Study on the metabolites of 1alpha,25-dihydroxyvitamin D4.

Article abstract of DOI:10.1016/S0039-128X(00)00208-7

Three major metabolites of 1alpha,25-dihydroxyvitamin D(4) were isolated from the bile of rat and the structures were elucidated on the basis of spectral data and the periodate oxidative cleavage of the diol structures of the metabolites. One of the metabolites was the known calcitroic acid. Another two metabolites were isomers and identified as 9,10-secoergosta-5,7,10(19)-triene-1alpha,3beta,24,25-tetrahydroxy-26-oic acid and 9,10-secoergosta-5,7,10(19)-triene-1alpha,3beta,24,25-tetrahydroxy-28-oic acid. It was found that 1alpha,25-dihydroxyvitamin D(4) is metabolized in a similar manner in vivo to that of 1alpha,25-dihydroxyvitamin D(2) but differently from 1alpha,25-dihydroxyvitamin D(3).

Full text of DOI:10.1016/S0039-128X(00)00208-7

Steroids 66 (2001) 93–97
Study on the metabolites of 1␣,25-dihydroxyvitamin D4
Yoji Tachibana*, Masahiro Tsuji
Pharmaceutical Research Center, Nisshin Flour Milling Co., Ltd., 5–3-1 Tsurugaoka, Ooi-machi, Iruma-gun, Saitama 356-0045, Japan
Received 31 May 2000; received in revised form 8 August 2000; accepted 14 August 2000
Abstract
Three major metabolites of 1␣,25-dihydroxyvitamin D were isolated from the bile of rat and the structures were elucidated on the basis
4
of spectral data and the periodate oxidative cleavage of the diol structures of the metabolites. One of the metabolites was the known calcitroic
acid. Another two metabolites were isomers and identified as 9,10-secoergosta-5,7,10(19)-triene-1␣,3␤,24,25-tetrahydroxy-26-oic acid and
9
,10-secoergosta-5,7,10(19)-triene-1␣,3␤,24,25-tetrahydroxy-28-oic acid. It was found that 1␣,25-dihydroxyvitamin D is metabolized in
4
a similar manner in vivo to that of 1␣,25-dihydroxyvitamin D but differently from 1␣,25-dihydroxyvitamin D . © 2001 Elsevier Science
2
3
Inc. All rights reserved.
Keywords: 1␣,25-Dihydroxyvitamin D ; Metabolite; Bile; Calcitroic acid; 26-Carboxy-1␣,24,25-trihydroxyvitamin D ; 28-Carboxy-1␣,24,25-trihydroxyvi-
4
4
tamin D4
1
. Introduction
Active forms of vitamin D [1], namely 1␣,25-dihy-
antiproliferative and cell-differentiating effects [9]. In order
to clarify the effect of the methyl group of 3 on the metab-
olism and the biological activities in vivo in comparison
with those of 1, we describe here the metabolic pathway of
3 by investigating the metabolites in the bile of rats by
means of spectral data and the periodate oxidative cleavage.
3
droxyvitamin D (1) and its derivatives have been widely
3
studied for their versatile biological activities [2]. However,
less is known about the effect of metabolism of vitamin D
derivatives on their biological activities. Most of the me-
tabolism studies have been performed with the natural hor-
mone (1) [3]. Very little is known about the metabolism of
the naturally occurring 1␣,25-dihydroxyvitamin D2 (2)
which has equal biological activities to 1 in mammals [4,5].
As to the metabolism of 1␣,25-dihydroxyvitamin D4
2
. Experimental procedures
2.1. General methods and equipment
(
22,23-dihydro-1␣,25-dihydroxyvitamin D ) (3) which has
2
an extra 24S methyl group on the side chain of 1, no study
has been hitherto reported. In the course of our continuous
study on the vitamin D derivatives [6], we synthesized 2 [7]
and 3 [8] to investigate the effect of the double bond
NMR spectra were recorded on a JEOL FX-400 NMR
and a Varian UNITY 500 spectrometer. Chemical shift are
given in ppm relative to that of Me Si (␦ ϭ 0) in CDCl .
