Syntheses and biological evaluation of novel 2α-substituted 1α,25-dihydroxyvitamin D3 analogues

Article abstract of DOI:10.1016/S0960-894X(00)00189-X

Novel 2α-substituted 1α,25-dihydroxyvitamin D₃ analogues were efficiently synthesized and their biological activities were evaluated. 2α-Methyl-1α25-dihydroxyvitamin D₃ (2), whose unique biological activities were previously reported

Full text of DOI:10.1016/S0960-894X(00)00189-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1129±1132
Syntheses and Biological Evaluation of Novel 2ꢀ-Substituted
1ꢀ,25-Dihydroxyvitamin D3 Analogues
Yoshitomo Suhara, a Ken-ichi Nihei, a Hirokazu Tanigawa, a Toshie Fujishima, a
Katsuhiro Konno, a Kimie Nakagawa, b Toshio Okano b and Hiroaki Takayama a,*
aFaculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan
bDepartment of Hygienic Sciences, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan
Received 14 February 2000; accepted 16 March 2000
AbstractÐNovel 2a-substituted 1a,25-dihydroxyvitamin D3 analogues were eciently synthesized and their biological activities
were evaluated. 2a-Methyl-1a,25-dihydroxyvitamin D3 (2), whose unique biological activities were previously reported, was mod-
i®ed to 2a-alkyl (ethyl and propyl) and 2a-hydroxyalkyl (hydroxymethyl, hydroxyethyl, and hydroxypropyl) analogues 3±7 by
elongation of the alkyl chain and/or introduction of a terminal hydroxyl group. 2a-Hydroxypropyl-1a,25-dihydroxyvitamin D3 (7)
exhibited an exceptionally potent calcium-regulating e€ect and a unique activity pro®le. # 2000 Elsevier Science Ltd. All rights
reserved.
The growing interest in 1a,25-dihydroxyvitamin D3 (1),
the hormonally active form of vitamin D3, has promp-
ted numerous e€orts to synthesize vitamin D analogues
as potential therapeutic agents.1 A majority of them are
modi®ed in the side chain or in the A-ring.2 A number of
non-steroidal vitamin D mimics have been synthesized
quite recently.3
2a-methyl substitution with 20-epimerization, i.e., double
modi®cation, produced a much more potent analogue.5
These results prompted us to design new analogues with
further modi®cation of the 2a-methyl group. We antici-
pated that if the 2a-methyl group were replaced by a
longer 2a-alkyl or the 2a-hydroxyalkyl group, the result-
ing analogues would provide insight into the biological
signi®cance of the 2a-methyl substitution. We report here
the synthesis of several new analogues, 3±7, and their
biological evaluation.
In order to investigate the A-ring conformation±activity
relationships, we synthesized all the possible A-ring dia-
stereomers of 2-methyl-1,25-dihydroxyvitamin D3 and
found that the 2a-methyl isomer (2) showed higher
potency than 1 in terms of VDR binding anity, elevation
of rat serum Ca concentration, and induction of HL-60
cell di€erentiation.4 Furthermore, the combination of this
The analogues were synthesized according to the method
developed by Trost and co-workers using palladium-
catalyzed coupling of the A-ring enyne synthon with the
CD-ring portion.6 To introduce 2a-substituents into 1,
*Corresponding author. Tel.: +81-426-85-3713; fax: +81-426-85-3714; e-mail: hi-takay@pharm.teikyo-u.ac.jp
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00189-X

1130
Y. Suhara et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1129±1132
the synthesis of the A-ring portion was started with a d-
xylose derivative (8), as shown in Scheme 1. Selective
protection of the C-5 primary alcohol of 8 with a piva-
loyl group, followed by oxidation with PCC gave the
ketone 9 in 98% yield. Conversion of the ketone to an
exomethylene group by means of the Wittig reaction, and
subsequent treatment with 1 N HCl/BnOH a€orded the
alcohol 10 in 58% yield along with the b-isomer (32%
yield). The C-2 hydroxyl group of 10 was inverted via the
Mitsunobu reaction to give the secondary alcohol 11,
corresponding to the 1a-hydroxyl group of vitamin D
analogues, in 84% yield. After protection of the alcohol
moiety of 11 with a methoxymethyl (MOM) group, the
exomethylene group was selectively converted to the 3b-
hydroxymethyl compound 12 in high yield by hydro-
boration with 9-BBN. Protection of the primary hydroxyl
group with a tert-butyldimethylsilyl (TBS) group a€orded
the TBS ether 13 in 99% yield. Thus, modi®cation of the
C-2 and C-3 hydroxyl groups of the d-xylose derivative,
which correspond to the C-1 hydroxyl group and C-2
alkyl or hydroxyalkyl group of the vitamin D analogues,
respectively, was accomplished.
