Highly antiproliferative, low-calcemic, side-chain ketone analogs of the hormone 1α,25-dihydroxyvitamin D3

Article abstract of DOI:10.1016/j.bmc.2005.06.031

A series 2a-4b of seven new side-chain ketone analogs of calcitriol (1) have been prepared. Unexpectedly, several of these 24- and 25-tert-butyl ketones, even though lacking the classical side-chain tertiary hydroxyl group, are considerably more antiproli

Full text of DOI:10.1016/j.bmc.2005.06.031

Bioorganic & Medicinal Chemistry 13 (2005) 5569–5580
Highly antiproliferative, low-calcemic, side-chain ketone analogs
of the hormone 1a,25-dihydroxyvitamin D3
Gary H. Posner,a,* Hyung Jin Kim,a Mehmet Kahraman,a Heung Bae Jeon,a
Byung Chul Suh,a Hongbin Li,a Patrick Dolanb and Thomas W. Kenslerb
aDepartment of Chemistry, The Johns Hopkins University, Baltimore, MD 21218, USA
bDivision of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University,
Baltimore, MD 21205, USA
Received 21 March 2005; revised 17 June 2005; accepted 17 June 2005
Available online 20 July 2005
Abstract—A series 2a–4b of seven new side-chain ketone analogs of calcitriol (1) have been prepared. Unexpectedly, several of these
24- and 25-tert-butyl ketones, even though lacking the classical side-chain tertiary hydroxyl group, are considerably more antipro-
liferative in vitro than the hormone calcitriol (1) even at physiologically relevant low nanomolar concentrations and are less calcemic
than calcitriol (1) in vivo. In addition, ketone analog 19-nor-2a is not significantly less calcemic in vivo than 19-methylene analog 2a.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
hydroxylase enzyme.5,6 Hoffman-La Roche researchers
showed that incorporating a 16,17-double bond into
an analog generally promotes antiproliferative activity.7
Reddy and colleagues8 showed that such a 16-ene 24-ke-
tone 25-OH metabolite is even more antiproliferatively
potent than its 24-CH2 non-ketone parent, and we have
similarly observed the high antiproliferative activity of a
16-ene 25-ketone analog.9 However, as far as we know,
there are no reported examples of any 16,17-saturated
side-chain tert-butyl ketone analogs of calcitriol (1).
Finally, DeLuca and colleagues10 have reported recently
a biologically active abbreviated side-chain analog hav-
ing no side-chain heteroatom.
For chemotherapy of various human illnesses, medicinal
organic chemists are designing and preparing analogs
that are at least as potent as, but less calcemic than,
the natural hormone 1a,25-dihydroxyvitamin D3 (1, cal-
citriol).1–4 Currently, approximately eight such analogs
of calcitriol (1) are regularly used as drugs that promote
healthier living.4 A continuing chemical challenge, how-
ever, is to determine which small structural changes and
which simple functional group alterations in the large
natural hormone molecule will elicit desirable biological
responses. Understanding such chemical structure–
biological activity relationships (SAR) is important for
rational design of new analogs as potential drug candi-
dates.3,4 Toward this goal, we have developed a series
of calcitriol analogs that lack the natural 25-OH group
widely thought to be necessary for high biological activ-
ity.1–3 Specifically, we rationally designed and prepared
some side-chain sulfone5 and sulfoximine6 analogs such
that these functional groups might act as H-bond accep-
tors (rather than as H-bond donors like the natural
25-OH group) toward the vitamin D receptor (VDR);
several of these new analogs are potent, selective, and
low-calcemic inhibitors of the human CYP24
In this study, we describe a series of seven new 16,17-sat-
urated analogs (2a–4b) of calcitriol (1) in which two
minimal changes have been made in the side chain: (1)
the natural side-chain tertiary hydroxyl group is re-
placed by a methyl group (forming a terminal tert-butyl
group) and (2) a ketone carbonyl group is placed at
either C-24 or C-25.
2. Results
2.1. Chemistry
Synthesis of new analogs 2a, 19-nor-2a, and 3a is out-
lined in Scheme 1, starting from previously reported
side-chain iodides 511 and 6.12 As noted before in the
Keywords: Vitamin D; Calcitriol; Antiproliferative.
*
Corresponding authors. Tel.: +1 410 516 4670; fax: +1 410 516
8420; e-mail: ghp@jhu.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.031

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G. H. Posner et al. / Bioorg. Med. Chem. 13 (2005) 5569–5580
16-ene series,12 so now in the 16,17-saturated series,
Horner–Wadsworth–Emmons (HWE) coupling of the
A-ring nucleophile with 8,24- and 8,25-diketones 9 and
10 proceeds regiospecifically at only C-8, the less-hin-
dered ketone carbonyl group. Synthesis of new enone
analog 2b is summarized in Scheme 2, featuring
Scheme 1.

G. H. Posner et al. / Bioorg. Med. Chem. 13 (2005) 5569–5580
5571
Scheme 2.
successful HWE coupling of triene aldehyde 14 without
compromising the essential but sensitive conjugated tri-
ene unit. Synthesis of new analog 3b is outlined in
Scheme 3, and synthesis of analogs 4a and 4b is shown
in Scheme 4.
generalizations is that there is little observed difference
in antiproliferative activity between the pair of saturated
versus a,b-unsaturated ketones 2a versus 2b. 24,24-
Difluorinated analog 4a and 24,24-dimethylated analog
4b, however, are much less antiproliferative than calci-
triol (1) and than the corresponding 24-unsubstituted
analog 2a (data not shown); thus, both electron-with-
drawing groups (i.e., F) and a sterically hindered 25-car-
bonyl as in analog 4b diminish antiproliferative activity
considerably.
2.2. Biology
Our standard in vitro murine keratinocyte assay12 indi-
cates that the 25-oxo analogs 2a and 2b as well as the 24-
oxo analogs 3a and 3b are much more antiproliferative
than natural calcitriol (1) as is especially notable at
physiologically relevant low nanomolar concentrations
(Fig. 1). Noteworthy is that analog 3a, differing only
slightly from natural calcitriol (1) by being a 24-ketone
and by having a methyl group in place of the classical
25-OH group, is desirably much more antiproliferative
than calcitriol (1). Noteworthy also for SAR
Our standard in vivo mouse urine assay12 shows that at
least 10 times higher doses of the saturated ketone ana-
logs 2a and 3a are required to cause the same level of uri-
nary calcium excretion as the natural hormone calcitriol
(1, Fig. 2). When dosed at the same level as calcitriol (1),
ketone 2a was much less calcemic than calcitriol (1).
