Synthesis of 21-nor-22-oxa-1Iα,25-dihydroxyvitamin D3 derivatives in quest of a drug with low calcemic activity

Article abstract of DOI:10.1016/j.tetlet.2004.08.160

Two 21-nor-22-oxa-1α,25-dihydroxyvitamin D₃ derivatives have been synthesized in quest of a drug with lower calcemic activity than Maxacalcitol, 22-oxa-1α,25-dihydroxyvitamin D₃, being used as antihyperparathyroidism and antipsoriatic drug. Of two 21-nor products obtained, the product carrying one carbon elongated side chain with diethylcarbinol moiety has been found to exhibit comparable differentiation-inducing activity to Maxacalcitol with much lower exhibition of calcemic activity.

Full text of DOI:10.1016/j.tetlet.2004.08.160

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 7837–7841
Synthesis of 21-nor-22-oxa-1a,25-dihydroxyvitamin D3
derivatives in quest of a drug with low calcemic activity
Hitoshi Shimizu,a,* Kazuki Shimizu,b Yasushi Uchiyama,b Atsuko Sugita,b
Tetsuhiro Mikami,a Tsuyoshi Yamauchi,a Masahiro Katoa and Kazumi Morikawab
aSynthetic Technology Research Department, Chugai Pharmaceutical Co., Ltd, 5-5-1 Ukima, Kita-Ku, Tokyo 115-8543, Japan
bChemistry Research Department I and Pharmaceutical Research Department II, Chugai Pharmaceutical Co., Ltd,
1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
Received 29 July 2004; revised 23 August 2004; accepted 27 August 2004
Abstract—Two 21-nor-22-oxa-1a,25-dihydroxyvitamin D3 derivatives have been synthesized in quest of a drug with lower calcemic
activity than Maxacalcitol, 22-oxa-1a,25-dihydroxyvitamin D3, being used as antihyperparathyroidism and antipsoriatic drug. Of
two 21-nor products obtained, the product carrying one carbon elongated side chain with diethylcarbinol moiety has been found
to exhibit comparable differentiation-inducing activity to Maxacalcitol with much lower exhibition of calcemic activity.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Owing to a wide range of activities in addition to its role
in calcium homeostasis (calcemic activity), 1a,25-dihydr-
oxyvitamin D3 1, known as Calcitriol, and its derivatives
have received a great deal of attention from both
biological and synthetic points of view.1 In our extensive
synthetic studies in this field, we prepared a 22-oxa-ana-
logue of Calcitriol 1 named as Maxacalcitol 2 which was
found to exhibit higher differentiation-inducing activity
than Calcitriol 1 with lower calcemic activity2 and, on
the basis of this finding, we have developed a practical
antihyperparathyroidism and antipsoriatic drug with
low calcemic activity.3,4 During the investigation we
have also found that calcemic activity is greatly reduced
compared with the reduction of differentiation-inducing
activity in other Calcitriol analogues, 3a and 3b, which
are lacking 21-methyl functionality.5,6 In this regard,
the Leo group in Denmark reported an interesting
observation that the 21-epimer of 22-oxa-1a,25-dihydr-
oxyvitamin D3 carrying one carbon elongated side chain
with diethylcarbinol moiety named as Lexacalcitol 4
enhances significantly both calcemic and differentia-
tion-inducing activities7,8 though its calcemic activity
seemed to be somewhat higher than for practical pur-
poses.9 Since it is essential to enforce the differentia-
tion-inducing activity or to reduce the calcemic activity
compared with Maxacalcitol 2 for the development of
a new drug having better drug profile, we were very
interested in the above findings, namely, a greater reduc-
tion in the calcemic activity relative to the differentia-
tion-inducing activity observed in the 21-nor
compounds, 3a and 3b, and a greater amplification on
both calcemic and differentiation-inducing activities in
Lexacalcitol 4, the 21-epimer with elongated side chain.
