Synthesis and biological activities of the two C(23) epimers of 1α,23,25-trihydroxy-24-oxo-19-nor-vitamin D3: Novel analogs of 1α,23(S),25-trihydroxy-24-oxo-vitamin D3, a natural metabolite of 1α,25- dihydroxyvitamin D3

Article abstract of DOI:10.1016/S0039-128X(99)00110-5

In a previous report, we indicated that 1α,23(S),25-trihydroxy-24- oxovitamin D₃ [1α,23(S),25(OH)₃-24-oxo-D₃], a natural metabolite of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] is almost equipotent to 1α,25(OH)₂D₃ in suppressing parathyroid hormone (PTH) secretion (Lee et al., 1997. Biochemistry 36, 9429-9437). Also, 1α,23(S),25(OH)₃-24-oxo-D₃ has been shown to possess only weak in vivo calcemic actions. Thus, vitamin D₃ analogs structurally related to 1α,23(S),25(OH)₃-24-oxo-D₃ may have therapeutic value. Furthermore, biologic activity studies of various synthetic analogs of 1α,25(OH)₂D₃ showed that the removal of carbon-19 (C- 19) reduces the calcemic activity of 1α,25(OH)₂D₃. Therefore, in an attempt to produce vitamin D₃ analogs with a better therapeutic index, we synthesized C(23) epimers of 1α,23,25(OH)₃-24-oxo-19-nor-vitamin D₃ [1α,23,25(OH)₃-24-oxo-19-nor-D₃]. The two epimers were compared to 1α,25(OH)₂-19-nor-D₃ and 1α,25(OH)₂D₃ in their ability to generate biologic activities in several in vitro assay systems. In the assay measuring the suppression of parathyroid hormone (PTH) secretion in bovine parathyroid cells, 1α,23(S),25(OH)₃-24-oxo-19-nor-D₃ was as potent as 1α,25(OH)₂-19- nor-D₃ but was less potent than 1α,25(OH)₂D₃. In the same assay 1α,23(R),25(OH)₃-24-oxo-19-nor-D₃ exhibited greater potency than 1α,23(S),25(OH)₃-24-oxo-19-nor-D₃. In the assays measuring the ability of vitamin D compounds to inhibit clonal growth and to induce differentiation of human promyelocytic leukemia (HL-60) cells, 1α,23(S),25(OH)₃-24-oxo-19-nor- D₃ was less potent than 1α,25(OH)₂-19-nor-D₃ but was equipotent to 1α,25(OH)₂D₃. More importantly, in the same assays, 1α,23(R),25(OH)₃-24- oxo-19-nor-D₃ was more potent than 1α,23(S),25(OH)₃-24-oxo-19-nor-D₃ and was equipotent to 1α,25(OH)₂-19-nor-D₃. Also, the vitamin D receptor- mediated transcriptional activity of 1α,23(R),25(OH)₃-24-oxo-19-nor-D₃ was almost equal to that of 1α,25(OH)₂-19-nor-D₃, but higher than that of 1α,23(S),25(OH)₃-24-oxo-19-nor-D₃. This finding explains in part the greater in vitro biologic activities of 1α,23(R),25(OH)₃-24-oxo-19-nor-D₃. In summary, our results indicate that 1α,23(R),25(OH)₃-24-oxo-19-nor-D₃ and to a lesser extent 1α,23(S),25(OH)₃-24-oxo-19-nor-D₃ are potent 19-nor vitamin D₃ analogs, which suppress PTH secretion in bovine parathyroid cells and strongly inhibit clonal growth and induce differentiation of HL-60 cells in vitro. (C) 2000 Elsevier Science Inc.

Full text of DOI:10.1016/S0039-128X(99)00110-5

Steroids 65 (2000) 252–265
Synthesis and biological activities of the two C(23) epimers of
1␣,23,25-trihydroxy-24-oxo-19-nor-vitamin D3: novel analogs of
1␣,23(S),25-trihydroxy-24-oxo-vitamin D3, a natural metabolite of
1␣,25-dihydroxyvitamin D3
Nancy E. Leea, Paul G. Williardb, Alex J. Brownc, Moray J. Campbelld, H. Phillip Koefflere,
Sara Pelegf, D. Sunita Raog, G. Satyanarayana Reddyg,*
aDepartment of Chemistry, Simmons College, Boston, MA 02115, USA
bDepartment of Chemistry, Brown University, Providence, RI 02912, USA
cRenal Division, Washington University School of Medicine, St. Louis, MI 63130, USA
dDepartment of Medicine, The University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK
eDivision of Hematology/Oncology, Cedars–Sinai Medical Center/UCLA School of Medicine, Los Angeles, CA 90048, USA
fDepartment of Medical Specialties, University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA
gDepartment of Pediatrics, Women and Infants’ Hospital of Rhode Island, Brown University School of Medicine, 101 Dudley Street, Providence,
RI 02905, USA
Received 16 July 1999; received in revised form 8 November 1999; accepted 23 November 1999
Abstract
In a previous report, we indicated that 1␣,23(S),25-trihydroxy-24-oxovitamin D3 [1␣,23(S),25(OH)3-24-oxo-D3], a natural metabolite
of 1␣,25-dihydroxyvitamin D3 [1␣,25(OH)2D3] is almost equipotent to 1␣,25(OH)2D3 in suppressing parathyroid hormone (PTH) secretion
(Lee et al., 1997. Biochemistry 36, 9429–9437). Also, 1␣,23(S),25(OH)3-24-oxo-D3 has been shown to possess only weak in vivo calcemic
actions. Thus, vitamin D3 analogs structurally related to 1␣,23(S),25(OH)3-24-oxo-D3 may have therapeutic value. Furthermore, biologic
activity studies of various synthetic analogs of 1␣,25(OH)2D3 showed that the removal of carbon-19 (C-19) reduces the calcemic activity
of 1␣,25(OH)2D3. Therefore, in an attempt to produce vitamin D3 analogs with a better therapeutic index, we synthesized C(23) epimers
of 1␣,23,25(OH)3-24-oxo-19-nor-vitamin D3 [1␣,23,25(OH)3-24-oxo-19-nor-D3]. The two epimers were compared to 1␣,25(OH)2-19-
nor-D3 and 1␣,25(OH)2D3 in their ability to generate biologic activities in several in vitro assay systems. In the assay measuring the
suppression of parathyroid hormone (PTH) secretion in bovine parathyroid cells, 1␣,23(S),25(OH)3-24-oxo-19-nor-D3 was as potent as
1␣,25(OH)2-19-nor-D3 but was less potent than 1␣,25(OH)2D3. In the same assay 1␣,23(R),25(OH)3-24-oxo-19-nor-D3 exhibited greater
potency than 1␣,23(S),25(OH)3-24-oxo-19-nor-D3. In the assays measuring the ability of vitamin D compounds to inhibit clonal growth and
to induce differentiation of human promyelocytic leukemia (HL-60) cells, 1␣,23(S),25(OH)3-24-oxo-19-nor-D3 was less potent than
1␣,25(OH)2-19-nor-D3 but was equipotent to 1␣,25(OH)2D3. More importantly, in the same assays, 1␣,23(R),25(OH)3-24-oxo-19-nor-D3
was more potent than 1␣,23(S),25(OH)3-24-oxo-19-nor-D3 and was equipotent to 1␣,25(OH)2-19-nor-D3. Also, the vitamin D receptor-
mediated transcriptional activity of 1␣,23(R),25(OH)3-24-oxo-19-nor-D3 was almost equal to that of 1␣,25(OH)2-19-nor-D3, but higher than
that of 1␣,23(S),25(OH)3-24-oxo-19-nor-D3. This finding explains in part the greater in vitro biologic activities of 1␣,23(R),25(OH)3-24-
oxo-19-nor-D3. In summary, our results indicate that 1␣,23(R),25(OH)3-24-oxo-19-nor-D3 and to a lesser extent 1␣,23(S),25(OH)3-24-
oxo-19-nor-D3 are potent 19-nor vitamin D3 analogs, which suppress PTH secretion in bovine parathyroid cells and strongly inhibit clonal
growth and induce differentiation of HL-60 cells in vitro. © 2000 Elsevier Science Inc. All rights reserved.
Keywords: 1␣,25(OH)2D3; 1␣,25(OH)2-19-nor-D3; 1␣,23(R),25(OH)3-24-oxo-19-nor-D3; 1␣,23(S),25(OH)3-24-oxo-19-nor-D3
* Corresponding author. Tel.: ϩ1-401-274-1122; fax: ϩ1-401-453-7571.
E-mail address: sreddy@wihri.org (G.S. Reddy)
0039-128X/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(00)00110-5

