Synthesis and biological activities of new 1α,25-dihydroxy-19-norvitamin D3 analogs with modifications in both the A-ring and the side chain

Bioorganic & Medicinal Chemistry 14 (2006) 4277–4294

Synthesis and biological activities of new

1a,25-dihydroxy-19-norvitamin D₃analogs with modiﬁcations

in both the A-ring and the side chain

Masato Shimizu,^a,*Yukiko Miyamoto,^bEmi Kobayashi,^bMika Shimazaki,^b

Keiko Yamamoto,^bWolfgang Reischl^cand Sachiko Yamada^b

^aLaboratory of Medicinal Chemistry, School of Biomedical Science, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai,

Chiyoda-ku, Tokyo 101-0062, Japan

^bInstitute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai,

Chiyoda-ku, Tokyo 101-0062, Japan

^cDepartment of Organic Chemistry, University of Vienna, A-1090 Vienna, Austria

Received 22 December 2005; revised 21 January 2006; accepted 23 January 2006

Available online 24 February 2006

Abstract—In a series of studies on structure–activity relationships of 2-substituted 19-norvitamin D analogs, we found that 1a,25-

dihydroxy-19-norvitamin D₃analogs with 2b-hydroxyethoxy or 2E-hydroxyethylidene moieties show strong binding aﬃnity for

the vitamin D receptor (VDR) as well as marked transcriptional activity. To further examine the eﬀects of side chain structure

on the activity of 2-substituted 19-norvitamin D analogs, we have synthesized new 19-norvitamin D₃analogs with modiﬁcations

in both the A-ring at the C(2) position and the side chain. The side chains of these analogs contained a double bond between

C(22) and C(23) or an oxygen atom at C(22). The biological activity of the analogs was evaluated in vitro. All the side chain-mod-

iﬁed analogs were less active than 1a,25-dihydroxyvitamin D₃1e and the parent compounds 3–6e possessing a natural 20R-conﬁg-

uration in binding to the VDR, but, except for the (20R)-22-oxa analogs 3–6d, were signiﬁcantly more potent in transcriptional

activity. Of the side-chain-modiﬁed analogs 4 and 5, the 2b-hydroxyethoxy- and 2E-hydroxyethylidene-22,24-diene-24a,26a,27a-tri-

homo analogs showed markedly higher transcriptional activity (25- and 17.5-fold, respectively) compared with 1e. Elongation of the

side chain at the C-24, C-26, and C-27 positions and introduction of a 22,24-diene moiety strongly increased transcriptional activity,

as seen in the 20-epi analogs 3–6f.

1. Introduction

regions of certain genes, such as those encoding osteopon-

tin, osteocalcin, and 1a,25-D₃24-hydroxylase (CYP24).¹

1a,25-Dihydroxyvitamin D₃[1a,25-(OH)₂D₃] (1e) exerts

pleiotropic eﬀects, including regulation of bone mineral-

ization and calcium homeostasis as well as regulation of

cell proliferation and diﬀerentiation, and has immuno-

modulatory activity.¹1a,25-(OH)₂D₃exerts most of its

eﬀects via the vitamin D receptor (VDR), which belongs

to the large superfamily of nuclear receptors. The VDR

has been shown to heterodimerize with the retinoid X

receptor (RXR) and binds to vitamin D response

elements (VDREs), which are located in promoter

1a,25-Dihydroxy-19-norvitamin D₃[1,25-(OH)₂-19-nor-

VD] 2e is a highly stable compound because of its lack

of the labile conjugated triene moiety characteristic of

vitamin D; it possesses the desirable properties of low

calcemic activity but preserved cell diﬀerentiation activity.^2,3

Introduction of a substituent into the C(2) position

dramatically changes the biological activity of vitamin

D derivatives.^4–12During the last decade, a number of

2-substituted vitamin D analogs have been synthesized

and studied intensively for biological activity.¹³(20S)-

2-Methylene-1a,25-dihydroxy-19-norvitamin D₃(2MD,

Fig. 1), developed by DeLuca and co-workers, shows

preferential activity on bone compared with intestine

and also stimulates bone formation in ovariectomized

rats without causing toxic hypercalcemia.^14,15ED-71

Keywords: 19-Norvitamian D; Side-chain-modiﬁed vitamin D analog;

Vitamin D receptor; Transcriptional activity.

*

Corresponding author. Tel.: +81 03 5280 8117; fax: +81 03 5280

8005; e-mail: shimizu.mr@tmd.ac.jp

doi:10.1016/j.bmc.2006.01.061

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M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

R'

activity, and such modiﬁcations are of key importance

in modulating the biological activities of vitamin D.

To elucidate further the SAR of 19-norvitamin D analogs,

we focused our attention on examining the eﬀect of side

chain structure on the biological activities of 2-substitut-

ed analogs. Limited biological data are available for

vitamin D analogs with structural modiﬁcations on both

the A-ring and the side chain,^18–21although such modi-

ﬁcations could provide additional information regarding

the SAR of vitamin D. Biological activity is markedly

aﬀected by the size, chemical properties (hydrophilicity

or hydrophobicity), and position of the substituent, as

well as by the stereochemistry of substitution. From

the active space group concept proposed by Yamamoto

et al.,^22,23analogs bearing a double bond at C(22), an

oxygen atom at the C(22) position or a 22,24-diene moi-

ety are predicted to have high potency in cell diﬀerenti-

ation and transcriptional activities but weak calcemic

activity. Methylation at C(26) and C(27) is also expected

to increase biological activity. 22-Oxa-1,25-(OH)₂D₃

(Maxacalcitol) is currently marketed for the treatment

of psoriasis and 22,24-diene-24,26,27-trihomo-1,25-

(OH)₂D₃(EB-1089) is under development as an antican-

cer drug (Fig. 1).

R'

C

D

19

5

R

4

A

3

HO

OH

1

HO

OH

HO

OH

2

O

1: R = CH

2

OH

2: R = H

2

3

4

(steroid numbering)

R'

HO

OH

HO

OH

5 (E-isomer)

6 (Z-isomer)

OH

20

O

OH

a:

d:

OH

R' =

b:

c:

f:

O

Here, we report the synthesis and biological activities of

a new class of highly modiﬁed (20R)- and (20S)-19-nor-

vitamin D analogs 3–6 with hydroxyethoxy or hydroxy-

ethylidene groups at C(2) and a double bond or oxygen

atom at C(22).

OH

e:

R'

2. Results and discussion

2.1. Synthesis

HO

OH

HO

OH

HO

OH

For the synthesis of both A-ring- and side-chain-modi-

ﬁed 1a,25-(OH)₂-19-nor VD analogs 3–6, Wittig–Horn-

er coupling of the A-ring phosphine oxides 7 or 8 with

the protected 25-hydroxy Grundmann’s ketones 22a–d

was employed.

O

OH

EB-1089 (R' = b)

Maxacalcitol (R' = c)

ED-71 (R' = e)

2MD (R' = f)

Figure 1. Structures of 1a,25-dihydroxyvitamin D₃and its analogs.

(Fig. 1), which contains a 2b-hydroxypropoxyl group,

also increases bone mass.¹⁶Both 2MD and ED-71 are

promising therapeutic candidates for the treatment of

osteoporosis and are currently in phase II and phase

III clinical trials, respectively. In our examination of

the structure–activity relationships (SAR) of the

A-ring-modiﬁed 19-norvitamin D analogs, we synthe-

sized 19-norvitamin D analogs containing hydroxyeth-

oxy or hydroxyethylidene moieties at the C(2) position

and demonstrated that these analogs show strong

binding aﬃnity for the VDR and enhanced transcrip-

tional activity.^7,8We also synthesized novel 2,2-disubsti-

(3R,5R)-A-ring phosphine oxide 7 (based on sugar num-

bering) was prepared from ^D-glucose as reported,⁷and

we synthesized the A-ring synthon 8 with a hydroxyethy-

lidene group at the C(4) position as shown in Figure 2.

The cyclohexanone derivative 9, which was synthesized

from ^D-glucose in 26% yield,⁷was reduced with sodium

borohydride (NaBH₄) to give 10 (ca. 2:1 diastereomeric

mixture, quantitative yield). Benzylation of 10 with ben-

zyl bromide aﬀorded the benzyl ether 11 (85%), which

was selectively hydrolyzed by treatment with a mixture

of aqueous acetic acid in THF to yield the 4-hydroxy

compound 12 (80%). Swern oxidation of 12 gave the 4-

keto derivative 13 (quantitative yield) as a single com-

pound. Cyanomethylation of 13 with diethyl(cyano-

methyl)phosphonate [(EtO)₂P(O)CH₂CN] aﬀorded 14

(96%) as an approximate 2:1 diastereomeric mixture,

which on reduction with diisobutylaluminum hydride

(DIBAL-H) followed by NaBH₄gave the hydroxyethyl-

idene derivative 16 in excellent yield. Protection of the

free hydroxyl group of 16 with tert-butyldimethylsilyl

chloride (TBSCl) followed by hydrogenolysis of the

tuted 19-norvitamin

D

analogs; among these,

(20S)-2-methyl-2-hydroxy-1a,25-dihydroxy-19-norvi-

tamin D is as potent as 2MD in inducing osteoclast

formation.^9,17

Up to now, more than 3000 vitamin D analogs have

been synthesized. Most of the structural modiﬁcations

of 1e occur in the side chain, followed by A-ring

alterations.¹³Some of these analogs have low calcemic

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

4279

P(O)Ph₂

OTBS

P(O)Ph₂

2

6

1

4

5

3

OTBS

TBSO

OTMS

7

8

(sugar numbering)

O

OR'

1) NaBH₄

1) DMSO, (COCl)₂

2) BnBr

3) aq. AcOH

OTBS

TBSO

2) (EtO)₂P(O)CH₂CN

n-BuLi

OTBS

OTMS

TBSO

OR

10: R = TMS; R' = H (quant)

11: R = TMS; R' = Bn (85%)

12: R = H; R' = Bn (80%)

9

OR'

OBn

1) DIBAL-H

2) NaBH₄

DMSO

3) TBSCl, Imid.

4) H

(COCl)₂

OTBS

TBSO

OTBS

TBSO

₂/Pd-C

R

13: R = O (quant)

14: R = CHCN (96%)

15: R = CHO; R' = Bn (93%)

16: R = CH₂OH; R' = Bn (98%)

17: R = CH₂OTBS; R' = Bn (91%)

18: R = CH₂OTBS; R' = H (91%)

R

O

1) CH CO Me

2

i) TsCl

TMS , n-BuLi

8 (63%)

ii) Ph₂PLi

OTBS 2) DIBAL-H

OTBS

TBSO

OTBS

TBSO

iii) H₂O₂

20: R = CO₂Me (93%)

21: R = CH₂OH (85%)

19 (quant)

Figure 2. Synthetic scheme of the A-ring phosphine oxides with the hydroxyethylidene moiety.

benzyl ether 17 catalyzed by 10 wt % palladium on

carbon gave 18, which upon Swern oxidation yielded

the 1-keto compound 19 (quantitative yield) as the sole

product. Transformation of 19 to the desired phosphine

oxide 8 was accomplished according to the procedures

reported by DeLuca and co-workers.⁴

group with chlorotriethylsilane (TESCl) yielded 22d

(94% from 25). The same sequence of reaction with

(20S)-20-ol 23 aﬀorded the (20S)-22-oxa derivatives

22c and 22⁰c.

Eight 2-hydroxyethoxy-19-norvitamin D analogs 3a–d

and 4a–d bearing four diﬀerent side chains were synthe-

sized according to the sequence developed in Figure 4.

The protected 25-hydroxy Grundmann’s ketones 22a–d

were treated with the A-ring phosphine oxide 7 (ca.

2:1 isomeric mixture) in the presence of n-butyl lithium

to aﬀord derivatives with the 19-norvitamin D structures

28a–d (52–88%) as a mixture of diastereomers in the ra-

tios depicted in Figure 4. Deprotection of the TMS-pro-

tecting group in 28a and 28c with aqueous acetic acid in

THF gave 2-hydroxy compounds 29a (98%) and 29c

(72%), whereas under the same conditions 28d yielded

the mono-ol 29d (27%) accompanied by the 2,25-dihy-

droxy compound 30d (65%) as the major product. The

derivative 28b possessing a 22,24-diene-24,26,27-triho-

mo group is not stable in the above acidic conditions

and was found to undergo dehydration to aﬀord a side

product containing a conjugated triene group in the side

chain. Silyl-protected 28b was treated with tetrabutyl-

ammonium ﬂuoride (TBAF) to yield the tetra-ol 29b

(97%), which was reprotected regioselectively by TBSCl

in the presence of triethylamine to give 30b (66%).

Protected 25-hydroxy Grundmann’s ketones 22a and

22b (based on steroid numbering) possessing 22-ene or

22,24-diene moieties were prepared as reported.^24–26

(20S)- or (20R)-22-Oxa C/D-ring ketones 22c or 22d

were synthesized from the 20-hydroxy compound 23 de-

rived from vitamin D₂as illustrated in Figure 3. Brieﬂy,

the tosylate 24 with a 5-carbon fragment was prepared

from a standard Grignard reaction of 4-hydroxy-2-buta-

none, tosylation of the resulting diol, and then protec-

tion by tert-butyldimethyl triﬂuoromethanesulfonate

(TBSOTf). Williamson’s etheriﬁcation of (20R)-23 with

24 in the presence of a large excess of sodium hydride

aﬀorded (20R)-22-oxa ketone 25 (86%). Attempts to

alkylate 23 with the corresponding O-silyl-protected

25-hydroxylated bromide or iodide led mainly to the

recovery of 23 with a low yield of the desired C/D-ring

ketones (approx. 20%). Deprotection of TBS protecting

groups with p-toluenesufonic acid, followed by tetrapro-

pylammonium perruthenate (Pr₄NRuO₄)-catalyzed oxi-

dation of the diol 26, then protection of the 25-hydroxy

4280

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

OTES

22

24

20

25

23

OMOM

8

O

22b

22a

O

OH

24

OTBS

OR'

1) TsO

Pr RuO

4 4

NaH

2) TsOH, Et N

3

OR

OTBS

23 (20S & 20R)

25: R = R' = TBS (S: 82%; R: 86%)

26: R = R' = H (S: 98%; R: 97%)

O

S O

R

O

OH

OR

OTES

MOMCl

or TESCl

O

27 (S: 98%; R: 98%)

22c: R = MOM (85%)

22'c: R = TES (77%)

22d (quant)

1) MeMgBr

(64%)

TsO

OH

TBSOTf TsO

Et N

HO

OTBS

2) TsCl, Pyr.

(97%)

3

O

24: (87%)

Figure 3. Synthetic scheme of the 25-hydroxy Grundmann’s ketones.

R

aq. AcOH

or 1) TBAF, Et N

Br(CH ) OTBS

2 2

7, n-BuLi

22a, b, c, d

NaH

3

2) TBSCl, Et N

3

OTBS

OTMS

TBSO

OR''

R''O

OH

29a: R' = MOM; R'' = TBS (98%)

29b: R' = R'' = H (97%)

29c: R' = MOM; R'' = TBS (72%)

29d: R' = TES; R'' = TBS (27%)

30b: R' = R'' = TBS (66%)

28a: R' = MOM (ca. 5:4; 56%)

28b: R' = TES (ca. 3:2; 52%)

28c: R' = MOM (ca. 2:1; 78%)

28d: R' = TES (ca. 3:2; 88%)

30d: R' = H; R'' = TBS (65%)

R

a:

3a, 4a: R' = H (83%)

3b, 4b: R' = H (91%)

OR'

CSA

3c, 4c: R' = H (76%)

3d, 4d: R' = H (91%)

OR'

b:

OTBS

TBSO

O

c:

d:

OR'

OTBS

31a: R' = MOM (83%)

31b: R' = TBS (71%)

31c: R' = MOM (70%)

31d: R' = H (71%)

O

Figure 4. Synthetic scheme of the A-ring- and side-chain-modiﬁed 2-hydroxyethoxy-19-norvitamin D analogs.