In order to know the effect of these two factors on the
improvement of the drug profile, we synthesized two
21-nor-22-oxa-1a,25-dihydroxyvitamin D3 derivatives,
21-nor-Maxacalcitol 5 and 21-nor-Lexacalcitol 6, for
biological evaluation. We wish to report here our pre-
liminary result providing a promising clue to the control
of calcemic activity and differentiation-inducing activity
for the improvement of the drug profile (Fig. 1).
The synthesis started from the known tetracyclic keto-
ne3a 7 which was first transformed into the primary alco-
hol 9, regio- and stereoselectively, in excellent overall
yield via the exo-methylene 8 by Wittig methylenation
followed by hydroboration–oxidation reaction. The
etherification of the primary alcohol 9 with an appropriate
alkyl halide or a sulfonate, such as 2-methyl-2-tri-
ethylsiloxybutyl bromide and 2-methyl-2-triethylsiloxy-
butyl tosylate, under standard Williamson conditions
completely failed to give the desired product. The
Keywords: Vitamin D3 derivative; 22-Oxavitamin D3 derivatives;
Separation of activity; Calcemic activity; Differentiation-inducing
activity.
*
Corresponding author. Tel.: +81 0339686072; fax: +81
0339686265; e-mail: shimizuhts@chugai-pharm.co.jp
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.08.160

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H. Shimizu et al. / Tetrahedron Letters 45 (2004) 7837–7841
which on the photolysis under the conditions established
for the 21-methyl analogues3 furnished 21-nor-Maxa-
calcitol 5 through concomitant photo-cycloreversion
and thermal hydrogen migration.1 Yield of the final step
was 22%13 (Scheme 1).
R
O
OH
HO
OH
Synthesis of 21-nor-Lexacalcitol 6 utilized the cycload-
duct 13 formed in the synthesis of 21-nor-Maxacalcitol
5 shown above. Thus, treatment of the adduct 13 with
potassium tert-butoxide induced retro-Michael cleavage
giving rise to an excellent yield of the primary alcohol
17. In contrast to the above mentioned etherification
reaction between the primary alcohol 9 and the c,c,c-
trisubstituted primary electrophiles, the Williamson
etherification between the alcohol 17 and the d,d,d-
trisubstituted primary bromide 18 proceeded without
difficulty to generate the desired ether 19 in good yield.
This was apparently due to the one-carbon elongation in
the electrophile, which lessened the steric congestion to
allow the standard SN2pathway giving rise to the ether
HO
OH
Calcitriol 1
OH
Maxacalcitol 2:R=Me
21-nor-Maxacalcitol 5:R=H
R
O
X
OH
HO
OH
HO
OH
OH
21-nor-Calcitriol 3a:X=CH2
21-oxa-21-nor-Calcitriol 3b:X=O
Lexacalcitol 4:R=Me
21-nor-Lexacalcitol 6:R=H
Figure 1.
O
TBSO
i
ii
observed difficulty in the Williamson synthesis was
apparently due to the steric effect of the methyl and
the O-protecting groups on the tertiary center of the
alkylating agents, which shields the nucleophilic attack
of the steroidal primary alcohol 9 in the SN2pathway. 10
We had, therefore, to take a rather circuitous route.
Thus, the alcohol 9 was reacted with N,N-dimethylacryl-
amide in THF in the presence of sodium hydride to ini-
tiate a Michael 1,4-addition3c to give rise to the ether 10.
