Synthesis, biological activity, and conformational analysis of four seco-D-15,19-bisnor-1α,25-dihydroxyvitamin D analogues, diastereomeric at C17 and C20

J . Med. Chem. 1999, 42, 3539-3556

3539

Syn th esis, Biologica l Activity, a n d Con for m a tion a l An a lysis of F ou r

seco-D-15,19-bisn or -1r,25-Dih yd r oxyvita m in D An a logu es, Dia ster eom er ic a t C17

a n d C20

Xiaoming Zhou, Gui-Dong Zhu, Dirk Van Haver, Maurits Vandewalle, Pierre J . De Clercq,*

Annemieke Verstuyf,^†and Roger Bouillon^†

Laboratory for Organic Synthesis, Department of Organic Chemistry, University of Gent, Krijgslaan 281 (S4),

B-9000 Gent, Belgium, and Laboratory of Experimental Medicine and Endocrinology, Catholic University of Leuven,

Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium

Received December 30, 1998

The synthesis of four CD-ring-modified 19-nor-1R,25-dihydroxyvitamin D₃derivatives lacking

C15, referred to as 6C analogues, and diastereomeric at C17 and C20 is described. The synthesis

involves an Ireland-Claisen rearrangement of a 3-methyl-substituted ester of (1R)-3-methyl-

2-cyclohexen-1-ol as the key step, followed by elaboration of the side chain, transformation

into a C8 cyclohexanone derivative, and final Wittig-Horner coupling with the 19-nor A-ring

phosphine oxide. Despite possessing a more flexible side chain than the parent hormone,

biological evaluation showed an unexpected superagonistic antiproliferative and prodifferen-

tiating activity (10-50 times higher as compared to that of 1R,25(OH)₂D₃) for the diastereomer

with the “natural” configuration at C17 and C20. The other diastereomers exhibit a 25-90%

decrease in activity. All four analogues show a decreased binding affinity (45% or less), and

their calcemic activity is 4-400 times less than that of 1R,25(OH)₂D₃. The conformational

behavior of their side chain was studied using molecular mechanics calculations, and the result

is presented as volume maps. A relative activity volume was determined by subtraction of the

volume map of the least active analogue from the volume map of the most active one. This

shows three regions corresponding to preferred orientations in space of the side chain of the

active analogue. One of these regions was found to overlap with the region that is preferentially

occupied by the most active of the four diastereomeric 22-methyl-substituted 1R,25(OH)₂D₃

analogues.

Ta ble 1. Biological Activities of 1-5^a

In tr od u ction

binding

differentiation

HL-60

calcium

The discovery that 1R,25-dihydroxyvitamin D₃(1;

further abbreviated as 1,25(OH)₂D₃), the hormonally

active form of vitamin D, possesses, next to its classical

calciotropic activity,¹immunosuppressive activity²and

is also effective in the inhibition of cellular proliferation

and in the induction of cellular differentiation³has

resulted in a very active search for analogues in which

the calcemic activity and the other activities are dis-

sociated.⁴Indeed, the therapeutic utility of the natural

hormone in the treatment of certain cancers and skin

diseases is severely limited because effective doses

provoke hypercalcemia. Notable successes have already

been recorded in the discovery of analogues that have

such dissociated activity. A few illustrative examples

are 20-epi-1,25(OH)₂D₃(2),^5a19-nor-1,25(OH)₂D₃(3),⁶

a 16-ene-23-yne derivative developed by Hoffmann-La

Roche (4),⁷and 22-oxa-1,25(OH)₂D₃(5).⁸The latter

compound was the first analogue in which the cell-

differentiating and calcemic activities of vitamin D were

separated. It has a substantially higher cell-differentiat-

ing activity than the natural hormone 1, but it has low

calcemic activity. The comparison of some relevant

biological activities for compounds 1-5 shown in Table

entry VDR (pig) DBP (human)

Ca serum (mice)

1

2

3

4

5

100

88

30

85

18

100

0.2

100

3000

90

950

197

100

800

0.3

8

48

0

0.3

a

The activities are presented as relative values, the reference

value of 1,25(OH)₂D₃(1) being defined as 100. Further details

about the methodology are given in the Experimental Section. All

data resulted from evaluation in this laboratory (Leuven).

1 illustrates the possibility of discrimination of the

various actions of vitamin D.

The activity of vitamin D originates from a genomic

pathway wherein the hormone binds to the intracellular

vitamin D receptor (VDR) which regulates gene tran-

scription and the synthesis of new proteins that are

more directly responsible for the biological response.⁹

Hence, the VDR-1,25(OH)₂D₃complex, and in particu-

lar its conformation, plays a crucial role in vitamin D

activity. Structure-function studies have demonstrated

that the combined presence of a polar group in the

terminal portion of the side chain, such as a (25R)- or

(24R)-hydroxyl group, and a 1R-hydroxyl group is es-

sential for activity. In this respect the structure of 1,-

25(OH)₂D₃can be regarded as one consisting of a central

rigid hydrophobic part, the CD-bicyclic ring system, to

* Corresponding author. Fax: +32-9-264.49.98. E-mail: pierre.

declercq@rug.ac.be.

^†Catholic University of Leuven.

Published on Web 08/21/1999

3540 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Zhou et al.

Ch a r t 1

Ta ble 2. Biological Activities of 6a -d ^a

binding

differentiation

calcium

entry

VDR (pig)

100

1.7

DBP (rat)

HL-60

ICA (chick)

1

100

0.45

67

0.25

100

1.3

100

11900 (2000)

(8)

100

25

50

50 (100)

(0.1)

6a

6b

6c

6d

33

2000 (100)

1 (3)

a

The activities are presented as relative values, the reference

value of 1,25(OH)₂D₃(1) being defined as 100. Data taken from

reference 11b. Values in parentheses have been obtained in this

laboratory (Leuven).

Ch a r t 2

which are connected two flexible moieties, i.e., the side

chain at C17 and the seco-B,A-ring part that is attached

to the C-ring via a diene in which the 6,7-bond is freely

rotatable. Up to 1995 the search for new analogues has

mostly concentrated on derivatives possessing structural

changes in the flexible parts of the molecule with a

strong preference for the side chain; i.e., more than 278

were recorded in the nonpatent literature in the period

1985-1995.⁴Among these, several possess the inverted

stereochemistry at C20, such as the 20-epi derivative

2. Analogue 2 has been reported to be several orders of

magnitude more effective than 1,25(OH)₂D₃in the

inhibition of cellular proliferation and induction of cell

differentiation using the human histiocytic lymphoma

cell line U937.⁵These observations have led to the claim

that no vitamin D analogue study would be complete

without an evaluation of both C20-epimeric series.^5bAs

a logical corollary, theoretical studies have been directed

at establishing a link between biological activity and

the conformational profile of the side chain (vide infra).¹⁰

In this context an interesting study of the conforma-

tion-function relationship of vitamin D has been re-

cently reported based on the conformational profile of

the side chain of the four 22-methyl-substituted dia-

stereomers 6a -d (Chart 1).¹¹A few relevant biological

activities are shown in Table 2, which indicate a

pronounced differential behavior among these four

analogues. Conformational studies further showed each

of the four derivatives to exhibit preferred domains in

space that are occupied by the side chain. Through

further comparison with 1 and 2, preferred side-chain

conformations of 1,25(OH)₂D₃and its 20-epimer for

binding to the VDR were proposed.

In the present paper we report on the synthesis, the

biological activity, and the conformational study of the

four vitamin D analogues 8a -d (Chart 2), which are

structurally characterized by the absence of the five-

membered D-ring. Whereas such analogues can be

formally identified as 14,16-seco-15-nor analogues, we

rather prefer to focus on the remaining six-membered

C-ring of the central part and will further use “6C

analogues” as the general term. In a preliminary com-

munication we have already reported on the synthesis

of 6C analogues 7a , 8a , 9a , and 10a ,¹²which show

variations in the structure of the side chain, i.e., the

Diastereomeric Dihydroxyvitamin D Analogues

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3541

Ch a r t 3

natural (7, 8) and the 23-yne (9, 10) side chains, and of

the A-ring, i.e., the natural (7, 9) and the corresponding

19-nor (8, 10) A-rings. Rather than the R,S-stereono-

menclature, which would vary following the priority

convention, for the sake of clarity and consistency, the

a -d designation as shown in Chart 2 will be used

throughout the paper for identification of the stereo-

chemical relationship. Also, carbon atoms of the vitamin

D skeleton will be referred to according to the conven-

tional steroid numbering.

This comprehensive study has led to two rather

remarkable results: (1) in one diastereomeric series, i.e.,

the a series, analogues possess a superagonistic activity

as compared to 1, which is not only unexpected but also

highly unusual in view of the greater flexibility of the

D-seco derivative; (2) the important differences in activi-

ties observed in the four diastereomeric series a -d can

be related to relative differences in the conformational

profile of the corresponding side chain.

The reason 19-nor derivatives (cf. 8) were developed

has a fundamental origin that is related to the well-

known thermal equilibrium between vitamin D and

previtamin D (eq 1). The latter process constitutes one

absence of the ∆^10,19exocyclic double bond as in the 19-

nor derivatives 3, 8, and 10 prevents the further

rearrangement to the corresponding previtamin. The 19-

nor-1,25(OH)₂D₃previtamin derivative has been shown

to be devoid of vitamin D activity.¹⁶

Syn th esis

The synthesis of the analogues follows a modular

strategy based on the following three stages (Chart 3).

(i) Establishment of an adequately functionalized six-

membered C-ring 12 via the Ireland-Claisen rear-

rangement¹⁷of the (Z)-enolate silyl ether derived from

cyclohexenyl ester 11: The creation of stereocenter C13

of 12 in the required configuration depends on the

stereogenicity of stereocenter C8 in precursor 11, while

the configuration at C17 in 12 is established during the

reaction. (ii) The obtained 3-substituted cyclohexene

derivative 12 is further transformed into the C8 cyclo-

hexanone derivative 13 in which the desired side chain

has been constructed. (iii) The attachment of the A-ring,

in its normal or 19-nor form, is performed using Lyth-

goe’s strategy¹⁸involving Wittig-Horner reaction of

phosphine oxide 14¹⁹or 15.^6,20Final removal of the

protecting groups then leads to the analogues 7-10.

The synthesis of the 6C analogue 8a is shown in

Scheme 1. It follows the above modular strategy and

has been the subject of a preliminary communication.¹²

The route centers on the Ireland-Claisen rearrange-

ment of ester 18.²¹The latter is obtained via esterifi-

cation of commercially available (R)-(+)-citronellic acid

(17; 98% ee) with (R)-3-methyl-2-cyclohexen-1-ol (16).

The synthesis of the latter proceeds via a sequence that

has been previously developed for (R)-2-cyclohexenol

and involves the enzymatic kinetic resolution of racemic

2-iodo-3-methyl-2-cyclohexen-1-ol as the key step (lipase

PS, vinyl acetate, toluene, rt).²¹The Ireland-Claisen

rearrangement is performed on the (Z)-silyl ketene

acetal obtained from 18 through the use of HMPA as

cosolvent (LDA, THF-HMPA). Thermal rearrangement

of the corresponding TBDMS ether (18 h) leads, after

acid workup, to a mixture of diastereomeric acids 19a

and 19b (ratio >10:1; 58% yield), that could not be

separated at this stage. After reduction with lithium

of the two pericyclic reactions that intervene in the

biosynthesis of vitamin D. The first process is the

photochemically induced B-ring opening of 7,8-didehy-

drocholesterol (known as DHC or provitamin) which

leads to previtamin D.¹³A subsequent 1,7-sigmatropic

shift involving the 19-methyl hydrogens and position 9

leads to vitamin D in a folded conformation (not shown)

characterized by the s-cis geometry at the 6,7-bond. Fast

rotation at that bond eventually generates vitamin D

in the preferred s-trans conformation, which also cor-

responds to the one that is usually depicted. The

thermally induced equilibrium mixture of vitamin D and

previtamin D contains the vitamin form as the major

isomer (88:12 at 80 °C).¹⁴This is the result of subtle

conformational preferences related to the unfavorable

position of the previtamin’s ∆⁸bond in the trans-fused

hydrindane system; hence, the vitamin form is pre-

ferred.¹⁵In the 6C analogues, however, one may expect

the previtamin form, possessing a more substituted

trienic moiety, to become more stable (eq 2). As will be

shown later, this is indeed the case. Obviously, the

3542 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Zhou et al.

Sch em e 1^a

Sch em e 2^a

a

(a) DCC, DMAP, CH₂Cl₂(a : 98%; c: 92%); (b) LDA, THF-

HMPA, TBDMSCl, (a , b: 78%; c, d : 64%); (c) LiAlH₄, THF (a , b:

90%; c, d : 94%); (d) (i) Py‚SO₃, Et₃N, DMSO, CH₂Cl₂(a : 81%; c:

97%), (ii) DBU, CH₂Cl₂, rt; (e) NaBH₄, MeOH (b: 95%; d : 88%).

a

(a) DCC, DMAP, CH₂Cl₂(96%); (b) LDA, THF-HMPA, TB-

hydroxide, and sodium borohydride to give 23a (67%

yield), followed by protection as the triethylsilyl (TES)

ether (92% yield). (3) Oxygenation of the cyclohexene

ring of 24a is effected by reaction with 9-BBN, followed

by hydrogen peroxide (95% yield). This resulted in a

diastereomeric mixture (46:54) of alcohols 25a that was

not further separated. Subsequent oxidation with PDC

gave ketone 26a in 80% yield. The introduction of the

19-nor A-ring was performed via Wittig-Horner reac-

tion and led, after removal of the protecting groups by

treatment with tetrabutylammonium fluoride (TBAF),

to analogue 8a in good yield.

The synthesis of the other analogues 8b-d proceeded

via a functionalized truncated side chain. The therefore

required homochiral esters 28a and 28c (Scheme 2)

were obtained via esterification of 16 with acids 27a and

27c, respectively. (S)-4-Benzyloxy-3-methylbutanoic acid

(27a ) was obtained from methyl (R)-3-hydroxy-2-meth-

ylpropionate²³in 70% yield (99% ee), via tosylate

displacement with sodium cyanide in DMSO, followed

by nitrile hydrolysis with potassium hydroxide. The

same reaction sequence starting from methyl (S)-3-

DMSCl, reflux, 16 h (58%); (c) LiAlH₄, THF (86%); (d) TsCl,

pyridine (95%); (e) LiAlH₄, THF (95%); (f) Hg(OAc)₂, NaOH;

NaBH₄(67%); (g) Et₃SiCl, DMAP, imidazole, DMF (92%); (h)

9-BBN, H₂O₂(95%); (i) PDC (80%); (j) 14 or 15, n-BuLi, THF, -78

°C (with 14: 88%; with 15: 85%); (k) Bu₄NF, THF, rt (7a , 8a :

93%).

aluminum hydride (LAH), the alcohol 20a was obtained

in pure form after HPLC separation. The stereochemical

assignment is in line with a boatlike transition state

that has been shown to prevail when the (Z)-enolate

silyl ether is involved in the rearrangement of a six-

membered cyclohexene derivative.²²The same method

has been used in the other series and will be discussed

in detail later, including the establishment of the

assignment via iodolactone formation.

The further conversion of alcohol 20a into cyclohex-

anone 26a involves three stages. (1) After conversion

of the primary alcohol into the corresponding tosylate

21a , the latter is reduced with LAH to yield 22a (90%

overall). (2) The introduction of the 25-hydroxyl group

is performed via selective reaction of the trisubstituted

side-chain double bond using mercuric acetate, sodium

Diastereomeric Dihydroxyvitamin D Analogues

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3543

Ta ble 3. ¹H NMR Data (500 MHz, CDCl₃) of 32a -d :

Sch em e 3^a

3

Chemical Shift of H17 and Coupling Constant J _H17,H20

a

b

c

d

δ (ppm)

J (Hz)

2.55

6.8

2.85

2.9

2.82

2.9

2.47

7.5

hydroxy-2-methylpropionate led to the enantiomeric

acid 27c. As reported in a previous communication,¹²

27a can also be obtained via treatment of the expensive

(S)-3-methyl-γ-butyrolactone with benzyl alcohol under

basic conditions.²⁴

After esterification of 16 with homochiral acid 27a

(92% yield), ester 28a was subjected to the Ireland-

Claisen rearrangement sequence (LDA, THF-HMPA;

TBDMSCl). This led to a 78% yield of acids 29a and

29b (ratio 14:1, respectively). The further sequence

involved LAH reduction and HPLC separation of the

resulting alcohols 30a and 30b.

