Synthesis and biological activities of a new series of secosteroids: vitamin D phosphonate hybrids

Article abstract of DOI:10.1016/S0039-128X(00)00148-3

By a structural combination of phosphonate and bisphosphonate moieties with the vitamin D skeleton a series of new vitamin D analogs was synthesized. Derivatives with 24β-hydroxy- or 24-keto groups exerted considerable vitamin D activities in vitro while the hypercalcemic potentials were significantly reduced as compared to 1α,25-dihydroxyvitamin D₃ (calcitriol). Whereas the 24-hydroxy analogs did not influence bone formation in vivo in dosages below the hypercalcemic threshold, the 24-ketones were found to induce synthesis of new bone matrix in non-hypercalcemic doses. Vitamin D bisphosphonate hybrids, on the other hand, which did not elicit substantial vitamin D activities in vitro and tend to decrease serum calcium levels in vivo clearly induced osteoid formation in rats, indicating a mechanism of action different to calcitriol. Copyright

Full text of DOI:10.1016/S0039-128X(00)00148-3

Steroids 66 (2001) 257–266
Synthesis and biological activities of a new series of secosteroids:
vitamin D phosphonate hybrids
Andreas Steinmeyer*, Katica Schwarz, Martin Haberey, Gernot Langer, Herbert Wiesinger
Preclinical Drug Research, Schering AG, Institute of Medicinal Chemistry, Muellerstrasse 178, D-13342 Berlin, Germany
Abstract
By a structural combination of phosphonate and bisphosphonate moieties with the vitamin D skeleton a series of new vitamin D analogs
was synthesized. Derivatives with 24␤-hydroxy- or 24-keto groups exerted considerable vitamin D activities in vitro while the hypercal-
cemic potentials were significantly reduced as compared to 1␣,25-dihydroxyvitamin D3 (calcitriol). Whereas the 24-hydroxy analogs did
not influence bone formation in vivo in dosages below the hypercalcemic threshold, the 24-ketones were found to induce synthesis of new
bone matrix in non-hypercalcemic doses. Vitamin D bisphosphonate hybrids, on the other hand, which did not elicit substantial vitamin D
activities in vitro and tend to decrease serum calcium levels in vivo clearly induced osteoid formation in rats, indicating a mechanism of
action different to calcitriol. © 2001 Elsevier Science Inc. All rights reserved.
Keywords: Steroids; Vitamin D; Phosphonate; Hybrid; Bone; Osteoporosis
1. Introduction
The present study describes the combination of phospho-
nate and bisphosphonate substructures with the vitamin D
skeleton and the arising biologic activities.
Osteoporosis is a major disease of elderly people.
Women after the menopause as well as men of older age
could suffer from bone loss. Very often fractures are the
consequence of this disease frequently resulting in long
term hospitalization of the patients.
2. Experimental
It is known for a while that vitamin D derivatives exert
activities on bone [1]. The natural metabolite of vitamin D3,
1␣,25-dihydroxyvitamin D3 (calcitriol), stimulates bone
turnover by inducing osteoclastic bone resorption as well as
the formation of new bone by increasing osteoblastic activ-
ity [2] and anabolic effect. Vitamin D derivatives, however,
are not extensively being used for treatment of osteoporosis
due to the risk of hypercalcemia by the active dose range.
Bisphosphonates belong to another series of compounds
causing distinct effects on bone. These stable mimics of
pyrophosphate bind tightly to the bone mineral hydroxyap-
atite and inhibit osteoclastic activity of bone resorption [3].
Several bisphosphonates are in clinical use [4]. Due to the
extremely long half-lives of bisphosphonates the conse-
quences of long term treatment with these compounds are
not clear as yet.
2.1. Chemistry
For the first time vitamin D derivatives possessing
phosphorus atoms in the side chain were synthesized by
W. Dauben [5] (Scheme 1). It turned out that the incor-
poration of the phosphine oxide unit and the phosphonate
structure remarkably reduced vitamin D activity in vitro
and in vivo. Since only a few such derivatives have been
described, we initiated a new study of vitamin D phos-
phonate hybrids.
The synthesis of our first series 25-phosphonates took
advantage of the known Barton-Hesse procedure starting
from vitamin D2 which was converted to C-22 aldehyde
1 according to the literature [6,7] (Scheme 2). Triplet
sensitized photochemical rearrangement of the triene sys-
tem easily yielded aldehyde 2 with the natural triene
geometry. Wadsworth-Emmons reaction using methyl
(dimethoxyphosphinyl)acetate gave access to the unsat-
urated ester 3 which was reacted with deprotonated dial-
* Corresponding author. Tel.: ϩ49-30-4681-1785; fax: ϩ49-30-4689-
1785.
E-mail address: andreas.steinmeyer@schering.de (A. Steinmeyer).
0039-128X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(00)00148-3

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A. Steinmeyer et al. / Steroids 66 (2001) 257–266
consequence of a complexation effect between the two
phosphorus containing groups. Deprotection was performed
smoothly with Dowex௡ resin producing the surprisingly
stable dihydroxy enol phosphate derivative 17.
For comparison the corresponding analog to 17 with two
phosphonate units was synthesized. Thus, the ester 3 was
reduced with diisobutylaluminum hydride in toluene and
subsequently the allylic alcohol 18 was reoxidized to the
aldehyde 19 using pyridinium chlorochromate (Scheme 4).
Wadsworth-Emmons reaction using tetraethyl methylenebis-
phosphonate in the presence of sodium hydride afforded the
diene phosphonate 20 which could be converted in a Mi-
chael addition with diethyl methylphosphonate to the sym-
metrically substituted vitamin D bisphosphonate derivative
21. Cleavage of the silyl ethers was performed as usual
yielding the diol 22.
Finally, the aldehyde 19 was reacted with tetraethyl
methylenebisphosphonate applying Knoevenagel conditions
[8] giving the terminally substituted bisphosphonate deriv-
ative 23 which was deprotected as demonstrated earlier
furnishing the hybrid analog 24 (Scheme 5).
Scheme 1
kyl methylphosphonates affording the ␤-ketophospho-
nates 4, 5, and 6, which were desilylated with acidic
Dowex ion exchange resin furnishing the vitamin D de-
rivatives 7, 8, and 9. Furthermore, the keto groups in the
bissilylated ␤-ketophosphonates 4, 5, and 6 were selec-
tively reduced under Luche conditions. Subsequently, the
silyl groups were cleaved and the diastereomeric alcohols
were separated by HPLC.
In addition, synthesis of structures with two phosphorus
bearing units in the side chain were attempted. The ␤-ke-
tophosphonate 4 was deprotonated with lithium diisopro-
pylamide followed by quenching of the anion with diethyl
chlorophosphate (Scheme 3). By this reaction the enol phos-
phate 16 was formed as single isomer with respect to the
double bond configuration. Probably this selectivity is the
2.1.1. General remarks
1H Nuclear magnetic resonance (NMR) spectra: General
Electric QE 300 (tetramethylsilane as internal standard);
Scheme 2.