As expected, the reaction of the alcohol 9 with the less
hindered electrophile proceeded without difficulty to af-
ford the ether 10 in a satisfactory yield. When an acry-
late ester or methyl vinyl ketone was used in place of
the amide as a Michael acceptor, an undesired second-
ary reaction took place under the conditions to give a
complex mixture. By following the standard conditions
established in the synthesis of vitamin D3 deriva-
tives,1d,6a the ether 10 obtained was treated with NBS
in hexane to give the allyl bromide 11, which was treated
with c-collidine in toluene to generate the 1,3-diene 12
accompanied by inseparable by-products. The crude
diene 12 was first treated with 4-phenyl-1,2,4-triazo-
line-3,5-dione (PTAD)11 to give the adduct 13 for puri-
fication. The adduct 13 after purification was then
heated in DMI at 140ꢁC to regenerate the diene 12 by
retro-Diels–Alder reaction. Overall yield of the diene
12 from the ether 10 through a four-step sequence was
13%. At this point, transformation of the tertiary amide
12 into the tertiary alcohol 15 was carried out though it
was found to be not an easy task. It required two steps
and the cerium reagent12 was essential in each of the
steps so as to have the nucleophilic reaction proceed.3c
Thus, the reaction of the reagent, prepared in situ from
methylmagnesium bromide and cerium chloride in THF,
was treated with the amide 12 and gave the methyl ke-
tone 14, which was again treated with the same cerium
reagent to afford the tertiary alcohol 15. Deprotection
of 15 under standard conditions3 afforded the triol 16,
TBSO
8
7
O
NMe2
OH
O
N
TBSO
iv
iii
TBSO
9
10
N
TBSO
TBSO
O
vi
O
N
Ph
v
Br
TBSO
TBSO
12
11
NMe2
O
O
O
TBSO
O
vii
ix
viii
12
N
TBSO
O
N
O
14
NPh
13
O
OH
RO
xi
21-nor-Maxacalcitol 5
RO
15: R=TBS
16: R=H
x
Scheme 1. Reagents and conditions: (i) PPh3CH3Br, t-BuOK, THF,
90%; (ii) 9-BBN, THF, then 30% H2O2, 3M aq NaOH, quant; (iii)
N,N-dimethylacrylamide, NaH, THF, 72%; (iv) NBS, AIBN, hexane;
(v) c-collidine, toluene; (vi) 4-phenyl-1,2,4-triazoline-3,5-dione,
CH2Cl2, 19% (three steps); (vii) DMI, 140ꢁC, 69%; (viii) MeMgBr,
CeCl3, THF; (ix) MeMgBr, CeCl3, THF, 68% (two steps); (x) TBAF,
THF, 70%; (xi) hm, THF, 22%.

H. Shimizu et al. / Tetrahedron Letters 45 (2004) 7837–7841
7839
OH
OH
OH
TBSO
i
RO
13
iii
i
17
N
TBSO
O
N
RO
NPh
17
O
22: R=TBS
23: R=H
HO
OH
ii
O
24
Br
OTES
OTES
iii
18
Scheme 3. Reagents and conditions: (i) DMI, 140ꢁC, 73%; (ii) TBAF,
THF, 68%; (iii) hm, THF, 8%.
ii
19
OR2
gave the triol 23 on deprotection. Photolysis of 23 under
the same conditions above furnished the target primary
alcohol 24 in 8% yield13 (Scheme 3).
O
R1O
v
21-nor-Lexacalcitol 6
Having obtained the desired 21-demethylated com-
pounds, 21-nor-Maxacalcitol 5 and 21-nor-Lexacalcitol
6, in hand, we next examined their biological activities
with respect to calcemic and differentiation-inducing
activities in comparison to their corresponding 21-
methyl derivatives, Maxacalcitol 2 and Lexacalcitol 4,
using Calcitriol 1 as standard. Calcemic activity was
evaluated on the basis of in vivo calcium ion level in
mice, while intrinsic activity as a vitamin D was evalu-
ated on the basis of the affinities to chick vitamin D
receptor (VDR) and to rat vitamin D binding protein
(DBP) and differentiation-inducing activity in HL-60
cell, which are compiled in Table 1. For brevity, the
relationship between calcemic activity and differentia-
tion-inducing activity (HL-60) in two compounds, in
addition to the 21-methyl compounds and the presumed
metabolite, was also depicted in Figure 2 as a relative
diagram based on Calcitriol 1 as standard. As seen in
the figure, both 21-nor-Maxacalcitol 5 and 21-nor-Lex-
acalcitol 6 exhibited no significant calcemic activities
which were almost the same as the primary alcohol 24,
while 21-methyl compounds 2 and 4, in particular the
latter, exhibited a considerable extent of activity higher
R1O
20: R1=TBS, R2=TES
21: R1,R2=H
iv
Scheme 2. Reagents and conditions: (i) t-BuOK, THF, 83%; (ii) t-
BuOK, 18-crown-6, THF, 73%; (iii) DMI, 140ꢁC, 71%; (iv) TBAF,
THF, 70%; (v) hm, THF, 8%.
