Synthesis and structure-activity relationships of 16-ene-22-thia-1α,25-dihydroxy-26,27-dimethyl-19-norvitamin D3 analogs having side chains of different sizes

Available online at www.sciencedirect.com

Bioorganic & Medicinal Chemistry 16 (2008) 1796–1815

Synthesis and structure–activity relationships of 16-ene-22-

thia-1a,25-dihydroxy-26,27-dimethyl-19-norvitamin D₃

analogs having side chains of diﬀerent sizes

Hajime Takaku,^aYukiko Miyamoto,^aShiori Asami,^aMika Shimazaki,^a,b

Sachiko Yamada,^bKeiko Yamamoto,^cNobuyuki Udagawa,^d

Hector F. DeLuca^eand Masato Shimizu^a,*

^aLaboratory of Medicinal Chemistry, School of Biomedical Science, Tokyo Medical and Dental University,

2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan

^bInstitute of Biomaterial and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai,

Chiyoda-ku, Tokyo 101-0062, Japan

^cLaboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-tamagawagakuen,

Machida-shi, Tokyo 194-8543, Japan

^dDepartment of Biochemistry, Matsumoto Dental University, Nagano 399-0781, Japan

^eDepartment of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA

Received 8 October 2007; revised 31 October 2007; accepted 2 November 2007

Available online 6 November 2007

Abstract—We have synthesized eight novel 16-ene-22-thia-26,27-dimethyl-19-norvitamin D₃analogs 1–5 bearing side chains of dif-

ferent sizes, in combination with 20S- and 20R-isomers. The target compounds were prepared by Wittig-Horner reaction of A-ring

phosphine oxide with 16-ene-22-thia-25-hydroxy Grundmann’s ketones having diﬀerent sized side chains, which were derived from

the S-phenyloxycarbonyl derivative 13 as key intermediates. The binding aﬃnity to the vitamin D receptor (VDR), VDR-mediated

transcriptional activity, and osteoclast-inducing activity of synthetic 22-thia-19-norvitamin D analogs 1–5 were investigated. The

(20S)-22-thia-19,24-dinorvitamin D analog 1a is as active as the natural hormone 1a,25-dihydroxyvitamin D₃(1a,25-(OH)₂D₃)

in terms of biological activities tested in vitro. The analogs 2a and 3a exhibited almost the same potency as 1a,25-(OH)₂D₃in

binding to the VDR, were about 20 times more potent than 1a,25-(OH)₂D₃in terms of transcriptional activity, and 3a was

approximately 100 times as potent as 1a,25-(OH)₂D₃in eliciting osteoclast formation. The biological activities of (20S)-22-

thia compounds were more potent (by more than 10-fold) than those of the corresponding 20R-counterparts, but the activity

of (20R)-compounds 1b, 2b, and 3b in stimulating the formation of osteoclasts was similar to that of 1a,25-(OH)₂D₃, and the

24-dihomo- and trihomo-analogs 4a and 5a showed low transcriptional activity. These results suggest that elongation of the side

chain in 22-thia analogs by up to one carbon can be stably accommodated in the VDR ligand binding pocket.

1. Introduction

is an important hormone in the regulation of calcium

and phosphorus homeostasis and bone mineralization.¹

In addition, 1a,25-(OH)₂D₃plays a role in controlling dif-

ferentiation and growth inhibition of a variety of cell

types including malignant cells and keratinocytes, and

has immunomodulatory activity on B and T cells.^1,2

Vitamin D₃, originally discovered as an antirachitic fac-

tor, is converted in the liver to 25-hydroxyvitamin D₃

and then further hydroxylated in the kidney to an active

metabolite, 1a,25-dihydroxyvitamin D₃[1a,25-(OH)₂

D₃]. Currently it is well recognized that 1a,25-(OH)₂D₃

1a,25-(OH)₂D₃has been used for the treatment of osteo-

porosis, rickets, secondary hyperparathyroidism, psoria-

sis, and renal osteodystrophy. However, application of

1a,25-(OH)₂D₃as an anticancer drug induces signiﬁcant

hypercalcemia. Analogs having weak calcemic activity

while retaining strong cell diﬀerentiation and antiprolifer-

Keywords: 16-Ene-22-thia-19-norvitamin D; VDR binding aﬃnity;

Transcriptional activity; Osteoclast formation; Structure–activity

relationships.

*

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

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

doi:10.1016/j.bmc.2007.11.013

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

1797

ation activities have been developed, and a number of

1a,25-(OH)₂D₃analogs have been synthesized.^3–5Ana-

logs exhibiting lower calcemic activity while retaining or

showing improved cell diﬀerentiation activity have cer-

tain structural characteristics: (1) deletion of the 19-exo-

methylene group,⁶(2) introduction of heteroatoms such

as oxygen or sulfur at the C-22 position,^3,7,8and (3) incor-

poration of a double bond at the C-16 or C-22 position.^5,9

Among them, 1a,25-dihydroxy-22-oxavitamin D₃(maxa-

calcitol)³and 1a,25-dihydroxy-19-norvitamin D₂(pari-

calcitol)¹⁰have been shown to have desirably low

calcemic activity, and have been developed for clinical

treatment of psoriasis and secondary hyperparathyroid-

ism. On the other hand, structural modiﬁcations enhanc-

ing the transcriptional activity and cell diﬀerentiation

activity include inversion of stereochemistry at the C-20

position and homologation at C-24 or at C-26 and C-

27.^11,12KH1060 shows extremely strong transcriptional

activity and dramatically enhanced cell diﬀerentiation po-

tency in comparison with 1a,25-(OH)₂D₃.¹³It is also reported

that 22-oxavitamin D analogs with a 16,17-double bond is

susceptible to oxidation due to a newly generated allylic cen-

ter at C-20, and are assumed to exhibit reduced calcemic

activity due to faster oxidative metabolism in vivo.^14,15

ried out employing Wittig-Horner coupling of 22-thia-

C/D-ring Grundmann’s ketones with the A-ring phos-

phine oxide in a convergent manner. The C/D-ring syn-

thons (26, 39–42) with a normal- or epi-conﬁguration

(S- or R-) at the C-20 position (steroid numbering) were

prepared via key reaction of O-phenyl chlorothionofor-

mate with the 16-alcohol 10 or 12, which was derived

from the known 20-keto derivative 6,²³as illustrated in

Figures 2 and 3.

The 20-oxo compound 6, which was obtained from vita-

min D₂in ﬁve steps according to published proce-

dures,²³was reduced with sodium borohydride

(NaBH₄) to obtain epimeric alcohols 7a and 7b at C-

20 in a ratio of ca. 8:1.^24,25The reduction of 6 with

metallic potassium by reﬂuxing with 2-propanol aﬀor-

ded a slight excess of (20S)-alcohol 7b relative to

(20R)-7a (7a:7b = ca. 4:5).^26,27These alcohols were sep-

arated by column chromatography. Dehydration of 7 is

dependent on the conﬁguration of the C-20 center.

Treatment of (20R)-7a with POCl₃in pyridine pro-

ceeded stereospeciﬁcally to provide the known

17(20)Z-ethylidene derivative 8,^25,28whereas similar

treatment of (20S)-7b gave the E-ethylidene 9. Compar-

ison with an authentic 8 on the basis of spectral data

conﬁrmed the designated 17(20)Z-ethylidene structure.

Stereoselective hydroxylation of the Z-ethylidene 8 with

selenium dioxide in the presence of tert-butyl hydroper-

oxide (TBHP) under Sharpless conditions aﬀorded an

allylic 16a-hydroxyl (OH) compound 10.^29,30Oxidation

of 10 under Swern conditions gave the corresponding

16-keto compound 11, which upon reduction with

NaBH₄aﬀorded a mixture of allylic alcohols in which

the 16b-OH isomer 12 predominated (10:12 = ca.

1:8).²⁹The stereochemistry of 10 was unambiguously as-

signed on the basis of NMR spectroscopy and mecha-

nistic considerations of compounds with similar

structural features. The 18-methyl group cis to the

16b-OH moiety appeared at 0.17 ppm lower ﬁeld than

the 18-methyl trans to the 16a-OH group. It is well

known that an axial proton resonates at a higher ﬁeld

than the equatorial counterpart. Upﬁeld shift of an axial

16a-proton in 12 by ca. 0.09 ppm provides supporting

evidence for the stereochemistry. Introduction of both

the 16,17-double bond and side chains bearing a sulfur

atom at the C-22 position was accomplished by employ-

ing an eﬃcient method described by the Chugai group.⁷

Reaction of the16a-alcohol 10 with O-phenyl chloro-

thionoformate resulted in formation of O-phenyl

thiocarbonate, which underwent [3,3]-sigmatropic rear-

rangement to aﬀord exclusively a homoallylic (20S)-S-

phenyloxycarbonyl derivative 13a as a sole product,^7,31

whereas under the same conditions the epimeric 16b-

alcohol 12 was converted stereoselectively to the 20-epi

compound 13b. This reaction is highly dependent on

the conﬁguration of the OH group at the C-16 center.

In the reaction of the 16a-alcohol 10 with PhOC(S)Cl,

initially formation of a 16a-thiocarbonate, followed by

a [3,3]-shift, would take place through the less hindered

a-face to produce (20S)-S-phenyloxycarbonyl derivative

13a. 16-Ene-22-thia-C/D-ring ketones 26, 39–42 having

side chains of diﬀerent sizes were synthesized by elonga-

tion reaction of the thiol carbonates 13 with bromoester

We have reported that structural modiﬁcations at the C-2

position of the A-ring, or on both the A-ring and the side

chain, produced more than 100 synthetic 19-norvitamin

D₃analogs, some of which have interesting biological

proﬁles.^16–22Among them, we found that (20S)-2a-

methyl-2b-hydroxy-1a,25-dihydroxy-19- norvitamin D₃

showed equivalent binding aﬃnity to the VDR and li-

gand-dependent transcriptional activity (30 fold) com-

pared with the natural ligand 1a,25-(OH)₂D₃.^18,20,22As

a continuation of our studies of structural modiﬁcations

of the A-ring and/or the side chain and the structure–

activity relationships (SAR) of 19-norvitamin D analogs,

we have designed highly modiﬁed analogs of 1a,25-dihy-

droxy-19-norvitamin D₃containing an anticalcemic

16,17-double bond and a 22-thia group, and also a power-

ful antiproliferative 26,27-dimethyl group. We also be-

came interested in the eﬀect of the anticalcemic and

antiproliferative groups, the length of the side chain,

and the stereochemistry of C-20 center on the biological

activities of the 19-norvitamin D analogs.

We report herein the synthesis of 16-ene-22-thia-1a,25-

dihydroxy-26,27-dimethyl-19-norvitamin D analogs 1–

5 using a strategy based on a convergent approach using

the Wittig-Horner reaction of the A-ring phosphine

oxide with the 25-hydroxy-Grundmann’s ketones

(Fig. 1). We also examine the biological activity of the

synthetic 22-thia-19-nor derivatives on the aﬃnity of

binding to the VDR, in activating gene transcription,

and eﬀects on osteoclast formation.

2. Results and discussion

2.1. Chemistry

The synthesis of the 16-ene-22-thia-19-norvitamin D

analogs 1–5 bearing diﬀerent-sized side chains was car-

1798

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

OH

22

24

25

OH

20

O

(CH₂)_n

OH

16

19

3

1

HO

OH

HO

OH

HO

OH

1α,25-(OH)₂D₃

22-Oxa- or 16-ene-22-oxa-

1α,25-(OH)₂D₃analogs

KH1060

(CH₂)_n

OH

S

(CH₂)_n

1a: n=1

2a: n=2

3a: n=3

1b: n=1

2b: n=2

3b: n=3

4a: n=4

5a: n=5

HO

OH

HO

OH

Figure 1. The structures of 1a,25-(OH)₂D₃and its analogs.

20

O

OH

18

17

POCl₃

SeO₂

TBHP

NaBH₄

Vitamin D₂

16

8

11

OTBS

6

7a (20R)

7b (20S)

8 (Z-isomer)

9 (E-isomer)

S

OPh

S

CO₂Me

(CH₂)_n

1) Br(CH₂)_nCO₂Et

KOH, MeOH

O

PhOC(S)Cl

OH

2) CH₂N₂

OTBS

10

13a

14a: n = 1

15a: n = 2

16a: n = 3

17a: n = 4

18a: n = 5

DMSO, (COCl)₂

16-ketone (11)

NaBH₄

S

CO₂Me

S

OPh

(CH₂)_n

1) Br(CH₂)_nCO₂Et

KOH, MeOH

O

PhOC(S)Cl

OH

2) CH₂N₂

OTBS

12

13b

14b: n = 1

15b: n = 2

16b: n = 3

Figure 2. Synthesis of 16-ene-sulfanyl-carboxylic acid methyl esters.

derivatives bearing alkyl chains of diﬀerent lengths. The

reaction of 13a with ﬁve bromoesters Br(CH₂)_nCOOEt

(n = 1–5) under alkaline solvolysis conditions, and sub-

sequent methylation by diazomethane, gave sulfanyl

carboxylic acid methyl esters 14a–18a in good yields.

The 20R-epimers 14b–16b were prepared from 13b in a

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

1799

OH

S

(CH₂)n

p-TsOH

or TBAF

EtMgBr

or EtLi

14-18

OH

OTBS

19: n = 1

20: n = 2

21: n = 3

22: n = 4

23: n = 5

24: n = 1

27: n = 2

1) DMSO, (COCl)₂

2) TESCl

1) MOMCl

2) TBAF

O

S⁺

OR'

OR

S

(CH₂)n

DMSO, (COCl)₂

or PDC

S

O

OR

OH

31: R = TBS; R' = MOM (n = 2)

32: R = TBS; R' = MOM (n = 3)

33: R = TBS; R' = MOM (n = 4)

34: R = TBS; R' = MOM (n = 5)

35: R = H; R' = MOM (n = 2)

36: R = H; R' = MOM (n = 3)

37: R = H; R' = MOM (n = 4)

38: R = H; R' = MOM (n = 5)

25: R = H (n = 1)

28: R = H (n = 2)

26: R = TES (n = 1)

39: R = MOM (n = 2)

40: R = MOM (n = 3)

41: R = MOM (n = 4)

42: R = MOM (n = 5)

29: R2 = OH, H

30: R2 = O

R2

a = 20S; b = 20R

Figure 3. Synthesis of 16-ene-22-thia-C/D ring ketones.

synthetic sequence similar to that of the (20S)-

counterparts.

38a. Oxidation of 35a–38a under Swern conditions or

by pyridinium dichromate (PDC) gave the C/D-ring

kentones 39a–42a. The (20R)-methyl esters 14b–16b

were converted to the corresponding C/D-ring ketones

26b, 39b, and 40b utilizing the same sequence of reac-

tions as described for the 20S-series compounds.

The (20S)-methyl ester 14a (n = 1) was treated with ethyl

magnesiumbromide, aﬀording the tertiary alcohol 19a,

which was deprotected with para-toluenesulfonic acid

(p-TsOH) or tetrabutylammonium ﬂuoride (TBAF) to

provide the diol 24a. The oxidation of 24a under Swern

conditions, followed by protection of the 25-hydroxyl

group with chlorotriethylsilane (TESCl), gave the C/D-

ring synthon 26a. Since more than 20% of a mono-ethyl-

ated ketone was recovered after ethylation of 14 by

Grignard reagent, we employed ethyllithium (EtLi) in-

stead of EtMgBr in the following reaction. Ethylation

of 15a (n = 2) with freshly prepared EtLi aﬀorded 20a

in an excellent yield, and removal of the tert-butyl-

dimethyl silyl (TBS) protecting group of 20a by TBAF

gave 27a. The Swern oxidation of 27a was unsuccessful,

leading to unexpected diene compounds 29 and 30, and

the desired ketone 28a was not produced. Although the

exact mechanism responsible for generating the un-

wanted dienes is unclear, they may be produced via

the ﬁve-membered ring oxosulfonium ion as an interme-

diate, resulting in the intramolecular cyclization reaction

of the sulfur atom to the activated tertiary alcohol group

by treatment of 27a with DMSO/(COCl)₂(Fig. 3). It was

considered that the side reaction would be avoided by

protecting the free 25-OH group in 27. The 25-OH

group in 20a–23a was protected by chloromethyl methyl

ether (MOMCl), aﬀording methoxymethyl (MOM)

ethers 31a–34a, which upon deprotection of the silyl

ether group by TBAF yielded the C(8)-alcohols 35a–

The synthesis of 16-ene-22-thia-19-norvitamin D ana-

logs 1–5 bearing side chains of diﬀerent sizes was carried

out by Wittig-Horner reaction as shown in Figure 4: the

coupling of C/D-ring ketones 26, 39–42 with the conju-

gate base of A-ring allylic phosphine oxide 43 obtained

by the reported method,³²followed by cleavage of both

silyl and MOM ether groups with camphor sulfonic acid

(CSA), gave the desired compounds 1–5 with a 20S- or

20R-conﬁguration.

2.2. Biological activity

We have synthesized eight new 22-thia-19-norvitamin D

analogs with high structural modiﬁcations in both the

D-ring and the side chain. The side chains of these ana-

logs have the sulfur atom at C-22, methylation at C-26

and C-27, and diﬀerent-sized side chains in combination

with epimerization at C-20. The biological activity of

these analogs 1–5 was evaluated in vitro.