Since the available amount of 30b was too small for

further synthetic work and since variation of the condi-

tions of the Ireland-Claisen rearrangement did not lead

to substantial differences in the diastereomeric ratio,

we had to recourse to the following sequence for obtain-

ing larger amounts of 30b. After oxidation of 30a with

sulfur trioxide pyridine complex in DMSO and dichlo-

romethane, the obtained aldehyde 31a was isomerized

in base (DBU, CH₂Cl₂, rt, 62 h) to yield an equilibrium

mixture of 31a and 31b (ratio 55:45, respectively). After

reduction of the crude mixture (sodium borohydride,

methanol, -10 °C) to the corresponding alcohols, pure

30b was obtained through HPLC separation.

Using the same reaction conditions Ireland-Claisen

rearrangement of diastereomeric ester 28c led to 29c

and 29d in a 29:1 ratio, respectively (64% yield). After

LAH reduction and HPLC purification, the alcohol 30c

was obtained in 94% yield. In the same way as described

above the diastereomeric alcohol 30d was obtained after

reduction of the mixture of aldehydes 31c and 31d (ratio

63:37) that resulted from the basic equilibration of 31c.

The stereochemical assignment of alcohols 30a -d

rests on the ¹H NMR analysis of the lactones 32a -d

that were obtained upon treatment of the acids 29 with

iodine in acetonitrile. In the case of reaction of the

alcohols 29a and 29b, the major isomer (32a ) shows no

NOE between Me-18 and H17, whereas a significant

NOE is present in the other isomer (32b). In the case

of 29c and 29d structural assignment rests on similar

NOE experiments (Table 3) of the two iodo lactones 32c

and 32d . Whereas 32c was directly obtained from 29c,

32d resulted from iodolactonization of 29d that was

obtained through J ones oxidation of 30d .

a

(a) TsCl, pyridine (a : 80%; b: 100%; d : 78%); (b) LiAlH₄, THF

(a : 95%; b: 80%; c: 89% from 30; d : 80%); (c) 9-BBN, H₂O₂,

NaOH, EtOH (a : 95%; b: 84%; c: 95%; d : 94%); (d) t-BuPh₂SiCl,

DMAP, imidazole, DMF (a : 95%; b: 93%; c: 90%; d : 79%); (e)

Pd(OH)₂, cyclohexene, EtOH (a : 85%; b: 88%; c: 90%; d : 85%).

tosylation (tosyl chloride, pyridine, 0 °C) also led to a

minor fraction of the trans-disubstituted tetrahydrofu-

ran cyclization products 34a and 34d , respectively (ca.

20%).²⁵

The cyclohexenes 35a -d are further transformed into

the corresponding alcohols 38a -d via a sequence in-

volving: (i) treatment with 9-BBN and basic hydrogen

peroxide to a mixture of diastereomeric alcohols 36R and

36â, which were not separated at this stage; (ii) protec-

tion of the secondary alcohol in 36 as tert-butyldiphe-

nylsilyl (TBDPS) ether; (iii) debenzylation of 37 using

palladium hydroxide on carbon (Pearlman’s catalyst) in

cyclohexene-ethanol. The separation of alcohols 38R

and 38â was successful in the case of the a -c series

(HPLC). Although the stereogenicity at C8 is of no

further importance since eventually the secondary

alcohol will be oxidized, the R,â-structural assignment

was possible via difference NOE NMR. Upon irradiation

of the methyl group at C13 a strong NOE is observed

for the proton at C8 in the case of 38R (a -c) while no

NOE signal is observed in the case of 38â.

As shown in Scheme 4, in the case of the b and c

series the â-alcohols 38âb and 38âc were transformed

into the corresponding iodides 39b and 39c (triph-

enylphosphine, iodine). In a following step, part of the

side chain was introduced via a nickel-mediated conju-

gate addition.²⁶The required Ni(0) complex was conve-

niently prepared in situ by simple heating to 60 °C of 1

equiv of nickel(II) chloride‚6H₂O with 5 equiv of zinc

powder in the presence of ethyl acrylate in pyridine.²⁷

Addition of iodides 39b and 39c to the complex led to

esters 40b and 40c in high yield. Subsequent Grignard

reaction with excess methylmagnesium bromide led to

41 and after silyl deprotection to 42 in high yield.

The further transformation of cyclohexenes 30 into

the corresponding alcohols 38 is shown in Scheme 3.

The reductive removal of the primary hydroxyl group

proceeds via the corresponding tosylate 33 (LAH, THF).

It is worth mentioning that in the case of 30a and 30d

3544 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Zhou et al.

Sch em e 4^a

Sch em e 5^a

a

(a)I₂, PPh₃, imidazole (b, c: 94%);(b)NiCl₂, Zn, H₂CdCHCO₂Et,

pyridine (b: 88%; c: 91%); (c) MeMgBr, THF (b: 86%; c: 95%);

(d) Bu₄NF, THF (b: 100%; c: 90%); (e) PDC, CH₂Cl₂(b: 94%; c:

86%); (f) Et₃SiCl, DMAP, imidazole, DMF (b: 82%); (g) (i) 15,

n-BuLi, THF, -78 °C, (ii) Bu₄NF, THF (b: 100%; c: 85%).

a

(a) TsCl, pyridine (d : 93%); (b) HCtCC(CH₃)₂OEE, NaH,

DMSO (d : 57%); (c) Bu₄NF, THF (d : 94%); (d) H₂, 5% Rh/Al₂O₃,

EtOAc (d : 90%); (e) PDC, CH₂Cl₂(d : 90%); (f) 15, n-BuLi, THF,

-78 °C; (g) Bu₄NF, THF (8d : 92% from 44); (h) PDC, CH₂Cl₂(a :

85%); (i) 14, n-BuLi, THF, -78 °C (a : 89%); (j) PPTS, CH₂Cl₂, rt;

(k) Bu₄NF, THF (9a : 92% over 2 steps).

Further oxidation gave hydroxy ketones 43b and 43c.

In the b series, the hydroxyl group was protected as TES

ether 44b in view of the subsequent Wittig-Horner

reaction with phosphine oxide 15. Full deprotection

using TBAF eventually led to 8b and the corresponding

Z-isomer (vide infra). In the c series Wittig-Horner

condensation was directly performed on the free alcohol

43c and led after desilylation to 8c.

The analogue 8d was obtained employing a different

strategy (Scheme 5). The following steps were involved

starting from the mixture of 38Rd and 38âd : (i)

substitution of the corresponding tosylate 45 with the

sodium salt derived from the ethoxyethyl ether of

2-methyl-3-butyn-2-ol (NaH, DMSO);²⁸(ii) desilylation

(TBAF, THF) of 46d ; (iii) hydrogenation of 47d using

5% Rh/Al₂O₃in ethyl acetate (90%) to alcohol 48d .

Subsequent oxidation with PDC led to a mixture of 49d

and deprotected 43d . The latter was silylated in situ

using triethylsilyl triflate to 44d and further condensed

with the anion derived from phosphine oxide 15. After

full deprotection analogue 8d was obtained in high yield

(95%).

It is appropriate here to comment upon the stereose-

lectivity of the Wittig-Horner reaction, which is strongly

(or even exclusively) in favor of the formation of the

E-isomer. Only in the case of 44b a substantial amount

of the Z-isomer was isolated (ratio E/Z 7:3). Assignment

of the E-configuration is based on the analysis of the

¹H NMR spectral data. There is an almost perfect

correspondence between the data (shift and coupling

patterns) that are related to the A,seco-B,C-ring part

of the molecule in the case of 8a , 8c, and 8d (see

Experimental Section). Although spin decoupling ex-

periments did not allow for the unambiguous assign-

ment of the protons at C1 and/or C3 and at C4 and/or

C10, the assignments that are given in the Experimen-

tal Section are based on the comparison with the

corresponding spectral data that were obtained for 1R,-

25-(OH)₂-2-methylene-19-norvitamin D₃and that have

been discussed in detail.²⁹Quite surprisingly, NOE

difference spectroscopy involving the vinylic proton at

C7 did not allow for the distinction between the E- and

Z-configuration of the 7,8-double bond. However, the

downfield resonance of the equatorial (â) proton at C9

relative to the resonance of the proton at C14 is in

accord with the E-configuration. This observation, to-

gether with the proven structure of the previtamin

derivative 51 (vide infra), leaves no doubt about the

preferred formation of the E-derivative in the coupling

reaction. In the case of 8b the structural identification

rests on the comparison of the shift values observed for

protons at C9 and C14 in both isomers (see Experimen-

tal Section). The preferred formation of the 7,8E-double

bond is in line with the stereoselectivity that is observed

in the Wittig-Horner reaction of the anion derived from

phosphine oxide 14 with the intact trans-fused perhy-

drindanones (Chart 4).¹⁹Although the different steric

environment of the carbonyl (cf. C9 and C14) is probably

responsible for the latter observed selectivity, the above

results suggest that the quaternary substitution at the

farther removed center C13 could also be important. The

reason this center is determining in the preferred

formation of the E-derivatives (and less so in the case

of the b series) certainly deserves further study.

Diastereomeric Dihydroxyvitamin D Analogues

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3545

Ta ble 4. Biological Activities of 8a -d ^a

Ch a r t 4

binding

calcium

cell differentiation and

proliferation

VDR DBP

Ca serum

(mice)

entry (pig) (human) HL-60 MCF-7 keratinocytes

1

100

45

8

10

9

100

3

0

100

1000

75

10

70

100

2000

75

40

90

100

5000

60

80

85

100

13

0.25

25

8a

8b

8c

8d

a

The activities are presented as relative values, the reference

value of 1,25(OH)₂D₃(1) being defined as 100.

8b-d were e10% compared to that of 1,25(OH)₂D₃

()100% activity). Although 8a possesses a lower binding

affinity for the VDR and exhibits a greater flexibility

than the natural hormone, this analogue demonstrates

superagonistic antiproliferative and prodifferentiating

effects (10-50-fold higher than that of 1,25(OH)₂D₃).

Derivatives 8b-d were less potent than the parent

compound in inhibiting cell proliferation or stimulating

cell differentiation. Interestingly derivative 8c, the C20

epimer of the “natural” analogue 8a , showed decreased

biological activity (50-100 times). This finding is in

contrast with what has been observed in the case of 20-

epi-1,25(OH)₂D₃(2) that shows a 30-fold⁵increase in

cell-differentiating activity. The calcemic activity of all

these analogues was decreased compared to that of 1,-

25(OH)₂D₃, in particular 8b and 8c demonstrated the

lowest calcemic effects (400 times less calcemic than 1,-

25(OH)₂D₃).

The vitamin/previtamin D₃-like equilibrium (see eq

2) was followed for derivative 9a . The synthesis of the

latter is summarized in Scheme 5. It involves, starting

from 38âa , tosylation to 45âa , introduction of the 23-

yne side chain (46âa ), desilylation to 47âa , and PDC

oxidation to yield cyclohexanone 50a . The latter is the

substrate for the classical Wittig-Horner reaction,

which gave, after deprotection, the desired derivative

9a . The 1,7-sigmatropic shift which generates 51 from

Con for m a tion a l An a lysis

As mentioned in the Introduction, the seco-steroid 1

possesses three different parts:³¹a central rigid trans-

fused CD-ring system to which are connected two

flexible entities, the side chain and the six-membered

A-ring attached to C8 via the s-trans-diene portion of

the seco-B-ring. So, in a rough approximation the role

of the central hydrophobic part of the molecule may

consist in isolating the two flexible parts, each carrying

one of the hydroxyl groups, i.e., the 1R-OH and 25- or

24-OH groups, that are essential for recognition by the

receptor protein. Presumably the relative orientation of

these two polar moieties relative to the central parts

which itself probably fits in a hydrophobic pocket of the

receptor proteinsis essential in determining the phar-

macophoric pattern of the molecule.

In view of its flexibility, uncovering the active shape

of the natural hormone is a very difficult task. Indeed,

it has been well-established that flexible ligands can

undergo substantial geometrical distortions away from

their calculated or X-ray structure in order to achieve

a suitable binding conformation.³²Since a greater

preorganization of the active geometry is expected to

result in enhanced binding, a logical step in the search

for new active analogues is the development of mol-

ecules with reduced mobility in either one (or both) of

the flexible parts. With regard to the side-chain portion

of the molecule, this basically consists of constraining

one or several of the five essentially free rotatable

carbon-carbon bonds along the C17-C25 chain. Fol-

lowing this concept 23-yne (e.g. 9, 10) and aryl-

substituted derivatives have been conceived.³³In the

same context, the synthesis, the biological activity, and

the conformational analysis of the four possible 22-

1

9a (eq 3) was followed by H NMR (acetone-d₆, 24 °C).

Starting from pure 9a a 1:1 ratio was observed after

325 h; after 2 months the observed ratio amounted to

23:77 in favor of the previtamin derivative. Following

the decrease in concentration over a period of 7 half-

life values reveals a linear relation from which k ) 3.5

× 10^-7s^-1is computed.³⁰We further note that the

pattern observed for the olefinic proton at C9 in 51 (δ

5.67, m, W_1/2) 8-9 Hz) provides an indirect indication

of the E-geometry of the 7,8-double bond in 9a (cf.

stereoselectivity of the Wittig-Horner reaction).

Biologica l Eva lu a tion

The biological evaluation of analogues 8a -d has

included: (1) determination of binding affinity for the

porcine intestinal VDR and (2) for the human vitamin

D binding protein (hDBP), (3) antiproliferative activity

in vitro on MCF-7 and keratinocyte cell lines and cell-

differentiating activity in vitro on HL-60 cell lines, and

(4) calcemic activity in vivo in vitamin D-replete normal

NMRI mice. Results are given in Table 4. The 6C

analogues 8a -d demonstrated low (3% as compared to

1,25(OH)₂D₃) or no binding affinity for hDBP. Analogue

8a showed 45% of the binding activity for pig VDR,

whereas the affinities of the diastereomeric derivatives

3546 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Zhou et al.

F igu r e 1. Stereoviews of the volume maps representing the conformational behavior of the side chain of 1,25(OH)₂D₃(1) and 6C

analogues 8a -d (truncated model); colored balls indicate positions of O25; energy window: 20 kJ /mol. (Top) Viewing direction

and 1,25(OH)₂D₃(1) with color code. (Middle and bottom) Four diastereomeric 6C analogues 8a -d : middle left, 17R,20R analogue

8a ; bottom left, 17R,20S analogue 8c; middle right, 17S,20S analogue 8d ; bottom right, 17S,20R analogue 8b.

methyl-substituted analogues 6a -d , diastereomeric at

C20 and C22, have been recently reported.¹¹Among

these four, analogue 6c was found to possess the highest

binding affinity for the VDR and a substantially higher

potency than 1,25(OH)₂D₃(1) to induce differentiation

of HL-60 cells.