N.E. Lee et al. / Steroids 65 (2000) 252–265
253
1. Introduction
In a recent report, we indicated that 1␣,23(S),25-trihy-
droxy-24-oxovitamin D3 [1␣,23(S),25(OH)3-24-oxo-D3], a
natural metabolite of 1␣,25(OH)2D3 is almost equipotent to
its parent in suppressing PTH secretion [17]. This finding
has therapeutic significance as 1␣,23(S),25(OH)3-24-
oxo-D3 has been shown previously that it only generates
minimal in vivo calcemic activity when compared to
1␣,25(OH)2D3 [18]. Therefore, in an attempt to reduce even
further the minimal calcemic effects of 1␣,23(S),25(OH)3-
24-oxo-D3, we performed the present study 1) to synthesize
the C(23) epimers of 1␣,23,25(OH)3-24-oxo-19-nor-D3;
and 2) to compare these epimers to 1␣,25(OH)2D3 and the
analog 1␣,25(OH)2-19-nor-D3 in their ability to generate
the following biologic activities: a) to suppress PTH secre-
tion in primary cultures of bovine parathyroid cells, b) to
inhibit clonal growth of human promyelocytic leukemia
cells (HL-60 cells), c) to induce differentiation of HL-60
cells [as assessed by the expression of CD11b surface pro-
tein (a differentiation marker)], and d) to induce growth
hormone production in rat osteosarcoma cells (ROS 17/2.8)
transfected with an osteocalcin VDRE/growth hormone
gene contruct. The structures of 1␣,25(OH)2D3,
1␣,23(S),25(OH)3-24-oxo-D3, 1␣,25(OH)2-19-nor-D3 and
C(23) epimers of 1␣,23,25(OH)3-24-oxo-19-nor-D3 are
shown in Fig. 1.
The role of the seco-steroid hormone, 1␣,25-dihy-
droxyvitamin D3 [1␣,25(OH)2D3] in the normal develop-
ment of the skeleton and maintenance of calcium homeosta-
sis is well-established [1]. In addition to its role in mineral
homeostasis, 1␣,25(OH)2D3 has been shown to induce dif-
ferentiation of myeloid leukemia cells [2–7]. Other biologic
activities include its role in immune suppression, treatment
of the hyperproliferative skin disorders, and suppression of
parathyroid hormone (PTH) secretion [1,8]. However, doses
of 1␣,25(OH)2D3 required to obtain therapeutically useful
effects produced severe hypercalcemia [9]. This complica-
tion of hypercalcemia has limited the use of 1␣,25(OH)2D3
as a therapeutic agent. Therefore, efforts have been directed
toward synthesizing analogs of 1␣,25(OH)2D3 that possess
selectively noncalcemic actions such as differentiation of
leukemic cells without producing hypercalcemia [6,10,11].
To date, hundreds of synthetic analogs of 1␣,25(OH)2D3
have been synthesized and tested for their non-calcemic
actions [12]. Biologic activity studies of various synthetic
analogs of 1␣,25(OH)2D3 have shown that removal of car-
bon-19 (19-nor) reduces the potency of the calcemic actions
of 1␣,25(OH)2D3 whereas the potency of the non-calcemic
actions of the hormone are retained or enhanced [12–16].
Fig. 1. Structures of 1␣,25(OH)2D3, 1␣,25(OH)2-19-nor-D3, 1␣,23,25(OH)3-24-oxo-D3, and the two C(23) epimers of 1␣,23,25(OH)3-24-oxo-19-nor-D3.

254
N.E. Lee et al. / Steroids 65 (2000) 252–265
2. Materials and methods
desired iodine 6 as a colorless oil (1.1g. 97%). Rf ϭ 0.65
(25% ethyl acetate/hexanes).
1H NMR (400 MHz, CDCl3): ␦ 4.10 (br s, 1H), 3.33 (d,
J ϭ 9.5 Hz, 1H), 3.19 (m, 1H), 1.95–0.8 (m, 20H). MS m/e
(rel int): 321 (Mϩ, 30), 177 (76), 135 (28), 111 (100), 95
(42), 81 (40), 67 (25), 55 (27).
2.1. General
Melting points were determined by using a Thomas–
Hoover apparatus and are uncorrected. Infrared (IR) spectra
were recorded with a Perkin-Elmer 1600 Series FTIR spec-
trophotometer. Optical rotations were measured with a Per-
kin–Elmer 241 polarimeter in a 1-dm cell. 1H NMR spectra
were recorded on either Bruker WM-250 MHz or Bruker
AM-400 MHz spectrometers using TMS (0.0 ppm) as an
internal standard. 13C NMR spectra were recorded on a
Bruker AM-400 MHz spectrometer at 100 MHz by using
CDCl3 (77 ppm) as an internal standard. Mass spectra were
obtained on routine intermediates with a Kratos MS 80RFA
mass spectrometer under EI or CI conditions and for the
final products on a Hewlett-Packard 5985B mass spectrom-
eter. Thin layer chromatography (TLC) was performed on
EM Science precoated silica gel 60F-254 glass-supported
plates with 0.25-mm thickness. Spots were visualized by
either ultraviolet light, exposure to iodine, or spraying with
a 5% solution of phosphomolybdic acid (PMA) in ethanol.
Flash chromatography was performed with ICN (70–220
mesh) of Kieselgel (230–400 mesh). Preparative thin layer
chromatography was performed on Analtech Silica Gel GF
plates (20 cm ϫ 20 cm, 1000 ␮m thickness). Diethyl ether
and THF were distilled from sodium/benzophenone ketyl
under nitrogen. All the solvents and reagents used in the
experiments were purified and dried according to the meth-
ods described by Perrin et al. [19]. All reactions were run
under an inert atmosphere of argon. High-performance liq-
uid chromatography (HPLC) was performed with a Waters
Model 600E chromatograph equipped with a Waters pho-
todiode array detector (Model 990) to monitor UV-absorb-
ing material at 265 nm. Crystalline 1␣,25(OH)2D3 was a gift
from Dr Milan R. Uskokovic of Hoffmann–La Roche, Nut-
ley, NJ, USA and 1␣,25(OH)2-19-nor-D3 was a gift from Dr
Hector F. DeLuca of University of Wisconsin, Madison,
WI, USA.
2.3. De-A,B-22-benzenesulphonyl-23,24-dinorcholestan-8-
ol (7a)
The iodide 6 (1.1 g, 3.4 mmol) was dissolved in anhy-
drous DMF (10 ml) under an argon atmosphere and sodium
benzenesulfinic acid (1.12 g, 6.83 mmol) was added in one
portion. The suspension eventually became a solution and
was stirred overnight. The reaction was quenched with a
saturated NH4Cl solution and extracted with ether (2 ϫ 50
ml). The ether layer was washed with water (2 ϫ 25 ml) and
brine. Drying (MgSO4) and removal of the ether gave a
thick oil that was chromatographed with 20% ethyl acetate/
hexanes to give the desired sulfone 7a as a colorless oil
(1.07 g, 94%). Rf ϭ 0.14 (25% ethyl acetate/hexanes).
1H NMR (400 MHz, CDCl3): ␦ 7.88 (d, J ϭ 6.2 Hz, 2H),
7.63 (m, 1H), 7.54 (m, 2H), 4.02 (br s, 1H), 3.12 (d, J ϭ
14.2 Hz, 1H), 2.82 (dd, J ϭ 14.2, 9.6 Hz, 1H), 2.05 (m, 1H),
1.92 (d, J ϭ 13.3 Hz, 1H), 1.17 (d, J ϭ 8.8 Hz, 3H), 0.87
(s, 3H). IR (neat, cm): 3530.3, 1296.2, 1143.4. 13C NMR
(100 MHz, CDCl3): ␦ 140.25, 133.43, 129.16, 127.76,
68.89, 61.84, 55.74, 52.43, 42.02, 40.08, 33.43, 31.86,
26.93, 22.25, 19.87, 17.22, 13.21.
2.4. De-A,B-22-benzenesulphonyl-23,24-dinorcholestan-8-
benzyloxymethyl ether (7b)
To a solution of the alcohol 7a (230 mg, 0.68 mmol) in
CH2Cl2 (5 ml), Hunig’s base (0.24 ml, 1.4 mmol) and
freshly distilled benzyl chloromethyl ether (0.14 ml, 0.9
mmol) was added at room temperature. The reaction was
stirred overnight during which time it changed from a col-
orless solution to an orange solution. When all of the alco-
hol was reacted, as monitored by TLC, the reaction was
quenched with water and extracted with ether (2 ϫ 20 ml).
The ether layer was washed with 0.1 N HCl, brine, and dried
(MgSO4). Filtration and removal of ether gave a thick oil
that was chromatographed with 10% ethyl acetate/hexanes
to give the protected alcohol 7b as a white solid (312 mg,
100%). Rf ϭ 0.55 (25% ethyl acetate/hexanes).
2.2. Chemical synthesis of C(23) epimeric mixture of
1␣,23,25(OH)3-24-oxo-19-nor-D3 De-A,B-22-iodo-23,24-
dinorcholestan-8-ol (6)
To a solution of Lythgoe’s diol 5 (750 mg, 3.54 mmol)
in benzene (50 ml), triphenylphosphine (930 mg, 3.54
mmol), imidazole (725 mg, 10.6 mmol), and iodine (897
mg, 3.54 mmol) were added at room temperature. The
reaction became two phased with a white gel-like substance
at the bottom of the flask. The mixture was stirred vigor-
ously for 3 h. The reaction was quenched with water and
extracted with benzene. The benzene layer was washed with
0.5N HCl (2 ϫ 50 ml), 5% NaSO3 and brine. Drying
(MgSO4) and concentration gave a thick oil that was chro-
matographed with 25% ethyl acetate/hexanes to afford the
1H NMR (400 MHz, CDCl3): ␦ 7.91 (m, 2H), 7.60 (m,
3H), 7.31 (m, 5H), 4.76 (d, J ϭ 6.9 Hz, 1H), 4.66 (d, J ϭ
6.9 Hz, 1H), 4.58 (s, 2H), 3.93 (br s, 1H), 3.17 (d, J ϭ 14.2
Hz, 1H), 2.84 (dd, J ϭ 14.2, 9.6 Hz, 1H), 1.19 (d, J ϭ 6.6
Hz, 3H), 0.88 (s, 3H). IR (neat, cm): 1305.1, 1149.1. 13C
NMR (100 MHz, CDCl3): 140.38, 138.03, 133.45, 129.20,
128.36, 127.84, 127.81, 127.57, 93.64, 74.59, 69.25, 61.94,
55.89, 52.32, 42.31, 40.29, 32.05, 30.29, 27.08, 22.56,
19.99, 17.78, 13.16. HRMS (CI, isobutane): Calculated for

