13-Methyl-substituted des-C,D analogs of (20S)-1α,25-dihydroxy-2-methylene-19-norvitamin D3 (2MD): Synthesis and biological evaluation

Bioorganic & Medicinal Chemistry 17 (2009) 1747–1763

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Bioorganic & Medicinal Chemistry

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13-Methyl-substituted des-C,D analogs of (20S)-1a,25-dihydroxy-2-methylene-

19-norvitamin D₃(2MD): Synthesis and biological evaluation

Katarzyna Plonska-Ocypa ^a,b, Rafal R. Sicinski ^a,b, Lori A. Plum ^a, Pawel Grzywacz ^a, Jadwiga Frelek ^c,

Margaret Clagett-Dame ^a, Hector F. DeLuca ^a,

*

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

^bDepartment of Chemistry, University of Warsaw, ul. Pasteura 1, 02-093 Warsaw, Poland

^cInstitute of Organic Chemistry, Polish Academy of Sciences, ul. Kasprzaka 44/52, 01-224 Warsaw, Poland

a r t i c l e i n f o

a b s t r a c t

Article history:

Analogs of (20S)-1a,25-dihydroxy-2-methylene-19-norvitamin D₃(2, 2MD), substituted at C-13 but lack-

Received 24 September 2008

Revised 19 November 2008

Accepted 23 November 2008

Available online 10 December 2008

ing both C and D rings, were prepared in convergent syntheses, starting with the chiral ester 14 and the

phosphine oxide 29. Two of the synthesized vitamins (11 and 32) were analogs in which the 13-methyl

group constituted a substituent of an unsaturated fragment, that is, C(13)–C(17) double bond, whereas in

the two other cases (12 and 13), the methyl group belonged to a ternary carbon stereogenic center. The

aim of these studies was to further explore extensive modiﬁcations in the ‘upper’ part of the vitamin D

skeleton in the hope of ﬁnding biologically active analogs of potential therapeutic value.

The commercial (R)-(ꢀ)-methyl-3-hydroxy-2-methylpropionate (14) was converted in six steps to

alcohol 18, the vitamin D side chain fragment. Its subsequent three-step transformation led to aldehyde

20 which was subjected to the Still–Gennari HWE reaction with anion derived from ester 21. The

Keywords:

Vitamin D analogs

19-Norvitamin D

des-C,D-Steroids

Calcemic activity

obtained

a

,b-unsaturated esters 22 and 23 served as convenient starting compounds to the syntheses

-unsaturated (28) and saturated (39 and 40), required

of the corresponding chiral acyclic aldehydes, b,

c

for the ﬁnal Wittig–Horner coupling with the anion of the phosphine oxide 29. After hydroxyl deprotec-

tion, the synthesized vitamin D analogs 11–13 and 32 were puriﬁed and biologically tested. Only the

(13R,20S)-analog 12 retained substantial, although 30 times lower than 1a,25-(OH)₂D₃, binding ability

to the full-length rat recombinant vitamin D receptor (VDR). This analog was also very effective in differ-

entiation of HL-60 cells, and it exerted signiﬁcant transcriptional activity (2 times and 15 times less

potent, respectively, as compared to the native hormone). The in vivo tests showed that all synthesized

vitamin D analogs were devoid of calcemic activity.

1. Introduction

In the course of our structure–activity studies in the vitamin D

ﬁeld, we discovered one of the most promising structural modiﬁca-

In addition to its well known, classical role in calcium and phos-

phorus homeostasis,^1,2

,25-dihydroxyvitamin D₃, [1 ,25-

tions of the A ring: a ‘shift’ of the exocyclic methylene moiety from

C-10 to C-2. Ten years ago we described the synthesis of 1 ,25-

dihydroxy-2-methylene-19-norvitamin analogs which were

1a

a

(OH)₂D₃, calcitriol, 1; Fig. 1] exhibits a broad spectrum of biological

actions. For example, this native hormone possesses signiﬁcant cel-

lular differentiation activity and a strong immunomodulatory ef-

fect.^3,4Among thousands of structural analogs of calcitriol

prepared and biologically tested to date, some exhibit an interest-

ing separation of calcemic and differentiation activities.⁵Consider-

ing the fact that strong calcemic activity limits the therapeutic

potential of vitamin D compounds in non-classical uses, such selec-

tivity may be of considerable importance.

D

characterized by signiﬁcant biological potency, enhanced dramati-

cally in compounds with an ‘unnatural’ (20S)-conﬁguration.⁶Ana-

log 2 (2MD) seemed to be most interesting since it was shown that

in ovariectomized rats this compound can produce a signiﬁcant in-

crease in bone density.⁷2MD is currently undergoing Phase II clin-

ical testing as a potential drug for osteoporosis. Several compounds

related to 2MD (differing mostly in the side chain structure) have

been synthesized in our laboratory⁸and some of them displayed

a selective pattern of biological activities.⁹

Removal of the C and D rings constitutes another interesting

modiﬁcation of the vitamin D skeleton. First compounds (retiferols

3 and 4) lacking the C,D-substructure were obtained 13 years

* Corresponding author. Tel.: +1 608 262 1620; fax: +1 608 262 7122.

E-mail address: deluca@biochem.wisc.edu (H.F. DeLuca).

doi:10.1016/j.bmc.2008.11.082

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K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

OH

HO

OH

HO

OH

HO

R

1

2

3: R = H

4: R = OH

OH

CF₃

F₃C

R

OH

R

X

HO

OH

HO

OH

5

6: R = Me, X = CH₂

7: R = Et, X = CH₂

10

8: R = Me, X = O

9: R = Et, X = O

20

17

20

S

OH

13

14

R

13

S

8

HO

OH

HO

OH

HO

OH

11

12

13

Figure 1. Chemical structures of 1a,25-dihydroxyvitamin D₃(calcitriol, 1) and analogs.

ago.¹⁰One year later, des-C,D vitamins possessing an additional

ring E were described¹¹and during the following years syntheses

of des-C,D 19-norvitamin D₃derivatives (5–9) were also re-

ported.^12,13Very recently, we synthesized and biologically tested

a des-C,D analog of 2MD (10), which retained some transcriptional

activity and binding afﬁnity to the vitamin D receptor (VDR) albeit

decreased by two orders of magnitude in comparison with the

analogous vitamins possessing intact C,D rings. Considering that

the structure of this analog is characterized by extremely high ﬂex-

ibility, which should allow this molecule to accommodate easily to

the active sites of the ligand binding domain (LBD) of the VDR, the

poor binding ability of 10 is puzzling. Since it is known that C,D-

rings of 2MD are surrounded in its crystalline complex with the

LBD of the VDR,¹⁴by strongly hydrophobic amino acids, insufﬁ-

cient hydrophobic interactions of analog 10 with the receptor are

one potential explanation for this result. To verify this hypothesis,

we decided to prepare the des-C,D vitamins substituted with

groups (such as alkyls), which could increase the hydrophobic

interaction of the molecule with VDR.

As a continuation of our search for biologically active 2-methy-

lene-19-norvitamin D compounds, we decided to synthesize the

analogs 11–13 which are characterized by the lack of the C,D-rings

and a presence of one methyl group at C-13.

2. Results

2.1. Synthesis

Our synthesis began with commercial (R)-(ꢀ)-methyl-3-hydro-

xy-2-methylpropionate (14, Scheme 1), and it focused on the prep-

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

1749

ClMg

16

BOMO

OTs

BOMO

HO

a, b, c

COOCH₃

d

14

15

17

18

e, b

h

f, g

OTBS

OH

O

HO

20

19

O

OCH₂CF₃

MeOOC

P

OCH₂CF₃

OTBS

21

MeOOC

HOH₂C

j

i

BOM = -CH₂OCH₂Ph

OTBS

22:

23:

Z

E

-isomer

24:

25:

Z

-isomer

E-isomer

Scheme 1. Synthesis of unsaturated allylic alcohols 24 and 25. Reagents: (a) PhCH₂OCH₂Cl, CH₂Cl₂; (b) LiAlH₄, THF; (c) p-TsCl, DMAP, Et₃N, CH₂Cl₂; (d) 16, Li₂CuCl₄, THF; (e)

MCPBA, CH₂Cl₂; (f) TBSOTf, CH₂Cl₂; (g) Pd/C, H₂, AcOEt; (h) TPAP, NMO, CH₂Cl₂; (i) 21, KHMDS, 18-crown-6, THF; (j) DIBALH, CH₂Cl₂/toluene.

OTBS

a, b

OTBS

HOH₂C

24

26

OTBS

c

OTBS

d

28

27

O

OH

OPPh₂

e

OTBS

OH

TBSO

OTBS

29

f

+

TBSO

OTBS

HO

OH

HO

OH

30: 7

31: 7

E

Z

11

32

Scheme 2. Synthesis of des-C,D vitamins 11 and 32. Reagents: (a) p-TsCl, DMAP, Et₃N CH₂Cl₂; (b) LiAlH₄, THF; (c) TBAF, THF; (d) TPAP, NMO, CH₂Cl₂; (e) 29, PhLi, THF; (f) HF/

MeCN, THF; HPLC separation.

aration of the corresponding aldehydes required for Wittig–Horner

coupling with the phosphine oxide 29 (Scheme 2), obtained by us

previously.⁶Reduction of the BOM-protected hydroxy ester with

LiAlH₄and the tosylation of the formed alcohol furnished the tos-

ylate 15 that reacted with Grignard reagent 16 to give an oleﬁnic

compound 17. Epoxidation of the latter compound and the follow-

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K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

ing reduction of the formed isomeric epoxides gave alcohol 18. Its

tertiary hydroxy group was protected as TBS ether and then the

primary hydroxyl deprotected by hydrogenation yielding hydroxy

ether 19. Subsequent oxidation with PDC provided aldehyde 20

that was treated with the anion derived from phosphono ester

21, which in turn was prepared from commercial reagents

(F₃CCH₂O)₂POCH₂COOCH₃and Br(CH₂)₂OTBS. The attempted

Still–Gennari HWE oleﬁnation reaction proved to be successful –

a mixture of Z- and E-isomeric unsaturated esters 22 and 23

(1.7:1 ratio) was formed in quantitative yield. The assignment of

conﬁguration of the products followed from an analysis of their

¹H NMR spectra: signal of oleﬁnic proton in E-isomer 23 was

deshielded by 0.92 ppm. Since HPLC separation of these geometri-

cal isomers was difﬁcult, they were subjected to the DIBALH reduc-

tion and the formed isomeric allylic alcohols 24 and 25 could be

easily separated by column chromatography. The prevailing Z-iso-

separated by HPLC. Value of the vicinal coupling constant

(14.9 Hz) between vinylic 7- and 8-protons, found in the ¹H NMR

spectra of the main vitamin D products, indicated their trans-rela-

tionship and allowed to easy assignment of the double bond con-

ﬁgurations of compounds 30 and 11. Unfortunately, the proton

magnetic resonance spectrum of the isomeric 7Z-isomer 12, iso-

lated in microgram quantities, was more complicated due to signal

overlapping. Therefore, the conﬁgurational assignment was based

on the literature data (formation of isomeric 7E- and 7Z-vitamins

D was described previously^15–17) and comparison with analogous

vitamins obtained in our laboratory (results to be published).

The allylic alcohol 25 served as a starting compound for the

preparation of the saturated aldehydes 39 and 40 (Scheme 3).

Hydrogenation of this compound provided the mixture of diaste-

reomeric saturated alcohols 33 and 34. These products were sepa-

rated by HPLC and the following synthetic steps were executed

separately with both diastereomers. For further characterization

of isomers, the corresponding diols 35 and 36 were also obtained.

Conﬁguration of the new stereogenic center in the synthesized

alcohols was established by a series of chemical transformations

(described later) and analysis of circular dichroism (CD) data.

The synthetic path leading to the aldehyde 39 started from

tosylation of the (R,S)-alcohol 33 and the reduction of the tosylate.

Selective hydroxyl deprotection in the obtained diether 37 fol-

lowed by oxidation provided the saturated aldehyde 39 which

was subjected to Wittig–Horner coupling with the anion generated

from 29. Removal of the silyl protecting groups in the product 41

mer 24 was used for the preparation of the b,c-unsaturated alde-

hyde 28 (Scheme 2). Thus, tosylation of the allylic alcohol and

subsequent reduction of the formed tosylate provided unsaturated

diether 26. Then, primary hydroxyl was selectively deprotected

and the alcohol 27 oxidized to the desired aldehyde 28. Wittig–

Horner reaction of this compound with lithium phosphinoxy carb-

anion derived from the phosphine oxide 29 gave a mixture of pro-

tected vitamin D analogs. The main product was identiﬁed as 7E-

isomer 30 contaminated with a minute amount of its 7Z-counter-

part 31. Removal of the silyl protecting groups was performed with

hydroﬂuoric acid and the ﬁnal vitamin D analogs 11 and 32 were

S

OTBS

a

OTBS

HOH₂C

TBSO

CH₂OH

25

33: R = TBS (

R,

S

)

34: R = TBS (S,S

)

b

OR

b

35: R = H (R,S)

36: R = H (S,S)

33, 34

c, d

OTBS

b, e

39: (

40: (S,S

R

,

S

)

37: (

38: (S,S

R

,

S

)

O

OTBS

OPPh₂

f

S

OH

OTBS

g

TBSO

OTBS

29

TBSO

OTBS

HO

OH

41: (

R

,

S

S)

)

12: (

R,S)

42: (S

13: (S,S)

Scheme 3. Synthesis of des-C,D vitamins 12 and 13. Reagents: (a) PtO₂, H₂, MeOH; HPLC separation; (b) TBAF, THF; (c) p-TsCl, DMAP, Et₃N, CH₂Cl₂; (d) LiAlH₄, THF; (e) TPAP,

NMO, CH₂Cl₂; (f) 29, PhLi, THF; (g) HF/MeCN, THF; HPLC separation.

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

1751

gave the ﬁnal (13R,20S)-vitamin D analog 12. An analogous syn-

thetic sequence, as the conversion of 33 to 12, was used for the

transformation of the isomeric alcohol 34 into the (13S,20S)-vita-

min D compound 13.

The literature data indicate that in the case of

c

-lactones a con-

formation of their penta-membered rings is the determining factor

for the sign of the CD bands.^18,19The ‘ring-chirality rule’ introduced

by Legrand and Bucourt²⁰states that a sign of the n–p^*Cotton ef-

fect can be directly correlated with a value of the torsion angle O–

2.2. Determination of stereochemistry at C-13

C(@O)–C –C_b(h, Fig. 2). Thus, a positive (negative) value of angle h

a

corresponds to a negative (positive) sign of the Cotton effect. Con-

formation and this angle, in turn, are dependent on the substitu-

tion of the lactone ring. It was also nicely demonstrated in our

case. We carried out MM⁺force ﬁeld calculations (HYPERCHEM 7.0,

Hypercube, Inc) for the diastereomeric lactones 45 and 46 and,

for comparison, their parent compounds with a free tertiary hydro-

xyl. We then performed conformational searches in every series

(described in Section 5) involving rotations of the lactone ring tor-

sions and the torsions from their alkyl substituents. The results of

these molecular mechanics studies unequivocally showed that, for

the lactones possessing an R-conﬁguration of the alkoxyalkyl (or

As a method allowing us to establish the absolute conﬁgura-

tions at the newly created stereogenic centers of the synthesized

compounds, we used the CD data of the

c-lactones 45 and 46

(Scheme 4) prepared from the unsaturated esters 22 and 23.