In contrast to the case of 6, where the introduction of

an extraneous 22-methyl group reduces the mobility of

the side chain, which is reflected by a rather well-

defined and restricted occupation volume for the side

chain,^11athe present case deals with 6C analogues 8

that, by virtue of the absence of the trans-fused five-

membered D-ring, exhibit a greater flexibility than the

natural hormone. In this respect, the superagonistic

activity of 8a compared to 1 is remarkable. The develop-

ment of analogues exhibiting profound structural changes

in the CD-portion of the molecule was inspired by the

search for derivatives that would exhibit a dissociation

in the calcemic and other activities.³⁴However, the

discovery of analogues possessing superagonistic activ-

ity was unexpected. Interestingly, the result of the

biological evaluation (Table 4) suggests that analogue

8a may undergo the necessary geometrical changes to

achieve a suitable binding conformation and that this

particular geometry is not available for the diastereo-

meric derivatives 8b-d (or at least substantially less).

This hypothesis led us to examine in detail the confor-

mational profile of the side chain in 8a -d using mo-

lecular mechanics calculations. In this context, the

Riverside group has used the dot map concept for

defining the region in space that is available for occupa-

tion by the flexible side chain.¹⁰This has revealed in

the case of the couples 1 and 2 and the 20-epimers of 5

substantial differences in conformational behavior, which

might be determining in the rationalization of the

observed differences in biological activity. In the same

context, we have recently reported on the use of volume

maps in the conformational analysis of vitamin D

Diastereomeric Dihydroxyvitamin D Analogues

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3547

F igu r e 2. Stereoviews of the relative activity volumes determined for 8a and 6c and of the volume map of 22-oxa-1,25(OH)₂D₃

(5): left, view as used in Figure 1; right, view similar to the one used in refs 11a and 38. (Top) Relative activity volume resulting

from subtraction of the 6C analogue 8c from 8a (volume maps of 8a and 8c shown in Figure 1). (Middle) Relative activity volume

resulting from subtraction of the 22-methyl-substituted derivative 6d from 6c (volume maps of 6c and 6d not shown). (Bottom)

22-Oxa-1,25(OH)₂D₃(5) with indication of the A-, EA-, and F-regions (see refs 11a and 38).

analogues.³⁵In particular, volume maps constitute an

extension of the dot map procedure with enhanced

possibility for visualization of the conformational be-

havior of the side chain. Figure 1 shows the volume

maps resulting from the analysis of the side chain of

1,25(OH)₂D₃(1) and of derivatives 8a -d taking into

account all local minimum energy conformations found

within 20 kJ /mol of the global minimum. In these maps

the molecule is represented using a gray ball-and-stick

model corresponding to the global minimum energy form

viewed along an axis perpendicular to the C13-C18

bond in line with C9 in front (Figure 1, top left). Typical

of the procedure is (1) the replacement of the dots used

to indicate the positions of O25 by balls to create an

3548 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Zhou et al.

occupation volume for the side chain and (2) the use of

colors to identify particular energy windows. Within the

used color palette (yellow-brown) the four different

nuances correspond to energy windows of 0-5, 5-10,

10-15, and 15-20 kJ /mol, respectively, the brightest

color indicating the most stable forms (Figure 1, top

right). It is immediately apparent that the flexibility of

analogues 8a -d (1013, 1375, 1408, and 1037 conforma-

tions, respectively) compared to, for example, the parent

hormone 1 (366 conformations; Figure 1, top) is much

greater and that their side chain can occupy regions in

space not accessible to 1 (at least within the given

energy window). Further inspection of the four maps

reveals the pseudo-enantiomeric relationship that exists

between 8a and 8d and between 8c and 8b, respectively.

tion could be responsible for the observed biological

activity and could thus correspond to the orientation of

the side chain in the active conformation of the com-

pound.³⁷

In conclusion, we have synthesized four conforma-

tionally flexible derivatives (8a -d ) of 1,25(OH)₂D₃(1),

lacking C15 and C19 and diastereomeric at C17 and

C20. Biological evaluation showed an unexpected su-

peragonistic antiproliferative and prodifferentiating

activity for diastereomer 8a with the “natural” config-

uration at C17 and C20. The conformational behavior

of the side chain of 8a -d was studied using molecular

mechanics calculations, and the result is presented as

volume maps. From the analysis of the relative activity

volume obtained by subtraction of the volume map of

8c from 8a , we suggest an orientation of the side chain

of 8a which might correspond to the active conformation

of the compound.

We have also proposed a volume subtraction proce-

dure for the determination of a relative activity volume,

a region in space corresponding to the preferred occupa-

tion of the side chain of the more active analogue of a

pair as compared to the inactive one.³⁶To determine a

relative activity volume for 8a , the volume map of the

least active 8c was subtracted from the active 8a (see

Experimental Section for details). This resulted in the

reduced volume map presented in Figure 2 in two

stereoscopic views (top)³⁷and which consists of 177

conformations of 8a (17% of the original 1013 conforma-

tions (0-20 kJ /mol), 72 mol % according to a Boltzmann

distribution at 298 K). It shows that within the given

energy window the side chain of 8a can occupy three

regions in space which are not or less accessible to the

side chain of 8c. Subtraction of the volume maps of

respectively 8b and 8d from the one of 8a led to similar

reduced volumes showing the same three regions,

although less well-defined (not shown).

Exp er im en ta l Section

Syn th esis. All air-sensitive reactions were run under Ar

or N₂atmosphere, and reagents were added through septa

using oven-dried syringes. Et₂O and THF were distilled from

benzophenone ketyl prior to use. Pyridine, Et₃N, i-Pr₂NH, CH₃-

CN, DMF, HMPA, and DMSO were distilled from CaH₂.

Hexane was distilled from Na, and CH₂Cl₂was distilled from

P₂O₅. TLC were run on glass plates precoated with silica gel

(Merck UV 254 Å). Column and flash chromatography was

performed on silica gel (Merck, 70-325 or 230-400 mesh), and

HPLC separations were performed on Bio-Sil D 90-10, 10-

µm columns (Bio-Rad) of 1 × 25 cm and 2.2 × 25 cm. IR spectra

were obtained on a Perkin-Elmer 1600 series FTIR spectro-

photometer using KBr plates with product film or solution in

CH₂Cl₂. NMR spectra were recorded in CDCl₃(unless other-

wise stated) on a Bruker AM-360 or AM-500 spectrometer and

are reported relative to CDCl₃(δ 7.26) as an internal standard.

Mass spectra were recorded on a Finnigan 4000 or HP-5988

spectrometer at 70 eV. Optical rotations were measured on a

Perkin-Elmer 241 polarimeter.

(2R,3R)-3,7-Dim eth yl-2-((1S)-1-m eth yl-2-cycloh exen yl)-

6-octen oic Acid (19a ) a n d (2S,3R)-3,7-Dim eth yl-2-((1S)-

1-m et h yl-2-cycloh exen yl)-6-oct en oic Acid (19b ). To a

stirred solution of (R)-(+)-citronellic acid (17; 0.98 g, 5.76

mmol) in CH₂Cl₂(50 mL) were added (R)-3-methyl-2-cyclo-

hexen-1-ol (16, 84% ee; 0.64 g, 5.76 mmol), DCC (2.96 g, 14.4

mmol), and DMAP (0.73 g, 6 mmol) at 0 °C under N₂

atmosphere. After 5 min the reaction mixture was allowed to

warm to room temperature and was stirred for 18 h. EtOH (4

mL) and AcOH (4 mL) were added at 0 °C. The mixture was

stirred at 0 °C for 20 min and at room temperature for 1 h.

Et₂O was added, and the formed white solid was removed by

filtration. The filtrate was evaporated under reduced pressure,

and the residual liquid was dissolved in Et₂O. The solution

was washed with H₂O, dried over anhydrous MgSO₄, and

evaporated under reduced pressure. The crude material was

chromatographed on silica gel (hexanes-EtOAc 40:1) to give

18 (1.521 g, 96%).

In applying the same procedure to the case of the 22-

methyl-substituted derivatives 6, we found that sub-

traction of the volume in space available to the inactive

analogue 6d from the space available for occupation by

the side chain of the active analogue 6c led to a rather

reduced volume that could correspond to the orientation

of the side chain in the active conformation of the

compound. Stereoscopic images of the so-obtained rela-

tive activity volume of 6c, formed by 38 conformations

(18% of the original 210 conformations (0-20 kJ /mol),

88 mol % according to a Boltzmann distribution at 298

K) are shown in Figure 2 (middle).

In a recent study of side-chain analogues, Yamada

and co-workers³⁸found a strong correlation between the

occupation of a region, denoted by the authors as the

EA-region (see Figure 2, bottom), and the cell-dif-

ferentiating potency of the analogue, the side chains of

almost all potent analogues being distributed around

this EA-region. They also defined a so-called F-region

(more or less in front of the EA-region when viewed

along the direction shown in Figure 2 on the right) that

is typically occupied by the 25-oxygen of analogues

possessing moderately high cell-differentiating activity

and low calcemic activity, such as 22-oxa-1,25(OH)₂D₃

(5). The conformational profile of the latter analogue is

also shown in Figure 2 (bottom). Comparison of the

reduced volume map of 8a with the reduced volume map

of 6c and with the F-region shown for 5 suggests that,

of the three populations of side-chain conformations

found in the reduced volume map of 8a , the F-popula-

To a stirred solution of i-Pr₂NH (456 µL, 3.27 mmol) in THF

(10 mL) was added n-BuLi (2.45 M solution in hexane, 1.33

mL, 3.27 mmol) at -15 °C under N₂atmosphere. The reaction

mixture was stirred for 20 min, HMPA (3 mL) was added, and

the mixture was cooled to -78 °C. A solution of ester 18 (0.77

g, 2.92 mmol) in THF (2 mL) was slowly added, and after 10

min the reaction mixture was allowed to warm to -50 °C and

stirred for 20 min. Solid TBDMSCl (491 mg, 3.27 mmol) was

added, and stirring was continued for 20 min. The reaction

mixture was allowed to warm to room temperature, stirred

for 3 h, and then refluxed for 16 h. After cooling an aqueous

5% HCl solution (15 mL) was added; the mixture was stirred

at room temperature for 1 h and extracted twice with Et₂O.

The combined extracts were washed with H₂O, dried over

Diastereomeric Dihydroxyvitamin D Analogues

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3549

anhydrous MgSO₄, and evaporated under reduced pressure.

The residual oil was chromatographed on silica gel (hexanes-

EtOAc, 25:1 to 5:1) to give an unseparable mixture of 19a and

19b (>10:1; 448 mg, 58%): R_f(hexanes-EtOAc, 5:1) 0.35; IR

(film) 2930, 1704, 1462, 1381, 1285, 1253, 1202 cm^-1; ¹H NMR

(360 MHz) δ 5.62 (1 H, m), 5.41 (1 H, d, J ) 11.0 Hz), 5.09 (1

H, t, J ) 6.9 Hz), 2.22 (1 H, d, J ) 5.9 Hz), 2.08 (1 H, m), 1.90

(3 H, m), 1.68 (3 H, s), 1.58 (3 H, s), 1.62 (7 H, m), 1.10 (3 H,

s), 1.02 (3 H, d, J ) 6.8 Hz) ppm; MS m/z 264 (M⁺, 5), 249 (1),

221 (1), 208 (5), 154 (15), 109 (15), 96 (100). Anal. Calcd for

0.88 (3 H, d, J ) 7.0 Hz), 0.80 (3 H, d, J ) 7.1 Hz) ppm; MS

m/z 252 (M⁺, 1), 95 (100). Anal. Calcd for C₁₇H₃₂O: C, 80.95;

H, 12.70. Found: C, 81.10; H, 12.95.

(3S)-3-Meth yl-3-((1R,2R)-6-tr ieth ylsilyloxy-1,2,6-tr im -

eth ylh ep tyl)cycloh exa n on e (26a ). To a solution of alcohol

23a (50 mg, 0.2 mmol) in dry DMF (3 mL) were successively

added Et₃SiCl (90 mg, 0.6 mmol), imidazole (54 mg, 0.8 mmol),

and DMAP (10 mg). The reaction mixture was stirred at room

temperature for 20 h and then extracted with Et₂O. The

combined extracts were washed with an aqueous 5% HCl

solution, dried over anhydrous MgSO₄, and evaporated under

reduced pressure. The residue was chromatographed on silica

gel (hexanes-EtOAc, 98:2) to give 24a (67 mg, 92%).

To a solution of cyclohexene 24a (121 mg, 0.33 mmol) in

THF (8 mL) was added 9-BBN (0.5 M solution in THF, 6.6

mL, 3.3 mmol), and the reaction mixture was refluxed for 30

h. EtOH (1 mL), a 6 M NaOH solution (0.8 mL), and a 30%

H₂O₂solution (1.6 mL) were added; the mixture was heated

at 50 °C for 1 h and then extracted with Et₂O. The combined

extracts were washed with an aqueous 5% HCl solution, dried

over anhydrous MgSO₄, and evaporated under reduced pres-

sure. The residue was chromatographed on silica gel (hex-

anes-EtOAc, 98:2) to give 25a (121 mg, 95%).

A mixture of cyclohexanol 25a (121 mg, 0.34 mmol) and PDC

(480 mg, 1.2 mmol) in CH₂Cl₂(9 mL) was stirred at room

temperature for 20 h and then filtered through a short pad of

silica gel. The crude product was purified by HPLC (hexanes-

EtOAc, 20:1) to give 26a (97 mg, 80%): IR (film) 2958, 1722,

1461, 1381, 1282, 1234, 1042 cm^-1; ¹H NMR (500 MHz) δ 2.28

(1 H, d, J ) 13.2 Hz), 2.26 (2 H, m), 2.06 (1 H, dt, J ) 13.2,

1.6 Hz), 1.90 (1 H, m), 1.83 (1 H, m), 1.67 (3 H, m), 1.18 (6 H,

s), 0.93 (9 H, t, J ) 8.0 Hz), 0.90 (3 H, d, J ) 6.9 Hz), 0.86 (3

H, s), 0.79 (3 H, d, J ) 7.2 Hz), 0.56 (6 H, q, J ) 8.0 Hz) ppm;

MS m/z 354 (M⁺, 10), 353 (5), 173 (30), 111 (60), 55 (100).

Cou p lin g Rea ction of Cycloh exa n on es 26a , 43c, 44b,

44d , a n d 50a w ith P h osp h in e Oxid es 14 a n d 15sGen er a l

P r oced u r e. To a solution of A-ring phosphine oxide 14 (15)

(0.134 mmol) in dry THF (1.5 mL) was dropwise added n-BuLi

(2.5 M solution in hexane, 48 µL, 0.120 mmol) at -78 °C under

N₂atmosphere. The formed dark red solution was stirred at

-78 °C for 1 h after which a solution of the C8 cyclohexanone

(0.034 mmol) in dry THF (0.50 mL) was added. The still red

solution was stirred at -78 °C for 2 h and was then allowed

to warm to room temperature. The reaction mixture was

directly loaded onto a silica gel column, and the reaction

product was further purified by HPLC to give the A-ring

coupled product.

C

₁₇H₂₈O₂: C, 77.27; H, 10.61. Found: C, 77.37; H, 10.43.

(2R,3R)-3,7-Dim eth yl-2-((1S)-1-m eth yl-2-cycloh exen yl)-

6-octen -1-ol (20a ). To a suspension of LiAlH₄(302 mg, 7.95

mmol) in THF (10 mL) was added a solution of the above acids

19a and 19b (420 mg, 1.59 mmol) in THF (5 mL), and the

reaction mixture was refluxed for 48 h. Excess LiAlH₄was

destroyed by addition of an aqueous 5% HCl solution, and the

mixture was repeatedly extracted with Et₂O. The combined

extracts were washed with H₂O, dried over anhydrous MgSO₄,

and evaporated under reduced pressure. The residue was

chromatographed on silica gel (hexanes-EtOAc, 20:1 to 10:1)

to afford a mixture of 20a and 20b (>10:1; 344 mg, 86%) which

was further separated by HPLC (hexanes-EtOAc, 6:1) to give

pure 20a : R_f(hexanes-EtOAc, 5:1) 0.35; IR (film) 3339, 2928,

2871, 1454, 1376, 1028 cm^-1; ¹H NMR (500 MHz) δ 5.67 (1 H,

dt, J ) 10.1, 3.5 Hz), 5.53 (1 H, dt, J ) 10.1, 1.8 Hz), 5.12 (1

H, tt, J ) 6.8, 1.6 Hz), 3.78 (1 H, m), 3.72 (1 H, m), 2.06 (1 H,

m), 1.93 (2 H, m), 1.88 (1 H, tt, J ) 7.8, 7.8 Hz), 1.70 (2 H. m),

1.68 (3 H, s), 1.61 (2 H, m), 1.59 (3 H, s), 1.50 (1 H, m), 1.40 (1

H, ddd, J ) 10.5, 4.5, 1.2 Hz), 1.33 (1 H, m), 1.28 (2 H, m),

1.13 (1 H, m), 1.13 (3 H, d, J ) 7.0 Hz), 1.01 (3 H, s) ppm; MS

m/z 250 (M⁺, 5), 232 (3), 219 (10), 137 (40), 95 (100).