4
3
Mass spectra were obtained from a JEOL DX-303, a Shi-
madu Qp-5000 and a Hitachi M-2000 instrument. UV spec-
tra were taken on a JASCO UBest-50 spectrometer. HPLC
was performed with a Shimadu SPD-10A, a Waters 600 E
and a Hitachi 655 liquid chromatograph with a UV detector
(265 nm). Radioactivity was measured with an Aloka LSC-
3500 liquid scintillation counter. Solvents were evaporated
in nitrogen stream. Male Wistar strain rats (Japan SLC)
were housed in wire cages and given free access to food and
water. The animals were used after 2 months when the body
weight had reached 300–430 g. ␤-Gluconidase and arylsul-
fatase were purchased from Tokyo Hormone Co., Ltd.
(C22-C23) and the methyl group at C-24 position on the
side chain on the biological activities to compare with those
of 1 (Fig. 1A). Interestingly, it was found that the introduc-
tion of a (S)-methyl group at C-24 position of 1 (compound
3
) strongly increases the affinity for vitamin D binding
protein (DBP), transactivation potencies on target genes
without changing the affinity for vitamin D receptor (VDR),
*
Corresponding author. Tel.: ϩ81-492-67-3990; fax: ϩ81-492-67-
3
0
951.
E-mail address: tachibanay@mail.ni-net.co.jp (Y. Tachibana).
039-128X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(00)00208-7

9
4
Y. Tachibana, M. Tsuji / Steroids 66 (2001) 93–97
umn: Inertsil SIL (5 ␮m, GL. Schience Co., Ltd., Saitama),
ϫ 250 mm; detector: UV, 265 nm; solvent: chloroform/
6
ethanol (30:1 v/v)) to give 2 mCi of 4 (0.33%). Specific
radioactivity: 16.4 Ci/mmol. Radiochemical purity: 99%.
The UV, NMR and HPLC spectra were completely identical
with those of cold authentic 3.
2
.3. Isolation of the metabolites (5, 8 and 9)
Rats were anesthetized by intraabdominal injection of
pentbarbital (25 mg/kg of body weight) under light ether
anesthesia. The bile duct was then cannulated with polyeth-
ylene tubing. After the abdominal incision was closed with
sutures and wound clipping, the animals were kept in re-
straining cages (Bollman cages).
For the isolation of the metabolites, the bile duct cannu-
lated six rats were dosed (2.6 mg/10 ␮Ci/kg) with a 30%
ethanol solution (0.26 mg/␮Ci/0.25 ml) prepared from 3 and
4
. The bile samples from all rats were collected for 24 h
after the dose and pooled (204 ml). After the addition of 1
M acetic acid-sodium acetate buffer (20 ml, pH 5.0), ␤-glu-
3
3
curonidase (320 ϫ 10 units) and arylsulfatase (130 ϫ 10
units) to the bile (204 ml), the mixture was incubated at
37°C for 3 h and adjusted to pH 4 with 1 N HCl and then
pH 3 with 0.1 M citric acid Na- HCl buffer (pH 3.0, 20 ml).
The mixture was extracted with chloroform/methanol (1:1
v/v, 120 ml) and centrifuged (3000ϫ g, 10 min). Water
layer was reextracted in the same way as described above.
The combined chloroform solution was dried over sodium
sulfate and concentrated to yield 20 mg of the extract. The
extract (20 mg) was subjected to Sephadex LH-20 (2.5 ϫ 80
mm) chromatography eluted with chloroform/hexane/meth-
anol (15:4:1 v/v) (fractions, 0–140) and chloroform/meth-
anol (1:1 v/v) (fractions, 141–200) at a flow rate of 1 ml/min
Fig. 1. Structures of 1␣,25-dihydroxyvitamin D (1), 1␣,25-dihydroxyvi-
3
3
tamin D (2), 1␣,25-dihydroxyvitamin D (3), [1␤- H]-1␣,25-dihydroxyvi-
2
4
tamin D (4), calcitroic acid (5), periodate cleavage products (6 and 7) of
4
methyl esters of M2 (8) and M3 (9).
