Synthesis and Biological Activity of 2,22-Dimethylene Analogues of 19-Norcalcitriol and Related Compounds

Article abstract of DOI:10.1021/acs.jmedchem.0c00580

Continuing our search for Vitamin D analogues, we explored the modification of the steroidal side chain and inserted a methylene moiety in position C-22 together with either lengthening the side chain or introducing a ring at the terminal end. Our conform

Full text of DOI:10.1021/acs.jmedchem.0c00580

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Article
Synthesis and Biological Activity of 2,22-Dimethylene
Analogues of 19-Norcalcitriol and Related Compounds
Izabela Sibilska-Kaminski, Rafal R. Sicinski, Lori A. Plum, and Hector F. Deluca
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00580 • Publication Date (Web): 08 Jun 2020
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Synthesis and Biological Activity of 2,22-Dimethylene Analogues
of 19-Norcalcitriol and Related Compounds
a
b
Izabela K. Sibilska-Kaminski , Rafal R. Sicinski ,
a
a,*
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Lori A. Plum , Hector F. DeLuca
aDepartment of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison,
WI 53706, USA;
bDepartment of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
*
To whom correspondence should be addressed:
Dr. Hector F. DeLuca
Department of Biochemistry
University of Wisconsin
433 Babcock Drive
Madison, WI 53706
Telephone: 608-262-1620
Fax: 608-262-7122
Email: deluca@biochem.wisc.edu
Keywords: Vitamin D analogues; 2MD; Wittig-Horner coupling; Vitamin D receptor; Bone
calcium mobilization; Intestinal calcium transport
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ABSTRACT: Continuing our search for vitamin D analogues we explored modification of the
steroidal side chain and inserted a methylene moiety in position C-22 together with either
lengthening the side chain or introducing a ring at the terminal end. Our conformational studies
confirmed that the presence of a methylene group attached to C-22 restricts conformational
flexibility of the side chain which can result in changes in biological characteristics of a molecule.
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All synthesized 1α,25-dihydroxy-2,22-dimethylene-19-norvitamin D analogues proved equal to
3
calcitriol in their ability to bind to VDR and most of them exert significantly higher differentiation
and transcriptional activity than calcitriol. The most active compounds were characterized by the
presence of an elongated side chain or 26,27-dimethylene bridge. The synthetic strategy was based
on the Wittig-Horner coupling of the known A-ring phosphine oxide with the corresponding
Grundmann ketones prepared from a 20-epi-Inhoffen-Lythgoe diol derived from vitamin D2.
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INTRODUCTION
Physiological actions of 1α,25-dihydroxyvitamin D [calcitriol, 1α,25-(OH) D , 1, Figure
3
2
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1
) are exerted through the vitamin D receptor (VDR),1-3 a member of the steroid receptor gene
family of transcription factors.4
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Figure 1. Chemical structure of 1α,25-dihydroxyvitamin D3 (calcitriol, 1), (20S)-1α,25-
dihydroxyvitamin D (2), the previously synthesized compounds 3-7, and 2,22-dimethylene analogues
3
described in this work 8-13.
Superagonistic analogs of calcitriol have been attractive targets of synthetic efforts because
of their potential use as drugs for osteoporosis and other bone disorders.5-8 Almost three decades
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ago Binderup and collaborators established that epimerization at C-20 in the calcitriol molecule
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enhances VDR affinity. Moreover, 20-epi calcitriol (2) showed a 2–5 fold increased calcemic
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activity in comparison with calcitriol 1. It was also proved that the presence of an additional
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(
22R)-methyl group (compound 3) further elevates biological potency. An even more dramatic
increase in activity was observed for 2MD (4),12-16 the 2-methylene-19-nor analogue of calcitriol
discovered by us in 1998,17 and characterized by an inverted configuration at C-20. Next,
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preparation of 2MD homologues 5 and 6, which both proved to be very calcemic, confirmed an
observation that elongation of the steroidal side chain at C-26 and C-27 causes an enhancement of
calcemic activity of vitamin D.19,20 Further modification of the 2MD side chain achieved in our
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laboratory was based on the earlier studies and involved introduction of an additional 22-methyl
substituent. All possible diastereomers at C-20 and C-22 were synthesized and according to the
expectations (see below), (20S,22R)-22-methyl analog 7 had the highest biological activity, both
in vitro and in vivo. Taking these facts into account, we considered modification of the 2MD
molecule at C-22 by substituting it with a methylene group (analog 9) and coupled this structural
change with side chain elongation (analogs 10-13). In addition, the importance of the 25-hydroxyl
group was readdressed by elimination of it (analog 8).
The concept of vitamin D side-chain modification was developed by many research groups,
both in industry and academia. These combined efforts resulted in the preparation of many calcitriol
analogs of promising biological properties.22-24 In 2000 Moras group solved a crystalline structure
of the VDR(LBD)-1 complex and proved that 25-hydroxyl group of the natural hormone creates
25
hydrogen bonds with His-305 and His-397. This valuable findings allowed scientists to perform
docking experiments of the structurally modified calcitriol analogs to the VDR. However, crystal
structures of the VDR complexes with 20-epi-calcitriol analogs possessing different side chains,
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published one year later, indicated - on the contrary to the general belief that different receptor
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conformations would be induced - conserved ligand-binding pocket with the same histidine
residues anchoring terminal side-cahain hydroxyl present in the examined steroidal ligands. Thus,
these crystallographic structures provided surprising information that the aliphatic side chain of the
vitamin D compounds can sometimes be forced to adopt energetically unfavorable conformations.
Since steroidal ligand must change its side-chain conformation to fit into the ligand binding pocket
and anchor to the respective amino acids, energy expenditure associated with this process may be
relevant to its biological activity.
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As a consequence, these studies led to the realization that conformation of flexible steroidal
side chain and the spatial position of its terminal hydroxyl group, play an important role in the
binding of the modified vitamin D molecule to VDR and subsequent physiological actions. First
“
dot maps” presenting conformational flexibility of the vitamin D side chain by showing positions
of the terminal 25-oxygen in the energetically favorable conformers was reported by Okamura and
his collaborators.27,28 Later, an interesting structure-function hypothesis was formulated by
Yamada and collaborators in a series of publications.11,29-32 In their concept of „active space
region”, Japanese scientists postulated that VDR affinity and other biological functions (cell
differentiation, transactivation potency) of calcitriol analogs can be predicted by conformational
analysis of their side chains. Thus, the most stable conformers are identified and location of their
2
5-oxygen can be assigned to the one of the spatial regions (EG, G, EA and A). The authors
analyzed the conformation of over 50 potent calcitriol analogs and they proved that this method
can be especially useful for compounds possessing additional side chain substituents (for instance
at C-22) causing severe restriction of the side chain mobility due to hampered rotation around the
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0,22-bond. Therefore, in the following years, several research groups, especially these led by
Yamada33-34 and Mourino,35-39 developed syntheses of various conformationally restricted vitamin
D analogs.
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Applied to the analogs described in this paper, the “active space group concept” predicted the
proposed analogs would exert significant biological activity and thus, the syntheses pursued.
RESULTS AND DISCUSSION
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Conformational analysis. Since the presence of a sp carbon atom 22 in our planned
structures of 22-methylene 2MD analogs obviously confines their side chain rotation, we
performed systematic conformational searches21,40 of the corresponding model 8-methylene des-
A,B-compounds possessing side chains analogous to those present in vitamins 9-13. Then,
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according to a protocol described by us previously in detail, three-dimensional „dot maps” were
created indicating the spatial regions potentially occupied by the 25-oxygen present in the
energetically preferred conformations of the examined compounds (Figure 1S, Supporting
Information). Since the results of structural analysis in all cases gave similar results, indicating that
the planned 22-methylene analogs would exert high biological activity, comparable to the parent
2
MD, their synthesis was undertaken and described in this work.
Chemistry. Synthesis of the target vitamins 8-13 was achieved by applying Lythgoe’s
strategy41-43 based on the Wittig-Horner reaction. A-Ring phosphine oxide 14 (Figure 2), described
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by us previously , was coupled with corresponding Grundmann ketones 15a-f which in turn were
obtained from protected hydroxy aldehyde 16 (Scheme 1S, Supporting Information).