Introduction of a conjugated C@C double bond as in
Scheme 3.

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G. H. Posner et al. / Bioorg. Med. Chem. 13 (2005) 5569–5580
Scheme 4.
Figure 2. Effect of vitamin D3 analogs on calcium levels in rat urine.
Figure 1. Dose–response effects of analogs on keratinocyte
proliferation (96 h).
tionality produces analog 3a that is substantially more
antiproliferative in vitro than the natural hormone even
at physiologically relevant nanomolar concentrations;
and (2) simply removing the one carbon exocyclic 19-
methylene group produces analog 19-nor-2a that is not
significantly less calcemic than 19-methylene analog 2a.
These important SAR generalizations may help design
even more potent and safe new analogs of calcitriol (1).
enone 2b does not alter calcemic activity very much (Fig.
2). Surprisingly, based on DeLucaÕs observations that
19-nor analogs usually are much less calcemic than their
19-methylene versions,7,13 the 19-nor version of 25-ke-
tone 2a (i.e., 19-nor-2a) does not have significantly less
calcemic activity than its 19-methylene counterpart 2a.
4. Experimental
3. Conclusions
Unless otherwise noted, reactions were run in flame-
dried round-bottomed flasks under an atmosphere of
ultra-high-purity (UHP) argon. Diethyl ether (ether)
and tetrahydrofuran (THF) were distilled from sodium
Two unexpected observations arise from this work: (1)
simply replacing the natural 25-OH group in calcitriol
(1) by a –CH3 group and incorporating a 24-ketone func-

G. H. Posner et al. / Bioorg. Med. Chem. 13 (2005) 5569–5580
5573
benzophenone ketyl prior to use. Methylene chloride
(CH2Cl2) was distilled from calcium hydride prior to
use. All other compounds were purchased from Aldrich
Chemical Co. and used without further purification.
Analytical thin-layer chromatography (TLC) was con-
ducted with silica gel 60 F254 plates (250 lm thickness;
Merck). Column chromatography was performed using
short path silica gel (particle size <230 mesh), flash sil-
ica gel (particle size 400–230 mesh), or Florisil (200
mesh). Yields are not optimized. Purity of products
was judged to be >95% based on their chromatograph-
ic homogeneity. High-performance liquid chromatogra-
phy (HPLC) was carried out with a Rainin HPLX
system equipped with two 25 mL/min preparative
pump heads using Rainin Dynamax 10 · 250 mm
A 15-mL round-bottomed flask was charged with TES-
tert-butyl ketone 7 (0.06 g, 0.2 mmol) dissolved in 5 mL
of distilled THF. Tetrabutylammonium fluoride (TBAF
1 M solution in THF, 440 lL, 0.4 mmol) was added to
the reaction flask, and this solution was left to stir at
room temperature for 7 h. The resulting mixture
quenched with 2 mL of water, extracted with ethyl ace-
tate (3· 25 mL), dried over MgSO4, concentrated, and
purified using silica gel column chromatography (20%
ethyl acetate/hexanes) to give a colorless oil (34 mg,
25
1
80%): ½aꢁ +20.4 (c 2.0, CHCl3); H NMR (400 MHz,
D
CDCl3) d 4.05 (m, 1H), 2.51–2.35 (m, 2H), 1.98–1.94
(m, 1H), 1.89–1.68 (m, 4H), 1.56–1.29 (m, 11H), 1.12
(s, 9H), 0.91 (s, 3H), 0.86 (d, J = 6.4 Hz, 3H); 13C
NMR (100 MHz, CDCl3) d 216.5, 69.3, 56.5, 52.5,
44.2, 41.8, 40.3, 34.9, 33.5, 33.3, 29.9, 27.0, 26.4, 22.5,
18.4, 17.4, 13.5; IR (neat, cmꢀ1) 3516, 2935, 2870,
1703, 1477, 1365, 1067, 990; HRMS m/z [M+Na] calcd
317.2450 for C19H34O2Na+, found 317.2436.
˚
(semipreparative) columns packed with 60 A silica gel
(8 lm pore size), either as bare silica or as C-18-bonded
silica. Melting points were measured using a Mel-Temp
metal-block apparatus and were uncorrected. Nuclear
magnetic resonance (NMR) spectra were obtained
either on a Varian XL-400 spectrometer, operating at
A flame-dried 25 mL flask equipped with a magnetic stir
bar, and a septum along with argon (Ar) ballon was
charged with the alcohol tert-butyl ketone (0.035 g,
0.11 mmol) and dissolved in 2 mL freshly distilled
CH2Cl2. Then, to this solution, PDC (125 mg,
0.32 mmol) was added. The resulting mixture was
allowed to stir at room temperature for 8 h. The mixture
were filtered with Celite, concentrated, and purified
using silica gel chromatography (30% ethyl acetate/hex-
1
400 MHz for H and 100 MHz for 13C, or on a Varian
XL-500 spectrometer, operating at 500 MHz for 1H
and 125 MHz for 13C. Chemical shifts are reported in
parts per million (ppm, d) downfield from tetrameth-
ylsilane. Infrared (IR) spectra were obtained using a
Perkin-Elmer 1600 FT-IR spectrometer. Absorption
bands are reported in wave numbers (cmꢀ1). Low-
and high-resolution mass spectra (LRMS and HRMS)
were obtained with electronic or chemical ionization
(EI or CI) either (1) at Johns Hopkins University on
a VG Instruments 70-S spectrometer run at 70 eV for
EI and run with ammonia (NH3) as a carrier gas for
CI or (2) at the Ohio State University on a Finnigan-
MAT CH5, a Finnigan-MAT 731, or a VG Instru-
ments 70-VSE spectrometer run at 70 eV for EI and
run with methane (CH4) for CI.
anes) to give diketone 9 as a colorless oil (31 mg, 90%):
25
D
½aꢁ +11.5 (c 0.01, CHCl3); 1H NMR (400 MHz,
CDCl3) d 2.47–2.41 (m, 3H), 2.27–2.19 (m, 2H), 2.10
(d, J = 12.8 Hz, 1H), 2.02–1.86 (m, 4H), 1.76–1.16 (m,
6H), 1.18 (s, 9H), 0.92 (d, J = 6.0 Hz, 3H), 0.61 (s,
3H); 13C NMR (100 MHz, CDCl3) d 216.19, 211.94,
61.88, 56.59, 49.82, 44.18, 40.89, 38.89, 35.11, 33.20,
29.80, 27.37, 26.41, 23.97, 19.02, 18.51, 12.45; IR (neat,
cmꢀ1) 2958, 2872, 1708, 1474, 1364, 1223, 1068; HRMS
m/z [M+Na] calcd 315.2294 for C19H32O2Na+, found
315.2276.