2-Hydroxy-19-norvitamin D derivatives 29a,c,d and 30b

were allowed to react with (2-bromoethoxy)-tert-buty-

ldimethylsilane in the presence of excess sodium hydride,

and then all the protecting groups were removed by

treatment with camphor sulfonic acid (CSA) to aﬀord

the 2-hydroxyethoxy-19-norvitamin D analogs 3a–d

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

4281

and 4a–d in good yields. All C(2)-epimeric pairs were

separated by HPLC. The C(2)-conﬁguration of 2-

hydroxyethoxy analogs was determined as reported.^7,8

2-hydroxyethylidene-19-norvitamin D analogs 5a, c, d

and 6a, c, d (74–96%) as geometrical isomers. All 1:1

mixtures of E- and Z-isomers in 5 and 6 were separated

by HPLC. 19-Norvitamin D analogs 5b and 6b with

elongated side chains were prepared by the reaction of

the phosphine oxide 8 possessing the desired 2-hydrox-

yethylidene group at C(2) with the C/D-ring ketone

22b, followed by deprotection of 38b with TBAF. The

coupling yield of 38b (E:Z = ca. 6:1; 27%) was signiﬁ-

cantly low, probably as a result of the bulkiness of the

side chain of the C/D-ring part, and the E-isomer was

the major product.⁶

(E)- and (Z)-1a,25-Dihydroxy-2-(2-hydroxyethylidene)-

19-norvitamin D₃analogs 5a–d and 6a–d were synthe-

sized as illustrated in Figure 5. Wittig–Horner reaction

of 22’c with the A-ring synthon 7 gave 19-norvitamin

D derivative 32c (ca. 2:1; 59%) as a mixture of diastereo-

mers, and deprotection of the TMS-protecting group of

32c aﬀorded 33c (79%). The 2-hydroxy compounds 29a,

33c, and 30d obtained above were oxidized to the 2-keto

derivatives 34a, 34c, and 34d, respectively, in excellent

yield. Cyanomethylation of 2-ketones 34a, c, d with

(EtO)₂P(O)CH₂CN gave 35a (85%), 35c (96%), and

35d (97%) as approximately 1:1 mixtures of E- and Z-

isomers. 2-(2-Hydroxyethylidene) derivatives 37a, c, d

were obtained in reasonable yields by two-step reduc-

tions of cyanomethyl derivatives 35a, c, d with

DIBAL-H followed by NaBH₄. The MOM or silyl-pro-

tecting groups were cleaved by CSA to give the desired

2.2. Biological activity

Analogs 3a–d to 6a–d, with the structural modiﬁcations

described above, are expected to show dramatic changes

in activity pattern compared with the parent compounds

3–6e. We evaluated the aﬃnity of the 19-norvitamin D

analogs (3–12) for the recombinant rat VDR

ligand-binding domain (LBD)²⁷and VDR-mediated

R

(EtO₂)₂P

O

CH₂CN

1) 7, n-BuLi

29a

33c

30d

DMSO

22'c

2) aq. AcOH

(COCl)₂

n-BuLi

OTBS

TBSO

OR''

O

34a: R' = MOM (97%)

34c: R' = H (81%)

32c: R' = TES; R'' = TMS

(ca. 2:1; 59%)

34d: R' = TES (98%)

33c: R' = R'' = H (79%)

R

1) DIBAL-H

2) NaBH₄

5a, 6a: R' = H (96%)

5c, 6c: R' = H (74%)

5d, 6d: R' = H (96%)

CSA

OTBS

CN

TBSO

R''

35a: R' = MOM (ca. 1:1; 85%)

35c: R' = H (ca. 1:1; 96%)

35d: R'= TES (ca. 1:1; 97%)

36a: R' = MOM; R'' = CHO (94%)

36c: R' = H; R'' = CHO

36d: R' = TES; R'' = CHO (87%)

37a: R' = MOM; R'' = CH₂OH (87%)

37c: R' = H; R'' = CH₂OH (25%)

37d: R' = TES; R'' = CH₂OH (81%)

R

a:

OR'

8, n-BuLi

TBAF

b:

c:

d:

5b, 6b: R' = H (80%)

22b

O

OR'

OTBS

TBSO

OR'

38b: R' = TES (E : Z = ca. 6:1; 27%)

Figure 5. Synthetic scheme of the A-ring- and side-chain-modiﬁed 2-hydroxyethylidene-19-norvitamin D analogs.

4282

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

transcriptional activity was tested in COS-7 cells. The

results obtained are shown in Table 1.

change to the high-energy b-conformation before it can

be accommodated in the VDR LBP. This might explain

the decreased VDR-binding aﬃnity for the Z-isomer.

Introduction of a double bond at C(22) had variable and

weak eﬀects on VDR binding, although most of the

22,24-diene and 22-oxa analogs showed decreased bind-

ing to the VDR as compared with the parent com-

pounds 3–6e. Replacement of C(22)–H₂by oxygen

reduced both VDR aﬃnity and transcriptional activity,

which were further decreased by epimerization at

C(20). The two isomeric pairs 3/4 and 5/6 exhibited

distinct biological proﬁles. In spite of structural modiﬁ-

cation of the side chain, analogs containing 2b-hydroxy-

ethoxy or 2E-hydroxyethylidene substituents at C(2)

displayed enhanced binding aﬃnity for the VDR as well

as enhanced gene-activating properties compared with

their counterparts. Analogs 4a, 5a, and 5b had VDR-

binding aﬃnity similar to that of 1e. Comparison of

the two isomers 3 and 4 showed that the E-isomer 3 is

more potent than the Z-isomer 4. 1a,25-(OH)₂D₃in

solution exists as a mixture of two equilibrating A-ring

chair conformations, the a-conformation with an axial

1a-OH and an equatorial 3b-OH and the b-conforma-

tion with an equatorial 1a-OH and an axial 3b-OH.²⁸

In the crystal structure of VDR LBD complexed with

1a,25-(OH)₂D₃and its derivatives, the native ligand

docked in the ligand-binding pocket (LBP) adopts the

b-conformation at the A-ring.^29–33In the solution state,

the E-isomer 3 predominantly takes the b-conformation,

whereas the Z-isomer 4 prefers the a-conformation. The

low-energy A-ring a-conformation of the Z-isomer must

It should be noted that for all analogs tested transcrip-

tional activity was ampliﬁed relative to VDR aﬃnity.

The transcriptional activity of analogs containing

22,24-diene groups was higher than that of the parent

compounds 3–6e and similar to that of the 20-epi ana-

logs 3–6f. In contrast, the transcriptional activity of

the (20R)-22-oxa analogs 3–6d was weaker than that

of the parent compounds 3–6e, in accordance with the

eﬀect on receptor binding. In the crystal structure of

the VDR LBD/1a,25-(OH)₂D₃complex, the aliphatic

side chain of 1a,25-(OH)₂D₃exhibits the gauche (ꢀ)

conformation with the C16–C17–C20–C22 torsion angle

close to ꢀ30ꢁ.^29,32Analogs with 22-ene or (20S)-22-oxa

moieties take a similar gauche (ꢀ) conformation in the

free state and can be accommodated in the LBP in their

major conformations. In the transcriptional assay, the

diene-trihomo analogs 3–6b showed the highest potency

among the test compounds. Elongation of the side chain

increases hydrophobic interaction with the amino acid

residues lining the LBP. When accommodated in the

VDR LBD, the side chain of the diene-trihomo analogs

3–6b takes a compact conformation in the LBP, forming

hydrogen bonds with His305 and His397, as observed in

the crystal structures of VDR/KH1060 or VDR/seocalc-

itol complexes.^30,31Docking studies of the diene-trih-

omo analogs 4b and 5b using the docking software

FlexX (Tripos, St. Louis) indicate that the terminal

hydroxyl groups of the substituents at C(2) can form a

hydrogen bond with the backbone carbonyl group of

Asp144. Additional hydrophobic interactions of methyl

groups at the terminal carbons 26 and 27 with Phe422,

Leu414, and Leu227 are observed. Such additional

interactions with the LBD around the A-ring and the

side chain could stabilize the transcriptionally active

receptor conformation.

Table 1. Relative VDR aﬃnity and transcriptional activity of A-ring-

and side-chain-modiﬁed 19-norvitamin D analogs^a

Compound

VDR aﬃnity^b

Transcription^c

1e

3a

3b

3c

3d

3e

3f

1

1.7

0.27

0.04

0.02

0.1^d

1.0^d

0.94

0.14

0.17

0.03

1.0^d

5.0^d

0.96

0.99

0.48

0.12

2.0^d

1.6^d

0.05

0.04

0.006

0.0007

0.007^d

0.02^d

5.6

2.5

0.13

3.5^d

10.0^d

3.1

Comparison of the 22-oxa analogs having a diﬀerent

conﬁguration at C(20) showed that the (20S)-22-oxa

analogs 3–6c have more potent transcriptional activity

than the (20R)-22-oxa analogs 3–6d, in spite of the struc-

tural modiﬁcation at C(2). The active space group

concept^22,23suggests that the side chain of the (20S)-

22-oxa analogs is located in a region showing strong

transcriptional activity (or cell diﬀerentiating activity).

The side chain of the (20S)-22-oxa analogs occupies a

narrow spatial region and these analogs can be docked

in the VDR LBP with a small energy loss.

4a

4b

4c

4d

4e

4f

25.0

2.8

1.25

7.0^d

30.0^d

11.7

17.5

25.0

0.26

2.0^d

12.5^d

1.4

5a

5b

5c

5d

5e

5f

3. Conclusion

6a

6b

6c

6d

6e

6f

2.8

0.6

We have described the synthesis and biological evalua-

tion of 16 novel A-ring- and side-chain-modiﬁed 19-nor-

vitamin D analogs. Our present data demonstrate that

structural modiﬁcations on the side chain reduce the

aﬃnity for the VDR of nearly all analogs examined

and that, the gene-activating activity of the analogs,

except for the (20R)-22-oxa compounds 3–6d, was

similar to or stronger than that of the parent compounds

0.22

0.3^d

4.2^d

^aActivities are shown as % of that of 1e.

^bRat vitamin D receptor ligand-binding domain.

^cActivity was assessed in terms of ED₅₀

^dRef. 7 and 8.

.

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

4283

3–6e. In addition, introduction of a 22,24-diene-

24,26,27-trihomo group into the side-chain enhanced

transcriptional activity to a similar extent as that caused

by C(20) epimerization. A more extensive biological

evaluation is in progress in our laboratory.

0.88 (each 9H, s), 1.02 (3H, d, J = 6.6 Hz), 1.20 (6H,

s), 2.80 (1H, m), 3.37 (3H, s), 3.54 (1H, m), 3.80 (1H,

m), 3.87 (1H, m), 4.73 (2H, s), 5.33 (2H, m), 5.81 (1H,

d, J = 11.1 Hz), 6.10 (1H, d, J = 11.1 Hz). 2b-Isomer

1

(minor): H NMR (CDCl₃) d: 0.04–0.06 (12H, s), 0.12

(9H, s), 0.54 (3H, s), 0.86, 0.89 (each 9H, s), 1.02 (3H,

d, J = 6.6 Hz), 1.02 (6H, s), 2.80 (1H, m), 3.37 (3H, s),

3.60 (1H, m), 3.80 (1H, m), 3.93 (1H, m), 4.73 (2H, s),

5.33 (2H, m), 5.78 (1H, d, J = 11.2 Hz), 6.13 (1H, d,

J = 11.2 Hz).

4. Experimental

NMR spectra were obtained on a Bruker ARX-400

spectrometer, operating at 400 MHz for H. Chemical

1

shifts are reported in parts per million (ppm, d) down-

ﬁeld from tetramethylsilane as an internal standard (d

0 ppm) for H NMR. Abbreviations used are: singlet

4.2. 24a,26a,27a-Trihomo-1a-[(tert-butyldimethylsilyl)oxy]-

2-[(trimethylsilyl)oxy]-22,24-diene-25-[(triethylsilyl)oxy]-

19-norvitamin D₃tert-butyldimethylsilyl ether (28b)

1

(s), doublet (d), triplet (t), multiplet (m), aromatic

(arom), broad signal (br). Low- and high-resolution

mass spectra (MS and HRMS) were obtained with elec-

tronic ionization (EI) on a JEOL JMS-AX505HA spec-

trometer run at 70 eV for EI; m/z values are given with

relative intensities in parentheses. UV spectra were ob-

tained on a Beckmann DU-7500 spectrophotometer. A

mixture of diastereomers were separated by HPLC

equipped with a Model PU-980 pump, a Rhedyne Mod-

el 7125 injector and a Model MD-910 multiwavelength

UV detector from Jasco (Tokyo, Japan). Column chro-

matography was carried out on silica gel (Wakogel C-

200), unless otherwise indicated. All reactions, unless

speciﬁcally mentioned, were conducted under an atmo-

sphere of argon gas. Yields are not optimized.

The same procedure as described above, but using 7

(206.0 mg, 0.394 mmol, a mixture of ca. 2:1) and 22b

(101.8 mg, 0.235 mmol) gave 28b (106.4 mg, 52% based

on 22b) as a mixture of two isomers in a ca. 3:2 ratio.

MS m/z (%): 872 (no M⁺), 740 (33), 683 (7), 608 (65), 551

(17), 505 (43), 459 (18), 324 (31), 149 (100), 75 (99).

HRMS m/z: 740.5422 (M⁺ꢀTBSOH) (Calcd for

1

C₄₄H₈₀O₃Si₃: 740.5415). 2a-Isomer (major): H NMR

(CDCl₃) d: 0.039, 0.051, 0.059, 0.064 (each 3H, s, 4·

Si–Me), 0.119 (9H, s, 3· Si–Me), 0.57 (3H, s, H-18),

0.56 (6H, q, J = 7.9 Hz, 3· SiCH₂), 0.82 (6H, t,

J = 7.5 Hz, H-26a, 27a), 0.87, 0.88 (each 9H, s, 2· Si–

t-Bu), 0.94 (9H, t, J = 7.9 Hz, 3· SiCH₂CH₃), 1.06

(3H, d, J = 6.6 Hz, H-21), 2.80 (1H, m, H-9), 3.53

(1H, m, H-2), 3.80 (1H, m, H-3), 3.88 (1H, m, H-1),

5.52 (1H, d, J = 15.2 Hz, H-24a, overlapped with H-

22), 5.81 (1H, d, J = 11.1 Hz, H-7), 5.94 (1H, dd,

J = 14.9, 10.4 Hz, H-23), 6.05 (1H, dd, J = 15.2,

10.4 Hz, H-24), 6.10 (1H, d, J = 11.1 Hz, H-6). 2b-Iso-

In the following experiments, the numbering of the

compounds 7–21 and 24 corresponding to the A-ring

part of the 19-norvitamin D was expressed based on

the IUPAC nomenclature of organic chemistry. The

nomenclature of the compounds 3 to 6a–d and

22–38 was expressed on the basis of the steroidal

numbering.

1

mer (minor): H NMR (CDCl₃) d: 0.039, 0.051, 0.059,

0.064 (each 3H, s, 4· Si–Me), 0.124 (9H, s, 3· Si–Me),

0.56 (3H, s, H-18), 0.56 (6H, q, J = 7.9 Hz, 3· SiCH₂),

0.82 (6H, t, J = 7.5 Hz, H-26a, 27a), 0.86, 0.89 (each

9H, s, 2· Si–t-Bu), 0.94 (9H, t, J = 7.9 Hz, 3·

SiCH₂CH₃), 1.06 (3H, d, J = 6.6 Hz, H-21), 2.80 (1H,

m, H-9), 3.59 (1H, m, H-2), 3.80 (1H, m, H-3), 3.94

(1H, m, H-1), 5.52 (1H, d, J = 15.2 Hz, H-24a, over-

lapped with H-22), 5.78 (1H, d, J = 11.1 Hz, H-7), 5.94

(1H, dd, J = 14.9, 10.4 Hz, H-23), 6.05 (1H, dd,

J = 15.2, 10.4 Hz, H-24), 6.13 (1H, d, J = 11.1 Hz, H-6).

4.1. 1a-[(tert-Butyldimethylsilyl)oxy]-2-[(trimethylsilyl)oxy]-

22-ene-25-[(methoxymethyl)oxy]-19-norvitamin D₃tert-

butyldimethylsilyl ether (28a)

To a stirred solution of 7 (268.2 mg, 0.407 mmol, a mix-

ture of ca. 2:1) in dry THF (2 mL) at ꢀ78 ꢁC was added

slowly n-BuLi (261 lL, 0.407 mmol, 1.56 M solution in

hexane), and the resulting dark orange solution was stir-

red for 15 min. To this colored solution was added a

solution of 22a (87.5 mg, 0.271 mmol) in dry THF

(1 mL), the reaction mixture was stirred for 2 h at

ꢀ78 ꢁC, quenched with saturated NH₄Cl and extracted

with AcOEt. The AcOEt layer was washed with brine

dried over MgSO₄, and evaporated in vacuo. The resi-

due was puriﬁed by chromatography on silica gel

(10 g) using 2% AcOEt in hexane to aﬀord 28a

(116.1 mg, 56% based on 22a) as a mixture of two iso-

mers in a ca. 5:4 ratio, and 10% AcOEt in hexane to give

the unreacted starting material 22a (11.6 mg, 13%).

4.3. 1a-[(tert-Butyldimethylsilyl)oxy]-2-[(trimethylsilyl)oxy]-

22-oxa-25-[(methoxymethyl)oxy]-19-norvitamin D₃tert-

butyldimethylsilyl ether (28c)

The same procedure as described above, but using 7

(244.4 mg, 0.38 mmol, a mixture of ca. 2:1) and 22c

(79.8 mg, 0.19 mmol) gave 28c (111.8 mg, 78% based

on 22c) as a mixture of two isomers in a ca. 3:2 ratio.

MS m/z (%): 766 (M⁺, 12), 704 (15), 634 (33), 572 (47),

515 (12), 75 (100). HRMS m/z: 766.5442 (Calcd for

C₄₂H₈₂O₆Si₃: 766.5419). 2a-Isomer (major) ¹H NMR

(CDCl₃) d: 0.03–0.08 (12H, s, 4· Si–Me), 0.13 (9H,

Si–Me₃), 0.52 (3H, s, H-18), 0.87, 0.88 (each 9H, s, 2·

Si–t-Bu), 1.16 (3H, d, J = 6.0 Hz, H-21), 1.25 (6H, s,

H-26, 27), 2.78 (1H, m, H-9), 3.21 (1H, m, H-20), 3.33

MS m/z (%): 762 (M⁺, 18), 700 (28), 630 (39), 568 (57),

511 (18), 465 (25), 309 (36), 147 (35), 109 (56), 75 (100).

