds to the lowest energy and corresponds to the interaction
conformations of greater similarity to those adopted experimentally.
Figure 7. Docking between 2 (conformation 2) and: A) ethanol, B) butylamine, C) 1,4-
diaminobutane.
Figure 8. Docking between 2 (conformation 2) and: A) ethanolamine, B) 1,4-butanediol.
It has been observed that in the cases of docking with ethanol, butylamine and 1,4-
diaminobutane, nucleophiles are far away from the catalytic serine in the most likely

interaction conformation what could explain the lack of reactivity (Figure 7). When
ethanolamine was used as nucleophile, the hydroxyl group of the nucleophile is very close
to the oxygen of the carbonyl group of the acylated glycyrrhetinic acid interacting through
hydrogen bond. Therefore, the amino group is placed at an appropriate distance to react.
This would allow a greater stabilization of the tetrahedral intermediate allowing this
nucleophile to approximate enough to the acyl enzyme complex and to react (Figure 8A).
In addition, the same hydrogen bond stabilization occurs in the case of 1,4-butanediol
finding both hydroxyl groups of nucleophile very close to carbonyl moiety of 2, which
might explain the observed reactivity (Figure 8B).
2.3. Biological evaluation
We first evaluated cytotoxicity of GA and compounds 1, 2 and 4a-d at concentrations
ranging from 1 to 200 µM. Each one was dissolved in dimethylsulfoxide (DMSO) and
diluted in culture medium. The maximum concentration of DMSO tested (1%) exhibited no
cytotoxicity for Vero cells. After 24 h incubation at 37 °C, cell viability was determined
(Figure 9A).
The derivatives showed a CC50 value higher than 200 µM, except for compound 4d which
exhibited a CC50 value of 190.2 µM (Figure 8A). All compounds exhibited CC50 values
higher than that corresponding to GA (61 µM). Hence, all compounds tested were no
cytotoxic for Vero cells.

Figure 9. Cytotoxicity of: (A) glycyrrhetinic acid (GA) (□) and compounds 2 (△), 4a (○), 4b (▲),
4c (◇) and 4d (●); (B) stereoisomers of 4d: 4d(S) (◒) and 4d(R) (◓). Vero cells were grown in 96-
well cell plates and treated or not with the compounds (1-200 µM). After 24 h incubation at 37 °C,
cell viability was measured by means of a MTT colorimetric assay.
Then, to evaluate a potential antiviral action of all compounds, first we performed a
qualitative screening. Compounds 2, 4a-c and 5 showed no anti-HSV-1 activity. As
expected, 50 µM GA inhibited HSV-1-induced cytopathic effect in a 75% with respect to
infected untreated controls, whereas no protection of cellular damage was observed in the
presence of 10 µM.[32] Derivative 4d showed a similar reduction of cytopathic effect at 50
µM and, besides, 10 µM of compound 4d protected 25% of infected cells, concentration at
which GA had already lost the antiviral activity.
To evaluate whether this protection correlated with a decrease in viral infectivity, viral
yields belonging to those wells where protection was observed were harvested and titrated
in Vero cells. Compound 4d was able to prevent HSV-1 multiplication when added after
infection in a dose-dependent manner, exhibiting an EC50 of 4.95 µM. In this case, the
selectivity index (SI = CC50/EC50) was 38.38 (Figure 10). Taking into account that
compound 4d was tested as a mixture of the stereoisomers, we performed the racemic
resolution of (±)-1-amino-2-propanol.[33, 34]
Once the two pure enantiomers of (±)-1-amino-2-propanol were obtained, the reaction of 2
with (S)-(+)-1-amino-2-propanol and (R)-(-)-1-amino-2-propanol was carried out. The
corresponding stereoisomeric alkanolamides, which denominated 4d(S) and 4d(R), were
obtained with 75% yield.
In order to investigate if the antiviral activity would be ascribed to one of the two
stereoisomers of the molecule, after Vero cell infection with HSV-1 (m.o.i. = 0.1), different
concentrations of the stereoisomeric mixture, 4d(S) and 4d(R) were added and, after 24 h
incubation at 37 ºC, supernatants were titrated in Vero cells. As expected, the
stereoisomeric mixture followed a dose-response curve with a EC50 of 6.2 µM (Figure
10A). Noteworthy, 4d(R) slightly restrained viral replication starting from a concentration
of 25 µM, while 4d(S) was the stereoisomer that gathered most of the anti-HSV-1 activity,

