Synthesis, biological evaluation, and conformational analysis of A-ring diastereomers of 2-methyl-1,25-dihydroxyvitamin D3 and their 20-epimers: Unique activity profiles depending on the stereochemistry of the A-ring and at C-20

J . Med. Chem. 2000, 43, 4247-4265

4247

Syn th esis, Biologica l Eva lu a tion , a n d Con for m a tion a l An a lysis of A-Rin g

Dia ster eom er s of 2-Meth yl-1,25-d ih yd r oxyvita m in D₃a n d Th eir 20-Ep im er s:

Un iqu e Activity P r ofiles Dep en d in g on th e Ster eoch em istr y of th e A-Rin g a n d

a t C-20

Katsuhiro Konno,^†Toshie Fujishima,^†Shojiro Maki,^†Zhaopeng Liu,^†Daishiro Miura,^‡Manabu Chokki,^‡

Seiichi Ishizuka,^‡Kentaro Yamaguchi,^|Yukiko Kan, Masaaki Kurihara,^#Naoki Miyata,^#Connie Smith,^§

Hector F. DeLuca,^§and Hiroaki Takayama*^,†

Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, J apan,

Teijin Institute for Bio-Medical Research, Teijin Ltd., Hino, Tokyo 191-8512, J apan, Chemical Analytical Center,

Chiba University, Inage-ku, Chiba 263-8522, J apan, Suntory Institute for Bioorganic Research, Shimamoto,

Osaka 618-8503, J apan, National Institute of Health Sciences, Setagaya, Tokyo 158-8501, J apan, and Department of

Biochemistry, College of Agricultural and Life Sciences, 420 Henry Mall, University of Wisconsin-Madison,

Madison, Wisconsin 63706

Received J une 19, 2000

All eight possible A-ring diastereomers of 2-methyl-1,25-dihydroxyvitamin D₃(2) and 2-methyl-

20-epi-1,25-dihydroxyvitamin D₃(3) were convergently synthesized. The A-ring enyne synthons

19 were synthesized starting with methyl (S)-(+)- or (R)-(-)-3-hydroxy-2-methylpropionate (8).

This was converted to the alcohol 14 as a 1:1 epimeric mixture in several steps. After having

been separated by column chromatography, each isomer led to the requisite A-ring enyne

synthons 19 again as 1:1 mixtures at C-1. Coupling of the resulting A-ring enynes 20a -h

with the CD-ring portions 5a ,b in the presence of a Pd catalyst afforded the 2-methyl analogues

2a -h and 3a -h in good yield. In this way, all possible A-ring diastereomers were synthesized.

The synthesized analogues were biologically evaluated both in vitro and in vivo. The potency

was highly dependent on the stereochemistry of each isomer. In particular, the RRâ-isomer 2g

exhibited 4-fold higher potency than 1R,25-dihydroxyvitamin D₃(1) both in bovine thymus

VDR binding and in elevation of rat serum calcium concentration and was twice as potent as

the parent compound in HL-60 cell differentiation. Furthermore, its 20-epimer, that is, 20-

epi-RRâ 3g, exhibited exceptionally high activities: 12-fold higher in VDR binding affinity,

7-fold higher in calcium mobilization, and 590-fold higher in HL-60 cell differentiation, as

compared to 1R,25-dihydroxyvitamin D₃(1). Accordingly, the double modification of 2-methyl

substitution and 20-epimerization resulted in unique activity profiles. Conformational analysis

1

of the A-ring by H NMR and an X-ray crystallographic analysis of the RRâ-isomer 2g are also

described.

In tr od u ction

Many of the analogues synthesized so far are altered

in the side chain, providing many useful analogues with

high potency or selective activity.⁴For example, 24,24-

difluoro-1R,25-dihydroxyvitamin D₃is the first analogue

that exhibits higher potency than 1.⁵22-Oxa-1R,25-

dihydroxyvitamin D₃(OCT),⁶26,27-dihomo-1R,25-dihy-

droxyvitamin D₃,⁷and 20-epi-1R,25-dihydroxyvitamin

D₃⁸show more potent activity in cell differentiation than

in calcemic effects. Notably, the combination of these

three side chain modifications together with 24-homolo-

gation led to a highly potent analogue, 20-epi-22-oxa-

1R,25-dihydroxy-24,26,27-trihomovitamin D₃(KH-1060).⁸

In recent years, modification of the A-ring has attracted

much attention because it can afford useful analogues

exhibiting unique activity profiles as well: for example,

2â-(3-hydroxypropoxy)-1R,25-dihydroxyvitamin D₃(ED-

71),⁹1R,25-dihydroxy-19-norvitamin D₃,¹⁰and 1-hy-

droxymethyl-25-hydroxyvitamin D₃.¹¹Modification of

the CD-ring has also been investigated, giving a highly

active analogue.¹²It is noteworthy that nonsteroidal

vitamin D mimics, i.e., compounds showing vitamin D

1R,25-Dihydroxyvitamin D₃(1) is a hormonally active

metabolite of vitamin D₃and functions as a regulator

of calcium and phosphorus homeostasis.¹The discovery

by Suda et al. that 1 exhibits a variety of biological

activities such as cell differentiation and proliferation,

besides the classical functions, stimulated renewed

interest in both the chemistry and biology of 1.²Over

the past two decades, extensive studies have focused on

the physiological roles of 1, and a large number of

analogues have been synthesized and biologically evalu-

ated to investigate the structure-activity relation-

ships (SARs) and to develop potential therapeutic

agents.³

* To whom correspondence should be addressed. Tel: +81-426-85-

3713. Fax: +81-426-85-3714. E-mail: hi-takay@pharm.teikyo-u.ac.jp.

^†Teikyo University.

^‡Teijin Ltd.

^|Chiba University.

Suntory Institute for Bioorganic Research.

#

National Institute of Health Sciences.

^§University of Wisconsin-Madison.

Published on Web 10/03/2000

4248 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22

Konno et al.

F igu r e 1. Conformational equilibrium of 1R,25-dihydroxy-

vitamin D₃.

F igu r e 2. Deter m in a tion of a bsolu te ster eoch em istr y a t

th e C-3 p osition .

Consequently, 2R-methyl-1R,25-dihydroxyvitamin D₃

(RRâ-isomer) showed higher potency than 1.^26aThis

remarkable effect of the 2-methyl substitution and the

results of the 20-epimerization previously reported

prompted us to further synthesize all possible diaster-

eomers of 2-methyl-20-epi-1,25-dihydroxyvitamin D₃(3),

modified both in the A-ring and in the side chain.

Biological evaluation of these double-modified analogues

gave rise to an exceptionally highly active analogue.^26b

Herein, we report in detail the synthesis of all eight

possible diastereomers of 2-methyl-1,25-dihydroxy-

vitamin D₃(2) and its 20-epimers (3). The results of

biological evaluation and conformational analysis, in-

cluding an X-ray crystallographic analysis, of these

analogues are also described.

activity without having seco-steroidal vitamin D struc-

ture, have been found quite recently.¹³

1R,25-Dihydroxyvitamin D₃(1) exerts its functions

through interaction with its specific receptor, the vita-

min D receptor (VDR),¹⁴belonging to the nuclear

receptor superfamily.¹⁵The binding of ligand 1 to VDR

triggers the whole sequence of biological responses:

conformational change, heterodimerization with the

retinoid X receptor (RXR), binding to DNA, and tran-

scriptional activities. Therefore, in recent years, this

specific interaction has been the focus of attention with

respect to structure-function relationships.¹⁶In par-

ticular, interaction of the side chain with VDR is well-

documented,¹⁷and Yamada et al. showed that potential

side chain structures can be predicted by conformational

analysis using molecular mechanics.¹⁸Furthermore,

differential interaction of compounds having the natural

side chain and their 20-epimers has been demon-

strated.¹⁹In contrast, however, little is known about the

conformation-activity relationships of the A-ring. Oka-

mura et al. reported that the A-ring of 1 equilibrates

between two chair conformers, the R- and â-form (Figure

Resu lts a n d Discu ssion

Ch em istr y. For the synthesis of analogues modified

both in the A-ring and in the side chain, the convergent

method is undoubtedly advantageous over the classical

steroidal approach, because it can be more effective and

flexible. Separate preparation of the requisite two units,

followed by coupling, would produce a wide variety of

analogues. Several convergent methods have been re-

ported since 1981,²⁷and of these, the procedure reported

by Trost et al. using palladium-catalyzed coupling of the

A-ring enyne synthon 4 with the CD-ring portion (5)

seems to be the most useful (Scheme 1).²⁸In this

method, cyclization of the A-ring and coupling with the

CD-ring proceeds in a single step, facilitating the

synthesis of the A-ring moiety and its modification.

Preparations of both the 20-natural- and 20-epi-CD-

ring portions, 5a ²⁸and 5b,^26b,crespectively, have been

reported. The A-ring enyne synthon 4a can be synthe-

sized in several different ways, but they do not seem to

be applicable to analogue synthesis.²⁹Therefore, we

have developed a practical method for the synthesis of

4a , which is versatile and applicable to a wide variety

of A-ring analogues including diastereomers. It is based

on introduction of an acetylene unit and a vinyl group

into the epoxy-aldehyde (or its equivalent) precursor 6a

derived from 3-buten-1-ol (7a ). The epimeric mixture

obtained at the introduction of each unit is separable

by chromatography, and thus all the A-ring diastereo-

mers are available. Indeed, we have synthesized all the

A-ring diastereomers of 1 and its 20-epimers.^26cUtiliz-

ing this method, the 2-methyl-substituted A-ring enyne

4b may be synthesized starting from methyl 3-hydroxy-

2-methylpropionate (8) through the corresponding

3-buten-1-ol derivative 7b and the epoxy-aldehyde

precursor 6b. Preparation of 7b from 8 should be

straightforward. Since both enantiomers of 8 are com-

1

1), a in 1:1 ratio as determined by H NMR analysis,²⁰

and they proposed that the â-form, in which the 1R-

hydroxyl occupies the equatorial position, may be re-

sponsible for biological activity.²¹This conformational

equilibrium of the A-ring was confirmed by ¹³C NMR

analysis.²²Recent studies on the 1-alkyl-1,25-dihydroxy-

vitamin D₃analogues by Yamada et al. seemed to

support this proposal,²³but DeLuca et al. concluded

that, on the contrary, the axial orientation of the 1R-

hydroxy group is essential for biological activity from

studies with 1R,25-dihydroxy-19-norvitamin D₃ana-

logues.²⁴Quite recently, Norman et al. proposed a three-

dimensional model of the ligand binding domain of the

human VDR based on the X-ray crystallographic atomic

coordinates of that of the rat R1-thyroid receptor and

suggested that the optimal shape of a ligand is one

where the plane of the A-ring in relation to the CD-ring

is at some intermediate angle (0-90°).²⁵

In view of the results of A-ring modification, as well

as the conformation-activity relationships in the A-ring

as stated above, we have synthesized all eight possible

diastereomers of 2-methyl-1,25-dihydroxyvitamin D₃(2),

demonstrating that the potency of the analogues varies

depending on the configuration not only of the C-1 and

C-3 hydroxy groups but also of the 2-methyl group.

A-Ring Diastereomers of Methyldihydroxyvitamin D₃

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4249

Sch em e 1

Sch em e 2

mercially available, all the enantiomers would be easily

accessible in the same way.

troduced by the reaction of 13 with ethynyltrimethyl-

silane/BuLi-BF₃etherate, giving a 1:1 mixture of the

alcohols 14a (less polar) and 14b (more polar) in 85%

yield. These isomers were readily separable by silica gel

column chromatography, and the absolute configuration

at C-3 (the vitamin D numbering) of each isomer was

determined by the ¹H NMR analysis of its MTPA

esters.³⁰The results are shown in Figure 2. Thus, the

less polar isomer 14a has the (3R)-configuration (3â-

isomer), whereas the more polar isomer 14b has the

(3S)-configuration (3R-isomer). The optical purity of

each isomer was also determined by this analysis.

Protection of the 3-OH in the 3â-isomer 14a (Scheme

3) with THP to give 15, followed by treatment with

Conversion of methyl (S)-(+)-3-hydroxy-2-methylpro-

pionate (8a ) (Scheme 2) to the requisite 3-buten-1-ol

derivative 12 was accomplished in excellent yield in a

conventional manner: protection of the primary alcohol

by TBDPSCl, then DIBAL-H reduction of the ester 9 to

give the alcohol 10, followed by Swern oxidation, and

finally the Wittig reaction of the resultant aldehyde 11.

Racemization of the aldehyde 11 occurred to the extent

of 5-10%, and the optical purity of all intermediates

after this stage is 80-90%. Treatment of the olefin 12

thus obtained with mCPBA afforded the epoxide 13 as

a 1:1 mixture in 97% yield. The acetylene unit was in-

4250 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22

Konno et al.

Sch em e 3

Sch em e 4

Sch em e 5

TBAF, furnished the primary alcohol 16, which was

then oxidized by the Swern procedure to give the

aldehyde 17 in excellent yield. When another protective

group (MEM, MOM) was used instead of THP, epimer-

ization occurred at C-2 of the aldehyde 17, i.e., only the

THP protective group suppressed the epimerization. The

reaction of 17 with vinylmagnesium bromide in the

presence of CeCl₃afforded a 1:1 mixture of the dia-

steromeric allyl alcohols 18 in 92% yield. After removal

of the THP protecting group, the resulting mixture of

the requisite A-ring enyne synthons 19a ,b was readily

separated by silica gel column chromatography. The

relative stereochemistry of the 1,3-diol in each isomer

was determined by ¹³C NMR analysis of its acetonides.³¹

The results are shown in Scheme 4. The less polar

isomer 19a was shown to be 1,3-anti, in other words,

the Rââ-isomer (the Greek letters denote the configura-

tions of C-1, -2, and -3, respectively, in the vitamin D

numbering), and the more polar isomer 19b was 1,3-

syn, the âââ-isomer. In the same way, the other 2â-Me

isomers 19c,d were obtained from the 3R-isomer 14b

(Scheme 5). Furthermore, starting from the enantio-

meric 8b and following exactly the same sequence of

reactions from 8a to 19a -d , the corresponding enanti-

omers 19e-h were synthesized (Scheme 6). Thus, we

have accomplished the synthesis of all eight possible

diastereomers of 2-Me-substituted A-ring enyne syn-

thons.

Coupling of the above A-ring enynes with the CD-ring

portions proceeded smoothly, as described in the litera-

ture.²⁸Scheme 7 shows the results for the case of the

Rââ-isomer 19a . The diol 19a was protected by TBS to

afford 20a , which was coupled with the natural-CD-ring

portion 5a by using palladium catalysis, followed by

A-Ring Diastereomers of Methyldihydroxyvitamin D₃

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4251

Sch em e 6

Sch em e 7

deprotection with camphorsulfonic acid (CSA) in MeOH,

and the product was finally purified by recycling HPLC

to give the 2â-Me analogue 2a (Rââ-isomer) in good

yield. On the other hand, coupling of 20a with the 20-

epi-CD-ring portion 5b afforded the corresponding 20-

epi analogue 3a (Rââ-isomer). In the same manner, the

rest of the analogues (2b-h and 3b-h ) were synthe-

sized. Thus, we have synthesized a total of sixteen 2-Me-

substitued analogues.

logues 2a -h , however, showed a similar characteristic

¹H NMR pattern and sign of optical rotation, but they

are not correlated to the stereochemistry of the diaster-

eomers (Table 1). The stereochemistry of these ana-

logues was unambiguously assigned by means of well-

established procedures as indicated above.

The analogue 2g (RRâ-isomer) gave a crystal suitable

for X-ray crystallographic analysis. The ORTEP drawing

and the molecular packing along the c axis are shown

in Figures 3 and 4, respectively. These results confirmed

the A-ring stereochemistry determined by spectroscopic

methods as described above. Both the molecular struc-

ture and the conformation are quite similar to those of

1R,25-dihydroxyvitamin D₃(1).³³The A-ring exists

exclusively in the â-form, in which the C-1 and C-3

hydroxyl groups occupy equatorial and axial positions,

respectively. Stabilization of this conformer in the solid

state may be due to the formation of a hydrogen bond

Posner and co-workers found that, in diastereomeric

pairs differing only by inversions of stereochemistry at

positions 1-3, but not in the CD-ring or in the side

1

chain, the H NMR signals of C-18 and C-19 showed a

characteristic pattern of chemical shifts that is related

to the sign of optical rotation of the compound. By means

of these criteria, they tentatively assigned the stereo-

chemistry of the diastereomers compared with that of

closely related vitamin D analogues.³²The 2-Me ana-

4252 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22

Konno et al.

Ta ble 1. Characteristic Physical Constants (selected ¹H NMR

data and optical rotation values) of the Synthesized Analogues

2a -h

mechanics calculation. First, the conformations of 2a ,g,

both of which have natural OH configurations, were

1

analyzed by H NMR (Figure 5). As a solvent, CDCl₃

stereochemistry

¹H NMR (δ)

was used because virtually no solvent dependency was

observed among CDCl₃, CD₃OD, and DMSO-d₆. COSY

and TOCSY experiments allowed assignment of all

A-ring signals. The conformational equilibrium was

deduced from the vicinal coupling constant between

H(3R)-H(4â) by the method based on the data reported

for cyclohexanol protons (J _ax,ax) 11.1 Hz, J _eq,eq) 2.7

Hz),³⁴which is widely accepted for analysis of the A-ring

of vitamin D compounds.^20,23In the case of 2g, the

signals of H(4R) and H(4â) both appeared as doublets

of doublets (dd), and the J _3R-4âvalue of 7.7 Hz indicated

a ratio of 60:40 in favor of the R-form. This was

supported by the NOESY experiment. NOEs of H(2â)-

H(4â) and 2-Me-H(4R) were observed, which suggested

that both chair conformers, R- and â-form, respectively,

compd

C-1

C-2

C-3

[R]_D

C-18

C-19

2f

â

R

â

R

â

R

â

R

â

R

â

R

â

R

â

R

â

R

â

R

+27.3

-35.2

+87.0

-44.8

+58.0

-47.7

+41.1

-11.2

0.54

0.55

0.54

0.56

0.54

0.55

0.53

0.55

5.35, 5.01

5.37, 5.02

5.23, 4.98

5.25, 5.01

5.31, 5.05

5.30, 5.07

5.28, 5.01

5.28, 5.02

2a

2e

2b

2c

2h

2g

2d

1

were involved. When the H NMR spectrum of 2g was

measured at -50 °C in CDCl₃, broadening of the A-ring

signals, in particular the 2-Me signal, was apparent,

again supporting the above conclusion. In the case of

2a , however, the signal of H(4â) was a doublet of

doublets (dd), whereas that of H(4R) was a broad doublet

owing to allylic coupling with H(6). The conformational

equilibrium of 2a deduced from the J _3R-4âvalue of 4.8

Hz showed a ratio of 25:75 in favor of the â-form. In

this case, the NOESY experiment did not give clear

results, while the allylic coupling between H(4R) and

H(6) suggested a major contribution of the â-form in the

conformational equilibrium of 2a .

On the basis of the preliminary results with 2a ,g, the

¹H NMR spectra of all the synthesized analogues 2a -h

and 3a -h were analyzed. The ¹H NMR spectra were

taken in CDCl₃-D₂O to exclude the coupling of H(1) and

H(3) with OH protons in these cases. Analyses by COSY

and decoupling experiments allowed the assignment of

all A-ring signals and the coupling constants. There was

no marked difference in chemical shifts between each

pair of the 20-natural and 20-epi-isomers, except for the

chemical shifts due to the 20-methyl groups. The ¹H

NMR spectral data of the A-ring protons of 2a -h are

summarized in Table 2. In addition to the above analysis

using vicinal coupling constants between H(3)-H(4),

those of H(1)-H(2) and H(2)-H(3) were utilized. In

every case, the A-ring is in equilibrium between two

chair conformations, the R- and â-forms (Figure 6), as

indicated by the analysis of the coupling patterns of H(2)

and H(3). In this situation, all H(3) signals should

appear as doublets of triplets (dt) because the coupling

constant due to H(2)-H(3) is the same as one of those

from H(3)-H(4R) and H(3)-H(4â). Likewise, the H(2)

signals should appear as doublets of doublets of quartets

(ddq) for 1,3-trans compounds, and triplets of quartets

(tq) for 1,3-cis compounds. The conformational equilibria

were deduced from the vicinal coupling constants based

F igu r e 3. ORTEP drawing of the crystal structure of 2R-

methyl-1R,25-dihydroxyvitamin D₃(2g).

