Synthesis and antiproliferative activity of novel steroidal dendrimer conjugates

Article abstract of DOI:10.1016/j.steroids.2013.09.001

We describe the synthesis of steroidal dendrimer conjugates of first and second generation with tetramethylene core and 5-hydroxy-isophtalic acid dimethyl ester as branching unit modified to incorporate ethynylestradiol or 17α-estradiol as terminal units. The steroidal dendrimer conjugates, the free drug (steroids) and dendrimer were tested against a panel of cancer cell lines (CEM, MCF7, HeLa) and normal human fibroblast (BJ). The steroidal dendrimer conjugates of first generation exhibited cytotoxic activity and induced apoptosis in chronic leukemia (CEM) as resultant activation of caspase cascade which is mainly provoked in G2/M arrested cells.

Full text of DOI:10.1016/j.steroids.2013.09.001

Steroids 78 (2013) 1254–1262
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and antiproliferative activity of novel steroidal dendrimer
conjugates
Nancy E. Magaña-Vergara a, Lucie Rárová b, Delia Soto-Castro a, Norberto Farfán c, Miroslav Strnad b,
,
⇑
Rosa Santillan a,
⇑
a Departamento de Química, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740, México D.F. 07000, Mexico
b Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacky´ University, Šlechtitelu˚ 11, CZ-783 71 Olomouc, Czech Republic
c Facultad de Química, Departamento de Química Orgánica, Universidad Nacional Autónoma de México, México D.F. 04510, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 May 2013
Received in revised form 1 August 2013
Accepted 1 September 2013
Available online 21 September 2013
We describe the synthesis of steroidal dendrimer conjugates of first and second generation with tetra-
methylene core and 5-hydroxy-isophtalic acid dimethyl ester as branching unit modified to incorporate
ethynylestradiol or 17a-estradiol as terminal units. The steroidal dendrimer conjugates, the free drug
(steroids) and dendrimer were tested against a panel of cancer cell lines (CEM, MCF7, HeLa) and normal
human fibroblast (BJ). The steroidal dendrimer conjugates of first generation exhibited cytotoxic activity
and induced apoptosis in chronic leukemia (CEM) as resultant activation of caspase cascade which is
mainly provoked in G2/M arrested cells.
Keywords:
Steroidal dendrimer conjugates
Antiproliferative activity
Ethynylestradiol
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
contrary, the covalent conjugation, with or without the assistance
of target moieties, improves the selective drug accumulation, in-
More than 11 million people are diagnosed with cancer each
year and the incidence of this disease is projected to rise continu-
ously to 16 million by 2020 [1]. For this reason, the discovery of
new active drugs and the development of delivery devices capable
of improving the therapeutic index of biologically active molecules
and decreasing unwanted side effects [2] are of crucial importance.
Dendrimers are synthetic macromolecules possessing well de-
fined branching architectures in nanometric size that can be easily
tailored to endow specific properties [3–7]. They have attracted
great attention due to their potential in the delivery of anticancer
drugs because their high multivalency enhances cellular interac-
tions and promotes a faster endocytosis [8–11]. As drug delivery
systems, three strategies [12–14] have been employed: (i) forma-
tion of dendrimer networks around the drug; (ii) drug encapsula-
tion inside the dendrimer mediated by electrostatic and van der
Walls interactions; (iii) drug ‘‘conjugation’’ by covalent attachment
or electrostatic binding at the periphery. However, several reports
suggest [13,14] that, when a drug is encapsulated or electrostati-
cally attached to a dendrimer, it may be released prematurely in
the body as a result of a small pH change. Consequently, the ob-
served therapeutic effect is lower than one would expect. On the
creases the circulation time in the body, favoring a sustained liber-
ation [2,12,14] and inducing apoptosis [15–17].
Synthetic estrogens, such as 17a-ethynylestradiol and 17a-
estradiol have been used to prevent or reduce menopause symp-
toms, as oral contraceptives, in the treatment of alopecia, and in
neurodegenerative disorders such as Alzheimer and Parkinson dis-
eases [18]. Interestingly steroidal compounds have also shown
antiproliferative activity and the ability to induce apoptosis [19].
In particular, the cytotoxicity of 17
kemia Jurkat T cells has been attributed to apoptosis, mainly in-
duced in G2/M-arrested cells [20], while 17 -ethynylestradiol
a-estradiol toward human leu-
a
has been evaluated with promising results as inhibitor of human
prostate [21] and colon cancer [22]. The combination of dendritic
compounds with steroidal derivatives has given rise to new archi-
tectures with a wide-range of biological effects when used as drug
delivery systems [23–26], including the treatment of malaria [27]
and in vitro in the lung inflammatory process [28].
With the aim to exploit the high drug payload of dendrimers
without loss of monodispersity and well defined structure, to
explore the possibility to enhance the cytotoxic activity of
17a-ethynylestradiol and 17a-estradiol, and bearing in mind the
advantages of covalent conjugation in drug delivery, four new ste-
roidal dendrimer conjugates were synthetized in this work. The
dendrimers were prepared using a flexible tetramethylene core,
and 5-hydroxy-isophtalic acid dimethyl ester as branching unit
⇑
Corresponding authors. Tel.: +52 555 747 3725; fax: +52 555 747 3389 (R.
Santillan). Tel.: +420 585 634 850; fax: +420 585 634 870 (M. Strnad).
E-mail addresses: miroslav.strnad@upol.cz (M. Strnad), rsantill@cinvestav.mx (R.
Santillan).
modified to incorporate
4 units of 17a-ethynylestradiol or
0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.09.001

N.E. Magaña-Vergara et al. / Steroids 78 (2013) 1254–1262
1255
17a-estradiol for first generation (8 and 9) and 8 units for the sec-
2.2.3. 1,4-Bis(3,5-bis(bromomethyl)phenoxy)butane (4)
ond (10 and 11). Their preliminary biological assays show that all
new dendrimers and dendrimeric conjugates of G1 are noncytotox-
ic against normal fibroblastic cells (BJ), but the conjugates of first
generation are able to induce apoptosis in leukemia cancer cells
(CEM) displaying a higher cytotoxic activity than the free drug.