Thus, hydrogenation of these compounds provided a mixture

of saturated esters 43 and 44 which were separated by HPLC.

The less polar isomer 43 was treated with 10-camphorsulfonic

acid and, according to our expectations, it underwent smooth

conversion to the corresponding lactone 45. Analogously, the

more polar ester 44 furnished the more polar, stereoisomeric

lactone 46.

hydroxyalkyl) substituent at C , their strongly energetically pre-

a

OTBS

MeOOC

OTBS

22:

23:

Z-isomer

E

-isomer

a

O

OTBS

O

OTBS

43

44

less polar isomer

more polar isomer

b

O

S

OTBS

O

R

S

45

46

less polar isomer

CD: isomer R,S

more polar isomer

CD: isomer S,S

c

S

OTBS

HOH₂C

R

S

OH

35

36

more polar isomer

less polar isomer

Scheme 4. Synthesis of lactones 45 and 46 aimed at establishing of conﬁgurations at stereogenic centers in the obtained stereoisomeric compounds. Reagents: (a) PtO₂, H₂,

AcOEt; HPLC separation; (b) CAS, MeOH; (c) LiAlH₄, THF.

1752

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

mer 12 more pronounced afﬁnity was detected, albeit still de-

R

creased 30 times compared to 1, whereas the potency of the

(13S,20S)-vitamin D analog 13 proved to be lower by two orders

of magnitude in comparison with the native hormone. The next as-

say constituted measuring the compound ability to induce differ-

entiation of human promyelocyte HL-60 cells into monocytes.

Only two of the tested analogs (12 and 13) were found active;

however, in this case the difference in their potency was dramatic:

analog 12 exhibited only twofold decreased activity, whereas iso-

mer 13 proved to be 100 times less active with respect to the par-

ent hormone. A similar pattern of the relative potencies of the des-

C,D vitamins was discovered when their transcription activity was

established, indicated in the 24-hydroxylase (CYP-24) promoter

driving luciferase reporter gene system. Thus, analogs 12 and 13

β

θ

O

θ

β

R

(S)-lactone: C.D. (+)

(R)-lactone: C.D. (-)

Figure 2. Determination of a sign of the n–p^*Cotton effect for 3-substituted

-lactones by a ‘ring-chirality rule’. The compounds are shown in their energetically

c

preferred ring conformations; hydrogen atoms are omitted for clarity.

were 15 and 450 times, respectively, less active than 1a,25-

ferred ring conformations are characterized by a positive torsional

angle h (ca. 20°), whereas in the case of their counterparts with an

S-conﬁguration of substituents, the preferred ring conformations

have a negative value of this angle (ca. ꢀ20°). The steric energy dif-

ferences (ca. 1.35 kcal/mol) between the global minimum confor-

mations and the lowest energy structures with the ‘inverted’

rings and the opposite h values indicate that at room temperature

the prevailing conformations amount to more than 90% of the

population.

(OH)₂D₃, whereas the remaining tested compounds 11 and 32 were

found inactive in this regard. We tested two des-C,D ring analogs,

that is, compounds 10 and 13 in vivo for their ability to activate

intestinal calcium transport and bone calcium mobilization (serum

calcium) (Table 2). Even when tested at very high doses, neither

compound had signiﬁcant activity in raising serum calcium at

the expense of one or in stimulating intestinal calcium transport,

while 1a,25-(OH)₂D₃(compound 1) had activity at all dose levels

and gave an excellent dose-dependent response.

In an effort to corroborate the stereochemical conclusions made

on the basis of the ‘ring-chirality rule’, the ECD spectra of lactones

45 and 46 were calculated by means of the time-dependent density

functional theory (TDDFT). For a given molecular conformation,

TDDFT provides a detailed picture of the electronic structure,

which in turn is the key to a quantitative treatment of chiroptical

properties.²¹Thus, comparing the computed CD spectra to the

measured ones, we can infer conclusions on the stereochemistry

of the lactones studied here. In particular, we can assign the abso-

lute conﬁguration of the crucial C-3 carbon atom for compounds 45

and 46, which provides an independent test of the ‘ring-chirality

rule’.

To obtain the simulated CD spectra, the lowest energy conform-

ers of 45 and 46, calculated by the MM⁺force ﬁeld, were used

(Fig. 2). To reduce calculation times the O-TBDMS group we re-

placed by OH group. No signiﬁcant contributions of the O-TBDMS

molecular moiety was expected to the overall rotational strengths.

Next, the rotatory strength was calculated for each conformer at

the B3LYP/6-31++G(D,P) level. As can be seen in Figure 3, the

experimental and simulated CD spectra are in an excellent agree-

ment thus providing an independent (conclusive) evidence for

the absolute conﬁguration determination. Building on the results

of conformational analysis, the experimentally developed ‘ring-

chirality rule’ and the TDDFT calculations we can conclude that

the absolute conﬁguration of the synthesized lactones—3R for the

less polar compound 45 and 3S for the isomeric 46 (Fig. 3) is estab-

lished with high degree of conﬁdence. Each lactone 45 and 46 was

then subjected to reduction with LiAlH₄and the isolated products

were found identical in all respects with the previously obtained

(Scheme 3) diols 35 and 36.

3. Discussion

The structure–activity studies in the vitamin D ﬁeld still attract

attention of numerous research laboratories. Among the approxi-

mate three thousand analogs of the natural hormone 1a,25-

(OH)₂D₃synthesized to date, a vast majority is characterized by

an intact hydrindane system of two steroidal C and D rings. There-

fore, an examination of the biological effect of removing these two

rings seemed to be of signiﬁcant interest. Considering that the A-

ring modiﬁcation, consisting of a shift of the exocyclic methylene

group from C-10 to C-2, exerts a positive inﬂuence on the VDR

binding and transcriptional activity of the analogs,⁶we decided

to synthesize an analog of 2MD lacking its C and D rings. Recently

we synthesized compound 10 with such a structure and it turned

out that biological activity of this analog is two logs smaller in

comparison to the native hormone.²²In continuation these studies,

synthesis of its analogs substituted at C-13 with the methyl group

was undertaken and is reported in this paper. Construction of the

aliphatic chain, representing the vitamin D side chain (carbons

20–27) and attached carbons 17, 13, 14 and 8, began from the com-

mercially available hydroxy ester 14. This substrate was efﬁciently

transformed into unsaturated esters 22 and 23 which in turn

served as suitable starting compounds for the synthesis of all pre-

pared vitamin D analogs.

A comparison of the in vitro activities of compounds described

in this work with the corresponding potencies of the unsubstituted

at C-13 analog 10, obtained by us previously,²²indicated that an

introduction of a double bond between C(13) and C(17) resulted

in signiﬁcant decrease of the compounds’ activities, especially

drastic in the case of the 7Z-isomer 32. The presence of the addi-

tional methyl group as a substituent of the saturated chain proved

to be beneﬁcial only in the case of the 13R-conﬁguration (analog

12) at the newly created stereogenic center.

2.3. Biological evaluation

The described assignment and correlation of conﬁgurations al-

lowed us also to assign conﬁgurations of the methyl substituents

in the vitamin D compounds 12 and 13. All synthesized des-C,D

4. Conclusions

analogs of (20S)-1a,25-dihydroxy-2-methylene-19-norvitamin D₃

were tested for their ability to bind rat vitamin D receptor (Table

1). The 7E-vitamin 11 with a C(13)–C(17) double bond was 600

times less potent than the natural hormone in displacement of

Further structure–activity studies performed on analogs of 2MD

lacking the C and D rings showed that such a drastic modiﬁcation

of the basic vitamin D skeleton is promising and can provide

compounds of pharmaceutical interest. However, introduction of

radiolabeled 1

a,25-(OH)₂D₃from the receptor protein, and its 7Z-

isomer 32 was completely devoid of activity. For the (13R,20S)-iso-

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

1753

Table 1

VDR binding properties,^aHL-60 differentiating activities,^band transcriptional activities of the vitamin D compounds 10–13 and 32

Compound

Compd no.

VDR binding

HL-60 differentiation

EC₅₀Ratio

24-OHase transcription

EC₅₀Ratio

K_i

Ratio

OH

1

1.0 ꢁ 10^ꢀ10

M

1

3.0 ꢁ 10^ꢀ9

M

1

2.0 ꢁ 10^ꢀ10

M

1

HO

OH

10

5.0 ꢁ 10^ꢀ8

M

80

3.0 ꢁ 10^ꢀ7

M

100

5.0 ꢁ 10^ꢀ8

M

250

HO

OH

11

6.0 ꢁ 10^ꢀ8

M

600

>10^ꢀ6

M

>333

>10^ꢀ7

M

>500

HO

OH

32

>>10^ꢀ6

M

>>10000

>10^ꢀ6

M

>333

>>10^ꢀ7

M

>>500

HO

OH

12

3.0 ꢁ 10^ꢀ8

M

30

6.0 ꢁ 10^ꢀ9

M

2.0

3.0 ꢁ 10^ꢀ9

M

15

HO

OH

(continued on next page)

1754

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

Table 1 (continued)

Compound

Compd no.

VDR binding

HL-60 differentiation

24-OHase transcription

EC₅₀Ratio

K_i

Ratio

EC₅₀

Ratio

OH

13

1.0 ꢁ 10^ꢀ8

M

100

3.0 ꢁ 10^ꢀ7

M

100

9.0 ꢁ 10^ꢀ8

M

450

HO

OH

a

Competitive binding of 1

a,25-(OH)₂D₃(1) and the synthesized vitamin D analogs to the full-length recombinant rat vitamin D receptor. The experiments were carried out

in duplicate on two different occasions. The K_ivalues are derived from dose–response curves and represent the inhibition constant when radiolabeled 1

present at 1 nM and a K_dof 0.2 nM is used. The binding ratio is the average ratio of the analogs K_ito the K_ifor 1 ,25-(OH)₂D₃.

,25-(OH)₂D₃and the synthesized vitamin D analogues. Differentiation state was determined by

a

,25-(OH)₂D₃is

a

b

Induction of differentiation of HL-60 promyelocytes to monocytes by 1

a

measuring the percentage of cells reducing nitro blue tetrazolium (NBT). The experiment was repeated in duplicate two times. The ED₅₀values are derived from dose-

response curves and represent the analogs concentration capable of inducing 50% maturation. The differentiation activity ratio is the average ratio of the analogs ED₅₀to the

ED₅₀for 1a,25-(OH)₂D₃.

c

Transcriptional assay in rat osteosarcoma cells stably transfected with a 24-hydroxylase gene reporter plasmid. The ED₅₀values are derived from dose-response curves

and represent the analogs concentration capable of increasing the luciferase activity 50%. The lucerifase activity ratio is the average ratio of the analogs ED₅₀to the ED₅₀for

1a,25-(OH)₂D₃.

dm^ꢀ3were examined in cells with the path length of 0.1 or 1 cm.

¹H nuclear magnetic resonance (NMR) spectra were recorded at

200, 400 and 500 MHz with Varian Unity plus spectrometer as well

as Bruker Instruments DMX-400 and DMX-500 Avance console

spectrometers in deuteriochloroform. ¹³C nuclear magnetic reso-

nance (NMR) spectra were recorded at 50 and 100 MHz with a Var-

ian Unity plus and Bruker Instrument DMX-400 spectrometers in

deuteriochloroform. Chemical shifts (d) are reported downﬁeld

from internal Me₄Si (d 0.00). Electron impact (EI) mass spectra

were obtained with a Micromass AutoSpec (Beverly, MA) instru-

ment. High-performance liquid chromatography (HPLC) was per-

formed on a Waters Associates liquid chromatograph equipped

with a Model 6000A solvent delivery system, a Model U6K Univer-

sal injector, and a Model 486 tunable absorbance detector. THF was

freshly distilled before use from sodium benzophenone ketyl under

argon.

Figure 3. Experimental and simulated CD spectra of lactones 45: (exp. ꢃ–ꢃ–ꢃ–),

(DFT –––) and 46: (exp. ꢃꢃꢃ), (DFT ——).

5.1. (R)-Toluene-4-sulfonic acid 3-benzyloxymetoxy-2-methyl-

propyl ester (15)

a rigid moiety, namely the trisubstituted double bond C(13)@

C(17), in the long aliphatic fragment consisting of carbons 8, 14,

13, 17 and steroidal side chain, practically abolishes the biological

activity of such analogs. Conﬁguration of the methyl substituents

at C-13 proved to be of crucial importance: the R-substitution sig-

niﬁcantly enhanced the activity, whereas S-alkyl did not seem to

play any role. These ﬁndings offer clear suggestions regarding the

direction of other modiﬁcations in the related molecules. It is likely

that attaching a longer or branched 13R-alkyl substituent will fur-

ther increase the transcriptional potency and VDR afﬁnity of des-

C,D vitamin D compounds. Also, the inﬂuence of a double alkyl

substitution at C-13 on biological activity of the analogs should

be examined.

To a solution of R-(ꢀ)-methyl-3-hydroxy-2-methylpropionate

(4 mL, 4.26 g, 36 mmol) in anhydrous CH₂Cl₂(30 mL) was added

N,N-diisopropylethylamine (11.8 mL, 8.75 g, 60 mmol) at room

temperature. The mixture was cooled to ꢀ78 °C and benzyl chloro-

methyl ether (5.6 mL, 6.29 g, 40 mmol) was added dropwise via

cannula. The cooling bath was removed and the reaction mixture

was stirred at room temperature for 16 h. Then tetrabutylammo-

nium iodide (50 mg) and benzyl chloromethyl ether (2 mL,

3.15 g, 20 mmol) was added. The mixture was stirred at room tem-

perature for 3 h, poured into water, and extracted with methylene

chloride. The combined organic layers were dried (Na₂SO₄) and

concentrated under reduced pressure. The residue was chromato-

graphed on silica gel using hexane/AcOEt (9:1) as an eluent to give

(2R)-3-benzyloxymetoxy-2-methyl-propionic acid methyl ester

5. Experimental

(8.29 g, 97%) as a colorless oil; ½a _D²⁴

ꢂ

ꢀ3 (c 0.17, CHCl₃); ¹H NMR

Chemistry: Ultraviolet (UV) absorption spectra were recorded

with a Perkin–Elmer Lambda 3B UV–vis spectrophotometer in eth-

anol and hexane. Circular dichroism (CD) spectra were recorded

between 185 and 300 nm at room temperature with a JASCO J-

715 spectropolarimeter in acetonitrile solutions. The solutions

with concentrations in the range of 0.8 ꢁ 10^ꢀ4–1.2 ꢁ 10^ꢀ3mol/

(500 MHz, CDCl₃) d 1.19 (3H, d, J = 7.1 Hz, CH–CH₃), 2.77 (1H, m,

CH–CH₃), 3.64 (1H, dd, J = 9.4, 5.4 Hz, one of CH₂–CH), 3.70 (3H,

s, CH₃O), 3.78 (1H, dd, J = 9.4, 7.8 Hz, one of CH₂–CH), 4.57 (2H, s,

OCH₂O), 4.74 (2H, s, CH₂Ph), 7.29 (1H, m, Ar-H_para), 7.35 (4H, m,

Ar-H_ortho,meta); ¹³C NMR (125 MHz) d 13.91 (CH₃), 39.99 (CH–

CH₃), 51.70 (CH₃O), 69.22 and 69.60 (CH₂CH and CH₂–Ph), 94.50

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

1755

Table 2

Inability of the des-C,D-analogs of (20S)-1

a,25-dihydroxy-2-methylene-19-norvitamin D₃to support intestinal calcium transport and bone calcium mobilization in vitamin D-

deﬁcient rats on a low-calcium diet^a

Compound

Compd

no.