We ﬁrst tested the binding aﬃnity for the recombinant

rat VDR ligand-binding domain (LBD)³³and VDR-

mediated transcriptional activity in COS-7 cells. The re-

sults are summarized in Table 1 and the activities were

shown as percentages of that of the natural hormone,

1800

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

OR'

S

(CH₂)_n

OR

S

(CH₂)_n

+

1) LHMDS or nBuLi

2) CSA

P(O)Ph₂

OTBS

O

TBSO

26: n = 1

39: n = 2

40: n = 3

41: n = 4

42: n = 5

RO

OR

43

44: R = TBS; R' = TES (n = 1)

45: R = TBS; R' = MOM (n = 2)

46: R = TBS; R' = MOM (n = 3)

47: R = TBS; R' = MOM (n = 4)

48: R = TBS; R' = MOM (n = 5)

1 - 5: R = R' = H (n = 1-5)

a = 20S; b = 20R

Figure 4. Synthesis of 16-ene-22-thia-19-norvitamin D₃analogs bearing side chains of diﬀerent sizes.

1a,25-(OH)₂D₃. The amino acid sequence of rat VDR

LBD shows approximately 93% similarity with that of

human VDR LBD, and the rat VDR is four amino acids

shorter than the human counterpart. Rat VDR LBD

binds the natural hormone with similar aﬃnity to that

of the full-length VDR. In the evaluation of VDR-bind-

ing aﬃnity, the length of the side chain in pairs of three

analogs 1–3 did not inﬂuence the aﬃnity. The stereo-

chemistry at C-20 appeared critical: the analogs 1a–3a

with a 20S-conﬁguration showed binding potency simi-

lar to that of 1a,25-(OH)₂D₃, and the 20R-epimers 1b–

3b were 10- to 20-fold less potent than the corresponding

20S-epimers. The VDR-mediated transcriptional activ-

ity of the eight 22-thia-19-norvitamin D analogs was

tested by luciferase assay with the mouse osteopontin

vitamin D response element (VDRE) in COS-7 cells.

The relative activities of new analogs 1–5 were assessed

in terms of the ED₅₀values calculated from their dose-

response curves. Among the compounds bearing 20S-

conﬁgurations, the 24-nor analog 1a was comparable

in potency to the natural ligand. The analog 2a, with a

normal-sized side chain (containing three atoms between

the C-20 and C-25 positions) and carrying a 26,27-di-

methyl group, and the 24-homo analog 3a were approx-

imately 20 times more active than 1a,25-(OH)₂D₃in

stimulating transcriptional activity. The 24-dihomo-

and 24-trihomo analogs 4a and 5a displayed markedly

(50- to 100-fold) decreased transcriptional activity.

Epimerization at the C-20 position decreased transcrip-

tional potency relative to the 20S-epimers 10- to 20-fold

in accordance with the eﬀect on VDR-binding aﬃnity.¹⁹

D 1a may reﬂect the hydrogen bond of the terminal

25-OH moiety with histidine 305 (His305) and His397

due to the sulfur atom with a large van der Waals ra-

dius. The X-ray crystallographic data for human or

rat VDR LBD complexed with 1a,25-(OH)₂D₃and its

analogs including the 20-epimers revealed that the 25-

OH groups contact the same amino acid residues

His305 and His397 within the LBP.^35–37This interaction

is crucial for stimulating VDR responses. Elimination of

the 25-OH group lowers the VDR binding aﬃnity to

approximately 1/1000,³⁸and it is well known that hered-

itary vitamin D-resistant rickets (HVDRR) is caused by

a missense mutation of VDR which results in the

replacement of histidine with glutamine (Gln) at amino

acid 305 (His305Gln VDR).³⁹Quite recently, we suc-

ceeded in determining the crystal structure of rat VDR

LBD in complex with an analog of 1a having a substitu-

ent at the A-ring (data not shown).³¹Analysis of the

crystal structure of the rat VDR LBD/ligand complex

revealed contacts of the 25-OH group of the ligand with

the LBP-lining amino acid residues His305 and His397:

the distances between the oxygen atoms and His305 and

˚

His397 were 2.65 and 2.82 A, respectively. The 22-thia-

analogs 2a and 3a are about 20 times more eﬀective than

1a,25-(OH)₂D₃. Elongation of the side chain by up to

two carbons compared with 1a,25-(OH)₂D₃can be

accommodated in the LBP, forming hydrogen bonds

with the same two histidines, and increases the hydro-

phobic interactions with the amino acid residue lining

the LBP.⁴⁰Methylation at C-26 and C-27 has similar

eﬀects to this elongation.¹²The extremely lower

transcriptional activity of the 24-dihomo- and 24-triho-

mo-analogs 4a and 5a might be explained by interfer-

ence with formation of the active conformation of the

VDR/ligand complex due to the longer side chains

containing the large sulfur atom at C-22. Upon ligand

binding in the VDR LBP, the VDR changes its confor-

mation to the transcriptionally active form, in which

helix 12 (H12) located in the C-terminal region seals

the LBP and forms a transactivation function 2 (AF-2)

surface.^41,42Coactivator proteins bind this hydrophobic

AF-2 surface through the leucine (Leu) x x Leu Leu

(LxxLL, x = any amino acids) motif in their nuclear

receptor interacting domain.^36,44It is known for the

It

should

be

noted

that

(20R)-22-thia-1a,

25-dihydroxyvitamin D₃analogs without the 16,17-dou-

ble bond have high biological activities compared with

their 20S-counterparts.⁸The 24-nor analog 1a showed

almost equivalent activity with respect to the natural

ligand in terms of VDR binding aﬃnity and transcrip-

tional activity. It is reported that deletion of the methy-

lene group of 1a,25-(OH)₂D₃in the side chain results in

about a 10-fold reduction of HL-60 diﬀerentiating po-

tency.³⁴This might be due to the lack of hydrogen

bonds between the terminal 25-OH group and the amino

acids lining the ligand binding pocket (LBP).^35,36The

preserved biological activities of 22-thia-24-norvitamin

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

1801

Table 1. Relative VDR aﬃnity and transcriptional activity of 16-ene-

22-thia-19-norvitamin D analogs^a

estrogen receptor that when ligands bearing a bulky side

chain are accommodated in the LBP, H-12 cannot adopt

the active conformation. These ligands act as antago-

nists.^43–45

Compound

VDR aﬃnity

Transcription

1a,25-(OH)₂D₃

100

60

100^b

104^b

9^b

1a

1b

2a

2b

3a

3b

4a

5a

Next, we examined the eﬀects of 22-thia-19-norvitamin

D analogs 1–3 on osteoclast formation in a mouse co-

culture system, and the results are shown in Figure 5.

Treatment of the culture with the 22-thia analogs re-

sulted in a dose-dependent increase in the number of

osteoclasts formed. 1a,25-(OH)₂D₃and 22-thia analogs

1–3 induced multinucleated, tartrate-resistant acid phos-

phatase (TRAP, a marker enzyme of osteoclasts)-posi-

tive cells typical of osteoclasts. 1a,25-(OH)₂D₃and its

analogs caused a dose-dependent increase in the number

of osteoclasts. The 22-thia-24-nor analogs 1a and 1b

showed similar activity to 1a,25-(OH)₂D₃regardless of

the conﬁguration of the C-20 center. Analogs 2a and

2b strongly stimulated osteoclast formation at 10^ꢀ10M

and were more active than the natural ligand. The 24-

homo analog 3a was more than 10 times as potent as

1a,25-(OH)₂D₃in stimulating osteoclast formation,

whereas its 20-epimer 3b exhibited almost the same

activity as the natural hormone. Eﬀects of (20S)-22-thia

analogs on osteoclast-inducing activity correlate with

the transcriptional activities. The transcriptional activity

of analogs 1b, 2b, and 3b having the 20R-conﬁguration

was about 10% as potent as that of their corresponding

counterparts, and quite diﬀerent from their eﬀects on

osteoclastogenesis.

6

100

4

2000^b

27^b

100

8

2000^b

20^b

1^b

2^b

N.D.

N.D

^aActivities are shown as percentages of that of 1a,25-(OH)₂D₃.

^bActivity was assessed in terms of ED₅₀

.

300

250

200

150

100

50

1α,25-(OH)₂D₃

1a

1b

0

Conc. (nM)

250

200

150

100

50

3. Conclusion

1α,25-(OH)₂D₃

2a

The synthesis of newly designed hybrid analogs having

the sulfur atom at C-22, the 16,17-double bond, 26,27-

dimethyl groups, and diﬀerent-sized side chains, has

been achieved successfully. The isomeric pairs, (20S)-

and (20R)-22-thia-19-norvitamin D analogs 1–3, showed

distinct biological proﬁles, and the 20S-isomers were

more active than the 20R-isomers in terms of VDR

binding and transcriptional activity. 20S-Isomers were

comparable in potency to 1a,25-(OH)₂D₃in terms of

binding aﬃnity for the VDR, and 1-20 times more active

than 1a,25-(OH)₂D₃in stimulating transcriptional activ-

ity. The most promising analog is 3a, in which incorpo-

ration of a sulfur atom at C-22 is combined with

homologation at C-24, C-26 and C-27. We have demon-

strated some synthetic 19-norvitamin D analogs with

various substituents at the C-2 position, and among

them, 2b-hydroxyethoxy-, or 2a-methyl-2b-hydroxy-

19-norvitamin D derivatives have powerful biological

activities. The synthesis of the current 22-thia analogs

in combination with C-2 substituents, which is expected

to yield some interesting biological properties, is now in

progress.

2b

0

Conc. (nM)

250

200

150

100

50

1α,25-(OH)₂D₃

3a

3b

0

4. Experimental

Conc. (nM)

¹H NMR spectra were obtained on a Bruker ARX-400

spectrometer, operating at 400 MHz for H. Chemical

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

1

Figure 5. Eﬀects of 1a,25-(OH)₂D₃and its derivatives on osteoclast

formation in co-culture.

1802

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

ﬁeld from tetramethylsilane as an internal standard (d

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

ture was acidiﬁed with 2 N HCl and extracted with

AcOEt. The AcOEt layer was washed with brine, dried

over anhydrous MgSO₄, and evaporated to dryness.

The residue was puriﬁed by column chromatography

on silica gel (30 g) with hexane to give 8 (975.6 mg,

78%).

1

(s), doublet (d), triplet (t), multiplet (m), broad signal

(br). Low- and high-resolution mass spectra (LR-MS

and HR-MS) were obtained with electronic ionization

(EI) on a JEOL JMS-AX505HA spectrometer run at

70 eV for EI; m/z values are given with relative intensi-

ties in parentheses. UV spectra were obtained on a Beck-

mann DU-7500 spectrophotometer. A mixture of

diastereomers was separated by HPLC equipped with

a Model PU-980 pump, a Rheodyne Model 7125 injec-

tor, and a Model MD-910 multiwavelength UV detector

from JASCO. Column chromatography was carried out

on silica gel (Wako Pure Chem. Ind. Ltd. Wakogel C-

200). All reactions were conducted under an atmosphere

of argon gas. Yields were not optimized.

¹H NMR (CDCl₃) d: 0.01, 0.02 (each 3H, s, 2· Si–Me),

0.89 (9H, s, Si-t-Bu), 1.11 (3H, s, H-18), 1.64 (3H, dt,

J = 7.1, 2.0 Hz, H-21), 4.07 (1H, m, H-18), 5.04 (1H,

qt, J = 7.1, 2.0 Hz, H-20). ¹³C NMR (CDCl₃) d: ꢀ4.9,

ꢀ4.6, 13.2, 18.1, 18.2, 19.8, 24.0, 26.0 (3·), 30.6, 34.6,

38.3, 44.4, 52.9, 69.9, 112.5, 151.1. MS m/z (%): 294

(M⁺, 6), 279 (4), 237 (100), 163 (20), 162 (60), 161

(85), 75 (75). HRMS m/z 294.2385 (Calcd for

C₁₈H₃₄OSi: 294.2379).

4.1. (20R)- and (20S)-Alcohols (7a) and (7b)

4.3. 17(20)E-Ethylidene (9)

4.1.1. Method I. To a stirred, ice-cooled solution of 6

(1.90 g, 6.118 mmol) in methanol (MeOH, 20 mL) was

added sodium borohydride (NaBH₄, 231.9 mg,

6.130 mmol). After being stirred for 1 h at the same tem-

perature, the mixture was poured into ice-water and ex-

tracted with AcOEt. The organic phase was washed with

brine, dried over anhydrous magnesium sulfate

(MgSO₄), and evaporated in vacuo. The residue was

chromatographed on silica gel (70 g) with 2% AcOEt

in hexane to give (20R)-7a (1.67 g) and (20S)-7b

(208 mg) in a total yield of 98% (ca. 7a:7b = 8:1).

Following the same procedure as described above, treat-

ment of 7b (453.1 mg, 1.45 mmol) with POCl₃(337.8 lL,

3.62 mmol) gave 9 (402.3 mg, 94%).

¹H NMR (CDCl₃) d: 0.02 (6H, s, 2· Si–Me), 0.89 (9H, s,

Si-t-Bu), 0.98 (3H, s, H-18), 1.52 (3H, d, J = 6.7 Hz, H-

21), 4.07 (1H, m, H-8), 4.97 (1H, qt, J = 6.7, 2.5 Hz, H-

20). MS m/z (%): 294 (M⁺, (3), 279 (3), 237 (100), 163

(18), 162 (41), 161 (76), 75 (76).

4.4. 17(20)E-Ethylidene-16a-ol (10)

4.1.2. Method II. A solution of 6 (750.5 mg, 2.42 mmol)

in anhydrous 2-propanol (2-PrOH, 65 mL) was stirred

under reﬂux and metallic potassium (6.6 g, 169 mmol)

was added in small pieces in a period of 0.5 h, and the

mixture was reﬂuxed for another 1 h. The mixture was

cooled to room temperature, poured into ice-water,

and extracted with AcOEt. The organic extracts were

washed with brine, dried over anhydrous MgSO₄, and

evaporated to dryness. The residue was puriﬁed by col-

umn chromatography on silica gel with 2% AcOEt in

hexane to aﬀord (20R)-7a (293.3 mg, 39%) and (20S)-

7b (357.6 mg, 47%). (ca. 7a:7b = 4:5).

To a stirred suspension of selenium dioxide (177.5 mg,

1.600 mmol) in methylene chloride (CH₂Cl₂, 5 mL) at

0 ꢁC was added dropwise 70 wt % tert-butyl hydroper-

oxide in H₂O (552 lL, 5.696 mmol). Stirring was contin-

ued for 1 h, and a solution of 8 (942.3 mg, 3.199 mmol)

in CH₂Cl₂(10 mL) was added dropwise. The reaction

mixture was stirred for 4 h at ambient temperature

and poured into ice-water with 10% sodium hydroxide

(NaOH). After extraction with AcOEt, the organic

phase was washed with brine, dried over anhydrous

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

ﬁed by column chromatography on silica gel (30 g) with

5% AcOEt in hexane to aﬀord 10 (847.0 mg, 85%).

(20R)-7a ¹H NMR (CDCl₃) d: ꢀ0.003, 0.01 (each 3H, s,

2· Si–Me), 0.89 (9H, s, Si-t-Bu), 1.01 (3H, s, H-18), 1.12

(3H, d, J = 6.1 Hz, H-21), 3.73 (1H, m, H-20), 4.01 (1H,

m, H-8). MS m/z (%): 312 (M⁺, 1), 255 (19), 237 (100),

161 (38), 75 (72). HRMS m/z: 310.2487 (Calcd for

¹H NMR (CDCl₃) d: 0.01, 0.02 (each 3H, s, 2· Si–Me),

0.88 (9H, s, Si-t-Bu), 1.10 (3H, s, H-18), 1.72 (3H, dd,

J = 7.2, 1.0 Hz, H-21), 4.10 (1H, m, H-8), 4.44 (1H, m,

H-16), 5.51 (1H, qd, J = 7.2, 1.2 Hz, H-20). MS m/z

(%): 310 (M⁺, 5), 292 (5), 253 (37), 235 (100), 178 (24),

161 (89), 160 (75). HRMS m/z: 310.2337 (Calcd for

C₁₈H₃₄O₂Si: 310.2328).

1

C₁₈H₃₆O₂Si: 312.2485). (20S)-7b H NMR (CDCl₃) d:

ꢀ0.003, 0.01 (each 3H, s, 2· Si–Me), 0.88 (9H, s, Si-t-

Bu), 0.91 (3H, s, H-18), 1.20 (3 H, d, J = 6.2 Hz, H-

21), 3.68 (1H, m, H-20), 4.02 (1H, m, H-8). MS m/z

(%): 312 (M⁺, 1), 255 (5), 237 (14), 161 (18), 75 (100).

HRMS m/z: 310.2470 (Calcd for C₁₈H₃₆O₂Si: 312.2485).