Compound 3 (1.28 g, 3.53 mmol) was dissolved in a 2:1 solution
of HBr: H2SO4 (100 mL), The mixture was heated at 100–110 °C for
1 h, allowed to cool and diluted with H2O (100 mL). The aqueous
solution was extracted with CH2Cl2 (3 Â 150 mL) and the com-
bined organic layers were washed with saturated aqueous NaHCO3
solution (100 mL), dried (MgSO4), filtered and concentrated to
yield a brown solid. The crude product was purified by column
chromatography using hexane to obtain 1.84 g (3.01 mmol, 84%)
of 4 as a white crystalline solid. M.p. 126–128 °C.
2. Experimental
1H NMR (CDCl3, 300 MHz) d(ppm): 6.99 (2H, brs, H-6), 6.85 (4H,
2.1. General
brs,H-4), 4.42 (8H, s, H-7), 4.04 (4H, m, H-2), 1.98 (4H, m, H-1).13
C
The 1H and 13C NMR spectra were recorded on JEOL 400 and
500, and Bruker 300 using CDCl3, DMSO-d6 as solvent. Chemical
shifts are reported in parts per million (ppm) relative to internal
TMS. Mass spectra were recorded with an Agilent Technologies
MS TOF using the ESI(+) technique. IR spectra were recorded using
a Perkin–Elmer Spectrum GX FTIR spectrometer.
All reagents were commercially available. THF was refluxed
over sodium/benzophenone and distilled under reduced pressure
in a nitrogen atmosphere prior to use. Column chromatography
was carried out with silica gel (70–230 mesh). 5-Hydroxy-iso-
phthalic acid dimethyl ester (1) was synthesized following the lit-
erature [29]. Compounds 2 and 4 have been reported in the
literature, however, using the method described herein the yields
were optimized [30,31]. Unambiguous NMR spectral assignment
was attained using one and two dimensional spectra.
NMR (CDCl3, 75 MHz) d(ppm): 159.3 (C-3), 139.6 (C-5), 121.8 (C-6),
115.2 (C-4), 67.5 (C-2), 32.9 (C-7), 25.8 (C-1). IR (
m
max/cmÀ1): 2928,
2871, 1593, 1452, 1388, 1328, 1296 (C-O), 1166 (C–O–C), 1055,
1019, 940, 870, 848, 732, 697, 671, 648 y 633 (C–Br).
2.2.4. 1,4-Bis(3,5-bis(3,5-bis(carboxymethyl)phenoxymethyl)
phenoxy) butane (5)
5-Hydroxy-isophthalic acid dimethyl ester (1) (0.13 g,
0.62 mmol) and potassium carbonate (0.30 g, 2.17 mmol) were
stirred in acetonitrile, followed by slow addition of 1,4-bis(3,5-
bis(bromomethyl)phenoxy)butane (0.10 g, 0.16 mmol) and tetra-
butylammonium fluoride (1 mg) of. The mixture was refluxed
72 h, filtered and rinsed with CH2Cl2. The solvent was removed un-
der reduced pressure to obtain 0.15 g (0.13 mmol, 82%) of com-
pound 5 as a beige solid. M.p. 176–177 °C.
1H NMR (CDCl3, 400 MHz) d(ppm): 8.27 (4H, s, H-11), 7.81 (8H,
s, H-9), 7.08 (8H, s, H-7), 6.96 (4H, s, H-4), 5.10 (2H, s, H-6), 4.08
(4H, brs, H-2), 3.92 (24H, s, COOMe), 2.00 (4H, brs, H-1). 13C
NMR (CDCl3, 100 MHz) d(ppm): 166.1 (O@COMe), 159.7 (C-3),
158.7 (C-8), 138.1 (C-5), 131.9 (C-10), 123.4 (C-11), 120.1 (C-9),
118.5 (C-6), 113.3 (C-4), 70.2 (C-7), 67.6 (C-2), 52.5 (COOMe),
2.2. Synthesis of compounds
2.2.1. 1,4-Bis(3,5-bis(carboxymethyl)phenoxy)butane (2)
5-Hydroxy-isophthalic acid dimethyl ester (1) (4.29 g,
20.41 mmol) and potassium carbonate (8.29 g, 59.98 mmol) were
stirred in acetonitrile, followed by slow addition of 1,4-dibromobu-
tane (1.1 mL, 9.26 mmol) and 18-crown-6 (1 mg). The mixture was
heated at 60 °C during 48 h, filtered and rinsed with CH2Cl2. The
solvent was removed under reduced pressure to obtain 4.30 g
(9.06 mmol, 98%) of compound 2 as beige solid. M.p. 152–153 °C,
in agreement with the literature [24].
26.0 (C-1). IR (m
max/cmÀ1): 2954, 1721 (O–C@O), 1597, 1431,
1339, 1311, 1239 (C-O), 1170, 1113, 1064, 1043, 1000, 907, 875,
836, 792, 752, 721, 682, 564. HR-ESI-TOF-MS (m/z): calcd for C60-
H58O22[M+Na]+: 1153.3317; found: 1153.3315.
2.2.5. 1,4-Bis(3,5-bis(3,5-bis(hydroxymethyl)phenoxymethyl)
phenoxybutane (6)
1H NMR (CDCl3, 400 MHz) d(ppm): 8.24 (2H, t, J = 1.5 Hz, H-6),
7.71 (4H, d, J = 1.5 Hz, H-4), 4.11 (4H, m, H-2), 3.91 (12H, s, H-8),
2.01 (4H, m, H-1). 13C NMR (CDCl3, 100 MHz) d(ppm): 166.2 (C-
7), 159.0 (C-3), 131.8 (C-5), 123.0 (C-6), 119.8 (C-4), 68.0 (C-2),
A solution of 5 (0.10 g, 0.09 mmol) in dry THF was added drop-
wise to
a magnetically stirred suspension of LiAlH4 (0.04 g,
1.05 mmol) in dry THF under N2. The reaction was stirred 24 h at
room temperature, and quenched by the slow addition of a solu-
tion of ammonium chloride (10 mL) and ethyl acetate (40 mL) with
cooling. The aluminum salts were filtered and the solvents were
then removed under reduced pressure to give 0.04 g (0.05 mmol,
53%) of compound 6 as a white solid. M.p. 146–147 °C.
52.4 (C-8), 25.8 (C-1). IR (m
max/cmÀ1): 2951, 2852, 2360, 1725
(O–C@O), 1596, 1453, 1433, 1337, 1312, 1238 (C–O), 1111 (C-O),
1050, 1018, 1000, 902, 875, 753, 671, 628. HR-ESI-TOF-MS (m/z):
calcd for C24H26O10[M+H]+: 475.1604; found: 475.1583.