3
2
.2. Synthesis of [1␤- H]-1␣,25-dihydroxyvitamin D (4)
4
Label compound (4) (16.4 Ci/mmol) (radiochemical pu-
rity: 99% by HPLC analysis) was synthesized at Daiichi
Chemical Pharmaceutical Co., Ltd.
To an acetonitrile solution (20 ml) of 3 (60 mg), Dess-
Martin reagent (60 mg) was added. The mixture was stirred
for 2 h at room temperature. Ethyl acetate was added and the
resulting mixture was washed with saturated aqueous
Na S O , NaHCO and brine. The organic layer was dried
(
0.5 ml/fraction) to give the three major radioactive com-
ponents, M1 (fractions 30–60) and M2, M3 (fractions 150–
80). After the solvent of more polar fractions containing
2
2
3
3
1
(
MgSO ) and evaporated. The residue was purified by col-
4
M2 and M3 was removed, the residue was dissolved in 1 ml
of methanol and excess of etheral diazomethane was added.
The mixture was stirred at 4°C for 18 h. The solvent was
evaporated and the residue was chromatographed on Seph-
adex LH-20 (13 ϫ 430 mm). Elution with chloroform/
hexane (13:7 v/v) afforded methyl esters of M2 and M3.
The respective methyl ester derivatives were further purified
by reversed phase HPLC (column: Capcell Pak C18 (0.5
␮m, Shiseido Co., Ltd., Tokyo), 4.6 ϫ 250 mm; solvent:
acetonitrile/0.01% acetic acid (2:3 v/v); flow rate: 1 ml/min)
umn chromatography on silica gel (chloroform/ethyl acetate
4:1 v/v)) to afford 48 mg of 1-oxo-25-hydroxyprevitamin
(
D as a pale yellow oil.
4
1
H-NMR ␦: 0.71 (3H, s, 18-CH ), 0.90 (3H, d, J ϭ 6.8
3
Hz, 28-CH ), 0.97 (3H, d, J ϭ 6.4 Hz, 21-CH ), 1.16, 1.18
3
3
(
each 3H, s, 26-CH , 27-CH ), 1.79 (3H, s, 19-CH ), 4.15
3 3 3
(1H, m, 3-H), 5.49 (1H, m, 9-H), 6.04, 6.11 (2H, ABq, J ϭ
1
1.7 Hz, 6-H, 7-H).
To an aqueous solution (5 ml) of NaB[ H ] (0.5 Ci,
3
4
5
0Ci/mmol), the dioxane solution (2 ml) of 1-oxo-25-hy-
(
(
(
M2: t 37 min, M3: t 36 min) and normal phase HPLC
droxyprevitamin D (16 mg) was added. The mixture was
R R
4
column: Inertsil SIL, solvent: dichloromethane/methanol
19:1 v/v); flow rate: 1 ml/min) (M2: t 14 min, M3: t 16
stirred at room temperature for 1 h. After the addition of
saturated aqueous NH Cl solution (2 ml), the mixture was
R
R
4
extracted with ethyl acetate, dried (MgSO ) and concen-
min) to yield each 40␮g of methyl esters of M2 and M3,
respectively. The less polar fractions containing M1 was
treated similarly as described above to give 40 ␮g of methyl
ester of M1.
4
trated. To the residue, ethanol (5 ml) was added. The solu-
tion was refluxed with stirring for 1 h. The solution was
concentrated and the residue was purified by HPLC (col-

Y. Tachibana, M. Tsuji / Steroids 66 (2001) 93–97
95
2
.4. Periodate oxidation of methyl esters of M2 and M3
An acetone solution (50 ␮l) of methyl ester of M2 (4 ␮g)
and 10% aqueous NaIO (20 ␮l) was stirred at room tem-
4
perature for 3 h. The mixture was extracted with ethyl
acetate. After the solvent was evaporated, the residue was
purified by HPLC (column: Inertsil SIL; solvent: dichlo-
romethane/methanol (19:1 v/v); flow rate: 1 ml/min) to give
6
. Similar treatment of the methyl ester of M3 gave 7 (Fig.