4.5. 17(20)E-Ethylidene-16-one (11)

To a stirred solution of oxalyl chloride [(COCl)₂,

314 lL, 3.599 mmol] in dry CH₂Cl₂(3 mL) at ꢀ78 ꢁC

was added dimethylsulfoxide (DMSO, 509 lL,

7.173 mmol) in dry CH₂Cl₂(1 mL). After being stirred

for 10 min, a solution of 10 (845.6 mg, 2.723 mmol) in

dry CH₂Cl₂(7 mL) was added dropwise. The reaction

mixture was stirred at ꢀ78 ꢁC for 15 min, and triethyl-

amine (Et₃N, 2.09 mL, 14.995 mmol) was added. The

4.2. 17(20) Z-Ethylidene (8)

To

a stirred, ice-cooled solution of 7a (1.33 g,

4.281 mmol) in anhydrous pyridine (15 mL) was added

phosphoryl chloride (POCl₃, 0.8 mL, 8.583 mmol). The

mixture was stirred for 1 h at 0 ꢁC and for 21 h at ambi-

ent temperature, poured into ice-water. The whole mix-

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

1803

entire mixture was stirred at ꢀ78 ꢁC for 10 min, and at

0 ꢁC for 30 min, quenched with ice water, and extracted

with CH₂Cl₂. The CH₂Cl₂extract was washed with

brine, and dried over anhydrous MgSO₄, solvent was

evaporated in vacuo, and. the residue was chromato-

graphed on silica gel (25 g) with 5% AcOEt in hexane

to give 11 (792.8 mg, 94%).

126.2, 126.9, 129.7 (2·), 151.4, 154.8, 170.3. MS m/z

(%): 446 (M⁺, 2), 431 (1), 389 (3), 292 (10), 235 (100),

161 (80), 160 (66). HRMS m/z: 446.2299 (Calcd for

C₂₅H₃₈O₃SSi: 446.2311).

4.8. (20R)-16-Ene-thionocarbonic acid O-phenyl ester

(13b)

¹H NMR (CDCl₃) d: 0.02, 0.05 (each 3H, s, 2· Si–Me),

0.90 (9H, s, Si-t-Bu), 1.25 (3H, s, H-18), 1.83 (3H, d,

J = 7.5 Hz, H-21), 4.13 (1H, m, H-8), 6.43 (1H, q,

J = 7.5 Hz, H-20). ¹³C NMR (CDCl₃) d: ꢀ4.9, ꢀ4.6,

13.3, 18.0, 20.6 (2·) 34.4, 35.0, 38.4, 49.3, 69.7, 73.7,

118.5. MS m/z (%): 308 (M⁺, 12), 293 (3), 251 (100).

HRMS m/z: 294.2165 (Calcd for C₁₈H₃₂O₂Si: 308.2172).

Following the same procedure as mentioned above,

treatment of 12 (289.2 mg, 0.931 mmol) with PhOC(S)Cl

(258 lL, 1.865 mmol) gave 13b (313.0 mg, 75%).

¹H NMR(CDCl₃) d: 0.026, 0.033 (each 3H, s, 2· Si–Me),

0.89 (9H, s, Si-t-Bu), 1.07 (3H, s, H-18), 1.61 (3H, d,

J = 6.9 Hz, H-21), 4.04 (1H, q, J = 6.9 Hz, H-20), 4.10

(1H, m, H-8), 5.72 (1H, m, H-16), 7.14–7.40 (5H, m,

arom-H). ¹³C NMR (CDCl₃) d: ꢀ5.0, ꢀ4.6, 18.16,

18.22, 19.3, 22.9, 26.0 (3·), 31.2, 34.7, 35.5, 38.6, 47.0,

54.8, 69.1, 121.6 (2·), 126.2, 126.8, 129.7 (2·), 151.4,

153.7, 170.4. MS m/z (%): 446 (M⁺, 3), 431 (1), 389

(4), 293 (17), 235 (13), 161 (100). HRMS m/z: 446.2318

(Calcd for C₂₅H₃₈O₃SSi: 446.2311).

4.6. 17(20)E-Ethylidene-16b-ol (12)

To a solution of 11 (144.7 mg, 0.469 mmol) in MeOH

(1 mL) at 0 ꢁC were successively added cerium (III) chlo-

ride heptahydrate (17.5 mg, 0.047 mmol) and NaBH₄

(17.7 mg, 0.469 mmol). After being stirred at 0 ꢁC for

30 min, the mixture was poured into ice-water and ex-

tracted with AcOEt. The organic phase was washed with

brine, dried over anhydrous MgSO₄, and evaporated to

dryness. The residue was puriﬁed by column chromatog-

raphy on silica gel (Wacogel C-300, 5 g) with 2% AcOEt

in hexane to give 10 (16.7 mg) and 12 (126.4 mg) in a to-

tal yield of 98%.

4.9. (20S)-16-Ene-sulfanyl-acetic acid methyl ester (14a)

To a stirred solution of 13a (740.5 mg, 1.658 mmol) in

10% KOH in MeOH (10 mL) at ambient temperature

was added ethyl bromoacetate (0.55 mL, 4.978 mmol).

After being stirred for 4 h, the reaction mixture was

poured into ice-water containing 2 N HCl, and extracted

with AcOEt. The organic extracts were washed with

brine, dried over anhydrous MgSO₄, and evaporated

in vacuo. The resulting residue was dissolved in CH₂Cl₂

(10 mL), cooled to 0 ꢁC, and diazomethane in ether

(about 0.5 M in ether, 10 mL) was added. Upon stirring

for 30 min at the same temperature, solvent was evapo-

rated in vacuo. The residue was chromatographed on

silica gel (20 g) with 3% AcOEt in hexane to aﬀord

14a (661.0 mg, quantitative yield).

¹H NMR (CDCl₃) d: 0.02 (6H, s, 2· Si–Me), 0.90 (9H, s,

Si-t-Bu), 1.27 (3H, s, H-18), 1.71 (3H, dd, J = 7.2,

1.6 Hz, H-21), 4.04 (1H, m, H-8), 4.35 (1H, m, H-16),

5.44 (1 H, qd, J = 7.2, 1.6 Hz, H-20). ¹³C NMR (CDCl₃)

d: ꢀ4.9, ꢀ4.6, 13.3, 18.1, 18.2, 21.0, 26.0 (3·), 34.4, 34.6,

37.9, 44.2, 48.6, 69.2, 75.0, 118.2, 151.5. MS m/z (%): 310

(M⁺, 5), 292 (9), 253 (17), 235 (51), 178 (11), 161 (100),

160 (28). HRMS m/z: 310.2299 (Calcd for C₁₈H₃₄O₂Si:

310.2328).

4.7. (20S)-16-Ene-thionocarbonic acid O-phenyl ester

(13a)

¹H NMR (CDCl₃) d: 0.02 (6H, s, 2· Si–Me), 0.88 (9H, s,

Si-t-Bu), 1.12 (3H, s, H-18), 1.36 (3H, d, J = 7.0 Hz, H-

21), 2.28 (1H, m), 3.16 (2H, s, H-23), 3.58 (1H, q,

J = 7.0 Hz, H-20), 3.71 (3H, s, OMe), 4.07 (1H, m, H-

8), 5.64 (1H, m, H-16). ¹³C NMR (CDCl₃) d: ꢀ5.0,

ꢀ4.6, 18.1, 18.2, 19.3, 21.9, 26.0 (3·), 31.0, 33.1, 34.7,

35.7, 38.6, 46.8, 52.5, 55.5, 69.2, 125.9, 155.1, 171.6.

MS m/z (%): 398 (M⁺, 35), 383 (15), 341 (28), 309 (9),

292 (9), 235 (92), 161 (100), 160 (48). HRMS m/z:

398.2307 (Calcd for C₂₁H₃₈O₃SSi: 398.2311).

To a stirred solution of 10 (630.2 mg, 2.029 mmol) in dry

pyridine (6 mL) at 0 ꢁC was added O-phenyl chloroth-

ionoformate [PhOC(S)Cl, 337 lL, 2.436 mmol] and the

mixture was stirred for 3.5 h. An additional PhOC(S)Cl

(220 lL, 1.590 mmol) was added and stirring was con-

tinued for another 2.5 h at 0 ꢁC. The reaction mixture

was poured into ice-water containing 2 N HCl and then

extracted with AcOEt. The AcOEt layer was washed

with brine and dried over anhydrous MgSO₄. Solvent

was evaporated to dryness, and the residue was chro-

matographed on silica gel (30 g) with 3% AcOEt in hex-

ane to yield 13a (772.5 mg, 85%).

4.10. (20R)-16-Ene-sulfanyl-acetic acid methyl ester (14b)

Following the same procedure as described above, treat-

ment of 13b (345.3 mg, 0.773 mmol) with ethyl bromo-

acetate (258 lL, 2.327 mmol), followed by methylation

with ethereal diazomethane, aﬀorded 14b (296.3 mg,

96%).

¹H NMR (CDCl₃) d: 0.026, 0.030 (each 3H, s, 2· Si–

Me), 0.89 (9H, s, Si-t-Bu), 1.19 (3H, s, H-18), 1.54

(3H, d, J = 6.9 Hz, H-21), 2.31 (1H, m, H-15), 4.07

(1H, q, J = 6.9 Hz, H-20), 4.10 (1H, m, H-8), 5.71

(1H, m, H-16), 7.14–7.39 (5 H, m, arom H). ¹³C NMR

(CDCl₃) d: ꢀ5.0, ꢀ4.6, 18.1, 18.2, 19.7, 23.4, 26.0 (3·),

31.3, 34.7, 35.4, 38.9, 47.3, 55.3, 69.1, 121.6 (2·),

¹H NMR (CDCl₃) d: 0.02, 0.03 (each 3H, s, 2· Si–Me),

0.89 (9H, s, Si-t-Bu), 1.05 (3H, s, H-18), 1.46 (3H, d,

J = 7.0 Hz, H-21), 2.26 (1H, m), 3.16, 3.24 (each 1H,

d, J = 14.7 Hz, H-23), 3.47 (1H, q, J = 7.0 Hz, H-20),

1804

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

3.72 (3H, s, OMe), 4.07 (1H, m, H-8), 5.59 (1H, m, H-

16). ¹³C NMR (CDCl₃) d: ꢀ5.0, ꢀ4.6, 18.2 (2·), 19.6,

22.0, 26.0 (3·), 31.1, 32.5, 34.7, 35.6, 37.1, 47.0, 52.5,

54.8, 69.1, 125.4, 154.7, 171.1. MS m/z (%): 398 (M⁺,

37), 383 (16), 341 (24), 292 (7), 235 (62), 161 (100), 160

(33). HRMS m/z: 398.2298 (Calcd for C₂₁H₃₈O₃SSi:

398.2311).

(1H, m, H-8), 5.61 (1H, m, H-16). ¹³C NMR (CDCl₃)

d: ꢀ5.0, ꢀ4.7, 18.11, 18.16, 19.4, 22.3, 24.8, 25.9 (3·),

30.4, 30.9, 33.2, 34.7, 35.7, 37.5, 46.7, 51.7, 55.5, 69.2,

125.0, 156.0, 173.8. MS m/z (%): 426 (M⁺, 20), 411 (9),

369 (10), 292 (10), 235 (100), 161 (86), 160 (70). HRMS

m/z: 426.2629 (Calcd for C₂₃H₄₂O₃SSi: 426.2624).

4.14. (20R)-16-Ene-sulfanyl-butyric acid methyl ester

(16b)

4.11. (20S)-16-Ene-sulfanyl-propionic acid methyl ester

(15a)

Following the same procedure as described above, treat-

ment of 13b (331.2 mg, 0.741 mmol) with ethyl 4-bromo-

butyrate (338 lL, 2.362 mmol), followed by methylation

with ethereal diazomethane, aﬀorded 16b (292.2 mg, 92%).

Following the same procedure as described above, treat-

ment of 13a (331.9 mg, 0.763 mmol) with ethyl 3-bromo-

propionate (292 lL, 2.278 mmol), followed by

methylation with ethereal diazomethane, aﬀorded 15a

(262.4 mg, 86%).

¹H NMR (CDCl₃) d: 0.02, 0.03 (each 3H, s, 2· Si–Me),

0.89 (9H, s, Si-t-Bu), 1.04 (3H, s, H-18), 1.44 (3H, d,

J = 7.0 Hz, H-21), 2.24 (1H, m, H-15), 2.40–2.60 (4H,

m, H-23, 24a), 3.30 (1H, q, J = 7.0 Hz, H-20), 3.67

(3H, s, OMe), 4.09 (1H, m, H-8), 5.53 (1H, m, H-16).

¹³C NMR (CDCl₃) d: ꢀ5.0, ꢀ4.6, 18.2 (2·), 19.6, 22.5,

25.0, 26.0 (3·), 29.9, 31.1, 33.2, 34.8, 35.8, 36.3, 47.0,

51.8, 54.9, 69.2, 124.2, 155.7, 173.9. MS m/z (%): 426

(M⁺, 24), 411 (11), 369 (9), 292 (5), 235 (70), 161

(100), 160 (44). HRMS m/z: 426.2617 (Calcd for

C₂₃H₄₂O₃SSi: 426.2624).

¹H NMR (CDCl₃) d: 0.01 (6H, s, 2· Si–Me), 0.88 (9H, s,

Si-t-Bu), 1.12 (3H, s, H-18), 1.35 (3H, d, J = 7.0 Hz, H-

21), 2.27 (1H, m, H-15), 2.57 (2H, m, H-24), 2.68 (2H,

m, H-23), 3.41 (1H, q, J = 7.0 Hz, H-20), 3.68 (3H, s,

–OMe), 4.07 (1H, m, H-8), 5.61 (1H, m, H-16). ¹³C

NMR (CDCl₃) d: ꢀ5.0, ꢀ4.7, 18.1, 18.2, 19.5, 22.3,

26.0 (3·), 26.2, 31.0, 34.69, 34.73, 35.7, 37.9, 46.8,

51.9, 55.5, 69.2, 125.3, 155.8, 172.8. MS m/z (%): 412

(M⁺, 17), 355 (10), 292 (6), 235 (100), 161 (76), 160

(53). HRMS m/z: 412.2449 (Calcd for C₂₂H₄₀O₃SSi:

412.2467).

4.15. (20S)-16-Ene-sulfanyl-pentanoic acid methyl ester

(17a)

4.12. (20R)-16-Ene-sulfanyl-propionic acid methyl ester

(15b)

Following the same procedure as described above, treat-

ment of 13a (115.4 mg, 0.258 mmol) with ethyl 5-bro-

movalerate (122.7 lL, 0.775 mmol), followed by

methylation with ethereal diazomethane, aﬀorded 17a

(110.0 mg, 97%).

Following the same procedure as described above, treat-

ment of 13b (207.2 mg, 0.464 mmol) with ethyl 3-bromo-

propionate (178 lL, 1.388 mmol), followed by

methylation with ethereal diazomethane, aﬀorded 15b

(142.2 mg, 74%).

¹H NMR (CDCl₃) d: 0.030 (6H, s, 2· Si–Me), 0.89

(9H, s, Si-t-Bu), 1.13 (3H, s, H-18), 1.35 (3H, d,

J = 7.0 Hz, H-21), 2.32 (2H, t, J = 7.5 Hz, 24aa), 2.43

(2H, t, J = 7.2 Hz, H-23), 3.39 (1H, m, H-20), 3.67

(3H, s, –CO₂Me), 4.08 (1H, m, H-8), 5.61 (1H, m, H-

16). MS m/z (%): 440 (M⁺, 16), 383 (9), 292 (9), 235

(100), 161, (69), 160 (83). HRMS m/z: 440.2766 (Calcd

for C₂₄H₄₄O₃SSi: 440.2780).

¹H NMR (CDCl₃) d: 0.02, 0.03 (each 3H, s, 2· Si–Me),

0.89 (9H, s, Si-t-Bu), 1.04 (3H, s, H-18), 1.47 (3H, d,

J = 7.0 Hz, H-21), 2.24 (1H, m, H-15), 2.58 (2H, m, H-

24), 2.72 (2 H, m, H-23), 3.32 (1H, q, J = 7.0 Hz, H-20),

3.69 (3H, s, OMe), 4.08 (1H, m, H-8), 5.54 (1H, m, H-

16). ¹³C NMR (CDCl₃) d: ꢀ5.0, ꢀ4.6, 18.2 (2·), 19.6,

22.5, 25.5, 26.0 (3·), 31.0, 34.7, 35.0, 35.8, 36.5, 47.0,

51.9, 54.8, 69.2, 124.4, 155.4, 172.8. MS m/z (%): 412

(M⁺, 8), 355 (5), 292 (3), 235 (100), 161 (82), 160 (63).

HRMS m/z: 412.2457 (Calcd for C₂₂H₄₀O₃SSi: 412.2467).

4.16. (20S)-16-Ene-sulfanyl-hexanoic acid methyl ester

(18a)

Following the same procedure as described above, treat-

ment of 13a (82.7 mg, 0.185 mmol) with ethyl 6-bromo-

hexanoate (99.8 lL, 0.555 mmol), followed by

methylation with ethereal diazomethane, aﬀorded 18a

(74.1 mg, 88%).

4.13. (20S)-16-Ene-sulfanyl-butyric acid methyl ester

(16a)

Following the same procedure as described above, treat-

ment of 13a (124.5 mg, 0.279 mmol) with ethyl 4-bromo-

butyrate (127 lL, 0.887 mmol), followed by methylation

with ethereal diazomethane, aﬀorded 16a (118.5 mg,

99%).

¹H NMR(CDCl₃) d: 0.03 (6H, s, 2· Si–Me), 0.89 (9H, s,

Si-t-Bu), 1.13 (3H, s, H-18), 1.35 (3H, d, J = 7.0 Hz, H-

21), 2.29 (1H, m), 2.32 (2H, t,J = 7.5 Hz), 2.42 (2H, t,

J = 7.3 Hz, H-23), 3.37 (1H, q, J = 7.0 Hz, H-20), 3.67

(3H, s, CO₂Me), 4.08 (1H, m, H-8), 5.61 (1H, m, H-

16). MS m/z (%): 454 (M⁺, 3), 439 (1), 397 (6), 365 (1),

292 (3), 235(61), 217 (9), 161 (50), 160 (47), 75 (100).

