Synthesis of glycoside derivatives of hydroxyanthraquinone with ability to dissolve and inhibit formation of crystals of calcium oxalate. Potential compounds in kidney stone therapy

Article abstract of DOI:10.1016/j.ejmech.2009.11.042

Synthesis of glycosyl derivatives of hydroxyanthraquinones (6-10) potentially useful for kidney stone therapy is presented. These compounds were analyzed as inhibitors of calcium oxalate crystals formation as well as substances with the ability of dissolving crystalline calcium oxalate. In addition, the effect of the compounds obtained on real kidney stones was analyzed by ex vivo tests. The tests on L929 and A545 cell lines have shown that the compounds obtained were not cytotoxic.

Full text of DOI:10.1016/j.ejmech.2009.11.042

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European Journal of Medicinal Chemistry 45 (2010) 1001–1007
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of glycoside derivatives of hydroxyanthraquinone with ability to
dissolve and inhibit formation of crystals of calcium oxalate. Potential compounds
in kidney stone therapy
a
b
b
c
c
´
´
Anna Fra˛ckowiak , Przemys1aw Skibinski , Wies1aw Gawe1 , Ewa Zaczynska , Anna Czarny ,
,
Roman Gancarz a
*
a Medicinal Chemistry and Microbiology Department, Wroclaw University of Technology, Poland
b Chair and Department of Analytical Chemistry, Wroclaw Medical University, Poland
c Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of glycosyl derivatives of hydroxyanthraquinones (6–10) potentially useful for kidney stone
therapy is presented. These compounds were analyzed as inhibitors of calcium oxalate crystals formation
as well as substances with the ability of dissolving crystalline calcium oxalate. In addition, the effect of
the compounds obtained on real kidney stones was analyzed by ex vivo tests. The tests on L929 and A545
cell lines have shown that the compounds obtained were not cytotoxic.
Received 21 June 2009
Received in revised form
16 November 2009
Accepted 20 November 2009
Available online 26 November 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Anthraquinone derivatives
Calcium oxalate crystals
Kidney stones
Cytotoxicity
1. Introduction
lucidin. It was found that this compound induces mutations, which
very often leads to carcinogenic changes [2–5]. Another plant
Nephrolithiasis is caused by formation of stones in kidneys.
There are several types of kidney stones differing in chemical
composition. Among them calcium oxalate stones are the most
common (70–80% of all stone cases). The incidence and prevalence
of kidney stone formation may be affected by genetic, nutritional
and environmental factors [1]. Mostly the kidney stones are
removed by ureteroscopic fragmentation using laser, ultrasonic or
mechanical forms of energy to fragment the larger stones or in
percutaneous nephrolithotomy and using a urethral stent.
rich in anthraquinone glycosides and used in kidney therapy is Aloe
Vera [6].
In the literature one can find information on other plants such
as: Berberis vulgaris-Q, Dioscorea Q, Calcarea carb 200, Ocimum
sanctum, Syzygium cumini L., Amblica officinalis, Citrus aurantifolia,
Citrus sinensie extracts of which have kidney stone dissolving
properties. Some authors have suggested that such action is due to
the presence of proteins and sugars, which are able to form
complexes with calcium ions [7,8].
There is no drug able to dissolve kidney stones. Today, most of
the drugs used are diuretic, antiseptic and antibacterial but none of
them is able to dissolve the kidney stones. Several years ago, two
plant preparations, ‘‘Rubinex’’ and ‘‘Rubiolizyna’’, obtained from
Radix Rubiae tinctorum L. were used for that purpose. It was
reported that their properties were due to the complexing ability of
hydroxyanthraquinone derivatives to calcium ions. The use of Radix
R. tinctorum L. extract has been withdrawn due to the presence of
Since in recent years many data have appeared indicating that
sugars have high potency for calcium binding [7,8], we decided to
synthesize glycosides of hydroxyanthraquinones, compounds
combining the properties of hydroxyanthraquinones and sugars, in
the hope that such compounds can be potentially used in kidney
stone therapy.