N.E. Lee et al. / Steroids 65 (2000) 252–265
255
C19H27O3S (M - CH2OCH2Ph) 335.1681, Found 335.1688.
2.7. De-A,B-24-oxo-25-hydroxy-cholestan-8-
benzyloxymethyl ether (9)
[␣]20D: ϩ53.5 (c ϭ 2.8, CHCl3).
Oxalyl chloride (0.21 ml, 2.4 mmol) was dissolved in
CH2Cl2 (10 ml) and cooled to Ϫ78°C. DMSO (0.31 ml, 4.8
mmol) in CH2Cl2 (0.5 ml) was added dropwise and the
reaction mixture was stirred for 1 h. The alcohol 8b (500
mg, 1.2 mmol) in CH2Cl2 (10 ml) was added dropwise using
a dropping funnel over an hour. A white precipitate was
observed as the addition took place. After stirring at Ϫ78°C
for 2 h and at Ϫ40°C for 15 min, it was recooled to Ϫ78°C
and triethylamine (1.2 ml, 8.4 mmol) was added dropwise.
The reaction was allowed to warm up to Ϫ10°C on its own
and stirred at this temperature until reaction was complete
(about 5 h). Water was added to the reaction and was
extracted with CH2Cl2 (3 ϫ 30 ml). Organic layer was
washed with 0.1 N HCl, brine, and dried (MgSO4). Filtra-
tion and removal of CH2Cl2 gave a yellow oil that was
chromatographed with 15% ethyl acetate/hexanes to give
two products. The minor product (91 mg, 16%) corre-
sponded to 24-oxo-25-thiomethoxy methyl ether. Rf ϭ 0.8
(25% ethyl acetate/hexanes). The major product was the
expected 24-oxo-25-hydroxy compound 9 (355 mg, 71%).
Rf ϭ 0.6 (25% ethyl acetate/hexanes).
2.5. De-A,B-22-benzenesulphonyl-24,25-diol-cholestan-8-
benzyloxymethyl ether (8a)
To a solution of the sulfone 7b (500 mg, 1.1 mmol) in
THF (5 ml), diisopropylamine (0.62 ml, 4.4 mmol) and
tetramethylethylene diamine (0.5 ml) were added. After
cooling the reaction to Ϫ20°C, n-BuLi (2.5 M in hexanes,
1.7 ml, 4.4 mmol) was added slowly in a dropwise manner
over 10 min. The reaction turned yellow as the anion formed
and was allowed to stir for 15 min. 2-Methyl-3,4-epoxy-2-
butan-2-ol (220 mg, 2.2 mmol) in THF (2 ml) was added
dropwise over 15 min. The reaction was stirred for 5 h and
was quenched with a saturated NH4Cl solution (10-ml) and
extracted with ethyl acetate (3 ϫ 20 ml). The organic layer
was washed with brine (15 ml) and dried (MgSO4). Filtra-
tion and removal of the ethyl acetate gave an oil that was
chromatographed with 35% ethyl acetate/hexanes to give
the recovered starting material (50 mg) and the desired
product 8a (500 mg, 81%). Rf ϭ 0.05 (25% ethyl acetate/
hexanes).
1H NMR (250 MHz, CDCl3): ␦ 7.90 (m, 2H), 7.62 (m,
3H), 7.34 (m, 5H), 4.69 (m, 3H), 4.56 (s, 2H), 3.89 (m, 1H),
3.52 (d, J ϭ 9.2 Hz, 1H), 2.47 (d, J ϭ 4.8 Hz, 1H), 0.66 (s,
3H). IR (neat, cm): 3476.2, 1284.7, 1141.4. HRMS (FAB):
Calculated for C32H46O6S (M ϩ H) 559.3093, Found
559.3091.
1H NMR (400 MHz, CDCl3): ␦ 7.35 (m, 4H), 7.29 (m,
1H), 4.79 (d, J ϭ 6.9 Hz, 1H), 4.68 (d, J ϭ 6.9 Hz, 1H),
4.61 (m, 2H), 3.96 (m, 1H), 3.83 (s, OH), 2.51 (m, 2H). IR
(neat, cm): 3481.9, 1705.7. 13C NMR (100 MHz, CDCl3): ␦
215.01, 138.14, 128.39, 127.88, 127.58, 93.69, 76.20,
74.85, 69.23, 56.45, 52.40, 42.14, 40.59, 35.04, 32.37,
30.49, 29.79, 27.23, 26.56, 22.78, 18.41, 17.96, 13.41.
HRMS (EI): Calculated for C18H31O3 (M - CH2OCH2Ph)
295.2273, Found 295.2286. [␣]20D: ϩ50.2 (c ϭ 1.4,
CHCl3).
2.6. De-A,B-24,25-dihydroxy-cholestan-8-benzyloxymethyl
ether (8b)
To a solution of the sulfone 8a (780 mg, 1.4 mmol) in
methanol (20 ml) at 0°C, sodium phosphate dibasic
(Na2HPO4) (5.95g, 42 mmol) and 5% sodium amalgam (9.4
g, 2.1 mmol) were added. The mixture was stirred vigor-
ously for 1 h at 0°C. When the reaction was complete, as
indicated by TLC, it was diluted with methanol and filtered
through a pad of celite. Methanol was removed and the
residue was diluted with brine (10 ml) and extracted with
ethyl acetate (3 ϫ 30 ml). Drying (MgSO4), filtration and
removal of ethyl acetate gave an oil that was chromato-
graphed with 30% ethyl acetate/hexanes to give the pure
diol 8b (500 mg, 86%). Rf ϭ 0.2 (25% ethyl acetate/
hexanes).
2.8. De-A,B-24-oxo-23,25-dihydroxy cholestan-8-
benzyloxymethyl ether (10)
A solution of LDA was prepared by adding n-BuLi
(2.5M in hexanes, 0.28 ml, 0.72 mmol) to a solution of
diisopropylamine (0.135 ml, 0.96 mmol) in THF (2 ml) at
Ϫ20°C. After stirring for 40 min, a solution of the ketone 9
(50 mg, 0.12 mmol) in THF (2 ml) was added dropwise over
20 min. Trimethylsilyl chloride (0.12 ml, 0.96 mmol) was
added after 5 min. The reaction was allowed to warm up to
0°C on its own and was stirred at this temperature for 3.5 h.
At this point TLC showed disappearance of all the starting
material and a new spot was observed with a very high Rf
corresponding to the silyl enol ether. A saturated NaHCO3
solution was added to the reaction and was extracted with
pentane (3 ϫ 20 ml). The organic layer was washed with
brine, dried (MgSO4), filtered, and concentrated in vacuo.
The resulting oil was placed under vacuum overnight and
was used in the next step without further purification. Rf ϭ
0.75 (5% ethyl acetate/hexanes). The crude silyl enol ether
was dissolved in CH2Cl2 (5 ml) and cooled to 0°C. m-
1H NMR (250 MHz, CDCl3): ␦ 7.30 (m, 5H), 4.79 (d,
J ϭ 6.9 Hz, 1H), 4.68 (d, J ϭ 6.9 Hz, 1H), 4.61 (s, 2H), 3.96
(m, 1H), 3.53 (m, 1H). IR (neat, cm) 3425.2. 13C NMR (100
MHz, CDCl3): Major isomer: ␦ 138.17, 128.38, 127.89,
127.57, 93.71, 78.83, 74.96, 73.16, 69.22, 56.69, 52.42,
42.13, 40.63, 35.27, 32.71, 30.53, 28.11, 27.27, 26.59,
23.28, 22.80, 18.49, 17.98, 13.42. HRMS (FAB): Calcu-
lated for C18H33O2 (M - OBOM) 281.2480, Found
281.2481.