HRMS m/z: 454.2928 (Calcd for C₂₅H₄₆O₃SSi:

454.2937).

¹H NMR (CDCl₃) d: 0.03 (6H, s, 2· Si–Me), 0.89 (9H, s,

Si-t-Bu), 1.13 (3H, s, H-18), 1.35 (3H, d, J = 7.0 Hz, H-

21), 1.89 (2H, t, J = 7.2 Hz, H-24), 2.28 (1H, m, H-15),

2.43, 2.47 (each 2H, t, J = 7.2 Hz, H-23, 24a), 3.38

(1H, q, J = 7.0 Hz, H-20), 3.67 (3H, s, OMe), 4.08

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

1805

4.17. (20S)-16-Ene-22-thia-24-nor-26,27-dimethyl-8-ol

tert-butyldimethylsilyl ether (19a)

16). MS m/z (%): 396 (M⁺, 5), 339 (2), 324 (4), 292 (4),

267 (6), 235 (60), 161 (69), 160 (39), 75 (100).

To a stirred solution of 14a (661.0 mg, 1.658 mmol) in

dry tetrahydrofuran (THF, 5 mL) at 0 ꢁC was added

4.19. (20S)-16-Ene-22-thia-26,27-dimethyl-8-ol tert-

butyldimethylsilyl ether (20a)

ethylmagnesium

bromide

(EtMgBr,

4.97 mL,

4.974 mmol, 1.0 M solution in THF). After 3 h stirring,

an additional EtMgBr (1.66 mL, 1.66 mmol) was added

and the mixture was stirred for 2 h. The entire reaction

mixture was poured into ice-water containing 2 N HCl

and extracted with AcOEt. The organic layer was

washed with brine, dried over anhydrous MgSO₄, and

evaporated to dryness. The residue was puriﬁed by col-

umn chromatography on silica gel (20 g) with 2%

AcOEt in hexane to aﬀord 19a (494.0 mg, 70%) and

the ethyl-ketone (154.2 mg, 23%).

To a stirred solution of 15a (262.4 mg, 0.639 mmol) in

dry THF (2 mL) at ꢀ78 ꢁC was added ethyllithium

(EtLi, 1.28 mL, 1.920 mmol, about 1.5 M solution in

ether). After being stirred for 1 h, the reaction mixture

was quenched with saturated ammonium chloride

(NH₄Cl), poured into ice-water, and extracted with

AcOEt. The organic phase was washed with brine, dried

over anhydrous MgSO₄, and evaporated to dryness. The

residue was puriﬁed by column chromatography on sil-

ica gel (15 g) with 8% AcOEt in hexane to give 20a

(274.9 mg, 98%).

1

19a: H NMR (CDCl₃) d: 0.03 (6H, s, 2· Si–Me), 0.87

(6H, t, J = 7.4 Hz, H-26a, 27a), 0.89 (9H, s, Si-t-Bu),

1.15 (3H, s, H-18), 1.38 (3H, d, J = 7.0 Hz, H-21), 2.56

(2H, s, H-23), 3.37 (1H, q, J = 7.0 Hz, H-20), 4.08

(1H, m, H-8), 5.63 (1H, m, H-16). ¹³C NMR (CDCl₃)

d: ꢀ5.0, ꢀ4.6, 8.2 (2·), 18.1, 18.2, 19.6, 22.7, 26.0

(3·), 31.0 (3·), 34.7, 35.8, 39.5, 41.6, 46.9, 55.5,

69.2, 74.0, 125.3, 156.3. MS m/z (%): 426 (M⁺, 8),

408 (20), 393 (2), 351 (2), 325 (11), 309 (5), 292 (9),

235 (91), 217 (17), 193 (8), 161 (100), 160 (57). HRMS

m/z: 426.2990 (Calcd for C₂₄H₄₆O₂SSi: 426.2988).

¹H NMR (CDCl₃) d: 0.023, 0.025 (each 3H, s, 2· Si–

Me), 0.856, 0.861 (each 3H, t, J = 7.5 Hz, H-26a, 27a),

0.89 (9H, s, Si-t-Bu), 1.14 (3H, s, H-18), 1.37 (3H, d,

J = 7.0 Hz, H-21), 1.69 (4H, m, H-26, 27), 2.29 (1H,

m, H-15), 2.49 (2H, m, H-23), 3.43 (1H, q, J = 7.0 Hz,

H-20), 4.08 (1H, m, H-8), 5.62 (1 H, m, H-16). ¹³C

NMR (CDCl₃) d: ꢀ5.0, ꢀ4.7, 7.96, 8.02, 18.1 (2·),

19.6, 22.3, 25.5, 25.9 (3·), 30.9, 31.1, 34.7, 35.7, 37.7,

37.8, 46.8, 55.5, 69.2, 74.9, 77.5, 125.3, 156.2. MS m/z

(%): 440 (M⁺, 9), 422 (10), 365 (3), 292 (5), 235 (100),

161 (82), 160 (61). HRMS m/z: 440.3146 (Calcd for

C₂₅H₄₈O₂SSi: 440.3144).

1

Ethyl-ketone: H NMR (CDCl₃) d: 0.022, 0.024 (each

3H, s, 2· Si–Me), 0.89 (9H, s, Si-t-Bu), 1.08 (3H, t,

J = 7.3 Hz, H-26), 1.12 (3H, s, H-18), 1.35 (3H, d,

J = 7.0 Hz, H-21), 2.61 (2H, m, H-25), 3.18 (2H, m,

H-23), 3.39 (1H, q, J = 7.0 Hz, H-20), 4.08 (1H, m,

H-8), 5.64 (1H, m, H-16). MS m/z (%): 396 (M⁺,

35), 381 (6), 339 (8), 324 (32), 292 (9), 267 (37), 235

(73), 161 (100), 160 (52).

4.20. (20R)-16-Ene-22-thia-26,27-dimethyl-8-ol tert-

butyldimethylsilyl ether (20b)

Following the same procedure as described above, treat-

ment of 15b (142.2 mg, 0.346 mmol) with EtLi (0.69 mL,

1.035 mmol, 1.5 M solution in ether) aﬀorded 20b

(141.1 mg, 93%).

4.18. (20R)-16-Ene-22-thia-24-nor-26,27-dimethyl-8-ol

tert-butyldimethylsilyl ether (19b)

¹H NMR (CDCl₃) d: 0.02, 0.03 (each 3H, s, 2· Si–Me),

0.86 (6H, t, J = 7.5 Hz, H-26a, 27a), 0.89 (9H, s, Si-t-

Bu), 1.05 (3H, s, H-18), 1.46 (3H, d, J = 6.9 Hz, H-21,

overlapped with H-26, 27), 2.25 (1H, m, H-15), 2.51

(2H, m, H-23), 3.34 (1H, q, J = 6.9 Hz, H-20), 4.09

(1H, m, H-8), 5.54 (1H, m, H-16). ¹³C NMR (CDCl₃)

d: ꢀ5.0, ꢀ4.6, 8.0 (2·), 18.2, 19.6, 22.5, 24.8, 26.0 (3·),

31.04, 31.11, 31.12, 34.8, 35.9, 36.5, 38.0, 47.0, 54.9,

69.2, 75.0, 77.4, 124.2, 155.6. MS m/z (%): 440 (M⁺,

15), 422 (11), 407 (2), 365 (3), 292 (3), 235 (42), 161

(100), 160 (30). HRMS m/z: 440.3157 (Calcd for

C₂₅H₄₈O₂SSi: 440.3144).

Following the same procedure as described above, treat-

ment of 14b (355.7 mg, 0.892 mmol) with EtMgBr

(2.68 mL, 2.68 mmol) aﬀorded 19b (264.9 mg, 70%)

and the ethyl-ketone (97 mg, 27%).

1

19b: H NMR (CDCl₃) d: 0.02, 0.03 (each 3H, s, 2· Si–

Me), 0.86, 0.88 (each 3H, t, J = 7.2 Hz, H-26a, 27a), 0.89

(9H, s, Si-t-Bu), 1.04 (3H, s, H-18), 1.45 (3H, d,

J = 7.0 Hz, H-21), 2.57, 2.63 (each 1H, d,J = 12.7 Hz,

H-23), 3.31 (1H, q, J = 7.0 Hz, H-20), 4.09 (1H, m, H-

8), 5.55 (1H, m, H-16). ¹³C NMR (CDCl₃) d: ꢀ5.0,

4.6, 8.22, 8.26, 18.2 (2·), 19.6, 22.7, 26.0 (3·), 31.0,

31.07, 31.11, 34.7, 35.9, 37.7, 40.6, 47.0, 54.8, 69.1,

73.8, 124.5, 155.6. MS m/z (%): 426 (M⁺, 7), 408 (6),

351 (2), 325 (5), 309 (15), 292 (6), 235 (79), 217 (7),

193 (4), 161 (46), 160 (58), 75 (100). HRMS m/z:

426.2973 (Calcd for C₂₄H₄₆O₂SSi: 426.2988). Ethyl-ke-

4.21. (20S)-16-Ene-22-thia-24-homo-26,27-dimethyl-8-ol

tert-butyldimethylsilyl ether (21a)

Following the same procedure as described above, treat-

ment of 16a (470.3 mg, 1.102 mmol) with EtLi

(2.204 mL, 3.306 mmol, 1.5 M solution in ether) aﬀor-

ded 21a (498.3 mg, 99%).

1

tone: H NMR (CDCl₃) d: 0.018, 0.024 (each 3H, s,

2· Si–Me), 0.88 (9H, s, Si-t-Bu), 1.08 (3H, t,

J = 7.4 Hz, H-26), 1.03 (3H, s, H-18), 1.42 (3H, d,

J = 6.9 Hz, H-21), 2.53–2.74 (2H, m, H-25), 3.18, 3.23

(each 1H, d, J = 14.0 Hz, H-23), 3.32 (1H, q,

J = 6.9 Hz, H-20), 4.08 (1H, m, H-8), 5.58 (1H, m, H-

¹H NMR (CDCl₃) d: 0.03 (6H, s, 2· Si–Me), 0.86 (6H, t,

J = 7.5 Hz, H-26a, 27a), 0.89 (9H, s, Si-t-Bu), 1.13 (3H,

s, H-18), 1.35 (3H, d, J = 7.0 Hz, H-21), 1.46 (4H, q,

1806

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

J = 7.5 Hz, H-26, 27), 2.28 (1H, m, H-15), 2.44 (2H, t,

J = 7.0 Hz, H-23), 3.39 (1H, q, J = 7.0 Hz, H-20), 4.08

(1H, m, H-8), 5.62 (1H, m, H-16). ¹³C NMR (CDCl₃)

d: ꢀ5.0, ꢀ4.6, 7.97, 8.01, 18.2, 19.5, 22.5, 23.7, 26.0

(3·), 30.96, 31.17, 31.24, 31.9, 34.8, 35.8, 37.76, 37.84,

46.8, 55.5, 69.2, 74.7, 77.5, 124.9, 156.2. MS m/z (%):

454 (M⁺, 8), 436 (5), 379 (2), 292 (5), 235 (100), 161

(100), 160 (65). HRMS m/z: 436.3214 (M⁺ꢀH₂O) (Calcd

for C₂₆H₄₈OSSi: 436.3195).

161 (100), 160 (64). HRMS m/z: 482.3630 (Calcd for

C₂₈H₅₄O₂SSi: 482.3614).

4.25. (20S)-16-Ene-22-thia-24-nor-26,27-dimethyl-8-ol

(24a)

A mixture of 19a (541.0 mg, 1.268 mmol), p-toluenesul-

fonic

acid

monohydrate

(p-TsOH,

723.4 mg,

3.803 mmol) in MeOH (5 mL) was stirred for 5 h at

ambient temperature, poured into ice-water, and ex-

tracted with AcOEt. The organic phase was washed with

5% sodium bicarbonate (NaHCO₃), and brine, and then

dried over anhydrous MgSO₄. Evaporation of the sol-

vent gave the residue, which was puriﬁed by column

chromatography on silica gel (20 g) with 20% AcOEt

in hexane to yield 24a (328.8 mg, 83%).

4.22. (20R)-16-Ene-22-thia-24-homo-26,27-dimethyl-8-ol

tert-butyldimethylsilyl ether (21b)

Following the same procedure as described above, treat-

ment of 16b (292.3 mg, 0.685 mmol) with EtLi (1.37 mL,

2.055 mmol, 1.5 M solution in ether) aﬀorded 21b

(306.1 mg, 98%).

¹H NMR (CDCl₃) d: 0.87 (6H, t, J = 7.5 Hz, H-26a,

27a), 1.19 (3H, s, H-18), 1.39 (3H, d, J = 7.0 Hz, H-

21), 2.33 (1H, m), 2.57 (2H, s, H-23), 3.39 (1H, q,

J = 7.0 Hz, H-20), 4.18 (1H, m, H-8), 5.67 (1H, m, H-

16). ¹³C NMR (CDCl₃) d: 8.19, 8.22, 17.9, 19.0, 22.6,

30.5, 31.0, 31.1, 34.0, 35.5, 39.4, 41.5, 46.5, 54.9, 69.3,

74.0, 125.4, 156.0. MS m/z (%): 312 (M⁺, 4), 294 (6),

276 (16), 178 (13), 160 (73), 145 (100). HRMS m/z:

312.2112 (Calcd for C₁₈H₃₂O₂S: 312.2123).

¹H NMR (CDCl₃) d: 0.02, 0.03 (each 3H, s, 2· Si–Me),

0.86 (6H, t, J = 7.5 Hz, H-26a, 27a), 0.89 (9H, s, Si-t-

Bu), 1.05 (3H, s, H-18), 1.44 (3H, d, J = 7.0 Hz, H-21),

1.45 (4H, q, J = 7.5 Hz, H-26, 27), 2.24 (1H, m, H-15),

2.47 (2H, m, H-23), 3.30 (1H, q, J = 7.0 Hz, H-20),

4.09 (1H, m, H-8), 5.53 (1 H, m, H-16). ¹³C NMR

(CDCl₃) d: ꢀ5.0, ꢀ4.6, 8.0 (2·), 18.2, 19.6, 22.7, 23.9,

26.0 (3·), 31.05, 31.18, 31.24 (2·), 34.8, 35.9, 36.4,

37.9, 47.0, 54.9, 69.2, 74.7, 77.4, 124.0, 155.9. MS m/z

(%): 454 (M⁺, 7), 436 (3), 379 (2), 292 (5), 235 (100),

161 (73), 160 (65). HRMS m/z: 454.3325 (Calcd for

C₂₆H₅₀O₂SSi: 454.3301).

4.26. (20R)-16-Ene-22-thia-24-nor-26,27-dimethyl-8-ol

(24b)

Following the same procedure as described above, treat-

ment of 19b (286.4 mg, 0.671 mmol) with p-TsOH

(255.3 mg, 1.342 mmol) aﬀorded 24b (181.5 mg, 84%).

4.23. (20S)-16-Ene-22-thia-24,24-dihomo-26,27-dimethyl-

8-ol tert-butyldimethylsilyl ether (22a)

Following the same procedure as described above, treat-

ment of 17a (89.9 mg, 0.204 mmol) with EtLi (0.544 mL,

0.816 mmol, 1.5 M solution in ether) aﬀorded 22a

(86.3 mg, 90%).

¹H NMR (CDCl₃) d: 0.86, 0.88 (each 3H, t, J = 7.5 Hz,

H-26a, 27a), 1.07 (3H, s, H-18), 1.46 (3H, d, J = 7.0 Hz,

H-21), 2.26 (1H, m), 2.57, 2.63 (each 1H, d, J = 12.7 Hz,

H-23), 3.31 (1H, q, J = 7.0 Hz, H-20), 4.18 (1H, m, H-8),

5.59 (1H, m, H-16). ¹³C NMR (CDCl₃) d: 8.20, 8.25,

17.9, 19.0, 22.6, 30.6, 31.0, 31.1, 34.0, 35.7, 37.6, 40.5,

46.7, 54.2, 69.2, 73.8, 124.5, 155.3. MS m/z (%): 312

(M⁺, 8), 294 (2), 276 (15), 178 (7), 160 (71), 145 (100).

¹H NMR (CDCl₃) d: 0.03 (6H, s, 2· Si–Me), 0.85 (6H, t,

J = 7.5 Hz, H-26a, 27a), 0.89 (9H, s, Si-t-Bu), 1.13 (3H,

s, H-18), 1.36 (3H, d, J = 6.9 Hz, H-21), 1.46 (4H, q,

J = 7.5 Hz, H-26, 27), 2.28 (1H, m), 2.44 (2H, t,

J = 7.4 Hz, H-23), 3.38 (1H, q, J = 6.9 Hz, H-20), 4.08

(1H, m, H-8), 5.61 (1 H, m, H-16). MS m/z (%): 468

(M⁺, 7), 450 (3), 393 (2), 292 (8), 235 (100), 161 (82),

160 (71). HRMS m/z: 468.3470 (Calcd for C₂₇H₅₂OSSi:

468.3457).