with an EC50 value of 1.92 µM and a SI > 104.19. None of both stereoisomers were
cytotoxic for Vero cells (Figure 10B).
Figure 10. Antiviral activity of compounds 4d (○), 4d(S) (□) and 4d(R) (△). Vero cells grown in
96-well plates were infected with a m.o.i = 0.1 of HSV-1 KOS strain (A) and HSV-1 B2006 strain
(B), and treated or not with the three compounds. After 24 h of incubation at 37 °C, virus was
collected and titrated in Vero cells.
ACV and related nucleoside analogues can successfully mitigate HSV infections, but the
emergence of HSV-1 populations resistant to ACV has created a barrier for treatment,
especially in the case of immunocompromised patients. Thus, we evaluated the antiviral
action of the stereoisomeric mixture and the two stereoisomers against HSV-1 ACV-
resistant strain (B2006). We observed the same profiles of dose-dependent inhibition, with
an EC50 of 8.67 µM for compound 4d and 9.89 µM for 4d(S), and a slight inhibition for the
4d(R) (Figure 10B).
3. Materials and methods
3.1. General
Solvents and Chemicals were purchased from Merck Argentina and Sigma-Aldrich
Argentina and used without further purification. Lipase from Candida rugosa (CRL) (905
U/mg solid) was purchased from Sigma Chemical Co.; Candida antarctica lipase B (CAL
B): Novozym 435 (7400 PLU/g), Lipase from Rhizomucor miehei (LIP): Lipozyme® RM

1M, Novozymes (7800 U/g), Candida antarctica lipase B (CAL B immo plus): Purolite
(9550 PLU/g) and lipase from Thermomyces lanuginosus (TLL): Sigma Aldrich (3000 U/g)
were generous gifts of Novozymes Spain; lipase from Rhizopus oryzae (ROL) is a
heterologous lipase, which was produced by Chemical Engineering Department of
Barcelona Autonomous University; Carica papaya lipase (CPL) is the remaining solid
fraction of papaya latex, after wash off of proteases using distilled water. CPL is a naturally
immobilized enzyme and was a generous gift of Dr. Georgina Sandoval, CIATEJ, Mexico.
E/S: enzyme amount in mg/substrate amount in mg. Enzymatic reactions were carried out
on MaxQ 4000 Thermo Scientific Co. digital incubator shaker, at the corresponding
temperature and 200 rpm. To monitor the reaction progress aliquots were withdrawn and
analyzed by TLC performed on commercial 0.2 mm aluminum-coated silica gel plates
(F254) and visualized by immersion in an aqueous solution of (NH4)6Mo7O24.4H2O (0.04
M), Ce(SO4)2 (0.003 M) in concentrated H2SO4 (10%). % Conversion was determined
using a Waters 1515 HPLC equipped with an isocratic pump, manual injector and Waters
2414 refractive index detector was used. The reactions were monitored employing a C-18
Phenomenex Phenogel column 5 M 10E5A, 300 x 7.8 mm and eluting with AcCN:H2O
60:40-acetic acid 1% at 1.00 mL/min. Melting points were measured in a Fisher Johns
apparatus and are uncorrected. Optical rotation values were measured in a CHCl3 solution
1
13
with a Perkin Elmer-343 automatic digital polarimeter at 25 °C. H NMR and C NMR
spectra were recorded at room temperature in CDCl3 as solvent using a Bruker AM-500
1
13
NMR instrument operating at 500.14 MHz and 125.76 MHz for H and C respectively.
1
The H NMR spectra are referenced with respect to the residual CHCl3 proton of the
13
solvent CDCl3 at  = 7.26 ppm. Coupling constants are reported in Hertz (Hz). C NMR
spectra were fully decoupled and are referenced to the middle peak of the solvent CDCl3 at
 = 77.0 ppm. Splitting patterns are designated as: s, singlet; d, doublet; t, triplet; q,
quadruplet; qn, quintet; dd, double doublet, etc. High Resolution Mass Spectrometry was
recorded with Thermo Scientific EM/DSQ II – DIP. The results were within ±0.02% of the
theoretical values.
3.2. Chemical acetylation