A. Steinmeyer et al. / Steroids 66 (2001) 257–266
259
Scheme 3.
infrared (IR) spectra: Perkin Elmer PE 621; UV spectra:
Perkin Elmer Lambda 3; mass spectra (MS): Finnigan TSQ
700. Combustion analysis were carried out by Schering
analytical department. TLC analyses were performed on
Merck 60 F254 silica gel plates.
Tetrahydrofuran and diethyl ether were distilled over
sodium/benzophenone prior to use. All other solvents
were purchased as p.a. (pro analysi) quality and were
dried over molecular sieves. All reactions were run under
positive argon pressure. Unless noted otherwise, ‘usual
work-up’ indicates quenching of the reaction mixture
with sodium chloride solution, extraction with ethyl ac-
etate, washing of the organic layer with either sodium
bicarbonate solution or dilute hydrochloric acid and so-
dium chloride solution, drying over sodium sulfate, and
evaporation of the solvent. Purification of crude materials
was performed by chromatography on silicagel using
ethyl acetate/hexane as eluents.
Scheme 4.
Scheme 5.

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A. Steinmeyer et al. / Steroids 66 (2001) 257–266
2.2. (5Z,7E)-(1S,3R)-1,3-bis[[(1,1-dimethylethyl)
dimethylsilyl]oxy]-9,10-secopregna-5,7,10(19)-triene-20-
carbaldehyde 2
mmol) in 1 ml tetrahydrofuran was added and stirring
was continued for 1 h. After usual work-up and
purification 376 mg (76.3%) 4 were isolated as colorless
foam.
IR (KBr): 3390, 2960, 1680, 1650, 1250, 1030 cmϪ1
.
A solution of (5E,7E)-(1S,3R)-1,3-bis[[(1,1-dimethyleth-
yl)dimethylsilyl]oxy]-9,10-secopregna-5,7,10(19)-triene-
20-carbaldehyde 1 (7.5 g, 13.1 mmol) [7] in 200 ml toluene
in a Pyrex flask was treated with anthracene (2.0 g, 11.2
mmol) and triethylamine (690 mg, 6.8 mmol). The solution
was irradiated for 30 min with a mercury high pressure lamp
while nitrogen was passed through the vessel. After filtra-
tion the solvent was removed in vacuo and the residue was
purified by chromatography giving 7.1 g (94.7%) 2 as col-
orless foam.
NMR (300 MHz, CDCl3): ␦ ϭ 0.05 ppm (s, 12H); 0.54
(s, 3H); 0.86 (s, 18H); 1.10 (d, 3H); 3.16 (s, 3H); 3.71 (s,
3H); 3.75 (s, 3H); 4.16 (m, 1H); 4.36 (m, 1H); 4.82 (s, 1H);
5.16 (s, 1H); 6.01 (d, 1H); 6.07 (d, 1H); 6.23 (d, 1H); 6.72
(dd, 1H).
MS (CI) m/z: 721 (MϩH, 45), 606 (100), 200 (21).
Analysis calculated for C39H69O6PSi2: C, 64.96; H, 9.64.
Found: C, 64.91; H, 9.59.
IR (KBr): 3390, 2950, 1720, 1240, 1050 cmϪ1
.
2.5. Diethyl [(5Z,7E,22E)-(1S,3R)-1,3-bis[[(1,1-
dimethylethyl)dimethylsilyl]oxy]-24-oxo-24a-homo-9,10-
secochola-5,7,10(19),22-tetraen-24a-yl]phosphonate 5
NMR (300 MHz, CDCl3): ␦ ϭ 0.05 ppm (s, 12H); 0.55
(s, 3H); 0.88 (s, 18H); 1.11 (d, 3H); 2.37 (m, 1H); 4.18 (m,
1H); 4.37 (m, 1H); 4.84 (s, 1H); 5.17 (s, 1H); 6.00 (d, 1H);
6.22 (d, 1H); 9.58 (d, 1H).
MS (CI) m/z: 573 (MϩH, 100), 500 (62), 248 (58).
Analysis calculated for C34H60O3Si2: C, 71.27; H, 10.55.
Found: C, 71.10; H, 10.50.
Diethyl methylphosphonate (867 mg, 5.7 mmol) was
reacted with ester 2 (1.2 g, 1.9 mmol) in analogy to the
protocol described for compound 4. After usual work-up
and purification 1.05 g (73.8%) 5 were isolated as colorless
foam.
IR (KBr): 3380, 2960, 1690, 1660, 1250, 1050 cmϪ1
.
2.3. Ethyl (5Z,7E,22E)-(1S,3R)-1,3-bis[[(1,1-
dimethylethyl)dimethylsilyl]oxy]-9,10-secochola-
5,7,10(19),22-tetraen-24-oate 3
NMR (300 MHz, CDCl3): ␦ ϭ 0.06 ppm (s, 12H); 0.57
(s, 3H); 0.89 (s, 18H); 1.11 (d, 3H); 1.33 (t, 6H); 3.19 (d,
2H); 4.13 (q, 4H); 4.16 (q, 2H); 4.17 (m, 1H); 4.37 (m, 1H);
4.84 (s, 1H); 5.19 (s, 1H); 6.01 (d, 1H); 6.18 (d, 1H); 6.23
(d, 1H); 6.81 (dd, 1H).
MS (CI) m/z: 749 (MϩH, 100), 633 (45), 176 (52).
Analysis calculated for C41H73O6PSi2: C, 65.73; H, 9.82.
Found: C, 65.68; H, 9.76.
Sodium hydride (455 mg, 11.4 mmol, 60% in mineral
oil) was suspended in 40 ml tetrahydrofuran. At 0°C
methyl (dimethoxyphosphinyl)acetate (2.69 g, 14.8
mmol) in 5 ml tetrahydrofuran was slowly added and the
mixture was stirred for 30 min. The aldehyde 2 (1.8 g, 3.1
mmol) in 20 ml tetrahydrofuran was added and stirring
was continued for 1 h. After usual work-up and
purification 1.4 g (70.8%) 3 were isolated as colorless
foam.
2.6. Bis(1-methylethyl) [(5Z,7E,22E)-(1S,3R)-1,3-
bis[[(1,1-dimethylethyl)dimethylsilyl]oxy]-24-oxo-24a-
homo-9,10-secochola-5,7,10(19),22-tetraen-24a-
yl]phosphonate 6
IR (KBr): 3380, 2940, 1650, 1250, 1050 cmϪ1
.
