26- and 27-Methyl groups of 2-substituted, 19-nor-1α,25- dihydroxylated vitamin D compounds are essential for calcium mobilization in vivo

Article abstract of DOI:10.1016/j.bioorg.2013.01.001

Twelve new analogs of 19-nor-1α,25-dihydroxyvitamin D₃ 6-17, were prepared by a multi-step procedure from known alcohols 18 and 19. We have examined the influence of removing two methyl groups located at C-25, as well as the 25-hydroxy group, o

Full text of DOI:10.1016/j.bioorg.2013.01.001

Bioorganic Chemistry 47 (2013) 9–16
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
26- and 27-Methyl groups of 2-substituted, 19-nor-1a,25-dihydroxylated
vitamin D compounds are essential for calcium mobilization in vivo
⇑
Pawel Grzywacz, Lori A. Plum, Margaret Clagett-Dame, Hector F. DeLuca
Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 September 2012
Available online 9 February 2013
Twelve new analogs of 19-nor-1a,25-dihydroxyvitamin D3 6–17, were prepared by a multi-step proce-
dure from known alcohols 18 and 19. We have examined the influence of removing two methyl groups
located at C-25, as well as the 25-hydroxy group, on the biological in vitro and in vivo biological activity.
Surprisingly, removal of the 26- and 27-methyl groups from either the 2
a-methyl or 2-methylene-19-
Keywords:
Analogs of 19-nor-1
D3
26- and 27-Methyl groups
Bone mobilization
nor-1 ,25-dihydroxyvitamin D3 reduced vitamin D receptor binding, HL-60 differentiation, and 25-
a
a,25-dihydroxyvitamin
hydroxylase transcription in vitro only slightly to moderately (compounds 6–13). However, these com-
pounds were devoid of in vivo bone mobilization activity and had markedly reduced activity on intestinal
calcium transport. The analogs 14–17 with a 2b-methyl substitution had little or no activity in vitro and
in vivo as expected from previous work.
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
2. Results and discussion
1a,25-Dihydroxyvitamin D3 (1,25-(OH)2D3, calcitriol, 1; Fig. 1),
2.1. Chemistry
the hormonally active form of vitamin D3, possesses a broad spec-
trum of biological activities. Its function consists of regulation of
calcium homeostasis [1–4], immunomodulation [5,6], cell differen-
tiation and antiproliferation [7–10]. Potential therapeutic applica-
tions of 1,25-(OH)2D3, which are associated with the inhibition of
cell growth and with the stimulation of cell differentiation, are
however hampered by the danger of hypercalcemia. This has re-
sulted in attempts at modification of its structure to separate calce-
mic activities from other biological responses [11,12]. We have
focused on side chain modifications [13,14]. In 2010, we reported
the biological properties of 2-methyl and 2-methylene-19,26-dinor
analogs 2–5 (Fig. 1) with a fixed 25-hydroxy group [15]. The study
clearly shows that removal of one methyl from carbon-25 of the
19-norvitamin D3 compounds markedly reduces the calcemic
activity from either bone or intestine. The lack of that methyl
group had little impact on VDR binding, HL-60 differentiation
and in vitro transcription. These results suggest that in vivo calce-
mic activity might be further reduced by the elimination of both
26- and 27-methyl groups. We now report several new analogs
(6–17) produced by removing two 26,27-methyl groups, as well
as 25-hydroxy group, from the 2-carbon-substituted 19-nor-1,25-
(OH)2D3 and its 20S isomer.
