Synthesis and antiproliferative activity of side-chain unsaturated and homologated analogs of 1,25-dihydroxyvitamin D(2). (24E)-(1S)-24-Dehydro-24a-homo-1,25-dihydroxyergocalciferol and congeners.

Article abstract of DOI:10.1016/S0039-128X(02)00038-7

A series of analogs of 1,25-dihydroxyergocalciferol (1-4) was synthesized and screened for their antiproliferative activity in vitro. The structure of new analogs was designed based on biological activity of the previously obtained side-chain modified analogs of vitamin D(2) and D(3). The analogs were obtained by the Julia olefination of C(22)-vitamin D sulfone 11 with side-chain aldehyde 15. The analogs were tested for their antiproliferative activity against the cells of human breast cancer lines T47D and MCF7 as well as human and mouse leukemia lines, HL-60 and WEHI-3, respectively. Analog 2 (PRI-1907) showed the strongest antiproliferative activity out of the present series of analogs of 1,25-dihydroxyvitamin D(2) with the mono homologated and double unsaturated side chain. The activity of 2 was 3-150 times stronger, depending on the cell line, than that of 1,25-dihydroxycholecalciferol (calcitriol), used as standard. Copyright 2002 Elsevier Science Inc.

Full text of DOI:10.1016/S0039-128X(02)00038-7

Steroids 67 (2002) 789–798
Synthesis and antiproliferative activity of side-chain unsaturated
and homologated analogs of 1,25-dihydroxyvitamin D2
(24E)-(1S)-24-Dehydro-24a-homo-1,25-dihydroxyergocalciferol
and congenersଝ
a
Michał Chodynski , Joanna Wietrzykb,1, Ewa Marcinkowskab, Adam Opolskib,
´
Wiesław Szelejewskia, Andrzej Kutnera,∗
Pharmaceutical Research Institute, 8 Rydygiera, 01-793 Warszawa, Poland
a
b
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Weigl, 53-114 Wrocław, Poland
Received 23 January 2002; received in revised form 2 April 2002; accepted 10 April 2002
Abstract
A series of analogs of 1,25-dihydroxyergocalciferol (1–4) was synthesized and screened for their antiproliferative activity in vitro. The
structure of new analogs was designed based on biological activity of the previously obtained side-chain modified analogs of vitamin D2
and D3. The analogs were obtained by the Julia olefination of C22-vitamin D sulfone 11 with side-chain aldehyde 15. The analogs were
tested for their antiproliferative activity against the cells of human breast cancer lines T47D and MCF7 as well as human and mouse
leukemia lines, HL-60 and WEHI-3, respectively. Analog 2 (PRI-1907) showed the strongest antiproliferative activity out of the present
series of analogs of 1,25-dihydroxyvitamin D2 with the mono homologated and double unsaturated side chain. The activity of 2 was
3–150 times stronger, depending on the cell line, than that of 1,25-dihydroxycholecalciferol (calcitriol), used as standard. © 2002 Elsevier
Science Inc. All rights reserved.
Keywords: Vitamin D2 analogs; C22-Vitamin D synthon; Antiproliferative activity; Breast cancer cell lines MCF7 and T47D; Human promyelocytic leukemia
cell line HL-60; Mouse leukemia cell line WEHI-3
1. Introduction
side-chain, using our vitamin D C-22 synthon approach
[14]. The analog was 50–200 times more potent than
1,25-(OH)2D3 in regulation of cell growth and differentia-
tion in a number of cancer cell lines and in animal models
[15]. However, the analog still retained [16] substantial
calcemic activity at the level of 50% that of 1,25-(OH)2D3.
This might strongly limit the possible therapeutic applica-
tion of this compound in clinical anticancer treatment, due
to development of hypercalcemia in patients [12].
In our continuous search for vitamin D compounds
with anticancer potential [17] we have previously
obtained [18,19] vitamin D3 dihomo analogs monounsatu-
rated in the side-chain with lowered calcemic and enhanced
cell-differentiating activity [20]. A few years ago we first ob-
tained side-chain homoanalogs [21] of 1,25-dihydrovitamin
D2 [1,25-(OH)2D2], but its biological potential was not fully
explored at that time. Vitamin D2 and analogs are thought
to be generally less toxic than the respective vitamin D3
compounds.
Vitamin D hormone (1,25-dihydroxyvitamin D3, cal-
citriol, 1,25-(OH)2D3 [1,2]) was recently found to enhance
the antitumor activity of paclitaxel, a microtubule-disrupting
agent, in human cancer cells both in vitro and in vivo in
a synergistic schedule-independent manner [3]. Combined
effects [4,5] of 1,25-(OH)2D3 and cytostatics (anthracycline
antibiotics [6,7], platinum drugs [8]) or antiestrogens [9,10]
were also reported. It is now the common thought that
combined therapy based on vitamin D might have a clinical
significance in the treatment of patients with various solid
tumors [11,12].
Ernst Binderup et al. [13] obtained vitamin D3 ana-
log (seocalcitol, EB 1089) with extended and unsaturated
ଝ
This work was presented in part at the 11th Workshop on vitamin D,
N∗ashville, TN, 27 May–1 June 2000.
Corresponding author. Tel.: +48-22-633-8555; fax: +48-22-633-8296.
E-mail address: akutner@mercury.ci.uw.edu.pl (A. Kutner).
1
Based on these findings and with the aid of molec-
ular dynamics modeling [22], we have now designed a
J.W. is a stipendist of the Annual stipends for Young Scientists, the
Foundation for Polish Science (FNP), 2001–2002.
0039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(02)00038-7

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M. Chodyn´ski et al. / Steroids 67 (2002) 789–798
homologated analog of 1,25-(OH)2D2 with the conjugated
unsaturation in the side-chain.
m, 3-H), 4.17 (1H, m, 1-H), 4.48 (1H, m, 9-H), 5.28 (2H,
m, 6-H and 7-H), 7.4 (5H, m, Ar–H), 9.6 (1H, s, 22-H).
In the present paper, we described synthesis of this com-
pound, as well as of a series of its 26,27-congeners, from
vitamin D C22-synthon by using our previously developed
[23,24] convergent strategy. Moreover, we present their an-
tiproliferative activity in vitro against the cells of various
human and murine tumor cell lines.
2.3. (7E)-(1S,3R)-4ꢀ-Phenyl-6,9-[1ꢀ,2ꢀ]epi[1ꢀ,2ꢀ,4ꢀ]
triazolo-1,3-bis[(t-butyldimethyl-silyl)oxy]-22,23-dinor-
9,10-secochola-5(10),7-dien-24-oxy-3ꢀ,5ꢀ-dion (7)
Imidazole (0.64 g, 9.40 mmol) and t-butyldimethylsilyl
chloride (1.28 g, 8.49 mmol) were added to a solution of
diol 6 (1.96 g, 3.77 mmol) in 20 ml of DMF. This mixture
was stirred under nitrogen at RT for 24 h. Extraction with
a mixture of PE40-65/ethyl acetate (4/1, v/v) resulted in di-
silylated aldehyde 7 (2.89 g, 98% yield) as a colorless oil.