Bone Ca mobilization and intestinal Ca transport

Dose level Bone (serum Ca increase compared to

Intestine (increase in S/M ratio compared to

vehicle)

l

g/kg

pmol/rat/

day

Serum

Ca

Serum Ca increase compared to

vehicle

bodyweight

OH

1

0

4

—

0.2

0.7

2.1

87

260

780

4.5

5.3

5.9

0.5

1.3

1.9

4.2

2.8

4.2

HO

OH

19

7020

7.9

3.0

5.9

ND

10

0

4.1

3.9

4.2

4.3

—

1.9

5.7

22.8

1040

3120

12,480

ꢀ0.2

0.1

0.2

HO

OH

13

0

4.3

4.1

4.2

ꢀ0.2

ꢀ0.1

ꢀ0.2

ꢀ0.1

ꢀ0.5

0.4

5.5

55

165

2340

23,400

70,200

ꢀ0.5

ꢀ0.9

HO

OH

a

Weanling male rats were maintained on a 0.47% Ca diet for 1 week and then switched to a low-calcium diet containing 0.02% Ca for an additional 3 weeks. During the last

week, they were dosed daily with the appropriate vitamin D compound for 7 consecutive days. All doses were administered intraperitoneally in 0.1 mL propylene glycol/

ethanol (95:5). Controls received the vehicle. Determinations were made 24 h after the last dose. There were 5–6 rats per group.

(OCH₂O), 127.63, 127.84 and 128.33 (Ar_{ortho,meta,para}), 137.61

(Ar_ipso); MS (EI) m/z (relative intensity) no M⁺, 207 (M⁺ꢀOCH₃, 2),

131 (34), 120 (64), 91 (100); HRMS (ESI) exact mass calcd for

C₁₃H₁₈O₄Na (M⁺+Na) 261.1103, measured 261.1110.

128.49 (Ar_{ortho,meta,para}), 137.58 (Ar_ipso); MS (EI) m/z (relative

intensity) no M⁺, 180 (8), 120 (100), 108 (95), 89 (72); HRMS

(ESI) exact mass calcd for C₁₂H₁₈O₃Na (M⁺+Na) 233.1154, mea-

sured 233.1158.

A solution of the obtained ester (13.3 g, 56 mmol) in anhydrous

THF (55 mL) was added dropwise to the suspension of lithium alu-

minum hydride (3.2 g, 84 mmol) in anhydrous THF (250 mL) at

0 °C. The cooling bath was removed and the reaction was stirred

at room temperature for 20 h, quenched with cold water and ex-

tracted with AcOEt. The solvents were removed in vacuum and

the crude oil was puriﬁed by silica gel chromatography using hex-

ane/AcOEt (8:2) as an eluent to afford an oily I-3-benzyloxymeth-

To the mixture of the obtained diol (11.0 g, 50 mmol), DMAP

(178 mg, 1.46 mmol) and triethylamine (28 mL, 200 mmol) in

anhydrous CH₂Cl₂(100 mL) was added tosyl chloride (14.25 g,

75 mmol) at 0 °C. The reaction mixture was allowed to warm up

to room temperature and the stirring was continued overnight.

Then the mixture was diluted with CH₂Cl₂(100 mL) and it was

washed with saturated aq solution of NaHCO₃, dried (Na₂SO₄)

and concentrated under reduced pressure. The residue was chro-

matographed on a silica gel using hexane/AcOEt (7:3) as an eluent

oxy-2-methyl-propan-1-ol (11.1 g, 95%); ½a _D²⁴

ꢀ3 (c 0.17, CHCl₃);

ꢂ

¹H NMR (500 MHz, CDCl₃) d 0.92 (3H, d, J = 7.1 Hz, CH–CH₃), 2.02

(1H, m, CH–CH₃), 2.39 (1H, s, OH), 3.54 (1H, dd, J = 9.4, 7.6 Hz,

one of CH₂–CH), 3.60 (d, J = 9.4 Hz, CH₂OH), 3.65 (1H, dd, J = 9.4,

4.8 Hz, one of CH₂–CH), 4.6 (2H, s, OCH₂O), 4.75 (2H, s, CH₂Ph),

7.30 (1H, m, Ar-H_para), 7.35 (4H, m, Ar-H_ortho,meta); ¹³C NMR

(125 MHz) d 13.61 (CH₃), 35.62 (CH–CH₃), 67.19 (CH₂OH), 69.58

(CH₂CH), 72.38 (CH₂–Ph), 94.79 (OCH₂O), 127.82, 127.90 and

to give the oily tosylate 15 (18.8 g, 99%); ½a _D²⁴

ꢂ

ꢀ5 (c 0.15, CHCl₃); ¹H

NMR (500 MHz, CDCl₃) d 0.94 (3H, d, J = 7.1 Hz, CH–CH₃), 2.09 (1H,

m, CH–CH₃), 2.42 (3H, s, CH₃Ph), 3.42 (1H, dd, J = 9.4, 6.6 Hz, one of

CH₂–CH), 3.47 (1H, dd, J = 9.4, 5.1 Hz, one of CH₂–CH), 3.97 (1H, dd,

J = 9.4, 5.8 Hz, one of CH₂–OTs), 4.03 (1H, dd, J = 9.4, 5.8 Hz, one of

CH₂–OTs), 4.51 (2H, s, OCH₂O), 4.65 (2H, s, CH₂Ph), 7.30 (7H, br m,

Ar-H), 7.78 (2H, d, J = 8.2 Hz, Ar-H_orthofrom tosyl); ¹³C NMR

1756

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

(125 MHz) d 13.58 (CH₃), 21.60 (Ph-CH₃), 33.45 (CH–CH₃), 68.61

(CH₂CH), 69.27 (CH₂OTs), 71.96 (CH₂–Ph), 94.56 (OCH₂O), 127.68,

127.82, 128.36, 129.75, 132.6, 137.58 and 144.66 (Ar); MS (EI) m/

z (relative intensity) no M⁺, 257(M⁺ꢀOCH₂Ph, 65), 245 (55), 227

(81), 86 (100); HRMS (ESI) exact mass calcd for C₁₉H₂₄O₅SNa

(M⁺+Na) 387.1242, measured 387.1252.

CH), 3.46 (1H, dd, J = 10.8, 6.0 Hz, one of CH₂–CH), 4.60 (2H, s,

OCH₂O), 4.76 (2H, s, CH₂Ph), ca. 7.3 (5H, m, Ar-H); HRMS (ESI) ex-

act mass calcd for C₁₇H₂₈O₃Na (M⁺+Na) 303.1936, measured

303.1947.

5.4. (S)-6-(tert-Butyldimethylsilyloxy)-2,6-dimethyl-heptan-1-

ol (19)

5.2. (S)-1-Benzyloxymethoxy-2,6-dimethyl-hept-5-en (17)

To a solution of alcohol 18 (1.08 g, 3.9 mmol) and 2,6-lutidine

(0.9 mL, 7.7 mmol) in anhydrous CH₂Cl₂(21 mL) at 0 °C was drop-

wise added tert-butyldimethylsilyl triﬂate (1.46 mL, 6.1 mmol).

The solution was stirred at 0 °C for 1.5 h and poured into water.

The organic layer was separated, the water phase was extracted

with CH₂Cl₂. The combined extracts were washed with diluted

HCl, dried (MgSO₄) and evaporated. The oily residue was chro-

matographed on silica gel using hexane/AcOEt (9:1) as an eluent

to give the oily [(S)-6-benzyloxymethoxy-1,1,5-trimetyl-hexyl-

4-Chloro-2-methyl-2-butane (15.5 mL, 14.4 g, 138 mmol) was

added dropwise to the stirred magnesium turnings (6.75 g,

225 mmol) in anhydrous THF (465 mL) under argon at 0 °C. The

stirring was continued at 0 °C for 1 h. The cooling bath was re-

moved and the mixture was stirred at room temperature for addi-

tional 1.5 h. The mixture was then cooled to ꢀ78 °C and the formed

Grignard reagent 16 was added via cannula to a solution of the tos-

ylate 15 (10 g, 27.5 mmol) in anhydrous THF (70 mL). Then solu-

tion of Li₂CuCl₄[previously prepared from LiCl (1.36 g,

32.1 mmol), CuCl₂(2.17 g, 16.1 mmol) and CH₂Cl₂(160 mL)] was

added. The cooling bath was removed and the reaction was stirred

at room temperature for 17 h. The mixture was extracted with

CH₂Cl_2., the organic layer was washed with NH₄Cl and NaHCO₃,

dried (Na₂SO₄) and evaporated. The residue was chromatographed

on silica gel using hexane/AcOEt (7:3) as an eluent to give the oily

oxy]-tert-butyldimethylsilane (1.4 g, 100%);

½

a ²_D⁴

ꢂ

ꢀ4 (c 0.19,

CHCl₃); ¹H NMR (200 MHz, CDCl₃) d 0.06 [6H, s, Si(CH₃)₂], 0.85

(9H, s, Si–t-Bu), 0.94 (3H, d, J = 6.6 Hz, CH–CH₃), 1.17 [6H, s,

C(CH₃)₂], 1.74 (1H, m, CH–CH₃), 3.36 (1H, dd, J = 9.3, 6.6 Hz, one

of OCH₂–CH), 3.46 (1H, dd, J = 9.3, 6.1 Hz, one of OCH₂CH), 4.60

(2H, s, O–CH₂–O), 4.76 (2H, s, CH₂–Ph), 7.30 (5H, m, Ar-H); ¹³C

NMR (50 MHz)

d

ꢀ1.84 [Si(CH₃)₂], 17.31 (CH–CH₃), 18.31

oleﬁn 17 (5.65 g, 78%); ½a _D²⁴

ꢂ

+2 (c 0.24, CHCl₃); ¹H NMR (400 MHz,

[SiC(CH₃)₃], 21.80 (CH₂CH₂CH₂), 26.05 [SiC(CH₃)₃], 29.96 and

30.09 [C(CH₃)₂], 33.69 (CH–CH₃), 34.36 (CH₂CH₂CH₂), 45.49

(CH₂CH₂CH₂), 69.43 (CH₂–Ph), 73.66 [C(CH₃)₂], 73.84 (OCH₂CH),

94.98 (OCH₂O), 127.86, 128.11 and 128.62 (Ar_{ortho,meta,para}),

138.21 (Ar_ipso); HRMS (ESI) exact mass calcd for C₂₃H₄₂O₃SiNa

(M⁺+Na) 417.2801, measured 417.2805.

CDCl₃) d 0.94 (3H, d, J = 6.6 Hz, CH–CH₃), 1.18 and 1.46 (1H and 1H,

each m), 1.60 and 1.68 [3H and 3H, each s, @C(CH₃)₂], 1.87 (1H, m,

CH–CH₃), 2.05 (2H, m, @CCH₂), 3.37 (1H, dd, J = 9.4, 6.8 Hz, one of

CH₂–CH), 3.44 (1H, dd, J = 9.4, 5.8 Hz, one of CH₂–CH), 4.60 (2H, s,

OCH₂O), 4.76 (2H, s, CH₂Ph), 5.10 (1H, br t, J ꢄ 7 Hz, CH@C), 7.30

(1H, m, Ar-H_para), 7.34 (4H, m, Ar-H_ortho,meta); ¹³C NMR (125 MHz)

d 16.96 (CH-CH₃), 17.53 (one of CH₃C@), 25.60 (one of CH₃C@),

32.92 (CH–CH₃), 33.57 (CH₂CH₂CH), 69.27 (CH₂–Ph), 73.37

(CH₂CH), 94.64 (OCH₂O), 124.49 (C–CH₃), 127.52, 127.77, 128.28

(Ar_{ortho,meta,para}), 137.95 [@C(CH₃)₂]; MS (EI) m/z (relative intensity)

262 (M⁺, 22), 232.2 (65), 154.1 (100); HRMS (ESI) exact mass calcd

for C₁₇H₂₆O₂Na (M⁺+Na) 285.1830, measured 285.1837.

To a solution of the formed diether (0.5 g, 1.27 mmol) in ethyl

acetate (25 mL) was added Pd/C (10%, 380 mg) at room tempera-

ture. The reaction mixture was hydrogenated for 3 h under the

hydrogen pressure of 10 MPa. Then the mixture was ﬁltered and

the solvent was evaporated under reduced pressure. The oily resi-

due was applied on a silica Sep-Pak cartridge (5 g) and washed

with hexane/AcOEt (9:1) to give an oily alcohol 19 (272 mg,

78%); ½a ²_D⁴

ꢂ

ꢀ5.3 (c 0.93, CHCl₃); ¹H NMR (400 MHz, CDCl₃) d 0.06

5.3. (S)-7-Benzyloxymethoxy-2,6-dimethyl-heptan-2-ol (18)

[6H, s, Si(CH₃)₂], 0.85 (9H, s, Si–t-Bu), 1.09 (3H, d, J = 7.1 Hz, CH–

CH₃), 1.17 [6H, s, C(CH₃)₂], 1.63 (1H, m, CH₃CH), 3.42 (1H, dd,

J = 6.6, 10.5 Hz, one of CH₂OH), 3.51 (1H, dd, J = 5.8, 10.5 Hz, one

of CH₂OH); ¹³C NMR (100 MHz) d ꢀ2.07 [Si(CH₃)₂], 16.54 (CH–

CH₃), 18.09 [SiC(CH₃)₃], 21.56 (CH₂–CH₂–CH₂), 25.82 [SiC(CH₃)₃],

29.76 and 29.87 [2 ꢁ C(CH₃)₂], 33.69 (CH₂CH₂CH₂), 35.78 (CH–

CH₃), 45.28 (CH₂CH₂CH₂), 68.78 (CH₂OH), 73.45 [C(CH₃)₂]; HRMS

(ESI) exact mass calcd for C₁₅H₃₄O₂SiNa (M⁺+Na) 297.2226, mea-

sured 297.2191.

