Catalytic asymmetric synthesis and anticancer effects of the novel non-calcemic analog of vitamin D, 2α-fluoro-19-nor-22-oxa-1α,25-dihydroxyvitamin D3 in metastatic lung carcinoma

Article abstract of DOI:10.1016/j.jfluchem.2007.03.002

1α,25-Dihydroxyvitamin D₃ (1α,25-D₃) has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, the major limitation to its clinical use is that it causes hypercalcemia. Therefore, vitamin D analogs with potent cell regulatory effects but with weaker calcemic effects than 1α,25-D₃ are required. Among them, 22-oxa-1α,25-D₃ and 19-nor-1α,25-D₃ have anti-cancer effects with relatively low calcemic effects. Modifications at the C-2α position of the A-ring also produced analogs with a unique biological profile. Not only the side-chain but also the A-ring modification thus generates a unique analog with potent cell regulatory effects and low calcemic activity as well. We report here that the hybrid 1α,25-D₃ analog, synthesized via the highly regio- and stereo-selective ring opening 2α-fluorination and catalytic asymmetric carbonyl-ene cyclization, with 2α-fluoro, 19-nor, and 22-oxa modification exhibits unique cell regulatory activities against the development of metastatic lung carcinoma.

Full text of DOI:10.1016/j.jfluchem.2007.03.002

Journal of Fluorine Chemistry 128 (2007) 654–667
www.elsevier.com/locate/fluor
Catalytic asymmetric synthesis and anticancer effects of the novel
non-calcemic analog of vitamin D, 2a-fluoro-19-nor-22-oxa-1a,
25-dihydroxyvitamin D3 in metastatic lung carcinoma
b
c
d
Kimie Nakagawa a, Toshio Okano a, , Keiichi Ozono , Shigeaki Kato , Noboru Kubodera ,
*
Shiho Ohba e, Yoshimitsu Itoh e, Koichi Mikami e,
*
a Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe 658-8558, Japan
b Department of Developmental Medicine, Pediatrics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
c Institute of Molecular and Cellular Bioscience, University of Tokyo, Tokyo 113-0032, Japan
d Chugai Pharmaceutical Co. Ltd., Tokyo 104-8301, Japan
e Department of Applied Chemistry, Tokyo Institute of Technology, Tokyo 152-8552, Japan
Received 6 December 2006; received in revised form 1 March 2007; accepted 5 March 2007
Available online 12 March 2007
Abstract
1a,25-Dihydroxyvitamin D3 (1a,25-D3) has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, the major
limitation to its clinical use is that it causes hypercalcemia. Therefore, vitamin D analogs with potent cell regulatory effects but with weaker
calcemic effects than 1a,25-D3 are required. Among them, 22-oxa-1a,25-D3 and 19-nor-1a,25-D3 have anti-cancer effects with relatively low
calcemic effects. Modifications at the C-2a position of the A-ring also produced analogs with a unique biological profile. Not only the side-chain
but also the A-ring modification thus generates a unique analog with potent cell regulatory effects and low calcemic activity as well. We report here
that the hybrid 1a,25-D3 analog, synthesized via the highly regio- and stereo-selective ring opening 2a-fluorination and catalytic asymmetric
carbonyl-ene cyclization, with 2a-fluoro, 19-nor, and 22-oxa modification exhibits unique cell regulatory activities against the development of
metastatic lung carcinoma.
# 2007 Published by Elsevier B.V.
Keywords: 2a-F-19-nor-22-oxa-1a,25-D3; Non-calcemic; Lung cancer; Metastasis
1. Introduction
suppression of gene transcription [9]. The broad distribution of
VDR in many tissues and the fact that 1a,25-D3 modulates
proliferation and differentiation of normal and malignant cells
make this hormone a potentially useful agent for the treatment of
diseases such as cancer [10], psoriasis [11,12], and immune
disorders [13]. However, the major limitation to its clinical use is
that it causes hypercalcemia [14]. Therefore, vitamin D analogs
with potent cell regulatory effects but with weaker calcemic
effects than 1a,25-D3 are required [15].
The chemical synthesis of analogs is generally carried out for
a variety of reasons; one is to increase potency, and another is to
increase selectivity and further to diminish toxicity. The long
history of the syntheses of analogs of 1a,25-D3 has yielded a
number of important compounds that have been clearly deve-
loped for therapeutic use; 1a,25-D3 itself, 25-hydroxyvitaminD3
(25-OH-D3), 1a-hydroxyvitamin D3 (1a-OH-D3), 1a-hydro-
xyvitamin D2 (1a-OH-D2), 22-oxa-1a,25-D3, 19-nor-1a,25-D2,
The physiologically active form of vitamin D3, 1a,25-D3 [1],
plays a key role in the regulation of calcium homeostasis in
mammals (Fig. 1). 1a,25-D3 also exerts cell regulatory effects in
target cells [2–4]. The exact mechanism of action of 1a,25-D3 in
target cells has not been clarified, but it is well accepted that
1a,25-D3 first binds to the nuclear vitamin D receptor (VDR) [5],
a member of the superfamily of steroid receptors [6,7]. The
1a,25-D3-liganded VDR then heterodimerizes with the retinoid
X receptor (RXR) [8] and subsequently this complex binds to the
vitamin D-responsiveelements (VDREs) in thepromoter regions
of the primary responding genes, leading to either activation or
* Corresponding authors.
E-mail addresses: t-okano@kobepharma-u.ac.jp (T. Okano),
mikami.k.ab@m.titech.ac.jp (K. Mikami).
0022-1139/$ – see front matter # 2007 Published by Elsevier B.V.
doi:10.1016/j.jfluchem.2007.03.002

K. Nakagawa et al. / Journal of Fluorine Chemistry 128 (2007) 654–667
655
explosively been investigated. 2-Fluoro-1a,25-dihydroxyvita-
min D3 (2-F-1a,25-D3) was previously synthesized, however,
only in the 2b form from steroidal 1,2a-epoxy 3b-ol because of
the steric and electronic effects of 19-methyl group and A-ring
(Eq. (1)) [30]; the corresponding 1,2b-epoxy 3b-ol gave the 1a-
fluorinated regioisomer (Eq. (2)) not the 2a-F stereoisomer [31].
Therefore, the introduction of 2a-F group requires an acyclic
epoxyalcohol precursor rather than the cyclic one for the highly
regio- and stereo-selective ring opening 2a-fluorination (Fig. 2);
We have thus developed a selective fluorinating reagent, group
IV metal fluorides/ammonium hydrogen fluoride [32,33]. In the
subsequent asymmetric carbonyl-ene cyclization catalyzed by
(6-Br-)BINOL-Ti complexes [34,35], olefinic Z/E-geometrical
control ofA-ring canbetranslated, onthe basisofthe‘‘molecular
symmetry’’, into the a/b-configurational control of fluorine
group at C-2, respectively.
During the course of our investigation of 2a-F-1a,25-D3, we
have focused on the 19-nor and 22-oxa derivatives because they
appear to reduce the hypercalcemic activity of 1a,25-D3
(Fig. 1): 2a-F-19-nor-22-oxa-1a,25-D3 [38] as well as 19-nor-
22-oxa-1a,25-D3 [39]. First, we evaluated their biological
properties in terms of binding affinity for VDR and serum
vitamin D-binding protein (DBP), and transcriptional activity
on the vitamin D target gene. 2a-F-19-nor-22-oxa-1a,25-D3
had transcriptional activity on rat 25-hydroxyvitamin D3-24-
hydroxylase gene as equipotent as 1a,25-D3 and two-fold as
strong as 22-oxa-1a,25-D3. 2a-F-19-nor-22-oxa-1a,25-D3 is
the most potent analog on VDR-dependent transcriptional
activity [38].
Fig. 1. Chemical structures of 22-oxa-1a,25-D3 and 2a-substituted 19-nor-22-
oxa-1a,25-D3 analogs referred to in this report.
and calcipotriol [16–22]. The selectivity or reduced toxicity
achieved in these cases is largely the result of rapid metabolic
inactivation rather than selective activity of the analog at the
molecular level. There remains, therefore, a great demand for
analogs of 1a,25-D3 that are selective in their activities, that are
safe as much as they do not cause hypercalcemia, and that retain
high potency.
We have further developed the animal model of anti-cancer
effects of vitamin D analogs, a highly metastatic model using
Organofluorine compounds have attracted much attention,
because of their great importance as biologically active agents,
which exhibit specific biological properties [23–29]. Therefore,
the selective fluorination to modify biological activity has
Lewis lung carcinoma (LLC) cells expressing green fluorescent
protein (GFP) [41]. LLC has been an important tumor model for
studies of metastasis and angiogenesis and neoadjuvant
chemotherapy. A major advantage of GFP-expressing LLC

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K. Nakagawa et al. / Journal of Fluorine Chemistry 128 (2007) 654–667
Fig. 2. Synthetic strategy for 2a-F-19-nor-22-oxa-1a,25-D3 based on 2a-fluorination of acyclic epoxyalcohol and catalytic asymmetric carbonyl-ene cyclization.
(LLC-GFP) cells is that imaging requires no preparative
procedures. The method is, therefore, suitable for visualizing
live tissue during tumor progression. In addition, GFP labeling
is extremely effective for measuring the number and volume of
metastatic nodules in target organs. It has previously been
reported that VDR knockout (VDRꢀ/ꢀ) mice with corrected
hypocalcemia and hypervitaminosis D being fed a high calcium
and vitamin D-deficient diet can be used as an experimental
model for screening the anti-cancer effects of new vitamin D
analogs in vivo [42]. Our studies using the experimental model
of LLC-GFP cells injected wild-type (VDR+/+) and VDRꢀ/ꢀ
mice, have shown that 22-oxa-1a,25-D3 reduced the metastasis
of lung cancer cells without increasing the serum calcium
concentration [41]. The weaker calcemic effect is mainly
attributed to its short half-life in the blood stream [40].
However, high dose administration induced hypercalcemia in
VDR+/+ mice.
stereoselective introduction of fluorine atom was achieved via
the highly regio- and stereo-selective ring opening 2a-
fluorination of chiral epoxy alcohol mediated by Lewis acid
metal fluoride/ammonium hydrogen fluoride ((iv) in Fig. 3)
[32,33]. The use of Hf, which has large ionic radii, leads to 2-
selective ring opening fluorination. Asymmetric induction of 3-
OH group was accomplished by the asymmetric carbonyl-ene
cyclization catalyzed by BINOL-Ti catalyst ((viii) in Fig. 3)
[34,35]. The geometrical (Z/E) and enantioselectivities over the
newly formed olefin and 3-OH group depend on the chirality
matching of the 6,60-dibromo-1,10-bi-2-naphtol (6,60-Br2-
BINOL) ligand and 1-OH group. Finally, 2a-F-19-nor-22-
oxa-1a,25-D3 was synthesized via Lythgoe coupling [36,37]
and subsequent deprotection of the silyl group (Fig. 3).
2.2. Effect on anti-proliferative activity and differentiation-
inducing activity in HL-60 cells
The purpose of the present study was to examine the
effectiveness and weak calcemic effect even in high dose of 2a-
F-19-nor-22-oxa-1a,25-D3 in the preclinical treatment of
animal model of metastatic lung cancer using LLC-GFP cells.
Their in vitro and in vivo biological activities were compared
with those of 22-oxa-1a,25-D3 which is known to be a less
calcemic and more potent against VDR-mediated gene
expression than a natural hormone 1a,25-D3. 22-Oxa and
19-nor analogs of 1a,25-D3 are known to hardly bind to serum
DBP which functions as a carrier protein of vitamin D
derivatives in blood circulation. In our previous report, it is
shown that 2a-F-19-nor-22-oxa-1a,25-D3 has extremely weak
DBP binding potency [38]. In contrast, it has VDR binding
potency approximately four times as strong as that of 19-nor-
22-oxa-1a,25-D3 and much higher than that of 22-oxa-1a,
2. Results and discussion
2.1. Catalytic asymmetric synthesis of 2a-fluoro-19-nor-
22-oxa-1a,25-dihydroxyvitamin D3 A-ring analog
2a-Fluorinated (2a-F) [38] A-ring analog was synthesized
according to the scheme shown in Fig. 3. The key step for the