1
B).
3
3
3
. Results and discussion
.1. Structures of the metabolites (M1, M2 and M3)
.1.1. Spectroscopic analysis
Active form of vitamin D (3) together with its tritium
Fig. 2. Mass spectrum and mass fragmentation pattern of the methyl ester
of the metabolite (M2).
4
3
labeled compound [1␤- H]-1␣,25-(OH) D (4) synthesized
2
4
from 3 in three steps (oxidation of the hydroxyl group at C-1
position of 3 with Dess-Martin reagent, reduction with
peaks at m/z 414 and 396 due to the elimination of 58 mass
3
NaB[ H ] and thermal rearrangement of the tritiated previ-
4
units (CH CH CHO) and the loss of water from the parent
3
2
tamin D by heating) according to the method described in
ϩ
4
peak 490 (M ) (Fig. 3).
the literature [10] were orally administered to Wistar rats.
The bile collected was treated with ␤-glucuronidase and
arylsulfatase and extracted by the procedure described by
Bligh and Dyer [11]. The organic phase of the extract was
purified by Sephadex LH-20 chromatography to yield three
main metabolites (M1, M2 and M3). The metabolites after
methylation with diazomethane were further purified by
Sephadex LH-20 chromatography, reversed phase and nor-
mal phase HPLC to provide the methyl esters of M1, M2
and M3 in the molar ratio of 1:1:1, respectively. Mass, UV
13
As shown by mass spectral study, C-NMR spectra of
the methyl esters of M2 and M3 also showed 29 carbon
signals showing that the metabolites retained the original
skelton of 3. The spectra (DEPT and HMQC) revealed that
the signal due to the tertiary carbon atom at C-24 position (␦
4
5.26) of 3 disappeared and newly formed signals ascrib-
able to quarternary carbon atom appeared at ␦ 79.58 and ␦
2.49 for methyl esters of M2 and M3. Accordingly, it is
8
suggested that M2 and M3 were hydroxylated at C-24
position. The spectra show the existence of methyl ester
1
and H-NMR spectra of the methyl ester of M1 were com-
groups at ␦ 52.92 and ␦ 52.99 (OCH ), and carbonyl group
3
pletely agreed with those of the methyl ester of calcitroic
acid (5) reported in the literature [12].
UV spectra of the methyl esters of M2 and M3 exhibited
absorbance maximum at 265 nm and minimum at 228 nm
showing that they have the 5,6-cis-triene chromophore of
the D vitamins.
at ␦ 176.49 and ␦ 176.18 for methyl esters of M2 and M3,
respectively (Table 1) [13].
Mass spectra of the methyl esters of M2 and M3 showed
the same molecular ion peak (m/z 490) indicating that the
metabolites are isomers and there are additional hydroxy
group and carboxy group derived from the oxidation of
methyl group of the intact molecule of 3 (Fig. 2 and Fig. 3).
The peak at m/z 287 in methyl esters of M2 and M3 implies
that secosteroid nucleus of 3 has remained unchanged. This
finding demonstrates that new metabolites are formed as a
result of change occurring only on the side chain of their
parent 3. The presence of characteristic mass fragments of
the methyl ester of M2 at m/z 386, 368 and 351 shows thaϩt
the fragments are produced from the loss of water from M
followed by a loss of 104 (CH CH(OH)(COOCH )) or 103
3
3
ϩ
(
CH C(OH)(COOCH ) ) mass units (Fig. 2). This suggests
3 3
that the carboxylation occurred at the C-26 position for M2.
The mass spectrum of the methyl ester of M3 exhibited
Fig. 3. Mass spectrum and mass fragmentation pattern of the methyl ester
of the metabolite (M3).