1H NMR (DMSO-d6, 300 MHz) d(ppm): 7.18 (2H, brs, H-6), 7.08
(4H, brs, H-11), 6.85 (4H, brs, H-4), 6.82 (8H, brs, H-9), 5.04 (8H,
brs, H-7), 4.44 (16H, brs, H-12), 4.05 (4H, brs, H-2), 2.48 (8H, brs,
OH), 1.87 (4H, brs, H-1). 13C NMR (DMSO-d6, 75 MHz) d(ppm):
159.2 (C-3), 158.7 (C-8), 144.4 (C-10), 139.4 (C-5), 118.9 (C-6),
117.3 (C-11), 113.2 (C-4), 111.3 (C-9), 69.3 (C-7), 67.6 (C-2), 63.3
2.2.2. 1,4-Bis(3,5-bis(hydroxymethyl)phenoxy)butane (3)
A solution of tetraester 2 (1.52 g, 40.00 mmol) in dry THF was
added dropwise to a magnetically stirred suspension of LiAlH4
(3.00 g, 6.32 mmol) in dry THF under N2. The reaction was stirred
24 h at room temperature, and quenched by the slow addition of
a solution of ammonium chloride (10 mL) and ethyl acetate
(40 mL) with cooling. The aluminum salts were filtered and the sol-
vents were then removed under reduced pressure to give 1.52 g
(4.19 mmol, 56%) of compound 3 as a white solid. M.p. 147–149 °C.
1H NMR (DMSO-d6, 300 MHz) d(ppm): 6.82 (2H, s, H-6), 6.73
(4H, s, H-4), 4.42 (8H, s, H-7), 3.99 (4H, brs, H-2), 3.35 (OH), 1.84
(4H, brs, H-1).13C NMR (DMSO-d6, 75 MHz) d(ppm): 159.0 (C-3),
144.3 (C-5), 116.9 (C-6), 111.0 (C-4), 67.4 (C-2), 63.2 (C-7), 25.9
(C-12), 25.8 (C-1). IR (m
max/cmÀ1): 3317 (OH), 2918, 2850, 2359,
1720, 1595, 1451, 1375, 1294, 1256, 1154, 1110, 1021 (C-O), 843,
801, 752, 684. HR-APCI-TOF-MS (m/z): calcd for C52H58O14[-
M+Na]+: 929.3724; found: 929.3720.
2.2.6. 1,4-Bis(3,5-bis(3,5-bis(chloromethyl)phenoxymethyl)
phenoxybutane (7)
To a magnetically stirred solution of 6 (0.75 g, 0.83 mmol) in dry
CH2Cl2 was added dry pyridine (0.59 mL, 7.28 mmol), followed by
dropwise addition of SOCl2 (0.52 mL, 7.28 mmol) under N2. The
reaction was stirred 48 h at room temperature, and quenched by
the slow addition of water. The organic phase was extracted with
(C-1). IR (
1297, 1265, 1168, 1064, 1027, 979, 918, 841, 701, 677, 607. HR-
m
max/cmÀ1): 3291, 3170, 2943, 2873, 2853, 1595, 1449,
ESI-MS (m/z) calcd for
385.1609.
C
20H26O6[M+Na]+: 385.1627; found:

1256
N.E. Magaña-Vergara et al. / Steroids 78 (2013) 1254–1262
CH2Cl2, and dried over Na2SO4. The product was purified by col-
umn chromatography using hexane:ethyl acetate (85:15) to give
0.58 g (0.55 mmol, 67%) of 7, as a white solid. M.p. 107–109 °C.
1H NMR (CDCl3, 500 MHz) d(ppm): 7.06 (2H, brs, H-6), 7.01 (4H,
brs, H-11), 6.95 (8H, brs, H-9), 6.94 (4H, brs, H-4), 5.04 (8H, brs, H-
purified by column chromatography using EtOAc/hexane to give
0.56 g (0.18 mmol, 75%) of 10 as a pale yellow solid. M.p. 175–
177 °C.
1H NMR (CDCl3, 500 MHz) d(ppm): 7.18 (8H, d, J = 8.6 Hz, H-1’),
7.06 (4H, s, H-11), 7.05 (2H, s, H-6), 6.99 (8H, s, H-9), 6.93 (4H, s, H-
4), 6.75 (8H, dd, Jo = 8.6, Jm = 2.5 Hz,H-2’), 6.68 (8H, d, J = 2.5 Hz, H-
4’), 5.01 (8H, s H-7), 4.97 (16H, s, H-12), 4.05 (4H, brs, H-2), 2.85
(16H, m, H-6’), 2.59 (8H, s, H-20’), 0.86 (24H, s, Me-18’). 13C NMR
(CDCl3, 125 MHz) d(ppm): 159.6 (C-3), 159.2 (C-8), 156.7 (C-3’),
139.3 (C-10), 138.8 (C-5), 138.1 (C-5’), 132.9 (C-10’), 126.5 (C-1’),
118.8 (C-11), 114.9 (C-4’), 113.23 (C-9), 113.16 (C-4), 112.4 (C-
2’), 87.6 (C-19’), 79.9 (C-17’), 74.2 (C-20’), 69.9 (C-7), 69.7 (C-12),
67.6 (C-2), 49.5 (C-14’), 47.2 (C-13’), 43.6 (C-9’), 39.4 (C-8’), 39.0
(C-16’), 32.8 (C-12’) 29.9 (C-6’), 27.3 (C-7’), 26.4 (C-11’), 26.0 (C-
7), 4.53 (16H, brs, H-12), 4.07 (4H, brs, H-2), 1.99 (4H, brs, H-1). 13
C
NMR (CDCl3, 125 MHz) d(ppm): 159.6 (C-3), 159.2 (C-8), 139.4 (C-
10),138.5 (C-5), 121.3 (C-11), 118.5 (C-6), 115.0 (C-9), 113.2 (C-4),
69.9 (C-7), 67.6 (C-2), 45.8 (C-12), 26.0 (C-1). IR (m
max/cmÀ1): 2957,
2876 (C–H), 1729, 1594(C@C), 1259 (C–O eter), 1454, 1326, 1300,
1259, 1158, 1043, 958, 842, 708 (C-Cl), 674, 583. MALDI TOF MS
(m/z) calcd for C52H50Cl8O6[M+H]+: 1051.1194; found: 1050.1887.