Oleﬁn 17 (3.2 g, 12.2 mmol) was dissolved in anhydrous CH₂Cl₂

(60 mL) and NaHCO₃(1.6 g, 18.4 mmol) was added. Then 3-chloro-

peroxybenzoic acid (60%, 12.8 g, 36.6 mmol) was added at room

temperature with stirring. The stirring was continued for 24 h,

and the mixture was diluted with ether, shaked with water and

2M NaOH. The organic layer was washed with water and saturated

NH₄Cl, dried (Na₂SO₄) and evaporated. The residue was chromato-

graphed on silica gel using hexane/AcOEt (9:1) as an eluent to give

the oily mixture of (2S)-1-benzyloxymethoxy-2,6-dimethyl-5,6-

epoxy-heptanes (2.5 g, 74%); ¹H NMR (500 MHz, CDCl₃) d 0.96

(3H, d, J = 6.7 Hz, CH–CH₃), 1.25 (1H, m), 1.27 and 1.31 [3H and

3H, each s, C(CH₃)₂], 1.5–1.7 (3H, br m), 1.79 (1H, m, CH–CH₃),

2.73 (1H, m, CH₂CHO), 3.45 (2H, br m, CH₂–CH), 4.60 (2H, s,

OCH₂O), 4.76 (2H, s, CH₂Ph), 7.29 (1H, m, Ar-H_para), 7.34 (4H, d,

J = 4.3 Hz, Ar-H_ortho,meta).

5.5. (S)-6-(tert-Butyldimethylsilyloxy)-2,6-dimethyl-heptanal

(20)

To a solution of NMO (0.3 g, 2.6 mmol) in CH₂Cl₂(11 mL) were

added 4 Å molecular sieves (1.65 g) and the mixture was stirred at

room temperature for 15 min. Then was added TPAP (30 mg,

0.08 mmol) and a solution of the alcohol 19 (0.3 g, 1.09 mmol) in

CH₂Cl₂(1.2 mL). The resulted dark mixture was stirred for

30 min, ﬁltered through a silica Sep-Pak (5 g) and evaporated.

The oily residue was dissolved in hexane, applied on a silica Sep-

Pak cartridge (5 g) and washed with hexane/AcOEt (98:2) to give

To a solution of the formed epoxides (3.05 g, 10.8 mmol) in

anhydrous ether (110 mL) at 0 °C was added lithium aluminum hy-

dride (2.0 g, 53 mmol). The cooling bath was removed and the

reaction was stirred at room temperature for 18 h. Then the

reaction was quenched with cold water and NH₄Cl aq and ex-

tracted with CH₂Cl₂. The solvents were removed under reduced

pressure and the crude oil was chromatographed on a silica gel

using hexane/AcOEt (9:1) as an eluent to give an oily alcohol

an oily aldehyde 20 (253 mg, 85%); ½a _D²⁴

ꢂ

+14.6 (c 0.88, CHCl₃); ¹H

NMR (200 MHz, CDCl₃) d 0.06 [6H, s, Si(CH₃)₂], 0.85 (9H, s, Si–t-

Bu), 0.92 (3H, d, J = 6.8 Hz, CH–CH₃), 1.17 [6H, s, C(CH₃)₃], 2.35

(1H, m, CH₃CH), 9.62 (1H, d, J = 1.9 Hz, CHO); ¹³C NMR (50 MHz)

18 (2.6 g, 86%); ½a _D²⁴

ꢂ

ꢀ4 (c 0.19, CHCl₃); ¹H NMR (200 MHz,

d

ꢀ2.08 [Si(CH₃)₂], 13.25 (CH–CH₃), 18.07 [SiC(CH₃)₂], 21.58

CDCl₃) d 0.94 (3H, d, J = 6.5 Hz, CH–CH₃), 1.20 [6H, s, (CH₃)₂COH],

1.75 (1H, m, CH–CH₃), 3.38 (1H, dd, J = 10.8, 6.6 Hz, one of CH₂–

(CH₂–CH₂–CH₂), 25.80 [SiC(CH₃)₃], 29.76 and 29.80 [2 ꢁ C(CH₃)₂],

31.02 (CH₂CH₂CH₂), 44.96 (CH₂CH₂CH₂), 46.34 (CH–CH₃), 73.24

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

1757

[C(CH₃)₂], 205.31 (CHO); HRMS (ESI) exact mass calcd for

eluted was Z-isomer 24 (115 mg) and its E-isomer 25 (77.5 mg,

C₁₅H₃₂O_2SiNa (M⁺+Na) 295.2069, measured 295.2090.

overall yield 92%) was obtained in the later fractions.

Compound 24 (Z-isomer): ½a _D²⁴

ꢂ

+5.6 (c 1.0, CHCl₃), ¹H NMR

5.6. 2-[P,P-Bis(2⁰,2⁰,2⁰-triﬂuoroethyl)phosphono]-4-(tert-

butyldimethylsilyloxy)-butyric acid methyl ester (21)

(500 MHz, CDCl₃) d 0.04 and 0.08 [6H and 6H, each s, 2 ꢁ Si(CH₃)₂],

0.84 and 0.90 (9H and 9H, each s, 2 ꢁ Si–t-Bu), 0.93 (3H, d,

J = 6.6 Hz, CH-CH₃), 1.15 and 1.16 [3H and 3H, each s, OC(CH₃)₂],

2.30 (2H, t, J = 5.4 Hz, CH₂C@C), 2.43 (1H, m, CH–CH₃), 3.73 (2H,

m, CH₂OTBS), 4.12 (2H, m, CH₂OH), 5.08 (1H, d, J = 9.8 Hz, C@CH);

¹³C NMR (50 MHz) d ꢀ5.32 and ꢀ1.89 [Si(CH₃)₂], 18.09 and 18.30

[2 ꢁ SiC(CH₃)₃], 21.61 (CH–CH₃), 22.30 (CH₂), 26.02 and 26.05

[2 ꢁ SiC(CH₃)₃], 29.89 and 30.11 [C(CH₃)₂], 32.16 (CH–CH₃), 38.42

(CH₂), 39.19 (CH₂C@), 45.29 (CH₂), 61.02 (CH₂OTBS), 64.92

(CH₂OH), 73.66 [OC(CH₃)₂], 129.42 (CH@C), 136.82 (CH@C); HRMS

(ESI) exact mass calcd for C₂₅H₅₄O₃Si₂Na (M⁺+Na) 481.3509, mea-

sured 481.3531.

To a suspension of NaH (60%, 730 mg; washed with hexane) in

anhydrous DMF (6.6 mL) at 0 °C was slowly added a solution of

(F₃CCH₂O)₂POCH₂COOCH₃(5 g, 15.7 mmol) in anhydrous DMF

(6.6 mL). The mixture was stirred at room temperature for 1.5 h

and a solution of Br(CH₂)₂OTBS (8.4 mL, 9.3 g, 39.2 mmol) was

added in a freshly distilled HMPA (6.8 mL, 39.2 mmol). After stir-

ring at room temperature for 48 h, the reaction mixture was di-

luted with ethyl acetate and poured into water. The organic

phase was separated and the water layer was extracted with ethyl

acetate. The combined organic extracts were washed with water,

dried (MgSO₄) and evaporated. The oily residue was puriﬁed by

column chromatography on silica gel using hexane/AcOEt

(98.5:1.5) as an eluent to give a semicrystalline product 21 (2.3 g,

30%); ¹H NMR (200 MHz, CDCl₃) d 0.03 [6H, s, Si(CH₃)₂], 0.87 [9H, s,

Si–t-Bu], 0.92 (3H, d, J = 6.8 Hz, CH–CH₃), 2.08 (1H, m, one of

CH₂CH₂CH), 2.21 (1H, m, one of CH₂CH₂CH), 3.39 and 3.44 (1H

and 1H, each dd, J = 10.5, 3.5 Hz, PCHC@O), 3.60 and 3.72 (1H

and 1H, each m, CH₂OTBS), 3.77 (3H, COOCH₃), 4.42 (4H, m,

2 ꢁ CH₂OP); HRMS (ESI) exact mass calcd for C₁₅H₂₈F₆O₆SiP

(M+H⁺) 477.1314, measured 477.1297.

Compound 25 (E-isomer): ½a _D²⁴

ꢂ

ꢀ7.1 (c 1.10, CHCl₃), ¹H NMR

(400 MHz, CDCl₃) d 0.04 and 0.08 [6H and 6H, each s, 2 ꢁ Si(CH₃)₂],

0.83 and 0.90 (each 9H, each s, 2 ꢁ Si–t-Bu), 0.94 (3H, d, J = 6.6 Hz,

CH–CH₃), 1.15 [6H, s, OC(CH₃)₂], 2.32 (2H, m, CH₂C@C), 2.42 (1H,

m, CH–CH₃), 3.69 (2H, m, CH₂OTBS), 3.98 (2H, m, CH₂OH), 5.25

(1H, d, J = 10.0 Hz, C@CH); ¹³C NMR (50 MHz) d ꢀ5.47 and ꢀ2.09

[Si(CH₃)₂], 18.09 and 18.30 [2 ꢁ SiC(CH₃)₃], 21.07 (CH–CH₃),

22.15 (CH₂–CH₂–CH₂), 25.81 and 25.90 [2 ꢁ SiC(CH₃)₃], 29.71 and

29.93 [C(CH₃)₂], 32.24 (CH–CH₃), 32.50 (CH₂), 38.08 (CH₂C@),

45.18 (CH₂), 63.34 (CH₂OTBS), 68.58 (CH₂CO), 73.41 [C(CH₃)₂],

134.97 (CH@C), 136.28 (CH@C); HRMS (ESI) exact mass calcd for

C₂₅H₅₄O₃Si₂Na (M⁺+Na) 481.3509, measured 481.3528.

5.7. (Z)- and (E)-(S)-8-(tert-Butyldimethylsilyloxy)-2-[2⁰-(tert-

butyldimethylsilyloxy)ethyl]-4,8-dimethyl-non-2-enoic acid

methyl esters (22 and 23)

5.9. (E)-(S)-1,9-Bis-(tert-butyldimethylsilyloxy)-3,5,9-

trimethyl-dec-3-ene (26)

To a solution of phosphono ester 21 (1 g, 2.12 mmol) and 18-

crown-6 (2.5 g, 92.5 mmol) in anhydrous THF (50 mL) at ꢀ30 °C

was dropwise added KHMDS (0.5 M in toluene, 4.25 mL,

2.12 mmol). After stirring for 15 min the mixture was cooled to

ꢀ40 °C and a solution of the aldehyde 20 (288 mg, 1.06 mmol)

in anhydrous THF (6.3 mL) was added. The mixture was stirred

for 2 h at ꢀ40 °C, 1 h at ꢀ30 °C, 1 h at ꢀ20 °C, 1 h at 0 °C and ﬁ-

nally for 18 h at room temperature. Saturated NH₄Cl was added

and the mixture was extracted with ethyl acetate. The organic

phase was dried (MgSO₄) and evaporated. The oily residue was

puriﬁed by column chromatography on silica gel using hexane/

AcOEt (95:5) as an eluent to give a mixture of isomeric esters

22 and 23 (1.7:1; 512 mg, 100%); HRMS (ESI) exact mass calcd

for C₂₆H₅₄O₄Si₂Na (M⁺+Na) 509.3458, measured 509.3445; ¹H

NMR (200 MHz, CDCl₃) selected signals of Z-isomer 22: d 0.97

(3H, d, J = 6.6 Hz, CH–CH₃), 2.44 (2H, m, CH₂C@C), 3.66 (2H, t,

J = 7.2 Hz, CH₂OTBS), 3.72 (3H, s, COOCH₃), 5.70 (1H, d,

J = 10.2 Hz, C@CH); selected signals of E-isomer 23: d 1.00 (3H,

d, J = 6.6 Hz, CH–CH₃), 2.56 (2H, t, J = 7.2 Hz, CH₂C@C), 3.62 (2H,

t, J = 7.2 Hz, CH₂OTBS), 3.72 (3H, s, COOCH₃), 6.62 (1H, d,

J = 10.2 Hz, C@CH).

To a solution of the alcohol 24 (68 mg, 0.14 mmol), Et₃N (65 lL,

0.50 mmol) and catalytic quantity of DMAP in anhydrous CH₂Cl₂

(5 mL) at 0 °C was added solution of TsCl (48 mg, 0.17 mmol) in

anhydrous CH₂Cl₂(2.8 mL). The mixture was stirred at room tem-

perature for 2 h, and at 6 °C for 48 h, and the solvents were re-

moved in vacuo. The residue was redissolved in anhydrous THF

(8 mL), cooled to 0 °C and LiAlH₄(230 mg, 6.0 mmol) was then

added. After stirring at 6 °C for 18 h, water was added and the mix-

ture was extracted with ethyl acetate. Organic extracts were

washed with water, dried (MgSO₄) and evaporated. An oily residue

was puriﬁed on a silica Sep-Pak (2 g). Elution with hexane/AcOEt

(98:2) gave an oily diether 26 (37.2 mg, overall yield 57%); ¹H

NMR (400 MHz, CDCl₃)

d 0.04 and 0.05 [each 6H, each s,

2 ꢁ Si(CH₃)₂], 0.84 and 0.89 (each 9H, each s, 2 ꢁ Si–t-Bu), 0.90

(3H, d, J = 6.6 Hz, CH–CH₃), 1.16 [6H, s, OC(CH₃)₂], 1.61 (3H, d,

J = 1.5 Hz, CH₃–C@C), 2.18 (2H, t, J = 7.2 Hz, CH₂C@C), 2.42 (1H,

m, CH–CH₃), 3.65 (2H, t, J = 7.2 Hz, CH₂OTBS), 5.25 (1H, d,

J = 10.0 Hz, C@CH); ¹³C NMR (50 MHz)

d

ꢀ5.26 and ꢀ2.08

[Si(CH₃)₂], 16.65 (CH₃C@C), 18.08 and 18.34 [2 ꢁ SiC(CH₃)₃],

21.20 (CH–CH₃), 22.18 (CH₂), 25.83 and 25.96 [2 ꢁ SiC(CH₃)₃],

29.72 and 29.90 [C(CH₃)₂], 32.36 (CH–CH₃), 38.36 (CH₂), 43.14

(CH₂), 45.16 (CH₂C@), 62.68 (CH₂OTBS), 73.50 [OC(CH₃)₂], 127.90

(CH@C), 133.39 (CH@C); HRMS (ESI) exact mass calcd for

C₂₅H₅₄O₂Si₂Na (M⁺+Na) 465.3560, measured 465.3544.

