Synthetic conjugates of genistein affecting proliferation and mitosis of cancer cells

Article abstract of DOI:10.1016/j.bmc.2010.11.024

This paper describes the synthesis and antiproliferative activity of conjugates of genistein (1) and unsaturated pyranosides. Constructs linking genistein with a sugar moiety through an alkyl chain were obtained in a two-step synthesis: in a first step ge

Full text of DOI:10.1016/j.bmc.2010.11.024

Bioorganic & Medicinal Chemistry 19 (2011) 295–305
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthetic conjugates of genistein affecting proliferation and mitosis
of cancer cells
Aleksandra Rusin a, , Jadwiga Zawisza-Puchałka b, Katarzyna Kujawa a, Agnieszka Gogler-Pigłowska a,
⇑
c
c
a
b
b
´
´
Joanna Wietrzyk , Marta Switalska , Magdalena Głowala-Kosinska , Aleksandra Gruca , Wiesław Szeja ,
Zdzisław Krawczyk a,b, Grzegorz Grynkiewicz d
a Department of Tumor Biology, Maria Sklodowska-Curie Memorial Cancer Center and the Institute of Oncology, Wybrzeze AK 15, 44-100 Gliwice, Poland
b Silesian University of Technology, Krzywoustego 8, 44-100 Gliwice, Poland
c Institute of Immunology and Experimental Therapy Polish Academy of Sciences, Weigla 12, 53-114, Poland
d Pharmaceutical Research Institute, Rydygiera 8, 01-793 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes the synthesis and antiproliferative activity of conjugates of genistein (1) and unsat-
urated pyranosides. Constructs linking genistein with a sugar moiety through an alkyl chain were
obtained in a two-step synthesis: in a first step genistein was converted into an intermediate bearing
Received 16 September 2010
Revised 4 November 2010
Accepted 8 November 2010
Available online 11 November 2010
an
x-hydroxyalkyl substituent, containing two, three or five carbon atoms, at position 7, while the second
step involved Ferrier glycosylation reaction, employing glycals. Antiproliferative activity of several
genistein derivatives was tested in cancer cell lines in vitro. The most potent derivative, Ram-3 inhibited
the cell cycle, interacted with mitotic spindles and caused apoptotic cell death. Neither genistein nor the
sugar alone were able to influence the mitotic spindle organization. Our results indicate, that conjugation
of genistein with certain sugars may render the interaction of derivatives with new molecular targets.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Genistein glycoconjugates
Antiproliferative activity
Cell cycle
Mitotic spindle
Apoptosis
1. Introduction
concentration one order of magnitude lower than a parent com-
pound, or even affecting a new target.7 Current paper is a continu-
Genistein (1) is a phytochemical with widely demonstrated
beneficial effects for human health.1 Due to remarkable similarity
in chemical structure to estrogen and its ability for binding to
the estrogen receptor genistein is classified as a natural selective
estrogen receptor modulator.2,3 Genistein aglycone might be a
new potential therapy for the management of postmenopausal
osteoporosis in humans combining a powerful bone-forming as
well as an antiresorptive activity.4 In addition, hundreds of scien-
tific publications report its wide range of nonhormonal activities,
many of which have profound implications for cell function.
Among the targets inhibiting cancer cell growth topoisomerase II,
tyrosine kinases, the transforming growth factor-b (TGFb) signal-
ing pathway and nuclear transcription factor NF-kB are indicated.5
Genistein may be also useful in treatment of inherited metabolic
diseases, such as mucopolysaccharidoses, due to influencing an
epidermal growth factor-dependent pathway.6
ation of our previous work8 on glycosylation as an option for
enhancement antiproliferative potential of genistein due to target-
ing mitotic spindles.
In plants, in which genistein is produced, it exists mainly in a
glycosylated form, genistin9 which increases isoflavonoid solubil-
ity and stability.10 Although the main form of consumed genistein
is its glycoside, genistin, and the only form transported through
enterocytes to blood vessels in human is aglycon,11,12 there is no
significant difference between bioavailability of genistein con-
sumed in the form of aglycon or a glycoside, because of efficient
deconjugation of the latter in the digestive tract.12 After absorp-
tion, genistein is metabolized mostly to glucuronate and sulfate
conjugates.13
While it is well documented that aglycon form of genistein can
affect various cellular processes, much less is known about the bio-
logical activity of its glycoderivatives. Genistein glucuronides have
been shown to have weak estrogenic activity and capability of
in vitro activation of human natural killer cells14,15 and genistein
7-O-b-maltosides and 7-O-b-maltotriosides showed potent inhibi-
In order to enhance the pharmacological potential of genistein
several groups functionalized the molecule and found among
plenty of synthetic derivatives several, which were active at
tory effects on histamine release from rat peritonea mast cells.16
A
highly relevant were findings that several semi-synthetic genistein
glycosides could exert significant antiproliferative activity against
different cancer cell lines.8,17,18
⇑
Corresponding author. Tel.: +48 32 278 98 44; fax: +48 32 278 98 40.
E-mail address: arusin@io.gliwice.pl (A. Rusin).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.024

296
A. Rusin et al. / Bioorg. Med. Chem. 19 (2011) 295–305
Preliminary estimation of the structure–activity relationship led
2.1.2.2. Procedure for preparation 7-O-(5-hydroxypentyl)geni-
stein. Tetrabutylammonium salt of genistein 2 (10 mM) and 5-
bromopentyl acetate (11 mM) in 50 mL of dry DMF were stirred
at temperature 50 °C for 24 h. After the completion of reaction,
the mixture was diluted with 250 mL of ice water to form yellow
solid. Recrystallization of the solid from 100 mL hot methanol gave
acylated product (3c). This acylated product (5 mM) and magne-
sium shavings in 50 mL of dry methanol were stirred at tempera-
ture 40 °C for 24 h. After the completion of reaction, the mixture
was neutralized with an Amberlyst 15 followed by filtration. The
filtrate was distilled to form white solid. Recrystallization of the so-
lid from 100 mL hot methanol gave product 3d.
5-Hydroksy-7-(5-hydroxypentyl)-3-(4-hydroksyphenyl)chro-
men-4-one (3d). Obtained after recrystallization as a white solid,
in 88% yield; mp 154–157 °C; 1H NMR d (DMSO-d6): 1.42–1.50
(m; 4H; 2 ꢀ –CH2–); 1.71–1.76 (m; 2H; –CH2–); 3.42 (q; 2H;
J = 5.1 Hz; –CH2OH–); 4.09 (t; 2H; J = 6.6 Hz; –CH2O–); 4.40 (t;
1H; J = 5.1 Hz; –CH2OH); 6.39, 6.64 (2 ꢀ d; 2H; J = 2.2 Hz; H-6,
H-8); 6.83 (d; 2H; J = 8.7 Hz; H-30, H-50); 7.39 (d; 2H; J = 8.7 Hz;
H-20, H-60); 8.40 (s; 1H; H-2); 9.62 (s; 1H; 4’-OH); 12.95 (s; 1H;
5-OH). HRMS (ESI) Calcd C20H20O6 [M+Na]+ 379.1152. Found
379.1163.
to the conclusion that lipophilic glycosides are much more active
than hydrophilic ones, however, the lipophilicity is not the only
parameter influencing the activity of a molecule.17 There is a strong
suggestion that the sugar moiety plays a key role for the biological
activity of the genistein derivative, since one of the described glyco-
sides, named G21, affected microtubule array, similarly to mitotic
poisons.8 In the presented study we report the synthesis of new
compounds linking genistein with an unsaturated pyranoside
through an alkyl chain and describe their antiproliferative activity
against cancer cell lines, corroborating the working hypothesis, that
unsaturated sugar is a carrier of certain structural features reflected
in antiproliferative activity of genistein conjugates.