F igu r e 4. Molecular packing in the unit cell of the crystal of

2R-methyl-1R,25-dihydroxyvitamin D₃(2g).

on the data reported for cyclohexanol protons (J _ax,ax

)

11.1 Hz, J _eq,eq) 2.7 Hz)³⁴as stated above. In every case,

one was favored over the other, but the extent varied

depending on the stereochemistry. Consequently, in the

cases of 1,3-cis compounds (b, c, e, and h ), the form in

which both hydroxyl groups occupied axial positions was

predominant because of hydrogen bond formation be-

tween these groups. On the other hand, the form in

network involving the C-1, C-3, and C-25 hydroxyl

groups and water molecules included in the crystal.

To gain information on the A-ring conformation-

activity relationships, the solution conformations of the

A-ring of the synthesized analogues were examined by

means of ¹H NMR analyses together with molecular

A-Ring Diastereomers of Methyldihydroxyvitamin D₃

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4253

F igu r e 5. Con for m a tion a l equ ilibr ia a n d obser ved NOEs of 2a ,g.

Ta ble 2. ¹H NMR Data of A-Ring Protons of the Synthesized

Analogues 2a -h ^a

the experimental data, which well-supported the ¹H

NMR analyses. Thus, the orientations of the two hy-

droxyl groups and the 2-methyl group affect the equi-

librium ratio, and as a result, the equilibrium is biased

to various extents in a direction that depends on the

A-ring stereochemistry.

compd H(1)

H(2)

H(3)

4.03

dt

(9.2) (9.2, 2.6, 7.0) (4.8, 2.6) (13.6, 2.7) (13.6, 4.8)

H(4R)

2.52

dd^b

H(4â)

2.42

dd

2a

2b

2c

2d

2e

2f

4.01 1.78

ddq

d

4.17 1.90

tq

3.90

dt

2.50

2.59

dd

dd^b

Biologica l Eva lu a tion . The biological activities of

the synthesized analogues were evaluated in compari-

son with those of 1R,25-dihydroxyvitamin D₃(1). The

results are summarized in Tables 3 and 4. It is well-

recognized that the 1R,3â-configuration is crucial for

potent vitamin D activity and epimerization of each

hydroxyl group dramatically decreases the activity by

about 100 times for 1â and 10 times for 3R.³⁶In addition

to that, the configuration of the 2-methyl group was

found considerably to alter the activity. Consequently,

the potency was highly dependent on the stereo-

chemistry of the A-ring substituents. Thus, the RRâ-

isomer 2g showed much higher activity than the parent

compound 1 among the 20-natural analogues. Most

interestingly, the combination of A-ring modification

and 20-epimerization, double modification, brought

about a remarkable alteration of the potency, and the

20-epi-RRâ-isomer 3g exhibited exceptionally high po-

tency.

d

(2.4) (2.4, 7.0)

3.90 2.00

d

(5.9) (5.9, 7.0)

4.26 1.87

d

(3.4, 2.4) (14.0, 2.4) (14.0, 3.4)

3.65

dt

2.34

dd

2.64

dd

tq

(3.3, 6.0) (13.7, 6.0) (13.7, 3.3)

3.81

dt

2.24

dd

2.66

dd

ddq

(3.3) (8.4, 3.3, 7.0) (4.3, 8.4) (13.2, 8.4) (13.2, 4.3)

4.17 1.92

tq

3.90

dt

2.58

dd

2.50

d

dd^b

(2.7) (2.7, 7.0)

4.04 1.86

(4.0, 2.7) (14.0, 4.0) (14.0, 2.7)

4.06

dt

2.42

dd

2.51

dd

d

ddq

(8.4) (8.4, 2.9, 7.0) (5.9, 2.9) (13.6, 5.9) (13.6, 2.9)

2g

2h

4.30 1.92

ddq

3.84

dt

2.67

dd

2.23

dd

d

(3.3) (7.7, 3.3, 7.2) (4.0, 7.7) (13.2, 4.0) (13.2, 7.7)

3.96 2.12

tq

(4.4) (5.1, 7.0)

3.72

dt

2.65

dd

2.36

dd

d

(3.0, 5.1) (13.9, 3.0) (13.9, 5.1)

a

b

The spectra were recorded at 400 MHz in CDCl₃-D₂O. Each

signal was observed as a broad doublet because of long-range

coupling with H(6). The vicinal coupling constants between H(3)

and H(4) were recorded in decoupling experiments designed to

exclude the coupling with H(6).

Binding affinity to the VDR was examined using

bovine thymus.³⁷Among the 20-natural analogues, most

of the 1R-hydroxy compounds (2a ,e,g) exhibited signifi-

cant to high VDR affinity, whereas the 1â-hydroxy

analogues (2b,d ,f,h ) had virtually no affinity, as ex-

pected, in accordance with previous findings. Further-

more, a comparison of the 2-epimeric pairs of these 1R-

compounds revealed that the 2R-methyl isomers 2e,g

showed much higher potency than the corresponding 2â-

methyl isomers 2c,a , respectively. Accordingly, the 2R-

methyl group enhances the VDR affinity, whereas the

2â-methyl group decreases the affinity. Thus, the RRâ-

isomer 2g showed 4-fold higher affinity than 1R,25-

dihydroxyvitamin D, while the RâR-isomer 2c had

virtually no affinity, despite having a 1R-configuration.

Compared to the 20-natural counterparts, each 20-epi-

analogue showed much higher VDR affinity, ranging

from 3- to 10-fold higher. In particular, the 20-epi-RRâ-

isomer 3g showed 12-fold higher affinity, and the 2â-

which the 2-Me group occupied the equatorial position

was favored in 1,3-trans compounds (a , d , f, and g)

because of 1,3-diaxial interaction between 2-Me and

H(4). However, the equilibrium ratio varies even within

the 1,3-cis and the 1,3-trans groups: for example, the

ratio in the isomer e is 85:15, but it is only 60:40 in the

isomer c. This should be due to the disposition of the

2-methyl group; in the favored R-conformer, it is equato-

rial in e but axial in c. In these cases, calculation of

energy difference and estimation of the ratio would be

valuable. Therefore, molecular mechanics calculation

using MM2* was carried out with model compounds

lacking the side chain.³⁵As expected, the two chair

conformers are the most stable conformers in each

isomer, and the calculated values of energy difference

and equilibrium ratio at 25 °C are shown in parentheses

in Figure 6. These results were in good agreement with

1

F igu r e 6. A-Ring co n f o r mati o n al equilibria of the synthesized analogues. The numbers show the equilibrium ratio deduced from the H NMR analysis. The calculated ratio at 25

°C and energy difference values (kcal/mol) are shown in parentheses.

A-Ring Diastereomers of Methyldihydroxyvitamin D₃

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4255

Ta ble 3. Relative Potency of the Synthesized Analogues with

Respect to 1R,25-Dihydroxyvitamin D₃(1)^a

Ta ble 4. Intestinal Calcium Transport (ICT) and Bone

Calcium-Mobilizing (BCM) Activities of the 2-Methyl Analogues

in Vitamin D-Deficient Rats on a Low-Calcium Diet^a

compd^b

VDR^c

Ca^d

HL-60^e

DBP^f

amount

(pmol/day/

7 days)

ICT serosal/

mucosal ratio

(mean ( SEM) (mean ( SEM)

BCM serum Ca

in mg/100 mL

1R,25-(OH)₂VD₃(1)

2a (Rââ)

100

13

<0.1

0.3

0.8

4

<0.1

400

<0.1

160

<0.1

7

100

2

100

10

1.5

3

100

79

1000

21

1300

45

1200

68

compd

2b (âââ)

NT^g

NT

400

NT

115

NT

19

2c (RâR)

none (control)

0

260

500

260

500

260

500

3.8 ( 0.28

5.0 ( 0.20**

4.3 ( 0.27

4.3 ( 0.16

4.2 ( 0.32

3.9 ( 0.17

4.0 ( 0.32

3.3 ( 0.21

4.1 ( 0.26

2d (ââR)

1

5.5 ( 0.12***

4.1 ( 0.20

2e (RRR)

13

2a (Rââ)

2f (âRR)

0.5

200

1

2600

1

5.0 ( 0.09^‡

5.0 ( 0.10^‡

4.9 ( 0.17^‡

5.2 ( 0.19***

5.4 ( 0.08***

2g (RRâ)

2b (âââ)

2c (RâR)

2h (âRâ)

200

3a (20-epi-Rââ)

3b (20-epi-âââ)

3c (20-epi-RâR)

3d (20-epi-ââR)

3e (20-epi-RRR)

3f (20-epi-âRR)

3g (20-epi-RRâ)

3h (20-epi-âRâ)

<0.3

6

none (control)

0

260

500

260

500

260

500

2.8 ( 0.21

4.9 ( 0.25***

3.0 ( 0.44

3.4 ( 0.21

3.8 ( 0.05

5.0 ( 0.73***

3.1 ( 0.29

3.0 ( 0.22

3.8 ( 0.11

6.8 ( 0.25***

4.1 ( 0.13

3.8 ( 0.12

4.2 ( 0.31

4.1 ( 0.18

3.9 ( 0.14

3.9 ( 0.09

190

730

1

59000

3

1

17

144

NT

655

NT

2d (ââR)

<0.1

1200

<0.1

2e (RRR)

2f (âRR)

a

b

The potency of 1 is normalized to 100. The Greek letters

denote the configurations at C-1, C-2, and C-3, respectively.

^cBinding affinity to bovine thymus vitamin D receptor. Increas-

d

none (control)

0

260

65

260

500

1000

4.4 ( 0.31

4.3 ( 0.12

ing effect on serum calcium level in normal rats. ^eDifferentiation-

inducing effect on HL-60 cells. ^fBinding affinity to calf serum

1

8.8 ( 0.32***

9.3 ( 0.52***

7.3 ( 0.60***

7.0 ( 1.10***

7.2 ( 0.99***

5.5 ( 0.15**

6.0 ( 0.08***

7.8 ( 0.25***

4.4 ( 0.06

2g (RRâ)

g

vitamin D binding protein. NT, not tested.

2h (âRâ)

4.3 ( 0.10

methyl-20-epi analogue 3a (20-epi-Rââ) showed compa-

rable potency to 1. The affinity of 3g is as strong as that

of (22R)-22-methyl-20-epi-1R,25-dihydroxyvitamin D₃,

which has the highest VDR binding affinity so far

known.^18bThe VDR binding affinity of 20-epi-1R,25-

dihydroxyvitamin D₃relative to that of 1R,25-dihy-

droxyvitamin D₃(normalized to 100) is reported to be

120 for chick intestinal VDR and 500 for bovine thymus

VDR.⁸Therefore, the double modification of 2-methyl

substitution and 20-epimerization seemed to have ad-

ditive effects on VDR binding.

The rank order of potency for HL-60 cell differentia-

tion³⁸among each series of compounds 2a -h and 3a -h

was essentially parallel to that of VDR binding. Thus,

among the 20-natural analogues, 2g was twice as potent

as 1R,25-dihydroxyvitamin D₃(1), 2a ,e showed signifi-

cant activity, and the others had only weak activity.

However, 20-epimerization enhanced the potency far

more than expected from the change of VDR binding

affinity. Most notably, the potency of the 20-epi-RRâ-

isomer 3g is 590 times higher than that of 1, which is

comparable in activity to KH-1060, the most potent

analogue reported to date.⁸Compared to the 36-fold

relative enhancement of 20-epi-1R,25-dihydroxyvitamin

D₃vs 1R,25-dihydroxyvitamin D₃,^26cthat of 20-epi-RRâ

3g vs RRâ 2g is much greater, approximately 295 times.

Therefore, this effect should be due to the combination

of 2R-methyl introduction and 20-epimerization, the

double modification. Again, 2â-methyl substitution de-

creased the activity, as seen in the analogue 3a , the

2-epimer of 3g, but the compound still showed 26 times

higher potency than 1.

none (control)

0

130

260

130

260

500

2.3 ( 0.49

4.4 ( 0.15

5.4 ( 0.19^‡

5.5 ( 0.26^‡

4.6 ( 0.10

4.4 ( 0.16

4.9 ( 0.18

5.0 ( 0.17

1

3.6 ( 0.13^†

4.9 ( 0.33***

3.8 ( 0.32^†

4.6 ( 0.57***

4.2 ( 0.57*

3.9 ( 0.38*

3a (20-epi-Rââ)

3b (20-epi-âââ)

none (control)

1

3c (20-epi-RâR)

0

260

500

130

260

500

4.3 ( 0.48

4.7 ( 0.12

6.0 ( 0.17***

4.4 ( 0.09

4.3 ( 0.04

4.4 ( 0.05

4.4 ( 0.08

4.3 ( 0.13

4.4 ( 0.08

6.3 ( 0.39***

5.6 ( 0.69**

5.4 ( 0.99**

5.5 ( 0.70**

5.5 ( 0.43**

5.0 ( 0.56

3d (20-epi-ââR)

3f (20-epi-âRR)

4.9 ( 0.35

none (control)

1

3e (20-epi-RRR)

0

260

500

50

100

500

1000

2.7 ( 0.34

4.6 ( 0.16

5.1 ( 0.24***

4.4 ( 0.19^‡

4.0 ( 0.17

6.1 ( 0.25***

4.6 ( 0.19

4.6 ( 0.04

3g (20-epi-RRâ)

3h (20-epi-âRâ)

4.3 ( 0.70^‡

5.2 ( 0.60***

4.4 ( 0.25^‡

3.4 ( 0.34

5.8 ( 0.21***

7.8 ( 0.23***

4.8 ( 0.15

4.7 ( 0.13

a

Weanling male rats were maintained on a vitamin D-deficient

and 0.47% Ca diet for 1 week and then switched to a vitamin

D-deficient and low-calcium diet containing 0.02% Ca for an

additional 3 weeks. During the last week, they were dosed daily

with a vitamin D analogue for 7 consecutive days. All doses were

administered intraperitoneally in 0.1 mL of propylene glycol/

ethanol (95:5). The control group received the vehicle. Determina-

tions were made 24 h after the last dose. There were 5-6 rats/

group. Statistical analysis was done by the use of Student’s t-test.

Differences from control in each panel: ***p < 0.001, **p < 0.005,

^‡p ) 0.005, *p ) 0.01, ^†p < 0.025.

the potency of the 20-epi-Rââ-isomer 3a was comparable

to that of 1. Intestinal calcium transport and bone

calcium mobilization were also examined by using

vitamin D-deficient rats.⁴⁰The results, shown in Table

4, were in good agreement with those for elevation of

rat serum calcium concentration. Among the 20-natural

analogues, only the RRâ-isomer 2g showed activity of

4-5 times higher than that of 1, and the other isomers

had virtually no activity. In these cases, 20-epimeriza-

tion had no enhancing effect on the activity; only the

A similar trend to those of VDR binding and HL-60

cell differentiation was found in calcemic activity. In the

elevation of serum calcium concentration using normal

SD male rats,³⁹the RRâ-isomer 2g also exhibited 4-fold

higher potency than 1, whereas its 2-methyl epimer 2a

had quite low activity. Again, 20-epimerization en-

hanced the potency, but in this case, the extent was not

as much as for HL-60 differentiation: the 20-epi-RRâ-

isomer 3g showed 6.5-fold higher potency than 1 and

4256 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22

Konno et al.

20-epi-RRâ-isomer 3g was as active as 2g and all other

when it binds to the receptor. The configuration of the

2-methyl group seems to influence the potency because

the isomers bearing the 2R-methyl group are much more

potent than their counterparts, as seen in RRâ g vs Rââ

a and in RRR e vs RâR c. Therefore, the 2R-methyl group

may have a specific favorable interaction with the

receptor, for example, a hydrophobic interaction. A steric

effect is also plausible: that is, there may be a hydro-

phobic pocket in the receptor into which only the 2R-

methyl group can fit well. Quite recently, a crystal

structure of a mutant VDR in complex with 1R,25-

dihydroxyvitamin D₃⁴⁴and three-dimensional modeling

of the VDR ligand binding domain⁴⁵have been reported.

Interestingly, the experimental data show that there is

an additional space near position 2 of the A-ring, which

could accommodate the 2-methyl group. Further studies

are required to elucidate the A-ring conformation-

activity relationship and the significance of the 2R-

methyl substitution.⁴⁶

isomers showed very little activity.

In binding to the vitamin D binding protein (DBP)

using fetal calf serum DBP,⁴¹the 1â-isomers showed

high affinity, while the 1R-isomers had only poor affin-

ity, which was consistent with previous findings (Table

3).⁴²Moreover, all the 20-epi analogues were very poor

ligands for DBP, showing approximately 300 times less

affinity than 1. These results imply that 20-epimeriza-

tion remarkably decreases the DBP binding activity

irrespective of the A-ring stereochemistry. This may be

related in part to the high potency of the 20-epi

analogues in HL-60 cell differentiation.

It is noteworthy that 20-epi-ââR 3d was significantly

active in HL-60 cell differentiation despite having a 1â-

hydroxy configuration, which should impart low or no

activity. Compared to 1R,25-dihydroxyvitamin D₃, 3d

showed 7% VDR binding affinity, 2-fold greater HL-60

differentiation activity, and about one-fifth of the calcium-

mobilizing activity. Since all the compounds epimeric

at either one of the 1, 2, 3, and 20 positions, 20-epi-RâR

3c, 20-epi-âRR 3f, 20-epi-âââ 3b, and ââR 2d , respec-

tively, exhibited virtually no activity, the combination

of all these configurations is essential for activity. It is

plausible that these effects arise from rotation or

turnover of the molecule due to having opposite con-

figurations at all those positions, so that it can mimic

the 20-epi-RRâ 3g or RRâ 2g.

We also examined transactivation of target genes in

transfected cells and modulation of cell-surface CD11b

antigen expression in HL-60 cells in order to gain more

insight into the biological action of these 2-methyl

analogues.⁴³As already reported, the rank orders of the

transcriptional potencies were essentially parallel to

those of the VDR binding affinity and HL-60 cell

differentiation: that is, 20-epi-RRâ 3g was the most

potent and exhibited exceptionally high activity, and

RRâ 2g and 20-epi-Rââ 3a showed activity comparable

to or greater than that of 1.^43aIn further evaluation of

biological activities, we surprisingly found that the

structural requirement for inducing apoptosis of HL-

60 cells was clearly different from that for inducing

differentiation of these cells. In contrast to the require-

ment of the 1R- and 3â-hydroxyl groups for differentia-

tion activity, compounds bearing the 1â-hydroxyl group

together with the 3R- or 3â-hydroxyl group were potent

stimulators of apoptosis of these cells.^43bTherefore,

these findings provided useful information not only for

structure-function studies of 1R,25-dihydroxyvitamin

D₃analogues but also for the development of therapeutic

agents for the treatment of leukemia and other cancers.

Con clu sion s

In this study, we have synthesized all possible A-ring

diastereomers of 1R,25-dihydroxyvitamin D₃and its 20-

epimers utilizing our newly developed procedure for

synthesis of A-ring enyne synthon. The advantage of

this procedure is that all possible A-ring diastereomers

are readily accessible in the same way, which is in

marked contrast to the known procedures. Biological

evaluation of all these analogues demonstrated that the

activities are highly dependent on the configuration not

only of the C-1 and C-3 hydroxyl groups but also of the

2-methyl group, providing unique activity profiles. 2R-

Methyl substitution with 1R,3â-hydroxyl groups in-

creased the VDR binding, calcium mobilization, and HL-

60 cell differentiation activities, and further modification

by 20-epimerization enhanced the potencies. Thus, we

found the exceptionally potent analogue 3g, which is

as potent as KH-1060, the most potent analogue re-

ported to date. Moreover, the analogue 3d exhibited

significant activities, despite having a 1â-configuration.

Further evaluation of the biological activities of all the

2-methyl analogues led to the finding that the structural

requirement for inducing apoptosis of HL-60 cells was

clearly different from that for inducing differentiation

of these cells. These results show that the synthesis of

all possible diastereomers is an effective approach to

fully understanding SARs. The information provided by

this study should cast light on the physiological roles

of vitamin D and its SAR for various biological activities.

Exp er im en ta l Section

Gen er a l. Melting points were determined by using a

Yanagimoto hot-stage apparatus and are uncorrected. NMR

spectra were recorded on a J EOL GSX-400 or a Bruker DMX-

500 spectrometer. Chemical shifts are expressed in ppm

relative to tetramethylsilane. Mass spectra (MS) and high-

resolution mass spectra (HRMS) were recorded on a J EOL

J MS D-300 mass spectrometer. Infrared spectra were recorded

One of the objectives of this study was to gain

information about the A-ring conformation-activity

relationship, as mentioned in the Introduction. Modi-

fication by the 2-methyl group was intended to alter the

equilibrium between the R- and â-conformers so that

we could examine which conformer contributes more to

the activity. Unfortunately, however, this study does not

permit any decisive conclusion. The most potent isomer

g favored the R-conformer, whereas the second most

potent a favored the â-conformer, and the energy

difference between the two conformers was not large:

a 60:40 ratio for g and a 75:25 ratio for a . In this

situation, the A-ring may adopt either conformation

on a J asco FT/IR-8000 spectrometer and are expressed in cm^-1

.