POPh2
a: R =
b: R =
OTES
R
H
TBSO
OTBS
OTES
OTES
H
c: R =
e: R =
d: R =
f: R =
O
OTES
OTES
14
15
Figure 2. The building blocks used for the synthesis of target compounds.
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For the preparation of 16 we used slightly modified literature procedures; the synthesis is
described in the Supporting Information. The availability of this compound allowed us to carry out
its reactions with the different Grignard reagents RMgBr (Scheme 1), generated from the
corresponding bromides A-F; their preparation is described in Supporting Information. The formed
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2-alcohols (steroidal numbering) 17a-f were obtained in a very good yield as mixtures of 22-
epimers (with 20R,22R-isomers prevailing). Their oxidation with Dess-Martin periodinane
provided single 22-keto compounds 18a-18f. The obtained ketones were then subjected to Wittig
methylenation and in the formed products 19a-19f hydroxyl groups were deprotected by treatment
with tetrabutylammonium fluoride. The resulting hydrindanol 20a and the diols 20b’-20f’ were
subjected to oxidation with PDC to give the corresponding Grundmann ketone analog 15a and
2
1b’-21f’. The latter series of compounds, containing a 25-hydroxyl group, were silylated and the
5-OTES derivatives 15b-15f were efficiently obtained. Synthesis of the desired C/D-building
2
blocks 15a-15f allowed their coupling with the A-ring phosphine oxide 14. Removal of the silyl
protecting groups in the formed 19-norvitamin D compounds 22a-22f furnished the target 2,22-
dimethylene-19-nor-1α,25-(OH) D compounds 8-13.
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Biological Evaluation. All of the newly synthesized 22-methylenated vitamins (8-13) were
examined for biological activity, using both in vitro and in vivo testing, and the results were
compared to 1α,25-(OH) D (1) and 2MD (4). The data presented here (Table 1) show that
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introduction of a 22-methylene moiety did not dramatically affect the binding affinity of the ligands
to the full-length recombinant rat receptor unless it was coupled with removal of the 25-hydroxyl
group.
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OH
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OTES
OTES
OTES
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17a - 17f
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OTES
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0b’ - 20f'
a: R =
b: R =
c: R =
d: R =
b': R =
c': R =
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OH
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OTES
OTES
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OTES
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OTES
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OTES
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POPh2
15a - 15f
TBSO
OTBS
TBSO
OTBS
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Scheme 1. (a) Mg, bromide A-F, THF (17a: 92%; 17b: 98%; 17c: 99%; 17d: 98%; 17e: 95%; 17f: 94%);
(
b) Dess-Martin periodinane, CH Cl (18a: 91%; 18b: 96%; 18c: 92%; 198: 97%; 18e: 89%; 18f: 87%); (c)
2 2
+
-
Ph P CH Br , t-BuOK, THF (19a: 92%; 19b: 97%; 19c: 97%; 19d: 75%; 19e: 80%; 19f: 68%); (d) TBAF,
3
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THF (20a: 97%; 20b’: 96%; 20c’: 96%; 20d’: 86%; 20e’: 95%; 20f’: 91%); (e) PDC, CH Cl (15a: 87%;
2
2
21b’: 90%; 21c’: 88%; 21d’: 89%; 21e’: 91%; 21f’: 90%); (f) TESOTf, 2,6-lutidine, CH Cl (15b: 98%;
2
2
1
2
5c: 95%; 15d: 99%; 15e: 84%; 15f: 91%); (g) n-BuLi, THF (22a: 96%; 22b: 75%; 22c: 92%; 22d: 85%;
2e: 94%; 22f: 88%); (h) TBAF, THF (8: 71%; 9: 77%; 10: 50%; 11: 53%; 12: 43%; 13: 50%).
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Elimination of the 25-hydroxyl group resulted in nearly two logs less binding affinity. Testing in
cultured cells provided more distinction amongst the compounds. All 22-methylene analogues,
with the exception of 8, proved to be more potent than the natural hormone 1 in the cell-based
assays. Compounds 10 and 12 showed the highest ability to elicit cellular differentiation of human
promyelocytic HL-60 cells into monocytes, exceeding that of 2MD twice, and 1α,25-(OH) D 50-
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fold. The transcriptional activity of the synthesized compounds was examined in ROS cells
harboring a luciferase gene driven by the 24-hydroxylase (CYP24A1) promoter. The 24-homo
analog 10 as well as compound 13 were the most potent at inducing transcription being 20 times
as active as the native hormone 1. The remaining analogues with the exception of 25-desoxy-
compound 8, induced activation of the CYP24A1 gene similar or slightly more than 1α,25-(OH) D
2
3
but less than 2MD (4). Overall, the in vitro findings were as predicted with elongation of the side
chain resulting in more potent analogues and removal of the 25-hydroxyl group diminishing VDR
binding and cellular activities
In contrast to the in vitro testing, all 22-methylene analogues, showed increased efficacy compared
to the native hormone in intestinal calcium transport (Figure 3-8). While intestinal activity saturated
with 780 pmol of 1 α,25(OH) D , it continued to increase in animals administered the 22-methylene
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analogues. The explanation for these differences in efficacy is the topic of a subsequent
investigation
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a
b
Table 1. Relative VDR Binding Activities, HL-60 Differentiating Activities, and Transcriptional
c
Activities of the Vitamin D Hormone (1), 2MD (4) and the Vitamin D Analogues 8-13.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HL-60b
24OHasec
transcription
VDR Bindinga
Compd
Structure
Comp.
No.
differentiation
K ratio
i
ED ratio
ED ratio
50
50
H
OH
H
1
4
1
1
1
1
HO
OH
H
OH
H
25
29
HO
OH
H
H
8
9
0.01
0.5
1
0.1
0.05
2.5
20
HO
OH
H
OH
H
4
HO
OH
OH
H
H
1
0
50
HO
OH
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11
1
10
6.7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
H
H
1
1
2
3
0.5
50
29
6.7
20
HO
OH
OH
H
H
1
HO
OH
aCompetitive binding of 1α,25-(OH)
rat vitamin D receptor. The experiments were carried out in duplicate on two different occasions. The K
derived from the dose-response curves and represent the inhibition constant when radiolabeled 1α,25-(OH)
present at 1 nM and a K of 0.2 nM is used. The numbers shown in the Table are expressed as the average ratio of the
D
3
(1) and the synthesized vitamin D analogues to the full-length recombinant
values are
is
2
i
2
D
3
d
b
1α,25-(OH)
1α,25-(OH)
2
D
D
3
K
i
to the K
i
for the analogue. Induction of differentiation of HL-60 promyelocytes to monocytes by
(1) and the synthesized vitamin D analogues. Differentiation state was determined by measuring the
percentage of cells reducing nitro blue tetrazolium (NBT). The experiment was repeated in duplicate two times. The
ED50 values are derived from the dose-response curves and represent the analogue concentration capable of inducing
2 3
0% maturation. The numbers shown in the Table are expressed as the average ratio of the 1α,25-(OH) D ED50 to
the ED50 for the analogue. Transcriptional assay in rat osteosarcoma cells stably transfected with a 24-hydroxylase
gene reporter plasmid. The ED50 values are derived from dose-response curves and represent the analogue
concentration capable of increasing the luciferase activity by 50%. The numbers shown in the Table are expressed as
2
3
5
c
the average ratio of the 1α,25-(OH)
2
D
3
ED50 to the ED50 for the analogue.
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2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Bone Calcium Mobilization
Intestinal Calcium Transport
* #
12
14
12
1
0
8
6
4
2
0
1
0
8
6
4
2
0
*
*
*
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
*
Vehicle
87pmol
780 pmol
87pmol
780pmol
Vehicle
87pmol
780 pmol
87pmol
780pmol
compound 1 compound 1 compound 8 compound 8
compound 1 compound 1 compound 8 compound 8
Figure 3. Bone calcium mobilization and intestinal calcium transport of 1α,25-(OH) D (1) and the synthesized 22-methylene compound 8. Each
group contains 5 to 7 rats. Statistical significance (p <0.05) is denoted by an asterisk (*) (comparison to vehicle) or a pound sign (#) (comparison to
α,25-(OH) D ).