4.1. tert-Butyl C,D-ring ketone 9
A 10-mL round-bottomed flask was charged with diiso-
propylamine (207 lL, 1.41 mmol distilled over calcium
hydride prior to use) and 2 mL of distilled THF. This
solution was cooled to ꢀ78 ꢁC, and n-BuLi (1.0 ml of
1.6 M solution in hexane, 1.37 mmol) was added via
syringe. Pinacolone (174 lL, 1.33 mmol dried over
potassium carbonate and activated molecular sieve
for 24 h immediately prior to use) was dissolved in
1 mL of distilled THF and cooled to ꢀ78 ꢁC at which
point it was added to the reaction flask via cannula.
The reaction was left to stir for 30 min. Hexamethyl-
phosphoramide (HMPA, 300 lL) was then added via
syringe, and the reaction mixture was allowed to stir
for an additional 15 min. A solution of iodide 5
(0.09 g, 0.19 mmol) in 2 mL of THF was cooled to
ꢀ78 ꢁC and added to the reaction mixture via cannula.
The reaction mixture was stirred at ꢀ78 ꢁC for 2 h and
to warm to room temperature slowly. The resulting yel-
low mixture quenched with 2 mL of water, extracted
with ethyl acetate (3· 25 mL), dried over MgSO4, con-
centrated, and purified using silica gel column chroma-
tography (10% ethyl acetate/hexanes) to give a colorless
oil 7 (0.06 g, 80%).
4.2. tert-Butyl C,D-ring ketone 10
A 10-mL round-bottomed flask was charged with diiso-
propylamine (322 lL, 2.29 mmol distilled over calcium
hydride prior to use) and 2 mL of distilled THF. This
solution was cooled to ꢀ78 ꢁC, and n-BuLi (1.39 mL
of 1.6 M solution in hexane, 2.23 mmol) was added via
syringe. Pinacolone (272 lL, 2.17 mmol dried over
potassium carbonate and activated molecular sieve for
24 h immediately prior to use) was dissolved in 1 mL
of distilled THF and cooled to ꢀ78 ꢁC at which point
it was added to the reaction flask via cannula. The reac-
tion was left to stir for 30 min. Hexamethylphosphora-
mide (HMPA, 300 lL) was then added via syringe,
and the reaction mixture was allowed to stir for an addi-
tional 15 min.
A solution of iodide 6 (140 mg,
0.31 mmol) in 2 mL of THF was cooled to ꢀ78 ꢁC and
added to the reaction mixture via cannula. The reaction
mixture was stirred at ꢀ78 ꢁC for 2 h and to warm to
room temperature slowly. The resulting yellow mixture
quenched with 2 mL of water, extracted with ethyl ace-
tate (3· 25 mL), dried over MgSO4, concentrated, and

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G. H. Posner et al. / Bioorg. Med. Chem. 13 (2005) 5569–5580
purified using silica gel column chromatography (4%
ethyl acetate/hexanes) to give 8 as a colorless oil
(130 mg, 97%).
in 1 mL freshly distilled THF, and cooled down to
ꢀ78 ꢁC in a dry ice bath. The solution of C,D-ring ke-
tone was gently transferred into the flask containing
the phosphine oxide anion at ꢀ78 ꢁC over several min-
utes. After the addition was complete, the deep red color
persisted and the mixture was allowed to stir at ꢀ78 ꢁC
for 8 h, during that time it was visually checked. On
observation of the light yellow color, the reaction was
quenched at ꢀ78 ꢁC by adding 3 mL of pH 7 buffer
and allowed to come to room temperature. The mixture
was then rinsed into a separatory funnel with ethyl ace-
tate and extracted with ethyl acetate (3· 25 mL). The
combined extracts were washed with water (1· 25 mL)
and brine solution, dried over MgSO4, filtered, and puri-
fied using silica gel chromatography (5% ethyl acetate/
hexanes) to give a colorless oil (40 mg, 55%).
A 15-mL round-bottomed flask was charged with TES-
tert-butyl ketone 8 (130 mg, 0.30 mmol) dissolved in
5 mL of distilled THF. Tetrabutylammonium fluoride
(TBAF, 1 M solution in THF, 369 lL, 0.36 mmol) was
added to the reaction flask, and this solution was left
to stir at room temperature for 7 h. The resulting mix-
ture quenched with 2 mL of water, extracted with ethyl
acetate (3· 25 mL), dried over MgSO4, concentrated,
and purified using silica gel column chromatography
(10% ethyl acetate/hexanes) to give a colorless oil
25
D
(80 mg, 84%): ½aꢁ +20.8 (c 0.65, CHCl3); 1H NMR
(400 MHz, CDCl3) d 4.04 (m, 1H), 2.42–2.39 (m, 2H),
1.97–1.94 (m, 1H), 1.84–1.76 (m, 3H), 1.59–1.15 (m,
8H), 1.10 (s, 9H), 0.90 (s, 3H), 0.89 (d, J = 7.6 Hz,
3H); 13C NMR (100 MHz, CDCl3) d 216.1, 66.3, 56.3,
52.5, 44.0, 41.8, 40.3, 36.8, 35.3, 35.1, 33.5, 27.1, 26.3,
22.4, 20.2, 18.4, 17.3, 13.5; IR (neat, cmꢀ1) 3358, 2932,
2872, 1701, 1365, 1152, 1023; HRMS m/z [M+Na] calcd
331.2607 for C20H36O2Na+, found 331.2590.