1H NMR (CDCl3) δ: 0.04 (6H, s, Si–CH3), 0.10 (6H, s,
Si–CH3), 0.83 (3H, s, 18-CH3), 0.87 (9H, s, Si–C–CH3),
0.90 (9H, s, Si–C–CH3), 1.15 (3H, d, 21-CH3), 1.83 (3H, s,
19-CH3), 2.64 (1H, m, 14-H), 3.17 (1H, m, 11-H), 4.00 (1H,
m, 3-H), 4.17 (1H, m, 1-H), 4.48 (1H, m, 9-H), 5.28 (2H,
m, 6-H and 7-H), 7.4 (5H, m, Ar–H), 9.6 (1H, s, 22-H).
2. Experimental
2.1. Synthesis
Sodium amalgam was prepared by using the method
adopted from Organic Syntheses. Tetrahydrofuran (THF)
was distilled over sodium benzophenone ketyl. Infrared
(IR) spectra were recorded on a Perkin-Elmer Model
1725X FT-IR spectrophotometer as films of oily substances
or CHCl3 solutions of crystalline substances. Ultravio-
let (UV) spectra were taken on a Shimadzu Model 160A
UV–VIS spectrophotometer in the solvents indicated. Nu-
clear magnetic resonance (1H and 13C NMR) spectra were
recorded at 200 MHz on a Varian Gemini 2000 spectro-
meter, at 400 MHz on a Bruker AM 400 spectrometer, and
at 500 MHz on a Bruker AM 500 spectrometer in the sol-
vents indicated, downfield from internal tetramethylsilane
(TMS) as standard. Electron impact mass spectrometry
spectra (EIMS) were recorded on a Finnigan MAT Model
8200 spectrometer. Column flash chromatography was per-
formed on silica gel Si 60 (230–400 mesh, Merck) and on
LiChroprep RP-18 (25–40 mm, Merck). High-performance
liquid chromatography (HPLC) separations were performed
using a Knauer Instrument Model 64, Hibar Si 60 col-
umn, 5 ␮m, 4 cm × 25 cm (Merck), Si 100 column, 10 ␮m,
10 cm × 25 cm and 22 cm × 25 cm.
2.4. (7E)-(1S,3R)-4ꢀ-Phenyl-6,9-[1ꢀ,2ꢀ]epi[1ꢀ,2ꢀ,4ꢀ]
triazolo-1,3-bis[(t-butyldimethylsilyl)oxy]-22,23-dinor-
9,10-secochola-5(10),7-diene-24-ol-3ꢀ,5ꢀ-dion (8)
Sodium borohydride (0.26 g, 6.9 mmol) was added to a so-
lution of aldehyde 7 (4.13 g, 5.5 mmol) in a mixture of 50 ml
of methylene chloride and 5 ml of MeOH. This mixture was
stirred under nitrogen at RT for 15 min and washed with wa-
ter. Extraction and removal of the solvents resulted in alcohol
8 (4.04 g, 97% yield) as a colorless foam. 1H NMR (CDCl3)
δ: 0.04 (6H, s, Si–CH3), 0.10 (6H, s, Si–CH3), 0.80 (3H, s,
18-CH3), 0.87 (9H, s, Si–C–CH3), 0.90 (9H, s, Si–C–CH3),
1.05 (3H, d, 21-CH3), 1.83 (3H, m, 19-CH3), 2.61 (1H, m,
14-H), 3.15 (1H, m, 11-H), 3.41 (1H, m, 22-H), 3.66 (1H,
m, 22-H), 4.00 (1H, m, 3-H), 4.17 (1H, m, 1-H), 4.47 (1H,
m, 9-H), 5.26 (2H, m, 6-H and 7-H), 7.4 (5H, m, Ar–H).
2.2. (7E)-(1S,3R)-4ꢀ-Phenyl-6,9-[1ꢀ,2ꢀ]epi[1ꢀ,2ꢀ,4ꢀ]
triazolo-1,3-dihydroxy-22,23-dinor-9,10-secochola-
5(10),7-dien-24-oxy-3ꢀ,5ꢀ-dion (6)
2.5. (5Z,7E)-(1S,3R,20S)-1,3-
[bis(t-Butyldimethylsilyl)oxy]-22,23-dinor-
9,10-secochola-5,7,10(19)-triene-24-ol (9)
A gentle stream of ozone was purged with stirring through
the solution of adduct 5 (3.2 g, 5.1 mmol) in 50 ml of methy-
lene chloride and 0.5 ml of pyridine at −79 ◦C for 17 min.
The mixture was flushed with nitrogen. Zinc powder (2.8 g)
and acetic acid were added and the reaction mixture was al-
lowed to warm up to RT. The mixture was heated at 40 ◦C for
10 min. After extraction and solvent removal, 2.67 g of yel-
low foam was obtained. A silica gel chromatography separa-
tion using a solvent system of methylene chloride/methanol
(96/4, v/v) resulted in aldehyde 6 as a light yellow foam
(1.53 g, 55%). A sample for analytical purposes was crys-
tallized from a mixture of n-hexane and ethyl acetate to give
fine colorless needles: m.p. 162–164 ◦C. 1H NMR (CDCl3)
δ: 0.84 (3H, s, 18-CH3), 1.16 (3H, d, 21-CH3), 1.93 (3H, s,
19-CH3), 2.64 (1H, m, 14-H), 3.17 (1H, m, 11-H), 4.00 (1H,
A solution of KOH (2.3 g, 41 mmol) in 10 ml MeOH was
added to a solution of alcohol 8 (1.9 g, 2.5 mmol) in 5 ml
MeOH. The mixture was stirred under nitrogen in a pres-
surized reaction vessel at 95 ◦C for 48 h. Next, the mixture
was cooled down to RT and diluted with 15 ml of MeOH.
Then, the mixture was purged with oxygen at 5 ◦C for 24 h.
Finally, the reaction mixture was concentrated under re-
duced pressure and extracted with methylene chloride. A sil-
ica gel column chromatography separation using a mixture
PE 40-65/EtOAc (93/7, v/v) resulted in alcohol 9 (0.86 g,
60% yield) as colorless oil. Additional flash chromatogra-
phy on silica gel resulted in analytical sample of alcohol 9
as a colorless oil. IR (film): 3463, 2951, 2856, 1603, 1471,
1360, 1257, 1075, 991, 907, 836 cm−1; UV (EtOH) λmax
:

M. Chodyn´ski et al. / Steroids 67 (2002) 789–798
791
1
264.6 nm; H NMR (CDCl3) δ: 0.48 (3H, s, 18-CH3), 0.83
(18H, s, Si–C–CH3), 1.06 (3H, d, 21-CH3), 4.19 (1H, m,
3-H), 4.37 (1H, m, 1-H), 4.86 (1H, d, 19Z-H), 5.18 (1H, d,
19E-H), 6.05 (1H, d, 7-H), 6.24 (1H, d, 6-H); MS (relative
intensity) m/z: 574 (M+, 65), 559 (12), 442 (95), 427 (24),
248 (100), 192 (22), 73 (91), 57 (86), 43 (96).