(2R,3R)-3,7-Dim eth yl-2-((1S)-1-m eth yl-2-cycloh exen yl)-

6-octen e (22a ). To a solution of alcohol 20a (250 mg, 1 mmol)

in pyridine (15 mL) was added TsCl (572 mg, 3 mmol). The

light yellow solution was stirred at room temperature for 17

h and then poured into ice water. The mixture was repeatedly

extracted with Et₂O, and the combined extracts were washed

with an aqueous 5% HCl solution and H₂O, dried over

anhydrous MgSO₄, and evaporated under reduced pressure.

The residue was chromatographed on silica gel (hexanes-

EtOAc, 50:1 to 36:1) to give tosylate 21a (>95% yield)

contaminated with TsCl.

To a suspension of LiAlH₄(342 mg, 9 mmol) in THF (30

mL) was added a solution of the above tosylate 21a in THF (5

mL), and the reaction mixture was refluxed for 2 h. Excess

LiAlH₄was destroyed by addition of EtOH, and the mixture

was repeatedly extracted with Et₂O. The combined extracts

were washed with an aqueous 5% HCl solution and H₂O, dried

over anhydrous MgSO₄, and evaporated under reduced pres-

sure. The residue was chromatographed on silica gel (hexane)

to give 22a (222 mg, 95%): IR (film) 2925, 2865, 1647, 1453,

(1R,3R)-5-((Z,2E)-2-((3S)-3-((1R,2R)-6-Hyd r oxy-1,2,6-tr i-

m e t h ylh e p t yl)-3-m e t h ylcycloh e xylid e n e )e t h ylid e n e )-

cycloh exa n e-1,3-d iol (8a ). Cyclohexanone 26a was coupled

with phosphine oxide 15 according to the above general

procedure. The crude product was purified by HPLC (hex-

anes-EtOAc, 200:1) to give protected 8a (85%).

1

1378 cm^-1; H NMR (500 MHz) δ 5.58 (1 H, dt, J ) 10.2, 3.5

Hz), 5.45 (1 H, dm, J ) 10.2 Hz), 5.12 (1 H, br t, J ) 7.0 Hz),

2.02 (1 H, m), 1.92 (2 H, m), 1.85 (1 H, tt, J ) 15.4, 7.9 Hz),

1.69 (1 H, m), 1.68 (3 H, s), 1.59 (3 H, s), 1.56 (2 H, m), 1.46 (1

H, m), 1.35 (1 H, m), 0.93 (3 H, s), 0.88 (3 H, d, J ) 6.8 Hz),

0.78 (3 H, d, J ) 7.1 Hz) ppm; MS m/z 234 (M⁺, 1), 57 (100).

To a solution of protected 8a (52 mg, 0.07 mmol) in THF (2

mL) was added TBAF (1 M solution in THF, 1.12 mL, 1.12

mmol); the mixture was stirred at room temperature for 14 h

and then directly loaded onto a silica gel column (CH₂Cl₂-

MeOH, 20:1). The product was further purified by HPLC (CH₂-

Cl₂-MeOH, 20:1) to give 8a (26 mg, 93%): UV (MeOH) λ_max

(6R,7R)-2,6,7-Tr im eth yl-7-((1S)-1-m eth yl-2-cycloh exe-

n yl)h ep ta n -2-ol (23a ). To a suspension of Hg(OAc)₂(346 mg,

1.09 mmol) in H₂O (1.25 mL) and THF (1.25 mL) was added

a solution of alkene 22a (212 mg, 0.906 mmol) in THF (2.5

mL), and the reaction mixture was stirred at room tempera-

ture for 2 h. A 3 M NaOH solution (1.09 mL) followed by a 1

M NaBH₄solution in 3 M NaOH (1.09 mL) were added; the

mixture was stirred for 10 min and then repeatedly extracted

with Et₂O. The combined extracts were washed with brine,

dried over anhydrous MgSO₄, and evaporated under reduced

pressure. The residue was chromatographed on silica gel

(hexanes-EtOAc, 20:1 to 5:1) to give alcohol 23a (154 mg,

249.5 nm; IR (film) 3382, 2935, 1615, 1454, 1380, 1048 cm^-1

;

¹H NMR (500 MHz) δ 6.26 (1 H, d, J ) 11.2 Hz, H-6), 5.94 (1

H, d, J ) 11.2 Hz, H-7), ∼4.09 (2 H, m, H-1 and H-3), 2.67 (1

H, dd, J ) 13.3, 3.8 Hz, H-10â), 2.49 (1 H, dd, J ) 13.3, 3.7

Hz, H-4R), 2.43 (1 H, m, H-9â), 2.29 (1 H, dd, J ) 13.3, 7.6

Hz, H-10R), 2.19 (1 H, dd, J ) 13.3, 6.6 Hz, H-4â), 2.04 (1 H,

d, J ) 13.0 Hz, H-14â), 2.00 (1 H, m), 1.90 (1 H, m), 1.86 (1 H,

m), 1.84 (1 H, d, J ) 13.0 Hz, H-14R), 1.70 (1 H, m), 1.56 (3 H,

m), 1.21 (6 H, s), 0.89 (3 H, d, J ) 6.9 Hz), 0.77 (3 H, d, J )

7.3 Hz), 0.76 (3 H, s) ppm; MS m/z 392 (M⁺, 1).

(1R)-3-Meth yl-2-cycloh exen yl (3S)-4-Ben zyloxy-3-m e-

t h ylb u t a n oa t e (28a ) a n d (1R)-3-Met h yl-2-cycloh exen yl

(3R)-4-Ben zyloxy-3-m eth ylbu ta n oa te (28c). Esterification

of (R)-3-methyl-2-cyclohexen-1-ol (16, 80% ee) with (S)-(-)-4-

benzyloxy-3-methylbutanoic acid (27a , 99% ee) was carried out

1

67%): IR (film) 3364, 2963, 2867, 1462, 1379, 1153 cm^-1; H

NMR (500 MHz) δ 5.58 (1 H, dt, J ) 10.3, 3.5 Hz), 5.46 (1 H,

dm, J ) 10.3 Hz), 1.93 (2 H, m), 1.70 (1 H, m), 1.58 (4 H, m),

1.50-1.34 (5 H, m), 1.26 (2 H, m), 1.21 (6 H, s), 0.94 (3 H, s),

3550 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Zhou et al.

as described for the preparation of 18. Column chromatogra-

phy on silica gel (hexanes-EtOAc, 75:1 to 25:1) followed by

HPLC (hexanes-EtOAc, 25:1) gave 28a (98%) as a colorless

oil. In an analogous way esterification of 16 with (R)-(-)-4-

benzyloxy-3-methylbutanoic acid (27c, 99% ee) afforded 28c

4.51 (1 H, ABd, J ) 11.9 Hz), 3.80 (1 H, ddd, J ) 12.0, 8.2, 3.7

Hz), 3.73 (1 H, ddd, J ) 12.0, 8.1, 5.6 Hz), 3.70 (1 H, ddd, J )

11.4, 5.8, 3.2 Hz), 3.54 (1 H, ddd, J ) 11.4, 8.0, 5.4 Hz), 2.08

(1 H, m), 1.90 (2 H, m), 1.49 (1 H, ddd, J ) 5.7, 3.6, 2.1 Hz),

1.13 (3 H, d, J ) 7.4 Hz), 1.03 (3 H, s) ppm; MS m/z 288 (M⁺,

1), 257 (1), 191 (1), 176 (5), 161 (5), 135 (4), 121 (7), 107 (8), 95

(92%). Data of 28a : R_f(hexanes-EtOAc, 5:1) 0.50; [R]_D

)

(100). Data of 30c: R_f(hexanes-EtOAc, 2:1) 0.42; [R]_D

)

+86.7 (c 0.96, CHCl₃); IR (film) 3067, 3030, 2924, 2864, 1726,

1672, 1496, 1453, 1377, 1249, 1181, 1097, 1026 cm^-1; ¹H NMR

(500 MHz) δ 7.33 (5 H, m), 5.44 (1 H, m), 5.25 (1 H, m), 4.50

(2 H, s), 3.36 (1 H, dd, J ) 9.2, 5.9 Hz), 3.31 (1 H, dd, J ) 9.2,

7.2 Hz), 2.48 (1 H, dd, J ) 15.0, 5.9 Hz), 2.32 (1 H, m), 2.14 (1

H, dd, J ) 15.0, 8.0 Hz), 1.95 (2 H, m), 1.74 (2 H, m), 1.70 (3

H, s), 1.65 (2 H, m), 0.99 (3 H, d, J ) 6.8 Hz) ppm; MS m/z

302 (M⁺, 2), 273 (2), 247 (1), 207 (12), 192 (3), 181 (2), 149 (3),

111 (88), 95 (50), 91 (100). Data of 28c: R_f(hexanes-EtOAc,

5:1) 0.50; [R]_D) +100.4 (c 1.01, CHCl₃); IR (film) 3056, 3029,

2934, 2865, 1727, 1671, 1496, 1454, 1378, 1248, 1111, 1096

cm^-1; ¹H NMR (500 MHz) δ 7.33 (5 H, m), 5.43 (1 H, m), 5.24

(1 H, m), 4.51 (1 H, ABd, J ) 12.4 Hz), 4.49 (1 H, ABd, J )

12.4 Hz), 3.35 (1 H, dd, J ) 9.1, 5.9 Hz), 3.30 (1 H, dd, J )

9.1, 6.8 Hz), 2.47 (1 H, dd, J ) 12.0, 6.1 Hz), 2.31 (1 H, m),

2.15 (1 H, dd, J ) 14.9, 7.9 Hz), 1.94 (2 H, m), 1.74 (2 H, m),

1.70 (3 H, s), 1.65 (2 H, m), 0.99 (3 H, d, J ) 6.8 Hz) ppm; MS

m/z 302 (M⁺, 2), 273 (2), 247 (1), 207 (12), 192 (3), 181 (2), 149

(3), 111 (88), 95 (50), 91 (100). Anal. Calcd for C₁₉H₂₆O₃: C,

75.46; H, 8.67. Found: C, 75.43; H, 8.80.

-2.53, [R]₃₆₅) -8.59 (c 0.40, CHCl₃); IR (film) 3420, 3056,

3016, 2956, 2938, 2865, 1453, 1363, 1273, 1092, 1045, 1027

cm^-1; ¹H NMR (500 MHz) δ 7.33 (5 H, m), 5.62 (1 H, ddd, J )

10.2, 3.5, 3.5 Hz), 5.39 (1 H, d, J ) 10.2 Hz), 4.57 (1 H, ABd,

J ) 12.4 Hz), 4.52 (1 H, ABd, J ) 12.4 Hz), 3.74 (1 H, dd, J )

10.8, 3.1 Hz), 3.56 (1 H, dd, J ) 10.1, 9.2 Hz), 3.45 (1 H, dd, J

) 8.7, 4.0 Hz), 3.35 (1 H, dd, J ) 8.7, 3.6 Hz), 2.15 (1 H, m),

1.92 (2 H, m), 1.60 (4 H, m), 1.09 (3 H, d, J ) 7.3 Hz), 1.03 (3

H, s) ppm; MS m/z 288 (M⁺, 1), 257 (1), 191 (6), 176 (5), 161

(5), 135 (4), 121 (7), 107 (8), 95 (100).

Isom er iza tion Sequ en ce of Alcoh ol 30a to 30b a n d 30c

to 30d . To a solution of 30a (300 mg, 1.04 mmol) in DMSO (8

mL) and CH₂Cl₂(4 mL) were added Et₃N (527 µL) and sulfur

trioxide pyridine complex (497 mg, 3.13 mmol) at -15 °C. After

stirring for 1.5 h at -10 to -4 °C, the reaction mixture was

poured into Et₂O and brine. The organic phase was dried over

anhydrous MgSO₄and evaporated under reduced pressure.

The residue was purified by HPLC (hexanes-EtOAc, 10:1) to

afford aldehyde 31a (242 mg, 81%) as a colorless oil. In an

analogous way 30c gave aldehyde 31c (97%).

(2R,3S)-4-Ben zyloxy-3-m eth yl-2-((1S)-1-m eth yl-2-cyclo-

h exen yl)bu ta n oic Acid (29a ) a n d (2R,3R)-4-Ben zyloxy-

3-m eth yl-2-((1S)-1-m eth yl-2-cycloh exen yl)bu ta n oic Acid

(29c). The Ireland-Claisen rearrangement of ester 28a was

carried out according to the procedure described for 18. The

crude material was purified by HPLC (hexanes-EtOAc, 5:1)

to give a mixture of 29a and 29b (ratio 14:1, 78%) as a colorless

oil. In an analogous way 28c gave a mixture of 29c and 29d

(ratio 29:1, 64%). Data of 29a : R_f(hexanes-EtOAc, 5:1) 0.23;

IR (film) 3500-3200, 3056, 3015, 2931, 2862, 1700, 1646, 1615,

1451, 1421, 1369, 1256, 1200, 1154, 1097, 1026 cm^-1; ¹H NMR

(500 MHz) δ 7.33 (5 H, m), 5.63 (1 H, ddd, J ) 9.0, 4.8, 2.7

Hz), 5.40 (1 H, m), 4.50 (2 H, s), 3.52 (1 H, dd, J ) 9.4, 3.8

Hz), 3.42 (1 H, dd, J ) 9.4, 6.1 Hz), 2.39 (1 H, d, J ) 4.3 Hz),

2.18 (1 H, m), 1.94 (1 H, m), 1.90 (1 H, ddd, J ) 8.7, 5.6, 2.7

Hz), 1.14 (3 H, d, J ) 7.2 Hz), 1.11 (3 H, s) ppm; MS m/z 302

(M⁺, 1), 253 (1), 221 (3), 206 (4), 167 (2), 149 (8), 111 (12), 95

(95), 91 (100). Data of 29c: R_f(hexanes-EtOAc, 5:1) 0.23; IR

(film) 3500-2500, 3056, 3027, 2935, 2873, 1704, 1496, 1454,

1416, 1366, 1256, 1206, 1102 cm^-1; ¹H NMR (500 MHz) δ 7.33

(5 H, m), 5.64 (1 H, ddd, J ) 10.1, 7.7, 3.2 Hz), 5.43 (1 H, br

d, J ) 10.3 Hz), 4.55 (1 H, ABd, J ) 13.0 Hz), 4.51 (1 H, ABd,

J ) 13.0 Hz), 3.46 (1 H, dd, J ) 9.1, 5.7 Hz), 3.40 (1 H, dd, J

) 9.1, 5.6 Hz), 2.53 (1 H, d, J ) 3.2 Hz), 2.19 (1 H, m), 1.93 (2

H, m), 1.77 (1 H, m), 1.64 (3 H, m), 1.13 (3 H, s), 1.05 (3 H, d,

J ) 7.0 Hz) ppm; MS m/z 302 (M⁺, 1), 253 (1), 221 (3), 206 (4),

167 (2), 149 (8), 111 (12), 95 (95), 91 (100).

