2-Methylene 19-nor-25-dehydro-1α-hydroxyvitamin D3 26,23-lactones: Synthesis, biological activities and molecular basis of passive antagonism

Available online at www.sciencedirect.com

Bioorganic & Medicinal Chemistry 16 (2008) 457–473

2-Methylene 19-nor-25-dehydro-1a-hydroxyvitamin D₃

26,23-lactones: Synthesis, biological activities and molecular

basis of passive antagonism

Nobuko Yoshimoto,^a,cYuka Inaba,^a,b,cSachiko Yamada,^b,*Makoto Makishima,^b

Masato Shimizu^aand Keiko Yamamoto^c,*

^aSchool of Biomedical Sciences, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan

^bDepartment of Biochemistry, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan

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

Machida, Tokyo 194-8543, Japan

Received 21 August 2007; revised 10 September 2007; accepted 11 September 2007

Available online 14 September 2007

Abstract—To investigate the molecular mechanism of vitamin D receptor (VDR) antagonists having no structurally bulky group

interfering with helix 12 of the ligand-binding domain of the VDR, we have synthesized four diastereomers at C(20) and C(23)

of 19-nor-1a-hydroxyvitamin D₃25-methylene-26,23-lactone bearing a 2MD-type A-ring. All four analogs showed signiﬁcant

VDR aﬃnity. Transactivation was tested by using Cos7 cells and HEK293 cells. In both types of cells, LAC67a showed little trans-

activation potency and inhibited the activation induced by the natural hormone concentration-dependently, indicating that LAC67a

works as an antagonist for the VDR in these cells. LAC67b, LAC82a and LAC82b similarly acted as VDR antagonists in Cos7 cells,

but in HEK293 cells they behaved as potent VDR agonists. Docking of four lactones into the VDR–LBD, followed by structural

analysis, demonstrated that each lactone lacks the hydrophobic interaction with helix12 necessary for maintaining the active con-

formation of the VDR, indicating that these lactones are passive-type antagonists. Furthermore, each docking structure explained

the characteristic transactivation proﬁles of the four lactones. On the basis of our present ﬁndings, we suggest that the ligand acts as

an agonist if there are appropriate coactivators in the cells to bind to the looser VDR–ligand complex, and as an antagonist if there

are no such appropriate coactivators. The molecular basis of the passive antagonism is discussed in detail.

1a,25-Dihydroxyvitamin D₃[1,25-(OH)₂D₃, 1] plays an

important role in regulation of calcium and bone metab-

olism, cellular diﬀerentiation and proliferation, and im-

mune responses.¹The majority of these actions are

mediated by the vitamin D receptor (VDR), which is a

member of the nuclear receptor superfamily and func-

tions as a ligand-dependent transcriptional factor.^1,2

Upon ligand binding, the VDR undergoes conforma-

tional change to form the AF2 surface to allow binding

of a coactivator.³In the absence of the ligand, a core-

pressor binds to the AF2 surface.^3,4

Two types of nuclear receptor antagonists have been re-

ported. The selective estrogen receptor modulators

(SERM), 4-hydroxytamoxifen and raloxifene, constitute

the ﬁrst group of antagonists, which have a bulky substi-

tuent that creates spatial restriction with residues on he-

lix 12 (H12) of estrogen receptor a in the active

conformation and prevents the H12 from occupying

the correct position on the AF2 surface.^5,6Ligands of

this type are called active antagonists. The other group

of antagonists has no such bulky substituent. For exam-

ple, progesterone works as an antagonist of the mineral-

ocorticoid receptor (MR), although this compound is

easily accommodated in the ligand-binding pocket

(LBP).⁷Recently, Bledsoe et al. have solved and re-

ported the X-ray crystal structure of the MR ligand-

binding domain (LBD) complexed with progesterone,

in which progesterone interacts poorly with helix 3

(H3) and helix 10/11 (H10/11) of MR–LBD.⁸This infe-

rior interaction results in insuﬃcient interactions among

Keywords: Vitamin D receptor; Antagonist; Partial agonist; Transac-

tivation; Nuclear receptor; Vitamin D; Passive antagonist.

*

Corresponding authors. Tel./fax: +81 42 721 1580; e-mail addresses:

yamada.vd@image.ocn.ne.jp; yamamoto@ac.shoyaku.ac.jp

doi:10.1016/j.bmc.2007.09.017

458

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

H3, helix 5 (H5), H10/11, and H12 mediated by the li-

gands, so that the correct AF2 surface cannot form,

and thus progesterone behaves as an antagonist.^7,8Li-

gands of this type are known as passive antagonists.

antagonist. These are analogs of TEI compounds having

methylene lactone on the side chain. The 2-methylene

19-nor structure of the A-ring was selected because the

19-nor structure is known to be more stable than the

conjugated triene structure of the hormone and is easily

synthesized. Insertion of a methylene group into C(2)

was expected to increase the VDR aﬃnity and the bio-

logical activity, as in the case of 2MD compound.¹²

Since no VDR antagonist with a 20-epi-conﬁguration

had yet been reported, LAC82a and LAC82b were also

designed to evaluate the eﬀects of the stereochemistry

at C(20). In this report, we describe the synthesis and

biological activities of four diastereomers of vitamin

D₃derivatives having methylene lactone on the side

chain (LAC67a, LAC67b, LAC82a, and LAC82b), and

their interaction with the receptor. In addition, the

molecular basis of these lactones acting as passive antag-

onists was investigated by docking analysis.

Thousands of vitamin D analogues have been synthe-

sized to date. Most of them act as VDR agonists,

whereas only two known types act as VDR antagonists.

As active antagonists, ZK compounds such as

ZK168281 2 have been developed by the Schering group

(Chart 1).⁹These compounds have a bulky ester group

at the side chain terminal that causes spatial restriction

with H12 of the active conformation of the VDR. The

other antagonists are TEI compounds, such as

TEI9647 3b and TET9648 3a, developed by the Teijin

group.^10,11They have a methylene lactone on the side

chain, and no bulky structure directed toward H12. In

the course of our studies to develop VDR antagonists

for treatment of metabolic bone disease such as Paget

disease^11band to investigate the molecular basis of the

VDR antagonism, we designed and synthesized four

diastereomers of 2-methylene 19-nor-25-dehydro-1a-

hydroxyvitamin D₃26,23-lactone (LAC67a, LAC67b,

LAC82a, and LAC82b) as the candidates of passive

1. Synthesis

Four methylene lactone compounds (LAC67a, LAC67b,

LAC82a, and LAC82b) were synthesized using the Wit-

tig–Horner reaction of Grundmann’s ketone derivative

9/10 with A-ring phosphine oxide 4g derived from (ꢀ)-

quinic acid, followed by 2-methylenation and subse-

quent methylene lactonization (Scheme 1).

OH

O

OEt

OH

Phosphine oxide 4g was derived from (ꢀ)-quinic acid in

59% overall yield by a modiﬁcation of DeLuca’s meth-

od. Conversion of (ꢀ)-quinic acid to methyl ester 4a

and conversion of keto compound 4c to phosphine

oxide 4g have been reported by DeLuca’s group^13a

and Shimizu’s group,^13brespectively. We reduced

methyl ester 4a with NaBH₄instead of DIBAL¹⁴to give

4b, which was oxidatively cleaved to give keto com-

pound 4c. Phosphine oxide 4g was combined with

Grundmann’s ketone derivative 9, derived from vitamin

D₂, to give the 23-cyanide 11 at 80% yield. Removal of

the protecting group followed by oxidation of the 2-hy-

droxyl group of 13 aﬀorded the corresponding 2-keto

compound 15, which was then treated with Wittig re-

agent to give the 2-methylene compound 17. The cyano

group of 17 was reduced by DIBAL to give the aldehyde

19. This aldehyde was reacted with an organo-chro-

mium complex, prepared from allylic bromide and

low-valent chromium (II) derived from CrCl₃by reduc-

tion with LiAlH₄, to give the lactone derivative 21.¹⁵

This method is a one-step allylation–lactonization reac-

tion that was developed by Kittaka’s group.¹⁶Methy-

lene lactone 21 was obtained as a 2:3 mixture in terms

of the stereochemistry at C23. Since treatment of lactone

21 with n-Bu₄NF gave a complex mixture of various

compounds, TBDMS groups protecting the two hydro-

xyl groups were removed by acid-hydrolysis to give a

mixture of LAC67a and LAC67b, which were then sep-

arated by HPLC. Stereochemistry at C(23) of LAC67a

and LAC67b was determined by the Kusumi–Mosher

method¹⁷described below.

HO

OH

HO

OH

1:1,25-(OH)₂D₃

2:ZK168281

O

HO

OH

HO

OH

3a:TEI9648

3b:TEI9647

O

HO

OH

HO

OH

LAC67a:20R,23R

LAC82a:20S,23R

LAC67b:20R,23S

LAC82b:20S,23S

LAC82a and LAC82b were synthesized by the same pro-

cedure as LAC67a and LAC67b (Scheme 1).

Chart 1. Structures of 1,25-(OH)₂D₃and its analogs.

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

459

O

HOOC OH

HO OH

MeOOC OH

TBSO OTBS

HOH₂C OH

TBSO OTBS

a

b

c

d

TBSO

OTBS

OH

D-(-)-Quinic Acid

4a (2 steps 87%)

4b (93%)

4c (quant.)

COOMe

O

OH

e

f

TBSO

OTBS

OTMS

TBSO

OTBS

OTMS

TBSO

OTBS

OTMS

4d (99%)

4e (90%)

4f (99%)

g

O

PPh₂

CN

TBSO

OTBS

OTMS

CN

OTs

CN

4g (3 steps 82%)

h

i

VD₂

j

TBSO

OTBS

OX

OH

O

11:20R (X=TMS; 80%)

12:20S (X=TMS; 75%)

13:20R (X=H; 86%)

14:20S (X=H; 83%)

7:20S (98%)

8:20R (99%)

5:20S

6:20R

9:20R (92%)

10:20S (96%)

k

CN

CHO

O

Br

COOMe

l

n

o

TBSO

OTBS

TBSO

OTBS

TBSO

OTBS

X

15:20R (X=O; 96%)

16:20S (X=O; 99%)

17:20R (X=CH₂; 94%)

18:20S (X=CH₂; 89%)

19:20R (90%)

20:20S (87%)

21:20R (83%)

22:20S (82%)

m

O

p

+

HO

OH

HO

OH

LAC67a:20R

LAC82a:20S

LAC67b:20R

LAC82b:20S

Scheme 1. Synthetic scheme of lactones. Reagents: (a) 1—MeOH, p-TsOH; 2—TBSCl, Et₃N; (b) NaBH₄; (c) NaIO₄; (d) TMSCl, imidazole; (e)

Me₃SiCH₂CO₂Me, LDA; (f) DIBAL-H; (g) 1—p-TsCl, n-BuLi, then Ph₂PH, n-BuLi; 2—10% H₂O₂; (h) KCN, DMSO; (i) TPAP, NMO, Molecular

Sieves 4A; (j) n-BuLi; (k) AcOH; (l) (COCl)₂, DMSO, Et₃N; (m) MeP⁺Ph₃Br^ꢀ, n-BuLi; (n) DIBAL-H; (o) CrCl₃, LiAlH₄; (p) CSA, MeOH.

460

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

2. Determination of stereochemistry at C(23)

by 1,25-(OH)₂D₃(1) concentration-dependently, indi-

cating they can work as VDR antagonists. Interestingly,

both the EC₅₀and IC₅₀of LAC67b in the transactiva-

tion assay were lower than those of LAC67a, in agree-

ment with their VDR aﬃnity.

Stereochemistry at C(23) of each lactone was determined

by the Kusumi–Mosher method (Scheme 2). Methylene

lactone 21 was reduced by DIBAL to give a mixture of

23,26-diols 23a and 23b, which were separable by silica

gel column chromatography. The primary alcohol of

each compound was selectively protected as the pivalate.

Then, the pivalate esters (25a and 25b) were converted to

the corresponding S- and R-MTPA esters of the 23-hy-

droxyl group (27a and 28a; 27b and 28b). Chemical shift

diﬀerences between the S-MTPA ester and the corre-

sponding R-MTPA ester are shown in Figure 1a and

b. This analysis clearly indicated that the 23,26-diol

23a has a 23R-conﬁguration. This diol 23a was con-

verted to the corresponding lactone LAC67a by oxida-

tion with MnO₂, followed by removal of the TBDMS

group. HPLC analysis showed that the methylene lac-

tone derived from 23a co-migrated with LAC67a. Based

on these analyses we determined that LAC67a and

LAC67b have a 23R- and 23S-conﬁguration, respec-

tively. In 20-epi-compounds, each stereochemistry at

C(23) of LAC82a and LAC82b was determined to be

R and S, respectively, according to the same method

(Fig. 1c and d).