2.8. Ethyl 3-formyl-2E-butenoate (15)
Triethyl phosphonoacetate (12, 670 mg, 3 mmol) was
added to NaH (150 mg, 3.6 mmol) in dry DMF and stirred at
0 ◦C for 1 h. Pyruvic aldehyde dimethyl acetal 13 (444 mg,
3.6 mmol) was added and stirring was continued for 24 h
at RT. Extraction with ethyl acetate resulted in acetal 14
as a colorless oil (395 mg, 70%). IR (CHCl3): 2984, 2937,
1719, 1661, 1446, 1367, 1324, 1220, 1157, 1106, 1061,
969, 875 cm−1; 1H NMR (CDCl3) δ: 1.29 (3H, t, J = 7 Hz,
CH2CH3), 1.88 (3H, d, J = 2.0 Hz, 3-CH3, cis-isomer),
2.11 (3H, d, J = 1.5 Hz, 3-CH3, trans-isomer), 3.32 [6H,
s, 4-H, trans-isomer], 3.42 [6H, s, 4-H, cis-isomer], 4.18
(2H, q, J = 7 Hz, CH2CH3), 4.61 [1H, s, (CH3O)2CH],
6.02 (1H, d, J = 1.5 Hz, 2-H). This oil was stirred for 3 h
in CH2Cl2/HCl 3 N. The organic layer was concentrated to
afford crude 15 (280 mg). A silica gel flash chromatography
separation using CH2Cl2/hexane (1/1, v/v) provided pure 15
(200 mg, 67%) as a colorless oil. IR (CHCl3): 2987, 2835,
2.6. (5Z,7E)-(1S,3R)-1,3-bis[(t-Butyldimethylsilyl)oxy]-
24-(p-toluenesulfonyloxy)-22,23-dinor-9,10-secochola-
5,7,10(19)-triene (10)
p-Toluenesulfonyl chloride (382 mg, 2 mmol) was added
to a solution of 520 mg (0.905 mmol) of alcohol 9 in 20 ml
of methylene chloride. Triethylamine (4 ml) and catalytic
amount of 4,4-dimethylaminopyridine (DMAP) were added
and the solution was stirred at 20 ◦C for 28 h. Saturated aque-
ous solution of NaHCO3 was added (5 ml) and the mixture
was vigorously stirred for 1 h. A silica gel flash chromato-
graphy separation resulted in tosylate 10 (540 mg, 90%) as
a colorless oil. IR (film): 2952, 2856, 1605, 1471, 1360,
1719, 1702, 1396, 1352, 1297, 1164, 1041, 878, 828 cm−1
;
1257, 1175, 1075, 948, 908, 807 cm−1; UV (EtOH) λmax
:
UV (EtOH) λmax: 228.2 nm; 1H NMR (CDCl3) δ: 1.36 (3H,
1
263.2 nm; H NMR (CDCl3) δ: 0.41 (3H, s, 18-CH3), 0.84
(18H, s, Si–C–CH3), 0.91 (3H, d, 21-CH3), 2.42 (3H, s,
Ar–CH3), 4.22 (1H, m, 3-H), 4.38 (1H, m, 1-H), 4.76 (1H,
d, 19Z-H), 5.18 (1H, d, 19E-H), 5.92 (1H, d, 7-H), 6.26 (1H,
d, 6-H), 7.48 and 7.78 (2H, d, Ar–H); MS (relative inten-
sity) m/z: 728 (M+, 38), 713 (8), 596 (74), 464 (55), 248
(85), 155 (61), 105 (65), 91 (69), 75 (100).
=
t, J = 7 Hz, CH2CH3), 2.18 (3H, d, J = 1.5 Hz, CCH3),
4.28 (2H, q, J = 7 Hz, CH2CH3), 6.49 (1H, d, J = 1.5 Hz,
=
CH), 9.56 (1H, s, CHO).
2.9. Ethyl (5Z,7E,23aE)-(1S,3R)-1,3-
bis[(t-butyldimethylsilyl)oxy)-22-phenylsulfonyl-
23-hydroxy-23a-methyl-23a,23b-dihomo-9,10-secochola-
5,7,10(19),23-tetraene-24-oate (16)
2.7. (5Z,7E)-(1S,3R)-1,3-bis[(t-Butyldimethylsilyl)oxy]-
24-phenylsulfonyl-22,23-dinor-9,10-secochola-
5,7,10(19)-triene (11)
A solution of n-BuLi (130 ␮l, 1.6 M, 0.210 mmol) was
added with stirring to a solution of sulfone 11 (135 mg,
0.193 mmol) in 1 ml of THF at −70 ◦C. After 15 min a so-
lution of 50 ␮l (0.380 mmol) of aldehyde 15. [IR (CHCl3):
2987, 2835, 1720, 1702, 1369, 1352, 1298, 1165, 1041,
Lithium bromide dihydrate (500 mg, 4 mmol) and lithium
carbonate (300 mg, 4 mmol) were added to a solution of to-
sylate 10 (920 mg, 1.26 mmol) in 10 ml of DMF. The mixture
was stirred at 80 ◦C for 20 min. Sodium benzenesulfonate
(1.4 g, 8.5 mmol) was added and stirring was continued for
2.5 h. Ethyl acetate extraction and flash chromatography sep-
aration on a silica gel resulted in sulfone 11 (740 mg, 88%)
as a colorless oil. IR (film): 2953, 2856, 1471, 1447, 1305,
878 cm−1; UV (EtOH) λmax: 228.2 nm; H NMR (CDCl3)
1
δ: 1.38 (3H, t, J = 7 Hz, OEt), 2.18 (3H, s, 4-CH3), 4.29
(2H, q, J = 7 Hz, OEt), 6.52 (1H, s, 2-H), 9.58 (1H, s,
CHO)] was added. The mixture was stirred for 15 min at
−70 ◦C. A THF extraction and silica gel chromatography
separation resulted in ester 16 (145 mg, 88%) as colorless
oil. For analytical purposes ester 16 (mixture of isomers) was
isolated by silica gel chromatography as a colorless oil. IR
(CHCl3): 3484, 3068, 2952, 2857, 1716, 1654, 1472, 1447,
1257, 1144, 1085, 989, 908, 836 cm−1; UV (EtOH) λmax
:
264.4 nm; 1H NMR (CDCl3) δ: 0.05 [12H, br s, 2Si (CH3)2],
0.51 (3H, s, 18-CH3), 0.88 (18H, br s, 2t-BuSi), 1.20 (3H, d,
J = 6.4 Hz, 21-CH3), 2.88 and 3.16 (2H, dd, J = 4.3 Hz,
24-CH2), 4.19 (1H, m, 3-H), 4.37 (1H, m, 1-H), 4.83 (1H,
d, J = 2.4 Hz, 19Z-H), 5.16 (1H, d, J = 2.4 Hz, 19E-H),
5.97 (1H, d, J = 11.7 Hz, 7-H), 6.21 (1H, d, J = 11.7 Hz,
6-H), 7.58 and 7.91 (5H, m, Ar–H), 0.39 (3H, s, 18-CH3),
0.85 (18H, s, Si–C–CH3), 1.10 (3H, d, 21-CH3), 4.11 (1H,
m, 3-H), 4.34 (1H, m, 1-H), 4.72 (1H, d, 19Z-H), 5.29 (1H,
d, 19E-H), 5.91 (1H, d, 7-H), 6.27 (1H, d, 6-H), 7.69 and
7.92 (5H, m, Ar–H); MS (relative intensity) m/z: 698 (M+,
51), 683 (11), 566 (95), 551 (17), 248 (100), 192 (18), 73
(82); HRMS calculated for C40H66O4SSi2: 698.4221; found:
698.4279.