K. Nakagawa et al. / Journal of Fluorine Chemistry 128 (2007) 654–667
657
Fig. 3. Synthesis of 2a-F-19-nor-22-oxa-1a,25-D3. (i) ꢁ–CO2Me, EtAlCl2, CH2Cl2, r.t.; (ii) DIBAL-H, CH2Cl2, ꢀ78 8C; (iii) Ti(OiPr)4 (20 mol%), (ꢀ)-
Diethyltartrate (DET) (24 mol%), TBHP, MS4A, CH2Cl2, ꢀ30 8C; (iv) HfF4, Bu4NH2F3 (1.0 equiv. each), THF, r.t.; (v) triisopropylsilyltriflate (TIPSOTf),
2,6-lutidine, CH2Cl2, 0 8C; (vi) HF/Py, Et3N, THF, r.t.; (vii) pyridinium chlorochromate (PCC), MS3A, CH2Cl2, r.t.; (viii) (R)-6,60-Br2-BINOL-Ti (10 mol%),
CH2Cl2, r.t.; (ix) TIPSOTf, 2,6-lutidine, CH2Cl2, 0 8C; (x) Li, NH3, tBuOH, Et2O, ꢀ78 8C to r.t.; (xi) N-chlorosuccineimide (NCS), Me2S, CH2Cl2, ꢀ20 8C; (xii)
Ph2PLi, THF, ꢀ78 8C to r.t.; (xiii) H2O2, CH2Cl2, r.t.; (xiv) nBuLi, THF, ꢀ78 8C to r.t.; (xv) tetrabutylammonium fluoride (TBAF), THF, r.t.
25-D3. Moreover, we reported that the 2a-F-19-nor-1a,25-D3
shows the significant activity in transactivation [38]. In the
present study, we examined the anti-cancer effects of 2a-F-19-
nor-22-oxa-1a,25-D3. Effects of 19-nor analogs on cell cycle
phase distribution and cell-surface CD11b antigen expression
in HL-60 cells was measured using FACS analysis. Table 1
indicates the relative activity of the analogs compared with 50%
effective concentration (EC50) of 1a,25-D3. All the 19-nor-22-
oxa analogs have weak effects on cell cycle G1 arrest and
differentiation in HL-60 cells. Relative potency of 2a-F-19-
nor-22-oxa-1a,25-D3 compared to 22-oxa-1a,25-D3 was 51%
in the G1 arrest-inducing activity and 23% in differentiation-
inducing activity. Effects of 2a-F-19-nor-22-oxa-1a,25-D3
were less potent than 1a,25-D3 and 22-oxa-1a,25-D3.
2.3. Transactivations on target genes
A summary of the transcriptional effectiveness EC50 of all the
analogs evaluated is reported for the MG-63 cells in Table 1. The
order of the transcriptional activities on a rat 25(OH)D3-24-
hydroxylasegenepromotercontainingtwoVDREsintransfected
MG-63 cells for the 19-nor-22-oxa-1a,25-D3 analogs is 22-oxa-
1a,25-D3 > 2a-F-19-nor-22-oxa-1a,25-D3 > 1a,25-D3 > 19-
nor-22-oxa-1a,25-D3. 2a-F-19-nor-22-oxa-1a,25-D3 exhibited
Table 1
Biological activities of 19-nor-22-oxa-1a,25(OH)2D3 analogs
Compound
Rat serum DBP*
HL-60 cells#
Diff. CD11b
Transcriptional potency (MG-63 cells)#
Cell cycle G1 arrest
Rat 24OHase
VDR-GAL
RXRa-GAL
VDR/SRC-1
1a,25-D3
22-oxa-1a,25-D3
100
100
85
100
172
44
100
270
20
100
730
220
280
690
100
243
31
100
23
0.3
19-nor-22-oxa-1a,25-D3
2a-Me-19-nor-22-oxa-1a,25-D3
2a-F-19-nor-22-oxa-1a,25-D3
N.R.
N.R.
N.R.
12
15
10
52
60
93
46
23
51
180
43
209
N.R.: not reached to 50% displacement of [3H]-25(OH)D3 or [3H]-1a,25-D3; Not; not tested.
*
Ref. [25].
#
All results are expressed as percentage activity (at 50% of the dose response) in comparison with 1a,25-D3 (=100% activity).

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K. Nakagawa et al. / Journal of Fluorine Chemistry 128 (2007) 654–667
potencynearly asequipotentas 22-oxa-1a,25-D3 andtwo-foldas
strong as 1a,25-D3. To determine whether transcriptional
potency is associated with binding potency to VDR, we
cotransfected an one-hybrid plasmid inserted with a human
VDR cDNA connected with GAL-DBD and a luciferase reporter
plasmid containing GAL-BS into MG-63 cells and treated the
cells with various concentrations of 1a,25-D3 and the analogs.
We found that 2a-F-19-nor-22-oxa-1a,25-D3 had potent in situ
VDR-binding activity as equipotent as 22-oxa-1a,25-D3 and
seven-fold as strong as 1a,25-D3. Moreover, we contrasted the
ability of 1a,25-D3, 22-oxa-1a,25-D3 and 19-nor-22-oxa-1a,25-
D3 analogsto promote VDR interaction with RXRa and the p160
coactivators SRC-1 in the mammalian hybrid luciferase assay in
MG-63 cells. All the 19-nor-22-oxa-1a,25-D3 analogs had less
potent as 22-oxa-1a,25-D3 on VDR/RXR heterodimer-depen-
dent transcriptional activity on RXR-GAL4 system. However, in
VDR/SRC-1-GAL4 system, 2a-F-19-nor-22-oxa-1a,25-D3
manifested a 10-fold and two-fold increase in potency over
22-oxa-1a,25-D3 and 1a,25-D3, respectively. On the other hand,
2a-F-19-nor-22-oxa-1a,25-D3 is equally equipotent to 22-oxa-
1a,25-D3 and 1a,25-D3. These results clearly indicate that 2a-F
is a structural motif to enhance VDR binding and VDR/SRC-1
interaction potency and increase biological activity at the
transcriptional level.
interactions of VDR with SRC-1 as well as 2MD, which is
19-nor and 2-substituted analog. The existence of multiple
VDR coactivators may allow target cells to respond to different
stimuli in a more specific manner. By comparing analog 2a-F-
19-nor-22-oxa-1a,25-D3 with 22-oxa-1a,25-D3, or 19-nor-22-
oxa-1a,25-D3, we found a more profound effect on stabilization
of the ligand within the receptor as a result of 2a-F
introduction. Fluorine, being the most electronegative ion,
may increase the overall negative charge on the surface of the
receptor, though inside the ligand binding domain, acting as a
recruitment enhancer for coactivators. While our findings focus
primarily on the potency of 2a-F-19-nor-22-oxa-1a,25-D3,
they may well provide avenues to discover new vitamin D leads
that possess highly potent and cell-specific anti-cancer effects.
2.4. Effect of 2a-F-19-nor-22-oxa-1a,25-D3 on serum
calcium
The in vivo calcemic effects of 2a-F-19-nor-22-oxa-1a,25-
D3 were assessed by monitoring serum calcium levels after oral
injection. The dose–response effect of each analog was
calculated at the time when the effect was maximum (after
24 h) and expressed as the dosage required to elevate the serum
calcium level by 1 mg/dL (Fig. 4). 22-oxa-1a,25-D3 causes
mild hypercalcemia with insignificant decrease in body weight
(data not shown). In contrast, the serum calcium remained
nearly normal over a period of 72 h after oral injection in 2a-F-
19-nor-22-oxa-1a,25-D3 100 mg/kg administration group. The
results are in agreement with other in vivo studies using a
maximum dose of 1 mg/kg body weight of 22-oxa-1a,25-D3
[41]. Therefore, high concentration of 22-oxa-1a,25-D3 could
not be used for in vivo studies as it may cause calcemic side
effects at >10 mg/kg. However, we found that higher doses (10
and 100 mg/kg) of 2a-F-19-nor-22-oxa-1a,25-D3 singly
injected did not cause hypercalcemia suggesting that high
doses of 2a-F-19-nor-22-oxa-1a,25-D3 may not cause adverse
side effects. Initial characterization of these analogs reveals a
significantly stronger activity than that of 1a,25-D3; in
particular, 2a-F-19-nor-22-oxa-1a,25-D3 display a transactiva-
tion potency even or over that of 22-oxa-1a,25-D3 and 19-nor-
22-oxa-1a,25-D3 while having a significantly lower calcemic
index than 22-oxa-1a,25-D3. Thus, 2a-F-19-nor-22-oxa-
1a,25-D3 is excellent target for further analysis and develop-
ment of the therapeutic agent of cancers.
2a-F-19-nor-22-oxa-1a,25-D3 displays low affinity for
serum DBP as compared with 1a,25-D3. These characteristics
are likely to influence its pharmacokinetic properties and thus
its actions in vivo. In this report, we focused upon the molecular
mechanisms that underlie the enhanced potency of 2a-F-19-
nor-22-oxa-1a,25-D3. This potency is manifested in cell culture
at the level of endogenous gene expression as well as
transcription, despite the fact that both 2a-F-19-nor-22-oxa-
1a,25-D3 and 1a,25-D3 bind equally well to VDR. Indeed, this
property is a characteristic of many chemically related classes
of vitamin D analogs, as herein shown using VDR-GAL4 and
VDR/SRC-1 hybrid luciferase assays. Among three 19-nor-22-
oxa-1a,25-D3 analogs we tested, the strongest effects on VDR/
SRC-1 interaction was only limited to 2a-F-19-nor-22-oxa-
1a,25-D3. Although 22-oxa-1a,25-D3 has potent antiprolifera-
tive effects, it shows even weaker effect on SRC-1 binding to
VDR than 1a,25-D3. It has been reported that 22-oxa-1a,25-D3
selectively induces VDR to interact with TIF2 but not with
SRC-1 or AIB1/ACTR [42]. These results suggest that high
potency of 2a-F-19-nor-22-oxa-1a,25-D3 may be due to the
ability of 2a-F modification to induce unique VDR conforma-
tions that favor enhanced interaction with coactivator such as
SRC-1. These findings provide a molecular explanation for the
increased potency of 2a-F-19-nor-22-oxa-1a,25-D3 and
together with previous studies may explain the potency of
other vitamin D analogs that exhibit similar properties.
Previously it has been reported that 2-methylene-19-nor-
(20S)-1,25-dihydroxyvitamin D3 (2MD) is a highly potent
analog of 1a,25-D3 whose actions are mediated through the
VDR. 2MD is more potent in promoting interaction with RXR
and the coactivators SRC-1 and DRIP205 [43]. Therefore,
differences in VDR structure in the presence of 2a-F-19-nor-
22-oxa-1a,25-D3 may be responsible for the enhanced
2.5. Effects of 1a,25-D3 and 2a-F-19-nor-22-oxa-1a,25-D3
on cell growth and metastatic activity of LLC-GFP cells in
vitro
We examined the effects of 1a,25-D3, 22-oxa-1a,25-D3 and
2a-F-19-nor-22-oxa-1a,25-D3 on the proliferation and invad-
ing activity of lung cancer LLC-GFP cells in vitro. As shown in
Fig. 5a, treatment with 10ꢀ7 M of 1a,25-D3, 22-oxa-1a,25-D3,
19-nor-22-oxa-1a,25-D3 and 2a-F-19-nor-22-oxa-1a,25-D3
for 3 days resulted a significant arrest of the cell cycle at
G0/G1 in LLC-GFP cells. The order of potency of the cell cycle
arrest inducing activities for these analogs in LLC-GFP cells is