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N.E. Lee et al. / Steroids 65 (2000) 252–265
Chloroperbenzoic acid (50%, 50 mg, 0.15 mmol) in CH2Cl2
(5 ml) was added very slowly using a dropping funnel over
30 min. The reaction was stirred at 0°C for 4 h and then
quenched with a saturated NaHCO3 solution and extracted
with CH2Cl2 (2 ϫ 20 ml). The organic layer was washed
with brine and concentrated in vacuo. The resulting residue
was dissolved in acetonitrile (3 ml) and treated with 0.5 N
HCl (7–10 drops). After stirring for 30 min, the reaction
mixture was neutralized and acetonitrile was removed. The
remaining residue was dissolved in ethyl acetate (50 ml) and
was washed with brine (3 ml). Drying (MgSO4), filtration,
and concentration gave the crude oil that was chromato-
graphed using 20% ethyl acetate/hexanes to give the desired
dihydroxyketone 10 (40 mg, 78% for 2 steps). Rf ϭ 0.35
(25% ethyl acetate/hexanes).
0.16 mmol) in absolute ethanol (2 ml), 10% palladium on
carbon (20 mg) was added. A balloon was filled with hy-
drogen gas and was connected to the reaction flask via an
adapter. The reaction mixture was flushed with hydrogen
gas several times. The balloon was filled again with hydro-
gen gas and the reaction mixture was stirred overnight under
an atmospheric pressure of hydrogen. When the deprotec-
tion was complete, as monitored by TLC, the reaction mix-
ture was filtered through a pad of celite. Ethanol was re-
moved, and the residue was chromatographed using 20%
ethyl acetate/hexanes to give the alcohol 11b (53 mg, 95%),
Rf ϭ 0.52 (25% ethyl acetate/hexanes).
1H NMR (400 MHz, CDCl3): ␦ 4.28 (dd, J ϭ 10.5, 2.6
Hz, H23-major), 4.25 (dd, J ϭ 7.3, 4.8 Hz, H23-minor), 4.05
(m, 1Hg), 1.49 (s, 3H-major), 1.48 (s, 3H-minor), 1.45 (s,
3H-major ϩ minor), 1.42 (s, 3H-minor), 1.40 (s, 3H-major),
1.26 (s, 3H-major ϩ minor), 0.97 (d, J ϭ 6.6 Hz, 3H-
minor), 0.94 (s, 3H-major), 0.92 (s, 3H-minor), 0.90 (d, J ϭ
6.4 Hz, 3H major). IR (neat, cm): 3520, 1737.8. 13C NMR
(100 MHz, CDCl3): major isomer: ␦ 213.86, 100.16, 79.39,
71.95, 69.28, 57.01, 52.59, 42.03, 40.46, 36.22, 33.57,
31.32, 29.38, 27.05, 26.23, 25.69, 24.48, 17.81, 17.42,
13.59. HRMS (EI): Calculated for C20H33O4 (M - CH3)
337.2379, Found: 337.2374.
1H NMR (400 MHz, CDCl3): ␦ 7.34 (m, 5H), 4.79 (d,
J ϭ 6.9 Hz, 1H), 4.67 (m, 2H), 4.60 (s, 2H), 3.96 (m, 1H),
2.92 (d, J ϭ 8.0 Hz, OH-minor isomer), 2.89 (d, J ϭ 7.9 Hz,
OH-major isomer), 2.63 (s, OH-minor isomer), 2.57 (s,
OH-major isomer). IR (neat, cm): 3439.6, 1709.7. 13C NMR
(100 MHz, CDCl3): major isomer: ␦ 217.08, 138.15,
128.39, 128.01, 128.68, 93.68, 77.20, 74.67, 71.03, 69.23,
57.09, 52.50, 42.40, 40.89, 40.64, 34.87, 32.52, 30.51,
27.72, 27.62, 27.42, 22.78, 17.96, 13.48. HRMS (FAB):
Calculated for C18H31O3 (M - OBOM) 295.2273, Found
295.2261.
2.11. De-A,B-24-oxo-23,25-(isopropylidenedioxy)-
cholestan-8-one (12)
2.9. De-A,B-24-oxo-23,25-(isopropylidenedioxy)-
cholestan-8-benzyloxymethyl ether (11a)
Oxalyl chloride (0.025 ml, 0.284 mmol) was dissolved in
CH2Cl2 (1 ml) and cooled to Ϫ78°C. DMSO (0.037 ml,
0.57 mmol) in CH2Cl2 (0.4 ml) was added dropwise and the
reaction mixture was stirred for 45 min. The alcohol 11b (50
mg, 0.142 mmol) in CH2Cl2 (1 ml) was added slowly in a
dropwise manner over 15 min. The reaction mixture was
allowed to warm up to Ϫ40°C on its own over 2.5 h and was
kept at this temperature for 15 min. After the mixture was
recooled to Ϫ78°C, triethylamine (0.16 ml, 1.14 mmol) was
added dropwise. The reaction mixture was allowed to warm
to Ϫ10°C on its own and was stirred at this temperature for
30 min. Water was added to the reaction mixture and was
extracted with CH2Cl2 (2 ϫ 20 ml). The organic layer was
washed with brine, dried (MgSO4), filtered and CH2Cl2 was
removed to give an oil that was chromatographed with 25%
ethyl acetate/hexanes to give the ketone 12 (47 mg, 96%).
Rf ϭ 0.54 (25% ethyl acetate/hexanes).
To a solution of the diol 10 (190 mg, 0.43 mmol) in
anhydrous DMF (4 ml), 2,2-dimethoxypropane (2 ml) was
added, followed by pyridinium p-toluenesulfonate (30 mg,
0.1 mmol). The reaction mixture was stirred for 2 days and
worked up by diluting with water (30 ml) and extracting
with ether (3 ϫ 20 ml). The organic layer was dried
(MgSO4), filtered and concentrated to give an oil that was
chromatographed using 10% ethyl acetate/hexanes to give
the ketal 11a (160 mg, 78%). Rf ϭ 0.86 (25% ethyl acetate/
hexanes).
1H NMR (400 MHz, CDCl3): ␦ 7.33 (m, 5H), 4.79 (d,
J ϭ 6.9 Hz, 1H), 4.68 (d, J ϭ 6.9 Hz, 1H), 4.61 (s, 2H), 4.31
(m, 1H23), 3.97 (m, 1 Hg), 1.52 (s, 3H), 1.48 (s, 3H), 1.44
(s, 3H), 1.29 (s, 3H). IR (neat, cm): 1737.0. 13C NMR (100
MHz, CDCl3): major isomer: ␦ 213.89, 138.13, 128.36,
127.87, 127.55, 100.15, 93.65, 79.39, 74.89, 72.02, 69.17,
57.04, 52.39, 42.26, 40.64, 36.27, 34.36, 31.49, 30.49,
29.39, 27.16, 26.24, 25.69, 25.07, 22.76, 17.95, 13.45.
HRMS (EI): Calculated for C28H41O5 (M - CH3): 457.2954,
Found 457.2942.
1H NMR (400 MHz, CDCl3): ␦ 4.29 (m, 1H-C23 major ϩ
minor), 2.45 (m, 1H), 2.26 (m, 2H), 1.04 (d, J ϭ 6.6 Hz,
3H-C21-minor), 0.97 (d, J ϭ 6.1 Hz, 3H-C21-major), 0.66 (s,
3H-C18-major), 0.64 (s, 3H-C18-minor). IR (neat, cm):
1737.4, 1714.5. 13C NMR (100 MHz, CDCl3): major iso-
mer: ␦ 213.64, 211.82, 100.19, 79.40, 71.83, 61.92, 56.99,
49.95, 40.91, 38.99, 36.19, 31.53, 29.33, 27.35, 26.21,
25.67, 25.06, 23.98, 19.05, 17.96, 12.58. HRMS (CI, isobu-
tane): Calculated for C21H35O4 (M ϩ H) 351.2535, Found
351.2522.
2.10. De-A,B-24-oxo-23,25-(isopropylidenedioxy)-
cholestan-8-ol (11b)
To a solution of de-A,B-24-oxo-23,25-(isopropyli-
denedioxy)-cholestan-8-(benzyloxy) methyl ether (75 mg,

