A Simple Method for Preparation of 18α-Glycyrrhetinic Acid and Its Derivatives

Article abstract of DOI:10.1023/A:1024961503987

Treatment of 18β-glycyrrhizic acid with a methanolic solution of HCl resulted in 1:1 mixture of methyl esters of 18α- and 18β- glycyrrhetinic acids. Benzoylation of the mixture led to methyl esters of 3-benzoyl-18α-glycyrrhetinic acid and 3-benzoyl-18β-glycyrrhetinic acid, which were separated by chromatography on silica gel. 18α- Glycyrrhetinic acid was prepared by alkaline hydrolysis of methyl 3-benzoyl-18α-glycyrrhetinate and was further used for the syntheses of 3-keto-18α-glycyrrhetinic acid and methyl esters of 18α- glycyrrhetinic acid and 3-keto-18α-glycyrrhetinic acid.

Full text of DOI:10.1023/A:1024961503987

Russian Journal of Bioorganic Chemistry, Vol. 29, No. 4, 2003, pp. 390–394. Translated from Bioorganicheskaya Khimiya, Vol. 29, No. 4, 2003, pp. 429–433.
Original Russian Text Copyright © 2003 by Ignatov, Prokof’ev, Ipatova, Timofeev, Medvedeva, Misharin.
A Simple Method for Preparation of 18a-Glycyrrhetinic Acid
and Its Derivatives
D. V. Ignatov*, Yu. I. Prokof’ev*, O. M. Ipatova*, V. P. Timofeev**,
, 1
N. V. Medvedeva*, and A. Yu. Misharin*
*Orekhovich Research Institute of Biomedicinal Chemistry, Russian Academy of Medical Sciences,
Pogodinskaya ul. 10, Moscow, 119992 Russia
**Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, GSP Moscow, 117984 Russia
Received June 14, 2002; in final form, September 10, 2002
Abstract—Treatment of 18β-glycyrrhizic acid with a methanolic solution of HCl resulted in 1 : 1 mixture of
methyl esters of 18α- and 18β-glycyrrhetinic acids. Benzoylation of the mixture led to methyl esters of 3-ben-
zoyl-18α-glycyrrhetinic acid and 3-benzoyl-18β-glycyrrhetinic acid, which were separated by chromatography
on silica gel. 18α-Glycyrrhetinic acid was prepared by alkaline hydrolysis of methyl 3-benzoyl-18α-glycyrrhe-
tinate and was further used for the syntheses of 3-keto-18α-glycyrrhetinic acid and methyl esters of 18α-gly-
cyrrhetinic acid and 3-keto-18α-glycyrrhetinic acid.
Key words: 18α-glycyrrhetinic acid, terpenoids
1
INTRODUCTION
medicinal plants. Unlike the corresponding 18β-isomer
(II), it is commercially unavailable.A simple method of
preparation of (IV)–(VI), (VIII), and (IX) from the
commercial preparation of sodium 18β-glycyrrhizate
(glycyrrhizin purchased fromVital-Chem Zhuhai Com-
pany, Ltd) is described below.
A crystalline product was prepared by splitting off
the carbohydrate residue from glycyrrhizin (I) (2 N
HCl in methanol, 6 h, 80°ë). 1H NMR spectrum of the
product indicates that it consists of two methyl esters at
the ratio of 1 : 1 (Fig. 1a). The spectrum of one of the
components completely coincides with that of the
methyl ester of 18β-glycyrrhetinic acid (III) (Fig. 1b),
whereas the spectrum of the second product points out
to the presence of seven C-methyl groups, one methoxy
group, and β-hydroxyl in position 3. In addition, H12 of
this compound resonates at stronger field than those in
(II) and (III). This spectrum completely coincided with
that of (V) (Fig. 1c) and was identical to that of methyl
18α-glycyrrhetinate described in [11].