K. Nakagawa et al. / Journal of Fluorine Chemistry 128 (2007) 654–667
659
Fig. 4. Effects of 2a-F-19-nor-22-oxa-1a,25-D3 on serum calcium. Mice were
divided into several groups of six mice, each receiving oral administration of 1,
10 or 100 mg/kg of 1a,25-D3 or its analogs in 2 mL/kg of 0.1% Triton X-100
solution. The resulting increases in serum calcium were measured at 24 h after
administration by the o-cresolphthalein complexone (OCPC) method. The
relative activity of each analog with respect to 1a,25-D3 was evaluated as
follows: the dose–response effect of each analogue was calculated at the time
when the effect was maximum and expressed as the dosage required to elevate
the serum calcium level by 1 mg/dL.
Fig. 5. Effects of 2a-F-19-nor-22-oxa-1a,25-D3 on LLC-GFP cell growth and
metastasis in vitro. (a) Cell cycle phase distribution in 1a,25-D3, 22-oxa-1a,25-
D3, 19-nor-22-oxa-1a,25-D3 or 2a-F-19-nor-22-oxa-1a,25-D3 treated-LLC-
GFP cells at 72 h. (b) The capacity of 1a,25-D3, 22-oxa-1a,25-D3, 19-nor-
22-oxa-1a,25-D3 or 2a-F-19-nor-22-oxa-1a,25-D3 treated-LLC-GFP cells to
transverse a Matrigel-coated filter for 24 h. Each bar represents the mean ꢂ S.E.
**,##: P < 0.01 and ***,###: P < 0.001 (n = 6).
22-oxa-1a,25-D3 > 2a-F-19-nor-22-oxa-1a,25-D3 = 1a,25-
D3 > 19-nor-22-oxa-1a,25-D3.
2.6. Effects of continuous treatment with 2a-F-19-nor-22-
oxa-1a,25-D3 on the development of lung metastases in the
LLC-GFP cell injected VDR+/+ mice and VDRꢀ/ꢀ mice fed
a high calcium and vitamin D-deficient diet
A prior study showing reduced random migration by LLC-
GFP cells after 3 days of incubation with 1a,25-D3
determined the effect of increasing concentrations of
1a,25-D3 on tumor cell invasiveness through reconstituted
basement membrane-coated filters [44]. The vehicle-treated
LLC-GFP cells readily transversed the matrix-coated
membranes. In contrast, incubation with 1a,25-D3 and these
analogs caused a dose-dependent decline in the invasive
capability of the tumor cells. The order of potency of the
anti-metastatic activities for these analogs in LLC-GFP
cells is 22-oxa-1a,25-D3 > 2a-F-19-nor-22-oxa-1a,25-D3 =
1a,25-D3 > 19-nor-22-oxa-1a,25-D3. 2a-F-19-nor-22-oxa-
1a,25-D3 had potent anti-invading activity in LLC-GFP
cells nearly as equipotent as 22-oxa-1a,25-D3 and sig-
nificantly as strong as 19-nor-22-oxa-1a,25-D3 (Fig. 5b). The
modifications upon the A-ring with 2a-F increased the anti-
metastatic activity in LLC-GFP cells compared with 19-nor-
22-oxa-1a,25-D3. We therefore believe that the structural
modification reported for 2a-F-19-nor-22-oxa analogs could
serve as basis for the development of therapeutic agents for
the metastatic cancers that can mimic the low calcemic
effects while retaining the strong anti-metastatic capability.
The number of mice that developed lung metastases was
analyzed by measuring the lung weight, nodule count and GFP
mRNA expression in the lung by quantitative RT-PCR. VDRꢀ/ꢀ
mice exhibit hypocalcemia and extremely high serum levels of
1a,25-D3 [45]. However, we previously reported that feeding
these animals a high calcium and vitamin D-deficient diet
resulted in the complete elimination of 1a,25-D3 and correction
of calcium in the serum of both VDR+/+ and VDRꢀ/ꢀ mice [44].
Our recent report suggests that VDRꢀ/ꢀ mice with corrected
hypocalcemia and hypervitaminosis D can be used as an
experimental model for screening the anti-cancer effects of new
vitamin D analogs in vivo [41,44]. In this study using this
experimental model, 2a-F-19-nor-22-oxa-1a,25-D3 was admi-
nistered by an osmotic minipump for 3 weeks. LLC-GFP cells
were injected into VDR+/+ mice receiving a high dose of 2a-F-
19-nor-22-oxa-1a,25-D3 (50 mg/kg/day) and these animals
subsequently showed insignificant hypercalcemia. VDRꢀ/ꢀ
mice receiving a high dose of 2a-F-19-nor-22-oxa-1a,25-D3
and similarly injected with tumor cells did not show any

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K. Nakagawa et al. / Journal of Fluorine Chemistry 128 (2007) 654–667
Fig. 6. Effects of continuous treatment with 2a-F-19-nor-22-oxa-1a,25-D3 on the development of lung metastases in LLC-GFP cell-injected VDR+/+ mice and
VDRꢀ/ꢀ mice fed a high calcium and vitamin D-deficient diet. 2a-F-19-nor-22-oxa-1a,25-D3 (10 or 50 mg/kg/day) or vehicle was infused continuously by an osmotic
minipump implanted s.c. on the same day as the LLC-GFP cell injection. Serum and lungs were collected on 3 weeks after the cell injection. (a and c) Serum calcium
concentration and GFP mRNA expression of the lung in LLC-GFP cell injected VDR+/+ mice. (b and d) Serum calcium concentration and GFP mRNA expression of
the lung in LLC-GFP cell injected VDRꢀ/ꢀ mice. Each bar represents the mean ꢂ S.E. (n = 10). ***: P < 0.001 significant difference from vehicle treated animals.
##: P < 0.01 and ###: P < 0.001 significant difference from 2a-F-19-nor-22-oxa-1a,25-D3 (10 mg/kg/day) treated animals.
significant change in serum calcium levels when compared with
the vehicle-treated control group (Fig. 6a and b).
therapies. Among the recently discovered vitamin D3 analogs,
EB1089 (1a,25-dihydroxy-22,24-diene-24,26,27-trihomovita-
min D3), QW-1624F2-2 (1-hydroxymethyl-16-ene-24,24-F2-
26,27-bishomo-25-hydroxyvitamin D3) and 22-oxa-1a,25-D3
might find clinical use [46]. More recently, we studied the
effectiveness of 22-oxa-1a,25-D3 on metastatic lung cancer.
Low dose (1 mg/kg/day) of 22-oxa-1a,25-D3 showed effec-
tiveness similar to that of 1a,25-D3 in continuous treatment of
metastatic lung tumors in VDR+/+ and VDRꢀ/ꢀ mice without
hypercalcemia. However, high dose (10 mg/kg/day) adminis-
tration of 22-oxa-1a,25-D3 caused hypercalcemia in VDR+/+
mice [41]. The conclusion regarding the reduced-calcemic,
synthetic vitamin D analogs including 22-oxa-1a,25-D3 is that
while their effectiveness appears sufficiently promising for
clinical trials, their hypercalcemia-related toxicity remains
problematic.
The number of mice that developed lung metastases was
analyzed by measuring the GFP mRNA expression in the lung
by quantitative RT-PCR. This parameter was significantly
lower in the 2a-F-19-nor-22-oxa-1a,25-D3-treated group than
in the untreated group among VDR+/+ mice and VDRꢀ/ꢀ mice
fed a high calcium and vitamin D-deficient diet (Fig. 6c and d).
The anti-tumor effect of 2a-F-19-nor-22-oxa-1a,25-D3 in
VDR+/+ mice and VDRꢀ/ꢀ mice were dosed dependently. This
result indicates that 2a-F-19-nor-22-oxa-1a,25-D3 can act
directly on cancer cells under conditions without calcemic
activity and other actions of 2a-F-19-nor-22-oxa-1a,25-D3 in
the host. These results indicate that 2a-F-19-nor-22-oxa-1a,25-
D3 is less calcemic and more effective than 22-oxa-1a,25-D3 in
suppressing metastatic tumor growth of LLC-GFP cells. Its
anti-tumor efficacy combined with non-calcemic effects (at 10
higher dose than 22-oxa-1a,25-D3) suggest that higher
concentrations of 2a-F-19-nor-22-oxa-1a,25-D3 could be used.
Because dose-limiting hypercalcemia of the vitamin D
analogs limits the therapeutic potential, identification of low-
calcemic analogs is critical in vitamin D3-mediated anticancer
Therefore, we invented A-ring and side-chain hybrid
vitamin D analogs that retain the potent transcriptional activity
on vitamin D target genes and anti-cancer effects but are much
less likely to raise serum calcium in vivo. 2a-F-19-nor-22-oxa-
1a,25-D3 is a new non-calcemic vitamin D analog in A-ring
and side-chain modified analogs. In the present study, we

