New highly calcemic 1α,25-dihydroxy-19-norvitamin D3 compounds with modified side chain: 26,27-dihomo- and 26,27-dimethylene analogs in 20S-series

Article abstract of DOI:10.1016/S0039-128X(01)00156-8

New highly potent 2-substituted (20S)-1α,25-dihydroxy-19-norvitamin D₃ analogs with elongated side chain were prepared by Wittig-Horner coupling of A-ring phosphine oxide with the corresponding protected (20S)-25-hydroxy Grundmann's ketones. Biologic evaluation in vitro and in vivo of the synthesized compounds was accomplished. All the synthesized vitamins possessing a 25-hydroxylated saturated side chain were slightly less active (3-5X) than 1α,25-dihydroxyvitamin D₃ in binding to the porcine intestinal vitamin D receptor and significantly more potent (12-150X) in causing differentiation of HL-60 cells. In vivo, 2-methylene-26,27-dihomo and 2α-methyl-26,27-dimethylene analogs were at least 10 times more active, and 2α-methyl-26,27-dihomo compound at least 5 times more active than the vitamin D hormone both in stimulating intestinal calcium transport and bone calcium mobilization (serum calcium increase). It was also established that a 260 pmol dose of the corresponding 2β-methyl analogs had a similar effect on intestinal calcium transport and a much more pronounced effect on bone calcium mobilization as the same dose of 1α,25-dihydroxyvitamin D₃. Copyright

Full text of DOI:10.1016/S0039-128X(01)00156-8

Steroids 67 (2002) 247–256
New highly calcemic 1␣,25-dihydroxy-19-norvitamin D3 compounds
with modified side chain: 26,27-dihomo- and 26,27-dimethylene
analogs in 20S-series
Rafal R. Sicinski1, Jean M. Prahl, Connie M. Smith, Hector F. DeLuca*
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA
Received 26 February 2001; received in revised form 25 June 2001; accepted 6 July 2001
Abstract
New highly potent 2-substituted (20S)-1␣,25-dihydroxy-19-norvitamin D3 analogs with elongated side chain were prepared by
Wittig-Horner coupling of A-ring phosphine oxide with the corresponding protected (20S)-25-hydroxy Grundmann’s ketones. Biologic
evaluation in vitro and in vivo of the synthesized compounds was accomplished. All the synthesized vitamins possessing a 25-hydroxylated
saturated side chain were slightly less active (3–5X) than 1␣,25-dihydroxyvitamin D3 in binding to the porcine intestinal vitamin D receptor
and significantly more potent (12–150X) in causing differentiation of HL-60 cells. In vivo, 2-methylene-26,27-dihomo and 2␣-methyl-
26,27-dimethylene analogs were at least 10 times more active, and 2␣-methyl-26,27-dihomo compound at least 5 times more active than
the vitamin D hormone both in stimulating intestinal calcium transport and bone calcium mobilization (serum calcium increase). It was also
established that a 260 pmol dose of the corresponding 2␤-methyl analogs had a similar effect on intestinal calcium transport and a much
more pronounced effect on bone calcium mobilization as the same dose of 1␣,25-dihydroxyvitamin D3. © 2002 Elsevier Science Inc. All
rights reserved.
Keywords: Vitamin D analogs; Vitamin D receptor; 19-Norvitamin D compounds; Differentiation of HL-60 cells; Calcemic activity
1. Introduction
exomethylene moiety resulted in considerable suppression
of calcemic activity of 2a and, interestingly, preservation of
its cell differentiating abilities. Our further investigation of
structure-activity relationship of the 19-norvitamin D mol-
ecules focused on the modification of the ring A at C-2 [19]
and establishing the possible role of A-ring conformation.
Recently, we have found that introduction of an exometh-
ylene or 1␣-methyl substituent at C-2 into the 19-nor struc-
ture 2a unexpectedly results in the analogs possessing sig-
nificant calcemic activity [20], enhanced dramatically in
compounds with ‘unnatural’ (20S) side chain configuration.
There is an obvious interest in synthesizing vitamin D
analogs more potent than 1a in terms of calciotropic activity
because of their potential use in the treatment of such
common bone diseases as osteoporosis [21]. Thus, we be-
came interested in the further exploration of similar vitamin
D analogs and examining the effect of side chain structure
on the activity of 2-methylene and 2-methyl substituted
analogs.
1␣,25-Dihydroxyvitamin D3 (1␣,25-(OH)2D3, calcitriol,
1a; Fig. 1), the most active hormonal form of vitamin D3,
regulates calcium homeostasis in animals and humans [1–4]
by promoting intestinal calcium transport as well as miner-
alization and resorption of bones. In addition to these classic
functions, 1a plays a much wider biologic role: it affects the
immune system and regulates cellular differentiation and
proliferation of a variety of normal and malignant cells
[5–14]. These observations stimulated great synthetic ef-
forts aimed at preparation of the vitamin D analogs charac-
terized by dissociation of cell differentiation and calcemic
activities [1,5,6,15]. Such interesting separation was found
in the 1␣,25-dihydroxy-19-norvitamin D3 2a, synthesized a
decade ago in our laboratory [16–18]. Deletion of 19-
* Corresponding author. Tel.: ϩ1-608-262-1620; fax: ϩ1-608-262-
7122.
E-mail address: deluca@biochem.wisc.edu (H.F. DeLuca).
1Present address: Department of Chemistry, University of Warsaw, ul.
Pasteura 1, 02–093 Warsaw, Poland.
Elongation of the side chain at carbons 26 and 27 usually
causes a 2–5 fold increase in HL-60 differentiating potency
[22–24], increasing also to some extent affinity for VDR
0039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(01)00156-8

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R.R. Sicinski et al. / Steroids 67 (2002) 247–256
2.2. (20S)-25-[(triethylsilyl)oxy]-des-A,B-26,27-
dihomocholestan-8-one (17)
To a solution of (20S)-25-hydroxy Grundmann’s ketone
analog 16 (18.5 mg, 0.06 mmol) in anhydrous CH2Cl2 (60
␮l) was added 2,6-lutidine (17.4 ␮l, 0.15 mmol) and trieth-
ylsilyl trifluoromethanesulfonate (20.3 ␮l, 0.09 mmol). The
mixture was stirred at room temperature under argon for 1 h.
Benzene was added and water, and the organic layer was
separated, washed with sat. CuSO4 and water, dried
(MgSO4) and evaporated. The oily residue was redissolved
in hexane and applied on a silica Sep-Pak cartridge (2 g).
Elution with hexane (10 ml) gave a small quantity of less
polar compounds; further elution with hexane/ethyl acetate
(9:1) provided the silylated ketone. Final purification was
achieved by HPLC (10-mm X 25-cm Zorbax-Sil column, 4
ml/min) using hexane/ethyl acetate (95:5) solvent system.
Pure protected hydroxy ketone 17 (16.7 mg, 66%) was
Fig. 1. Chemical structure of 1␣,25-dihydroxyvitamin D3 (calcitriol) and
its analogs.
[25] as well as calcemic activity of vitamin D hormone
analogs [26]. Epimerization at carbon 20 has a much more
profound effect on HL-60 differentiating potency of the
vitamin D compound, increasing it 20–30 times [24,27,28].