5.8. (Z)- and (E)-(S)-8-(tert-Butyldimethylsilyloxy)-2-[2⁰-(tert-

butyldimethylsilyloxy)ethyl]-4,8-dimethyl-non-2-en-1-ol (24

and 25)

5.10. (E)-(S)-9-(tert-Butyldimethylsilyloxy)-3,5,9-trimethyl-

To a stirred solution of the isomeric esters 22 and 23 (1.7:1;

250 mg, 0.52 mmol) in toluene/CH₂Cl₂(2:1, 8.6 mL) was added at

ꢀ78 °C diisobutylaluminium hydride (1.5 M in toluene, 2.3 mL,

3.3 mmol). Stirring was continued at ꢀ78 °C for 1.5 h, and the reac-

tion was quenched by addition of 2 M potassium sodium tartrate

and diluted HCl. The mixture was extracted with ethyl acetate,

the organic extracts were washed with water, dried (MgSO₄) and

evaporated. The oily residue was chromatographed on silica gel

using hexane/AcOEt (99:1?95:5) as an eluent: the ﬁrst product

dec-3-en-1-ol (27)

To a solution of diether 26 (26 mg, 58

(6 mL) was added at room temperature tetrabutylammonium ﬂuo-

ride (1.0 M in THF, 190 L, 190 mol). The mixture was stirred un-

der argon at room temperature for 18 h, poured into brine, and

extracted with ethyl acetate. Organic extracts were washed with

brine, dried (MgSO₄), and evaporated. The oily residue was puriﬁed

on a silica Sep-Pak (2 g). Elution with hexane/AcOEt (9:1) gave an

lmol) in anhydrous THF

l

1758

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

oily alcohol 27 (19 mg, 100%); ½a _D²⁴

ꢂ

+5 (c 0.5, CHCl₃), ¹H NMR

¹³C NMR (100 MHz) d ꢀ5.05, ꢀ4.88 and ꢀ2.06 [3 ꢁ Si(CH₃)₂],

(500 MHz, CDCl₃) d 0.04 [6H, s, Si(CH₃)₂], 0.84 (9H, s, Si–t-Bu),

0.90 (3H, d, J = 6.6 Hz CH–CH₃), 1.16 [6H, s, OC(CH₃)₂], 1.63 (1H,

s, CH₃C@C), 2.23 (2H, t, J = 6.2 Hz, CH₂C@C), 2.25 (1H, m, CH–

CH₃), 5.02 (1H, d, J = 9.2 Hz, C@CH); ¹³C NMR (50 MHz) d ꢀ2.21

[Si(CH₃)₂], 15.80 (CH₃C@C), 18.01 [SiC(CH₃)₃], 21.16 (CH–CH₃),

22.15 (CH₂), 25.96 [SiC(CH₃)₃], 29.60 and 29.75 [C(CH₃)₂], 32.43

(CH–CH₃), 38.15 (CH₂), 42.59 (CH₂), 45.03 (CH₂C@C), 59.95

(CH₂OH), 73.45 [OC(CH₃)₂], 129.36 (CH@C), 135.25 (CH@C); HRMS

(ESI) exact mass calcd for C₁₉H₄₀O₂SiNa (M⁺+Na) 351.2696, mea-

sured 351.2690.

16.42 (C-12), 18.11 [3 ꢁ SiC(CH₃)₃], 21.23 (C-20), 22.24 (C-23),

25.75, 25.82 and 25.86 [3 ꢁ SiC(CH₃)₃], 29.79 and 30.03 (C-26

and C-27), 32.47 (C-21), 38.42 and 47.20 (C-4 and C-10), 38.88

(C-22), 43.24 (C-24), 45.22 (C-14), 71.73 and 72.30 (C-1 and C-3),

73.32 (C-25), 106.34 (C@CH₂), 127.03 (C-6), 131.47 (C-7), 132.06

(C-8), 132.44 (C-17), 133.28 (C-5), 152.86 (C-2), 160.55 (C-13);

HRMS (ESI) exact mass calcd for C₁₄H₇₈O₃Si₃Na (M⁺+Na)

713.5151, measured 713.5157.

To a solution of protected vitamins 30 and 31 (3.5 mg, 5

in THF (120 L) and methanol (120 L) was added 46% HF/MeCN

(1:9, 60 L) at room temperature. After stirring for 7 h, a satu-

lmol)

l

5.11. (S)-9-(tert-Butyldimethylsilyloxy)-3,5,9-trimethyl-dec-3-

enal (28)

rated NaHCO₃was added. The mixture was extracted with

CH₂Cl₂, organic extracts were washed with brine, dried (MgSO₄)

and evaporated. The residue was ﬁrst puriﬁed on a silica Sep-

Pak (2 g). Elution with hexane/ethyl acetate (1:1) gave an oily

mixture of deprotected vitamins 11 and 32 (1.2 mg, 70%). Separa-

tion of the isomers was achieved by HPLC (9.4 mm ꢁ 25 cm Zor-

bax-Sil column, 4 mL/min) using a hexane/2-propanol (8.5:1.5)

solvent system. Pure vitamin D analog 11 was collected at R_V

To a solution of NMO (22 mg, 120 lmol) in CH₂Cl₂(0.8 mL)

were added 4 Å molecular sieves (120 mg) and the mixture was

stirred at room temperature for 15 min. Then was added TPAP

(2.7 mg, 7.3

lmol) and a solution of the alcohol 27 (27.5 mg,

80 mol) in CH₂Cl₂(35

l

lL). The resulted dark mixture was stirred

for 1 h, ﬁltered through a silica Sep-Pak (2 g) and evaporated. The

oily residue was dissolved in hexane, applied on a silica Sep-Pak

cartridge (2 g) and washed with hexane/AcOEt (99.8:0.2) to give

35 mL, and isomeric compound 32 (37 lg) was collected at R_V

33 mL. Purity of both vitamins was checked by reversed-phase

HPLC (9.4 mm ꢁ 25 cm, Eclipse XDB-C18 column, 4 mL/min)

using a methanol/water (85:15) solvent system. Vitamin D com-

pounds 11 and 32 gave sharp peaks at R_V21 and 22 mL,

respectively.

an unstable b,c-unsaturated aldehyde 28 (22.3 mg, 83%).

An analytical sample of the product was obtained after HPLC

(10 mm ꢁ 25 cm Zorbax-Sil column, 4 mL/min) puriﬁcation using

a hexane/ethyl acetate (98:2) solvent system. Pure aldehyde 28

was collected at R_V32 mL; ¹H NMR (400 MHz, CDCl₃) d 0.04 [6H,

s, Si(CH₃)₂], 0.84 (9H, s, Si–t-Bu), 0.90 (3H, d, J = 6.6 Hz, CH–CH₃),

1.17 [6H, s, OC(CH₃)₂], 1.74 (1H, s, CH₃C@C), 2.25 (1H, m, CH–

CH₃), 3.10 (2H, m, CH₂C@C), 5.24 (1H, d, J = 9.8 Hz, C@CH), 9.58

(1H, J = 2.2 Hz, CHO).

5.12.2. (20S)-1a,25-Dihydroxy-2-methylene-8(12),14(17)-diseco-

13(17)-dehydro-9,11,15,16,18,19-hexanorvitamin D₃(11)

UV (in EtOH) k_max237.5, 244.5, 253.0 nm; ¹H NMR

(400 MHz, CDCl₃) d 0.92 (3H, d, J = 6.7 Hz, 21-H₃), 1.20 (6H, s,

26- and 27-H₃), 1.60 (3H, br s, 12-H₃), 2.27 (1H, dd, J = 13.2,

6.6 Hz, 4b-H), 2.35 (1H, dd, J = 13.0, 7.6 Hz, 10

dd, J = 13.2, 4.2 Hz, -H), 2.73 (1H, dd, J = 13.0, 4.2 Hz,

10b-H), 2.75 (2H, d, J = 6.9 Hz, 14-H₂), 4.48 (2H, m, 1b- and

-H), 4.94 (1H, d, J = 9.1 Hz, 17-H), 5.09 and 5.11 (each 1H,

each s, C@CH₂); 5.69 (1H, dt, J = 14.9, 6.9 Hz, 8-H), 6.04 (1H,

d, J = 10.8 Hz, 6-H), 6.30 (1H, dd, J = 14.9, 10.8 Hz, 7-H); HRMS

(ESI) exact mass calcd for C₂₂H₃₆O₃(M⁺+Na) 371.2562, mea-

sured 371.2559.

a-H), 2.56 (1H,

5.12. (1R,3R)-5-[(2⁰E,5⁰E)-(S)-11⁰-Hydroxy-5⁰,7⁰,11⁰-trimethyl-

dodeca-2⁰,5⁰-dienylidene]-2-methylene-cyclohexane-1,3-diol

(30) and (1R,3R)-5-[(2⁰Z,5⁰E)-(S)-11⁰-hydroxy-5⁰,7⁰,11⁰-trimethyl-

dodeca-2⁰,5⁰-dienylidene]-2-methylene-cyclohexane-1,3-diol

(31)

4a

3a

To a solution of phosphine oxide 29 (113 mg, 194

anhydrous THF (1.9 mL) at ꢀ78 °C was slowly added phenyllithium

(1.8 M in cyclohexane, 108 L, 194 mol) under argon with stir-

lmol) in

l

5.12.3. (7Z)-(20S)-1a,25-Dihydroxy-2-methylene-8(12),14(17)-

ring. The solution turned deep orange. The mixture was stirred at

diseco-13(17)-dehydro-9,11,15,16,18,19-hexanorvitamin D₃

ꢀ78 °C for 20 min, and a precooled (ꢀ78 °C) solution of the alde-

(32)

hyde 28 (22 mg, 60

lmol) in anhydrous THF (370

lL) was slowly

UV (in EtOH) k_max238.5, 245.5 nm; ¹H NMR (600 MHz, CDCl₃) d

0.92 (3H, d, J = 6.6 Hz, 21-H₃), 1.20 (6H, s, 26- and 27-H₃), 1.61 (3H,

br s, 12-H₃), 2.31 (1H, dd, J = 13.0, 7.1 Hz, 4b-H), 2.34 (2H, m, 14-

added. The mixture was stirred at ꢀ78 °C under argon for 3 h

and at 6 °C for 16 h. Ethyl acetate and water were added, and the

organic phase was separated, washed with brine, dried (MgSO₄)

and evaporated. The oily residue was puriﬁed on a silica Sep-Pak

cartridge (2 g). Elution with hexane/AcOEt (99.9:0.1) gave a mix-

ture of protected vitamin D analogs 30 and 31 (15.5 mg, 30%). Main

product was identiﬁed as (1R,3R)-1,3-bis-(tert-butyldimethylsilyl-

oxy)-5-[(2⁰E,5⁰E)-(S)-11⁰-(tert-butyldimethylsilyloxy)-5⁰,7⁰,11⁰-tri-

methyl-dodeca-2⁰,5⁰-dienylidene]-2-methylene-cyclohexane (30).

H₂), 2.41 (1H, dd, J = 13.2, 7.1 Hz, 10

4.0 Hz, 4 -H), 2.72 (1H, dd, J = 13.2, 4.0 Hz, 10b-H), 4.50 (2H, m,

1b- and 3 -H), 4.93 (1H, d, J = 9.3 Hz, 17-H), 5.11 (2H, s, C@CH₂),

a-H), 2.61 (1H, dd, J = 13.0,

a

5.48 (1H, m, 8-H), 6.33 (2H, m, 6- and 7-H).

5.13. (2R,4S)- and (2S,4S)-8-(tert-Butyldimethylsilyloxy)-2-[2⁰-

(tert-butyldimethylsilyloxy)ethyl]-4,8-dimethyl-nonan-1-ol (33

and 34)

5.12.1. (20S)-1a,25-Bis-(tert-butyldimethylsilyloxy)-2-

methylene-8(12),14(17)-diseco-13(17)-dehydro-9,11,15,16,18,

19-hexanorvitamin D₃tert-butyldimethylsilyl ether (30)

UV (in hexane) k_max237.5, 244.5, 253.0 nm; ¹H NMR (400 MHz,

To a solution of the alcohol 25 (99 mg, 0.2 mmol mmol) in

anhydrous methanol (4 mL) was added PtO₂(30 mg) at room tem-

perature. The mixture was stirred for 3 days in hydrogen atmo-

sphere, and each day three portions of PtO₂(30 mg) were added.

The mixture was ﬁltered, the solvent evaporated and the oily res-

idue was puriﬁed on a silica Sep-Pak (2 g). Elution with hexane/

AcOEt (95:5) gave an oily mixture of isomeric alcohols 33 and 34

(1:1.3, 172 mg, 72%). The isomers were separated by HPLC

(9.4 mm ꢁ 25 cm Zorbax-Sil column, 4 mL/min) using a hexane/

ethyl acetate (95:5) solvent system. (2R,4S)-Alcohol 33 was col-

CDCl₃)

d 0.027, 0.038, 0.052 and 0.063 [each 3H, each s,

4 ꢁ (SiCH₃)₂], 0.050 [6H, s, (SiCH₃)₂], 0.85, 0.87 and 0.89 [each

9H, each s, 3 ꢁ Si–t-Bu], 0.91 (3H, d, J = 6.7 Hz, 21-H₃), 1.16 (6H,

s, 26- and 27-H₃), 1.76 (3H, 12-H₃), 2.29–2.48 (3H, br m), 2.72

(2H, d, J = 6.9 Hz, 14-H₂), 4.42 (2H, m, 1b- and 3a-H), 4.93 and

4.97 (each 1H, each s, C@CH₂), 5.56 (1H, dt, J = 14.9, 6.9 Hz, 8-H),

5.91 (1H, d, J = 10.8 Hz, 6-H), 6.25 (1H, dd, J = 14.9, 10.8 Hz, 7-H);

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

1759

lected at R_V45 mL, whereas (2S,4S)-alcohol 34 was collected at R_V

HRMS (ESI) exact mass calcd for

369.2801, measured 369.2803.

C

₁₉H₄₂O₃SiNa (M⁺+Na)

50 mL.

Compound 33: ½a ²_D⁴

ꢂ

+3.6 (c 0.96, CHCl₃); ¹H NMR (400 MHz,

CDCl₃) d 0.05 and 0.08 [each 6H, each s, 2 ꢁ Si(CH₃)₂], 0.85 and

0.90 (each 9H, each s, 2 ꢁ Si–t-Bu), 0.87 (3H, d, J = 6.6 Hz, CH–

CH₃), 1.17 [6H, s, C(CH₃)₂], 1.74 (1H, m, CH–CH₃), 3.38 (1H, dd,

J = 11.0, 7.1 Hz, one of CH₂OH), 3.59 (1H, dd, J = 11.0, 3.4 Hz, one

of CH₂OH), 3.66 (1H, m, one of CH₂OSiTBS), 3.78 (1H, m, one of

CH₂OSiTBS); ¹³C NMR (50 MHz) d ꢀ5.49 and ꢀ2.07 [Si(CH₃)₂],

18.09 and 18.21 [SiC(CH₃)₃], 19.86 (CH–CH₃), 21.50 (CH₂–CH₂–

CH₂), 25.85 [SiC(CH₃)₃], 29.78 and 29.78 [2 ꢁ C(CH₃)₂], 30.10

(CH–CH₃), 36.39 (CH₂), 37.05 (CHCH₂–OH), 37.76 (CH₂), 39.53

(CH₂), 45.30 (CH₂), 61.86 (CH₂OTBS), 66.05 (CH₂OH), 73.49

[C(CH₃)₂]; HRMS (ESI) exact mass calcd for C₂₅H₅₆O₃Si₂Na

(M⁺+Na) 483.3666, measured 483.3670.