2
3
1
2
3
Bone Calcium Mobilization
Intestinal Calcium Transport
1
1
2
0
8
6
4
2
0
14
1
1
2
0
8
6
4
2
0
*
#
*
*
*
*
*
Vehicle
87 pmol
780 pmol
87pmol
780 pmol
Vehicle
87pmol
780pmol
87pmol
780pmol
compound 1 compound 1 compound 9 compound 9
compound 1 compound 1 compound 9 compound 9
Figure 4. Bone calcium mobilization and intestinal calcium transport of 1α,25-(OH) D (1) and the synthesized 22-methylene compound 9. Each
group contains 5 to 7 rats. Statistical significance (p <0.05) is denoted by an asterisk (*) (comparison to vehicle) or a pound sign (#) (comparison to
1α,25-(OH) D ).
2
3
2
3
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9
Bone Calcium Mobilization
Intestinal Calcium Transport
12
* #
14
12
10
8
6
4
*
#
1
0
8
6
4
2
0
*
*
*
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
2
0
Vehicle
87 pmol
780 pmol
87pmol
780pmol
Vehicle
87pmol
compound 1 compound 1 compound compound
10 10
780 pmol
87pmol
780pmol
compound 1 comound 1 compound compound
10
1
0
Figure 5. Bone calcium mobilization and intestinal calcium transport of 1α,25-(OH) D (1) and the synthesized 22-methylene compound 10. Each
group contains 5 to 7 rats. Statistical significance (p <0.05) is denoted by an asterisk (*) (comparison to vehicle) or a pound sign (#) (comparison to
α,25-(OH) D ).
2
3
1
2
3
Bone Calcium Mobilization
Intestinal Calcium Transport
12
14
12
10
8
6
4
*
#
1
0
8
6
4
2
0
*
#
*
*
*
*
2
0
Vehicle
87pmol
compound 1 compound 1 compound compound
11
780pmol
87pmol
780pmol
Vehicle
87pmol
compound 1 compound 1 compound compound
11 11
780pmol
87pmol
780pmol
1
1
Figure 6. Bone calcium mobilization and intestinal calcium transport of 1α,25-(OH) D (1) and the synthesized 22-methylene compound 11. Each
group contains 5 to 7 rats. Statistical significance (p <0.05) is denoted by an asterisk (*) (comparison to vehicle) or a pound sign (#) (comparison to
α,25-(OH) D ).
2
3
1
2
3
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Bone Calcium Mobilization
Intestinal Calcium Transport
12
14
12
10
8
6
4
*
#
* #
1
0
8
6
4
2
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
*
*
*
2
0
Vehicle
87 pmol
780 pmol
87 pmol
780 pmol
Vehicle
87pmol
780pmol
87pmol
780pmol
compound 1 compound 1 compound compound
12
compound 1 compound 1 compound compound
12 12
1
2
Figure 7. Bone calcium mobilization and intestinal calcium transport of 1α,25-(OH) D (1) and the synthesized 22-methylene compound 12. Each
group contains 5 to 7 rats. Statistical significance (p <0.05) is denoted by an asterisk.
2
3
Bone Calcium Mobilization
Intestinal Ca Transport
* #
1
1
2
0
8
6
4
2
0
14
1
10
8
2
*
#
*
6
4
2
0
Vehicle
87pmol
compound 1 compound 1 compound compound
13
780pmol
87pmol
780pmol
Vehicle
87pmol
780pmol
87pmol
780pmol
compound 1 compound 1 compound compound
13 13
1
3
Figure 8. Bone calcium mobilization and intestinal calcium transport of 1α,25-(OH) D (1) and the synthesized 22-methylene compound 13. Each
group contains 5 to 7 rats. Statistical significance (p <0.05) is denoted by an asterisk (*) (comparison to vehicle) or a pound sign (#) (comparison to
α,25-(OH) D ).
2
3
1
2
3
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9
Bone calcium mobilization activity of the analogues provided more separation amongst the
2
2-methylene analogues. Compounds 10 and 12 with elongated side chains in the 20R-
configuration were the most potent which is consistent with the results obtained in vitro. The analog
with the five-membered ring, compound 13, as well as compound 11, also show increased bone
calcium mobilization activity compared to the native hormone. The remainder of the 22-methylene
analogues were in the same potency range as the native hormone.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Overall, two new analogs, compounds 10 and 12, were generated that possess increased biological
potency compared to both 1α,25-(OH) D and 2MD. Both of these compounds have elongated
2
3
sides chains which has previously been shown to increase biological activity. The reason for this
heightened activity is unknown.
CONCLUSIONS
As predicted by the “active space region” model, the addition of a 22-methylene group
coupled with side chain elongation resulted in analogues with more potent cellular differentiation,
cellular transactivation, intestinal calcium transport and bone calcium mobilization activities.
Interestingly, the addition of a five-membered ring also increased biological potency, but not quite
to the same extent as aliphatic extension (13 vs 10 and 12). This difference is most noticeable in
the cell differentiaton assay, which might suggest that flexibility of the side chain is important for
eliciting cellular differentiation of human promyelocytic HL-60 cells into monocytes.
The results of the in vivo testing in bone indicate only analogues 10-13 are more potent than
the native hormone. These results indicate that a simple 22-methylene addition (compound 9)
without chain elongation or ring addition is not sufficient to cause differential bone calcium
mobilization activity compared to 1α,25-(OH) D . Both a 22-methylene moiety as well as chain
2
3
elongation are necessary for increasing in vivo potency in the bone. In the intestine on the other
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1
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5
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5
5
5
5
5
6
hand, all analogues were more potent than 1α,25-(OH) D . Thus, all biological activity, with the
2
3
exception of the bone activity, is predicted by the active space region concept.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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0
EXPERIMENTAL SECTION
Chemistry. Ultraviolet (UV) absorption spectra were obtained on a Shimadzu UV-1800
UV spectrophotometer in 100% EtOH. All nuclear magnetic resonance spectra were recorded in
deuterochloroform using Bruker DMX-400 (400 MHz) and Bruker DMX-500 (500 MHz).
Chemical shifts () are reported in parts per million relative to (CH ) Si ( 0.00) as an internal
3
4
standard. Abbreviations used are singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m). High
resolution mass spectra were registered on LCT (TOF) or Mass Quattro LC spectrometers. High-
performance liquid chromatography (HPLC) was performed on a Waters Associates liquid
chromatograph equipped with a model 6000A solvent delivery system, model U6K Universal
injector, and model 486 tunable absorbance detectors. Solvents were dried and distilled following
standard procedures.
The purity of final compounds was determined by HPLC, and they were judged at least
9
9% pure. Two HPLC columns (9.4 mm × 25 cm Zorbax-Sil and 9.4 mm × 25 cm Zorbax Eclipse
XDB-C18) were used as indicated in Table 2 (Supporting Information). The purity and identity of
1
the synthesized vitamins were additionally confirmed by inspection of their H NMR and high-
resolution mass spectra.
Preparation of the aldehyde 16 and the alkyl bromides A-F has been described in Supporting
Information.
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9
(
20R,22R)- and (20R,22S)-8β-[(Triethylsilyl)oxy]-des-A,B-cholestan-22-ols (17a). A solution of
isopentyl magnesium bromide, prepared from 1-bromo-3-methylbutane (A, 393 µL, 3.28 mmol)
and magnesium powder (78 mg, 3.21 mmol) in anhydrous THF (6 mL) as described in Supporting
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Information, was added to the solution of the aldehyde 16 (212 mg, 653 µmol) in anhydrous THF
o
(3 mL) at -78 C under argon. After 30 min a cooling bath was removed, and the mixture was
allowed to reach room temperature. Reaction was quenched by addition of an aqueous NH Cl
4
solution, extracted with ethyl acetate, dried (Na SO ) and concentrated. The resulting product was
2
4
applied on a silica Sep-Pak cartridge and eluted with hexane/ethyl acetate (95:5) to give product
1
7a (226 mg, 92%) as a colorless oil.