A 15-mL round-bottomed flask was charged with the
coupled compound (20 mg, 0.02 mmol) dissolved in
5 mL of distilled THF. Tetrabutylammonium fluoride
(TBAF, 1 M solution in THF, 148 lL, 0.1 mmol) was
added to the reaction flask, and this solution was left
to stir at room temperature for 7 h. The resulting mix-
ture quenched with 2 mL of water, extracted with ethyl
acetate (3· 25 mL), dried over MgSO4, concentrated,
and purified using silica gel column chromatography
(25% ethyl acetate/hexanes) to give 2a and 2a0
(11 mg, 83%) as a mixture of diastereomers. The
diastereomeric mixture was separated by an HPLC
using Chiral OD [semipreparative (1· 25 cm), flow
rate = 2.5 mL/min] eluted with 2.5% isopropyl alcohol
in hexanes to afford 4 mg of 2a (1a , 3b) and 1.3 mg
of 2a0 (1b , 3a) in 55 and 17% yield. The retention time
A flame-dried 25-mL flask equipped with a magnetic stir
bar, and a septum along with an Ar ballon was charged
with the alcohol tert-butyl ketone (80 mg, 0.25 mmol)
and dissolved in 2 mL freshly distilled CH2Cl2. Then,
to this solution, PDC (273 mg, 0.72 mmol) was added.
The resulting mixture was allowed to stir at room tem-
perature for 8 h. The mixture were filtered with Celite,
concentrated, and purified using silica gel chromatogra-
phy (10% ethyl acetate/hexanes) to give diketone 10 as a
25
D
colorless oil (79 mg, 99%): ½aꢁ +7.6 (c 0.75, CHCl3). 1H
for 2a is 103.15 min and 2a0 is 121.28 min. Data for 2a
25
D
NMR (400 MHz, CDCl3) d 2.44 (m, 3H), 2.29–2.16 (m,
2H), 2.13–2.08 (m, 1H), 2.03–1.80 (m, 2H), 1.77–1.25
(m, 12H), 1.12 (s, 9H), 0.97 (d, J = 6.4 Hz, 3H), 0.62
(s, 3H); 13C NMR (100 MHz, CDCl3) d 216.0, 212.1,
62.0, 56.3, 49.9, 44.1, 40.9, 38.9, 36.6, 35.4, 35.3, 27.4,
26.4, 24.0, 20.1, 19.0, 18.6, 12.4; IR (neat, cmꢀ1) 2958,
2873, 1711, 1478, 1367, 1073; HRMS m/z [M+Na] calcd
329.2451 for C20H34O2Na+, found 329.2469.
(1a, 3b): ½aꢁ
+20.3 (c 0.1, CHCl3); 1H NMR
(400 MHz, CDCl3) d 6.36 (d, J = 11.2 Hz, 1H), 5.99
(d, J = 11.2 Hz, 1H), 5.31 (s, 1H), 4.98 (s, 1H), 4.41
(m, 1H), 4.21 (m, 1H), 2.80 (dd, J = 12.0, 4.0 Hz,
1H), 2.58 (dd, J = 12.0, 2.8 Hz, 1H), 2.42 (m, 2H),
2.29 (dd, J = 13.6, 6.4 Hz, 1H), 2.03–1.82 (m, 5H),
1.66–1.62 (m, 2H), 1.53–1.51 (m, 3H), 1.48–1.23 (m,
10H), 1.11 (s, 9H), 0.92 (d, J = 6.4 Hz, 3H), 0.515 (s,
3H); 13C NMR (100 MHz, CDCl3) d 216.2, 147.6,
143.3, 132.8, 125.0, 116.9, 111.8, 70.8, 66.9, 56.3,
56.2, 45.9, 45.3, 44.1, 42.8, 40.4, 36.9, 36.0, 35.5,
29.1, 27.6, 26.4, 23.6, 22.2, 20.4, 18.8, 12.0; IR (neat,
cmꢀ1) 3374, 2949, 2360, 1705, 1464, 1366, 1054;
4.3. 25(O) Analogs (+)-2a and (+)-2b0
Racemic phosphine oxide ( )-11 and enantiomerically
pure C,D-ring ketone (+)-10, each was separately azeo-
tropically dried with anhydrous benzene (4· 1 mL) on a
rotary evaporator and held under vacuum (ca.
0.1 mmHg) for at least 48 h prior to use. A flame-dried
10-mL recovery flask equipped with a magnetic stir
bar, a septum along with an Ar balloon was charged
with racemic phosphine oxide ( )-11 (70 mg, 0.12 mmol)
which was dissolved in 2 mL freshly distilled THF. The
flask was cooled down to ꢀ78 ꢁC in a dry ice bath. To
this solution, n-BuLi (82 lL, 0.12 mmol, 1.6 M solution
in hexanes) was added dropwise over several minutes,
during which time a deep red color was developed and
persisted. This mixture was allowed to stir at ꢀ78 ꢁC
for an additional 10 min. Meanwhile, a flame-dried 10-
mL recovery flask equipped with a magnetic stir bar,
and a septum along with an Ar balloon was charged
with C,D-ring ketone 10 (33 mg, 0.1 mmol), dissolved
HRMS m/z [M+Na] calcd 465.3339 for C29H46O3Na+,
25
found 465.3310. Data for 2a0 (1b, 3a): ½aꢁ +3.9 (c 0.1,
D
CHCl3); 1H NMR (400 MHz, CDCl3) d 6.39 (d,
J = 11.2 Hz, 1H), 6.05 (d, J = 11.2 Hz, 1H), 5.32 (s,
1H), 5.00 (s, 1H), 4.44 (m, 1H), 4.21 (m, 1H), 2.82
(dd, J = 12.0, 3.6 Hz, 1H), 2.61 (dd, J = 13.2, 4.0 Hz,
1H), 2.44 (m, 3H), 2.30 (dd, J = 13.2, 7.6 Hz, 1H),
2.04–1.82 (m, 5H), 1.68–1.63 (m, 2H), 1.55–1.49 (m,
3H), 1.47–1.25 (m, 10H), 1.31 (s, 9H), 0.94 (d,
J = 6.4 Hz, 3H), 0.54 (s, 3H); 13C NMR (100 MHz,
CDCl3) d 216.2, 247.2, 143.3, 132.6, 125.0, 117.0,
112.6, 94.4, 71.4, 66.8, 56.3, 56.2, 45.9, 45.5, 42.8,
40.4, 36.9, 36.0, 35.5, 29.0, 27.6, 26.4, 23.6, 22.3,
20.4, 18.8, 12.0; IR (neat, cmꢀ1) 3368, 2951, 1705,
1465, 1054; HRMS m/z [M+Na] calcd 465.3339 for
C29H46O3Na+, found 465.3332.