2.2.7. 17a-Ethynylestradiol-dendrimer conjugate of first generation
(8)
1), 22.9 (C-15’), 12.8 (Me-18’). IR (
m
max/cmÀ1): 3285, 2930, 2868,
17a-Ethynylestradiol (0.19 g, 0.64 mmol) and potassium car-
1598, 1496, 1454, 1291, 1231, 1145, 1050, 1020, 843, 786, 571.
MALDI TOF MS (m/z): calcd for C
3171.7355; found: 3171.7646.
bonate (0.30, 2.17 mmol) were stirred in acetonitrile. To this solu-
tion were added 4 (0.10 g, 0.16 mmol) and tetrabutylammonium
fluoride (1 mg). The reaction was refluxed 3 days, and rinsed with
CH2Cl2. The solvent was removed under reduced pressure and the
crude product was purified by column chromatography using
EtOAc in hexane to give 0.18 g (0.12 mmol, 74%) of 8 as a pale yel-
low solid. M.p. 153–155 °C.
212H234O22[M+Na+NH3]+:
2.2.10. 17a-Estradiol-dendrimer conjugate of second generation (11)
17 -estradiol (0.045 g, 0.17 mmol) and potassium carbonate
a
(0.07 g, 0.50 mmol) were stirred in acetonitrile. To this solution
were added the compound 7 (0.02 g, 0.019 mmol) and tetrabutyl-
ammonium fluoride (1 mg). The reaction was refluxed 48 h, fil-
tered and rinsed with water and hexane to obtain 0.05 g
(0.017 mmol, 89%) of 11 as a pale yellow solid. M.p. 169–171 °C.
1H NMR (CDCl3, 300 MHz) d(ppm): 7.19 (8H, d, J = 6.9 Hz, H-1’),
7.07 (4H, s, H-11), 7.03 (2H, s, H-6), 6.99 (8H, s, H-9), 6.94 (4H, s, H-
4), 6.75 (8H, d, J = 6.9 Hz, H-2’), 6.69 (8H, s, H-4’), 5.02 (8H, s, H-7),
4.05 (4H, brs, H-2), 2.82 (16H, m, H-6’), 0.68 (24H, s, Me-18’). 13C
NMR (CDCl3, 75 MHz) d(ppm): 159.5 (C-3), 156.7 (C-3’), 139.2 (C-
5), 138.1 (C-5’), 133.1 (C-10’), 126.4 (C-1’), 118.4 (C-4), 114.9 (C-
4’), 112.9 (C-6), 112.3 (C-2’), 80.1 (C-17’), 69.8 (C-7), 67.5(C-2),
47.8 (C-14’), 45.6 (C-13’), 43.7 (C-9’), 39.1 (C-8’), 32.5 (C-12’),
31.5 (C-16’), 30.0 (C-6’), 28.1 (C-7’), 26.3 (C-11’), 26.0 (C-1), 24.3
1H NMR (CDCl3, 500 MHz) d(ppm): 7.20 (4H, d, J = 8.6 Hz, H-1’),
7.05 (2H, s, H-6), 6.92 (4H, s, H-4), 6.76 (4H, dd, Jo = 8.6 Hz,
Jm = 2.7 Hz, H-2’), 6.70 (4H, d, J = 2.7 Hz, H-4’), 4.98 (8H, s, H-7),
4.05 (4H, brs, H-2), 2.60 (4H, brs, H-20’), 0.87 (12H, s, Me-18’).
13C NMR (CDCl3, 125 MHz) d(ppm): 159.5 (C-3), 156.7 (C-3’),
139.2 (C-5), 138.1 (C-5’), 132.9 (C-10’), 126.5 (C-1’), 118.5 (C-4),
114.9 (C-4’), 112.9 (C-6), 112.4 (C-2’), 87.6 (C-19’), 79.9 (C-17’),
74.2 (C-20’), 69.9 (C-7), 67.6 (C-2), 49.5 (C-14’), 47.2 (C-13’), 43.6
(C-9’), 39.4 (C-8’), 39.0 (C-16’), 32.8 (C-12’) 29.9 (C-6’), 27.3 (C-
7’), 26.4 (C-11’), 26.0 (C-1), 22.9 (C-15’), 12.8 (Me-18’). IR (mmax
/
cmÀ1): 3282 („C–H), 2923, 2855, 2161 (C„C), 1716, 1662, 1601,
1496, 1456, 1377, 1289, 1231, 1159, 1058, 1021, 844, 734, 624.
HR-APCI-TOF-MS (m/z): calc for C100H114O10[M+Na]+: 1497.8309;
found: 1497.8284.
(C-15’), 17.1 (C18’). IR (m
max/cmÀ1): 3451(O-H), 2928, 2864 (C-H),
1598 (C@C), 1496, 1453, 1376, 1295, 1233, 1155, 1036, 846, 788,
710, 573. MALDI TOF MS (m/z): calcd for C196H234O22[M+Na]+:
2962.7089; found: 2962.8896.
2.2.8. 17a-Estradiol-dendrimer conjugate of first generation (9)
To a mixture of 17a-estradiol (0.18 g, 0.66 mmol), and potas-
sium carbonate (0.30 g, 2.17 mmol) in acetonitrile was added 4
(0.10 g, 0.16 mmol) and tetrabutylammonium fluoride (1 mg).
The reaction was refluxed 3 days, the solvent was removed under
reduced pressure and the crude product was purified by column
chromatography using methanol/CH2Cl2 to give 0.18 g (0.13 mmol,
80%) of 9 as a pale yellow solid. M.p. 154–156 °C.