Moreover, 20-epi compounds show 2–5 fold increased cal-
cemic activity [29] as well as slightly enhanced affinity for
VDR [28]. A similar tendency was also found in the 19-nor
series, since 20-epi analogs such as 2b [30], 3b and 4b [20]
were proved to be significantly more calcemic than their
20R counterparts 2a, 3a and 4a. Therefore, in search of
enhancing calcemic activity of vitamin D, we have decided
to synthesize 20S compounds homologated at C-26 and
C-27, namely, 26,27-dihomo analog 7 (Fig. 2) and 26,27-
dimethylene compound 11 as well as their dihydroderiva-
tives: 2␣-methyl- (8, 14) and 2␤-methyl-substituted 19-
norvitamins (9, 15).
1
eluted at RV 37 ml as a colorless oil: H NMR (CDCl3) ␦
0.573 (6H, q, J ϭ 7.9 Hz, 3ϫ SiCH2), 0.639 (3H, s, 18-H3),
0.825 (6H, t, J ϭ 7.5 Hz, 26- and 27-CH3), 0.861 (3H, d,
J ϭ 6.1 Hz, 21-H3), 0.949 (9H, t, J ϭ 7.9 Hz, 3ϫ
SiCH2CH3), 2.45 (1H, dd, J ϭ 11.4, 7.6 Hz, 14␣-H).
2.3. (20S)-25-[(triethylsilyl)oxy]-des-A,B-26,27-
dimethylene-cholestan-8-one (19)
The protected compound 19 was obtained from (20S)-
25-hydroxy Grundmann’s ketone analog 18 in the same way
as 17. The silylated ketone 19 was purified by HPLC
(10-mm X 25-cm Zorbax-Sil column, 4 ml/min) using hex-
ane/ethyl acetate (95:5) solvent system and it was eluted at
2. Experimental
1
RV 39 ml (46% yield) as a colorless oil: H NMR (CDCl3)
␦ 0.576 (6H, q, J ϭ 7.9 Hz, 3ϫ SiCH2), 0.638 (3H, s,
18-H3), 0.865 (3H, d, J ϭ 6.1 Hz, 21-H3), 0.949 (9H, t, J ϭ
7.9 Hz, 3ϫ SiCH2CH3), 2.45 (1H, dd, J ϭ 11.4, 7.5 Hz,
14␣-H).
2.1. General
Ultraviolet (UV) absorption spectra were recorded with a
Hitachi Model 60–100 UV-vis spectrometer in the solvent
1
noted. H nuclear magnetic resonance (NMR) spectra were
2.4. (20S)-1␣,25-dihydroxy-2-methylene-26,27-dihomo-19-
norvitamin D3 (7)
recorded at 500 MHz with a Bruker AM-500 FT spectrom-
eter in deuteriochloroform. Chemical shifts (␦) are reported
downfield from internal Me4Si (␦ 0.00). Low- and high-
resolution mass spectra were recorded at 70 eV on a Kratos
DS-50 TC instrument equipped with a Kratos DS-55 data
system. Samples were introduced into the ion source main-
tained at 120–250°C via a direct insertion probe. High-
performance liquid chromatography (HPLC) was performed
on a Waters Associates liquid chromatograph equipped with
a Model 6000A solvent delivery system, a Model 6 UK
Universal injector, a Model 486 tunable absorbance detec-
tor, and a differential R 401 refractometer. THF was freshly
distilled before use from sodium benzophenone ketyl under
argon. Bicyclic ketones 16 and 18 were purchased from
Tetrionics, Inc. (Madison, WI). These compounds were
obtained by a procedure analogous to that described for
other (20S)-25-hydroxy Grundmann’s ketones [30].
To a solution of phosphine oxide 20 (9.1 mg, 15.6 ␮mol)
in anhydrous THF (150 ␮l) at 0°C was slowly added n-BuLi
(2.5 M in hexanes, 7 ␮l, 17.5 ␮mol) under argon with
stirring. The solution turned deep orange. It was stirred for
10 min at 0°C, then cooled to Ϫ78°C and a precooled
(Ϫ78°C) solution of protected hydroxy ketone 17 (16.5 mg,
39.0 ␮mol) in anhydrous THF (300 ϩ 100 ␮l) was slowly
added. The mixture was stirred under argon at Ϫ78°C for
1.5 h and at 0°C for 19 h. Water and ethyl acetate were
added, and the organic phase was washed with brine, dried
(MgSO4) and evaporated. The residue was dissolved in
hexane and applied on a silica Sep-Pak cartridge, and
washed with hexane/ethyl acetate (99.7:0.3, 20 ml) to give
slightly impure 19-norvitamin derivative 6 (ca. 4 mg). The
Sep-Pak was then washed with hexane/ethyl acetate (96:4,

R.R. Sicinski et al. / Steroids 67 (2002) 247–256
249
Fig. 2. Chemical structure of the synthesized vitamin D compounds and a scheme of the synthesis.
10 ml) to recover some unchanged C,D-ring ketone (con-
taminated with 14␤-isomer), and with ethyl acetate (10 ml)
to recover diphenylphosphine oxide 20 (ca. 6 mg) that was
subsequently purified by HPLC (10-mm X 25-cm Zorbax-
Sil column, 4 ml/min) using hexane/2-propanol (9:1) sol-
vent system; pure compound 20 (5.1 mg) was eluted at RV

250
R.R. Sicinski et al. / Steroids 67 (2002) 247–256
36 ml. The protected vitamin 6 was further purified by
HPLC (6.2-mm X 25-cm Zorbax-Sil column, 4 ml/min)
using hexane/ethyl acetate (99.9:0.1) solvent system. Pure
compound 6 (3.6 mg, 30% yield; 67% yield considering the
recovery of unreacted 20) was eluted at RV 19 ml as a
pound 10 (10.1 mg, 42% yield; 74% yield considering the
recovery of unreacted 20) was eluted at RV 27 ml as a
colorless oil: UV (in hexane) ␭
244.0, 252.5, 262.5 nm;
max
1H NMR (CDCl3) ␦ 0.027, 0.048, 0.067, and 0.080 (each
3H, each s, 4ϫ SiCH3), 0.544 (3H, s, 18-H3), 0.575 (6H, q,
J ϭ 7.9 Hz, 3ϫ SiCH2), 0.854 (3H, d, J ϭ 6.1 Hz, 21-H3),
0.866 and 0.896 (9H and 9H, each s, 2ϫ Si-t-Bu), 0.947
(9H, t, J ϭ 7.9 Hz, 3ϫ SiCH2CH3), 1.99 (2H, m), 2.18 (1H,
dd, J ϭ 12.8, 8.6 Hz, 4␤-H), 2.34 (1H, dd, J ϭ 13.2, 2.7 Hz,
10␤-H), 2.46 (1H, dd, J ϭ 12.8, 4.4 Hz, 4␣-H), 2.51 (1H,
dd, J ϭ 13.2, 6.0 Hz, 10␣-H), 2.82 (1H, br d, J ϭ 12 Hz,
9␤-H), 4.42 (2H, m, 1␤- and 3␣-H), 4.92 and 4.97 (1H and
1H, each s, ϭ CH2), 5.84 and 6.22 (1H and 1H, each d, J ϭ
11.2 Hz, 7- and 6-H); MS m/z (relative intensity) 784 (Mϩ,
8), 755 (Mϩ - Et, 4), 727 (Mϩ - t-Bu, 6), 652 [100], 520
[31], 366 [49], 199 [23].