2. Results and discussion
mutagenic
1,3-dihydroxy-2-hydroxymethyl-9,10-anthraquinone
(lucidin). 1 kg of Radix R. tinctorum L. contains about 70 mg of
2.1. Chemistry
The best known glycosylation donors for aromatic compounds
are acetates, halides, trichloroacetimidates and thioglycosides
* Corresponding author. Tel.: þ48 71 320 3972; fax: þ48 71 328 4064.
E-mail address: roman.gancarz@pwr.wroc.pl (R. Gancarz).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.042

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1002
A. Fra˛ckowiak et al. / European Journal of Medicinal Chemistry 45 (2010) 1001–1007
[9–13]. Bromides in turn are the glycosyl donors most frequently
described in the literature [13].
In the present work we have prepared five glycosides in which
the sugar parts were glucose and galactose, whereas the aglycones
were: 1,2-dihydroxy-9,10-anthraquinone, 1,4-dihydroxy-9,10-
anthraquinone, 1,2,5,8-tetrahydroxy-9,10-anthraquinone.
The acetylation of glucose yielded the mixture of anomers in
which more than 80% was alfa anomer. The identification of alfa
and beta anomers was based on their chemical schifts and coupling
constants 3JHH of anomeric proton. The next steps i.e. bromination
and coupling gave the beta anomer of final product.
All syntheses proceeded very easily except that involving 1,4-
dihydroxy-9,10-anthraquinone. In this compound the two hydroxyl
groups are in such a position that a hydrogen bond with the
carbonyl group can be formed. This makes a hydroxyl group less
suitable for glycosylation.
We have selected a four-step method of preparing glycosides of
hydroxyanthraquinones consisting of acetylation of sugar
compounds, their bromination, coupling with the hydroxyan-
thraquinone and finally removal of the acetyl groups [14–19]. All
compounds presented in this paper were prepared by this method
(Scheme 1).
Anthraquinone derivatives like: ruberythric acid, alizarin, qui-
nizarin, purpuric acid, rubiadin, purpurin present in Radix R.
tinctorum L. possess the ability to dissolve kidney stones as well as
to prevent their deposition in the urinary tract [20,21]. Such
properties are believed to be connected with the ability of
hydroxyanthraquinones to form complexes with calcium ions.
Most of these compounds can form glycosides. The sugar
component increases the solubility of the molecule as well as
increases its complexing properties due to the presence of
hydroxyl groups or additional carboxylic groups in the case of
glucuronic acid [22–24].
The compounds obtained (1–10) are presented in Table 1.
The glycosides obtained (6–10) and the corresponding agly-
cones were analyzed as substances able to form complexes with
calcium ions. The measurements were performed by conducto-
metric titration (ability to inhibit calcium oxalate formation) and
CH2OH
CH2OAc
CH2OAc
O
O
O
H
H
H
H
H
OAc
H
Ac2O
H
H
OAc
H
OAc
H
H
+
H
OH
OH
OAc
HO
AcO
AcO
OH
OAc
OAc
H
H
H
HBr/CH3COOH
CH2OAc
O
H
H
H
OAc
H
Br
AcO
OAc
H
O
Ag2O/quinoline
HO
OH
O
O
CH2OAc
O
H
O
H
H
OAc
OH
major isomer β
NaOH
O
H
AcO
OAc
H
O
O
CH2OH
O
H
O
H
H
OH
H
OH
HO
OH
H
Scheme 1. The synthesis of glycoside of hydroxyanthraquinones.

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A. Fra˛ckowiak et al. / European Journal of Medicinal Chemistry 45 (2010) 1001–1007
1003
Table 2
flame photometry (ability to dissolve calcium oxalate taken as
a model for kidney stones).
Results of conductometric titration.