2. Material and methods
2.1. Synthesis of genistein derivatives
2.1.1. General
The reagents (chemicals), all being of A.R. grade, were pur-
chased from Across Organic. Genistein (1) (>96%) provided by
Pharmaceutical Research Institute (Warsaw, Poland) was further
refined by recrystallization from ethanol, which afforded pale
yellow needles, mp: 298–299 °C.
2.1.2.3. General procedure for synthesis of 2,3-unsaturated
pyranosides. To a solution of glycal (4, 5, 6) (0.2 mM) in anhy-
drous acetonitrile (40 mL) the aglycone (3a, 3b, 3d) (0.2 mM) and
anhydrous InCl3 (0.02 mM) was added. The contents were stirred
at temperature 25 °C for 2 h and the reaction monitored by TLC
(CHCl3/MeOH = 10:1 (v/v)). The reaction mixture was quenched
by the addition of aqueous sodium hydrogen carbonate (10%,
25 mL), extracted with dichloromethane (3 ꢀ 25 mL), dried over
anhydrous sodium sulfate, filtered and washed with dichlorometh-
ane, and the combined organic extract was concentrated under
vacuum. The residue was purified by column chromatography
(CHCl3) on silica gel to obtain the product.
Reactions and the resulted products were monitored by thin-
layer chromatography (TLC) on Merck pre-coated silica gel F254
plates with separated compounds visualized at 254 nm under a
UV lamp. Melting points (uncorrected) were determined with
MTT-2 model. ESI mass spectra were obtained on a Mariner System
5304 mass spectrometer, and 1H NMR spectra were recorded in
DMSO-d6 or CDCl3 on a Varian (300 MHz) spectrometer with sol-
vent signals allotted as internal standard. Sonication was per-
formed in a ultrasonic cleaner Sonorex Super with irradiation
delivered at 35 kHz and 500 W. Hydroxyalkylgenistein intermedi-
ates were recrystallised from methanol. Glycoconjugates were
purified by column chromatography (Silica Gel 60, 70–230 mesh,
Merck) and eluted with chloroform. Chromatographic fractions
were pooled according to TLC analysis and solvents were evapo-
rated under diminished pressure in rotary evaporators.
5-Hydroksy-7-[(4,6-di-O-acetyl-2,3-dideoxy-a-D-erythro-hex-2-
enopyranosyl)-2-O-ethyl]-3-(4-hydroksyphenyl)chromen-4-one (7a).
Obtained after chromatography as colorless syrup, in 49% yield; 1H
NMR(CDCl3) d: 2.10 (s; 3H; AcO);2.12 (s; 3H; AcO);3.9–3.97 (m; 1H;
H-5); 4.08–4.32 (m; 6H; 2 ꢀ –CH2O–; H-6a, H-6b); 5.15 (br s; 1H; H-
1); 5.36 (dd; 1H; J = 9.7 Hz; J = 1.3 Hz; H-4); 5.86–5.95 (m; 2H; H-2,
H-3); 6.41, 6.38 (2 ꢀ d; 2H; J = 2.3 Hz; H-8g, H-6g); 6.61 (br s; 1H; 40-
OHg);6.85(d; 2H;J = 8.6 Hz;H-30g, H-50g);7.34 (d;2H;J = 8.6 Hz;H-
20g, H-60g); 7.85 (s; 1H; H-2g); 12.83 (s; 1H; 5-OHg). HRMS (ESI)
Calcd C27H26O11 [M+Na]+ 549.1367. Found 549.1369.
2.1.2. Chemistry
2.1.2.1. General procedure for preparation 7-O-x-hydroxyalkyl-
genistein. Tetrabutylammonium salt of genistein (2) (10 mM)
and alcohol (2-bromo-1-ethanol or 3-bromo-1-propanol, 11 mM)
in 50 mL of dry DMF were stirred at temperature 50 °C for 24 h.
After the completion of reaction, the mixture was diluted with
250 mL of ice water to form yellow solid. Recrystallization of the
solid from 100 mL hot methanol gave product 3a or 3b.
5-Hydroksy-7-(2-hydroxyethyl)-3-(4-hydroksyphenyl)chromen-4-
one (3a). Obtained after recrystallization as a white solid, in 88%
yield; mp 219–221 °C; 1H NMR d (DMSO-d6): 3.70–3.78 (m; 2H;
–CH2O–); 4.11 (t; 2H; J = 9.6 Hz; –CH2O–); 4.97 (br s; 1H; –
CH2OH); 6.40, 6.65 (2 ꢀ d; 2H; J = 2.2 Hz; H-6, H-8); 6.83 (d; 2H;
J = 8.7 Hz; H-30, H-50); 7.39 (d; 2H; J = 8.7 Hz; H-20, H-60); 8.40 (s;
1H; H-2); 9.63 (br s; 1H; 40-OH); 12.96 (s; 1H; 5-OH). HRMS
(ESI) Calcd C17H14O6 [M+Na]+ 337.0682. Found 337.0666.
5-Hydroksy-7-(3-hydroxypropyl)-3-(4-hydroksyphenyl)chromen-
4-one (3b). Obtained after recrystallization as a white solid, in 89%
yield; mp 201–203 °C; 1H NMR d (DMSO-d6): 1.84–1.92 (m; 2H; –
CH2–); 3.54–3.59 (m; 2H; –CH2O–); 4.16 (dd; 2H; J = 6.2 Hz,
J = 6.4 Hz; –CH2OH); 4.62 (t; 1H; J = 6.4 Hz; –CH2OH); 6.39, 6.65
(2 ꢀ d; 2H; J = 2.2 Hz; H-6, H-8); 6.83 (d; 2H; J = 8.6 Hz; H-30, H-
50); 7.39 (d; 2H; J = 8.6 Hz; H-20, H-60); 8.41 (s; 1H; H-2); 9.64 (br
s; 1H; 40-OH); 12.96 (s; 1H; 5-OH). HRMS (ESI) Calcd C18H16O6
[M+Na]+ 351.0839. Found 351.0833.
5-Hydroksy-7-[(4,6-di-O-acetyl-2,3-dideoxy-a-D-erythro-hex-2-
enopyranosyl)-3-O-propyl]-3-(4-hydroksyphenyl)chromen-4-one (7b).
Obtained after chromatography as colorless syrup, in 65% yield;
1H NMR (CDCl3) d: 2.04–2.19 (m; 2H; –CH2–); 2.09 (s; 3H; AcO);
2.11 (s; 3H; AcO); 3.71 (dt; 1H; J = 9.9 Hz; J = 6.1 Hz; –CH2O–);
3.98 (dt; 1H; J = 9.9 Hz; J = 6.1 Hz; –CH2O); 4.04–4.16 (m; 3H; H-
5; –CH2O–); 4.17 (dd; 1H; J = 12.1 Hz; J = 2.6 Hz; H-6b); 4.25
(dd; 1H; J = 12.1 Hz; J = 5.3 Hz; H-6a); 5.06 (br s; 1H; H-1); 5.33
(dd; 1H; J = 9.7 Hz; J = 1.5 Hz; H-4); 5.85 (ddd; 1H; J = 10.2 Hz;
J = 2.4 Hz; J = 1.8 Hz; H-3); 5.91 (dd; 1H; J = 10.2 Hz; J = 1.1 Hz;
H-2); 6.37, 6.40 (2 ꢀ d; 2H; J = 2.4 Hz; H-6g, H-8g); 6.61 (br s;
1H; 40OHg); 6.85 (d; 2H; J = 8.6 Hz; H-30g, H-50g); 7.34 (d; 2H;
J = 8.6 Hz; H-20g, H-60g); 7.84 (s; 1H; H-2g); 12.83 (s; 1H; 5-OHg).
HRMS (ESI) Calcd C28H28O11 [M+Na]+ 563,1523. Found 563.1531.
5-Hydroksy-7-[(4,6-di-O-acetyl-2,3-dideoxy-a-D-erythro-hex-2-eno-
pyranosyl)-5-O-pentyl]-3-(4-hydroksyphenyl)chromen-4-one (7d).