colorless oil: UV (in hexane) ␭
244.0, 252.5, 262.5 nm;
max
1H NMR (CDCl3) ␦ 0.026, 0.048, 0.066, and 0.079 (each
3H, each s, 4ϫ SiCH3), 0.544 (3H, s, 18-H3), 0.570 (6H, q,
J ϭ 7.9 Hz, 3ϫ SiCH2), 0.821 (6H, t, J ϭ 7.5 Hz, 26- and
27-CH3), 0.849 (3H, d, J ϭ 6.7 Hz, 21-H3), 0.864 and 0.896
(9H and 9H, each s, 2ϫ Si-t-Bu), 0.946 (9H, t, J ϭ 7.9 Hz,
3ϫ SiCH2CH3), 1.99 (2H, m), 2.18 (1H, dd, J ϭ 12.6, 8.2
Hz, 4␤-H), 2.34 (1H, dd, J ϭ 13.0, 2.9 Hz, 10␤-H), 2.46
(1H, dd, J ϭ 12.6, 4.3 Hz, 4␣-H), 2.51 (1H, dd, J ϭ 13.0,
6.2 Hz, 10␣-H), 2.82 (1H, br d, J ϭ 12 Hz, 9␤-H), 4.43 (2H,
m, 1␤- and 3␣-H), 4.92 and 4.97 (1H and 1H, each s, ϭ
CH2), 5.84 and 6.22 (1H and 1H, each d, J ϭ 11.2 Hz, 7-
and 6-H); MS m/z (relative intensity) 786 (Mϩ, 15), 757
(Mϩ - Et, 22), 729 (Mϩ - t-Bu, 5), 654 [100], 522 [15], 366
[43], 201 [31].
(a) Protected vitamin 10 was hydrolyzed using the AG
50W-X4 resin in methanol as described for 6. The products
were separated by HPLC (6.2-mm X 25-cm Zorbax-Sil
column, 4 ml/min) using hexane/2-propanol (9:1) solvent
system and the following analytically pure 2-methylene-19-
norvitamins were isolated: 1␣-hydroxy-25-dehydrovitamin
12 (17% yield) was collected at RV 13 ml, 1␣-hydroxy-25-
methoxyvitamin 13 (19% yield) was collected at RV 16 ml
and 1␣,25-dihydroxyvitamin 11 (51% yield) was collected
at RV 21 ml.
Protected vitamin 6 (3.5 mg) was dissolved in benzene
(150 ␮l) and the resin (AG 50W-X4, 40 mg; prewashed
with methanol) in methanol (550 ␮l) was added. The mix-
ture was stirred at room temperature under argon for 14 h,
diluted with ethyl acetate/ether (1:1, 4 ml) and decanted.
The resin was washed with ether (8 ml) and the combined
organic phases washed with brine and saturated NaHCO3,
dried (MgSO4) and evaporated. The residue was purified by
HPLC (6.2-mm X 25-cm Zorbax-Sil column, 4 ml/min)
using hexane/2-propanol (9:1) solvent system. Analytically
pure 2-methylene-19-norvitamin 7 (1.22 mg, 62%) was col-
11: UV (in EtOH) ␭max 243.5, 252.0, 262.0 nm; 1H NMR
(CDCl3) ␦ 0.551 (3H, s, 18-H3), 0.859 (3H, d, J ϭ 6.6 Hz,
21-H3), 1.95–2.05 (2H, m), 2.30 (1H, dd, J ϭ 13.5, 8.4 Hz,
10␣-H), 2.33 (1H, dd, J ϭ 13.3, 6.3 Hz, 4␤-H), 2.58 (1H,
dd, J ϭ 13.3, 4.0 Hz, 4␣-H), 2.82 (1H, br d, J ϭ 12 Hz,
9␤-H), 2.85 (1H, dd, J ϭ 13.5, 4.4 Hz, 10␤-H), 4.48 (2H, m,
1␤- and 3␣-H), 5.09 and 5.11 (1H and 1H, each s, ϭ CH2),
5.89 and 6.36 (1H and 1H, each d, J ϭ 11.3 Hz, 7- and 6-H);
MS m/z (relative intensity) 442 (Mϩ, 100), 424 [47], 406
[15], 339 [34], 287 [27], 271 [42], 269 [36], 251 [26]; exact
mass calcd for C29H46O3 442.3447, found 442.3442.
12: UV (in EtOH) ␭max 243.5, 251.5, 262.0 nm; 1H NMR
(CDCl3) ␦ 0.542 (3H, s, 18-H3), 0.847 (3H, d, J ϭ 6.5 Hz,
21-H3), 1.93–2.07 (4H, m), 2.18–2.25 (2H, m), 2.26–2.36
(4H, m), 2.58 (1H, dd, J ϭ 13.3, 3.9 Hz, 4␣-H), 2.82 (1H,
br d, J ϭ 13 Hz, 9␤-H), 2.85 (1H, dd, J ϭ 13.3, 4.5 Hz,
10␤-H), 4.48 (2H, m, 1␤- and 3␣-H), 5.09 and 5.11 (1H and
1H, each s, ϭ CH2), 5.32 (1H, m, w/2 ϭ 7 Hz, 24-H), 5.88
and 6.36 (1H and 1H, each d, J ϭ 11.1 Hz, 7- and 6-H); MS
m/z (relative intensity) 424 (Mϩ, 100), 406 [7], 339 [16],
287 [16], 271 [24], 269 [17], 251 [12]; exact mass calcd for
C29H44O2 424.3341, found 424.3343.
lected at RV 21 ml as a white solid: UV (in EtOH) ␭
243.5, 252.0, 262.0 nm; H NMR (CDCl3) ␦ 0.550 (3H, s,
max
1
18-H3), 0.855 (3H, d, J ϭ 6.8 Hz, 21-H3), 0.860 (6H, t, J ϭ
7.5 Hz, 26- and 27-CH3), 2.00 (3H, m), 2.30 (1H, dd, J ϭ
13.3, 8.6 Hz, 10␣-H), 2.33 (1H, dd, J ϭ 13.3, 6.3 Hz, 4␤-H),
2.58 (1H, dd, J ϭ 13.3, 3.9 Hz, 4␣-H), 2.82 (1H, br d, J ϭ
12 Hz, 9␤-H), 2.85 (1H, dd, J ϭ 13.3, 4.7 Hz, 10␤-H), 4.48
(2H, m, 1␤- and 3␣-H), 5.09 and 5.11 (1H and 1H, each s,
ϭ CH2), 5.89 and 6.36 (1H and 1H, each d, J ϭ 11.3 Hz, 7-
and 6-H); MS m/z (relative intensity) 444 (Mϩ, 100), 426
[35], 408 [11], 397 [19], 379 [32], 341 [31], 287 [32], 273
[43], 269 [28], 251 [22]; exact mass calcd for C29H48O3
444.3603, found 444.3602.
2.5. (20S)-1␣,25-dihydroxy-26,27-dimethylene-2-
methylene-19-norvitamin D3 (11)
Protected vitamin 10 was obtained by Wittig-Horner
coupling of the corresponding Grundmann’s ketone 19
(17.8 mg, 42.3 ␮mol) and phosphine oxide 20 (17.7 mg,
30.4 ␮mol) performed as described for preparation of 6.
