Synthesis and antiviral activities of spacer-linked 1-thioglucuronide analogues of glycyrrhizin

Article abstract of DOI:10.3762/bjoc.8.79

The influenza virus infection remains a significant threat to public health and the increase of antiviral resistance to available drugs generates an urgent need for new antiviral compounds. Starting from the natural, antivirally active compound glycyrrhizin, spacerbridged derivatives were generated with improved antiviral activity against the influenza A virus infection. Simplified analogues of the triterpene saponin glycyrrhizin containing 1-thio-β-D-glucuronic acid residues have been prepared in good yields by alkylation of 3-amino and 3-thio derivatives of glycyrrhetinic acid with a 2-iodoethyl 1-thio-β-D-glucopyranosiduronate derivative. The spacer-connected 3-amino derivatives were further transformed into N-acetylated and N-succinylated derivatives. The deprotected compounds containing these carboxylic acid appendices mimic the glycon part of glycyrrhizin as well as the hemisuccinate derivative of glycyrrhetinic acid, carbenoxolone. Antiviral activities of the compounds were determined in a biological test based on influenza A virus-infected cells, wherein the 3-(2-thioethyl)-N-acetylamino- and 3-(2-thioethyl)-thio-linked glucuronide derivatives were effective inhibitors with IC₅₀ values as low as 54 μM.

Full text of DOI:10.3762/bjoc.8.79

Synthesis and antiviral activities of spacer-linked
1-thioglucuronide analogues of glycyrrhizin
Christian Stanetty1,§, Andrea Wolkerstorfer2,§, Hassan Amer1,
Andreas Hofinger1, Ulrich Jordis3, Dirk Claßen-Houben2 and Paul Kosma*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 705–711.
1Department of Chemistry, University of Natural Resources and Life
Sciences-Vienna, Muthgasse 18, A-1190 Vienna, Austria, 2Savira
Pharmaceuticals GmbH, Veterinärplatz 1, A-1210 Vienna, Austria and
3Institute of Applied Synthetic Chemistry, Vienna University of
Technology, Getreidemarkt 9, A-1060 Vienna, Austria
doi:10.3762/bjoc.8.79
Received: 09 March 2012
Accepted: 12 April 2012
Published: 08 May 2012
Email:
This article is part of the Thematic Series "Synthesis in the
glycosciences II".
Paul Kosma* - paul.kosma@boku.ac.at
* Corresponding author
Guest Editor: T. K. Lindhorst
§ These authors contributed equally to this work
© 2012 Stanetty et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
antiviral activity; carbenoxolone; glycyrrhizin; influenza A virus;
thioglycoside; triterpene
Abstract
The influenza virus infection remains a significant threat to public health and the increase of antiviral resistance to available drugs
generates an urgent need for new antiviral compounds. Starting from the natural, antivirally active compound glycyrrhizin, spacer-
bridged derivatives were generated with improved antiviral activity against the influenza A virus infection. Simplified analogues of
the triterpene saponin glycyrrhizin containing 1-thio-β-D-glucuronic acid residues have been prepared in good yields by alkylation
of 3-amino and 3-thio derivatives of glycyrrhetinic acid with a 2-iodoethyl 1-thio-β-D-glucopyranosiduronate derivative. The
spacer-connected 3-amino derivatives were further transformed into N-acetylated and N-succinylated derivatives. The deprotected
compounds containing these carboxylic acid appendices mimic the glycon part of glycyrrhizin as well as the hemisuccinate deriva-
tive of glycyrrhetinic acid, carbenoxolone. Antiviral activities of the compounds were determined in a biological test based on
influenza A virus-infected cells, wherein the 3-(2-thioethyl)-N-acetylamino- and 3-(2-thioethyl)-thio-linked glucuronide derivatives
were effective inhibitors with IC50 values as low as 54 µM.