Ultraviolet spectra were recorded with a Hitachi 200-10

spectrophotometer. Optical rotations were determined by using

a J asco DIP-370 digital polarimeter. Elemental analyses were

carried out in the Microanalytical Laboratory, Faculty of

Pharmaceutical Sciences, University of Tokyo, and were within

0.3% of the theoretical values. Recycling preparative HPLC

was performed on a Waters LC equipped with a 510 HPLC

A-Ring Diastereomers of Methyldihydroxyvitamin D₃

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4257

pump and 484 tunable absorbance detector. Analytical HPLC

was performed on a Waters M600E LC system equipped with

a 490E tunable absorbance detector. Analyses were conducted

by using reversed-phase HPLC (Lichrosorb RP-18 column, 4

× 250 mm, 1.0 mL/min, acetonitrile:water ) 60:40) and

retention times (t_R) are expressed in minutes. Crystallographic

data were collected on a Rigaku AFC7S diffractometer with

graphite-monochromated Cu KR radiation.

Meth yl (S)-3-[(ter t-Bu tyld ip h en ylsilyl)oxy]-2-m eth yl-

p r op ion a te (9). To a solution of methyl (S)-3-hydroxy-2-

methylpropionate (10.4 g, 88 mmol) and imidazole (13.2 g, 194

mmol) in dry CH₂Cl₂(20 mL) was added TBDPSCl (25 mL,

97 mmol) at 0 °C with stirring. After the ice bath was removed,

the reaction mixture was stirred at room temperature for 1 h.

The reaction mixture was diluted with water and the whole

was extracted with CH₂Cl₂. The organic layer was washed with

brine, dried over magnesium sulfate and filtered. Evaporation

of the filtrate gave a crude mixture, from which 9 (31.4 g) was

separated by silica gel column chromatography (2% ethyl

acetate-n-hexane) as a colorless oil in quantitative yield: [R]_D

+13.8 (c ) 1.52, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 1.04 (9

H, s), 1.15 (3 H, d, J ) 7.0 Hz), 2.72 (1 H, d, quint, J ) 7.0, 5.8

Hz), 3.67 (3 H, s), 3.73 (1 H, dd, J ) 9.8, 6.4 Hz), 7.35-7.44 (6

H, m), 7.64-7.68 (4 H, m); ¹³C NMR (100 MHz, CDCl₃) δ 13.5

(q), 19.2 (s), 26.7 (q), 42.2 (d), 51.5 (q), 65.9 (t), 127.7 (d), 129.7

(d), 133.5 (s), 135.6 (d), 175.3 (s); IR (neat) 1741 cm^-1; MS m/z

356 (M)⁺, 325 (M - OMe)⁺; HRMS m/z (M)⁺calcd 356.1808

for C₂₁H₂₈O₃Si, found 356.1796.

stirring n-BuLi (1.6 M solution in n-hexane, 5.2 mL, 9.3 mmol)

under argon at 0 °C. The resulting mixture was stirred for 20

min, then a solution of 11 (1.2 g, 3.7 mmol) in THF (15 mL)

was added to it. The reaction mixture was further stirred for

15 min and then for 45 min at room temperature. Saturated

NH₄Cl (aq) was added to the mixture, and the whole was

extracted with ethyl acetate. The organic layer was washed

with brine, dried over magnesium sulfate, filtered and con-

centrated. The crude product was purified by silica gel column

chromatography (2% ethyl acetate-n-hexane) to give 12 (1.1

g) as a colorless oil in 92% yield: [R]_D+12.1 (c ) 0.90, CHCl₃);

¹H NMR (400 MHz, CDCl₃) δ 1.03 (3 H, d, J ) 7.0 Hz), 1.05 (9

H, s), 2.39 (1 H, m), 3.49 (1 H, dd, J ) 9.7, 6.7 Hz), 3.57 (1 H,

dd, J ) 9.7, 6.1 Hz), 5.01 (3 H, m), 7.35-7.44 (6 H, m), 7.65-

7.68 (4 H, m); ¹³C NMR (100 MHz, CDCl₃) δ 16.2 (q), 19.3 (s),

26.9 (q), 40.2 (d), 68.5 (t), 114.1 (t), 127.6 (d), 129.5 (d), 134.0

(s), 135.7 (d), 141.4 (d); IR (neat) 1472, 1428 cm^-1; MS m/z

t

267 (M - Bu)⁺; HRMS m/z (M - Bu)⁺calcd for C₁₇H₁₉OSi

267.1205, found 267.1202.

(2S,3RS)-1-[(ter t-Bu tyld ip h en ylsilyl)oxy]-2-m eth yl-3,4-

ep oxybu ta n e (13). To a solution of 12 (1.0 g, 3.1 mmol) in

dry CH₂Cl₂(25 mL) was added mCPBA (1.4 g, 7.4 mmol) under

argon. The resultant solution was stirred for 15 min at 0 °C,

then further stirred overnight after removal of the ice bath.

The whole mixture was extracted with ethyl acetate after the

addition of water. The organic layer was washed with brine

and dried over magnesium sulfate. Evaporation of the solvent

afforded a residue, from which 13 (1.1 g) was separated by

silica gel column chromatography (2% Et₂O-n-hexane) as a

colorless oil in quantitative yield: ¹H NMR (400 MHz, CDCl₃)

δ 0.99 (3 H, d, J ) 6.8 Hz), 1.05 (9 H × 3/5, s), 1.07 (9 H × 2/5,

s), 1.58 (1 H, m), 2.54 (1 H × 2/5, dd, J ) 5.0, 2.7 Hz), 2.60 (1

H × 3/5, dd, J ) 5.0, 2.7 Hz), 2.73 (1 H × 2/5, dd, J ) 5.0, 4.3

Hz), 2.76 (1 H × 3/5, dd, J ) 5.0, 4.3 Hz), 2.85 (1 H × 3/5, ddd,

J ) 7.0, 4.3, 2.7 Hz), 2.97 (1 H × 2/5, ddd, J ) 7.0, 4.3, 2.7

Hz), 3.62 (1 H, dd, J ) 6.7, 3.4 Hz), 3.70 (1 H, dd, J ) 7.0, 5.0

Hz), 7.36-7.45 (6 H, m), 7.64-7.70 (4 H, m); IR (neat) 1472,

(R)-3-[(ter t-Bu t yld ip h en ylsilyl)oxy]-2-m et h ylp r op a n -

1-ol (10). To a solution of 9 (3.54 g, 9.93 mmol) dissolved in

dry toluene was added DIBAL-H (1.0 M in toluene, 19.8 mL)

at -78 °C under argon. Stirring was continued for 1 h at -78

°C, then ethyl acetate was added to the reaction mixture to

destroy the excess reagent. The mixture was washed with 0.5

N HCl (aq) and then with brine, dried over magnesium sulfate

and filtered. The solvent was removed under reduced pressure

to give a residue, from which 10 (2.90 g) was separated by

silica gel column chromatography (4-10% ethyl acetate-n-

hexane) as a colorless oil in 90% yield: [R]_D+1.5 (c ) 1.34,

CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 0.83 (3 H, d, J ) 7.0

Hz), 1.06 (9 H, s), 1.99 (1 H, m), 2.58 (1 H, m), 3.60 (1 H, dd,

J ) 10.1, 7.6 Hz), 3.68 (2 H, m), 3.72 (1 H, dd, J ) 10.1, 4.6

Hz), 7.37-7.46 (6 H, m), 7.67-7.69 (4 H, m); ¹³C NMR (100

MHz, CDCl₃) δ 13.2 (q), 19.2 (s), 26.7 (q), 37.6 (d), 67.5 (t),

68.6 (t), 127.8 (d), 129.8 (d), 133.20 (s), 133.21 (s), 135.57 (d),

135.58 (d); IR (neat) 3383, 1472, 1428 cm^-1; MS m/z 271 (M -

t

1427, 1294 cm^-1; MS m/z 283 (M - Bu)⁺; HRMS m/z (M -

^tBu)⁺; calcd for C₁₇H₁₉O₂Si 283.1154, found 283.1167.

(2S,3R)-1-[(ter t-Bu tyldiph en ylsilyl)oxy]-2-m eth yl-6-(tr i-

m eth ylsilyl)h ex-5-yn -3-ol (14a ) a n d (2S,3S)-1-[(ter t-Bu -

t yld ip h en ylsilyl)oxy]-2-m et h yl-6-(t r im et h ylsilyl)h ex-5-

yn -3-ol (14b). Under an argon atmosphere, ethynyltrimethyl-

silane (9.4 mL, 67 mmol) was dissolved in THF, and n-BuLi

(1.6 M in n-hexane solution, 36 mL, 57 mmol) was added to

the mixture at 0 °C. The whole was stirred for 20 min then

cooled to -78 °C. The resulting mixture was added to a stirred

solution of 13 (6.47 g, 19 mmol) in THF at -78 °C. The reaction

mixture was stirred at -78 °C for 50 min after addition of BF₃‚

Et₂O (4.7 mL, 38 mmol). The reaction was quenched by the

addition of satd NH₄Cl (aq), and the whole was extracted with

ethyl acetate. The organic layer was washed with brine, dried

over magnesium sulfate, filtered and concentrated. This crude

residue was purified by silica gel column chromatography (5%

ethyl acetate-n-hexane) to give 14a (3.42 g, 41%, less polar)

and 14b (4.33 g, 52%, more polar), both as colorless oils.

t

^tBu)⁺; HRMS m/z (M - Bu)⁺calcd for C₁₆H₁₉O₂Si 271.1155,

found 271.1159.

(S)-3-[(ter t-Bu t yld ip h en ylsilyl)oxy]-2-m et h ylp r op a -

n a l (11). A solution of oxalyl chloride (1.8 mL, 21 mmol) in

CH₂Cl₂was added to a solution of DMSO (3.0 mL, 44 mmol)

in CH₂Cl₂, and the mixture was stirred at -78 °C for 1 h under

an argon atmosphere. The resulting mixture was transferred

to a solution of 10 (3.40 g, 10 mmol) in CH₂Cl₂at -78 °C and

the mixture was stirred for 30 min. Subsequently, Et₃N (11.5

mL) was added to the mixture followed by stirring for 1 h,

while the temperature was elevated from -78 to 0 °C. The

reaction mixture was extracted with ethyl acetate after the

addition of water. The organic phase was washed with brine,

dried over magnesium sulfate and filtered. The solvent was

removed under reduced pressure to give a residue, from which

11 (3.37 g) was separated by silica gel column chromatography

(4% ethyl acetate-n-hexane) as a colorless oil in 99% yield:

[R]_D+4.0 (c ) 1.28, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 1.04

(9 H, s), 1.10 (3 H, d, J ) 7.0 Hz), 2.56 (1 H, m), 3.87 (1 H,

ddd, J ) 10.0, 6.1, 4.8 Hz), 7.37-7.46 (6 H, m), 7.63-7.67 (4

H, m), 9.77 (1 H, d, J ) 1.5 Hz); ¹³C NMR (100 MHz, CDCl₃)

δ 13.2 (q), 19.2 (s), 26.8 (q), 41.9 (d), 67.6 (t), 127.8 (d), 129.8

(d), 133.07 (s), 133.12 (s), 135.9 (d), 204.4 (d); IR (neat) 1732,

14a : [R]_D+5.0 (c ) 0.54, CHCl₃); ¹H NMR (400 MHz, CDCl₃)

δ 0.15 (9 H, s), 0.91 (3 H, d, J ) 7.1 Hz), 1.07 (9 H, s), 1.96 (1

H, m), 2.46 (1 H, dd, J ) 10.6, 6.4 Hz), 2.54 (1 H, dd, J ) 10.6,

4.9 Hz), 2.84 (1 H, d, J ) 3.1 Hz), 3.67 (1 H, dd, J ) 10.4, 6.4

Hz), 3.75 (1 H, m), 3.79 (1 H, dd, J ) 10.4, 4.3 Hz), 7.37-7.46

(6 H, m), 7.65-7.68 (4 H, m); ¹³C NMR (100 MHz, CDCl₃) δ

0.1 (q), 13.5 (q), 19.1 (s), 26.7 (t), 26.8 (q), 39.2 (d), 67.6 (t),

73.6 (d), 86.9 (s), 103.6 (s), 127.7 (d), 129.8 (d), 133.0 (s), 133.6

(d), 135.6 (d); IR (neat) 3491, 2179 cm^-1; MS m/z 423 (M -

t

Me)⁺, 365 (M - TMS)⁺, 308 (M - TMS - Bu)⁺; HRMS m/z

(M - Me)⁺calcd for C₂₅H₃₅O₂Si₂423.2243, found 423.2209.

14b: [R]_D-10.4 (c ) 0.58, CHCl₃); ¹H NMR (400 MHz,

CDCl₃) δ 0.14 (9 H, s), 1.00 (3 H, d, J ) 7.0 Hz), 1.06 (9 H, s),

1.92 (1 H, m), 2.42 (1 H, dd, J ) 10.1, 7.0 Hz), 2.50 (1 H, dd,

J ) 10.1, 4.3 Hz), 2.84 (1 H, d, J ) 3.1 Hz), 3.67 (1 H, dd, J )

10.2, 6.4 Hz), 3.75 (1 H, dd, J ) 10.2, 4.2 Hz), 3.97 (1 H, dd, J

) 10.4, 4.3 Hz), 7.37-7.46 (6 H, m), 7.65-7.68 (4 H, m); ¹³C

NMR (100 MHz, CDCl₃) δ 0.1 (q), 10.1 (q), 19.2 (s), 26.1 (t),

t

1472, 1428 cm^-1; MS m/z 326 (M)⁺, 269 (M - Bu)⁺; HRMS

m/z (M)⁺calcd for C₂₀H₂₆O₂Si 326.1708, found 326.1705.

(R )-4-[(t er t -Bu t yld ip h e n ylsilyl)oxy]-3-m e t h ylb u t -1-

en e (12). To a suspension of methyltriphenylphosphonium

bromide (2.2 g, 7.4 mmol) in THF (15 mL) was added with

4258 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22

Konno et al.

26.9 (q), 38.3 (d), 68.1 (t), 72.2 (d), 86.8 (s), 103.8 (s), 127.8 (d),

129.7 (d), 129.8 (d), 133.0 (s), 133.2 (s), 135.6 (d), 135.6 (d); IR

(400 MHz, CDCl₃) δ 0.99 (3 H × 1/2, d, J ) 6.7 Hz), 1.01 (3 H

× 1/2, d, J ) 7.0 Hz), 1.52 (4 H, m), 1.74 (2 H, m), 1.99 (1 H ×

1/2, t, J ) 2.7 Hz), 2.00 (1 H × 1/2, t, J ) 2.7 Hz), 2.16 (1 H,

m), 2.33 (1 H × 1/2, m), 2.38 (1 H × 1/2, ddd, J ) 17.1, 6.1, 2.8

Hz), 2.56 (1 H × 1/2, ddd, J ) 17.1, 4.0, 2.8 Hz), 2.63 (1 H ×

1/2, ddd, J ) 17.1, 4.0, 2.8 Hz), 2.72 (1 H × 1/2, ddd, J ) 17.1,

7.0, 2.8 Hz), 3.48 (3 H × 1/2, m), 3.70 (2 H, m), 3.98 (3 H ×

1/2, m), 4.70 (1 H, m); ¹³C NMR (100 MHz, CDCl₃) δ 13.9 (q),

14.3 (q), 19.6 (t), 21.2 (t), 22.5 (t), 23.5 (t), 25.0 (t), 25.2 (t),

30.9 (t), 31.1 (t), 37.5 (d), 39.0 (d), 62.8 (t), 64.6 (t), 65.2 (t),

70.0 (d), 70.1 (d), 77.1 (d), 80.8 (s), 80.9 (s), 81.8 (d), 100.0 (d),

t

(neat) 3484, 2176 cm^-1; MS m/z 381 (M - Bu)⁺; HRMS m/z

t

(M - Bu)⁺calcd for C₂₂H₂₉O₂Si₂381.1706, found 381.1714.

Gen er a l P r oced u r e for Syn th eses of MTP A Ester s. A

solution of each of the above alcohols dissolved in dry CH₂Cl₂

was treated with DMAP (2 equiv) and (R)- or (S)-MTPACl (2

equiv) at room temperature under an argon atmosphere. The

reaction solution was purified by preparative TLC (10% ethyl

acetate-n-hexane) without pretreatment to afford the corre-

sponding MTPA ester.

(S)-MTP A ester of 14a : ¹H NMR (400 MHz, CDCl₃) δ 0.116

(9 H, s), 0.820 (3 H, d, J ) 7.0 Hz), 1.048 (9 H, s), 2.186 (1 H,

q, J ) 6.1 Hz), 2.588 (1 H, dd, J ) 11.0, 6.7 Hz), 2.751 (1 H,

dd, J ) 11.0, 6.7 Hz), 3.486 (1 H, dd, J ) 10.3, 5.4 Hz), 3.537

(1 H, dd, J ) 10.3, 5.8 Hz), 3.57 (3 H, s), 5.317 (1 H, dd, J )

10.3, 6.7 Hz), 7.28-7.45 (9 H, m), 7.49-7.54 (2 H, m), 7.59-

7.65 (4 H, m).

(R)-MTP A ester of 14a : ¹H NMR (400 MHz, CDCl₃) δ

0.075 (9 H, s), 0.949 (3 H, d, J ) 7.0 Hz), 1.051 (9 H, s), 2.254

(1 H, q, J ) 6.7 Hz), 2.552 (1 H, dd, J ) 11.6, 6.1 Hz), 2.745 (1

H, dd, J ) 11.6, 5.2 Hz), 3.42 (3 H, s), 3.564 (1 H, dd, J )

10.7, 5.8 Hz), 3.643 (1 H, dd, J ) 10.7, 6.5 Hz), 5.272 (1 H, dd,

J ) 10.6, 5.8 Hz), 7.28-7.45 (9 H, m), 7.50-7.56 (2 H, m),

7.59-7.65 (4 H, m).

101.2 (d); IR (neat) 3310, 3021, 2176, 1375, 1248, 1046 cm^-1

.

(2R,3R)-2-Met h yl-3-[(2-t et r a h yd r op yr a n yl)oxy]h ex-5-

yn a l (17). A solution of oxalyl chloride (0.8 mL, 9.3 mmol) in

CH₂Cl₂(5 mL) was added to a solution of DMSO (1.3 mL, 19

mmol) in CH₂Cl₂(5 mL), and the mixture was stirred at -78

°C for 1 h under an argon atmosphere. The resulting mixture

was transferred to a solution of 16 (1.04 g, 4.9 mmol) in CH₂Cl₂

(20 mL) at -78 °C and the mixture was stirred for 30 min.