N.E. Lee et al. / Steroids 65 (2000) 252–265
257
2.12. 1-[(tert-butyldimethylsilyl)oxy]-23,25-
(isopropylidenedioxy)-24-oxo-19-nor-vitamin D3 tert-
butyldimethylsilyl ether (14)
67.42, 67.22, 56.90, 56.36, 45.98, 44.65, 42.19, 40.96,
40.48, 37.23, 33.13, 28.89, 27.77, 27.75, 27.64, 23.43,
22.28, 18.14, 12.15. minor isomer: ␦ 216.07, 142.85,
131.22, 123.82, 115.38, 73.29, 71.04, 67.42, 67.22, 56.94,
56.19, 45.89, 44.65, 42.19, 40.80, 40.37, 35.42, 33.13,
29.68, 27.89, 27.75, 27.64, 23.43, 22.32, 20.06, 11.96.
To a solution of the phosphine oxide 13 (24 mg, 0.042
mmol) in dry THF (0.5 ml) at Ϫ78°C, n-BuLi (1.1 M in
hexanes, 0.05 ml, 0.055 mmol) was added dropwise. The
reaction became dark orange to red in color from a colorless
solution. After 5 min, the ketone 12 (10 mg, 0.028 mmol) in
THF (0.3 ml) was slowly added dropwise. The red color
disappeared gradually and became yellow by the end of
addition. The reaction was stirred at Ϫ78°C for 1.5 h and
then warmed to room temperature over 4 h. The solvent was
removed and the residue was partitioned between ether and
saturated NaHCO3 solution. The organic layer was washed
with brine, dried (MgSO4) and concentrated to give an oil
that was chromatographed on silica gel with 5% ethyl ace-
tate/hexanes to give the desired coupled triene 14 (4.8 mg,
80%) and unreacted ketone 12 (7 mg). Rf ϭ 0.9 (25% ethyl
acetate/hexanes).
2.14. Analysis of 19-nor vitamin D3 analog 4 by HPLC
A total of 2.4 mg of the 19-nor vitamin D3 analog 4 was
subjected to HPLC under the chromatographic conditions
described in the legend to Fig. 4. We observed two UV
absorbing peaks, a major (peak 1) and a minor (peak 2) peak
(Fig. 4). The elution volume of each UV absorbing peak
was as follows: peak 1, 334–348 ml and peak 2, 350–370
ml. The HPLC effluent corresponding to each UV absorbing
peak was collected separately and rechromatographed on
the same HPLC run described above to obtain each indi-
vidual isomer in the pure form.
1H NMR (400 MHz, CDCl3): major isomer, ␦ 6.17 and
5.80 (d, J ϭ 11.4 Hz, 2H-C6,7), 4.32 (m, 1H- C23), 4.08 (m,
2H-C1,3), 2.82 (d, J ϭ 11.5 Hz, 1H-C9), 0.96 (d, J ϭ 6.2 Hz,
3H-C21), 0.87 (s, 9H, t-Bu), 0.86 (s, 9H, t-Bu), 0.57 (s,
3H-C18), 0.07 (s, 3H, SiMe2), 0.06 (s, 3H, SiMe)2, 0.005 (s,
6H, SiMe2). IR (neat, cm): 1738.7. 13C NMR (100 MHz,
CDCl3): major isomer, ␦ 213.88, 140.60, 133.77, 121.71,
116.25, 100.18, 79.42, 72.01, 68.09, 67.96, 56.93, 56.24,
45.98, 45.74, 43.70, 40.66, 36.80, 36.37, 32.14, 29.41,
28.66, 27.56, 26.25, 25.85, 25.83, 25.70, 25.07, 23.38,
22.21, 18.13, 18.09, 18.05, 12.13, Ϫ4.67, Ϫ4.76, Ϫ4.80,
Ϫ4.93.
2.15. PTH secretion in primary cultures of bovine
parathyroid cells
2.15.1. Cell culture
Bovine parathyroid glands, obtained from a local
slaughterhouse and transported to the laboratory in cold
PBS, were digested with collagenase as previously de-
scribed [20] and seeded at a density of 80 000 cells/cm2
in DMEM/Ham’s F-12 (1:1) containing 4% heat-inacti-
vated newborn calf serum, 15 mM HEPES, 100 IU/ml
penicillin, 100 ␮g/ml streptomycin 5 ␮g/ml insulin, 2
mM glutamine, and 1% nonessential amino acids. After
24h, cells were placed in medium containing 0.1% bovine
serum albumin (BSA) and 5 ␮g/ml transferrin in place of
the serum. Cells were grown to confluency (6 days) in
this serum-free medium.
2.13. 1␣,23(R and S),25-trihydroxy-24-oxo-19-nor-vitamin
D3 (4)
To a solution of the protected 19-nor vitamin D3 14 (4.8
mg) in methanol (1 ml) and CH2Cl2 (0.1 ml), Dowex
50WX-400 resin (90 mg) was added. The mixture was
stirred vigorously in the dark for 14 h. The reaction mixture
was then filtered and concentrated in vacuo to give a residue
that was chromatographed with 100% ethyl acetate to give
the pure 19-nor vitamin D3 analog 4 (2.5 mg, 86%). Rf ϭ
0.16 (75% ethyl acetate/hexanes).
2.15.2. Analysis of PTH secretion
Parathyroid cell cultures were prepared as described
above and grown for 4 days in serum-free medium. The
cells were then treated for 3 days with the vitamin D3
compounds shown in Fig. 1 at concentrations ranging from
10Ϫ11 to 10Ϫ7 M with daily changes of the medium. Steady
state PTH secretion was determined by washing the cells
three times with Dulbeccco’s PBS and then placing them in
serum-free medium for 3 h. The medium was collected,
centrifuged at 4°C, and analyzed for PTH using CH9 anti-
body as described previously [20]. The cell monolayers
were dissolved in 0.1 N NaOH and assayed for protein by
the method of Bradford [21] using a kit from Bio-Rad
Laboratories (Richmond, CA, USA). PTH secretion is ex-
pressed as picograms of PTH per milligram of cell protein.
All statistical analysis was undertaken using the analysis of
variance (ANOVA) and student’s t-test.
1H NMR (400 MHz, CDCl3): ␦ 6.31 and 5.85 (d, J ϭ
11.4 Hz, 2H-C6 & 7), 4.71 (m, 1H-C23-major), 4.64 (m,
1H-C23-minor), 4.12 and 4.04 (m, 2H-C1 & 3), 2.97 (d, J ϭ
7.8 Hz, OH-C23 minor), 2.92 (d, J ϭ 7.8 Hz, OH-C23-
major), 2.81 (dd, J ϭ 12.3, 3.3 Hz, 1H-C9), 2.74 (dd, J ϭ
13.2, 3.7 Hz, 1H), 2.63 (s, OH25-minor), 2.56 (s, OH25-
major), 2.48 (dd, J ϭ 13.4, 3.3 Hz, 1H), 2.23 (m, 2H), 1.44
and 1.42 (s, 6H, C26 & 27), 1.09 (d, J ϭ 6.6 Hz, 3H-C21-
minor), 1.06 (d, J ϭ 6.5 Hz, 3H-C21-major), 0.59 (s, 3H-
C18-major), 0.56 (s, 3H-C18-minor). IR (neat, cm): 3398.1,
1718.7. 13C NMR (100 MHz, CDCl3): major isomer: ␦
217.03, 142.76, 131.28, 123.78, 115.45, 77.21, 71.04,