K. Nakagawa et al. / Journal of Fluorine Chemistry 128 (2007) 654–667
661
demonstrated that 2a-F-19-nor-22-oxa-1a,25-D3 reduces the
development of lung metastases without calcemic activity. The
mechanisms by which 2a-F-19-nor-22-oxa-1a,25-D3 inhibits
tumor growth are not fully understood. In this study, 2a-F-19-
nor-22-oxa-1a,25-D3 induced growth arrest at G0/G1 in LLC-
GFP cells. Moreover, 2a-F-19-nor-22-oxa-1a,25-D3 signifi-
cantly inhibited tumor invasive activity. We previously
indicated that 1a,25-D3 and 22-oxa-1a,25-D3-treated LLC-
GFP cells decreased metastatic activity and mRNA expression
of MMP-2 and MMP-9, a family of proteases capable of
degrading extracellular matrix and basement membrane
components including collagens under physiological condi-
tions [41]. Therefore, 2a-F-19-nor-22-oxa-1a,25-D3 may
mediate the expression of MMP-2 and MMP-9 genes in
LLC-GFP cells. Obviously, the mechanisms underlying the
anti-cancer effects of 2a-F-19-nor-22-oxa-1a,25-D3 are com-
plex and further investigation within this area is required in
order to obtain a better understanding.
oxa-1a,25-D3, achieved with significantly less toxicity in terms
of hypercalcemia and weight loss than 1a,25-D3 and 22-oxa-
1a,25-D3, suggest that 2a-F-19-nor-22-oxa-1a,25-D3 may
offer a therapeutic option in advanced lung carcinoma.
3. Conclusion
We have demonstrated that the hybrid analog with 2a-
fluoro, 19-nor, and 22-oxa modification of 1a,25-D3 exhibits
unique cell regulatory activities and clearly identified the
structural motifs responsible for non-calcemic and anti-
metastatic activity. We synthesized the 2a-F-19-nor-22-oxa-
1a,25-D3 analog via the highly regio- and stereo-selective
epoxide opening 2a-fluorination and catalytic asymmetric
carbonyl-ene cyclization, and examined the effect of the 2a-F-
analog as a prophylactic for the treatment of lung metastasis.
The 2a-F-analog did not cause hypercalcemia or a significant
increase in mortality. Toxicity from hypercalcemia has been
the major deterrent in the use of vitamin D analogs in clinical
trial. Lung cancer patients have a very poor prognosis. It is
therefore important to develop a therapeutic agent with anti-
proliferative and anti-metastatic effects, but which has
minimal side-effects. The 2a-F-analog has potential useful-
ness in the treatment of these tumors and should be
incorporated in clinical trials. These findings will provide
useful information not only for the development of therapeutic
agents for treatment of lung cancer and other metastatic
cancers but also for studies on the structure-function relation-
ship of 1a,25-D3.
In vivo experiments that monitor serum calcium suggest that
2a-F-19-nor-22-oxa-1a,25-D3 less calcemic than 22-oxa-
1a,25-D3. In continuous administration of 2a-F-19-nor-22-
oxa-1a,25-D3 using minipump, 2a-F-19-nor-22-oxa-1a,25-D3
did not induce any adverse effects. The body weight of the mice
remained normal and serum calcium levels did not cause any
side effects. The in vivo efficacy data suggest that 2a-F-19-nor-
22-oxa-1a,25-D3 is more effective than 22-oxa-1a,25-D3 in
suppressing metastatic lung tumors. Its antitumor efficacy
combined with non-calcemic effects suggested that higher
concentrations of 2a-F-19-nor-22-oxa-1a,25-D3 could be used
for cancer therapy. The differences in the effectiveness between
2a-F-19-nor-22-oxa-1a,25-D3 and 22-oxa-1a,25-D3 could be
due to differential catabolism or differential liganded-VDR
interaction with coactivators. It is possible that 2a-F-19-nor-
22-oxa-1a,25-D3 might be hard to be metabolized and strongly
promote the interaction of the VDR with potential facilitators of
transactivation such as SRC-1 in cells.
Our recent report suggests that VDRꢀ/ꢀ mice with corrected
hypocalcemia and hypervitaminosis D can be used as an
experimental model to screen the anti-cancer effects of new
vitamin D analogs in vivo [44]. In this study using this
experimental model, 2a-F-19-nor-22-oxa-1a,25-D3 was admi-
nistered for 3 weeks using an osmotic minipump. The LLC-
GFP cells injected VDRꢀ/ꢀ mice receiving a high dose of 2a-F-
19-nor-22-oxa-1a,25-D3 (50 mg/kg/day) did not show hyper-
calcemia and significantly decreased the tumor growth. This
result clearly indicates that 2a-F-19-nor-22-oxa-1a,25-D3 can
act directly on cancer cells under conditions without calcemic
activity or other actions of 2a-F-19-nor-22-oxa-1a,25-D3 in the
host. The high dose of 2a-F-19-nor-22-oxa-1a,25-D3 does not
cause hypercalcemia and loss of body weight in VDR+/+ mice.
In the previous reports, it has been suggested that mice treated
with 1a,25-D3 experienced a severe loss of thymocytes, and
that thymic atrophy resulted as a consequence of 1a,25-D3-
induced hypercalcemia [47,48]. Therefore, 2a-F-19-nor-22-
oxa-1a,25-D3 is highly effective in inhibiting the metastatic
tumor growth without hypercalcemia. In this experiment, our
findings of marked anti-metastatic effects of 2a-F-19-nor-22-
4. Experimental
4.1. Catalytic asymmetric synthesis of 2a-fluoro-19-nor-
22-oxa-1a,25-dihydroxyvitamin D3 A-ring analog
4.1.1. (2S, 3R)-5-[2-(Benzyloxy)ethyl]-2-fluoro-5-hexene-
1,3-diol (1)
Ten millilitres test tube with argon inlet was charged with
hafnium (IV) tetrafluoride (0.130 g, 0.51 mmol) and THF
(2.0 mL) at 0 8C. To the suspension was added tetrabutylam-
monium
dihydrogentrifluoride
(H2F3NBu4)
(0.166 g,
0.55 mmol) in THF (0.5 mL) and stirred for 10 min at that
temperature. (2R,3R)-5-[2-(Benzyloxy)ethyl]-2,3-epoxy-5-en-
1-hexanol (96% ee) (0.130 g, 0.53 mmol) was added dropwise
and stirred for 10 min and then the reaction mixture was
warmed up to room temperature. After stirring for 24 h at that
temperature, the reaction mixture was quenched with sat.
sodium hydrogencarbonate. After usual workup, the residue
was purified by silica-gel chromatography to give (2S,3R)-5-[2-
(benzyloxy)ethyl]-2-fluoro-5-hexene-1,3-diol (1) and (2R,3S)-
5-[2-(benzyloxy)ethyl]-3-fluoro-5-hexene-1,2-diol (0.132 g,
94%) as the regioisomer in a ratio of 78:22.
4.1.1.1. (2S, 3R)-5-[2-(Benzyloxy)ethyl]-2-fluoro-5-hexene-
1,3-diol (1). 19F NMR (376 MHz, CDCl3) d ꢀ197.1 (dtd,
J = 47.0, 24.8, 9.0 Hz, 1F); 1H NMR (400 MHz, CDCl3) d 2.19
(dd, J = 14.4, 2.0 Hz, 1H), 2.39 (t, J = 6.4 Hz, 2H), 2.48 (d,