HRMS m/z: 762.5474 (Calcd for C₄₃H₈₂O₅Si₃:

762.5470). 2a-Isomer (major): ¹H NMR (CDCl₃) d:

0.04–0.06 (12H, s), 0.12 (9H, s), 0.55 (3H, s), 0.87,

4284

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

(1H, m, H-23), 3.36 (3H, s, OMe), 3.53 (1H, m, H-2),

3.66 (1H, m, H-23), 3.80 (1H, m, H-3), 3.87 (1H, m,

H-1), 4.70 (2H, s, OCH₂O), 5.82 (1H, m, d,

J = 11.2 Hz, H-7), 6.09 (1H, d, J = 11.2 Hz, H-6). 2b-

¹H NMR (CDCl₃) d: 0.06–0.01 (12H), 0.56 (3H, s), 0.87,

0.88 (each 9H, s, 2· Si–t-Bu), 1.02 (3H, d, J = 6.6 Hz, H-

21), 1.20 (6H, s, H-26, 27), 2.80 (1H, m, H-9), 3.37 (3H,

s, OMe), 3.51 (1H, m, H-2), 3.90 (1H, m, H-3), 3.99 (1H,

m, H-1), 4.73 (2H, s, OCH₂O), 5.33 (2H, m, H-22, 23),

5.79 (1H, d, J = 11.1 Hz, H-7), 6.15 (1H, d,

J = 11.1 Hz, H-6). 2b-Isomer: ¹H NMR (CDCl₃) d:

0.06–0.10 (12H, 4· Si–Me), 0.54 (3H, s, H-18), 0.86,

0.90 (each 9H, s, 2· Si–t-Bu), 1.02 (3H, d, J = 6.6 Hz,

H-21), 1.20 (6H, s, H-26, 27), 2.80 (1H, m, H-9), 3.37

(3H, s, OMe), 3.59 (1H, m, H-2), 3.99 (2H, m, H-1, 3),

4.73 (2H, s, OCH₂O), 5.33 (2H, m, H-22, 23), 5.79

(1H, d, J = 11.2 Hz, H-7), 6.18 (1H, d, J = 11.2 Hz, H-

6).

1

Isomer (minor) H NMR (CDCl₃) d: 0.03–0.08 (12H,

s, 4· Si–Me), 0.12 (9H, Si–Me₃), 0.51 (3H, s, H-18),

0.86, 0.89 (each 9H, s, 2· Si–t-Bu), 1.16 (3H, d,

J = 6.0 Hz, H-21), 1.25 (6H, s, H-26, 27), 2.78 (1H, m,

H-9), 3.21 (1H, m, H-20), 3.33 (1H, m, H-23), 3.36

(3H, s, OMe), 3.60 (1H, m, H-2), 3.66 (1H, m, H-23),

3.80 (1H, m, H-3), 3.92 (1H, m, H-1), 4.70 (2H, s,

OCH₂O), 5.80 (1H, m, d, J = 11.2 Hz, H-7), 6.10 (1H,

d, J = 11.2 Hz, H-6).

4.4. 20-epi-1a-[(tert-Butyldimethylsilyl)oxy]-2-[(trimethylsi-

lyl)oxy]-22-oxa-25-[(triethylsilyl)oxy]-19-norvitamin D₃

tert-butyldimethylsilyl ether (28d)

4.6. 1a- [(tert-Butyldimethylsilyl)oxy]-2-hydroxy-22-oxa-

25-[(methoxymethyl)oxy]-19-norvitamin D₃tert-butyldi-

methylsilyl ether (29c)

The same procedure as described above, but using 7

(212.0 mg, 0.321 mmol, a mixture of ca. 2:1) and 22d

(85.0 mg, 0.214 mmol) gave 28d (158.8 mg, 88% based

on 22d) as a mixture of two isomers in a ca. 3:2 ratio.

In a same way, deprotection of 28c (63.2 mg, 0.13 mmol,

ca. 3:2 isomeric mixture) in aq AcOH in THF aﬀorded

29c (41.4 mg, 72%) as a mixture of two isomers in a

ca. 2:1 ratio.

MS m/z (%): 836 (no M⁺), 704 (10), 647 (3), 618 (7), 572

(19), 486 (20), 469 (13), 383 (17), 309 (19), 75 (100).

HRMS m/z: 704.5032 (M⁺ꢀTBSOH) (Calcd for

MS m/z (%): 694 (M⁺, 3), 632 (5), 575 (4), 505 (2), 443

(12), 75 (100). HRMS m/z: 694.5002 (Calcd for

1

C₄₀H₇₆O₄Si₃: 704.5051). 2a-Isomer (major): H NMR

C₃₉H₇₄O₆Si₂: 694.5024). 2a-Isomer: H NMR (CDCl₃)

(CDCl₃) d: 0.04–0.06 (12H, 4· Si–Me), 0.13 (9H, s, 3·

Si–Me), 0.56 (3H, s, H-18), 0.57 (6H, q, J = 7.9 Hz, 3·

SiCH₂), 0.87, 0.88 (each 9H, s, 2· Si–t-Bu), 0.94 (9H,

t, J = 7.9 Hz, 3· SiCH₂CH₃), 1.08 (3H, d, J = 5.9 Hz,

H-21), 1.21, 1.23 (each 3H, s, H-26, 27), 2.80 (1H, m,

H-9), 3.26 (1H, m, H-20), 3.32, 3.69 (each 1H, m, H-

23), 3.53 (1H, m, H-2), 3.80 (1H, m, H-3), 3.89 (1H,

m, H-1), 5.79 (1H, d, J = 11.1 Hz, H-7), 6.11 (1H, d,

J = 11.1 Hz, H-6). 2b-Isomer (minor): ¹H NMR

(CDCl₃) d: 0.04–0.06 (12H, 4· Si–Me), 0.12 (9H, s, 3·

Si–Me), 0.54 (3H, s, H-18), 0.57 (6H, q, J = 7.9 Hz, 3·

SiCH₂), 0.86, 0.89 (each 9H, s, 2· Si–t-Bu), 0.94 (9H,

t, J = 7.9 Hz, 3· SiCH₂CH₃), 1.08 (3H, d, J = 5.9 Hz,

H-21), 1.21, 1.23 (each 3H, s, H-26, 27), 2.80 (1H, m,

H-9), 3.26 (1H, m, H-20), 3.32, 3.69 (each 1H, m, H-

23), 3.59 (1H, m, H-2), 3.80 (1H, m, H-3), 3.93 (1H,

m, H-1), 5.77 (1H, d, J = 11.2 Hz, H-7), 6.14 (1H, d,

J = 11.2 Hz, H-6).

d: 0.06–0.09 (12H, s, 4· Si–Me), 0.52 (3H, s, H-18),

0.87, 0.88 (each 9H, s, 2· Si–t-Bu), 1.16 (3H, d,

J = 6.0 Hz, H-21), 1.24 (6H, s, H-26, 27), 2.78 (1H, m,

H-9), 3.22 (1H, m, H-20), 3.32 (1H, m, H-23), 3.35

(3H, s, OMe), 3.50 (1H, m, H-2), 3.66 (1H, m, H-23),

3.92 (1H, m, H-3), 3.99 (1H, m, H-1), 4.70 (2H, s,

OCH₂O), 5.80 (1H d, J = 11.1 Hz, H-7), 6.14 (1H, d,

J = 11.1 Hz, H-6). 2b-Isomer: ¹H NMR (CDCl₃) d:

0.06–0.09 (12H, s, 4· Si–Me), 0.51 (3H, s, H-18), 0.86,

0.89 (each 9H, s, 2· Si–t-Bu), 1.16 (3H, d, J = 6.0 Hz,

H-21), 1.24 (6H, s, H-26, 27), 2.78 (1H, m, H-9), 3.22

(1H, m, H-20), 3.32 (1H, m, H-23), 3.35 (3H, s, OMe),

3.58 (1H, m, H-2), 3.66 (1H, m, H-23), 3.99 (2H, m,

H-1, 3), 4.70 (2H, s, OCH₂O), 5.80 (1H, m, d,

J = 11.1 Hz, H-7), 6.17 (1H, d, J = 11.1 Hz, H-6).

4.7. 20-epi-1a-[(tert-Butyldimethylsilyl)oxy]-2,25-dihy-

droxy-22-oxa-19-norvitamin D₃tert-butyldimethylsilyl

ether (29d) and 20-epi-1a-[(tert-butyldimethylsilyl)oxy]-2-

hydroxy-22-oxa-25-[(triethylsilyl)oxy]-19-norvitamin D₃tert-

butyldimethylsilyl ether (30d)

4.5. 1a-[(tert-Butyldimethylsilyl)oxy]-2-hydroxy-22-ene-25-

[(methoxymethyl)oxy]-19-norvitamin D₃tert-butyldimeth-

ylsilyl ether (29a)

In

a same way, deprotection of 28d (153.0 mg,

A solution of 28a (60.0 mg, 0.0786 mmol, ca. 5:4 isomer-

ic mixture) in THF/AcOH/H₂O (v/v/v, 8:8:1, 4.25 mL)

was stirred at ambient temperature for 18 h, and diluted

with AcOEt. The organic phase was successively washed

with 5% NaHCO₃and brine and dried over Na₂SO₄.

After evaporation of the solvent, the resulting residue

was puriﬁed by chromatography on silica gel (6 g) using

5% AcOEt in hexane to aﬀord 29a (53.4 mg, 98%) as a

mixture of two isomers in a ca. 5:4 ratio.

0.183 mmol, ca. 3:2 isomeric mixture) in aq AcOH in

THF aﬀorded 29d (77.1 mg, 65%) and 30d (37 0 mg,

27%) as a mixture of two isomers in a ca. 3:2 ratio,

respectively.

Compound 29d: MS m/z (%): 650 (M⁺, 2), 632 (8), 546

(6), 489 (8), 443 (10), 357 (8), 265 (22), 113 (30), 75

(100). HRMS m/z: 650.4776 (Calcd for C₃₇H₇₀O₅Si₂:

1

650.4762). 2a-Isomer: H NMR (CDCl₃) d: 0.06–0.10

(12H, 4· Si–Me), 0.56 (3H, s, H-18), 0.87, 0.88 (each

9H, s, 2· Si–t-Bu), 1.14 (3H, d, J = 5.9 Hz, H-21),

1.22, 1.24 (each 3H, s, H-26, 27), 2.80 (1H, m, H-9),

3.28 (1H, m, H-20), 3.46 (1H, m, H-23), 3.51 (1H, m,

MS m/z (%): 690 (M⁺, 6), 628 (9), 571 (7), 501 (5), 439

(29), 309 (11), 237 (11), 109 (63) 75 (100). HRMS m/z:

690.5084 (Calcd for C₄₀H₇₄O₅Si₃: 690.5075). 2a-Isomer:

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

4285

H-2), 3.59 (1H, s, OH), 3.85 (1H, m, H-23), 3.92 (1H, m,

H-3), 4.00 (1H, m, H-1), 5.78 (1H, d, J = 11.1 Hz, H-7),

6.16 (1H, d, J = 11.1 Hz, H-6). 2b-Isomer: ¹H NMR

(CDCl₃) d: 0.06–0.10 (12H, 4· Si–Me), 0.54 (3H, s, H-

18), 0.86, 0.89 (each 9H, s, 2· Si–t-Bu), 1.14 (3H, d,

J = 5.9 Hz, H-21), 1.23, 1.24 (each 3H, s, H-26, 27),

2.80 (1 H, m, H-9), 3.28 (1H, m, H-20), 3.46 (1H, m,

H-23), 3.59 (1H, m, H-2), 3.85 (1H, m, H-23), 4.00

(2H, m, H-1, 3), 5.78 (1H, d, J = 11.2 Hz, H-7), 6.19

(1H, d, J = 11.2 Hz, H-6).

H-18), 0.87 (6H, t, J = 7.4 Hz, H-26a, 27a), 1.05 (3H,

d, J = 6.6 Hz, H-21), 1.55, 1.56 (each 2H, q,

J = 7.4 Hz, H-26, 27), 2.43 (2H, m, H-4), 2.80 (1H, m,

H-9), 3.07 (1H, dd, J = 13.2, 4.9 Hz, H-10), 3.48 (1H,

dd, J = 8.8, 3.0 Hz, H-2), 3.67 (1H, m, H-3), 4.09 (1H,

m, H-1), 5.53 (1H, d, J = 15.2 Hz, H-24a, overlapped

with H-22), 5.83 (1H, d, J = 11.1 Hz, H-7), 5.98 (1H,

dd, J = 15.0, 10.3 Hz, H-23), 6.15 (1H, dd, J = 15.2,

10.3 Hz, H-24), 6.29 (1H, d, J = 11.1 Hz, H-6).

4.9. 24a,26a,27a-Trihomo-1a-[(tert-butyldimethylsilyl)oxy]-

2-hydroxy-22,24-diene-25-[(triethylsilyl)oxy]-19-norvita-

min D₃tert-butyldimethylsilyl ether (30b)

Compound 30d: MS m/z (%): 764 (M⁺, 1), 707 (1), 632

(4), 575 (2), 546 (3), 489 (4), 443 (5), 357 (20), 265

(11), 103 (31), 75 (100). HRMS m/z: 764.5643 (Calcd

for C₄₃H₈₄O₅Si₃: 764.5627). 2a-Isomer: ¹H NMR

(CDCl₃) d: 0.06–0.10 (12H, 4· Si–Me), 0.56 (3H, s, H-

18), 0.56 (6H, q, J = 7.9 Hz, 3· SiCH₂), 0.87, 0.88 (each

9H, s, 2· Si–t-Bu), 0.94 (9H, t, J = 7.9 Hz, 3·

SiCH₂CH₃), 1.09 (3H, d, J = 5.9 Hz, H-21), 1.21, 1.23

(each 3H, s, H-26, 27), 2.81 (1H, m, H-9), 3.26 (1H,

m, H-20), 3.32 (1H, m, H-23), 3.51 (1H, m, H-2), 3.70

(1H, m, H23), 3.91 (1H, m, H-3), 4.01 (1H, m, H-1),

5.78 (1H, d, J = 11.1 Hz, H-7), 6.18 (1H, d,

J = 11.1 Hz, H-6). 2b-Isomer: ¹H NMR (CDCl₃) d:

0.06–0.10 (12H, 4· Si–Me), 0.55 (3H, s, H-18), 0.56

(6H, q, J = 7.9 Hz, 3· SiCH₂), 0.86, 0.89 (each 9H, s,

2· Si–t-Bu), 0.94 (9H, t, J = 7.9 Hz, 3· SiCH₂CH₃),

1.09 (3H, d, J = 5.9 Hz, H-21), 1.21, 1.23 (each 3H, s,

H-26, 27), 2.81 (1H, m, H-9), 3.26 (1H, m, H-20), 3.32

(1H, m, H-23), 3.59 (1H, m, H-2), 3.70 (1H, m, H-23),

4.01 (2H, m, H-1, 3), 5.78 (1H, d, J = 11.2 Hz, H-7),

6.20 (1H, d, J = 11.2 Hz, H-6).

To a stirred solution of 29b (48.0 mg, 0.105 mmol, ca.

3:2 isomeric mixture) in dry DMF (1 mL) were added

Et₃N (117 lL, 0.840 mmol), tert-butyldimethylsilyl chlo-

ride (63.9 mg, 0.424 mmol), and 4-(dimethylamino)pyri-

dine (6.4 mg, 0.052 mmol) at ambient temperature.

After 2 h stirring, the reaction mixture was poured into

ice water, and extracted with AcOEt. Organic extracts

were washed with brine dried over Na₂SO₄, and evapo-

rated in vacuo. The residue was puriﬁed by chromatog-

raphy on silica gel (5 g) using 5% AcOEt in hexane to

yield 30b (55.3 mg, 66%) as a mixture of two isomers

in a ca. 3:2 ratio.

MS m/z (%): 800 (no M⁺), 668 (6), 611 (2), 536 (3), 479

(12), 386 (6), 149 (100), 75 (79). HRMS m/z: 668.5021

(M⁺ꢀTBSOH) (Calcd for C₄₁H₇₂O₃Si₂: 668.5020).

2a-Isomer: ¹H NMR (CDCl₃) d: 0.06–0.10 (18H, 6·

Si–Me), 0.57 (3H, s, H-18), 0.87 (6H, t, J = 7.5 Hz, H-

26a, 27a), 0.87–0.92 (27H, 3· Si–t-Bu), 1.06 (3H, d,

J = 6.6 Hz, H-21), 1.54, 1.55 (each 2H, q, J = 7.5 Hz,

H-26, 27), 2.80 (1H, m, H-9), 3.51 (1H, m, H-2), 3.92

(1H, m, H-3), 4.00 (1H, m, H-1), 5.53 (1H, d,

J = 15.3 Hz, H-24a), 5.55 (1H, dd, J = 15.2, 8.5 Hz, H-

22), 5.79 (1H, d, J = 11.1 Hz, H-7), 5.98 (1H, dd,

J = 15.2, 10.4 Hz, H-23), 6.15 (1H, dd, J = 15.3,

10.4 Hz, H-24), 6.18 (1H, d, J = 11.1 Hz, H-6). 2b-Iso-

4.8. 24a,26a,27a-Trihomo-1a,2a,25-trihydroxy- and 24a,

26a,27a-trihomo-1a,2b,25-trihydroxy-22,24-diene-19-nor-

vitamin D₃(29b)

A mixture of 28b (55 mg, 0.063 mmol, ca. 3:2 isomeric

mixture), Et₃N (20 lL), and tetrabutylammonium ﬂuo-

ride (504 lL, 0.504 mmol, 1.0 M solution in THF) in

dry THF (1 mL) was stirred at ambient temperature

for 4 h. The mixture was poured into ice water and

extracted with AcOEt. The organic phase was washed

with brine and dried over MgSO₄. Removal of the sol-

vent in vacuo aﬀorded the residue, which was puriﬁed

by chromatography on silica gel (5 g) with 2% MeOH

in AcOEt to yield 29b (28.0 mg, 97%) as a mixture of

two isomers in a ca. 3:2 ratio.