with DIBAL-H, 2,4,6-trimethylbenzenesulfonyl chloride
(TmCl) and lithium hexamethyldisilazide (LiHMDS) in
high yield. The acetylene unit was introduced by the
reaction of 15 with (trimethylsilyl)acetylene/n-BuLi-
ꢀ
BF3 OEt2 in THF to give the alcohol 16 in 80% yield.
Removal of both silyl protecting groups (TMS and
TBS) by tetrabutylammonium ¯uoride (TBAF), and
subsequent manipulation of the protecting groups by
selective pivaloylation, removal of MOM group and silyl-
ation a€orded the fully protected triol 17 in good yield.
Finally, further protecting group manipulation through
18 gave the desired A-ring synthon 19 in high yield.
Elongation of the 2a-substituent of the A-ring synthon
was carried out in a conventional manner as shown in
Scheme 2. Sequential sulfonylation, cyanide addition and
reduction of the alcohol 18 furnished the single-carbon-
elongated alcohol 20 in good overall yield, and this was
protected to give the A-ring synthon 21. The alcohol 20
was further converted to the 2a-ethyl derivative through
the tosylate 22. In the same manner, the double-carbon-
elongated synthons 25 and 26 were prepared from 22±24.
The requisite A-ring synthons were obtained from the
d-xylose derivative 13. Hydrogenolysis of the benzyl
group of 13, followed by ring opening by means of the
Wittig reaction a€orded 14 in good yield. This was
converted to the epoxide 15 by sequential treatment
Finally, palladium-catalyzed coupling of the A-ring
synthons 19, 21, 23, 25 and 26 with the CD-ring portion
27, followed by deprotection with camphorsulfonic acid
(CSA) in MeOH gave the 2a-alkyl and 2a-hydroxyalkyl
analogues 3±77 as shown in Scheme 3. Thus, we have
Scheme 1. (a) PvCl, Py, 86%; (b) PCC, MS 4A, CH2Cl2, 98%; (c) Ph3P+CH3Br , KHMDS, THF, 85%; (d) 1 N HCl, BnOH, 1,4-dioxane/toluene,
58%; (e) p-nitrobenzoic acid, Ph3P, DEAD, THF; (f) 10 mM NaOH, H2O/MeOH, 84% (2 steps); (g) MOMCl, DPIEA, TBAl, CH2Cl2, 88%; (h) 9-
BBN, THF, then 3 M NaOH, 30% H2O2, 85%; (i) TBSCl, imidazole, DMF, 99%; (j) H2, Pd(OH)2, EtOH; (k) Ph3P+CH33Br , LiHMDS, THF,
79% (2 steps); (l) DIBAL-H CH2Cl2, 84%; (m) TmCl, DMAP, CH2Cl2; (n) LiHMDS, THF, 86% (2 steps); (o) TMSCꢁCH, n-BuLi, BF3ꢀ OEt2,
THF, 80%; (p) TBAF, THF, 99%; (q) PvCl, Py/CH2Cl2, 85%; (r) PPTS, t-BuOH, 74%; (s) TBSOTf, 2,6,-lutidine, CH2Cl2, 99%; (t) DIBAL-H,
CH2Cl2, 96%; (u) TBSCL, imidazole, DMF, 88%.

Y. Suhara et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1129±1132
1131
Scheme 2. (a) TmCL, DMAP, Ch2Cl2; (b) NaCN, DMSO, 61±81% (2 steps); (c) DIBAL-H, CH2Cl2, 85±89%; (d) NaBH4, MeOH, 82±98%; (e)
TBSCl, imidazole, DMF, 78±83%; (f) LAH, Et2O, 80% (2 steps).
synthesized ®ve 2a-substituted 1a,25-dihydroxyvitamin
D3 analogues.