In a typical procedure, glycyrrhetinic acid (100 mg) was heated at room temperature with
acetic anhydride (2 mL) and pyridine (1 mL) for 16 h. After completion of the reaction, the
mixture was partitioned between saturated solution ammonium chloride (10 mL) and
methylene chloride (10 mL). The aqueous phase was extracted with methylene chloride (3
x 10 mL). The combined organic layers were washed with saturated solution of sodium
chloride (5 x 10 mL), dried (MgSO4), and the solvent was evaporated. The residue was
purified by column chromatography (silica gel) employing mixtures of hexane/EtOAc as
eluent (7:3–1:9). Yield 98%.
3.3. Enzymatic reaction with alkanolamines. General procedure.
To a solution of 3-acetylglycyrrhetinic acid (2) (50 mg) in acetonitrile (5 mL), CAL B (500
mg) and the corresponding alkanolamine (1.2 eq) was added. The mixture was shaken at
200 rpm and 55 °C. Once the reaction was finished (48 h), the enzyme was filtered off and
the solvent evaporated under reduced pressure. The residue was purified by column
chromatography (silica gel) employing mixtures of hexane-EtOAc as eluent (4:1 - 1:4).
Reuse experiments: the filtered and washed enzyme was used in the next enzymatic
esterification under the same reaction conditions.
3-Acetylglycyrrhetinic acid (2): Yield 98% of pure compound as a white solid; mp 311–
312 °C (ref. 28: 310-313 °C); []D25 +162.8° (c 1.0, CHCl3) (ref. 28: []D25 + 163.3° (c 1.0,
CHCl3)). 1H NMR (CDCl3, 500 MHz)  5.70 (s, 1H, H-12), 4.52 (dd, J = 4.6, 11.7 Hz, 1H,
H-3), 2.80 (dt, J = 3.8, 13.8 Hz, 1H, H-1a), 2.37 (s, 1H, H-9), 2.18 (dd, J = 3.4, 12.8 Hz,
1H, H-18), 2.05 (s, 3H, -OCOCH3), 1.37 (s, 3H, CH3-29), 1.23 (s, 3H, CH3-26), 1.16 (s,
3H, CH3-25), 1.13 (s, 3H, CH3-28), 0.88 (s, 6H, CH3-23, CH3-24), 0.83 (s, 3H, CH3-27);
13C NMR (CDCl3, 125.76 MHz)  200.7 (C-11), 179.2 (C-30), 171.4 (C-31), 170.4 (C-13),
128.0 (C-12), 80.8 (C-3), 61.6 (C-9), 54.8 (C-5), 48.2 (C-18), 45.4 (C-20), 43.6 (C-14),
43.1 (C-8), 41.0 (C-19), 38.6 (C-10), 37.9 (C-1), 37.6 (C-4), 36.8 (C-22), 32.5 (C-7), 31.7
(C-17), 30.9 (C-21), 28.3 (C-16), 28.2 (C-15), 27.9 (C-29), 26.3 (C-27), 26.2 (C-2), 23.4-
23-1 (C-23, C-24), 21.1 (C-32), 18.5 (C-28), 17.2 (C-6), 16.5 (C-26), 16.3 (C-25). HRMS:
[M + Na]+ Calcd. C32H48NaO5 535.3399. Found: C32H48NaO5 535.3405.