Compound
The end point of the
D
EP
l]
sample curve [
m
l]
[
m
3. Complexing properties and biological investigation
None (standard)
1,2-dihydroxy-9,10-anthraquinone
2-(b-D-glucopyranosyloxy)-1-hydroxy-9,10-
270
230
190
0
40
80
3.1. Inhibition of calcium oxalate crystal formation
anthraquinone (6)
1,4-dihydroxy-9,10-anthraquinone
1-(b-D-glucopyranosyloxy)-4-hydroxy-9,10-
anthraquinone (7)
The inhibition of calcium oxalate crystal formation was carried
out by conductometric titration of calcium chloride with sodium
oxalate in the absence (standardization curve (1)) and in the
presence of the compound examined (sample curve (2)) [25,26].
The effect of the inhibition of calcium oxalate formation was
240
210
30
60
1,2,5,8-tetrahydroxy-9,10-anthraquionone
180
90
90
180
2-(
9,10-anthraquinone (8)
2-( -galactopyranosyloxy)-1-hydroxy-9,10- 200
anthraquinone (9)
2-( -galactopyranosyloxy)-1,5,8-trihydroxy- 100
9,10-anthraquinone (10)
b
-D
-glucopyranosyloxy)-1,5,8-trihydroxy-
expressed as
without the presence of one of the compounds studied in the solu-
tion. The higherthe EP, the bettertheinhibitingpropertiesofcrystal
DEP, i.e. the shift of the titration end point (EP) in and
b-D
70
b
-D
170
D
formation of such a compound. The results are presented in Table 2.
The presence of glycoside part increases the complexing ability
D
EP ¼ the end point of the standardization curve ꢀ the end point of the sample
curve.
of the final compound which is manifested by increased
biggest effect was observed after glycosylation of 1,2-dihydroxy-
9,10-anthraquinone ( EP was changed from 40 l to 80 l after
DEP. The
D
m
m
The results are presented in Table 3. They express the
glycosylation). However the highest dissolving properties were
observed for 1,2,5,8-tetrahydroxy-9,10-anthraquinone and its
glycosides (8,10). It leads to the conclusion that the higher the
number of hydroxyl groups in the aromatic region the better
complexing properties of the compound.
percentage increase of total concentration of calcium ions in the
solution studied, compared with saturated calcium oxalate
solution.
The results obtained indicate that all the compounds studied
and their glycosides have good properties of coordination of
calcium ions. It was also found that the presence of the sugar
fragment increases the complexing ability, compared with that of
the pure aglycones.
3.2. Dissolution of calcium oxalate
The best complexing properties, measured by flame photom-
etry, expressed as the increase of calcium concentration in solution
of the compounds studied, compared with saturated calcium
Flame photometry was the method chosen to estimate the
ability to dissolve calcium oxalate treated as model kidney stones
[25,26].
Table 1
The structures of the compounds synthetized.
R
R
8
O
1
8
9
1
R
2
R
7
7
6
12
11
13
14
2
3
A
5
B
C
4
10
R
R
3
6
R
R
4
O
5
Number
of the
Name of the compound
R1
R2
R3
R4
R5
R6
R7
R8
compound
1
2
3
4
5
6
7
8
9
10
2-(2,3,4,6-tetra-O-acetyl-
b
-
D
-glucopyranosyloxy)-1-
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
O-AC-glucose
O-AC-glucose
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
hydroxy-9,10-anthraquinone
2-(2,3,4,6-tetra-O-acetyl- -glucopyranosyloxy)-1,5,8-
trihydroxy-9,10-anthraquinone
1-(2,3,4,6-tetra-O-acetyl-
hydroxy-9,10-anthraquinone
2-(2,3,4,6-tetra-O-acetyl-
hydroxy-9,10-anthraquinone
2-(2,3,4,6-tetra-O-acetyl- -galactopyranosyloxy)-1,5,8-
trihydroxy-9,10-anthraquinone
2-( -glucopyranosyloxy)-1-hydroxy-9,10-
anthraquinone
1-( -glucopyranosyloxy)-4-hydroxy-9,10-
anthraquinone
2-( -glucopyranosyloxy)-1,5,8-trihydroxy-9,10-
anthraquinone
2-( -galactoopyranosyloxy)-1-hydroxy-9,10-
anthraquinone
2-( -galactoopyranosyloxy)-1,5,8-trihydroxy-9,10-
anthraquinone
b
-D
OH
H
OH
H
b
-D
-glucopyranosyloxy)-4-
-galactopyranosyloxy)-1-
O-AC-
glucose
H
b
-D
O-AC-galactose
O-AC-galactose
O-glucose
H
H
H
b
-D
H
OH
H
OH
H
b
-D
H
b
-D
O-glucose
H
H
b
-D
O-glucose
O-galactose
O-galactose
H
H
H
OH
H
OH
H
b-D
b
-D
OH
OH

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A. Fra˛ckowiak et al. / European Journal of Medicinal Chemistry 45 (2010) 1001–1007
Table 3
Results of flame photometry – calcium oxalate test.