Subsequently, Et₃N (5.0 mL) was added to the mixture

followed by stirring for 1 h, while the temperature was allowed

to rise from -78 to 0 °C. The reaction mixture was extracted

with ethyl acetate after the addition of water. The organic

phase was washed with brine, dried over magnesium sulfate

and filtered. The solvent was removed under reduced pressure

to give a residue, from which 17 (989 mg) was separated by

silica gel column chromatography (20% ethyl acetate-n-

hexane) as a colorless oil in 96% yield: ¹H NMR (400 MHz,

CDCl₃) δ 1.12 (3 H × 1/2, d, J ) 7.2 Hz), 1.15 (3 H × 1/2, d, J

) 7.2 Hz), 1.57 (4 H, m), 1.76 (2 H, m), 2.02 (1 H × 1/2, t, J )

2.8 Hz), 2.04 (1 H × 1/2, t, J ) 2.8 Hz), 2.44 (1 H × 1/2, ddd,

J ) 17.4, 5.1, 2.8 Hz), 2.57 (1 H × 1/2, ddd, J ) 17.1, 6.1, 2.8

Hz), 2.38 (1 H × 1/2, ddd, J ) 17.1, 6.1, 2.8 Hz), 2.56 (1 H ×

1/2, ddd, J ) 17.4, 5.5, 2.8 Hz), 2.73 (1 H × 1/2, ddd, J ) 17.4,

6.7, 2.6 Hz), 2.82 (1 H × 1/2, dquint, J ) 2.1, 7.0 Hz), 2.90 (1

H × 1/2, dquint, J ) 2.1, 7.0 Hz), 3.53 (1 H, m), 3.81 (1 H ×

1/2, m), 3.96 (1 H, m), 4.10 (1 H × 1/2, dd, J ) 11.8, 5.6 Hz),

4.70 (1 H × 1/2, m), 4.82 (1 H × 1/2, m), 9.82 (1 H × 1/2, d, J

) 1.5 Hz), 9.85 (1 H × 1/2, d, J ) 2.1 Hz); ¹³C NMR (100 MHz,

CDCl₃) δ 10.3 (q), 10.5 (q), 19.4 (t), 19.6 (t), 21.0 (t), 23.3 (t),

25.2 (t), 25.4 (t), 30.6 (t), 30.7 (t), 49.0 (d), 49.7 (d), 62.7 (t),

62.9 (t), 70.5 (d), 71.3 (d), 74.2 (d), 79.7 (s), 80.1 (s), 97.0 (d),

100.6 (d), 203.7 (d), 204.1 (d); IR (neat) 2942, 2876, 2174, 1724

(S)-MTP A ester of 14b: ¹H NMR (400 MHz, CDCl₃) δ

0.116 (9 H, s), 0.861 (3 H, d, J ) 7.0 Hz), 1.067 (9 H, s), 2.276

(1 H, q, J ) 6.1 Hz), 2.565 (1 H, dd, J ) 10.6, 5.8 Hz), 2.703 (1

H, dd, J ) 10.6, 6.1 Hz), 3.46 (3 H, s), 3.491 (1 H, m), 3.532 (1

H, m), 5.487 (1 H, dd, J ) 9.8, 5.8 Hz), 7.28-7.46 (9 H, m),

7.49-7.56 (2 H, m), 7.60-7.69 (4 H, m).

(R)-MTP A ester of 14b: ¹H NMR (400 MHz, CDCl₃) δ

0.124 (9 H, s), 0.797 (3 H, d, J ) 6.7 Hz), 1.056 (9 H, s), 2.172

(1 H, q, J ) 6.7 Hz), 2.652 (1 H, t, J ) 6.7 Hz), 2.708 (1 H, m),

3.339 (1 H, m), 3.441 (1 H, m), 3.58 (3 H, s), 5.457 (1 H, dd, J

) 10.3, 6.1 Hz), 7.28-7.46 (9 H, m), 7.49-7.55 (2 H, m), 7.61-

7.65 (4 H, m).

(4R,5S)-6-[(ter t-Bu tyld ip h en ylsilyl)oxy]-5-m eth yl-4-[(2-

tetr a h yd r op yr a n yl)oxy]-1-(tr im eth ylsilyl)h ex-1-yn e (15).

To a solution of 14a (2.15 g, 4.9 mmol) and DHP (0.76 mL, 8.3

mmol) in dry CH₂Cl₂(10 mL) was added PPTS (160 mg, 0.64

mmol) at room temperature, and the resulting mixture was

allowed to stand overnight. The mixture was diluted with

ether, then washed with brine and dried over magnesium

sulfate. Evaporation of the solvent gave a residue, from which

15 (2.51 g) was separated by silica gel column chromatography

(1% ethyl acetate-n-hexane) as a colorless oil in 98% yield:

¹H NMR (400 MHz, CDCl₃) δ 0.127 (9 H × 1/2, s), 0.135 (9 H

× 1/2, s), 0.95 (3 H, d, J ) 7.0 Hz), 1.058 (9 H × 1/2, s), 1.061

(9 H × 1/2, s), 1.52 (4 H, m), 1.74 (2 H, m), 2.13 (1 H, m), 2.38

(1 H × 1/2, dd, J ) 17.1, 7.3 Hz), 2.46 (1 H × 1/2, dd, J ) 17.1,

4.6 Hz), 2.54 (1 H × 1/2, dd, J ) 17.1, 5.5 Hz), 2.66 (1 H × 1/2,

dd, J ) 17.1, 5.8 Hz), 3.45 (1 H, m), 3.64 (2 H, m), 3.77 (1 H,

m), 3.90 (1 H × 1/2, dt, J ) 7.3, 4.9 Hz), 4.02 (1 H × 1/2, m),

4.66 (1 H × 1/2, t, J ) 3.1 Hz), 4.86 (1 H, dd, J ) 4.3, 2.7 Hz),

7.35-7.44 (6 H, m), 7.65-7.70 (4 H, m); ¹³C NMR (100 MHz,

CDCl₃) δ -0.05 (q), 0.003 (q), 12.8 (q), 13.2 (q), 19.2 (s), 19.3

(t), 19.7 (t), 22.3 (t), 24.1 (t), 25.4 (t), 25.5 (t), 26.8 (q), 26.9 (q),

30.7 (t), 30.8 (t), 38.2 (d), 40.0 (d), 62.1 (t), 62.6 (t), 65.4 (t),

65.8 (t), 76.3 (d), 77.7 (d), 85.8 (s), 98.3 (d), 99.3 (d), 104.9 (s),

127.7 (d), 129.7 (d), 133.9 (s), 134.0 (s), 135.7 (d); IR (neat)

2176, 1362, 1250, 1113, 1078, 1028 cm^-1; MS m/z 522 (M)⁺,

507 (M - Me)⁺; HRMS m/z (M)⁺calcd for C₃₁H₄₆O₃Si₂

522.2986, found 522.2986.

cm^-1

.

(3RS,4S,5R)-4-Meth yl-5-[(2-tetr ah ydr opyr an yl)oxy]oct-

1-en -7-yn -3-ol (18). A suspension of CeCl₃(595 mg, 2.4 mmol)

in dry THF (7 mL) was stirred at 0 °C for 2 h under argon

atmosphere. To the mixture was added at -78 °C a solution

of vinylmagnesium bromide (1.0 M in THF, 2.25 mL, 6.0

mmol), and the mixture was further stirred for 2 h at -78 °C.

A solution of 17 (190 mg, 0.90 mmol) in THF (5 mL) was

introduced into the thus prepared mixture. Stirring was

continued for 2 h, then the reaction was quenched by the

addition of satd NH₄Cl (aq). Extraction with ethyl acetate

followed by evaporation of the solvent afforded a residue, from

which 18 (197 mg) was separated by silica gel column chro-

matography (20% ethyl acetate-n-hexane) as a colorless oil

in 92% yield: ¹H NMR (400 MHz, CDCl₃) δ 0.85 (3 H × 1/4, d,

J ) 7.0 Hz), 0.88 (3 H × 1/4, d, J ) 7.3 Hz), 0.90 (3 H × 1/4,

d, J ) 7.0 Hz), 0.93 (3 H × 1/4, d, J ) 7.0 Hz), 1.52 (4 H, m),

1.73 (2 H, m), 2.01 (1 H, m), 2.17 (1 H, m), 2.40 (1 H × 1/2, m),

2.59 (1 H, m), 2.68 (1 H × 1/4, d, J ) 3.7 Hz), 2.80 (1 H × 1/2,

m), 2.88 (1 H × 1/4, d, J ) 4.6 Hz), 3.37 (1 H × 1/4, d, J ) 4.3

Hz), 3.52 (1 H, m), 3.73 (1 H × 3/4, m), 3.80 (1 H × 1/4, d, J )

4.3 Hz), 3.97 (1 H × 5/4, m), 4.13 (1 H × 1/4, m), 4.19 (1 H ×

1/4, m), 4.51 (1 H × 1/4, m), 4.70 (1 H, m), 4.81 (1 H × 1/4, m),

5.24 (2 H, m), 5.88 (1 H, m); ¹³C NMR (100 MHz, CDCl₃) δ 9.8

(q), 10.7 (q), 12.4 (q), 13.4 (q), 19.4 (t), 19.5 (t), 20.2 (t), 21.5

(t), 22.8 (t), 23.1 (t), 23.5 (t), 24.9 (t), 25.1 (t), 25.2 (t), 25.3 (t),

30.8 (t), 31.0 (t), 31.1 (t), 39.8 (d), 41.1 (d), 41.8 (d), 42.2 (d),

62.5 (t), 62.8 (t), 63.8 (t), 65.6 (t), 69.7 (d), 70.0 (d), 70.0 (d),

70.1 (d), 70.3 (d), 72.1 (d), 75.5 (d), 76.4 (d), 76.9 (d), 77.2 (d),

80.5 (d), 80.6 (s), 80.8 (s), 81.5 (s), 81.5 (d), 98.1 (d), 100.3 (d),

100.5 (d), 101.5 (d), 114.0 (t), 114.5 (t), 115.6 (t), 116.1 (t), 139.1

(2S,3R)-2-Met h yl-3-[(2-t et r a h yd r op yr a n yl)oxy]h ex-5-

yn -1-ol (16). To a stirred solution of 15 (1.13 g, 2.2 mmol) in

THF (20 mL) was added TBAF (1.0 M THF solution, 8.8 mL,

8.8 mmol), and the resulting mixture was stirred at room

temperature for 4 h. Water was added, and the mixture was

extracted with ethyl acetate. The organic phase was washed

with brine, dried over magnesium sulfate and filtered. Evapo-

ration of the filtrate gave a residue, from which 16 (450 mg)

was separated by silica gel column chromatography (20% ethyl

acetate-n-hexane) as a colorless oil in 96% yield: ¹H NMR

A-Ring Diastereomers of Methyldihydroxyvitamin D₃

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4259

(d), 139.5 (d), 139.9 (d); IR (neat) 3451, 3309, 2120, 1642, 1217,

J ) 2.8 Hz), 2.33 (1 H, ddd, J ) 16.8, 6.1, 2.8 Hz), 2.45 (1 H,

d, J ) 4.6 Hz), 2.48 (1 H, ddd, J ) 16.8, 5.2, 2.8 Hz), 2.77 (1

H, d, J ) 3.7 Hz), 4.19 (2 H, m), 5.23 (1 H, dt, J ) 10.1, 1.5

Hz), 5.23 (1 H, dt, J ) 10.7, 1.5 Hz), 5.34 (1 H, dt, J ) 17.1,

1.5 Hz), 5.92 (1 H, ddd, J ) 17.1, 10.4, 5.8 Hz); ¹³C NMR (100

MHz, CDCl₃) δ 10.4 (q), 24.3 (t), 40.6 (d), 70.4 (d), 76.7 (d),

81.2 (s), 115.9 (t), 139.8 (d); IR (neat) 3567, 3303, 2977, 2917,

1182, 1074 cm^-1

.

(3R,4R,5R)-4-Meth yloct-1-en -7-yn e-3,5-d iol (19a ) a n d

(3S,4R,5R)-4-Meth yloct-1-en -7-yn e-3,5-d iol (19b). To a so-

lution of 18 (315 mg, 1.3 mmol) in methanol (10 mL) was added

TsOH‚H₂O (25 mg, 0.13 mmol), and the mixture was allowed

to stand at room temperature for 1 h. After evaporation of

methanol, brine was added to the mixture and the whole was

extracted with ether. The organic layer was dried over

magnesium sulfate, filtered and concentrated. The crude

mixture was purified by silica gel column chromatography

(10% ethyl acetate-n-hexane) to give 19a (79 mg, 39%, less

polar) and 19b (75 mg, 37%, more polar) each as a colorless

oil.

2120, 1649 cm^-1

.

The following four compounds (19e-h ) were prepared by

the same procedures as described above, starting from 8b

instead of 8a .

(3R,4S,5S)-4-Meth yloct-1-en -7-yn e-3,5-d iol (19e): [R]_D

-12.7 (c ) 0.70, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 0.83 (3

H, d, J ) 7.3 Hz), 1.83 (1 H, m), 2.08 (1 H, t, J ) 2.8 Hz), 2.41

(1 H, ddd, J ) 16.8, 6.7, 2.8 Hz), 2.58 (1 H, ddd, J ) 16.8, 3.7,

2.8 Hz), 2.78 (1 H, brs), 3.35 (1 H, brs), 3.75 (1 H, ddd, J )

8.5, 6.7, 3.7 Hz), 4.12 (1 H, m), 5.20 (1 H, dq, J ) 10.2, 1.5

Hz), 5.27 (1 H, dq, J ) 17.4, 2.7 Hz), 5.87 (1 H, ddd, J ) 17.4,

10.1, 7.3 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 12.7 (q), 25.4 (t),

42.5 (d), 70.9 (d), 73.9 (d), 78.0 (d), 80.6 (s), 117.0 (t), 138.3

19a : [R]_D-20.2 (c ) 0.58, CHCl₃); ¹H NMR (400 MHz,

CDCl₃) δ 0.90 (3H, d, J ) 7.0 Hz), 1.95 (1H, dquint, J ) 2.8,

7.0 Hz), 2.08 (1H, t, J ) 2.8 Hz), 2.43 (1H, ddd, J ) 17.1, 7.0,

2.8 Hz), 2.54 (1H, ddd, J ) 17.1, 4.6, 2.8 Hz), 2.72 (1H, d, J )

5.5 Hz), 2.96 (1H, d, J ) 4.6 Hz), 3.79 (1H, dd, J ) 7.0, 4.6

Hz), 4.44 (1H, dtt, J ) 7.0, 5.5, 1.5 Hz), 5.23 (1H, dt, J ) 10.7,

1.5 Hz), 5.32 (1H, dt, J ) 17.1, 1.5 Hz), 5.94 (1H, ddd, J )

17.1, 10.7, 5.5 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 11.6 (q), 25.5

(t), 41.3 (d), 70.9 (q), 72.6 (d), 74.3 (d), 80.6 (s), 115.6 (t), 138.2

(d); IR (neat) 3387, 3308, 3083, 2120, 1644 cm^-1

.

(3S,4S,5S)-4-Meth yloct-1-en -7-yn e-3,5-d iol (19f): [R]_D

-30.3 (c ) 0.70, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 0.90 (3

H, d, J ) 7.3 Hz), 1.96 (1 H, dquint, J ) 7.3, 2.8 Hz), 2.07 (1

H, t, J ) 2.8 Hz), 2.42 (1 H, ddd, J ) 16.8, 7.0, 2.8 Hz), 2.54

(1 H, ddd, J ) 16.8, 4.6, 2.8 Hz), 2.73 (1 H, brs), 2.96 (1 H,

brs), 3.79 (1 H, q, J ) 6.7 Hz), 4.43 (1 H, m), 5.23 (1 H, dt, J

) 10.7, 1.5 Hz), 5.32 (1 H, dt, J ) 17.1, 1.5 Hz), 5.93 (1 H,

ddd, J ) 17.1, 10.7, 5.2 Hz); ¹³C NMR (100 MHz, CDCl₃) δ

11.6 (q), 25.6 (t), 41.4 (d), 71.0 (d), 72.7 (d), 74.5 (d), 80.5 (s),

115.6 (t), 138.3 (d); IR (neat) 3384, 3308, 3086, 3015, 2120,

(d); IR (neat) 3384, 3308, 3015, 2120, 1644 cm^-1

.

19b: [R]_D+9.1 (c ) 0.68, CHCl₃); ¹H NMR (400 MHz, CDCl₃)

δ 0.83 (3 H, d, J ) 7.0 Hz), 1.83 (1 H, m), 2.07 (1 H, t, J ) 2.8

Hz), 2.41 (1 H, ddd, J ) 16.8, 6.7, 2.8 Hz), 2.58 (1 H, ddd, J )

16.8, 4.0, 2.8 Hz), 2.88 (1 H, brs), 3.41 (1 H, brs), 3.74 (1 H,

m), 4.14 (1 H, t, J ) 7.0 Hz), 5.19 (1 H, dq, J ) 10.4, 0.9 Hz),

5.27 (1 H, dq, J ) 17.1, 1.2 Hz), 5.88 (1 H, ddd, J ) 17.1, 10.4,

7.3 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 12.7 (q), 25.4 (t), 42.2

(d), 70.8 (q), 73.9 (d), 78.0 (d), 80.6 (s), 117.0 (t), 139.3 (d); IR

1644 cm^-1

.

(neat) 3387, 3308, 3083, 2120, 1643 cm^-1

.

(3R,4S,5R)-4-Meth yloct-1-en -7-yn e-3,5-d iol (19g): [R]_D

+26.9 (c ) 0.68, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 1.01 (3

H, d, J ) 7.3 Hz), 1.86 (1 H, m), 2.03 (1 H, t, J ) 2.8 Hz), 2.33

(1 H, ddd, J ) 16.8, 6.1, 2.8 Hz), 2.48 (1 H, ddd, J ) 16.8, 7.9,

2.4 Hz), 2.49 (1 H, d, J ) 2.4 Hz), 2.79 (1 H, d, J ) 3.4 Hz),

4.17 (2 H, m), 5.23 (1 H, dt, J ) 10.4, 1.5 Hz), 5.33 (1 H, dt, J

) 17.1, 1.5 Hz), 5.92 (1 H, ddd, J ) 17.1, 10.4, 5.8 Hz); ¹³C

NMR (100 MHz, CDCl₃) δ 10.4 (q), 24.3 (t), 40.5 (d), 70.3 (d),

76.7 (d × 2), 81.2 (s), 115.8 (t), 138.8 (d); IR (neat) 3385, 3308,

Gen er a l P r oced u r e for Syn th eses of Aceton id es. Each

of the above enyne compounds (5 mg) dissolved in acetone (0.4

mL) was treated with dimethoxypropane (0.1 mL) and CSA

(1.5 mg, 0.2 equiv) at room temperature. Evaporation of the

solvent afforded a residue, from which the corresponding

acetonide was purified by silica gel column chromatography

(5% ethyl acetate-n-hexane).

Aceton id e of 19a : ¹H NMR (400 MHz, CDCl₃) δ 0.90 (3

H, d, J ) 7.0 Hz), 1.39 (3 H, s), 1.40 (3 H, s), 1.86-1.92 (1 H,

m), 2.01 (1 H, t, J ) 2.8 Hz), 2.44 (1 H, ddd, J ) 17.4, 6.1, 2.8

Hz), 2.48 (1 H, ddd, J ) 17.4, 5.5, 2.8 Hz), 3.49 (1 H, dt, J )

7.6, 5.8 Hz), 4.43 (1 H, ddt, J ) 6.1, 5.2, 1.5 Hz), 5.17 (1 H, dt,

J ) 10.7, 1.2 Hz), 5.26 (1 H, dt, J ) 17.4, 1.2 Hz), 5.79 (1 H,

ddd, J ) 17.4, 10.7, 6.1 Hz); ¹³C NMR (100 MHz, CDCl₃) δ

12.9 (q), 24.1 (q), 25.2 (q), 29.7 (t), 39.8 (d), 69.7 (s), 70.6 (d),

73.0 (d), 81.0 (d), 100.9 (s), 115.8 (t), 139.6 (t).

Aceton id e of 19b: ¹H NMR (400 MHz, CDCl₃) δ 0.82 (3

H, d, J ) 6.7 Hz), 1.45 (3 H, s), 1.49 (3 H, s), 1.51-1.61 (1 H,

m), 2.01 (1 H, t, J ) 2.7 Hz), 2.42 (1 H, ddd, J ) 17.4, 5.5, 2.7

Hz), 2.52 (1 H, ddd, J ) 17.4, 4.0, 2.7 Hz), 3.68 (1 H, ddd, J )

10.1, 5.8, 4.0 Hz), 3.91 (1 H, ddt, J ) 10.1, 7.3, 1.5 Hz), 5.24 (1

H, dt, J ) 7.3, 1.5 Hz), 5.29 (1 H, dd, J ) 17.4, 1.5 Hz), 5.76

(1 H, ddd, J ) 17.4, 10.1, 7.3 Hz); ¹³C NMR (100 MHz, CDCl₃)

δ 12.2 (q), 19.7 (q), 29.7 (t), 30.0 (q), 39.8 (d), 69.7 (s), 70.6 (d),

73.0 (d), 81.0 (d), 100.9 (s), 115.8 (t), 135.6 (t).

3085, 2120, 1644 cm^-1

.

(3S,4S,5R)-4-Meth yloct-1-en -7-yn e-3,5-d iol (19h ): [R]_D

-24.3 (c ) 0.84, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 0.93 (3

H, d, J ) 7.0 Hz), 1.85 (1 H, ddq, J ) 2.8, 2.4, 7.0 Hz), 2.04 (1

H, t, J ) 2.8 Hz), 2.37 (1 H, ddd, J ) 16.8, 6.7, 2.8 Hz), 2.41

(1 H, d, J ) 3.4 Hz), 2.50 (1 H, ddd, J ) 16.8, 7.0, 2.4 Hz),

2.97 (1 H, d, J ) 2.4 Hz), 4.10 (1 H, ddt, J ) 10.1, 7.0, 2.4 Hz),

4.44 (1 H, m), 5.20 (1 H, dt, J ) 10.7, 1.5 Hz), 5.29 (1 H, dt, J

) 17.4, 1.5 Hz), 5.90 (1 H, ddd, J ) 17.4, 10.7, 6.7 Hz); ¹³C

NMR (100 MHz, CDCl₃) δ 4.7 (q), 24.8 (t), 40.3 (d), 70.4 (d),

74.0 (d), 76.8 (d), 81.0 (s), 114.9 (t), 139.4 (d); IR (neat) 3424,

3308, 3018, 2120, 1640 cm^-1

.