5.16. (3R,5S)-1,9-Bis-(tert-butyldimethylsilyloxy)-3,5,9-

trimethyl-decane (37)

To a solution of the alcohol 33 (32 mg, 70

290 mol) and catalytic quantity of DMAP in anhydrous CH₂Cl₂

(155 L) at 0 °C was added TsCl (30 mg, 106 mol). The mixture

lmol), Et₃N (38 lL,

l

was stirred at room temperature for 18 h, and the solvents were re-

moved in vacuo. The residue was redissolved in anhydrous THF

(4 mL), cooled to 0 °C and LiAlH₄(88 mg, 2.35 mmol) was then

added. After stirring at 6 °C for 6 h, water was added and the mix-

ture was extracted with ethyl acetate. Organic extracts were

washed with water, dried (MgSO₄) and evaporated. An oily residue

was puriﬁed on a silica Sep-Pak (2 g). Elution with hexane/AcOEt

Compound 34: ½a ²_D⁴

ꢂ

ꢀ3.2 (c 1.16, CHCl₃); ¹H NMR (500 MHz,

CDCl₃) d 0.055 and 0.087 [each 6H, each s, 2 ꢁ Si(CH₃)₂], 0.85 and

0.91 (each 9H, each s, 2 ꢁ Si–t-Bu), 0.86 (3H, d, J = 6.6 Hz, CH–

CH₃), 1.17 [6H, s, C(CH₃)₂], 3.41 (1H, m, one of CH₂OH), 3.57 (1H,

m, one of CH₂OH), 3.65 (1H, m, one of CH₂OSiTBS), 3.78 (1H, m,

(98:2) gave an oily diether 37 (21.4 mg, overall yield 70%); ½a _D²⁴

ꢂ

+4.2 (c 0.50, CHCl₃); ¹H NMR (400 MHz, CDCl₃) d 0.046 and 0.057

[each 6H, each s, 2 ꢁ Si(CH₃)₂], 0.86 (9H, s, Si–t-Bu), 0.90 (9H, s,

Si–t-Bu and 2 ꢁ 3H, CH–CH₃), 1.18 [6H, s, C(CH₃)₂], 3.64 (2H, m,

CH₂OTBS); ¹³C NMR (50 MHz) d ꢀ5.24 and ꢀ2.05 [Si(CH₃)₂],

18.12 and 18.32 [SiC(CH₃)₃], 19.48 (CH–CH₃), 19.58 (CH–CH₃),

21.61 (CH₂–CH₂–CH₂), 25.86 and 25.98 [2 ꢁ SiC(CH₃)₃], 26.80

(CH–CH₃), 29.76 and 29.91 [2 ꢁ C(CH₃)₂], 30.01 (CH–CH₃), 38.36

(CH₂), 40.83 (CH₂), 44.91 (CH₂), 45.36 (CH₂), 61.38 (CH₂OTBS),

73.57 [C(CH₃)₂]; HRMS (ESI) exact mass calcd for C₂₅H₅₆O₂Si₂Na

(M⁺+Na) 467.3717, measured 467.3733.

one of CH₂OSiTBS); ¹³C NMR (50 MHz)

d

ꢀ5.50 and ꢀ2.07

[Si(CH₃)₂], 18.08 and 18.21 [SiC(CH₃)₃], 19.70 (CH–CH₃), 21.55

(CH₂–CH₂–CH₂), 25.61 and 25.83 [2 ꢁ SiC(CH₃)₃], 26.90 (CH–CH₃),

29.73 and 29.89 [2 ꢁ C(CH₃)₂], 29.98 (CH–CH₃), 35.40 (CH₂),

37.24 (CHCH₂–OH), 37.76 (CH₂), 39.31 (CH₂), 45.28 (CH₂), 61.92

(CH₂OSiTBS), 66.93 (CH₂OH), 73.46 [C(CH₃)₂]; HRMS (ESI) exact

mass calcd for C₂₅H₅₆O₃Si₂Na (M⁺+Na) 483.3666, measured

483.3664.

5.17. (3S,5S)-1,9-Bis-(tert-butyldimethylsilyloxy)-3,5,9-

trimethyl-decane (38)

5.14. (2R)-2-[(2⁰S)-6⁰-(tert-Butyldimethylsilyloxy)-2⁰,6⁰-

dimethyl-heptyl]-butane-1,4-diol (35)

Tosylation of the alcohol 34 (34 mg, 74 lmol) and the tosylate

To a solution of diether 33 (15 mg, 33

(3.8 mL) was added at room temperature tetrabutylammonium

ﬂuoride (1.0 M in THF, 125 L, 125 mol). The mixture was stirred

l

mol) in anhydrous THF

reduction were performed analogously to the process described

above for the isomeric alcohol 33. After analogous work-up of

the reaction mixture the product was puriﬁed on a silica Sep-Pak

(2 g). Elution with hexane/AcOEt (98:2) gave an oily diether 38

(23.7 mg, overall yield 72%).

l

under argon at room temperature for 1.5 h, poured into brine, and

extracted with ethyl acetate. Organic extracts were washed with

brine, dried (MgSO₄), and evaporated. The oily residue was puri-

ﬁed on a silica Sep-Pak (0.5 g). Elution with hexane/AcOEt (6:4)

Compound 38: ½a ²_D⁴

ꢂ

ꢀ1.1 (c 1.15, CHCl₃); ¹H NMR (400 MHz,

CDCl₃) d 0.055 and 0.066 [each 6H, each s, 2 ꢁ Si(CH₃)₂], 0.85 and

0.89 (each 9H, each s, 2 ꢁ Si–t-Bu), 0.82 (3H, d, J = 6.4 Hz, CH–

CH₃), 0.84 (3H, d, J = 6.5 Hz, CH–CH₃), 1.17 [6H, s, C(CH₃)₂], 3.64

gave an oily diol 35 (11 mg, 100%); ½a _D²⁴

ꢂ

+11.0 (c 0.45, CHCl₃); ¹H

NMR (200 MHz, CDCl₃) d 0.05 [6H, s, Si(CH₃)₂], 0.84 (9H, s, Si–t-

Bu), 0.89 (3H, d, J = 6.4 Hz, CH–CH₃), 1.17 [6H, s, OC(CH₃)₂], 3.44

(1H, dd, J = 10.7, 7.1 Hz, one of CHCH₂OH), 3.6–3.9 (3H, br m,

CH₂CH₂OH and one of CHCH₂OH); ¹³C NMR (50 MHz) d ꢀ2.05

[Si(CH₃)₂], 18.12 [SiC(CH₃)₃], 19.86 (CH–CH₃), 21.51 (CH₂–CH₂–

CH₂), 25.85 [SiC(CH₃)₃], 29.78 [C(CH₃)₂], 30.15 (CH–CH₃), 36.62

(CH₂), 36.73 (CHCH₂–OH), 37.76 (CH₂), 39.48 (CH₂), 45.29 (CH₂),

61.24 (CH₂OH), 66.25 (CH₂OH), 73.48 [OC(CH₃)₂]; HRMS (ESI) ex-

act mass calcd for C₁₉H₄₂O₃SiNa (M⁺+Na) 369.2801, measured

369.2798.

(2H, m, CH₂OTBS); ¹³C NMR (50 MHz)

d

ꢀ5.23 and ꢀ2.04

[Si(CH₃)₂], 18.11 and 18.33 [SiC(CH₃)₃], 20.14 (CH–CH₃), 20.38

(CH–CH₃), 21.60 (CH₂–CH₂–CH₂), 25.86 and 25.99 [2 ꢁ SiC(CH₃)₃],

29.82 and 29.87 [2 ꢁ C(CH₃)₂], 30.03 (CH–CH₃), 36.40 (CH₂),

37.52 (CH₂), 39.87 (CH₂), 45.39 (CH₂), 61.46 (CH₂OTBS), 73.57

[C(CH₃)₂]; HRMS (ESI) exact mass calcd for C₂₅H₅₆O₂Si₂Na

(M⁺+Na) 467.3717, measured 467.3737.

5.18. (3R,5S)-9-(tert-Butyldimethylsilyloxy)-3,5,9-trimethyl-

decanal (39)

5.15. (2S)-2-[(2⁰S)-6⁰-(tert-Butyldimethylsilyloxy)-2⁰,6⁰-

dimethyl-heptyl]-butane-1,4-diol (36)

To a solution of diether 37 (21.4 mg, 48

(7 mL) was added tetrabutylammonium ﬂuoride (1.0 M w THF,

75 L, 75 mol) at room temperature. The mixture was stirred un-

lmol) in anhydrous THF

Deprotection of a primary hydroxyl in isomeric diether 34

l

(12 mg, 26

l

mol) was performed analogously to the process de-

der argon at room temperature for 18 h, poured into brine and ex-

tracted with ethyl acetate. The combined organic extracts were

washed with brine, dried (MgSO₄) and evaporated. The oily prod-

uct was puriﬁed on a silica Sep-Pak (2 g). Elution with hexane/

AcOEt (9:1) gave an oily (3R,5S)-9-(tert-butyldimethylsilyloxy)-

scribed above for 33. Pure diol 36 (9 mg, 100%) was eluted from

a Sep-Pak cartridge with hexane/ethyl acetate (6:4); ½a _D²⁴

ꢂ

+7.0 (c

0.35, CHCl₃); ¹H NMR (200 MHz, CDCl₃) d 0.06 [6H, s, Si(CH₃)₂],

0.84 (9H, s, Si–t-Bu), 0.86 (3H, d, J = 6.4 Hz, CH–CH₃), 1.17 [6H, s,

OC(CH₃)₂], 3.46 (1H, dd, J = 10.7, 7.1 Hz, CHCH₂OH), 3.6–3.8 (3H,

br m, CH₂CH₂OH and one of CHCH₂OH); ¹³C NMR (50 MHz) d

ꢀ2.05 [Si(CH₃)₂], 18.11 [SiC(CH₃)₃], 19.72 (CH–CH₃), 21.55

(CH₂CH₂–CH₂), 25.84 [SiC(CH₃)₃], 29.77 [C(CH₃)₂], 30.02 (CH–

CH₃), 35.60 (CH₂), 36.89 (CHCH₂–OH), 37.92 (CH₂), 39.30 (CH₂),

45.30 (CH₂), 61.29 (CH₂OH), 67.13 (CH₂OH), 73.47 [OC(CH₃)₂];

3,5,9-trimethyl-decan-1-ol (16.5 mg, 100%); ½a _D²⁴

ꢂ

+6.6 (c 0.34,

CHCl₃); ¹H NMR (400 MHz, CDCl₃) d 0.06 [6H, s, Si(CH₃)₂], 0.86

(9H, s, Si–t-Bu), 0.84 (3H, d, J = 6.8 Hz, CH–CH₃), 0.87 (3H, d,

J = 6.8 Hz, CH–CH₃), 1.17 [6H, s, C(CH₃)₂], 3.68 (2H, m, CH₂OH);

¹³C NMR (100 MHz) d ꢀ2.06 [Si(CH₃)₂], 18.11 [SiC(CH₃)₃], 19.43

(CH–CH₃), 19.56 (CH–CH₃), 21.60 (CH₂–CH₂–CH₂), 25.85

1760

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

[SiC(CH₃)₃], 26.91 (CH–CH₃), 29.78 and 29.90 [2 ꢁ C(CH₃)₂], 30.02

(CH–CH₃), 38.43 (CH₂), 40.87 (CH₂), 44.91 (CH₂), 45.33 (CH₂),

61.20 (CH₂OH), 73.54 [C(CH₃)₂]; HRMS (ESI) exact mass calcd for

C₁₉H₄₂O₂SiNa (M⁺+Na) 453.2852, measured 453.2845.

(1.8 M in cyclohexane, 57 lL, 102 lmol) under argon with stirring.

The solution turned deep orange. The mixture was stirred at

ꢀ78 °C for 20 min and a precooled (ꢀ78 °C) solution of the alde-

hyde 39 (11 mg, 34 lmol) in anhydrous THF (210 lL) was slowly

To a solution of NMO (14 mg, 0.12 mmol) in CH₂Cl₂(0.5 mL) were

added 4 Å molecular sieves (75 mg) and the mixture was stirred at

room temperature for 15 min. Then was added TPAP (1 mg,

added. The mixture was stirred at ꢀ78 °C under argon for 3 h

and at 6 °C for 16 h. Ethyl acetate and water were added, and the

organic phase was separated, washed with brine, dried (MgSO₄)

and evaporated. The oily residue was puriﬁed on a silica Sep-Pak

cartridge (2 g). Elution with hexane/AcOEt (99.8:0.2) gave a pro-

tected vitamin D analog 41 (2.8 mg, 12%).

2.66

lmol) anda solutionof theobtainedalcohol(16.5 mg, 50

lmol)

in CH₂Cl₂(100

lL). The resulted dark mixture was stirred for 20 min,

ﬁltered through a silica Sep-Pak (2 g) and evaporated. The oily resi-

due was dissolved in hexane, applied on a silica Sep-Pak cartridge

(2 g) and washed with hexane/AcOEt (98:2) to give an oily aldehyde

5.20.1. (13R,20S)-1a,25-Bis-(tert-butyldimethylsilyloxy)-2-

methylene-8(12),14(17)-diseco-9,11,15,16,18,19-

hexanorvitamin D₃tert-butyldimethylsilyl ether (41)

39 (11.2 mg, 68%); ½a _D²⁴

ꢂ

+4.0 (c 0.15, CHCl₃); ¹H NMR (400 MHz,

CDCl₃) d 0.06 [6H, s, Si(CH₃)₂], 0.85 (9H, s, Si–t-Bu), 0.87 (3H, d,

J = 6.8 Hz, CH–CH₃), 0.93 (3H, d, J = 6.5 Hz, CH–CH₃), 1.17 [6H, s,

C(CH₃)₂], 2.11 (1H, m, CH–CH₃), 2.24 (1H, ddd, J = 15.8, 7.6, 2.2 Hz,

one of CH₂CHO), 2.35 (1H, ddd, J = 15.8, 5.7, 2.2 Hz, one of CH₂CHO),

9.75 (1H, t, J = 2.2 Hz, CHO); ¹³C NMR (100 MHz) d ꢀ2.09 [Si(CH₃)₂],

18.08 [SiC(CH₃)₃], 19.30 (CH–CH₃), 19.62 (CH–CH₃), 21.48 (CH₂–

CH₂–CH₂), 25.60 (CH–CH₃), 25.82 [SiC(CH₃)₃], 29.94 (CH–CH₃),

29.75 and 29.89 [2 ꢁ C(CH₃)₂], 38.12 (CH₂), 44.53 (CH₂), 45.23

(CH₂), 51.80 (CH₂CHO), 73.44 [C(CH₃)₂], 203.16 (CHO); HRMS (ESI)

exact mass calcd for [C₁₈H₃₇O₂Si]⁺313.2563, measured 313.2558.