N-(3-acetylglycyrrhetinoyl)ethanolamine (4a): Yield 83 % of pure compound as a white
solid; mp 255–256 °C; []D25 158.8 (c 1.0, CHCl3). 1H NMR (CDCl3, 500 MHz)  5.61 (s,
1H, H-12), 4.46 (dd, J = 4.8, 11.7 Hz, 1H, H-3), 3.61 (m, 3H, -HNCHaHbCH2OH), 3.40 (dt,
J = 5.5, 14.0 Hz, 1H, -HNCHaHbCH2OH), 2.72 (dt, J = 3.8, 13.8 Hz, 1H, H-1a), 2.33 (s,
1H, H-9), 2.15 (dd, J = 3.5, 12.6 Hz, 1H, H-18), 2.01 (s, 3H, -OCOCH3), 1.33 (s, 3H, CH3-
29), 1.13 (s, 3H, CH3-26), 1.11 (s, 3H, CH3-25), 1.08 (s, 3H, CH3-28), 0.83 (s, 6H, (CH3-
23, CH3-24), 0.77 (s, 3H, CH3-27); 13C NMR (CDCl3, 125.76 MHz)  200.7 (C-11), 172.2
(C-30), 171.1 (C-31), 170.6 (C-13), 127.7(C-12), 80.7 (C-3), 62.3 (-HNCH2CH2OH), 61.8
(C-9), 55.1 (C-5), 48.9 (C-18), 45.6 (C-20), 43.5 (C-14), 42.4 (C-8), 40.4 (C-19), 39.5 (-
HNCH2CH2OH), 38.9 (C-10), 38.3 (C-1), 38.1 (C-4), 37.1 (C-22), 32.8 (C-7), 31.9 (C-17),
30.4 (C-21), 28.9 (C-16), 28.5 (C-15), 28.1 (C-29), 26.6 (C-27), 26.3 (C-2), 23.6 (C-23),
23.4 (C-24), 21.4 (C-32), 18.5 (C-28), 17.4 (C-6), 16.8 (C-26), 16.5 (C-25). HRMS: [M +
Na]+ Calcd. C34H53NNaO5 578.3821 Found: C34H53NNaO5 578.3817.
N-(3-acetylglycyrrhetinoyl)butanolamine (4b): Yield 76.0 % of pure compound as a
25
1
white solid; mp > 300 °C; []D +160.3° (c 1.0, CHCl3). H NMR (CDCl3, 500 MHz) 
5.65 (s, 1H, H-12), 4.49 (dd, J = 4.6, 11.7 Hz, 1H, H-3), 3.61 (t, J = 5.9 Hz, 3H, -
HNCHaHb(CH2)2CH2OH), 3.26 (q, J = 5.9 Hz, 1H, -HNCHaHb(CH2)2CH2OH), 2.75 (dt, J
= 3.8, 13.6 Hz, 1H, H-1a), 2.36 (s, 1H, H-9), 2.22 (dd, J = 3.0, 12.0 Hz, 1H, H-18), 2.04 (s,
3H, -OCOCH3), 1.35 (s, 3H, CH3-29), 1.13 (s, 3H, CH3-26), 1.10 (s, 3H, CH3-25), 1.07 (s,
13
3H, CH3-28), 0.86 (s, 6H, s, CH3-23, CH3-24), 0.77 (s, 3H, CH3-27); C NMR (CDCl3,
125.76 MHz)  201.1 (C-11), 172.1 (C-30), 171.0 (C-31), 170.4 (C-13), 127.6 (C-12), 80.6
(C-3), 62.1 (-HN(CH2)3CH2OH), 61.7 (C-9), 55.0 (C-5), 48.8 (C-18), 45.5 (C-20), 43.3 (C-
14), 42.3 (C-8), 40.2 (C-19), 39.3 (HNCH2(CH2)3OH), 38.8 (C-10), 38.2 (C-1), 38.0 (C-4),
37.0 (C-22), 32.7 (C-7), 31.8 (C-17), 30.3 (C-21), 29.7 (-HNCH2CH2CH2CH2OH), 29.1 (C-
15), 28.8 (C-16), 28.0 (C-29), 26.5 (C-27), 26.4 (C-2), 26.1 (-HNCH2CH2CH2CH2OH),
23.5 (C-23), 23.2 (C-24), 21.3 (C-32), 18.4 (C-28), 17.3 (C-6), 16.7 (C-26), 16.4 (C-25).
HRMS: [M + Na]+ Calcd. C36H57NNaO5 606.4134 Found: C36H57NNaO5 606.4130.
N-(3-acetylglycyrrhetinoyl)-1-amino-2-propanol (4c): Yield 68 %; white solid, mp 249–
1
251 °C; []D2511.323(c 1.0, CHCl3). H NMR (CDCl3, 500 MHz)  5.65 (s, 1H, H-12),