(3R ,4R ,5R )-3,5-Bis[(t er t -b u t yld im e t h ylsilyl)oxy]-4-

m eth yloct-1-en -7-yn e (20a ). To a solution of 19a (58 mg,

0.38 mmol) in CH₂Cl₂(5 mL) was added 2,6-lutidine (0.18 mL,

1.5 mmol) and subsequently TBSOTf (0.34 mL, 1.5 mmol) at

0 °C. The mixture was stirred for 1 h, water was added to the

solution, and the whole was extracted with ethyl acetate. The

organic phase was washed with brine, dried over magnesium

sulfate and filtered. The solvent was removed under reduced

pressure to give a residue, from which 20a (141 mg) was

separated by silica gel column chromatography (2% ethyl

acetate-n-hexane) as a colorless oil in 98% yield: [R]_D+4.6

(c ) 1.30, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 0.01 (3 H, s),

0.05 (3 H, s), 0.07 (3 H, s), 0.11 (3 H, s), 0.88 (3 H, d, J ) 7.0

Hz), 0.89 (9 H, s), 0.90 (9 H, s), 1.78 (1 H, dquint, J ) 4.9, 7.0

Hz), 1.93 (1 H, t, J ) 2.8 Hz), 2.26 (1 H, ddd, J ) 16.8, 7.0, 2.8

Hz), 2.40 (1 H, ddd, J ) 16.8, 4.3, 2.8 Hz), 3.86 (1 H, dt, J )

7.0, 4.3 Hz), 4.11 (1 H, ddt, J ) 7.3, 5.8, 1.8 Hz), 5.09 (1 H, dt,

J ) 7.0, 4.3 Hz), 5.14 (1 H, dt, J ) 17.4, 1.8 Hz), 5.84 (1 H,

ddd, J ) 17.4, 10.1, 7.3 Hz), 5.94 (1 H, ddd, J ) 17.1, 10.7, 5.5

Hz); ¹³C NMR (100 MHz, CDCl₃) δ -4.7 (q), -4.6 (q), -4.2 (q),

-3.8 (q), 9.7 (q), 18.12 (s), 18.23 (s), 23.7 (t), 25.87 (q), 25.94

(3R,4R,5S)-4-Meth yloct-1-en -7-yn e-3,5-d iol (19c) a n d

(3S,4R,5S)-4-Meth yloct-1-en -7-yn e-3,5-d iol (19d ). These

compounds were obtained by the same procedures as described

above for 19a ,b, starting from 14b instead of 14a .

19c: [R]_D+20.1 (c ) 0.74, CHCl₃); ¹H NMR (400 MHz,

CDCl₃) δ 0.93 (3 H, d, J ) 7.0 Hz), 1.85 (1 H, qt, J ) 7.0, 2.4

Hz), 2.04 (1 H, t, J ) 2.8 Hz), 2.35 (1 H, ddd, J ) 16.8, 6.7, 2.4

Hz), 2.37 (1 H, t, J ) 2.8 Hz), 2.50 (1 H, ddd, J ) 16.8, 7.0, 2.4

Hz), 2.94 (1 H, t, J ) 2.4 Hz), 4.11 (1 H, tt, J ) 7.0, 2.4 Hz),

4.44 (1 H, m), 5.20 (1 H, dt, J ) 10.7, 1.5 Hz), 5.28 (1 H, dq,

J ) 17.4, 1.5 Hz), 5.90 (1 H, ddd, J ) 17.4, 10.7, 4.8 Hz); ¹³C

NMR (100 MHz, CDCl₃) δ 4.8 (q), 24.9 (t), 40.4 (d), 70.5 (q),

74.0 (d), 76.8 (d), 81.0 (s), 114.9 (t), 139.4 (d); IR (neat) 3385,

3300, 2978, 2912, 2120, 1649 cm^-1

.

19d : [R]_D-18.1 (c ) 0.73, CHCl₃); ¹H NMR (400 MHz,

CDCl₃) δ 1.01 (3 H, d, J ) 7.3 Hz), 1.86 (1 H, m), 2.03 (1 H, t,

4260 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22

Konno et al.

(q), 46.1 (d), 69.6 (d), 72.0 (d), 75.5 (d), 82.6 (s), 115.2 (t), 140.9

(d); IR (neat) 3310, 3018, 2957, 2932, 2120, 1642, 1520, 1472,

1215, 837, 752, 669 cm^-1; MS m/z 382 (M)⁺, 367 (M - Me)⁺,

325 (M - ^tBu)⁺; HRMS m/z (M)⁺calcd for C₂₁H₄₂O₂Si₂

382.2724, found 382.2725.

NMR (100 MHz, CDCl₃) δ -4.64 (q), -4.56 (q), -4.2 (q), -3.8

(q), 9.7 (q), 18.14 (s), 18.25 (s), 23.8 (t), 25.89 (q), 25.96 (q),

46.1 (d), 69.6 (d), 72.0 (d), 75.5 (d), 82.6 (s), 115.2 (t), 140.9

(d); IR (neat) 3310, 3019, 2957, 2932, 2120, 1644, 1520, 1472,

1256, 1215, 837, 752, 669 cm^-1; MS m/z 382 (M)⁺, 367 (M -

t

Me)⁺, 325 (M - Bu)⁺; HRMS m/z (M)⁺calcd for C₂₁H₄₂O₂Si₂

The following seven compounds (20b-h ) were prepared by

the same procedure as described for 20a .

382.2724, found 382.2727.

(3R ,4S ,5R )-3,5-Bis[(t er t -b u t yld im e t h ylsilyl)oxy]-4-

m eth yloct-1-en -7-yn e (20g): [R]_D+10.3 (c ) 1.15, CHCl₃);

¹H NMR (400 MHz, CDCl₃) δ 0.03 (3 H, s), 0.06 (3 H, s), 0.07

(3 H, s), 0.08 (3 H, s), 0.76 (3 H, d, J ) 7.0 Hz), 0.889 (9 H, s),

0.891 (9 H, s), 1.90 (1 H, m), 1.97 (1 H, t, J ) 2.8 Hz), 2.38 (2

H, m), 4.01 (2 H, m), 5.10 (1 H, dq, J ) 10.1, 0.9 Hz), 5.13 (1

H, dq, J ) 17.1, 0.9 Hz), 5.74 (1 H, ddd, J ) 17.1, 10.1, 7.6

Hz); ¹³C NMR (100 MHz, CDCl₃) δ -4.6 (q), -4.5 (q), -4.0 (q),

-3.4 (q), 9.5 (q), 18.15 (s), 18.22 (s), 25.5 (t), 25.9 (q), 26.0 (q),

43.8 (d), 70.1 (d), 70.6 (d), 76.0 (d), 81.6 (s), 115.9 (t), 140.0

(d); IR (neat) 3310, 3021, 2958, 2930, 2121, 1524, 1256, 1215

cm^-1; MS m/z 382 (M)⁺, 367 (M - Me)⁺, 325 (M - ^tBu)⁺; HRMS

m/z (M)⁺calcd for C₂₁H₄₂O₂Si₂382.2724, found 382.2724.

(3S ,4R ,5R )-3,5-Bis[(t er t -b u t yld im e t h ylsilyl)oxy]-4-

m eth yloct-1-en -7-yn e (20b): [R]_D-10.4 (c ) 0.96, CHCl₃);

¹H NMR (400 MHz, CDCl₃) δ 0.02 (3 H, s), 0.057 (3 H, s), 0.063

(3 H, s), 0.11 (3 H, s), 0.78 (3 H, d, J ) 7.0 Hz), 0.86 (6 H, s),

0.90 (12 H, s), 1.89 (1 H, dquint, J ) 5.5, 7.0 Hz), 1.93 (1 H, t,

J ) 2.8 Hz), 2.26 (1 H, ddd, J ) 16.8, 7.0, 2.8 Hz), 2.39 (1 H,

ddd, J ) 16.8, 4.0, 2.8 Hz), 3.97 (1 H, ddd, J ) 6.7, 5.2, 4.0

Hz), 4.12 (1 H, ddt, J ) 6.7, 6.4, 1.2 Hz), 5.09 (1 H, dq, J )

10.4, 1.2 Hz), 5.16 (1 H, dq, J ) 17.1, 1.5 Hz), 5.75 (1 H, ddd,

J ) 17.1, 10.4, 6.1 Hz); ¹³C NMR (100 MHz, CDCl₃) δ -4.9

(q), -4.7 (q), -4.2 (q), -3.0 (q), 9.8 (q), 18.11 (s), 18.13 (s), 23.6

(t), 25.87 (q), 25.89 (q), 45.0 (d), 69.5 (d), 71.4 (d), 74.9 (d), 82.5

(s), 115.2 (t), 139.6 (d); IR (neat) 3310, 3018, 2957, 2932, 2120,

1520, 1256, 1215 cm^-1; MS m/z 382 (M)⁺, 367 (M - Me)⁺, 325

(3S ,4S ,5R )-3,5-Bis[(t er t -b u t yld im e t h ylsilyl)oxy]-4-

m eth yloct-1-en -7-yn e (20h ): [R]_D-0.3 (c ) 1.85, CHCl₃);

¹H NMR (400 MHz, CDCl₃) δ 0.01 (3 H, s), 0.049 (3 H, s), 0.051

(3 H, s), 0.08 (3 H, s), 0.89 (18 H, s), 0.92 (3 H, d, J ) 7.0 Hz),

1.85 (1 H, m), 1.96 (1 H, t, J ) 2.8 Hz), 2.39 (2 H, dd, J ) 6.7,

2.8 Hz), 3.88 (1 H, ddd, J ) 6.6, 6.1, 4.0 Hz), 4.08 (1 H, m),

5.10 (1 H, dq, J ) 10.4, 0.9 Hz), 5.14 (1 H, dq, J ) 17.4, 0.9

Hz), 5.81 (1 H, ddd, J ) 17.4, 10.4, 7.0 Hz); ¹³C NMR (100

MHz, CDCl₃) δ -4.8 (q), -4.6 (q), -4.0 (q), -3.8 (q), 9.4 (q),

18.15 (s), 18.24 (s), 25.5 (t), 25.91 (q), 25.94 (q), 43.9 (d), 70.0

(d), 70.9 (d), 75.3 (d), 81.8 (s), 115.3 (t), 140.8 (d); IR (neat)

3310, 3020, 2957, 2932, 2121, 1520, 1256, 1215 cm^-1; MS m/z

t

(M - Bu)⁺; HRMS m/z (M)⁺calcd for C₂₁H₄₂O₂Si₂382.2724,

found 382.2719.

(3R ,4R ,5S )-3,5-Bis[(t er t -b u t yld im e t h ylsilyl)oxy]-4-

m eth yloct-1-en -7-yn e (20c): [R]_D+3.5 (c ) 2.06, CHCl₃);

¹H NMR (400 MHz, CDCl₃) δ 0.01 (3 H, s), 0.049 (3 H, s), 0.051

(3 H, s), 0.08 (3 H, s), 0.89 (18 H, s), 0.92 (3 H, d, J ) 7.0 Hz),

1.89 (1 H, dquint, J ) 4.0, 6.7 Hz), 1.95 (1 H, t, J ) 2.8 Hz),

2.38 (2 H, dd, J ) 5.8, 2.8 Hz), 3.88 (1 H, ddd, J ) 6.4, 6.1, 4.0

Hz), 5.10 (1 H, dq, J ) 10.4, 0.9 Hz), 5.14 (1 H, dq, J ) 17.4,

0.9 Hz), 5.81 (1 H, ddd, J ) 17.4, 10.4, 7.0 Hz); ¹³C NMR (100

MHz, CDCl₃) δ -4.8 (q), -4.6 (q), -4.0 (q), -3.9 (q), 9.7 (q),

9.4 (q), 18.14 (s), 18.23 (s), 25.5 (t), 25.90 (q), 25.93 (q), 46.1

(d), 70.1 (d), 71.0 (d), 75,2 (d), 81.8 (s), 115.3 (t), 140.8 (d); IR

(neat) 3081, 2957, 2124, 1646 cm^-1; MS m/z 382 (M)⁺, 367 (M

t

367 (M - Me)⁺, 325 (M - Bu)⁺; HRMS m/z (M - Me)⁺calcd

for C₂₀H₃₉O₂Si₂367.2488, found 367.2487.

(5Z,7E )-(1S ,2R ,3R )-2-Me t h y l-9,10-seco-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (2a ). To a mixture of Pd₂(dba)₃‚

CHCl₃(13 mg, 0.012 mmol), PPh₃(30 mg, 0.12 mmol) and Et₃N

(1.5 mL) was added a solution of 5a (44 mg, 0.12 mmol) and

20a (61 mg, 0.16 mmol) in toluene (3 mL). The resulting

mixture was stirred at room temperature for 10 min and

subsequently heated to reflux for 6 h. Water was added to the

cooled reaction mixture, and the whole was extracted with

ether. The organic layer was washed with brine, dried over

magnesium sulfate and filtered. Evaporation of the filtrate

afforded a residue, from which the silylated vitamin was

separated by silica gel column chromatography (10% ethyl

acetate-n-hexane) as a colorless oil. The product thus obtained

was dissolved in methanol (3 mL) and treated with CSA (28

mg, 0.12 mmol). The resulting mixture was stirred for 16 h at

room temperature. The reaction mixture was poured into satd

NaHCO₃(aq) and the whole was extracted with ether. The

extract was washed with brine, dried over magnesium sulfate

and filtered. The solvent was evaporated under reduced

pressure to give a residue, from which 2a (20 mg) was

separated by silica gel column chromatography (50% ethyl

acetate-n-hexane) as a white solid in 41% yield. Further

purification for biological evaluation was conducted by using

reversed-phase recycle HPLC (YMC-Pack ODS column, 20 ×

150 mm, 9.0 mL/min, acetonitrile:water ) 70:30). Analytical

HPLC was conducted by using reversed-phase HPLC (Li-

chrosorb RP-18 column, 4 × 250 mm, 1.0 mL/min, acetonitrile:

water ) 60:40) to give 2a (t_R) 18.28 min) as a single

homogeneous peak: [R]_D-35.2 (c ) 0.46, CHCl₃); UV (EtOH)

λ_max263 nm, λ_min227 nm; ¹H NMR (400 MHz, CDCl₃-D₂O) δ

0.55 (3 H, s), 0.94 (3 H, d, J ) 6.3 Hz), 1.15 (3 H, d, J ) 7.0

Hz), 1.22 (6 H, s), 1.78 (1 H, ddq, J ) 9.2, 2.6, 7.0 Hz), 2.42 (1

H, dd, J ) 13.6, 4.8 Hz), 2.52 (1 H, br. d, J ) 13.6 Hz), 2.82 (1

H, dd, J ) 11.9, 3.7 Hz), 4.01 (1 H, d, J ) 9.2 Hz), 4.03 (1 H,

dt, J ) 4.8, 2.6 Hz), 5.02 (1 H, t, J ) 1.8 Hz), 5.37 (1 H, t, J )

1.8 Hz), 6.03 (1 H, d, J ) 11.2 Hz), 6.35 (1 H, d, J ) 11.2 Hz);

¹H NMR (500 MHz, CDCl₃) δ 0.55 (3 H, s), 0.94 (3 H, d, J )

6.5 Hz), 1.15 (3 H, d, J ) 6.9 Hz), 1.22 (6 H, s), 1.78 (1 H, ddq,

J ) 9.4, 2.6, 6.9 Hz), 2.42 (1 H, dd, J ) 13.9, 4.8 Hz), 2.53 (1

H, br. d, J ) 13.7 Hz), 2.82 (1 H, dd, J ) 11.9, 3.9 Hz), 4.02 (2

t

- Me)⁺, 325 (M - Bu)⁺; HRMS m/z (M)⁺calcd for C₂₁H₄₂O₂-

Si₂382.2724, found 382.2723.

(3S ,4R ,5S )-3,5-Bis[(t er t -b u t yld im e t h ylsilyl)oxy]-4-

m eth yloct-1-en -7-yn e (20d ): [R]_D-5.8 (c ) 0.79, CHCl₃);

¹H NMR (400 MHz, CDCl₃) δ 0.03 (3 H, s), 0.06 (3 H, s), 0.07

(3 H, s), 0.08 (3 H, s), 0.76 (3 H, d, J ) 7.0 Hz), 0.889 (9 H, s),

0.892 (9 H, s), 1.91 (1 H, dquint, J ) 3.7, 7.0 Hz), 1.97 (1 H, t,

J ) 2.8 Hz), 2.38 (2 H, m), 4.02 (2 H, m), 5.09 (1 H, dt, J )

10.4, 0.9 Hz), 5.13 (1 H, dt, J ) 17.1, 0.9 Hz), 5.73 (1 H, ddd,

J ) 17.1, 10.1, 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ -4.6

(q), -4.5 (q), -4.0 (q), -3.4 (q), 9.1 (q), 18.1 (s), 18.2 (s), 23.8

(t), 25.86 (q), 25.95 (q), 43.7 (d), 70.1 (d), 70.6 (d), 76.0 (d), 81.5

(s), 115.9 (t), 140.0 (d); IR (neat) 3316, 3079, 2957, 2932, 2887,

2859, 2122, 1645, 1472, 1462, 1254, 1075, 837, 775 cm^-1; MS

m/z 382 (M)⁺, 367 (M - Me)⁺, 325 (M - ^tBu)⁺; HRMS m/z (M)⁺

calcd for C₂₁H₄₂O₂Si₂382.2724, found 382.2724.

(3R ,4S ,5S )-3,5-Bis[(t er t -b u t yld im e t h ylsilyl)oxy]-4-

m eth yloct-1-en -7-yn e (20e): [R]_D+11.3 (c ) 1.05, CHCl₃);

¹H NMR (400 MHz, CDCl₃) δ 0.01 (3 H, s), 0.06 (3 H, s), 0.06

(3 H, s), 0.11 (3 H, s), 0.78 (3 H, d, J ) 7.0 Hz), 0.86 (9 H, s),

0.90 (9 H, s), 1.88 (1 H, dquint, J ) 5.5, 6.7 Hz), 1.93 (1 H, t,

J ) 2.8 Hz), 2.26 (1 H, ddd, J ) 16.8, 7.0, 2.4 Hz), 4.12 (1 H,

ddt, J ) 6.7, 5.2, 1.8 Hz), 5.09 (1 H, dq, J ) 10.4, 1.2 Hz), 5.15

(1 H, dq, J ) 17.1, 1.2 Hz), 5.75 (1 H, ddd, J ) 17.1, 10.4, 6.7

Hz); ¹³C NMR (100 MHz, CDCl₃) δ -4.9 (q), -4.7 (q), -4.2 (q),

-4.1 (q), 9.9 (q), 18.13 (s), 18.15 (s), 23.5 (t), 25.88 (q), 25.91

(q), 45.0 (d), 69.5 (d), 71.4 (d), 75.0 (d), 82.5 (s), 115.2 (t), 139.6

(d); IR (neat) 3310, 3020, 2957, 2932, 2121, 1520, 1256, 1215

cm^-1; MS m/z 382 (M)⁺, 367 (M - Me)⁺, 325 (M - ^tBu)⁺; HRMS

m/z (M)⁺calcd for C₂₁H₄₂O₂Si₂382.2724, found 382.2719.