1386, 1300, 1215, 1143, 1082, 836 cm−1; UV (EtOH) λmax
:
264.6, 218.8 nm.
2.10. (5Z,7E,24E)-(1S,3R)-1,3-
bis[(t-Butyldimethylsilyl)oxy]-22-phenylsulfonyl-
24-methyl-24a-homo-9,10-secocholesta-
5,7,10(19),24-tetraen-23,25-diol (17)
A solution of CH3MgBr in ethyl ether (150 ␮l, 3 M,
0.45 mmol) was added to a solution of ester 16 (145 mg,
0.17 mmol) in 2 ml of THF. The mixture was stirred for 2 h
at 20 ◦C. A THF extraction and silica gel chromatography

792
M. Chodyn´ski et al. / Steroids 67 (2002) 789–798
separation resulted in dihydroxysulfone 17 (99 mg, 70%) as
Alcohols 22, 23 and 24 were prepared by the method
described for the synthesis of 21, in yields 50, 67 and 52%,
respectively.
a colorless oil. IR (CHCl3): 3484, 3068, 2953, 2856, 1645,
1471, 1447, 1384, 1302, 1255, 1141, 1082, 909, 835 cm−1
;
UV (EtOH) λmax: 265.4, 215.2 nm.
2.15. (24E)-(1S)-24-Dehydro-24a-homo-
1,25-dihydroxyergocalciferol (1)
2.11. (5Z,7E,24E)-(1S,3R)-1,3-
bis[(t-Butyldimethylsilyl)oxy]-22-phenylsulfonyl-
24-methyl-24a,26,27-trihomo-9,10-secocholesta-
5,7,10(19),24-tetraen-23,25-diol (18)
A solution of tetrabutylammonium fluoride in THF
(250 ␮l, 1 M, 0.25 mmol) was added to a solution of alcohol
21 (40 mg, 0.06 mmol) in 0.5 ml of THF. The mixture was
stirred under argon at 60 ◦C for 1.5 h. A THF extraction,
silica gel chromatography filtration and preparative HPLC
separation in reversed phases (a Hibar RP-18 column, 5 ␮m,
Merck, H2O/CH3CN, 45/55 v/v) resulted in triol 1 (20 mg,
74%) as a colorless oil. IR (CHCl3): 3344, 2947, 2927,
Dihydroxysulfone 18 was obtained from ester 16 (100 mg,
0.12 mmol) by the method described for 17 by using ethyl-
magnesium bromide (200 ␮l, 3 M, 0.6 mmol). A THF ex-
traction and silica gel filtration resulted in a mixture 18
(32 mg, 32% yield) as colorless oil. UV (EtOH) λmax: 265.6,
215.6 nm.
2869, 1636, 1445, 1382, 1216, 1149, 1054, 963, 756 cm−1
;
1
UV (EtOH) λmax: 264.2, 237.8 nm; H NMR (CDCl3) δ:
0.57 (3H, s, 18-CH3), 1.05 (3H, d, J = 6.6 Hz, 21-CH3),
1.40 (6H, s, 26,27-CH3), 1.97 (3H, s, 28-CH3), 4.23 (1H, m,
3-H), 4.43 (1H, m, 1-H), 5.00 (1H, bs, 19Z-H), 5.33 (1H, bs,
19E-H), 5.51 (1H, s, 24a-H), 5.52 (1H, dd, J1 = 15.6 Hz,
J2 = 8.6 Hz, 22-H), 5.93 (1H, d, J = 15.8 Hz, 23-H), 6.01
(1H, d, J = 11.3 Hz, 7-H), 6.38 (1H, d, J = 11.3 Hz, 6-H);
13C NMR (CDCl3) δ: 12.251 (18), 13.253 (28), 20.754 (21),
22.227 (15), 23.578 (11), 27.769 (16), 29.074 (9), 31.276
and 31.413 (26 and 27), 40.386 (12), 40.462 (20), 42.861
(2), 45.260 (4), 45.958 (13), 56.405 (17), 56.511 (14),
66.897 (3), 70.829 (1), 71.118 (25), 111.916 (19), 117.215
(7), 125.050 (6), 133.006 (23), 133.143 (5), 135.451 (24a),
135.633 (24), 136.847 (22), 143.164 (8), 147.764 (10); MS
(relative intensity) m/z: M+ 440 (6), 422 (11), 404 (21),
386 (9), 285 (15), 269 (13), 251 (14), 135 (56), 107 (100),
59 (6); HRMS calculated for C29H44O3: 440.3290; found:
440.3284 and calculated for C29H44O3–H2O: 422.3185;
found: 422.3180.
2.12. (5Z,7E,24E)-(1S,3R)-1,3-
bis[(t-Butyldimethylsilyl)oxy]-22-phenylsulfonyl-
24-methyl-24a-homo-26,27-diethyl-9,10-secocholesta-
5,7,10(19),24-tetraen-23,25-diol (19)
Dihydroxysulfone 19 was obtained from ester 16 by using
the method described for 17. Ester 16 (105 mg, 0.15 mmol)
was dissolved in n-propylmagnesium bromide (0.5 ml, 2 M,
1.0 mmol) and stirred at ambient temperature for 2 h. A THF
extraction and silica gel filtration resulted in a mixture 19
(70 mg, 53% yield) as colorless oil. UV (EtOH) λmax: 265,
215 nm.
2.13. (5Z,7E,24E)-(1S,3R)-1,3-
bis[(t-Butyldimethylsilyl)oxy]-22-phenylsulfonyl-
24-methyl-24a-homo-26,27-di-n-propyl-9,10-secocholesta-
5,7,10(19),24-tetraen-23,25-diol (20)
Dihydroxysulfone 20 was obtained from ester 16 by using
the method described for 17. n-Butyl lithium (370 ␮l, 1.6 M
in hexane, 0.59 mmol) was added, with stirring, to a solution
of ester 16 (98 mg, 0.12 mmol) in 0.5 ml THF at −20 ◦C.
The mixture was stirred at 0 ◦C for 1 h. A THF extraction
and silica gel filtration resulted in a mixture 20 (43 mg, 41%
yield) as colorless oil. UV (EtOH) λmax: 265, 215 nm.