Table 1. Relative potency of the synthesized analoguesa
Compound
VDRb DBPc
binding binding
HL-60
celld
anity anity di€erentiation
Ca
mobilizatione
The results of biological evaluation are summarized in
Table 1, in comparison with those of 1a,25-dihydroxy-
vitamin D3 (1) and 2a-methyl-1a,25-dihydroxyvitamin
D3 (2). First of all, we examined the receptor binding in
an assay using bovine thymus VDR.8 The 2a-alkyl
analogues 3 and 4 showed much lower binding anity
than the 2a-methyl analogue 2; in other words, the short-
est-chain analogue has the highest anity among the 2a-
alkyl analogues. In contrast, the 2a-hydroxypropyl ana-
logue 7, bearing the most elongated chain, showed the
highest binding anity, 3-fold higher than that of 1,
among the 2a-hydroxyalkyl analogues.9 Therefore, the
mode of binding of the 2a-hydroxyalkyl analogues may
be di€erent from that of the 2a-alkyl analogues. Steric
hindrance of the 2a-alkyl group and hydrogen bonding
ability of the 2a-hydroxyalkyl group are presumably
involved in the di€erence of the mode of binding. Okano et
al. previously reported the VDR binding anity of 2b-
alkyl (methyl, ethyl, propyl, butyl, pentyl, and hexyl) and
2b-hydroxyalkyl (hydroxypropyl, hydroxybutyl, hydro-
xypentyl, and hydroxyhexyl) analogues.10 In contrast with
those compounds, some of the 2a-substituted analogues
showed signi®cantly higher binding anity than 1a,25-
dihydroxyvitamin D3.
1 (1a,25-(OH)2VD3)
2 (2a-methyl)
3 (2a-ethyl)
100
400
40
20
20
100
44
48
21
95
74
362
100
444
106
44
10
86
100
658
68
636
124
372
4 (2a-propyl)
5 (2a-hydroxymethyl)
6 (2a-hydroxyethyl)
7 (2a-hydroxypropyl)
70
300
240
50,866
aThe potency of 1a,25-(OH)2VD3 is normalized to 100 in each case.
bBovine thymus.
cRat serum.
dCell di€erentiation was assessed in terms of expression of antigen
CD11b.
eRat serum Ca level.
The anity for vitamin D binding protein (DBP)11 was
tested using rat serum DBP. The binding anity was
decreased in the 2a-alkyl analogues including 2, whereas
it was largely retained or even increased in the 2a-
hydroxyalkyl analogues. In particular, the analogue 7
showed very high anity for DBP, being similar in that
respect to the b-isomer (2b-hydroxypropoxy-1a,25-
dihydroxyvitamin D3: ED-71).12 This result implies that
Scheme 3.

1132
Y. Suhara et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1129±1132
there is no discrimination between the a- and b-isomers
of the 2-hydroxypropyl analogues in DBP binding.
References and Notes
1. Ettinger, R. A.; DeLuca, H. F. Adv. Drug Res. 1996, 28, 269.
2. Bouillon, R.; Okamura, W. H.; Norman, A. W. Endocri.
Rev. 1995, 16, 200.
3. Boehm, M. F.; Fitzgerald, P.; Zou, A.; Elgort, M. G.; Bis-
cho€, E. D.; Mere, L.; Mais, D. E.; Bissonnette, R. P.; Hey-
man, R. A.; Nadzan, A. M.; Reichman, M.; Allegretto, E. A.
Chem. Biol. 1999, 6, 265.
4. Konno, K.; Maki, S.; Fujishima, T.; Liu, Z.-P.; Miura, D.;
Chokki, M.; Takayama, H. Bioorg. Med. Chem. Lett. 1998, 8,
151.
The rank order of potency for inducing di€erentiation
in HL-60 cells13 was parallel to that of VDR binding;
that is, as the chain length became longer, the potency
decreased in 2a-alkyl analogues, whereas it increased in
the 2a-hydroxyalkyl analogues. Only compounds 2 and
7 showed much higher activity than 1a,25-dihydrox-
yvitamin D3 in this experiment.