4.50 (dd, J = 4.6, 11.7 Hz, 1H, H-3), 3.49-3.58 (m, 3H, CH3CH(NH)CH2OH-), 2.76 (dt, J =
3.7, 13.6 Hz, 1H, H-1a), 2.35 (s, 1H, H-9), 2.18 (d, J = 4.6, 12.3, 1H, Hz, H-18), 2.03 (s,
3H, -OCOCH3), 1.35 (s, 3H, CH3-29), 1.23 (s, 3H, CH3-26), 1.13 (s, 3H, CH3-25), 1.11 (d,
J = 7.1 Hz, 3H, CH3CH(CH2OH)NH-), 0.86 (s, 6H, CH3-23, CH3-24), 0.78 (s, 3H, CH3-
27); 13C NMR (CDCl3, 125.76 MHz)  200.8 (C-11), 181.8 (C-30), 177.1 (C-31), 171.1 (C-
13), 127.9 (C-12), 80.6 (C-3), 66.5 (CH3CH(CH2OH)NH-), 61.7 (C-9), 54.9 (C-5), 48.5 (C-
18), 45.4 (C-20), 44.2 (CH3CH(CH2OH)NH-), 43.3 (C-8, C-14), 41.7 (C-19), 38.7 (C-10),
38.0 (C-1), 37.9 (C-4), 36.9 (C-22), 32.6 (C-7), 31.8 (C-17), 31.4 (C-21), 29.7 28.7 (C-16),
28.6 (C-15), 28.0 (C-29), 26.5 (C-27), 26.4 (C-2), 23.5 (C-23), 23.2 (C-24), 22.6
(CH3CH(CH2OH)NH-), 21.3 (C-32), 18.6 (C-28), 17.3 (C-6), 16.6 (C-26), 16.4 (C-25).
HRMS: [M + Na]+ C36H57NNaO5 606.4134 Found: C36H57NNaO5 606.4139.
N-(3-acetylglycyrrhetinoyl)-2-amino-1-propanol (4d): Yield 73 %; white solid, mp 253–
25
1
254 °C; []D 15.135 (c 1.0, CHCl3). H NMR (CDCl3, 500 MHz)  5.69 (s, 1H, H-12),
4.51 (dd, J = 4.6, 11.7 Hz, 1H, H-3), 3.92 (m, 2H, CH3CH(OH)CHaHbNH-), 3.46 (dd, J =
3.0, 1H, 6.7 Hz, CH3CH(OH)CHaHbNH-), 2.80 (dt, J = 3.7, 13.6 Hz, 1H, H-1a), 2.36 (s,
1H, H-9), 2.18 (dd, J = 3.4, 12.2 Hz, 1H, H-18), 2.05 (s, 3H, -OCOCH3), 1.36 (s, 3H, CH3-
29), 1.21 (s, 3H, CH3-26), 1.16 (s, 3H, CH3-25), 1.13 (d, J = 7.1 Hz, 3H,
CH3CH(CH2OH)NH-), 0.88 (s, 6H, CH3-23, CH3-24), 0.82 (s, 3H, CH3-27); 13C NMR
(CDCl3, 125.76 MHz)  201.1 (C-11), 183.4 (C-30), 172.1 (C-31), 171.5 (C-13), 127.3 (C-
12), 80.8 (C-3), 66.0 (CH3CH(OH)CH2NH-), 61.5 (C-9), 54.7 (C-5), 48.6 (C-18), 46.6
(CH3CH(OH)CH2NH-), 45.3 (C-20), 44.7 (C-14), 43.2 (C-8), 42.0 (C-19), 38.5 (C-10),
37.9 (C-1), 37.8 (C-4), 36.7 (C-22), 32.4 (C-7), 31.6 (C-17), 31.4 (C-21), 28.7 (C-16), 28.4
(C-15), 27.7 (C-29), 26.3 (C-27), 26.2 (C-2), 23.2 (C-23), 23.0 (C-24), 21.3 (C-32), 20.1
(CH3CH(OH)CH2NH-), 18.4 (C-28), 17.1 (C-6), 16.3 (C-26), 16.1 (C-25). HRMS: [M +
Na]+ C36H57NNaO5 606.4134 Found: C36H57NNaO5 606.4128.
N-(3-acetylglycyrrhetinoyl)-4-hydroxy-1-butanol (5): Yield 86 %; white solid, mp 293
°C; []D25 + 152.3 ° (c 1.0, CHCl3). 1H NMR (CDCl3, 500 MHz)  5.69 (s, 1H, H-12), 4.52
(dd, J = 4.6, 11.7 Hz, 1H, H-3), 4.18 (t, J = 6.4 Hz, 2H, -COOCH2(CH2)2CH2OH), 3.72 (t, J
= 6.5 Hz, 2H, -COOCH2(CH2)2CH2OH), 2.79 (dt, J = 3.5, 13.6 Hz, 1H, H-1a), 2.36 (s, 1H,

H-9), 2.18 (dd, J = 3.0, 12.0 Hz, 1H, H-18), 2.05 (s, 3H, -OCOCH3), 1.37 (s, 3H, CH3-29),
1.22 (s, 3H, CH3-26), 1.16 (s, 3H, CH3-25), 1.13 (s, 3H, CH3-28), 0.88 (s, 6H, s, CH3-23,
CH3-24), 0.83 (s, 3H, CH3-27); 13C NMR (CDCl3, 125.76 MHz)  200.1 (C-11), 174.7 (C-
30), 171.0 (C-31), 169.2 (C-13), 128.2 (C-12), 80.6 (C-3), 64.7 (-COOCH2(CH2)2CH2OH),
62.5 (-COOCH2(CH2)2CH2OH), 61.7 (C-9), 55.0 (C-5), 48.2 (C-18), 45.4 (C-20), 43.7 (C-
14), 43.2 (C-8), 40.9 (C-19), 38.8 (C-10), 38.1 (C-1), 37.7 (C-4), 36.9 (C-22), 32.7 (C-7),
31.9
(C-17),
31.8
(-COOCH2CH2CH2CH2OH),
31.0
(C-21),
29.6
(-
COOCH2CH2CH2CH2OH), 28.5 (C-16), 28.4 (C-15), 28.1 (C-29), 26.5 (C-27), 26.4 (C-2),
23.6 (C-23), 23.4 (C-24), 21.3 (C-32), 18.7 (C-28), 17.4 (C-6), 16.7 (C-26), 16.4 (C-25).
HRMS: [M + Na]+ Calcd. C36H56NaO6 607.3975 Found: C36H56NaO6 607.3981.
3.4. Molecular modeling
CAL
B
structure
was
downloaded
from
RCSB
Protein
DataBank(http://www.rcsb.org/pdb/), PDB code for CAL B is 1TCA. All substrates were
minimized using semiempirical AM1 method with the algorithm Polak-Ribiere in
Hyperchem. The docking calculations were carried out with Autodock 4.2 program [35].
The Autodock 4.2 method was applied considering all the rotatable bonds for substrates 1
and 2, while all the protein was considered stiff. For the location and extent of the 3D area,
the search space is defined by specifying a center, the number of points in each dimension,
and points between spaces to focus the search space in the active site of the enzyme, it was
taken as grid box center the coordinates x, y, z of a water molecule close to Ser105.
3.5. Drug screening
3.5.1 Cytotoxicity assay
The cell viability was determined by using the cleavage of the tetrazolium salt MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The absorbance of each well
was measured on a Eurogenetics MPR-A 4i microplate reader. Results were expressed as a
percentage of absorbance of treated cells with respect to untreated ones. The CC50 was
defined as the concentration of compound that caused 50% reduction in cell viability.
3.5.2. Antiviral qualitative screening