Separation of compound 10 was achieved by Sep-Pak fil-
tration and its final purification was performed by HPLC
(6.2-mm X 25-cm Zorbax-Sil column, 4 ml/min) using
hexane/ethyl acetate (99.9:0.1) solvent system. Pure com-
13: UV (in EtOH) ␭max 243.5, 252.0, 262.0 nm; 1H NMR
(CDCl3) ␦ 0.553 (3H, s, 18-H3), 0.858 (3H, d, J ϭ 6.5 Hz,
21-H3), 1.95–2.05 (2H, m), 2.30 (1H, dd, J ϭ 13.3, 8.3 Hz,
10␣-H), 2.33 (1H, dd, J ϭ 13.4, 6.0 Hz, 4␤-H), 2.58 (1H,
dd, J ϭ 13.4, 3.8 Hz, 4␣-H), 2.82 (1H, br d, J ϭ 13 Hz,
9␤-H), 2.85 (1H, dd, J ϭ 13.3, 4.4 Hz, 10␤-H), 3.13 (3H, s,
OCH3), 4.48 (2H, m, 1␤- and 3␣-H), 5.09 and 5.11 (1H and
1H, each s, ϭ CH2), 5.89 and 6.36 (1H and 1H, each d, J ϭ

R.R. Sicinski et al. / Steroids 67 (2002) 247–256
251
11.2 Hz, 7- and 6-H); MS m/z (relative intensity) 456 (Mϩ,
54), 424 [27], 406 [12], 339 [16], 287 [13], 271 [41], 99
[100]; exact mass calcd for C30H48O3 456.3603, found
456.3603.
21-H3), 0.860 (6H, t, J ϭ 7.4 Hz, 26- and 27-CH3), 1.143
(3H, d, J ϭ 6.9 Hz, 2␤-CH3), 2.34 (1H, dd, J ϭ 13.7, 3.3 Hz,
4␤-H), 2.43 (1H, br d, J ϭ 13.7 Hz, 4␣-H), 2.80 (1H, dd,
J ϭ 12 and 4 Hz, 9␤-H), 3.08 (1H, dd, J ϭ 12.8, 4.1 Hz,
10␤-H), 3.50 (1H, m, w/2 ϭ 26 Hz, 1␤-H), 3.90 (1H, m,
w/2 ϭ 11 Hz, 3␣-H), 5.87 and 6.26 (1H and 1H, each d, J ϭ
11.2 Hz, 7- and 6-H); MS m/z (relative intensity) 446 (Mϩ,
39), 428 [46], 410 [12], 399 [30], 381 [17], 289 [37], 273
[50], 271 [31], 253 [27], 69 [100]; exact mass calcd for
C29H50O3 446.3760, found 446.3740.
(b) Protected vitamin 10 (2.0 mg) was dissolved in an-
hydrous THF (250 ␮l) and treated with tetrabutylammo-
nium fluoride (1.0 M in THF, 40 ␮l). The mixture was
stirred under argon at room temperature for 16 h and ex-
tracted with ethyl acetate and ether. The organic extracts
were combined and washed with saturated NaHCO3 and
brine, dried (MgSO4) and evaporated. The residue was
dissolved in hexane/2-propanol (1:1), filtered through a sil-
ica Sep-Pak, and purified by HPLC (6.2-mm X 25-cm
Zorbax-Sil column, 4 ml/min) using hexane/2-propanol
(9:1) solvent system. Pure 2-methylene-19-norvitamin 11
(0.7 mg, 62%) was collected at RV 21 ml.
2.7. (20S)-1␣,25-Dihydroxy-26,27-dimethylene-2␣- and
(20S)-1␣,25-dihydroxy-26,27-dimethylene-2␤-methyl-19-
norvitamin D3 (14 and 15)
Vitamins 14 and 15 were prepared by hydrogenation of
2-methylene compound 11, performed analogously as de-
scribed above for 26,27-dihomo analog 7. The reaction
products were first purified by filtration through silica Sep-
Pak. Then compounds were purified by HPLC (6.2 mm X
25 cm Zorbax-Sil column, 4 ml/min) using hexane/2-pro-
panol (9:1) solvent system. The mixture (ca. 1:1) of 2-meth-
yl-19-norvitamins 14 and 15 (39% yield) gave a single peak
at RV 23 ml. Final separation of both epimers was achieved
by reversed-phase HPLC (6.2-mm X 25-cm Zorbax-ODS
column, 2 ml/min) using methanol/water (85:15) solvent
system. 2␤-Methyl vitamin 15 was collected at RV 19 ml
and its 2␣-epimer 14 at RV 24 ml.
2.6. (20S)-1␣,25-Dihydroxy-2␣- and (20S)-1␣,25-
dihydroxy-2␤-methyl-26,27-dihomo-19-norvitamin D3 (8
and 9)
Tris(triphenylphosphine)rhodium (I) chloride (2.3 mg,
2.5 ␮mol) was added to dry benzene (2.5 ml) presaturated
with hydrogen. The mixture was stirred at room temperature
until a homogeneous solution was formed (ca. 45 min). A
solution of vitamin 7 (1.0 mg, 2.3 ␮mol) in dry benzene (0.5
ml) was then added and the reaction was allowed to proceed
under a continuous stream of hydrogen for 4.5 h. A new
portion of the catalyst (2.3 mg, 2.5 ␮mol) was added and
hydrogen was passed for additional 1 h. Benzene was re-
moved under vacuum, the residue was redissolved in hex-
ane/ethyl acetate (1:1, 2 ml) and applied on Waters silica
Sep-Pak. A mixture of 2-methyl vitamins was eluted with the
same solvent system (20 ml). The compounds were further
purified by HPLC (6.2 mm X 25 cm Zorbax-Sil column, 4
ml/min) using hexane/2-propanol (9:1) solvent system. The
mixture (ca. 1:1) of 2-methyl-19-norvitamins 8 and 9 (0.37 mg,
37%) gave a single peak at RV 23 ml. Separation of both
epimers was achieved by reversed-phase HPLC (6.2-mm X
25-cm Zorbax-ODS column, 2 ml/min) using methanol/water
(85:15) solvent system. 2␤-Methyl vitamin 9 was collected at
RV 21 ml and its 2␣-epimer 8 at RV 27 ml.
14: UV (in EtOH) ␭max 242.5, 251.0, 261.5 nm; 1H NMR
(CDCl3) ␦ 0.534 (3H, s, 18-H3), 0.853 (3H, d, J ϭ 6.6 Hz,
21-H3), 1.134 (3H, d, J ϭ 6.8 Hz, 2␣-CH3), 2.13 (1H, ϳ t,
J ϳ 12 Hz, 4␤-H), 2.22 (1H, br d, J ϭ 13 Hz, 10␤-H), 2.60
(1H, dd, J ϭ 12.8, 4.6 Hz, 4␣-H), 2.80 (2H, m, 9␤- and
10␣-H), 3.61 (1H, m, w/2 ϭ 23 Hz, 3␣-H), 3.96 (1H, m,
w/2 ϭ 11 Hz, 1␤-H), 5.82 and 6.37 (1H and 1H, each d, J ϭ
11.3 Hz, 7- and 6-H); MS m/z (relative intensity) 444 (Mϩ,
84), 426 [53], 289 [36], 271 [58], 253 [19]; exact mass calcd
for C29H48O3 444.3603, found 444.3602.