The convergent synthesis of new analogs 6–17 was based on the
Lythgoe type Horner-Wittig olefination reaction [16], which was
successfully utilized by us earlier for preparation of the vitamin D
compounds2–5 [15]. This approach required using new Grundmann
type ketones 31–34 (Scheme 1), which we decided to prepare by
attaching a three-carbon fragment to iodides (20 and 21) in one step,
based on an efficient method [17] introduced by Mourino in the vita-
min D field [18]. The needed compounds (20 and 21) were easily ob-
tained from the known alcohols 18 and 19 [13]. The 22-hydroxy
group of the compound 18 was selectively tosylated and then con-
verted into the corresponding iodide 20. To improve the efficacy of
the synthesis of iodide 21 the primary alcohol 19 was treated with
triphenylphosphine, iodine and imidazole [19]. The introduction of
side chains was realized via Ni(0) catalyzed oxidative addition [17]
of iodides (20 and 21) to iso-propyl acrylate, which provided iso-pro-
pyl esters 22 and 23. The removal of the tert-butyldimethylsilyl pro-
tective group from the ether 23 under acidic conditions gave the
secondary alcohol 24. The iso-propyl esters (22 and 24) were treated
with lithium aluminum hydride to produce diols 25 and 26. The pri-
mary hydroxy groups of compounds (25 and 26) were selectively
protected as tert-butyldimethylsilyl ethers 27 and 28. The same
25-hydroxy groups of diols (25 and 26) were selectively converted
into tosylates, which were further reduced with lithium aluminum
hydride to afford the C,D-ring fragments 29 and 30, possessing alkyl
side chains. The secondary alcohols 27–30were oxidizedwith pyrid-
inium dichromate in the presence of pyridinium para-toluenesulfo-
nate to the corresponding ketones 31–34. The Horner-Wittig
⇑
Corresponding author. Fax: +1 608 262 7122.
E-mail address: deluca@biochem.wisc.edu (H.F. DeLuca).
0045-2068/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2013.01.001

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P. Grzywacz et al. / Bioorganic Chemistry 47 (2013) 9–16
Fig. 1. Chemical structures of 1a,25-dihydroxyvitamin D3 (calcitriol, 1) and 19-nor analogs 2–17.
Scheme 1. Synthesis of the Grundmann ketones 31–34. Reagents and yields: (i) (1) TsCl, DMAP, Et3N, CH2Cl2; (2) KI, acetone, (20 85%); (ii) (1) I2, Ph3P, imidazole, CH2Cl2;
(2) 19, CH2Cl2, (21 100%); (iii) (1) Zn, pyridine, iso-propyl acrylate, NiCl2.6H2O; (2) 20 or 21, pyridine, (22 83%, 23 82%); (iv) aqueous HF, THF, MeCN, (24 82%); (v) (1) LiAlH4,
THF; (2) aqueous KNa tartrate, (25 94%, 26 96%); (vi) TBSCl, Et3N, CH2Cl2, (27 95%, 28 94%); (vii) (1) TsCl, DMAP, Et3N, CH2Cl2; (2) LiAlH4, THF; (3) aqueous KNa tartrate, (29
90%, 30 84%); (viii) PDC, PPTS, CH2Cl2, (31 89%, 32 90%, 33 95%, 34 82%).
reaction between the corresponding C,D-fragments (31–34) and the
anion, generated from the phosphine oxide 35 by phenyllithium,
produced the protected vitamin D compounds 36–39 (Scheme 2).
Thesilyl-protective groupswere cleavedin thepresenceofhydroflu-
oric acid and after the final purification by HPLC the target vitamin D
analogs 6–9 were obtained. The homogenous catalytic hydrogena-

P. Grzywacz et al. / Bioorganic Chemistry 47 (2013) 9–16
11
Scheme 2. Syntheses of the vitamin D analogs 6–17. Reagents and yields: (i) (1) 35, PhLi, THF; (2) 31, 32, 33 or 34, THF, (36 88%, 37 80%, 38 91%, 39 79%); (ii) aqueous HF, THF,
MeCN, (6 79%, 7 87%, 8 75%, 9 77%); (iii) H2, (Ph3P)3RhCl, PhH, (10 36%, 11 26%, 12 38%, 13 40%, 14 31%, 15 22%, 16 34%, 17 35%).
tion of 2-methylene compounds 6–9, in the presence of tris(triphen-
ylphosphine)rhodium(I) chloride, provided approximately an equi-
molar mixture of 2-methyl-19-norvitamins 10–17, which were
easily separated by HPLC.