Obtained after chromatography as colorless syrup, in 48% yield;
1H NMR (CDCl3) d: 1.51–1.64 (m; 2H; –CH2–); 1.66–1.76 (m; 2H;
–CH2–); 1.79–1.89 (m; 2H; –CH2–); 2.10 (s; 3H; AcO); 2.11 (s;
3H; AcO); 3.55 (dt; 1H; J = 9.7 Hz; –CH2O–); 3.83 (dt; 1H;
J = 9.7 Hz; –CH2O–); 4.02 (dd; 2H; –CH2O–); 4.09–4.18 (m; 1H;

A. Rusin et al. / Bioorg. Med. Chem. 19 (2011) 295–305
297
H-5); 4.20 (dd; 1H; J = 12.1 Hz; J = 2.6 Hz; H-6b); 4.27 (dd; 1H;
J = 12.1 Hz; J = 5.1 Hz; H-6a); 5.05 (br s; 1H; H-1); 5.33 (dd; 1H;
J = 9.7 Hz; J = 1.5 Hz; H-4); 5.85 (ddd; 1H; J = 11.2 Hz; J = 1.5 Hz;
J = 1.8 Hz; H-3); 5.91 (d; 1H; J = 11.2 Hz; H-2); 6.35,6.38 (2 ꢀ d;
2H J = 2.2 Hz; H-8g, H-6g); 6.86 (d; 2H; J = 8.6 Hz; H-30g; H-50g);
7.36 (d; 2H; J = 8.6 Hz; H-20g; H-60g); 7.84 (s; 1H; H-2g); 12.80
(s; 1H; 5-OHg). HRMS (ESI) Calcd C30H32O11 [M+Na]+ 591.1837.
Found 591.1841.
(dt; 1H; J = 9.9 Hz; J = 6.2 Hz; –CH2O–); 3.91–3.95 (m; 2H; H-50, –
CH2O–); 3.99 (ddd; 1H; J = 1.9 Hz, J = 5.4 Hz, J = 9.5 Hz, H-5);
4.09–4.27 (m; 7H; H-4, H-6a, H-6b, H-6a0, H-6b0; –CH2O–); 4.57
(d; 1H; J = 7.9 Hz; H-10); 5.00 (br s; 1H; H-1); 5.00 (dd; 1H;
J = 10.4 Hz; J = 3.5 Hz; H-30); 5.20 (dd; 1H; J = 10.4 Hz; J = 7.9 Hz;
H-20); 5.38 (dd; 1H; J = 3.5 Hz; J = 1.0 Hz; H-40); 5.61 (br s; 1H;
40-OHg); 5.75 (ddd; 1H; J = 10.4 Hz; J = 2.8 Hz; J = 2.2 Hz; H-3);
6.12 (dd; 1H; J = 10.4 Hz; H-2); 6.36, 6.40 (2 ꢀ d; 2H; J = 2.4 Hz;
H-6g, H-8g); 6.89 (d; 2H; J = 8.6 Hz; H-30g; H-50g); 7.39 (d; 2H;
J = 8.6 Hz; H-20g, H-60g); 7.86 (s; 1H; H-2g); 12.80 (s; 1H; 5g-OH).
LRMS (ESI) Calcd C40H44O19 [M+Na]+ 851.2374. Found 851.4.
5-Hydroksy-7-[(4-O-acetyl-2,3,6-trideoxy-
pyranosyl)-2-O-ethyl]-3-(4-hydroksyphenyl)chromen-4-one
a
-
L
-erythro-hex-2-eno-
(8a).
Obtained after chromatography as colorless syrup, in 54% yield;
1H NMR (CDCl3) d: 1.25 (d; 3H; J = 6.22 Hz; H-6); 2.10 (s; 3H;
AcO); 3.90–4.26 (m; 5H; H-5, 2 ꢀ –CH2O–); 5.06–5.10 (m; 2H; H-
1, H-4); 5.86–5.89 (m; 2H; H-2, H-3); 6.23 (br s; 1H; 40-OHg);
6.39, 6.42 (2 ꢀ d; 2H; J = 2.4 Hz; H-8g, H-6g); 6.86 (d; 2H
J = 8.6 Hz; H-30g, H-50g); 7.35 (d; 2H; J = 8.6 Hz; H-2’g, H-60g);
7.86 (s; 1H; H-2g); 12.83 (s; 1H; 5-OHg). HRMS (ESI) Calcd
5-Hydroksy-7-[(4-O-(2,3,4,6-tetra-O-acetyl-b-
D-galactopyrano-
syl)-6-O-acetyl-2,3-dideoxy- -erythro-hex-2-enopyranosyl)-5-O-
a
-D
pentyl]-3-(4-hydroksyphenyl)chromen-4-one (9d). Obtained after
chromatography as colorless syrup, in 53% yield; 1H NMR (CDCl3)
d: 1.49–1.58 (m; 2H; –CH2–); 1.63–1.70 (m; 2H; –CH2–); 1.77–
1.88 (m; 2H; –CH2–); 1.98 (s; 3H; AcO); 2.05 (s; 3H; AcO); 2.08
(s; 3H; AcO); 2.12 (s; 3H; AcO); 2.16 (s; 3H; AcO); 3.51 (dt; 1H;
J = 9.5 Hz; J = 6.3 Hz; CH2O); 3.66 (m; 1H; H-50); 3.79 (dt; 1H;
J = 9.5 Hz; J = 6.3 Hz; CH2O); 3.87–4.23 (m; 8H; H-5, –CH2O–, H-
6a, H-6b, H-6a0, H-6b0, H-4); 4.58 (d; 1H; J = 8.1 Hz; H-10); 4.96–
5.03 (m; 2H; H-1; H-30); 5.22 (dd; 1H; J = 10.3 Hz, J = 8.1 Hz; H-
20); 5.39 (dd; 1H; J = 3.7 Hz; J = 1.0 Hz; H-40); 5.77 (ddd; 1H;
J = 10.3 Hz; J = 2.2 Hz; J = 2.5 Hz; H-3); 6.09–6.13 (m; 2H; H-2; 40-
OHg); 6.35, 6.38 (2 ꢀ d; 2H; J = 2.2 Hz; H-6g; H-8g); 6.88 (d; 2H;
J = 8.7 Hz; H-30g, H-50g); 7.37 (d; 2H; J = 8.7 Hz; H-20g, H-60g);
7.85 (s; 1H; H-2g); 12.81 (s; 1H; 5g-OH). LRMS (ESI) Calcd
C
25H14O9 [M+Na]+ 491.1313. Found 491.1324.
5-Hydroksy-7-[(4-O-acetyl-2,3,6-trideoxy-a-L-erythro-hex-2-eno-
pyranosyl)-3-O-propyl]-3-(4-hydroksyphenyl)chromen-4-one (8b).
Obtained after chromatography as colorless syrup, in 86% yield;
1H NMR (CDCl3) d: 1.20 (d; 3H; J = 6.0 Hz; H-6); 2.05–2.14 (m;
2H; –CH2–); 2.09 (s; 3H; AcO); 3.68 (dt; 1H; J = 9.9 Hz; J = 6.2 Hz;
–CH2O–); 3.91–4.01 (m; 2H; H-5, –CH2O–); 4.11–4.17 (m; 2H; –
CH2O–); 4.99 (br s; 1H; H-1); 5.05 (dd; 1H; J = 9.3 Hz; J = 1.5 Hz;
H-4); 5.22 (br s; 1H; 40-OHg); 5.80 (ddd; 1H; J = 10.8 Hz;
J = 2.4 Hz; J = 1.5 Hz; H-3); 5.87 (d; 1H; J = 10.8 Hz; H-2); 6.39,
6.41 (2 ꢀ d; 2H; J = 2.1 Hz; H-8g, H-6g); 6.89 (d; 2H; J = 8.7 Hz;
H-30g, H-50g); 7.39 (d; 2H; J = 8.7 Hz; H-20g, H-60g); 7.86 (s; 1H;
H-2g); 12.83 (s; 1H; 5-OHg). HRMS (ESI) Calcd C26H26O9 [M+Na]+
505.1469. Found 505.1485.
C
42H48O19 [M+Na]+ 879.2686. Found 879.4.