2.16. (24E)-(1S)-24-Dehydro-24a,26,27-trihomo-
1,25-dihydroxyergocalciferol (2)
Triol 2 (5 mg, 57% yield) was obtained from 22 (13 mg,
0.019 mmol) by using tetrabutylammonium fluoride (50 ␮l,
1 M, 0.050 mmol) in analogy to the method described for the
preparation of 1 from 21 in the form of a colorless oil. IR
(CHCl3): 3358, 2947, 2870, 1646, 1440, 1384, 1216, 1148,
1055, 964, 758 cm−1; UV (EtOH) λmax: 264.4, 238.2 nm,
1H NMR (CDCl3) δ: 0.57 (3H, s, 18-CH3), 0.89 (6H, t, J =
7.3 Hz, 26ꢀ,27ꢀ-CH3), 1.06 (3H, d, J = 6.6 Hz, 21-CH3),
1.63 (4H, t, J = 7.3 Hz, 26,27-CH2), 1.95 (3H, s, 28-CH3),
4.23 (1H, m, 3-H), 4.43 (1H, m, 1-H), 5.00 (1H, bs, 19Z-H),
5.31 (1H, s, 24a-H), 5.33 (1H, bs, 19E-H), 5.49 (1H, dd,
J1 = 8.4 Hz, J2 = 15.7 Hz, 22-H), 5.96 (1H, d, J =
15.7 Hz, 23-H), 6.02 (1H, d, J = 11.4 Hz, 7-H), 6.39 (1H,
d, J = 11.4 Hz, 6-H); MS (relative intensity) m/z: 468 (M+,
4), 450 (10), 432 (7), 287 (19), 269 (17), 225 (7), 209 (9),
175 (11), 155 (21), 135 (100), 107 (35), 91 (32), 81 (21),
2.14. (5Z,7E,22E,24E)-(1S,3R)-1,3-
bis[(t-Butyldimethylsilyl)oxy]-24a-homo-9,10-seco-
ergosta-5,7,10(19),24-tetraen-25-ol (21)
Powdered Na2HPO4 (10 mg) and 1 ml of a saturated so-
lution of Na2HPO4 in methanol were added under argon
to a solution of dihydroxysulfone 17 (99 mg, 0.12 mmol)
in 0.5 ml of methanol, followed by sodium amalgam (5%,
0.6 g). The mixture was stirred under argon at RT for 2 h.
A hexane/toluene (1/1, v/v) extraction and silica gel chro-
matography separation resulted in alcohol 21 (40 mg, 50%)
as a colorless oil. IR (CHCl3): 3360, 2952, 1643, 1472, 1471,
1254, 1076, 835 cm−1; UV (EtOH) λmax: 264.6, 237.8 nm.

M. Chodyn´ski et al. / Steroids 67 (2002) 789–798
793
57 (76); HRMS calculated for C31H48O3: 468.3604; found:
468.3601.
by courtesy of Professor Spik and Dr Mazurier (Laboratory
of Biological Chemistry USTL, Lille, France). The cells
were plated in 96-well plates (Costar, USA) at a density
of 5 × 103 cells per well and cultured in an opti-MEM or
in RPMI-1640 medium supplemented with 2 mM glutamine
(Gibco, Warsaw, Poland), streptomycin (50 ␮g/ml) (Jelfa,
Jelenia Góra, Poland), penicillin (50 U/ml) (Jelfa, Jelenia
Góra, Poland) and 5% fetal calf serum (Gibco, Grand Is-
land, USA) 24 h before addition of the tested compounds.
Then, the cells were cultured, in 37 ◦C, in humid atmosphere
saturated with 5% CO2.
2.17. (24E)-(1S)-24-Dehydro-24a-homo-26,27-
diethyl-1,25-dihydroxyergocalciferol (3)
Triol 3 was obtained from 23 (40 mg, 0.055 mmol) by us-
ing tetrabutylammonium fluoride (150 ␮l, 1 M, 0.150 mmol)
with the method described for the preparation of 1 from
21. Lyophylization of the HPLC eluent resulted in triol 3
as a white powder (12 mg, 44% yield). IR (CHCl3): 3370,
2956, 2872, 1644, 1455, 1378, 1216, 1054, 964, 758 cm−1
;
1
UV (EtOH) λmax: 264.0, 237.8 nm; H NMR (CDCl3) δ:
0.57 (3H, s, 18-CH3), 0.91 (6H, t, J = 7.3 Hz, 26ꢀꢀ-CH3,
27ꢀꢀ-CH3), 1.06 (3H, d, J = 6.6 Hz, 21-CH3), 1.94 (3H, bs,
28-CH3), 4.23 (1H, m, 3-H), 4.43 (1H, m, 1-H), 5.00 (1H,
bs, 19Z-H), 5.32 (2H, bs, 24a-H, 19E-H), 5.48 (1H, dd, J1 =
8.4 Hz, J2 = 15.5 Hz, 22-H), 5.93 (1H, d, J = 15.5 Hz,
23-H), 6.00 (1H, d, J = 11.4 Hz, 7-H), 6.38 (1H, d, J =
11.4 Hz, 6-H); MS (relative intensity) m/z: 496 (M+, 5), 478
(13), 460 (10), 435 (14), 301 (26), 269 (24), 251 (22), 175
(12), 135 (64), 107 (25), 95 (35), 81 (22), 71 (100); HRMS
calculated for C33H52O3: 496.3917; found: 496.3881.
2.19.2. Analogs
Samples of analogs 1–4 were stored in the amber am-
poules under argon at −20 ◦C. The amount of each analog in
the ampoule was determined by the UV spectrophotometry
(Carl Zeiss spectrophotometer, Jena, Germany) at 265 nm.
Analogs were dissolved in absolute ethanol to the concen-
tration of 10−4 M and, subsequently, diluted in the culture
medium to reach the required concentrations (ranging from
1000 to 0.01 nM).
2.19.3. MTT assay
The MTT assay was performed after exposure of cells to
varying concentrations of analogs tested for 120 h. The MTT
solution (20 ␮l) was added to each well for the last 3–4 h of
incubation [MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide, Sigma, St. Louis, MO; stock solution:
5 mg/ml]. The viable cells reduced in their mitochondria a
pale yellow MTT to a navy blue formazan. After the incu-
bation time was completed, a sample of 80 ␮l of the lysing
mixture was added to each well (lysing mixture: 225 ml
dimethylformamide, 67.5 g sodium dodecyl sulphate, both
from Sigma, St. Louis, MO, USA, and 275 ml of distilled
water). After the formazan crystals were dissolved (24 h),
the optical densities of the samples were measured on a
Multiskan RC photometer (Labsystems, Helsinki, Finland)
at 570 nm.
2.18. (24E)-(1S)-24-Dehydro-24a-homo-
26,27-di-n-propyl-1,25-dihydroxyergocalciferol (4)
Triol 4 was obtained from 24 (17 mg, 0.023 mmol) by us-
ing tetrabutylammonium fluoride (60 ␮l, 1 M, 0.060 mmol)
with the method described for the preparation of 1 from 21.