Most of the new analogues exhibited higher calcium-
mobilizing activity14 than 1; in particular, the analogue 7
showed approximately 500 times higher potency, and this
remarkably high activity is unique among vitamin D ana-
logues reported to date. Therefore, this 2a-hydroxypropyl
analogue 7 may provide clues to achieving separation of
the biological functions of vitamin D. In the case of the
hydroxyalkyl series, the longer the chain, the higher the
activity. This rank order of potency is parallel to that of
VDR binding anity, implying that the calcium-regulat-
ing e€ect of the 2a-hydroxyalkyl analogues may involve
interaction with VDR. In the alkyl series, in which VDR
binding anity decreased with increasing chain length,
the 2a-propyl analogue 4 showed anomalously high cal-
cium-mobilizing activity, comparable to that of 2. This
result is not easy to rationalize in terms of a VDR-depen-
dent mechanism, and therefore, the analogue 4 may exert
its calcium-regulating e€ect through a distinct mechanism
from the other analogues.
5. Fujishima, T.; Liu, Z.-P.; Miura, D.; Chokki, M.; Ishizuka, S.;
Konno, K.; Takayama, H. Bioorg. Med. Chem. Lett. 1998, 8,
2145.
6. Trost, B. M.; Dumas, J.; Villa, M. J. Am. Chem. Soc. 1992,
114, 9836.
7. 3: 1H NMR (400 MHz, CDCl3±D2O/TMS) d 0.53 (3H, s), 0.94
(d, 3H, J=6.6 Hz), 0.95 (3H, t, J=7.4 Hz), 1.21 (6H, s), 2.24 (1H,
dd, J=8.0, 12.8 Hz), 2.66 (1H, dd, J=4.4, 12.8 Hz), 2.83 (1H, m),
3.89 (1H, dt, J=4.4, 8.0 Hz), 4.39 (1H, d, J=2.2 Hz), 4.99 (1H, d,
J=2.2 Hz), 5.27 (1H, d, J=1.5 Hz), 6.00 (1H, d, J=11.4 Hz), 6.40
(1H, d, J=11.4 Hz) ; HREIMS 444.3604 calcd for C29H48O3
1
(M+) 444.3603. 4: H NMR (400 MHz, CDCl3±D2O/TMS) d
0.53 (3H, s), 0.94 (3H, d, J=6.4 Hz), 1.01 (3H, t, J=6.8 Hz), 1.21
(6H, s), 2.24 (1H, dd, J=8.4, 13.2 Hz), 2.66 (1H, dd, J=4.0, 13.2
Hz), 2.83 (1H, m), 3.88 (1H, dt, J=4.4, 8.4 Hz), 4.36 (1H, d,
J=3.3 Hz), 4.99 (1H, d, J=1.8 Hz), 5.26 (1H, d, J=1.8 Hz), 6.00
(1H, d, J=11.4 Hz), 6.40 (1H, d, J=11.4 Hz); HREIMS 458.3755
calcd for C30H50O3 (M+) 458.3760. 5: 1H NMR (400 MHz,
CDCl3±D2O/TMS) d 0.53 (3H, s), 0.94 (3H, d, J=6.8 Hz), 1.21
(6H, s), 2.31 (2H, dd, J=9.9, 13.0 Hz), 2.67 (1H, dd, J=4.6, 13.0
Hz), 2.84 (1H, m), 3.96 (1H, dd, J=4.6, 11.2 Hz), 4.05 (1H, dd,
J=4.6, 11.2 Hz), 4.24 (1H, dt, J=4.6, 9.9 Hz), 4.46 (1H, d, J=2.9
Hz), 5.02 (1H, d, J=1.8 Hz), 5.29 (1H, d, J=1.8 Hz), 5.98 (1H, d,
J=11.0 Hz), 6.45 (1H, d, J=11.0 Hz); HREIMS m/z 428.3287
calcd for C28H44O3 (M+ H2O) 428.3290. 6: 1H NMR (400
MHz, CDCl3±D2O/TMS) d 0.53 (3H, s), 0.94 (3H, d, J=6.4 Hz),
1.21 (6H,s), 2.26 (1H, dd, J=8.0, 13.0 Hz), 2.66 (1H, dd, J=4.0,
13.0 Hz), 2.83 (1H, m), 3.79 (2H, m), 3.94 (1H, dt, J=4.0, 8.0 Hz),
4.37 (1H, d, J=2.2 Hz), 5.02 (1H, d, J=1.8 Hz), 5.30 (1H, bs),
6.01 (1H, d, J=11.2 Hz), 6.40 (1H, d, J=11.2 Hz); HREIMS m/z
Thus, the activity pro®les of the synthesized analogues
are high structure-sensitive, in that even a single-carbon
chain di€erence greatly alters the pro®le. Consequently,
these analogues should be useful for studies on the
action mechanism of vitamin D, and also as lead com-
pounds for developing therapeutic agents. Further stu-
dies, however, are needed to elucidate fully the activity
pro®les and modes of action of these analogues.