Interestingly, LAC82a and LAC82b showed signiﬁcant

agonistic activity that is 34% and 43% eﬃcacy compared

with the hormone (1), respectively (Fig. 3b), and their

EC₅₀values were 6 and 10 nM. These results clearly

indicate that LAC82a and LAC82b are also partial ago-

nists. Inversion of stereochemistry from 20R to 20S en-

hanced transactivation in terms of both eﬃcacy and

potency (EC₅₀). As expected, both compounds reduced

the transactivation induced by hormone (1) to their

own eﬃcacy of maximal activation (Fig. 3d). LAC82b

with the 23S-conﬁguration showed stronger antagonis-

tic activity than LAC82a with the 23R-conﬁguration,

in agreement with LAC67b and TEI9647 3b with the

23S-conﬁguration.^10,21We found that 20-epimerization

(from LAC67a to LAC82a; from LAC67b to LAC82b)

enhanced both agonistic and antagonistic activities.

To evaluate the eﬀects of cell type, we performed the

same transactivation assay using HEK293 cells. As also

observed in Cos7 cells, LAC67a had little transactiva-

tion potency, whereas LAC67b showed signiﬁcant ago-

nistic activity (Fig. 4a). LAC67a, but not LAC67b,

signiﬁcantly reduced the transactivation stimulated by

1,25-(OH)₂D₃(Fig. 4c). These results indicate that

LAC67a acts almost as an antagonist in HEK293 cells,

whereas LAC67b acts as a partial agonist with potent

activity. The 20-epimer lactones LAC82a and LAC82b

showed strong transactivation potency (Fig. 4b). As ex-

pected, they showed little antagonistic activity (Fig. 4d),

indicating that both behave almost as full agonists in

HEK293 cells.

3. Biological activities

Biological activities in vitro are summarized in Table 1.

Binding aﬃnity for the VDR was evaluated by compet-

itive-binding assay using rat recombinant VDR–LBD

prepared in our laboratory (Fig. 2).¹⁸The binding aﬃn-

ity of LAC67b was 1/7 as potent as the natural hormone

1, while that of the 23R-isomer LAC67a was 1/200 as

potent as the hormone 1. Interestingly, both 20-epimers

LAC82a and LAC82b showed VDR-binding potency

intermediate (1/50) between LAC67a and LAC67b.

These results indicated that all four lactones are VDR

ligands having signiﬁcant aﬃnity. The ﬁnding that

LAC67b with a 23S-conﬁguration showed stronger

VDR aﬃnity than its 23R-isomer LAC67a is in agree-

ment with the properties of the TEI compounds,

TEI9647 3b with a 23S-conﬁguration having stronger

aﬃnity than its 23R-epimer TEI9648 3a.^10,19However,

this was not the case for synthetic 20-epi compounds be-

cause LAC82a and LAC82b had almost the same VDR

aﬃnity.

4. Receptor interaction

Docking analysis was performed by the procedure de-

scribed in the Experimental section. Figure 5a shows

the X-ray crystal structure of VDR–LBD complexed

with 1,25-(OH)₂D₃.²²Figure 5c–f show the docking

models of LAC67a, LAC67b, LAC82a, and LAC82b

with the VDR–LBD, and their superposition is shown

in Figure 5b. As shown in Figure 5, all of these four ana-

logs, LAC67a, LAC67b, LAC82a, and LAC82b, were

accommodated in the VDR–LBP where their hydroxyl

groups at the 1a- and 3b-positions form pincer-type

hydrogen bonds with Ser237 and Arg274, and Tyr143

and Ser278, respectively. Furthermore, the carbonyl

group at C(26) forms a pincer-type hydrogen bond with

His305 and His397, as in the case of 1,25-(OH)₂D₃1.

Diﬀerences from 1,25-(OH)₂D₃1 were found in hydro-

phobic interactions between amino acid residues on

H12, including the tail of H11 and the ligand. While

the terminal C(26)-methyl group of 1,25-(OH)₂D₃1 di-

rectly interacted with residues Val418, Phe422 on H12

and Tyr401 on H11 (Fig. 5a), the terminal methylene

group of LAC67a is rather too distant from these three

residues to interact directly (Fig. 5c). This docking mode

The ability of methylene lactones to induce transcription

of a vitamin D-responsive gene was tested using a rat

osteopontin luciferase reporter gene assay system in

Cos7 cells.²⁰It is well known that, in this assay, 1,25-

(OH)₂D₃(1) increases luciferase activity in a concentra-

tion-dependent manner. As shown in Figure 3a,

LAC67a and LAC67b activated the VDR in a concen-

tration-dependent manner, but their eﬃcacy of maximal

activation was only 14% and 11% of that of the hor-

mone (1), respectively, indicating that both lactones

are extremely weak partial VDR agonists. Antagonistic

activity was evaluated by inhibition of VDR stimulation

by 10 nM 1,25-(OH)₂D₃(1). As shown in Figure 3c,

both compounds inhibited the transactivation induced

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

461

OPiv

OH

O

OH

c

(S)-MTPA

27a:20R (78%)

29a:20S (quant.)

b

OPiv

OH

25a:20R (27%)

26a:20S (45%)

TBSO

OTBS

d

OPiv

O

(R)-MTPA

23a:20R (27%)

24a:20S (32%)

28a:20R (85%)

30a:20S (42%)

a

21:20R

22:20S

+

OH

OPiv

O

c

(S)-MTPA

b

OPiv

27b:20R (71%)

29b:20S (quant.)

OH

25b:20R (44%)

26b:20S (24%)

TBSO

OTBS

d

OPiv

O

(R)-MTPA

23b:20R (23%)

24b:20S (30%)

28b:20R (79%)

30b:20S (47%)

e

f

23a

21a:23R

(83%)

LAC67a

(65%)

f

24b

22b:23S

LAC82b

(87%)

(51%)

Scheme 2. Synthesis of MTPA esters. Reagents: (a) DIBAL-H; (b) PivCl, pyridine; (c) R-MTPACl, Et₃N, DMAP; (d) S-MTPACl, Et₃N, DMAP; (e)

MnO₂; (f) CSA, MeOH.

-0.04

a

+0.09

b

-0.04

O

-0.01

O

+0.13

+0.01

OPiv

+0.08

-0.03

+0.17

-0.07

MTPA

+0.02

MTPA plane

27b-28b

27a-28a

c

+0.04

OPiv

d

-0.1

+0.03

O

OPiv

+0.04

-0.15

-0.16

-0.15

+0.07

+0.06

O

MTPA

-0.02

+0.02

MTPA plane

29a-30a

29b-30b

Figure 1. Determination of stereochemistry at C(23). Chemical shift diﬀerences.

462

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

Table 1. Biological activities of synthetic lactone compounds

VDR binding^aEC₅₀(M)

Cos7^b

Transact.^dEC₅₀(M)

HEK293^c

Inhibit.^eIC₅₀(M)

Transact.^dEC₅₀(M)

Inhibit.^eIC₅₀(M)

1,25-(OH)₂D₃(1)

LAC67a

LAC67b

1 · 10^ꢀ10

2 · 10^ꢀ8

7 · 10^ꢀ10

5 · 10^ꢀ9

5 · 10^ꢀ10

—

1 · 10^ꢀ9

2 · 10^ꢀ7

1 · 10^ꢀ9

2 · 10^ꢀ8

1.5 · 10^ꢀ8

—

1.5 · 10^ꢀ7

2.5 · 10^ꢀ8

6 · 10^ꢀ9

1 · 10^ꢀ6

3 · 10^ꢀ7

1 · 10^ꢀ7

3 · 10^ꢀ7

ND

LAC82a

LAC82b

1 · 10^ꢀ8

ND

^aCompetitive binding of 1,25-(OH)₂D₃(1) and synthetic lactones to the rat vitamin D receptor. The EC₅₀values are derived from dose–response

curves and represent the analog concentration required for 50% displacement of the radio-labeled 1,25-(OH)₂D₃from the receptor protein. The

experiments were carried out in duplicate.

^bTransactivation was evaluated by dual luciferase assay using a full-length human VDR expression plasmid (pCMX-hVDR), a reporter plasmid

containing three copies of the mouse osteopontin VDRE (SPPx3-TK-Luc), and the internal control plasmid containing sea pansy luciferase

expression constructs (pRL-CMV) in Cos7 cells as described previously.²⁰

^cTransactivation potencies were evaluated using a full-length hVDR expression plasmid (pCMX-hVDR) and a reporter plasmid containing three

copies of the mouse osteopontin VDRE (SPPx3-TK-Luc) in HEK293 cells as described previously.³¹

^dAgonistic activity. The EC₅₀values are derived from dose–response curves and represent the analog concentration capable of inducing 50%

maximal transactivation response. All experiments were carried out in triplicate.

^eAntagonistic activity. The IC₅₀values are derived from dose–response curves and represent the analog concentration capable of reducing 50%

maximal transactivation response.

chain and Val418, Phe422, and Tyr401 at the C-terminal

of the VDR is within 4.7 A.^22,26–30These results indicate

˚

120

100

1,25D3

that intimate interaction is needed in order to form a

transcriptionally active conformation of the VDR.

Docking of synthetic lactones into the VDR–LBD and

structural analysis yielded insights into the molecular

basis of VDR passive antagonism.

LAC67a

LAC67b

LAC82a

LAC82b

80

60

40

20

As shown in Figure 5, the hydroxyl groups at the 1a-

and 3b-positions of the four lactones form pincer-type

hydrogen bonds with Ser237 and Arg274, and Tyr143

and Ser278, respectively, and the carbonyl group at

C(26) forms a pincer-type hydrogen bond with His305

and His397, as is the case for 1,25-(OH)₂D₃1. Con-

struction of these complete six hydrogen bonds is one

of the most important reasons why lactones can bind

to the VDR. However, docking models of the four lac-

tones demonstrated that hydrophobic interaction with

the C-terminal of the VDR is not enough for full agonist

action. In the case of LAC67a, all of the three residues

Tyr401, Val418, and Phe422 are far from the side chain

of this ligand, as shown in Figure 5c, indicating the loos-

er packing of H12 including the tail of H11. We con-

cluded that the lack of these hydrophobic interactions

is the reason why LAC67a works as an antagonist both

in Cos7 as well as HEK293 cells. The docking model of

LAC67b indicates that LAC67b has, in addition to six

complete hydrogen bonds, appropriate hydrophobic

interactions with Tyr401 and Val418, but not Phe422.

This proper interaction with Tyr401 and Val418 would

be one reason why LAC67b works as a potent agonist

in HEK293 cells. HEK293 cells, but not Cos7 cells,

might have an appropriate coactivator that can bind

to the AF2 surface of the VDR docked with LAC67b.

0

10^-1110^-1010^-910^-810^-710^-610^-5

Concentration (M)

Figure 2. Competitive-binding assay of synthetic lactones.

suggests that LAC67a can bind to VDR but cannot

form a transcriptionally active conformation in which

H12 folds back ligand-dependently to form the AF2-

surface. In the complex of LAC67b, Tyr401 and

Val418 occupy the appropriate positions, but Phe422

is located far from the ligand (Fig. 5d).