15: UV (in EtOH) ␭max 242.5, 251.0, 261.5 nm; 1H NMR
(CDCl3) ␦ 0.547 (3H, s, 18-H3), 0.859 (3H, d, J ϭ 6.8 Hz,
21-H3), 1.143 (3H, d, J ϭ 6.8 Hz, 2␤-CH3), 2.34 (1H, dd,
J ϭ 13.7, 3.3 Hz, 4␤-H), 2.43 (1H, br d, J ϭ 13.7 Hz, 4␣-H),
2.80 (1H, br d, J ϭ 12 Hz, 9␤-H), 3.08 (1H, dd, J ϭ 12.9,
4.4 Hz, 10␤-H), 3.50 (1H, m, w/2 ϭ 25 Hz, 1␤-H), 3.90
(1H, m, w/2 ϭ 12 Hz, 3␣-H), 5.87 and 6.26 (1H and 1H,
each d, J ϭ 11.3 Hz, 7- and 6-H); MS m/z (relative inten-
sity) 444 (Mϩ, 75), 426 [59], 289 [34], 271 [59], 253 [18];
exact mass calcd for C29H48O3 444.3603, found 444.3611.
8: UV (in EtOH) ␭max 242.5, 251.0, 261.0 nm; 1H NMR
(CDCl3) ␦ 0.534 (3H, s, 18-H3), 0.851 (3H, d, J ϳ 7 Hz,
21-H3), 0.858 (6H, t, J ϭ 7.5 Hz, 26- and 27-CH3), 1.133
(3H, d, J ϭ 6.9 Hz, 2␣-CH3), 2.13 (1H, ϳ t, J ϳ 12 Hz,
4␤-H), 2.23 (1H, br d, J ϭ 13.4 Hz, 10␤-H), 2.60 (1H, dd,
J ϭ 13.1, 4.4 Hz, 4␣-H), 2.80 (2H, m, 9␤- and 10␣-H), 3.61
(1H, m, w/2 ϭ 26 Hz, 3␣-H), 3.96 (1H, m, w/2 ϭ 13 Hz,
1␤-H), 5.82 and 6.37 (1H and 1H, each d, J ϭ 11.2 Hz, 7-
and 6-H); MS m/z (relative intensity) 446 (Mϩ, 53), 428
[46], 410 [12], 399 [35], 381 [17], 289 [35], 273 [48], 271
[30], 253 [24], 69 [100]; exact mass calcd for C29H50O3
446.3760, found 446.3758.
2.8. Measurement of intestinal calcium transport and
bone calcium mobilization
Twenty day old weanling male rats from the low vitamin
D colony were purchased from the Sprague-Dawley Co.
(Indianapolis, IN) and fed the vitamin D-deficient diet [31],
containing 0.47% calcium and 0.3% phosphorus for 1 week.
9: UV (in EtOH) ␭max 242.5, 251.0, 261.0 nm; 1H NMR
(CDCl3) ␦ 0.546 (3H, s, 18-H3), 0.855 (3H, d, J ϭ 6.8 Hz,

252
R.R. Sicinski et al. / Steroids 67 (2002) 247–256
Table 1
VDR binding propertiesa and HL-60 differentiating activitiesb of 2-substituted side-chain modified (20S)-1␣,25-dihydroxy-19-norvitamin D3 analogs
Compound
Compd.
no.
VDR Binding
ED50 (M)
HL-60 Differentiation
ED50 (M)
Binding
ratio
Activity
ratio
1␣,25-(OH)2D3
2-Methylene-26,27-dihomo-19-nor-
(20S)-1␣,25-(OH)2D3
2␣-Methyl-26,27-dihomo-19-nor-(20S)-
1␣,25-(OH)2D3
2␤-Methyl-26,27-dihomo-19-nor-(20S)-
1␣,25-(OH)2D3
1a
7
8.7 ϫ 10Ϫ10
4.3 ϫ 10Ϫ9
3.1 ϫ 10Ϫ9
4.8 ϫ 10Ϫ9
2.7 ϫ 10Ϫ9
2.9 ϫ 10Ϫ8
1.5 ϫ 10Ϫ8
3.5 ϫ 10Ϫ9
2.3 ϫ 10Ϫ9
1
4.0 ϫ 10Ϫ9
2.6 ϫ 10Ϫ11
6.0 ϫ 10Ϫ11
1.1 ϫ 10Ϫ10
3.6 ϫ 10Ϫ11
4.1 ϫ 10Ϫ9
4.3 ϫ 10Ϫ9
4.4 ϫ 10Ϫ11
3.2 ϫ 10Ϫ10
1
154
67
0.20
0.28
0.18
0.32
0.03
0.06
0.25
0.38
8
9
36
2-Methylene-26,27-dimethylene-19-
nor-(20S)-1␣,25-(OH)2D3
11
12
13
14
15
111
0.98
0.93
91
2-Methylene-26,27-dimethylene-24-
dehydro-19-nor-(20S)-1␣-OH-D3
2-Methylene-26,27-dimethylene-25-
methoxy-19-nor-(20S)-1␣-OH-D3
2␣-Methyl-26,27-dimethylene-19-nor-
(20S)-1␣,25-(OH)2D3
2␤-Methyl-26,27-dimethylene-19-nor-
(20S)-1␣,25-(OH)2D3
12.5
a Competitive binding of 1␣,25-(OH)2D3 and the synthesized vitamin D analogs to the porcine intestinal vitamin D receptor. The experiments were carried
out in triplicate on two different occasions. The ED50 values are derived from dose-response curves and represent the analog concentration required for 50%
displacement of the radiolabeled 1␣,25-(OH)2D3 from the receptor protein. Binding ratio is the ratio of the ED50 for 1␣,25-(OH)2D3 to the analog average
ED50
.
b Induction of differentiation of HL-60 promyelocytes to monocytes by 1␣,25-(OH)2D3 and the synthesized vitamin D analogs. Differentiation state was
determined by measuring the percentage of cells reducing nitro blue tetrazolium (NBT). The experiment was repeated three times. The values ED50 are
derived from dose-response curves and represent the analog concentration capable of inducing 50% maturation. Differentiation activity ratio is the ratio of
the ED50 for 1␣,25-(OH)2D3 to the analog average ED50
.
They were then switched to the reduced calcium diet (0.02%
Ca) for an additional 2 weeks. These animals have no
detectable levels of 25-OH-D3 or 1␣,25-(OH)2D3 in their
plasma as measured by methods described previously [32].
For this first experiment, the indicated rats received a single
i.v. dose of the indicated compound in 0.05 ml of ethanol (data
not shown). In the other experiment, the rats were given the
indicated doses of compounds in 0.1 ml of (95:5) 1,2-pro-
panediol/ethanol by intraperitoneal (i.p.) injection each day for
7 days. In the first experiment, the rats were euthanized at
various times after the dose (data not shown). In the second
experiment, they were sacrificed 24 h after the last dose. The
rats were sacrificed under ether anesthesia by decapitation,
their blood and intestines were collected and used immediately
to determine calcium transport activity and serum calcium
concentration. Calcium was measured in the presence of 0.1%
lanthanium chloride by means of a Perkin-Elmer atomic ab-
sorption spectrometer Model 3110. Intestinal calcium transport
was determined by the everted intestinal sac method using the
proximal 10 cm of intestine as described earlier [31]. Statistical
analysis was by the Student’s t-test [33]. Intestinal calcium
transport is expressed as serosal:mucosal ratio of calcium in the
sac to the calcium in the final incubation medium or S/M. Bone
calcium mobilization represents the rise in serum calcium of
the rats maintained on a very low calcium diet. In that mea-
surement, the rise in serum calcium must arise from bone and
hence is a determination of bone calcium mobilization.