Glycyrrhizin (I) (18β-glycyrrhizic acid) is a biolog-
ically active compound from the liquorice root (Glycyr-
rhiza uralensis). This compound is widely used in med-
icine for treatment of liver pathologies and also is a part
of many therapeutic agents [1–8]. Recently, we discov-
ered that 18β-glycyrrhizic acid (I), its basic metabolite,
18β-glycyrrhetinic acid (II), and methyl 18β-glycyr-
rhetinate (III) activate the FXR nuclear receptor in the
cells of human hepatoblastoma of the Hep G2 line and
effectively regulate the gene transcription of the most
important proteins of lipid metabolism [9]. Of particu-
lar interest is the fact that (I)–(III) exert different
effects on the mRNA level of the target genes of the
FXR receptor: cholesterol 7α-hydroxylase, lecithin–
cholesterol acyltransferase, phospholipid-transferring
protein, apolipoproteins A1 and C3, and mRNA of
FXR itself. Therefore, derivatives of glycyrrhetinic acid
can be regarded as potential regulators of lipid metabo-
lism, and their activity would probably depend on the
structural peculiarities of their molecules.
Three independent processes obviously proceed in
the course of glycyrrhizin (I) treatment with HCl in
methanol: cleavage of glycoside bound, etherification
of carboxyl group, and isomerization at C18. The last
process should involve 11,13(18)-dien-11-ol as an
intermediate product. In this connection, we studied the
isomerization of methyl 18β-glycyrrhetinate (III) in
HCl/MeOH and [2H]Cl/MeO[2H] mixtures. When
The goal of this study is the preparation of several
derivatives of 18α-glycyrrhetinic acid (IV) for their
further examination as agonists of the FXR receptor.
RESULTS AND DISCUSSION
18α-Glycyrrhetinic acid (IV) is formed from (I) and
(II) under rigorous conditions of action of strong acids
or bases [10] and is a minor component of extracts of
1
heated in HCl/MeOH, (III) isomerized, and H NMR
spectrum of the resulting mixture of isomers (III) and
(V) completely coincided with that given in Fig. 1a.
Partial deuterium exchange proceeded under heating of
(III) in [2H]Cl/MeO[2H] solution. 1H NMR spectrum of
1
Corresponding author; phone: +7 (095) 246-3375; e-mail:
misharin@ibmh.msk.su
1068-1620/03/2904-0390$25.00 © 2003 MAIK “Nauka /Interperiodica”

A SIMPLE METHOD FOR PREPARATION
391
29
30
CH3
COOY
CH3
CH3
COOY
19
20
21
H
H
12
18
O
O
22
11
25CH3 26 CH3
CH3
13 17
14 16
CH3 CH3
CH3
1
28
9
2
CH315
10
8
7
3
5
27
4
6
X
CH3
X
CH3
CH3
CH3
24
23
(I) X = GlcAβ1
GlcA – D-glucuronic acid
(II) X = HO; Y = H
2GlcAβ1-, Y = H
(IV) X = HO, Y = H
(V) X = HO; Y = CH3
(VI) X = BzO; Y = CH3
(III) X = HO; Y = CH3
(VIII) X = O; Y = H
(IX) X = O; Y = CH3
(VII) X = BzO; Y = CH3
the partially deuterated product indicated H–D
A separation of the mixture of 18α- and 18β-glycyr-
exchange at C12 (Fig. 2) and C18 (changes in the area rhetinic acids by crystallization from ethanol had been
of poorly resolved multiplets within the range 1.25– reported [12] during the preparation of our paper to
publication. We repeated this procedure and isolated
18α-glycyrrhetinic acids (IV) from the products of
basic hydrolysis of the mixture of (III) and (V) in 20%
yield [or 40% from the content of 18α-isomer (V) in the
racemic mixture].
The oxidation of (IV) by the treatment with CrO3–
Py complex resulted in ketoacid (VIII) in 80% yield.
Methylation of acids (IV) and (VIII) with the diazo-
methane excess in ether gave the corresponding methyl
esters (V) and (IX) in quantitative yields. Thus, 18α-
glycyrrhetinic acid (IV) and its derivatives (V), (VI),
(VIII), and (IX) were prepared from glycyrrhizic acid
(I).
2.05 ppm are not shown in Fig. 2). This result correlates
with the formation of intermediate protonated
11,13(18)-dien-11-ol. The absence of deuterium
exchange at C9 (Fig. 2) under these conditions
excluded the formation of alternative 9(11),12-dien-11-
ol during the isomerization.
Methyl 18α- and 18β-glycyrrhetinates (V) and (III)
exhibited similar chromatographic mobilities at
HPTLC and HPLC under all the examined conditions.