2.1.2.4. Procedure for synthesis of n-propyl-2,3-unsaturated
pyranoside (10). To a solution of rhamnal (5) (0.2 mM) in anhy-
drous dichloromethane (40 mL) 1-propanol (0.2 mM) and BF3ꢁEt2O
(0.02 mM) was added. The contents were stirred at temperature
ꢂ25 °C to 10 °C for 0,5 h and the reaction monitored by TLC (tolu-
ene/AcOEt = 1:1 (v/v)). The reaction mixture was quenched by the
addition of aqueous sodium hydrogen carbonate (10%, 25 mL), ex-
tracted with dichloromethane (3 ꢀ 25 mL), dried over anhydrous
sodium sulfate, filtered and washed with water, and the combined
organic extract was concentrated under vacuum. The residue was
purified by column chromatography (90:1 petroleum ether/AcOEt)
on silica gel to obtain the product 10 as colorless syrup, in 50% yield.
5-Hydroksy-7-[(4-O-acetyl-2,3,6-trideoxy-a-L-erythro-hex-2-eno-
pyranosyl)-5-O-pentyl]-3-(4-hydroksyphenyl)chromen-4-one (8d).
Obtained after chromatography as colorless syrup, in 55% yield;
1H NMR (CDCl3) d: 1.24 (d; 3H; J = 6.3 Hz; H-6); 1.53–1.74 (m;
4H; 2 ꢀ –CH2–); 1.80–1.90 (m; 2H; –CH2–); 2.10 (s; 3H; AcO);
3.53 (dt; 1H; J = 9.6 Hz; J = 6.1 Hz; –CH2O–); 3.82 (dt; 1H;
J = 9.6 Hz; J = 6.6 Hz; –CH2O–) 3.96–4.05 (m; 3H; H-5, –CH2O–);
4.97 (br s; 1H; H-1); 5.06 (dd; 1H; J = 9.3 Hz; J = 1.5 Hz; H-4);
5.22 (br s; 1H; 40-OHg); 5.81 (ddd; 1H; J = 10.5 Hz; J = 2.4 Hz;
J = 1.5 Hz; H-3); 5.87 (d; 1H; J = 10.5 Hz; H-2); 6.37, 6.39 (2 ꢀ d;
2H; J = 2.2 Hz; H-8g, H-6g); 6.89 (d; 2H; J = 8.4 Hz; H-3;g, H-50g);
7.40 (d; 2H; J = 8.4 Hz; H-20g, H-60g); 7.85 (s; 1H; H-2g); 12.82
(s; 1H; 5-OHg). HRMS (ESI) Calcd C28H30O9 [M+Na]+ 533.1782.
Found 533.1806.
Propyl 4-O-acetyl-2,3,6-trideoxy-a-L-erythro-heks-2-en-pyrano-
side (10). 1H NMR (CDCl3) d: 0,95 (t; 3H; J = 7.3 Hz; –CH3); 1.22
(d; 3H; J = 6.3 Hz; H-6); 1.59–1.74 (m; 2H; –CH2–); 2.08 (s; 3H;
AcO); 3.46 (dt; 1H; J = 9.5 Hz; –CH2O–); 3.72 (dt; 1H; J = 9.5 Hz –
CH2O–); 3.98 (dq;1H; J = 9.2 Hz, J = 6,3 Hz; H-5); 4.96 (br s; 1H;
H-1); 5.05 (dd; 1H; J = 9.2 Hz; H-4); 5.81 (ddd; 1H; J = 10.3 Hz;
H-3); 5.84 (d; 1H; J = 10.3 Hz; H-2).
5-Hydroksy-7-[(4-O-(2,3,4,6-tetra-O-acetyl-b-
D-galactopyrano-
syl)-6-O-acetyl-2,3-dideoxy- -erythro-hex-2-enopyranosyl)-2-O-
a
-D
ethyl]-3-(4-hydroksyphenyl)chromen-4-one (9a). Obtained after
chromatography as colorless syrup, in 67% yield; 1H NMR (CDCl3)
d: 1.98 (s; 3H; AcO); 2.05 (s; 3H; AcO); 2.08 (s; 3H; AcO); 2.12
(s; 3H; AcO); 2.16 (s; 3H; AcO); 3.89–4.35 (m; 11H; H-4, H-5, H-
50, H-6a, H-6b, H-6a0, H-6b0; 2 ꢀ CH2O); 4.58 (d; 1H; J = 8.2 Hz;
H-10); 5.02 (dt; 1H; J = 10.6 Hz; J = 3.5 Hz; H-30); 5.09 (d; 1H;
J = 1.1 Hz; H-1); 5.21 (dd; 1H; J = 10.6 Hz; J = 8.2 Hz; H-20); 5.29
(br s; 1H; 40-OHg); 5.39 (m; 1H; H-40); 5.79 (dt; 1H; J = 10.3 Hz;
J = 2.3 Hz; H-3); 6.13 (dd; 1H; J = 10.3 Hz; J = 1.1 Hz; H-2); 6.38,
6.42 (2 ꢀ d; 2H; J = 2.4 Hz; H-6g, H-8g); 6.91 (d; 2H; J = 8.8 Hz;
H-30g; H-50g); 7.41 (d; 2H; J = 8.8 Hz; H-20g, H-60g); 7.86 (s; 1H;
H-2g); 12.82 (s; 1H; 5g-OH). LRMS (ESI) Calcd C39H42O19 [M+Na]+
837.2218. Found 837.4.
2.2. Biological activity
2.2.1. Cells
Human, A-549 (lung), LNCaP, DU145 and PC3 (prostate), MCF-7
and SKBR-3 (breast), HCT116, HT-29, LOVO and Caco-2 (colon),
AGS (stomach) cancer and T98G (glioblastoma) cell lines were
obtained from American Type Culture Collection (Rockville,
Maryland, USA). A-549, LNCaP, MCF-7, SKBR-3, HT-29 cancer and
T98G cell lines are being maintained in the Institute of Immunol-
ogy and Experimental Therapy, Wroclaw, Poland. DU145, PC3,
LOVO, Caco-2 and AGS cell lines are being maintained in Maria
Sklodowska-Curie Memorial Cancer Center and the Institute of
Oncology, Gliwice, Poland.
5-Hydroksy-7-[(4-O-(2,3,4,6-tetra-O-acetyl-b-
D-galactopyrano-
syl)-6-O-acetyl-2,3-dideoxy- -erythro-hex-2-enopyranosyl)-3-O-
a
-D
propyl]-3-(4-hydroksyphenyl)chromen-4-one (9b). Obtained after
chromatography as colorless syrup, in 78% yield; 1H NMR (CDCl3)
d: 1.98 (s; 3H; AcO); 2.05 (s; 3H; AcO); 2.07 (s; 3H; AcO); 2.11
(s; 3H; AcO); 2.15 (s; 3H; AcO); 2.07–2.13 (m; 2H; –CH2–); 3.66
LNCaP and HT-29 cells were cultured in RPMI 1640+Opti-MEM
(1:1) medium (Gibco, Scotland, UK) supplemented with 2 mM
L-glutamine, 1 mM sodium pyruvate and 5% fetal bovine serum
(all from Sigma–Aldrich Chemie GmbH, Steinheim, Germany).