2.9. Measurement of cellular differentiation
Human leukemia HL-60 cells, originally obtained from
ATTC, were plated at 2 ϫ 105 cells per plate, incubated in
Eagle’s modified medium as described previously [34]. The
compounds tested were added in the indicated concentrations
in 0.05 ml of ethanol so that the ethanol concentration never
exceeded 1%. The incubation was carried out for 4 days and at
the end of the 4th day, superoxide production was measured by
nitro blue tetrazolium (NBT) reduction. The number of cells
containing intracellular black-blue formazan deposits was de-
termined by light microscopy using a hemacytometer. At least
200 cells were counted in duplicate per determination. Percent-
age differentiation represents percentage cells providing NBT
reduction appearance. The results were plotted on semilog
paper, and relative differentiation activities of the analogs were
determined by comparison of the compound concentrations
capable of inducing 50% maturation according to the assay.
This method is described in detail elsewhere [34]. The exper-
iment was repeated 3 times and the results are reported as the
mean Ϯ SD.
2.10. Measurement of binding to the porcine intestinal
vitamin D receptor
Porcine intestinal nuclear extract was prepared as de-
scribed earlier [35].

R.R. Sicinski et al. / Steroids 67 (2002) 247–256
253
Table 2
Support of intestinal calcium transport and bone calcium mobilization by 2-substituted side-chain modified (20S)-1␣,25-dihydroxy-19-norvitamin D3
analogs in vitamin D-deficient rats on a low-calcium dieta
Compound
Compd.
no.
Amount
(pmol)
Ca Transport S/M
(mean Ϯ SEM)
Serum Ca
(mean Ϯ SEM)
None (control)
1␣,25-(OH)2D3
0
260
15
32
32
65
15
32
32
65
2.7 Ϯ 0.3b
4.7 Ϯ 0.2b
1a
7
7.2 Ϯ 0.6c
5.6 Ϯ 0.2c
1
1
2-Methylene-26,27-dihomo-
19-nor-(20S)-1␣,25-(OH)2D3
2␣-Methyl-26,27-dihomo-
19-nor-(20S)-1␣,25-(OH)2D3
2-Methylene-26,27-dimethylene-
19-nor-(20S)-1␣,25-(OH)2D3
2␣-Methyl-26,27-dimethylene-
19-nor-(20S)-1␣,25-(OH)2D3
4.0 Ϯ 0.4d
5.3 Ϯ 0.1d
2
2
8.2 Ϯ 0.6d
7.3 Ϯ 0.4d
1
1
5.8 Ϯ 0.4e
5.9 Ϯ 0.2e
2
2
8
8.4 Ϯ 0.8e
9.3 Ϯ 0.2e
1
1
5.6 Ϯ 0.6f
5.4 Ϯ 0.2f
2
2
11
14
5.3 Ϯ 0.5f
6.4 Ϯ 0.2f
1
1
7.9 Ϯ 1.0g
6.9 Ϯ 0.5g
2
2
9.0 Ϯ 1.0g
9.0 Ϯ 0.3g
None (control)
1␣,25-(OH)2D3
0
260
65
260
65
260
65
260
65
3.6 Ϯ 0.4b
5.0 Ϯ 0.1b
1a
9
5.0 Ϯ 0.4c
6.3 Ϯ 0.2c
1
1
2␤-Methyl-26,27-dihomo-
19-nor-(20S)-1␣,25-(OH)2D3
2-Methylene-26,27-dimethylene-
24-dehydro-19-nor-(20S)-1␣-OH-D3
2-Methylene-26,27-dimethylene-
25-methoxy-19-nor-(20S)-1␣-OH-D3
2␤-Methyl-26,27-dimethylene-
19-nor-(20S)-1␣,25-(OH)2D3
4.7 Ϯ 0.6d
5.0 Ϯ 0.0d
2
2
5.2 Ϯ 0.6d
9.9 Ϯ 0.3d
1
1
3.5 Ϯ 0.4e
5.3 Ϯ 0.2e
2
2
12
13
15
3.6 Ϯ 0.5e
5.8 Ϯ 0.2e
1
1
5.5 Ϯ 0.8f
5.7 Ϯ 0.1f
2
2
4.3 Ϯ 0.5f
10.8 Ϯ 0.3f
1
1
4.9 Ϯ 0.6g
5.8 Ϯ 0.2g
2
2
260
5.4 Ϯ 0.7g
9.5 Ϯ 0.1g
a Weanling male rats were maintained on a 0.47% Ca diet for 1 week and then switched to a low-calcium diet containing 0.02% Ca for an additional 3
weeks. During the last week, they were dosed daily with the appropriate vitamin D compound for 7 consecutive days. All doses were administered
intraperitoneally in 0.1 ml propylene glycol/ethanol (95:5). Controls received the vehicle. Determinations were made 24 h after the last dose. There were at
least 6 rats per group. Statistical analysis was done by Student’s t-test. Statistical data: serosal/mucosal (S/M), panel 1, b from c, d2, e2, g1, and g2, P Ͻ 0.001,
b from d1, NS, b from e1, f1 and f2, P ϭ 0.001; panel 2, b from c, d1, d2, f1, g1, and g2, P Ͻ 0.05, b from e1, e2, and f2, NS; serum calcium, panel 1, b from
c, e1, and f1, P Ͻ 0.05, b from d1 and f1, NS, b from d2 and f2, P ϭ 0.005, b from e2 and g2, P Ͻ 0.001; panel 2, b from c and g1, P Ͻ 0.01, b from d1
and e1, NS, b from d2, f2, and g2, P Ͻ 0.001, b from e2 and f1, P ϭ 0.05.
It was diluted and 0.1 mg of protein (200 fmol of binding
activity) in 100 ␮l was used in each tube. 10 000 Cpm of
1␣,25-(OH)2[26,27-3H] D3 (160 Ci/mmol) was added in 2.0
␮l of ethanol. To this was added either standard radioinert
1␣,25-(OH)2D3 at various concentrations or the indicated
analog at various concentrations in 5 ␮l ethanol. The mix-
ture was incubated at room temperature for 4 h on a shaker
and then 100 ␮l of hydroxyapatite (50% slurry) added. The
sample was vortexed at 5 min intervals for 15 min on ice.
The hydroxyapatite was then washed 3 times by adding 0.5
ml TE 5% Triton X100, centrifuging at 200ϫ g for 5 min
and aspirating the supernatant. The radioactivity bound to
the hydroxyapatite was determined by liquid scintillation
counting in Bio-Safe II scintillation fluid. The values are
plotted versus concentration of analog or standard. Each
value represents triplicate values. The displacement exper-
iments were carried out in triplicate on two different occa-
sions.
synthesized by us previously, a choice of the synthetic
method, as Wittig-Horner coupling, was obvious. Tertiary
hydroxy group in 16 and 18 was silylated and the protected
Grundmann ketones 17 and 19 were coupled with the con-
jugate base of phosphine oxide 20 to give protected 2-meth-
ylene-19-norvitamin D analogs 6 and 10, respectively.