G. H. Posner et al. / Bioorg. Med. Chem. 13 (2005) 5569–5580
5575
4.4. 19-nor-25(O) Analog (+)-2a
azeotropically dried with anhydrous benzene (4·
1 mL) on a rotary evaporator and held under vacuum
(ca. 0.1 mmHg) for at least 48 h prior to use. A flame-
dried 10-mL recovery flask equipped with a magnetic
stir bar, a septum along with an Ar balloon was
charged with enantiomerically pure A-ring phosphine
oxide (ꢀ)-11 (50 mg, 0.08 mmol) which was dissolved
in 2 mL freshly distilled THF. The flask was cooled
down to ꢀ78 ꢁC in a dry ice bath. To this solution,
n-BuLi (48 lL, 0.08 mmol, 1.6 M solution in hexanes)
was added dropwise over several minutes, during which
time a deep red color was developed and persisted.
This mixture was allowed to stir at ꢀ78 ꢁC for an addi-
tional 10 min. Meanwhile, a flame-dried 10-mL recov-
ery flask equipped with a magnetic stir bar, and a
septum along with an Ar balloon was charged with
enantiomerically pure C,D-ring ketone (+)-9 (22 mg,
0.075 mmol), dissolved in 1 mL freshly distilled THF,
and cooled down to ꢀ78 ꢁC in an dry ice bath. The
solution of C,D-ring ketone was gently transferred into
the flask containing the phosphine oxide anion at
ꢀ78 ꢁC via cannula over several minutes. After the
addition was complete, the deep red color persisted
and the mixture was allowed to stir at ꢀ78 ꢁC for
9 h, during that time it was visually checked. On obser-
vation of the light yellow color, the reaction was
quenched at ꢀ78 ꢁC by adding 3 mL of pH 7 buffer
and allowed to come to room temperature. The mix-
ture was then rinsed into a separatory funnel with ethyl
acetate and extracted with ethyl acetate (3· 25 mL).
The combined extracts were washed with water (1·
25 mL) and brine solution, dried over MgSO4, filtered,
and purified using silica gel chromatography (5% ethyl
acetate/hexanes) to give a colorless oil coupled product
(13 mg, 35%).
19-nor Phosphine oxide (+)-11 and enantiomerically
pure C,D-ring ketone (+)-10, each was separately azeo-
tropically dried with anhydrous benzene (4· 1 mL) on
a rotary evaporator and held under vacuum (ca.
0.1 mmHg) for at least 48 h prior to use. A flame-dried
10-mL recovery flask equipped with a magnetic stir
bar, a septum along with an Ar balloon was charged with
phosphine oxide (+)-11 (60 mg, 0.12 mmol) which was
dissolved in 2 mL freshly distilled THF. The flask was
cooled down to ꢀ78 ꢁC in a dry ice bath. To this solution
was added n-BuLi (81 lL, 0.12 mmol, 1.42 M solution in
hexanes) dropwise over several minutes, during which
time a deep red color was developed and persisted. This
mixture was allowed to stir at ꢀ78 ꢁC for an additional
10 min. Meanwhile, a flame-dried 10-mL recovery flask
equipped with a magnetic stir bar, a septum along with
an Ar balloon was charged with C,D-ring ketone (+)-
10 (27 mg, 0.08 mmol), dissolved in 1 mL freshly distilled
THF, and cooled down to ꢀ78 ꢁC in a dry ice bath. The
solution of C,D-ring ketone was gently transferred into
the flask containing the phosphine oxide anion at
ꢀ78 ꢁC via cannula over several minutes. After the addi-
tion was complete, the deep red color persisted and the
mixture was allowed to stir at ꢀ78 ꢁC for 8 h, during
which time it was visually checked. On observation of
the light yellow color, the reaction was quenched at
ꢀ78 ꢁC by adding 3 mL of pH 7 buffer and allowed to
come to room temperature. The mixture was then rinsed
into a separatory funnel with ethyl acetate and extracted
with ethyl acetate (3· 25 mL). The combined extracts
were washed with water (1· 25 mL) and brine solution,
dried over MgSO4, filtered, and purified using silica gel
chromatography (2% ethyl acetate/hexanes) to give a
colorless oil (8 mg, 15%).
A 15-mL round-bottomed flask was charged with the
coupled product (13 mg, 0.02 mmol) dissolved in
5 mL of distilled THF. Tetrabutylammonium fluoride
(TBAF 1 M solution in THF, 59 lL, 0.06 mmol) was
added to the reaction flask, and this solution was left
to stir at room temperature for 7 h. The resulting mix-
ture quenched with 2 mL of water, extracted with ethyl
acetate (3· 25 mL), dried over MgSO4, concentrated,
and purified using silica gel column chromatography
(25% ethyl acetate/hexanes) to give 24(O)TB 3 (7 mg,
82%) as a colorless oil. The product was separated
by an HPLC using Chiral OD [semipreparative (1·
A 15-mL round-bottomed flask was charged with the
coupled compound (8 mg, 0.02 mmol) dissolved in
5 mL of distilled THF. Tetrabutylammonium fluoride
(TBAF, 1 M solution in THF, 148 lL, 0.1 mmol) was
added to the reaction flask, and this solution was left
to stir at room temperature for 7 h. The resulting mixture
quenched with 2 mL of water, extracted with ethyl ace-
tate (3· 25 mL), dried over MgSO4, concentrated, and
purified using silica gel column chromatography (25%
ethyl acetate/hexanes) to give 19- nor-2a (4 mg, 85%):
25
D
½aꢁ +15.2 (c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3)
d 6.31 (d, J = 11.6 Hz, 1H), 5.85 (d, J = 11.2 Hz, 1H),
4.41 (m, 1H), 4.05 (m, 1H), 2.81–2.71 (m, 2H), 2.50–
2.18 (m, 5H), 2.11–1.22 (m, 18H), 1.13 (s, 9H), 0.94 (d,
J = 6.8 Hz, 3H), 0.53 (s, 3H); 13C NMR (100 MHz,
CDCl3) d 216.2, 143.2, 131.0, 123.9, 115.2, 67.4, 67.2,
56.3, 56.2, 45.8, 44.7, 42.2, 40.4, 37.2, 36.9, 36.0, 35.5,
29.7, 28.9, 27.6, 26.4, 23.5, 22.2, 20.4, 18.8, 12.0; IR
(neat, cmꢀ1) 3500, 2926, 1706, 1364, 1049; HRMS m/z
[M+Na] calcd 453.3339 for C28H46O3Na+, found
453.3351; UV (CH2Cl2) kmax = 255 nm (e = 29,530).