20-epi-Compounds, LAC82a and LAC82b, also showed

moderate contacts with Tyr401 and Val418 and weak

contact with Phe422 (Fig. 5e and f). Compared with

LAC67a and LAC67b, C(22)H₂of the 20-epi-lactones

LAC82a and LAC82b interacts more intimately with

Val300. This close contact, similar to that observed in

the complex of 20-epi-1,25-(OH)₂D₃, might be the rea-

son why 20-epi-compounds have more potent activity

than 20-normal compounds.^23–25

This implies that LAC67b might work as a tissue- or

cell-selective agonist for the VDR. Recently, we have

clariﬁed the level of CYP24A1 mRNA in several cell

types treated with LAC67a and LAC67b by real time

PCR, and suggested that LAC67b might act as a selec-

tive VDR modulator (SVDRM).³¹SVDRMs would

5. Discussion

Almost all of the X-ray crystal structures of the VDR–

LBD complexed with a ligand revealed that the distance

between the terminal alkyl group of the vitamin D side

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

463

1.2

1

a

b

3

0.8

0.6

0.4

0.2

0

2

1

0

D

8

vehicle

10

9

8

7

6

5

10

9

8

7

6 5

vehicle D 10

9

8

7

6

5

10

9

8

7

6 5

8

Lac67a

-log [M]

Lac82a

Lac67b

Lac82b

-log [M]

1.2

1

c

d

3

2

0.8

0.6

0.4

1

0.2

0

D

8

D

8

vehicle

10

9

8

7

6

5

10

9

8

7

6

5

vehicle

10

9

8

7

6

5

10

9

8

7

6 5

Lac82a

Lac82b

Lac67a

Lac67b

+

1,25-(OH)₂D₃

-log [M]

Figure 3. Transactivation of synthetic lactones in Cos7 cells. The activities were evaluated by dual luciferase assay using a full-length human VDR

expression plasmid (pCMX-hVDR), a reporter plasmid containing three copies of the mouse osteopontin VDRE (SPPx3-TK-Luc), and the internal

control plasmid containing sea pansy luciferase expression constructs (pRL-CMV) in Cos7 cells as described previously.^20,25(a) agonistic activity of

LAC67a and LAC67b, (b) agonistic activity of LAC82a and LAC82b, (c) antagonistic activity of LAC67a and LAC67b in the presence of 10 nM 1,25-

(OH)₂D₃, (d) antagonistic activity of LAC82a and LAC82b in the presence of 10 nM 1,25-(OH)₂D₃.

act like as SERMs. Certain constituents in each cell are

important for determining cell speciﬁcity. For example,

the antagonistic function of ZK159222 has been shown

to depend on the cell-speciﬁc ratio between VDR and

RXR proteins.³²Kato’s group have reported that deple-

tion of serum from the culture medium converted

TEI9647 3b from an antagonist to an agonist of VDR-

mediated transactivation, whereas it retained antagonis-

tic activity in the presence of serum.³³Taken together

with our previous report, these ﬁndings indicate that

LAC67a is a VDR antagonist while LAC67b might be

a SVDRM.³¹

The mechanism of the antagonistic activity of the meth-

ylene lactone compound TEI9647 3b has been reported

by the Ishizuka and Norman group. They reported that

the importance of Cys403 and Cys410 of human VDR

for expression of the antagonistic activity of TEI9647

3b suggests a mechanism involving Michael-type addi-

tion of these cysteines to the methylene lactone.^34,35

On the other hand, the same group, using a ligand ex-

change assay, demonstrated that TEI9647 3b bound to

VDR is freely exchanged with 1,25-(OH)₂D₃

1

in vitro.³⁶Since LAC67b has completely the same struc-

ture at the side chain as TEI9647 3b, the mechanism of

its antagonistic action is thought to be quite similar to

that of the latter. LAC67b acts as a VDR antagonist

in Cos7 cells transfected with human VDR, whereas this

compound functions as a potent VDR agonist in

HEK293 cells transfected with human VDR. The latter

fact that LAC67b can function as a potent VDR agonist

suggests that this compound does not form a covalent

bond with the human VDR. Therefore, we suggest that

methylene lactones such as LAC67b and TEI9647 3b in-

hibit the transactivation as passive antagonists, and not

via covalent bond formation.

Docking models of LAC82a and LAC82b show six com-

plete hydrogen bonds and moderate interactions with

Tyr401 and Val418, but not Phe422. Since these hydro-

phobic interactions are insuﬃcient, the activity of these

compounds might depend on the cellular environment.

Therefore, the ligand would act as an agonist if there

are appropriate coactivators in that cell, whereas it

would act as an antagonist if proper proteins are lack-

ing. These observations demonstrate that the four syn-

thetic lactones are passive antagonists.

464

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

50

a

b

3000

2000

1000

40

30

20

10

0

vehicle

D

8

9

8

7

6

9

8

7

6

vehicle

D

8

9

8

7

6

9

8

7

6

Lac67a

-log [M]

Lac82a

-log [M]

Lac67b

Lac82b

1.2

1

c

d

40

30

20

0.8

0.6

0.4

10

0

0.2

0

vehicle

D

8

7

6

8

7

6

vehicle

D

8

7

6

8

7

6

Lac67a

Lac82a

Lac67b

Lac82b

+

1,25-(OH)₂D₃

-log [M]

Figure 4. Transactivation of four methylene lactones in HEK293 cells. Transactivation potencies were evaluated using a full-length hVDR expression

plasmid (pCMX-hVDR) and a reporter plasmid containing three copies of the mouse osteopontin VDRE (SPPx3-TK-Luc) in HEK293 cells as

described previously.³¹(a) agonistic activity of LAC67a and LAC67b, (b) agonistic activity of LAC82a and LAC82b, (c) antagonistic activity of

LAC67a and LAC67b in the presence of 10 nM 1,25-(OH)₂D₃, (d) antagonistic activity of LAC82a and LAC82b in the presence of 10 nM 1,25-

(OH)₂D₃.

6. Conclusions

were recorded on a JASCO FT/IR-300E spectrometer.

UV spectra were recorded on a BECKMAN DU7500

spectrophotometer. All air and moisture sensitive reac-

tions were carried out under argon atmosphere.

We synthesized four methylene lactone analogs and

evaluated their aﬃnity for the VDR as well as their ago-

nistic and antagonistic activities. Based on data for their

biological activities and a docking study, we concluded

that these methylene lactones are passive antagonists

lacking hydrophobic interactions with H12 of the

VDR. We have already synthesized and reported an-

other type of antagonist having a bulky substituent of

the adamantine ring at the side chain. Further experi-

ments are now in progress to study the molecular mech-

anism of VDR antagonism using these two types of

synthetic antagonist.

7.1. (3R,5R)-3,5-Bis{tert-butyl(dimethyl)silyl]oxy}-1-

(hydroxymethyl)-1,4-cyclohexanediol (4b)

To a solution of methyl ester 4a (10.4 g, 24 mmol) in

EtOH (60 mL) was added NaBH₄(2.8 g, 75 mmol),

and the mixture was stirred at 0 ꢁC for 5 h. The reaction

was quenched with H₂O and brine at 0 ꢁC and was ex-

tracted with AcOEt. The organic layer was washed with

brine, dried over MgSO₄, and evaporated. The residue

was chromatographed on silica gel (40 g, hexane/

AcOEt = 17:3) to give alcohol 4b (9.1 g, 93.3%) as a col-

orless solid.

7. Experimental

¹H NMR spectra were recorded in CDCl₃at 400 MHz

and chemical shifts are reported as d units relative to tet-

ramethylsilane or solvent signal as an internal standard.

¹³C NMR spectra were recorded at 100 MHz. High and

low resolution mass spectra were obtained on a JEOL

JMS-AX505HA spectrometer at 70 eV. Relative intensi-

ties are given in parentheses in low mass. IR spectra

4b ¹H NMR d 0.11 and 0.12 and 0.15 and 0.16 (each 3H,

s, SiMe), 0.90 and 0.91 (each 9H, s, t-Bu), 1.34 (1H, dd,

J = 12.9, 10.9 Hz), 1.50 (1H, dd, J = 14.5, 2.4 Hz), 2.00

(2H, m), 2.12 (1H, dd, J = 8.2, 4.7 Hz), 2.28 (1H, d,

J = 2.9 Hz), 3.33 (2H, m, CH₂OH), 3.41 (1H, dd,

J = 11.0, 4.7 Hz), 4.11 (1H, ddd, J = 10.7, 9.0, 4.7 Hz),

4.32 (1H, m), 4.53 (1H, br s, OH).

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

465

Figure 5. Docking models of LAC67a, LAC67b, LAC82a and LAC82b into the VDR–LBD. Hydrogen bonds and hydrophobic interactions are

depicted as dotted red and green lines, respectively. (a) 1,25-(OH)₂D₃and VDR–LBD complex. 26-Methyl group of 1,25-(OH)₂D₃makes intimate

van der Waals contacts with Tyr401 (Y401), Val418 (V418), and Phe422 (F422). (b) Superposition of four docking models. LAC67a, LAC67b,

LAC82a and LAC82b in the VDR–LBP are drawn in blue, cyan, green, and yellow color, respectively. (c) LAC67a and VDR–LBD complex. (d)

LAC67b and VDR–LBD complex. (e) LAC82a and VDR–LBD complex. f. LAC82b and VDR–LBD complex.

7.2. (3R,5R)-3,5-Bis{tert-butyl(dimethyl)silyl]oxy}-4-

hydroxycyclohexanone (4c)

brine, dried over MgSO₄, and evaporated. The residue

was chromatographed on silica gel (30 g, hexane/

AcOEt = 23:2) to give ketone 4c (900.0 mg, quant.) as

a colorless solid.

To a solution of alcohol 4b (951.6 mg, 2.34 mmol) in

MeOH (30 mL) was added sodium periodate-saturated

water (8 mL), and the mixture was stirred at 0 ꢁC for

3 h. The reaction was poured into brine and was ex-

tracted with AcOEt. The organic layer was washed with

4c ¹H NMR d 0.06 (6H, s, SiMe · 2), 0.08 and 0.09 (each

3H, s, SiMe), 0.85 and 0.90 (each 9H, s, t-Bu), 2.25 (1H,

m), 2.45 (1H, m), 2.60 (1H, m), 2.77 (1H, dd, J = 14.4,

466

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

3.3 Hz), 3.80 (1H, m, H-4), 4.27 (2H, m, H-3, 5). ¹³C

NMR d ꢀ5.1, ꢀ4.94, ꢀ4.88, ꢀ4.7, 17.9, 18.0, 25.6 (3

carbons), 25.7 (3 carbons), 44.3, 46.0, 69.4, 70.0, 72.6,

207.3.

7.6. (3S)-3-[(7aR)-7a-Methyl-4-oxooctahydro-1H-inden-

1-yl]butanenitrile (10)

Compound 10 was obtained from 8 by the same proce-

dure as described for 9 (yield 95.8%) as a colorless solid.

7.3. (3R)-3-[(4S,7aR)-4-Hydroxy-7a-methyloctahydro-1H-

inden-1-yl]butanenitrile (7)

10 ¹H NMR d 0.62 (3H, s, H-18), 1.08 (3H, d,

J = 6.6 Hz, H-21), 2.49 (1H, dd, J = 11.6, 7.7 Hz, H-

14). ¹³C NMR d 12.9, 19.0, 19.7, 24.0, 24.4, 27.1, 32.0,

38.6, 40.8, 49.5, 54.8, 61.6, 118.5, 211.2. MS m/z (%):

219 (M⁺, 65), 204 (100), 191 (28), 176 (80), 163 (45),

124 (58), 96 (65).

To a solution of tosylate 5 (130.1 mg, 0.36 mmol) in dry

DMSO (500 lL) was added KCN (46.5 mg, 0.71 mmol),

and the mixture was stirred at 70 ꢁC for 1.5 h. The reac-

tion was quenched with H₂O at 0 ꢁC and was extracted

with AcOEt. The organic layer was washed with brine,

dried over MgSO₄, and evaporated. The residue was

chromatographed on silica gel (5 g, hexane/

AcOEt = 1:1) to give cyanide 7 (78.1 mg, 98.2%) as a

colorless solid.

7.7. (3R)-3-{(1R,3R,7E)-1,3-Bis{tert-butyl(dimethyl)silyl]-

oxy}-2-[(trimethylsilyl)oxy]-9,10-secoestra-5,7-dien-17-

yl}butanenitrile (11)

To a solution of phosphine oxide 4 (1.18 g, 1.80 mmol, a

2:3 mixture of diastereomers at C(4)) in dry THF

(10 mL) at ꢀ78 ꢁC was added slowly n-BuLi (1.59 M

hexane solution, 1.1 mL, 1.79 mmol), and the resulting

dark orange solution was stirred for 15 min. To this col-

ored solution was added a solution of ketone 9

(258.7 mg, 1.18 mmol) in dry THF (5 mL), the reaction

mixture was stirred for 1.5 h at same temperature, and

then the mixture was allowed to warm to 0 ꢁC for 2 h.

The reaction was quenched with saturated NH₄Cl aq

solution at 0 ꢁC and was extracted with AcOEt. The or-

ganic layer was washed with brine, dried over MgSO₄,

and evaporated. The residue was chromatographed on

silica gel (20 g, hexane/AcOEt = 19:1) to give compound

11 (683.7 mg, 79.8%, a 2:3 mixture of C(2) epimers) as

colorless oil.

7 ¹H NMR d 0.96 (3H, s, H-18), 1.15 (3H, d, J = 6.6 Hz,

H-21), 2.25 (1H, dd, J = 16.7, 6.9 Hz, H-22), 2.35 (1H,

dd, J = 16.7, 3.8 Hz, H-22), 4.09 (1H, m, H-8). ¹³C

NMR d 13.8, 17.5, 19.3, 22.6, 24.8, 27.2, 33.2, 33.7,

40.2, 42.1, 52.5, 55.3, 69.1, 119.1. MS m/z (%): 221

(M⁺, 15), 206 (45), 188 (15), 163 (15), 135 (20), 125

(20), 111 (100).

7.4. (3S)-3-[(4S,7aR)-4-Hydroxy-7a-methyloctahydro-1H-

inden-1-yl]butanenitrile (8)

Compound 8 was obtained from 6 by the same proce-

dure as described for 7 (yield 98.5%) as a colorless

solid.

8 ¹H NMR d 0.94 (3H, s, H-18), 1.07 (3H, d, J = 6.7 Hz,

H-21), 2.43 (2H, m, H-22), 4.09 (1H, m, H-8). ¹³C NMR

d 14.0, 17.5, 19.7, 22.4, 24.2, 27.1, 31.9, 33.6, 40.1, 41.8,

52.4, 55.0, 69.1, 118.9. IR (neat) 3493, 2930, 2871, 2248,

1454, 1167, 991, 954 cm^ꢀ1. MS m/z (%): 221 (M⁺, 20),

206 (60), 188 (15), 163 (15), 135 (35), 125 (25), 111

(100), 93 (30). HRMS calcd for C₁₄H₂₃ON 221.1780,

found 221.1781.