1

mer: H NMR (CDCl₃) d: 0.06–0.10 (18H, 6· Si–Me),

0.56 (3H, s, H-18), 0.87 (6H, t, J = 7.5 Hz, H-26a,

27a), 0.87–0.92 (27H, 3· Si–t-Bu), 1.06 (3H, d,

J = 6.6 Hz, H-21), 1.54, 1.55 (each 2H, q, J = 7.5 Hz,

H-26, 27), 2.80 (1H, m, H-9), 3.59 (1H, m, H-2), 4.00

(2H, m, H-1, 3), 5.53 (1H, d, J = 15.3 Hz, H-24a), 5.55

(1H, dd, J = 15.2, 8.5 Hz, H-22), 5.79 (1H, d,

J = 11.1 Hz, H-7), 5.98 (1H, dd, J = 15.2, 10.4 Hz, H-

23), 6.14 (1H, d, J = 11.1 Hz, H-6), 6.15 (1H, dd,

J = 15.3, 10.3 Hz, H-24).

MS m/z (%): 458 (M⁺, 33), 440 (95), 422 (14), 404 (16),

386 (52), 318 (40), 289 (90), 237 (44), 149 (100). HRMS

m/z: 458.3383 (Calcd for C₂₉H₄₆O₄: 458.3396). 2a-Iso-

mer: ¹H NMR (CDCl₃) d: 0.57 (3H, s, H-18), 0.87

(6H, t, J = 7.4 Hz, H-26a, 27a), 1.05 (3H, d,

J = 6.6 Hz, H-21), 1.55, 1.56 (each 2H, q, J = 7.4 Hz,

H-26, 27), 2.62 (1H, dd, J = 12.8, 4.1 Hz, H-4), 2.80

(1H, m, H-9), 2.89 (1H, dd, J = 14.7, 4.3 Hz, H-10),

3.53 (1H, dd, J = 8.2, 2.9 Hz, H-2), 3.79 (1H, m, H-3),

4.09 (1H, m, H-1), 5.53 (1H, d, J = 15.2 Hz, H-24a,

overlapped with H-22), 5.80 (1H, d, J = 11.1 Hz, H-7),

5.98 (1H, dd, J = 15.0, 10.3 Hz, H-23), 6.15 (1H, dd,

J = 15.2, 10.3 Hz, H-24), 6.37 (1H, d, J = 11.1 Hz,

H-6). 2b-Isomer: ¹H NMR (CDCl₃) d: 0.57 (3H, s,

4.10. 1a-[(tert-Butyldimethylsilyl)oxy]-2-[(tert-butyldimeth-

ylsilyl)oxy-ethoxy]-22-ene-25-[(methoxymethyl)oxy]-19-nor-

vitamin D₃tert-butyldimethylsilyl ether (31a)

A suspension of 29a (44.3 mg, 0.064 mmol, ca. 5:4 iso-

meric mixture), NaH (77.0 mg, 1.925 mmol, 60% disper-

sion in mineral oil), and (2-bromoethoxy)-tert-

butyldimethylsilane (69 lL, 0.320 mmol) in dry DMF

(1 mL) was stirred vigorously for 20 h at 0 ꢁC, and the

reaction mixture was poured into ice water and then

extracted with AcOEt/hexane (v/v, 1:1). The organic

phase was washed with brine and dried over MgSO₄.

4286

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

Following evaporation of the solvent in vacuo, the resi-

due was puriﬁed by chromatography on silica gel (10 g)

using 2% AcOEt in hexane to aﬀord 31a (45.0 mg, 83%)

as a mixture of two isomers in a ca. 1:1 ratio.

790.5798 (M⁺ꢀCH₃OCH₂OH) (Calcd for C₄₅H₈₆O₅Si₃:

790.5783).

4.13. 20-epi-1a-[(tert-Butyldimethylsilyl)oxy]-2-[2-(tert-

butyldimethylsilyl)oxy]-ethoxy]-22-oxa-25-hydroxy-19-

norvitamin D₃tert-butyldimethylsilyl ether (31d)

¹H NMR (CDCl₃) d: 0.05–0.09 (18H, 6· Si–Me), 0.54,

0.56 (ca. 1:1) (3H, s, H-18), 0.86–0.91 (27H, 3· Si–t-

Bu), 1.02 (3H, d, J = 6.6 Hz, H-21), 1.24 (6H, s, H-26,

27), 2.80 (1H, m, H-9), 3.19, 3.28 (ca. 1:1) (1H, m, H-

2), 3.37 (3H, s, OMe), 3.5–4.1 (7H, m, OCH₂CH₂O,

H-1, 3), 4.73 (2H, s, OCH₂O), 5.32 (2H, m, H-22, 23),

5.77, 5.80 (ca. 1:1) (1H, d, J = 11.0 Hz, H-7), 6.12,

6.14 (ca. 1:1) (1H, d, J = 11.0 Hz, H-6). MS m/z (%):

848 (no M⁺), 786 (1), 716 (4), 654 (8), 610 (8),

553 (5), 522 (10), 465 (12), 75 (100). HRMS m/z:

786.5856 (M⁺ꢀMeOCH₂OH) (Calcd for C₄₆H₈₆O₄Si₃:

786.5834).

Following the same procedure as described above, treat-

ment of 29d (73.5 mg, 0.113 mmol, ca. 3:2 isomeric mix-

ture) with the bromide (118lL, 0.550 mmol) yielded 31d

(65.0 mg, 71%) as a mixture of two isomers in a ca. 3:2

ratio.

¹H NMR (CDCl₃) d: 0.05–0.07 (18 H, 6· Si–Me), 0.54,

0.55 (ca. 2:3) (3H, s, H-18), 0.86–0.89 (27H, 3· Si–t-Bu),

1.13 (3H, d, J = 5.5 Hz, H-21), 1.23, 1.24 (each 3H, s, H-

26, 27), 2.80 (1H, m, H-9), 3.2–4.1 (10H, m, OCH_2-

CH₂O, H-1, 2, 3, 20, 23), 5.77 (1H, H-7), 6.14 (1H, H-

6). MS m/z (%): 808 (no M⁺), 790 (1), 676 (4), 658 (5),

572 (6), 526 (5), 397 (18), 233 (74), 75 (100). HRMS

m/z: 790.5766 (M⁺ꢀH₂O) (Calcd for C₄₅H₈₆O₅Si₃:

790.5783).

4.11. 24a,26a,27a-Trihomo-1a-[2-(tert-butyldimethylsilyl)-

oxy]-2-[2-(tert-butyldimethylsilyl)oxy]-ethoxy-22,24-diene-

25-[(tert-butyldimethylsilyl)oxy]-19-norvitamin D₃tert-butyl-

dimethylsilyl ether (31b)

Following the same procedure as described above, treat-

ment of 30b (52.4 mg, 0.065 mmol, ca. 3:2 isomeric mix-

ture) with the bromide (68 lL, 0.317 mmol) yielded 31b

(44.7 mg, 71%) as a mixture of two isomers in a ca. 3:2

ratio.

4.14. 1a,25-Dihydroxy-2a-(2-hydroxyethoxy)- and 1a,25-

dihydroxy-2b-(2-hydroxyethoxy)-22-ene-19-norvitamin

D₃(3a and 4a)

A mixture of 31a (45.0 mg, 0.053 mmol, ca. 1:1 isomeric

mixture) and (ꢀ)-10-camphor sulfonic acid (73.8 mg,

0.318 mmol) in dry MeOH (1 mL) was stirred at ambi-

ent temperature for 2 h. The reaction mixture was

poured into 5% NaHCO₃and extracted with AcOEt.

The organic phase was washed with brine, dried over

MgSO₄, and evaporated in vacuo. The residue was chro-

matographed on silica gel (4 g) using 2% MeOH in

AcOEt to give the isomeric mixture of 3a and 4a

(20.3 mg, 83%). The mixture was separated by HPLC

(YMC-Pack ODS-AM SH-342-5, 150 · 20 mm, 25%

H₂O in MeOH) to aﬀord pure 3a (8.3 mg) and 4a

(7.9 mg), respectively.

¹H NMR (CDCl₃) d: 0.05–0.10 (24H, 8· Si–Me), 0.55,

0.57 (ca. 2:3) (3 H, s, H-18), 0.85–0.92 (42H, 4· Si–

t-Bu, H-26a, 27a), 1.05 (3H, d, J = 6.6 Hz, H-21), 1.54,

1.55 (each 2H, d, J = 7.5 Hz, H-26, 27), 2.80 (1H, m,

H-9), 3.18–4.45 (7 H, m, OCH₂CH₂O, H-1, 3), 5.52

(1H, d, J = 15.1 Hz, H-24a), 5.54 (1H, dd, J = 15.0,

8.6 Hz, H-22), 5.78, 5.81 (ca. 3:2) (1H, d, J = 11.2 Hz,

H-7), 5.97 (1H, dd, J = 15.0, 10.4 Hz, H-23), 6.11, 6.14

(ca. 2:3) (1H, d, J = 11.2 Hz, H-6), 6.15 (1H, dd,

J = 15.1, 10.4 Hz, H-24). MS m/z (%): 958 (no M⁺),

826 (1), 769 (2), 649 (14), 651 (11), 562 (12), 519 (24),

233 (100). HRMS m/z: 826.6146 (M⁺ꢀTBSOH) (Calcd

for C₄₉H₉₀O₄Si₃: 826.6147).

Compound 3a: ¹H NMR (CDCl₃) d: 0.57 (3H, s, H-18),

1.04 (3H, d, J = 6.6 Hz, H-21), 1.20 (6H, s, H-26, 27),

2.62 (1H, dd, J = 13.5, 4.5 Hz, H-4), 2.67 (each 1H, br

s, 2· OH), 2.79 (1H, m, H-9), 2.86 (1H, dd, J = 14.4,

4.9 Hz, H-10), 3.03 (1H, br s, OH), 3.33 (1H, dd,

J = 8.0, 2.8 Hz, H-2), 3.68–3.83 (4H, m, OCH₂CH₂O),

3.94 (1H, m, H-3), 4.15 (1H, m, H-1), 5.38 (2H, m, H-

22, 23), 5.82 (1H, d, J = 11.2 Hz, H-7), 6.33 (1H, d,

J = 11.2 Hz, H-6). UV k_max(EtOH): 244 (e 27,400),

252 (e 32,000), 261 (e 21,700) nm. MS m/z (%): 462

(M⁺, 55), 444 (58), 426 (43), 408 (22), 346 (32), 317

(68), 299 (39), 255 (69), 237 (76), 133 (100). HRMS

m/z: 462.3348 (Calcd for C₂₈H₄₆O₅: 462.3345).

4.12. 1a-[(tert-Butyldimethylsilyl)oxy]-2-[2-(tert-butyldim-

ethylsilyl)oxy]-ethoxy]-22-oxa-25-[(methoxymethyl)oxy]-

19-norvitamin D₃tert-butyldimethylsilyl ether (31c)

Following the same procedure as described above, treat-

ment of 29c (41.4 mg, 0.57 mmol, ca. 2:1 isomeric mix-

ture) with the bromide (781.4 lL, 3.64 mmol) yielded

31c (35.0 mg, 70%) as a mixture of two isomers in a

ca. 3:1 ratio.

¹H NMR (CDCl₃) d: 0.04–0.07 (18H, s, 6· Si–Me), 0.51,

0.52 (ca. 1:3) (3H, s, H-18), 0.85–0.95 (27H, s, 3· Si–

t-Bu), 1.16 (3H, d, J = 6.0 Hz, H-21), 1.25 (6H, s, H-

26, 27), 2.79 (1H, m, H-9), 3.21 (1H, m, H-23), 3.33

(1H, m, H-20), 3.36 (3H, s, OMe), 3.60–3.90 (5H, m,

OCH₂CH₂), 3.93 (1H, m), 4.03 (1H, m), 4.71 (2H, s,

OCH₂O), 5.79, 5.81 (ca. 1:3) (1H, m, d, J = 11.1 Hz,

H-7), 6.11, 6.13 (ca. 3:1) (1H, d, J = 11.1 Hz, H-6).

MS m/z (%): 852 (no M⁺), 790 (1), 720 (3), 658 (4),

614 (4), 557 (2), 526 (4), 469 (5), 75 (100). HRMS m/z:

Compound 4a: ¹H NMR (CDCl₃) d: 0.56 (3H, s, H-18),

1.04 (3H, d, J = 6.6 Hz, H-21), 1.20 (6H, s, H-26, 27),

2.34 (1H, d, J = 14.2 Hz, H-4), 2.48 (1H, dd, J = 14.2,

2.0 Hz, H-4), 2.62 (1H, br s, OH), 2.79 (1H, m, H-9),

3.07 (1H, dd, J = 13.2, 3.8 Hz, H-10), 3.28 (1H, dd,

J = 8.7, 2.7 Hz, H-2), 3.42 (1H, br s, OH), 3.67 (1H,

m, H-1), 3.75–3.87 (4 H, m, OCH₂CH₂O), 4.17 (1H,

m, H-3), 5.39 (2H, m, H-22, 23), 5.84 (1H, d,

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

4287

J = 11.2 Hz, H-7), 6.27 (1H, d, J = 11.2 Hz, H-6). UV

k_max(EtOH): 243 (e 27,700), 251 (e 32,300), 261 (e

21,600) nm. MS m/z (%): 462 (M⁺, 41), 444 (44), 426

(37), 408 (17), 346 (39), 317 (55), 299 (29), 255 (59),

237 (75), 133 (100). HRMS m/z: 462.3362 (Calcd for

C₂₈H₄₆O₅: 462.3345).

J = 11.2 Hz, H-6). UV k_max(EtOH): 243 (e 29,600),

251 (e 34,500), 261 (e 23,200) nm. MS m/z (%): 466

(M⁺, 39), 448 (30), 430 (13), 362 (14), 345 (12), 317

(13), 237 (9), 133 (20), 113 (50), 69 (100). HRMS m/z:

466.3267 (Calcd for C₂₇H₄₆O₆: 466.3294).

Compound 4d: ¹H NMR (CDCl₃) d: 0.55 (3H, s, H-18),

1.13 (3H, d, J = 5.9 Hz, H-21), 1.22, 1.24 (each 3H, s, H-

26, 27), 2.34 (1H, d, J = 14.1 Hz, H-4), 2.48 (1H, dd,

J = 14.1, 2 Hz, H-4), 2.67 (1H, br s, OH), 2.79 (1H, m,

H-9), 3.07 (1H, dd, J = 13.4, 3.8 Hz, H-10), 3.27 (2H,

m, H-2, 20), 3.45 (1H, m, H-23), 3.56 (1H, s, OH),

3.64–3.87 (6H, m, OCH₂CH₂O, H-1, 23), 4.17 (1H, m,

H-3), 5.82 (1H, d, J = 11.2 Hz, H-7), 6.27 (1H, d,

J = 11.2 Hz, H-6). UV k_max(EtOH): 243 (e 32,500),

251 (e 37,900), 261 (e 25,100) nm. MS m/z (%): 466

(M⁺, 28), 448 (22), 430 (11), 362 (9), 345 (9), 317 (9),

237 (11), 133 (19), 113 (43), 69 (100). HRMS m/z:

466.3300 (Calcd for C₂₇H₄₆O₆: 466.3294).

4.15. 1a,25-Dihydroxy-2a-(2-hydroxyethoxy)- and 1a,25-

dihydroxy-2b-(2-hydroxyethoxy)-22-oxa-19-norvitamin

D₃(3c and 4c)

Following the same procedure as described above, treat-

ment of 31c (25.1 mg, 0.039 mmol, ca. 3:1 isomeric mix-

ture) with (ꢀ)-10-camphor sulfonic acid (73.3 mg,

0.32 mmol) gave a mixture of 3c and 4c (13.8 mg,

76%), which was separated by the reversed-phase HPLC

to give pure 3c (6.23 mg) and 4c (1.83 mg), respectively.

Compound 3c: ¹H NMR (CDCl₃) d: 0.54 (3H, s, H-18),

1.19 (3H, d, J = 6.0 Hz, H-21), 1.23, 1.24 (each 3H, s, H-

26, 27), 2.19 (2H, m), 2.62 (1H, dd, J = 13.3, 4.6 Hz, H-

4), 2.80 (1H, m, H-9), 2.86 (1H, dd, J = 14.5.0, 5.0 Hz,

H-10), 3.25 (1H, m, H-20), 3.35 (1H, dd, J = 7.9,

2.9 Hz, H-2), 3.49 (1H, m, H-23), 3.75–3.90 (5H, m,

OCH₂CH₂OH, OH), 3.95 (1H, m, H- 3), 4.15 (1H, m,

H-1), 5.82 (1H, d, J = 11.2 Hz, H-7), 6.32 (1H, d,

J = 11.2 Hz, H-6). MS m/z (%): 466 (M⁺, 13), 448 (18),

430 (10), 412 (2), 317 (10), 237 (9), 133 (18), 113 (36),

69 (100). HRMS m/z: 466.3282 (Calcd for C₂₇H₄₆O₆:

466.3294). UV k_max(EtOH): 243, 251, 261 nm.

4.17. 24a,26a,27a-Trihomo-1a,25-dihydroxy-2a-(2- hydroxy-

ethoxy)- and 24a,26a,27a-trihomo-1a,25-dihydroxy-2b-(2-

hydroxyethoxy)-22, 24-diene-19-norvitamin D₃(3b and 4b)

A mixture of 31b (42.0 mg, 0.044 mmol, ca. 3:2 isomeric

mixture), Et₃N (30 lL), and tetrabutylammonium ﬂuo-

ride (0.350 mL, 0.350 mmol, 1.0 M solution in THF)

in dry THF (1 mL) was stirred at ambient temperature

for 5 h. The mixture was poured into ice water and

extracted with AcOEt. The organic phase was washed

with brine, dried over Na₂SO₄, and evaporated in vacuo.