(3S ,4S ,5S )-3,5-Bis[(t er t -b u t yld im e t h ylsilyl)oxy]-4-

m eth yloct-1-en -7-yn e (20f): [R]_D-4.9 (c ) 1.30, CHCl₃);

¹H NMR (400 MHz, CDCl₃) δ 0.01 (3 H, s), 0.05 (3 H, s), 0.07

(3 H, s), 0.10 (3 H, s), 0.88 (3 H, d, J ) 7.0 Hz), 0.89 (9 H, s),

0.90 (9 H, s), 1.78 (1 H, m), 1.93 (1 H, t, J ) 2.8 Hz), 2.26 (1

H, ddd, J ) 16.8, 7.0, 2.8 Hz), 2.40 (1 H, ddd, J ) 16.8, 4.3,

2.8 Hz), 3.85 (1 H, dt, J ) 7.0, 4.3 Hz), 4.11 (1 H, ddt, J ) 7.3,

5.8, 1.5 Hz), 5.10 (1 H, dq, J ) 10.1, 0.6 Hz), 5.14 (1 H, dq, J

) 17.4, 0.9 Hz), 5.84 (1 H, ddd, J ) 17.4, 10.1, 7.3 Hz); ¹³C

A-Ring Diastereomers of Methyldihydroxyvitamin D₃

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4261

H, m), 5.02 (1 H, t, J ) 1.8 Hz), 5.37 (1 H, t, J ) 1.8 Hz), 6.03

(1 H, d, J ) 11.3 Hz), 6.35 (1 H, d, J ) 11.7 Hz); ¹H NMR (500

MHz, CD₃OD) δ 0.57 (3 H, s), 0.96 (3 H, d, J ) 6.5 Hz), 1.07

(3 H, d, J ) 6.9 Hz), 1.17 (6 H, s), 1.76 (1 H, ddq, J ) 9.5, 2.6,

6.9 Hz), 2.37 (1 H, dd, J ) 14.0, 5.6 Hz), 2.42 (1 H, br. d, J )

14.0 Hz), 2.85 (1 H, dd, J ) 12.5, 4.5 Hz), 3.95 (1 H, d, J ) 9.0

Hz), 3.98 (1 H, dt, J ) 5.6, 2.6 Hz), 4.97 (1 H, t, J ) 2.2 Hz),

5.38 (1 H, t, J ) 1.9 Hz), 6.08 (1 H, d, J ) 11.9 Hz), 6.27 (1 H,

d, J ) 11.0 Hz); ¹H NMR (400 MHz, DMSO-d₆) δ 0.50 (3 H, s),

0.90 (3 H, d, J ) 6.5 Hz), 0.94 (3 H, d, J ) 6.8 Hz), 1.04 (6 H,

s), 1.58 (1 H, ddq, J ) 9.8, 2.7, 6.8 Hz), 2.24 (2 H, m), 2.77 (1

H, dd, J ) 13.3, 5.2 Hz), 3.76 (1 H, d, J ) 9.0 Hz), 3.81 (1 H,

dt, J ) 7.0, 3.3 Hz), 4.76 (1 H, dd, J ) 2.7, 1.2 Hz), 5.26 (1 H,

dd, J ) 2.7, 1.8 Hz), 5.97 (1 H, d, J ) 11.6 Hz), 6.14 (1 H, d,

J ) 11.2 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 11.9 (q), 13.8 (q),

18.7 (q), 20.7 (t), 22.2 (t), 23.6 (t), 27.6 (t), 29.0 (t), 29.2 (q),

29.3 (q), 36.0 (d), 36.3 (t), 40.4 (t), 43.6 (t), 44.4 (t), 44.6 (d),

45.9 (s), 56.3 (d), 56.5 (d), 70.9 (d), 71.1 (s), 74.7 (d), 111.0 (t),

117.0 (d), 124.9 (d), 133.4 (s), 143.1 (s), 147.5 (s); MS m/z 430

(M)⁺412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺, 376 (M - 3H₂O)⁺;

HRMS m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447, found 430.3450.

(5Z ,7E )-(1S ,2S ,3S )-2-M e t h y l-9,10-s ec o-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (2e): t_R) 27.11 min; [R]_D+87.0

1

(c ) 0.30, CHCl₃); UV (EtOH) λ_max265 nm, λ_min227 nm; H

NMR (400 MHz, CDCl₃-D₂O) δ 0.54 (3 H, s), 0.93 (3 H, d, J

) 6.4 Hz), 1.22 (6 H, s), 1.22 (3 H, d, J ) 7.0 Hz), 1.92 (1 H,

tq, J ) 2.7, 7.0 Hz), 2.50 (1 H, br. d, J ) 14.0 Hz), 2.58 (1 H,

dd, J ) 14.0, 4.0 Hz), 2.85 (1 H, dd, J ) 12.2, 3.4 Hz), 3.90 (1

H, dt, J ) 4.0, 2.7 Hz), 4.17 (1 H, d, J ) 2.7 Hz), 4.98 (1 H, d,

J ) 1.8 Hz), 5.23 (1 H, d, J ) 1.8 Hz), 6.03 (1 H, d, J ) 11.0

Hz), 6.48 (1 H, d, J ) 11.0 Hz); ¹³C NMR (100 MHz, CDCl₃) δ

12.0 (q), 13.5 (q), 18.7 (q), 20.8 (t), 22.2 (t), 23.4 (t), 27.6 (t),

29.0 (t), 29.1 (q), 29.3 (q), 36.1 (d), 36.4 (t), 40.2 (d), 40.5 (t),

44.4 (t), 44.9 (t), 45.9 (s), 56.3 (d), 56.5 (d), 71.1 (s), 72.5 (d),

78.7 (d), 114.1 (t), 126.3 (d), 130.9 (d), 143.2 (s), 147.2 (s); MS

m/z 430 (M)⁺, 412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺, 376 (M -

3H₂O)⁺; HRMS m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447, found

430.3446.

(5Z ,7E )-(1R ,2S ,3S )-2-M e t h y l-9,10-s ec o-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (2f): t_R) 18.61 min; [R]_D+27.3

1

(c ) 0.10, CHCl₃); UV (EtOH) λ_max264 nm, λ_min228 nm; H

NMR (400 MHz, CDCl₃-D₂O) δ 0.54 (3 H, s), 0.93 (3 H, d, J

) 6.4 Hz), 1.12 (3 H, d, J ) 7.0 Hz), 1.22 (6 H, s), 1.86 (1 H,

ddq, J ) 8.4, 2.9, 7.0 Hz), 2.42 (1 H, dd, J ) 13.6, 5.9 Hz),

2.51 (1 H, dd, J ) 13.6, 2.9 Hz), 2.82 (1 H, dd, J ) 12.2, 2.4

Hz), 4.04 (1 H, d, J ) 8.4 Hz), 4.06 (1 H, dt, J ) 5.9, 2.9 Hz),

5.01 (1 H, t, J ) 1.8 Hz), 5.35 (1 H, t, J ) 1.8 Hz), 6.00 (1 H,

d, J ) 11.2 Hz), 6.36 (1 H, d, J ) 11.2 Hz); MS m/z 430 (M)⁺,

412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺, 376 (M - 3H₂O)⁺; HRMS

m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447, found 430.3447.

The following seven compounds (2b-h ) were prepared by

the same procedure as described for 2a .

(5Z,7E )-(1R ,2R ,3R )-2-Me t h y l-9,10-seco-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (2b): t_R) 28.05 min; [R]_D-44.8

1

(c ) 0.30, CHCl₃); UV (EtOH) λ_max264 nm, λ_min226 nm; H

NMR (400 MHz, CDCl₃-D₂O) δ 0.56 (3 H, s), 0.94 (3 H, d, J

) 6.2 Hz), 1.22 (6 H, s), 1.23 (3 H, d, J ) 7.0 Hz), 1.90 (1 H,

tq, J ) 2.4, 7.0 Hz), 2.50 (1 H, br. d, J ) 14.0 Hz), 2.59 (1 H,

dd, J ) 14.0, 3.4 Hz), 2.85 (1 H, dd, J ) 12.1, 3.4 Hz), 3.90 (1

H, dd, J ) 3.4, 2.4 Hz), 4.17 (1 H, d, J ) 2.4 Hz), 5.01 (1 H, d,

J ) 1.8 Hz), 5.25 (1 H, d, J ) 2.1 Hz), 6.09 (1 H, d, J ) 11.4

Hz), 6.48 (1 H, d, J ) 11.4 Hz); ¹³C NMR (100 MHz, CDCl₃) δ

11.9 (q), 13.9 (q), 18.8 (q), 20.7 (t), 22.3 (t), 23.6 (t), 27.6 (t),

29.1 (t), 29.2 (q), 29.3 (q), 36.1 (d), 36.4 (t), 40.1 (t), 40.4 (t),

44.4 (t), 45.1 (t), 45.9 (s), 56.3 (d), 56.5 (d), 71.1 (s), 72.6 (d),

78.9 (d), 114.3 (t), 117.0 (d), 126.3 (d), 131.1 (s), 143.2 (s), 147.4

(s); MS m/z 430 (M)⁺, 412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺, 376

(M - 3H₂O)⁺; HRMS m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447,

found 430.3446.

(5Z ,7E )-(1S ,2S ,3R )-2-M e t h y l-9,10-s ec o-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (2g): t_R) 19.60 min; mp 136 °C

(recrystallization from ethyl acetate); [R]_D+41.1 (c ) 0.20,

1

CHCl₃); UV (EtOH) λ_max265 nm, λ_min226 nm; H NMR (400

MHz, CDCl₃-D₂O) δ 0.53 (3 H, s), 0.93 (3 H, d, J ) 6.4 Hz),

1.08 (3 H, d, J ) 7.0 Hz), 1.22 (6 H, s), 1.92 (1 H, ddq, J ) 7.7,

3.3, 7.2 Hz), 2.23 (1 H, dd, J ) 13.2, 7.7 Hz), 2.67 (1 H, dd, J

) 13.2, 4.0 Hz), 2.82 (1 H, dd, J ) 12.5, 4.0 Hz), 3.84 (1 H, dt,

J ) 4.0, 7.4 Hz), 4.30 (1 H, d, J ) 3.3 Hz), 5.01 (1 H, d, J ) 1.8

Hz), 5.28 (1 H, dd, J ) 1.8, 1.1 Hz), 6.01 (1 H, d, J ) 11.4 Hz),

1

6.39 (1 H, d, J ) 11.4 Hz); H NMR (500 MHz, CDCl₃) δ 0.53

(3 H, s), 0.93 (3 H, d, J ) 6.5 Hz), 1.08 (3 H, d, J ) 6.9 Hz),

1.22 (6 H, s), 1.92 (1 H, ddq, J ) 7.8, 3.5, 6.9 Hz), 2.24 (1 H,

dd, J ) 13.3, 7.7 Hz), 2.67 (1 H, dd, J ) 13.5, 4.0 Hz), 2.82 (1

H, dd, J ) 12.8, 4.4 Hz), 3.84 (1 H, m), 4.31 (1 H, dd, J ) 3.5,

4.2 Hz), 5.01 (1 H, d, J ) 1.6 Hz), 5.28 (1 H, br. s), 6.01 (1 H,

d, J ) 11.3 Hz), 6.39 (1 H, d, J ) 11.2 Hz); ¹H NMR (500 MHz,

CD₃OD) δ 0.56 (3 H, s), 0.96 (3 H, d, J ) 6.5 Hz), 1.03 (3 H, d,

J ) 6.9 Hz), 1.16 (6 H, s), 1.79 (1 H, ddq, J ) 8.1, 3.5, 6.9 Hz),

2.17 (1 H, dd, J ) 13.2, 8.2 Hz), 2.59 (1 H, dd, J ) 13.3, 4.1

Hz), 2.86 (1 H, dd, J ) 12.4, 3.9 Hz), 3.72 (1 H, dt, J ) 4.2, 8.1

Hz), 4.22 (1 H, d, J ) 3.5 Hz), 4.89 (1 H, d, J ) 2.5 Hz), 5.22

(1 H, d, J ) 1.8 Hz), 6.08 (1 H, d, J ) 11.2 Hz), 6.32 (1 H, d,

(5Z ,7E )-(1S ,2R ,3S )-2-M e t h y l-9,10-s ec o-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (2c): t_R) 18.98 min; [R]_D+58.0

1

(c ) 0.83, CHCl₃); UV (EtOH) λ_max261 nm, λ_min226 nm; H

NMR (400 MHz, CDCl₃-D₂O) δ 0.54 (3 H, s), 0.94 (3 H, d, J

) 6.4 Hz), 1.05 (3 H, d, J ) 7.0 Hz), 1.22 (6 H, s), 2.00 (1 H,

tq, J ) 5.9, 7.0 Hz), 2.34 (1 H, dd, J ) 13.7, 6.0 Hz), 2.64 (1 H,

dd, J ) 13.7, 3.3 Hz), 2.83 (1 H, dd, J ) 12.2, 4.0 Hz), 3.65 (1

H, dt, J ) 3.3, 6.0 Hz), 3.90 (1 H, d, J ) 5.9 Hz), 5.05 (1 H, d,

J ) 1.5 Hz), 5.31 (1 H, d, J ) 1.5 Hz), 6.02 (1 H, d, J ) 11.4

Hz), 6.41 (1 H, d, J ) 11.4 Hz); ¹³C NMR (100 MHz, CDCl₃) δ

11.9 (q), 14.9 (q), 18.7 (q), 20.7 (t), 22.2 (t), 23.5 (t), 27.6 (t),

29.0 (t), 29.2 (q), 29.3 (q), 36.0 (d), 36.4 (t), 40.5 (t), 42.7 (t),

44.2 (d), 44.4 (t), 45.9 (s), 56.3 (d), 56.5 (d), 71.1 (s), 73.3 (d),

77.9 (d), 113.5 (t), 117.0 (d), 125.0 (d), 131.9 (s), 143.2 (s), 146.1

(s); MS m/z 430 (M)⁺, 412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺, 376

(M - 3H₂O)⁺; HRMS m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447,

found 430.3447.

1

J ) 11.0 Hz); H NMR (500 MHz, DMSO-d₆) δ 0.49 (3 H, s),

0.84 (3 H, d, J ) 6.9 Hz), 0.90 (3 H, d, J ) 6.4 Hz), 1.04 (6 H,

s), 1.73 (1 H, ddq, J ) 6.8, 3.7, 6.4 Hz), 2.05 (1 H, dd, J )

13.3, 7.1 Hz), 2.45 (1 H, dd, J ) 13.3, 3.4 Hz), 2.78 (1 H, dd, J

) 13.3, 4.6 Hz), 3.60 (1 H, dt, J ) 4.2, 7.1 Hz), 4.13 (1 H, d, J

) 3.4 Hz), 4.76 (1 H, d, J ) 2.6 Hz), 5.16 (1 H, d, J ) 2.0 Hz),

5.98 (1 H, d, J ) 11.3 Hz), 6.18 (1 H, d, J ) 11.2 Hz); ¹³C NMR

(100 MHz, CDCl₃) δ 11.9 (q), 12.4 (q), 18.7 (q), 20.7 (t), 20.7

(t), 22.2 (t), 23.5 (t), 27.6 (t), 29.0 (q), 29.2 (q), 29.3 (q), 36.0

(d), 36.4 (t), 40.5 (t), 43.5 (t), 44.2 (t), 44.4 (d), 45.9 (s), 56.3

(d), 56.5 (d), 71.1 (s), 71.7 (d), 76.1 (d), 75.5 (d), 113.2 (t), 117.0

(d), 124.9 (d), 133.0 (s), 143.3 (s), 146.7 (s); MS m/z 430 (M)⁺,

412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺, 376 (M - 3H₂O)⁺; HRMS

m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447, found 430.3449. Anal.

(C₂₈H₄₆O₃‚H₂O) C, H.

(5Z,7E )-(1R ,2R ,3S )-2-Me t h y l-9,10-seco-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (2d ): t_R) 18.39 min; [R]_D-11.2

1

(c ) 0.43, CHCl₃); UV (EtOH) λ_max265 nm, λ_min226 nm; H

NMR (400 MHz, CDCl₃-D₂O) δ 0.55 (3 H, s), 0.94 (3 H, d, J

) 6.4 Hz), 1.11 (3 H, d, J ) 7.0 Hz), 1.22 (6 H, s), 1.87 (1 H,

ddq, J ) 8.4, 3.3, 7.0), 2.24 (1 H, dd, J ) 13.2, 8.4 Hz), 2.66 (1

H, dd, J ) 13.2, 4.3 Hz), 2.82 (1 H, dd, J ) 11.8, 3.6 Hz), 3.81

(1 H, dt, J ) 4.3, 8.4 Hz), 4.26 (1 H, d, J ) 3.3 Hz), 5.02 (1 H,

d, J ) 1.7 Hz), 5.28 (1 H, d, J ) 1.7 Hz), 6.02 (1 H, d, J ) 11.4

Hz), 6.40 (1 H, d, J ) 11.4 Hz); ¹³C NMR (100 MHz, CDCl₃) δ

11.9 (q), 12.8 (q), 18.8 (q), 20.8 (t), 22.3 (t), 23.6 (t), 27.6 (t),

29.0 (t), 29.2 (q), 29.3 (q), 36.0 (d), 36.4 (t), 40.5 (t), 44.1 (t),

44.4 (t), 44.5 (d), 45.9 (s), 56.3 (d), 56.5 (d), 71.1 (s), 71.6 (d),

76.1 (d), 113.7 (t), 117.0 (d), 124.9 (d), 133.0 (s), 143.3 (s), 146.8

(s); MS m/z 430 (M)⁺, 412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺, 376

(M - 3H₂O)⁺; HRMS m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447,

found 430.3445.

(5Z,7E )-(1R ,2S ,3R )-2-Me t h y l-9,10-seco-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (2h ): t_R) 20.06 min; [R]_D-47.7

1

(c ) 0.84, CHCl₃); UV (EtOH) λ_max262 nm, λ_min226 nm; H

NMR (400 MHz, CDCl₃-D₂O) δ 0.55 (3 H, s), 0.94 (3 H, d, J

) 6.4 Hz), 1.02 (3 H, d, J ) 7.0 Hz), 1.22 (6 H, s), 2.12 (1 H,

tq, J ) 5.1, 7.0 Hz), 2.36 (1 H, dd, J ) 13.9, 5.1 Hz), 2.65 (1 H,

dd, J ) 13.9, 3.0 Hz), 2.83 (1 H, dd, J ) 12.5, 4.3 Hz), 3.72 (1

4262 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22

Konno et al.

H, dt, J ) 3.0, 5.1 Hz), 3.96 (1 H, d, J ) 4.4 Hz), 5.07 (1 H, d,

J ) 1.7 Hz), 5.30 (1 H, d, J ) 1.7 Hz), 6.05 (1 H, d, J ) 11.3

Hz), 6.42 (1 H, d, J ) 11.3 Hz); ¹³C NMR (100 MHz, CDCl₃) δ

11.9 (q), 14.9 (q), 18.7 (q), 20.7 (t), 22.2 (t), 23.6 (t), 27.6 (t),

29.0 (t), 29.2 (q), 36.1 (d), 36.3 (t), 40.4 (t), 42.0 (t), 43.2 (d),

44.4 (t), 45.9 (s), 56.3 (d), 56.5 (d), 71.1 (s), 73.3 (d), 78.5 (d),

114.4 (t), 116.9 (d), 125.2 (d), 131.8 (s), 143.2 (s), 143.2 (s), 145.7

(s); MS m/z 430 (M)⁺, 412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺, 376

(M - 3H₂O)⁺; HRMS m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447,

found 430.3447.

H, t, J ) 1.8 Hz), 6.01 (1 H, d, J ) 11.6 Hz), 6.36 (1 H, d, J )

11.6 Hz); MS m/z 430 (M)⁺, 412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺,

376 (M - 3H₂O)⁺; HRMS m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447,

found 430.3445.

(5Z,7E)-(1S,2S,3R,20S)-2-Met h yl-9,10-seco-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (3g): t_R) 17.62 min; UV (EtOH)

1

λ_max266 nm, λ_min226 nm; H NMR (400 MHz, CDCl₃) δ 0.53

(3 H, s), 0.85 (3 H, d, J ) 6.7 Hz), 1.08 (3 H, d, J ) 6.8 Hz),

1.21 (6 H, s), 2.23 (1 H, dd, J ) 13.4, 7.9 Hz), 2.67 (1 H, dd, J

) 13.4, 4.0 Hz), 2.83 (1 H, m), 3.83 (1 H, ddd, J ) 7.9, 4.4, 4.0

Hz), 4.29 (1 H, d, J ) 3.3 Hz), 5.01 (1 H, d, J ) 1.8 Hz), 5.28

(1 H, m), 6.01 (1 H, d, J ) 11.3 Hz), 6.39 (1 H, d, J ) 11.3 Hz);

MS m/z 430 (M)⁺, 412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺, 376 (M

- 3H₂O)⁺; HRMS m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447, found

430.3443.

The following eight compounds (3a -h ) were prepared by

the same procedure as described for 2a using 5b instead of

5a .