4.27. (20S)-16-Ene-22-thia-24-nor-26,27-dimethyl-8-one

(25a)

To a stirred solution of (COCl)₂(187 lL, 2.144 mmol) in

dry CH₂Cl₂(2 mL) at ꢀ78 ꢁC was added DMSO (303 lL,

4.270 mmol) in dry CH₂Cl₂(0.5 mL). After being stirred

for 10 min, a solution of 24a (304.7 mg, 0.975 mmol) in

dry CH₂Cl₂(3 mL) was added dropwise. The reaction

mixture was stirred at ꢀ78 ꢁC for 10 min, and Et₃N

(1.36 mL, 9.757 mmol) was added. The entire mixture

was stirred at ꢀ78 ꢁC for 15 min, and at 0 ꢁC for

30 min, quenched with ice water, and extracted with

CH₂Cl₂. The CH₂Cl₂extract was washed with brine,

dried over anhydrous MgSO₄, and evaporated to dryness.

The residue was chromatographed on silica gel (15 g) with

15% AcOEt in hexane to give 25a (251.2 mg, 83%).

4.24. (20S)-16-Ene-22-thia-24,24,24-trihomo-26,27-

dimethyl-8-ol tert-butyldimethylsilyl ether (23a)

Following the same procedure as described above, treat-

ment of 18a (144.8 mg, 0.318 mmol) with EtLi

(0.849 mL, 1.274 mmol, 1.5 M solution in ether) aﬀor-

ded 23a (99.9 mg, 82%).

¹H NMR (CDCl₃) d: 0.03 (6H, s, 2· Si–Me), 0.85 (6H, t,

J = 7.5 Hz, H-26a, 27a), 0.89 (9H, s, Si-t-Bu), 1.13 (3H,

s, H-18), 1.36 (3H, d, J = 7.0 Hz, H-21), 2.29 (1H, m),

2.43 (2H, t, J = 7.3 Hz, H-23), 3.37 (1H, q, J = 7.0 Hz,

H-20), 4.08 (1H, m, H-8), 5.61 (1H, m, H-16). MS m/z

(%): 482 (M⁺, 11), 464 (3), 292 (5), 235 (98), 217 (8),

¹H NMR (CDCl₃) d: 0.87, 0.88 (each 3H, t, J = 7.5 Hz,

H-26a, 27a), 0.97 (3H, s, H-18), 1.45 (3H, d, J = 7.0 Hz,

H-21), 2.54, 2.57 (each 1H, d, J = 12.9 Hz, H-23), 2.87

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

1807

(1H, dd, J = 10.5, 6.3 Hz, H-14), 3.46 (1H, q, J = 7.0 Hz,

H-20), 5.59 (1H, m, H-16). ¹³C NMR (CDCl₃) d: 8.4

(2·), 18.4, 22.0, 24.3, 27.6, 31.3 (2·), 34.6, 40.0, 40.8,

41.3, 53.6, 64.0, 74.2, 125.8, 153.8, 210.9. MS m/z (%):

310 (M⁺, 13), 292 (49), 209 (29), 177 (100), 176 (50),

161 (27). HRMS m/z: 310.1944 (Calcd for C₁₈H₃₀O₂S:

310.1966).

¹H NMR (CDCl₃) d: 0.60 (6H, q, J = 7.9 Hz, 3· Si–

CH₂CH₃), 0.86 (3H, s, H-18), 0.867, 0.873 (each 3H, t,

J = 7.3 Hz, H-26a, 27a), 0.96 (9H, t, J = 7.9 Hz, 3· Si–

CH₂CH₃), 1.46 (3H, d, J = 7.0 Hz, H-21), 2.54, 2.58

(each 1H, d, J = 11.6 Hz, H-23), 2.90 (1H, dd,

J = 10.6, 6.4 Hz), 3.32 (1H, q, J = 7.0 Hz, H-20), 5.50

(1H, m, H-16). ¹³C NMR (CDCl₃) d: 7.2 (x 3), 7.5

(3·), 8.3, 8.4, 17.8, 22.1, 24.3, 27.4, 32.1 (2·), 34.7,

37.9, 39.3, 40.7, 54.0, 62.9, 78.4, 123.9, 153.5, 211.0.

4.28. (20R)-16-Ene-22-thia-24-nor-26,27-dimethyl-8-one

(25b)

4.31. (20S)-16-Ene-22-thia-26,27-dimethyl-8,25-diol

(27a)

The Swern oxidation of 24b (162.3 mg, 0.519 mmol) was

carried out according to the same procedure described

for the preparation of 25a to give 25b (125.6 mg, 78%).

A mixture of 20a (140.7 mg, 0.319 mmol), Et₃N (50 lL,

0.359 mmol), and tetrabutylammonium ﬂuoride

(0.957 mL 0.957 mmol, 1.0 M solution in THF) in dry

THF (1.5 mL) was stirred at 50 ꢁC for 70 h. The mixture

was poured into ice water and extracted with AcOEt.

The organic phase was washed with brine and dried over

anhydrous MgSO₄. Removal of the solvent in vacuo

aﬀorded the residue, which was puriﬁed by chromatog-

raphy on silica gel (8 g) with 8% MeOH in AcOEt to

yield 27a (94.0 mg, 90%).

¹H NMR (CDCl₃) d: 0.85 (3H, s, H-18), 0.88, 0.89 (each

3H, t, J = 7.5 Hz, H-26a, 27a), 1.48 (3H, d, J = 7.0 Hz,

H-21), 2.60, 2.62 (each 1H, d, J = 13.2 Hz, H-23), 2.91

(1H, dd, J = 10.6, 6.6 Hz), 3.35 (1H, q, J = 7.0 Hz, H-

20), 5.56 (1H, m, H-16). ¹³C NMR (CDCl₃) d: 8.20,

8.23, 17.7, 22.5, 24.2, 27.4, 30.98, 31.14, 34.8, 38.5,

40.5, 40.6, 53.9, 62.8, 74.0, 124.6, 153.3, 210.9. MS m/z

(%): 310 (M⁺, 16), 292 (24), 209 (29), 177 (78), 176

(100), 161 (63).

¹H NMR (CDCl₃) d: 0.858, 0.861 (each 3H, t,

J = 7.5 Hz, H-26a, H-27a), 1.19 (3H, s, H-18), 1.38

(3H, d, J = 7.0 Hz, H-21), 1.47 (4H, q, J = 7.4 Hz, H-

26, H-27), 2.34 (1H, m, H-15), 2.49 (2H, m, H-23),

3.43 (1H, q, J = 7.0 Hz, H-20), 4.13 (1H, m, H-8), 5.66

(1H, m, H-16).

4.29. (20S)-16-Ene-22-thia-24-nor-25-(triethylsilyl)oxy-

26,27-dimethyl-8-one (26a)

To a stirred solution of 25a (38.7 mg, 0.125 mmol) in

dry N,N-dimethylformamide (DMF, 0.8 mL) were

added imidazole (50.9 mg, 0.748 mmol) and chlorotri-

ethylsilane (TESCl, 62.8 lL, 0.374 mmol) at 0 ꢁC and

the mixture was stirred for 5 h at ambient temperature.

Additional imidazole (50.9 mg) and TESCl (62.8 lL)

were added, the whole mixture was stirred for 16 h

at ambient temperature, poured into ice water, and

then extracted with AcOEt: hexane (1:1, v/v). The or-

ganic phase was washed with brine, dried over anhy-

drous MgSO₄, and evaporated to dryness. The

residue was puriﬁed by column chromatography on

silica gel (6 g) with 5% AcOEt in hexane to give 26a

(35.4 mg, 67%).

4.32. 16,20-Diene-22-thia-26,27-dimethyl-8,25-diol (29)

and 16,20-diene-22-thia-25-hydroxy-26,27-dimethyl-8-

one (30)

To a stirred solution of (COCl)₂(54.7 lL, 0.627 mmol)

in dry CH₂Cl₂(1 mL) at ꢀ78 ꢁC was added a solution

of DMSO (88.7 lL, 1.250 mmol) in dry CH₂Cl₂

(0.5 mL). After being stirred for 10 min, a solution of

27a (93 mg, 0.285 mmol) in dry CH₂Cl₂(3 mL) was

added dropwise. The reaction mixture was stirred for

15 min at ꢀ78 ꢁC, and Et₃N (397.2 lL, 2.850 mmol)

was added. The whole mixture was stirred at ꢀ78 ꢁC

for 15 min and at 0 ꢁC for 30 min, quenched with ice

water, and extracted with CH₂Cl₂. The CH₂Cl₂extract

was washed with brine, dried over MgSO₄, and concen-

trated in vacuo. The residue was puriﬁed by chromatog-

raphy on silica gel (8 g) using 10% AcOEt in hexane to

aﬀord 30 (32.2 mg, 35%), and 15% AcOEt in hexane

to give 29 (55.3 mg, 60%).

¹H NMR(CDCl₃) d: 0.58 (6H, q, J = 7.9 Hz, 3· Si–

CH₂CH₃), 0.85, 0.86 (each 3H, t, J = 7.3 Hz, H-26a,

27a), 0.95 (9H, t, J = 8.0 Hz, 3· Si–CH₂CH₃), 0.97

(3H, s, H-18), 1.42 (3H, d, J = 7.0 Hz, H-21), 2.50,

2.53 (each 1H, d, J = 11.7 Hz, H-23), 2.87 (1H, dd,

J = 10.6, 6.4 Hz), 3.43 (1H, q, J = 7.0 Hz, H-20), 5.57

(1H, m, H-16). ¹³C NMR (CDCl₃) d: 7.2 (3·), 7.4

(3·), 8.3, 8.5, 18.0, 21.5, 24.1, 27.3, 31.9, 32.3, 34.4,

39.3, 40.1, 40.6, 53.4, 63.9, 78.5, 125.1, 153.8, 210.9.

MS m/z (%): 424 (M⁺, 1), 395 (3), 338 (8), 292 (10),

263 (1), 201 (100), 177 (14). HRMS m/z: 424.2842 (Calcd

for C₂₄H₄₄O₂SSi: 424.2831).

29: ¹H NMR (CDCl₃) d: 0.86, 0.88 (each 3 H, t,

J = 7.5 Hz, H-26a, 27a), 1.24 (3H, s, H-18), 1.50

(4H, q, J = 7.5 Hz, H-26, H-27), 2.36 (1H, m, H-15),

4.19 (1H, m, H-8), 5.00, 5.32 (each 1H, s, H-21),

6.05 (1H, m, H-16). MS m/z (%): 324 (M⁺, 84), 306

(41), 288 (28), 277 (31), 210 (56). 30 ¹H NMR

(CDCl₃) d: 0.87, 0.89 (each 3H, t, J = 7.5 Hz, H-26a,

27a), 1.00 (3H, s, H-18), 1.51 (4H, q, J = 7.5 Hz, H-

26, 27), 2.53 (1H, m, H-15), 2.96 (1H, dd, J = 10.9,

6.5 Hz), 5.03, 5.29 (each 1H, s, H-21), 5.98 (1H, m,

H-16). MS m/z (%): 322 (M⁺, 67), 304 (69), 275

(54), 233 (23), 208 (51).

4.30. (20R)-16-Ene-22-thia-24-nor-25-(triethylsilyl)oxy-

26,27-dimethyl-8-one (26b)

Following the same procedure as described above, treat-

ment of 25b (21.3 mg, 0.069 mmol) with TESCl

(69.1 lL, 0.412 mmol) in the presence of imidazole

(56.0 mg, 0.823 mmol) aﬀorded 26b (17.7 mg, 61%).

1808

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

4.33. (20S)-16-Ene-22-thia-25-(methoxymethyl)oxy-

26,27-dimethyl-8-ol tert-butyldimethylsilyl ether (31a)

m, H-23), 3.39 (3H, s, OMe, overlapped with H-20),

4.08 (1H, m, H-8), 4.65 (2H, s, OCH₂O), 5.61 (1H, m,

H-16). ¹³C NMR (CDCl₃) d: ꢀ5.0, ꢀ4.6, 7.93, 7.96,

18.17, 18.21, 19.5, 22.5, 23.6, 26.0 (3·), 28.0 (2·), 31.0,

31.8, 34.8, 34.9, 35.8, 37.8, 46.8, 55.5, 55.9, 69.2, 80.9,

90.6, 124.9, 156.2. MS m/z (%): 498 (M⁺, 4), 466 (2),

436 (7), 292 (5), 235 (100), 161 (61), 160 (61). HRMS

m/z: 498.3575 (Calcd for C₂₈H₅₄O₃SSi: 498.3563).

To a stirred solution of 20a (366.9 mg, 0.832 mmol) in

dry CH₂Cl₂(3 mL) were added N,N-diisopropylethyl-

amine (ⁱPro₂NEt, 725 lL, 4.160 mmol) and chloro-

methyl methyl ether (MOMCl, 158 lL, 2.080 mmol) at

0 ꢁC, the mixture was stirred at amb. temperature for

6 h, poured into a mixture of 2 N HCl and ice water,

and then extracted with CH₂Cl₂. The organic layer

was washed with 5% NaHCO₃and brine, dried over

anhydrous MgSO₄, and evaporated to dryness. The res-

idue was puriﬁed by column chromatography on silica

gel (22 g) with 3% AcOEt in hexane to aﬀord 31a

(309.1 mg, 77%).

4.36. (20R)-16-Ene-22-thia-24-homo-25-(methoxymethyl)

oxy-26,27-dimethyl-8-ol tert-butyldimethylsilyl ether (32b)

Following the same procedure as described above, treat-

ment of 21b (290.1 mg, 0.638 mmol) with MOMCl

(121 lL, 1.593 mmol) in the presence of ⁱPro₂NEt

(556 lL, 3.192 mmol) aﬀorded 32b (294.8 mg, 93%).

¹H NMR (CDCl₃) d: 0.021, 0.025 (each 3H, s, 2· Si–

Me), 0.83 (6H, t, J = 7.5 Hz, H-26a, H-27a), 0.88 (9H,

s, Si-t-Bu), 1.15 (3H, s, H-18), 1.37 (3H, d, J = 7.0 Hz,

H-21), 2.28 (1H, m, H-15), 2.42 (2H, m, H-23), 3.38

(3H, s, –OMe), 3.40 (1H, q, J = 7.0 Hz, H-20), 4.08

(1H, m, H-8), 4.65 (2H, s, OCH₂O), 5.62 (1H, m, H-

16). ¹³C NMR (CDCl₃) d: ꢀ5.0, ꢀ4.6, 8.0 (2·), 18.2,

19.6, 22.4, 25.2, 26.0 (4·), 27.9, 28.0, 31.0, 34.7, 35.6,

35.8, 37.9, 46.9, 55.6, 55.9, 69.2, 81.0, 90.6, 125.0,

156.1. MS m/z (%): 484 (M⁺, 3), 452 (2), 422 (5), 407

(1), 365 (1), 292 (4), 235 (100), 161 (63), 160 (67). HRMS

m/z: 484.3429 (Calcd for C₂₇H₅₂O₃SSi: 484.3406).

¹H NMR (CDCl₃) d: 0.02, 0.03 (each 3H, s, 2· Si–Me),

0.83 (6H, t, J = 7.4 Hz, H-26a, 27a), 0.89 (9H, s, Si-t-

Bu), 1.05 (3H, s, H-18), 1.44 (3H, d, J = 7.0 Hz, H-21),

1.51 (4H, q, J = 7.4 Hz, H-26, 27), 2.24 (1H, m, H-15),

2.45 (2H, m, H-23), 3.30 (1H, q, J = 7.0 Hz, H-20),

3.39 (3H, s, OMe), 4.08 (1 H, m, H-8), 4.65 (2H, s,

OCH₂O), 5.53 (1H, m, H-16). ¹³C NMR (CDCl₃) d:

ꢀ5.0, ꢀ4.6, 8.0 (2·), 18.2 (2·), 19.6, 22.7, 23.8, 26.0

(3·), 28.0 (2·), 31.1, 31.2, 34.8, 35.0, 35.9, 36.5, 47.0,

54.9, 55.9, 69.2, 81.0, 90.6, 124.0, 155.9. MS m/z (%):

498 (M⁺, 4), 466 (2), 436 (9), 292 (4), 235 (74), 161

(73), 160 (48), 75 (100). HRMS m/z: 498.3559 (Calcd

for C₂₈H₅₄O₃SSi: 498.3563).

4.34. (20R)-16-Ene-22-thia-25-(methoxymethyl)oxy-

26,27-dimethyl-8-ol tert-butyldimethylsilyl ether (31b)

4.37. (20S)-16-Ene-22-thia-24,24-dihomo-25-(methoxy-

methyl)oxy-26,27-dimethyl-8-ol tert-butyldimethylsilyl

ether (33a)

Following the same procedure as described above, treat-

ment of 20b (113.7 mg, 0.258 mmol) with MOMCl

(39 lL, 0.513 mmol) in the presence of ⁱPro₂NEt

(180 lL, 1.033 mmol) yielded 31b (124.0 mg, 99%).

Following the same procedure as described above, treat-

ment of 22a (57.4 mg, 0.122 mmol) with MOMCl

(37.1 lL, 0.488 mmol) in the presence of ⁱPro₂NEt

(170.3 lL, 0.978 mmol) yielded 33a (60.8 mg, 97%).