25 cm), tR = 38.5 min] eluted with 7% IPA in hexanes
25
to afford 4 mg of 24(O)TB 3: ½aꢁ +29.6 (c 3.0,
D
CHCl3); 1H NMR (400 MHz, CDCl3) d 6.36 (d,
J = 11.2 Hz, 1H), 5.99 (d, J = 11.2 Hz, 1H), 5.32 (s,
1H), 4.99 (s, 1H), 4.42 (m, 1H), 4.22 (m, 1H), 2.81
(dd, J = 12.0, 4.0 Hz, 1H), 2.59 (dd, J = 12.0, 2.8 Hz,
1H), 2.52–2.28 (m, 5H), 2.20–1.20 (m, 16H), 1.13 (s,
9H), 0.91 (d, J = 6.4 Hz, 3H), 0.53 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 216.5, 147.5, 143.1, 132.9, 124.9,
117.0, 111.8, 70.8, 66.8, 56.4, 56.2, 45.8, 45.2, 44.2,
42.8, 40.4, 35.7, 33.2, 30.2, 29.0, 27.5, 26.4, 23.5,
22.2, 18.6, 14.6, 12.0; IR (neat, cmꢀ1) 3356, 2949,
2872, 1703, 1465, 1364, 1054, 754; HRMS m/z
[M+Na] calcd 451.3182 for C28H44O3Na+, found
451.3178.
4.5. 24(O)TB analog (+)-3a
Enantiomerically pure A-ring phosphine oxide (ꢀ)-11
and C,D-ring ketone (+)-9, each was separately

5576
G. H. Posner et al. / Bioorg. Med. Chem. 13 (2005) 5569–5580
4.6. Cyano-C,D-ring ketone 13
dissolved in 1 mL anhydrous dichloromethane to give
ca. 0.05 M solution. Then the flask was cooled down
to ꢀ78 ꢁC. To this well-stirred solution, DIBAL
(0.077 mmol, 51.6 mL, 1.5 M solution in toluene) was
added via syringe at this temperature and the mixture
was then allowed to stir at ꢀ78 ꢁC for 1 h. This reaction
mixture was diluted with ether (25 mL), and 1 N solu-
tion of HCl (ca. 1 mL) was added and stirred for a
few minutes. The reaction mixture was then rinsed into
a separatory funnel with ethyl acetate and was extracted
with ethyl acetate (4· 10 mL). The combined extracts
were washed with water (10 mL) and brine solution
(10 mL), dried over Na2SO4, and filtered. The filtrate
was concentrated in vacuo to give the crude product that
was purified by flash column chromatography eluted
with 5% ethyl acetate in hexanes in the presence of 1%
triethylamine affording 25 mg of 23-aldehyde 14 in
83% yield.
A flame-dried 10-mL recovery flask equipped with a
magnetic stir bar, and a septum along with an Ar bal-
loon was charged with C-8 alcohol (50 mg, 0.22 mmol)
dissolved in 2.2 mL freshly distilled CH2Cl2. To this
solution, PDC (170 mg, 0.45 mmol) and 0.22 g of
oven-dried Celite were added in one portion at room
temperature. The resulting mixture was allowed to stir
at room temperature for about 12 h. The mixture was
directly purified by column chromatography eluted with
first 20% ethyl acetate in hexanes to afford 43 mg of
1
ketone 13 in 83% yield: H NMR (CDCl3, 400 MHz) d
2.52 (dd, J = 7.6 Hz, 1H), 2.41–2.21 (m, 4H), 2.12–2.01
(m, 2H), 1.99–1.74 (m, 4H), 1.69–1.54 (m, 2H), 1.37–
1.25 (m, 1H), 1.2 (d, J = 6.8 Hz, 3H), 0.67 (s, 3H); 13C
NMR (CDCl3, 100 MHz) d 211.1, 118.4, 61.5, 54.9,
49.5, 40.7, 38.4, 33.1, 27.3, 24.6, 23.7, 19.2, 18.9, 12.5;
HRMS m/z [M+Na] calcd 242.1515 for C14H21NO,
found 242.1519.
A flame-dried 10-mL recovery flask equipped with a
magnetic stir bar, and a septum along with an Ar bal-
loon was charged with KOtBu (3.5 mg, 0.031 mmol)
and 0.5 mL freshly distilled THF. Then, the flask was
cooled down to ꢀ78 ꢁC in a dry ice bath. To this solu-
tion, dimethylphosphonate 15 (6.4 mg, 0.031 mmol)
was added as a solution in 0.5 mL freshly distilled
THF. After 30 min, 0.5 mL of THF solution of 23-alde-
hyde 14 (15 mg, 0.026 mmol) was transferred into the
flask via cannula over several minutes at ꢀ78 ꢁC. After
the addition was complete, the mixture was gradually
warmed up to room temperature and then stirred for
about 4 h. The reaction was quenched by adding 2 mL
distilled water and then rinsed into a separatory funnel
with ethyl acetate. The mixture was extracted with ethyl
acetate (3· 10 mL). The combined extracts were washed
with water (10 mL), and brine solution (10 mL), dried
over Na2SO4, and filtered. The filtrate was concentrated
in vacuo to give the crude product that was purified by
flash column chromatography eluted with 5% ethyl
acetate in hexanes to afford 12 mg of trans-olefin as
4.7. 23-Enone analogs (+)-2b and 2b0
Phosphine oxide ( )-11 and C,D-ring ketone 29, each was
separately azeotropically dried with anhydrous benzene
(4· 1 mL) on a rotary evaporator and held under vacuum
(ca. 0.1 mmHg) for at least 48 h prior to use. A flame-
dried 10-mL recovery flask equipped with a magnetic stir
bar, a septum along with an Ar balloon was charged with
phosphine oxide ( )-11 (60 mg, 0.1 mmol) and dissolved
in ca 1.2 mL freshly distilled THF to give ca. 0.1 M solu-
tion, and the flask was cooled down to ꢀ78 ꢁC in a dry ice
bath. To this solution, n-BuLi (70 lL, 0.11 mmol, 1.6 M
solution in hexanes) was added dropwise over several
minutes, during which time a deep red color was devel-
oped and persisted. This mixture was allowed to stir at
ꢀ78 ꢁC for an additional 10 min. Meanwhile, a flame-
dried 10-mL recovery flask equipped with a magnetic stir
bar, and a septum along with an Ar balloon was charged
with C,D-ring ketone 13 (15 mg, 0.068 mmol), dissolved
in 0.5 mL freshly distilled THF, and cooled down to
ꢀ78 ꢁC in a dry ice bath. The solution of C,D-ring ketone
13 was transferred dropwise into the flask containing the
phosphine oxide anion at ꢀ78 ꢁC via cannula over several
minutes. After the addition was complete, the deep red
color persisted and the mixture was allowed to stir at
ꢀ78 ꢁC for 2 h, during that time it was visually checked.