The residue was chromatographed on silica gel (5 g)

with 2% MeOH in AcOEt to yield the isomeric mixture

of 3b and 4b (20.0 mg, 91%). The mixture was separated

Compound 4c: ¹H NMR (CDCl₃) d: 0.53 (3H, s, H-18),

1.19 (3H, d, J = 6.0 Hz, H-21), 1.23, 1.24 (each 3H, s,

H-26, 27), 2.35 (1 H, d, J = 15.0 Hz, H-4), 2.48 (1H,

dm, J = 15.0 Hz, H-4), 2.80 (1H, m, H-9), 3.07 (1H, d,

J = 13.2, 3.4 Hz, H-10), 3.25 (1H, m, H-20), 3.32 (1H,

dd, J = 8.0, 2.8 Hz, H-2), 3.50 (1H, m, H-23), 3.65–

3.90 (6H, m, OCH₂CH₂OH, OH, H-3, 23), 4.17 (1H,

m, H-1), 5.85 (1H, d, J = 11.2 Hz, H-7), 6.27 (1H, d,

J = 11.2 Hz, H-6). MS m/z (%): 466 (M⁺, 10), 448 (10),

430 (8), 412 (3), 317 (10), 237 (13), 133 (21), 113 (31),

69 (100). HRMS m/z: 466.3311 (Calcd for C₂₇H₄₆O₆:

466.3294). UV k_max(EtOH): 243, 251, 260 nm.

by

HPLC

(YMC-Pack

ODS-AM

SH-342-5,

150 · 20 mm, 20% H₂O in MeOH) to give pure 3b

(9.4 mg) and 4b (8.3 mg), respectively.

1

Compound 3b: H NMR (CDCl₃) d: 0.57 (3H, s, H-

18), 0.86 (6H, t, J = 7.4 Hz, H-26a, 27a), 1.04 (3H, d,

J = 6.6 Hz, H-21), 1.54, 1.56 (each 2H, q, J = 7.4 Hz,

H-26, 27), 2.61 (1H, dd, J = 13.4, 4.4 Hz, H-4), 2.72

(1H, br s, OH), 2.80 (1H, m, H-9), 2.86 (1H, dd,

J = 14.4, 4.8 Hz, H-10), 3.12 (1H, br s, OH), 3.32

(1H, dd, J = 8.0, 2.5 Hz, H-2), 3.48 (1H, br s, OH),

3.67–3.81 (4H, m, OCH₂CH₂O), 3.93 (1H, m, H-3),

4.15 (1H, m, H-1), 5.53 (1H, d, J = 15.3 Hz, H-24a,

overlapped with H-22), 5.81 (1H, d, J = 11.1 Hz, H-

7), 5.97 (1H, dd, J = 15.0, 10.3 Hz, H-23), 6.14 (1H,

dd, J = 15.3, 10.3 Hz, H-24), 6.33 (1H, d,

J = 11.1 Hz, H-6). UV k_max(EtOH): 235 (e 44,000),

243 (e 44,200), 251 (e 38,000), 261 (e 23,800) nm. MS

m/z (%): 502 (M⁺, 11), 484 (62), 466 (33), 448 (7),

386 (17), 333 (40), 237 (29), 149 (100), 133 (43), 93

(49). HRMSs m/z: 502.3658 (Calcd for C₃₁H₅₀O₅:

502.3658).

4.16. 20-epi-1a,25-Dihydroxy-2a-(2-hydroxyethoxy)- and

1a,25-dihydroxy-2b-(2-hydroxyethoxy)-22-oxa-19-norvita-

min D₃(3d and 4d)

Following the same procedure as described above, treat-

ment of 31d (63.0 mg, 0.078 mmol, ca. 3:2 isomeric mix-

ture) with (ꢀ)-10-camphor sulfonic acid (108.5 mg,

0.467 mmol) gave a mixture of 3d and 4d (33.0 mg,

91%), which was separated by the reversed-phase HPLC

to give pure 3d (13.9 mg) and 4d (10.3 mg), respectively.

Compound 3d: ¹H NMR (CDCl₃) d: 0.55 (3H, s, H-18),

1.12 (3H, d, J = 5.9 Hz, H-21), 1.22, 1.23 (each 3H, s, H-

26, 27), 2.60 (1H, dd, J = 13.4, 4.5 Hz, H-4), 2.79 (1H,

m, H-9), 2.86 (1H, dd, J = 14.5, 4.8 Hz, H-10), 3.26

(1H, m, H-20), 3.31 (1H, dd, J = 8.1, 2.7 Hz, H-2),

3.45 (1H, m, H-23), 3.57 (1H, s, OH), 3.66–3.86 (5H,

m, OCH₂CH₂O, H-23), 3.92 (1H, m, H-3), 4.14 (1H,

m, H-1), 5.79 (1H, d, J = 11.2 Hz, H-7), 6.33 (1H, d,

Compound 4b: ¹H NMR (CDCl₃) d: 0.56 (3H, s, H-18),

0.86 (6H, t, J = 7.5 Hz, H-26a, 27a), 1.05 (3H, d,

J = 6.6 Hz, H-21), 1.55, 1.56 (each 2H, q, J = 7.5 Hz,

H-26, 27), 2.34 (1H, d, J = 14 Hz, H-4), 2.47 (1H, d,

J = 14, 2 Hz, H-4), 2.64 (1H, br s, OH), 2.80 (1H, m,

H-9), 3.07 (1H, dd, J = 13.2, 4.0 Hz, H-10), 3.27 (1H,

4288

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

dd, J = 8.7, 2.6 Hz, H-2), 3.64–3.86 (5H, m, OCH_2-

CH₂O, H-1), 4.16 (1H, m, H-3), 5.53 (1H, d,

J = 15.3 Hz, H-24a, overlapped with H-22), 5.83 (1H,

d, J = 11.1 Hz, H-7), 5.97 (1H, dd, J = 15.0, 10.3 Hz,

H-23), 6.15 (1H, dd, J = 15.3, 10.3 Hz, H-24), 6.26 (1H,

d, J = 11.1 Hz, H-6). UV k_max(EtOH): 235 (e 44,000),

243 (e 44,500), 251 (e 38,900), 261 (e 24,000) nm. MS

m/z (%): 502 (M⁺, 13), 484 (78), 466 (39), 448 (7), 386

(13), 333 (48), 237 (27), 149 (100), 133 (46), 93 (49).

HRMS m/z: 502.3664 (Calcd for C₃₁H₅₀O₅: 502.3658).

AcOEt. The organic phase was successively washed with

5% NaHCO₃and brine, dried over MgSO₄, and evapo-

rated in vacuo. The residue was puriﬁed by chromatog-

raphy on silica gel (5 g) using 6% AcOEt in hexane to

give 33c (69.3 mg, 79%) as a mixture of two isomers in

a ca. 3:2 ratio.

MS m/z (%): 650 (M⁺, 2), 632 (10), 546 (7), 489 (7), 357

(55), 75 (100). HRMS m/z: 650.4754 (Calcd for

1

C₃₇H₇₀O₅Si₂: 650.4762). 2a-Isomer: H NMR (CDCl₃)

d: 0.06–0.09 (12H, s, 4· Si–Me), 0.53 (3H, s, H-18), 0.87,

0.88 (each 9H, s, 2· Si–t-Bu), 1.19 (3H, d, J = 5.9 Hz,

H-21), 1.24, 1.25 (each 3H, s, H-26, 27), 2.79 (1H, m, H-

9), 3.25 (1H, m, H-20), 3.45–3.60 (2H, m, H-2, 23), 3.84

(1H, m, H-23), 3.92 (1H, m, H-3), 3.99 (1H, m, H-1),

5.80 (1H, m, d, J = 11.1 Hz, H-7), 6.14 (1H, d,

J = 11.1 Hz, H-6). 2b-Isomer: ¹H NMR (CDCl₃) d:

0.06–0.09 (12H, s, 4· Si–Me), 0.52 (3H, s, H-18), 0.86,

0.89 (each 9H, s, 2· Si–t-Bu), 1.19 (3H, d, J = 5.9 Hz,

H-21), 1.24, 1.25 (each 3H, s, H-26, 27), 2.79 (1H, m, H-

9), 3.25 (1H, m, H-20), 3.45–3.60 (2H, m, H-2, 23), 3.84

(1 H, m, H-23), 3.99 (2H, m, H-1, 3), 5.80 (1H, m, d,

J = 11.1 Hz, H-7), 6.17 (1H, d, J = 11.1 Hz, H-6).

4.18. 1a-[(tert-Butyldimethylsilyl)oxy]-2-[(trimethylsilyl)oxy]-

22-oxa-25-[(triethylsilyl)oxy]-19-norvitamin D₃tert-butyl-

dimethylsilyl ether (32c)

To a stirred solution of 7 (222.0 mg, 0.34 mmol, a mix-

ture of ca. 2:1) in dry THF (1 mL) at ꢀ78 ꢁC was added

n-BuLi (215.2 lL, 0.34 mmol, 1.56 M solution in hex-

ane) and the resulting dark orange solution was stirred

for 15 min. To this cooled solution was added a solution

of 22⁰c (61.2 mg, 0.17 mmol) in dry THF (0.5 + 0.2 mL),

and the whole mixture was stirred at ꢀ78 to ꢀ20 ꢁC for

2.5 h. The reaction mixture was quenched with saturated

NH₄Cl and extracted with AcOEt. The organic extract

was washed with brine and 5% NaHCO₃, dried over

MgSO₄, and evaporated in vacuo. The residue was puri-

ﬁed by chromatography on silica gel (10 g) using 2%

AcOEt in hexane to aﬀord 32c (39.8 mg, 59% based on

22⁰c) as a mixture of two isomers in a ca. 2:1 ratio,

15% AcOEt in hexane to give the unreacted

starting material 22⁰c (36.1 mg, 33%), and 40% AcOEt

in hexane to aﬀord the unreacted starting material 7

(123.8 mg).

4.20. 1a-[(tert-Butyldimethylsilyl)oxy]-2-oxo-22-ene-25-

[(methoxymethyl)oxy]-19-norvitamin D₃tert-butyldimeth-

ylsilyl ether (34a)

To a stirred solution of oxalyl chloride (7.8 lL,

0.088 mmol) in dry CH₂Cl₂(1 mL) at ꢀ78 ꢁC was added

a solution of DMSO (12.4 lL, 0.175 mmol) in dry

CH₂Cl₂(0.2 mL). After being stirred for 5 min, a solu-

tion of 29a (50.4 mg, 0.073 mmol, ca. 5:4 isomeric mix-

ture) in dry CH₂Cl₂(1.2 mL) was added dropwise. The

reaction mixture was stirred for 15 min at ꢀ78 ꢁC, and

Et₃N (51 lL, 0.365 mmol) was added. The whole mix-

ture was stirred at ꢀ78 ꢁC for 40 min and at 0 ꢁC for

20 min, quenched with ice water, and extracted with

CH₂Cl₂. The CH₂Cl₂extract was washed with brine,

dried over MgSO₄, and concentrated in vacuo. The res-

idue was puriﬁed by chromatography on silica gel (5 g)

using 5% AcOEt in hexane to aﬀord 34a (49.0 mg,

97%) as a single compound.

MS m/z (%): 836 (no M⁺), 704 (7), 647 (2), 618 (5), 572

(12), 486 (13), 469 (7), 383 (9), 309 (10), 75 (100). HRMS

m/z: 704.5066 (M⁺ꢀTESOH) (Calcd for C₄₀H₇₆O₄Si₄:

704.5051). 2a-Isomer (major) ¹H NMR (CDCl₃) d:

0.056, 0.063 (each 6H, s, 2· Si–Me), 0.124 (9H, Si–

Me₃), 0.52 (3H, s, H-18), 0.57 (6H, q, J = 8.0 Hz, 3·

Si–CH₂CH₃), 0.87, 0.88 (each 9H, s, 2· Si–t-Bu), 0.94

(9H, t, J = 8.0 Hz, 3· Si–CH₂CH₃), 1.16 (3H, d,

J = 6.0 Hz, H-21), 1.22 (6H, s, H-26, 27), 2.80 (1H, m,

H-9), 3.20 (1H, m, H-20), 3.35 (1H, m, H-23), 3.54

(1H, m, H-2), 3.67 (1H, m, H-23), 3.80 (1H, m, H-3),

3.88 (1H, m, H-1), 5.83 (1H, m, d, J = 11.0 Hz, H-7),

¹H NMR (CDCl₃) d: 0.055, 0.065, 0.069, 0.094 (each 3H,

s, 4· Si–Me), 0.56 (3H, s, H-18), 0.88, 0.89 (each 9H, s, 2·

Si–t-Bu), 1.03 (3H, d, J = 6.6 Hz, H-21), 1.20 (6H, s, H-

26, 27), 2.44 (1H, dd, J = 13.3, 8.9 Hz), 2.52 (1H, dd,

J = 14.2, 3.8 Hz), 2.69 (2H, m), 2.81 (1H, m, H-9), 3.37

(3H, s, OMe), 4.35 (1H, dd, J = 6.4, 4.2 Hz), 4.55 (1H,

dd, J = 8.7, 5.5 Hz), 4.73 (2H, s, OCH₂O), 5.34 (2H, m,

H-22, 23), 5.80 (1H, d, J = 11.2 Hz, H-7), 6.34 (1H, d,

J = 11.2 Hz, H-6). MS m/z (%): 688 (no M⁺), 631 (5),

599 (16), 569 (100), 437 (22), 325 (17), 109 (81), 75 (52).

HRMS m/z: 569.3848 (M⁺ꢀBu–MeOCH₂OH) (Calcd

for C₃₄H₅₇O₃Si₂: 569.3846).

1

6.09 (1H, d, J = 11.0 Hz, H-6). 2b-Isomer (minor) H

NMR (CDCl₃) d: 0.036, 0.044 (each 6H, s, 2· Si–Me),

0.120 (9H, Si–Me₃), 0.53 (3H, s, H-18), 0.57 (6H, q,

J = 8.0 Hz, 3· Si–CH₂CH₃), 0.86, 0.89 (each 9H, s, 2·

Si–t-Bu), 0.94 (9H, t, J = 8.0 Hz, 3· Si–CH₂CH₃), 1.16

(3H, d, J = 6.0 Hz, H-21), 1.22 (6H, s, H-26, 27), 2.80

(1H, m, H-9), 3.20 (1H, m, H-20), 3.35 (1H, m, H-23),

3.59 (1H, m, H-2), 3.67 (1H, m, H-23), 3.80 (1H, m,

H-3), 3.94 (1H, m, H-1), 5.80 (1H, m, d, J = 11.0 Hz,

H-7), 6.12 (1H, d, J = 11.0 Hz, H-6).

4.19. 1a-[(tert-Butyldimethylsilyl)oxy]-2,25-dihydroxy-22-

oxa-19-norvitamin D₃tert-butyldimethylsilyl ether (33c)

4.21. 1a-[(tert-Butyldimethylsilyl)oxy]-2-oxo-22-oxa-25-

hydroxy-19-norvitamin D₃tert-butyldimethylsilyl ether

(34c)

A solution of 32c (111.8 mg, 0.13 mmol, ca. 2:1 isomeric

mixture) in THF/AcOH/H₂O (4.25 mL, 8:8:1, v/v/v) was

stirred at ambient temperature for 22 h and diluted with

The Swern oxidation of 33c (69.3 mg, 0.1 mmol, ca. 2:1

isomeric mixture) was carried out according to the same

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

4289

procedure described for the preparation of 34a to give

34c (50.8 mg, 81%) as a single compound.

0.120 (each 3H, s, 4· Si–Me), 0.56 (3H, s, H-18), 0.84,

0.92 (each 9H, s, 2· Si–t-Bu), 1.02 (3H, d, J = 6.6 Hz,

H-21), 1.20 (6H, s, H-26, 27), 2.80 (1H, m, H-9), 3.12

(1H, m, H-10), 3.37 (3H, s, OMe), 4.46 (1H, m, H-1),

4.73 (2H, s, OCH₂O), 4.99 (1H, m, H-3), 5.33 (2H, m,

H-22, 23), 5.47 (1H, d, J = 1.8 Hz, C@CH), 5.82 (1H,

d, J = 11.1 Hz, H-7), 6.18 (1H, d, J = 11.1 Hz, H-6).

Z-Isomer: ¹H NMR (CDCl₃) d: 0.065, 0.075, 0.111,

0.133 (each 3H, s, 4· Si–Me), 0.55 (3H, s, H-18), 0.84,

0.92 (each 9H, s, 2· Si–t-Bu), 1.02 (3H, d, J = 6.6 Hz,

H-21), 1.20 (6H, s, H-26, 27), 2.80 (1H, m, H-9), 2.99

(1H, m, H-10), 3.37 (3H, s, OMe), 4.57 (1H, m, H-3),

4.73 (2H, s, OCH₂O), 5.04 (1H, m, H-1), 5.33 (2H, m,

H-22, 23), 5.47 (1H, d, J = 1.8 Hz, C@CH), 5.78 (1H,

d, J = 11.1 Hz, H-7), 6.31 (1H, d, J = 11.1 Hz, H-6).

¹H NMR (CDCl₃) d: 0.05, 0.09 (each 3H, s, Si–Me), 0.07

(6H, s, 2· Si–Me), 0.53 (3H, s, H-18), 0.87, 0.89 (each

9H, s, 2· Si–t-Bu), 1.19 (3H, d, J = 6.0 Hz, H-21),

1.23, 1.24 (each 3H, s, H-26, 27), 2.44 (1 H, dd,

J = 13.0, 8.7 Hz), 2.54 (1H, dd, J = 14.1, 3.9 Hz), 2.65

(1H, dd, J = 13.0, 5.4 Hz), 2.70 (1H, dd, J = 14.1,

6.6 Hz), 2.82 (1H, m, H-9), 3.49 (1H, m, H-20), 3.25,

3.84 (each 1H, m, H-23), 4.36 (1H, m, H-3), 4.53 (1H,

m, H-1), 5.82 (1H, m, d, J = 11.2 Hz, H-7), 6.33 (1H,

d, J = 11.2 Hz, H-6). MS m/z (%): 648 (M⁺, 2), 591

(5), 573 (100), 441 (32). HRMS m/z: 648.4621 (Calcd

for C₃₇H₆₈O₅Si₂: 648.4605).