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K. Nakagawa et al. / Journal of Fluorine Chemistry 128 (2007) 654–667
J = 14.4 Hz, 1H), 2.80 (bs, 1H), 3.25 (bs, 1H), 3.63 (t,
J = 6.4 Hz, 2H), 3.85 (dd, J = 24.8, 4.0 Hz, 2H), 3.93–4.00 (m,
1H), 4.24–4.40 (ddt, J = 47.2, 6.8, 4.0 Hz, 1H), 4.51 (s, 2H),
4.98 (bs, 1H), 4.99 (bs, 1H), 7.29–7.35 (m, 5H); 13C NMR
(101 MHz, CDCl3) d 35.6, 40.6 (d, 3J = 3.1 Hz), 62.2 (d,
2J = 21.4 Hz), 68.6 (d, 2J = 25.4 Hz), 69.2, 73.2, 94.8 (d,
1J = 173.8 Hz), 114.9, 127.8, 127.9, 128.4, 137.8, 142.8.
(101 MHz, CDCl3) d 11.9, 12.6, 18.0, 18.1, 18.2, 35.2, (d,
3
2J = 22.2 Hz), 36.3, 64.5 (d, J = 9.9 Hz), 68.9, 72.3, 74.6 (d,
2J = 20.7 Hz), 93.3 (d, 1J = 173.8 Hz), 113.4, 127.6, 127.8,
128.4, 138.6, 143.0.
4.1.3. (2S,3R)-5-[2-(Benzyloxy)ethyl]-2-fluoro-3-
triisopropylsilyloxy-5-hexen-1-ol (3)
To a solution of triethylamine (0.28 mL, 2.0 mmol) and
HF/Py (0.048 mL, 2.0 mmol) in THF (2.5 mL) was added
(4R,5S)-2-[2-(benzyloxy)ethyl]-5-fluoro-4,6-bis(triisopropyl-
silyloxy)-1-hexene (2) (0.240 g, 0.413 mmol) at 0 8C. After
stirring for 22 h at room temperature, methoxytrimethylsilane
(TMSOMe, 0.5 mL) was added to the reaction mixture at 0 8C
and stirred for 2 h. After THF was evaporated under reduced
pressure, the residue was purified by silica-gel chromato-
graphy to give (2S,3R)-5-[2-(benzyloxy)ethyl]-2-fluoro-3-
triisopropylsilyloxy-5-hexen-1-ol (3) (0.116 g, 66%): 19F
NMR (376 MHz, CDCl3) d ꢀ193.3 (dddd, J = 50.4, 39.9,
4.1.1.2. (2R,3S)-5-[2-(Benzyloxy)ethyl]-3-fluoro-5-hexene-
1,2-diol. 19F NMR (376 MHz, CDCl3) d ꢀ190.4 (dddd,
J = 56.0, 46.6, 20.7, 10.2 Hz, 1F); 1H NMR (400 MHz, CDCl3)
d 2.36–2.54 (m, 4H), 2.96 (bs, 1H), 3.08 (bs, 1H), 3.59 (t,
J = 6.8 Hz, 2H), 3.65–3.73 (br, 3H), 4.51 (s, 2H), 4.50–4.68
(dddd, J = 56.0, 9.2, 6.0, 3.2 Hz, 1H), 4.94 (s, 1H), 4.96 (s, 1H),
7.29–7.35 (m, 5H); 13C NMR (101 MHz, CDCl3) d 36.2, 38.0
(d, 2J = 20.7 Hz), 62.6 (d, 3J = 5.4 Hz), 68.8, 72.7 (d,
2J = 23.7 Hz), 73.0, 92.0 (d, 1J = 173.1 Hz), 114.1, 127.7,
127.9, 128.5, 138.2, 142.1.
1
26.3, 14.7 Hz, 1F); H NMR (400 MHz, CDCl3) d 1.08 (bs,
4.1.2. (4R,5S)-2-[2-(Benzyloxy)ethyl]-5-fluoro-4,6-
bis(triisopropylsilyloxy)-1-hexene (2)
21H), 1.61 (br, 1H) 2.30 (dd, J = 14.8, 10.0 Hz, 1H), 2.35 (t,
J = 6.8 Hz, 2H), 2.44 (dd, J = 14.8, 6.8 Hz, 1H), 3.58 (t,
J = 6.8 Hz, 2H), 3.88 (bdd, J = 26.0, 12.4 Hz, 1H), 4.01 (ddd,
J = 24.4, 12.4, 6.0 Hz, 1H), 4.41 (dddd, J = 17.6, 12.4, 5.2,
2.4 Hz 1H), 4.43–4.58 (dtd, J = 47.2, 6.0, 2.4 Hz, 1H), 4.51 (s,
2H), 4.89 (bs, 1H), 4.91 (bs, 1H), 7.29–7.35 (m, 5H); 13C NMR
To a solution of (2S,3R)-5-[2-(benzyloxy)ethyl]-2-fluoro-5-
hexene-1,3-diol (1) and (2R,3S)-5-[2-(benzyloxy)ethyl]-3-
fluoro-5-hexene-1,2-diol (2.53 g, 9.43 mmol) in CH2Cl2
(19 mL) was added 2,6-lutidine (2.86 mL, 24.5 mmol) and
triisopropylsilyltriflate (TIPSOTf) (6.59 mL, 24.5 mmol) at
0 8C. After stirring for 1.5 h at that temperature, the reaction
mixture was quenched with sat. sodium hydrogencarbonate.
After usual workup, the residue was purified by silica-gel
chromatography to give (4R,5S)-2-[2-(benzyloxy)ethyl]-5-
fluoro-4,6-bis(triisopropylsilyloxy)-1-hexene (2) and (4S,5R)-
2-[2-(benzyloxy)ethyl]-4-fluoro-5,6-bis(triisopropylsilyloxy)-
1-hexene (5.17 g, 94%). Regioisomers could be separated by
silica-gel chromatography.
3
(101 MHz, CDCl3) d 12.5, 18.0, 36.1, 41.6 (d, J = 9.2 Hz),
61.3 (d, 2J = 22.1 Hz), 68.8, 72.0 (d, 2J = 20.6 Hz), 73.0, 94.7
(d, 1J = 175 Hz), 114.2, 127.6, 127.7, 128.3, 138.2, 142.1; IR
(neat) 3350, 2946, 2868, 1647, 1497, 1456, 1365, 1249, 1207,
1100, 1067, 1015, 998, 884, 737, 681 cmꢀ1; [aD24] = ꢀ4.36
(ca. 1.28, CHCl3).
4.1.4. (2R,3R)-5-[2-(Benzyloxy)ethyl]-2-fluoro-3-
triisopropylsilyloxy-5-hexenal (4)
˚
To a suspension of powdered molecular sieves 3 A (MS
˚
3 A, 3.0 g) and pyridinium chlorochromate (1.30 g,
4.1.2.1. (4R,5S)-2-[2-(Benzyloxy)ethyl]-5-fluoro-4,6-bis(trii-
sopropylsilyloxy)-1-hexene (2). 19F NMR (376 MHz, CDCl3)
d ꢀ191.3 (dtd, J = 45.5, 25.2, 23.7 Hz, 1F); 1H NMR
(400 MHz, CDCl3) d 1.08 (bs, 42H), 2.35 (d, J = 8.0 Hz,
2H), 2.38 (t, J = 6.8 Hz, 2H), 3.60 (t, J = 6.8 Hz, 2H), 3.97 (dd,
J = 22.8, 4.8 Hz, 2H), 4.34 (dtd, J = 18.4, 8.0, 2.0 Hz, 1H), 4.53
(s, 2H), 4.48–4.64 (dtd, J = 48.0, 4.8, 2.0 Hz, 1H), 4.92 (bs,
2H), 7.33–7.36 (m, 5H); 13C NMR (101 MHz, CDCl3) d 12.0,
12.7, 18.0, 18.0, 18.1, 18.2, 36.4, 41.0 (d, 3J = 8.4 Hz), 62.1 (d,
2J = 25.4 Hz), 68.9, 71.5 (d, 2J = 20.6 Hz), 73.1, 96.3 (d,
1J = 177.0 Hz), 114.0, 127.6, 127.7, 128.4, 138.4, 142.6; IR
(neat) 2946, 2870, 1647, 1464, 1383, 1365, 1249, 1102, 1069,
6.0 mmol) in CH2Cl2 (60 mL) was added (2S,3R)-5-[2-
(benzyloxy)ethyl]-2-fluoro-3-triisopropylsilyloxy-5-hexen-1-
ol (3) (1.28 g, 3.02 mmol) in CH2Cl2 (3.0 mL) in the water
bath. After stirring for 1 h, ether, celite and florisil were added
and stirred for 10 min. Then the resultant suspension was
filtered through a column of celite and florisil. After
evaporation under reduced pressure, the resultant residue
was purified by silica-gel chromatography to give (2R,3R)-5-
[2-(benzyloxy)ethyl]-2-fluoro-3-triisopropylsilyloxy-5-hexe-
nal (1.02 g, 80%): 19F NMR (376 MHz, CDCl3) d ꢀ206.2
(ddd, J = 49.3, 21.8, 3.8 Hz, 1F); 1H NMR (300 MHz,
CDCl3); d 1.08 (bs, 21H), 2.30 (d, J = 6.4Hz, 2H), 2.40
(d, J = 14.4 Hz, 1H), 2.57 (dd, J = 14.4, 10.8 Hz, 1H), 3.58 (t,
J = 6.4 Hz, 2H), 4.45–4.54 (m, 1H), 4.51 (s, 2H), 4.76 (d,
J = 48.8 Hz, 1H), 5.01 (s, 1H), 7.32–7.35 (m, 5H), 9.70 (d,
J = 5.2 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 12.2, 17.8,
1015, 998, 907, 894, 797, 735, 683 cmꢀ1
.
4.1.2.2. (4S, 5R)-2-[2-(Benzyloxy)ethyl]-4-fluoro-5,6-bis(trii-
sopropylsilyloxy)-1-hexene. 19F NMR (376 MHz, CDCl3) d
ꢀ188.5 (ddt, J = 46.6, 41.0, 15.0 Hz, 1F); 1H NMR (400 MHz,
CDCl3) d 1.07–1.09 (m, 42H), 2.39 (ddd, J = 34.0, 14.8, 6.8 Hz,
1H), 2.48 (t, J = 7.2 Hz, 2H), 2.64 (ddd, J = 25.2, 14.8, 10.0 Hz,
1H). 3.65 (t, J = 7.2 Hz, 2H), 3.80–3.85 (m, 2H), 4.20–4.28 (m,
1H), 4.53 (s, 2H), 4.84–4.99 (ddt, J = 43.2, 10.4, 2.0 Hz, 1H),
4.97 (bs, 1H), 5.00 (bs, 1H), 7.30–7.37 (m, 5H); 13C NMR
2
17.9, 36.0, 40.1, 68.5, 72.9, 73.6 (d, J = 19.2 Hz), 96.3 (d,
1J = 185 Hz), 115.6, 127.5, 127.5, 127.5, 127.6, 128.3, 138.2,
2
147.8, 198.9 (d, J = 37.5 Hz); IR (neat) 2948, 2870, 1744,
1647, 1458, 1363, 1114, 1000, 884, 735 cmꢀ1; [aD24] = +17.6
(ca. 1.87, CHCl3).