On observation of the light yellow color, the reaction
was quenched at ꢀ78 ꢁC by adding of 3 mL of pH 7 buffer
and allowed to come to room temperature. The mixture
was then rinsed into a separatory funnel with ethyl acetate
and extracted with ethyl acetate (4· 25 mL). The com-
bined extracts were washed with water (25 mL) and brine
solution (25 mL), dried over Na2SO4, and filtered. The fil-
trate was concentrated in vacuo to give the crude product
that was purified by column chromatography eluted with
25% ethyl acetate in hexanes in the presence of 1% trieth-
ylamine to afford 34 mg of the coupled product in 85%
yield in a 3.8:1 ratio as determined by 1H NMR.
1
determined by H NMR in 70% yield.
This product (12 mg, 0.018 mmol) was charged into a
5 mL argon purged polypropylene vial equipped with
a magnetic stir bar, a septum along with a cap and
dissolved in 1.0 mL acetonitrile to give ca. 0.02 M
solution. To this well-stirred solution, HF (1.8 mmol,
49% aqueous solution) was added via syringe at room
temperature and the mixture was then allowed to stir
at room temperature for 2 h. TLC showed the comple-
tion of the reaction. This reaction mixture was diluted
with ether (25 mL) and saturated solution of NaHCO3
was added until no more carbon dioxide was liber-
ated. The reaction mixture was then rinsed into a
separatory funnel with ethyl acetate and was extracted
with ethyl acetate (4· 10 mL). The combined extracts
were washed with water (10 mL) and brine solution
(10 mL), dried over Na2SO4, and filtered. The filtrate
was concentrated in vacuo to give the crude product
as a mixture of diastereomers in 4:1 ratio (as deter-
mined by 1H NMR of the crude reaction mixture).
The crude was purified by column chromatography
eluted with 100% ethyl acetate in the presence of 1%
This coupled product (30 mg, 0.051 mmol) was charged
into 5-mL round-bottomed flask equipped with a mag-
netic stir bar, and a septum along an Ar balloon and

G. H. Posner et al. / Bioorg. Med. Chem. 13 (2005) 5569–5580
5577
TEA to afford 5.2 mg of a diastereomeric mixture of
2b and 2b0 in 66% yield. This analog 2b was purified
by an HPLC [semipreparative (1· 25 cm) Chiracel
OD, tR = 48.2 min] eluted with 5% isopropyl alcohol
temperature. The mixture solution was stirred over-
night and then passed through a 2 cm pad of flash sil-
ica gel and washed with EtOAc. The filtrate was
concentrated and subjected to column chromatogra-
phy with EtOAc/hexanes (1:4) as eluent to give
1
in hexanes to afford 2.2 mg of 2b: H NMR (CDCl3,
400 MHz) d 6.95–6.88 (m, 1H), 6.49 (d, J = 15.2 Hz,
1H), 6.37 (d, J = 11.2 Hz, 1H), 6.01 (d, J = 11.6 Hz,
1H), 5.33–5.32 (m, 1H), 5.00–4.99 (m, 1H), 4.45–4.01
(m, 1H), 4.25–4.22 (m, 1H), 2.84 (dd, J = 12.0,
4.4 Hz, 1H), 2.60 (dd, J = 13.2, 5.0 Hz, 1H), 2.34–
2.29 (m, 2H), 2.07–1.89 (m, 6H), 1.68–1.49 (m,
17H), 1.35–1.25 (m, 4H), 0.95 (d, J = 6.4 Hz, 3H),
0.55 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 204.3,
147.6, 146.2, 142.9, 132.9, 125.7, 124.9, 117.1, 111.8,
70.8, 66.8, 56.2, 56.0, 45.9, 45.2, 42.8, 42.7, 40.3,
39.3, 36.0, 29.0, 27.6, 26.2, 23.5, 22.2, 19.1, 12.0; IR
(neat, cmꢀ1) 3369, 2949, 2872, 1684, 1619, 1437,
1366, 1055, 908; HRMS m/z [M+Na] calcd 463.3183
for C30H45NO3Na+, found 463.3193; UV (MeOH)
11 mg (90%) of the desired C,D-ring ketone 18 as a
25
D
colorless oil: ½aꢁ +30.8 (c 0.5, CHCl3); 1H NMR
(400 MHz, CDCl3) d 6.74 (dd, J = 15.2, 9.2 Hz, 1H),
6.42 (dd, J = 15.2, 8.0 Hz, 1H), 2.46 (dd, J = 11.2,
7.6 Hz, 1H), 2.18–2.32 (m, 3H), 2.00–2.12 (m, 2H),
1.91 (m, 1H), 1.46–1.78 (m, 5H), 1.27 (m, 1H), 1.14
(s, 9H), 1.12 (d, J = 6.4 Hz, 3H), 0.67 (s, 3H); 13C
NMR (100 MHz, CDCl3)
d 211.5, 204.6, 151.5,
122.3, 61.7, 55.3, 49.9, 42.9, 40.9, 39.7, 38.8, 27.5,
26.2, 24.0, 19.5, 19.1, 12.7; IR (neat, cmꢀ1) 2961,
2872, 1713, 1688, 1622, 1477, 1366, 1233, 1076, 989,
950, 860; HRMS m/z [M+Na] calcd 313.2138 for
C19H30O2Na+, found 313.2132.
k
max = 265 nm (e = 15,864).
4.10. 22-Ene-24(O)TB (+)-3b
4.8. 22-Ene-24 ketone silyl ether 17
A solution of 50 mg (0.086 mmol) of enantiomerically
pure phosphine oxide (ꢀ)-11 in 1.5 mL of anhydrous
THF was cooled to ꢀ78 ꢁC and treated with 54 lL
(0.086 mmol, 1.6 M in hexanes) of n-BuLi under argon
atmosphere. The mixture turned deep reddish color
and was stirred for 15 min at ꢀ78 ꢁC. To the solution,
a precooled (ꢀ78 ꢁC) solution of 10 mg (0.034 mmol)
of the enantiomerically pure C,D-ring ketone (+)-18 in
1.5 mL of anhydrous THF via cannula was added drop-
wise. The reaction kept going until the reddish-orange
color faded to yellow (about 3 h). The reaction was
quenched by adding 1.0 mL of pH 7 buffer at ꢀ78 ꢁC,
and then warmed to room temperature, extracted with
EtOAc (3· 20 mL), washed with brine, dried over
MgSO4, and concentrated. The residue was subjected
to column chromatography with EtOAc/hexanes (1:10)
as eluent to afford 4 mg (18%) of the coupled product
as a colorless oil.