1

11 (a 2:3 mixture of C(2) epimers) H NMR d 0.0–0.1

(12H, s, SiMe · 4), 0.120, 0.125 (2:3) (9H, s, TMS),

0.56, 0.57 (2:3) (3H, s, H-18), 0.854, 0.867 (2:3) and

0.873, 0.891 (3:2) (each 9H, s, t-Bu), 1.17 (3H, d,

J = 6.6 Hz, H-21), 2.80 (1H, m, H-9), 3.54, 3.59 (3:2)

(1H, m, H-2), 3.80 (1H, m, H-3), 3.88, 3.92 (3:2) (1H,

m, H-1), 5.79, 5.82 (2:3) (1H, d, J = 11.0 Hz, H-7),

6.09, 6.12 (3:2) (1H, d, J = 11.0 Hz, H-6). IR (neat)

2953, 2856, 2245, 1620, 1471, 1251, 1095, 837,

775 cm^ꢀ1. MS m/z (%): 659 (M⁺, 20), 528 (30), 527

(70), 470 (90), 424 (80), 380 (50), 306 (30), 147 (30), 73

(60). HRMS calcd for C₃₇H₆₉O₃NSi₃659.4585, found

659.4597.

7.5. (3R)-3-[(7aR)-7a-Methyl-4-oxooctahydro-1H-inden-

1-yl]butanenitrile (9)

To a solution of cyanide 7 (286.6 mg, 1.30 mmol) in dry

CH₂Cl₂(3 mL) were added Molecular Sieves 4A

(150 mg) and N-methylmorpholine N-oxide (NMO,

1.26 g, 10.8 mmol) at room temperature. After 10 min,

tetrapropylammonium perruthenate (TPAP, 24.6 mg,

0.070 mmol) was added to the mixture, and the reaction

mixture was stirred for 2 h at same temperature. The

mixture was chromatographed on silica gel (20 g, hex-

ane/AcOEt = 1:1) to give ketone 9 (260.5 mg, 91.5%)

as a colorless solid.

7.8. (3S)-3-{(1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]-

oxy}-2-[(trimethylsilyl)oxy]-9,10-secoestra-5,7-dien-17-yl}

butanenitrile (12)

Compound 12 was obtained from 10 by the same proce-

dure as described for 11 (yield 74.7%, a 2:3 mixture of

C(2) epimers) as colorless oil.

1

9 ¹H NMR d 0.67 (3H, s, H-18), 1.21 (3H, d, J = 6.6 Hz,

H-21), 2.52 (1H, dd, J = 11.7, 7.6 Hz, H-14). ¹³C NMR

d 12.6, 19.1, 19.4, 23.9, 24.8, 27.4, 33.2, 38.6, 40.8, 49.7,

55.1, 61.6, 118.6, 211.2. IR (neat) 2957, 2875, 2244,

1710, 1463, 1383, 1233 cm^ꢀ1. MS m/z (%): 219 (M⁺,

70), 204 (80), 179 (25), 176 (90), 163 (60), 124 (40), 96

(45).

12 (a 2:3 mixture of C(2) epimers) H NMR d ꢀ0.1–0.1

(12H, s, SiMe · l4), 0.119, 0.123 (2:3) (9H, s, TMS),

0.54, 0.55 (2:3) (3H, s, H-18), 0.85, 0.86 (2:3) and 0.87,

0.89 (3:2) (each 9H, s, t-Bu), 1.09 (3H, d, J = 6.6 Hz,

H-21), 2.80 (1H, m, H-9), 3.54, 3.59 (3:2) (1H, m, H-

2), 3.80 (1H, m, H-3), 3.88, 3.92 (3:2) (1H, m, H-1),

5.79, 5.82 (2:3) (1H, d, J = 11.1 Hz, H-7), 6.08, 6.11

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

467

(3:2) (1H, d, J = 11.1 Hz, H-6). IR (neat) 2954, 2928,

2856, 2247, 1729, 1471, 1251, 1095, 837 cm^ꢀ1. MS m/z

(%): 659 (M⁺, 10), 602 (8), 527 (35), 470 (40), 424 (38),

309 (58), 256 (52), 236 (55), 138 (60), 75 (100). HRMS

calcd for C₃₇H₆₉O₃NSi₃659.4585, found 659.4579.

pound 13 (308.1 mg, 0.53 mmol, a 2:3 mixture of C(2)

epimers) in dry CH₂Cl₂(2.5 mL) was added to the mix-

ture, and the reaction mixture was stirred for 15 min at

same temperature. Then, to this solution was added

Et₃N (363 lL, 2.62 mmol), and mixture was stirred at

ꢀ78 ꢁC for 30 min and at 0 ꢁC for 1 h. The reaction

was quenched with H₂O at 0 ꢁC and was extracted with

CH₂Cl₂. The organic layer was washed with brine, dried

over MgSO₄, and evaporated. The residue was chro-

matographed on silica gel (10 g, hexane/AcOEt = 9:1)

to give ketone 15 (294.8 mg, 96.0%) as a colorless oil.

7.9. (3R)-3-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]-

oxy}-2-hydroxy-9,10-secoestra-5,7-dien-17-yl)butaneni-

trile (13)

A solution of compound 11 (532.6 mg, 0.81 mmol, a 2:3

mixture of C(2) epimers) in AcOH/THF/H₂O (8:8:1,

17 mL) was stirred at room temperature for 22.5 h.

The reaction was quenched with 5% NaHCO₃aq solu-

tion at 0 ꢁC and was extracted with AcOEt. The organic

layer was washed with brine, dried over MgSO₄, and

evaporated. The residue was chromatographed on silica

gel (20 g, hexane/AcOEt = 49:1 then 19:1) to give com-

pound 11 (38.4 mg, 7.2%, a 2:3 mixture of C(2) epimers)

and compound 13 (colorless oil, 405.6 mg, 85.5%, a 2:3

mixture of C(2) epimers).

1

15 H NMR d 0.05 and 0.08 (each 3H, s, SiMe), 0.06

(6H, s, SiMe · 2), 0.57 (3H, s, H-18), 0.86 and 0.88 (each

9H, s, t-Bu), 1.18 (3H, d, J = 6.6 Hz, H-21), 2.83 (1H, m,

H-9), 4.35 (1H, dd, J = 6.4, 4.2 Hz, H-3), 4.53 (1H, dd,

J = 8.7, 5.5 Hz, H-1), 5.81 (1H, d, J = 11.1 Hz, H-7),

6.33 (1H, d, J = 11.1 Hz, H-6). ¹³C NMR d ꢀ5.00,

ꢀ4.95, ꢀ4.7, ꢀ4.6, 12.3, 18.3, 18.5, 19.6, 22.3, 23.4,

25.0, 25.9 (3 carbons), 26.0 (3 carbons), 27.7, 28.9,

37.7, 40.3, 41.6, 45.9, 46.7, 55.3, 56.2, 74.5, 74.8, 116.4,

119.1, 124.5, 129.5, 142.3, 208.7. IR (neat) 2952, 2929,

2856, 2245, 1738, 1470, 1254, 1098, 836 cm^ꢀ1. MS m/z

(%): 585 (M⁺, 2), 570 (3), 528 (100), 396 (62), 230 (40),

75 (62), 73 (32). HRMS calcd for C₃₄H₅₉O₃NSi₂

585.4033, found 585.4055.

1

13 (a 2:3 mixture of C(2) epimers) H NMR d 0.0–0.2

(12H, s, SiMe · 4), 0.55, 0.56 (2:3) (3H, s, H-18), 0.84,

0.86 (2:3) and 0.87, 0.89 (3:2) (each 9H, s, t-Bu), 1.17

(3H, d, J = 6.6 Hz, H-21), 2.80 (1H, m, H-9), 3.51,

3.58 (3:2) (1H, m, H-2), 3.91, 3.99 (3:2) (1H, m, H-3),

3.99 (1H, m, H-1), 5.79 (1H, d, J = 11.2 Hz, H-7),

6.14, 6.17 (3:2) (1H, d, J = 11.2 Hz, H-6). IR (neat)

3481, 2953, 2930, 2857, 2246, 1717, 1471, 1254, 1090,

837, 779 cm^ꢀ1. MS m/z (%): 587 (M⁺, 5), 530 (7), 438

(5), 398 (100), 306 (15), 165 (10), 129 (10), 75 (40), 73

(18).

7.12. (3S)-3-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]-

oxy}-2-oxo-9,10-secoestra-5,7-dien-17-yl)butanenitrile

(16)

Compound 16 was obtained from 14 by the same proce-

dure as described for 15 (yield 99.2%) as a colorless oil.

1

7.10. (3S)-3-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]-

oxy}-2-hydroxy-9,10-secoestra-5,7-dien-17-yl)butaneni-

trile (14)

16 H NMR d 0.04 and 0.08 (each 3H, s, SiMe), 0.05

(6H, s, SiMe · 2), 0.55 (3H, s, H-18), 0.86 and 0.88 (each

9H, s, t-Bu), 1.09 (3H, d, J = 6.7 Hz, H-21), 2.82 (1H, m,

H-9), 4.35 (1H, dd, J = 6.4, 4.2 Hz, H-3), 4.53 (1H, dd,

J = 8.6, 5.5 Hz, H-1), 5.81 (1H, d, J = 11.2 Hz, H-7),

6.33 (1H, d, J = 11.2 Hz, H-6). ¹³C NMR d ꢀ5.1,

ꢀ5.0, ꢀ4.8, ꢀ4.7, 12.3, 18.2, 18.4, 19.6, 22.2, 23.4,

24.9, 25.81 (3 carbons), 25.85 (3 carbons), 27.6, 28.8,

34.0, 37.6,40.2, 45.8, 46.6, 55.2, 56.1, 74.4, 74.7, 116.3,

119.0, 124.5, 129.4, 142.2, 208.5. MS m/z (%): 528

(M⁺ꢀC(CH₃)₃, 100), 510 (4), 396 (42), 378 (12), 325

(15), 259 (10), 147 (12), 73 (45).

Compound 14 was obtained from 12 by the same proce-

dure as described for 13 (yield 82.6%, a 2:3 mixture of

C(2) epimers) as colorless oil.

1

14 (a 2:3 mixture of C(2) epimers) H NMR d ꢀ0.1–0.2

(12H, s, SiMe · 4), 0.53, 0.55 (2:3) (3H, s, H-18), 0.85,

0.86 (2:3) and 0.87, 0.89 (3:2) (each 9H, s, t-Bu), 1.09

(3H, d, J = 6.6 Hz, H-21), 2.80 (1H, m, H-9), 3.51,

3.58 (3:2) (1H, m, H-2), 3.91, 3.99 (3:2) (1H, m, H-3),

3.99 (1H, m, H-1), 5.80 (1H, d, J = 11.2 Hz, H-7),

6.13, 6.16 (3:2) (1H, d, J = 11.2 Hz, H-6). IR (neat)

7.13. (3R)-3-((1R,3R,7E)-1,3-Bis{tert-butyl(dimethyl)

silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)

butanenitrile (17)

3555, 2952, 2855, 2246, 1462, 1254, 1085, 836 cm^ꢀ1

.

MS m/z (%): 587 (M⁺, 3), 530 (8), 438 (10), 398 (100),

380 (10), 306 (20), 257 (10), 236 (15), 129 (15), 73 (25).

HRMS calcd for C₃₄H₆₁O₃NSi₂587.4190, found

587.4196.

To a suspension of methyltriphenylphosphonium bro-

mide (527.3 mg, 1.48 mmol) in dry THF (1.5 mL) at

0 ꢁC was added slowly n-BuLi (1.59 M hexane solution,

923 lL, 1.47 mmol), and the resulting yellow solution

was stirred for 1 h. To this colored solution was added

a solution of ketone 15 (285.9 mg, 0.49 mmol) in dry

THF (2 mL), the reaction mixture was stirred at 0 ꢁC

for 1 h, and then at room temperature for 1 h. The reac-

tion was quenched with H₂O at 0 ꢁC and was extracted

with AcOEt. The organic layer was washed with brine,

dried over MgSO₄, and evaporated. The residue was

chromatographed on silica gel (10 g, hexane/

7.11. (3R)-3-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]

oxy}-2-oxo-9,10-secoestra-5,7-dien-17-yl)butanenitrile

(15)

To a solution of oxalyl chloride (2.0 M CH₂Cl₂solution,

315 lL, 0.63 mmol) in dry CH₂Cl₂(1 mL) was added a

solution of DMSO (90 lL, 1.27 mmol) in dry CH₂Cl₂

(500 lL) at ꢀ78 ꢁC. After 10 min, a solution of com-

468

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

AcOEt = 19:1) to give 2-methylene 17 (268.4 mg, 94.2%)

as a colorless oil.