To a solution of 31a (230 mg, 0.8 mmol) in CH₂Cl₂(20 mL)

was added DBU (915 µL, 5.9 mmol). The reaction mixture was

stirred at room temperature for 62 h and concentrated under

reduced pressure, and the residue was dissolved in Et₂O. The

solution was washed with brine, dried over anhydrous MgSO₄,

and evaporated under reduced pressure. The residue was

chromatographed on silica gel to give a mixture of 31a and

the epimeric aldehyde 31b (ratio 55:45, 225 mg, 98%). In an

analogous way 31c led to a mixture of aldehydes 31c and 31d

(ratio 63:37, 100%).

To a solution of the aldehyde mixture 31a and 31b (240 mg,

0.9 mmol) in MeOH (4.5 mL) was portionwise added NaBH₄

(136 mg, 3.6 mmol) at -10 °C, and stirring was continued for

15 min. Brine was added, and the mixture was extracted with

Et₂O. The combined organic extracts were dried over anhy-

drous MgSO₄and evaporated under reduced pressure. The

residual material was purified by HPLC (hexanes-EtOAc, 5:1)

to give 30a and 30b (227 mg, 95%) which were further

separated to give pure 30b. In an analogous way 31c and 31d

led to a mixture of 30c and 30d (88%) and further separation

to pure 30d . Data of 30b: R_f(hexanes-EtOAc, 2:1) 0.42; [R]_D

) +24.7, [R]₃₆₅) +79.0 (c 0.33, CHCl₃); IR (film) 3410, 3062,

3011, 2926, 2857, 1649, 1598, 1456, 1362, 1090, 1044 cm^-1

;

¹H NMR (500 MHz) δ 7.33 (5 H, m), 5.62 (1 H, ddd, J ) 9.0,

3.0, 4.5 Hz), 5.46 (1 H, br d, J ) 10.2 Hz), 4.57 (1 H, ABd, J )

12.0 Hz), 4.52 (1 H, ABd, J ) 12.0 Hz), 3.70 (1 H, m), 3.59 (1

H, m), 3.49 (1 H, dd, J ) 8.8, 4.1 Hz), 3.40 (1 H, dd, J ) 9.1,

3.0 Hz), 2.19 (1 H, dd, J ) 7.8, 3.7 Hz), 1.90 (2 H, m), 1.60 (2

H, m), 1.50 (2 H, m), 1.39 (1 H, m), 1.06 (3 H, d, J ) 7.3 Hz),

1.00 (3H, s) ppm; MS m/z 288 (M⁺, 1), 257 (1), 191 (6), 176 (5),

161 (5), 135 (4), 121 (7), 107 (8), 95 (100). Data of 30d : R_f

(hexanes-EtOAc, 2:1) 0.42; [R]_D) +15.88, [R]₃₆₅) +54.90 (c

0.59, CHCl₃); IR (film) 3400, 3062, 3011, 2929, 2857, 1593,

1455, 1362, 1095, 1045 cm^-1; ¹H NMR (500 MHz) δ 7.33 (5 H,

m), 5.60 (1 H, ddd, J ) 10.4, 4.5, 2.8 Hz), 5.00 (1 H, br d, J )

10.4 Hz), 4.55 (1 H, ABd, J ) 11.9 Hz), 4.50 (1 H, ABd, J )

11.9 Hz), 3.74 (1 H, m), 3.72 (1 H, dd, J ) 6.3, 3.1 Hz), 3.70 (1

H, dd, J ) 6.5, 3.2 Hz), 3.53 (1 H, ddd, J ) 7.0, 4.3, 1.1 Hz),

2.10 (1 H, s), 1.90 (2 H, m), 1.58 (4 H, m), 1.17 (3 H, d, J ) 7.4

Hz), 1.00 (3 H, s) ppm; MS m/z 288 (M⁺, 1), 257 (1), 191 (6),

176 (5), 161 (5), 135 (4), 121 (7), 107 (8), 95 (100).

(2R,3S)-4-Ben zyloxy-3-m eth yl-2-((1S)-1-m eth yl-2-cyclo-

h exen yl)b u t a n -1-ol (30a ) a n d (2R,3R)-4-Ben zyloxy-3-

m eth yl-2-((1S)-1-m eth yl-2-cycloh exen yl)bu ta n -1-ol (30c).

To a suspension of LiAlH₄(628 mg, 16.56 mmol) in THF (40

mL) was added a solution of the mixture of acids 29a and 29b

(14:1; 1.0 g, 3.31 mmol) in THF (20 mL), and the reaction

mixture was refluxed for 43 h. Excess LiAlH₄was destroyed

by addition of an aqueous 5% HCl solution, and the mixture

was repeatedly extracted with Et₂O. The combined extracts

were washed with H₂O and brine, dried over anhydrous

MgSO₄, and evaporated under reduced pressure. The residue

was chromatographed on silica gel to afford a mixture of 30a

and 30b (0.91 g, 95%) which was further separated by HPLC

(hexanes-EtOAc, 5:1) to give pure 30a . In an analogous way

the mixture of acids 29c and 29d led to 30c and 30d (96%),

and further HPLC separation gave pure 30c. Data of 30a : R_f

(hexanes-EtOAc, 2:1) 0.42; [R]_D) +25.2, [R]₃₆₅) +78.6 (c

3.98, CHCl₃); IR (film) 3440, 3088, 3064, 3011, 2929, 2865,

1489, 1463, 1448, 1363, 1205, 1097, 1069, 1027 cm^-1; ¹H NMR

(500 MHz) δ 7.33 (5 H), m), 5.62 (1 H, ddd, J ) 9.0, 4.4, 3.0

Hz), 5.43 (1 H, d, J ) 10.2 Hz), 4.56 (1 H, ABd, J ) 11.9 Hz),

(2S,3R)-4-Ben zyloxy-3-m eth yl-2-((1S)-1-m eth yl-2-cyclo-

h exen yl)bu ta n oic Acid (29d ). To a stirred, ice-cooled solu-

tion of alcohol 30d (40 mg, 0.14 mmol) in acetone (1 mL) was

added dropwise J ones reagent (162 µL) until the mixture

turned red-brown. Excess reagent was destroyed by the

addition of an aqueous 10% NaHSO₃solution, and the mixture

Diastereomeric Dihydroxyvitamin D Analogues

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3551

was extracted with Et₂O. The combined extracts were dried

over anhydrous MgSO₄and evaporated under reduced pres-

sure. The residue was purified by HPLC (hexanes-EtOAc, 5:1)

to give 29d (25 mg, 60%) as a colorless oil. Alcohol 30b gave

in a similar way acid 29b. Data of 29d : R_f(hexanes-EtOAc,

5:1) 0.23; IR (film) 3500-2500, 3056, 3027, 2935, 2873, 1704,

1496, 1454, 1416, 1366, 1256, 1206, 1102 cm^-1; ¹H NMR (500

MHz) δ 7.33 (5 H, m), 5.74 (1 H, br d, J ) 10.3 Hz), 5.63 (1 H,

dt, J ) 10.3, 3.5 Hz), 4.49 (1 H, ABd, J ) 12.0 Hz), 4.45 (1 H,

ABd, J ) 12.0 Hz), 3.64 (1 H, ABd, J ) 9.3, 3.4 Hz), 3.38 (1 H,

ABd, J ) 9.3, 6.9 Hz), 2.36 (1 H, d, J ) 5.2 Hz), 2.20 (1 H, m),

1.95 (1 H, m), 1.93 (1 H, m), 1.68 (1 H, m), 1.64 (1 H, m), 1.63

(1 H, m), 1.19 (3 H, d, J ) 7.0 Hz) 1.12 (3 H, s) ppm; MS m/z

302 (M⁺, 1), 253 (1), 221 (3), 206 (4), 167 (2), 149 (8), 111 (12),

95 (95), 91 (100).

CHCl₃); IR (film) 3086, 3056, 3013, 2956, 2928, 2859, 1496,

1454, 1364, 1100, 1028 cm^-1; ¹H NMR (500 MHz) δ 7.33 (5 H,

m), 5.60 (1 H, dt, J ) 10.2, 3.6 Hz), 5.46 (1 H, ddd, J ) 9.0,

3.3, 2.0 Hz), 4.52 (1 H, ABd, J ) 12.0 Hz), 4.48 (1 H, ABd, J

) 12.0 Hz), 3.28 (1 H, dd, J ) 9.2, 7.3 Hz), 3.21 (1 H, dd, J )

9.2, 7.0 Hz), 2.20 (1 H, ddd, J ) 11.0, 7.0, 1.5 Hz), 1.91 (2 H,

m), 1.63 (1 H, m), 1.61 (2 H, m), 1.31 (1 H, m), 0.99 (3 H, s),

0.84 (3 H, d, J ) 6.9 Hz), 0.75 (3 H, d, J ) 7.3 Hz) ppm; MS

m/z 272 (M⁺, 1), 181 (7), 163 (4), 148 (2), 107 (5), 95 (100).

Data of 35c: R_f(hexanes-EtOAc, 5:1) 0.66; [R]_D) +14.5, [R]₃₆₅

) +38.5 (c 0.90, CHCl₃); IR (film) 3056, 3016, 2956, 2930, 2862,

1489, 1453, 1378, 1363, 1098, 1028 cm^-1; ¹H NMR (500 MHz)

δ 7.33 (5 H, m), 5.61 (1 H, dt, J ) 10.0, 3.6 Hz), 5.46 (1 H, dd,

J ) 10.0, 1.0 Hz), 4.53 (1 H, ABd, J ) 12.1 Hz), 4.49 (1 H,

ABd, J ) 12.1 Hz), 3.26 (1 H, dd, J ) 9.2, 7.3 Hz), 3.19 (1 H,

dd, J ) 9.2, 6.9 Hz), 2.15 (1 H, ddd, J ) 11.0, 7.0, 1.7 Hz),

1.93 (2 H, m), 1.62 (2 H, m), 1.56 (2 H, m), 1.37 (1 H, m), 0.96

(3 H, s), 0.86 (3 H, d, J ) 6.9 Hz), 0.78 (3 H, d, J ) 7.3 Hz)

ppm; MS m/z 272 (M⁺, 1), 257 (1), 215 (1), 191 (1), 181 (6),

163 (3), 107 (5), 95 (100). Data of 35d : R_f(hexanes-EtOAc,

5:1) 0.66; [R]_D) -10.57, [R]₃₆₅) -41.76 (c 0.56, CHCl₃); IR

(film) 3056, 3015, 2954, 2930, 2862, 1646, 1600, 1454, 1374,

(3R,3a R,7S,7a S)-3-((1S)-2-Ben zyloxy-1-m eth yleth yl)-7-

iod o-3a -m eth ylp er h yd r oben zo[b]fu r a n -2-on e (32a ). A so-

lution of acid 29a (45.3 mg, 0.15 mmol) and I₂(76.2 mg, 0.30

mmol) in CH₃CN (0.5 mL) was stirred in the dark at room

temperature for 24 h. Et₂O was added, and the organic layer

was washed with a saturated aqueous Na₂SO₃solution (2×)

and a saturated aqueous NaHCO₃solution (2×), dried over

anhydrous MgSO₄, and evaporated under reduced pressure.

Column chromatography on silica gel afforded iodo lactone 32a

(55.4 mg, 86%) as a light yellow oil. The iodo lactones 32b-d

were obtained in a similar way. Data of 32a : R_f(hexanes-

EtOAc, 5:1) 0.36; IR (film) 3060, 2964, 2933, 2863, 1770, 1454,

1

1364, 1096, 1026 cm^-1; H NMR (500 MHz) δ 7.33 (5 H, m),

5.57 (1 H, dt, J ) 10.1, 4.0 Hz), 5.43 (1 H, dd, J ) 10.1, 1.2

Hz), 4.51 (1 H, ABd, J ) 12.1 Hz), 4.47 (1 H, ABd, J ) 12.1

Hz), 3.61 (1 H, dd, J ) 9.1, 3.5 Hz), 3.13 (1 H, dd, J ) 9.1, 8.5

Hz), 2.11 (1 H, m), 1.90 (2 H, m), 1.59 (2 H, m), 1.57 (2 H, m),

1.30 (1 H, m), 1.05 (3 H, d, J ) 6.9 Hz), 0.96 (3 H, s), 0.77 (3

H, d, J ) 7.5 Hz) ppm; MS m/z 272 (M⁺, 1), 219 (1), 181 (6),

163 (4), 148 (2), 123 (4), 107 (4), 95 (100), 91 (86).

1

1358, 1094, 1032 cm^-1; H NMR (500 MHz) δ 7.30 (5 H, m,),

4.52 (1 H, ABd, J ) 11.9 Hz), 4.49 (1 H, ABd, J ) 11.9 Hz),

4.30 (1 H, d, J ) 7.8 Hz), 4.08 (1 H, m), 3.74 (1 H, dd, J ) 9.2,

5.3 Hz), 3.57 (1 H, dd, J ) 9.2, 5.3 Hz), 2.55 (1 H, d, J ) 6.8

Hz), 2.25 (1 H, m), 2.19 (1 H, m), 1.94 (2 H, m), 1.60 (1 H, m),

1.43 (1 H, m), 1.24 (3 H, s), 1.13 (3 H, d, J ) 7.0 Hz) ppm; MS

m/z 428 (M⁺, 1), 386 (2), 337 (6), 280 (8), 221 (2), 195 (22), 153

(20), 135, (4), 91 (100).

(2S,3R)-3-((1S,3S)-3-ter t-Bu t yld ip h en ylsilyloxy-1-m e-

th ylcycloh exyl)-2-m eth ylbu ta n -1-ol (38a ), (2S,3S)-3-

((1S,3S)-3-ter t-Bu tyldiph en ylsilyloxy-1-m eth ylcycloh exyl)-

2-m eth ylbu ta n -1-ol (38b), (2R,3R)-3-((1S,3S)-3-ter t-Bu tyl-

d ip h en ylsilyloxy-1-m et h ylcycloh exyl)-2-m et h ylb u t a n -

1-ol (38c), a n d (2R,3S)-3-((1S,3S)-3-ter t-Bu tyld ip h en ylsi-

lyloxy-1-m eth ylcycloh exyl)-2-m eth ylbu ta n -1-ol (38d ). To

a solution of 35a (0.150 g, 0.55 mmol) in THF (14 mL) was

added 9-BBN (0.5 M solution in THF, 11 mL, 5.5 mmol), and

the mixture was refluxed for 48 h. The organoboranes were

oxidized by successively adding EtOH (2.65 mL), 6 N NaOH

(1.36 mL), and 30% H₂O₂(2.66 mL). The reaction mixture was

heated at 55 °C for 1.5 h and was then extracted with Et₂O.

The combined organic extracts were washed with an aqueous

5% HCl solution, H₂O, and brine, dried over anhydrous MgSO₄,

and evaporated under reduced pressure. The residual material

was purified by HPLC (hexanes-EtOAc, 5:1) to afford the

mixture of alcohols 36a (â/R diastereomeric ratio 1.5:1, 0.152

g, 95%) as a colorless oil. In an analogous way 35b gave 36b

(â/R diastereomeric ratio 1.3:1, 84%), 35c gave 36c (â/R

diastereomeric ratio 2:1, 95%), and 35d gave 36d (â/R diaster-

eomeric ratio 2:1, 94%).

To a solution of 36a (0.145 g, 0.50 mmol) in DMF (3.0 mL)

were successively added TBDPSCl (0.21 mL, 0.80 mmol),

imidazole (0.053 g, 0.77 mmol), and DMAP (5 mg); the mixture

was stirred at room temperature for 16 h and was then diluted

with Et₂O. The organic phase was washed with 5% HCl, H₂O,

and brine, dried over anhydrous MgSO₄, and evaporated under

reduced pressure. The residual material was separated by

HPLC (hexanes-EtOAc, 25:1) to give 37a (0.245 g, 93%). In

an analogous way 36b gave 37b (93%), 36c gave 37c (90%),

and 36d gave 37d (76%).