258
N.E. Lee et al. / Steroids 65 (2000) 252–265
Fig. 2. Scheme for the synthesis of De-A,B-24-oxo-23,25-(isopropylidenedioxy)-cholestan-8-one (11).
2.16. Colony formation of HL-60 cells in soft agar
agar system as described earlier [22]. The lower layer con-
tained 0.5% agar in which various concentrations [10Ϫ11
–
The ability of all the vitamin D3 compounds shown in
Fig. 1 to inhibit cell proliferation of HL-60 myeloid leuke-
mia cells was determined by soft agar colony assay. In this
experiment, the HL-60 cells were seeded in a two-layer soft
10Ϫ7 M] of the vitamin D compounds were mixed and the
upper layer contained 0.3% agar in which the HL-60 cells
(5 ϫ 103 cells/plate) were mixed. The assay was performed
in 24 well plates, the total volume in each well being 400 ␮l.

N.E. Lee et al. / Steroids 65 (2000) 252–265
259
Fig. 3. Scheme for the synthesis of 1␣,23,25(OH)3-24-oxo-19-nor-D3 (4) by coupling A-ring phosphine oxide 12 with CD ring ketone 11.
The HL-60 cells were incubated with the test compounds
for 10 days at 37°C in a humidified atmosphere containing
5% CO2 in air. The colonies containing 40 or more cells
were then counted under an inverted microscope. The ex-
periment was repeated thrice using duplicate dishes for all
experimental points. All statistical analysis was undertaken
using the student’s t-test.
(DMEM) and 10% FCS. Forty-eight hours later, the cells
were transfected with plasmid (2 ␮g/dish) containing the
vitamin D response element (VDRE) from the human os-
teocalcin gene (GGTGACTCACCGGGTGAACGGGGG-
CATT) [23]. This response element was inserted upstream
of the thymidine kinase promotor/growth hormone fusion
gene. All transfections were performed by the DEAE dex-
tran method [24], and the cells were treated for 1 min with
10% dimethyl sulfoxide. Ligands (at the indicated concen-
trations) in serum-free medium were added for 1 h imme-
diately after transfection. The control samples were treated
with vehicle only. The cells were then washed twice in
phosphate-buffered saline (PBS) and added to DMEM with
10% fetal calf serum (FCS). Medium samples for measure-
ments of growth hormone were collected 24 h after ligand
treatment. Growth hormone production from the reporter
gene was measured by a radioimmunoassay (RIA) as de-
scribed by the manufacturer (Nichols Institute, San Juan
Capistrano, CA, USA).
2.17. Expression of CD11b in HL-60 cells
The ability of all the vitamin D3 compounds shown in
Fig. 1 to differentiate HL-60 cells was assessed by analysis
of expression of CD11b protein, a differentiation marker.
Mid-exponentially growing HL-60 cells (2 ϫ 105/ml) were
treated for 3 days with different concentrations (10Ϫ9 and
10Ϫ8 M) of the vitamin D compounds and then analyzed by
fluorescence activated cell sorting (FACS) for expression of
CD11b. The HL-60 cells were incubated with a saturating
concentration of murine CD11b antibody (Carpenteria, CA,
USA) for 45 min on ice, followed by incubation with a goat
antimouse FITC-conjugated secondary antibody. A FITC-
conjugated isotype control was used. Immunofluorescence
was analyzed with a Becton-Dickinson FACScan flow cy-
tometer using LYSIS II software. The results are expressed
as % CD11b positive cells. All statistical analysis was
undertaken using the student’s t-test.
3. Results
3.1. Synthesis and separation of two C(23) epimers of
1␣,23,25(OH)3-24-oxo-19-nor-D3 (4)
C(23) epimeric mixture of 1␣,23,25(OH)3-24-oxo-19-
nor-D3 was synthesized by coupling A-ring phosphine ox-
ide 13 with CD ring ketone 12 using Lythgoe’s Horner–
Wittig method [25]. The 19-nor A-ring phosphine oxide 13
was prepared from quinic acid, as previously described by
Perlman et al. [26].
2.18. Transcriptional assay in ROS 17/2.8 cells
To test the vitamin D receptor-mediated transcriptional
activities of the 19-nor vitamin D3 compounds, ROS 17/2.8
cells were plated in 35-mm dishes at a density of 3 ϫ
105/dish in Dulbecco’s modified essential medium
The appropriate CD ring ketone 12 was synthesized from

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N.E. Lee et al. / Steroids 65 (2000) 252–265
Lythgoe’s diol 5 as shown in Fig. 2. Selective iodination of
the primary alcohol with triphenylphosphine and iodine
gave 6 in 97% yield. Displacement of the iodide with
sodium salt of benzene sulfinic acid in DMF resulted in 7a
with a yield of 94%. Initially, TBDMS group was used as
the protecting group for the C-8 alcohol. However, the
selective deprotection of the TBDMS group in later steps
was proven to be unsuccessful under the standard condi-
tions. The deprotection required the use of HF that removed
all the protecting groups. As a result, benzyloxymethyl ether
7b (BOM) was chosen as a suitable protecting group for the
C-8 alcohol. The deprotection of the BOM group proceeded
under normal hydrogenation conditions and it survived the
sodium-almagam reduction step that was required in remov-
ing the sulfone group.
The 23,25-dihydroxy-24-oxo side chain was introduced
to the CD ring sulfone using the method reported by
Takayama [27]. Coupling of the sulfone 7b with 2-methyl-
3,4-epoxy-butan-2-ol [28] gave 8a as shown in Fig. 2.
Reductive removal of the sulfone with sodium-almagam,
followed by Swern oxidation of the C-24 alcohol gave the
ketone 9. The ␣-hydroxylation of the ketone 9 was accom-
plished by oxidizing the silyl enol ether with MCPBA to
give 2:1 mixture of C- 23 epimers 10. The dihydroxy group
at C-23 and C-25 was protected as a ketal and the BOM
group was removed by hydrogenation to give the alcohol
11b. The C-8 hydroxy group was oxidized to the ketone
using Swern’s oxidation [29] to give CD ring ketone 12.
Coupling of A ring phosphine oxide 13 with the CD-
ketone 12 (Fig. 3) was successful using Lythgoe’s condi-
tions. Thus, phosphine oxide 13 was treated with n-BuLi at
Ϫ78°C to form the anion, followed by addition of ketone 12
gave the coupled product. Deprotection of the ketal and
TBDMS groups using Dowex acidic resin gave the desired
19-nor analog 4 in 69% yield for the two steps. The 19-nor
analog 4 was isolated as a mixture of diastereomers,
epimeric at C-23. The ratio of the two diastereomers was
approximately 2:1 as determined by the 1H NMR spectrum.
The two diastereomers were separated using the HPLC
system described in Fig. 4 and the individual quantities of
the two diastereomers obtained were about 1.6 mg, the
major compound and 0.8 mg, the minor compound. Thus,
we obtained each individual diastereomer in pure form to
study their biologic activities.
Fig. 4. HPLC separation of the two C(23) epimers of synthetic
1␣,23,25(OH)3-24-oxo-19-nor-D3: HPLC was performed on Zorbax–Sil
column (9.4 mm ϫ 25 cm) that eluted with hexane: 2-propanol (95:5 v/v)
at
a flow rate of 2 ml/min. The UV absorption spectra of
1␣,23(R),25(OH)3-24-oxo-19-nor-D3 (1) and 1␣,23(S),25(OH)3-24-oxo-
19-nor-D3 (2) are shown in the insert.
shows two peaks for the C-23 proton at 4.70 and 4.64 with
a ratio of 2:1, respectively. We assigned the major isomer
that is more downfield with chemical shift of 4.70 ppm the
R configuration and the minor isomer with chemical shift at
4.64 ppm the S configuration.
3.2. Effect of 1␣,25(OH)2D3 and 19-nor vitamin D3
compounds on PTH secretion in cultured bovine
parathyroid cells
The streochemistry at C-23 was determined by compar-
ing our 1H NMR data with Takayama and co-workers [27].
We followed Takayama’s synthesis in preparing the same
side chain resulting in two C(23) epimers of
1␣,23,25(OH)3-24-oxo-19-nor-D3. On the basis of Takaya-
ma’s assignment of R to C(23) by X-ray diffraction analysis
of the major isomer, we also assigned our major isomer the
The biologic activities of 1␣,23,25(OH)3-24-oxo-19-
nor-D3 epimers in parathyroid cells were determined by
comparing the potencies of these compounds to those of
1␣,25(OH)2D3 and 1␣,25(OH)2-19-nor-D3 in suppressing
PTH secretion. Parathyroid cells were incubated with vari-
ous concentrations of 1␣,23(S),25(OH)3-24-oxo-19-nor-D3,
1␣,23(R),25(OH)3-24-oxo-19-nor-D3, 1␣,25(OH)2-19-nor-
D3, and 1␣,25(OH)2D3 for 3 days. The steady state rate of
PTH secretion was then measured during a 3 h period. As
shown in Fig. 5, 1␣,25(OH)2D3, 1␣,25(OH)2-19-nor-D3 and
both epimers of 1␣,23,25(OH)3-24-oxo-19-nor-D3 de-
1
R configuration. Takayama reported the H NMR chemical
shift of the C-23 proton of the major isomer with the R
configuration to be more downfield at 4.72 ppm compared
to that of the minor isomer with the S configuration that
1
resonates at 4.66 ppm. The H NMR data of compound 4