9
6
Y. Tachibana, M. Tsuji / Steroids 66 (2001) 93–97
Table 1
1
3
C-NMR spectral data for 3, the methyl esters of M2 and M3
Position
Group
3
M2
M3
(methyl ester)
(methyl ester)
ppm(␦)
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
4
5
6
7
8
CH
CH2
CH
CH2
C
CH
CH
C
CH2
C
CH2
CH2
C
CH
CH2
CH2
CH
CH3
CH2
CH
CH3
CH2
CH2
CH
C
70.81
42.84
66.86
45.26
70.89
42.84
66.85
45.25
70.73
42.70
66.80
45.13
147.47
124.95
116.92
143.18
28.99
132.77
23.51
40.26
45.81
55.74
22.16
27.36
56.18
11.94
111.79
35.96
18.83
29.64
29.04
—
147.64
125.04
116.97
143.29
29.10
132.81
23.59
40.44
45.90
56.29
22.28
27.56
56.31
12.00
111.76
36.61
19.15
34.82
27.85
45.26
—
148.64
125.02
117.01
143.18
29.32
132.82
23.56
40.42
45.89
56.21
22.25
27.59
56.28
11.99
111.80
36.24
18.88
32.56
28.71
—
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
Fig. 4. (A) Mass spectrum and mass fragmentation pattern of the periodate
cleavage product (6) of the methyl ester of M2, (B) Mass spectrum and
mass fragmentation pattern of the periodate cleavage product (7) of the
methyl ester of M3.
were present not metabolized. No signal due to the hydroxy-
1
3
methyl group was observed as predicted from the C-NMR
spectra. The results are summarized in Table 2.
It is suggested that the metabolites (M2 and M3) have the
vicinal diol structures from the results described above. The
oxidative cleavage of the methyl esters of M2 and M3 with
79.58
75.59
—
20.57
20.40
52.92
176.49
82.49
74.02
24.75
24.83
—
C
73.65
26.13
27.27
14.81
—
CH3
CH3
CH3
NaIO was carried out to afford the expected products (6
4
and 7) whose structures were supported by the mass spectra
(Fig. 4).
OCH3
52.99
176.18
CO
—
Based on the evidences described above, it is concluded
that the metabolites (M2, M3) are finally identified as 9,10-
secoergosta-5,7,10(19)-triene-1␣,3␤,24,25-tetrahydroxy-
26-oic acid (8) and 9,10-secoergosta-5,7,10(19)-triene-
1␣,3␤,24,25-tetrahydroxy-28-oic acid (9) although the
stereochemistry at C-24 position is not determined.
1
In the H-NMR spectra four methyl groups were ob-
served showing one of the methyl groups at C-26 (C-27) or
8 position of five methyl groups of 3 was metabolized to
2
carboxylic acid since the methyl groups at C-18 and C-21
Table 2
1
H-NMR spectral data for 3, the methyl esters of M2 and M3
Position
Group
3
M2
M3
(methyl ester)
(methyl ester)
ppm(␦)
1
3
6
7
8
9
H
H
H
H
CH3
CH2
4.43 (m)
4.30 (m)
6.02, 6.39
(ABq, J ϭ 11.2 Hz)
0.54 (s)
4.42 (m)
4.31 (m)
6.02, 6.36
(ABq, J ϭ 11.2 Hz)
0.54 (s)
4.43 (m)
4.30 (m)
6.00, 6.69
(ABq, J ϭ 11.2 Hz)
0.54 (s)
1
1
5.01 (s)
5.00 (s)
5.00 (s)
5
.33 (s)
0.94
d, J ϭ 5.9 Hz)
5.32 (s)
0.92
(d, J ϭ 5.9 Hz)
—
1.43 (s)
5.32 (s)
0.93
(d, J ϭ 5.9 Hz)
1.20 (s)
1.32 (s)
2
1
CH3
(
26
27
28
CH3
CH3
CH3
1.16 (s)
1.17 (s)
0.90
1.20 (s)
—
(d, J ϭ 6.8 Hz)
OCH3
—
3.82 (s)
3.82 (s)

Y. Tachibana, M. Tsuji / Steroids 66 (2001) 93–97
97
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