Therefore, we made no attempts of preparative separa-
tion of the mixture of isomers (III) and (V). For the iso-
lation of 18α-isomer, we first treated the mixture of
methyl esters (III) and (V) with benzoyl chloride in
pyridine. The resulting 3-benzoates of (III) and (V)
were satisfactorily separated by HPTLC in a 24 : 2 : 1
hexane–ethyl acetate–acetic acid mixture. The com-
pound with higher mobility (Rf 0.36) was identical to
methyl 3-benzoyl-18β-glycyrrhetinate (VII), whereas
the compound with a lower mobility (Rf 0.31) corre-
sponded to 18α-isomer (VI). (VI) was isolated from
the isomer mixture by the preparative column chroma-
tography on silica gel in a 24 : 2 : 1 hexane–ethyl ace-
tate–acetic acid system in 32% yield from the starting
sodium 18β-glycyrrhizate (I).
EXPERIMENTAL
1
Spectra of H NMR were recorded on a AMX III-
400 spectrometer (Bruker) in deuterochloroform.
Chemical shifts and spin–spin coupling constants (J)
are given in ppm and Hz, respectively. The δ CHCl3
value was taken to be 7.25 ppm. The resonances were
assigned on the basis of results published in [11, 12].
HPTLC was performed on the precoated HPTLC
Kieselgel F254 (Merck) plates and on the same plates
preliminarily treated with 2% solution of AgNO3 in
acetonitrile and activated for 40 min at 100°ë. The
detection was performed in UV-light (254 nm) and by
spraying with 3% solution of ammonium molybdate in
5% sulfuric acid followed by heating. Melting points
were determined in capillary tubes.
Chemical reagents were from Aldrich and Merck.
Solutions HCl/ëH3OH and [2H]Cl/ëH3O[2H] were
prepared by the treatment of calculated amounts of
acetyl chloride with ëH3OH or ëH3O[2H]. An ether
Methyl and benzoyl groups were removed from
(VI) by boiling with 4 N NaOH for 2 h in a of water–
propyl alcohol–ethylene glycol mixture. The isomer-
ization at C18 was no higher than 6% under these con-
ditions according to the ratio of integral intensities of
resonances at 5.51 and 5.62 ppm [H12 in 18α- and 18β-
glycyrrhetinic acids (IV) and (II), respectively]. After
two recrystallizations from acetone, 18α-glycyrrhetinic
acids (IV) containing no admixture of 18β-isomer was
isolated in 84% yield from (VI).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 4 2003

392
IGNATOV et al.
(a)
(b)
(c)
5
4
3
2
1 ppm
1
Fig. 1. H NMR spectra of methyl esters of glycyrrhetinic acid: (a) a mixture after the treatment of the acid (I) with HCl in methanol;
(b) (III); and (c) (V).
solution of diazomethane and ërO3–Py complex were s, H12); 7.36 (2 H, t, J 7.0, Bz); 7.49 (1 H, t, J 7.0, Bz);
7.97 (2 H, d, J 7.0, Bz).
prepared by the standard procedures [13, 14]. Sodium
salt of 18β-glycyrrhizic acid (I) and 18β-glycyrrhetinic
acid (II) were purchased from Vital-Chem Zhuhai
Company, Ltd (China) and from Sigma, respectively.
Methyl 18b-glycyrrhetinate (III) was prepared by
the treatment of 18β-glycyrrhetinic acids (II) with
diazomethane. For its 1H NMR spectrum, see Fig. 1b.
Methyl 3-benzoyl-18a-glycyrrhetinate (VI).
Methanol (250 ml) was added to a solution of sodium
glycyrrhizate (I) (10.0 g, 11.25 mmol), and the reaction
mixture was stirred for 5 min with Dowex-50 (ç+) ion-
exchange resin (120 ml). The resin was filtered off and
washed with isopropanol (200 ml). The combined fil-
trate was evaporated, and the residue was recrystallized
from a 10 : 10 : 1 acetone–water–acetic acid mixture.
After drying in a vacuum, white crystals of18β-glycyr-
rhizic acid (I) (8.1 g, 10.12 mmol, 90%) were obtained.