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A. Rusin et al. / Bioorg. Med. Chem. 19 (2011) 295–305
A-549 cells in RPMI 1640+Opti-MEM (1:1) medium (both from
Gibco, Scotland, UK) supplemented with 2 mM -glutamine and
5% fetal bovine serum (all from Sigma–Aldrich Chemie GmbH,
Steinheim, Germany). MCF-7 and SKBR-3 cells in Eagle medium
stained with propidium iodide (PI) (100 lg/mL). The DNA content
L
was analyzed using Becton Dickinson FACSCanto cytometer (BD
Company, USA) to monitor the cell cycle changes. Experiments
were repeated at least twice.
(IIET, Wroclaw, Poland) supplemented with 2 mM L-glutamine,
1.0 mM sodium pyruvate, 1% amino acid, 0.8 mg/L of insulin and
10% fetal bovine serum (all from Sigma–Aldrich Chemie GmbH,
Steinheim, Germany). T98G cells in Eagle medium (IIET, Wroclaw,
2.2.6. Mitotic index
Cells, collected as described above, were cytospinned, fixed
with cold methanol, stained with solution of DAPI (3 lM). Slides
Poland) supplemented with 2 mM
L-glutamine, 1.0 mM sodium
were mounted in DAKOÒFluorescent Mounting Medium (Dako,
USA) and examined under an ECLIPSE E800 Nikon microscope
using an objective 40ꢀ. Mitotic cells were counted among 1000
cells. Experiments were repeated three times.
pyruvate, 1% amino acid and 10% fetal bovine serum (all from
Sigma–Aldrich Chemie GmbH, Steinheim, Germany). DU145, PC3,
LOVO, Caco-2, AGS cells were cultured in RPMI 1640 medium
(Sigma–Aldrich, Germany) supplemented with 10% fetal bovine
serum (MP Biomedicals, LLC). HCT116 cells were cultured McCoy0s
5A medium (Sigma–Aldrich, Germany) supplemented with 10%
fetal bovine serum (MP Biomedicals, LLC, United States).
All culture media were supplemented with 100 units/mL
penicillin, and 100 lg/mL streptomycin (both from Polfa Tarchomin
S.A., Warsaw, Poland) and all cell lines were grown at 37 °C with 5%
CO2 humidified atmosphere.
2.2.7. Tubulin immunostaining
Cells growing on 8-well chamber slides (Lab-Tek PermanoxÒ
slides, Nalgen Nunc International, Rochester, NY, USA) were trea-
ted with the tested compounds for 24 h. Cells were fixed in ice-cold
methanol (ꢂ20 °C) for 10 min, air-dried, immediately washed in
PBS and blocked with normal goat serum (NGS) (1%; Vector Labo-
ratories, Burlingame, CA, USA) for 0.5 h in a humidified chamber at
room temperature. Then, the slides were treated for one hour with
mouse anti-b-tubulin (1:50, v/v; Sigma–Aldrich, Germany). After
subsequent three rinses in PBS, cells were incubated for 0.5 h with
goat anti-mouse FITC-conjugated secondary antibody (Sigma–
Aldrich, Germany), rinsed several times in PBS, and counterstained
2.2.2. Cytotoxicity assays
Cell viability was estimated using an MTT (3-[4,5-dimethylthi-
azol-2-y]2,5-diphenyltetrazolium bromide) assay (Sigma–Aldrich,
Germany), according to the supplier0s protocol or SRB (sulforhoda-
mine B) assay. Shortly, 24 h before addition of the tested com-
pounds, the cells were plated in 96-well plates (Sarstedt,
Germany) at the density of 2 ꢀ 103 cells per well. Assays were per-
formed after 72 h of continuous exposure to varying concentra-
tions of the tested agents. Each compound in each concentration
was tested in triplicate in a single experiment, which was repeated
at least three times. Viability of cells was expressed as a percentage
versus vehicle control (DMSO treatment). IC50 was defined as a
concentration of a drug that decreased cell viability by 50%.
with solution of DAPI (3 lM). Slides were mounted in DAKO
ÒFluorescent Mounting Medium (Dako, USA).
2.2.8. Analysis of spindle abnormalities
Ram-3 treated cells immunofluorescently stained for tubulin
were examined under the ECLIPSE E800 Nikon microscope using
an oil immersion objective 100ꢀ. For each treatment 100 of mitotic
cells were analyzed for abnormalities. Experiments were repeated
twice. Photographs of representative spindles were taken with
Hamamatsu C5810 camera (Hamamatsu Photonics, Japan).
2.2.3. MTT assay
After 72 h treatment with tested agents the medium was aspi-
2.2.9. In vitro tubulin polymerization assay
rated and cells were incubated for 3 h at 37 °C with 0.5 mg/mL
MTT solution (50 lL) in Dulbecco modified essential medium
(DMEM) (Sigma–Aldrich, Germany) without phenol red. Then the
medium was aspired, insoluble crystals of formazan were solubi-
lized in 2-propanol/HCl solution, and optical density (k = 570 nm)
was determined in a microplate reader BioTek Synergy II (BioTek
Instruments, USA).
The effects of Ram-3 on tubulin polymerization were investi-
gated using the HTS-Tubulin Polymerization Assay Kit CytoDYNA-
MIX Screen™ 1 (CDS01, Cytoskeleton Inc. Denver, CO, USA),
according to the manufacturer protocol. Tubulin dimers (>97% pur-
ity) were suspended with 100 ll of G-PEM buffer (80 mM PIPES,
2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP, pH 6.9) plus 5% glycerol.
Tests were run in the absence (vehicle control used as a reference)
or presence of the test compounds (10
lM, 20 lM and 50 lM
2.2.4. SRB assay
Ram-3 and 3 M paclitaxel) in pre-warmed (37 °C) 96-well plate.
l
The cells were attached to the bottom of plastic wells by fixing
them with cold 50% TCA (trichloroacetic acid, Sigma–Aldrich
Chemie GmbH, Steinheim, Germany) on the top of the culture
medium in each well. The plates were incubated at 4 °C for 1 h
and then washed five times with tap water. The cellular material
fixed with TCA was stained with 0.4% sulforhodamine B (SRB, Sig-
ma–Aldrich Chemie GmbH, Steinheim, Germany) dissolved in 1%
acetic acid (POCH, Gliwice, Poland) for 30 min. Unbound dye was
removed by rinsing (4ꢀ) in 1% acetic acid. The protein-bound
dye was extracted with 10 mM unbuffered Tris base (Sigma–
Aldrich Chemie GmbH, Steinheim, Germany) for determination of
the optical density (k = 540 nm) in a microplate reader Multiskan
RC photometer (Labsystems, Helsinki, Finland).
The polymerization of tubulin was followed by measuring the in-
crease in absorbance at 340 nm as a function of time (every minute
for 1 h) at 37 °C. The experiments were repeated twice.
2.2.10. Apoptosis detection
2.2.10.1. Agarose gel electrophoresis/DNA ladder analysis. Cells
treated with Ram-3 or untreated control were collected and
washed with PBS. Cells were incubated overnight at 56 °C with
50 mM Tris (pH 7.5), 100 mM EDTA, 1% SDS, 150 mM NaCl,
1 mg/mL proteinase K, and next treated with phenol/ chloroform
to extract DNA. DNA was precipitated with isopropanol/sodium
acetate, treated with 0.1 mg/mL RNaseA, and then separated in a
1.8% agarose/ethidium bromide gel in 0.5ꢀTBE (90 mM Tris,
64.6 mM boric acid, 2.5 mM EDTA, pH 8.3). After electrophoresis
DNA was visualized by UV light and photographed.
2.2.5. Cell cycle analysis
Cells in subconfluent proliferating cultures were incubated with
the tested compounds continuously for indicated period of time.