¹H NMR (CDCl₃) d: 0.02, 0.03 (each 3H, s, 2· Si–Me),

0.84 (6H, t, J = 7.5 Hz, H-26a, 27a), 0.89 (9H, s, Si-t-

Bu), 1.05 (3H, s, H-18), 1.46 (3H, d, J = 7.0 Hz, H-21),

2.25 (1H, m, H-15), 2.47 (2H, m, H-23), 3.32 (1H, q,

J = 7.0 Hz, H-20), 3.39 (3H, s, OMe), 4.09 (1H, m, H-

8), 4.65 (2H, s, OCH₂O), 5.53 (1H, m, H-16). ¹³C

NMR (CDCl₃) d: ꢀ5.0, ꢀ4.6, 8.0 (2·), 18.2, 19.6, 22.6,

24.5, 26.0 (4·), 27.9 (2·), 31.0, 34.8, 35.8, 35.9, 36.5,

47.0, 54.9, 55.9, 69.2, 81.0, 90.6, 124.0, 155.7. MS m/z

(%): 484 (M⁺, 7), 452 (3), 422 (11), 407 (1), 365 (2),

292 (15), 235 (100), 161 (56), 160 (86). HRMS m/z:

484.3417 (Calcd for C₂₇H₅₂O₃SSi: 484.3406).

¹H NMR (CDCl₃) d: 0.02 (6H, s, 2· Si–Me), 0.82 (6H, t,

J = 7.5 Hz, H-26a, 27a), 0.89 (9H, s, Si-t-Bu), 1.13 (3H,

s, H-18), 1.35 (3H, d, J = 7.0 Hz, H-21), 1.51 (4H, q,

J = 7.5 Hz, H-26, 27), 2.44 (2H, t, J = 7.3 Hz, H-23),

3.39 (3 H, s, –CH₂OCH₃, overlapped with H-20), 4.08

(1 H, m, H-8), 4.65 (2H, s, –CH₂OCH₃), 5.61 (1 H, m,

H-16). MS m/z (%): 512 (M⁺, 6), 480 (3), 450 (9), 292

(3), 235 (100), 161 (83), 160 (58). HRMS m/z: 512.3749

(Calcd for C₂₉H₅₆O₃SSi: 512.3719).

4.35. (20S)-16-Ene-22-thia-24-homo-25-(methoxy-

methyl)oxy-26,27-dimethyl-8-ol tert-butylodimethylsilyl

ether (32a)

4.38. (20S)-16-Ene-22-thia-24,24-24-trihomo-25-(methoxy-

methyl)oxy-26,27-dimethyl-8-ol tert-butyldimethylsilyl

ether (34a)

Following the same procedure as described above, treat-

ment of 21a (469.4 mg, 1.032 mmol) with MOMCl

(235 lL, 3.094 mmol) in the presence of ⁱPro₂NEt

(1.08 mL, 6.200 mmol) gave 32a (492.1 mg, 96%).

Following the same procedure as described above, treat-

ment of 23a (66.0 mg, 0.137 mmol) with MOMCl

(31.1 lL, 0.409 mmol) in the presence of ⁱPro₂NEt

(142.9 lL, 0.820 mmol) gave 34a (70.5 mg, 98%).

¹H NMR (CDCl₃) d: 0.03 (6H, s, 2· Si–Me), 0.83 (6H, t,

J = 7.5 Hz, H-26a, 27a), 0.89 (9H, s, Si-t-Bu), 1.13 (3H,

s, H-18), 1.35 (3H, d, J = 7.0 Hz, H-21), 1.51 (4H, q,

J = 7.5 Hz, H-26, 27), 2.28 (1H, m, H-15), 2.41 (2H,

¹H NMR (CDCl₃) d: 0.03 (6H, s, 2· Si–Me), 0.83 (6H, t,

J = 7.5 Hz, H-26a, 27a), 0.89 (9H, s, Si-t-Bu), 1.13 (3H,

s, H-18), 1.36 (3H, d, J = 7.0 Hz, H-21), 2.29 (1H, m),

2.43 (2H, t, J = 7.3 Hz, H-23), 3.39 (3H, s, –CH₂OCH₃,

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

1809

overlapped with H-20), 4.08 (1H, m, H-8), 4.65 (2H, s, –

CH₂OCH₃), 5.61 (1H, m, H-16). MS m/z (%): 526 (M⁺,

8), 494 (4), 464 (9), 292 (4), 235 (100), 217 (6), 161 (100),

160 (56). HRMS m/z: 526.3884 (Calcd for C₃₀H₅₈O₃SSi:

526.3876).

34.0, 34.9, 35.5, 37.7, 46.4, 55.0, 55.9, 69.3, 80.9, 90.6,

125.0, 155.9.MS m/z (%): 384 (M⁺, 2), 322 (3), 304 (2),

160 (61), 145 (100). HRMS m/z: 384.2695 (Calcd for

C₂₂H₄₀O₃S: 384.2698).

4.42. (20R)-16-Ene-22-thia-24-homo-25-(methoxy-

methyl)oxy-26,27-dimethyl-8-ol (36b)

4.39. (20S)-16-Ene-22-thia-25-(methoxymethyl)oxy-

26,27-dimethyl-8-ol (35a)

Following the same procedure as described above, treat-

ment of 32b (294.8 mg, 0.591 mmol) with TBAF

(1.77 mL, 1.773 mmol) yielded 36b (222.5 mg, 98%).

A mixture of 31a (309.1 mg, 0.638 mmol), Et₃N (100 lL,

0.717 mmol), and TBAF (1.914 mL, 1.914 mmol, 1.0 M

solution in THF) in dry THF (3 mL) was stirred at

50 ꢁC for 91 h. The mixture was poured into ice water

and extracted with AcOEt. The organic phase was

washed with brine and dried over anhydrous MgSO₄.

Removal of the solvent in vacuo aﬀorded the residue,

which was puriﬁed by chromatography on silica gel

(15 g) with 30% AcOEt in hexane to yield 35a

(207.9 mg, 88%).

¹H NMR (CDCl₃) d: 0.83 (6H, t, J = 7.5 Hz, H-26a,

27a), 1.08 (3H, s, H-18), 1.46 (3H, d, J = 7.0 Hz, H-

21), 1.52 (4H, q, J = 7.5 Hz, H-26, 27), 2.29 (1H, m,

H-15), 2.45 (2H, m, H-23), 3.31 (1H, q, J = 7.0 Hz, H-

20), 3.39 (3H, s, OMe), 4.18 (1H, m, H-8), 4.65 (2H, s,

OCH₂O), 5.56 (1H, m, H-16). ¹³C NMR (CDCl₃) d:

7.9 (2·), 17.9, 18.9, 22.5, 23.7, 28.00, 28.02, 30.5, 31.1,

34.1, 35.0, 35.6, 36.4, 46.6, 54.3, 55.9, 69.3, 80.9, 90.6,

124.0, 155.6. MS m/z (%): 384 (M⁺, 1), 352 (1), 322

(2), 304 (1), 178 (6), 160 (58), 145 (100). HRMS m/z:

384.2686 (Calcd for C₂₂H₄₀O₃S: 384.2698).

¹H NMR (CDCl₃) d: 0.83 (6H, t, J = 7.5 Hz, H-26a, H-

27a), 1.19 (3H, s, H-18), 1.38 (3H, d, J = 7.0 Hz, H-21),

2.33 (1H, m, H-15), 2.42 (2H, m, H-23), 3.39 (3H, s, –

OMe, overlapped with H-20), 4.18 (1H, m, H-8), 4.65

(2H, s, OCH₂O), 5.66 (1H, m, H-16). ¹³C NMR (CDCl₃)

d: 8.0 (x 2), 17.9, 18.9, 22.2, 25.1, 27.9, 28.0, 30.4, 34.0,

35.4, 35.5, 37.8, 46.5, 55.0, 55.9, 69.3, 80.9, 90.6, 125.0,

155.9. MS m/z (%): 370 (M⁺, 4), 338 (3), 308 (6), 178

(16), 160 (62), 145 (100). HRMS m/z: 370.2570 (Calcd

for C₂₁H₃₈O₃S: 370.2542).

4.43. (20S)-16-Ene-22-thia-24,24-dihomo-25-(methoxy-

methyl)oxy-26,27-dimethyl-8-ol (37a)

Following the same procedure as described above, treat-

ment of 33a (62.8 mg, 0.122 mmol) with TBAF (489 lL,

0.489 mmol) gave 37a (46.3 mg, 95%).

4.40. (20R)-16-Ene-22-thia-25-(methoxymethyl)oxy-

26,27-dimethyl-8-ol (35b)

¹H NMR (CDCl₃) d: 0.83 (6H, t, J = 7.5 Hz, H-26a,

27a), 1.18 (3H, s, H-18), 1.37 (3H, d, J = 7.0 Hz, H-

21), 1.51 (4H, q, J = 7.5 Hz, H-26, 27), 2.33 (1H, m),

2.44 (2H, t, J = 7.2 Hz, H-23), 3.39 (3H, s, –CH₂OCH₃,

overlapped with H-20), 4.17 (1H, m, H-8), 4.65 (2H, s, –

CH₂OCH₃), 5.65 (1H, m, H-16). MS m/z (%): 398 (M⁺,

6), 336 (8), 178 (20), 160 (60), 145 (100). HRMS m/z:

398.2828 (Calcd for C₂₃H₄₂O₃S: 398.2855).

Following the same procedure as described above, treat-

ment of 31b (120.0 mg, 0.247 mmol) with TBAF

(742 lL, 0.742 mmol) gave 35b (87.5 mg, 95%).

¹H NMR (CDCl₃) d: 0.84 (6H, t, J = 7.5 Hz, H-26a,

27a), 1.09 (3H, s, H-18), 1.47 (3H, d, J = 7.0 Hz, H-

21), 2.29 (1H, m, H-15), 2.44 (2H, m, H-23), 3.32 (1H,

q, J = 7.0 Hz, H-20), 3.39 (3H, s, OMe), 4.17 (1H, m,

H-8), 4.66 (2H, s, OCH₂O), 5.56 (1H, m, H-16). ¹³C

NMR (CDCl₃) d: 7.9 (2·), 17.9, 18.9, 22.5, 24.5, 27.9

(2·), 30.5, 34.0, 35.6, 35.7, 36.4, 46.6, 54.3, 55.9, 69.2,

80.9, 90.6, 123.9, 155.5. MS m/z (%): 370 (M⁺, 2), 338

(5), 308 (4), 178 (21), 160 (78), 145 (100). HRMS m/z:

370.2558 (Calcd for C₂₁H₃₈O₃S: 370.2542).

4.44. (20S)-16-Ene-22-thia-24,24,24-trihomo-25-(methoxy-

methyl)oxy-26,27-dimethyl-8-ol (38a)

Following the same procedure as described above, treat-

ment of 34a (73.4 mg, 0.139 mmol) with TBAF (557 lL,

0.557 mmol) gave 38a (52.7 mg, 92%).

¹H NMR (CDCl₃) d: 0.82 (6H, t, J = 7.5 Hz, H-26a,

27a), 1.18 (3H, s, H-18), 1.37 (3H, d, J = 7.0 Hz, H-

21), 1.51 (4H, q, J = 7.5 Hz, H-26, 27), 2.33 (1H, m),

2.43 (2H, t, J = 7.3 Hz, H-23), 3.39 (3H, s, –CH₂OCH₃,

overlapped with H-20), 4.18 (1H, m, H-8), 4.65 (2H, s, –

CH₂OCH₃), 5.64 (1H, m, H-16). MS m/z (%): 412 (M⁺,

9), 380 (4), 350 (11), 178 (32), 161 (73), 160 (32), 145

(100). HRMS m/z: 412.3008 (Calcd for C₂₄H₄₄O₃S:

412.3011).

4.41. (20S)-16-Ene-22-thia-24-homo-25-(methoxy-

methyl)oxy-26,27-dimethyl-8-ol (36a)

Following the same procedure as described above, treat-

ment of 32a (476.3 mg, 0.955 mmol) with TBAF

(2.86 mL, 2.864 mmol) gave 36a (360.0 mg, 98%).

¹H NMR (CDCl₃) d: 0.83 (6H, t, J = 7.5 Hz, H-26a,

27a), 1.18 (3H, s, H-18), 1.37 (3H, d, J = 7.0 Hz, H-

21), 1.51 (4H, q, J = 7.5 Hz, H-26, 27), 2.33 (1H, m,

H-15), 2.41 (2H, m, H-23), 3.39 (3H, s, OMe, over-

lapped with H-20), 4.17 (1 H, m, H-8), 4.65 (2H, s,

OCH₂O), 5.61 (1H, m, H-16). ¹³C NMR (CDCl₃) d:

7.91, 7.94, 17.9, 18.8, 22.3, 23.5, 28.0 (2·), 30.4, 31.7,

4.45. (20S)-16-Ene-22-thia-25-(methoxymethyl)oxy-

26,27-dimethyl-8-one (39a)

A

mixture of 35a (98.7 mg, 0.266 mmol), Celite

(400.8 mg), and pyridinium dichromate (PDC,

200.4 mg, 0.533 mmol) in dry CH₂Cl₂(2.5 mL) was stir-

1810

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

red for 3 h at ambient temperature and a reaction mix-

ture was applied to the top of the silica gel column

(15 g). The column was eluted with 50% AcOEt in hex-

ane to give 39a (75.3 mg, 77%).

(84). HRMS m/z: 382.2531 (Calcd for C₂₂H₃₈O₃S:

382.2542).

4.48. (20R)-16-Ene-22-thia-24-homo-25-(methoxymethyl)

oxy-26,27-dimethyl-8-one (40b)

¹H NMR (CDCl₃) d: 0.829, 0.832 (each 3H, t,

J = 7.5 Hz, H-26a, 27a), 0.97 (3H, s, H-18), 1.44 (3H,

d, J = 7.0 Hz, H-21), 2.29 (1H, m), 2.42 (2H, m), 2.87

(1H, dd, J = 10.6, 6.4 Hz, H-15), 3.38 (3H, s, OMe),

3.47 (1H, q, J = 7.0 Hz, H-20), 4.63, 4.65 (each 1H, d,

J = 7.4 Hz, OCH₂O), 5.57 (1H, m, H-16). ¹³C NMR

(CDCl₃) d: 7.97, 7.99, 18.1, 21.5, 24.1, 24.6, 27.3,

27.92, 27.96, 34.3, 35.5, 38.4, 40.6, 53.4, 55.9, 63.9,

80.9, 90.6, 125.2, 153.6, 210.9. MS m/z (%): 368 (M⁺,

5), 336 (6), 306 (17), 176 (100), 161 (90). HRMS m/z:

368.2408 (Calcd for C₂₁H₃₆O₃S: 368.2385).

The Swern oxidation of 36b (210.4 mg, 0.547 mmol) was

carried out according to the same procedure described

for the preparation of 36a to give 40b (178.5 mg, 85%).

¹H NMR (CDCl₃) d: 0.84 (6H, t, J = 7.5 Hz, H-26a,

27a), 0.86 (3H, s, H-18), 1.47 (3H, d, J = 7.0 Hz, H-

21), 1.52 (4H, q, J = 7.5 Hz, H-26, 27), 2.91 (1H, dd,

J = 10.6, 6.5 Hz, H-14), 3.35 (1H, q, J = 7.0 Hz, H-20),

3.39 (3H, s, OMe), 4.66 (2H, s, OCH₂O), 5.53 (1H, m,

H-16). ¹³C NMR (CDCl₃) d: 8.2 (2·), 17.9, 22.5, 23.9,

24.5, 27.6, 28.2 (2·), 31.4, 35.0, 35.2, 37.6, 40.9, 54.2,

56.1, 63.1, 81.1, 90.8, 124.3, 153.8, 211.2. MS m/z (%):

382 (M⁺, 7), 350 (9), 320 (26), 177 (100), 176 (33).

HRMS m/z: 382.2523 (Calcd for C₂₂H₃₈O₃S: 382.2542).

4.46. (20R)-16-Ene-22-thia-25-(methoxymethyl)oxy-

26,27-dimethyl-8-one (39b)

Following the same procedure as described above, treat-

ment of 35b (48.7 mg, 0.131 mmol) with PDC (98.9 mg,

0.263 mmol) and Celite (200 mg) aﬀorded 39b (39.0 mg,

81%).

4.49. (20S)-16-Ene-22-thia-24,24-dihomo-25-(methoxy-

methyl)oxy-26,27-dimethyl-8-one (41a)

The Swern oxidation of 37a (46.3 mg, 0.116 mmol) was

carried out according to the same procedure described

for the preparation of 36a to give 41a (42.9 mg, 93%).

¹H NMR (CDCl₃) d: 0.84 (6H, t, J = 7.5 Hz, H-26a,

27a), 0.86 (3H, s, H-18), 1.48 (3H, d, J = 7.0 Hz, H-

21), 2.90 (1H, m, H-14), 3.39 (3H, s, OMe, overlapped

with H-20), 4.66 (2H, s, OCH₂O), 5.53 (1H, m, H-16).

¹³C NMR (CDCl₃) d: 8.2 (2·), 17.9, 22.5, 24.5, 24.8,

27.6, 28.1 (2·), 35.0, 35.9, 37.6, 40.9, 54.2, 56.1, 63.2,

81.1, 90.8, 124.3, 153.7, 211.1. MS m/z (%): 368 (M⁺,

5), 336 (3), 306 (20), 176 (100), 161 (90). HRMS m/z:

368.2397 (Calcd for C₂₁H₃₆O₃S: 368.2385).