UV (in hexane) k_max236.0, 243.5, 252.0 nm; ¹H NMR (500 MHz,

CDCl₃; vitamin D numbering) d 0.027, 0.038, 0.064 and 0.066 [each

3H, each s, 4 ꢁ Si(CH₃)₂], 0.057 [6H, s, (SiCH₃)₂], 0.82 (3H, d, J = 6.4

Hz, 20-H₃), 0.83 (3H, d, J = 6.5 Hz, 13-H₃), 0.85, 0.87 and 0.89

[3 ꢁ 9H, each s, 3 ꢁ Si–t-Bu], 1.17 (6H, s, 26- and 27-H₃), 1.92 (1H,

m, 21-H), 2.03 (2H, m, 14-H₂), 2.15 (1H, dd, J = 13.2, 7.7 Hz, 4b-H),

2.40 (3H, m, 4a-H, 10a- and 10b-H), 4.42 (2H, m, 1b- and 3a-H),

4.93 and 4.96 (each 1H, each s, C@CH₂), 5.60 (1H, dt, J = 14.6,

7.4 Hz, 8-H), 5.90 (1H, d, J = 10.8 Hz, 6-H), 6.22 (1H, dd, J = 14.6,

10.8 Hz, 7-H); HRMS (ESI) exact mass calcd for C₄₀H₈₀O₃Si₃Na

(M⁺+Na) 715.5313, measured 715.5329.

5.19. (3S,5S)-9-(tert-Butyldimethylsilyloxy)-3,5,9-trimethyl-

decanal (40)

5.21. (1R,3R)-1,3-Bis-(tert-butyldimethylsilyloxy)-5-[(E)-

(5⁰S,7⁰S)-11⁰-(tert-butyldimethylsilyloxy)-5⁰,7⁰,11⁰-trimethyl-

dodec-2⁰-enylidene]-2-methylene-cyclohexane (42)

Selective deprotection of the primary hydroxyl group in the

diether 38 (27.5 mg, 62 lmol) was performed analogously to the

process described above for the isomeric compound 37. After anal-

ogous work-up of the reaction mixture the oily product was puri-

ﬁed on a silica Sep-Pak (2 g). Elution with hexane/AcOEt (9:1) gave

an oily (3S,5S)-9-(tert-butyldimethylsilyloxy)-3,5,9-trimethyl-dec-

Wittig–Horner reaction of the aldehyde 40 (14.5 mg, 44

with the anion generated from the phosphine oxide 29 (77 mg,

132 mol) was performed analogously to the process described

above for the isomeric compound 39. After analogous work-up of

the reaction mixture the oily product was puriﬁed on a silica

Sep-Pak (2 g). Elution with hexane/AcOEt (98:2) gave a protected

vitamin D analog 42 (8.8 mg, 30%). This product was further puri-

ﬁed by HPLC (9.4 mm ꢁ 25 cm Zorbax-Sil column, 4 mL/min) using

a hexane/2-propanol (99.9:0.1) solvent system. Pure protected

vitamin D analog 42 was collected at R_V13 mL.

lmol)

l

an-1-ol (20.7 mg, 100%); ½a _D²⁴

ꢂ

ꢀ4.6 (c 0.78, CHCl₃); ¹H NMR

(400 MHz, CDCl₃) d 0.066 [6H, s, Si(CH₃)₂], 0.84 (3H, d, J = 6.8 Hz,

CH–CH₃), 0.86 (9H, s, Si–t-Bu), 0.87 (3H, d, J = 6.8 Hz, CH–CH₃),

1.17 [6H, s, C(CH₃)₂], 3.68 (2H, m, CH₂OH); ¹³C NMR (100 MHz) d

ꢀ2.06 [Si(CH₃)₂], 18.10 [SiC(CH₃)₃], 19.19 (CH–CH₃), 19.20 (CH–

CH₃), 21.53 (CH₂–CH₂–CH₂), 25.84 [SiC(CH₃)₃], 26.96 (CH–CH₃),

29.78 and 29.87 [2 ꢁ C(CH₃)₂], 30.01 (CH–CH₃), 37.36 (CH₂),

39.90 (CH₂), 44.90 (CH₂), 45.35 (CH₂), 61.25 (CH₂OH), 73.53

[C(CH₃)₂]; HRMS (ESI) exact mass calcd for C₁₉H₄₂O₂SiNa

(M⁺+Na) 453.2852, measured 453.2852.

5.21.1. (13S,20S)-1a,25-Bis-(tert-butyldimethylsilyloxy)-2-

methylene-8(12),14(17)-diseco-9,11,15,16,18,19-

Oxidation of the obtained alcohol (15.5 mg, 47

l

mol) was per-

hexanorvitamin D₃tert-butyldimethylsilyl ether (42)

formed analogously to the process described above for the isomeric

compound. After analogous work-up of the reaction mixture the

oily product was puriﬁed on a silica Sep-Pak (2 g). Elution with

hexane/AcOEt (98:2) gave an oily aldehyde 40 (15.4 mg, 95%);

UV (in hexane) k_max236.0, 243.0, 252.0 nm; ¹H NMR (400 MHz,

CDCl₃; vitamin D numbering) d 0.027 and 0.037 [each 3H, each s,

4 ꢁ Si(CH₃)₂], 0.059 and 0.064 [each 6H, each s, Si(CH₃)₂], 0.85,

0.87 and 0.89 [3 ꢁ 9H, each s, 3 ꢁ Si–t-Bu], 0.85–0.88 (6H, over-

lapped with Si–t-Bu, 13- and 20-H₃), 1.18 (6H, s, 26- and 27-H₃),

½

a ²_D⁴

ꢂ

ꢀ10.6 (c 0.72, CHCl₃); ¹H NMR (400 MHz, CDCl₃) d 0.06 [6H,

s, Si(CH₃)₂], 0.85 (9H, s, Si–t-Bu), 0.87 (3H, d, J = 6.6 Hz, CH–CH₃),

0.95 (3H, d, J = 6.2 Hz, CH–CH₃), 1.18 [6H, s, C(CH₃)₂], 2.10–2.22

(2H, br m, CH–CH₃and one of CH₂CHO), 2.39 (1H, m, one of

CH₂CHO), 9.76 (1H, t, J = 2.3 Hz, CHO); ¹³C NMR (100 MHz) d

ꢀ2.08 [Si(CH₃)₂], 18.09 [SiC(CH₃)₃], 19.97 (CH–CH₃), 20.55 (CH–

CH₃), 21.40 (CH₂–CH₂–CH₂), 25.67 (CH–CH₃), 25.82 [SiC(CH₃)₃],

29.77 and 29.88 [2 ꢁ C(CH₃)₂], 30.01 (CH–CH₃), 38.18 (CH₂),

44.80 (CH₂), 45.27 (CH₂), 50.95 (CH₂CHO), 73.45 [C(CH₃)₂],

203.20 (CHO); HRMS (ESI) exact mass calcd for [C₁₈H₃₇O₂Si]⁺

313.2563, measured 313.2549.

1.83 (1H, m, 21-H), 2.39 (4H, m), 4.41 (2H, m, 1b- and 3a-H),

4.93 and 4.96 (each 1H, each s, C@CH₂), 5.58 (1H, dt, J = 14.7,

7.4 Hz, 8-H), 5.90 (1H, d, J = 10.7 Hz, 6-H), 6.23 (1H, dd, J = 14.7,

10.7 Hz, 7-H); HRMS (ESI) exact mass calcd for C₄₀H₈₀O₃Si₃Na

(M⁺+Na) 715.5313, measured 715.5341.

5.22. (1R,3R)-5-[(E)-(5⁰R,7⁰S)-11⁰-Hydroxy-5⁰,7⁰,11⁰-trimethyl-

dodec-2⁰-enylidene]-2-methylene-cyclohexane-1,3-diol (12)

To a solution of protected vitamin 41 (2.8 mg, 3 lmol) in THF

(1 mL) and acetonitrile (0.5 mL) was added 46% HF/MeCN (1:9,

1 mL) at room temperature. After stirring for 6 h, a saturated NaH-

CO₃was added. The mixture was extracted with CH₂Cl₂, organic

extracts were washed with brine, dried (MgSO₄) and evaporated.

The residue was ﬁrst puriﬁed on a silica Sep-Pak (0.5 g). Elution

with hexane/ethyl acetate (1:1) gave deprotected vitamin 12

5.20. (1R,3R)-1,3-Bis-(tert-butyldimethylsilyloxy)-5-[(E)-(5⁰R,7⁰S)-

11⁰-(tert-butyldimethylsilyloxy)-5⁰,7⁰,11⁰-trimethyl-dodec-2⁰-

enylidene]-2-methylene-cyclohexane (41)

To a solution of phosphine oxide 29 (60 mg, 102

lmol) in anhy-

drous THF (1 mL) at ꢀ78 °C was slowly added phenyllithium

(387 lg, 22%). Puriﬁcation of this compound was achieved by HPLC

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

1761

(9.4 mm ꢁ 25 cm Zorbax RX-Sil column, 3 mL/min) using a hex-

ane/2-propanol (85:15) solvent system. Vitamin D analog was col-

lected at R_V25 mL. Further puriﬁcation was performed by

reversed-phase HPLC (9.4 mm ꢁ 25 cm, Eclipse XDB-C18 column,

3 mL/min) using a methanol/water (85:15) solvent system. Pure

vitamin D compound 12 was collected at R_V19 mL.

[2 ꢁ SiC(CH₃)₃], 29.95 and 30.08 [C(CH₃)₂], 31.09 (CH–CH₃), 36.09

(CH₂), 36.83 (CH₂), 38.10 (CH₂), 40.13 (C–COOCH₃), 45.42 (CH₂),

51.53 (OCH₃), 61.17 (CH₂OTBS), 73.69 [OC(CH₃)₂], 177.21 (C@O);

HRMS (ESI) exact mass calcd for C₂₆H₅₆O₄Si₂Na (M⁺+Na)

511.3615, measured 511.3596.

Compound 44: ½a ²_D⁴

ꢂ

ꢀ1.4 (c 0.9, CHCl₃); ¹H NMR (400 MHz,

CDCl₃) d 0.034 and 0.057 [each 6H, each s, 2 ꢁ Si(CH₃)₂], 0.85 and

0.89 (each 9H, each s, 2 ꢁ Si–t-Bu), ca. 0.88 (3H; overlapped with

Si–t-Bu; CH–CH₃), 1.17 [6H, s, OC(CH₃)₂], 2.61 (1H, m, CHCOOCH₃),

3.58 (2H, m, CH₂OTBS), 3.66 (3H, s, COOCH₃); ¹³C NMR (125 MHz) d

ꢀ5.19 and ꢀ1.85 [Si(CH₃)₂], 18.30 and 18.51 [SiC(CH₃)₃], 19.87

(CH–CH₃), 21.65 (CH₂), 26.08 and 26.16 [2 ꢁ SiC(CH₃)₃], 30.12

and 30.16 [C(CH₃)₂], 30.99 (CH–CH₃), 35.45 (CH₂), 36.87 (CH₂),

37.34 (CH₂), 40.09 (C–COOCH₃), 45.44 (CH₂), 51.60 (OCH₃), 61.27

(CH₂OTBS), 73.68 [OC(CH₃)₂], 177.41 (C@O); HRMS (ESI) exact

mass calcd for C₂₆H₅₆O₄Si₂Na (M⁺+Na) 511.3615, measured

511.3606.

5.22.1. (13R,20S)-1a,25-dihydroxy-2-methylene-8(12),14(17)-

diseco-9,11,15,16,18,19-hexanorvitamin D₃(12, 13MeLP)

UV (in EtOH) k_max233.5, 242.0, 251.5 nm; ¹H NMR (500 MHz,

CDCl₃; vitamin D numbering) d 0.83 (3H, d, J = 6.4 Hz, 20-H₃), 0.84

(3H, d, J = 6.4 Hz, 13-H₃), 1.22 (6H, s, 26- and 27-H₃), 2.06 (2H,

m, CH₂), 2.26 (1H, dd, J = 13.1, 7.0 Hz, 10

J = 13.2, 7.4 Hz, 4b-H), 2.57 (1H, dd, J = 13.2, 4.1 Hz, 4

(1H, dd, J = 13.2, 4.0 Hz, 10b-H), 4.49 (2H, m, 1b- and 3

a

-H), 2.40 (1H, dd,

-H), 2.70

-H), 5.10

a

(2H, s, C@CH₂); 5.69 (1H, dt, J = 14.8, 7.3 Hz, 8-H), 6.04 (1H, d,

J = 10.8 Hz, 6-H), 6.30 (1H, dd, J = 14.8, 10.8 Hz, 7-H); HRMS (ESI)

exact mass calcd for C₂₂H₃₈O₃Na (M⁺+Na) 373.2719, measured

373.2730.

5.25. (R)-3-[(S)-6⁰-(tert-Butyldimethylsilyloxy)-2,6-dimethyl-

heptyl]-dihydro-furan-2-one (45)

5.23. (1R,3R)-5-[(E)-(5⁰S,7⁰S)-11⁰-hydroxy-5⁰,7⁰,11⁰-trimethyl-

dodec-2⁰-enylidene]-2-methylene-cyclohexane-1,3-diol (13)

To a solution of the ester 43 (5 mg, 10

was added at room temperature 10-camphorsulfonic acid (20 mg,

86 mol) and the mixture was stirred for 1 h, poured into brine

lmol) in MeOH (1.2 mL)

Deprotection of hydroxyl groups in vitamin D compound 42

l

(1.7 mg, 2.4

l

mol) was performed analogously to the process de-

and extracted with ethyl acetate. The combined organic extracts

were washed with brine, dried (MgSO₄) and evaporated. The oily

product was puriﬁed on a silica Sep-Pak (0.5 g). Elution with hex-

scribed above for the isomeric compound 41. After analogous

work-up of the reaction mixture the product was puriﬁed on a sil-

ica Sep-Pak (0.5 g). Elution with hexane/AcOEt (1:1) gave an oily

vitamin D analog 13. This product was further puriﬁed by HPLC

(9.4 mm ꢁ 25 cm Zorbax RX-Sil column, 4 mL/min) using a hex-

ane/2-propanol (85:15) solvent system. Pure vitamin D analog 13

ane/AcOEt (95:5) gave an oily lactone 45 (3.3 mg, 85%); ½a _D²⁴

ꢀ11 (c

ꢂ

0.4, CHCl₃); CD

D

e

(k_max) ꢀ0.6 (218.8); ¹H NMR (500 MHz, CDCl₃) d

0.06 [6H, s, Si(CH₃)₂], 0.85 (9H, s, Si–t-Bu), 0.94 (3H, d, J = 7.0 Hz,

CH–CH₃), 1.18 [6H, s, OC(CH₃)₂], 1.86–1.97 (2H, m, CH–CH₃and

one of 4-H₂), 2.41 (1H, m, one of 4-H₂), 2.57 (1H, dq, J = 9.0,

4.5 Hz, 3-H), 4.18 (1H, dt, J = 7.0, 9.0 Hz, one of 5-H₂), 4.35 (1H,

dt, J = 3.0, 9.0 Hz, one of 5-H₂); ¹³C NMR (125 MHz) d ꢀ2.27

[Si(CH₃)₂], 18.58 [SiC(CH₃)₃], 19.97 (CH–CH₃), 21.37 (CH₂), 25.82

[SiC(CH₃)₃], 29.57 [C(CH₃)₂], 29.83 (CH₂), 30.98 (CH-CH₃), 36.42

(CH₂), 38.14 (CH₂), 45.20 (CH-CH₂), 45.26 (CH₂), 66.60 (CH₂O),

73.63 [OC(CH₃)₂], 180.10 (C@O); HRMS (ESI) exact mass calcd for

C₁₉H₃₈O₃SiNa (M⁺+Na) 342.2590, measured 342.2599.