Introduction
The triterpene saponin glycyrrhizin (GL) and its aglycon antiviral properties [1,2]. The antiviral activities have been
glycyrrhetinic acid (GA) are the main triterpene components of reported to be directed against a broad spectrum of viruses
licorice roots and harbor various pharmacological activities, comprising herpes-, corona-, alpha-, and flaviviruses, HIV,
including antitumor, anti-inflammatory, antioxidant and Epstein–Barr virus, influenza A virus (IAV), vaccinia and
705

Beilstein J. Org. Chem. 2012, 8, 705–711.
Figure 1: Structure of glycyrrhizin (GL), carbenoxolone (CBX), and spacer analogues.
Results and Discussion
polio type I viruses as examples [3-8]. In particular anti-
influenza virus activities have been described, although the Synthesis of 3-amino derivatives
underlying mechanisms of action are diverse [9-12]. In order to Commercially available methyl (2,3,4-tri-O-acetyl-D-glucopy-
evaluate the potential antiviral properties of glycyrrhizin deriva- ranosyl)uronate bromide (1) was first reacted with potassium
tives, we set out to modify the chemical environment in the thioacetate in DMF to furnish the known 1-thioacetyl deriva-
vicinity of ring A of glycyrrhetinic acid. The envisaged modifi- tive 2 in 82% yield [16]. Subsequent treatment of 2 with sodium
cations should assess the impact of acidic groups, which reside methoxide, under controlled conditions at low temperature
at the glucuronic acid residues in the β-(1→2)-linked disaccha- (−60 °C → −45 °C), provided the corresponding glucuronyl
ride unit of GL, with respect to antiviral properties (Figure 1).
1-thiol 3 in 89% yield [17,18]. Reaction of 3 with an excess of
1,2-dibromoethane (3–4 equiv) in DMF in the presence of
Previously, ring A modified derivatives of glycyrrhetinic acid sodium hydride, with the strict exclusion of oxygen, afforded
were prepared containing 3-amino, 3-thio and 1-thio groups, the 2-bromoethyl 1-thioglycoside 4 in 80% yield. Under these
allowing for an extension of the oleanolic acid unit with spacer conditions formation of the bis-substitution product 5 (3%) and
groups as well as facile covalent attachment of carboxyalkyl the disulfide oxidation product of 1-thiol 3 was observed in only
groups by alkylation or acylation reactions [13,14]. For the very minor quanities. The corresponding 2-iodoethyl derivative
compounds included in this investigation, the size of the spacer 6 was prepared by a Finkelstein reaction from the bromoethyl
group was selected to closely match the formal distances 1-thioglycoside 4 in 95% yield (Scheme 1).
between the A-ring and the carboxylic acid function present in
the glucopyranosiduronic residues of the parent compound The previously reported 3β-amino derivative of the diphenyl-
glycyrrhizin (GL) and in the hemisuccinate moiety of the methyl ester of glycyrrhetinic acid 7 [19] was then used for the
glycyrrhetinic acid derivative carbenoxolone (CBX) [15] introduction of the spacer-extended 1-thio-glucopyra-
(Figure 1). For the sake of clarity, the nomenclature and nosiduronate residue. Coupling of the 3β-amino derivative 7
numbering for the triterpene system as used throughout this with two equivalents of the 2-iodoethyl glycoside 6 in DMF in
report is illustrated in Figure 1. In order to enhance resistance the presence of Hünig base proceeded smoothly to provide the
against enzymatic cleavage by glucuronidases, the glucuronic alkylated amine 8 in 79% yield without the formation of a bis-
acid residue was introduced through a stable thioglycosidic alkylated product (Scheme 2), whereas the use of the corre-
linkage.
sponding bromide 4 resulted in a significantly slower reaction.
Scheme 1: Synthesis of methyl 2-haloethyl 1-thio-glucuronide derivatives: (a) 1 M NaOMe, MeOH, −60 °C to −45 °C, 89% ; (b) Br(CH2)2Br, NaH,
DMF, −5 °C (80% for 4, 3% for 5); (c) NaI, acetone, 4 °C, 16 h, 95%.