These silylated derivatives were hydrolyzed using a slurry
of AG 50W-X4 cation-exchange resin in methanol, success-
fully used by us for hydroxyl deprotection in many similar
vitamin D analogs [19,20]. In the case of the 26,27-dihomo
compound the removal of silyl groups provided vitamin 7 in
62% yield, however, analogous hydrolysis in the 26,27-
dimethylene series yielded, in addition to the desired 11,
also minor products being the result of tertiary 25-hydroxyl
elimination (12) and etherification (13). In the latter case a
use of tetrabutylammonium fluoride for silyl group removal
was advantageous, providing compound 11 in 65% yield.
Selective hydrogenation of the A-ring exomethylene group
in vitamins 7 and 11 was achieved in a homogeneous
hydrogenation process, catalyzed by tris(triphenylphos-
phine)-rhodium(I) chloride (Wilkinson’s catalyst), giving
the corresponding pairs of 2␣- and 2␤-methyl derivatives
(8, 9 and 14, 15, respectively) in ca. 40% yield. Assignment
of the configuration at C-2 in these compounds was based
3. Results
3.1. Synthetic studies
Considering an access to the CD-ring synthons 16 and 18
as well as availability of the A-ring fragment 20 [20],
1
on analysis of their H NMR spectra indicating close sim-

254
R.R. Sicinski et al. / Steroids 67 (2002) 247–256
Fig. 3. Conformational equilibrium in ring A of 2-methylene-19-norvitamins (a) and the preferred, energy minimized A-ring conformations of the synthesized
2␤-methyl (b) and 2␣-methyl (c) analogs. The corresponding percentage populations of conformers are calculated for model compounds lacking side chain
(data taken from Ref. [20]) and given for room temperature (25°C).
ilarity to those of the previously synthesized 2-methyl-19-
norvitamins [20].
(20S)-1␣,25-(OH)2D3, synthesized in the present inves-
tigation, proved to be approximately 100 times more
active than the natural hormone. 2␤-Methyl compounds 9
and 15 were 3–8 times less active than the corresponding
2␣-epimers 8 and 14, whereas 24-dehydro and 25-me-
thoxy analog (12 and 13) showed similar activity as
1␣,25-(OH)2D3. The most active in vitro vitamins (7, 8,
11 and 14), when tested in vivo in rats, showed also the
highest calcemic activity. Especially potent were 2-meth-
ylene analog in 26,27-dihomo series (7) and 2␣-methyl-
26,27-dimethylene compound 14. Their calcemic activity
appeared to be at least 10 times higher than that of
1␣,25-(OH)2D3 and undoubtedly these compounds be-
long to the most potent vitamin analogs tested in our
laboratory. Compounds 12 and 13 showed bone calcium
mobilization activity similar or higher than 1␣,25-
(OH)2D3 while having little activity in the intestine.
Rather unexpectedly, 2␤-methyl substituted 19-norvita-
mins 9 and 15 were found to be more calcemic than the
hormone 1, being particularly effective in bone calcium
mobilization.
3.2. Biologic evaluation
The synthesized vitamins were tested for their ability
to bind the porcine intestinal vitamin D receptor (VDR).
The effects of the experimental compounds have been
compared to that of 1␣,25-(OH)2D3 whose dose was
adjusted to provide less than 50% of a maximal response
based on many prior experiments determining dose re-
sponse curves in vivo. The results presented in Table 1
show that, with an exception of significantly less effec-
tive analogs 12 and 13, all obtained compounds were
only slightly (3–5 times) less potent than the natural
hormone 1a in displacement of the radiolabeled 1␣,25-
(OH)2D3 from the receptor protein. Also, cellular activity
of these compounds has been assessed by studying their
ability to induce differentiation of human promyelocytic
HL-60 cells into monocytes. All the side chain homolo-
gated 2-methylene and 2␣-methyl analogs of 19-nor-

R.R. Sicinski et al. / Steroids 67 (2002) 247–256
255
[3] DeLuca HF. The vitamin D story: a collaborative effort of basic
science and clinical medicine. FASEB J 1988;2:224–36.
[4] Ikekawa N. Structures and biological activities of vitamin D metab-
olites and their analogs. Med Res Rev 1987;7:333–66.
[5] Bouillon R, Okamura WH, Norman AW. Structure-function relation-
ships in the vitamin D endocrine system. Endocrine Rev 1995;16:
200–57.
[6] Walters MR. Newly identified actions of the vitamin D endocrine
system. Endocrine Rev 1992;13:719–64.
[7] Shabahang M, Buffan A, Nolla J, Schumaker L, Brenner R, Buras R,
Nauta R, Evans SRT. The effect of 1,25-dihydroxyvitamin D3 on the
growth of soft tissue sarcoma cells as mediated by the vitamin D
receptor. Ann Surg Oncol 1996;3:144–9.
[8] Evans S, Houghton A, Schumaker L, Brenner R, Buras R, Davoodi F,
Nauta R, Shabahang M. Vitamin D receptor and growth inhibition by
1,25-dihydroxyvitamin D3 in human malignant melanoma cell lines.
J Surg Res 1996;61:127–33.
[9] Connor RI, Rigby WFC. 1,25-Dihydroxyvitamin D3 inhibits produc-
tive infection of human monocytes by HIV-1. Biochem Biophys Res
Commun 1991;176:852–9.
[10] Yu XP, Hustmyer FG, Garvey WT, Manolagas SC. Demonstration of
a 1,25-dihydroxyvitamin D3-responsive protein in human lympho-
cytes: immunologic crossreactivity and inverse regulation with the
vitamin D receptor. Proc Natl Acad Sci USA 1991;88:8347–51.
[11] Suda T, Shinki T, Takahashi N. The role of vitamin D in bone and
intestinal cell differentiation. Annu Rev Nutr 1990;10:195–211.
[12] Suda T. The role of 1␣,25-dihydroxyvitamin D3 in the myeloid cell
differentiation. Proc Soc Exp Biol Med 1989;191:214–20.
[13] Ostrem VK, DeLuca HF. The vitamin D-induced differentiation of
HL-60 cells: structural requirements. Steroids 1987;49:73–102.
[14] Tsoukas CD, Provvedini DM, Manolagas SC. 1,25-Dihydroxyvita-
min D3: a novel immunoregulatory hormone. Science 1984;224:
1438–40.
[15] Koeffler HP, Amatruda T, Ikekawa N, Kobayashi Y, DeLuca HF.
Induction of macrophage differentiation of human normal and leuke-
mic myeloid stem cells by 1,25-dihydroxyvitamin D3 and its fluori-
nated analogs. Cancer Res 1984;44:5624–8.
[16] Perlman KL, Swenson RE, Paaren HE, Schnoes HK, DeLuca HF.
Novel synthesis of 19-nor-vitamin D compounds. Tetrahedron Lett
1991;32:7663–6.
[17] DeLuca HF, Schnoes HK, Perlman KL, Sicinski RR, Prahl JM.
Wisconsin Alumni Research Foundation, U.S.A. 19-Norvitamin D
compounds. Eur Pat Appl EP 387,077. 1990 Sept 12.; Chem Abstr
1991;144,185853 u.