Vero cells grown in 96-well plates were infected with HSV-1 (KOS strain) with a
multiplicity of infection (m.o.i) of 0.1 and, after virus adsorption, cells were treated or not
with different concentrations of the synthesized derivatives, in a range where no
cytotoxicity was observed. After 24 h of incubation at 37 °C in 5% CO2 atmosphere,
cytopathic effect was evaluated under microscope and the protective effect of each
compound was established as a percentage with respect to infected untreated cells.
3.5.3. Viral titration
Vero cells grown in 24-well plates were infected with serial 10-fold dilutions of viral yields
and incubated for 1 h at 37 ºC. Residual inocula were replaced by maintenance medium
with 0.7% of methylcellulose. After 72 h of incubation at 37 ºC, cells were fixed with
formaldehyde 10%, stained with Crystal Violet, and plaque forming units were counted.
The EC50 was defined as the concentration of compound that caused 50% reduction in viral
yield.
4. Conclusions
In this work we described the chemoenzymatic synthesis of new derivatives of
glycyrrhetinic acid. Scheme 2 shows the performed reactions and the obtained products.
The CAL B lipase showed selective activity catalyzing the synthesis of alkanolamides (4)
and hydroxyesters (5) only from 3-acetyl glycyrrhetinic acid (2), which was previously
obtained through a chemical method. Lipase catalysis was not observed in the reaction of
neither glycyrrhetinic acid (1) with any nucleophile nor 2 with alcohols, amines and
diamines. The enzymatic approach shows interesting advantages: the reaction is simple, it
is worked at low temperature, the products are easily isolated by filtration and solvent
evaporation and shows low environmental impact.
In order to explain the experimental results, molecular modeling studies were also carried
out. We have determined that the presence of an additional hydroxyl group in the
nucleophile is essential to reactivity. The hydrogen bond interaction between the
nucleophile and the acyl enzyme complex favors the reaction as in the case of
alkanolamines and diols.
In addition, we have evaluated the effect of the synthesized derivatives as antiviral agents.

There is an urgent need to explore new and effective antiviral drugs. The present work has
investigated the antiviral effect of five synthesized GA derivatives. From all compounds
assayed, only compound 4d showed antiviral activity against HSV-1 and, furthermore,
against an HSV-1 ACV-resistant strain, in vitro conditions. We observed that most of the
activity was present in the 4d(S) estereoisomer, while the 4d(R) estereoisomer alone did
not produced a significant effect on viral replication.
Scheme 2. Reactions and products from glycyrrhetinic acid.
Supporting Information. Spectral data for compounds 2, 4a-d and 5 associated with this
article are supplied.
Acknowledgments