4.22. 20-epi-1a-[(tert-Butyldimethylsilyl)oxy]-2-oxo-22-oxa-

25-[(triethylsilyl)oxy]-19-norvitamin D₃tert-butyldimethyl-

silyl ether (34d)

4.24. 1a-[(tert-Butyldimethylsilyl)oxy]-2-cyanomethylene-

22-oxa-25-hydroxy-19- norvitamin D₃tert-butyldimeth-

ylsilyl ether (35c)

The Swern oxidation of 30d (60.7 mg, 0.079 mmol, ca.

3:2 isomeric mixture) was carried out according to the

same procedure described for the preparation of 34a

to aﬀord 34d (59.5 mg, 98%) as a single compound.

Following the same procedure as described above, treat-

ment of 34c (50.8 mg, 0.07 mmol)with diethyl (cyanom-

ethyl)phosphonate (24.0 lL, 0.14 mmol) aﬀorded 35c

(45.9 mg, E:Z = ca. 1: 1, 96%).

¹H NMR (CDCl₃) d: 0.057, 0.066, 0.070, 0.097 (each

3H, s, 4· Si–Me), 0.56 (3H, s, H-18), 0.57 (6H, q,

J = 7.9 Hz, 3· SiCH₂), 0.87, 0.89 (each 9H, s, 2· Si–t-

Bu), 0.94 (9H, t, J = 7.9 Hz, 3· SiCH₂CH₃), 1.09 (3H,

d, J = 5.9 Hz, H-21), 1.21, 1.23 (each 3H, s, H-26, 27),

2.45 (1H, dd, J = 13.2, 8.7 Hz), 2.52 (1H, dd, J = 14.0,

4.0 Hz), 2.67 (1H, dd, J = 13.2, 5.3 Hz), 2.72 (1 H, dd,

J = 14.0, 6.6 Hz), 2.83 (1H, m, H-9), 3.26 (1H, m, H-

20), 3.32, 3.70 (each 1H, m, H-23), 4.36 (1H, dd,

J = 6.3, 4.2 Hz), 4.55 (1H, dd, J = 8.7, 5.5 Hz), 5.79

(1H, d, J = 11.0 Hz, H-7), 6.37 (1H, d, J = 11.0 Hz, H-

6). MS m/z (%): 762 (no M⁺), 705 (8), 573 (12), 487

(22), 355 (12), 103 (51), 75 (100). HRMS m/z: 705.4740

(M⁺ꢀBu) (Calcd for C₃₉H₇₃O₅Si₃: 705.4766).

MS m/z (%): 671 (M⁺, 8), 653 (12), 614 (10), 596 (80),

569 (100), 521 (21), 464 (15), 410 (5). HRMS m/z:

671.4775 (Calcd for C₃₉H₆₉NO₄Si₂: 671.4765). E-Iso-

1

mer: H NMR (CDCl₃) d: 0.05, 0.06, 0.09, 0.11 (each

3H, s, 4· Si–Me), 0.54 (3H, s, H-18), 0.83, 0.92 (each

9H, s, 2· Si–t-Bu), 1.19 (3H, d, J = 6.0 Hz, H-21),

1.24, 1.25 (6H, s, H-26, 27), 2.30, 2.36 (each 1H, d,

J = 14.0 Hz), 2,80 (1H, m, H-9), 3.10 (1H, m, H-10),

3.25 (1H, m, H-20), 3.50, 3.85 (each 1H, m, H-23),

4.44 (1H, m, H-1), 4.98 (1H, t, J = 2.8 Hz, H-3), 5.47

(1H, m, C@CH), 5.83 (1H, m, d, J = 11.2 Hz, H-7),

6.17 (1H, d, J = 11.2 Hz, H-6). Z-Isomer: ¹H NMR

(CDCl₃) d: 0.06, 0.07, 0.10, 0.13 (each 3H, s, 4· Si–

Me), 0.52 (3H, s, H-18), 0.83, 0.92 (each 9H, s, 2· Si–

t-Bu), 1.19 (3H, d, J = 6.0 Hz, H-21), 1.25, 1.27 (6H, s,

H-26, 27), 2.60 (1H, m), 2.82 (1H, m, H-9), 2.98 (1H,

m, H-10), 3.25 (1H, m, H-20), 3.50, 3.85 (each 1H, m,

H-23), 4.55 (1H, m, H-1), 5.03 (1H, t, J = 2.8 Hz, H-

3), 5.47 (1H, m, C@CH), 5.79 (1H, d, J = 11.2 Hz, H-

7), 6.30 (1H, d, J = 11.2 Hz, H-6).

4.23. 1a-[(tert-Butyldimethylsilyl)oxy]-2-cyanomethylene-

22-ene-25-[(methoxymethyl)oxy]-19-norvitamin D₃tert-

butyldimethylsilyl ether (35a)

To a stirred solution of diethyl (cyanomethyl)phospho-

nate (32 lL, 0.197 mmol) in dry THF (1 mL) at

ꢀ40 ꢁC was added n-BuLi (126 lL, 0.197 mmol,

1.56 M solution in hexane). The mixture was stirred

for 15 min, and a solution of 34a (68.0 mg, 0.099 mmol)

in dry THF (1.2 mL) was added dropwise. Stirring was

continued for 2 h at ꢀ40 ꢁC after which time the reac-

tion mixture was quenched with saturated NH₄Cl and

extracted with AcOEt. The AcOEt layer was washed

with brine and dried over MgSO₄. Evaporation of the

solvent gave the residue, which was puriﬁed by chroma-

tography on silica gel (5 g) using 3% AcOEt in hexane to

aﬀord 35a (59.6 mg, 85%) as a mixture of E- and Z-iso-

mers in a ca. 1:1 ratio.

4.25. 20-epi-1a-[(tert-Butyldimethylsilyl)oxy]-2-cyano-

methylene-22-oxa-25-[(triethylsilyl)oxy]-19-norvitamin

D₃tert-butyldimethylsilyl ether (35d)

Following the same procedure as described above, treat-

ment of 34d (120.3 mg, 0.157 mmol) with diethyl

(cyanomethyl)phosphonate (51 lL, 0.315 mmol) aﬀor-

ded 35d (120.6 mg, E:Z = ca. 1:1, 97%).

MS m/z (%): 785 (M⁺, 2), 728 (8), 701 (12), 653 (6), 596

(9), 569 (16), 510 (17), 483 (11), 103 (66), 75 (100).

HRMS m/z: 785.5637 (Calcd for C₄₅H₈₃O₄NSi₃:

1

785.5630). E-Isomer: H NMR (CDCl₃) d: 0.05, 0.07,

MS m/z (%): 711 (M⁺, 5), 649 (18), 592 (61), 565 (76),

517 (20), 408 (26), 109 (92), 75 (99), 73 (100). HRMS

m/z: 711.5054 (Calcd for C₄₂H₇₃O₄Si₂: 711.5078).

E-Isomer: ¹H NMR (CDCl₃) d: 0.054, 0.065, 0.094,

0.10, 0.12 (each 3H, s, 4· Si–Me), 0.56 (3H, s, H-18),

0.56 (6H, q, J = 7.9 Hz, 3· SiCH₂), 0.84, 0.92 (each

9H, s, 2· Si–t-Bu), 0.93 (9H, t, J = 7.9 Hz, 3·

SiCH₂CH₃), 1.09 (3H, d, J = 5.9 Hz, H-21), 1.21, 1.23

4290

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

(each 3H, s, H-26, 27), 2.31, 2.37 (each 1H, br d,

J = 14 Hz, H-4), 2.80 (1H, m, H-9), 3.12 (1H, m, H-

10), 3.26 (1H, m, H-20), 3.32, 3.69 (each 1H, m, H-

23), 4.46 (1H, m, H-1), 4.99 (1H, m, H-3), 5.47 (1H, d,

J = 1.8 Hz, C@CH), 5.80 (1H, d, J = 11.1 Hz, H-7),

OCH₂O), 5.35 (2H, m, H-22, 23), 5.53 (1H, m, H-1),

5.79 (1H, d, J = 11.3 Hz, H-7), 6.17 (1H, m, C@CH),

6.31 (1H, d, J = 11.3 Hz, H-6), 10.16 (1H, d,

J = 7.9 Hz, CHO).

1

6.20 (1 H, d, J = 11.1 Hz, H-6). Z-Isomer: H NMR

Compound 37a: MS m/z (%): 716 (M⁺, 1), 584 (39), 522

(14), 491 (9), 147 (8), 109 (19), 75 (100). HRMS m/z:

584.4277 (M⁺ꢀTBSOH) (Calcd for C₃₆H₆₀O₄Si:

(CDCl₃) d: 0.06, 0.08, 0.11, 0.13 (each 3H, s, 4· Si–

Me), 0.56 (3H, s, H-18), 0.56 (6H, q, J = 7.9 Hz, 3·

SiCH₂), 0.84, 0.92 (each 9H, s, 2· Si–t-Bu), 0.94 (9H,

t, J = 7.9 Hz, 3· SiCH₂CH₃), 1.09 (3H, d, J = 5.9 Hz,

H-21), 1.21, 1.23 (each 3H, s, H-26, 27), 2.61 (1H, m,

H-4), 2.82 (1H, m, H-9), 2.99 (1H, m, H-10), 3.26 (1H,

m, H-20), 3.32, 3.70 (each 1H, m, H-23), 4.58 (1H,

ddd, J = 11.0, 5.9, 1.9 Hz, H-3), 5.04 (1H, m, H-1),

5.47 (1H, d, J = 1.9 Hz, C@CH), 5.77 (1H, d,

J = 11.2 Hz, H-7), 6.33 (1 H, d, J = 11.2 Hz, H-6).

1

584.4261). E-Isomer: H NMR (CDCl₃) d: 0.02, 0.06

(each 3H, s, 2· Si–Me), 0.08 (6H, s, 2· Si–Me), 0.56

(3H, s, H-18), 0.85, 0.92 (each 9H, s, 2· Si–t-Bu),

1.02 (3H, d, J = 6.6 Hz, H-21), 1.20 (6H, s, H-26, 27),

2.30 (2H, m, H-4), 2.80 (1H, m, H-9), 2.88 (1H, dd,

J = 12.7, 4.6 Hz, H-10), 3.37 (3H, s, OMe), 4.20, 4.30

(each 1H, m, CH₂OH), 4.37 (1H, dd, J = 9.5, 4.0 Hz,

H-1), 4.73 (2H, s, OCH₂O), 4.81 (1H, m, H-3), 5.33

(2H, m, H-22, 23), 5.72 (1H, m, C@CH), 5.85 (1H, d,

J = 11.1 Hz, H-7), 6.14 (1H, d, J = 11.1 Hz, H-6). Z-

Isomer: ¹H NMR (CDCl₃) d: 0.01, 0.08, 0.08, 0.09

(each 3H, s, 4· Si–Me), 0.55 (3H, s, H-18), 0.84, 0.93

(each 9 H, s, 2· Si–t-Bu), 1.02 (3H, d, J = 6.6 Hz, H-

21), 1.20 (6H, s, H-26, 27), 2.55 (1H, dd, J = 12.5,

4.9 Hz, H-4), 2.83 (2H, m, H-9, 10), 3.37 (3H, s,

OMe), 4.22 (1H, dd, J = 12.4, 7.0 Hz, CH₂OH), 4.30

(1H, dd, J = 12.4, 7.0 Hz, CH₂OH), 4.48 (1H, m, H-

3), 4.73 (2 H, s, OCH₂O), 4.86 (1H, m, H-1), 5.33

(2H, m, H-22, 23), 5.72 (1H, td, J = 7.0, 1.3 Hz,

C@CH), 5.80 (1H, d, J = 11.1 Hz, H-7), 6.25 (1H, d,

J = 11.1 Hz, H-6).

4.26. 1a-[(tert-Butyldimethylsilyl)oxy]-2-[2-(formyl)-ethyl-

idene]-22-ene-25-[(methoxymethyl)oxy]-19-norvitamin D₃

tert-butyldimethylsilyl ether (36a) and 1a-[(tert-butyl-

dimethylsilyl)oxy]-2-[2-(hydroxy)-ethylidene]-22-ene-25-

[(methoxymethyl)oxy]-19-norvitamin D₃tert-butyl-

dimethylsilyl ether (37a)

To a stirred solution of 35a (59.6 mg, 0.084 mmol, ca.

1:1 isomeric mixture) in dry toluene (1 mL) at ꢀ78 ꢁC

was added dropwise diisobutylaluminum hydride

(126 lL, 0.126 mmol, 1.0 M solution in hexane), and

the mixture was stirred for 1.5 h at the same tempera-

ture. The reaction mixture was diluted with hexane

and directly loaded onto silica gel column (5 g). The col-

umn was eluted with 5% AcOEt in hexane to aﬀord 36a

(56.0 mg, 94%) as a mixture of E- and Z-isomer in a ca.

1:1 ratio.

4.27. 1a-[(tert-Butyldimethylsilyl)oxy]-2-[2-(formyl)-ethyl-

idene]-22-oxa-25- hydroxy-19-norvitamin D₃tert-butyldi-

methylsilyl ether (36c) and 1a-[(tert-butyldimethylsilyl)oxy]-

2-[2-(hydroxy)-ethylidene]-22-oxa-25- hydroxy-19-

norvitamin D₃tert-butyldimethylsilyl ether (37c)

NaBH₄(3.6 mg, 0.094 mmol) was added to a solution of

36a (56.0 mg, 0.078 mmol, ca. 1:1 isomeric mixture) in

MeOH/THF (v/v, 2:1, 1.5 mL) at 0 ꢁC. After being stir-

red for 1 h at 0 ꢁC, the mixture was poured into ice

water, and extracted with AcOEt. The organic layer

was washed with brine, dried over MgSO₄, and evapo-

rated in vacuo. The residue was puriﬁed by chromatog-

raphy on silica gel (6 g) using 8% AcOEt in hexane to

give 37a (E-isomer, 26.3 mg, 47%, and Z-isomer,

22.7 mg, 40%). The total yield was 87%.

Following the same procedure as described above, treat-

ment of 35c (34.8 mg, 0.05 mmol, ca. 1:1 isomeric mix-

ture) with diisobutylaluminum hydride (100 lL,

0.10 mmol) gave 36c, then the treatment of 36c and

NaBH₄(1.9 mg, 0.05 mmol) yielded 37c (6.25 mg,

25%) as a mixture of two isomers in a ca. 1: 1 ratio.

1

Compound 36c: E-Isomer: H NMR (CDCl₃) d: 0.01,

0.07 (each 3H, s, 2· Si–Me), 0.09 (6H, s, 2· Si–Me),

0.54 (3H, s, H-18), 0.84, 0.92 (each 9H, s, 2· Si–t-Bu),

1.20 (3H, d, J = 6.0 Hz, H-21), 1.24, 1.25 (6H, s, H-26,

27), 2.40 (2H, m, H-4), 2.80 (1H, m, H-9), 3.06 (1H,

dd, J = 13.2, 4.9 Hz, H-10), 3.26 (1H, m, H-20), 3.50,

3.85 (each 1H, m, H-23), 4.57 (1H, m, H-1 or 3), 5.46

(1H, m, H-1 or 3), 5.86 (1H, m, d, J = 11.0 Hz, H-7),

6.15 (1H, dd, J = 8.0, 1.3 Hz, C@CH), 6.17 (1H, d,

J = 11.0 Hz, H-6), 10.18 (1H, d, J = 7.9 Hz, CHO). Z-

Compound 36a: MS m/z (%): 714 (M⁺, 9), 652 (13), 595

(20), 582 (13), 520 (34), 491 (23), 463 (14), 411 (17), 109

(33), 75 (100). HRMS m/z: 714.5074 (Calcd for

1

C₄₂H₇₄O₅Si₂: 714.5075). E-Isomer: H NMR (CDCl₃)

d: 0.01–0.10 (12H, 4· Si–Me), 0.57 (3H, s, H-18), 0.84,

0.92 (each 9H, s, 2· Si–t-Bu), 1.03 (3H, d, J = 6.6 Hz,

H-21), 1.20 (6H, s, H-26, 27), 2.42 (2H, m, H-4), 2.82

(1H, m, H-9), 3.05 (1H, m, H-10), 3.37 (3H, s, OMe),

4.56 (1H, m, H-1), 4.73 (2H, s, OCH₂O), 5.35 (2H, m,

H-22, 23), 5.46 (1H, m, H-3), 5.84 (1H, d, J = 11.4 Hz,

H-7), 6.16 (1H, m, C@CH), 6.19 (1H, d, J = 11.4 Hz,

H-6), 10.18 (1H, d, J = 7.9 Hz, CHO). Z-Isomer: ¹H

NMR (CDCl₃) d: 0.01–0.10 (12H, 4· Si–Me), 0.56

(3H, s, H-18), 0.84, 0.93 (each 9H, s, 2· Si–t-Bu), 1.03

(3H, d, J = 6.6 Hz, H-21), 1.20 (6H, s, H-26, 27), 2.65

(1H, m, H-4), 2.82 (1H, m, H-9), 3.00 (1H, m, H-10),

3.37 (3H, s, OMe), 4.70 (1H, m, H-3), 4.73 (2H, s,

1

Isomer: H NMR (CDCl₃) d: 0.02, 0.07, 0.099, 0.102

(each 3H, s, 4· Si–Me), 0.53 (3H, s, H-18), 0.84, 0.93

(each 9H, s, 2· Si–t-Bu), 1.20 (3H, d, J = 6.0 Hz, H-

21), 1.24, 1.25 (6H, s, H-26, 27), 2.24 (1H, m, H-4),

2.83 (1H, m, H-9), 2.99 (1H, m, H-10), 3.26 (1H, m,

H-20), 3.50, 3.85 (each 1H, m, H-23), 4.68 (1H, m, H-

1 or 3), 5.53 (1H, m, H-1 or 3), 5.80 (1H, m, d,

J = 11.0 Hz, H-7), 6.16 (1H, dd, J = 8.0, 1.5 Hz,

C@CH), 6.30 (1H, d, J = 11.0 Hz, H-6), 10.17 (1H, d,

J = 7.9 Hz, CHO).