K. Nakagawa et al. / Journal of Fluorine Chemistry 128 (2007) 654–667
663
4.1.5. (Z,1R,2S,3R)-5-[2-(Benzyloxy)ethylidene]-2-fluoro-
3-triisopropylsilyloxy-1-cyclohexanol. (trans, Z-exo) (5)
To a solution of (R)-BINOL-derived chiral titanium complex
[34,35] (0.0251 g, 0.045 mmol) in CH2Cl2 (2.5 mL) was added
(2R,3R)-5-[2-(benzyloxy)ethyl]-2-fluoro-3-(triisopropylsily-
loxy)-5-hexenal (4) (0.189 g, 0.45 mmol) at room temperature
under an argon atmosphere. After stirring for 24 h at that
temperature, the reaction mixture was poured into sat. sodium
hydrogencarbonate. After usual workup, the residue was
purified by silica-gel chromatography to give trans,Z-exo,
trans,E-exo, cis-endo, trans-endo, cis-exo (E/Z mixture)
(0.132 g, 94%) as regioisomers in a ratio of 71:8:11:6:3.
(Z,1R,2S,3R)-5-[2-(Benzyloxy)ethylidene]-2-fluoro-3-triiso-
propylsilyloxy-1-cyclohexanol (trans,Z-exo) (5) was separated
by silica-gel chromatography: 19F NMR (376 MHz, CDCl3); d
ꢀ192.4 (cis-exo, m, 1F), ꢀ195.3 (cis-exo, m, 1F), ꢀ197.8
(trans,Z-exo, bd, J = 43.6 Hz, 1F), ꢀ198.9 (trans,E-exo, bd,
J = 45.9 Hz, 1F), ꢀ199.9 (trans-endo, bd, J = 44.1 Hz, 1F),
1J = 184 Hz), 123.5, 127.5, 127.7, 128.3, 136.3, 138.3; IR
(neat) 2945, 2896, 2868, 1464, 1383, 1367, 1249, 1112, 1069,
1013, 998. 917, 884, 795, 758, 679 cmꢀ1; [aD27] = ꢀ9.49 (ca.
1.21, CHCl3).
4.1.7. (Z,3R,4S,5R)-2-[4-Fluoro-3,5-
bis(triisopropylsilyloxy)cyclohexylidene]ethanol (7)
A 25 mL, three necked, round-bottomed flask equipped with
a finger-type-condenser and a magnetic stirring bar and argon
inlet was charged with anhydrous liquid ammonia (ca.
0.18 mL) at ꢀ78 8C. To the solution were added lumps of
lithium (ca. 0.049 g, 7.0 mmol) and then (1R,2S,3R)-5-[(Z)-2-
(benzyloxy)ethylidene]-2-fluoro-1,3-bis(triisopropylsilyloxy)-
cyclohexane (6) (0.510 g, 0.880 mmol) in ether (0.88 mL) and
tert-butyl alcohol (0.33 mL) at that temperature. After stirring
for 15 min, methanol (1.0 mL) was added to the reaction
mixture. Then the mixture was warmed up to room temperature
until the excess ammonium evaporated. After usual workup, the
resultant residue was purified by silica-gel chromatography to
give (Z,3R,4S,5R)-2-[4-fluoro-3,5-bis(triisopropylsilyloxy)cy-
clohexylidene]-ethanol (7) (0.364 g, 85%): 19F NMR
1
ꢀ202.9 (cis-endo, bd, J = 50.4 Hz, 1F); H NMR (400 MHz,
CDCl3) d 1.04 (bs, 21H), 2.15 (d, J = 13.5 Hz, 2H), 2.55–2.66
(m, 2H), 4.02 (dd, J = 11.7, 6.6 Hz, 2H), 4.11–4.17 (m, 2H),
4.27 (d, J = 6.3 Hz, 1H), 4.38 (ddd, J = 34.5, 7.8, 3.0 Hz, 1H),
4.49 (s, 2H), 5.61 (t, J = 6.6 Hz, 1H), 7.33–7.35 (m, 5H); 13C
NMR (101 MHz, CDCl3) d 12.2, 17.8, 17.9, 33.6 (d,
3J = 5.4 Hz), 40.4 (d, 3J = 5.4 Hz), 65.9, 67.9 (d,
2J = 21.4 Hz), 68.8 (d, 2J = 16.9 Hz), 72.2, 95.7(d,
1J = 184 Hz), 124.2, 127.5, 127.7, 128.2, 135.5, 137.9; IR
(neat) 3302, 3034, 2946, 2868, 1497, 1464, 1383, 1365, 1249,
1
(376 MHz, CDCl3) d ꢀ198.6 (bs, 1F); H NMR (300 MHz,
CDCl3) d 1.05 (bs, 3H), 1.06 (bs, 3H), 1.07 (bs, 18H), 1.07 (bs,
18H), 1.24 (bs, 1H), 2.13 (dd, J = 13.8, 6.3 Hz, 1H), 2.36 (dd,
J = 13.8, 3.9 Hz, 1H), 2.45–2.55 (bd, J = 13.8 Hz, 1H), 2.50
(dd, J = 13.8, 3.9 Hz, 1H), 4.13 (dd, J = 12.0, 6.9 Hz, 1H), 4.18
(dd, J = 12.0, 6.9 Hz, 1H), 4.19–4.27 (m, 2H), 4.29–4.54 (ddd,
J = 49.5, 6.3, 2.4 Hz, 1H), 5.54 (t, J = 6.9 Hz, 1H); 13C NMR
(75 MHz, CDCl3) d 12.1, 12.2, 17.8, 33.0 (d, 3J = 6.1 Hz), 40.8
(d, 3J = 3.7 Hz), 69.1 (d, 2J = 16.9 Hz), 69.3 (d, 2J = 23.1 Hz),
94.7 (d, 1J = 183 Hz), 126.4, 135.9; IR (neat) 3242, 2948, 2896,
2870, 2362, 1464, 1383, 1247, 1118, 1071, 1041, 998, 917, 884,
758, 679 cmꢀ1; [aD27] = ꢀ3.55 (ca. 1.26, CHCl3).
1187, 1123, 1067, 1027, 998, 917, 882, 750, 698, 679 cmꢀ1
;
[aD27] = +7.70 (ca. 0.71, CHCl3); HPLC (Inertsil SIL, hexane/
ethyl acetate = 4:1, flow rate 0.8 mL/min, detection 254 nm
light); trans,Z-exo tR = 18.4 min; trans-endo tR = 15.6 min;
trans,E-exo tR = 15.0 min; cis-endo tR = 14.3 min; cis-exo
tR = 13.9 min; cis-exo tR = 9.4 min.
4.1.6. (Z,1S,2R,6R)-4-[2-(Benzyloxy)ethylidene]-1-fluoro-
2,6-bis(triisopropylsilyloxy)cyclohexane (6)
To a solution of (1R,2S,3R)-5-[(Z)-2-(benzyloxy)ethyli-
4.1.8. (Z,1S,2R,6R)-4-[2-Chloroethylidene]-1-fluoro-2,
6-bis(triisopropylsilyloxy)cyclohexane (8)
To a solution of N-chlorosuccimide (0.116 g, 0.87 mmol) in
CH2Cl2 (1.8 mL) was slowly added dimethyl sulfide (0.068 mL,
0.92 mmol) at 0 8C under argon. After stirring for 10 min at that
temperature, then mixture was cooled to ꢀ20 8C for 10 min, and
a solution of 2-[(3R,4S,5R)-4-fluoro-3,5-bis(triisopropylsilylox-
y)cyclohexylidene]ethanol (7) (0.089 g, 0.181 mmol) in CH2Cl2
(1.0 mL)was added. Afterstirringfor15 minatthat temperature,
the mixture was warmed up to room temperature for 1.5 h. After
usual workup, the residue obtained was purified by neutral
silica-gelchromatographytogive(Z,1S,2R,6R)-4-[2-chloroethy-
lidene]-1-fluoro-2,6-bis(triisopropylsilyloxy)cyclohexane (8)
(0.070 g, 76%): 19F NMR (376 MHz, CDCl3) d ꢀ200.0 (m,
dene]-2-fluoro-3-(triisopropylsilyloxy)-cyclohexanol
(5)
(0.372 g, 0.880 mmol) in CH2Cl2 (0.88 mL) was added 2,6-
lutidine (0.26 mL, 2.20 mmol) and triisopropylsilyltriflate
(TIPSOTf) (0.31 mL, 1.14 mmol) at 0 8C. After stirring for
1 h at that temperature, the reaction mixture was quenched with
sat. sodium hydrogencarbonate. After usual workup, the
resultant residue was purified by silica-gel chromatography
to give (1R,2S,3R)-5-[(Z)-2-(benzyloxy)ethylidene]-2-fluoro-
1,3-bis(triisopropylsilyloxy)-cyclohexane (6) (0.510 g, 100%):
1
19F NMR (376 MHz, CDCl3) d ꢀ199.5 (bs, 1F); H NMR
(400 MHz, CDCl3) d 1.05 (bs, 42H), 2.16 (dd, J = 14.0, 6.0 Hz,
1H), 2.36 (dd, J = 14.0, 4.4 Hz, 1H), 2.40 (dd, J = 14.0, 4.8 Hz,
1H), 2.53 (dt, J = 14.0, 4.4 Hz, 1H), 4.05 (d, J = 6.8 Hz, 2H),
4.19 (dddd, J = 21.2, 7.2, 4.4, 2.4 Hz, 1H), 4.25 (ddt, J = 12.0,
6.0, 4.4 Hz, 1H), 4.46 (ddd, J = 49.6, 6.0, 2.4 Hz, 1H), 4.46 (d,
J = 12.0 Hz, 1H), 4.50 (d, J = 12.0 Hz, 1H), 5.51 (t, J = 6.8 Hz,
1H), 7.28–7.35 (m, 5H); 13C NMR (101 MHz, CDCl3) d 12.2,
12.2, 12.3, 17.7, 17.9, 33.1 (d, 3J = 5.4 Hz), 40.5, 65.9, 68.9 (d,
2J = 16.9 Hz), 69.2 (d, 2J = 23.7 Hz), 71.9, 94.3 (d,
1
1F); H NMR (300 MHz, CDCl3) d 1.08 (bs, 42H), 2.16 (dd,
J = 14.0, 5.2 Hz, 1H), 2.45 (d, J = 6.0 Hz, 2H), 2.51 (dt, J = 14.0,
4.4 Hz, 1H), 4.04 (dd, J = 11.6, 7.6 Hz, 1H), 4.13 (dd, J = 11.6,
8.0 Hz, 1H), 4.20–4.28 (m, 2H), 4.42–4.56 (ddd, J = 49.2, 5.6,
2.0 Hz, 1H), 5.54 (t, J = 8.0 Hz, 1H).; 13C NMR (75 MHz,
CDCl3) d 12.2, 12.4, 18.0, 18.0, 18.0, 31.7 (d, 3J = 5.4 Hz), 39.9,
2
2
40.2, 68.8 (d, J = 17.7 Hz), 69.1 (d, J = 24.5 Hz), 94.0 (d,
1J = 184 Hz), 122.7, 138.3; IR (neat) 2948, 2870, 1464, 1386,