NMR (300 MHz, CDCl3): ␦ ϭ 0.05 ppm (s, 12H); 0.57
(s, 3H); 0.90 (s, 18H); 1.09 (d, 3H); 3.72 (s, 3H); 4.19 (m,
1H); 4.38 (m, 1H); 4.87 (s, 1H); 5.19 (s, 1H); 5.77 (d, 1H);
6.02 (d, 1H); 6.23 (d, 1H); 6.87 (dd, 1H).
MS (CI) m/z: 629 (MϩH, 53), 613 (28), 496 (78).
Analysis calculated for C37H64O4Si2: C, 70.64; H, 10.25.
Found: C, 70.69; H, 10.21.
Bis(1-methylethyl) methylphosphonate (240 mg, 1.3
mmol) was reacted with ester 2 (500 mg, 0.79 mmol) in
analogy to the protocol described for compound 4. After
usual work-up and purification, 476 mg (77.1%) 5 were
isolated as colorless foam.
IR (KBr): 3400, 2980, 1680, 1660, 1250, 1050 cmϪ1
.
NMR (300 MHz, CDCl3): ␦ ϭ 0.07 ppm (s, 12H); 0.58
(s, 3H); 0.89 (s, 18H); 1.12 (d, 3H); 1.33 (d, 12H); 3.16 (d,
2H); 4.18 (m, 1H); 4.38 (m, 1H); 4.72 (hept, 2H); 4.86 (s,
1H); 5.18 (s, 1H); 6.01 (d, 1H); 6.19 (d, 1H); 6.23 (d, 1H);
6.80 (dd, 1H).
2.4. Dimethyl [(5Z,7E,22E)-(1S,3R)-1,3-bis[[(1,1-
dimethylethyl)dimethylsilyl]oxy]-24-oxo-24a-homo-9,10-
secochola-5,7,10(19),22-tetraen-24a-yl]phosphonate 4
At Ϫ78°C dimethyl methylphosphonate (90 mg, 0.72
mmol) in 5 ml tetrahydrofuran was treated with n-butyl-
lithium (0.48 ml, 0.77 mmol, 1.6 M in hexane). After
stirring the solution for 30 min, ester 3 (430 mg, 0.68
MS (CI) m/z: 777 (MϩH, 30), 662 (62), 598 (100), 200
(35).
Analysis calculated for C43H77O6PSi2: C, 66.45; H, 9.99.
Found: C, 66.53; H, 9.92.

A. Steinmeyer et al. / Steroids 66 (2001) 257–266
261
2.7. Dimethyl [(5Z,7E,22E)-(1S,3R)-1,3-dihydroxy-24-oxo-
24a-homo-9,10-secochola-5,7,10(19),22-tetraen-24a-
yl]phosphonate 7
Analysis calculated for C31H49O6P: C, 67.86; H, 9.00.
Found: C, 67.85; H, 8.95.
2.10. Dimethyl [(5Z,7E,22E)-(1S,3R,24S)-1,3,24-
trihydroxy-24a-homo-9,10-secochola-5,7,10(19),22-
tetraen-24a-yl]phosphonate 13a and dimethyl
[(5Z,7E,22E)-(1S,3R,24R)-1,3,24-trihydroxy-24a-homo-
9,10-secochola-5,7,10(19),22-tetraen-24a-yl]phosphonate
13b
The bissilyl derivative 4 (35 mg, 4.9 mmol) was dis-
solved in 1 ml dichloromethane and 9 ml methanol. The
solution was treated with acidic Dowex® resin (350 mg) and
the mixture was stirred overnight. After filtration washing of
the filtrate with sodium bicarbonate solution and drying
over sodium sulfate the solvent was evaporated and the
residue was purified yielding 16 mg (66.9%) 7 as colorless
foam.
Ketone 4 (80 mg, 0.11 mmol) was dissolved in 2 ml
dichloromethane. At 0°C cerium trichloride heptahydrate
(52 mg, 0.14 mmol) and sodium borohydride (6 mg, mmol)
were added. After stirring for 15 min, the usual work-up
gave 61 mg (76%) 10 as diastereomeric mixture of 24-
alcohols which were treated with Dowex® resin in analogy
to the procedure described for compound 7 giving after
purification via HPLC 12 mg (15.2%) 13a and 11 mg
(13.8%) 13b as colorless foams.
IR (KBr): 3390, 2970, 1685, 1660, 1230, 1040 cmϪ1
.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.57 ppm (s, 3H); 1.10
(d, 3H); 3.18 (d, 2H); 3.68 (s, 3H); 3.74 (s, 3H); 4.17 (m,
1H); 4.37 (m, 1H); 4.96 (s, 1H); 5.28 (s, 1H); 6.00 (d, 1H);
6.09 (d, 1H); 6.35 (d, 1H); 6.77 (dd, 1H).
MS (CI) m/z: 493 (MϩH, 100), 475 (75), 457 (56), 156
(61).
Analysis calculated for C27H41O6P: C, 65.83; H, 8.39.
Found: C, 65.81; H, 8.45.
13a: IR (KBr): 3400, 2960, 1660, 1230, 1040 cmϪ1
.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.58 ppm (s, 3H); 1.02
(d, 3H); 3.21 (sbr, OH); 3.71 (s, 3H); 3.75 (s, 3H); 4.18 (m,
1H); 4.38 (m, 1H); 4.41 (m, 1H); 4.97 (s, 1H); 5.30 (s, 1H);
5.41 (dd, 1H); 5.58 (dd, 1H); 6.01 (d, 1H); 6.35 (d, 1H).
MS (CI) m/z: 495 (MϩH, 36), 477 (100), 459 (66), 441
(45), 168 (52).
2.8. Diethyl [(5Z,7E,22E)-(1S,3R)-1,3-dihydroxy-24-oxo-
24a-homo-9,10-secochola-5,7,10(19),22-tetraen-24a-
yl]phosphonate 8
Analysis calculated for C27H43O6P: C, 65.57; H, 8.76.
Found: C, 65.53; H, 8.71.
The bissilyl derivative 5 (80 mg, 0.11 mmol) was treated
with Dowex® resin in analogy to the procedure described
for compound 7 giving after purification 47 mg (84.5%) 8 as
colorless foam.
13b: IR (KBr): 3400, 2960, 1660, 1220, 1030 cmϪ1
.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.58 ppm (s, 3H); 1.03
(d, 3H); 3.28 (sbr, OH); 3.71 (s, 3H); 3.75 (s, 3H); 4.18 (m,
1H); 4.38 (m, 1H); 4.42 (m, 1H); 4.97 (s, 1H); 5.30 (s, 1H);
5.39 (dd, 1H); 5.55 (dd, 1H); 6.01 (d, 1H); 6.35 (d, 1H).
MS (CI) m/z: 495 (MϩH, 54), 477 (100), 459 (80), 441
(69), 168 (27).