2.3. Biological tests
2.3.1. Cell culture
Stock solutions (10 mmol/L) of dendrimer conjugate derivatives
were prepared by dissolving an appropriate quantity of each sub-
stance in DMF. Dulbecco’s modified Eagle’s medium (DMEM), fetal
1H NMR (CDCl3, 300 MHz) d(ppm): 7.20 (4H, d, J = 8.6 Hz, H-1’),
7.04 (2H, s, H-6), 6.92 (4H, s, H-4), 6.75 (4H, dd, Jo = 8.6 Hz,
Jm = 2.6 Hz, H-2’), 6.70 (4H, d, J = 2.6 Hz, H-4’), 4.99 (8H, s, H-7),
4.05 (4H, brs, H-2), 0.70 (12H, s, Me-18’). 13C NMR (CDCl3,
75 MHz) d(ppm): 159.5 (C-3), 156.7 (C-3’), 139.2 (C-5), 138.1 (C-
5’), 133.1 (C-10’), 126.4 (C-1’), 118.4 (C-4), 114.9 (C-4’), 112.9 (C-
6), 112.3 (C-2’), 80.1 (C-17’), 69.8 (C-7), 67.5(C-2), 47.8 (C-14’),
45.6 (C-13’), 43.7 (C-9’), 39.1 (C-8’), 32.5 (C-12’), 31.5 (C-16’),
30.0 (C-6’), 28.1 (C-7’), 26.3 (C-11’), 26.0 (C-1), 24.3 (C-15’), 17.1
bovine serum (FBS), trypsin, L-glutamine, penicillin and streptomy-
cin were purchased from Sigma (MO, USA). Calcein AM was ob-
tained from Molecular Probes (Invitrogen Corporation, CA, USA).
The cell lines for screening (T-lymphoblastic leukemia cell line
CEM, breast carcinoma cell line MCF-7, cervical carcinoma cell line
HeLa, and human fibroblasts BJ) were obtained from the American
Type Culture Collection (Manassas, VA, USA). All cell lines were
cultured in DMEM medium (Sigma, MO, USA), supplemented with
10% heat-inactivated fetal bovine serum, 2 mmol/L L-glutamine,
(C18’). IR (
m
max/cmÀ1): 3399 (OH), 2929, 2865, 1602,1496, 1231,
10,000 U penicillin and 10 mg/mL streptomycin. The cell lines were
maintained under standard cell culture conditions at 37 °C and 5%
CO2 in a humid environment. Cells were subcultured twice or three
times a week using the standard trypsinization procedure.
1156,1033, 609. HR-APCI-TOF MS (m/z): calcd for C92H114O10[-
M+Na]+: 1401.8304; found: 1401.8315.
2.2.9. 17a-Ethynylestradiol-dendrimer conjugate of second generation
(10)
2.3.2. Calcein AM assay
17
a
-Ethynylestradiol (0.56 g, 1.90 mmol) and potassium car-
Suspensions of the tested cell lines (ca. 1.0 Â 105 cells/mL) were
placed in 96-well microtiter plates and after 3 h of stabilization
(time zero) the compounds to be tested were added (in four
bonate (0.86 g, 6.3 mmol) were stirred in acetonitrile. To this solu-
tion was added 7 (0.25 g, 0.24 mmol) and tetrabutylammonium
fluoride (1 mg). The reaction was refluxed 3 days, the solvent
was removed under reduced pressure and the crude product was
20
lL aliquots) in serially diluted concentrations in dimethylform-
amide (DMF). Control cultures were treated with DMF alone, and

N.E. Magaña-Vergara et al. / Steroids 78 (2013) 1254–1262
1257
the final concentration of DMF in the incubation mixtures never
2.3.5. Western blotting
exceeded 0.6%. The test compounds were typically evaluated at
six 3-fold dilutions and the highest final concentration was gener-
ally 50 lM. After 72 h incubation, 100 lL Calcein AM solution
The cells were seeded into culture medium in 100-mm culture
dishes at a density of 1.5 Â 106 cells/mL and treated immediately
with dendrimer conjugate derivatives. DMF was used as a vehicle
for controls. After 24 h of treatment, the cells were washed three
times with cold PBS (10 mM, pH 7.4) and lysed in ice-cold RIPA
protein extraction buffer (20 mM Tris–HCl, pH 7.4, 5 mM EDTA,
2 mM EGTA, 100 mM NaCl, 2 mM NaF, 0.2% Nonidet P-40, 30 mM
PMSF, 1 mM DTT, 10 mg/mL of aprotinin and leupeptin). The lysate
was collected into a microfuge tube and incubated on ice for 1 h. It
was then cleared by centrifugation at 10,000g for 30 min at 4 °C
and the supernatant was collected. Proteins in lysates were quan-
tified by the Bradford method and diluted with Laemmli electro-
phoresis buffer. The proteins were then separated on 10% or 12%
SDS–polyacrylamide gels, transferred to nitrocellulose membranes
(Bio-Rad Laboratories, CA, USA) and stained with Ponceau S to
check equal protein loading. The membranes were blocked with
5% (w/v) non-fat dry milk and 0.1% Tween-20 in PBS for 2 h and
probed overnight with specific primary antibodies (Santa Cruz Bio-
technology, CA, USA). After washing in PBS and PBS with 0.1%
Tween-20, the membranes were probed with horseradish peroxi-
dase-conjugated secondary antibodies and visualized with West
Pico Supersignal chemiluminescent detection reagent (Thermo
Fisher Scientific, Rockford, USA). To confirm equal protein loading,
immunodetection was performed with anti-b-actin monoclonal
antibody (Santa Cruz Biotechnology, CA, USA). The experiments
were repeated three times. The protein expression in treated cells
was compared to untreated controls.
(Molecular Probes, Invitrogen, CA, USA) was added, and incubation
was continued for a further hour. The fluorescence of viable cells
was then quantified using a Fluoroskan Ascent instrument (Labsys-
tems, Finland). The percentage of surviving cells in each well was
calculated by dividing the intensity of the fluorescence signals
from the exposed wells by the intensity of signals from control
wells and multiplying by 100. These ratios were then used to con-
struct dose–response curves from which IC50 values, the concen-
trations of the respective compounds that were lethal to 50% of
the tumor cells, were calculated. The results obtained for selected
compounds are shown in Table 1.
2.3.3. Flow cytometric analysis of the cell cycle and apoptosis
CEM leukemia cancer cells were seeded in 100-mm culture
dishes, and immediately incubated with the test compounds. After
24 h, the cells were washed, fixed and stained in 0.1% [m/v] sodium
citrate, 0.1% [v/v] Triton X-100, 0.2 mg/mL RNase A and 10 lg/mL
propidium iodide in PBS. Their DNA contents were then assessed
with a flow cytometer (Cell Lab Quanta SC – MPL, Beckman Coulter,
CA USA), and the distribution of cells in subG1 (‘‘apoptotic cells’’),
G0/G1, S and G2/M peaks were quantified, by histogram analysis,
using MultiCycle AV software (Phoenix Flow Systems, CA, USA).