Compound
Concentration of
Ca2þ in sample
test [M]
D
Ca2þ [M]
The increase
of calcium
concentration [%]
1,2-dihydroxy-9,10-anthraquinone
9.10 ꢁ 10ꢀ4
2.20 ꢁ 10ꢀ3
7.60 ꢁ 10ꢀ4
2.00 ꢁ 10ꢀ3
4.90 ꢁ 10ꢀ3
5.60 ꢁ 10ꢀ3
1.60 ꢁ 10ꢀ3
1.90 ꢁ 10ꢀ3
5.40 ꢁ 10ꢀ4
1.83 ꢁ 10ꢀ3
3.90 ꢁ 10ꢀ4
1.66 ꢁ 10ꢀ3
4.53 ꢁ 10ꢀ3
5.23 ꢁ 10ꢀ3
1.23 ꢁ 10ꢀ3
1.53 ꢁ 10ꢀ3
246
595
205
2-(
1,4-dihydroxy-9,10-anthraquinone
1-( -glucopyranosyloxy)-4-hydroxy-9,10-anthraquinone (7)
1,2,5,8-tetrahydroxy-9,10-anthraquionone
b-D-glucopyranosyloxy)-1-hydroxy-9,10-anthraquinone (6)
b
-D
541
1324
1513
432
2-(
2-(
2-(
b
b
b
-D
-D
-D
-glucopyranosyloxy)-1,5,8-trihydroxy-9,10-anthraquinone (8)
-galactopyranosyloxy)-1-hydroxy-9,10-anthraquinone (9)
-galactopyranosyloxy)-1,5,8-trihydroxy-9,10-anthraquinone (10)
513
Concentration of Ca2þ in reference test ¼ 3.70 ꢁ 10ꢀ4 M.
D
Ca2þ ¼ Concentration of Ca2þ in sample test (2) ꢀ Concentration of Ca2þ in reference test (1).
oxalate solution, were observed in the case of 1,2,5,8-tetrahydroxy-
9,10-anthraquinone (1324%) and its glucoside (8) (1513%). The
worst complexing properties were noted for 1,4-dihydroxy-9,10-
anthraquinone and 1,2-dihydroxy-9,10-anthraquinone. In all cases
the addition of the sugar fragment improved the complexing
properties; the biggest influence was observed for 1,2-dihydroxy-
9,10-anthraquinone. In this case the value for the aglycone was
246%, whereas that for its glucoside (6) was 595%.
at 500 mg/ml, whereas products of their glycosylation (6,7,9) are
non-cytotoxic.
The results on both mouse fibroblast cultures L929 and human
lung cultures A549 are the same. Cell cultures after contact with the
glycosides did not show any damage, had proper morphologies and
showed good proliferation compared to the control cells.