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

4291

Compound 37c: MS m/z (%): 676 (M⁺, 2), 619 (5), 544

(37), 513 (11), 412 (7), 394 (9), 75 (100). HRMS m/z:

676.4904 (Calcd for C₃₉H₇₂O₅Si₂: 676.4918). E-Isomer:

¹H NMR (CDCl₃) d: 0.01–0.12 (12H, s, 4· Si–Me),

0.52 (3H, s, H-18), 0.82, 0.93 (each 9H, s, 2· Si–t-Bu),

1.19 (3H, d, J = 6.0 Hz, H-21), 1.23, 1.24 (6H, s, H-26,

27), 2.79 (1H, m, H-9), 3.04 (1H, m, H-10), 3.27 (1H,

m, H-20), 3.49, 3.83 (each 1H, m, H-23), 4.15–4.32

(2H, m, CH₂OH), 4.35 (1H, m, H-1), 4.81 (1H, m, H-

3), 5.59 (1H, m, C@CH), 5.88 (1H, m, d, J = 11.1 Hz,

H-7), 6.13 (1H, d, J = 11.1 Hz, H-6). Z-Isomer: ¹H

NMR (CDCl₃) d: 0.01–0.12 (12H, s, 4· Si–Me), 0.54

(3H, s, H-18), 0.82, 0.93 (each 9H, s, 2· Si–t-Bu), 1.19

(3H, d, J = 6.0 Hz, H-21), 1.23, 1.24 (6H, s, H-26, 27),

2.54 (1H, dd, J = 12.0, 4.5 Hz), 2.83 (2H, m, H-9, 10),

3.27 (1H, m, H-20), 3.49, 3.83 (each 1H, m, H-23),

4.15–4.32 (2H, m, CH₂OH), 4.47 (1H, m, H-1), 4.86

(1H, m, H-3), 5.59 (1H, m, C@CH), 5.82 (1H, m, d,

J = 11.1 Hz, H-7), 6.23 (1H, d, J = 11.1 Hz, H-6).

(33), 75 (100). HRMS m/z: 658.4830 (M⁺ꢀTBSOH)

(Calcd for C₃₉H₇₀O₄Si₂: 658.4813). E-Isomer: ¹H

NMR (CDCl₃) d: 0.01, 0.07 (each 3H, s, 2· Si–Me),

0.08 (6H, s, 2· Si–Me), 0.56 (3H, s, H-18), 0.56 (6H,

q, J = 7.9 Hz, 3· SiCH₂), 0.85, 0.92 (each 9H, s, 2·

Si–t-Bu), 0.94 (9H, t, J = 7.9 Hz, 3· SiCH₂CH₃), 1.09

(3H, d, J = 5.9 Hz, H-21), 1.21, 1.23 (each 3H, s, H-

26, 27), 2.31 (2H, m, H-4), 2.80 (1H, m, H-9), 2.88

(1H, dd, J = 12.6, 4.6 Hz, H-10), 3.26 (1H, m, H-20),

3.32, 3.70 (each 1H, m, H-23), 4.18, 4.31 (each 1H, m,

CH₂OH), 4.37 (1H, m, H-1), 4.82 (1H, m, H-3), 5.72

(1H, m, C@CH), 5.83 (1H, d, J = 11.0 Hz, H-7), 6.15

(1H, d, J = 11.0 Hz, H-6). Z-Isomer: ¹H NMR (CDCl₃)

d: 0.01, 0.07, 0.08, 0.10 (each 3H, s, 4· Si–Me), 0.56 (3H,

s, H-18), 0.57 (6H, q, J = 7.9 Hz, 3· SiCH₂), 0.84, 0.93

(each 9H, s, 2· Si–t-Bu), 0.94 (9H, t, J = 7.9 Hz, 3·

SiCH₂CH₃), 1.09 (3H, d, J = 5.9 Hz, H-21), 1.21, 1.23

(each 3H, s, H-26, 27), 2.55 (1H, dd, J = 12.5, 5.0 Hz,

H-4), 2.83 (2H, m, H-9, 10), 3.26 (1H, m, H-20), 3.32,

3.70 (each 1H, m, H-23), 4.22, 4.27 (each 1 H, m,

CH₂OH), 4.48 (1H, m, H-3), 4.86 (1H, m, H-1), 5.72

(1H, dt, J = 7.0, 1.4 Hz, C@CH), 5.79 (1H, d,

J = 11.1 Hz, H-7), 6.26 (1H, d, J = 11.1 Hz, H-6).

4.28. 20-epi-1a-[(tert-Butyldimethylsilyl)oxy]-2-[2-(formyl)-

ethylidene]-22-oxa-25-[(triethylsilyl)oxy]-19-norvitamin D₃

tert-butyldimethylsilyl ether (36d) and 20-epi-1a-[(tert-butyl-

dimethylsilyl)oxy]-2-[2-(hydroxy)-ethylidene]-22-oxa-25-[(tri-

ethylsilyl)oxy]-19-norvitamin D₃tert-butyldimethylsilyl ether

(37d)

4.29. 1a,25-Dihydroxy-2-[2-(hydroxy)-ethylidene]-22-ene-

19-norvitamin D₃(5a, E-isomer; 6a, Z-isomer)

Following the same procedure as described above, treat-

ment of 35d (77.0 mg, 0.098 mmol, a mixture of ca. 1:1)

with diisobutylaluminum hydride (147 lL, 0.147 mmol)

aﬀorded 36d, then the treatment of 36d and NaBH₄

(3.9 mg, 0.102 mmol) yielded 37d (E-isomer, 30.2 mg;

Z-isomer, 24.2 mg; 81%).

A mixture of the E-isomer 37a (26.3 mg, 0.037 mmol)

and

(ꢀ)-10-camphor

sulfonic

acid

(51.2 mg,

0.220 mmol) in dry MeOH (1 mL) was stirred at ambi-

ent temperature for 2 h. Cold 5% NaHCO₃was added,

and the mixture was extracted with AcOEt. The organic

phase was washed with brine and dried over MgSO₄.

Solvents were evaporated in vacuo, and the residue

was puriﬁed by chromatography on silica gel (5 g) using

2% MeOH in AcOEt to aﬀord 5a (15.6 mg, 96%). The

desired product was further puriﬁed by HPLC (YMC-

Pack ODS-AM SH-342-5, 150 · 20 mm, 20% H₂O in

MeOH) to give pure 5a (12.5 mg).

Compound 36d: MS m/z (%): 788 (M⁺, 5), 731 (5), 656

(8), 627 (7), 599 (4), 524 (5), 495 (3), 409 (5), 103 (42),

75 (100). HRMS m/z: 788.5649 (Calcd for C₄₅H₈₄O₅Si₃:

1

788.5627). E-Isomer: H NMR (CDCl₃) d: 0.01, 0.07,

0.09, 0.10 (each 3H, s, 4· Si–Me), 0.57 (3H, s, H-18),

0.56 (6H, q, J = 7.9 Hz, 3· SiCH₂), 0.84, 0.92 (each

9H, s, 2· Si–t-Bu), 0.94 (9H, t, J = 7.9 Hz, 3·

SiCH₂CH₃), 1.09 (3H, d, J = 5.9 Hz, H-21), 1.21, 1.23

(each 3H, s, H-26, 27), 2.42 (2H, m, H-4), 2.80 (1H,

m, H-9), 3.05 (1H, dd, J = 12.8, 5.3 Hz, H-10), 3.26

(1H, m, H-20), 3.32, 3.69 (each 1H, m, H-23), 4.57

(1H, m, H-1), 5.46 (1H, m, H-3), 5.83 (1H, d,

J = 11.1 Hz, H-7), 6.15 (1H, dd, J = 7.9, 1.1 Hz,

C@CH), 6.19 (1H, d, J = 11.1 Hz, H-6), 10.18 (1H, d,

J = 7.9 Hz, CHO). Z-Isomer: ¹H NMR (CDCl₃) d:

0.02, 0.08, 0.10, 0.11 (each 3H, s, 4· Si–Me), 0.56

(3H, s, H-18), 0.57 (6H, q, J = 7.9 Hz, 3· SiCH₂),

0.84, 0.93 (each 9H, s, 2· Si–t-Bu), 0.94 (9H, t,

J = 7.9 Hz, 3· SiCH₂CH₃), 1.09 (3H, d, J = 5.9 Hz,

H-21), 1.21, 1.23 (each 3H, s, H-26, 27), 2.65 (1H,

dd, J = 12.0, 4.8 Hz, H-4), 2.80 (1H, m, H-9), 3.00

(1H, m, H-10), 3.26 (1H, m, H-20), 3.32, 3.71 (each

1H, m, H-23), 4.69 (1H, m, H-3), 5.54 (1H, m, H-1),

5.78 (1H, d, J = 11.2 Hz, H-7), 6.16 (1H, dd, J = 7.9,

1.1 Hz, C@CH), 6.32 (1H, d, J = 11.1 Hz, H-6), 10.16

(1H, d, J = 7.9 Hz, CHO).

Deprotection using CSA was carried out as described

for (2E)-37a and 6a was obtained in 96% yield.

1

Compound 5a: H NMR (CD₃OD) d: 0.59 (3H, s, H-

18), 1.05 (3H, d, J = 6.6 Hz, H-21), 1.16 (6H, s, H-26,

27), 2.35 (1H, d, J = 14.0 Hz, H-4), 2.43 (1H, dd,

J = 14.0, 2.9 Hz, H-4), 2.86 (1H, m, H-9), 3.11 (1H, d,

J = 12.8, 5.0 Hz, H-10), 4.24 (2H, m, CH₂OH), 4.30

(1H, m, H-1), 4.83 (1H, m, H-3), 5.31, 5.42 (each 1H,

m, H-22, 23), 5.79 (1H, td, J = 6.9, 1.8 Hz, C@CH),

5.91 (1H, d, J = 11.1 Hz, H-7), 6.23 (1H, d,

J = 11.1 Hz, H-6). UV k_max(EtOH): 246 (e 37,000),

254 (e 42,000), 263 (e 27,800) nm. MS m/z (%): 444

(M⁺, 7), 426 (5), 408 (22), 390 (9), 372 (14), 281 (4),

263 (11), 252 (100), 147 (9), 109 (12). HRMS m/z:

444.3246 (Calcd for C₂₈H₄₄O₄: 444.3240).

1

Compound 6a: H NMR (CD₃OD) d: 0.61 (3H, s, H-

18), 1.04 (3H, d, J = 6.6 Hz, H-21), 1.16 (6H, s, H-26,

27), 2.65 (1H, dd, J = 12.4, 5.0 Hz, H-4), 2.85 (1H, m,

H-9), 2.93 (1H, d, J = 14.4, 3.0 Hz, H-10), 4.25 (2H,

m, CH₂OH), 4.39 (1H, m, H-3), 4.87 (1H, m, H-1),

5.31, 5.42 (each 1H, m, H-22, 23), 5.77 (1H, td,

Compound 37d: MS m/z (%): 790 (M⁺, 1), 772 (1), 733

(1), 658 (45), 627 (11), 526 (7), 508 (7), 376 (5), 103

4292

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

J = 6.9, 1.7 Hz, C@CH), 5.89 (1H, d, J = 11.1 Hz, H-7),

6.32 (1H, d, J = 11.1 Hz, H-6). UV k_max(EtOH): 246 (e

32,500), 254 (e 37,200), 263 (e 24,500) nm. MS m/z (%):

444 (M⁺, 10), 426 (5), 408 (23), 390 (27), 372 (91), 281

(54), 263 (79), 252 (57), 147 (86), 109 (100). HRMS m/

z: 444.3227 (Calcd for C₂₈H₄₄O₄: 444.3240).

3.58, 3.90 (each 1H, br s, 3· OH), 3.84 (1H, dt,

J = 9.4, 4.3 Hz, H-23), 4.08 (1 H, dd, J = 12.4, 5.2 Hz,

CH₂OH), 4.33 (2H, m, H-1, CH₂OH), 4.79 (1H, m, H-

3), 5.74 (1 H, m, C@CH), 5.84 (1H, d, J = 11.1 Hz, H-

7), 6.26 (1H, d, J = 11.1 Hz, H-6). UV k_max(EtOH):

246 (e 34,600), 254 (e 39,700), 263 (e 26,500) nm. MS

m/z (%): 448 (M⁺, 9), 430 (8), 412 (14), 394 (26), 376

(12), 308 (13), 263 (12), 131 (20), 113 (39), 69 (100).

HRMS m/z: 448.3188 (Calcd for C₂₇H₄₄O₅: 448.3189).

4.30. 1a,25-Dihydroxy-[2-(hydroxy)-ethylidene]-22-oxa-

19-norvitamin D₃(5c and 6c)

1

Following the same procedure as described above, treat-

ment of 37c (6.25 mg, 0.009 mmol) with CSA (22.2 mg,

0.036 mmol) yielded 5c and 6c (4.80 mg, 74%) in ca.

1:1 ratio, which was separated by the reversed-phase

HPLC to give pure 5c (1.77 mg) and 6c (1.85 mg),

respectively.

Compound 6d (Z-isomer): H NMR (CDCl₃) d: 0.57

(3H, s, H-18), 1.13 (3H, d, J = 5.9 Hz, H-21), 1.23,

1.24 (each 3H, s, H-26, 27), 2.68 (1H, dd, J = 12.6, 4.5

Hz, H-4), 2.81 (1H, m, H-9), 2.88 (1H, d, J = 14.2,

3.5 Hz, H-10), 3.28 (1H, m, H-20), 3.45, 3.84 (each

1H, m, H-23), 3.61 (1H, s, OH), 4.14 (1H, dd,

J = 12.5, 5.6 Hz, CH₂OH), 4.34 (1H, dd, J = 12.5, 8.4

Hz, CH₂OH), 4.44 (1H, m, H-3), 4.84 (1H, m, H-1),

5.75 (1H, m, C@CH), 5.82 (1H, d, J = 11.1 Hz, H-7),

6.38 (1H, d, J = 11.1 Hz, H-6). UV k_max(EtOH): 246

(e 32,300), 254 (e 37,100), 263 (e 24,600) nm. MS m/z

(%): 448 (M⁺, 7), 430 (7), 412 (14), 394 (25), 376 (12),

308 (13), 263 (12), 131 (21), 113 (39), 69 (100). HRMS

m/z: 448.3214 (Calcd for C₂₇H₄₄O₅: 448.3189).

Compound 5c: ¹H NMR (CDCl₃) d: 0.54 (3H, s, H-18),

1.19 (3H, d, J = 6.0 Hz, H-21), 1.24, 1.25 (each 3H, s, H-

26, 27), 2.44 (2H, m, H-4), 2.81 (1H, m, H-9), 3.12 (1H,

m, J = 13.1, 5.0 Hz, H-10), 3.26 (1H, m, H-20), 3.78

(1H, br s, OH), 3.49, 3.85 (each 1H, m, H-23), 4.23

(1H, dd, J = 12.4, 6.4 Hz, CH₂OH), 4.38 (1H, dd,

J = 12.4, 7.4 Hz), 4.44 (1H, m, H-1), 4.87 (1H, m, H-

3), 5.87 (1H, m, C@CH), 5.89 (1H, d, J = 11.2 Hz, H-

7), 6.30 (1H, d, J = 11.2 Hz, H-6). MS m/z (%): 448

(M⁺, 3), 430 (5), 412 (10), 394 (28), 376 (18), 308 (19),

263 (18), 131 (26), 113 (33), 69 (100). HRMS m/z:

448.3186 (Calcd for C₂₇H₄₄O₅: 448.3189). UV k_max

(EtOH): 246, 253, 263 nm.

4.32. 24a,26a,27a-Trihomo-1a-[(tert-butyldimethylsilyl)oxy]-

2-[2-(tert-butyldimethylsilyl)oxy]-ethylidene]-22,24-diene-

25- [(triethylsilyl)oxy]-19-norvitamin D₃tert-butyldimeth-

ylsilyl ether (38b)

To a stirred solution of 8 (119.5 mg, 0.164 mmol, a mix-

ture of two isomers) in dry THF (2 mL) at ꢀ78 ꢁC was

added dropwise n-BuLi (105 lL, 0.164 mmol, 1.56 M

solution in hexane), and the resulting dark orange solu-

tion was stirred for 15 min. To this colored solution was

added dropwise a solution of 22b (47.4 mg, 0.109 mmol)

in dry THF (1 mL), and the mixture was stirred for 2 h

at ꢀ78 ꢁC at which point it was quenched with saturated

NH₄Cl, and extracted with AcOEt. The AcOEt layer

was washed with brine and dried over MgSO₄. Evapora-

tion of the solvent in vacuo yielded the residue, which

was chromatographed on silica gel (10 g) with 1%

AcOEt in hexane to aﬀord 38b (27.7 mg, 27% based

on 22b) as a mixture of two isomers in a ca. 6:1 ratio,

and 5% AcOEt in hexane to give the unreacted starting

material 22b (27 mg, 57%).