calcium from bone was essentially eliminated by removal of the
26- and 27-methyl groups (Fig. 2). Although intestinal calcium
transport was not completely eliminated by removal of the two
terminal methyl groups, it was only significant at the higher doses
(100Â or more that of 1
a,25-(OH)2D3 1) (Fig. 3). Of some interest is
the fact that removal of the 25-hydroxy from the analogs that are
devoid of the 26- and 27-methyl groups (8, 9, 12, 13) actually in-
creases in vivo activity on intestine. The decrease in activity by a
hydroxyl on analogs of 1,25-(OH)2D3 having a shortened side chain
has been noted previously [27] but remains unexplained. Since
binding of the VDR appeared unchanged by the 25-hydroxyl in this
series, while in vivo activity on intestinal calcium transport is de-
creased, we suggest that the hydroxyl on the side chain is more
easily conjugated for excretion-reducing biological activity.
2.2. Biological evaluation
The removal of the 26- and 27-methyl groups from the side
chain of 19-nor-1
2-methylene (6–9) or 2
a
,25-dihydroxyvitamin D3 compounds having a
-methyl (10–13) substitution had little
a
or moderate impact on binding to the vitamin D receptor (VDR)
(Table 1). However, in two cell-based assays (differentiation of
HL-60 cells and transactivation of the CYP24A1 gene in rat bone
cells), activity levels were reduced up to 200Â (Table 1). As ex-
pected, 2b-methyl substitution of all derivatives (14–17) resulted
in a drastic reduction in all in vitro activities as previously noted
[13–15,20–24]. The 20S-configuration increased in vitro activity
of all analogs (7, 9, 11, 13, 15, 17) by 1 or 2 orders of magnitude
above the 20R counterparts (6, 8, 10, 12, 14, 16). This effect has
been noted previously with other analogs [13–15,24–26]. How-
ever, the ability of analogs (6–13) to support the mobilization of
3. Conclusions
Removal of the 26- and 27-methyl groups of either 2a-methyl
(6–9) or 2-methylene-19-nor-1,25-(OH)2D3 (10–13) has little or
moderate effect on in vitro binding to the VDR. However, in vivo
the absence of these methyl groups eliminated the ability to mobi-

Table 1
VDR binding propertiesa, HL-60 differentiating activitiesb, and transcriptional activitiesc of the vitamin D analogs 6–17.
Compound
Structure of a side chain (R)
Compd no.
VDR binding
HL-60 differentiation
EC50
24-OHase transcription
EC50
Ki
Ratio
Ratio
Ratio
1a,25-(OH)2D3
–
1
–
1 Â 10À10
M
M
1
1
2 Â 10À9
M
M
1
0.04
2 Â 10À10
M
M
1
1 Â 10À10
8 Â 10À11
7 Â 10À12
0.035
6
7
2 Â 10À10
3 Â 10À10
M
M
2
3
2 Â 10À8
3 Â 10À9
M
10
4 Â 10À9
M
20
M
1.5
5 Â 10À10
M
2.5
8
3 Â 10À10
3 Â 10À10
M
M
3
1 Â 10À7
5 Â 10À9
3 Â 10À8
M
M
M
50
2.5
15
4 Â 10À8
3 Â 10À9
6 Â 10À9
M
200
15
9
3
M
10
1 Â 10À9
M
10
M
30
11
1 Â 10À10
M
M
1
7 Â 10À9
M
3.5
7 Â 10À10
M
3.5
12
13
2 Â 10À9
M
20
2
2 Â 10À7
2 Â 10À8
M
M
100
10
5 Â 10À8
1 Â 10À8
M
250
50
2 Â 10À10
M
14
15
5 Â 10À8
5 Â 10À8
M
500
500
8 Â 10À7
1 Â 10À6
M
M
400
500
6 Â 10À7
6 Â 10À8
M
M
3000
300
M
16
17
ꢀ4 Â 10À6
M
ꢀ40,000
2 Â 10À5
5 Â 10À7
M
M
10,000
250
2 Â 10À6
3 Â 10À7
M
M
ꢀ10000
1 Â 10À7
M
1000
1500
a
Competitive binding of 1
a
,25-(OH)2D3 (1) and the synthesized vitamin D analogs to the full-length recombinant rat vitamin D receptor. The Ki values are derived from dose–response curves and represent the inhibition
,25-(OH)2D3 is present at 1 nM and a Kd of 0.2 nM is used. The binding ratio is the average ratio of the analog Ki to the Ki for 1 ,25-(OH)2D3.