Methyl of 3-benzoyl-18b-glycyrrhetinate (VII)
was prepared by the treatment of (III) with BzCl in
1
pyridine; H NMR spectrum: 0.80, 0.94, 1.04, 1.14,
1.15, 1.20, 1.29 (7 s, 3 H each, H28, H23, H24, H26,
H25, H29, and H27); 2.28 (1 H, s, H9); 2.73 (1 H, m,
H1); 3.62 (3ç, s, éëç3); 4.68 (1 H, m, H3); 5.62 (1 H,
18β-Glycyrrhizic acid (I) (7.2 g, 8.8 mmol) was
heated in 2 M solution of HCl in absolute methanol
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 4 2003

A SIMPLE METHOD FOR PREPARATION
393
(200 ml) in a hermetically closed flask for 8 h at 80°ë,
H12
H9
cooled, and evaporated to dryness. The residue was dis-
solved in boiling acetone (50 ml) and the hot solution
was poured out into water (1 l) under stirring. The mix-
ture was stirred for 1 h on an ice bath; the precipitate
was collected by filtration, recrystallized from a 4 : 1
acetone–water mixture, and dried in a vacuum. The
yield of white crystals of a mixture of methyl esters of
18β- (III) and 18α-glycyrrhetinic acids 3.8 g
(7.8 mmol, 86%); mp 231–236°C (with decomposi-
tion); for 1H NMR spectrum, see Fig. 1a.
#
*
#
*
*
#
#
*
5.68
5.60
5.52
δ, ppm
2.28
2.20
The resulting mixture of and (V) (3.5 g, 7.2 mmol)
was dissolved in anhydrous pyridine (30 ml), and BzCl
(3 ml, 26 mmol) was added to the mixture dropwise
under stirring on an ice bath. The reaction mixture was
stirred for 14 h at 20°ë, poured in a saturated solution
of NaHCO3 (500 ml) mixed with chopped ice (200 g),
stirred for 2 h at 20°ë, and extracted with ethyl acetate
(3 × 100 ml). The extract was dried over Na2SO4 and
evaporated. The residue was twice recrystallized from
1 : 1 acetone–methanol mixture. The mixture of (VI)
and (VII) was obtained as white crystals (mp 260–
262°ë), yield 2.0 g. An additional amount (1.2 g) of the
crystals (mp 256–261°C) was isolated from the com-
bined mother liquor by concentration and recrystalliza-
tion from 1 : 1 acetone–methanol mixture. The total
yield of the racemic mixture of benzoates of methyl
esters (VI) and (VII) was 3.2 g (75%); 1H NMR spec-
trum: 0.64 (s); 0.80 (s); 0.84 (s); 0.88 (s); 0.94 (s); 1.04
(s); 1.09 (s); 1.15 (s); 1.20 (s) (21 H totally, H28, H23,
H24, H26, H25, H29, and H27); 2.20 (s) and 2.28 (s) (1
H totally, H9, in 18α- and 18β-isomers); 2.67 (m) and
2.73 (m) (1 H totally, H1 in 18α- and 18β-isomers);
3.62 (3ç, Ò, éëç3); 4.68 (1 H, m, H3); 5.51 (broadened
s) and 5.62 (s) (1 H totally, H12 in 18α- and 18β-iso-
mers); 7.36 (2 H, t, J 7.0, Bz); 7.49 (1 H, t, J 7.0, Bz);
and 7.97 (2 H, d, J 7.0, Bz).
1
Fig. 2. H NMR spectra of products of the isomerization of
(III). The upper spectra and the bottom spectra corre-
sponded to the products formed after the treatment with
HCl/MeOH and [ H]Cl/MeO[ H], respectively. The reso-
nances from 18β-isomer and 18α-isomer were marked with
(*) and with (#), respectively.
2
2
18a-Glycyrrhetinic acids (IV) from methyl 3-
benzoyl-18a-glycyrrhetinate (VI). Methyl 3-benzoyl-
18α-glycyrrhetinate (VI) (0.06 g, 0.1 mmol) was boiled
in a stirred solution of NaOH (0.16 g, 40 mmol), ethyl-
ene glycol (1 ml), isopropanol (5 ml), and water (4 ml)
for 2 h, cooled, acidified with 10% ç2SO4 to pH 3.0,
and extracted with ethyl acetate (3 × 30 ml). The extract
was dried over Na2SO4 and evaporated. The product
was twice recrystallized from acetone. The yield of
1
(VI) 0.038 g (0.084 mmol, 84%); mp 360–363°C; H
NMR: 0.64, 0.88, 0.97, 1.08, 1.15, 1.20, and 1.29 (3 H
each, 7 s, H28, H23, H24, H26, H25, H29, and H27);
2.20 (1 H, s, H9); 2.62 (1 H, m, H1); 3.15 (1 H, m, H3);
and 5.52 (1 H, broadened s, H12).