7.16. (3S)-3-((1R,3R,7E)-1,3-Bis{tert-butyl(dimethyl)sil-

yl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)butanal

(20)

17 ¹H NMR d 0.02 and 0.05 and 0.06 and 0.08 (each 3H,

s, SiMe), 0.57 (3H, s, H-18), 0.86 and 0.89 (each 9H, s, t-

Bu), 1.18 (3H, d, J = 6.6 Hz, H-21), 2.82 (1H, m, H-9),

4.43 (2H, m, H-3, 1), 4.92 and 4.97 (each 1H, s,

–C@CH₂), 5.84 (1H, d, J = 11.2 Hz, H-7), 6.21 (1 H,

d, J = 11.2 Hz, H-6). ¹³C NMR d ꢀ4.9, ꢀ4.71, ꢀ4.66,

ꢀ4.64, 12.4, 18.36, 18.44, 19.7, 22.3, 23.4, 25.0, 25.98

(3 carbons), 26.04 (3 carbons), 27.8, 28.8, 34.1, 38.8,

40.4, 45.8, 47.8, 55.4, 56.2, 71.8, 72.7, 106.6, 116.7,

119.2, 122.4, 133.5, 140.4, 153.1. IR (neat) 2952, 2891,

Compound 20 was obtained from 18 by the same proce-

dure as described for 19 (yield 87.3%) as a colorless oil.

20 ¹H NMR d 0.02 and 0.04 and 0.06 and 0.07 (each 3H,

s, SiMe), 0.58 (3H, s, H-18), 0.86 and 0.89 (each 9H, s, t-

Bu), 0.95 (3H, d, J = 6.6 Hz, H-21), 2.26 (1H, ddd,

J = 15.9, 9.6, 3.4 Hz, H-22), 2.67 (1H, dd, J = 15.9, 3.1

Hz, H-22), 2.82 (1H, m, H-9), 4.42 (2H, m, H-3, 1),

4.92 and 4.97 (each 1H, s, –C@CH₂), 5.85 (1H, d,

J = 11.2 Hz, H-7), 6.20 (1H, d, J = 11.2 Hz, H-6), 9.75

(1 H, dd, J = 3.4, 1.0 Hz, H-23). ¹³C NMR d ꢀ4.9,

ꢀ4.7, ꢀ4.6 (2 carbons), 12.7, 18.4, 18.5, 20.0, 22.2,

23.5, 26.0 (3 carbons), 26.1 (3 carbons), 27.4, 28.8,

31.1, 38.8, 40.7, 45.8, 47.8, 50.3, 56.2, 56.3, 71.8, 72.7,

106.5, 116.7, 122.5, 133.4, 140.6, 153.1, 203.4.

2856, 2246, 1472, 1256 cm^ꢀ1

.

7.14. (3S)-3-((1R,3R,7E)-1,3-Bis{tert-butyl(dimethyl)sil-

yl] oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)butaneni-

trile (18)

Compound 18 was obtained from 16 by the same proce-

dure as described for 17 (yield 88.5%) as a colorless oil.

7.17. 5-[(2R)-2-((1R,3R,7E)-1,3-Bis{tert-butyl(dimethyl)-

silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)pro-

pyl]-3-methylenedihydro-2(3H)-furanone (21)

1

18 H NMR d 0.02 and 0.05 and 0.06 and 0.08 (each

3H, s, SiMe), 0.55 (3H, s, H-18), 0.86 and 0.89 (each

9H, s, t-Bu), 1.10 (3H, d, J = 6.7 Hz, H-21), 2.83 (1H,

m, H-9), 4.42 (2H, m, H-3, 1), 4.92 and 4.97 (each

1H, s, –C@CH₂), 5.85 (1H, d, J = 11.2 Hz, H-7),

6.20 (1H, d, J = 11.2 Hz, H-6). ¹³C NMR d ꢀ4.9,

ꢀ4.7, ꢀ4.6 (2 carbons), 12.6, 18.37, 18.45, 19.9,

22.2, 23.5, 24.5, 25.99 (3 carbons), 26.04 (3 carbons),

27.5, 28.7, 32.8, 38.8, 40.3, 45.5, 47.8, 55.0, 56.1,

71.8, 72.7, 106.6, 116.8, 119.0, 122.4, 133.5, 140.3,

153.1. IR (neat) 2934, 2885, 2857, 2247, 1472,

To a suspension of CrCl₃(74.8 mg, 0.47 mmol) in dry

THF (1 mL) was added slowly LiAlH₄(1.0 M solution

in THF, 235 lL, 0.24 mmol) at 0 ꢁC, and the mixture

was stirred at room temperature for 45 min. To the mix-

ture were added a solution of methyl 2-(bromo-

methyl)acrylate (34 lL, 0.28 mmol) in dry THF

(300 lL) and a solution of aldehyde 19 (68.9 mg,

0.12 mmol) in dry THF (750 lL) at room temperature,

and the resulting mixture was stirred at the same tem-

perature for 30 min. The reaction was quenched with

H₂O and 1N HCl at 0 ꢁC and was extracted with

AcOEt. The organic layer was washed with brine, dried

over MgSO₄, and evaporated. The residue was chro-

matographed on silica gel (5 g, hexane/AcOEt = 49:1)

to give methylene lactone 21 (amorphous solid,

63.6 mg, 82.7%, a 2:3 mixture of diastereomers at

C(23)) and 23-alcohol (a colorless oil, 4 mg, 5.8%).

1257 cm^ꢀ1

.

7.15. (3R)-3-((1R,3R,7E)-1,3-Bis{tert -butyl(dimethyl)sil-

yl] oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)butanal

(19)

To a solution of 2-methylene 17 (113.3 mg, 0.19 mmol)

in dry CH₂Cl₂(1 mL) was added a solution of DI-

BAL-H (1.01 M solution in toluene, 252 lL, 0.25 mmol)

at 0 ꢁC for 1.5 h. The reaction was quenched with 10%

potassium sodium tartrate aq solution at 0 ꢁC and was

extracted with CH₂Cl₂. The organic layer was washed

with brine, dried over MgSO₄, and evaporated. The res-

idue was chromatographed on silica gel (5 g, hexane/

AcOEt = 49:1) to give aldehyde 19 (102.3 mg, 90.0%)

as a colorless oil.

21 (a 2:3 mixture of C(23) epimers) ¹H NMR d ꢀ0.0–0.1

(12H, s, SiMe · 4), 0.557, 0.562 (2:3) (3H, s, H-18), 0.86

and 0.890, 0.894 (2:3) (each 9H, s, t-Bu), 1.02, 1.03 (3:2)

(3H, d, J = 6.3 Hz, H-21), 2.82 (1H, m), 3.06 (1H, m),

4.42 (2H, m, H-3, 1), 4.63 (1H, m, H-23), 4.91 and

4.97 (each 1H, s, –C@CH₂), 5.62 (1H, t, J = 2.4 Hz,

H-27), 5.83 (1H, d, J = 11.2 Hz, H-7), 6.21 (1H, d,

J = 11.2 Hz, H-6), 6.22 (1H, m, H-27). MS m/z (%):

654 (M⁺, 12), 597 (8), 522 (78), 465 (18), 366 (35), 251

(18), 197 (15), 75 (100), 73 (38).

19 ¹H NMR d 0.02 and 0.04 and 0.06 and 0.07 (each 3H,

s, SiMe), 0.59 (3H, s, H-18), 0.86 and 0.89 (each 9H, s, t-

Bu), 1.02 (3H, d, J = 6.5 Hz, H-21), 2.81 (1H, m, H-9),

4.42 (2H, m, H-3, 1), 4.91 and 4.97 (each 1H, s,

–C@CH₂), 5.83 (1H, d, J = 11.2 Hz, H-7), 6.20 (1H, d,

J = 11.2 Hz, H-6), 9.76 (1H, dd, J = 3.3, 1.2 Hz, H-23).

¹³C NMR d ꢀ4.9, ꢀ4.7, ꢀ4.6 (2 carbons), 12.3, 18.37,

18.45, 20.3, 22.4, 23.5, 25.98 (3 carbons), 26.04 (3 car-

bons), 28.1, 28.8, 32.1, 38.8, 40.6, 45.9, 47.8, 51.0,

56.40, 56.43, 71.8, 72.7, 106.5, 116.6, 122.5, 133.3,

140.8, 153.1, 203.6. MS m/z (%): 586 (M⁺, 12), 529

(10), 454 (100), 366 (34), 322 (12), 234 (12), 75 (32), 73

(30).

7.18. 5-[(2S)-2-((1R,3R,7E)-1,3-Bis{tert-butyl(dimethyl)-

silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)pro-

pyl]-3-methylenedihydro-2(3H)-furanone (22)

Compound 22 was obtained from 20 by the same proce-

dure as described for 21 (yield 81.8%, 2:3 mixture of

C(23) epimers) as amorphous solid.

22 (a 2:3 mixture of C(23) epimers) ¹H NMR d ꢀ0.1–0.1

(12H, s, SiMe · 4), 0.54, 0.55 (2:3) (3H, s, H-18), 0.859,

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

469

0.861 (3:2) and 0.89 (each 9H, s, t-Bu), 0.92, 0.95 (2:3)

(3H, d, J = 6.7 Hz, H-21), 2.81 (1H, m), 3.06 (1 H, m),

4.42 (2H, m, H-3, 1), 4.63 (1H, m, H-23), 4.92 and

4.97 (each 1H, s, –C@CH₂), 5.66 (1H, m, H-27), 5.84

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

6), 6.27 (1H, m, H-27).

–C@CH₂), 5.63 (1H, t, J = 2.4 Hz, H-27), 5.89 (1H, d,

J = 11.3 Hz, H-7), 6.23 (1H, t, J = 2.7 Hz, H-27), 6.36

(1H, d, J = 11.3 Hz, H-6). IR (neat) 3421, 2929, 2875,

1762 cm^ꢀ1. MS m/z (%): 426 (M⁺, 30), 390 (70), 375

(20), 285 (25), 251 (100), 197 (50), 157 (40), 105 (58).

HRMS calcd for C₂₇H₃₈O₄426.2770, found 426.2787.

UV (95% EtOH): k_max245, 253, 263 nm.

7.19. 5-{(2R)-2-[(1R,3R,7E)-1,3-Dihydroxy-2-methylene-

9,10-secoestra-5,7-dien-17-yl]propyl}-3-methylenedihy-

dro-2(3H)-furanone (LAC67a, b)

LAC82b (less polar) ¹H NMR d 0.57 (3H, s, H-18), 0.95

(3H, d, J = 6.6 Hz, H-21), 4.49 (2H, m, H-3, 1), 4.64

(1H, m, H-23), 5.09 and 5.12 (each 1H, s, –C@CH₂),

5.62 (1H, t, J = 2.4 Hz, H-27), 5.89 (1H, d,

J = 11.2 Hz, H-7), 6.23 (1H, t, J = 2.8 Hz, H-27), 6.34

(1H, d, J = 11.2 Hz, H-6). IR (neat) 3421, 2936, 2875,

1759 cm^ꢀ1. MS m/z (%): 426 (M⁺, 20), 408 (28), 390

(90), 375 (28), 285 (30), 251 (100), 197 (55), 157 (30),

105 (55). HRMS calcd for C₂₇H₃₈O₄426.2770, found

426.2759. UV (95% EtOH): k_max245, 253, 263 nm.

To a solution of methylene lactone 21 (26.0 mg,

0.040 mmol, 2:3 mixture of C(23) epimers) in MeOH

(1 mL) was added CSA (20.4 mg, 0.088 mmol) at room

temperature for 1.3 h. The reaction was quenched with

5% NaHCO₃at 0 ꢁC and was extracted with AcOEt.

The organic layer was washed with brine, dried over

MgSO₄, and evaporated. The residue was chromato-

graphed on silica gel (5 g, AcOEt) to give lactone ana-

logue LAC67 (5.7 mg, 54.0%, a 2:3 mixture of C(23)

epimers) as a colorless solid. The mixture was separated

by HPLC (LiChrosorb Si 60, Hibar RT 250-10, 7 lm,

hexane/AcOEt = 7:3, 5 mL/min) to give LAC67a

(2.7 mg) and LAC67b (2.1 mg). The purity of LAC67a

and LAC67b was proved to be about 100% by HPLC.

7.21. (6R)-6-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]-

oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)-2-meth-

ylene-1,4-heptanediol (23a, b)

To a solution of methylene lactone 21 (21.0 mg,

0.032 mmol) in dry toluene (1 mL) was added a solution

of DIBAL-H (1.01 M solution in toluene, 137 lL,

0.14 mmol) at 0 ꢁC for 1 h. The reaction was quenched

with 10% potassium sodium tartrate aq solution at

0 ꢁC and was extracted with AcOEt. The organic layer

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

rated. The residue was chromatographed on silica gel

(7 g, hexane/AcOEt = 4:1) to give 23,26-diol 23a (a col-

orless oil, 5.7 mg, 27.0%, less polar) and diol 23b (a col-

orless oil, 4.9 mg, 23.2%, more polar).