Lyophilization of the HPLC eluent resulted in triol 4 as a
white powder (4.8 mg, 41% yield). IR (CHCl3): 3358, 2954,
2870, 1643, 1456, 1378, 1215, 1054, 964 cm−1; UV (EtOH)
1
λmax: 264.0, 238.2 nm; H NMR (CDCl3) δ: 0.57 (3H, s,
18-CH3), 0.91 (6H, t, J = 6.3 Hz, 26ꢀꢀꢀ-CH3, 27ꢀꢀꢀ-CH3),
1.05 (3H, d, J = 6.6 Hz, 21-CH3), 1.95 (3H, bs, 28-CH3),
4.23 (1H, m, 3-H), 4.43 (1H, m, 1-H), 5.00 (1H, bs, 19Z-H),
5.33 (2H, bs, 24a-H, 19E-H), 5.49 (1H, dd, J1 = 8.4 Hz,
J2 = 15.4 Hz, 22-H), 5.94 (1H, d, J = 15.4 Hz, 23-H), 6.02
(1H, d, J = 11.4 Hz, 7-H), 6.38 (1H, d, J = 11.4 Hz, 6-H);
MS (relative intensity) m/z: 524 (M+, 26), 506 (29), 488
(12), 467 (33), 449 (56), 431 (48), 315 (89), 285 (35), 269
(89), 251 (71), 191 (28), 179 (53), 151 (52), 135 (100), 107
(25), 85 (94); HRMS calculated for C35H56O3: 524.4230;
found: 524.4220.
2.19.4. SRB assay
The details of the SRB assay were described by Ske-
han et al. [25]. The cells attached to the plastic were
fixed by gentle layering of cold 50% TCA (trichloroacetic
acid, Aldrich–Chemie, 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 a tap water. The
background optical density was measured in the wells filled
with the culture medium, without the cells. The cellular
material fixed with TCA was stained with 0.4% sulforho-
damine B (SRB, Sigma, Germany) dissolved in 1% acetic
acid (POCh, Gliwice, Poland) for 30 min. The unbound dye
was removed by rinsing four times with 1% acetic acid. The
protein-bound dye was extracted with 10 mM unbuffered
Tris base (POCh, Gliwice, Poland) for determination of opti-
cal density (at 540 nm) in a computer-interfaced, 96-well mi-
crotiter plate reader Multiskan RC photometer (Labsystems,
2.19. Antiproliferative assays in vitro
2.19.1. Cells
The human breast cancer cell lines T47D and MCF-7 as
well as the mouse leukemia cell line WEHI-3 were obtained
from the American Type Culture Collection (Rockville, MD)
and were maintained in the Cell Culture Collection of the In-
stitute of Immunology and Experimental Therapy, Wrocław,
Poland. The human promyelocytic leukemia HL-60 cell line
was obtained from the European Type Culture Collection

794
M. Chodyn´ski et al. / Steroids 67 (2002) 789–798
Helsinki, Finland). The results were calculated as an
ID30,40,50 (inhibitory dose 30, 40, 50)—the dose of tested
agent, which inhibits proliferation of cancer cells by 30,
40 and 50%. The ID values were calculated separately for
each experiment and the mean values S.D. were pre-
sented in the tables. Each compound in every concentration
was tested in triplicates in a single experiment, which was
repeated 3–7 times. A percent of the cell growth inhibition
for all experiments was calculated and presented in figures
with the respective standard deviations.
intermediate (Scheme 1). This practical synthesis of synthon
11 started from phenyltriazolinedione adduct 5 [26,27]. The
ergosterol side chain of adduct 5 was cleaved by ozonolysis
and subsequent reductive cleavage of the resulting ozonide
in 55% overall yield. Silylation and sodium borohydride
reduction of resulting aldehyde 6 resulted in C22-alcohol
in over 95% yield. The sulfone moiety was introduced at
C-22 by the usual method. The intermediate tosylate 10 was
converted into C22-bromide (not shown) and reacted with
sodium benzenesulfonate to give sulfone 11 in 88% yield.
For preparation of a side-chain fragment 15 (Scheme 2),
we used a Wittig–Horner methodology, applied by Curley
and Ticoras [28,29] for the total synthesis of retinoic acid
and its metabolites. Conjugated aldehyde–ester 15 was con-
veniently prepared in two steps from commercially available
components, without tedious distillation of the final prod-
uct. Thus, the Wittig reagent 12 was condensed with acetal
13 to give unsaturated ester 14 as a mixture of the geomet-
ric isomers. The biphasic acid hydrolysis of this ester re-
sulted in 15 in 67% overall yield. trans-Stereochemistry of
the product was confirmed by a chemical shift of aldehyde
proton in 1H NMR (δ = 9.56 ppm) compared to the known
shift of the cis-isomer (δ = 10.12 ppm). The Julia olefina-
tion of deprotonated sulfone 11 with aldehyde 15 resulted
in hydroxysulfones 16 in 88% yield (Scheme 3). Attempted
dehydroxy-desulfonylation of 16 resulted in a complicated
mixture of products. For this reason sulfone 16 was first re-
acted with Grignard or organolithium reagent to give a series
2.19.5. Statistical evaluation
Student’s t-test for independent samples was applied.
3. Results and discussion
The synthesis of analogs 1–4 (Fig. 1) followed our general
convergent strategy for the side-chain modified analogs of
vitamin D. In this strategy, a vitamin D analog is constructed
from vitamin D synthon and the respective side-chain
fragment. A C22-vitamin D synthon 11 (Scheme 1)
was used for our synthesis of analogs 1–4. Previously
[23], synthon 11 was obtained from a steroid precursor,
22,23-bisnor-5-cholenic acid, by the classical construction
of the vitamin D triene system, followed by Paaren–DeLuca
hydroxylation at C-1. In the present paper, an alternative
method is first described for the synthesis of this advanced
Fig. 1. (24E)-(1S)-24-Dehydro-24a-homo-1,25-dihydroxyergocalciferol (1) and its side-chain unsaturated homo analogs 2, 3, and 4.

M. Chodyn´ski et al. / Steroids 67 (2002) 789–798
795
Scheme 1. Synthesis of a C22-vitamin D synthon 11. Reagents and conditions: (i) O3, CH2Cl2, pyridine, −79 ◦C, 17 min; (ii) TBDMSCl, Im, DMF, RT,
24 h; (iii) NaBH4, MeOH, RT, 15 min; (iv) KOH, MeOH, 95 ◦C, 48 h; (v) p-TsCl, TEA, DMAP, CH2Cl2, 20 ◦C, 28 h; (vi) LiBr, Li2CO3, DMF, 80 ◦C,
20 min; C6H5SO3Na, 80 ◦C, 2.5 h.
of products 17–20 and then reacted with sodium amalgam in
buffered methanol giving vinyl alcohols 21–24. A fluoride
ion promoted desilylation of these alcohols resulted in a se-
ries of analogs 1–4. The all-trans geometry of the side chain,
resulting from the Julia olefination protocol, was confirmed
by the values of coupling constants J22,23 of side-chain pro-
for both the T47D and MCF-7 cell line in order to com-
pare the effect of analogs with that of calcitriol (Table 1).