Ben zyl (2S,3R)-2-Meth yl-3-((1S)-1-m eth yl-2-cycloh ex-

en yl)bu tyl Eth er (35a ), Ben zyl (2S,3S)-2-Meth yl-3-((1S)-

1-m et h yl-2-cycloh exen yl)b u t yl E t h er (35b ), Ben zyl

(2R,3R)-2-Met h yl-3-((1S)-1-m et h yl-2-cycloh exen yl)b u -

tyl Eth er (35c), a n d Ben zyl (2R,3S)-2-Meth yl-3-((1S)-1-

m eth yl-2-cycloh exen yl)bu tyl Eth er (35d ). To a solution of

alcohol 30a (0.63 g, 2.20 mmol) in pyridine (25 mL) was added

TsCl (1.25 g, 0.66 mmol) at 0 °C under N₂atmosphere The

mixture was stored in the refrigerator for 24 h, poured into

ice water, and extracted with Et₂O. The combined organic

extracts were washed with an aqueous 5% HCl solution, H₂O,

and brine, dried over anhydrous MgSO₄, and evaporated under

reduced pressure. Column chromatography on silica gel (hex-

anes-EtOAc, 5:1) gave tosylate 33a (0.77 g, 80%) and the

tetrahydrofuran derivative 34a (∼20%). In an analogous way

30b gave 33b (98%) and 30c gave 33c (97%), while 30d gave

33d (78%) and the tetrahydrofuran derivative 34d (∼20%).

To a suspension of LiAlH₄(0.464 g, 12.2 mmol) in THF (20

mL) was added a solution of 33a (0.6 g, 1.36 mmol) in THF (4

mL), and the reaction mixture was refluxed for 4 h. Excess

LiAlH₄was destroyed by the addition of EtOH, and the

mixture was extracted with Et₂O. The combined organic

extracts were washed with an aqueous 2.5% HCl solution, H₂O,

and brine, dried over anhydrous MgSO₄, and evaporated under

reduced pressure. The residual material was purified by HPLC

(hexanes-EtOAc, 8:1) to afford 35a (0.351 g, 95%) as a

colorless oil. In an analogous way 33b gave 35b (80% over the

two steps), 33c gave 35c (94%), and 33d gave 35d (80%). Data

Benzyl ether 37a (250 mg, 0.57 mmol) was dissolved in

EtOH (4.0 mL) and cyclohexene (2.0 mL). 20% Pd(OH)₂on

carbon (63 mg, 2.5:10 catalyst/substrate by weight) was added,

and the suspension was stirred under reflux for 6 h. The

catalyst was removed by filtration, and the filtrate was

concentrated under reduced pressure. The residual material

was separated by HPLC (hexanes-EtOAc, 5:1) to afford

alcohols 38âa and 38Ra (166 mg, 80%) as colorless oils. In an

analogous way 37b gave 38âb and 38Rb (88%), 37c gave 38âc

and 38Rc (90%), and 37d gave 38d as an unseparable mixture

of diastereomers (ratio 2:1; 85%). Data of 38âa : R_f(hexanes-

EtOAc, 5:1) 0.27; IR (film) 3362, 3071, 3049, 2932, 2858, 1472,

of 35a : R_f(hexanes-EtOAc, 5:1) 0.66; [R]_D) +48.5, [R]₃₆₅

)

+152.0 (c 0.46, CHCl₃); IR (film) 3088, 3063, 3012, 2958, 2929,

2869, 1496, 1454, 1366, 1099, 1028 cm^-1; ¹H NMR (500 MHz)

δ 7.33 (5 H, m), 5.59 (1 H, dt, J ) 10.2, 4.4 Hz), 5.44 (1 H, br

d, J ) 10.2 Hz), 4.50 (1 H, ABd, J ) 12.0 Hz), 4.46 (1 H, ABd,

J ) 12.0 Hz), 3.58 (1 H, dd, J ) 9.1, 3.7 Hz), 3.16 (1 H, dd, J

) 9.1, 8.5 Hz), 2.07 (1 H, m), 1.91 (2 H, m), 1.58 (2 H, m), 1.35

(1 H, ddd, J ) 9.3, 7.4, 1.9 Hz), 1.02 (3 H, d, J ) 6.9 Hz), 0.94

(3 H, s), 0.80 (3 H, d, J ) 7.4 Hz) ppm; MS m/z 272 (M⁺, 1),

257 (1), 215 (1), 191 (1), 181 (6), 163 (3), 107 (5), 95 (100). Data

of 35b: R_f(hexanes-EtOAc, 5:1) 0.66; [R]_D) +23.1 (c 0.32,

3552 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Zhou et al.

1463, 1428, 1377, 1362, 1111, 1070, 1030, 1008 cm^-1; ¹H NMR

(500 MHz) δ 7.66 (4 H, m), 7.42-7.34 (6 H, m), 3.75 (1 H, m),

3.63 (1 H, dd, J ) 10.3, 3.1 Hz), 3.20 (1 H, dd, J ) 9.8, 9.0

Hz), 1.82 (2 H, m), 1.52 (1 H, m), 1.43 (2 H, m), 1.31 (1 H, m),

1.20 (2 H, m), 1.04 (9 H, s), 0.98 (3 H, d, J ) 6.7 Hz), 0.67 (3

H, d, J ) 7.4 Hz), 0.56 (3 H, s) ppm; MS m/z 438 (M⁺, 1), 437

(M⁺-1, 1), 407 (1), 381 (2), 293 (2), 217 (3), 199 (40), 165 (15),

123 (21), 109 (34), 95 (100). Data of 38âb: R_f(hexanes-EtOAc,

5:1) 0.27; IR (film) 3367, 3066, 2932, 2855, 1458, 1428, 1383,

477 (1), 465 (50), 399 (2), 387 (2), 251 (2), 227 (4), 199 (100),

183 (14), 135 (36), 95 (76).

(3S)-3-((1S,2R)-6-Hyd r oxy-1,2,6-tr im eth ylh ep tyl)-3-m e-

th ylcycloh exan -1-on e (43b) an d (3S)-3-((1R,2S)-6-Hydr oxy-

1,2,6-tr im eth ylh ep tyl)-3-m eth ylcycloh exa n -1-on e (43c).

To a stirred solution of ester 40b (55 mg, 0.105 mmol) in dry

THF (1.5 mL) at 0 °C under N₂atmosphere was added

MeMgBr (3.0 M solution in Et₂O, 0.175 mL, 0.525 mmol) over

a period of 5 min. Stirring was continued for 2 h, and the

reaction mixture was quenched with a saturated aqueous NH₄-

Cl solution. After extraction with Et₂O, the combined extracts

were washed with brine, dried over anhydrous MgSO₄, and

concentrated under reduced pressure. The residual material

was purified by HPLC (hexanes-EtOAc, 5:1) to afford 41b (46

mg, 86%). In an analogous way 40c gave 41c (95%).

To a solution of 41b (32 mg, 0.063 mmol) in THF (1.0 mL)

was added TBAF (1 M solution in THF, 0.110 mL, 0.110 mmol)

at room temperature under N₂atmosphere. The reaction

mixture was stirred for 16 h and then directly loaded onto a

silica gel column (hexanes-EtOAc, 2:1). Further purification

by HPLC (isooctane-acetone, 1:1) afforded 42b (17 mg, 100%)

as a colorless oil. In an analogous way 41c gave 42c (90%).

A mixture of alcohol 42b (14.5 mg, 0.054 mmol) and PDC

(60 mg, 0.160 mmol) in CH₂Cl₂(1.5 mL) was stirred at room

temperature for 22 h, and the solution was filtered through a

pad of silica gel. HPLC purification (hexanes-EtOAc, 5:1) gave

43b (13.5 mg, 94%) as a colorless oil. In an analogous way

42c gave 43c (86%). Data of 43b: R_f(hexanes-EtOAc, 1:1)

0.38; [R]_D) -67.7 (c 0.20, CHCl₃); IR (film) 3410, 2963, 2923,

1705, 1456, 1421, 1380, 1354, 1154, 1072 cm^-1; ¹H NMR (500

MHz) δ 2.35 (1 H, d, J ) 13.4 Hz), 2.28 (2 H, m), 2.11 (1 H, d,

J ) 13.4 Hz), 1.88 (2 H, m), 1.74 (1 H, m), 1.69 (1 H, m), 1.53

(1 H, m), 1.45 (2 H, m), 1.32 (2 H, m), 1.27 (1 H, m), 1.22 (3 H,

s), 1.21 (3 H, s), 0.87 (3 H, s), 0.80 (3 H, d, J ) 6.8 Hz), 0.76 (3

H, d, J ) 7.2 Hz) ppm; MS m/z 268 (M⁺, 1), 253 (4), 235 (1),

201 (1), 169 (4), 140 (4), 123 (5), 111 (74), 83 (34), 69 (38), 55

1

1111, 1072, 1037 cm^-1; H NMR (500 MHz) δ 7.70 (4 H, m),

7.45-7.38 (6 H, m), 3.77 (1 H, ddd, J ) 12.0, 9.9, 4.3 Hz), 2.92

(2 H, m), 1.85 (1 H, br d, J ) 13.4 Hz), 1.84 (1 H, m), 1.57 (1

H, d, J ) 3.3 Hz), 1.54 (1 H, br d, J ) 4.4 Hz), 1.48 (1 H, m),

1.29 (1 H, m), 1.14 (1 H, dd, J ) 10.5, 13.2 Hz), 1.04 (9 H, s),

0.79 (3 H, s), 0.62 (3 H, d, J ) 6.8 Hz), 0.56 (3 H, d, J ) 7.2

Hz) ppm; MS m/z 438 (M⁺, 1), 381 (6), 351 (1), 293 (2), 199

(32), 165 (8), 135 (10), 95 (100). Data of 38âc: R_f(hexanes-

EtOAc, 5:1) 0.27; IR (film) 3351, 3071, 3047, 2932, 2858, 1470,

1428, 1389, 1373, 1111, 1075, 1037 cm^-1; ¹H NMR (500 MHz)

δ 7.67 (4 H, m), 7.42-7.34 (6 H, m), 3.74 (1 H, ddd, J ) 15.0,

8.8, 4.4 Hz), 3.33 (2 H, d, J ) 7.0 Hz), 1.86 (2 H, m), 1.50 (2 H,

m), 1.25 (1 H, m), 1.21 (3 H, m), 1.05 (9 H, s), 0.73 (3 H, d, J

) 6.8 Hz), 0.65 (3 H, d, J ) 7.2 Hz), 0.55 (3 H, s) ppm; MS m/z

438 (M⁺, 1), 437 (M⁺-1, 1), 407 (1), 381 (2), 293 (2), 217 (3),

199 (40), 165 (15), 123 (21), 109 (34), 95 (100).

Eth yl (5R,6S)-6-((1S,3S)-3-ter t-Bu tyld ip h en ylsilyloxy-

1-m eth ylcycloh exyl)-5-m eth ylh ep ta n oa te (40b) a n d Eth -

yl (5S,6R)-6-((1S,3S)-3-ter t-Bu tyld ip h en ylsilyloxy-1-m e-

th ylcycloh exyl)-5-m eth ylh ep ta n oa te (40c). To a solution

of alcohol 38âb (60 mg, 0.137 mmol) in THF (0.75 mL) were

successively added PPh₃(54 mg, 0.21 mmol), imidazole (29 mg,

0.42 mmol), and I₂(50 mg, 0.20 mmol) at -10 °C. The mixture

was allowed to warm to room temperature and stirred in the

dark for 29 h. The reaction mixture was cooled to 0 °C, a

saturated aqueous NaHCO₃solution was added, and the

mixture was extracted with Et₂O. The combined organic

extracts were washed with a saturated aqueous Na₂S₂O₃

solution and H₂O, dried over anhydrous MgSO₄, and concen-

trated under reduced pressure. Column chromatography on

silica gel (hexanes-EtOAc, 50:1) afforded 39b (75 mg, 94%)

as a light yellow oil. In an analogous way 38âc afforded 39c

(94%).

(100). Data of 43c: R_f(hexanes-EtOAc, 1:1) 0.38; [R]_D

)

-1.72, [R]₃₆₅) -75.0 (c 0.41, CHCl₃); IR (film) 3434, 2965,

2933, 1705, 1460, 1378, 1234, 1159, 1087, 1081 cm^-1; ¹H NMR

(500 MHz) δ 2.28 (2 H, m), 2.08 (1 H, m), 1.92 (1 H, m), 1.83

(1 H, ddd, J ) 11.5, 9.4, 5.1 Hz), 1.77 (1 H, dd, J ) 13.0, 6.1

Hz), 1.66 (2 H, dd, J ) 9.3, 4.3 Hz), 1.44 (2 H, m), 1.33 (3 H,

m), 1.21 (6 H, s), 0.86 (3 H, s), 0.80 (3 H, d, J ) 6.8 Hz), 0.78

(3 H, d, J ) 7.2 Hz) ppm; MS m/z 268 (M⁺, 1), 253 (2), 217 (1),

191 (1), 169 (3), 140 (40), 123 (6), 111 (100), 83 (38), 69 (40),

55 (100).

NiCl₂‚6H₂O (93 mg, 0.39 mmol) in pyridine (2.5 mL) was

treated under Ar atmosphere with Zn powder (126 mg, 1.93

mmol) and ethyl acrylate (0.20 mL, 1.93 mol) and then heated

at 65 °C for 30 min. The resulting brick-red mixture was cooled

to 20 °C, treated with iodide 39b (70 mg, 0.13 mmol) in

pyridine (1.0 mL), and stirred for 2 h. The mixture was poured

into EtOAc and filtered through Celite, and the pad was

washed with EtOAc. The combined filtrates were washed with

an aqueous 1 N HCl solution, H₂O, and brine, dried over

anhydrous MgSO₄, and concentrated under reduced pressure.