706

Beilstein J. Org. Chem. 2012, 8, 705–711.
Scheme 2: Synthesis of thioalkylglucuronide GA derivatives: (a) DMF, DIPEA, 45–50 °C, 16 h, 79%; (b) TEA, Ac2O, DCM, 0 °C, 2 h, 96%; (c)
anisole, TFA, DCM, rt, 3 h (75% for 10, 89% for 11); (d) 0.1 M NaOMe, MeOH, 0 °C (89% for 12, 97% for 14, 99% for 18); (e) 0.2 M NaOH, MeOH, rt,
3 h (91% for 13, ~quant. for 15, 99% for 19); (f) MeO2C(CH2)2COCl in DCM, DIPEA, 0 °C to rt, 90 min, then anisole, TFA, DCM, 0 °C, 3 h, 83% for
two steps.
At elevated temperature, elimination leading to the correspond- carbenoxolone analogue 16 [20,21]. Reaction of the 3-amino
ing hexenuronic acid derivative was observed (structure not group of 8 with the monomethyl ester of succinic acid chloride
shown). The secondary amino group in compound 8 was in dichloromethane in the presence of Hünig base was followed
subjected to further derivatization by N-acetylation (triethyl- by deblocking of the diphenylmethyl ester group to afford 17 in
amine, acetic anhydride), which gave compound 9 in 96% yield. a combined yield of 83%. The acetyl groups of 17 were
Deprotection of the ethyl 1-thio-glucuronide derivatives 8 and 9 removed under Zemplén conditions to afford the dimethyl ester
was achieved by acid-catalyzed cleavage of the diphenylmethyl 18. Alkaline hydrolysis of the ester groups eventually gave the
ester group with TFA/anisole (as carbocation scavenger), giving triacid derivative 19 in good yield. In contrast to the straightfor-
the monoacid derivatives 10 and 11 in 75% and 89% yield, res- ward chemical transformations, analysis of the NMR spectra of
pectively. Subsequent transesterification of 10 and 11 with this series of compounds was challenging and the N-acylated
methanolic NaOMe afforded the deacetylated methyl ester derivatives 9, 11, 14, 15 and 17–19 revealed complex spectra.
glucuronide derivatives 12 and 14, respectively, which were Whereas the secondary amides of 3β-amino-glycyrrhetinic acid
finally fully deprotected by hydrolysis of the methyl ester group derivatives displayed coherent NMR signals [22,23], the tertiary
with 0.2 M methanolic NaOH to furnish the diacid derivatives amides frequently exist as mixtures of cis/trans rotamers
13 and 15, in high overall yield.
leading to the duplication of signals [24,25]. The steric conges-
tion at position 3 exerted by the two adjacent methyl groups
A second series of compounds was designed as glucuronide- may additionally contribute to the restricted rotation of the
extended amide derivatives of the previously reported amide linkage. Thus, in the 1H NMR spectra of the N-acetyl-
707

Beilstein J. Org. Chem. 2012, 8, 705–711.
Figure 2: 400 MHz 1H NMR expansion plots of the carbohydrate region of compound 11, recorded at various temperatures.
and N-succinyl derivatives, recorded at room temperature, two 21 was performed in two steps as described for 10, which
sets of signals in an approximate 6:4 ratio were observed. Re- furnished the methyl ester derivative 22 and the glucuronic acid
versible coalescence of these signal groups was observed upon compound 23, respectively, in good yields (Scheme 3).