Compound 6c: ¹H NMR (CDCl₃) d: 0.54 (3H, s, H-18),

1.19 (3H, d, J = 6.0 Hz, H-21), 1.23, 1.24 (each 3H, s, H-

26, 27), 2.70 (1 H, dd, J = 12.8, 4.8 Hz, H-4), 2.82 (2H,

m, H-9, 10), 3.26 (1H, m, H-20), 3.49 (1H, m, H-23),

3.79 (1H, br s, OH), 3.85 (1H, m, H-23), 4.26 (1H, dd,

J = 12.6, 6.5 Hz, CH₂OH), 4.39 (1H, dd, J = 12.6,

7.2 Hz), 4.46 (1H, m, H-3), 4.88 (1H, m, H-1), 5.84

(1H, m, C@CH), 5.85 (1H, d, J = 11.2 Hz, H-7), 6.39

(1H, d, J = 11.2 Hz, H-6). MS m/z (%): 448 (M⁺, 3),

430 (6), 412 (17), 394 (38), 376 (19), 308 (27), 263 (21),

131 (29), 113 (36), 69 (100). HRMS m/z: 448.3162 (Calcd

for C₂₇H₄₄O₅: 448.3189). UV k_max(EtOH): 245, 254,

263 nm.

4.31. 20-epi-1a,25-Dihydroxy-[2-(hydroxy)-ethylidene]-

22-oxa-19-norvitamin D₃(5d and 6d)

¹H NMR (CDCl₃) d: 0.01–0.08 (18H, 6· Si–Me), 0.569

(6H, q, J = 7.9 Hz, 3· SiCH₂), 0.571 (3H, s, H-18), 0.83

(6H, t, J = 7.5 Hz, H-26a, 27a), 0.84, 0.90, 0.91 (each

9H, s, 3· Si–t-Bu), 0.94 (9H, t, J = 7.9 Hz, 3·

SiCH₂CH₃), 1.06 (3H, d, J = 6.6 Hz, H-21), 2.80 (1H,

m, H-9), 2.96, 3.04 (ca. 6:1) (1H, dd, J = 12.5, 4.7 Hz,

H-10), 4.24–4.47 (3H, m, H-1 or 3, CH₂OH), 4.77, 4.83

(ca. 6:1) (1H, m, H-1 or 3), 5.51 (1H, dd, J = 15.0,

8.2 Hz, H-22), 5.52 (1H, d, J = 15.3 Hz, H-24a), 5.60

(1H, m, C@CH), 5.86 (1H, d, J = 11.1 Hz, H-7), 5.94

(1H, dd, J = 15.0, 10.4 Hz, H-23), 6.05 (1H, dd,

J = 15.3, 10.4 Hz, H-24), 6.12 (1H, d, J = 11.1 Hz, H-

6). MS m/z (%): 940 (no M⁺), 826 (1), 883 (1), 808 (6),

751 (10), 676 (21), 544 (11), 75 (100). HRMS m/z:

808.6029 (M⁺ꢀTBSOH) (Calcd for C₄₉H₈₈O₃Si₃:

808.6041).

Following the same procedure as described above,

treatment of 37d (E-isomer, 43.6 mg, 0.055 mmol) with

CSA (76.8 mg, 0.331 mmol) yielded 5d (23.7 mg, 96%),

which was separated by the reversed-phase HPLC to

give pure 5d (20.1 mg), and treatment of 37d (Z-iso-

mer, 35.5 mg, 0.045 mmol) with CSA (62.5 mg,

0.269 mmol) gave 6d (19.3 mg, 96%), which was sepa-

rated by the reversed-phase HPLC to aﬀord pure 6d

(17.6 mg).

Compound 5d: ¹H NMR (CDCl₃) d: 0.54 (3H, s, H-18),

1.13 (3H, d, J = 5.9 Hz, H-21), 1.22, 1.23 (each 3H, s,

H-26, 27), 2.33, 2.43 (each 1H, br d, J = 13 Hz, H-4),

2.79 (1H, m, H-9), 3.12 (1H, d, J = 12.5, 4.4 Hz,

H-10), 3.26 (1H, m, H-20), 3.44 (1H, m, H-23), 3.51,

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

4293

4.33. 24a,26a,27a-Trihomo-1a,25-dihydroxy-2-[2-(hydro-

xy)-ethylidene]-22,24-diene-19-norvitamin D₃(5b and 6b)

and the mixture was vortexed 2–3 times. The mixture

was incubated for 1 h at room temperature. [³H]-

1a,25-(OH)₂D₃(speciﬁc activity, 6.62 TBq/mmol, ca.

5000 dpm) in 15 lL of EtOH was added, vortexed 2–3

times, and the whole mixture was then allowed to stand

at 4 ꢁC for 18 h. At the end of the second incubation,

200 lL of dextran-coated charcoal suspension (pur-

chased from Yamasa Shoyu) was added to bind any free

ligands (or to remove free ligands) and the sample was

vortexed. After 30 min at 4 ꢁC, bound and free [³H]-

1a,25-(OH)₂D₃were separated by centrifugation at

3000 rpm for 15 min at 4 ꢁC. Aliquots (500 lL) of the

supernatant were mixed with 9.5 mL of ACS-II scintilla-

tion ﬂuid (Amersham, Buckinghamshire, UK) and sub-

mitted for radioactivity counting. Each assay was

performed at least twice in duplicate.

A mixture of 38b (56 mg, 0.0595 mmol, ca. 6:1 isomeric

mixture), Et₃N (40 lL), and tetrabutylammonium ﬂuo-

ride (476 lL, 0.476 mmol, 1.0 M solution in THF) in

dry THF (1 mL) was stirred at ambient temperature

for 20 h. The mixture was poured into ice water and

extracted with AcOEt. The organic phase was washed

with brine and dried over Na₂SO₄. Evaporation of the

solvent in vacuo aﬀorded the residue, which was chro-

matographed on silica gel (5 g) with 2% MeOH in

AcOEt to yield a mixture of 5b and 6b (23.0 mg, 80%)

in a 10:1 ratio. The mixture was separated by HPLC

(LiChrosorb Si 60, Hibar RT 250-10, 250 · 10 mm, hex-

ane/CH₂Cl₂/MeOH 50:50:4) to give pure 5b (17.1 mg,

E-isomer) and 6b (1.9 mg, Z-isomer), respectively.

4.35. Transactivation assays

Compound 5b: ¹H NMR (CDCl₃) d: 0.56 (3H, s, H-18),

0.87 (6H, t, J = 7.5, H-26a, 27a), 1.05 (3H, d, J = 6.5 Hz,

H-21), 1.55, 1.56 (each 2H, q, J = 7.5 Hz, H-26, 27), 2.34

(1H, d, J = 13.3 Hz, H-4), 2.44 (1H, dd, J = 13.3, 2.0 Hz,

H-4), 2.81 (1H, m, H-9), 3.14 (1H, d, J = 12.5, 4.3 Hz, H-

10), 3.38, 3.74 (each 1H, br s, 2· OH), 4.09 (1H, dd,

J = 12.3, 5.2 Hz, CH₂OH), 4.34 (2 H, m, H-1, CH₂OH),

4.80 (1H, m, H-3), 5.53 (1H, d, J = 15.4 Hz, H-24a, over-

lapped with H-22), 5.75 (1H, m, C@CH), 5.88 (1H, d,

J = 11.1 Hz, H-7), 5.97 (1H, dd, J = 15.0, 10.4 Hz, H-

23), 6.15 (1H, dd, J = 15.4, 10.4 Hz, H-24), 6.27 (1 H,

d, J = 11.1 Hz, H-6). UV k_max(EtOH): 236 (e 47,300),

245 (e 48,000), 254 (e 43,200), 264 (e 27,200) nm. MS

m/z (%): 484 (M⁺, 7), 466 (15), 448 (17), 430 (44), 412

(34), 279 (33), 263 (25), 149 (100), 133 (35), 93 (38).

HRMS m/z: 484.3526 (Calcd for C₃₁H₄₈O₄: 484.3553).

COS-7 cells were grown in Dulbecco’s modiﬁed Eagle’s

medium (DMEM) supplemented with 5% fetal calf ser-

um (FCS). Cells were seeded on 24-well plates at a den-

sity of ꢁ2 · 10⁴per well. After 24 h, cells were

transfected with a reporter plasmid containing three

copies of the mouse osteopontin VDRE (5⁰-GGTTCAc-

gaGGTTCA, SPPx3-TK-LUC), a wild-type or mutant

hVDR expression plasmids [pCMX-hVDR or pSG5-

hVDR (D165-215)], and the internal control plasmid

containing sea pansy luciferase expression constructs

(pRL-CMV) by the lipofection method as described pre-

viously.³⁴After 4 h incubation, the medium was re-

placed with fresh DMEM containing 1% FCS

(HyClone, UT). The next day, the cells were treated with

either indicated concentration of 1,25-(OH)₂D₃, 19-nor-

vitamin D analogs, or ethanol vehicle and cultured for

24 h. Cells in each well were harvested with a cell lysis

buﬀer, and the luciferase activity was measured with a

luciferase assay kit (Tokyo Ink, Inc., Japan) according

to the manufacturer’s instructions. Transactivation mea-

sured by the luciferase activity was normalized with the

internal control. All experiments were done in triplicate.

Compound 6b: ¹H NMR (CDCl₃) d: 0.57 (3H, s, H-18),

0.87 (6H, t, J = 7.5 Hz, H-26a, 27a), 1.05 (3H, d,

J = 6.6 Hz, H-21), 1.55, 1.56 (each 2H, q, J = 7.5 Hz,

H-26, 27), 2.22 (1H, dd, J = 13.0, 9.7 Hz, H-4), 2.33

(1H, m, H-10), 2.70 (1H, dd, J = 13.0, 4.7 Hz, H-4),

2.82 (2H, m, H-9, 10), 4.25 (1H, dd, J = 12.6, 6.4 Hz,

CH₂OH), 4.38 (1 H, dd, J = 12.6, 7.3 Hz, CH₂OH),

4.46 (1H, m, H-3), 4.87 (1H, m, H-1), 5.54 (1H, d,

J = 15.3 Hz, H-24a, overlapped with H-22), 5.84 (2H,

m, H-7, C@CH), 5.98 (1H, dd, J = 15.0, 10.3 Hz, H-

23), 6.15 (1H, dd, J = 15.3, 10.3 Hz, H-24), 6.40 (1H,

d, J = 11.1 Hz, H-6). MS m/z (%): 484 (M⁺, 4), 466

(12), 448 (16), 430 (44), 412 (38), 279 (35), 263 (32),

149 (100), 133 (39), 93 (42). HRMS m/z: 484.3561 (Calcd

for C₃₁H₄₈O₄: 484.3553).

Supplementary data

Experimental details for preparation of compounds 10–

27 are available. Supplementary data associated with

this article can be found, in the online version, at

doi:10.1016/j.bmc.2006.01.061.

4.34. Vitamin D receptor-binding assay

References and notes

The rat VDR ligand-binding domain (LBD) (amino

acids 115–423) was expressed as an amino-terminal

His-tagged protein in Escherichia coli BL21 (DE3) pLys

S (Novagen). The supernatants were diluted—1000

times in 50 mM Tris buﬀer (100 mM KCl, 5 mM DTT,

and 0.5% CHAPS, pH 7.5) containing bovine serum

albumin (100 lg/mL) and were pipetted into glass cul-

ture tubes. A solution containing an increasing amount

of 1a,25-(OH)₂D₃or the synthetic analogs in 15 lL of

EtOH was added to the receptor solution in each tube

1. Feldman, D.; Glorrieux, F. H.; Pike, J. W. Eds.; Vitamin

D; Academic: New York, 1997.

2. Perlman, K. L.; Sicinski, R.; Schnoes, H. K.; DeLuca, H.

F. Tetrahedron Lett. 1990, 31, 1823.

3. Perlman, K. L.; Swenson, R. E.; Paaren, H. E.; Schnoes,

H. K.; DeLuca, H. F. Tetrahedron Lett. 1991, 32, 7663.

4. Sicinski, R. R.; Perlman, K. L.; DeLuca, H. F. J. Med.

Chem. 1994, 37, 3730.

5. Sicinski, R. R.; Prahl, J. M.; Smith, C. M.; DeLuca, H. F.

J. Med. Chem. 1998, 41, 4662.

4294

M. Shimizu et al. / Bioorg. Med. Chem. 14 (2006) 4277–4294

6. Sicinski, R. R.; Rotkiewicz, P.; Kolinski, A.; Sicinska, W.;

Prahl, J. M.; Smith, C. M.; DeLuca, H. F. J. Med. Chem.

2002, 45, 3366.

20. Plum, L. A.; Prahl, J. M.; Ma, X.; Sicinski, R. R.;

Gowlugari, S.; Clagett-Dame, M.; DeLuca, H. F. Proc.

Natl. Acad. Sci. U.S.A. 2004, 101, 6900.

7. Shimizu, M.; Iwasaki, Y.; Shibamoto, Y.; Sato, M.;

DeLuca, H. F.; Yamada, S. Bioorg. Med. Chem. Lett.

2003, 13, 809.

8. Shimizu, M.; Yamamoto, K.; Mihori, M.; Iwasaki, Y.;

Morizono, D.; Yamada, S. J. Steroid Biochem. Mol. Biol.

2004, 89–90, 75.

21. White, M. C.; Burke, M. D.; Peleg, S.; Brem, H.; Posner,

G. H. Bioorg. Med. Chem. 2001, 9, 1691–1699.

22. Yamamoto, K.; Sun, W. Y.; Ohta, M.; Hamada, K.;

DeLuca, H. F.; Yamada, S. J. Med. Chem. 1996, 39, 2727.

23. Yamada, S.; Yamamoto, K.; Masuna, H.; Ohta, M. J.

Med. Chem. 1998, 41, 1467.

9. Shimizu, M.; Iwasaki, Y.; Shimazaki, M.; Amano, Y.;

Yamamoto, K.; Reischl, W.; Yamada, S. Bioorg. Med.

Chem. Lett. 2005, 15, 1451.

10. Tsugawa, N.; Nakagawa, K.; Kurobe, M.; Ono, Y.;

Kubodera, N.; Ozono, K.; Okano, T. Biol. Pharm. Bull.

2000, 23, 66.

11. Kittaka, A.; Suhara, Y.; Takayanagi, H.; Fujishima, T.;

Kurihara, M.; Takayama, H. Org. Lett. 2000, 2, 2619.

12. Ono, K.; Yohida, A.; Saito, N.; Fujishima, T.; Honzawa,

S.; Suhara, Y.; Kishimoto, S.; Sugiura, T.; Waku, K.;

Takayama, H.; Kittaka, A. J. Org. Chem. 2003, 68, 7407.

13. Yamada, S.; Shimizu, M.; Yamamoto, K. Med. Res. Rev.

2003, 23, 89 (and references therein).

24. Fernandez, B.; Perez, J. A. M.; Granja, J. R.; Castedo, L.;

Mourino, A. J. Org. Chem. 1992, 57, 3173.

25. Fall, Y.; Vitale, C.; Mourino, A. Tetrahedron Lett. 2000,

41, 7337.

26. Posner, G. H.; Lee, J. K.; White, M. C.; Hutchings, R. H.;

Dai, H.; Kachinski, J. L.; Dolan, P.; Kensler, T. W. J.

Org. Chem. 1997, 62, 3299.

27. Strugnell, S. A.; Hill, J. J.; McCaslin, D. R.; Wieﬂing, B.

A.; Royer, C. A.; DeLuca, H. F. Arch. Biochem. Biophys.

1999, 364, 42.

28. Harvinga, E. Experientia 1973, 29, 1181.

29. Rochel, N.; Wurtz, J. M.; Mitschler, A.; Klaholz, B.;

Moras, D. Mol. Cell 2000, 5, 173.

14. Shevde, N. K.; Plum, L. A.; Clagett-Dame, M.; Yamam-

oto, H.; Pike, J. W.; DeLuca, H. F. Proc. Natl. Acad. Sci.

U.S.A. 2002, 99, 13487.

30. Ticchini-Valentini, G.; Rochel, N.; Wurts, J. M.; Mitsch-

ler, A.; Moras, D. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,

5491.

15. Yamamoto, H.; Shevde, N. K.; Warrier, A.; Plum, L. A.;

DeLuca, H. F. J. Biol. Chem. 2003, 278, 31756.

16. Kubodera, N.; Tsuji, N.; Uchiyama, Y.; Endo, K. J. Cell.

Biochem. 2003, 88, 286.

17. Submitted for publication.

18. Sicinski, R. R.; Prahl, J. M.; Smith, C. M.; DeLuca, H. F.

Steroid 2002, 67, 247.

19. Saito, N.; Masuda, M.; Matsunaga, T.; Saito, H.; Anzai,

M.; Takenouchi, K.; Miura, D.; Ishizuka, S.; Takimoto-

Kamimura, M.; Kittaka, A. Tetrahedron 2004, 60, 7951.

31. Tocchini-Valentini, G.; Rochel, N.; Wurtz, J. M.; Moras,

D. J. Med. Chem. 2004, 47, 1956.

32. Vanhooke, J. L.; Benning, M. M.; Bauer, C. B.; Pike, J.

W.; DeLuca, H. F. Biochemistry 2004, 43, 4101.

33. Eelen, G.; Verlinden, L.; Rochel, N.; Claessens, F.; De

Clercq, P.; Vandewalle, M.; Tocchini-Valentin, G.; Moras,

D.; Bouillon, R.; Verstuyf, A. Mol. Pharmacol. 2005, 67,

1566.

34. Choi, M.; Yamamoto, K.; Masuno, H.; Nakashima, K.;

Taga, T.; Yamada, S. Bioorg. Med. Chem. 2001, 9, 1721.

Article Doi

Synthesis and biological activities of new 1α,25-dihydroxy-19-norvitamin D3 analogs with modifications in both the A-ring and the side chain

DOI: 10.1016/j.bmc.2006.01.061

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Full text of DOI:10.1016/j.bmc.2006.01.061

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