The analogs were also screened against cells of the human
HL-60 and mouse WEHI-3 leukemia lines (Table 1). All
the analogs revealed strong antiproliferative activity against
these two targets with maximal growth inhibition of 76%
for HL-60 and 92% for WEHI-3 cells. The ID50 value was
estimated for both cell lines with the exception of analog
4 against WEHI-3 cells (Table 1). Analog 2 revealed the
strongest antiproliferative activity against all target cells ap-
plied. This effect was about 3, 8, and 150 times stronger than
that of calcitriol, for both WEHI-3, and MCF-7 as well as
for T47D and HL-60 cell lines, respectively. The observed
differences between inhibitory doses of calcitriol and 2 were
statistically significant. Thus, 2 is the most active analog out
of the series of side-chain modified analogues of vitamin
D tested. Analog 1 revealed activity weaker than that of 2
but generally stronger than that of calcitriol. Analog 4 did
not exhibit any antiproliferative activity in our experimental
protocol, with the exception of a weak growth inhibition of
the WEHI-3 cells (Table 1 and Fig. 2).
1
tons in the H NMR spectra.
Analogs 1–4 were examined for their antiproliferative ac-
tivity in vitro against cells of the human breast cancer lines
and human and mouse leukemia lines. The analogs revealed
antiproliferative activity against the human breast carcinoma
cell lines T47D and MCF-7 with maximal growth inhibi-
tion of 51 and 48%, respectively. This inhibitory effect was
dose dependent. The values ID40 and ID30 were calculated
Antiproliferative activity of the most active vitamin D2
analog 2 was comparable to that of the known vitamin D3
analog, EB 1089, of similar structure [12]. Based on the
known lower toxicity of vitamins D2, as compared to the
respective vitamins D3, it is anticipated that 2 might be
less calcemic in humans than EB 1089. Calcemic activity
of 2 and related new vitamin D2 analogs, both in vitro in
caco-2 cell lines and in animal models, will be reported
elsewhere.
Scheme 2. Synthesis of a side-chain fragment 15. Reagents and conditions:
(i) NaH, DMF, 0 ◦C, 1 h; (ii) 3 M HCl/CH2Cl2, RT, 3 h.

M. Chodyn´ski et al. / Steroids 67 (2002) 789–798
797
Scheme 3. Synthesis of (24E)-(1S)-24-dehydro-24a-homo-1,25-dihydroxyergocalciferol (1) and analogs 2, 3, and 4. Reagents and conditions: (i) n-BuLi,
THF, −70 ◦C, 15 min; (ii) CH3MgBr, THF, 20 ◦C, 2 h; (iii) Na/Hg, Na2HPO4, MeOH, RT, 2 h; (iv) TBAF, THF, 60 ◦C, 1.5 h.
Table 1
Inhibitory doses in vitro of vitamin D analogues 1–4 against the human and mouse leukemia (HL-60 and WEHI-3), and human breast carcinoma (T47D
and MCF-7) cell lines
Analog
Inhibitory dose (nM)
ID50
ID40, T47D
ID30, MCF-7
HL-60
WEHI-3
1,25-(OH)2D3
15.0
2.2
0.1
1.14
0.43 0.2
275.0
819.0
35.0
1.1
3.1
2.1∗
114.0
39.0
39.0
1.1
1
2
3
4
0.50∗
0.02∗
3.00
0.28
0.16
11.0
47.0
0.3
1.4∗
1.3∗
1.2
0.1
2.3
1.8
b
15.0
–
137.0
a
c
d
–
–
–
The symbol (∗) indicates statistically significant differences between the analogue and calcitriol (P < 0.05).
a Maximal growth inhibition, 43%.
b Maximal growth inhibition, 37%.
c Maximal growth inhibition, 20%.
d Maximal growth inhibition, 25%.
The mechanism of the antiproliferative effect in vitro of
the vitamin D analogs is not yet fully understood. It might
be related, however, to the effect in inducing the cell dif-
ferentiation [19,30]. Induction of apoptosis by the vitamin
D compounds has been also reported for breast cancer cells
[31]. Inhibition of the cell cycle in G1 phase is well docu-
mented for leukemic cells [32]. Deregulation of intracellular
signal transduction might also be considered [19,33,34].
References
[1] Jones G, Strugnell SA, DeLuca HF. Current understanding of the
molecular actions of vitamin D. Physiol Rev 1998;78:1193–231.
[2] Norman AW, Mena FR, Silva B. Structure function studies:
identification of vitamin D analogs for the ligand binding domains of
important proteins in the vitamin D-endocrine system. Rev Endocr
Metabol Disord 2001;2:229–38.
[3] Hershberger PA, Yu WD, Modzelewski RA, Reuger RM, Johnson
CS. Calcitriol (1,25-dihydroxycholecalciferol) enhances paclitaxel
antitumor activity in vitro and in vivo and accelerates paclitaxel
induced apoptosis. Clin Cancer Res 2001;7:1043–51.
Acknowledgments
[4] Siwin´ska A, Opolski A, Chrobak A, Wietrzyk J, Wojdat E, Kutner
A, et al. Potentiation of the antiproliferative effect in vitro of
doxorubicin, cisplatin and genistein by new analogues of vitamin D.
Anticancer Res 2001;21:1925–9.
This work was supported by the internal grant from the
Pharmaceutical Research Institute, Warsaw, Poland. Dr. M.
[5] Saito Y, Hatta H, Mano Y, Taira N, Shimizu T, Takeda K. vitamin
D3 analogue KH 1060 combined with TPA synergistically induces
mature macrophages in human myeloblastic leukemia ML-1 cells.
Anticancer Res 1999;19:1069–76.
[6] Ravid A, Rocker D, Machlenkin A, Rotem C, Hochman A,
Kessler-Icecson G, et al. 1,25-Dihydroxyvitamin D3 enhances the
´
Skarz˙ynski from the Pharmaceutical Research Institute is
acknowledged for recording the NMR spectra and Dr. W.
Danikiewicz from the Institute of Organic Chemistry of
the Polish Academy of Sciences for recording the mass
spectra.

798
M. Chodyn´ski et al. / Steroids 67 (2002) 789–798
susceptibility of breast cancer cells to doxorubicin-induced oxidative
damage. Cancer Res 1999;59:862–7.
analogues of calcitriol against various human normal and cancer cell
lines. Anticancer Res 1999;19:5217–22.
[7] Ketley NJ, Allen PD, Kelsey SM, Newland AC. Modulation of
idarubicin-induced apoptosis in human acute myeloid leukemia blats
by all-trans retinoic acid, 1,25-(OH)2 vitamin D3, and granulocyte-
macrophage colony-stimulating factor. Blood 1997;90:4578–87.
[8] Moffat KA, Johannes WU, Miller GJ. 1 Alpha,25-dihydroxyvitamin
D3 and platinum drugs act synergistically to inhibit the growth of
prostate cancer cell lines. Clin Cancer Res 1999;5:695–703.