(343

l

g, 40%) was collected at R_V25 mL.

5.23.1. (13S,20S)-1

a,25-dihydroxy-2-methylene-8(12),14(17)-

diseco-9,11,15,16,18,19-hexanorvitamin D₃(13, 13MeMP)

UV (in EtOH) k_max236.0, 242.5, 268.0 nm; ¹H NMR (500 MHz,

CDCl₃; vitamin D numbering) d 0.86 (3H, d, J = 6.4 Hz, 20-H₃), 0.87

(3H, d, J = 7.2 Hz, 13-H₃), 1.21 [6H, s, 26- and 27-H₃], 2.26 (1H, dd,

J = 13.1, 6.9 Hz, 10

(1H, dd, J = 13.1, 4.0 Hz, 4b-H), 2.71 (1H, dd, J = 13.2, 4.1 Hz, 10b-

H), 4.48 (2H, m, 1b- and 3 -H), 5.10 and 5.12 (each 1H, each s,

a-H), 2.39 (1H, dd, J = 13.2, 7.5 Hz, 4a-H), 2.56

a

5.26. (S)-3-[(S)-6-(tert-Butyldimethylsilyloxy)-2,6-dimethyl-

C@CH₂); 5.69 (1H, dt, J = 14.7, 7.4 Hz, 8-H), 6.04 (1H, d, J = 10.8 Hz,

6-H), 6.29 (1H, dd, J = 14.7, 10.8 Hz, 7-H); HRMS (ESI) exact mass

calcd for C₂₂H₃₈O₃Na (M⁺+Na) 373.2719, measured 373.2726.

heptyl]-dihydro-furan-2-one (46)

Conversion of the ester 44 (15 mg, 30 lmol) into lactone 46 was

performed analogously to the process described above for the iso-

meric compound 43. After analogous work-up of the reaction mix-

ture the product was puriﬁed on a silica Sep-Pak (0.5 g). Elution

with hexane/AcOEt (95:5) gave an oily lactone 46 (9 mg, 86%);

5.24. (2R,4S)- and (2S,4S)-8-(tert-Butyldimethylsilyloxy)-2-[2⁰-

(tert-butyldimethylsilyloxy)ethyl]-4,8-dimethyl-nonanoic acid

methyl esters (43 and 44)

½

a ²_D⁴

ꢂ

+15 (c 0.25, CHCl₃); CD

D

e

(k_max) ꢀ0.68 (217.4); ¹H NMR

To a stirred solution of the esters 22 and 23 (50 mg, 0.14 mmol)

in ethyl acetate (2 mL) was added Pd/C (10%, 25 mg) at room tem-

perature and the mixture was hydrogenated for 4.5 h at 10 MPa.

The mixture was ﬁltered, the solvent evaporated and the oily res-

idue was puriﬁed on silica Sep-Pak (2 g). Elution with hexane/

AcOEt (9:1) gave an oily mixture of products which were separated

by HPLC (9.4 mm ꢁ 25 cm Zorbax-Sil column, 4 mL/min) using a

hexane/ethyl acetate (99:1) solvent system. The isomeric esters

43 (11 mg, 22%) and 44 (10 mg, 20%) were collected at R_V29 and

30.5 mL, respectively.

(500 MHz, CDCl₃) d 0.06 [6H, s, Si(CH₃)₂], 0.85 (9H, s, Si–t-Bu),

0.90 (3H, d, J = 7.0 Hz, CH–CH₃), 1.17 [6H, s, OC(CH₃)₂], 1.72 (1H,

m, CH–CH₃), 1.89 and 2.39 (each 1H, each m, 4-H₂), 2.59 (1H, m,

3-H), 4.19 (1H, dt, J = 7.0, 9.5 Hz, one of 5-CH₂), 4.36 (1H, dt,

J = 2.5, 9.5 Hz, one of 5-H₂); ¹³C NMR (125 MHz) d ꢀ2.26 [Si(CH₃)₂],

18.31 [SiC(CH₃)₃], 18.80 (CH–CH₃), 21.75 (CH₂), 26.04 [SiC(CH₃)₃],

29.27 (CH₂), 29.95 [C(CH₃)₂], 31.16 (CH–CH₃), 37.53 (CH₂), 38.30

(CH–CH₂), 45.42 (CH₂), 66.46 (CH₂O), 73.63 [OC(CH₃)₂], 180.29

(C@O); HRMS (ESI) exact mass calcd for C₁₉H₃₈O₃SiNa (M⁺+Na)

342.2590, measured 342.2579.

Compound 43: ½a ²_D⁴

ꢂ

+1.4 (c 1.25, CHCl₃); ¹H NMR (400 MHz,

CDCl₃) d 0.036 and 0.054 [each 6H, each s, 2 ꢁ Si(CH₃)₂], 0.85 and

0.89 (each 9H, each s, 2 ꢁ Si–t-Bu), 0.88 (3H, d, J = 6.4 Hz, CH–

CH₃), 1.16 and 1.17 [each 3H, each s, OC(CH₃)₂], 2.65 (1H, m,

CHCOOCH₃), 3.60 (2H, m, CH₂OTBS), 3.66 (3H, s, COOCH₃); ¹³C

NMR (125 MHz) d ꢀ5.24 i ꢀ1.85 [Si(CH₃)₂], 18.30 and 18.50

[SiC(CH₃)₃], 19.43 (CH–CH₃), 21.64 (CH₂), 26.05 and 26.12

5.27. Reduction of the lactones 45 and 46 to the diols 35 and 36

5.27.1. (R)-2-[(S)-6-(tert-Butyldimethylsilyloxy)-2,6-dimethyl-

heptyl]-1,4-butanodiol (35)

To a suspension of LiAlH₄(50 mg, 1.33 mmol) in anhydrous THF

(800 lL) at 0 °C was added solution of the lactone 45 (8 mg,

1762

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

20

l

mol) in THF (200

l

L) and the mixture was stirred at 0 °C for

were treated with the compound tested so that the ﬁnal concentra-

tion of ethanol was less than 0.2%. Four days later, the cells were

harvested and a nitro blue tetrazolium (NBT) reduction assay

was performed. The percentage of differentiated cells was deter-

mined by counting a total of 200 cells and recording the number

that contained intracellular black-blue formazan deposits.²⁴The

experiment was repeated 2–3 times and the results are reported

as the mean. Veriﬁcation of differentiation to monocytic cells

was determined by measuring phagocytic activity (data not

shown).

1.5 h. Saturated Na₂SO₄was then added and the mixture was ex-

tracted with ethyl acetate. The combined organic extracts were

washed with water, dried (MgSO₄) and evaporated. The oily prod-

uct was puriﬁed on a silica Sep-Pak (0.5 g). Elution with hexane/

AcOEt (7:3) gave an oily diol 35 (5 mg, 62%).

5.27.2. (S)-2-[(S)-6-(tert-Butyldimethylsilyloxy)-2,6-dimethyl-

heptyl]-1,4-butanodiol (36)

Reduction of the lactone 46 (8 mg, 20 lmol) into diol 36 was

performed analogously to the process described above for the iso-

meric compound 45. After analogous work-up of the reaction mix-

ture the product was puriﬁed on a silica Sep-Pak (0.5 g). Elution

with hexane/AcOEt (7:3) gave an oily diol 36 (4 mg, 49%).

5.30. Transcriptional assay

Transcription activity was measured in ROS 17/2.8 (bone) cells

that were stably transfected with a 24-hydroxylase (24Ohase) gene

promoter upstream of a luciferase reporter gene.²⁵Cells were given

a range of doses. Sixteen hours after dosing the cells were har-

vested and luciferase activities were measured using a lumino-

meter. Each experiment was performed in duplicate 2–3 separate

times.

5.28. Measurement of binding to the full-length rat

recombinant vitamin D receptor (VDR)

Puriﬁed full-length rat recombinant receptor was prepared as

described earlier with a few modiﬁcations.²³The entire coding

region for the rat VDR was inserted into the p29 plasmid includ-

ing the ﬂexible insertion region (residues 165-211). During the

puriﬁcation of the full-length receptor, the eluate from the metal

afﬁnity column was dialyzed against the same buffer but at a pH

of 8.0 instead of 7.0 and 50 mM sodium phosphate was used in-

stead of 20 mM. The size of the SP-Sepharose Fast Flow column

was slightly different—1.5 ꢁ 17 cm and the salt gradient used for

elution of the VDR from this column linearly increased from 0 to

0.8 M phosphate buffer over a total volume of 300 mL. Fractions

judged pure by SDS–PAGE were combined and dialyzed against

25 mM EPPS at pH 8.5, containing 50 mM NaCl and 0.02%

NaN₃. Following dialysis, the protein was concentrated by ultra-

centrifugation to approximately 1.4 mg/mL. Aliquots of the puri-

ﬁed protein were ﬂash-frozen in liquid nitrogen and stored at

ꢀ80 °C until use. On the day of each binding assay, the protein

was diluted in TEDK₅₀(50 mM Tris, 1.5 mM EDTA, pH 7.4,

5 mM DTT, 150 mM KCl) with 0.1% Chaps detergent. The recep-

tor protein and ligand concentration were optimized such that

no more than 20% of the added radiolabeled ligand was bound

to the receptor. Unlabeled ligands were dissolved in ethanol

and the concentrations determined using UV spectrophotometry

5.31. Computational method

All calculations have been carried out on a simple PC endowed

with an AMD AthlonI 64X2 Dual core processor. The calculation of

optimized geometries and steric energies was done using the algo-

rithm from the MM⁺HYPERCHEM (release 7.0) software package

(Hypercube, Inc.). MM⁺is an all-atom force ﬁeld based on the

MM2 functional form. The procedure used for generation of the

respective conformers of the alkoxyalkyl (or hydroxyalkyl) substit-

uents and ﬁnding the global minimum structures of the lactones

was an analogous to that described previously by us for the vita-

min D side chain conformers²⁶and involved the Conformational

Search module of the ^HYPERCHEMprogram. Values of acyclic torsion

variation ( 10–180°) and ring torsion ﬂexing ( 10–70°) were cho-

sen to assure the accuracy of the conformer generation; in all con-

formational searches the number of iterations was set at the value

of 2000.

The calculations of electronic circular dichroism spectra were

done for lactones 45 and 46 with OH group instead of O-TBS group.

The real minimum energy conformers found by molecular

mechanics have been further fully optimized at the DFT/B3LYP/6-

31++G(D,P) level as implemented in ^GAUSSIAN03 package.²⁷To con-

ﬁrm the stability of calculated structures, the frequency calcula-

tions were performed at B3LYP/6-311++G(D,P) level. For the

stable structures, the rotational strengths were calculated at the

B3LYP/6-311++G(D,P) level. The rotatory strengths were calculated

using both the length and the velocity representations. The differ-

ences between the length and the velocity calculated values of the

rotatory strengths were quite small and for this reason only the

velocity rotatory strengths were taken into further consideration.

The CD were simulated by overlapping Gaussian functions for each

transition according to the procedure described by Diedrich and

Grimme.²⁸No correlation for the medium dielectric constant was

implemented.

[1a

,25(OH)₂D₃: molar extinction coefﬁcient

e = 18,200 and

k_max= 265.0 nm; the tested 19-norvitamin des-C,D compounds:

e

= 30,200 and k_max= 241.0 nm]. Radiolabeled ligand [³H-

1

a

,25(OH)₂D₃, ꢄ159 Ci/mmol] was added in ethanol at a ﬁnal

concentration of 1 nM. Radiolabeled and unlabeled ligands were

added to 100 L of the diluted protein at a ﬁnal ethanol concen-

tration of <10%, mixed and incubated overnight on ice to reach

binding equilibrium. The following day, 100 L of hydroxylapa-

l

tite slurry (50%) was added to each tube and mixed at 10-min

intervals for 30 min. The hydroxylapatite was collected by centri-

fugation and then washed three times with Tris–EDTA buffer

(50 mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-

100. After the ﬁnal wash, the pellets were transferred to scintil-

lation vials containing 4 mL of Biosafe II scintillation cocktail,

mixed and placed in a scintillation counter. Total binding was

determined from the tubes containing only radiolabeled ligand.

The displacement experiments were carried out in duplicate on

2–3 different occasions.

5.32. In vivo calcemic assays

Weanling male rats were maintained on a 0.47% Ca diet for 1

week and then switched to a low calcium diet containing 0.02%

Ca for an additional 3 weeks. During the last week, they were

dosed daily with the appropriate vitamin D compound for 7 con-

secutive days. All doses were administered intraperitoneally in

0.1 mL propylene glycol/ethanol (95:5). Controls received the vehi-

cle. Determinations were made 34 h after the last dose. There were

5–6 rats per group.

5.29. Measurement of cellular differentiation

Human promyelocytic leukemia (HL-60) cells were grown in

RPMI-1640 medium containing 10% fetal bovine serum at 37 °C

in the presence of 5% CO₂. HL-60 cells were plated at

1.2 ꢁ 10⁵cells/plate. Eighteen hours after plating, cells in duplicate

K. Plonska-Ocypa et al. / Bioorg. Med. Chem. 17 (2009) 1747–1763

1763

11. Sabbe, K.; D’Hallewyn, C.; De Clercq, P.; Vandewalle, M. Bioorg. Med. Chem. Lett.

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Acknowledgments

12. Barbiel, P.; Bauer, F.; Mohr, P.; Muller, M.; Pirson, W. Cyclohexanodiol

Derivatives. WO 1999, 99/43646.

13. Hu, Y.; Porco, J. A.; Labadie, J. W.; Gooding, O. W.; Trost, B. M. J. Org. Chem. 1998,

63, 4518.

14. Vanhooke, J. L.; Benning, M. M.; Bauer, C. B.; Pike, J. W.; DeLuca, H. F.

Biochemistry 2004, 43, 4101.

The work was supported by funds from the Wisconsin Alumni Re-

search Foundation. We gratefully acknowledge Jean Prahlfor carrying

out the binding studies and the HL-60 differentiation measurements;

and to Julia Zella for carrying the transcriptional studies.

15. Demin, S.; Van Haver, D.; Vandewalle, M.; De Clercq, P. J.; Bouillon, R.; Verstuyf,

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Article Doi

13-Methyl-substituted des-C,D analogs of (20S)-1α,25-dihydroxy-2-methylene-19-norvitamin D3 (2MD): Synthesis and biological evaluation

DOI: 10.1016/j.bmc.2008.11.082

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Article abstract of DOI:10.1016/j.bmc.2008.11.082

Full text of DOI:10.1016/j.bmc.2008.11.082

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