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 EC50 values are derived from dose–response curves and represent the analog concentration capable of inducing 50% maturation. The differentiation activity ratio is the average ratio of the analog EC50 to the
EC50 for 1 ,25-(OH)2D3.
Transcriptional assay in rat osteosarcoma cells stably transfected with a 24-hydroxylase gene reporter plasmid. The EC50 values are derived from dose–response curves and represent the analog concentration capable of
increasing the luciferase activity by 50%. The luciferase activity ratio is the average ratio of the EC50 for the analog to the EC50 for 1 ,25-(OH)2D3. All the experiments were carried out in duplicate on at least two different occasions.
constant when radiolabeled 1
a
a
b
a
a
c
a

P. Grzywacz et al. / Bioorganic Chemistry 47 (2013) 9–16
13
Fig. 2. Those compounds lacking a 25-hydroxy group can mobilize calcium from bone stores, but only at levels more than 1000 times that of the natural hormone. Rats were
made vitamin D-deficient, fed a diet nearly devoid of calcium and administered four intraperitoneal injections of the listed analogs prior to collecting blood for serum calcium
analysis.
lize bone calcium. Although this did not completely eliminate the
stimulation of intestinal calcium transport, it did decrease potency
by at least two orders of magnitude.
measured in chloroform using a Perkin–Elmer Model 343 polar-
imeter at 22 °C. Ultraviolet (UV) absorption spectra were re-
corded
with
a
Perkin–Elmer
Lambda
3B
UV–Vis
Converting the 2
a
-methyl to a 2b-methyl configuration in this
spectrophotometer in ethanol or hexane. 1H nuclear magnetic
resonance (NMR) spectra were recorded in deuteriochloroform
at 400 and 500 MHz with Bruker Instruments DMX-400 and
DMX-500 Avance console spectrometers. 13C nuclear magnetic
resonance (NMR) spectra were recorded in deuteriochloroform
at 100 and 125 MHz with the same Bruker Instruments. Chemical
shifts (d) in parts per million are quoted relative to internal Me4Si
(d 0.00). Numbers in parentheses following the chemical shifts in
the 13C NMR spectra refer to the number of attached hydrogens
as revealed by DEPT experiments. Electron impact (EI) mass spec-
tra were obtained with a Micromass AutoSpec (Beverly, MA)
instrument. High-performance liquid chromatography (HPLC)
was performed on a Waters Associates liquid chromatograph
equipped with a Model 6000A solvent delivery system, Model
U6K Universal injector and Model 486 tunable absorbance detec-
tor. The known alcohols 18 and 19 were prepared according to
the published procedures [13]. Solvents were dried and distilled
series of compounds (14–17) drastically reduces activity both
in vitro and in vivo. This has been noted in a previously prepared
group of both 19-nor compounds [28]. The 20S-modification in-
creases potency both in vivo and in vitro as previously noted in
other series of vitamin D analogs [29,30]. However, increased po-
tency is not the result of improved binding to the receptor. No
explanation can be provided for this finding but is significant in
designing analogs of 1a,25-(OH)2D3. The 25-hydroxyl group tradi-
tionally improves both binding to the receptor and both in vivo and
in vitro potency. However, if the 26- and 27-methyl groups are ab-
sent, the 25-hydroxyl actually reduces potency in vivo. This sug-
gests that the 25-hydroxyl requires surrounding hydrophobic
groups for expression of potency [31].