1
H NMR (400 MHz, CDCl ; signals of the main 20R,22R-isomer are listed) δ 0.555 (6H, q, J = 7.8
3
Hz, 3 × SiCH ), 0.801 (3H, d, J = 6.8 Hz, 21-H ), 0.889 (6H, d, J = 6.7 Hz, 26- and 27-H ), 0.922
2
3
3
(3H, s, 18-H ), 0.948 (9H, t, J = 7.8 Hz, 3 × SiCH CH ), 3.38 (1H, m, 22-H), 4.04 (1H, narr m, 8α-
3 2 3
1
3
H); C NMR (100 MHz, CDCl ) δ 4.9, 6.9, 11.1, 13.9, 17.7, 22.6, 22.8, 27.2, 28.1, 32.8, 34.6,
3
3
5.6, 38.9, 40.4, 41.9, 52.6, 53.1, 69.3, 72.8; HRMS (ESI) exact mass calculated for C H NO Si
24 50 2
+
(M + NH ) 412.3610, found 412.3611.
4
(20R,22R)- and (20R,22S)-8β,25-Bis[(triethylsilyl)oxy]-des-A,B-cholestan-22-ols (17b). A
solution of Grignard reagent, prepared from 1-bromo-3-methyl-3-[(triethylsilyl)oxy]butane (B;
1
.19 mL, 4.48 mmol) and magnesium powder (114 mg, 4.69 mmol) in anhydrous THF, was added
o
to the solution of the aldehyde 16 (308 mg, 950 µmol) in anhydrous THF (3.3 mL) at -78 C under
argon. After analogous procedure as described above for 17a, product 17b (489 mg, 98%) was
obtained as a colorless oil.
1
H NMR (500 MHz, CDCl ; signals of the main 20R,22R-isomer are listed) δ 0.554 and 0.577 (6H
3
and 6H, each q, J = 8.0 Hz, 6 × SiCH ), 0.813 (3H, d, J = 6.8 Hz, 21-H ), 0.914 (3H, s, 18-H ),
2
3
3
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
.944 and 0.947 (9H and 9H, each t, J = 8.0 Hz, 6 × SiCH CH ), 1.215 (6H, s, 26- and 27-H ), 3.79
2
3
3
1
3
(1H, m, 22-H), 4.04 (1H, narr m, 8α-H); C NMR (125 MHz, CDCl ) δ 4.9, 6.7, 6.9, 7.1, 11.2,
3
1
7
3.9, 17.7, 22.8, 27.1, 29.8, 29.8, 29.9, 34.6, 35.6, 39.1, 40.3, 41.9, 41.9, 52.5, 53.0, 69.3, 73.2,
+
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3.3; HRMS (ESI) exact mass calculated for C H O Si (M + Na ) 549.4135, found 549.4130.
3
0
62
3
2
(
20R,22R)- and (20R,22S)-8β,25-Bis[(triethylsilyl)oxy]-des-A,B-24-homocholestan-22-ols
17c). solution of Grignard reagent, prepared from 1-bromo-4-methyl-4-
(
A
[(triethylsilyl)oxy]pentane (C; 1.02 mL, 3.25 mmol) and magnesium powder (78 mg, 3.21 mmol)
in anhydrous THF (5.3 mL), was added to the solution of the aldehyde 16 (210 mg, 647 µmol) in
o
anhydrous THF (3 mL) at -78 C under argon. After analogous procedure as described above for
17a, product 17c (340 mg, 99%) was obtained as a colorless oil.
1
H NMR (400 MHz, CDCl ; signals of the main 20R,22R-isomer are listed) δ 0.554 and 0.566 (6H
3
and 6H, each q, J = 7.8 Hz, 6 × SiCH ), 0.800 (3H, d, J = 6.8 Hz, 21-H ), 0.922 (3H, s, 18-H ),
2
3
3
0
.942 and 0.962 (9H and 9H, each t, J = 7.8 Hz, 6 × SiCH CH ), 1.193 and 1.197 (3H and 3H, each
2 3
1
3
s, 26- and 27-H ), 3.86 (1H, m, 22-H), 4.04 (1H, narr m, 8α-H); C NMR (100 MHz, CDCl ) δ
3
3
4
.9, 6.8, 6.9, 7.1, 11.1, 13.9, 17.7, 21.3, 22.8, 27.2, 29.7, 29.9, 34.6, 35.6, 39.0, 40.4, 41.9, 45.2,
+
5
2.7, 53.1, 69.3, 72.6, 73.3; HRMS (ESI) exact mass calculated for C H O Si Na (M + Na)
31 64
3
2
563.4292, found 563.4293.
(20R,22R)- and (20R,22S)-8β,25-Bis[(triethylsilyl)oxy]-des-A,B-26,27-dihomocholestan-22-
ols (17d).
A
solution of Grignard reagent, prepared from 1-bromo-3-ethyl-3-
[(triethylsilyl)oxy]pentane (D; 1.32 mL, 4.28 mmol) and magnesium powder (103 mg, 4.24 mmol)
in anhydrous THF (6.3 mL), was added to the solution of the aldehyde 16 (278 mg, 858 µmol) in
o
anhydrous THF (3 mL) at -78 C under argon After analogous procedure as described above for
1
7a, product 17d (459 mg, 98%) was obtained as a colorless oil.
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1
H NMR (500 MHz, CDCl ; signals of the main 20R,22R-isomer are listed) δ 0.554 and 0.578 (6H
3
and 6H, each q, J = 7.9 Hz, 6 × SiCH ), 0.809 (3H, d, J = 6.6 Hz, 21-H ), 0.830 [6H, t, J = 7.4 Hz,
2
3
2
× C(CH CH )], 0.917 (3H, s, 18-H ), 0.947 (18H, t, J = 7.9 Hz, 6 × SiCH CH ), 3.78 (1H, m, 22-
2 3 3 2 3
1
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H), 4.04 (1H, narr m, 8α-H); C NMR (125 MHz, CDCl ) δ 4.9, 6.9, 7.0, 7.3, 8.3, 8.4, 11.2, 13.8,
3
1
7.7, 22.8, 27.1, 29.0, 31.6, 31.8, 34.6, 35.4, 39.1, 40.3, 41.9, 52.6, 53.0, 69.3, 73.5, 78.1; HRMS
+
+
(ESI) exact mass calculated for C H O Si (M + H ) 555.4628, found 549.4625.
32 67 3 2
(
20R,22R)- and (20R,22S)-8β,25-Bis[(triethylsilyl)oxy]-des-A,B-24,26,27-trihomocholestan-
2-ols (17e). A solution of Grignard reagent, prepared from 1-bromo-4-ethyl-4-
(triethylsilyl)oxy]hexane (E; 0.94 mg, 2.9 mmol) and magnesium powder (63 mg, 2.59 mmol) in
2
[
anhydrous THF (4.2 mL) was added to the solution of the aldehyde 16 (171 mg, 528 µmol) in
o
anhydrous THF (2 mL) at -78 C under argon. After analogous procedure as described above for
17a, the resulting product was applied on a silica Sep-Pak cartridge and eluted with hexane/ethyl
acetate (93:7) to give 17e (285 mg, 95%) as a colorless oil.
1
H NMR (400 MHz, CDCl ; signals of the main 20R,22R-isomer are listed) δ 0.553 and 0.573 (6H
3
and 6H, each q, J = 7.8 Hz, 6 × SiCH ), 0.800 (3H, d, J = 6.5 Hz, 21-H ), 0.824 [(6H, t, J = 7.4 Hz,
2
3
2
× C(CH CH )], 0.924 (3H, s, 18-H ), 0.945 and 0.949 (9H and 9H, each t, J = 7.8 Hz, 6 ×
2 3 3
1
3
SiCH CH ), 3.86 (1H, narr m, 22-H), 4.04 (1H, narr m, 8α-H); C NMR (100 MHz, CDCl ) δ 4.9,
2
3
3
6
.8, 6.9, 7.1, 7.3, 8.3, 11.1, 13.9, 17.7, 20.6, 22.9, 27.2, 31.4, 31.7, 34.6, 35.8, 39.0, 40.4, 41.9, 52.7,
+
5
3.1, 69.3, 72.5, 78.1; HRMS (ESI) exact mass calculated for C H O Si Na (M + Na) 591.4600,
3
3
68
3
2
found 591.4598.