[9] Abe-Hashimoto J, Kikuchi T, Matsumoto T, Nishii Y, Ogata E, Ikeda
K. Antitumor effect of 22-oxa-calcitriol, a noncalcemic analogue of
calcitriol, in athymic mice implanted with human breast carcinoma
and its synergism with tamoxifen. Cancer Res 1993;53:2534–7.
[10] Yanagihara K, Ito A, Toge T, Numoto M. Antiproliferative effects
of isoflavones on human cancer cell lines established from the
gastrointestinal tract. Cancer Res 1993;53:5815–21.
[11] van den Bemd G-JCM, Pols HAP, van Leeuwen JPTM. Anti-tumor
effects of 1,25-dihydroxyvitamin D3 and vitamin D analogs. Curr
Pharm Design 2000;6:717–32.
[12] Hansen CM, Hamberg KJ, Binderup E, Binderup L. Seocalcitol
(EB1089): a vitamin D analogue of anti-cancer potential. Back-
ground, design, synthesis, pre-clinical and clinical evaluation. Curr
Pharm Design 2000;6:803–28.
[13] Binderup E, Calverley MJ, Binderup L. Synthesis and biological
activity of 1a-hydroxylated vitamin D analogs with poly-unsaturated
side-chain. In: Norman AW, Bouillon R, Thomasset M, editors.
Vitamin D. Gene regulation, structure-function analysis and clinical
application. Proceedings of the Eight Workshop on vitamin D, Paris,
France. Berlin: Walter de Gruyter, 1991. p. 192–3.
[14] Kutner A, Perlman K, Sicin´ski RR, Schnoes HK, Phelps ME, DeLuca
HF. Vitamin D C-22 aldehydes. New key intermediates for the
synthesis of side-chain modified vitamin D analogues. Tetrahedron
Lett 1987;28:6129–32.
[15] Kissmeyer A-M, Binderup E, Binderup L, Mork Hansen C, Rastrup
Andersen N, Makin HLJ, et al. Metabolism of the vitamin D analog
EB 1089 by cultured human cells: redirection of hydroxylation site to
distal carbons of the side-chain. Biochem Pharmacol 1997;53:783–
93.
[16] Mork Hansen C, Maenpaa PH. EB 1089, a novel vitamin D analog
with strong antiproliferative and differentiation-inducing effects on
target cells. Biochem Pharmacol 1997;54:1173–9.
[21] Kutner A, Chodyn´ski M. Synthesis of side-chain homologated
analogs of 1,25-dihydroxycholecalciferol and 1,25-dihydroxyergo-
calciferol. Steroids 1991;56:311–5.
[22] Chodyn´ski M, Krupa M, Cholin´ski J, Wojciechowska W, Fitak H,
Zorgdrager J, et al. Synthesis and biological evaluation of active
metabolites of vitamin D3 and side-chain modified analogues of
vitamin D2. In: Norman AW, Bouillon R, Thomasset M, editors.
Vitamin D endocrine system. Structural, biological, genetic and
clinical aspects. Proceedings of the Eleventh Workshop on vitamin D,
Nashville, Tennessee, USA. Riverside: Printing and Reprographics
University of California, 2000. p. 85–8.
[23] Odrzywolska M, Chodyn´ski M, Halkes SJ, Velde van de J-P, Fitak H,
Kutner A. Convergent synthesis, chiral HPLC, and vitamin D recep-
tor affinity of analogs of 1,25-dihydroxycholecalciferol. Chirality
1999;11:249–55.
[24] Odrzywolska M, Chodyn´ski M, Zorgdrager J, Velde van de J-P,
Kutner A. Diastereoselective synthesis, binding affinity for vitamin
D receptor, and chiral stationary phase chromatography of hydroxy
analogs of 1,25-dihydroxycholecalciferol and 25-hydroxycholecal-
ciferol. Chirality 1999;11:701–6.
[25] Skehan P, Storeng R, Scudiero D. New colorimetric cytotoxicity assay
for anticancer-drug screening. J Natl Cancer Inst 1990;82:1107–12.
[26] Nerinckx W, De Clercq PJ, Couwenhoven C, Overbeek WRM, Halkes
SJ. An improved synthesis of 1␣-hydroxy vitamin D3. Tetrahedron
1991;47:9419–30.
[27] Vanmaele L, De Clercq PJ, Vandewalle M. An efficient synthesis of
1␣,25-dihydroxy vitamin D3. Tetrahedron 1985;41:141–4.
[28] Curley RW, Ticoras CJ. A convenient synthesis of the important
retinoid synthon. Ethyl trans-3-formyl-2-butenoate. Syn Comm
1986;16:627–31.
[29] Curley RW, Ticoras CJ. Stereospecific synthesis of the important
retinoid synthon ethyl trans-3-formyl-2-butenoate via direct two stage
oxidation of ethyl 3-methyl-2-butenoate. J Org Chem 1986;51:256–8.
[30] Elstner E, Linker-Israeli EM, Said J, Umiel T, de Vos S, Shintaku
PI, et al. 20-Epi-vitamin D3 analogues: a novel class of potent
inhibitors of proliferation and inducers of differentiation of human
breast cancer cells. Cancer Res 1995;55:2822–30.
[31] James SY, Mackay AG, Colston KW. Effects of 1,25-dihydroxy-
vitamin D3 and its analogues on induction of apoptosis in breast
cancer cells. J Steroid Biochem Mol Biol 1996;58:395–401.
[32] Wang QM, Jones JB, Studzinski GP. Cycline dependent kinase
inhibitor p27 as a mediator of the G1-S phase block induced by
1,25-dihydroxyvitamin D3. Cancer Res 1996;56:264–7.
[33] Marcinkowska E, Wie˛dłocha A, Radzikowski C. 1,25-Dihydroxy-
vitamin D3 induced activation and subsequent nuclear translocation
of MAPK is upstream regulated by PKC in HL-60 cells. Biochem
Biophys Res Commun 1997;241:419–26.
[17] Opolski A, Wietrzyk J, Siwin´ska A, Marcinkowska E, Chrobak A,
Kutner A, et al. Biological activity in vitro of side-chain modified
analogues of calcitriol. Curr Pharm Design 2000;6:755–66.
[18] Chodyn´ski M, Wojciechowska W, Halkes SJ, Velde van de JP,
Kutner A. Synthesis and in vitro evaluation of side chain-unsaturated
analogs of 24a,24b-dihomo-1,25-dihydroxycholecalciferol. Steroids
1997;62:546–53.
[19] Marcinkowska E, Kutner A, Radzikowski C. Cell differentiating
and antiproliferative activity of side-chain modified analogs of 1,25-
dihydroxyvitamin D3. J Steroid Biochem Molec Biol 1998;67:71–8.
[20] Opolski A, Wietrzyk J, Chrobak A, Marcinkowska E, Wojdat E,
Kutner A. Antiproliferative activity in vitro of side-chain modified
[34] Marcinkowska E, Wiedlocha A, Radzikowski C. Evidence that
phosphatidylinositol 3-kinase and p70S6K protein are involved in
differentiation of HL-60 cells induced by calcitriol. Anticancer Res
1998;18:3507–14.