19. Thermolysis of 19 in DMI at 140ꢁC induced a retro-
Diels–Alder reaction to leave the tetracyclic diene 20,
which was deprotected to give the triol 21. On photoly-
sis, the triol 21 furnished 21-nor-Lexacalcitol13 6. Yield
of the final step was 8% (Scheme 2).
Moreover, we also synthesized the primary alcohol 24,
which is presumed to be the common metabolite of both
21-nor-Maxacalcitol 5 and 21-nor-Lexacalcitol 6 for a
comparison in the biological evaluation. The synthesis
utilized the intermediate 17 in the synthesis of 21-nor-
Lexacalcitol 6. Thus, the primary alcohol 17 was heated
in DMI under the same conditions as above to induce a
retro-Diels–Alder reaction to give the diene 22, which
Table 1. Biological properties of Calcitriol 1, Maxacalcitol 2, Lexacalcitol 4, 21-nor-Maxacalcitol 5, 21-nor-Lexacalcitol 6, and the primary alcohol
24
VDRa (%)
DBPb (%)
HL-60c EC50 [M]
In vivo Ca (i.v.)d [mmol/L]
24h/48h (Mean SE, n = 5)
Vehicle
Calcitriol 1
1.34 0.02/1.32 0.04
1.80 0.15#/1.63 0.09#
1.42 0.03 e#/1.39 0.03e
1.77 0.14h#/2.27 0.16h#
1.35 0.04/1.30 0.04
1.36 0.05/1.34 0.02
1.37 0.03/1.33 0.03
100
100
7.73 · 10À9
1.82 · 10À9
1.19 · 10À11g
1.59 · 10À8
1.75 · 10À9
n.a.i
Maxacalcitol 2
Lexacalcitol 4
12.28
66.89
0.65
6.91
0.01
0.07
n.d.f
<0.20
<0.20
<0.20
5
6
24
a Affinity for chick vitamin D receptor.
b Affinity for rat vitamin D binding protein.
c Differentiation-inducing effect on HL-60 cells.
d Ionized calcium levels after 30lg/kg of analogues administration to ddY mice. The data were analyzed by unpaired StudentÕs t-test compared to
vehicle. Differences #p < 0.05 were considered significantly different.
e Vehicle: 1.33 0.05 (n = 5, 24h), 1.35 0.05 (n = 5, 48h), Calcitriol 1: 1.84 0.22 (n = 5, 24h), 1.77 0.13 (n = 5, 48h).
f Not determined.
g Calcitriol 1: 8.46 · 10À9
.
h Vehicle: 1.39 0.02( n = 5, 24h), 1.41 0.03 (n = 5, 48h), Calcitriol 1: 1.69 0.06 (n = 5, 24h), 1.63 0.04 (n = 5, 48h).
i No activity.

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H. Shimizu et al. / Tetrahedron Letters 45 (2004) 7837–7841
100000
10000
1000
100
try Research Department I, Chugai Pharmaceutical Co.,
In vivo Ca 48h
Ltd., and Dr. Fusao Makishima, Director, Pharmaceu-
tical Research Department II, Chugai Pharmaceutical
Co., Ltd., for their encouragement. Dr. Paul Langman,
Regulatory Affairs Department, Chugai Pharmaceutical
Co., Ltd., is also thanked for taking time to review this
manuscript.
In vitro HL-60 differentiation
inducing activity
#
#
#
10
References and notes
1
1. (a) Lythgoe, B. Chem. Soc. Rev. 1980, 9, 449; (b) Ikekawa,
N.; Fujimoto, Y. J. Syn. Org. Chem. Jpn. 1988, 46, 455; (c)
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Rev. 1995, 16, 200; (d) Zhu, G.-D.; Okamura, W. H.
Chem. Rev. 1995, 95, 1877; (e) Jankowski, P.; Marczak, S.;
Wicha, J. Tetrahedron 1998, 54, 12071; (f) Posner, G. H.;
Kahraman, M. Eur. J. Org. Chem. 2003, 3889.