664
K. Nakagawa et al. / Journal of Fluorine Chemistry 128 (2007) 654–667
1253, 1127, 1038, 1015, 998, 917, 884, 760, 681 cmꢀ1
;
(0.025 g, 33%): 19F NMR (376 MHz, CDCl3) d ꢀ197.7 (bs,
1F); 1H NMR (300 MHz, CDCl3) d 0.10 (s, 9H), 0.53 (s, 3H),
1.04 (bs, 6H), 1.05 (bs, 18H), 1.06 (bs, 18H), 1.16 (d,
J = 6.0 Hz, 3H), 1.50–1.57 (m, 7H), 1.60–1.64 (m, 2H), 1.66–
1.74 (td, J = 10.5, 2.1 Hz, 1H), 1.88 (bd, J = 11.7 Hz, 1H), 1.99
(m, 2H), 2.15 (dd, J = 14.1, 6.6 Hz, 1H), 2.39 (d, J = 14.4,
2.0 Hz, 1H), 2.40–2.64 (m, 2H), 2.77 (dd, J = 12.9, 3.9 Hz, 1H),
3.21 (bt, J = 6.3 Hz, 1H), 3.33 (bq, J = 6.3 Hz, 1H), 3.65 (bq,
J = 6.3 Hz, 1H), 4.17–4.27 (m, 2H), 4.32–4.50 (ddd, J = 49.2,
7.4, 2.1 Hz, 1H), 5.82 (d, J = 10.8 Hz, 1H), 6.19 (d,
J = 11.1 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 2.4, 12.2,
12.5, 17.9, 17.9, 19.2, 22.0, 23.1, 25.6, 30.3, 33.1, 39.7, 41.8,
[aD27] = +12.9 (ca. 1.36, CHCl3).
4.1.9. (Z,1S,2R,6R)-1-Fluoro-4-[2-
(diphenylphosphinyl)ethylidene]-2,6-
bis(triisopropylsilyloxy)cyclohexane (9)
To a solution of (Z,1S,2R,6R)-4-[2-chloroethylidene]-1-
fluoro-2,6-bis(triisopropylsilyloxy)cyclohexane (8) (0.100 g,
0.197 mmol) in THF (1.9 mL) was slowly added a freshly
prepared THF solution of lithium diphenylphosphide, until a
yellow color persisted at ꢀ78 8C under an argon. This was
quenched with water (0.8 mL), and the reaction mixture was
warmed up to room temperature. After THF was evaporated
under reduced pressure, the gummy residue was dissolved in
CH2Cl2 (4.0 mL), 12 drops of 30% hydrogen peroxide were
added and then stirred for 1 h at room temperature. After usual
workup, the resultant residue was purified by silica-gel
chromatography to give (Z,1S,2R,6R)-1-fluoro-4-[2-(diphenyl-
phosphinyl)ethylidene]-2,6-bis(triisopropylsilyloxy)cyclohex-
ane (9) (0.117 g, 88%): 19F NMR (376 MHz, CDCl3) d ꢀ199.1
(bs, 1F); 1H NMR (400 MHz, CDCl3) d 1.01 (bs, 6H), 1.06 (bs,
36H), 2.01 (bd, J = 10.8 Hz, 1H), 2.08 (bd, J = 10.8 Hz, 1H),
2.40–2.45(m, 2H), 3.13(dd, J = 14.8,7.6 Hz, 2H), 4.07–4.20(m,
2H), 4.31–4.45 (ddd, J = 49.6, 6.0, 2.0 Hz, 1H), 5.38 (dd,
J = 14.0, 7.2 Hz, 1H), 7.46 (td, J = 7.6, 2.0 Hz, 2H), 7.53 (bt,
J = 7.6 Hz, 4H), 7.71 (dd, J = 11.2, 7.6 Hz, 4H); 13C NMR
(101 MHz, CDCl3) d 12.2, 12.3, 12.3, 12.4, 18.0, 18.0, 30.5 (d,
3
2
44.5 (d, J = 2.5 Hz), 56.2, 57.4, 65.0, 69.4 (d, J = 17.0 Hz),
69.6 (d, 2J = 21.8 Hz), 73.2, 78.1, 95.5 (d, 1J = 184 Hz), 116.5,
122.9, 131.6, 141.5; IR (neat) 2948, 2870, 1464, 1381, 1367,
1325, 1251, 1218, 1112, 1038, 998, 967, 917, 884, 859, 839,
804, 758, 710, 681 cmꢀ1; [aD27] = +33.7 (ca. 1.26, CHCl3).
4.1.11. 2a-Fluoro-19-nor-1a,25-dihydroxy-22-oxavitamin
D3 (10)
To a solution of 2a-fluoro-19-nor-1a,3b-bis(triisopropylsi-
lyloxy)-25-(trimethylsilyloxy)-22-oxavitamin D3 (0.0025 g,
0.031 mmol) in THF (3.1 mL) was added a 1 M solution of
tetrabutylammonium fluoride (TBAF) in THF (0.31 mL,
3.1 mmol) at room temperature. After stirring for 24 h at that
temperature, the solution was evaporated under reduced
pressure. The residue was purified by neutral silica-gel
chromatography to give 2a-fluoro-19-nor-1a,25-dihydroxy-
22-oxavitamin D3 (10) (0.012 g, 92%): 19F NMR (376 MHz,
2
1JC-P = 70.4 Hz), 33.0, 40.7, 68.7 (d, JC-F = 16.8 Hz), 69.6 (d,
1
2
2JC-F = 21.4 Hz), 94.3 (d, JC-F = 185 Hz), 114.6(d, JC-P
=
3
3
1
9.2 Hz), 128.5 (d, JC-P = 2.3 Hz), 128.6 (d, JC-P = 2.3 Hz),
130.9 (d, 2JC-P = 8.5 Hz), 131.1 (d, 2JC-P = 8.4 Hz), 131.8 (d, 4JC-
P = 3.1 Hz), 131.9 (d, 4JC-P = 3.1 Hz), 132.3 (d, 1JC-P = 98.8 Hz),
CDCl3) d ꢀ201.0 (bd, J = 45.9 Hz, 1F); H NMR (400 MHz,
CDCl3) d 0.54 (s, 3H), 1.19 (d, J = 6.0 Hz, 3H), 1.23 (s, 3H),
1.24 (s, 3H), 1.56–1.60 (m, 11H), 1.86–1.89 (m, 1H), 1.96–2.02
(m, 2H), 2.17–2.22 (m, 1H), 2.36–2.39 (m, 1H), 2.62–2.72 (m,
1H), 2.78–2.82 (m, 1H), 3.24–3.27 (m, 1H), 3.46–3.51 (m, 1H),
3.82–3.87 (m, 1H), 4.09–4.23 (m, 2H), 4.49 (ddd, J = 49.6, 7.2,
2.8 Hz, 1H), 5.82 (d, J = 11.2 Hz, 1H), 6.35 (d, J = 11.2 Hz,
1H); 13C NMR (75 MHz, CDCl3) d 12.6, 18.7, 22.1, 23.0, 25.7,
1
3
132.9 (d, JC-P = 98.8 Hz), 137.1 (d, JC-P = 11.5 Hz); IR(neat)
2948, 2868, 1464, 1439, 1386, 1245, 1193, 1120, 1071, 1015,
998, 971, 917, 884, 851, 828, 754, 719, 683, 661 cmꢀ1
;
[aD27] = ꢀ12.7 (ca. 1.52, CHCl3).
3
4.1.10. 2a-Fluoro-19-nor-1a,3b-bis(triisopropylsilyloxy)-
25-(trimethylsilyloxy)-22-oxavitamin D3
28.7, 29.0, 29.2, 32.2, 39.5, 40.5 (d, J = 4.8 Hz), 41.4, 44.7,
56.1, 57.1, 65.6, 68.2 (d, 2J = 18.2 Hz), 68.5 (d, 2J = 21.8 Hz),
70.6, 79.0, 96.0 (d, 1J = 177 Hz), 115.9, 124.8, 129.4, 143.3; IR
(neat) 3320, 3052, 2936, 2878, 1622, 1446, 1379, 1348, 1267,
A solution of (Z,1S,2R,6R)-1-fluoro-4-[2-(diphenylphosphi-
nyl)ethylidene]-2,6-bis(triisopropylsilyloxy)cyclohexane (9)
(0.064 g, 0.095 mmol) in THF (1.9 mL) was cooled at
ꢀ78 8C and treated dropwise with 2.0 M n-butyllithium in
pentane (0.050 mL, 0.099 mmol) under an argon. After stirring
for 5 min, a solution of de-A,B-8-oxo-25-(trimethylsilyloxy)-
22-oxacholestane (0.037 g, 0.104 mmol) in THF (0.6 mL) was
added dropwise to the resultant deep red solution at ꢀ78 8C
over 10 min. The reaction mixture was gradually warmed up to
room temperature over about 20 h in the dark with stirring.
After the addition of a 2 N sodium potassium tartrate (0.6 mL)
and a 2 N potassium bicarbonate (0.6 mL), the reaction mixture
was extracted three times with ethyl acetate. The combined
organic layers were washed with brine and dried over
anhydrous sodium sulfate. After evaporation under reduced
pressure, the resultant residue was purified by neutral silica-gel
chromatography to give 2a-fluoro-19-nor-1a,3b-bis(triisopro-
1154, 1081, 1019, 965, 897, 866, 828, 795, 737, 702 cmꢀ1
;
[aD26] = +80.1 (ca. 0.87, CHCl3); HRMS m/z calcd. for
C25H41FO4 (M+) 424.4989, found 424.3004.
4.2. Cell culture
Human promyelocytic leukemia HL-60 cells were obtained
from Dr. Y. Seino of the Okayama University School of
Medicine. The cells were maintained in continuous culture in
RPMI-1640 medium (Nissui Seiyaku Co. Ltd., Tokyo, Japan)
supplemented with 10% fetal bovine serum (Gibco BRL, Grand
Island, NY, USA), 0.06 mg/mL kanamycin (Sigma).
Human osteosarcoma MG-63 cells, were maintained in
Dulbecco’s modification Eagle medium (Gibco BRL) supple-
mented with 1% penicillin, 1% streptomycin and 10% dextran-
coated charcoal-treated fetal calf serum (Gibco BRL).
pylsilyloxy)-25-(trimethylsilyloxy)-22-oxavitamin
D3