LAC67a (less polar) ¹H NMR d 0.58 (3H, s, H-18), 1.02

(3H, d, J = 6.5 Hz, H-21), 4.51 (2H, m, H-3, 1), 4.65

(1H, m, H-23), 5.09 and 5.12 (each 1H, s, –C@CH₂),

5.63 (1H, t, J = 2.4 Hz, H-27), 5.89 (1H, d,

J = 11.2 Hz, H-7), 6.23 (1H, t, J = 2.8 Hz, H-27), 6.35

(1H, d, J = 11.2 Hz, H-6). IR (neat) 3384, 2942, 2823,

1748 cm^ꢀ1. MS m/z (%): 426 (M⁺, 10), 390 (50), 375

(12), 285 (12), 252 (100), 250 (60), 197 (32), 105 (25).

HRMS calcd for C₂₇H₃₈O₄426.2770, found 426.2794.

UV (95%EtOH): k_max245, 254, 263 nm.

23a (less polar) ¹H NMR d 0.02 and 0.05 and 0.06 and 0.08

(each 3H, s, SiMe), 0.58 (3H, s, H-18), 0.85 and 0.90 (each

9H, s, t-Bu), 0.99 (3H, d, J = 6.5 Hz, H-21), 2.83 (1H, m,

H-9), 3.87 (1H, m, H-23), 4.12 (2H, s, H-26), 4.42 (2H, m,

H-3, 1), 4.92 and 4.97 (each 1H, s, –C@CH₂), 4.98 and

5.15 (each 1H, s, H-27), 5.84 (1H, d, J = 11.1 Hz, H-7),

6.22 (1H, d, J = 11.1 Hz, H-6). MS m/z (%): 658 (M⁺,

8), 640 (17), 526 (32), 508 (100), 454 (50), 366 (56), 234

(23), 147 (17), 75 (75), 73 (68).

1

LAC67b (more polar) H NMR d 0.57 (3H, s, H-18),

1.04 (3H, d, J = 6.3 Hz, H-21), 4.50 (2H, m, H-3, 1),

4.60 (1H, m, H-23), 5.10 and 5.12 (each 1H, s,

–C@CH₂), 5.63 (1H, t, J = 2.4 Hz, H-27), 5.89 (1H, d,

J = 11.5 Hz, H-7), 6.23 (1H, t, J = 2.8 Hz, H-27), 6.36

(1H, d, J = 11.5 Hz, H-6). IR (neat) 3452, 2923, 2853,

1759 cm^ꢀ1. MS m/z (%): 426 (M⁺, 10), 390 (80), 375

(20), 285 (20), 251 (100), 197 (42), 105 (40). HRMS calcd

for C₂₇H₃₈O₄426.2770, found 426.2750. UV (95%

EtOH): k_max245, 253, 263 nm.

23b (more polar) ¹H NMR d 0.02 and 0.05 and 0.06 and

0.08 (each 3H, s, SiMe), 0.56 (3H, s, H-18), 0.86 and

0.89 (each 9H, s, t-Bu), 1.01 (3H, d, J = 6.4 Hz, H-21),

2.82 (1H, m, H-9), 3.86 (1H, m, H-23), 4.13 (2H, s, H-

26), 4.42 (2H, m, H-3, 1), 4.92 and 4.97 (each 1H, s,

–C@CH₂), 5.00 and 5.17 (each 1H, s, H-27), 5.84 (1H,

d, J = 11.2 Hz, H-7), 6.21 (1H, d, J = 11.2 Hz, H-6).

MS m/z (%): 658 (M⁺, 10), 640 (18), 526 (45), 508

(100), 454 (63), 366 (65), 236 (32), 234 (30), 147 (22),

138 (21), 75 (92), 73 (90).

7.20. 5-{(2S)-2-[(1R,3R,7E)-1,3-Dihydroxy-2-methylene-

9,10-secoestra-5,7-dien-17-yl]propyl}-3-methylenedihy-

dro-2(3H)-furanone (LAC82a, b)

LAC82 was obtained from 22 by the same procedure as

described for LAC67 (yield 99.1%) as a colorless solid.

The mixture was separated by HPLC (LiChrosorb Si

60, Hibar RT 250-10, 7 lm, hexane/AcOEt = 7:3,

5 mL/min) to give LAC82a and LAC82b. The purity

of LAC82a and LAC82b was proved to be about

100% by HPLC.

7.22. (6S)-6-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]

oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)-2-meth-

ylene-1,4-heptanediol (24a, b)

1

LAC82a (more polar) H NMR d 0.56 (3H, s, H-18),

0.94 (3H, d, J = 6.4 Hz, H-21), 4.49 (2H, m, H-3, 1),

4.61 (1H, m, H-23), 5.10 and 5.11 (each 1H, s,

Compound 24 was obtained from 22 by the same proce-

dure as described for 23a and 23b (24a 31.8% more po-

lar, 24b 29.7% less polar) as colorless oil, respectively.

470

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

24a (more polar) ¹H NMR d 0.02 and 0.05 and 0.06 and

0.08 (each 3H, s, SiMe), 0.55 (3H, s, H-18), 0.86 and

0.89 (each 9H, s, t-Bu), 0.91 (3H, d, J = 6.7 Hz, H-21),

2.82 (1H, m, H-9), 3.86 (1H, m, H-23), 4.12 (2H, s, H-

26), 4.42 (2H, m, H-3, 1), 4.92 and 4.97 (each 1H, s,

–C@CH₂), 5.00 and 5.16 (each 1H, s, H-27), 5.84 (1H,

d, J = 11.1 Hz, H-7), 6.21 (1H, d, J = 11.1 Hz, H-6).

592 (45), 508 (42), 454 (92), 366 (95), 234 (30), 197

(20), 147 (22), 73 (100).

7.25. 2-((2R,4S)-4-{(4E)-4-[2-((3R,5R)-3,5-Bis{[tert-butyl

(dimethyl)silyl]oxy}-4-methylenecyclohexylidene)ethyli-

dene]-7a-methyloctahydro-1H-inden-1-yl}-2-hydroxypen-

tyl)-2-propenyl pivalate (26a)

1

24b (less polar) H NMR d 0.02 and 0.05 and 0.06 and

Compound 26a was obtained from 24a by the same pro-

cedure as described for 25a (yield 45.3%) as a yellow oil,

with the recovered 24a (34.7%).

0.08 (each 3H, s, SiMe), 0.58 (3H, s, H-18), 0.86 and

0.89 (each 9H, s, t-Bu), 0.91 (3H, d, J = 6.9 Hz, H-21),

2.82 (1H, m, H-9), 3.86 (1H, m, H-23), 4.11 (2H, s, H-

26), 4.42 (2H, m, H-3, 1), 4.92 (1H, s, –C@CH₂), 4.969

and 4.975 (each 1H, s, H-27, –C@CH₂), 5.14 (1H, s,

H-27), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.21 (1H, d,

J = 11.1 Hz, H-6).

1

26a H NMR d 0.03 and 0.05 and 0.07 and 0.08 (each

3H, s, SiMe), 0.55 (3H, s, H-18), 0.86 and 0.90 (each

9H, s, t-Bu), 1.23 (9H, s, –COC(CH₃)₃), 2.82 (1H, m,

H-9), 3.85 (1H, m, H-23), 4.43 (2H, m, H-3, 1), 4.53

and 4.58 (each 1H, d, J = 13.6 Hz, H-26), 4.92 and

4.97 (each 1H, s, –C@CH₂), 5.06 and 5.17 (each 1H, s,

H-27), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.21 (1H, d,

J = 11.1 Hz, H-6).

7.23. 2-((2R,4R)-4-{(4E)-4-[2-((3R,5R)-3,5-Bis{[tert-

butyl(dimethyl)silyl]oxy}-4-methylenecyclohexylidene)

ethylidene]-7a-methyloctahydro-1H-inden-1-yl}-2-hydroxy-

pentyl)-2-propenyl pivalate (25a)

7.26. 2-((2S,4S)-4-{(4E)-4-[2-((3R,5R)-3,5-Bis{[tert-butyl

(dimethyl)silyl]oxy}-4-methylenecyclohexylidene)ethyli-

dene]-7a-methyloctahydro-1H-inden-1-yl}-2-hydroxypen-

tyl)-2-propenyl pivalate (26b)

To a solution of 23,26-diol 23a (18.4 mg, 0.028 mmol)

in dry CH₂Cl₂(1 mL) were added dry pyridine

(9.5 lL, 0.12 mmol) and PivCl (4.5 lL, 0.036 mmol),

and the mixture was stirred at 0 ꢁC for 22.5 h. The

reaction was quenched with H₂O at 0 ꢁC and was ex-

tracted with CH₂Cl₂. The organic layer was washed

with brine, dried over MgSO₄, and evaporated. The

residue was chromatographed on silica gel (5 g, hex-

ane/AcOEt = 49:1 then 4:1) to give pivalate ester 25a

(a yellow oil, 5.5 mg, 26.5%) and 23,26-diol 23a

(5.6 mg, 30.4%).

Compound 26b was obtained from 24b by the same pro-

cedure as described for 25a (yield 23.8%) as a yellow oil,

with the recovered 24b (45.4%).

1

26b H NMR d 0.02 and 0.05 and 0.06 and 0.08 (each

3H, s, SiMe), 0.59 (3H, s, H-18), 0.86 and 0.90 (each

9H, s, t-Bu), 1.23 (9H, s, –COC(CH₃)₃), 2.82 (1H, m,

H-9), 3.88 (1H, m, H-23), 4.42 (2H, m, H-3, 1), 4.54

(2H, s, H-26), 4.92 and 4.97 (each 1H, s, –C@CH₂),

5.04 and 5.15 (each 1H, s, H-27), 5.84 (1H, d,

J = 11.0 Hz, H-7), 6.21 (1H, d, J = 11.0 Hz, H-6).

1

25a H NMR d 0.02 and 0.05 and 0.06 and 0.08 (each

3H, s, SiMe), 0.58 (3H, s, H-18), 0.85 and 0.90 (each

9H, s, t-Bu), 0.98 (3H, d, J = 6.4 Hz, H-21), 1.23 (9H,

s, –COC(CH₃)₃), 2.82 (1H, m, H-9), 3.88 (1H, m, H-

23), 4.42 (2H, m, H-3, 1), 4.55 (2H, s, H-26), 4.91 and

4.97 (each 1H, s, –C@CH₂), 5.04 and 5.14 (each 1H, s,

H-27), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.22 (1H, d,

J = 11.1 Hz, H-6). MS m/z (%): 742 (M⁺, 20), 724 (3),

640 (5), 610 (100), 592 (42), 508 (40), 454 (92), 366

(90), 234 (25), 197 (20), 147 (22), 73 (98).

7.27. (1R)-1-[(2R)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(di-

methyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)

propyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-butenyl

(2S)-3,3,3-triﬂuoro-2-methoxy-2-phenylpropanoate (27a)

To

0.0054 mmol) in dry CH₂Cl₂(100 lL) were added

Et₃N (7.5 lL, 0.054 mmol), DMAP (6.2 mg,

a

solution of pivalate ester 25a (4.0 mg,

7.24. 2-((2S,4R)-4-{(4E)-4-[2-((3R,5R)-3,5-Bis{[tert-butyl

(dimethyl)silyl]oxy}-4-methylenecyclohexylidene)ethyli-

dene]-7a-methyloctahydro-1H-inden-1-yl}-2-hydroxypen-

tyl)-2-propenyl pivalate (25b)

0.051 mmol), and a solution of R-MTPACl (5.1 lL,

0.027 mmol) in dry CH₂Cl₂(40 lL), the reaction mix-

ture was stirred for 15 min at room temperature. The

reaction was quenched with H₂O at 0 ꢁC and was ex-

tracted with CH₂Cl₂. The organic layer was washed with

brine, dried over MgSO₄, and evaporated. The residue

was chromatographed on silica gel (5 g, hexane/

AcOEt = 9:1) to give S-MTPA ester 27a (3.8 mg,

78.0%) as a colorless oil.

Compound 25b was obtained from 23b by the same pro-

cedure as described for 25a (yield 43.7%) as a yellow oil,

with the recovered 23b (8.2%).

1

25b H NMR d 0.02 and 0.05 and 0.06 and 0.08 (each

3H, s, SiMe), 0.55 (3H, s, H-18), 0.86 and 0.89 (each

9H, s, t-Bu), 1.00 (3H, d, J = 6.1 Hz, H-21), 1.23 (9H,

s, –COC(CH₃)₃), 2.82 (1H, m, H-9), 3.85 (1H, m, H-

23), 4.43 (2H, m, H-3, 1), 4.53 and 4.58 (each 1H, d,

J = 13.7 Hz, H-26), 4.92 and 4.97 (each 1H, s,

–C@CH₂), 5.06 and 5.17 (each 1H, s, H-27), 5.83 (1H,

d, J = 11.1 Hz, H-7), 6.21 (1H, d, J = 11.1 Hz, H-6).