¹H NMR (CDCl₃) d: 0.82 (6H, t, J = 7.5 Hz, H-26a,

27a), 0.96 (3H, s, H-18), 1.43 (3H, d, J = 7.0 Hz, H-

21), 1.51 (4H, q, J = 7.5 Hz, H-26, 27), 2.86 (1H, dd,

J = 10.6, 6.4 Hz), 3.39 (3H, s, –CH₂OCH₃), 3.44 (1H,

q, J = 7.0 Hz, H-20), 4.65 (2H, s, –CH₂OCH₃), 5.56

(1H, m, H-16). MS m/z (%): 396 (M⁺, 7), 364 (6), 334

(14), 176 (100). HRMS m/z: 396.2670 (Calcd for

C₂₃H₄₀O₃S: 396.2698).

4.47. (20S)-16-Ene-22-thia-24-homo-25-(methoxymethyl)

oxy-26,27-dimethyl-8-one (40a)

4.50. (20S)-16-Ene-22-thia-24,24,24-trihomo-25-(methoxy-

methyl)oxy-26,27-dimethyl-8-one (42a)

To a stirred solution of (COCl)₂(82 lL, 0.940 mmol)

in dry CH₂Cl₂(2 mL) at ꢀ78 ꢁC was added a solution

of DMSO (134 lL, 1.888 mmol) in dry CH₂Cl₂

(1 mL). After being stirred for 5 min, a solution of

36a (303.0 mg, 0.788 mmol) in dry CH₂Cl₂(3 mL)

was added dropwise. The reaction mixture was stirred

for 15 min at ꢀ78 ꢁC, and Et₃N (549 lL, 3.885 mmol)

was added. The whole mixture was stirred at ꢀ78 ꢁC

for 15 min and at 0 ꢁC for 30 min, quenched with ice

water, and extracted with CH₂Cl₂. The CH₂Cl₂

extract was washed with brine, dried over anhydrous

MgSO₄, and concentrated in vacuo. The residue was

puriﬁed by chromatography on silica gel (12 g)

using 10% AcOEt in hexane to aﬀord 40a (287.4

mg, 95%).

The Swern oxidation of 38a (94.6 mg, 0.234 mmol) was

carried out according to the same procedure described

for the preparation of 36a to give 42a (75.8 mg, 79%).

¹H NMR (CDCl₃) d: 0.82 (6H, t, J = 7.5 Hz, H-26a,

27a), 0.96 (3H, s, H-18), 1.43 (3H, d, J = 7.0 Hz, H-

21), 1.51 (4H, q, J = 7.5 Hz, H-26, 27), 2.86 (1H, dd,

J = 10.6, 6.4 Hz), 3.39 (3H, s, –CH₂OCH₃), 3.44 (1H,

q, J = 7.0 Hz, H-20), 4.65 (2H, s, –CH₂OCH₃), 5.56

(1H, m, H-16). MS m/z (%): 410 (M⁺, 4), 378 (4), 348

(12), 177 (100), 176 (75). HRMS m/z: 410.2874 (Calcd

for C₂₄H₄₂O₃S: 410.2855).

4.51. (20S)-1a-[(tert-Butyldimethylsilyl)oxy]-16-ene-22-

thia-25-[(triethylsilyl)oxy]-26,27-dimethyl-19,24-dinorvi-

tamin D₃tert-butyldimethylsilyl ether (44a)

¹H NMR (CDCl₃) d: 0.83 (6H, t, J = 7.5 Hz, H-26a,

27a), 0.96 (3H, s, H-18), 1.43 (3H, d, J = 7.0 Hz, H-

21), 1.51 (4H, q, J = 7.5 Hz, H-26, 27), 2.87 (1H, dd,

J = 10.6, 6.4 Hz, H-14), 3.38 (3H, s, OMe), 3.45 (1H,

q, J = 7.0 Hz, H-20), 4.65 (2H, s, OCH₂O), 5.56 (1H,

m, H-16). ¹³C NMR (CDCl₃) d: 7.9 (2·), 18.0, 21.5,

23.4, 24.0, 27.3, 27.9 (2·), 31.2, 34.2, 34.9, 38.3, 40.6,

53.3, 55.9, 63.8, 80.8, 90.5, 125.1, 153.5, 210.8. MS m/z

(%): 382 (M⁺, 31), 350 (44), 320 (50), 177 (100), 176

To a stirred solution of 43 (85.2 mg, 0.149 mmol) in dry

THF (0.7 mL) at ꢀ78 ꢁC was added slowly n-BuLi

(95.7 lL, 0.149 mmol, 1.56 M solution in hexane), and

the resulting dark orange solution was stirred for

15 min. To this colored solution was added a solution

of 26a (31.7 mg, 0.074 mmol) in dry THF (0.8 mL),

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

1811

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 anhydrous MgSO₄, and evaporated in vacuo. The

residue was puriﬁed by chromatography on silica gel

(5 g) using 2% AcOEt in hexane to aﬀord 44a

(22.8 mg, 48% based on 26a) and 10% AcOEt in hexane

to give the unreacted starting material 26a (8.5 mg,

27%).

H-20), 4.06, 4,11 (each 1H, m, H-1, 3), 4.64, 4.66 (each

1H, d, J = 7.2 Hz, OCH₂O), 5.60 (1H, m, H-16), 5.91

(1H, d, J = 11.2 Hz, H-7), 6.17 (1H, d,J = 11.2 Hz, H-

6). MS m/z (%): 720 (no M⁺), 528 (12), 471 (3), 396

(22), 339 (11), 264 (8), 75 (100).

4.54. (20R)-1a-[(tert-Butyldimethylsilyl)oxy]-16-ene-22-

thia-25-[(methoxymethyl)oxy]-26,27-dimethyl-19-norvita-

min D₃tert-butyldimethylsilyl ether (45b)

¹H NMR (CDCl₃) d: 0.046, 0.049, 0.055, 0.063 (each

3H, s, 4· Si–Me), 0.59 (6H, q, J = 7.9 Hz, 3· Si–

CH₂CH₃), 0.83 (3H, s, H-18), 0.85, 0.88 (each 9H, s,

2· Si-t-Bu), 0.95 (9H, t, J = 7.9 Hz, 3· Si–CH₂CH₃),

1.40 (3H, d, J = 7.0 Hz, H-21), 2.53 (2H, m, H-23),

2.80 (1H, m), 3.42 (1H, q, J = 7.0 Hz, H-20), 4.06 (1H,

m), 4.11 (1H, m), 5.60 (1H, m, H-16), 5.91 (1H, d,

J = 11.2 Hz, H-7), 6.17 (1H, d, J = 11.2 Hz, H-6). ¹³C

NMR (CDCl₃) d: ꢀ4.70, ꢀ4.62, ꢀ4.52, ꢀ4.43, 7.2, 7.5,

8.3, 8.4, 17.7, 18.3, 18.4, 21.7, 23.5, 26.0, 28.5, 29.6,

31.9, 32.3, 35.4, 36.9, 39.5, 40.1, 43.9, 46.3, 49.6, 59.1,

68.2, 68.3, 78.5, 116.6, 121.8, 125.1, 134.3, 140.0,

155.8. MS m/z (%): 776 (M⁺, 3), 747 (1), 719 (1), 644

(4), 615 (1), 528 (18), 471 (6), 396 (32), 339 (18), 201

(100). HRMS m/z: 776.5471 (Calcd for C₄₄H₈₄O₃SSi:

776.5449).

Following the same procedure as described above, treat-

ment of 43 (79.9 mg, 0.140 mmol) with 39b (25.8 mg,

0.070 mmol) in the presence of LHMDS (140 lL,

0.140 mmol, 1.0 M solution in THF) gave 45b

(25.6 mg, 51%).

¹H NMR (CDCl₃) d: 0.048, 0.062 (each 3H, s, 2· Si–

Me), 0.052 (6H, s, 2· Si–Me), 0.73 (3H, s, H-18), 0.84

(6H, t, J = 7.5 Hz, H-26a, 27a), 0.86, 0.88 (each 9H, s,

2· Si-t-Bu), 1.47 (3H, d, J = 7.0 Hz, H-21), 2.80 (1H,

m, H-9), 3.39 (3 H, s, OMe, overlapped with H-20),

4.07, 4.10 (each 1H, m, H-1, 3), 4.65 (2H, s, OCH₂O),

5.58 (1H, m, H-16), 5.91 (1H, d,J = 11.2 Hz, H-7),

6.17 (1H, d,J = 11.2 Hz, H-6). MS m/z (%): 720 (no

M⁺), 528 (19), 471 (5), 396 (30), 339 (17), 264 (13), 75

(100).

4.52. (20R)-1a-[(tert-Butyldimethylsilyl)oxy]-16-ene-22-

thia-25-[(triethylsilyl)oxy]-26,27-dimethyl-19,24-dinorvi-

tamin D₃tert-butyldimethylsilyl ether (44b)

4.55. (20S)-1a-[(tert-Butyldimethylsilyl)oxy]-16-ene-22-

thia-24-homo-25-[(methoxymethyl)oxy]-26,27-dimethyl-

19-norvitamin D₃tert-butyldimethylsilyl ether (46a)

Following the same procedure as described above, treat-

ment of 43 (69.4 mg, 0.122 mmol) with 26b (10.4 mg,

0.024 mmol) in the presence of n-BuLi (77.9 lL,

0.122 mmol) gave 44b (2.8 mg, 18%) and the unreacted

starting ketone 26b (5.5 mg, 53%).

Following the same procedure as described above, treat-

ment of 43 (89.5 mg, 0.157 mmol) with 40a (40.0 mg,

0.105 mmol) in the presence of LHMDS (157 lL,

0.157 mmol, 1.0 M solution in THF) gave 46a

(33.6 mg, 44%).

¹H NMR (CDCl₃) d: 0.05–0.07 (12H, 4· Si–Me), 0.59

(6H, q, J = 7.9 Hz, 3· Si–CH₂CH₃), 0.72 (3H, s, H-

18), 0.86, 0.88 (each 9H, 2· Si-t-Bu), 0.96 (9H, t,

J = 7.9 Hz, 3· Si–CH₂CH₃), 1.45 (3H, d, J = 7.0 Hz,

H-21), 2.56 (2H, s, H-23), 2.79 (1H, m), 3.33 (1H, q,

J = 7.0 Hz, H-20), 4.07, 4.10 (each 1H, m, H-1, 3),

5.56 (1H, m, H-16), 5.91 (1H, d, J = 11.2 Hz, H-7),

6.17 (1H, d, J = 11.2 Hz, H-6). MS m/z (%): 776 (M⁺,

5), 644 (11), 528 (38), 471 (11), 396 (66), 339 (31), 201

(100). HRMS m/z: 776.5449 (Calcd for C₄₄H₈₄O₃SSi:

776.5449).

¹H NMR (CDCl₃) d: 0.048, 0.061 (each 3H, s, 2· Si–

Me), 0.052 (6H, s, 2· Si–Me), 0.82 (6H, t, J = 7.5 Hz,

H-26a, 27a, overlapped with H-18), 0.86, 0.88 (each

9H, s, 2· Si-t-Bu), 1.41 (3H, d, J = 7.0 Hz, H-21), 1.51

(4H, q, J = 7.5 Hz, H-26, 27), 2.80 (1H, m, H-9), 3.38

(3H, s, OMe), 3.45 (1H, q, J = 7.0 Hz, H-20), 4.08

(2H, m, H-1, 3), 4.65 (2H, s, OCH₂O), 5.59 (1H, m,

H-16), 5.90 (1H, d,J = 11.2 Hz, H-7), 6.17 (1H, d,

J = 11.2 Hz, H-6). MS m/z (%): 734 (no M⁺), 528 (28),

471 (8), 396 (50), 339 (26), 264 (16), 75 (100).

4.56. (20R)-1a-[(tert-Butyldimethylsilyl)oxy]-16-ene-22-

thia-24-homo-25-[(methoxymethyl)oxy]-26,27-dimethyl-

19-norvitamin D₃tert-butyldimethylsilyl ether (46b)

4.53. (20S)-1a-[(tert-Butyldimethylsilyl)oxy]-16-ene-22-

thia-25-[(methoxymethyl)oxy]-26,27-dimethyl-19-norvita-

min D₃tert-butyldimethylsilyl ether (45a)

Following the same procedure as described above, treat-

ment of 43 (92.4 mg, 0.162 mmol) with 40b (41.3 mg,

0.108 mmol) in the presence of LHMDS (162. lL,

0.162 mmol, 1.0 M solution in THF) gave 46b

(22.5 mg, 28%).

Following the same procedure as described above, treat-

ment of 43 (125.8 mg, 0.220 mmol) with 39a (40.6 mg,

0.11 mmol) in the presence of lithium bis(trimethyl-

silyl)amide (LHMDS, 220 lL, 0.220 mmol, 1.0 M solu-

tion in THF) gave 45a (33.5 mg, 42%).

¹H NMR (CDCl₃) d: 0.05 (9H, s, 3· Si–Me), 0.06 (3H, s,

Si–Me), 0.72 (3H, s, H-18), 0.83 (6H, t, J = 7.5 Hz, H-

26a, 27a), 0.86, 0.88 (each 9H, s, 2· Si-t-Bu), 1.45 (3H,

d, J = 7.0 Hz, H-21), 1.52 (4H, q, J = 7.5 Hz, H-26,

27), 2.80 (1 H, m, H-9), 3.36 (1H, q, J = 7.0 Hz, H-

¹H NMR (CDCl₃) d: 0.046 (6H, s, 2· Si–Me), 0.05, 0.06

(each 3 , s, 2· Si–Me), 0.83 (3H, s, H-18) 0.85, 0.88 (each

9H, s, 2· Si-t-Bu), 1.42 (3H, d, J = 7.0 Hz, H-21), 2.80 (1

H, m, H-9), 3.38 (3H, s, OMe), 3.46 (1H, q, J = 7.0 Hz,

1812

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

20), 3.39 (3H, s, OMe), 4.09 (2H, m, H-1, 3), 4.65 (2H, s,

OCH₂O), 5.58 (1H, m, H-16), 5.91 (1H, d,J = 11.2 Hz,

H-7), 6.17 (1H, d, J = 11.2 Hz, H-6). MS m/z (%): 734

(no M⁺), 528 (57), 471 (16), 396 (86), 339 (38), 264

(20), 75 (100).

J = 7.0 Hz, H-20), 4.06, 4.13 (each 1H, m, H-1, 3),

5.63 (1H, m, H-16), 5.95 (1H, d, J = 11.3 Hz, H-7),

6.30 (1H, d, J = 11.3 Hz, H-6). ¹³C NMR (CDCl₃) d:

8.2 (2·), 17.7, 21.9, 23.6, 28.7, 29.7, 31.1 (2·), 35.3,

37.4, 39.9, 41.1, 42.4, 44.8, 49.7, 59.0, 67.4, 67.6, 74.0,

115.8, 123.9, 125.6, 131.8, 142.0, 155.5. MS m/z (%):

434 (M⁺, 2), 416 (11), 398 (11), 380 (9), 300 (100), 282

(32). HRMS m/z: 416.2766 (M⁺ꢀH₂O) (Calcd for

C₂₆H₄₀O₂S: 416.2749).

4.57. (20S)-1a-[(tert-Butyldimethylsilyl)oxy]-16-ene-

22-thia-24,24-dihomo-25-[(methoxymethyl)oxy]-26,27-

dimethyl-19-norvitamin D₃tert-butyldimethylsilyl

ether (47a)

4.60. (20R)-16-Ene-22-thia-26,27-dimethyl-1a,25-dihy-

droxy-19,24-dinorvitamin D₃(1b)

Following the same procedure as described above, treat-

ment of 43 (73.3 mg, 0.128 mmol) with 41a (29.5 mg,

0.074 mmol) in the presence of LHMDS (128 lL,

0.128 mmol, 1.0 M solution in THF) gave 47a

(26.8 mg, 48%).

Following the same procedure as described above, treat-

ment of 44b (4.4 mg, 0.0057 mmol) with (ꢀ)-10-cam-

phor sulfonic acid (7.9 mg, 0.034 mmol) gave 1b

(2.4 mg, 98%).

¹H NMR (CDCl₃) d: 0.05–0.06 (12H, 4· Si–Me), 0.820

(3H, s, H-18), 0.823 (6H, t, J = 7.5 Hz, H-26a, 27a),

0.86, 0.88 (each 9H, s, Si-t-Bu), 1.41 (3H, d, J = 7.0

Hz, H-21), 1.51 (4H, q, J = 7.5 Hz, H-26, 27), 2.80

(1H, m), 3.39 (3H, s, –CH₂OCH₃), 3.44 (1H, q, J = 7.0

Hz, H-20), 4.08 (2H, m, H-1, 3), 4.65 (2H, s,

–CH₂OCH₃), 5.60 (1H, m, H-16), 5.90 (1H, d,

J = 11.2 Hz, H-6), 6.16 (1H, d, J = 11.2 Hz, H-7).