N.E. Lee et al. / Steroids 65 (2000) 252–265
261
Fig. 5. Suppression of PTH secretion by 1␣,25(OH)2D3 and 19-nor vitamin D3 compounds: confluent cultures of bovine parathyroid cells were incubated
with the specified concentration of 1␣,25(OH)2D3, 1␣,25(OH)2-19-nor-D3, 1␣,23(R),25(OH)3-24-oxo-19-nor-D3 or 1␣,23(S),25(OH)3-24-oxo-19-nor-D3 for
72 h. The media was changed every 24 h and fresh concentration of the test analog was added each day. The cells were then washed, and steady state PTH
secretion was determined during a 3 h incubation. PTH was assessed by radioimmunoassay, and data were corrected for the total cell protein. The data are
expressed as mean Ϯ SD and represent combined data from two separate experiments. Values with the same character are significantly different from the
respective control (P Ͻ 0.05).
creased PTH secretion in a dose dependent manner. The two
compounds, 1␣,23(S),25(OH)3-24-oxo-19-nor-D3 and
1␣,25(OH)2-19-nor-D3 are equipotent and less active than
1␣,25(OH)2D3. For example, 1␣,23(S),25(OH)3-24-oxo-
19-nor-D3 and 1␣,25(OH)2-19-nor-D3 significantly (P Ͻ
0.05) decreased PTH secretion at a concentration of 1 ϫ
10Ϫ9 M whereas 1␣,25(OH)2D3 significantly (P Ͻ 0.05)
decreased PTH secretion at a concentration of 1 ϫ 10Ϫ11 M.
The compound, 1␣,23(R),25(OH)3-24-oxo-19-nor-D3 [sig-
nificantly (P Ͻ 0.05) decreased PTH secretion at a concen-
1␣,25(OH)2-19-nor-D3 allowed only 32 Ϯ 4% (mean Ϯ SE)
of colonies to grow compared to cells treated with
1␣,25(OH)2D3 where 85 Ϯ 2.5% of cells grew (P Ͻ 0.05).
Similarly, of the two epimers, the 1␣,23(R),25(OH)3-24-
oxo-19-nor-D3 was significantly more potent than the
1␣,23(S),25(OH)3-24-oxo-19-nor-D3. For example, at the
same dose of 1 ϫ 10Ϫ9 M, the 1␣,23(S),25(OH)3-24-oxo-
19-nor-D3 allowed 84 Ϯ 3.5% of colonies to grow. The
1␣,23(R),25(OH)3-24-oxo-19-nor-D3 significantly en-
hanced the potency (P Ͻ 0.05) allowing only 42 Ϯ 2.5% of
the colonies to grow.
tration of
1 ϫ
10Ϫ10 M] was more active than
1␣,23(S),25(OH)3-24-oxo-19-nor-D3 and 1␣,25(OH)2-19-
nor-D3 and less active than 1␣,25(OH)2D3.
3.4. Effect of 1␣,25(OH)2D3 and 19-nor vitamin D3
compounds on the expression of CD11b protein in HL-60
cells
3.3. Effect of 1␣,25(OH)2D3 and 19-nor vitamin D3
compounds on clonal growth of HL-60 cells
The HL-60 cells were cultured for 3 days with the vita-
min D3 compounds shown in Fig. 1 (1 ϫ 10Ϫ9 and 1 ϫ
10Ϫ8 M) and the expression of CD11b protein was tested.
Fig. 7 shows the effects of 1␣,25(OH)2D3 and 19-nor vita-
min D3 compounds on the expression of CD11b surface
protein by HL-60 cells. At 1 ϫ 10Ϫ9 M, 1␣,25(OH)2-19-
nor-D3 was significantly more active than 1␣,25(OH)2D3 in
inducing expression of CD11b protein on the cell surfaces
of HL-60 cells (P Ͻ 0.05). Almost 50% of the HL-60 cells
Fig. 6 shows the dose-response effects of all the vitamin
D3 compounds shown in Fig. 1 on clonal inhibition of
HL-60 cell growth in soft agar. It can be seen from Fig. 6
that all the vitamin D3 compounds tested exerted their
effects on HL-60 cell proliferation in a dose-dependent
manner. However, 1␣,25(OH)2-19-nor-D3 was significantly
more active than 1␣,25(OH)2D3 in inhibiting the clonal
growth of HL-60 cells. For example, at 1 ϫ 10Ϫ9 M the