heating a solution of the N-acetyl derivative 11 up to 100 °C in
DMSO-d6 and subsequent cooling to room temperature Antiviral activities
(Figure 2), supporting the assignment of two signal data sets for For biological testing the compounds were dissolved in DMSO
two rotamers. The orientation of the amide carbonyl group in and diluted in cell culture medium to a maximum final DMSO
the rotamers also had a pronounced effect on the NMR chem- concentration of 1%. For determination of the cytotoxicity of
ical shifts of carbon and proton signals in ring A, which led to a the compounds, MDCK cells were treated with compound
shift of H-3 to lower field (δ 4.47) and to a shielding of C-3 concentrations ranging from 3.1 µM to 250 µM for 48 h and
(δ 59.9) for the minor isomer, whereas the inverse effects were then cell viability was determined as a surrogate endpoint for
seen for the major rotamer (δ for H-3 at 3.36 and δ for C-3 at cytotoxicity. Except for the aglycons of GL, namely glycyrrhe-
66.7). Similar, but less-pronounced effects were observed for tinic acid (GA) and the hemisuccinate carbenoxolone (CBX),
the respective 1H and 13C signals at position 5 of ring A (see which showed CC50 values (half-maximal cytotoxic concentra-
Supporting Information File 1).
tion) of 7.4 µM and 17.8 µM, respectively, none of the com-
pounds tested were toxic at concentrations up to 250 µM. GL
was not toxic at 2500 µM (Table 1). The antiviral activity of the
Synthesis of 3-thio derivatives
Complementing the series of the 3β-amino series, related 3-thio compounds was determined by infecting MDCK cells with
derivatives were employed for the spacer elongation [14]. The influenza A H3N2 virus and treating them with different
previously reported 1,2-dehydro-3-thiol derivative 20 was concentrations of compounds at the same time. Most com-
subjected to alkylation with the 2-iodoethyl 1-thioglucuronide pounds prevented virus-induced cytopathicity and restored cell
compound 6 in the presence of K2CO3 to furnish the thioether- viability compared to virus-infected control cells. The antiviral
bridged glucuronide triterpene 21 in 60% yield. Deprotection of activity of glycyrrhizin (GL) against influenza and other viruses
708

Beilstein J. Org. Chem. 2012, 8, 705–711.
Scheme 3: Synthesis of 3-thioether-bridged glucuronide derivatives: (a) K2CO3, acetone, 60%; (b) 0.8 M NaOMe, MeOH, 71%; (c) 0.2 M NaOH,
MeOH, 95% .
reported in the literature was confirmed in this study resulting in (18) did not improve the activity; however, the compound was
an IC50 of 1026 µM (Table 1). GA and CBX did not show any still active with an IC50 value of 125 µM. The cleavage of both
antiviral activity at nontoxic concentrations. Compounds 12, 13, methyl ester groups of this compound resulted in compound 19
14, 15, 18 and 19, which possess a (1-thio-β-D-glucopyranosyl- and led to the loss of antiviral activity up to the highest concen-
uronic acid)ethylamino substituent at position 3 of GA did not tration tested (250 µM). Compound 16 containing only the
show toxicity at concentrations up to 250 µM, but showed N-succinyl substituent is a close analogue to CBX bearing an
enhanced antiviral activity compared to GL. Good activities amide instead of the ester group. Unlike CBX, which is toxic at
were found for compounds 12 and 18 with IC50 values of low concentrations, 16 was not toxic but inactive, indicating
220.4 μM and 125 µM, respectively, being between 4.7-fold to that the glucuronic acid moiety is associated with the antiviral
8-fold more active compared to the lead compound GL. The properties. This was further supported by the data obtained with
methyl ester 14 containing the N-acetylated spacer group was compound 23 containing a 1-thioethyl-linked glucuronic acid
approximately 14-fold more active compared to GL, reflected residue at position 3 of 1,2-dehydro-glycyrrhetinic acid. Com-
by an IC50 value of 72.1 µM. The free acid 15 displayed an pound 23 showed good activity with an even lower IC50 value
IC50 value in the same range as the methyl ester 14. Replace- of 54 µM compared to 15, and exhibited also no cytotoxicity at
ment of the N-acetyl group of 14 with a methylsuccinyl group the concentrations tested.