IR (KBr): 3400, 2960, 1690, 1640, 1250, 1050 cmϪ1
.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.56 ppm (s, 3H); 1.08
(d, 3H); 1.27 (t, 6H); 3.16 (d, 2H); 4.05 (q, 2H); 4.07 (q,
2H); 4.16 (m, 1H); 4.37 (m, 1H); 4.95 (s, 1H); 5.29 (s, 1H);
6.00 (d, 1H); 6.09 (d, 1H); 6.35 (d, 1H); 6.76 (dd, 1H).
MS (CI) m/z: 521 (MϩH, 37), 503 (85), 485 (61), 200
(100), 172 (72).
Analysis calculated for C27H43O6P: C, 65.57; H, 8.76.
Found: C, 65.58; H, 8.74.
Analysis calculated for C29H45O6P: C, 65.83; H, 8.39.
Found: C, 65.81; H, 8.45.
2.11. Diethyl [(5Z,7E,22E)-(1S,3R,24S)-1,3,24-trihydroxy-
24a-homo-9,10-secochola-5,7,10(19),22-tetraen-24a-
yl]phosphonate 14a and diethyl [(5Z,7E,22E)-
2.9. Bis(1-methylethyl) [(5Z,7E,22E)-(1S,3R)-1,3-
dihydroxy-24-oxo-24a-homo-9,10-secochola-5,7,10(19),22-
tetraen-24a-yl]phosphonate 9
(1S,3R,24R)-1,3,24-trihydroxy-24a-homo-9,10-secochola-
5,7,10(19),22-tetraen-24a-yl]phosphonate 14b
Ketone 5 (200 mg, 0.27 mmol) was dissolved in 5 ml
methanol. At 0°C cerium trichloride heptahydrate (127 mg,
0.34 mmol) and sodium borohydride (15 mg, 0.39 mmol)
were added. After stirring for 15 min, the usual work-up
was performed giving 190 mg (95%) 11 as diastereomeric
mixture of 24-alcohols which were treated with Dowex®
resin in analogy to the procedure described for compound 7
giving after purification via HPLC 36 mg (18.0%) 14a and
38 mg (19.1%) 14b as colorless foams.
The bissilyl derivative 6 (70 mg, 0.09 mmol) was treated
with Dowex® resin in analogy to the procedure described
for compound 7 giving after purification 34 mg (68.8%) 9 as
colorless foam.
IR (KBr): 3400, 2970, 1685, 1660, 1250, 1050 cmϪ1
.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.58 ppm (s, 3H); 1.10
(d, 3H); 1.30 (d, 12H); 3.10 (d, 2H); 4.18 (m, 1H); 4.38 (m,
1H); 4.68 (hept, 2H); 4.96 (s, 1H); 5.29 (s, 1H); 6.01 (d,
1H); 6.12 (d, 1H); 6.36 (d, 1H); 6.76 (dd, 1H).
MS (CI) m/z: 549 (MϩH, 100), 531 (81), 513 (62), 176
(29).
14a: IR (KBr): 3400, 2970, 1650, 1240, 1040 cmϪ1
.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.55 ppm (s, 3H); 1.02
(d, 3H); 1.31 (t, 6H); 4.07 (q, 2H); 4.09 (q, 2H); 4.17 (m,

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A. Steinmeyer et al. / Steroids 66 (2001) 257–266
1H); 4.40 (m, 1H); 4.96 (s, 1H); 5.29 (s, 1H); 5.40 (dd, 1H);
5.57 (dd, 1H); 6.01 (d, 1H); 6.36 (d, 1H).
MS (CI) m/z: 523 (MϩH, 61), 505 (100), 487 (80), 469
(79), 161 (50).
mmol, 1.6 M in hexane) was added at –78°C. After 30 min
ketone 5 (650 mg, 0.87 mmol) in 2 ml tetrahydrofuran was
introduced and stirring is continued for 30 min when diethyl
chlorophosphate (414 mg, 2.4 mmol) was added. The mix-
ture was allowed to reach 0°C and was stirred for 1 h. The
usual work-up and purification gave 624 mg (81.2%) di-
ethyl (5Z,7E,22E,24Z)-(1S,3R)-[1,3-bis[[dimethyl(1,1-dim-
ethylethyl)silyl]oxy]-24a-(diethoxy)phosphinyl-24a-homo-
9,10-secochola-5,7,10(19),22,24-pentaen-24a-yl]
Analysis calculated for C29H47O6P: C, 66.64; H, 9.06.
Found: C, 66.59; H, 9.01.
14b: IR (KBr): 3400, 2980, 1650, 1240, 1040 cmϪ1
.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.55 ppm (s, 3H); 1.02
(d, 3H); 1.30 (t, 6H); 4.07 (q, 2H); 4.09 (q, 2H); 4.17 (m,
1H); 4.38 (m, 1H); 4.96 (s, 1H); 5.29 (s, 1H); 5.39 (dd, 1H);
5.54 (dd, 1H); 6.01 (d, 1H); 6.36 (d, 1H).
phosphonate 16 as colorless foam.
16: IR (KBr): 3400, 2960, 1640, 1240, 1040 cmϪ1
.
MS (CI) m/z: 523 (MϩH, 50), 505 (100), 487 (69), 469
(41), 179 (36).
Analysis calculated for C29H47O6P: C, 66.64; H, 9.06.
Found: C, 66.61; H, 9.02.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.05 ppm (s, 12H); 0.56
(s, 3H); 0.86 (s, 18H); 1.07 (d, 3H); 1.28 (t, 6H); 1.31 (t,
6H); 4.07 (q, 4H); 4.17 (m, 1H); 4.22 (q, 4H); 4.37 (m, 1H);
4.84 (s, 1H); 5.15 (d, 1H); 5.18 (s, 1H); 5.96 (d, 1H); 6.00
(d, 1H); 6.22 (d, 1H); 6.28 (dd, 1H).
2.12. Bis(1-methylethyl) [(5Z,7E,22E)-(1S,3R,24S)-1,3,24-
trihydroxy-24a-homo-9,10-secochola-5,7,10(19),22-tetraen-
24a-yl]phosphonate 15a and bis(1-methylethyl)
[(5Z,7E,22E)-(1S,3R,24R)-1,3,24-trihydroxy-24a-homo-9,10-
secochola-5,7,10(19),22-tetraen-24a-yl]phosphonate 15b
MS (CI) m/z: 885 (MϩH, 100), 766 (82), 749 (60), 200
(44).
Analysis calculated for C45H82O9P2Si2: C, 61.06; H,
9.34. Found: C, 61.12; H, 9.38.
Bissilyl ether 16 (624 mg, 0.70 mmol) was treated with
Dowex® resin in analogy to the procedure described for
compound 7 giving after purification 323 mg (69.9%) 17 as
colorless foam.