2.3.4. Activities of caspases 3 and 7 (3/7)
Treated CEM cells were harvested by centrifugation and homog-
enized in an extraction buffer (10 mM KCl, 5 mM HEPES, 1 mM
EDTA, 1 mM EGTA, 0.2% CHAPS, plus protease inhibitors: aprotinin,
leupeptin, PMSF; pH 7.4) on ice for 20 min. The resulting homoge-
nates were clarified by centrifugation at 10000 Â g for 20 min at
4 °C. The protein contents of the samples were quantified by the
Bradford method and they were diluted to the equivalent protein
concentrations. Lysates were then incubated for 1 h with
100 mM Ac-DEVD-AMC as a substrate (Sigma–Aldrich) in an assay
buffer (25 mM PIPES, 2 mM EGTA, 2 mM MgCl2, 5 mM DTT, pH 7.3).
For negative controls, the lysates were supplemented with
100 mM Ac-DEVD-CHO (Sigma–Aldrich) as a caspase 3/7 inhibitor.
The fluorescence of the product was measured using a Fluoroskan
Ascent microplate reader (Labsystems, Finland) at 346/442 nm (ex/
em).
3. Results and discussion
3.1. Chemical synthesis and characterization
The dendrimeric structure consists of a core that determines the
shape and steric hindrance in the structure, promoting a spheri-
cally or ellipsoidal shape as function of generation. Noteworthy,
the PAMAM dendrimer at G1 with 8 NH2 groups (1.430 KDa and
22 Å) presents an irregular geometry and adopts a spherical shape
that increases the steric hindrance and the molecular rigidity in the
higher generations, complicating the drug payload at the periphery
[33]. Hence, we focused in the synthesis of dendrimeric structures
with tetramethylene core in order to promote an ellipsoidal shape
and minimize steric hindrance. We used dimethyl 5-hydrox-
yisophthalate as branching unit which undergoes modification of
the functional groups at the periphery via nucleophilic reactions
with the steroids.
Table 1
IC50 (lM) values obtained from the calcein AM assays with the tested cancer and
Following the synthetic route shown in Scheme 1, the dendri-
mer of first generation (tetramethyl ester 2) was obtained in high
yield (98%) by reaction of dimethyl 5-hydroxyisophthalate (1) with
1,4-dibromobutane in the presence of potassium carbonate, and
18-crown-6 as the phase transfer catalyst. Subsequent reduction
of 2 with LiAlH4 afforded tetraol 3 (56% yield) which was treated
with H2SO4/HBr to give tetrabromo derivative 4 (84% yield) as a so-
lid ready for coupling with the steroids or to the second iteration of
analogous transformations for the preparation of the second den-
drimer generation.
normal cell lines.
Compound
Cell linea
CEM
MCF7
HeLa
BJ
17
17
a
a
-ethynylestradiol
-estradiol
19.1 1.7
25.2 6.4
>50
>50
1.4 0.1
>50
>50
>50
G1
3
8
9
>50
3.5 1.3
2.1 0.2
>50
>50
>50
>50
>50
>50
>50
>50
>50
G2
Dendrimers of second generation (5, 6 and 7) were prepared by
repeating the synthetic procedures described for the first genera-
tion, to give the methyl ester 5 in high yield (82%). The ester was
reduced to give hydroxyl derivative 6, which was converted into
benzylic chlorides to give 7.
The structures of all compounds obtained in this work were
unambiguously established using 2D NMR experiments (1H and
13C, COSY, HETCOR, DEPT 90° and 135°), in conjunction with IR
and high resolution mass spectrometry.
6
10
11
>50
26.8 4.6
>50
>50
34.6 4.9
>50
>50
5.5 0.1
>50
3.3 1.2
5.1 1.3
>50
a
The cells were treated for 72 h with serial concentrations of the tested com-
pounds. The values quoted represent means SD, these were obtained from three
independent experiments performed in triplicate using 17 -ethynylestradiol, 17a-
estradiol, 3 and 6 as a control in chronic leukemia (CEM), breast adenocarcinoma
(MCF7) and, cervical carcinoma (HeLa) tumor cell lines and against normal human
fibroblasted cells (BJ).
a

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N.E. Magaña-Vergara et al. / Steroids 78 (2013) 1254–1262
Scheme 1. Synthesis of the dendrimeric structures of first and second generation.
The symmetry present in the molecules simplifies their NMR
hydroxyl group at positions 3 of both steroids for conjugation with
the dendrimer, without compromising binding affinity of the estro-
gen [34]. The covalently bound conjugates of first (8 and 9) and
second generation (10 and 11) were synthesized from estrogens
characterization. The structures were assigned based on the multi-
plicity, chemical shift and integration of the signals. Because the
dendrimers of G1 and G2 consist of a tetramethylene core and di-
methyl 5-hydroxyisophthalate as branching units, they have simi-
lar 1H and 13C NMR spectra. Nonetheless, there are characteristic
signals like the new methylene at 5.04 ppm in G2 dendrimers that
allow distinguishing one from the other.
The main difference between the two generations is the cou-
pling of one additional branching unit that results in a spectrum
with twice the intensity of the signals compared with the precur-
sor of G1.
Reduction of the ester groups to give G2 dendrimer with 8 OH
terminal groups (6) was corroborated by the signal at 4.44 (H-
12) ppm attributed to new methylene groups and the absence of
the carbonyl group in the 13C NMR spectrum. The presence of the
hydroxyl group in dendrimers 3 and 6 was demonstrated by IR
spectra which showed broad absorption bands at 3291 and 3317,
respectively. Substitution of the hydroxyl group in 6 by chlorine
17-a-ethynylestradiol and 17-a-estradiol and the corresponding
halogenated compound 4 or 7 which were linked via ether func-
tionalities, giving first and second generation conjugates in yields
of 74% and 86% (Scheme 2). It should be mentioned that a previous
publication has reported the synthesis of a dendrimeric conjugate
in which 12 molecules of the corticosteroid, methylprednisolone
(MP), were linked via ester to the PAMAM G4-OH dendrimer. In or-
der to obtain this conjugate, it was necessary to couple previously
glutaric acid to the steroid as spacer since the alternative route
incorporating the spacer to the dendrimer afforded lower conjuga-
tion ratio of MP, presumably due to steric hindrance at the periph-
ery [28]. In contrast, the steroidal conjugates 8, 9, 10 and 11,
synthesized in this work, are relatively non-hindered molecules
with high payload of steroid in the dendrimer.