MS m/z (%): 742 (M⁺, 20), 724 (5), 640 (3), 610 (92),

1

27a H NMR d 0.03 and 0.05 and 0.07 and 0.08 (each

3H, s, SiMe), 0.48 (3H, s, H-18), 0.86 and 0.89 (each

9H, s, t-Bu), 0.98 (3H, d, J = 6.3 Hz, H-21), 1.23 (9H,

s, –COC(CH₃)₃), 2.82 (1H, m, H-9), 3.51 (3H, s,

–OCH₃), 4.41 (2H, m, H-3, 1), 4.48 and 4.53 (each 1H,

d, J = 13.7 Hz, H-26), 4.92 and 4.97 (each 1H, s,

–C@CH₂), 4.93 and 5.05 (each 1H, s, H-27), 5.38 (1H,

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

471

m, H-23), 5.83 (1H, d, J = 11.1 Hz, H-7), 6.21 (1H, d,

J = 11.1 Hz, H-6), 7.38 (3H, m, Ph-3, 4, 5), 7.53 (2H,

m, Ph-2, 6).

m, H-23), 5.83 (1H, d, J = 11.0 Hz, H-7), 6.21 (1H,

d, J = 11.0 Hz, H-6), 7.38 (3 H, m, Ph-3, 4, 5), 7.52

(2H, m, Ph-2, 6).

7.28. (1R)-1-[(2R)-2-((1R,3R,7R)-1,3-Bis{[tert-butyl

(dimethyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-

17-yl)propyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-

butenyl (2R)-3,3,3-triﬂuoro-2-methoxy-2-phenylpropanoate

(28a)

7.31. (1R)-1-[(2S)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl

(dimethyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-

17-yl)propyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-

butenyl (2S)-3,3,3-triﬂuoro-2-methoxy-2-phenylpropano-

ate (29a)

In a similar manner to that for the synthesis of 27a from

25a, a crude product, which was obtained from 25a

(4.5 mg, 0.0061 mmol), Et₃N (8.4 lL, 0.061 mmol),

DMAP (6.7 mg, 0.055 mmol), and S-MTPACl (5.7 lL,

0.030 mmol) in dry CH₂Cl₂at room temperature for

15 min, was puriﬁed by chromatographed on silica gel

(5 g, hexane/AcOEt = 9:1) to give R-MTPA ester 28a

(5.0 mg, 84.8%) as a colorless oil.

Compound 29a was obtained from 26a by the same pro-

cedure as described for 27a (yield quant.) as a colorless

oil.

29a ¹H NMR d 0.03 and 0.05 and 0.06 and 0.08

(each 3H, s, SiMe), 0.53 (3H, s, H-18), 0.86 and

0.90 (each 9H, s, t-Bu), 1.21 (9H, s, –COC(CH₃)₃),

2.83 (1H, m, H-9), 3.54 (3H, s, –OCH₃), 4.42 (4

H, m, H-26, 3, 1), 4.85 (1H, s, H-27), 4.93 (1H,

s, –C@CH₂), 4.98 (2H, s, H-27, –C@CH₂), 5.34

(1H, m, H-23), 5.84 (1H, d, J = 11.0 Hz, H-7), 6.21

(1H, d, J = 11.0 Hz, H-6), 7.38 (3H, m, Ph-3, 4,

5), 7.53 (2H, m, Ph-2, 6).

1

28a H NMR d 0.03 and 0.05 and 0.07 and 0.08 (each

3H, s, SiMe), 0.40 (3H, s, H-18), 0.87 and 0.89 (each

9H, s, t-Bu), 1.23 (9H, s, –COC(CH₃)₃), 2.80 (1H, m,

H-9), 3.53 (3H, s, –OCH₃), 4.41 (2H, m, H-3, 1), 4.52

and 4.57 (each 1H, d, J = 13.7 Hz, H-26), 4.92 and

4.97 (each 1H, s, –C@CH₂), 5.00 and 5.12 (each 1H, s,

H-27), 5.42 (1H, m, H-23), 5.81 (1H, d, J = 11.0 Hz,

H-7), 6.20 (1H, d, J = 11.0 Hz, H-6), 7.37 (3H, m, Ph-

3, 4, 5), 7.53 (2H, m, Ph-2, 6).

7.32. (1R)-1-[(2S)-2-((1R,3R,7R)-1,3-Bis{[tert-butyl

(dimethyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-

17-yl)propyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-

butenyl (2R)-3,3,3-triﬂuoro-2-methoxy-2-phenylpropano-

ate (30a)

7.29. (1S)-1-[(2R)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)

silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)pro-

pyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-butenyl

(2S)-3,3,3-triﬂuoro-2-methoxy-2-phenylpropanoate (27b)

Compound 30a was obtained from 26a by the same pro-

cedure as described for 28a (yield 41.9%) as a colorless

oil.

1

Compound 27b was obtained from 25b by the same pro-

cedure as described for 27a (yield 70.6%) as a colorless

oil.

30a H NMR d 0.02 and 0.05 and 0.06 and 0.07 (each

3H, s, SiMe), 0.49 (3H, s, H-18), 0.86 and 0.89 (each

9H, s, t-Bu), 1.21 (9H, s, –COC(CH₃)₃), 2.81 (1H, m,

H-9), 3.52 (3H, s, –OCH₃), 4.42 (2H, m, H-3, 1), 4.52

(2H, s, H-26), 4.92 and 4.97 (each 1H, s, –C@CH₂),

5.01 and 5.13 (each 1H, s, H-27), 5.34 (1H, m, H-23),

5.82 (1H, d, J = 11.0 Hz, H-7), 6.20 (1H, d,

J = 11.0 Hz, H-6), 7.38 (3 H, m, Ph-3, 4, 5), 7.52 (2H,

m, Ph-2, 6).

1

27b H NMR d 0.02 and 0.05 and 0.06 and 0.08 (each

3H, s, SiMe), 0.51 (3H, s, H-18), 0.86 and 0.89 (each

9H, s, t-Bu), 1.22 (9H, s, –COC(CH₃)₃), 2.82 (1H, m,

H-9), 3.52 (3H, s, –OCH₃), 4.43 (2H, m, H-3, 1), 4.52

and 4.56 (each 1H, d, J = 13.7 Hz, H-26), 4.92 and

4.97 (each 1H, s, –C@CH₂), 5.01 and 5.14 (each 1H, s,

H-27), 5.32 (1H, m, H-23), 5.82 (1H, d, J = 11.1 Hz,

H-7), 6.21 (1H, d, J = 11.1 Hz, H-6), 7.39 (3H, m, Ph-

3, 4, 5), 7.53 (2H, m, Ph-2, 6).

7.33. (1S)-1-[(2S)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(di-

methyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)

propyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-bute-

nyl (2S)-3,3,3-triﬂuoro-2-methoxy-2-phenylpropanoate

(29b)

7.30. (1S)-1-[(2R)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)-

silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)pro-

pyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-butenyl

(2R)-3,3,3-triﬂuoro-2-methoxy-2-phenylpropanoate (28b)

Compound 29b was obtained from 26b by the same pro-

cedure as described for 27a (yield 99.8%) as a colorless

oil.

Compound 28b was obtained from 25b by the same pro-

cedure as described for 28a (yield 79.2%) as a colorless

oil.

1

29b H NMR d 0.04 and 0.05 and 0.07 and 0.08 (each

3H, s, SiMe), 0.29 (3H, s, H-18), 0.88 and 0.89 (each

9H, s, t-Bu), 1.23 (9H, s, –COC(CH₃)₃), 2.79 (1H, m,

H-9), 3.54 (3H, s, –OCH₃), 4.42 (2 H, m, H-3, 1), 4.53

and 4.58 (each 1H, d, J = 13.6 Hz, H-26), 4.93 and

4.97 (each 1H, s, –C@CH₂), 5.00 and 5.12 (each 1H, s,

H-27), 5.40 (1H, m, H-23), 5.80 (1H, d, J = 11.2 Hz,

H-7), 6.19 (1H, d, J = 11.2 Hz, H-6), 7.36 (3H, m, Ph-

3, 4, 5), 7.53 (2H, m, Ph-2, 6).

1

28b H NMR d 0.03 and 0.05 and 0.06 and 0.08 (each

3H, s, SiMe), 0.54 (3H, s, H-18), 0.86 and 0.90 (each

9H, s, t-Bu), 1.21 (9H, s, –COC(CH₃)₃), 2.82 (1H, m,

H-9), 3.53 (3H, s, –OCH₃), 4.42 (2H, m, H-3, 1), 4.43

(2H, s, H-26), 4.84 (1H, s, H-27), 4.92 (1H, s,

–C@CH₂), 4.97 (2H, s, H-27, –C@CH₂), 5.33 (1H,

472

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

7.34. (1S)-1-[(2S)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(di-

methyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)

propyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-butenyl

(2R)-3,3,3-triﬂuoro-2-methoxy-2-phenylpropanoate (30b)

7.38. (5S)-5-{(2S)-2-[(1R,3R,7E)-1,3-Dihydroxy-2-meth-

ylene-9,10-secoestra-5,7-dien-17-yl]propyl}-3-methylene-

dihydro-2(3H)-furanone (LAC82 b)

LAC82b was obtained from 22a by the same procedure

as described for LAC67 (yield 51.1%). The compound

was identiﬁed as LAC82b by HPLC analysis; Lichro-

spher Si 60, 5 lm, hexane/AcOEt = 1:1, ﬂow rate

2 mL/min, retention time 8.9 min.

Compound 30b was obtained from 26b by the same pro-

cedure as described for 28a (yield 46.5%) as a colorless oil.

1

30b H NMR d 0.03 and 0.05 and 0.07 and 0.08 (each

3H, s, SiMe), 0.44 (3H, s, H-18), 0.87 and 0.90 (each

9H, s, t-Bu), 1.23 (9H, s, –COC(CH₃)₃), 2.81 (1H, m,

H-9), 3.48 (3H, s, –OCH₃), 4.42 (2H, m, H-3, 1), 4.49

and 4.54 (each 1H, d, J = 13.5 Hz, H-26), 4.93 (2H, s,

H-27, –C@CH₂), 4.97 (1H, s, –C@CH₂), 5.06 (1H, s,

H-27), 5.37 (1H, m, H-23), 5.83 (1H, d, J = 11.1 Hz,

H-7), 6.20 (1H, d, J = 11.1 Hz, H-6), 7.39 (3H, m, Ph-

3, 4, 5), 7.53 (2H, m, Ph-2, 6).

7.38.1. Competitive-binding assay, rat VDR. The rat

VDR–LBD (amino acids 115–423) was expressed as an

amino-terminal His-tagged protein in Escherichia coli

BL21 (DE3) pLys S (Novagen).³⁷The cells were lysed

by sonication and the supernatants were diluted 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). This solution of crude rVDR–

LBD was pipetted into glass culture tubes. A solution

containing an increasing amount of 1a,25-(OH)₂D₃or

the synthetic analogs in 15 lL of EtOH was added to

the receptor solution in each tube and the mixture was

vortexed 2–3 times. The mixture was incubated for 1 h

at room temperature. [³H]-1,25-(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 dex-

tran-coated charcoal suspension (purchased from Ya-

masa Shoyu) was added to bind any free ligands (or

to remove free ligands) and the sample was vortexed.

After 30 min at 4 ꢁC, bound and free [³H]-1,25-

(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

scintillation ﬂuid (Amersham, Buckinghamshire,

U.K.) and submitted for radioactivity counting. Each

assay was performed at least twice in duplicate.

7.35. (5R)-5-[(2R)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(di-

methyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-

17-yl)propyl]-3-methylenedihydro-2(3H)-furanone (21a)

To a solution of diol 23a (11.4 mg, 0.017 mmol) in dry

CH₂Cl₂(2 mL) was added MnO₂(77.7 mg, 0.89 mmol),

and the mixture was stirred at room temperature for

76 h. The mixture was chromatographed on silica gel

(5 g, hexane/AcOEt = 4:1) to give methylene lactone

21a (9.4 mg, 83.1%).

1

21a H NMR d 0.02 and 0.05 and 0.07 and 0.08 (each

3H, s, SiMe), 0.57 (3H, s, H-18), 0.86 and 0.90 (each

9H, s, t-Bu), 1.02 (3H, d, J = 6.5 Hz, H-21), 4.43 (2H,

m, H-3, 1), 4.65 (1H, m, H-23), 4.91 and 4.97 (each

1H, s, –C@CH₂), 5.62 (1H, t, J = 2.4 Hz, H-27), 5.84

(1H, d, J = 11.2 Hz, H-7), 6.22 (1H, d, J = 11.2 Hz, H-

6), 6.23 (1H, t, J = 2.7 Hz, H-27).