Overall this series (6–17) illustrates the importance of the 26-
and 27-methyls (and their hydrophobic properties) of the vitamin
D analogs for a range of biological activities but especially its calce-
mic activities.
following standard procedures.
A designation ‘‘(volume + vol-
ume)’’, which appears in general procedures, refers to an original
volume plus a rinse volume.
4. Experimental section
All final vitamin D analogs synthesized by us gave single sharp
peaks on HPLC and they were judged at least 99% pure. Two HPLC
systems (straight- and reversed-phase) were employed as indi-
cated in Table 2 (Supporting Information).
4.1. Chemistry
Melting points (uncorrected) were determined on a Thomas-
Hoover capillary melting-point apparatus. Optical rotations were

14
P. Grzywacz et al. / Bioorganic Chemistry 47 (2013) 9–16
Fig. 3. All vitamin D analogs with an abbreviated side chain possess lower intestinal calcium transport activity compared to 1,25(OH)2D3. Ex-vivo measurements of gut
transport were made by preparing everted gut sacs from vitamin D-deficient rats given four intraperitoneal injections of the described compounds.
4.2. Procedure for the synthesis of compound 20
ture was stirred under argon at 0 °C for 15 min and then a solution
of the alcohol 19 (149 mg, 0.46 mmol) in anhydrous methylene
chloride (3 + 1 mL) was added. The reaction mixture was stirred
at 0 °C for 20 min and at room temperature for 18 h. It was washed
with water, dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy on silica (0 ? 3% ethyl acetate/hexane) to give the product
21 (201 mg, >96% yield).
To a stirred solution of the diol 18 (133 mg, 0.63 mmol), DMAP
(10 mg, 0.08 mmol) and triethylamine (250 lL, 182 mg, 1.8 mmol)
in anhydrous methylene chloride (5 mL), p-toluenesulfonyl chlo-
ride (180 mg, 0.94 mmol) was added at 0 °C. A cooling bath was re-
moved and the reaction mixture was stirred at room temperature
for 22 h. Methylene chloride was added and the mixture was
washed with water, dried over anhydrous Na2SO4 and concen-
trated under reduced pressure. The residue was purified by column
chromatography on silica (6 ? 12% ethyl acetate/hexane) to give
the primary tosylate (224 mg, 97% yield).
To a stirred solution of the tosylate (224 mg, 0.61 mmol) in
anhydrous acetone (20 mL), potassium iodide (3 g, 18 mmol) was
added. The reaction mixture was refluxed for 18 h. Water
(30 mL) was added to dissolve salts and the mixture was extracted
with ethyl acetate. Combined organic phases were washed with a
saturated aqueous Na2S2O3 solution, dried over anhydrous Na2SO4
and concentrated under reduced pressure. The residue was puri-
fied by column chromatography on silica (4 ? 6% ethyl acetate/
hexane) to give the iodide 20 (172 mg, 88% yield).
4.4. General procedure for the synthesis of compounds 22 and 23
To a stirred mixture of zinc powder (3.7–5.0 equiv), iso-propyl
acrylate (3.7–4.8 equiv) and anhydrous pyridine (4 mL), nickel
(II) chloride hexahydrate (1.0–1.2 equiv) was added at 50 °C. The
resulting mixture was warmed to 65 °C and stirred for 2 h. After
cooling to 0 °C, a solution of the iodide 20 or 21 (1 equiv) in anhy-
drous pyridine (2 + 1 mL) was added and the reaction mixture was
stirred at room temperature for 17 h. It was diluted with ethyl ace-
tate and the precipitate was filtered off through a pad of Celite. The
filtrate was washed with aqueous 5% solution of HCl and water,
dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The residue was purified by column chromatography
on silica (4 ? 6% ethyl acetate/hexane) to give the ester 22 or 23.