Table 1: Cytotoxic concentration 50% (CC50) of compound-treated uninfected cells and antiviral activities, shown as half-maximal inhibitory concen-
tration (IC50) values of glycyrrhizin (GL) analogues.
Compound
Substructure
CC50 [µM]
IC50 [µM]
GL
>2500
1026 ± 181
GA
7.4 ± 0.05
17.8 ± 2.3
nda
nda
CBX
12
>250
220.4 ± 41
709

Beilstein J. Org. Chem. 2012, 8, 705–711.
Table 1: Cytotoxic concentration 50% (CC50) of compound-treated uninfected cells and antiviral activities, shown as half-maximal inhibitory concen-
tration (IC50) values of glycyrrhizin (GL) analogues. (continued)
13
14
>250
>75
nd (>250)b
72.1 ± 19.1c
15
16
>250
>250
87 ± 7.9
nd (>250)b
18
>250
125 ± 15.9
19
23
>250
>250
nd (>250)b
54 ± 13
and not determined due to cytotoxicity; bnd (>250) no detectable antiviral activity up to 250 µM; capproximate value, since the compound precipitates
at concentrations >75 μM.
Conclusion
Acknowledgements
3-Amino and 3-thio derivatives of glycyrrhetinic acid served as The work was supported by the ZIT Zentrum für Innovation
versatile scaffolds for the attachment of glycosyl extensions und Technologie GmbH (Vienna Spot of Excellence: 182081).
through high-yielding alkylation reactions. The glucuronide We thank Michaela Mutzl, Barbara Butzendobler and Eva
derivatives linked via a thioethyl spacer group to the oleanolic Zingler for the performance of antiviral activity assays and
acid unit exhibited no toxicity at concentrations up to 250 μM David Baum and Karin Hofbauer for technical assistance in the
and significantly enhanced the anti-influenza virus activity of organic syntheses of precursors.
the natural triterpene glycoside glycyrrhizin.
References
1. Baltina, L. A. Curr. Med. Chem. 2003, 10, 155–171.
Supporting Information
2. Schwarz, S.; Csuk, R. Bioorg. Med. Chem. 2010, 18, 7458–7474.
doi:10.1016/j.bmc.2010.08.054
Supporting Information File 1
3. Lampis, G.; Deidda, D.; Pinza, M.; Pompei, R.
Experimental details for the preparation of compounds
2–19 and 21–23 as well as the biological assays.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-8-79-S1.pdf]
Antiviral Chem. Chemother. 2001, 12, 125–131.
4. Cinatl, J.; Morgenstern, B.; Bauer, G.; Chandra, P.; Rabenau, H.;
Doerr, H. W. Lancet 2003, 361, 2045–2046.
doi:10.1016/S0140-6736(03)13615-X
710

Beilstein J. Org. Chem. 2012, 8, 705–711.
5. Crance, J. M.; Scaramozzino, N.; Jouan, A.; Garin, D. Antiviral Res.
2003, 58, 73–79. doi:10.1016/S0166-3542(02)00185-7
6. Hattori, T.; Ikematsu, S.; Koito, A.; Matsushita, S.; Maeda, Y.;
Hada, M.; Fujimaki, M.; Takatsuki, K. Antiviral Res. 1989, 11, 255–261.
doi:10.1016/0166-3542(89)90035-1
License and Terms
This is an Open Access article under the terms of the
Creative Commons Attribution License
7. Harada, S. Biochem. J. 2005, 392, 191–199. doi:10.1042/BJ20051069
8. Lin, J.-C. Antiviral Res. 2003, 59, 41–47.
(http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
doi:10.1016/S0166-3542(03)00030-5
9. Pompei, R.; Paghi, L.; Ingianni, A.; Uccheddu, P. Microbiologica 1983,
6, 247–250.