Floating cells were collected and added to adherent cells, harvested
by trypsinization. Cells were washed with PBS and then fixed in ice-
2.2.10.2. Terminal deoxynucleotidyl transferase dUTP nick-end
labeling (TUNEL assay). TUNEL assay was performed according
to a manufacturer protocol (ROCHE) with slight modifications. In
brief, floating cells were harvested and pooled with adherent cells
cold ethanol (70%) for 30 min, treated with RNase (100 lg/mL) and

A. Rusin et al. / Bioorg. Med. Chem. 19 (2011) 295–305
299
detached form the bottom of a culture dish by trypsin. Cells were
cytospinned, fixed and then the slides were treated following the
section of a protocol for adherent cells. Apoptotic index was
counted in 500 nuclei. Experiments were repeated three times.
similar alkylation performed on well defined tetra-n-butyloammo-
nium salt of genistein (2),23 proceeded smoothly even with equimo-
lar amount of 3-bromopropanol (and 2-bromoethanol), affording
required product with very high regioselectivity. Synthesis of
5-hydroksypentyl derivative by the same procedure has failed,
because the substrate was consumed by competing intramolecular
cyclization reaction, which produced tetrahydrofuran. In this case,
5-bromopentyl acetate was used as alkylating reagent and subse-
quently the hydroxyl group was liberated through methanolysis.
The Lewis acid-catalyzed allylic rearrangement of glycals in the
presence of alcohols and other nucleophilic substrates (phenols) is
the method of choice for the synthesis of 2,3-unsaturated glyco-
sides. The reaction, as originally stated by Ferrier, involves interme-
diacy of a cyclic allylic oxocarbenium ion to which the nucleophile
adds preferentially in quasi-axial orientation. Typically, 2,3-unsatu-
rated O-glycosides are prepared by Ferrier rearrangement of acyloxy
glycals catalyzed by boron trifluoride diethyl etherate.24–27
2.2.11. Cell senescence analysis
Morphological features of cells, such as cell size increase and
accumulation of intracellular vesicles were observed under
phase contrast microscope. Senescence-associated-b-galactosidase
(b-gal) activity was chosen as a biochemical marker for replicative
senescence.19 In order to show b-gal activity cells were fixed for
5–10 min in 4% paraformaldehyde, washed and incubated with
fresh staining solution (1 mg/mL of X-gal diluted in the staining buf-
fer [40 mM citric acid/sodium phosphate pH6.0, 5 mM potassium
ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM
MgCl2]) for 12 h at 37 °C.
Owing to its significance in carbohydrate and natural product
chemistry, there has been a growing interest in identifying new
catalysts for the Ferrier reaction. The use of a number of reagents
for this transformation has appeared in the recent literature,
including titanium(IV) chloride (TiCl4),28 indium(III) chloride
(InCl3),29,30 indium(III) bromide (InBr3),31 acidic montmorillonite
K-10,32–34 tin(IV) chloride (SnCl4).35,36
3. Results and discussion
3.1. Chemistry
The synthesis of required compounds, designed through inspi-
ration with unusual biological activity of unsaturated genistein
glycoside G21,8 was accomplished according to general pathway
illustrated in Figures 1–3. Compounds 3a, 3b, 3d were the key
intermediates for the synthesis of the glycoconjugates.
The efficacy of selected catalysts: boron trifluoride etherate
(BF3ꢁEt2O), ytterbium(III) triflate (Yb(OTf)3), indium(III) chloride
(InCl3) was examined for the Ferrier rearrangement of 3b in reac-
tion with 5. In the presence of BF3ꢁEt2O an equimolar mixture of
In the first step, genistein was converted into an intermediate
bearing an
x-hydroxyalkyl substituent, containing two, three or
a,b-glycosides was formed. The ytterbium(III) triflate was inactive
five carbon atoms, at position 7, while the second step involved gly-
cosylation. Genistein quaternary ammonium salts (2) were applied
to complete the alkylation step. Resulting compounds (3a, 3b, 3d),
which contain aliphatic primary hydroxyl functionality, easily react
with acylated glycals (hex-1-enitols) under Lewis acid catalysis,
undergoing Ferrier rearrangement. The reaction is highly regio-
under our conditions. It was observed, that among mentioned cat-
alysts, InCl3 is superior in terms of the stereoselectivity and yield.
Thus, in the presence of InCl3 and acetonitrile as a solvent the reac-
tion of 3b with 5 proceeds at ambient temperature and millimolar
scale glycosylation is completed within less than 2 h, with equimo-
lar proportion of nucleophile, to give good yield of a-O-glycoside.
Glycosylation of remaining hydroxyalkyl-genistein derivatives
with glycals was performed under analogous conditions.
and stereo-selective, affording glycosides with
a-configuration.
Both synthetic steps are in principle very simple, but deserve some
comments, when applied to a multifunctional substrate like geni-
stein (1).
Several procedures are known, which were successfully applied
for alkylation of genistein.
Thus, regioselective alkylation of genistein was performed by
treatment of genistein with haloalkanes in the presence of base
like NaOH, K2CO3 in anhydrous acetone.20,21 Bromoalkyl deriva-
tives of genistein were obtained in reaction of 1 with 1,2- 1,3- or
1,4-dihaloalkanes under ultrasound irradiation.22
However, in our hands, reaction of genistein (1) with
3-bromopropanol invariably afforded a mixture of 7-O and 40-O-
hydroksyalkyl ethers, which are difficult to separate. In contrast,
3.2. Biological activity
3.2.1. Preliminary screening of the compounds for
antiproliferative activity and their influence on the cell cycle
Preliminary screening of new compounds for their antiprolifer-
ative activity was performed in a standard 72 h MTT assay on
HCT116 cell line. The data expressed as the concentration leading
to 50% inhibition of cell viability (IC50), for each genistein derivative
is summarized in Table 1. It can be seen that the derivatives named
Gen-5, Glu-5, Ram-2, Ram-3 and Ram-5, exhibited cytotoxic effect
Figure 1. Synthesis of hydroxyalkyl-genistein derivatives.

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A. Rusin et al. / Bioorg. Med. Chem. 19 (2011) 295–305
Figure 2. Synthesis of glycoconjugates, derivatives of genistein.
Figure 3. Synthesis of propyl glycoconjugate.
at the concentration significantly lower (IC50 below 10
lM), as
as 0 lM) roughly 50% of cells were in G1 phase of a cycle (high
compared to genistein (IC50 approx. 35 M). Cytotoxic assays
l
G1 peak), and around 24% of cells were in G2 or M phase (low
G2/M peak). After genistein treatment the shift of cells from
G1 to G2/M phase can be seen. Dose-dependent increase of cells ar-
rested in the G2/M was also observed after treatment of HCT116
cells with Glu-5, Ram-3 and Ram-5, with the most prominent
effect observed after treatment with Ram-3. Neither Ram-2 nor
Gen-5 influenced the cell cycle.
revealed that the sugar moiety was not toxic (Table 1). Also, the
treatment of cells with a combination of genistein and rhamnal
derivative did not evoke any synergistic effect (data not shown).
Five compounds: Gen-5, Glu-5, Ram-2, Ram-3 and Ram-5
showing highest activity in MTT assay were selected for further
analysis of cell cycle and determination of apoptosis with flow
cytometer.