(20R,22R)- and (20R,20S)-8β-[(Triethylsilyl)oxy]-24-{1’-[(triethylsilyl)oxy]cyclopentyl}-des-
A,B-25,26,27-trinorcholestan-22-ols (17f). A solution of Grignard reagent, prepared from 1-(2’-
bromoethyl)-1-[(triethylsilyl)oxy]cyclopentane (F; 1.514 g, 4.58 mmol) and magnesium turnings
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2
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6
(
119 mg, 4.89 mmol) in anhydrous THF (7 mL) was added to the solution of the aldehyde 16 (401
o
mg, 1.24 mmol) in anhydrous THF (4 mL) at -78 C under argon. After analogous procedure as
described above for 17a, product 17f (643 mg, 94%) was obtained as a colorless oil.
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H NMR (500 MHz, CDCl ; signals of the main 20R,22R-isomer are listed) δ 0.556 and 0.587 (6H
3
and 6H, each q, J = 7.9 Hz, 6 × SiCH ), 0.817 (3H, d, J = 6.8 Hz, 21-H ), 0.917 (3H, s, 18-H ),
2
3
3
1
3
0
.948 (18H, t, J = 7.9 Hz, 6 × SiCH CH ), 3.80 (1H, m, 22-H), 4.04 (1H, narr m, 8α-H); C NMR
2 3
(125 MHz, CDCl ) δ 4.9, 6.6, 6.9, 7.2, 11.2, 13.8, 17.7, 22.8, 23.5, 23.6, 27.1, 30.2, 34.6, 38.8,
3
3
9.3, 39.6, 39.9, 40.4, 41.9, 52.6, 53.0, 69.3, 73.4, 84.6; HRMS (ESI) exact mass calculated for
+
+
C H O Si (M + H ) 553.4472, found 553.4467
32
65
3
2
(20R)-8β-[(Triethylsilyl)oxy]-des-A,B-cholestan-22-one (18a). The mixture of the epimeric
alcohols 17a (226 mg, 570 µmol) and Dess-Martin periodinane (295 mg, 696 µmol) in anhydrous
methylene chloride (15 mL) was vigorously stirred at room temperature for 2 h. The reaction was
quenched with aqueous NaHCO solution, extracted with methylene chloride, dried (Na SO ) and
3
2
4
concentrated. The crude product was applied on a silica Sep-Pak cartridge and eluted with
hexane/ethyl acetate (98:2) to afford the ketone 18a (204 mg, 91%) as a colorless oil.
1
H NMR (500 MHz, CDCl ) δ 0.544 (6H, q, J = 7.8 Hz, 3 × SiCH ), 0.894 (6H, d, J = 6.4 Hz, 26-
3
2
and 27-H ), 0.900 (3H, s, 18-H ), 0.938 (9H, t, J = 7.8 Hz, 3 × SiCH CH ), 0.990 (3H, d, J = 7.0
3
3
2
3
1
3
Hz, 21-H ), 4.02 (1H, narr m, 8α-H); C NMR (125 MHz, CDCl ) δ 4.9, 6.9, 14.3, 17.0, 17.5, 22.4,
3
3
2
2.4, 25.6, 27.6, 32.2, 39.4, 39.7, 41.5, 47.1, 52.4, 53.2, 69.0, 215.8; HRMS (ESI) exact mass
+
calculated for C H NO Si (M + NH ) 412.3611, found 412.3615.
24 50
2
4
(20R)-8β,25-Bis[(triethylsilyl)oxy]-des-A,B-cholestan-22-one (18b). The oxidation of the
alcohols 17b (489 mg, 929 µmol) with Dess-Martin periodinane (475 mg, 1.12 mmol) in anhydrous
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methylene chloride (35 mL), performed as described above for 17a, afforded the ketone 18b (466
mg, 96%) as a colorless oil.
1
H NMR (500 MHz, CDCl ) δ 0.545 (6H, q, J = 8.0 Hz, 3 × SiCH ), 0.565 (6H, q, J = 7.9 Hz, 3 ×
3
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SiCH ), 0.904 (3H, s, 18-H ), 0.938 (9H, t, J = 8.0 Hz, 3 × SiCH CH ), 0.942 (9H, t, J = 7.9 Hz, 3
2
3
2
3
×
SiCH CH ), 0.998 (3H, d, J = 6.9 Hz, 21-H ), 1.204 (6H, s, 26- and 27-H ), 4.02 (1H, narr m,
2 3 3 3
1
3
8
α-H); C NMR (125 MHz, CDCl ) δ 4.9, 6.6, 6.9, 7.1, 14.2, 17.1, 17.5, 22.3, 25.6, 29.8, 30.0,
3
34.5, 36.7, 37.9, 39.3, 41.7, 47.3, 52.4, 53.4, 69.1, 72.4, 216.1; HRMS (ESI) exact mass calculated
+
+
for C H O Si (M + H ) 525.4159, found 525.4149.
3
0
61
3
2
(20R)-8β,25-Bis[(triethylsilyl)oxy]-des-A,B-24-homocholestan-22-one (18c). The oxidation of
the alcohols 17c (340 mg, 629.6 µmol) with Dess-Martin periodinane (330 mg, 778 µmol) in
anhydrous methylene chloride (20 mL), performed as described above for 17a, afforded the ketone
18c (313 mg, 92%) as a colorless oil.
1
H NMR (400 MHz, CDCl ) δ 0.549 and 0.556 (6H and 6H, each q, J = 7.8 Hz, 6 × SiCH ), 0.899
3
2
(
3H, s, 18-H ), 0.938 (18H, t, J = 7.8 Hz, 6 × SiCH CH ), 0.982 (3H, d, J = 6.9 Hz, 21-H ), 1.202
3 2 3 3
1
3
(6H, s, 26- and 27-H ), 4.02 (1H, narr m, 8α-H); C NMR (100 MHz, CDCl ) δ 4.9, 6.7, 6.9, 7.1,
3
3
1
4.4, 16.9, 17.5, 18.5, 22.3, 29.7, 34.5, 39.5, 42.3, 44.6, 46.9, 52.5, 53.2, 69.1, 73.3, 215.4; HRMS
+
(
ESI) exact mass calculated for C H O Si Na (M + Na) 561.4135, found 561.4138.
3
1
62
3
2
(20R)-8β,25-Bis[(triethylsilyl)oxy]-des-A,B-26,27-dihomocholestan-22-one
(18d).
The
oxidation of the alcohols 17d (447 mg, 807 µmol) with Dess-Martin periodinane (412 mg, 972
µmol) in anhydrous methylene chloride (30 mL), performed as described above for 17a, afforded
the crude product which was applied on a silica Sep-Pak cartridge and eluted with hexane/ethyl
acetate (96:4) to yield the ketone 18d (434 mg, 97%) as a colorless oil.
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2
2
2
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3
3
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4
4
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5
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6
1
H NMR (500 MHz, CDCl ) δ 0.545 and 0.575 (6H and 6H, each q, J = 7.9 Hz, 6 × SiCH ), 0.827
3
2
and 0.832 [3H and 3H, each t, J = 7.5 Hz, 2 × C(CH CH )], 0.904 (3H, s, 18-H ), 0.938 and 0.943
2
3
3
(9H and 9H, each t, J = 7.9 Hz, 6 × SiCH CH ), 0.995 (3H, d, J = 6.9 Hz, 21-H ), 4.02 (1H, narr
2 3 3
1
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
m, 8α-H); C NMR (125 MHz, CDCl ) δ 4.9, 6.9, 7.0, 7.3, 8.40, 8.44, 14.3, 17.1, 17.5, 22.3, 25.6,
3
3
1.5, 31.8, 34.5, 36.2, 39.5, 41.6, 47.3, 52.4, 53.4, 69.1, 78.4, 216.0; HRMS (ESI) exact mass
+
+
calculated for C H O Si (M + H ) 553.4462, found 553.4465.
32 65
3
2
(20R)-8β,25-Bis[(triethylsilyl)oxy]-des-A,B-24,26,27-trihomocholestan-22-one (18e). The
oxidation of the alcohols 17e (275 mg, 484 µmol) with Dess-Martin periodinane (250 mg, 589
µmol) in anhydrous methylene chloride (15 mL), performed as described above for 17a, afforded
the crude product which was applied on a silica Sep-Pak cartridge and eluted with hexane/ethyl
acetate (98:1) to yield the ketone 18e (245 mg, 89%) as a colorless oil.