[18] Perlman KL, Sicinski RR, Schnoes HK, DeLuca HF. 1␣,25-Dihy-
droxy-19-nor-vitamin D3, a novel synthetic vitamin D-related com-
pound with potential therapeutic activity. Tetrahedron Lett 1990;31:
1823–4.
[19] Sicinski RR, Perlman KL, DeLuca HF. Synthesis and biological
activity of 2-hydroxy and 2-alkoxy analogs of 1␣,25-dihydroxy-19-
norvitamin D3. J Med Chem 1994;37:3730–8.
[20] Sicinski RR, Prahl JM, Smith CM, DeLuca HF. New 1␣,25-dihy-
droxy-19-norvitamin D3 compounds of high biological activity: syn-
thesis and biological evaluation of 2-hydroxymethyl, 2-methyl, and
2-methylene analogues. J Med Chem 1998;41:4662–74.
[21] Nordin BEC, Morris HA. Osteoporosis and vitamin D. J Cell Bio-
chem 1992;49:19–25.
4. Discussion
Our previous results indicated that introduction of
2-methylene and 2␣-methyl substituent into (20S)-1␣,25-
dihydroxy-19-norvitamin D3 skeleton increases calcemic
potency of the analogs 3b and 4b [20]; preferential activity
on bone was found for the former compound. The presented
results show that elongation of the side chain in these
compounds can also result in the vitamins possessing ex-
ceptional calcemic activity. Similarly as (20S)-2␣-methyl
vitamin 4b, possessing normal size of the side chain, the
synthesized analogs with elongated side chain (8 and 14)
were characterized by exceptional potency, both on intesti-
nal calcium transport and bone calcium mobilization.
2-Methylene analogs with homologated side chain (7 and
11) also possessed very high potency in both assays, thus
showing somewhat different activity profile than 3b. An-
other interesting observation was the fact that 2␤-methyl
analogs 9 and 15 were also calcemic. Spectroscopic data
and molecular modeling indicate [20] that the A ring of
2-methylene-19-norvitamin consists of an equilibrium mix-
ture containing ca. 57% of equatorial 1␣-OH conformer
(Fig. 3a). We previously reported that 2␤-methyl substituted
19-norvitamins, having the A-ring conformational equilib-
rium shifted toward the form with an equatorial orientation
of 1␣-hydroxy group (Fig. 3b), showed very weak, if any,
biologic activity [20]. Although 2␤-methyl-19-norvitamins
9 and 15 are significantly less active than their 2␣-methyl
substituted counterparts 8 and 14, possessing the axial ori-
entation of their 1␣-OH in the strongly preferred chair
conformation of ring A (Fig. 3c), this difference is not so
drastic as in the case of the analogous pair 4b and 5b. These
findings point out the importance of 19-norvitamin D side
chain structure indicating that the problem how orientation
of the 1␣-substituent influences biologic activity can be
more complex than anticipated. Certainly, an extended side
chain affords considerable biologic activity even when the
1␣-OH is in the equatorial disposition.
Acknowledgments
We acknowledge the assistance of Rowland Randall in
recording the mass spectra. This work was supported in part
by funds from the National Foundation for Cancer Research
and the Wisconsin Alumni Research Foundation.
References
[22] Honda A, Mori Y, Otomo S, Ishizuka S, Ikekawa N. Effects of novel
26,27-dialkyl analogs of 1␣,25-dihydroxyvitamin D3 on differentia-
tion-inducing activity of human promyelocytic leukemia (HL-60)
cells in serum-supplemented or serum-free culture. Steroids 1991;56:
142–7.
[23] Ikekawa N. Chemical synthesis of vitamin D analogs with selective
biological activities: In: Norman AW, Schaefer K, Grigoleit HG,
Herrath D (Eds.), Vitamin D: Molecular, Cellular and Clinical En-
docrinology. Berlin: Walter de Gruyter, 1988. pp. 25–33.
[1] DeLuca HF, Burmester J, Darwish H, Krisinger J. Molecular mech-
anism of the action of 1,25-dihydroxyvitamin D3. In: Hansch C,
Sammes PG, Taylor JB (Eds.), Comprehensive Medicinal Chemistry
(Vol. 3). Oxford: Pergamon Press, Oxford, 1991. pp. 1129–43.
[2] Reichel H, Koeffler HP, Norman AW. The role of the vitamin D
endocrine system in health and disease. N Engl J Med 1989;320:980–
91.

256
R.R. Sicinski et al. / Steroids 67 (2002) 247–256
[24] Ikekawa N, Eguchi T, Hara N, Takatsuto S, Honda A, Mori Y,
Otomo S. 26,27-Diethyl-1␣,25-dihydroxyvitamin D3 and 24,24-
difluoro-1␣,25-dihydroxyvitamin D3. Chem Pharm Bull 1987;35:
4362–5.
[25] Eguchi T, Ikekawa N, Sumitani K, Kumegawa M, Higuchi S, Otomo
S. Effect on carbon lengthening at the side chain terminal of 1␣,25-
dihydroxyvitamin D3 for calcium regulating activity. Chem Pharm
Bull 1990;38:1246–9.
[26] Miyahara T, Harada M, Miyata M, Sugure A, Ikemoto Y, Takamura
T, Higuchi S, Otomo S, Kozuma H, Ikekawa N. Calcium regulating
activity of 26,27-dialkyl analogs of 1␣,25-dihydroxyvitamin D3. Cal-
cif Tissue Int 1992;51:218–22.
[29] Binderup L. Immunological properties of vitamin D analogues and
metabolites. Biochem Pharmacol 1992;43:1885–92.
[30] Paaren HE. Tetrionics, Inc., U.S.A. Preparation of 14-epi-19-nor-
vitamin D compounds with cell differentiation activity. US Patent
5936105. 1999 Aug 10.; Chem Abstr 131,144749x.
[31] Suda T, DeLuca HF, Tanaka Y. Biological activity of 25-hydroxy-
ergocalciferol in rats. J Nutr 1970;100:1049–52.
[32] Uhland-Smith A, DeLuca HF. 1,25-Dihydroxycholecalciferol ana-
logs cannot replace vitamin D in normocalcemic male rats. J Nutr
1993;123:1777–85.
[33] Snedecor GW, Cochran WG. Statistical Methods, 6th edition. Ames,
IA: Iowa State University Press, 1967.
[27] Calverley MJ, Binderup L. Synthesis and biological evaluation of
MC 1357, a new 20-epi-23-oxa-1␣,25-dihydroxy-vitamin D3 an-
alogue with potent non-classical effects. BioMed Chem Lett 1993;
3:1845–8.
[34] Ostrem VK, Lau WF, Lee SH, Perlman K, Prahl J, Schnoes HK,
DeLuca HF, Ikekawa N. Induction of monocytic differentiation of
HL-60 cells by 1,25-dihydroxyvitamin D analogs. J Biol Chem 1987;
262:14164–71.
[28] Binderup L, Latini S, Binderup E, Bretting C, Calverley M, Hansen
K. 20-Epi-vitamin D3 analogues: a novel class of potent regulators of
cell growth and immune responses. Biochem Pharmacol 1991;42:
1569–75.
[35] Dame MC, Pierce EA, Prahl JM, Hayes CE, DeLuca HF. Monoclonal
antibodies to the porcine intestinal receptor for 1,25-dihydroxyvita-
min D3: interaction with distinct receptor domains. Biochemistry
1986;25:4523–34.