¹H NMR (CDCl₃) d: 0.73 (3H, s, H-18), 0.87, 0.88 (each

3H, t, J = 7.5 Hz, H-26a, 27a), 1.46 (3H, d, J = 7.0 Hz,

H-21), 2.59, 2.63 (each 1H, d, J = 12.8 Hz, H-23), 2.77

(2H, m), 3.36 (1H, q, J = 7.0 Hz, H-20), 4.06, 4.13

(1H, m, H-1, 3), 5.61 (1H, m, H-16), 5.95 (1H, d,

J = 11.3 Hz, H-7), 6.31 (1H, d, J = 11.3 Hz, H-6). ¹³C

NMR (CDCl₃) d: 8.3, 17.4, 22.6, 23.7, 23.9, 28.8, 31.1,

35.5, 37.4, 38.9, 40.8, 42.4, 44.8, 49.6, 50.2, 58.1, 67.5,

67.6, 73.9, 115.6, 123.9, 124.8, 131.6, 142.2, 155.4. MS

m/z (%): 434 (M⁺, 2), 416 (8), 398 (12), 380 (8), 300

(100), 282 (50), 264 (48). HRMS m/z: 434.2859 (Calcd

for C₂₆H₄₂O₃S: 434.2855).

4.58. (20S)-1a-[(tert-Butyldimethylsilyl)oxy]-16-ene-22-

thia-24,24,24-trihomo-25-[(methoxymethyl)oxy]-26,27-

dimethyl-19-norvitamin D₃tert-butyldimethylsilyl ether

(48a)

Following the same procedure as described above, treat-

ment of 43 (82.0 mg, 0.1437 mmol) with 42a (29.5 mg,

0.072 mmol) in the presence of LHMDS (144 lL,

0.144 mmol, 1.0 M solution in THF) gave 48a

(15.0 mg, 27%).

4.61. (20S)-16-Ene-22-thia-26,27-dimethyl-1a,25-dihy-

droxy-19-norvitamin D₃(2a)

Following the same procedure as described above, treat-

ment of 45a (26.5 mg, 0.0367 mmol) with (ꢀ)-10-cam-

phor sulfonic acid (51.2 mg, 0.220 mmol) gave 2a

(14.6 mg, 89%).

¹H NMR (CDCl₃) d: 0.048, 0.061 (each 3H, s, 2· Si–

Me), 0.052 (6H, s, 2· Si–Me), 0.82 (3H, s, H-18), 0.82

(6H, t, J = 7.5 Hz, H-26a, 27a), 0.86, 0.88 (each 9H, s,

Si-t-Bu), 1.41 (3H, d, J = 7.0 Hz, H-21), 1.51 (4H, q,

J = 7.5 Hz), 2.80 (1H, m), 3.38 (3H, s, –CH₂OCH₃),

3.44 (1H, q, J = 7.0 Hz, H-20), 4.08 (2H, m, H-1, 3),

4.65 (2H, s, –CH₂OCH₃), 5.60 (1H, m, H-16), 5.90

(1H, d, J = 11.2 Hz, H-6), 6.16 (1H, d, J = 11.2 Hz,

H-7).

¹H NMR (CDCl₃) d: 0.83 (3H, s, H-18), 0.859, 0.861

(each 3H, t, J = 7.6 Hz, H-26a, H-27a), 1.43 (3H, d,

J = 7.0 Hz, H-21), 1.47 (4H, q, J = 7.6 Hz, H-26, 27),

2.78 (2H, m), 3.49 (1H, q, J = 7.0 Hz, H-20), 4.07. 4.13

(each 1H, m, H-1, 3), 5.62 (1H, m, H-16), 5.95 (1H, d,

J = 11.3 Hz, H-7), 6.30 (1H, d,J = 11.3 Hz, H-6). ¹³C

NMR (CDCl₃) d: 8.0 (2·), 17.7, 21.6, 23.9, 25.0, 28.7,

29.6, 31.0, 31.1, 35.2, 37.4, 37.8, 38.5, 42.4, 44.8, 49.7,

59.1, 67.3, 67.6, 75.0, 115.8, 123.8, 125.4, 131.8, 142.0,

155.4. MS m/z (%): 448 (M⁺, 1), 430 (11), 412 (7), 394

(2), 300 (100), 282 (23), 264 (10). HRMS m/z: 430.2893

(M⁺ꢀH₂O) (Calcd for C₂₇H₄₂O₂S: 430.2905).

4.59. (20S)-16-Ene-22-thia-26,27-dimethyl-1a,25-dihy-

droxy-19,24-dinorvitamin D₃(1a)

A mixture of 44a (22.2 mg, 0.029 mmol) and (ꢀ)-10-

camphor sulfonic acid (39.8 mg, 0.171 mmol) in MeOH

(1 mL) was stirred at ambient temperature for 1.5 h. The

reaction mixture was poured into 5% NaHCO₃and ex-

tracted with AcOEt. The organic phase was washed with

brine, dried over anhydrous MgSO₄, and evaporated in

vacuo. The residue was chromatographed on silica gel

(5 g) using 80% AcOEt in hexane to give 1a (12.2 mg,

98%).

4.62. (20R)-16-Ene-22-thia-26,27-dimethyl-1a,25-dihy-

droxy-19-norvitamin D₃(2b)

Following the same procedure as described above, treat-

ment of 45b (25.6 mg, 0.035 mmol) with (ꢀ)-10-cam-

phor sulfonic acid (49.5 mg, 0.213 mmol) gave 2b

(15.2 mg, 96%).

¹H NMR(CDCl₃) d: 0.84 (3H, s, H-18), 0.87, 0.88 (each

3H, t, J = 7.5 Hz, H-26a, 27a), 1.43 (3H, d, J = 7.0 Hz,

H-21), 2.56, (2H, m, H-23), 2.78 (2H, m), 3.44 (1H, q,

¹H NMR (CDCl₃) d: 0.74 (3H, s, H-18), 0.86 (6 H, t,

J = 7.5 Hz, H-26a, 27a), 1.47 (3H, d, J = 7.0 Hz, H-

21), 1.48 (4H, q, J = 7.5 Hz, H-26, 27), 2.78 (2H, m,

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

1813

H-9, 10), 3.39 (1 H, q, J = 7.0 Hz, H-20), 4.05 (1H, m,

H-1), 4.10 (1H, m, H-3), 5.60 (1H, m, H-16), 5.95 (1H,

d, J = 11.2 Hz, H-7), 6.30 (1H, d, J = 11.2 Hz, H-6).

¹³C NMR (CDCl₃) d: 8.0 (2·), 17.4, 22.4, 23.7, 25.0,

28.8, 29.7, 31.06, 31.13, 35.6, 37.4, 37.7, 37.9, 42.4,

44.8, 50.2, 58.2, 67.4, 67.6, 75.0, 115.5, 123.9, 124.5,

131.7, 142.2, 155.3. MS m/z (%): 448 (M⁺, 1), 430 (9),

412 (7), 394 (4), 300 (100), 282 (33), 249 (23). HRMS

m/z: 430.2879 (M⁺ꢀH₂O) (Calcd for C₂₇H₄₂O₂S:

430.2905). UV k_max(EtOH): 244, 252 (e 28650), 262 nm.

(17.2 mg, 99%), which was further puriﬁed by HPLC

(YMC-Pack ODS-AM SH-342-5AM, 32% H₂O/MeOH,

8 mL/min) to give pure 4a (5.2 mg).

¹H NMR (CDCl₃) d: 0.83 (3H, s, H-18), 0.85 (6H, t,

J = 7.5 Hz, H-26a, 27a), 1.41 (3H, d, J = 7.0 Hz, H-

21), 1.45 (4H, q, J = 7.5 Hz, H-26, 27), 2.77 (2H, m,

H-9, 10), 3.43 (1H, q, J = 7.0 Hz, H-20), 4.06, 4.13 (each

1H, m, H-1, 3), 5.60 (1 H, m, H-16), 5.95 (1H, d,

J = 11.2 Hz, H-6), 6.30 (1H, d, J = 11.2 Hz, H-7). ¹³C

NMR (CDCl₃) d: 8.0 (x 2), 17.6, 21.7, 23.1, 23.6, 28.7,

29.6, 30.2, 30.7, 31.2 (2·), 35.2, 37.4, 38.0, 38.3, 42.4,

44.8, 49.7, 59.0, 67.4, 67.6, 74.8, 115.8, 123.9, 125.1,

131.7, 142.1, 155.6. MS m/z (%): 476 (no M⁺), 440 (4),

300 (26), 282 (21), 264 (22), 95 (100). HRMS m/z:

440.3110 (M⁺ꢀ2H₂O) (Calcd for C₂₉H₄₄OS: 440.3113).

4.63. (20S)-16-Ene-22-thia-24-homo-26,27-dimethyl-1a, 25-

dihydroxy-19-norvitamin D₃(3a)

Following the same procedure as described above, treat-

ment of 46a (30.3 mg, 0.0412 mmol) with (ꢀ)-10-cam-

phor sulfonic acid (57.4 mg, 0.247 mmol) aﬀorded 3a

(18.6 mg, 97%).

4.66. (20R)-16-Ene-22-thia-24,24,24-trihomo-26,27-

dimethyl-1a,25-dihydroxy-19-norvitamin D₃(5a)

¹H NMR (CDCl₃) d: 0.83 (3H, s, H-18), 0.85 (6 H, t,

J = 7.5 Hz, H-26a, 27a), 1.41 (3H, d, J = 7.0 Hz, H-

21), 1.47 (4H, q, J = 7.5 Hz, H-26, 27), 2.77 (2H, m,

H-9, 10), 3.45 (1 H, q, J = 7.0 Hz, H-20), 4.05 (1H, m,

H-1), 4.12 (1H, m, H-3), 5.60 (1H, m, H-16), 5.95 (1H,

d, J = 11.2 Hz, H-7), 6.30 (1H, d,J = 11.2 Hz, H-6).

¹³C NMR (CDCl₃) d: 8.0 (2·), 17.6, 21.7, 23.6, 23.7,

28.7, 29.6, 31.1, 31.2, 31.4, 35.2, 37.4, 37.9, 38.4, 42.2,

44.8, 49.7, 59.0, 67.3, 67.6, 74.7, 115.8, 123.8, 125.1,

131.8, 142.1, 155.5. MS m/z (%): 462 (no M⁺), 444 (3),

426 (4), 408 (3), 300 (46), 282 (28), 264 (33), 143 (100).

HRMS m/z: 444.3066 (M⁺ꢀH₂O) (Calcd for

C₂₈H₄₄O₂S: 444.3062). UV k_max(EtOH): 244, 252,

262 nm.

Following the same procedure as described above, treat-

ment of 48a (15.0 mg, 0.0196 mmol) with (ꢀ)-10-cam-

phor sulfonic acid (27.4 mg, 0.1179 mmol) yielded 5a

(6.2 mg, 65%), which was further puriﬁed by HPLC [Hi-

bar RT 250-10 LiChrosorb Si 60 (7 lm), hexane/CHCl₃/

MeOH = 55/42/3 (v/v/v), 5 mL/min] to give pure 5a

(1.2 mg).

¹H NMR (CDCl₃) d: 0.83 (3H, s, H-18), 0.85 (6H, t,

J = 7.5 Hz, H-26a, 27a), 1.41 (3H, d, J = 7.0 Hz), 1.43

(4H, q, J = 7.5 Hz), 2.77 (2H, m, H-9, 10), 3.43 (1H,

q, J = 7.0 Hz, H-20), 4.06, 4.13 (each 1H, m, H-1, 3),

5.60 (1H, m, H-16), 5.95 (1H, d, J = 11.2 Hz, H-6),

6.30 (1H, d, J = 11.2 Hz, H-7). ¹³C NMR (CDCl₃) d:

8.0 (2·), 17.6, 21.7, 23.3, 23.6, 28.7, 29.7 (2·), 29.9,

30.7, 31.2 (2·), 35.2, 37.4, 38.3 (2·), 42.4, 44.8, 49.7,

59.1, 67.4, 67.6, 74.8, 115.8, 123.9, 125.1, 131.7, 142.2,

155.6. MS m/z (%): 490 (no M⁺), 472 (7), 454 (4), 300

(71), 282 (34), 264 (42), 55 (100). HRMS m/z: 472.3380

(M⁺ꢀH₂O) (Calcd for C₃₀H₄₈O₂S: 472.3375).

4.64. (20R)-16-Ene-22-thia-24-homo-26,27-dimethyl-1a,

25-dihydroxy-19-norvitamin D₃(3b)

Following the same procedure as described above, treat-

ment of 46b (22.5 mg, 0.031 mmol) with (ꢀ)-10-cam-

phor sulfonic acid (42.6 mg, 0.183 mmol) gave 3b

(13.7 mg, 97%).

4.67. Vitamin D receptor-binding assay

¹H NMR (CDCl₃) d: 0.73 (3H, s, H-18), 0.86 (6H, t,

J = 7.5 Hz, H-26a, 27a), 1.45 (3H, d, J = 7.0 Hz, H-

21), 1.46 (4H, q, J = 7.5 Hz, H-26, 27), 2.77 (2H, m,

H-9, 10), 3.36 (1H, q, J = 7.0 Hz, H-20), 4.06 (1H, m,

H-1), 4.13 (1H, m, H-3), 5.58 (1H, m, H-16), 5.95 (1H,

d, J = 11.3 Hz, H-7), 6.30 (1H, d,J = 11.3 Hz, H-6).

¹³C NMR (CDCl₃) d: 8.0 (2·), 17.4, 22.5, 23.7, 23.8,

28.8, 29.7, 31.16, 31.22, 31.4, 35.5, 37.4, 37.5, 37.9,

42.4, 44.8, 50.2, 58.2, 67.4, 67.6, 74.7, 115.5, 123.9,

124.3, 131.6, 142.2, 155.6. MS m/z (%): 462 (M⁺, 1),

444 (2), 426 (3), 408 (2), 300 (36), 282 (22), 264 (22),

143 (100). HRMS m/z: 444.3060 (M⁺ꢀH₂O) (Calcd for

C₂₈H₄₄O₂S: 444.3062). UV k_max(EtOH): 244, 252,

262 nm.

The rat recombinant VDR ligand-binding domain

(LBD) (amino acids 115–423) was expressed as an ami-

no-terminal His-tagged protein in E. coli BL21 (DE3)

pLys S (Novagen).³³The cells were lysed by sonication.

The supernatants were diluted approximately 1000 times

in 50 mM Tris buﬀer (100 mM KCl, 5 mM DTT, 0.5%

CHAPS, pH 7.5) containing bovine serum albumin

(100 lg/ml) and were pipetted into glass culture tubes.

A solution containing an increasing amount of 1a,25-

(OH)₂D₃or the synthetic analogs in 15łll of EtOH

was added to the receptor solution in each tube and

the mixture was vortexed 1–2 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

4.65. (20S)-16-Ene-22-thia-24,24-dihomo-26,27-dimethyl-

1a,25-dihydroxy-19-norvitamin D₃(4a)

Following the same procedure as described above, treat-

ment of 47a (26.8 mg, 0.0358 mmol) with (ꢀ)-10-cam-

phor sulfonic acid (49.8 mg, 0.2146 mmol) yielded 4a

1814

H. Takaku et al. / Bioorg. Med. Chem. 16 (2008) 1796–1815

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, U.K.) and

submitted for radioactivity counting. Each assay was

performed at least twice in duplicate.

4. Morisaki, M.; Koizumi, N.; Ikekawa, N.; Takeshita, T.;

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4.68. Transactivation assay

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COS-7 cells were grown in Dulbecco’s modiﬁed Ea-

gle’s medium (DMEM) supplemented with 5% fetal

calf serum (FCS). Cells were seeded on 24-well plates

at a density of ꢁ2 · 10⁴per well. After 24 h, cells

were transfected with a reporter plasmid containing

three copies of the mouse osteopontin VDRE (5⁰-

GGTTCAcgaGGTTCA, 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 previously.⁴⁶After 4-h incubation, the med-

ium was replaced with fresh DMEM containing 1%

FCS (HyClone, UT). The next day, the cells were

treated with either indicated concentration of 1a,25-

(OH)₂D₃, 19-norvitamin D analogs, or ethanol vehicle

and cultured for 24 h. Cells in each well were har-

vested with a cell lysis buﬀer, and the luciferase activ-

ity was measured with a luciferase assay kit (Tokyo

Ink, Inc., Japan) according to the manufacturer’s

instructions. Transactivation measured by the lucifer-

ase activity was normalized with the internal control.

All experiments were performed in triplicate.

4.69. Osteoclast diﬀerentiation assay

Bone marrow cells were obtained from tibiae of 5- to 8-

wk-old male mice of the ddY strain. Primary osteoblastic

cells were prepared from the calvariae of newborn ddY

mice as previously described.^47,48Brieﬂy, mouse bone

marrow cells (1.5 · 10⁵cell/well) and primary osteoclasts

(3 · 10³cells/well) were cocultured for 7 days in the pres-

ence of 1,25-(OH)₂D₃, 2MD or 19-norvitamin D analogs

(10^ꢀ8M to 10^ꢀ12M) in 0.3 ml of a-MEM (Sigma, St.

Louis, USA) supplemented with 10% FBS in 48-well

plates. Cells were replenished on day 3 with fresh medium.

Cells were then ﬁxed with 10% formaldehyde in PBS and

stained for tartrate-resistant acid phosphatase (TRAP) as

described.^48,49TRAP-positive multinucleated cells con-

taining three or more nuclei were counted as osteoclasts,

under microscopic examination. The results were ex-

pressed as means SEM of three cultures.

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Article Doi

Synthesis and structure-activity relationships of 16-ene-22-thia-1α,25-dihydroxy-26,27-dimethyl-19-norvitamin D3 analogs having side chains of different sizes

DOI: 10.1016/j.bmc.2007.11.013

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Full text of DOI:10.1016/j.bmc.2007.11.013

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