1
H NMR (400 MHz, CDCl ) δ 0.545 and 0.564 (6H and 6H, each q, J = 7.9 Hz, 6 × SiCH ), 0.826
3
2
(6H, t, J = 7.4 Hz, 26- and 27-H ), 0.901 (3H, s, 18-H ), 0.938 (18H, t, J = 7.9 Hz, 6 × SiCH CH ),
3 3 2 3
1
3
0
.985 (3H, d, J = 6.9 Hz, 21-H ), 4.02 (1H, narr m, 8α-H); C NMR (100 MHz, CDCl ) δ 4.9, 6.9,
3 3
7.0, 7.2, 8.2, 14.4, 16.9, 17.5, 17.8, 22.3, 25.6, 31.7, 34.5, 38.4, 39.8, 41.6, 42.3, 47.0, 52.5, 53.2,
+
+
6
5
9.1, 78.1, 215.4; HRMS (ESI) exact mass calculated for C H O Si (M + H ) 567.4624, found
33 67 3 2
67.4631.
(20R)-8β-[(Triethylsilyl)oxy]-24-{1’-[(triethylsilyl)oxy]cyclopentyl}-des-A,B-25,26,27-
trinorcholestan-22-one (18f). The oxidation of the alcohols 17f (643 mg, 1.232 mmol) with Dess-
Martin periodinane (628 mg, 1.48 mmol) in anhydrous methylene chloride (40 mL), performed as
described above for 17a, afforded the crude product which was applied on a silica Sep-Pak cartridge
and eluted with hexane/ethyl acetate (93:7) to yield the ketone 18f (558 mg, 87%) as a colorless
oil.
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H NMR (400 MHz, CDCl ) δ 0.549 and 0.581 (6H and 6H, each q, J = 7.9 Hz, 6 × SiCH CH ),
3
2
3
0
.913 (3H, s, 18-H ), 0.943 and 0.946 (9H and 9H, each t, J = 7.9 Hz, 3 × SiCH CH ), 0.999 (3H,
3 2 3
1
3
d, J = 6.9 Hz, 21-H ), 2.52 (1H, m), 2.63 (2H, m), 4.03 (1H, narr m, 8α-H); C NMR (100 MHz,
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CDCl ) δ 4.9, 6.5, 6.8, 7.1, 14.2, 16.9, 17.5, 22.2, 23.4, 25.6, 34.9, 37.3, 39.7, 39.9, 41.6, 47.2, 52.4,
3
+
5
.3, 69.0, 83.8, 215.7; HRMS (ESI) exact mass calculated for C H O Si Na (M + Na) 573.4135,
32 62
3
2
found 573.4147.
(
(
(
20R)-22-Methylene-8β-[(triethylsilyl)oxy]-des-A,B-cholestane (19a). Potassium tert-butoxide
347 mg, 3.1 mmol) was added to a stirred suspension of methyl triphenylphosphonium bromide
1.1 g, 3.1 mmol) in anhydrous THF (16 mL) at 0 °C. The mixture was warmed up to room
temperature and stirred for 30 min. A solution of the ketone 18a (204 mg, 517 µmol) in THF (3
mL) was added via cannula at 0 °C under argon. Cooling bath was removed and stirring was
continued at room temperature for 28 h. Water was added and the mixture was extracted with ethyl
acetate, dried (Na SO ) and concentrated. The residue was applied on a silica Sep-Pak cartridge
2
4
and eluted with hexane/diethyl ether (98:2) to afford compound 19a (187 mg, 92%).
1
H NMR (400 MHz, CDCl ) δ 0.544 (6H, q, J = 7.9 Hz, 3 × SiCH ), 0.897 (3H, s, 18-H ), ca. 0.91
3
2
3
(3H, 21-H ), ca. 0.92 (6H, d, J = 6.3 Hz, 26- and 27-H ), 0.941 (9H, t, J = 7.9 Hz, 3 × SiCH CH ),
3 3 2 3
1
.94 (2H, br t, J = 6.8 Hz), 2.17 (1H, m), 4.02 (1H, narr m, 8α-H), 4.63 and 4.76 (1H and 1H, each
1
3
s, =CH ); C NMR (100 MHz, CDCl ) δ 4.9, 6.9, 13.1, 17.7, 20.6, 22.5, 22.7, 22.8, 27.0, 28.3,
2
3
2
9.3, 34.7, 37.2, 39.5, 42.3, 43.5, 53.1, 54.0, 69.4, 107.9, 155.3; HRMS (ESI) exact mass calculated
+
for C H NOSi (M + NH ) 410.3818, found 410.3820.
25 52
4
(20R)-22-Methylene-8β,25-bis[(triethylsilyl)oxy]-des-A,B-cholestane (19b). Solution of
the ketone 18b (451 mg, 860 µmol) in THF (5 mL) was treated with the ylide generated from
methyl triphenylphosphonium bromide (1.831 g, 5.13 mmol) and potassium tert-butoxide (580 mg,
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2
2
2
2
2
2
2
3
3
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3
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3
3
3
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5
5
5
6
o
5
.16 mmol) in anhydrous THF (25 mL) at 45 C for 20 h. Heating bath was removed, mixture was
allowed to reach room temperature and water was added. Materials were extracted with ethyl
acetate, dried (Na SO ) and concentrated. The residue was applied on a silica Sep-Pak cartridge
2
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and eluted with hexane/diethyl ether (95:5) to afford compound 19b (435 mg, 97%).
1
H NMR (500 MHz, CDCl ) δ 0.544 and 0.571 (6H and 6H, each q, each J = 8.0 Hz, 6 × SiCH CH ),
3
2
3
0
.904 (3H, s, 18-H ), ca. 0.93 (3H, 21-H ), 0.942 and 0.949 (9H and 9H, each t, each J = 8.0 Hz, 6
3 3
× SiCH CH ), 1.224 (6H, s, 26- and 27-H ), 2.04 (2H, m), 2.19 (1H, m), 4.02 (1H, narr m, 8α-H),
2
3
3
13
4
.60 and 4.76 (1H and 1H, each br s, =CH ); C NMR (125 MHz, CDCl ) δ 4.9, 6.8, 6.9, 7.1, 13.0,
2 3
1
7.6, 20.4, 22.5, 25.5, 27.0, 29.9, 30.1, 34.7, 39.3, 42.2, 42.8, 43.9, 53.1, 53.9, 69.4, 73.2, 107.6,
+
+
155.3; HRMS (ESI) exact mass calculated for C H O Si (M + H ) 523.4366, found 523.4355.
3
1
63
2
2
(
20R)-22-Methylene-8β,25-bis[(triethylsilyl)oxy]-des-A,B-24-homocholestane (19c). Solution
of the ketone 18c (313 mg, 582 µmol) in THF (4 mL) was treated with the ylide generated from
methyl triphenylphosphonium bromide (1.24 g, 3.49 mmol) and potassium tert-butoxide (392 mg,
3
.50 mmol) in anhydrous THF (20 mL) at room temperature for 48 h. Water was added and the
mixture was extracted with ethyl acetate, dried (Na SO ) and concentrated. The residue was applied
2
4
on a silica Sep-Pak cartridge and eluted with hexane/diethyl ether (99:1) to afford compound 19c
(303 mg, 97%).
1
H NMR (400 MHz, CDCl ) δ 0.545 and 0.583 (6H and 6H, each q, J = 7.7 Hz, 6 × SiCH CH ),
3
2
3
0
2
.904 - 0.963 (24H, 6 × SiCH CH , 18- and 21-H ), 1.202 (6H, s, 26- and 27-H ), 1.92 (2H, m),
2 3 3 3
1
3
.17 (1H, m), 4.02 (1H, narr m, 8α-H), 4.66 and 4.78 (1H and 1H, each s, =CH ); C NMR (100
2
MHz, CDCl ) δ 4.9, 6.8, 6.9, 7.1, 13.1, 17.6, 20.5, 22.5, 22. 6, 26.9, 29.8, 30.0, 32.1, 34.7, 39.4,
3
42.2, 43.3, 45.2, 53.1, 53.9, 69.4, 73.4, 108.1, 154.9; HRMS (ESI) exact mass calculated for
+
C H O Si Na (M + Na) 559.4343, found 559.4343.