4.3. Procedure for the synthesis of compound 21
To a solution of triphenylphosphine (482 mg, 1.84 mmol) and
imidazole (250 mg, 3.68 mmol) in anhydrous methylene chloride
(15 mL), a solution of iodine (471 mg, 1.85 mmol) in anhydrous
methylene chloride (30 mL) was added at 0 °C. The reaction mix-
4.5. Procedure for the synthesis of compound 24
To a solution of the silyl ether 23 (94 mg, 0.22 mmol) in THF
(3 mL) and acetonitrile (3 mL), a solution of aqueous 48% HF in ace-

P. Grzywacz et al. / Bioorganic Chemistry 47 (2013) 9–16
15
tonitrile (1:9 ratio, 2 mL) was added and the resulting mixture was
4.10. General procedure for the synthesis of compounds 36–39
stirred at room temperature for 2 days. The reaction was quenched
with a saturated aqueous NaHCO3 solution and extracted with
ethyl acetate. Combined organic phases were washed with brine,
dried over anhydrous Na2SO4, concentrated under reduced pres-
sure. The residue was purified by column chromatography on silica
(3 ? 5% ethyl acetate/hexane) to give the recovered substrate 23
(9 mg, 0.02 mmol) and the secondary alcohol 24 (51 mg, 82%
yield).
To a stirred solution of the phosphine oxide 35 (1.4–3.0 equiv)
in anhydrous THF (700 L), a solution of phenyllithium (1.5–5.3
equiv) was added at À20 °C under argon. The mixture was stirred
for 30 min and then cooled to À78 °C. A pre-cooled (À78 °C) solu-
tion of the Grundmann’s type ketone 31, 32, 33 or 34 (1 equiv) in
l
anhydrous THF (200 + 100 lL) was added via cannula and the reac-
tion mixture was stirred for 5 h at À78 °C. Then the reaction mix-
ture was stirred at 4 °C for 19 h. Ethyl acetate (20 mL) was added
and the organic phase was washed with brine, dried over anhy-
drous Na2SO4 and concentrated under reduced pressure. The resi-
due was purified on a Waters silica Sep-Pak cartridge (0 ? 2%
ethyl acetate/hexane) to give the protected vitamin D compound
36, 37, 38 or 39.
4.6. General procedure for the synthesis of compounds 25 and 26
To a stirred solution of the ester 22 or 24 (1 equiv) in anhydrous
THF (4 mL), lithium aluminum hydride (5.8–11.3 equiv) was added
at 0 °C. A cooling bath was removed and the reaction mixture was
stirred at room temperature for 30 min. The excess hydride was
quenched by careful addition of aqueous 10% solution of potassium
sodium tartrate. The mixture was extracted with methylene chlo-
ride. Combined organic phases were washed with brine, dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The
residue was purified by column chromatography on silica
(6 ? 60% ethyl acetate/hexane) to give the diol 25 or 26.
4.11. General procedure for the synthesis of compounds 6–9
To a solution of the protected vitamin 36, 37, 38 or 39 in THF
(2 mL) and acetonitrile (2 mL), a solution of aqueous 48% HF in ace-
tonitrile (1:9 ratio, 1 mL) was added at 0 °C and the resulting mix-
ture was stirred at room temperature for 3 h. The reaction was
quenched with a saturated aqueous NaHCO3 solution and ex-
tracted with ethyl acetate. Combined organic phases were washed
with brine, dried over anhydrous Na2SO4, concentrated under re-
duced pressure. The residue was purified on a Waters silica Sep-
Pak cartridge (10 ? 30% ethyl acetate/hexane) to give the crude
products. Final purifications of the vitamin D compounds were per-
formed by straight phase HPLC, followed by reversed-phase HPLC
to give the analytically pure 19,26,27-trinorvitamin D analogs 6,
7, 8 or 9.
4.7. General procedure for the synthesis of compounds 27 and 28
To a solution of the diol 25 or 26 (1 equiv) and triethylamine (3–
4 equiv) in anhydrous methylene chloride (3 mL), tert-butyldi-
methylsilyl chloride (1.3 equiv) was added at room temperature
and the mixture was stirred for 18 h. The reaction was quenched
with water and extracted with ethyl acetate. Combined organic
phases were washed with brine, dried over anhydrous Na2SO4
and concentrated under reduced pressure. The residue was puri-
fied by column chromatography on silica (2% ethyl acetate/hexane)
to give the alcohol 27 or 28.