The formation of the steroidal dendrimer conjugates containing
(7) induced a
D
d of approximately 0.09 for the directly bonded
4 and 8 units of 17a-ethynylestradiol (8, 10), or 17a-estradiol (9,
methylene.
11) at the periphery was confirmed by the appearance of the ste-
roidal signals in the 1H NMR and 13C NMR spectra which were
unambiguously assigned using 2D experiments and are in agree-
ment with the shifts reported for the parent steroid [35,36]. The
aromatic protons of the dendrimer were assigned according to
Once dendrimers of first and second generation with haloge-
nated surface functionality were obtained, and considering the
advantages of steroidal payload in covalent conjugation, they were
coupled with the 17a-ethynylestradiol or 17a-estradiol using the

N.E. Magaña-Vergara et al. / Steroids 78 (2013) 1254–1262
1259
Scheme 2. Synthesis of two steroidal dendrimer conjugates derivatives of 17a-ethynylestradiol and 17a-estradiol of first (8 and 9) and second generation (10 and 11).
the integral and the coupling constants, and the signal shifted to
high frequencies at 4.98 (H-7) ppm in 8 and, at 5.02 (H-12) ppm
in 10 provided evidence for the formation of the conjugates.
The 13C NMR spectrum of 8 showed 27 different carbons which
were assigned using COSY, HETCOR and DEPT experiments. The
signal corresponding to the benzyl ether C-7 (–OCH2–) at
69.8 ppm confirms the formation of G1 steroidal dendrimer conju-
3.2. Biological evaluation
In order to evaluate the potential enhancement in cytotoxic
activity of 17 -ethynylestradiol and 17 -estradiol when they are
a
a
anchored to dendrimers of first and second generation, the steroi-
dal dendrimer conjugates of first (8 and 9) and second generation
(10 and 11), steroidal precursors, and hydroxyl terminated dendri-
mers (3 and 6) were screened against chronic leukemia (CEM),
breast adenocarcinoma (MCF7) and cervical carcinoma (HeLa) tu-
mor cell lines and against normal human fibroblast (BJ) as control
cells. The results are summarized in Table 1.
These results show the innocuity of all dendrimers, except for
dendrimer 6, against normal BJ cells. Also, the highest sensitivity
of CEM cell line to the tested compounds, allow us determine that
the cytotoxic activity in dendrimer conjugates is due to the steroi-
dal moiety, since dendrimers are non-cytotoxic. Compounds 8 and
9 were more effective against CEM at micromolar concentrations
as compared with their steroidal precursors. However, in contrast
to expectations, when the generation is increased, the higher pay-
load of steroidal residues does not have a favorable impact in cyto-
toxic activity, as can be seen in Table 1.
gates. The 13C NMR spectrum of G2 conjugate derivative of 17
a-
ethynylestradiol exhibits 32 different carbons and were assigned
as previously described.
For compounds 9 and 11 the expected 25 and 30 different car-
bons are observed in the 13C NMR spectrum and the signals for
both steroidal fragments were in agreement with the data reported
in the literature [31,32] and showed very slight variations. The sig-
nals corresponding to the aromatic ring of the dendrimer and the
newly formed ether linkage were also in agreement with those of
conjugates 8 and 10 previously discussed.
Additionally to NMR, the structure of the new dendrimeric con-
jugates was corroborated by using HRMS for 8 and 9 and MALDI
TOFF for 10 and 11 since their molecular weights fall in the upper
detection limit of the equipment used in HRMS.

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N.E. Magaña-Vergara et al. / Steroids 78 (2013) 1254–1262
With the aim to have an illustrative 3D image of conjugates
The action of caspase 3 that cleaves and inactivates the poly
ADP-ribose polymerase-1 (PARP) that is a nuclear enzyme involved
in DNA repair, led to increased DNA damage, followed by the DNA
fragmentation through uncontrolled endonuclease activity result-
ing in apoptosis [40,41]. We determined the activities of effector
caspases 3 and 7 (3/7) in CEM cells exposed to 8 or 9 using a fluo-
rogenic substrate, Ac-DEVD-AMC, and/or the caspase 3/7 inhibitor
Ac-DEVD-CHO. Cells were treated with a series of concentrations of
than can help to explain the decrease of activity as a consequence
of generation increase, possible conformations of steroidal dendri-
mer conjugates were explored by conformational search calcula-
tions in aqueous medium. The Monte-Carlo [37,38] algorithm in
Spartan 08 was used, where random changes were made in torsion
angles, respecting the steroidal stereochemistry during the search.
The conformers of minimum energy of 8, 9, 10 and 11 were opti-
mized at MMFFaq level [39]. Fig. 1 shows that the steroids are
more exposed in the first generation (8) than in the second (11),
the compounds exceeding their IC50 values: 5, 25 and 50
24 h. Conjugate 9 induced a ca. 3.7- and 4.3-fold increase in the
activity of caspase-3/7 after 24 h at 25 and 50 M compared with
lM for
whereby the steroids interact by
p
–p
stacking with the dendrimer
l
forming a very compact structure that precludes interaction with
cellular receptors.
These results suggest that our steroidal dendrimer conjugate of
first generation could potentially have therapeutic anticancer
applications against chronic leukemia and the second generation
does not show advantage due to the fact that the increase of den-
untreated controls. Compound 8 induced a ca. 2.5-fold increase
after 24 h at the highest tested concentration (Fig. 3). The observed
increase in the expression of caspase 3/7 (Fig. 3) in conjunction
with the DNA fragmentation data (Fig. 4) confirmed that both con-
jugates of first generation are able to induce apoptosis in leukemia
cancer cells (CEM) increasing the cytotoxic activity compared to
the free drug [20] suggesting that both conjugates have a common
mechanism of action in tumor cells.
drimer size promotes hydrophobic and
p–p interactions with the
steroidal moieties, inhibiting the interaction with cells.
In the context of the results reported herein (Table 1), we
decided to test G1conjugates 8 and 9 to determine how these con-
jugates disturbed the cell cycle and the possible pathway to induce
apoptosis in CEM cells by activities of caspase 3/7 and the Western
blot analysis.