0.1
2. Abe, E.; Miyaura, C.; Sakagami, H.; Takeda, M.; Konno,
K.; Yamazaki, T.; Yoshiki, S.; Suda, T. Proc. Natl. Acad.
Sci. U.S.A. 1981, 78, 4990.
3. (a) Murayama, E.; Miyamoto, K.; Kubodera, N.; Mori,
T.; Matsunaga, I. Chem. Pharm. Bull. 1986, 34, 4410; (b)
Kubodera, N.; Watanabe, H.; Kawanishi, T.; Matsumoto,
M. Chem. Pharm. Bull. 1992, 40, 1494; (c) Mikami, T.;
Iwaoka, T.; Kato, M.; Watanabe, H.; Kubodera, N.
Synth. Commun. 1997, 27, 2363.
Figure 2. Relative diagram between in vivo calcemic and differentia-
tion-inducing (HL-60) activities. HL-60 differentiation-inducing activ-
ity relative potency (%) = (Calcitriol EC50/analogue EC50) · 100.
In vivo calcemic activity (48h) relative potency (%) = ({analogueÕs
[Ca mmol/L] À VehicleÕs [Ca mmol/L]}/{CalcitriolÕs [Ca mmol/L] À
VehicleÕs [Ca mmol/L]}) · 100. #p < 0.05 compared to vehicle by
unpaired StudentÕs t-test.
4. Kubodera, N. J. Syn. Org. Chem. Jpn. 1996, 54, 139.
5. Morimoto, S.; Imanaka, S.; Koh, E.; Shiraishi, T.;
Nabata, T.; Kitano, S.; Miyashita, Y.; Nishii, Y.; Ogihara,
T. Biochemistry 1989, 19, 1143.
6. (a) Kubodera, N.; Miyamoto, K.; Ochi, K.; Matsunaga, I.
Chem. Pharm. Bull. 1986, 34, 2286; (b) Kubodera, N.;
Miyamoto, K.; Matsumoto, M.; Kawanishi, T.; Ohkawa,
H.; Mori, T. Chem. Pharm. Bull. 1992, 40, 648.
7. Brown, A. J.; Ritter, C. R.; Finch, J. L.; Morrissey, J.;
Martin, K. J.; Murayama, E.; Nishii, Y.; Slatopolsky, E.
J. Clin. Invest. 1989, 84, 728.
8. (a) Binderup, L.; Latini, S.; Binderup, E.; Bretting, C.;
Calverley, M.; Hansen, K. Biochem. Pharmacol. 1991, 42,
1569; (b) Niels, R. A.; Frants, A. B.; Gunnar, G. S.
BioMed. Chem. Lett. 1992, 2, 1713.
9. Kragballe, K.; Dam, T. N.; Hansen, E. R.; Baadsgaard,
O.; Larsen, F. G.; Sondergaard, J.; Axelsen, M. B. Acta
Derm. Venereol. 1994, 74, 398.
10. Cf. (a) Shimizu, H.; Shimizu, K.; Kubodera, N.; Yaku-
shijin, K.; Horne, D. A. Tetrahedron Lett. 2004, 45, 1347;
(b) Shimizu, H.; Shimizu, K.; Kubodera, N.; Yakushijin,
K.; Horne, D. A. Heterocycles 2004, 63, 1335.
than Calcitriol 1 indicating that the 21-methyl function-
ality, irrespective of its stereochemistry, plays an impor-
tant role in the exhibition of calcemic activity. The
differentiation-inducing activity, on the other hand,
was diminished in the demethyl compounds 5 and 6,
but still remained overwhelmingly in both Calcitriol 1
and Maxacalcitol 2, indicating the possibility of other
factors on side-chain moiety than the 21-methyl func-
tionality in the exhibition of differentiation-inducing
activity.
The present study has clearly demonstrated the crucial
role of the 21-methyl functionality on the exhibition of
calcemic activity irrespective of its stereochemistry. At
the same time, the present study suggested the impor-
tance of length and bulkiness of the side chain moiety
in the 21-nor-derivatives on the exhibition of differenti-
ation-inducing activity.