18a-Glycyrrhetinic acids (IV) from a mixture of
methyl 18a-glycyrrhetinate (V) and methyl 18b-
glycyrrhetinate (III). The mixture (1 : 1) of (V) and
(III) (0.97 g, 1 mmol of 18α-isomer (V), NaOH
(0.64 g, 160 mmol), water (4 ml), ethylene glycol
(1 ml), and isopropanol (5 ml) was boiled under stirring
for 2 h, cooled, acidified with 10% ç2SO4 to pH 3.0,
and extracted with ethyl acetate (3 × 30 ml). The extract
was dried over Na2SO4 and evaporated. The residue
was thrice recrystallized from ethanol. The yield of
(IV) 0.092 g (0.2 mmol, 20%); mp 360–363°ë (from
ethanol); 1H NMR spectrum was described above.
Methyl 3-benzoyl-18α-glycyrrhetinate (VI) was
isolated from the isomeric mixture (0.66 g, 1.1 mmol)
by chromatography on a column (3.5 × 120 cm) packed
with Silica gel L 40/100 and eluted with a 24 : 2 : 1 hex-
ane–ethyl acetate–acetic acid mixture, while monitor-
ing eluted fractions with TLC. The chromatographi-
cally homogeneous (VI) was isolated in yield of 0.18 g;
1
mp 263–268°ë (from acetone); H NMR spectrum:
0.64, 0.80, 0.84, 0.88, 0.94, 1.09, and 1.15 (3 H each, s,
H28, H23, H24, H26, H25, H29, and H27); 2.20 (1 H,
s, H9); 2.67 (1 H, m, H1); 3.62 (3ç, s, éëç3); 4.68 (1
H, m, H3); 5.51 (1 H, broadened s, H12); 7.36 (2 H, t,
J 7.0, Bz); 7.49 (1 H, t, J 7.0, Bz); 7.97 (2 H, d, J 7.0,
Bz). Fraction (0.2 g) with traces of the 18β-isomer was
also obtained. This fraction was rechromatographed
under the same conditions, and chromatographically
homogeneous product was recrystallized from acetone.
As a result, an additional amount (0.12 g) of (VI) was
3-Keto-18a-glycyrrhetinic acid (VIII). A solution
of 18α-glycyrrhetinic acid (IV) (0.092 g, 0.2 mmol) in
anhydrous ëç2ël2 (10 ml) was added to a solution of
ërO3–Py complex prepared from ërO3 (0.36 g) by the
procedure [13]. The reaction mixture was stirred at
room temperature for 1 h with the TLC monitoring of
oxidation, mixed with methanol (1 ml), treated with sil-
ica gel (1.0 g) after 15 min, and evaporated to dryness.
isolated. The total yield of (VI) was 32% from the start- Silica gel with the sorbed reaction products was trans-
ing glycyrrhizic acid (I). ferred onto a column (3 × 10 cm) with dry silica gel, and
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 4 2003

394
IGNATOV et al.
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1
mp 278°ë (with decomposition); H NMR spectrum:
0.67, 0.88, 0.97, 1.06, 1.09, 1.17, and 1.29 (3 H each,
7 s, H28, H23, H24, H26, H25, H29, and H27); 2.26
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Methyl esters of 18a-glycyrrhetinic acid (V) and
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solution of (V) or (VIII) (0.1 mmol) [14]. The reaction
was monitored by TLC. The solvent was evaporated,
and the resulting products were isolated by crystalliza-
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Methyl 18a-glycyrrhetinate (V): quantitative
yield; mp 264–266°ë (lit. mp 266–267°ë [11]); for 1H
NMR spectrum, see Fig. 1c.
Methyl 3-keto-18a-glycyrrhetinate (IX): quanti-
tative yield; mp 238–240°C; 1H NMR: 0.72, 0.84, 0.91,
1.06, 1.09, 1.16, and 1.21 (3 H each, 7 s, H28, H23,
H24, H26, H25, H29, and H27); 2.34 (1 H, s, H9); 2.78
(1 H, m, H1); 2.86 (1 H, m, H2); 3.68 (3 H, s, OCH3);
and 5.60 (1 H, broadened s, H12).
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ACKNOWLEDGMENTS
This study was supported by the Russian Founda-
tion for Basic Research, project nos. 00-04-48643 and
01-04-48504, and by the Moscow Government, project
no. GA-35/01 with OAO MKNT.
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 4 2003