Ketone 6 (190 mg, 0.24 mmol) was dissolved in 5 ml
methanol. At 0°C cerium trichloride heptahydrate (89 mg,
0.23 mmol) and sodium borohydride (10 mg, 0.27 mmol)
were added. After stirring for 15 min, the usual work-up
gave 178 mg (93.7%) 12 as diastereomeric mixture of 24-
alcohols which were treated with Dowex® resin in analogy
to the procedure described for compound 7 giving after
purification via HPLC 34 mg (17.8%) 15a and 33 mg
(17.4%) 15b as colorless foams.
17: IR (KBr): 3390, 2950, 1650, 1250, 1050 cmϪ1
.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.57 ppm (s, 3H); 1.07
(d, 3H); 1.29 (t, 6H); 1.31 (t, 6H); 4.03 (q, 2H); 4.07 (q, 2H);
4.18 (m, 1H); 4.21 (q, 2H); 4.24 (q, 2H); 4.38 (m, 1H); 4.96
(s, 1H); 5.17 (d, 1H); 5.29 (s, 1H); 5.99 (d, 1H); 6.00 (d,
1H); 6.29 (dd, 1H); 6.33 (d, 1H).
MS (CI) m/z: 657 (MϩH, 38), 639 (100), 621 (37), 172
(36).
15a: IR (KBr): 3390, 2980, 1640, 1250, 1040 cmϪ1
.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.56 ppm (s, 3H); 1.03
(d, 3H); 1.31 (d, 12H); 4.18 (m, 1H); 4.38 (m, 1H); 4.68
(hept, 2H); 4.95 (s, 1H); 5.28 (s, 1H); 5.40 (dd, 1H); 5.56
(dd, 1H); 6.00 (d, 1H); 6.36 (d, 1H).
MS (CI) m/z: 551 (MϩH, 61), 533 (100), 525 (80), 497
(79), 208 (36).
Analysis calculated for C33H54O9P2: C, 60.35; H, 8.29.
Found: C, 60.28; H, 8.22.
2.14. (5Z,7E,22E)-(1S,3R)-1,3-Bis[[(1,1-dimethyl-
ethyl)dimethylsilyl]oxy]-9,10-secochola-5,7,10(19),22-
tetraen-24-ol 18
Analysis calculated for C31H51O6P: C, 67.61; H, 9.33.
Found: C, 67.68; H, 9.40.
To a solution of ester 3 (750 mg, 1.2 mmol) in 20 ml in
toluene at –23°C diisobutylaluminum hydride (2.6 ml, 3.1
mmol, 1.2 M in toluene) was added. After stirring the
mixture for 30 min, 3 ml water/isopropanol (1:1) were
added and stirring was continued for 1 h. The precipitate
was removed by filtration and the solvent was evacuated in
vacuo. Purification of the residue gave 550 mg (76.8%) 18
as colorless foam.
15b: IR (KBr): 3390, 2990, 1640, 1250, 1050 cmϪ1
.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.55 ppm (s, 3H); 1.04
(d, 3H); 1.30 (d, 12H); 4.18 (m, 1H); 4.38 (m, 1H); 4.69
(hept, 2H); 4.95 (s, 1H); 5.29 (s, 1H); 5.39 (dd, 1H); 5.53
(dd, 1H); 6.00 (d, 1H); 6.35 (d, 1H).
MS (CI) m/z: 551 (MϩH, 69), 533 (100), 525 (77), 497
(54), 208 (40).
Analysis calculated for C31H51O6P: C, 67.61; H, 9.33.
Found: C, 67.57; H, 9.32.
IR (KBr): 3400, 2990, 1650, 1540, 1240, 1050 cmϪ1
.
NMR (300 MHz, CDCl3): ␦ ϭ 0.06 ppm (s, 12H); 0.58
(s, 3H); 0.88 (s, 18H); 1.07 (d, 3H); 4.08 (m, 2H); 4.18 (m,
1H); 4.38 (m, 1H); 4.87 (s, 1H); 5.18 (s, 1H); 5.57 (m, 2H);
6.01 (d, 1H); 6.24 (d, 1H).
MS (CI) m/z: 601 (MϩH, 45), 583 (100), 469 (67), 172
(49).
2.13. Diethyl [(5Z,7E,22E,24Z)-(1S,3R)-24a-
(diethoxyphosphinyl)-1,3-dihydroxy-24a-homo-9,10-
secochola-5,7,10(19),22,24-pentaen-24a-yl]phosphonate 17
Diisopropylamine (120 mg, 1.1 mmol) was dissolved in
20 ml tetrahydrofuran and n-butyllithium (0.68 ml, 1.1
Analysis calculated for C36H64O3Si2: C, 71.94; H, 10.73.
Found: C, 71.88; H, 10.70.

A. Steinmeyer et al. / Steroids 66 (2001) 257–266
263
2.15. (5Z,7E,22E)-(1S,3R)-1,3-Bis[[(1,1-dimethyl-
ethyl)dimethylsilyl]oxy]-9,10-secochola-5,7,10(19),22-
tetraen-24-al 19
the usual work-up followed by purification 112 mg 21
(61.9%) were obtained as colorless foam.
IR (KBr): 3400, 2990, 1650, 1640, 1250, 1050 cmϪ1
.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.06 ppm (s, 12H); 0.53
(s, 3H); 0.86 (s, 18H); 1.01 (d, 3H); 1.30 (t, 12H); 4.05 (m,
8H); 4.16 (m, 1H); 4.36 (m, 1H); 4.84 (s, 1H); 5.17 (s, 1H);
5.39 (m, 2H); 6.00 (d, 1H); 6.23 (d, 1H).
MS (CI) m/z: 885 (MϩH, 28), 839 (100), 732 (86), 595
(66), 581 (52), 186 (70).
Alcohol 18 (550 mg, 0.92 mmol) was dissolved in 10 ml
dichloromethane and treated with pyridinium chlorochro-
mate (0.88 g, 4.1 mmol). After stirring the mixture for 3 h,
diethyl ether was added. The precipitate was removed by
filtration and the solvent was evaporated in vacuo. Purifi-
cation of the residue gave 530 mg (96.4%) 19 as colorless
foam.
Analysis calculated for C46H86O8P2Si2: C, 62.41; H,
9.79. Found: C, 62.35; H, 9.75.
IR (KBr): 3400, 2950, 1700, 1680, 1250, 1050 cmϪ1
.
NMR (300 MHz, CDCl3): ␦ ϭ 0.07 ppm (s, 12H); 0.60
(s, 3H); 0.88 (s, 18H); 1.18 (d, 3H); 4.20 (m, 1H); 4.38 (m,
1H); 4.87 (s, 1H); 5.18 (s, 1H); 6.02 (d, 1H); 6.07 (dd, 1H);
6.23 (d, 1H); 6.72 (dd, 1H); 9.50 (d, 1H).