4.12. General procedure for the synthesis of compounds 10–17
Tris(triphenylphosphine)rhodium (I) chloride (1.0–1.1 equiv)
was added to dry benzene (8 mL) presaturated with hydrogen
(15 min). The mixture was stirred at room temperature until a
homogeneous solution was formed (ca. 25 min). A solution of the
analog 6, 7, 8 or 9 (1 equiv) in dry benzene (2 + 1 mL) was added
and the reaction was allowed to proceed under a continuous
stream of hydrogen for 4 h. The solvent was removed under re-
duced pressure, the residue was redissolved in hexane/ethyl ace-
tate (1:1) and applied on a Waters silica Sep-Pak cartridge (2 g).
A mixture of 2-methyl vitamins was eluted with the same solvent
system. A mixture of compounds was further purified by straight-
4.8. General procedure for the synthesis of compounds 29 and 30
To a stirred solution of the diol 25 or 26 (1 equiv), DMAP (0.2–
0.4 equiv) and triethylamine (3–4 equiv) in anhydrous methylene
chloride (3 mL), p-toluenesulfonyl chloride (1.4–1.5 equiv) was
added at 0 °C. A cooling bath was removed and the reaction mix-
ture was stirred at room temperature for 22 h. Methylene chloride
was added and the mixture was washed with a saturated aqueous
NaHCO3 solution, dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The residue was dissolved in anhydrous
THF (4 mL) and lithium aluminum hydride (60 mg, 1.6 mmol)
was added at 0 °C. A cooling bath was removed and the reaction
mixture was stirred at room temperature for 18 h. The excess hy-
dride was quenched by careful addition of aqueous 10% solution
of potassium sodium tartrate and the mixture was extracted with
methylene chloride. Combined organic phases were washed with
brine, dried over anhydrous Na2SO4 and concentrated under re-
duced pressure. The residue was purified by column chromatogra-
phy on silica (5% ethyl acetate/hexane) to give the alcohol 29 or 30.
phase HPLC. The separation of 2a-methyl analogs 10, 11, 12 or 13,
from the 2b-methyl ones 14, 15, 16 or 17, was achieved by re-
versed-phase HPLC.
5. Biological studies
5.1. In vitro studies
VDR binding, HL-60 differentiation and 24-hydroxylase tran-
scription assays were performed as previously described [28].
4.9. General procedure for the synthesis of compounds 31–34
5.2. In vivo studies
To a stirred solution of the alcohol 27, 28, 29 or 30 (1 equiv),
pyridinium p-toluenesulfonate (0.13–0.20 equiv) in anhydrous
methylene chloride (6 mL), pyridinium dichromate (4–5 equiv)
was added and the mixture was stirred at room temperature for
5 h. The resulting suspension filtered through a Waters silica
Sep-Pak cartridge (5 g), which was further washed with methylene
chloride. Solvents were evaporated under reduced pressure to give
the ketone 31, 32, 33 or 34.
Bone calcium mobilization and intestinal calcium transport
were performed as previously described [29,32]. Briefly, weanling
rats were made vitamin D-deficient by housing under lighting con-
ditions that block vitamin D production in the skin. In addition, the
animals were fed a diet devoid of vitamin D and alternating levels
of calcium. Experimental compounds were administered intraper-
itoneally once per day for four consecutive days. Twenty-four

16
P. Grzywacz et al. / Bioorganic Chemistry 47 (2013) 9–16
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Purity criteria, spectral data of the synthesized compounds;
representative figures with either the competitive binding curves
or dose–response curves derived from the binding, cellular differ-
entiation and transcriptional assays of the vitamin D analogs 6–
17. This material is available free of charge via the Internet at
http://.pubs.acs.org. Supplementary data associated with this arti-
cle can be found, in the online version, at http://dx.doi.org/
10.1016/j.bioorg.2013.01.001.
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