10.Utsunomiya, T.; Kobayashi, M.; Pollard, R. B.; Suzuki, F.
Antimicrob. Agents Chemother. 1997, 41, 551–556.
11.Wolkerstorfer, A.; Kurz, H.; Bachhofner, N.; Szolar, O. Antiviral Res.
2009, 83, 171–178. doi:10.1016/j.antiviral.2009.04.012
12.Michaelis, M.; Geiler, J.; Naczk, P.; Sithisarn, P.; Leutz, A.;
Doerr, H. W.; Cinatl, J., Jr. PLoS One 2011, 6, e19705.
doi:10.1371/journal.pone.0019705
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
(http://www.beilstein-journals.org/bjoc)
The definitive version of this article is the electronic one
which can be found at:
13.Claßen-Houben, D.; Kueenburg, B.; Kosma, P.; Jordis, U.; Stanetty, C.;
Szolar, O.; Wolkerstorfer, A. Antiviral Triterpene Derivatives. EP 2 258
713 A1, Dec 8, 2010.
doi:10.3762/bjoc.8.79
14.Amer, H.; Mereiter, K.; Stanetty, C.; Hofinger, A.; Czollner, L.;
Beseda, I.; Jordis, U.; Kueenburg, B.; Claßen-Houben, D.; Kosma, P.
Tetrahedron 2010, 66, 4390–4402. doi:10.1016/j.tet.2010.03.098
15.Jellinck, P. H.; Monder, C.; McEwen, B. S.; Sakai, R. R.
J. Steroid Biochem. Mol. Biol. 1993, 46, 209–213.
doi:10.1016/0960-0760(93)90296-9
16.Florio, P.; Thomson, R. J.; von Itzstein, M. Carbohydr. Res. 2000, 328,
445–448. doi:10.1016/S0008-6215(00)00228-7
17.MacDougall, J. M.; Zhang, X.-D.; Polgar, W. E.; Khroyan, T. V.; Toll, L.;
Cashman, J. R. J. Med. Chem. 2004, 47, 5809–5815.
doi:10.1021/jm049554t
18.Cao, H.; Yu, B. Tetrahedron Lett. 2005, 46, 4337–4340.
doi:10.1016/j.tetlet.2005.04.088
19.Beseda, I.; Czollner, L.; Shah, P. S.; Khunt, R.; Gaware, R.; Kosma, P.;
Stanetty, C.; del Ruiz-Ruiz, M. C.; Amer, H.; Mereiter, K.;
Da Cunha, T.; Odermatt, A.; Claßen-Houben, D.; Jordis, U.
Bioorg. Med. Chem. 2010, 18, 433–454.
doi:10.1016/j.bmc.2009.10.036
20.Ijichi, S.; Tamagaki, S. Chem. Lett. 2005, 34, 356–357.
doi:10.1246/cl.2005.356
21.Brieskorn, C. H.; Eschelbach, H. Arch. Pharm. 1979, 312, 752–762.
doi:10.1002/ardp.19793120907
22.Stanetty, C.; Czollner, L.; Koller, I.; Shah, P.; Gaware, R.;
Da Cunha, T.; Odermatt, A.; Jordis, U.; Kosma, P.; Claßen-Houben, D.
Bioorg. Med. Chem. 2010, 18, 7522–7541.
doi:10.1016/j.bmc.2010.08.046
23.Csuk, R.; Schwarz, S.; Siewert, B.; Kluge, R.; Ströhl, D.
Eur. J. Med. Chem. 2011, 46, 5356–5369.
doi:10.1016/j.ejmech.2011.08.038
24.Zhou, C. C.; Stewart, K. D.; Dhaon, M. K. Magn. Reson. Chem. 2005,
43, 41–46. doi:10.1002/mrc.1501
25.Stewart, W. E.; Siddall, T. H., III. Chem. Rev. 1970, 70, 517–551.
doi:10.1021/cr60267a001
711