(5Z,7E)-(1S,2R,3R,20S)-2-Met h yl-9,10-seco-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (3a ): t_R) 16.16 min; UV (EtOH)

1

λ_max263 nm, λ_min228 nm; H NMR (400 MHz, CDCl₃) δ 0.55

(5Z,7E)-(1R,2S,3R,20S)-2-Met h yl-9,10-seco-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (3h ): t_R) 17.68 min; UV (EtOH)

(3 H, s), 0.85 (3 H, d, J ) 6.4 Hz), 1.15 (3 H, d, J ) 6.7 Hz),

1.21 (6 H, s), 2.42 (1 H, dd, J ) 13.9, 4.9 Hz), 2.52 (1 H, br. d,

J ) 13.9 Hz), 2.82 (1 H, dd, J ) 11.9, 4.0 Hz), 4.02 (2 H, m),

5.02 (1 H, t, J ) 1.8 Hz), 5.37 (1 H, t, J ) 1.8 Hz), 6.03 (1 H,

d, J ) 11.3 Hz), 6.35 (1 H, d, J ) 11.3 Hz); MS m/z 430 (M)⁺,

412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺, 376 (M - 3H₂O)⁺; HRMS

m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447, found 430.3441.

(5Z,7E)-(1R,2R,3R,20S)-2-Met h yl-9,10-seco-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (3b): t_R) 16.81 min; UV (EtOH)

1

λ_max261 nm, λ_min226 nm; H NMR (400 MHz, CDCl₃) δ 0.55

(3 H, s), 0.85 (3 H, d, J ) 6.4 Hz), 1.02 (3 H, d, J ) 7.0 Hz),

1.22 (6 H, s), 1.83 (2 H, m), 2.00 (2 H, m), 2.11 (1 H, m), 2.27

(1 H, d, J ) 5.2 Hz), 2.36 (1 H, dd, J ) 14.0, 5.5 Hz), 2.65 (1

H, dd, J ) 13.7, 2.8 Hz), 2.77 (1 H, d, J ) 7.0 Hz), 2.84 (1 H,

dd, J ) 12.2, 4.3 Hz), 3.72 (1 H, m), 3.97 (1 H, t, J ) 4.9 Hz),

5.07 (1 H, d, J ) 2.1 Hz), 5.30 (1 H, d, J ) 2.1 Hz), 6.04 (1 H,

d, J ) 11.3 Hz), 6.43 (1 H, d, J ) 11.3 Hz); MS m/z 430 (M)⁺,

412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺, 376 (M - 3H₂O)⁺; HRMS

m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447, found 430.3445.

1

λ_max265 nm, λ_min226 nm; H NMR (400 MHz, CDCl₃) δ 0.55

(3 H, s), 0.85 (3 H, d, J ) 6.7 Hz), 1.22 (6 H, s), 1.23 (3 H, d,

J ) 7.3 Hz), 2.17 (1 H, d, J ) 4.3 Hz), 2.50 (1 H, br.d, J ) 14.0

Hz), 2.59 (1 H, dd, J ) 14.0, 3.7 Hz), 2.79 (1 H, d, J ) 7.6 Hz),

2.85 (1 H, dd, J ) 12.5, 4.9 Hz), 3.91 (1 H, m), 4.17 (1 H, m),

5.01 (1 H, d, J ) 2.1 Hz), 5.25 (1 H, d, J ) 1.8 Hz), 6.09 (1 H,

d, J ) 11.3 Hz), 6.48 (1 H, d, J ) 11.3 Hz); MS m/z 430 (M)⁺,

412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺, 376 (M - 3H₂O)⁺; HRMS

m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447, found 430.3446.

(5Z,7E)-(1S,2R,3S,20S)-2-Met h yl-9,10-seco-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (3c): t_R) 16.88 min; UV (EtOH)

X-r a y Cr ysta llogr a p h ic An a lysis of 2g. A colorless

prismatic crystal with dimensions of 0.20 × 0.12 × 0.32 mm³

was obtained by recrystallization from ethyl acetate. The

observed cell parameters are as follows: C₂₈H₄₈O₄, M_r

)

448.68, orthorhombic, P2₁2₁2₁; a ) 18.432(1), b ) 21.680(1), c

) 6.770(1) Å; V ) 2705.2(4) Å³, Z ) 4, Dx ) 1.102 g/cm³; λ(Cu

KR) ) 1.54178 Å, µ(Cu KR) ) 5.58 cm^-1; F(000) ) 992.00, room

temperature. The structure was solved by direct methods and

expanded using Fourier techniques. The non-hydrogen atoms

were refined anisotropically by full matrix least-squares

calculations. Hydrogen atoms were included but not refined.

R ) 0.054, R_w) 0.056 for 1861 reflections (I > 1.50σ(I)).

Further X-ray crystallographic data including bond lengths

and angles, H-atom coordinates, anisotropic thermal param-

eters, and structure factors (F_o- F_ctables) for this compound

are available as Supporting Information.

1

λ_max262 nm, λ_min228 nm; H NMR (400 MHz, CDCl₃) δ 0.54

(3 H, s), 0.85 (3 H, d, J ) 6.4 Hz), 1.06 (3 H, d, J ) 7.0 Hz),

1.22 (6 H, s), 2.12 (1 H, d, J ) 2.8 Hz), 2.34 (1 H, dd, J ) 14.7,

7.0 Hz), 2.60 (1 H, br.s), 2.64 (1 H, dd, J ) 13.4, 2.8 Hz), 2.84

(1 H, dd, J ) 11.6, 3.1 Hz), 3.65 (1 H, m), 3.90 (1 H, m), 5.05

(1 H, d, J ) 1.8 Hz), 5.30 (1 H, s), 6.02 (1 H, d, J ) 11.3 Hz),

6.41 (1 H, d, J ) 11.3 Hz); MS m/z 430 (M)⁺, 412 (M - H₂O)⁺,

394 (M - 2H₂O)⁺, 376 (M - 3H₂O)⁺; HRMS m/z (M)⁺calcd for

Bin d in g to Vita m in D Recep tor (VDR). Bovine thymus

1R,25-dihydroxyvitamin D₃receptor was obtained from Ya-

masa Biochemical (Chiba, J apan) and dissolved in 0.05 M

phosphate buffer (pH 7.4) containing 0.3 M KCl and 5 mM

dithiothreitol just before use. The receptor solution (500 µL,

0.23 mg protein) was pre-incubated with 50 µL of an ethanol

solution of 1R,25-dihydroxyvitamin D₃or an analogue at

various concentrations for 60 min at 25 °C. Then, the receptor

mixture was left to stand overnight with 0.1 nM [³H]-1R,25-

dihydroxyvitamin D₃at 4 °C. The bound and free [³H]-1R,25-

dihydroxyvitamin D₃were separated by treatment with dextran-

coated charcoal for 30 min at 4 °C, followed by centrifugation

at 3000 rpm for 10 min. The supernatant (500 µL) was mixed

with ACS-II (9.5 mL) (Amersham, England) and the radio-

activity was counted. The relative potency of the analogues

was calculated from their concentration needed to displace 50%

of [³H]-1R,25-dihydroxyvitamin D₃from its receptor compared

with the activity of 1R,25-dihydroxyvitamin D₃(assigned a

100% value).

C

₂₈H₄₆O₃430.3447, found 430.3447.

(5Z,7E)-(1R,2R,3S,20S)-2-Met h yl-9,10-seco-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (3d ): t_R) 16.29 min; UV (EtOH)

1

λ_max266 nm, λ_min228 nm; H NMR (400 MHz, CDCl₃) δ 0.54

(3 H, s), 0.85 (3 H, d, J ) 6.4 Hz), 1.10 (3 H, d, J ) 6.7 Hz),

1.22 (6 H, s), 1.68 (2 H, m), 1.85 (2 H, m), 1.98 (2 H, m), 2.24

(1 H, dd, J ) 13.4, 8.5 Hz), 2.65 (1 H, dd, J ) 13.4, 4.3 Hz),

2.82 (1 H, dd, J ) 12.2, 4.3 Hz), 3.81 (1 H, m), 4.27 (1 H, m),

5.02 (1 H, d, J ) 2.1 Hz), 5.28 (1 H, d, J ) 1.8 Hz), 6.02 (1 H,

d, J ) 11.3 Hz), 6.40 (1 H, d, J ) 11.3 Hz); MS m/z 430 (M)⁺,

412 (M - H₂O)⁺, 394 (M - 2H₂O)⁺, 376 (M - 3H₂O)⁺; HRMS

m/z (M)⁺calcd for C₂₈H₄₆O₃430.3447, found 430.3446.

(5Z,7E)-(1S,2S,3S,20S)-2-Met h yl-9,10-seco-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (3e): t_R) 24.60 min; UV (EtOH)

1

λ_max264 nm, λ_min226 nm; H NMR (400 MHz, CDCl₃) δ 0.53

(3 H, s), 0.85 (3 H, d, J ) 6.4 Hz), 1.21 (6 H, s), 1.22 (3 H, d,

J ) 7.0 Hz), 2.09 (1 H, d, J ) 4.6 Hz), 2.49 (1 H, br. d, J )

14.7 Hz), 2.58 (1 H, dd, J ) 14.0, 3.7 Hz), 2.80 (1 H, d, J ) 7.9

Hz), 2.85 (1 H, m), 3.91 (1 H, m), 4.17 (1 H, m), 4.98 (1 H, d,

J ) 2.1 Hz), 5.23 (1 H, d, J ) 1.8 Hz), 6.03 (1 H, d, J ) 11.3

Hz), 6.48 (1 H, d, J ) 11.3 Hz); MS m/z 430 (M)⁺, 412 (M -

H₂O)⁺, 394 (M - 2H₂O)⁺, 376 (M - 3H₂O)⁺; HRMS m/z (M)⁺

calcd for C₂₈H₄₆O₃430.3447, found 430.3447.

Bin d in g to Vita m in D Bin d in g P r otein (DBP ). Fetal calf

serum (FCS) was diluted 2500 times with 50 mM phosphate-

buffered saline (pH 7.0) and used as a source of DBP. The

incubation mixture used contained diluted FCS (200 µL), [³H]-

25-hydroxyvitamin D₃(480 pg, 50000 dpm/tube) dissolved in

100 µL of 50 mM phosphate-buffered saline containing 0.01%

Triton X-100 (pH 7.0), and various amounts of vitamin D₃

analogues (0-1 µg in 10 µL ethanol/tube) to be tested. After

overnight incubation at 4 °C, 500 µL of dextran-coated charcoal

suspension was added to each tube, and bound and free

vitamins were separated by vigorous mixing, followed by

(5Z,7E)-(1R,2S,3S,20S)-2-Met h yl-9,10-seco-5,7,10(19)-

ch olesta tr ien e-1,3,25-tr iol (3f): t_R) 16.93 min; UV (EtOH)

1

λ_max263 nm, λ_min228 nm; H NMR (400 MHz, CDCl₃) δ 0.53

(3 H, s), 0.85 (3 H, d, J ) 6.4 Hz), 1.13 (3 H, d, J ) 6.7 Hz),

1.21 (6 H, s), 1.69 (2 H, m), 1.84 (2 H, m), 1.98 (2 H, m), 2.41

(1 H, dd, J ) 13.7, 5.5 Hz), 2.51 (1 H, dd, J ) 13.4, 2.4 Hz),

2.82 (1 H, m), 4.05 (1 H, m), 5.01 (1 H, t, J ) 1.8 Hz), 5.35 (1

A-Ring Diastereomers of Methyldihydroxyvitamin D₃

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4263

centrifugation at 2000g for 10 min. Aliquots (500 µL) of the

supernatant were counted using a liquid scintillation counter.

Assa y for HL-60 Cell Differ en tia tion . Nitro blue tetra-

zolium (NBT) reducing activity was used as a cell differentia-

tion marker. HL-60 cells were cultured in RPMI-1640 medium

supplemented with 10% heat-inactivated FCS. Exponentially

proliferating cells were collected, suspended in fresh medium

and seeded in culture plates (Falcon, Becton Dickinson and

Co., Franklin Lakes, NJ ). Cell concentration at seeding was

adjusted to 2 × 10⁴cells/mL and the seeding volume was 1

mL/well. 1R,25-Dihydroxyvitamin D₃or an analogue dissolved

in ethanol was added to the culture medium at 0.1% volume

and culture was continued for 96 h at 37 °C in a humidified

atmosphere of 5% CO₂/air without medium change. The same

amount of vehicle was added to the control culture. NBT

reducing assay was performed according to the method of

Collins et al.³⁸Briefly, cells were collected, washed with PBS,

and suspended in serum-free medium. NBT/TPA solution

(dissolved in PBS) was added. Final concentrations of NBT

and TPA were 0.1% and 100 ng/mL, respectively. Then, the

cell suspensions were incubated at 37 °C for 25 min. After

incubation, cells were collected by centrifugation and resus-

pended in PBS. Cytospin smears were prepared, and the

counter-staining of nuclei was done with Kemechrot solution.

At least 500 cells/preparation were observed.

In Vivo Ca lciu m -Regu la tin g Assa y. Six-week-old normal

SD male rats were divided into several groups of five rats, each

receiving oral administration of 1.0, 10.0 or 100.0 µg/kg of

1R,25-dihydroxyvitamin D₃or an analogue in 2 mL/kg of 0.1%

Triton X-100 solution. The resulting increases in serum

calcium were measured at 8, 24, 48 and 72 h after administra-

tion by the o-cresolphthalein complexone method. The relative

activity of each analogue with respect to 1R,25-dihydroxy-

vitamin D₃was evaluated as follows: the dose-response effect

of each analogue was calculated at the time when the effect

was maximum and expressed as the dosage required to elevate

the serum calcium level by 1 mg/dL.

Mea su r em en t of In testin a l Ca lciu m Tr a n sp or t a n d

Bon e Ca lciu m Mobiliza tion . Male weanling SD rats were

maintained on a vitamin D-deficient diet containing normal

level of calcium and phosphorus (0.47% Ca, 0.3% P) for 7 days,

then were placed on a vitamin D-deficient and reduced calcium

diet (0.02% Ca, normal P) for the duration of the experiment.

After 21 days on the deficient diet, the animals were dosed

daily with the indicated analogues for 7 days. Each analogue

was suspended in 5% ethanol and 95% propylene glycol (0.1

mL) and injected intraperitoneally. The control groups received

the vehicle. There were 5 or 6 animals/group. The determina-

tions were made 24 h after the last dose. The rats were

sacrificed under ether anesthesia by decapitation; their blood

and intestines were collected and used immediately to deter-

mine calcium transport activity and serum calcium concentra-

tion. Calcium was measured in the presence of 0.1% lantha-

num chloride by means of a Perkin-Elmer atomic absorption

spectrometer model 3110. Intestinal calcium transport was

determined by the everted intestinal sac method using the

proximal 10 cm of intestine. Statistical analysis was done by

the use of Student’s t-test. Intestinal calcium transport is

expressed as serosal:mucosal ratio of calcium in the sac to the

calcium in the final incubation medium, or S/M. Bone calcium

mobilization represents the rise in serum calcium of the rats

maintained on a very-low-calcium diet. In that measurement,

the rise in serum calcium must arise from bone and hence is

a determination of bone calcium mobilization.

Refer en ces

(1) Ettinger, R. A.; DeLuca, H. F. The vitamin D endocrine system

and its therapeutic potential. Adv. Drug Res. 1996, 28, 269-

312.

(2) Miyaura, C.; Abe, E.; Kuribayashi, T.; Tanaka, H.; Konno, K.;

Nishii, Y.; Suda, T. 1R,25-Dihydroxyvitamin D₃induces dif-

ferentiation of human myeloid leukemia cells. Biochem. Biophys.

Res. Commun. 1981, 102, 937-943.

(3) Okamura, W. H.; Zhu, G. D. Chemistry and design: Structural

biology of vitamin D action. In Vitamin D; Feldman, Glorieux,

Pike, Eds.; Academic Press: New York, 1997; pp 939-971.

(4) Bouillon, R.; Okamura, W. H.; Norman, A. W. Structure-

function relationships in the vitamin

Endocr. Rev. 1995, 16, 200-257.

D endocrine system.

(5) (a) Yamada, S.; Ohmori, M.; Takayama, H. Synthesis of 24,24-

difluoro-1R,25-dihydroxyvitamin D₃. Chem Pharm. Bull. 1979,

27, 3196-3198. (b) Okamoto, S.; Tanaka, Y.; DeLuca, H. F.;

Kabayashi, Y.; Ikekawa, N. Biological activity of 24,24-difluoro-

1,25-dihydroxyvitamin D₃. Am. J . Physiol. 1982, 244, E159-

E163. (c) Kabakoff, B. D.; Kendrick, N. C.; Faber, D.; DeLuca,

H. F.; Yamada, S.; Takayama, H. Determination of biological

activity of 24,24-difluoro-1,25-dihydroxyvitamin D₃in the chick

using a new method for assessing intestinal calcium uptake.

Arch. Biochem. Biophys. 1982, 215, 582-588. (d) Konno, K.;

Ojima, K.; Hayashi, T.; Takayama, H. An alternative and

efficient synthesis of 24,24-difluoro-1R,25-dihydroxyvitamin D₃.

Chem. Pharm. Bull. 1992, 40, 1120-1124. (e) Ando, K.; Koike,

F.; Kondo, F.; Takayama, H. An improved synthesis of 24,24-

difluoro-1R,25-dihydroxyvitamin D₃from readily available vi-

tamin D₂. Chem. Pharm. Bull. 1995, 43, 189-192.

(6) (a) Murayama, E.; Miyamoto, K.; Kubodera, N.; Mori, T.;

Matsunaga, I. Synthetic studies of vitamin D₃analogues. VII.

Synthesis of 22-oxavitamin D₃analogues. Chem. Pharm. Bull.

1986, 34, 4410-4413. (b) Abe, J .; Nakano, T.; Nishii, Y.;

Matsumoto, T., Ogata, E.; Ikeda, K. A novel vitamin D₃analogue,

22-oxa-1,25-dihydroxyvitamin D₃, inhibits the growth of human

breast cancer in vitro and in vivo without causing hypercalcemia.

Endocrinology 1991, 129, 832-837.

(7) (a) Tanaka, Y.; DeLuca, H. F.; Kobayashi, Y.; Ikekawa, N.

26,26,26,27,27,27-Hexafluoro-1,25-dihydroxyvitamin D₃: a highly

potent, long-lasting analogue of 1,25-dihydroxyvitamin D₃. Arch.

Biochem. Biophys. 1984, 229, 348-354. (b) Inaba, M.; Okuno,

S.; Nishizawa, Y.; Yukioka, K.; Otani, S.; Matsui-Yuasa, I.;

Morisawa, S.; DeLuca, H. F.; Morii, H. Biological activity of

fluorinated vitamin D analogues at C-26 and C-27 on human

promyelocytic leukemia cells, HL-60. Arch. Biochem. Biophys.

1987, 258, 421-425.

(8) (a) Binderup, L.; Latini, S.; Binderup, E.; Bretting, C.; Calverley,

M. J .; Hansen, K. 20-Epi-vitamin D₃analogues: A novel class

of potent regulators of cell growth and immune responses.

Biochem. Pharmacol. 1991, 42, 1569-1575. (b) Dilworth, F. J .;

Calverley, M. J .; Makin, H. L.; J ones, G. Increased biological

activity of 20-epi-1,25-dihydroxyvitamin D₃is due to reduced

catabolism and altered protein binding. Biochem. Pharmacol.

1994, 47, 987-993.

(9) (a) Okano, T.; Tsugawa, N.; Masuda, S.; Takeuchi, A.; Kobayashi,

T.; Takita, Y.; Nishii, Y. Regulatory activities of 2â-(3-hydroxy-

propoxy)-1R,25-dihydroxyvitamin D₃, a novel synthetic vitamin

D₃derivative, on calcium metabolism. Biochem. Biophys. Res.

Commun. 1989, 163, 1444-1449. (b) Ono, Y.; Watanabe, H.;

Shiraishi, A.; Tanaka, S.; Higuchi, Y.; Sato, K.; Tsugawa, N.;

Okano, T.; Kobayashi, T.; Kubodera, N. Synthetic studies of

vitamin D analogues substituted at the 2â-position and their

preventive effects on bone mineral loss in ovariectomized rats.

Chem. Pharm. Bull. 1997, 45, 1626-1630.

(10) (a) Perlman, K. L.; Sicinski, R. R.; Schnoes, H. K.; DeLuca, H.

F. 1R,25-Dihydroxy-19-nor-vitamin D₃, a novel synthetic vitamin

D-related compound with potential therapeutic activity. Tetra-

hedron Lett. 1990, 31, 1823-1824. (b) Perlman, K. L.; Swenson,

R. E.; Paaren, H. E.; Schnoes, H. K.; DeLuca, H. F. Novel

synthesis of 19-nor-vitamin D compounds. Tetrahedron Lett.