7.36. (5S)-5-[(2S)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(di-

methyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-

yl) propyl]-3-methylenedihydro-2(3H)-furanone (22b)

7.38.2. Transfection and transactivation assay. Cos7 cells

were cultured in Dulbecco’s modiﬁed Eagle’s medium

(DMEM) supplemented with 5% fetal bovine serum

(FBS). Cells were seeded on 24-well plates at a density

of 2 · 10⁴per well. After 24 h, the cells were transfected

with a reporter plasmid containing three copies of the

mouse osteopontin VDRE (5⁰-GGTTCAcgaGGTTCA,

SPPx3-TK-Luc), a wild-type hVDR expression plasmid

(pCMX-hVDR), and the internal control plasmid con-

taining sea pansy luciferase expression constructs

(pRL-CMV) by the lipofection method as described

previously.^18,23,24After 4 h incubation, the medium

was replaced with fresh DMEM containing 5% char-

coal-treated FCS (HyClone, UT, USA). The next day,

the cells were treated with either the ligand or ethanol

vehicle and cultured for 24 h. Cells in each well were

harvested with a cell lysis buﬀer, and the luciferase activ-

ity was measured with a luciferase assay kit (Toyo Ink,

Inc., Japan). Transactivation measured by the luciferase

activity was normalized with the internal control. All

experiments were done in triplicate.

Compound 22b was obtained from 24b by the same pro-

cedure as described for 21a (yield 86.9%).

1

22b H NMR d 0.02 and 0.05 and 0.07 and 0.08 (each

3H, s, SiMe), 0.56 (3H, s, H-18), 0.86 and 0.90 (each

9H, s, t-Bu), 0.95 (3H, d, J = 6.5 Hz, H-21), 4.42 (2H,

m, H-3, 1), 4.64 (1H, m, H-23), 4.92 and 4.97 (each

1H, s, –C@CH₂), 5.62 (1H, t, J = 2.4 Hz, H-27), 5.84

(1H, d, J = 11.2 Hz, H-7), 6.21 (1H, d, J = 11.2 Hz, H-

6), 6.23 (1H, t, J = 2.8 Hz, H-27). MS m/z (%): 654

(M⁺, 3), 522 (10), 454 (12), 440 (12), 366 (15), 313

(40), 147 (20), 75 (100), 73 (70).

7.37. (5R)-5-{(2R)-2-[(1R,3R,7E)-1,3-Dihydroxy-2-meth-

ylene-9,10-secoestra-5,7-dien-17-yl]propyl}-3-methylen-

edihydro-2(3H)-furanone (LAC67a)

LAC67a was obtained from 21a by the same procedure

as described for LAC67 (yield 64.9%). The compound

was identiﬁed as LAC67a by HPLC analysis; Lichrosorb

Si 60, 5 lm, Hexane/AcOEt = 7:3, ﬂow rate 2 mL/min,

retention time 34.2 min.

7.38.3. Cell culture and cotransfection assay. Human

embryonic kidney (HEK) 293 cells were cultured in

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473

473

(b) Shimizu, M.; Iwasaki, Y.; Shibamoto, Y.; Sato, M.; DeLuca,

H. F.; Yamada, S. Bioorg. Med. Chem. Lett. 2003, 13, 809.

14. Ono, K.; Yoshida, A.; Saito, N.; Fujishima, T.; Honzawa,

S.; Suhara, Y.; Kishimoto, S.; Sugiura, T.; Waku, K.;

Takayama, H.; Kittaka, A. J. Org. Chem. 2003, 68, 7407.

15. Okuda, Y.; Nakatsukasa, S.; Oshima, K.; Nozaki, H.

Chem. Lett. 1985, 481.

16. Saito, N.; Saito, H.; Anzai, M.; Yoshida, A.; Fujishima,

T.; Takenouchi, K.; Miura, D.; Ishizuka, S.; Takayama,

H.; Kittaka, A. Org. Lett. 2003, 5, 4859.

17. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H.

J. Am. Chem. Soc. 1991, 113, 4092.

18. Shimizu, M.; Miyamoto, Y.; Kobayashi, E.; Shimazaki,

M.; Yamamoto, K.; Reischl, W.; Yamada, S. Bioorg. Med.

Chem. 2006, 14, 4277.

DMEM containing 5% FBS, 100 U/mL penicillin, and

100 lg/mL streptomycin at 37 ꢁC in a humidiﬁed atmo-

sphere containing 5% CO₂. Transfections in HEK293

cells were performed by the calcium phosphate coprecip-

itation assay as described previously.^31,38Eight hours

after transfection, compounds were added. Cells were

harvested after 16–24 h and were assayed for luciferase

and b-galactosidase activities using a luminometer and a

microplate reader (Molecular Devices, Sunnyvale, CA).

Luciferase data were normalized to the internal b-galacto-

sidase control and represent the means SD of triplicate

assays.

7.38.4. Graphical manipulations and ligand docking.

Graphical manipulations were performed using SYBYL

7.3 (Tripos, St. Louis). The atomic coordinates of the

human VDR–LBD (residues 118–427 D166–216) crystal

structure were retrieved from Protein Data Bank

(code: 2a-methyl-1,25-(OH)₂D₃,²⁹2HB8; 20-epi-1,25-

(OH)₂D₃,²⁶1IE9). LAC67a and LAC67b were docked

into the ligand-binding pocket of the VDR–LBD²⁹man-

ually by superposition with the 2a-methyl-1,25-(OH)₂D₃

at the A- to D-ring. LAC67a and LAC67b in the LBP of

the VDR–LBD were minimized on Tripos force ﬁeld

with 100 times iterations.

19. Saito, N.; Matsunaga, T.; Fujishima, T.; Anzai, M.; Saito,

H.; Takenouchi, K.; Miura, D.; Ishizuka, S.; Takayama,

H.; Kittaka, A. Org. Biomol. Chem. 2003, 1, 4396.

20. Yamamoto, K.; Masuno, H.; Choi, M.; Nakashima, K.;

Taga, T.; Ooizumi, H.; Umesono, K.; Sicinska, W.;

VanHooke, J.; DeLuca, H. F.; Yamada, S. Proc. Natl.

Acad. Sci. U.S.A. 2000, 97, 1467.

21. Ishizuka, S.; Miura, D.; Eguchi, H.; Ozono, K.; Chokki,

M.; Kamimura, T.; Norman, A. W. Arch. Biochem.

Biophys. 2000, 380, 92.

22. Rochel, N.; Wurtz, J. M.; Mitschler, A.; Klaholz, B.;

Moras, D. Mol. Cell 2000, 5, 173.

23. Yamamoto, K.; Sun, W. Y.; Ohta, M.; Hamada, K.;

DeLuca, H. F.; Yamada, S. J. Med. Chem. 1996, 39, 2727.

24. Masuno, H.; Yamamoto, K.; Wang, X.; Choi, M.;

Ooizumi, H.; Shinki, T.; Yamada, S. J. Med. Chem.

2002, 45, 1825.

25. Choi, M.; Yamamoto, K.; Itoh, T.; Makishima, M.;

Mangelsdorf, D. J.; Moras, D.; DeLuca, H. F.; Yamada,

S. Chem. Biol. 2003, 10, 261.

LAC82a and LAC82b were docked into the ligand-bind-

ing pocket of the VDR–LBD²⁶manually by superposi-

tion with the 20-epi-1,25-(OH)₂D₃at the A- to D-ring.

LAC82a and LAC82b in the LBP of the VDR–LBD were

minimized on Tripos force ﬁeld with 100 times iterations.

26. Tocchini-Valentini, G.; Rochel, N.; Wurtz, J. M.; Mits-

chler, A.; Moras, D. Proc. Natl. Acad. Sci. U.S.A. 2001,

98, 5491.

References and notes

27. Tocchini-Valentini, G.; Rochel, N.; Wurtz, J.-M.; Moras,

D. J. Med. Chem. 2004, 47, 1956.

1. Vitamin D Second Edition; Feldman, D., Pike, J. W.,

Glorieux, F. H., Eds.; Elsevier Academic Press, 2005.

2. DeLuca, H. F. J. Bone Miner. Metab. 1990, 8, 1.

3. Rosenfeld, M. G.; Lunyak, V. V.; Glass, C. K. Genes Dev.

2006, 20, 1405.

28. Vanhooke, J. L.; Benning, M. M.; Bauer, C. B.; Pike, J.

W.; DeLuca, H. F. Biochemistry 2004, 43, 4101.

29. Hourai, S.; Fujishima, T.; Kittaka, A.; Suhara, Y.;

Takayama, H.; Rochel, N.; Moras, D. J. Med. Chem.

2006, 49, 5199.

4. Hu, X.; Lazar, M. A. Nature 1999, 402, 93.

30. Vanhooke, J. L.; Tadi, B. P.; Benning, M. M.; Plum, L.

A.; DeLuca, H. F. Arch. Biochem. Biophys. 2007, 460, 161.

31. Inaba, Y.; Yamamoto, K.; Yoshimoto, N.; Matsunawa,

M.; Uno, S.; Yamada, S.; Makishima, M. Mol. Pharma-

col. 2007, 71, 1298.

32. Herdick, M.; Steinmeyer, A.; Carlberg, C. Chem. Biol.

2000, 7, 885.

33. Yamaoka, K.; Kim, M.; Takada, I.; Takeyama, K.;

Kamimura, T.; Kato, S. J. Steroid Biochem. Mol. Biol.

2006, 100, 177.

34. Takenouchi, K.; Sogawa, R.; Manabe, K.; Saitoh, H.;

Gao, Q.; Miura, D.; Ishizuka, S. J. Steroid Biochem. Mol.

Biol. 2004, 89–90, 31.

35. Ochiai, E.; Miura, D.; Eguchi, H.; Ohara, S.; Takenouchi,

K.; Azuma, Y.; Kamimura, T.; Norman, A. W.; Ishizuka,

S. Mol. Endocrinol. 2005, 19, 1147.

36. Bula, C. M.; Bishop, J. E.; Ishizuka, S.; Norman, A. W.

Mol. Endocrinol. 2000, 14, 1788.

37. Strugnell, S. A.; Hill, J. J.; McCaslin, D. R.; Wieﬂing, B.

A.; Royer, C. A.; DeLuca, H. F. Arch. Biochem. Biophys.

1999, 364, 42.

5. Brzozowski, A. M.; Pike, A. C. W.; Dauter, Z.; Hubbard,

R. E.; Bonn, T.; Engstrom, O.; Ohman, L.; Greene, G. L.;

Gustafsson, J.-A.; Carlquist, M. Nature 1997, 389, 753.

6. Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kushner,

P. J.; Agard, D. A.; Greene, G. L. Cell 1998, 95, 927.

7. Geller, D. S.; Farhi, A.; Pinkerton, N.; Fradley, M.;

Moritz, M.; Spitzer, A.; Meinke, G.; Tsai, F. T. F.; Sigler,

P. B.; Lifton, R. P. Science 2000, 289, 119.

8. Bledsoe, R. K.; Madauss, K. P.; Holt, J. A.; Apolito, C. J.;

Lambert, M. H.; Pearce, K. H.; Stanley, T. B.; Stewart, E.

L.; Trump, R. P.; Willson, T. M.; Williams, S. P. J. Biol.

Chem. 2005, 280, 31283.

9. Bury, Y.; Steinmeyer, A.; Carlberg, C. Mol. Pharmacol.

2000, 58, 1067.

10. Miura, D.; Manabe, K.; Ozono, K.; Saito, M.; Gao, Q.;

Norman, A.W.;Ishizuka, S. J. Biol.Chem. 1999, 274,16392.

11. (a) Ishizuka, S.; Kurihara, N.; Miura, D.; Takenouchi, K.;

Cornish, J.; Cundy, T.; Reddy, S. V.; Roodman, G. D. J.

Steroid Biochem. Mol. Biol. 2004, 89–90, 331; (b) Rood-

man, G. D.; Windle, J. J. J. Clin. Invest. 2005, 115, 200.

12. Sicinski, R. R.; Prahl, J. M.; Smith, C. M.; DeLuca, H. F.

J. Med. Chem. 1998, 41, 4662.

38. Kaneko, E.; Matsuda, M.; Yamada, Y.; Tachibana, Y.;

Shimomura, I.; Makishima, M. J. Biol. Chem. 2003, 278,

36091.

13. (a) Perlman, K. L.; Swenson, R. E.; Paaren, H. E.; Schnoes,

H. K.; DeLuca, H. F. Tetrahedron Lett. 1991, 32, 7663;

Article Doi

2-Methylene 19-nor-25-dehydro-1α-hydroxyvitamin D3 26,23-lactones: Synthesis, biological activities and molecular basis of passive antagonism

DOI: 10.1016/j.bmc.2007.09.017

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Full text of DOI:10.1016/j.bmc.2007.09.017

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