In conclusion, we have confirmed two structural factors
on the side chain of the 22-oxa-1a,25-dihydroxyvitamin
D3 system essential for the control of calcemic activity
and differentiation-inducing activity. Further work
along the side-chain modification of 21-nor-22-oxa-
1a,25-dihydroxyvitamin D3 derivatives as well as other
21-nor-1a,25-dihydroxyvitamin D3 derivatives on the
basis of the present study is currently in progress.
11. Kubodera, N.; Miyamoto, K.; Watanabe, H.; Kato, M.;
Sasahara, K.; Ochi, K. J. Org. Chem. 1992, 57, 5019.
12. Imamoto, T.; Takiyama, N.; Nakamura, K.; Hatajima, T.;
Kamiya, Y. J. Am. Chem. Soc. 1989, 111, 4392.
13. NMR spectra (270MHz for 1H and 67.8MHz for 13C
spectra, respectively) of the representative compounds: 5:
1H NMR: d 6.37 (d, J = 11.2Hz, 1H), 6.00 (d, J = 11.2Hz,
1H), 5.33 (s, 1H), 4.99 (s, 1H), 4.44 (m, 1H), 4.24 (m, 1H),
3.46–3.68 (m, 5H), 3.28–3.34 (m, 1H), 2.81–2.86 (m, 1H),
2.58–2.63 (m, 1H), 2.28–2.35 (m, 1H), 1.50–2.05 (m, 13H),
1.24–1.35 (m, 9H), 0.51 (s, 3H). 13C NMR: d 147.6, 142.6,
133.1, 124.9, 117.2, 111.8, 73.3, 70.9, 70.6, 68.8, 66.8, 55.8,
50.4, 45.3, 45.0, 42.9, 41.3, 39.1, 29.5, 29.1, 25.2, 23.3, 22.6,
12.6. Compound 6: 1H NMR: d 6.38 (d, J = 11.2Hz, 1H),
6.01 (d, J = 11.2Hz, 1H), 5.33 (s, 1H), 5.00 (s, 1H), 4.44
(m, 1H), 4.23 (m, 1H), 3.28–3.50 (m, 5H), 2.83–2.87 (m,
1H), 2.57–2.63 (m, 1H), 2.28–2.35 (dd, J = 13.4, 6.6Hz,
1H), 1.26–2.13 (m, 23H), 0.83–0.88 (m, 6H), 0.50 (s, 3H).
13C NMR: d 147.6, 142.8, 133.0, 124.9, 117.1, 111.8, 73.9,
Acknowledgements
We thank Dr. Kunio Ogasawara, Professor Emeritus,
Tohoku University, for his helpful suggestions. We also
thank Dr. Yutaka Miura, Group Manager, Synthetic
Technology Research Department, Chugai Pharmaceu-
tical Co., Ltd., Mr. Toshiro Kozono, Director, Chemis-

H. Shimizu et al. / Tetrahedron Letters 45 (2004) 7837–7841
7841
72.8, 71.8, 70.9, 66.9, 55.8, 50.3, 45.3, 45.1, 42.9, 39.1, 35.5,
31.0, 30.9, 29.1, 25.3, 23.9, 23.3, 22.6, 12.5, 7.9. Compound
24: 1H NMR: d 6.38 (d, 11.5Hz, 1H), 6.02(d, 11.5Hz,
1H), 5.33 (s, 1H), 5.00 (s, 1H), 4.41–4.47 (m, 1H), 4.23–
4.24 (m, 1H), 3.71–3.74 (m, 1H), 3.54–3.59 (m, 1H), 2.83–
2.89 (dd, J = 13.2, 3.6Hz, 1H), 2.57–2.63 (dd, J = 13.2,
3.6Hz, 1H), 2.28–2.36 (dd, J = 13.2, 6.6Hz, 1H), 1.10–2.08
(m, 16H), 0.53 (s, 3H). 13C NMR: d 147.7, 142.5, 124.9,
117.3, 111.8, 70.8, 66.9, 64.7, 55.9, 53.3, 45.3, 45.1, 42.9,
39.3, 29.1, 24.9, 23.3, 22.6, 12.5.