3.4. Conclusions
We have also analyzed the dissolving properties of the
compounds prepared in the case of natural kidney stones (obtained
from a patient during surgical treatment). The results are presented
Glycosylation of hydroxyanthraquinones gives derivatives (6–
10), which inhibit the formation and increase the solubility of the
model (calcium oxalate crystals) and of real kidney stones. The
glycosylation process increases solubility in water, improves
calcium complexation properties, and lowers cytotoxicity of the
studied compounds. The synthesized glycosyl derivatives of
hydroxyanthraquinones are potential drugs in kidney stone
therapy. In general it was observed that the increase of number of
hydroxyl groups in the aglycone is the main factor responsible for
in Table 4. The best properties in this case were shown by 2-(b-D-
glucopyranosyloxy)-1,5,8-trihydroxy-9,10-anthraquinone (8) (4833%).
The effect in the case on the real kidney stones is much higher than
in the case of the calcium oxalate model (1513%), which is probably
due to their different composition and different crystalline
structures.
good complexing properties. The
DEP and the D
Ca2þ obtained for
3.3. Biological investigation
1,2,5,8-tetrahydroxy-9,10-anthraquinone makes this compound
very attractive in kidney stone treatment. Further work is in
progress.
Since the aim of the work was to obtain compounds as potential
remedies in kidney stone therapy, it was necessary to evaluate their
cytotoxicity.
Cytotoxicity was investigated according to Polish standard (PN-
EN ISO 10993-5) on two cells lines, measuring in vitro growth of
mouse fibroblast L929 and human A549 cell line.
4. Experimental
4.1. General procedures
The cytotoxicity was defined as the highest dilution of test
samples that causes 50% or greater destruction of cells. The cells in
the in Eagle’s or DMEM medium served as control.
None of the glycosides studied showed a significant cytotoxic
effect. Glycosylation makes the compounds less cytotoxic even if
the aglycone itself has cytotoxic properties. 1,2-dihydroxy-9,10-
anthraquinone and 1,4-dihydroxy-9,10-anthraquinone are cytotoxic
All solvents and compounds (sugars and aglycons) were
supplied by ‘‘Sigma–Aldrich’’, real kidney stones were obtained
from a hospital. NMR was performed on a Bruker Avance DRX 300
(300 MHz).
ESI-MS was performed on molecular mass spectrometer
equipped wit a Fiunigan electrospray ionization source.
Table 4
Results of flame photometry – real kidney stones test.
Compound
Concentration of
Ca2þ in sample
test [M]
D
Ca2þ[M]
The increase
of calcium
concentration [%]
1,2-dihydroxy-9,10-anthraquinone
8.60 ꢁ 10ꢀ4
1.90 ꢁ 10ꢀ3
1.00 ꢁ 10ꢀ3
2.40 ꢁ 10ꢀ3
5.00 ꢁ 10ꢀ3
5.80 ꢁ 10ꢀ3
1.80 ꢁ 10ꢀ3
2.00 ꢁ 10ꢀ3
7.40 ꢁ 10ꢀ4
1.78 ꢁ 10ꢀ3
8.80 ꢁ 10ꢀ4
2.28 ꢁ 10ꢀ3
4.88 ꢁ 10ꢀ3
5.68 ꢁ 10ꢀ3
1.68 ꢁ 10ꢀ3
1.88 ꢁ 10ꢀ3
717
1583
833
2000
4167
4833
1500
1667
2-(
1,4-dihydroxy-9,10-anthraquinone
1-( -glucopyranosyloxy)-4-hydroxy-9,10-anthraquinone (7)
1,2,5,8-tetrahydroxy-9,10-anthraquionone
b-D-glucopyranosyloxy)-1-hydroxy-9,10-anthraquinone (6)
b
-D
2-(
2-(
2-(
b
b
b
-D
-D
-D
-glucopyranosyloxy)-1,5,8-trihydroxy-9,10-anthraquinone (8)
-galactopyranosyloxy)-1-hydroxy-9,10-anthraquinone (9)
-galactopyranosyloxy)-1,5,8-trihydroxy-9,10-anthraquinone (10)
Concentration of Ca2þ in reference test ¼ 1.20 ꢁ 10ꢀ4 M.
D
Ca2þ ¼ Concentration of Ca2þ in sample test (2) ꢀ Concentration of Ca2þ in reference test (1).