K. Nakagawa et al. / Journal of Fluorine Chemistry 128 (2007) 654–667
665
Cloned metastatic variant cells of Lewis lung carcinoma
(LLC) were kindly supplied by the Cell Resource Center for
Biomedical Research, Tohoku University. We previously
generated the GFP expression vector stable transfected LLC
cells (LLC-GFP cells) [21]. The medium used for culturing the
tumor cells and for all assays was RPMI1640 medium (Life
Technologies, Grand Island, NY) supplemented with L-
glutamine (0.29 mg/mL), kanamycin (0.06 mg/mL) and 10%
heat-inactive fetal bovine serum.
which is the product of the % fluorescence and the mean
fluorescence intensity, with 104 cells counted per treatment.
4.6. Transfection and luciferase activity assay
MG-63 cells, which are positive for VDR and RXR gene
expression, were transfected with 0.5 mg luciferase reporter
plasmid (pGVB2 vector, Toyo Ink Co., Ltd., Tokyo, Japan)
inserted with a rat 25(OH)D3-24-hydroxylase gene promoter
(ꢀ291/+9) including the two VDREs [49] and 0.25 mg of the
pRL-CMV vector (Toyo Ink Co., Ltd.) as an internal control
using the Tfx-50 reagent (Promega Corp., Tokyo, Japan). In the
mammalian two-hybrid luciferase assay, MG-63 cells were
transfected with 0.5 mg of a pM vector containing a human
RXRa cDNA connected to GAL-DBD (pM-RXRa) or
containing a steroid receptor coactivator-1 (SRC-1) [50] cDNA
connected to GAL-DBD (pM-SRC-1), 0.5 mg of a pVP16
vector (CLONTECH) containing a human VDR cDNA
connected to VP16 transactivation domain (pVP16-hVDR),
0.5 mg of pGVP2 vector containing GAL-BS and a pRL-CMV
vector as an internal control using the Tfx-50 reagent. The cells
were incubated with various concentrations of 1a,25-D3 or its
analogs for 2 days. The luciferase activities of the cell lysates
were measured with a luciferase assay system (Toyo Ink Co.,
Ltd.), according to the manufacturer’s instructions. Transacti-
vation measured by luciferase activity was standardized with
the luciferase activity of the same cells determined by the Sea
Pansy luciferase assay system as a control (Toyo Ink Co., Ltd.).
Each set of experiments was repeated at least three times, and
the results are presented in terms of fold-induction as
means ꢂ S.E.
4.3. HL-60 cells and synchronization of cell cycle at S
phase by excess amounts of thymidine
HL-60 cells were synchronized at S phase by the following
procedure: Cells (4 ꢃ 105 cells/mL) were cultured in 30 mL of
RPMI-1640 medium for 24 h and subsequently cultured for
16 h in RPMI-1640 medium supplemented with 2.5 mM
thymidine. After washing the cells with Ca, Mg-free phosphate
buffered saline [PBS(ꢀ)] twice, the synchronization of cell
cycle by the same manner was repeated, and the cells thus
obtained were used in the following experiments.
4.4. Flow cytometry
Cells (105 cells/well) were placed in 24-well tissue culture
plates and cultured for 3 days in RPMI-1640 medium with
1a,25-D3 or analogs under the same conditions as above. To
reduce the effects of contact inhibition, control cells were
adjusted to 60–70% confluence at the time of FACS analysis.
Each group of cells was collected and washed with PBS(ꢀ)
once. Then, the cells were resuspended in PBS(ꢀ) containing
0.2% Triton-X and 100 mg RNase, and incubated at 37 8C for
1 h. Cells were washed with PBS(ꢀ) and incubated with
0.5 mL of DNA-staining solution containing propidium iodide
(50 mg/mL) at 4 8C for 20 min. The cells were analyzed with a
flow cytometer equipped with an argon laser (488 nm, Becton
Dickinson FACScanTM) and cell cycle distribution was
analyzed by ModiFiT LT(Verity). Experiments were repeated
three times.
4.7. Animals
Female C57BL/6J mice (Clea Japan, Inc., Tokyo, Japan), 10
weeks of age, were used for all experiments. Mice were then fed
ad libitum distilled water and a chow diet (F-2, Clea Japan, Inc.,
Tokyo, Japan; ingredients: 1.2% calcium, 0.6% phosphorus and
1.08 IU vitamin D3/g). The mice were maintained under
specific pathogen-free conditions with a 12 h light, 12 h dark
cycle. This study was conducted in accordance with the
standards established by the Guidelines for the Care and Use of
Laboratory Animals of Kobe Pharmaceutical University.
VDRꢀ/ꢀ mice were generated by homologous gene targeting
as described previously [44]. Null mutant mice were obtained
by intercrossing a heterozygous VDR knockout female and a
heterozygous male. Mice were weaned at 3 weeks of age and
then fed ad libitum a high calcium and vitamin D-deficient diet
(Clea Japan, Inc.; ingredients: 2% calcium, 1.25% phosphorus,
0 IU vitamin D3/100 g, and 20% lactose). We used the wild-
type (VDR+/+) and VDRꢀ/ꢀ mice, 10 weeks of age.
4.5. Cell surface antigen expression analysis
Cells (105 cells/well) were placed in 24-well tissue culture
plates, and cultured for 3 days in RPMI-1640 medium with
1a,25-D3 or analogs under the same conditions as above. Each
group of cells was then collected and washed with PBS(ꢀ)
once. Then, the cells (2 ꢃ 105 cells) were resuspended in
100 mL diluent solution containing 1% bovine serum albumin
(BSA) and 1% sodium azide and incubated with 10 mL human
monoclonal FITC conjugated CD11b antibody (Sigma, USA)
and 10 mL human monoclonal PE conjugated CD14 antibody
(Sigma, USA) for 30 min at room temperature without light.
The cells were washed once with diluent solution and then fixed
in 300 mL of PBS(ꢀ) containing 2% paraformaldehyde.
Fluorescence was detected on a Becton Dickinson FACScanTM
at excitation wavelength of 490 nm and emission wavelength of
520 nm. Results were recorded as the mean fluorescence index,
4.8. In vivo calcium-regulating assay
Ten week-old normal C57BL/6J female mice were divided
into several groups of six mice, each receiving oral admin-
istration of 1, 10 or 100 mg/kg of 1a,25-D3 or its analogs in

666
K. Nakagawa et al. / Journal of Fluorine Chemistry 128 (2007) 654–667
2 mL/kg of 0.1% Triton X-100 solution. The resulting increases
in serum calcium were measured at 8, 24, 48 and 72 h after
administration by the o-cresolphthalein complexone (OCPC)
method. The relative activity of each analog with respect to
1a,25-D3 was evaluated as follows: the dose–response effect of
each analogue was calculated at the time when the effect was
maximum and expressed as the dosage required to elevate the
serum calcium level by 1 mg/dL.
reported as the number of cells/low power field enumerated
from triplicate chambers from each of four experiments.
4.11. Intravenous injection of LLC-GFP cells in VDRꢀ/ꢀ
mice with corrected hypocalcemia and hypervitaminosis D
and administration of 2a-F-19-nor-22-oxa-1a,25-D3
VDRꢀ/ꢀ mice were generated by homologous gene targeting
as described previously [45]. Mice were weaned at 3 weeks of
age then fed a high calcium and vitamin D-deficient diet (Clea
Japan, Inc.; ingredients: 2% calcium, 1.25% phosphorus, 0 IU
vitamin D3/100 g, and 20% lactose) ad libitum. We used wild-
type (VDR+/+) and VDRꢀ/ꢀ mice, 10 weeks of age, which were
fed a high calcium and vitamin D-deficient diet to completely
eliminate endogenous 1a,25-D3 and to correct serum calcium
levels. First, we confirmed that VDRꢀ/ꢀ mice showed extreme
hypervitaminosis D along with severe hypocalcemia when
compared to VDR+/+ mice. Feeding of the high calcium and
vitamin D-deficient diet resulted in the complete elimination of
1a,25-D3 both before and after 2a-F-19-nor-22-oxa-1a,25-D3
administration in VDR+/+ and VDRꢀ/ꢀ mice. Mice were injected
i.v. with a singledose of 5 ꢃ 105 LLC-GFP cells in a total volume
of 0.2 mL in RPMI1640 medium containing 10% FBS. There-
after, 2a-F-19-nor-22-oxa-1a,25-D3 was administered continu-
ously using an osmotic minipump (model 2 ML4 Alzet; Alza
Corp., Palo Alto, CA) implanted s.c. on the same day as the
inoculationwith LLC-GFP cells. An infusionrate of 10or 50 mg/
kg/24 h was chosen, and each minipump contained 2a-F-19-nor-
22-oxa-1a,25-D3 dissolved in 0.1% Tween-20 and 10% ethanol
todeliveracontinuousdoseforupto3weeksatarateof2.5 mL/h.
Untreated animals were implanted with a minipump containing
vehicle alone. Mice with tumor implants were sacrificed 3 weeks
laterandserumCalevel,lungweight,numberoflungnodulesand
GFP mRNA expression in the lung were measured.
4.9. Assessment of cell growth in vitro in LLC-GFP cells
The effects of 2a-F-19-nor-22-oxa-1a,25-D3 and 1a,25-D3
on the proliferation of LLC-GFP cells in vitro were assessed by
cell counting and cell cycle analysis. Cells were seeded at a
density of 2 ꢃ 105 cells/well in 6-well culture plates in
RPMI1640 medium containing 10% fetal bovine serum for
24 h. After 24 h, fresh medium containing 10% FBS with
vehicle (ethanol), 1a,25-D3 or 2a-F-19-nor-22-oxa-1a,25-D3
(10ꢀ7 M) was added to cultured cells, and incubation continued
for 3 days. 1a,25-D3 and 2aF-19-nor-22-oxa-1a,25-D3 were
dissolved in ethanol, the final concentration of ethanol in all
cultures not exceeding 0.1%. Cells were trypsinized at specific
time points, and an aliquot of cells was counted and the cell
cycle analyzed. For the analysis of the cell cycle, each group of
cells was collected and washed with PBS (ꢀ) once. Then the
cells were resuspended in PBS (ꢀ) containing 0.2% Triton-X
and 100 mg of RNase and incubated at 37 8C for 30 min. Cells
were washed with PBS (ꢀ) and incubated with 0.5 mL of DNA-
attaining solution containing propidium iodide (50 mg/mL) at
4 8C for 20 min. The cells were analyzed with a flow cytometer
equipped with an argon laser (488 nm, Becton Dickinson
FACScanTM) and the cell cycle distribution was analyzed using
ModiFit LT (Verity).
4.10. In vitro invasion of LLC-GFP cells through a
Matrigel-coated membrane
4.12. Imaging and tumor scoring
The capacity of tumor cells to traverse a basement
membrane-matrix-coated filter has been shown to be repre-
sentative of their invasiveness. Therefore, the capacity of tumor
cells to migrate through Matrigel-coated membranes was
measured. Nucleopore filters (8 mm pore size) were coated with
100 mL of a 1:20 dilution of Matrigel (Becton Dickinson,
Bedford, MA) and allowed to dry. Representative filters were
stained with crystal violet and, when examined microscopi-
cally, appeared to be evenly coated with Matrigel. Medium was
added to the lower compartment of each blind-well chemotactic
chamber. A dried, coated filter was placed over the lower
compartment. After reconstitution of Matrigel, 5 ꢃ 104 cells
were added to the upper compartment together with the same
concentration of 1a,25-D3 or 2a-F-19-nor-22-oxa-1a,25-D3
with which the cells had been preincubated with vehicle, 1a,25-
D3 or 2a-F-19-nor-22-oxa-1a,25-D3 (10ꢀ8 or 10ꢀ7 M) for 3
days. Chambers were incubated for 24 h, after which filters
were removed, wiped clean on the upper surface and fixed in
10% formalin. The number of cells on the lower surface of the
filter was counted under the fluorescent microscope. Data were
A Leica fluorescence stereomicroscope model MZ FL III
(Leica Microsystem, Inc., USA) equipped with a mercury 50 W
lamp power supply was used. Selective excitation of GFP was
produced through a D480/40 band-pass filter and 510 DCXR
dichroic mirror. Emitted fluorescence was collected through a
long-pass filter DC300F and digital camera system (Leica
Microsystem, Inc., USA). The numbers of metastatic nodules
on the surface of the lung were counted under the fluorescence
stereomicroscope. To quantify lung metastasis, the GFP-
expressing spots on the lungs were enumerated.
4.13. Real-time quantitative PCR
Lung tissues and LLC-GFP cells were homogenized in
ISOGEN (Nippon Gene, Tokyo Japan). Total RNAwas isolated
as specified by the manufacturer. The purified RNA was
reverse-transcribed with AMV reverse transcriptase (TaKaRa,
Japan). Quantitative analyses of gene expression were
conducted using GFP (50-CTGCTCTCTTGGGTCCACTGG-
30 and 50-CACCGCCTTGGCTTGTCACAT-30), and b-actin

K. Nakagawa et al. / Journal of Fluorine Chemistry 128 (2007) 654–667
667
(50-AGGCCCAGAGCAAGAGAGGTAT-30 and 50-CATGTC-
GTCCCAGTTGGTAACA-30)-specific primer sets, a Gen-
eAmp 5700 Sequence Detection System (PE Biosystems,
Foster City, CA) and the SYBR Green core reagent kit (PE
Biosystems, Foster City, CA).
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We acknowledge the excellent technical assistance provided
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Toda. This work was supported by the project for the Advanced
Research and Technology by Kobe Pharmaceutical University
and grants-in-aid received from the Japanese Ministry of
Education (06453133, 06555271, 10555319, 12672139,
13218129 and 15590083).
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