Flow cytometry analysis in PI stained cells shown the occur-
rence of apoptotic cells, after treatment with genistein Gen-5 and
Glu-5, seen on the histograms as a population on the left from
the G1 peak (Fig. 4). The analysis of histograms revealed also the
influence of genistein and some of derivatives on the cell cycle
(Fig. 4). In control cells (the first histogram in each set, marked
3.2.2. The effect of genistein derivatives on mitosis
In order to find whether a G2/M block of a cycle results from
arresting cells in G2 phase or in mitosis, we counted the mitotic in-
dex in cells treated for 24 h with 20
Ram-3 and Ram-5 and genistein (Table 2). Additionally, bearing
in mind that 20 M concentration of the tested derivatives exceeds
lM of Gen-5, Glu-5, Ram-2,
l

A. Rusin et al. / Bioorg. Med. Chem. 19 (2011) 295–305
301
Table 1
Comparison of cytotoxicity of new compounds in
HCT116 colon cancer cell line
a
Tested compound
IC50
(
lM)
Genistein (1)
Gen-2 (3a)
Gen-3 (3b)
Gen-5 (3d)
Lac-2 (2a)
Lac-3 (9a)
Lac-5 (9a)
Glu-2 (9d)
Glu-3 (7b)
Glu-5 (7d)
Ram-2 (8a)
Ram-3 (8b)
Ram-5 (8d)
Ram-P (10)
34.90 9.84
24.99 12.07
23.93 3.90
6.92 2.06
>20b
>20b
>20b
16.06 7.26
19.86 6.01
8.45 1.74
9.32 3.44
4.76 0.96
8.08 2.97
550 11.08
a
Concentration of a tested compound leading to
50% inhibition of cell proliferation (IC50 was
)
obtained in a standard 72 h MTT assay; mean val-
ues and standard deviations were calculated from
at least three independent experiments.
b
Cytotoxicity was not assessed above the con-
centration 20 lM due to low substance solubility
and its precipitation.
their IC50 at least twice, we used a 100 lM genistein. It can be seen
that three genistein derivatives (Glu-5, Ram-3, Ram-5), but not
genistein, blocked cells in mitosis, with the most profound effect
observed for Ram-3. However, the subtraction of mitotic index
from the fraction of cells stopped in G2/M phase in flow cytometry
analysis showed that all three compounds substantially blocked
cell cycle not only in mitosis but also in G2 phase. In contrast, gen-
istein used at high concentration (100 lM) caused cells to stop
entering the mitosis, and mitotic index dropped rapidly.
In our previous paper we found that the unsaturated genistein
disaccharide glycoside G21 blocked cells in mitosis due to induc-
tion of abnormal mitotic spindles,7 and the data presented above
suggested that Ram-3 (and two other compounds Glu-5 and
Ram-5 to a lower extent) could exhibit the same mode of action.
The analysis of Ram-3 treated HCT116 cells shown various abnor-
malities of the mitotic spindles, including multipolarity and lag-
ging chromosomes at the spindle poles (Fig. 5). Frequency of
abnormal spindles was dose-dependent (Table 3), exceeding 80%
Figure 4. Cell cycle phases distribution in HCT116 cells treated with different
genistein derivatives at growing concentration series for 24 h. Left—representative
histograms od cells with different DNA contents. G1—cells in G1 phase of a cycle;
G2/M—cells in G2 phase of a cycle or in mitosis. Right - mean values and standard
deviations from independent experiments. Apopt—apoptotic cells; G1—cells in G1
phase; S—cells in the S phase; G2—cells in G2 or M phase; Polypl—polyploidal cells.
after treatment with 20
treatment with 20 M and 50
phase microtubule array. At 20
ation, but at 50 M (the concentration ten times exceeding IC50),
l
M Ram-3. We also analyzed cells after
M Ram-3 for any changes of inter-
M Ram-3 we observed no alter-
l
l
l
l
Table 2
less dense and shortened microtubules were visible (not shown).
The difference between the concentration at which spindles were
affected and the concentration at which changes to interphase ar-
ray occurred is not unusual, as it is well recognized that microtu-
bule binding agents cause perturbations to mitotic spindles at
much lower concentration, than, when used for microtubule
depolimerization.37,38 The differences between dynamics of inter-
phase and mitotic spindle microtubules, correlating with different
sensitivity of those arrays to microtubule binding drugs are well
described phenomena. During mitosis microtubules show highly
dynamic instability, which is necessary for proper function of a
spindle, and any disturbance of this instability blocks the progres-
sion of mitosis from metaphase to anaphase without net depoly-
merization of microtubules.39–42
Mitotic index in HCT116 cells treated with different genistein derivatives for
24 h
Treatment
Mitotic index at 24 h treatmenta
Control
1.9 0.6
Genistein [20
lM; 100
lM]
1.8 0.4; 0.5 0.2
Gen-5 [20
Glu-5 [20
Ram-2 [20
Ram-3 [20
Ram-5 [20
l
M]
3
1.6
lM]
5.2 0.9
1.3 0.2
13.2 2.1
3.5 1.1
l
l
l
M]
M]
M]
a
Mean values and standard deviation were obtained from three inde-
pendent experiments. Mitotic index was counted in 1000 nuclei in each
treatment.
Although multiple molecular targets for antimitotic drugs are
known, including tubulins and various mitotic kinases, all of them,
either by interfering with the proper attachment of chromosomes
to the microtubules of mitotic spindle or altering the structure of a
spindle (i.e., change of the number of spindle poles), induce mitotic
checkpoint arrest of a cell cycle.43,44
In order to determine whether Ram-3 can directly interact with
microtubules, we performed microtubule polymerization in

302
A. Rusin et al. / Bioorg. Med. Chem. 19 (2011) 295–305
Figure 5. Spindle structure in Ram-3 treated HCT 116 cells. (A–C) Normal bipolar spindle (Type a); (D, E) bipolar spindle with large masses of lagging chromosomes at spidle
poles (Type b); (G–I) Tripolar spindle (Type c); (J–L) monopolar spindles (Type d).
Table 3
Spindle abnormalities in HCT116 cells after treatment with Ram-3 for 24 h
Ram-3 (
lM)
Normal spindles
Type aa (%)
Aberrant spindles
Type cc (%) Type dd (%)
Type bb (%)
Total (%)
0
5
10
20
94
85
44
16
3
6
8
3
1
14
38
40
0
6
7
7
5
1
15
42
3
0
3
6
0
0
3
2
0
0
1
1
6
15
56
84
3
6
8
3
a
b
c
Type a—normal bipolar spindle.
Type b—bipolar spindle with large mass of lagging chromosomes at spindle poles.
Type c—tripolar and multipolar spindles.
d
Type d—monopolar spindles. Aberrant spindles were counted among 100 mitotic cells in each treatment. Mean values and standard
deviations were obtained from two independent experiments.
cell-free system, using a standard assay (Fig. 6). In the presence of
the rate of tubulin polymerization increased dramatically as
paclitaxel (3
lM), the agent stimulating tubulin polymerization,
compared to the control, whereas Ram-3 inhibited tubulin

A. Rusin et al. / Bioorg. Med. Chem. 19 (2011) 295–305
303
Figure 6. Tubulin polymerization assay in cell-free system. Optical density of
tubulin solution measured spectrophotometrically at 340 nm in the absence of
drugs (control) or in the presence of 3 M paclitaxel (Ptx) and 10 M, 20 M and
50 M of Ram-3.
l
l
l
l
polymerization in a concentration dependent manner. This result
indicates, that Ram-3 can be classified as a new microtubule desta-
bilizing agent.
Figure 8. Apoptotic events after 24, 48, 72 h treatment of HCT116 cells with 20 lM
Ram-3. Control cells treated with 0.2% DMSO in culture medium. (A) Index of
TUNEL positive nuclei (apoptotic index) counted in 500 nuclei. Mean value and
standard deviation obtained from three independent experiments. (B) Internucle-
3.2.3. Cell death and senescence after Ram-3 treatment
Cell cycle arrest in mitosis strongly influences the fate of cells
treated with antimitotic drugs and loss of their proliferative poten-
tial. Usually, cells are not able to sustain prolonged mitotic arrest
and eventually escape from the mitosis, which is called mitotic
slippage. In this process cells decondense the chromatin, but
incomplete or improper division in many cases leads to tetra-
ploidy, often followed by cell death.45,46 The decrease of mitotic in-
dex from 13% after first day of treatment to 6% and 4% after the
second and third day of treatment, respectively indicates that cells,
initially blocked in mitosis by Ram-3 treatment, could gradually
escape from the block within the period of few days following mi-
totic arrest. Morphological observations during second and third
day of treatment shown many rounded cells, with membrane
blebbings (data not shown), suggesting massive apoptotic cell
death. Flow cytometry analysis revealed the presence of increased
fraction of both hypodiploid and tetraploid populations after 48
and 72 h of treatment (Fig. 7). Apoptotic death, suggested by
osomal DNA fragmentation (DNA laddering). Lane
1 (M)—marker; lane 2
(C)—control cells; lanes 3–5 (Ram-3 24, 48, 72 h) cells treated with Ram-3 for
indicated time.
morphological features and hypodiploidity was confirmed by
TUNEL assay and by internucleosomal DNA fragmentation
(Fig. 8). The apoptotic index calculated for cells exposed to
Ram-3 for 72 h reached 12%, what roughly corresponded to the
intensity of the mitotic block observed after 24 h treatment.