MS (CI) m/z: 599 (MϩH, 100), 568 (67), 466 (41), 187
(52).
2.18. Tetraethyl [(5Z,7E,22E)-(1S,3R)-1,3-dihydroxy-24-
nor-9,10-secocholesta-5,7,10(19),22-tetraene-26,27-
diyl]bisphosphonate 22
Bisphosphonate 21 (82 mg, 0.09 mmol) was treated with
Dowex® resin in analogy to the procedure described for
compound 7 giving after purification 26 mg (42.8%) 22 as
colorless foam.
Analysis calculated for C36H62O3Si2: C, 72.18; H, 10.43.
Found: C, 72.25; H, 10.50.
IR (KBr): 3390, 2980, 1650, 1640, 1250, 1050 cmϪ1
.
2.16. Diethyl (5Z,7E,22E,24E)-(1S,3R)-[1,3-bis[[(1,1-
dimethylethyl)dimethylsilyl]oxy]-24a-homo-9,10-
secochola-5,7,10(19),22,24-pentaen-24a-yl]phosphonate
20
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.56 ppm (s, 3H); 1.01
(d, 3H); 1.29 (t, 12H); 4.01 (m, 8H); 4.15 (m, 1H); 4.36 (m,
1H); 4.92 (s, 1H); 5.28 (s, 1H); 5.36 (m, 2H); 6.00 (d, 1H);
6.32 (d, 1H).
MS (CI) m/z: 657 (MϩH, 45), 639 (100), 621 (72), 470
(56), 193 (41).
Analysis calculated for C34H58O8P2: C, 62.18; H, 8.90.
Found: C, 62.23; H, 8.86.
Sodium hydride (100 mg, 2.8 mmol, 60% in mineral oil)
was suspended in 20 ml tetrahydrofuran at 0°C. Tetraethyl
methylenebisphosphonate (610 mg, 2.1 mmol) in 2 ml tet-
rahydrofuran were added. After 15 min aldehyde 19 (1.25 g,
2.1 mmol) in 5 ml tetrahydrofuran was introduced and
stirring was continued for 1 h at 20°C. After performing the
usual work-up followed by purification 890 mg 20 (58.2%)
were obtained as colorless foam.
2.19. Tetraethyl [(5Z,7E,22E)-(1S,3R)-1,3-bis[[(1,1-
dimethylethyl)dimethylsilyl]oxy]-24a-homo-9,10-
secochola-5,7,10(19),22,24-pentaene-24a,24a-
diyl]bisphosphonate 23
IR (KBr): 3400, 2960, 1640, 1610, 1240, 1040 cmϪ1
.
NMR (300 MHz, CDCl3): ␦ ϭ 0.06 ppm (s, 12H); 0.57
(s, 3H); 0.89 (s, 18H); 1.08 (d, 3H); 1.32 (t, 6H); 4.08 (q,
2H); 4.10 (q, 2H); 4.18 (m, 1H); 4.38 (m, 1H); 4.86 (s, 1H);
5.18 (s, 1H); 5.57 (dd, 1H); 5.95 (dd, 1H); 6.02 (d, 1H); 6.09
(dd, 1H); 6.24 (d, 1H); 7.07 (dd, 1H).
MS (CI) m/z: 733 (MϩH, 100), 600 (58), 595 (81), 200
(40).
Analysis calculated for C41H73O5PSi2: C, 67.17; H,
10.04. Found: C, 67.14; H, 10.05.
Titanium (IV) chloride (120 mg, 0.64 mmol) and tetra-
chloromethane (2.3 g, 16.6 mmol) were dissolved in 1.4 ml
tetrahydrofuran. At 0°C tetraethyl methylenebisphospho-
nate (93 mg, 0.32 mmol) and aldehyde 19 (200 mg, 0.33
mmol) in 1 ml tetrahydrofuran were added and stirring was
continued for 15 min. At that time N-methylmorpholine
(138 mg, 1.36 mmol) was introduced and the mixture was
stirred for 2.5 h at 20°C. After performing the usual
work-up followed by purification 161 mg 23 (55.5%) were
obtained as colorless foam.
2.17. Tetraethyl [(5Z,7E,22E)-(1S,3R)-1,3-bis[[(1,1-
dimethylethyl)dimethylsilyl]oxy]-24-nor-9,10-secocholesta-
5,7,10(19),22-tetraene-26,27-diyl]bisphosphonate 21
IR (KBr): 3400, 2950, 1660, 1620, 1250, 1050 cmϪ1
.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.07 ppm (s, 12H); 0.54
(s, 3H); 0.86 (s, 18H); 1.06 (d, 3H); 1.32 (t, 12H); 4.15 (m,
9H); 4.36 (m, 1H); 4.83 (s, 1H); 5.18 (s, 1H); 6.00 (d, 1H);
6.22 (dd, 1H); 6.23 (d, 1H); 7.10 (dd, 1H); 7.75 (dddd, 1H).
MS (CI) m/z: 869 (MϩH, 67), 731 (100), 600 (56), 217
(39).
To a solution of diethyl methylphosphonate (91 mg, 0.73
mmol) in 2 ml tetrahydrofuran at Ϫ78°C n-butyllithium
(0.45 ml, 0.73 mmol, 1.6 M in hexane) was added and the
mixture was stirred for 10 min when phosphonate 20 (150
mg, 0.20 mol) in 1 ml tetrahydrofuran was introduced.
Stirring was continued for 4 h at –78°C. After performing
Analysis calculated for C45H82O8P2Si2: C, 62.18; H,
9.51. Found: C, 62.20; H, 9.47.

264
A. Steinmeyer et al. / Steroids 66 (2001) 257–266
2.20. Tetraethyl [(5Z,7E,22E)-(1S,3R)-1,3-dihydroxy-24a-
homo-9,10-secochola-5,7,10(19),22,24-pentaene-24a,24a-
diyl]bisphosphonate 24
tives, cells were seeded at a density of 2 ϫ 105 cells/well in
96-well microtiter plates (Flow) in 200 ␮l RPMI 1640
without phenol red with 10% FCS. Compounds were grad-
ually diluted in ethanol and to the cells, final ethanol con-
centration not exceeding 0.1%. Controls were incubated
with ethanol only. After incubation for 96 h at 37°C 100 ␮l
of an NBT-TPA solution (final concentration: 1 mg/ml NBT
and 2 ϫ 10Ϫ7 mol/l TPA) was added to the cells and
incubated for 2 h at 37°C. The adhering cells were then
fixed with methanol for 15 min at room temperature and air
dried. Formation of formazan by adhering cells was quan-
tified [10] by solution of the intracellularily precipitated dye
in 100 ␮l KOH (2 mol/l) and 100 ␮l DMSO/well and
reading the optical density on an ELISA plate reader (SLT)
at 570 nm.