We thank UBA (UBACYT 20020130100212), CONICET (PIP 11220110100653) and
ANPCyT (PICT 2011-00595) for partial financial support. G.G.L, L.A. and A.B. are
Research Members of CONICET (Argentina).
References
[1] D.M. Biondi, C. Rocco, G. Ruberto, Dihydrostilbene Derivatives from Glycyrrhiza g
labra Leaves, J. Nat. Prod. 68(7) (2005) 1099-1102.
[2] A.-M.H. El-Refai, L.A. Sallam, H.A. El-Menoufy, H.A.S. Amin, Physiological and
chemical studies on the bioconversion of glycyrrhizin by Aspergillus niger NRRL 595, Mal
J Microbiol 8 (2012) 75-82.
[3] T. Nomura, T. Fukai, T. Akiyama, Chemistry of phenolic compounds of licorice
(Glycyrrhiza species) and their estrogenic and cytotoxic activities, Pure and applied
chemistry 74(7) (2002) 1199-1206.
[4] T.-C. Kao, M.-H. Shyu, G.-C. Yen, Glycyrrhizic acid and 18β-glycyrrhetinic acid
inhibit inflammation via PI3K/Akt/GSK3β signaling and glucocorticoid receptor activation,
J. Agric. Food Chem. 58(15) (2010) 8623-8629.
[5] D. Maitraie, C.-F. Hung, H.-Y. Tu, Y.-T. Liou, B.-L. Wei, S.-C. Yang, J.-P. Wang, C.-
N. Lin, Synthesis, anti-inflammatory, and antioxidant activities of 18β-glycyrrhetinic acid
derivatives as chemical mediators and xanthine oxidase inhibitors, Bioorg. Med. Chem.
17(7) (2009) 2785-2792.
[6] T. Ikeda, K. Yokomizo, M. Okawa, R. Tsuchihashi, J. Kinjo, T. Nohara, M. Uyeda,
Anti-herpes virus type 1 activity of oleanane-type triterpenoids, Biol. Pharm. Bull. 28(9)
(2005) 1779-1781.
[7] C. Serra, G. Lampis, R. Pompei, M. Pinza, Antiviral activity of new triterpenic
derivatives, Pharmacol. Res. 29(4) (1994) 359-366.
[8] S. Schwarz, S.D. Lucas, S. Sommerwerk, R. Csuk, Amino derivatives of glycyrrhetinic
acid as potential inhibitors of cholinesterases, Bioorg. Med. Chem. 22(13) (2014) 3370-
3378.
[9] R. Gaware, R. Khunt, L. Czollner, C. Stanetty, T. Da Cunha, D.V. Kratschmar, A.
Odermatt, P. Kosma, U. Jordis, D. Claßen-Houben, Synthesis of new glycyrrhetinic acid
derived ring A azepanone, 29-urea and 29-hydroxamic acid derivatives as selective 11β-
hydroxysteroid dehydrogenase 2 inhibitors, Bioorg. Med. Chem. 19(6) (2011) 1866-1880.

[10] C. Stanetty, L. Czollner, I. Koller, P. Shah, R. Gaware, T. Da Cunha, A. Odermatt, U.
Jordis, P. Kosma, D. Claßen-Houben, Synthesis of novel 3-amino and 29-hydroxamic acid
derivatives of glycyrrhetinic acid as selective 11β-hydroxysteroid dehydrogenase 2
inhibitors, Bioorg. Med. Chem. 18(21) (2010) 7522-7541.
[11] D.V. Kratschmar, A. Vuorinen, T. Da Cunha, G. Wolber, D. Classen-Houben, O.
Doblhoff, D. Schuster, A. Odermatt, Characterization of activity and binding mode of
glycyrrhetinic acid derivatives inhibiting 11β-hydroxysteroid dehydrogenase type 2, The J.
Steroid Biochem. Mol. Biol. 125(1) (2011) 129-142.
[12] K. Faber, Biotransformations in organic chemistry: a textbook, 6th Ed., Springer
Science & Business Media, 2011.
[13] J. Whitthall, P. Sutton, Practical Methods for Biocatalysis and Biotransformations 2,
John Wiley & Sons Ltd., New York, 2012.
[14] K. Buchholz, V. Kasche, U.T. Bornscheuer, Biocatalysts and enzyme technology, John
Wiley & Sons, 2012.
[15] G. Carrea, S. Riva, Organic synthesis with enzymes in non-aqueous media, John
Wiley & Sons, 2008.
[16] A. Baldessari, Lipases as catalysts in synthesis of fine chemicals, Lipases and
Phospholipases: Methods and Protocols (2012) 445-456.
[17] V. Gotor‐Fernández, E. Busto, V. Gotor, Candida antarctica lipase B: an ideal
biocatalyst for the preparation of nitrogenated organic compounds, Adv. Synt. Catal.
348(7‐8) (2006) 797-812.
[18] A. Cuetos, M. García-Ramos, E.-M. Fischereder, A. Díaz-Rodríguez, G. Grogan, V.
Gotor, W. Kroutil, I. Lavandera, Catalytic Promiscuity of Transaminases: Preparation of
Enantioenriched β-Fluoroamines by Formal Tandem Hydrodefluorination/Deamination,
Angew. Chem. Int. Ed. 55(9) (2016) 3144-3147.
[19] D. González-Martínez, V. Gotor, V. Gotor-Fernández, Application of Deep Eutectic
Solvents in Promiscuous Lipase-Catalysed Aldol Reactions, Eur. J. Org. Chem. 2016(8)
(2016) 1513-1519.
[20] G. García Liñares, P. Arroyo Mañez, A. Baldessari, Lipase‐Catalyzed Synthesis of
Substituted Phenylacetamides: Hammett Analysis and Computational Study of the
Enzymatic Aminolysis, Eur. J. Org. Chem. 2014(29) (2014) 6439-6450.