3
2
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(
20R)-22-Methylene-8β,25-bis[(triethylsilyl)oxy]-des-A,B-26,27-dihomocholestane
(19d).
Solution of the ketone 18d (419 mg, 759 µmol) in THF (4 mL) was treated with the ylide generated
from methyl triphenylphosphonium bromide (1.742 g, 4.88 mmol) and potassium tert-butoxide
o
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
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(481 mg, 4.28 mmol) in anhydrous THF (20 mL) at 45 C for 23 h. Heating bath was removed,
mixture was allowed to reach room temperature and water was added. Materials were extracted
with ethyl acetate, dried (Na SO ) and concentrated. The residue was applied on a silica Sep-Pak
2
4
cartridge and eluted with hexane/diethyl ether (97:3) to afford compound 19d (314 mg, 75%).
1
H NMR (500 MHz, CDCl ) δ 0.545 and 0.579 (6H and 6H, each q, J = 8.0 Hz, 6 × SiCH CH ),
3
2
3
0
.837 and 0.851 [3H and 3H, each t, J = 7.5 Hz, 2 × C(CH CH )], 0.905 (3H, s, 18-H ), ca. 0.93
2 3 3
(3H, 21-H ), 0.942 and 0.950 (9H and 9H, each t, J = 8.0 Hz, 6 × SiCH CH ), 1.94 (2H, m), 2.17
3
2
3
13
(1H, m), 4.02 (1H, narr m, 8α-H), 4.62 and 4.77 (1H and 1H, each br s, =CH ); C NMR (125
2
MHz, CDCl ) δ 4.9, 6.9, 7.3, 8.3, 8.4, 13.0, 17.6, 20.5, 22.5, 25.1, 27.0, 31.6, 31.9, 34.7, 36.5, 39.6,
3
4
2.2, 43.8, 531, 53.9, 69.4, 78.1, 107.7, 155.3; HRMS (ESI) exact mass calculated for C H O Si
33 67 2 2
+
+
(M + H ) 551.4679, found 551.4655.
(20R)-22-Methylene-8β,25-bis[(triethylsilyl)oxy]-des-A,B-24,26,27-trihomocholestane (19e).
Solution of the ketone 18e (245 mg, 432 µmol) in THF (2 mL) was treated with the ylide generated
from methyl triphenylphosphonium bromide (1.02 g, 2.86 mmol) and potassium tert-butoxide (323
mg, 2.87 mmol) n anhydrous THF (15 mL) at room temperature for 10 days. Water was added and
the mixture was extracted with ethyl acetate, dried (Na SO ) and concentrated. The residue was
2
4
applied on a silica Sep-Pak cartridge and eluted with hexane/diethyl ether (98:2) to afford
compound 19e (195 mg, 80%).
1
H NMR (500 MHz, CDCl ) δ 0.544 and 0.569 (6H and 6H, each q, J = 7.8 Hz, 6 × SiCH CH ),
3
2
3
0
.823 [6H, t, J = 7.4 Hz, 2 × C(CH CH )], 0.904 (3H, s, 18-H ), 0.929 (3H, d, J = 6.1 Hz, 21-H ),
2 3 3 3
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.941 and 0.944 (9H and 9H, each t, J = 7.8 Hz, 6 × SiCH CH ), 1.92 (2H, m), 2.17 (1H, m), 4.02
2
3
1
3
(1H, narr m, 8α-H), 4.65 and 4.78 (1H and 1H, each s, =CH ); C NMR (125 MHz, CDCl ) δ 4.9,
2
3
6
5
5
.9, 7.1, 7.3, 8.3, 8.4, 13.1, 17.6, 20.4, 21.9, 22.5, 27.0, 31.5, 31.8, 32.1, 34.7, 39.1, 39.4, 42.2, 43.5,
+
+
0
1
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4
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8
9
0
1
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4
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6
7
8
9
0
1
2
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4
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7
8
9
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9
0
1
2
3
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7
8
9
0
3.1, 53.9, 69.6, 78.2, 108.1, 155.0; HRMS (ESI) exact mass calculated for C H O Si (M + H )
3
4
69
2
2
65.4836, found 565.4828.
(20R)-22-Methylene-8β-[(triethylsilyl)oxy]-24-{1’-[(triethylsilyl)oxy]cyclopentyl}-des-A,B-
2
5,26,27-trinorcholestane (19f). Solution of the ketone 18f (491 mg, 892 µmol) in THF (4 mL)
was treated with the ylide generated from methyl triphenylphosphonium bromide (869 mg, 2.43
mmol) and potassium tert-butoxide (347 mg, 3.09 mmol) in anhydrous THF (25 mL), as it was
described for 18e, providing after the analogous work-up compound 19f (332 mg, 68%).
1
H NMR (400 MHz, CDCl ) δ 0.546 and 0.584 (6H and 6H, each q, J = 7.9 Hz, 6 × SiCH CH ),
3
2
3
0.909 (3H, s, 18-H ), 0.93 (3H, d, J = 6.15 Hz, 21-H ), 0.943 and 0.949 (9H and 9H, each t, each J
3
3
=
7.9 Hz, 6 × SiCH CH ), 2.08 (2H, m), 2.19 (1H, m), 4.02 (1H, narr m, 8α-H), 4.62 and 4.76 (1H
2
3
1
3
and 1H, each s, =CH ); C NMR (100 MHz, CDCl ) δ 4.9, 6.9, 7.1, 7.3, 8.3, 8.4, 13.1, 17.6, 20.4,
2
3
21.9, 22.5, 27.0, 31.5, 31.8, 32.1, 34.7, 39.1, 39.4, 42.2, 43.5, 53.1, 53.9, 69.4, 78.2, 108.1, 155.0;
+
+
HRMS (ESI) exact mass calculated for C H O Si (M + H ) 549.4440, found 549.4449.
3
3
64
2
2
(20R)-22-Methylene-des-A,B-cholestan-8β-ol (20a). To a solution of protected alcohol 19a (61.3
mg, 157.1 µmol) in THF (9 mL) was added tetrabutylammonium fluoride (1.0 M in THF; 760 μL,
7
60 μmol) at room temperature under argon. The stirring was continued for 20 h, brine was added
and the mixture was extracted with ethyl acetate. The organic extracts were dried (Na SO ) and
2
4
evaporated. The residue was purified on a silica Sep-Pak cartridge using hexane/ethyl acetate (8:2)
to give alcohol 20a (42 mg, 97%) as colorless oil.
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Journal of Medicinal Chemistry
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H NMR (400 MHz, CDCl ) δ 0.909 (6H, d, J = 6.7 Hz, 26- and 27-H ), 0.925 (3H, s, 18-H ), 0.944
3
3
3
(3H, d, J = 6.9 Hz, 21-H ), 1.95 (2H, m), 2.18 (1H, m), 4.02 (1H, narr m, 8α-H), 4.64 (1H, d, J =
3
1
3
1
2
.2 Hz, one of =CH ), 4.78 (1H, s, one of =CH ); C NMR (100 MHz, CDCl ) δ 13.1, 17.4, 20.5,
2 2 3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
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8
9
0
1
2
3
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8
9
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2
3
4
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7
8
9
0
2.0, 22.6, 22.8, 26.8, 28.2, 29.1, 3.6, 37.1, 39.0, 41.9, 43.5, 52.6, 53.7, 69.3, 108.1, 155.0; HRMS
+
(ESI) exact mass calculated for C H NO (M + NH ) 296.2954, found 296.2959.
1
9
38
4
(20R)-22-Methylene-des-A,B-cholestane-8β,25-diol (20b’). Solution of protected diol 19b (71.5
mg, 129.4 µmol) in THF (1 mL) was treated with tetrabutylammonium fluoride (1.0 M in THF;
42 μL, 742 μmol) as described for 19a. The crude product was purified on a silica Sep-Pak
7
cartridge using hexane/ethyl acetate (9:1) to give the diol 20b’ (39 mg, 96%) as colorless oil.