Column chromatography on silica gel and HPLC purification

(hexanes-EtOAc, 8:1) gave ester 40b (59 mg, 88%) as a

colorless oil. In an analogous way 39c gave 40c (91%). Data

of 40b: R_f(hexanes-EtOAc, 5:1) 0.61; [R]_D) -10.6 (c 0.15,

CHCl₃); IR (film) 3056, 2932, 2862, 1737, 1590, 1462, 1428,

1369, 1159, 1111, 1072 cm^-1; ¹H NMR (500 MHz) δ 7.67 (4 H,

m), 7.42-7.34 (6 H, m), 4.12 (2 H, q, J ) 7.1 Hz), 3.77 (1 H,

ddd, J ) 13.5, 9.2, 4.2 Hz), 2.08 (2 H, m), 1.84 (1 H, d, J )

13.4 Hz), 1.67 (1 H, m), 1.48 (2 H, m), 1.31 (1 H, m), 1.25 (3 H,

t, J ) 7.1 Hz), 1.18 (2 H, m), 1.05 (9 H, s), 0.82 (3 H, s), 0.67

(3 H, d, J ) 6.8 Hz), 0.56 (3 H, d, J ) 7.1 Hz) ppm; MS m/z

522 (M⁺, 1), 507 (1), 477 (2), 465 (60), 419 (3), 399 (3), 279 (3),

251 (3), 199 (100), 183 (14), 181 (18), 135 (30), 95 (48). Data of

40c: R_f(hexanes-EtOAc, 5:1) 0.61; [R]_D) +43.3 (c 0.25,

CHCl₃); IR (film) 3071, 2932, 2857, 1738, 1463, 1427, 1376,

1246, 1178, 1111, 1072 cm^-1; ¹H NMR (500 MHz) δ 7.67 (4 H,

m), 7.42-7.34 (6 H, m), 4.13 (2 H, q, J ) 7.3 Hz), 3.73 (1 H,

m), 2.25 (2 H, t, J ) 7.5 Hz), 1.84 (1 H, m), 1.66 (1 H, dd, J )

13.6, 6.8 Hz), 1.58 (1 H, m), 1.48 (1 H, m), 1.25 (3 H, t, J ) 7.2

Hz), 1.18 (3 H, m), 1.05 (9 H, s), 0.69 (3 H, d, J ) 6.7 Hz), 0.62

(3 H, d, J ) 7.2 Hz), 0.52 (3 H, s) ppm; MS m/z 522 (M⁺, 1),

(1R,3R)-5-((Z,2E)-2-((3S)-3-((1R,2S)-6-Hyd r oxy-1,2,6-tr i-

m e t h ylh e p t yl)-3-m e t h ylcycloh e xylid e n e )e t h ylid e n e )-

cycloh exa n e-1,3-d iol (8c). Cyclohexanone 43c was coupled

with phosphine oxide 15 according to the above general

procedure. The crude product was purified by HPLC (hex-

anes-EtOAc, 200:1) to give protected 8c (89%). Deprotection

using TBAF was carried out as described for 8a , and the

product was isolated by loading the crude reaction mixture

onto silica gel (hexane-acetone, 2:1) and further purification

by HPLC (hexane-acetone, 3:1) to afford analogue 8c (3.7 mg,

95%) as a white solid: R_f(hexanes-EtOAc, 1:1) 0.08; IR (film)

3386, 2954, 2923, 2720, 1595, 1462, 1426, 1380, 1359, 1154,

1067, 1051 cm^-1; ¹H NMR (500 MHz) δ 6.23 (1 H, d, J ) 11.2

Hz, H-6), 5.95 (1 H, d, J ) 11.2 Hz, H-7), 4.11 (1 H, m), 4.07

(1 H, m), 2.68 (1 H, dd, J ) 13.4, 3.8 Hz, H-10â), 2.49 (1 H,

dd, J ) 13.5, 3.6 Hz, H-4R), 2.46 (1 H, m, H-9â), 2.27 (1 H, dd,

J ) 13.4, 7.9 Hz, H-10R), 2.20 (1 H, dd, J ) 13.5, 6.6 Hz), 2.06

(1 H, d, J ) 13.4 Hz, H-14â), 2.00 (1 H, m), 1.91 (1 H, m), 1.84

(1 H, d, J ) 13.4 Hz, H-10R), 1.76 (1 H, dd, J ) 13.8, 7.0 Hz),

1.59 (1 H, m), 1.52 (1 H, m), 1.45 (4 H, m), 1.30 (2 H, m), 1.21

(6 H, s), 0.80 (3 H, d, J ) 6.8 Hz), 0.77 (3 H, s), 0.74 (3 H, d,

J ) 7.1 Hz) ppm; MS m/z 392 (M⁺, 1), 374 (8), 356 (7), 341 (1),

303 (1), 235 (5), 217 (8), 181 (6), 163 (18), 109 (30), 91 (36), 43

(100).

(1R,3R)-5-((Z,2E)-2-((3S)-3-((1S,2R)-6-Hyd r oxy-1,2,6-tr i-

m e t h ylh e p t yl)-3-m e t h ylcycloh e xylid e n e )e t h ylid e n e )-

cycloh exa n e-1,3-d iol (8b). To a solution of 43b (13 mg, 0.049

mmol) in DMF (750 µL) were successively added Et₃SiCl (22

Diastereomeric Dihydroxyvitamin D Analogues

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3553

mg, 0.147 mmol), imidazole (13 mg, 0.240 mmol), and DMAP

(3 mg). The mixture was stirred at room temperature for 16 h

and then diluted with Et₂O. The organic phase was washed

with an aqueous 5% HCl solution and H₂O, dried over

anhydrous MgSO₄, and concentrated under reduced pressure.

Column chromatography on silica gel gave silyl ether 44b (7.6

mg, 82% based on recovered 43b).

1121, 1082, 1054 cm^-1; ¹H NMR (500 MHz) δ 5.10 (1H, q, J )

5.44 Hz), 3.76 (1 H, m), 3.67 (1 H, m), 3.50 (1 H, m), 2.01 (2 H,

m), 1.89 (1 H, dd, J ) 16.3, 10.5 Hz), 1.75 (1 H, m), 1.65 (1 H,

m), 1.49 (3 H, s), 1.43 (3 H, s), 1.32 (3 H, d, J ) 5.2 Hz), 1.19

(3 H, t, J ) 7.0 Hz), 1.03 (3 H, d, J ) 6.8 Hz), 0.87 (3 H, s),

0.78 (3 H, d, J ) 7.3 Hz) ppm; MS m/z 338 (M⁺, 1), 323 (1),

294 (1), 249 (2), 233 (2), 199 (7), 151 (8), 123 (2), 109 (24), 73

(100).

Cyclohexanone 44b was coupled with phosphine oxide 15

according to the above general procedure to give a mixture of

protected 8b and (Z)-8b (ratio 7:3, 100%). The mixture was

deprotected using TBAF as described for 8c, and the products

were isolated by loading the crude reaction mixture onto silica

gel (hexane-acetone, 2:1) and further separation by HPLC

(hexane-acetone, 3:1) to afford 8b and (Z)-8b (100%) as white

solids. Data of 8b: R_f(hexanes-EtOAc, 1:1) 0.08; IR (film)

3386, 2964, 2923, 2872, 1595, 1431, 1380, 1354, 1149, 1113,

1068, 1051 cm^-1; ¹H NMR (500 MHz) δ 6.22 (1 H, d, J ) 11.3

Hz, H-6), 5.96 (1 H, d, J ) 11.3 Hz, H-7), 4.13 (1 H, m), 3.96

(1 H, m), 2.86 (1 H, dd, J ) 12.9, 3.6 Hz, H-10â), 2.43 (1 H, d,

J ) 13.1 Hz, H-4R), 2.37 (1 H, m, H-9â), 2.25 (1 H, d, J ) 13.0

Hz, H-14â), 2.21 (1 H, dd, J ) 13.1, 4.6 Hz, H-4â), 2.08 (2 H,

m), 1.87 (1 H, d, J ) 13.0 Hz, H-14R), 1.76 (1 H, dd, J ) 14.2,

6.3 Hz), 1.70 (1 H, m), 1.60 (1 H, m), 1.50 (1 H, dd, J ) 12.7,

4.9 Hz), 1.47 (1 H, m), 1.41 (1 H, m), 1.40 (1 H, d, J ) 6.8 Hz),

1.35 (1 H, m), 1.23 (3 H, s), 1.22 (3 H, s), 1.15 (2 H, m), 0.79 (3

H, d, J ) 6.8 Hz), 0.78 (3 H, s), 0.69 (3 H, d, J ) 7.2 Hz) ppm;

MS m/z 392 (M⁺, 1), 374 (6), 356 (8), 342 (2), 235 (4), 217 (4),

199 (6), 163 (16), 105 (20), 91 (34), 43 (100). Data of (Z)-8b:

R_f(hexanes-EtOAc, 1:1) 0.08; IR (film) 3386, 2964, 2923, 2872,

1595, 1380, 1149, 1113, 1068, 1051 cm^-1; ¹H NMR (500 MHz)

δ 6.21 (1 H, d, J ) 11.3 Hz), 6.08 (1 H, d, J ) 11.3 Hz), 4.13

(1 H, m), 4.09 (1 H, m), 2.52 (1 H, dd, J ) 12.8, 3.4 Hz), 2.40

(1 H, d, J ) 13.5 Hz), 2.15 (1 H, dd, J ) 12.8, 8.5 Hz), 2.10 (1

H, m), 1.98 (1 H, m), 1.92 (1 H, d, J ) 13.5 Hz), 1.76 (1 H, dd,

J ) 12.2, 8.3 Hz), 1.60 (1 H, m), 1.50 (1 H, dd, J ) 14.0, 5.7

Hz), 1.45 (1 H, dd, J ) 9.5, 5.1 Hz), 1.42 (1 H, d, J ) 9.1 Hz),

1.37 (1 H, m), 1.23 (3 H, s), 1.22 (3 H, s), 1.18 (1 H, m), 0.81 (3

H, d, J ) 6.6 Hz), 0.80 (3 H, s), 0.70 (3 H, d, J ) 7.2 Hz) ppm;

MS m/z 392 (M⁺, 1), 374 (6), 356 (8), 235 (4), 199 (6), 91 (34),

43 (100).

(3S)-3-((1R,2R)-6-(1-E t h oxyet h oxy)-1,2,6-t r im et h yl-4-

h ep t yn yl)-3-m et h ylcycloh exa n -1-ol (47a ) a n d (3S)-3-

((1S,2S)-6-(1-Eth oxyeth oxy)-1,2,6-tr im eth yl-4-h ep tyn yl)-

3-m eth ylcycloh exa n -1-ol (47d ). A solution of 38âa (98 mg,

0.223 mmol) and TsCl (107 mg, 0.558 mmol) in pyridine (1.5

mL) was stirred at room temperature for 19 h and then poured

into ice water. The mixture was extracted with Et₂O, and the

combined organic extracts were washed with an aqueous 2%

HCl solution, dried over anhydrous MgSO₄, and concentrated

under reduced pressure. The residual material was purified

by HPLC (hexanes-EtOAc, 25:1) to afford tosylate 45âa (120

mg, 91%). In an analogous way 38d (â/R diastereomeric

mixture) gave 45d (93%).

NaH (60%, 38 mg, 0.95 mmol) in dry DMSO (1.5 mL) was

stirred at 65 °C under N₂atmosphere for 2 h. To the formed

light-green solution was dropwise added the ethoxyethyl ether

of 2-methyl-3-butyn-2-ol (225 mg, 1.44 mmol) at room tem-

perature. The mixture was stirred for 45 min, and a solution

of tosylate 45âa (68 mg, 0.115 mmol) in DMSO (0.5 mL) was

added. Stirring was continued for 45 min, and the reaction

mixture was diluted with Et₂O. The organic phase was washed

with H₂O, dried over anhydrous MgSO₄, and concentrated

under reduced pressure. Column chromatography on silica gel

(hexanes-EtOAc, 50:1) gave 46âa (45 mg, 68%) as a colorless

oil. In an analogous way 45d gave 46d (9%) and desilylated

product 47d (48%).

(3S)-3-((1S,2S)-6-(1-Eth oxyeth oxy)-1,2,6-tr im eth ylh ep -

tyl)-3-m eth ylcycloh exa n -1-on e (49d ). Alkyne 47d (20 mg,

0.059 mmol) and 5% Rh on Al₂O₃(20 mg) in EtOAc (1.5 mL)

were stirred under H₂atmosphere (1 bar) at room temperature

for 4 h. The reaction mixture was filtered through silica gel,

and the product was purified by HPLC (hexanes-EtOAc, 5:1)

to afford 48d (18 mg, 90%).

A mixture of 48d (16 mg, 0.0467 mmol) and PDC (53 mg,

0.140 mmol) in CH₂Cl₂(1.5 mL) was stirred at room temper-

ature for 24 h. The reaction mixture was directly loaded onto

a silica gel column, and the product was further purified by

HPLC (hexanes-EtOAc, 5:1) to give cyclohexanone 49d (8.0

mg, 50%) and the deprotected product 43d (5.0 mg, 40%). Data

of 43d : R_f(hexanes-EtOAc, 1:1) 0.26; IR (film) 3404, 2969,

2944, 2882, 1707, 1459, 1383, 1237, 1159, 1086, 1043 cm^-1

;

¹H NMR (500 MHz) δ 2.28 (2 H, m), 2.15 (1 H, m), 1.88 (1 H,

m), 1.82 (1 H, m), 1.70 (1 H, m), 1.68 (1 H, m), 1.47-1.31 (6

H, m), 1.25 (2 H, m), 1.21 (6 H, s), 0.90 (3 H, d, J ) 6.8 Hz),

0.87 (3 H, s), 0.79 (3 H, d, J ) 7.2 Hz) ppm; MS m/z 268 (M⁺,

1), 253 (1), 250 (4), 249 (24), 199 (30), 140 (12), 111 (56), 55

(100).

(1R,3R)-5-((Z,2E)-2-((3S)-3-((1S,2S)-6-Hyd r oxy-1,2,6-tr i-

m e t h ylh e p t yl)-3-m e t h ylcycloh e xylid e n e )e t h ylid e n e )-

cycloh exa n e-1,3-d iol (8d ). A mixture of 43d (5.0 mg, 0.019

mmol), CF₃SO₃SiEt₃(8.6 mL, 0.038 mmol), and Et₃N (7.92 mL,

0.057 mmol) in CH₂Cl₂(0.5 mL) was stirred at room temper-

ature for 4 h. The reaction mixture was directly loaded onto a

silica gel column, and the product was further purified by

HPLC (hexanes-EtOAc, 10:1) to give 44d (5.0 mg, 70%) as a

colorless oil.

Cyclohexanone 44d was coupled with phosphine oxide 15

according to the above general procedure to give protected 8d

(100%). The compound was deprotected using TBAF as de-

scribed for 8a , and the product was isolated by loading the

crude reaction mixture onto silica gel (hexane-acetone, 2:1)

and further separation by HPLC (hexane-acetone, 3:1) to

afford 8d (92% from 44d ) as a white solid: R_f(hexanes-

EtOAc, 1:1) 0.08; IR (film) (hexane-acetone, 2.5:1) 0.30; IR

(film) 3358, 3036, 2964, 2929, 2872, 1600, 1456, 1431, 1380,

1

1154, 1087, 1050 cm^-1; H NMR (500 MHz) δ 6.26 (1 H, d, J

) 11.3 Hz, H-6), 5.95 (1 H, d, J ) 11.3 Hz, H-7), 4.09 (2 H, m,

H-1 and H-3), 2.65 (1 H, dd, J ) 13.3, 2.4 Hz, H-10â), 2.49 (1

H, dd, J ) 13.3, 3.8 Hz, H-4R), 2.31 (1 H, dd, J ) 13.3, 7.6 Hz,

H-10R), 2.19 (1 H, dd, J ) 13.3, 6.9 Hz, H-4â), 2.15 (1 H, d, J

) 13.1 Hz), 1.90 (1 H, d, J ) 13.1 Hz), 1.86 (1 H, m), 1.70 (1

H, m), 1.50 (2 H, m), 1.45 (1 H, m), 1.40 (1 H, m), 1.38 (2 H,

m), 1.22 (6 H, s), 0.89 (3 H, d, J ) 6.8 Hz), 0.76 (3 H, s), 0.72

(3 H, d, J ) 7.3 Hz) ppm; MS m/z 392 (M⁺, 2), 374 (3), 356 (4),

235 (4), 217 (8), 163 (14), 91 (30), 43 (100).

(3S)-3-((1R,2R)-6-(1-E t h oxyet h oxy)-1,2,6-t r im et h yl-4-

h ep tyn yl)-3-m eth ylcycloh exa n -1-on e (50a ). Alcohol 47a

was oxidized using PDC as described for 42c to give 50a (85%)

as a colorless oil: R_f(hexanes-EtOAc, 6:1) 0.62; [R]_D) +0.95,

[R]₃₆₅) -24.05 (c 0.42, CHCl₃); IR (film) 2974, 2935, 2877,

2233, 1713, 1456, 1379, 1313, 1252, 1160, 1122, 1081, 1053

1

cm^-1; H NMR (500 MHz) δ 5.10 (1 H, dq, J ) 4.9, 1.2 Hz),

3.68 (1 H, m), 3.50 (1 H, m), 2.26 (3 H, m), 2.09 (1 H, m), 1.90

(4 H, m), 1.68 (2 H, m), 1.50 (3 H, s), 1.43 (3 H, s), 1.33 (3 H,

d, J ) 5.2 Hz), 1.19 (3 H, t, J ) 7.0 Hz), 1.06 (3 H, d, J ) 6.7

Hz), 0.88 (3 H, s), 0.79 (3 H, d, J ) 7.2 Hz) ppm; MS m/z 336

(M⁺, 1), 321 (1), 292 (1), 263 (5), 247 (9), 221 (2), 191 (3), 177

(4), 149 (5), 135 (13), 111 (33), 73 (100).