1
460.3557 calcd for C29H48O4 (M+) 460.3553. 7: H NMR (400
MHz, CDCl3-D2O/TMS) d 0.53 (3H, s), 0.93 (3H, d, J=6.6 Hz),
1.21 (6H, s), 2.25 (1H, dd, J=8.0, 13.2 Hz), 2.66 (1H, dd, J=4.0,
13.2 Hz), 2.83 (1H, m), 3.71 (2H, t, J=5.6 Hz), 3.90 (dt, 1H,
J=4.0, 8.0 Hz), 4.38 (d, 1H, J=2.9 Hz), 5.00 (1H, d, J=1.8 Hz),
5.28 (1H, bs), 6.00 (1H, d, J=11.2 Hz), 6.40 (1H, d, J=11.2 Hz);
HREIMS 474.3709 calcd for C30H50O4 (M+) 474.3709.
8. Imae, Y.; Manaka, A.; Yoshida, N.; Ishimi, Y.; Shinki, T.;
Abe, E.; Suda, T.; Konno, K.; Takayama, H.; Yamada, S.
Biochim. Biophys. Acta. 1994, 1213, 302.
9. Posner et al. previously reported that a 2a-hydroxybutyl-
1a,25-dihydroxyvitamin D3 analogue had virtually no anity for
VDR. Posner, G. H.; Johnson, N. J. Org. Chem. 1994, 59, 7855.
10. (a) Ono, Y.; Watanabe, H.; Shiraishi, A.; Takeda, S.;
Higuchi, Y.; Sato, K.; Tsugawa, N.; Okano, T.; Kobayashi,
T.; Kubodera, N. Chem. Pharm. Bull. 1997, 45, 1626. (b) Tsu-
gawa, N.; Nakagawa, K.; Kurobe, M.; Ono, Y.; Kubodera, N;
Ozono, K.; Okano, T. Biol. Pharm. Bull. 2000, 23, 66.
11. Zhao, X. Y.; Eccleshall, T. R.; Krishnan, A. V.; Gross, C.;
Feldman, D. Mol. Endocrinol. 1997, 11, 366.
In this study, we have developed a synthetic route to A-
ring enyne synthon, which can be useful for 2a-substituted
vitamin D analogues, starting from D-xylose, and utilized
it for the synthesis of ®ve novel 2a-substituted vitamin D
analogues 3±7 by palladium-catalyzed convergent
method. In the light of the importance of 2-substituted
vitamin D analogues, as exempli®ed by ED-71,10,12 this
novel procedure has a great advantage, because it
should be applicable to a variety of 2a-substituted vita-
min D analogues. Biological evaluation of these analo-
gues demonstrated that they have unique activity
pro®les, and in particular, the 2a-hydroxypropyl analo-
gue 7 exhibited exceptionally potent calcium-regulating
activity. Further investigation along this line should
provide insight into the biological signi®cance of 2-sub-
stituted vitamin D analogues.
12. Okano, T.; Tsugawa, N.; Matsuda, S.; Takeuchi, A.;
Kobayashi, T.; Takita, Y.; Nishii, Y. Biochem. Biophys. Res.
Commun. 1989, 163, 1444.
Acknowledgements
13. Brackman, D.; Lund-Johansen, F.; Aarskog, D. J. Leuko-
cyte Biol. 1995, 58, 547.
14. Ishizuka, S.; Norman, A. W. J. Steroid Biochem. 1986, 25,
505.
This work was supported in part by a Grant-in-Aid
from the Ministry of Education, Science and Culture of
Japan.