262
N.E. Lee et al. / Steroids 65 (2000) 252–265
Fig. 7. Effects of 1␣,25(OH)2D3 and 19-nor vitamin D3 compounds on the
expression of CD11b antigens on HL-60 cells. The HL-60 cells were
treated for 3 days with 1␣,25(OH)2D3, 1␣,25(OH)2-19-nor-D3, or the
C(23) epimers of 1␣,23,25(OH)3-24-oxo-19-nor-D3 [10Ϫ8 and 10Ϫ9M] and
then analyzed by FACS for the expression of CD11b. Each value repre-
sents the mean Ϯ SE of three experiments with duplicate dishes.
Fig. 6. Effects of various doses of 1␣,25(OH)2D3 and 19-nor vitamin D3
compounds on the clonal growth of HL-60 cells. The HL-60 cells were
cultured with graded concentrations of either 1␣,25(OH)2D3, 1␣,25(OH)2-
19-nor-D3, 1␣,23(R),25(OH)3-24-oxo-19-nor-D3, or 1␣,23(S),25(OH)3-
24-oxo-19-nor-D3 [10Ϫ7–10Ϫ11M] for 10 days and colonies were counted
using an inverted microscope. The results are expressed as percentage of
control plates that do not contain the various vitamin D3 compounds. Each
value represents the mean Ϯ SE of three experiments with duplicate dishes.
of the vitamin D3 compounds had very little transcriptional
activity at 0.01 nM. Detectable transcriptional activity was
observed at 0.1 nM. Among the four vitamin D3 compounds,
1␣,25(OH)2D3 had the highest and 1␣,23(S),25(OH)3-24-oxo-
19-nor-D3 had the lowest transcriptional activities. The com-
pound, 1␣,23(R),25(OH)3-24-oxo-19-nor-D3 when compared
to 1␣,23(S),25(OH)3-24-oxo-19-nor-D3 was more active tran-
scriptionally even at 1 nM. The transcriptional activity of
1␣,23(R),25(OH)3-24-oxo-19-nor-D3 was slightly higher or
almost similar to 1␣,25(OH)2-19-nor-D3 at all concentrations
of the ligands. Thus, the higher transcriptional activity of
1␣,23(R),25(OH)3-24-oxo-19-nor-D3 when compared to
1␣,23(S),25(OH)3-24-oxo-19-nor-D3 correlated well with the
enhanced biologic potency of this epimer in terms of 1) sup-
pression of PTH hormone in bovine parathyroid cells; 2) in-
hibition of clonal growth of HL-60 cells and; 3) expression of
CD11b protein on HL-60 cells.
expressed CD11b protein when cultured in the presence of
1 ϫ 10Ϫ9M 1␣,25(OH)2-19-nor-D3, whereas only 30% of
the cells expressed CD11b protein when cultured in the
presence of an equimolar concentration of 1␣,25(OH)2D3.
The compound, 1␣,23(S),25(OH)3-24-oxo-19-nor-D3 in-
duced almost similar expression of CD11b protein on the
surfaces of HL-60 cells when compared to 1␣,25(OH)2D3.
The 1␣,23(R),25(OH)3-24-oxo-19-nor-D3 exhibited signif-
icantly higher biologic activity when compared to
1␣,23(S),25(OH)3-24-oxo-19-nor-D3 (P Ͻ 0.005) and was
equipotent to 1␣,25(OH)2-19-nor-D3 in inducing expression
of CD11b protein on HL-60 cells.
4. Discussion
3.5. Transcriptional activities of 1␣,25(OH)2D3 and 19-
nor-vitamin D3 compounds in ROS 17/2.8 cells
In our present study, we synthesized the C(23) epimers
of 1␣,23,25(OH)3-24-oxo-19-nor-D3 and studied their in
vitro biologic activities in several assay systems, and these
effects were compared with those of 1␣,25(OH)2-19-nor-D3
and 1␣,25(OH)2D3. We observed that even though
1␣,25(OH)2-19-nor-D3 was slightly less active than
1␣,25(OH)2D3 in reducing PTH secretion in primary cul-
tures of bovine parathyroid cells, both 1␣,25(OH)2-19-
nor-D3 and 1␣,23(S),25(OH)3-24-oxo-19-nor-D3 were al-
most equipotent in reducing PTH secretion. These biologic
activities of 19-nor vitamin D3 compounds are similar to our
To determine vitamin D receptor (VDR)-mediated trans-
activational activities, we assessed the abilities of all the
vitamin D3 compounds shown in Fig. 1 to induce growth
hormone production in ROS 17/2.8 cells transfected with an
osteocalcin VDRE/growth hormone gene contruct. Fig. 8
shows that the VDR-mediated transcriptional activity
through the osteocalcin VDRE was dose-dependent for
1␣,25(OH)2D3 and the 19-nor vitamin D3 compounds. Each

N.E. Lee et al. / Steroids 65 (2000) 252–265
263
most equipotent or slightly more active than 1␣,25(OH)2-
19-nor-D3.
We and other investigators have also demonstrated that
19-nor analogs are highly potent in suppressing the growth
of breast, prostrate and leukemia cells [13,15,30,31]. The
activities of 1␣,25(OH)2D3 and its analogs in regulating cell
growth and differentiation are primarily VDR mediated.
The higher VDR-mediated transciptional activity of
1␣,23(R),25(OH)3-24-oxo-19-nor-D3 when compared to
that of 1␣,23(S),25(OH)3-24-oxo-19-nor-D3 in inducing
growth hormone production in ROS 17/2.8 cells transfected
with an osteocalcin VDRE/growth hormone gene construct
provides in part an important explanation for its greater in
vitro activities of regulating cell growth and differentiation
and suppressing PTH secretion. At present, several different
factors seem to play a role in determining the biologic
effects of vitamin D analogs. Studies of Peleg and others
[32,33] have shown that 20-epi analogs of 1␣,25(OH)2D3
induce a different conformational change in the VDR that
results in the formation of tighter heterodimers with the
retinoid X receptor. This phenomenon leads to increased
transactivation of target genes. Studies have not yet deter-
mined whether the 19-nor vitamin D3 compounds produce a
conformational change in the nuclear VDR different from
that produced by 1␣,25(OH)2D3. Biologic activity of vita-
min D3 compounds can also be influenced by the rate of
cellular uptake and intracellular metabolism. Proteins
present in the culture medium can decrease the uptake of
vitamin D compounds by cultured cells [34]. The serum
vitamin D binding protein (DBP), which has high affinity
for all natural vitamin D metabolites, can reduce the bio-
logic activity of 1␣,25(OH)2D3 and its analogs by retarding
their cellular uptake [31,34]. Recently, Addo and Ray [35]
reported that 25OHD3 analogs with modifications at C-19
position possessed almost equal binding affinity for the
DBP when compared to 25OHD3 suggesting that C-19
modification of 25OHD3 does not reduce significantly its
binding to DBP. We tested the biologic activities of the
19-nor vitamin D3 compounds in the absence of FCS in the
culture medium to minimize the differences in cellular up-
take that could influence the biologic activity. Another key
factor in determining the biologic activity of vitamin D
compounds is their rate of metabolism. At present, the rates
of metabolism of the 19-nor vitamin D3 compounds have
not been examined.
Fig. 8. Transcriptional activity of the 19-nor vitamin D3 compounds in
ROS 17/2.8 cells. The cells were transfected by the DEAE-dextran method
with the fusion gene containing the osteocalcin VDRE attached to the
thymidine kinase/growth hormone reporter gene. Immediately after trans-
fection, the cells were treated with either 1␣,25(OH)2D3 or 19-nor vitamin
D3 compounds for 24 h in media containing 10% FCS. Culture medium
was assayed for growth hormone production by radioimmunoassay (Ni-
chols Institute). Each point of the dose-response curve is the mean of
duplicate transfections and the results shown are representative of four
transfection experiments.
earlier findings which indicated that 1␣,23(S),25(OH)3-24-
oxo-D3,
a
natural intermediary metabolite of
1␣,25(OH)2D3, was almost equipotent to 1␣,25(OH)2D3 in
suppressing PTH secretion [17]. In our present study, we
presented new information that the C-23 epimer with the
hydroxyl group in ‘R’ orientation [1␣,23(R),25(OH)3-24-
oxo-19-nor-D3], exhibited a higher biologic activity than the
epimer with the hydroxyl group in ‘S’ orientation
[1␣,23(S),25(OH)3-24-oxo-19-nor-D3] in bovine parathy-
roid cells. In HL-60 cells, unlike in parathyroid cells, the
19-nor compounds exhibited increased biologic activity.
1␣,25(OH)2-19-nor-D3 was significantly more potent than
1␣,25(OH)2D3 at inhibiting the clonal growth and inducing
differentiation of HL-60 cells. This finding suggests tissue
specific differences in biologic activities of 19-nor vitamin
D3 compounds. In HL-60 cells, unlike in bovine parathyroid
cells, the activity of 1␣,23(S),25(OH)3-24-oxo-19-nor-D3
when compared to 1␣,25(OH)2-19-nor-D3 was reduced.
However, it is of interest to note that in HL-60 cells also like
in bovine parathyroid cells 1␣,23(R),25(OH)3-24-oxo-19-
nor-D3 exhibited higher biologic activity when compared to
that of 1␣,23(S),25(OH)3-24-oxo-19-nor-D3, and was al-
In summary, our results indicate that 1␣,23(R),25(OH)3-
24-oxo-19-nor-D3 and to a lesser extent 1␣,23(S),25(OH)3-
24-oxo-19-nor-D3 are potent 19-nor vitamin D3 analogs,
which strongly inhibit clonal growth and induce differenti-
ation of HL-60 cells and suppress PTH secretion in para-
thyroid cells in vitro, and thus offer an appealing therapeutic
prospect. Our recent preliminary studies indicated that
1␣,25(OH)2-19-nor-D3 was metabolized via the C-24 oxi-
dation pathway in the perfused rat kidney, and we identified
1␣,23,25(OH)3-24-oxo-19-nor-D3 as a major metabolite of
1␣,25(OH)2-19-nor-D3. However, the stereochemistry of

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N.E. Lee et al. / Steroids 65 (2000) 252–265
class of potent inhibitors of proliferation and inducers of differenti-
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this metabolite has not been determined [36]. Based on our
previous work [17] that established 1␣,23(S),25(OH)3-24-
oxo-D3 as the natural metabolite of 1␣,25(OH)2D3,
1␣,23(S),25(OH)3-24-oxo-19-nor-D3 is most likely the nat-
ural epimer and 1␣,23(R),25(OH)3-24-oxo-19-nor-D3 is the
unnatural epimer.
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