[21] M.A. Zígolo, G. García Liñares, A. Baldessari, New cholic acid derivatives:
Biocatalytic synthesis and molecular docking study, Steroids 107 (2016) 10-19.
[22] G. García Liñares, M. Antonela Zígolo, L. Simonetti, S.A. Longhi, A. Baldessari,
Enzymatic synthesis of bile acid derivatives and biological evaluation against Trypanosoma
cruzi, Bioorg. Med. Chem. 23(15) (2015) 4804-4814.
[23] G. García Liñares, G. Parraud, C. Labriola, A. Baldessari, Chemoenzymatic synthesis
and biological evaluation of 2- and 3-hydroxypyridine derivatives against Leishmania
mexicana, Bioorg. Med. Chem. 20(15) (2012) 4614-4624.
[24] P.G. Quintana, G. García Liñares, S.N. Chanquia, R.M. Gorojod, M.L. Kotler, A.
Baldessari, Improved Enzymatic Procedure for the Synthesis of Anandamide and N‐Fatty
Acylalkanolamine Analogues: A Combination Strategy to Antitumor Activity, Eur. J. Org.
Chem. 2016(3) (2016) 518-528.
[25] S.N. Chanquia, N. Boscaro, L. Alche, A. Baldessari, G.G. Liñares, An Efficient
Lipase‐Catalyzed Synthesis of Fatty Acid Derivatives of Vanillylamine with Antiherpetic
Activity in Acyclovir‐Resistant Strains, Chem. Select 2(4) (2017) 1537-1543.
[26] M. Kukhanova, A. Korovina, S. Kochetkov, Human herpes simplex virus: life cycle
and development of inhibitors, Biochemistry (Moscow) 79(13) (2014) 1635-1652.
[27] D.W. Kimberlin, D.M. Coen, K.K. Biron, J.I. Cohen, R.A. Lamb, M. McKinlay, E.A.
Emini, R.J. Whitley, Molecular mechanisms of antiviral resistance, Antiviral Res. 26(4)
(1995) 369-401.
[28] I. Beseda, L. Czollner, P.S. Shah, R. Khunt, R. Gaware, P. Kosma, C. Stanetty, M.C.
del Ruiz-Ruiz, H. Amer, K. Mereiter, Synthesis of glycyrrhetinic acid derivatives for the
treatment of metabolic diseases, Bioorg. Med. Chem. 18(1) (2010) 433-454.
[29] A. Baldessari, C.P. Mangone, One-pot biocatalyzed preparation of substituted amides
as intermediates of pharmaceuticals, J. Mol. Cat. B: Enzymatic 11(4) (2001) 335-341.
[30] E.M. Rustoy, A. Baldessari, An efficient chemoenzymatic synthesis of the bactericide
lapyrium chloride, Eur. J. Org. Chem. 2005(21) (2005) 4628-4632.
[31] E.M. Rustoy, A. Baldessari, Chemoselective enzymatic preparation of N-
hydroxyalkylacrylamides, monomers for hydrophilic polymer matrices, J. Mol. Cat. B:
Enzymatic 39(1) (2006) 50-54.

[32] L. Wang, R. Yang, B. Yuan, Y. Liu, C. Liu, The antiviral and antimicrobial activities
of licorice, a widely-used Chinese herb, Acta Pharm. Sinica B 5(4) (2015) 310-315.
[33] D. Milijkovic, N. Sharaf El-Din and K. Gasi, Resolution the racemic 1-amino-2-
propanol, J. Serb. Chem. Soc 50(60) (1985) 277.
[34] L. Synoradzki, U. Bernaś, P. Ruśkowski, Tartaric acid and its O-acyl derivatives. Part
2. Application of tartaric acid and O-acyl tartaric acids and anhydrides. Resolution of
racemates, Org. Prep. Proc. Int. 40(2) (2008) 163-200.
[35] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J.
Olson, Automated docking using a Lamarckian genetic algorithm and an empirical binding
free energy function, J. Comp. Chem. 19(14) (1998) 1639-1662.

Docking
CAL B
H2NCH2CH(OH)CH3
AcCN, 25 °C, 48 h
Antiviral
activity
Glycyrrhetinic acid

Highlights:
 New derivatives of glycyrrhetinic acid were synthesized by chemoenzymatic way.
 A series of alkanolamides was prepared and totally spectroscopy characterized.
Alkanolamide derived from 1-amino-2-propanol showed antiviral activity.