1991, 32, 7663-7666.

(11) Posner, G. H.; Nelson, T. D.; Guyton, K. Z.; Kensler, T. W. New

vitamin D₃derivatives having unexpected antiproliferative

activity: 1-hydroxymethyl-25-hydroxyvitamin D₃homologues.

J . Med. Chem. 1992, 35, 3280-3287.

(12) (a) Sabbe, K.; D’Hlleweyn, C.; De Clercq, P. J .; Vandewalle, M.;

Bouillon, R.; Verstuyf, A. Synthesis of CD-ring modified 1R,25-

dihydroxyvitamin D analogues: E-ring analogues. Bioorg. Med.

Chem. Lett. 1996, 6, 1697-1702. (b) Zhu, G.-D.; Chen, Y.; Zhou,

X.; Vandewalle, M.; De Clercq, P. J .; Bouillon, R.; Verstuyf, A.

Synthesis of CD-ring modified 1R,25-dihydroxyvitamin D ana-

logues: C-ring analogues. Bioorg. Med. Chem. Lett. 1996, 6,

1703-1708. (c) Chen, Y.-J .; De Clercq, P. J .; Vandewalle. M.

Synthesis of new vitamin D₃analogues with a decalin-type CD-

ring. Tetrahedron Lett. 1996, 37, 9361-9364. (d) Wu, Y.;

D’Hlleweyn, C.; Van Haver, D.; De Clercq, P. J .; Vandewalle,

M. Synthesis of CD-ring modified 1R,25-dihydroxyvitamin D

Ack n ow led gm en t. This work was supported by a

grant-in-aid from the Ministry of Education, Science and

Culture of J apan and a grant from the Hayashi Memo-

rial Foundation for Female Natural Scientists.

Su p p or tin g In for m a tion Ava ila ble: X-ray crystallo-

graphic data and variable temperature ¹H NMR spectra for

2g. This material is available free of charge via the Internet

at http://pubs.acs.org.

4264 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22

Konno et al.

analogues: five-membered D-ring analogues. Bioorg. Med. Chem.

Lett. 1997, 7, 923-928. (e) Linclau, B.; De Clercq, P. J .;

Vandewalle, M. Synthesis of CD-ring modified 1R,25-dihy-

droxyvitamin D analogues: six-membered D-ring analogues.

Bioorg. Med. Chem. Lett. 1997, 7, 1461-1464. (f) Zhu, G.-D.;

Zhou, X.; Van Haver, D.; Vandewalle, M.; De Clercq, P. J .;

Verstuyf, A.; Bouillon, R. Synthesis, biological activity, and

conformational analysis of four seco-D-15,19-bisnor-1R,25-dihy-

droxyvitamin D analogues, diastereomeric at C17 and C20. J .

Med. Chem. 1999, 42, 3539-3556.

Fujishima, T.; Liu, Z.-P.; Miura, D.; Chokki, M.; Ishizuka, S.;

Konno, K.; Takayama, H. Synthesis and biological activity of

2-methyl-20-epi analogues of 1R,25-dihydroxyvitamin D₃. Bioorg.

Med. Chem. Lett. 1998, 8, 2145-2148. (c) Fujishima, T.; Konno,

K.; Nakagawa, K.; Kurobe, M.; Okano, T.; Takayama, H.

Efficient synthesis and biological evaluation of all A-ring dia-

stereomers of 1R,25-dihydroxyvitamin D₃and its 20-epimer.

Bioorg. Med. Chem. 2000, 8, 123-134.

(27) (a) Dai, H.; Posner, G. H. Synthetic approaches to vitamin D.

Synthesis 1994, 1383-1398. (b) Zhu, G.-D.; Okamura, W. H.

Synthesis of vitamin D (Calciferol) Chem. Rev. 1995, 95, 1877-

1952.

(13) Boehm, M. F.; Fitzgerald, P.; Zou, A.; Elgort, M. G.; Bischoff, E.

D.; Mere, L.; Mais, D. E.; Bissonnette, R. P.; Heyman, R. A.;

Nadzan, A. M.; Reichman, M.; Allegretto, E. A. Novel non-

secosteroidal vitamin D mimics exert VDR-modulating activities

with less calcium mobilization than 1,25-dihydroxyvitamin D₃.

Chem. Biol. 1999, 6, 265-275.

(14) (a) Darwish, H. M.; DeLuca, H. F. Recent advances in the

molecular biology of vitamin D action. Prog. Nucleic Acid Res.

Mol. Biol. 1996, 53, 321-344. (b) Pike, J . W. Vitamin D₃

receptors: Structure and function in transcription. Annu. Rev.

Nutr. 1991, 11, 189-216.

(15) (a) Evans, R. M. The steroid and thyroid hormone receptor

superfamily. Science 1988, 240, 889-895. (b) Umesono, K.;

Murakami, K. K.; Thompson, C. C.; Evans, R. M. Direct repeat

as selective elements for the thyroid hormone, retinoic acid, and

vitamin D₃receptors. Cell 1991, 65, 1255-1266.

(28) (a) Trost, B. M.; Dumas, J .; Villa, M. New strategies for the

synthesis of vitamin D metabolites via Pd-catalyzed reactions.

J . Am. Chem. Soc. 1992, 114, 9836-9845. (b) Trost, B. M.;

Dumas, J . New strategy for the total synthesis of 1R-hydroxy-

vitamin D derivatives. J . Am. Chem. Soc. 1992, 114, 1924-1925.

(29) (a) Tazumi, K.; Ogasawara, K. Enantioconvergent preparation

of the A-ring precursors of calciferol from either (R)- or (S)-

epichlorohydrin. J . Chem. Soc., Chem. Commun. 1994, 1903-

1904. (b) Trost, B. M.; Hanson, P. R. A practical asymmetric

synthesis of a 1,7-enyne A-ring synthon en route toward the total

synthesis of vitamin D₃analogues. Tetrahedron Lett. 1994, 35,

8119-8121. (c) Moriarty, R. M.; Kim, J .; Brumer III, H. A

general synthetic route to A-ring hydroxylated vitamin

D

analogues from pentoses. Tetrahedron Lett. 1995, 36, 51-54. (d)

Vrielynck, S.; Vandewalle, M.; Gar´ıa, A. M.; Mascaren˜as, J . L.;

Mourin˜o, A. A chemoenzymatic synthesis of A-ring key-inter-

mediates for 1R,25-dihydroxyvitamin D₃and analogues. Tetra-

hedron Lett. 1995, 36, 9023-9026. (e) Moriarty, R. M.; Kim., J .;

Brumer III, H. Toward a general synthesis of A-ring trihydrox-

ylated vitamin D analogues: synthesis of an A-ring synthon of

ED-71 from D-arabinose. Tetrahedron Lett. 1995, 36, 9265-9268.

(30) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. High-field

FT NMR application of Mosher’s method. The absolute configu-

rations of marine terpenoids. J . Am. Chem. Soc. 1991, 113,

4092-4096.

(16) Okamura, W. H.; Palenzuela, J . A.; Plumet, J .; Midland, M. M.

Vitamin D: Structure-function analyses and the design of

analogues. J . Cell. Biochem. 1992, 49, 10-18.

(17) Midland, M. M.; Plumet, J .; Okamura, W. H. Effect of C20

stereochemistry on the conformational profile of the side chains

of vitamin D analogues. Bioorg. Med. Chem. Lett. 1993, 3, 1799-

1804.

(18) (a) Yamamoto, K.; Ohta, M.; DeLuca, H. F.; Yamada, S. On the

side chain conformation of 1R,25-dihydroxyvitamin D₃respon-

sible for binding to the receptor. Bioorg. Med. Chem. Lett. 1995,

5, 979-984. (b) Yamamoto, K.; Sun, W.-Y.; Ohta, M.; Hamada,

K.; DeLuca, H. F.; Yamada, S. Conformationally restricted

analogues of 1R,25-dihydroxyvitamin D₃and its 20-epimer:

Compounds for study of the three-dimensional structure of

vitamin D responsible for binding to the receptor. J . Med. Chem.

1996, 39, 2727-2737. (c) Yamada, S.; Yamamoto, K.; Masuno,

H.; Ohta, M.; Conformation-function relationship of vitamin D:

conformational analysis predicts potential side-chain structure.

J . Med. Chem. 1998, 41, 1467-1475. (d) Yamamoto, K.; Ooizumi,

H.; Umesono, K.; Verstuyf, A.; Bouillon, R.; DeLuca, H. F.;

Shinki, T.; Suda, T.; Yamada, S. Three-dimensional structure-

function relationship of vitamin D: side-chain location and

various activities. Bioorg. Med. Chem. Lett. 1999, 9, 1041-1046.

(19) Liu, Y.-Y.; Collins, E. D.; Norman, A. W.; Peleg, S. Differential

interaction of 1R,25-dihydroxyvitamin D₃analogues and its 20-

epi homologues with the vitamin D receptor. J . Biol. Chem. 1997,

272, 3336-3345.

(20) (a) Wing, R. M.; Okamura, W. H.; Pirio, M. R.; Sine, S. M.;

Norman, A. W. Vitamin D in solution: conformations of vitamin

D₃, 1R,25-dihydroxyvitamin D₃, and dihydrotachysterol D₃.

Science 1974, 186, 939-941. (b) Wing, R. M.; Okamura, W. H.;

Rego, A.; Pirio, M. R.; Norman, A. W. Studies on vitamin D and

its analogues. VII. Solution conformations of vitamin D₃and

1R,25-dihydroxyvitamin D₃by high-resolution proton magnetic

resonance spectroscopy. J . Am. Chem. Soc. 1975, 97, 4980-4985.

(21) Okamura, W. H.; Norman, A. W.; Wing, R. M. Vitamin D:

Concerning the relationship between molecular topology and

biological function. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 4194-

4197.

(22) (a) Eguchi, T.; Kakinuma, K.; Ikekawa, N. Synthesis of 1R[19-

¹³C]-hydroxyvitamin D₃and ¹³C NMR analysis of the conforma-

tional equilibrium of the A-ring. Bioorg. Chem. 1991, 19, 327-

332. (b) Eguchi, T.; Ikekawa, N. Conformational analysis of

1R,25-dihydroxyvitamin D₃by nuclear magnetic resonance.

Bioorg. Chem. 1990, 18, 19-29.

(23) Ishida, H.; Shimizu, M.; Yamamoto, K.; Iwasaki, Y.; Yamada,

S. Syntheses of 1-alkyl-1,25-dihydroxyvitamin D₃. J . Org. Chem.

1995, 60, 1828-1833.

(24) Sicinski, R. R.; Prahl, J . M.; Smith, C. M.; DeLuca, H. F. New

1R,25-dihydroxy-19-norvitamin D₃compounds of high biological

activity: Synthesis and biological evaluation of 2-hydroxymethyl,

2-methyl, and 2-methylene analogues. J . Med. Chem. 1998, 41,

4662-4674.

(25) Norman, A. W.; Adams, D.; Collins, E. D.; Okamura, W. H.;

Fletterick, R. J . Three-dimensional model of the ligand binding

domain of the nuclear receptor for 1R,25-dihydroxy-vitamin D₃.

J . Cell. Biochem. 1999, 74, 323-333.

(31) (a) Rychnovsky, S. D.; Skalitzky, J . Stereochemistry of alternat-

ing polyol chains: ¹³C NMR analysis of 1,3-diol acetonides.

Tetrahedron Lett. 1990, 31, 945-948. (b) Rychnovsky, S. D.;

Rogers, B.; Yang, G. Analysis of two ¹³C NMR correlations for

determining the stereochemistry of 1,3-diol acetonides. J . Org.

Chem. 1993, 58, 3511-3515.

(32) (a) Posner, G. H.; Dai, H. 1-(Hydroxyalkyl)-25-hydroxyvitamin

D

₃analogues of calcitriol. 1. Synthesis. Bioorg. Med. Chem. Lett.

1993, 3, 1829-1834. (b) Posner, G. H.; Dai, H. Afarinkia, K.;

Murthy, N. N.; Guyton, K. Z.; Kensler, T. W. Asymmetric total

synthesis, X-ray crystallography, and preliminary biological

evaluation of 1-(1′-hydroxyethyl)-25-hydroxyvitamin D₃ana-

logues of natural calcitriol. J . Org. Chem. 1993, 58, 7209-7215.

(c) Posner, G. H.; White, M. C.; Dolan, P.; Kensler, T. W.;

Yukihiro, S.; Guggino, S. E. 1R,25-Dihydroxyvitamin D₃hybrid

analogues with structural changes at both the A-ring and the

C,D-ring side-chain. Bioorg. Med. Chem. Lett. 1994, 4, 2919-

2924. (d) Posner, G. H.; J ohnson, N. Stereocontrolled total

synthesis of calcitriol derivatives: 1,25-dihydroxy-2-(4′-hydroxy-

butyl)vitamin D₃analogues of an osteoporosis drug. J . Org.

Chem. 1994, 59, 7855-7861. (e) Posner, G. H.; Lee, J . K.; Li, Z.;

Takeuchi, K.; Guggino, S. E.; Dolan, P.; Kensler, T. W. 1R,25-

Dihydroxyvitamin D₃hybrid analogues with structural changes

at both the A-ring and the C,D-ring side-chain. II. Bioorg. Med.

Chem. Lett. 1995, 5, 2163-2168. (f) Posner, G. H.; Cho, C.-G.;

Anjeh, T. E. N.; J ohnson, N.; Horst, R. L.; Kobayashi, T.; Okano,

T.; Tsugawa, N. 2-Fluoroalkyl A-ring analogues of 1,25-dihy-

droxyvitamin D₃. Stereocontrolled total synthesis via intramo-

lecular and intermolecular Diels-Alder cycloadditions. Prelimi-

nary biological testing. J . Org. Chem. 1995, 60, 4617-4628. (g)

Posner, G. H.; Lee, J . K.; White, M. C.; Hutchings, R. H.; Dai,

H.; Kachinski, J . L.; Dolan, P.; Kensler, T. W. Antiproliferative

hybrid analogues of the hormone 1R,25-dihydroxyvitamin D₃:

design, synthesis, and preliminary biological evaluation. J . Org.

Chem. 1997, 62, 3299-3314. (h) Posner, G. H.; Lee, J . K.; Wang,

Q.; Peleg, S.; Burke, M.; Brem, H.; Dolan, P.; Kensler, T. W.

Noncalcemic, antiproliferative, transcriptionally active, 24-

fluorinated hybrid analogues of the hormone 1R,25-dihydroxy-

vitamin D₃. Synthesis and preliminary biological evaluation. J .

Med. Chem. 1998, 41, 3008-3014.

(33) Suwinska, K.; Kutner, A. Crystal and molecular structure of

1,25-dihydroxy cholecalciferol. Acta Crystallogr. 1996, B52, 550-

554.

(34) Anet, F. A. L. The use of remote deuteration for the determi-

nation of coupling constants and conformational equilibria in

cyclohexane derivatives. J . Am. Chem. Soc. 1962, 84, 1053-

1054.

(26) (a) Konno, K.; Maki, S.; Fujishima, T.; Liu, Z.-P.; Miura, D.;

Chokki, M.; Takayama, H. A novel and practical route to A-ring

enyne synthon for analogues: Synthesis of A-ring diastereomers

of 1R,25-dihydroxyvitamin D₃and 2-methyl-1,25-dihydroxy-

vitamin D₃. Bioorg. Med. Chem. Lett. 1998, 8, 151-156. (b)

(35) Calculated by Macro Model version 6.5 (Schroedinger, Inc.) on

an SGI O2 Workstation.

A-Ring Diastereomers of Methyldihydroxyvitamin D₃

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4265

(36) (a) Muralidharan, K. R.; de Lera, A. R.; Isaeff, S. D.; Norman,

A. W.; Okamura, W. H. Studies on the A-ring diastereomers of

1R,25-dihydroxyvitamin D₃. J . Org. Chem. 1993, 58, 1895. (b)

Norman, A. W.; Bouillon, R.; Farach-Carson, M. C.; Bishop, J .

E.; Zhou, L.-X.; Nemere, L.; Zhao, J .; Muralidharan, K. R.;

Okamura, W. H. Demonstration that 1â,25-dihydroxyvitamin

D₃is an antagonist of the nongenomic but not genomic biological

responses and biological profile of the three A-ring diastereomers

of 1R,25-dihydroxyvitamin D₃. J . Biol. Chem. 1993, 268, 20022-

20030.

(37) Imae, Y.; Manaka, A.; Yoshida, N.; Ishimi, Y.; Shinki, T.; Abe,

E.; Suda, T.; Konno, K.; Takayama, H.; Yamada, S. Biological

activities of 24-fluoro-1R,25-dihydroxyvitamin D-2 and its 24-

epimer. Biochim. Biophys. Acta 1994, 1213, 302-308.

(38) Collins, S. J .; Ruscetti, F. W.; Gallagher, R. E.; Gallo, R. C.

Normal functional characteristics of cultured human promyelo-

cytic leukemia cells (HL-60) after induction of differentiation by

dimethylsulfoxide. J . Exp. Med. 1979, 149, 969-974.

(39) Ishizuka, S.; Norman, A. W. The difference of biological activity

among four diasteroisomers of 1R,25-dihydroxycholecalciferol-

26,23-lactone. J . Steroid Biochem. 1986, 25, 505-510.

(40) (a) Uhland-Smith, A.; DeLuca, H. F. 1,25-Dihydroxycholecalcif-

erol analogues cannot replace vitamin D in normocalcemic male

rats. J . Nutr. 1993, 123, 1777-1785. (b) Suda, T.; DeLuca, H.

F.; Tanaka, Y. Biological activity of 25-hydroxyergocalciferol in

rats. J . Nutr. 1970, 100, 1049-1052.

(41) Honda, A.; Mori, Y.; Otomo, S.; Ishizuka, S.; Ikekawa, N. Effects

of novel 26,27-dialkyl analogues of 1R,25-dihydroxyvitamin D₃

on differentiation-inducing activity of human promyelocytic

leukemia (HL-60) cells in serum-supplemented or serum-free

culture. Steroids 1991, 56, 142.

(42) Bishop, J . E.; Collins, E. D.; Okamura, W. H.; Norman, A. W.

Profile of ligand specificity of the vitamin D binding protein for

1R,25-dihydroxyvitamin D₃and its analogues. J . Bone Miner.

Res. 1994, 8, 1277-1288.

(43) (a) Nakagawa, K.; Kurobe, M.; Ozono, K.; Konno, K.; Fujishima,

T.; Takayama, H.; Okano, T. Novel ring A stereoisomers of

2-methyl-1R,25-dihydroxyvitamin D₃and 2-methyl-20-epi-1R,25-

dihydroxyvitamin D₃: transactivation of target genes and

modulation of differentiation in human promyelocytic leukemia

(HL-60) cells. Biochem. Pharmacol. 2000, 59, 691-702. (b)

Nakagawa, K.; Kurobe, M.; Konno, K.; Fujishima, T.; Takayama,

H.; Okano, T. Structure specific control of differentiation and

apoptosis of human promyelocytic leukemic (HL-60) cells by

A-ring diastereomers of 2-methyl-1R,25-dihydroxyvitamin D₃

and its 20-epimer. Biochem. Pharmacol., in press.

(44) Rochel, N.; Wurtz, J . M.; Mitschler, A.; Klaholz, B.; Moras, D.

The crystal structure of the nuclear receptor for vitamin D bound

to its natural ligand. Mol. Cell 2000, 5, 173-179.

(45) Yamamoto, K.; Masuno, H.; Choi, M.; Nakashima, K.; Taga, T.;

Ooizumi, H.; Umesono, K.; Sicinska, W.; VanHooke, J .; DeLuca,

H. F.; Yamada, S. Three-dimensional modeling of and ligand

docking to vitamin D receptor ligand domain. Proc. Natl. Acad.

Sci. U.S.A. 2000, 97, 1467-1472.

(46) Suhara, Y.; Nihei, K.; Tanigawa, H.; Fujishima, T.; Konno, K.;

Nakagawa, K.; Okano, T.; Takayama, H. Syntheses and biologi-

cal evaluation of novel 2R-substituted 1R,25-dihydroxyvitamin

D₃analogues. Bioorg. Med. Chem. Lett. 2000, 10, 1129-1132.

J M000261J

Article Doi

Synthesis, biological evaluation, and conformational analysis of A-ring diastereomers of 2-methyl-1,25-dihydroxyvitamin D3 and their 20-epimers: Unique activity profiles depending on the stereochemistry of the A-ring and at C-20

DOI: 10.1021/jm000261j

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Full text of DOI:10.1021/jm000261j

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