Bisphosphonate 23 (68 mg, 0.08 mmol) was treated with
Dowex® resin in analogy to the procedure described for
compound 7 giving after purification 34 mg (68.0%) 24 as
colorless foam.
IR (KBr): 3400, 2980, 1660, 1630, 1250, 1050 cmϪ1
.
NMR (300 MHz, CD2Cl2): ␦ ϭ 0.56 ppm (s, 3H); 1.07
(d, 3H); 1.31 (t, 12H); 4.08 (m, 8H); 4.15 (m, 1H); 4.36 (m,
1H); 4.93 (s, 1H); 5.28 (s, 1H); 6.00 (d, 1H); 6.24 (dd, 1H);
6.33 (d, 1H); 7.00 (dd, 1H); 7.64 (dddd, 1H).
MS (CI) m/z: 641 (MϩH, 67), 623 (100), 605 (76), 468
(51), 183 (39).
Analysis calculated for C33H54O8P2: C, 61.86; H, 8.49.
Found: C, 61.92; H, 8.52.
2.21.4. Osteocalcin production in ROS 17/2.8
Osteoblast-like ROS 17/2.8 cells [11] were cultured with
5% FCS at 37°C in a humidified atmosphere at 5% CO2 in
the air. For incubation with 1␣,25-dihydroxyvitamin D3 and
derivatives, cells were seeded at a density of 105 cells/well
in 96-well microtiter plates in 200 ␮l DMEM and 2% FCS
for 72 h. The amount of osteocalcin produced was measured
with a sandwich ELISA.
2.21. Biologic methods and materials
2.21.1. Chemicals
1␣,25-Dihydroxy-[26,27-methyl-3H]cholecalciferol (180
Ci/mmol) was purchased from Amersham Buchler (Braun-
schweig, Germany). 1␣,25-Dihydroxyvitamin D3 (calcitriol)
was purchased from Solvay (Weesp, The Netherlands).
2.21.2. Receptor-binding affinity
2.21.5. Hypercalcemic and hypercalciuric activity in vivo
1␣,25-Dihydroxyvitamin D3 (2 ␮g/kg) or derivatives
were injected subcutaneously (s.c.) in 500 ␮l ethanol/0.9%
sodium chloride (40/60 v/v) to normal female Wistar rats
(150–170 g) housed in metabolism cages with free access to
a normal diet (Altromin) and water. Urine was collected for
a period of 0–16 h, then the animals were fed 1 mmol
calcium in 5 ml 6.5% Klucell (hydroxypropylcellulose) and
urine was collected for a second period of 16–22 h. Urine
volume of each fraction was measured for calculating ab-
solute calcium excretion. After 22 h animals were killed by
decapitation and blood was collected. The calcium concen-
tration in serum and urine was determined by complex
formation with 2-methylphenolphthalein. The dosages of
the derivatives which were equipotent to a dose of 2 ␮g/kg
calcitriol were determined.
A vitamin D receptor preparation was made from the
mucosa of normal pig intestine as described [9]. Briefly,
mucosa was homogenized in hypotonic TED buffer (Tris-
HCl 50 mmol/l, EDTA 1.5 mmol/l, dithiothreitol (DTT) 5
mmol/l) and centrifuged. The pellet was homogenized in
TEDK buffer (TED ϩ 0.3 mol/l KCl) and cleared by cen-
trifugation to yield the receptor preparation.
Receptor-binding affinity of the vitamin D derivatives
was assessed by displacement of [3H]-1␣,25-dihydroxyvi-
tamin D3 from the receptor preparation. To 250 ␮l of the
receptor preparation (1 mg/ml protein) was added 10 ␮l
each of a gradually diluted ethanolic solution of 1␣,25-
dihydroxyvitamin D3 or the derivatives and 10 ml (0.025
␮Ci) [3H]-1␣,25-dihydroxyvitamin D3; the solution was
incubated for 1 h at 4°C. Bound [3H]-1␣,25-dihydroxyvita-
min D3 was determined after removal of free tracer by the
addition of 250 ␮l 1% dextran-coated charcoal, 20 min
incubation at 4°C, and centrifugation. Relative-binding af-
finity (RBA) was calculated from the displacement curves
according to the equation RBA ϭ IC50 1␣,25-dihydroxyvi-
tamin D3 x 100/IC50 derivative, IC50 being the concentra-
tion at which 50% displacement of tracer was achieved.
2.21.6. Preliminary assessment of bone anabolic activity
1␣,25-Dihydroxyvitamin D3 or derivatives were injected
s.c. in analogy to the protocol described above to female
Wistar rats for 5 consecutive days. Urine is collected for
every 24 h. Serum samples are taken on day 1, 3 and 5. As
described above, calcium concentration in urine and serum
was determined. Osteocalcin concentration was determined
from serum by an ELISA. The animals were killed by
decapitation and bone samples were recovered (femur, ver-
tebra) and analyzed by means of thin sections of the trabec-
ular bone regions [12].
2.21.3. HL 60 cell differentiation
Human promyelocytic HL 60 cells were obtained from
the American Type Culture Collection and grown in RPMI
1640 with 10% heat-inactivated fetal calf serum (FCS) at
37°C in a humidified atmosphere at 5% CO2 in the air. For
incubation with 1␣,25-dihydroxyvitamin D3 or the deriva-

A. Steinmeyer et al. / Steroids 66 (2001) 257–266
265
Table 1
Table 2
RBA (%)
VDR
DR
HL 60
DR
ROS
DR
Ca
Dosage
[␮g/kd/d]
Hypercalcemia
Osteocalcin
Induction
Bone
Effects
Calcitriol
7
8
9
13b
14b
15b
17
22
24
100
10
10
17
5
5
5
30
0.7
0.3
1
22
58
32
31
1
5
100
160
18
670
5
80
1
300
Ͼ300
Ͼ300
1000
Calcitriol
Calcitriol
8
8
15b
15b
17
17
0.05
0.2
120
240
0.5
1
240
480
ϩϩ
ϩϩ
Ϫ
Ϫ
Ϫ
ϩ
Ϫ
Ϫ
ϩ
ϩϩ
Ϫ
Ϫ
Ϫ
ϩ
Ϫ
Ϫ
ϩ
ϩϩ
ϩ
ϩ
Ϫ
ϩ
ϩ
ϩ
210
150
32
Ͼ560
Ͼ560
n.d.
Ͻ300
Ͼ300*
ϾϾ2000*
ϾϾ6000*
n.d.
Ͼ1400
ϩ means elevation of parameter 10% above normal
ϩϩ means elevation of parameter 20% above normal
Average serum calcium level in rats is about 2.65 mol/l.