To a solution of silyl ether 46âa (50 mg, 0.087 mmol) in THF

(1.5 mL) was added TBAF (1 M solution in THF, 174 µL, 0.174

mmol). The reaction mixture was stirred at room temperature

for 10 h, directly loaded onto a silica gel column, and further

purified by HPLC (hexanes-EtOAc, 5:1) to give alcohol 47âa

(28.7 mg, 98%) as a colorless oil. In an analogous way 46d

gave 47d (94%). Data of 47âa : R_f(hexanes-EtOAc, 1:1) 0.48;

IR (film) 3420, 2977, 2933, 2867, 2233, 1462, 1378, 1253, 1158,

(1S,3R)-5-((Z,2E)-2-((3S)-3-((1R,2R)-6-Hyd r oxy-1,2,6-tr i-

m eth yl-4-h eptyn yl)-3-m eth ylcycloh exyliden e)eth yliden e)-

4-m eth ylen ecycloh exa n e-1,3-d iol (9a ). Cyclohexanone 50a

3554 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Zhou et al.

was coupled with phosphine oxide 14 according to the above

general procedure to give protected 9a (100%).

from its receptor compared with the activity of 1,25(OH)₂D₃

(assigned a 100% value).

Affin ity for h DBP . Binding of vitamin D metabolites and

analogues to hDBP was performed at 4 °C essentially as

described previously.⁴⁰[³H]1,25(OH)₂D₃and 1,25(OH)₂D₃or its

analogues were added in 5 µL of ethanol into glass tubes and

incubated with hDBP (0.18 µM) in a final volume of 1 mL (0.01

M Tris-HCl buffer and 0.154 M NaCl, pH 7.4) for 3 h at 4 °C.

Phase separation was then obtained by the addition of 0.5 mL

of cold dextran-coated charcoal.

P r om yelocytic Leu k em ia Cells HL-60. HL-60 cells were

seeded at 4 × 10⁴cells/mL in 25-cm²Falcon tissue chambers

using RPMI 1640 medium supplemented with 20% FCS and

gentamycin (50 µg/mL). Final volume was 5 mL. Cultures were

maintained at 37 °C in a humidified atmosphere of 5% CO₂in

air. One day later, 1,25(OH)₂D₃or analogues were added to

the cell culture in ethanol (final concentration < 0.2%). After

4 days of culture, the dishes were shaken to loosen adherent

cells. Cells were washed twice in RPMI, counted, and assayed

for differentiation markers (NBT reduction assay). Superoxide

production was measured as NBT reducing activity as de-

scribed previously.⁴¹HL-60 cells at 1 × 10⁶/mL were mixed

with an equal volume of freshly prepared solution of phorbol

12-myristate 13-acetate (200 ng/mL) and NBT (2 µg/mL) and

incubated for 30 min at 37 °C. The percentage of cells

containing black formazan deposits was determined using a

hemacytometer.

Br ea st Ca r cin om a Cells MCF -7. MCF-7 cells were cul-

tured in DMEM/nutrient mixture F12 (HAM) medium supple-

mented with 10% heat-inactivated FCS, glutamine (2 mM),

penicillin (100 units/mL), and streptomycin (0.1 mg/mL).

Cultures were maintained at 37 °C in humidified atmosphere

of 5% CO₂in air. MCF-7 cells were seeded at 5 × 10³cells/

well in the above-described medium in a 96-well microtiter

plate in a final volume of 0.2 mL/well. Triplicate cultures were

performed. After 24 h, 1,25(OH)₂D₃or analogues were added

in the appropriate concentrations for an incubation period of

72 h. Then, 1 µCi of [³H]thymidine was added to each well,

cells were harvested after 4-h incubation with a Packard

harvester, and radioactivity was measured by a Packard

Topcount system (Packard, Meriden).

Ker a tin ocytes. Human skin keratinocytes were isolated

and cultured using a modification of the method of Kitano and

Okada⁴²and was described previously.⁴³Briefly, foreskin was

soaked overnight at 4 °C in Dispase (20 units/mL). The

epidermis was peeled from the dermis and washed with

calcium- and magnesium-free solution for 20 min at 37 °C. The

cells were then suspended in keratinocyte medium (Gibco

Keratinocytes-SFM with insulin (5 µg/mL)) supplemented with

bovine pituitary extract (50 µg/mL; Gibco, Paisley, U.K.) and

epidermal growth factor (5 ng/mL; Gibco). The cells were

plated into culture flasks at 3 × 10⁶cells/175-cm²flask in a

final volume of 25 mL and cultured until 75% confluence.

Keratinocytes were then seeded in 96-well plates at a concen-

tration of 5 × 10³cells/0.1 mL of medium/well. The same

medium was used as described above but without insulin. After

1 day, serial dilutions of 1,25(OH)₂D₃or analogues, in ethanol

solution (<0.2% final concentration), were added to the cells

in a working volume of 0.1 mL. After an incubation period of

72 h, 1 µCi of [³H]thymidine (specific activity 35 Ci/mmol) was

added to each well for 2 h. Thereafter, cells were harvested

with the Packard Topcount system.

A solution of protected 9a (16.0 mg, 0.029 mmol) and PPTS

(18.5 mg) in CH₂Cl₂(3.5 mL) was stirred at room temperature

for 1 h, then directly loaded onto silica gel, and purified by

HPLC (hexanes-EtOAc, 5:1) to give ethoxyethyl-deprotected

9a (∼14.5 mg, 100%).

Ethoxyethyl-deprotected 9a was desilylated using TBAF as

described for 8a , and the products were isolated by loading

the crude reaction mixture onto silica gel (hexane-acetone,

2:1) and further separation by HPLC (hexane-acetone, 3:1)

to afford analogue 9a (100%) as a white solid: R_f(hexanes-

EtOAc, 1:1) 0.08; IR (film) 3384, 2955, 2923, 2869, 2232, 1664,

1641, 1619, 1448, 1432, 1371, 1233, 1156, 1051 cm^-1; ¹H NMR

(500 MHz) δ 6.33 (1 H, d, J ) 11.2 Hz), 6.05 (1 H, d, J ) 11.2

Hz), 5.33 (1 H, s), 4.99 (1 H, br s), 4.43 (1 H, m), 4.22 (1 H, m),

2.61 (1 H, dd, J ) 13.1, 4.9 Hz), 2.44 (1 H, m), 2.30 (1 H, dd,

J ) 12.8, 7.2 Hz), 2.26 (1 H, dd, J ) 14.0, 3.0 Hz), 2.06 (2 H,

m), 1.98 (4 H, m), 1.85 (2 H, m), 1.49 (6 H, s), 1.33 (2 H, m),

1.26 (3 H, m), 1.03 (3 H, d, J ) 6.8 Hz), 0.77 (3 H, s), 0.74 (3

H, d, J ) 7.2 Hz) ppm; MS m/z 400 (M⁺, 1), 382 (1), 342 (2),

311 (1), 255 (1), 227 (5), 209 (4), 151 (12), 133 (15), 91 (22), 43

(100).

P r ep a r a tion of P r evita m in 51 fr om An a logu e 9a . A

solution of 9a (5.2 mg) in acetone-d₆(1.5 mL) in a NMR tube

was kept in the dark under N₂atmosphere at 24 °C and the

1,7-sigmatropic hydrogen shift rearrangement was followed

1

by H NMR. After 58 days a 23:77 equilibrium mixture of 9a

and 51, respectively, was obtained. The solvent was removed,

and the products were separated by HPLC (RSil CN; hexane-

2-propanol-CH₃CN, 91:8:1) to give pure previtamin D ana-

logue 51: R_f(hexanes-EtOAc, 1:1) 0.08; IR (film) 3384, 2955,

2923, 2869, 2232, 1664, 1641, 1619, 1448, 1432, 1371, 1233,

1

1156, 1051 cm^-1; H NMR (500 MHz) δ 5.94 (1 H, br d, J )

12.2 Hz), 5.67 (1 H, br d, J ) 6.4 Hz), 5.64 (1 H, br d, J ) 12.2

Hz), 4.20 (1 H, m), 4.17 (1 H, m), 2.44 (1 H, dd, J ) 16.0, 4.4

Hz), 2.28 (1 H, dd, J ) 16.0, 2.8 Hz), 2.11 (2 H, m), 2.08 (1 H,

m), 2.02 (1 H, m), 1.98 (1 H, m), 1.83 (1 H, m), 1.72 (3 H, s),

1.50 (3 H, s), 1.49 (3 H, s), 1.04 (3 H, d, J ) 6.9 Hz), 0.82 (3 H,

s), 0.73 (3 H, d, J ) 7.3 Hz) ppm; MS m/z 400 (M⁺, 1), 382 (1),

342 (2), 311 (1), 255 (1), 227 (5), 209 (4), 151 (12), 133 (15), 91

(22), 43 (100).

Biologica l Eva lu a tion . All analogues published in the

literature (compounds 2-5, 6c, and 6d ) together with the 6C

analogues 8a -d were evaluated in our own laboratory (Leu-

ven) as described below.

Ma ter ia ls. 1,25(OH)₂D₃(1) was a gift of M. Uskokovic

(Hoffmann-La Roche, Nutley, NJ ) and J . P. van de Velde

(Duphar, Weesp, The Netherlands). [³H]1,25(OH)₂D₃(specific

activity 180 µCi/mmol) and [methyl-³H]thymidine (specific

activity 2 Ci/mmol) were purchased from Amersham (Buck-

inghamshire, U.K.). Culture media and gentamycin were

obtained from Gibco (Roskilde, Denmark). Penicillin and

streptomycin were from Boehringer Mannheim (Mannheim,

Germany). 4-Nitro blue tetrazolium (NBT) and phorbol 12-

myristate 13-acetate were purchased from Sigma (St. Louis,

MO). Fetal calf serum (FCS) was obtained from J RH Bio-

sciences (Sera-Lab, W. Sussex, U.K.). Human serum vitamin

D binding protein (hDBP) was prepared by affinity chroma-

tography as described previously.³⁹

Cells a n d An im a ls. The human promyelocytic leukemia

(HL-60) and breast carcinoma (MCF-7) cell lines were obtained

from the American Tissue Culture Co. (Rockville, MD).

Vitamin D-replete mice were obtained from the Proefdieren-

centrum of Leuven (Belgium).

In Vivo Activity of th e 1,25(OH)₂D₃An a logu es. The

hypercalcemic effect of the analogues was tested in vitamin

D-replete normal NMRI mice by daily subcutaneous injection

of 1,25(OH)₂D₃, its analogues, or the solvent during 7 consecu-

tive days, using serum calcium concentration as parameters.

Con for m a tion a l An a lysis a n d Molecu la r Mod elin g:

Volu m e Ma p s. Conformational analysis of the side chain of

compounds 1, 5, 6c, 6d , and 8a -d was carried out using the

MacroModel molecular modeling program of Still⁴⁴run on a

Digital VAXstation 4000-90A or SiliconGraphics Octane. Mo-

lecular mechanics calculations were carried out on model

compounds in which the A-ring and diene system up to C6

Affin ity for VDR. The affinity of the analogues of 1,25-

(OH)₂D₃to the VDR was evaluated by their ability to compete

with [³H]1,25(OH)₂D₃for binding to high-speed supernatant

from intestinal mucosa homogenates obtained from normal

pigs. The incubation was performed at 4 °C for 20 h, and phase

separation was obtained by addition of dextran-coated char-

coal. The relative affinity of the analogues was calculated from

their concentration needed to displace 50% of [³H]1,25(OH)₂D₃

Diastereomeric Dihydroxyvitamin D Analogues

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3555

(7) J ung, S. J .; Lee, Y. Y.; De Vos, S.; Elstner, E.; Norman, A. W.;

Green, J .; Uskokovic, M.; Koeffler, H. P. 1,25(OH)₂-16-Ene-

vitamin D₃is a Potent Antileukemic Agent with Low Potential

to Cause Hypercalcemia. Leuk. Res. 1994, 101, 713-718.

(8) 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.

(9) Haussler, M. R.; Whitfield, G. K.; Haussler, C. A.; Hsieh, J .;

Thompson, P. D.; Selznick, S. H.; Dominguez, C. E.; J urutka, P.

W. The Nuclear Vitamin D Receptor: Biological and Molecular

Regulatory Properties Revealed. J . Bone Miner. Res. 1998, 13,

325-349.

were replaced by an H atom. Only the most stable chair

conformation of the C-ring was taken into account. Rotations

with 60° increments were applied to the rotatable C-C bonds

of the side chain, while the 25-OH was rotated with increments

of 120°. The so-generated starting conformations were mini-

mized using the MM2* force field implementation of Macro-

Model, and the conformations within 20 kJ /mol of the mini-

mum energy form were retained. Using a PC computer

program all conformations of each compound were then

overlaid using C13 as a common origin (x, y, z ) 0), C14 was

positioned in the yz-plane (x ) 0), and C18 was made to

coincide with the positive y-axis (x, z ) 0). A line drawing was

generated of the minimum energy conformation, and the

position of O25 in each of the local energy minima within the

given energy window was represented by a dot to obtain dot

maps. The volume maps shown in Figures 1 and 2 were

generated by importing the above dot maps into the CS

Chem3D molecular modeling program,⁴⁵creating ball-and-

stick models using van der Waals radii of 0.7 and 0.8 Å for C

and O, respectively, and applying the appropriate color to O

according to the energy of the particular conformation.

Deter m in a tion of a Rela tive Activity Volu m e by Su b-

tr a ction . For subtraction of the dot maps of respectively 6d

from 6c and 8c from 8a , a PC computer program was used

which allowed for (1) the generation of a sphere centered

around each O25 position in the dot map of 6c (8a ) and (2)

the determination of the presence of a O25 position of 6d (8c)

in each of these spheres. For the procedure both dot maps were

oriented and overlaid using C13, C14, and C18 as above. In

case no O25 position of 6d (8c) was present in the sphere, the

particular O25 position of 6c (8a ) was retained. In case a O25

position of 6d (8c) was found in the sphere, the particular O25

position of 6c (8a ) was either (a) retained if the energy category

of the conformation of 6c (8a ) was lower (more stable) than

the energy category of the conformation of 6d (8c) or (b)

rejected if the energy category of the conformation of 6c (8a )

was equal or higher (less stable). For this, four energy

categories corresponding to 0-5, 5-10, 10-15, and 15-20 kJ /

mol, respectively, were used. The radius of the sphere was

varied so as to end up with 15-20% (arbitrarily chosen, but

found to give a workable reduced volume area) of the original

number of conformations (0-20 kJ /mol) of 6c and 8a , respec-

tively. The relative activity volumes shown in Figure 2 (top

and middle) were obtained by setting the radius of the sphere

to 4.1 Å in the case of 6c and to 1.65 Å in the case of 8a .

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Stereochemistry on the Conformational Profile of the Side

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25-Dihydroxyvitamin D₃and its 20-Epimer: Compounds for

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H. F.; Yamada, S. Side-chain conformation and activity of

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Norman, A. W., Bouillon, R., Thomasset, M., Eds.; University

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Tetrahedron Lett. 1991, 32, 7663-7666.

(21) (a) Takano, S.; Yamada, O.; Iida, H.; Ogasawara, K. Enantio-

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2-Phenylthio-2-cyclohexenol using the Same Lipase. Synthesis

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Asymmetry 1993, 4, 2323-2326. See also (c) Kawasaki, M.;

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Ack n ow led gm en t. We thank the FWO - Vlaan-

deren, G.0051.98, Ministerie voor Wetenschapsbeleid,

and THERAMEX S.A. for financial assistance to this

laboratory.

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J M980736V

Article Doi

Synthesis, biological activity, and conformational analysis of four seco-D-15,19-bisnor-1α,25-dihydroxyvitamin D analogues, diastereomeric at C17 and C20

DOI: 10.1021/jm980736v

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