Finally Western blot analyses were used to detect changes in
expression of apoptosis-related proteins in the CEM leukemia can-
cer cell line 24 h after treatment with steroidal dendrimer conju-
gate derivatives 8 and 9 (5, 25 and 50
lM). As shown in Fig. 4,
25 and 50 M treatments with 8 and 9 induced cleavage of PARP
l
To determine the phase in which the dendrimeric conjugates
induce apoptosis, CEM cells were incubated with steroidal dendri-
mer conjugates 8 and 9, flow cytometric analysis was used to
quantify the distribution of CEM cells in the different phases of
the cell cycle, and to determine the sub-G1 fraction as a marker
of the proportion of apoptotic cells. The results show that treat-
ment with 8 and 9 increased the proportions of S-phase cells in
all concentrations, along with reductions in proportions of G2/M
cells. In addition, the proportion of cells with sub-G1 amounts of
DNA (apoptotic cells) increased following treatment with the
tested conjugates 8 and 9, showing a threefold increase when trea-
after 24 h correlated with decreased levels of procaspase 3. In addi-
tion increased activity of caspase 3 was observed over the same
treatment time and concentrations for both compounds (Fig. 3).
Expression of a tumor suppressor protein p53 was observed in con-
trols of leukemia cancer cell line CEM and 8 caused its enhanced
expression after 24 h. The protein expression of p53 and Mdm2 in-
creased strongly following treatment with 25
50 M protein expression decreased. For both conjugates (8 and
9) at 50 M, after 24 h, a decreased expression of antiapoptotic
lM of 8, but at
l
l
proteins Mcl-1 and Bcl-2 was observed, indicating the commence-
ment of apoptosis. As reported previously, Mcl-1 is necessary for
cell viability and its decrease could be the cause of cell death in
CEM cells [32]. This antiapoptotic protein Mcl-1 has a crucial role
in regulating the apoptosis of T-cells [42]. It is known that apopto-
sis is mediated by caspase cascade activation and that caspase-3 is
an executioner protease that cleaves PARP, resulting in DNA degra-
dation and apoptotic death [32]. Our Western blot analysis demon-
strated the dose-dependent decrease of procaspase-3 and cleavage
ted with 9 50 lM relative to the untreated controls after 24 h
(Fig. 2). Thus, the tested steroidal dendrimer conjugate derivatives
of first generation effectively disturbed cell cycle and induced
apoptosis in CEM cells, via mitochondrial cytochrome c release
and resultant activation of caspase cascade, as evidenced by the
activities of caspases 3 and 7, which is mainly provoked in G2/
M-arrested cells [20].
Fig. 1. Minimum energy conformer optimized at the MMFFaq level in Spartan 08 (wavefunction Inc., CA) of 17-a estradiol dendrimer conjugate; a) first generation (9) and b)
second generation (11).

N.E. Magaña-Vergara et al. / Steroids 78 (2013) 1254–1262
1261
Fig. 2. Histograms, obtained by flow cytometric analysis, showing the distributions of CEM cells across the G0/G1, S, and G2/M phases of the cell cycle after 24 h treatment
with 8 and 9 and the sub-G1 fraction of cells (relative to untreated controls). The bars show the percentage of cells (%) in the corresponding phase.
Fig. 3. Increases in the activities of caspase 3 and 7 in CEM cells treated with 8 and 9 for 24 h, relative to untreated controls. The data shown represent averages from
experiments performed in triplicate.
In summary, we have prepared two generations of novel den-
drimers with flexible tetramethylene core and rigid dimethyl 5-
hydroxyisophthalate branching units. In order to test the utility
of these compounds, as platforms for improving the therapeutic in-
dex of biologically active molecules, we modified the surface of the
dendrimer with 17a-ethynylestradiol and 17a-estradiol to obtain
four novel steroidal dendrimer conjugates 8, 9, 10 and 11. The ste-
roidal dendrimer conjugates, the free drug and dendrimer were
tested against CEM, MCF7, HeLa cancer cell lines and BJ as normal
cells. Conjugates 8 and 9 confirmed the enhancement of the ste-
roids activity against CEM when they are conjugated to first gener-
ation of dendrimer. However in the case of second generation a
decrease in cytotoxic activity was observed. This could be attrib-
uted to unfavorable conformations in the steroidal moieties which
in the case of G2 lead to a folded structure while the four steroidal
fragments are available for cell interaction in G1, as determined by
conformational search.
The observation that conjugates of first generation (8, 9) are
able to induce apoptosis in leukemia cancer cells (CEM) and show
increased cytotoxic activity compared to the free drug, suggests
that both conjugates have a common mechanism of action in
CEM cells. The interesting anti-cancer activity of the prepared con-
jugates, suggest that the steroidal dendrimer conjugates 8 and 9
could be of potential use as new anticancer drugs.
Fig. 4. Western blot analysis of apoptosis-related proteins (PARP and procasapase-
3) in leukemia CEM cells treated with steroidal derivatives 8 and 9 compared to
their expression in untreated control cells. The expression of b-actin was monitored
as a protein loading control.
of PARP after 24 h treatment with steroidal dendrimer conjugates 8
and 9 in the leukemia cancer cell line CEM (Fig. 4).
Thus, the results confirm that these conjugates can induce cas-
pase-3 activated apoptosis (Fig. 3).

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[20] Jun DY, Park HS, Kim JS, Kim JS, Park W, Song BH, Kim HS, Taub D, Kim YH.
17 -Estradiol arrests cell cycle progression at G2/M and induces apoptotic cell
Acknowledgments
a
death in human acute leukemia Jurkat
2008;231:401–12.
T cells. Toxicol Appl Pharmacol
The authors thank ICyT for the scholarship to N.-E. Magaña-
Vergara. Thanks are given to Ma. T. Cortez for NMR spectra, G.
Cuéllar for mass spectra and Prof. Igor Alabugin for his suggestions
and critical reading of the manuscript. The Ministry of Education,
Youth and Sports of the Czech Republic (grant No. MSM
6198959216) and the Centre of the Region Haná for Biotechnolog-
ical and Agricultural Research (grant No. ED0007/01/01). Financial
support from CONACYT is gratefully acknowledged.
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human colon cancer cell line in vitro. Anticancer Res 1992;12:1327–30.
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Appendix A. Supplementary data
The spectroscopic characterization of all compounds 1H, 13C
NMR, MS (ESI) data are given. Supplementary data associated with
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