Average osteocalcin level in rats is about 50 ng/ml.
Bone effects are analyzed by a visual scoring system.
RBA ϭ Relative binding affinity, IC50 calcitriol/IC50 test compound ϫ
100
DR (HL 60, ROS 17/2.8) ϭ dose ratio, EC50 test compound/EC50
calcitriol
DR (Ca) ϭ dose derivative equipotent to 2 ␮g/kg calcitriol/2 ␮g/kg
calcitriol
In the vitamin D bisphosphonate hybrid class only the
enol phosphate 17 effectively bound to the VDR and ex-
erted very moderate vitamin D-like activity in HL 60 and
ROS 17/2.8. The other hybrid compounds (28, 30) did not
show any VDR binding affinity and were nearly inactive in
the in vitro assays. Upon administering these compounds to
rats, for the first time decreases in serum calcium levels
were detected. Thus, it seemed that the vitamin D bisphos-
phonate hybrids have distinct activity profiles different from
normal vitamin D analogs.
* Serum calcium levels are reduced as compared to control
n.d. ϭ not determined
3. Results
For the biologic evaluation several in vitro assays have
been applied. Bone activities of vitamin D derivatives are
believed to be genomic effects expressed via binding of the
ligand to the nuclear vitamin D receptor (VDR). Thus, the
binding affinities of the vitamin D analogs to the porcine
vitamin D receptor (VDR) were determined [13]. A prom-
inent assay reflecting vitamin D functional activity in vitro
is the induction of differentiation of the promyelocytic hu-
man leukemia cell line HL 60 to monocytes/macrophages
[13]. As a surrogate system for the bone anabolic activity
the rat osteosarcoma cell line ROS 17/2.8 was considered
[14]. In this cellular system vitamin D analogs induce the
synthesis of the bone matrix protein osteocalcin which is
believed to reflect osteoblastic bone formation. On the other
hand, the major side effect of vitamin D analogs, the induc-
tion of hypercalcemia, was investigated in vivo in intact
Wistar rats [13].
Regarding the 25-phosphonates it became clear that the
binding affinities to the VDR were largely reduced as com-
pared to calcitriol. In vitro the 24-ketones 7, 8, and 9 and the
24␤-hydroxy analogs 13b, 14b, and 15b showed moderate
activities (Table 1) whereas the 24␣-hydroxy derivatives
13a, 14a, and 15a were much less active (data not shown),
a behavior which was already observed in other structural
classes before [7]. The considerably high abilities of keto-
phosphonate 7 and 24␤-hydroxyphosphonate 15b to induce
osteocalcin synthesis in ROS 17/2.8 cells indicated strong
effects on bone matrix formation. On the other hand, all
derivatives are much less prone to induce hypercalcemia
than calcitriol. Thus, in the 25-phosphonate series deriva-
tives were identified with a certain degree of dissociation
between the vitamin D activity in vitro and undesired hy-
percalcemia in vivo.
In order to address the question whether the vitamin D
phosphonate hybrid analogs really affect bone as target
organ, preliminary in vivo studies were performed with
␤-ketophosphonate 8, 24␤-hydroxy analog 15b and the enol
phosphate 17 in comparison with calcitriol. Hence, intact
Wistar rats which were fed a normal diet were treated s.c.
with the analogs for 5 consecutive days. Calcium excretion
as well as calcium and osteocalcin levels in serum were
determined. The effect on bone was then examined by bone
histomorphometry of certain representative sections of the
tibia. Since additional experiments will be conducted to
validate these first results, only trends are outlined in this
report (Table 2). The reference compound, calcitriol, in-
duced bone formation significantly. During the short treat-
ment period (5 days) only unmineralized osteoid matrix is
generated. Upon longer treatment periods mineralization of
newly formed bone matrix can be observed. However, the
bone anabolic effect of calcitriol was accompanied by
strong elevation of calcium levels in urine and serum, thus
limiting the therapeutic usefulness remarkably. The 24␤-
hydroxy analog 15b which showed a certain degree of
dissociation between bone formation in vitro (induction of
osteocalcin synthesis in ROS 17/2.8 cells) and induction of
hypercalcemia in vivo did induce bone formation in the
absence of calcitropic effects in the described model. On the
other hand, the 24-ketophosphonate 8 and the enol phos-
phate 17 did not influence calcium and osteocalcin levels at
all in the dosages applied. Nevertheless, formation of new
osteoid could be detected in a significant number of animals.

266
A. Steinmeyer et al. / Steroids 66 (2001) 257–266
4. Discussion
References
[1] Tilyard MW, Spears GFS, Thomson J, Dovey S. Treatment of post-
menopausal osteoporosis with calcitriol or calcium. New Engl J Med
1992;326:357–62
[2] Suda T, Shinki T, Takahashi N. The role of vitamin D in bone and
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[4] Russell RGG, Rogers MJ. Bisphosphonates: From the laboratory to
the clinic and back again. Bone 1999;25:97–106.
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Bishop JE. 25-Phosphorus analogs of vitamin D3. Tetrahedron Lett
1991;32:4643–6.
The introduction of phosphonate and bisphosphonate
units into the vitamin D side chain resulted in different
activity profiles. Analogs which have 24␤-hydroxy groups
in the side chain showed considerable vitamin D activities
in vitro. Induction of hypercalcemia was less pronounced
than for calcitriol. However, in the animal model reflecting
bone formation, no clear dissociation between desired bone
anabolic activity and harmful hypercalcemia was visible.
The 24␣-hydroxy derivatives, on the other hand, were con-
siderably less active.
The corresponding 24-keto analogs also showed vitamin D
function in vitro, while hypercalcemic potentials were remark-
ably reduced in comparison with calcitriol. Furthermore, these
derivatives stimulated bone matrix formation in dosages which
did not increase calcium levels, indicating the existence of a
therapeutic window for treatment of bone diseases.
Finally, vitamin D bisphosphonate hybrids turned out to
be rather inactive in terms if vitamin D actions in vitro.
These compounds, however, induced osteoid formation in
vivo without elevating calcium levels. It could be assumed
that these analogs act on bone by a different mechanism as
compared to calcitriol for instance.
In summary, vitamin D compounds with phosphorus
containing moieties were synthesized. Depending on the
side chain substructures vitamin D phosphonate hybrids
behave as normal vitamin D analogs with a certain level of
dissociation or exert selective activity on bone. Since the
data still have preliminary character, further studies are
necessary to evaluate the full potential of these novel vita-
min D derivatives.
[6] Andrews DR, Barton DHR, Hesse RH, Pechet MM. Synthesis of
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Acknowledgments
The technical skills of Mr. Boehme and Mr. Piechowiak
are gratefully acknowledged. Biologic testing was per-
formed in the departments of Experimental Dermatology
and Special Pharmacology of Schering AG.