Ram-3 treatment led to elimination of substantial fraction of
HCT116 cells by apoptosis on one hand, and to the loss of the pro-
liferative potential of remaining cells on the other. Increase of tet-
raploid and polyploid cells fraction after 48 h and 72 h of treatment
was accompanied by the decrease of the mitotic index, and the
presence of cells with senescent phenotype (Fig. 9). Many cells
were enlarged, and shown positive perinuclear staining of the sites
of b-galactosidase activity (31 4%).
It is known that mitotic slippage activates checkpoint machin-
ery in tetraploidized cells, which inhibits progression of the cell cy-
cle at G1 and phase, dependent on the tumor suppressor protein
p53.47 Thus we decided to compare the rate of apoptotic cell death
in two isogenic HCT116 cell lines, differing their p53 status:
HCT116 p53ꢂ/ꢂ and HCT116 p53+/+. In general, the cytotoxicity
of Ram-3 against two lines was similar, and IC50 was
6.21 1.13 lM and 4.76 0.96 lM, respectively. The similar apop-
totic index (15% for p53ꢂ/ꢂ and 12% for p53+/+) indicates that
HCT116 cells treated with Ram-3 may enter apoptosis regardless
of the p53 status.
3.2.4. Antiproliferative activity of Ram-3 against cancer cell
lines of different origin
Antiproliferative potential of Ram-3 and genistein was assessed
in additional 11 human cell lines of different origin: glioblastoma,
breast, colon, prostate, lung and stomach cancers (Table 4). In all
tested lines we observed stronger inhibition of cell growth by
Ram-3 than by genistein. The susceptibility of different cell lines
to Ram-3 differed to some extent, but we did not observe any or-
gan specific activity of Ram-3. We also tested the influence of
Ram-3 on the cell cycle in three cell lines, in addition to
HCT116: AGS, A549 and DU145, observing cell cycle arrest in
G2/M phase. In selected cell lines the fraction of cells in G2/M be-
Figure 7. DNA content in HCT116 cells treated with Ram-3 for 48 and 72 h.
Left—representative histograms od cells with different DNA contents. G1—cells in G1
phase of a cycle; G2/M—cells in G2 phase of a cycle or in mitosis. Right—mean values
and standard deviations from independent experiments. Apopt—apoptotic cells;
G1—cells in G1 phase; S—cells in the
Polypl—polyploidal cells.
S phase; G2—cells in G2 or M phase;
fore and after treatment with 20 lM Ram-3 increased: in AGS cell

304
A. Rusin et al. / Bioorg. Med. Chem. 19 (2011) 295–305
that the derivative Gen-5, obtained through attachment of five
C-atoms side chain to C-7 of genistein, which exhibits similar lipo-
philicity and cytotoxicity as Ram-3 and, does not affect microtu-
bule network. The lack of toxicity of a sugar moiety (Ram-P), and
significantly higher toxicity of Ram-3, as compared with genistein,
and the unique ability of the glycoconjugate to affect microtubules
of a spindle indicate, that lipophilic sugar in Ram-3 does not act
simply as a carrier, delivering genistein into cells. The interaction
of Ram-3 with microtubules should be also discussed in the light
of the latest paper by Mukherjee et al.,48 which shows that geni-
stein is able to depolymerize interphase microtubules through
binding to a unique site of tubulin. In this context, it seems possi-
ble, that proper derivatization of genistein could enhance genistein
intrinsic capability of interaction with microtubules, and concom-
ittantly change this activity of genistein, which blocks cells in G2
phase and abolishes their entry into mitosis.
The interference with microtubules seems to be dependent both
on the sugar moiety and on the length of a spacer between geni-
stein and a sugar residue. Neither Ram-2 nor Ram-5, which differ
from Ram-3 only by the length of a spacer, are able to influence the
structure of the mitotic spindle. Another indication on the role of
spacer linking the sugar moiety and genistein for the biological
activity comes from the comparison of activity of G21 and a series
of Lac derivatives (Lac-2, Lac-3, Lac-5), in which the sugar moiety,
the same as in G21 is linked to genistein via a C2–C5 spacer. In this
case, extension of the distance between the sugar and aglycon led
to loss of the antimitotic activity of Lac derivatives, suggesting, that
the size of a molecule may affect its interaction with tubulin.
3.3. Conclusions
Figure 9. Senescent phenotype of HCT116 cells after 24 h treatment with Ram-3.
Arrows indicate enlarged cells with blue, perinuclear staininig of the sites of
b-galactosidase activity.
Genistein exhibits activity towards multiple molecular targets
and reveals potential for affinity TuneUp by derivatization. In our
experiments, among genistein modified at C7 by different mono
and di-unsaturated sugars, several compounds appeared more ac-
tive than a parent compound in preliminary screening for inhibi-
tion of cancer cell proliferation. The most potent compound,
Ram-3 was capable of influencing the mitotic spindle and inducing
apoptosis. For other active genistein glycoconjugates the molecular
targets remain to be revealed. We can not exclude their action on
the protein targets inhibited by genistein, such as tyrosine kinases
and topoisomerases, or new ones, not affected by genistein. Based
on our previous8 and presently reported results, it is evident that
glycoconjugates of genistein containing unsaturated pyranoside
moiety, belong to this part of the chemical structure space, which
contains islands of pharmacological activity, potentially medici-
nally useful and applicable for drug development.
line—from 24.3 0>0.2% to 39.7 2.2%, in A549—from 21.9 3% to
31.6 4.9% and in Du145—from 27.6 0.1% to 46.1 8%.
3.2.5. Relevance of structural features of glycoconjugates for
their biological activity
Data obtained so far neither allow to depict a single param-
eter which could determine structure–activity relationship, nor
unequivocally indicate molecular targets of genistein glycocon-
jugates responsible for their antiproliferative and antimitotic
activity.
The increase of antiproliferative potential of genistein deriva-
tives with the increase of lipophilicity is visible, however the lipo-
philicity is not the only parameter which influences the activity of
the compounds. The mode of action of compounds with similar
IC50 may differ substantially, depending on the type of derivatiza-
tion. The role of a sugar moiety as a structural element essential for
antimitotic properties of G21 and Ram-3 is emphasized by the fact,
Acknowledgments
This work was supported by a grant of Polish Ministry of
Science and Higher Education PBZ MNiL-1/1/2005. We would like
to acknowledge MSc Katarzyna Ludwik for the study of Ram-3 sta-
bility and MSc Anna Rusek and Ms. Krystyna Klyszcz for excellent
technical assistance.
Table 4
Cytotoxicity of Ram-3 and genistein in a panel of cancer cell lines of different origin
Cell line
Organ origin
Ram-3 IC50
(
lM)
Genistein IC50 (lM)
SKBR-3a
MCF-7a
HT-29a
Caco-2b
LoVob
Breast
Breast
Colon
Colon
28.02 6.89
8.88 0.75
36.14 4.69
23.3 3.23
9.85 1.66
16.74 10.34
14.45 5.1
17.98 6.09
10.7 2.27
14.73 2.23
3.05 0.7
96.70 4.6
62.71 15.7
52.78 9.1
>100
15.88 2.4
89.41 20.11
47.29 11.78
30.65 11.15
65.17 12.3
43.09 7.5
41.67 10.4
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