1
H NMR (500 MHz, CDCl ) δ 0.928 (3H, s, 18-H ), 0.959 (3H, d, J = 6.9 Hz, 21-H ), 1.24 (6H, s,
3
3
3
2
6- and 27-H ), 2.04 (2H, m), 2.21 (1H, m), 4.07 (1H, narr m, 8α-H), 4.65 (1H, d, J = 1.2 Hz, one
3
1
3
of =CH ), 4.81 (1H, s, one of =CH ); C NMR (125 MHz, CDCl ) δ 13.0, 17.3, 20.3, 21.9, 25.4,
2
2
3
2
6.8, 29.2, 29.3, 33.5, 38.9, 41.8, 41.9, 43.7, 52.5, 53.5, 69.2, 70.9, 108.4, 154.3; HRMS (ESI)
+
+
exact mass calculated for C H O (M + H ) 295.2637, found 295.2636.
1
9
35
2
(20R)-22-Methylene-des-A,B-24-homocholestane-8β,25-diol (20c’). Solution of protected diol
9c (109 mg, 203.3 µmol) in THF (1 mL) was treated with tetrabutylammonium fluoride (1.0 M in
1
THF; 1.16 mL, 1.16 mmol) as described for 19a. The crude product was purified on a silica Sep-
Pak cartridge using hexane/ethyl acetate (8:2) to give the diol 20c’ (60 mg, 96%) as colorless oil.
1
H NMR (400 MHz, CDCl ) δ 0.926 (3H, s, 18-H ), 0.943 (3H, d, J = 6.9 Hz, 21-H ), 1.233 (6H,
3
3
3
s, 26- and 27-H ), 1.96 (2H, m), 2.18 (1H, m), 4.02 (1H, narr m, 8α-H), 4.68 and 4.82 (1H and 1H,
3
1
3
each s, =CH ); C NMR (100 MHz, CDCl ) δ 13.1, 17.2, 20.5, 22.9, 23.2, 26.8, 29.7, 29.8, 32.2,
2
3
33.9, 39.7, 42.6, 43.2, 45.2, 53.4, 54.1, 69.5, 71.3, 108.2, 154.8; HRMS (ESI) exact mass calculated
+
for C H O Na (M + Na) 331.2613, found 331.2613.
2
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(
20R)-22-Methylene-des-A,B-26,27-dihomocholestan-8β,25-diol (20d’). Solution of protected
diol 19d (73 mg, 132 µmol) in THF (1 mL) was treated with tetrabutylammonium fluoride (1.0 M
in THF; 756 μL, 756 μmol) as described for 19a providing the diol 20d’ (37 mg, 86%) as colorless
0
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7
8
9
0
oil.
1
H NMR (500 MHz, CDCl ) δ 0.873 and 0.878 [3H and 3H, each t, J = 7.5 Hz, 2 × C(CH CH )],
3
2
3
0
.928 (3H, s, 18-H ), 0.958 (3H, d, J = 6.9 Hz, 21-H ), 1.97 (2H, m), 2.21 (1H, m), 4.07 (1H, narr
3 3
1
3
m, 8α-H), 4.67 (1H, narr m, one of =CH ), 4.82 (1H, s, one of =CH ); C NMR (125 MHz, CDCl )
2
2
3
δ 7.7, 7.8, 13.0, 17.3, 20.4, 21.9, 24.6, 26.8, 30.9, 33.6, 36.3, 39.0, 41.9, 43.7, 52.5, 53.6, 69.3, 74.8,
+
+
1
08.4, 154.5; HRMS (ESI) exact mass calculated for C H O (M + H ) 323.2950, found
21 39 2
323.2949.
(20R)-22-Methylene-des-A,B-24,26,27-trihomocholestane-8β,25-diol (20e’). Solution of
protected diol 19e (165 mg, 292.5 µmol) in THF (1.5 mL) was treated with tetrabutylammonium
fluoride (1.0 M in THF; 1.68 mL, 1.68 mmol) as described for 19a providing the diol 20e’ (92 mg,
9
5%) as colorless oil.
1
H NMR (500 MHz, CDCl ) δ 0.865 [6H, t, J = 7.5 Hz, 2 × C(CH CH )], 0.923 (3H, s, 18-H ),
3
2
3
3
0
4
2
.942 (3H, d, J = 6.8 Hz, 21-H ), 1.94 (2H, m), 2.18 (1H, m), 4.07 (1H, narr m, 8α-H), 4.67 and
3
1
3
.81 (1H and 1H, each s, =CH ); C NMR (125 MHz, CDCl ) δ 7.7, 7.8, 13.0, 17.3, 20.4, 21.5,
2
3
1.9, 26.8, 30.97, 31.0, 31.8, 33.6, 38.3, 38.9, 41.9, 43.4, 52.5, 53.6, 69.3, 74.7, 108.4, 154.4;
+
HRMS (ESI) exact mass calculated for C H NO (M + NH ) 354.3367, found 354.3369.
2
2
44
2
4
(20R)-22-Methylene-24-(1’-hydroxycyclopentyl)-des-A,B-25,26,27-trinorcholestan-8β-ol
20f’). Solution of protected diol 19f (216 mg, 394.4 µmol) in THF (2 mL) was treated with
(
tetrabutylammonium fluoride (1.0 M in THF; 2.25 μL, 2.25 mmol) as described for 19a providing
the diol 20f’ (114 mg, 91%) as colorless oil.
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Journal of Medicinal Chemistry
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H NMR (400 MHz, CDCl ) δ 0.928 (3H, s, 18-H ), 0.959 (3H, d, J = 6.7 Hz, 21-H ), 2.04 (2H,
3
3
3
1
3
m), 2.11 (1H, m), 4.06 (1H, narr m, 8α-H), 4.65 and 4.81 (1H and 1H, each s, =CH ); C NMR
2
(100 MHz, CDCl ) δ 12.9, 17.3, 20.3, 21.9, 25.9, 26.7, 33.5, 39.6, 39.7, 39.7, 41.9, 43.7, 52.5, 53.5,
3
+
+
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
3
9.2, 82.4, 102.3, 108.2, 154.6; HRMS (ESI) exact mass calculated for C H O (M + H )
21 37 2
21.2789, found 321.2785.
(20R)-22-Methylene-des-A,B-cholestan-8-one (15a). The alcohol 20a (81 mg, 291 µmol) and
pyridinium dichromate (468 mg, 2.17 mmol) in anhydrous methylene chloride (5 mL) was stirred
vigorously at room temperature overnight. The reaction mixture was then filtered through a pad of
Celite (washed with methylene chloride) and the solvents were removed under reduced pressure.
The crude product was applied on a silica Sep-Pak cartridge and eluted with hexane/diethyl ether
(98:2) to yield the ketone 15a (70 mg, 87%) as a colorless oil.
1
H NMR (400 MHz, CDCl ) δ 0.615 (3H, s, 18-H ), 0.927 (6H, d, J = 6.6 Hz, 26- and 27-H ), 0.977
3
3
3
(3H, d, J = 6.9 Hz, 21-H ), 2.43 (1H, dd, J = 11.5, 7.7 Hz, 14α-H), 4.68 (1H, narr m, one of =CH ),
3
2
1
3
4
.80 (1H, d, J = 1.5 Hz, one of =CH ); C NMR (100 MHz, CDCl ) δ 12.0, 18.6, 20.4, 22.6, 22.8,
2 3
24.1, 27.1, 28.2, 29.0, 37.0, 37.6, 41.9, 49.9, 53.7, 61.9, 108.6, 154.4, 212.0; HRMS (ESI) exact
+
mass calculated for C H NO (M + NH ) 294.2797, found 294.2798.
1
9
36
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(20R)-25-Hydroxy-22-methylene-des-A,B-cholestan-8-one (21b’). The solution of diol 20b’ (38
mg, 129 µmol) in anhydrous methylene chloride (2.5 mL) was treated with pyridinium dichromate
(194 mg, 516 µmol) as described for 20a. The crude product was applied on a silica Sep-Pak
cartridge and eluted with hexane/diethyl ether (75:25) to yield the ketone 21b’ (35 mg, 90%) as a
colorless oil.
1
H NMR (400 MHz, CDCl ) δ 0.619 (3H, s, 18-H ), 0.995 (3H, d, J = 6.9 Hz, 21-H ), 1.273 (6H,
3
3
3
s, 26- and 27-H ), 2.43 (1H, dd, J = 11.5, 7.8 Hz, 14α-H), 4.70 (1H, narr m, one of =CH ), 4.84
3
2
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