To a solution of the phosphate 15 (80 mg, 0.38 mmol) in
THF (5 mL), potassium tert-butoxide (43 mg, 0.38 mg)
was added at 0 ꢁC. After stirring for 1 h at 0 ꢁC, a solu-
tion of the aldehyde (+)-16 (60 mg, 0.18 mmol) in THF
(2 mL) was added via cannula at rt. Then, the mixture
was stirred for 4 days at rt. The resulting mixture was
quenched with water (3 mL), extracted with EtOAc
(3· 25 mL), dried over MgSO4, and concentrated. The
residue was subjected to column chromatography with
EtOAc/hexanes (1:15) as eluent to afford 50 mg (68%)
25
D
of the desired ketone 17 as a colorless oil: ½aꢁ +73.4
(c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) d 6.75
(dd, J = 15.2, 9.2 Hz, 1H), 6.39 (dd, J = 15.2, 0.8 Hz,
1H), 4.03 (m, 1H), 2.24 (m, 1H), 1.93 (dt, J = 12.4,
2.8 Hz, 1H), 1.82 (m, 1H), 1.50–1.69 (m, 4H), 1.13–
1.41 (m, 6H), 1.14 (s, 9H), 1.05 (d, J = 6.4 Hz, 3H),
0.94 (s, 3H), 0.935 (t, J = 8.0 Hz, 9H), 0.54 (q,
J = 8.0 Hz, 6H); 13C NMR (100 MHz, CDCl3) d 204.8,
152.9, 121.8, 69.3, 55.5, 52.9, 42.8, 42.4, 40.6, 39.7,
34.6, 27.4, 26.2, 23.0, 19.2, 17.7, 13.8, 6.9, 4.9; IR (neat,
cmꢀ1) 2953, 2875, 1725, 1690, 1623, 1457, 1366, 1234,
1166, 1081, 1018, 725; HRMS m/z [M+Na] calcd
429.3159 for C25H46O2SiNa+, found 429.3161.
The coupled product (4 mg, 0.0060 mmol) was dissolved
in 2 mL of anhydrous EtOH, and to the solution, 50 lL
of 49% aq HF was added. The resulting mixture was
stirred 2 h at room temperature, and then quenched with
5 mL of satd NaHCO3 solution. The solution was stir-
red for 10 min, and then extracted with EtOAc (3·
20 ml), washed with brine, dried over MgSO4, and con-
centrated. The residue was subjected to column chroma-
4.9. 22-Ene-24 ketone C,D-ring ketone 18
tography with EtOAc as eluent to give 2 mg (93%) of the
25
D
To a solution of silyl ether 17 (46 mg, 0.11 mmol) in
THF (3 mL), 0.35 mL (0.35 mmol) of a 1.0 M solution
of TBAF in THF was added at rt, and then it was
stirred overnight at rt. The reaction mixture was
quenched with water (2 mL), extracted with EtOAc
(3·, 20), washed with brine, dried over MgSO4, and
concentrated. The residue was subjected to column
chromatography with EtOAc/hexanes (1:3) as eluent
to give 12 mg (37%) of the desired alcohol as a color-
less oil.
desired product 3b as a colorless oil: ½aꢁ +65.0 (c 0.10,
CHCl3); 1H NMR (400 MHz, CDCl3) d 6.78 (dd,
J = 15.2, 9.2 Hz, 1H), 6.41 (d, J = 15.2 Hz, 1H), 6.37
(d, J = 11.2 Hz, 1H), 6.01 (d, J = 11.2 Hz, 1H), 5.32
(m, 1H), 4.99 (m, 1H), 4.43 (m, 1H), 4.23 (m, 1H),
2.83 (dd, J = 11.6, 3.2 Hz, 1H), 2.60 (dd, J = 13.2,
3.2 Hz, 1H), 2.20–2.34 (m, 2H), 1.89–2.04 (m, 4H),
1.43–1.76 (m, 6H), 1.14–1.41 (m, 5H), 1.15 (s, 9H),
1.09 (d, J = 6.8 Hz, 3H), 0.58 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 189.1, 152.4, 147.6, 142.6, 133.1,
124.9, 122.1, 117.2, 111.8, 70.8, 66.8, 56.1, 55.3, 46.1,
45.3, 42.8, 40.3, 40.2, 29.7, 29.0, 27.4, 26.2, 23.5, 22.3,
19.6, 12.3; IR (neat, cmꢀ1) 3401, 2955, 2926, 2872,
1684, 1653, 1617, 1558, 1546, 1507, 1457, 1079.
To a solution of the alcohol (12 mg, 0.041 mmol) in
CH2Cl2 (4 mL), 120 mg of oven-dried Celite and
PDC (120 mg, 0.33 mmol) was added at room

5578
G. H. Posner et al. / Bioorg. Med. Chem. 13 (2005) 5569–5580
4.11. Difluoro ethyl ester 19
To a solution of ketone (30 mg, 0.065 mmol) in THF
(5.0 mL), 0.20 mL (0.20 mmol) of a 1.0 M solution of
TBAF in THF was added, and then it was stirred at
0 ꢁC for 1 h and stirred overnight at room temperature.
The reaction mixture was quenched with water (5 mL),
extracted with EtOAc (3· 20 mL), washed with brine,
dried over MgSO4, concentrated in vacuo, and then
purified by column chromatography (25% EtOAc/hex-
anes) to give 21.0 mg (93%) of alcohol as a colorless oil.
A
suspension of activated zinc dust (98.5 mg,
1.51 mmol) and ethyl bromodifluoroacetate (0.19 mL,
1.51 mmol) in THF (5 mL) was refluxed for 20 min
and then cooled to 0 ꢁC. To this, the solution of the
aldehyde (+)-16 (100 mg, 0.30 mmol) in THF (5 mL)
was added. The reaction mixture was warmed to room
temperature, followed by refluxing for 20 min, and then
cooled to room temperature. The reaction mixture was
poured into 1 M KHSO4 and extracted with EtOAc
(3· 20 mL), washed with brine, dried over MgSO4, con-
centrated in vacuo, and then purified by column chro-
matography (10% EtOAc/hexanes) to give 84.5 mg
(62%) of a 1:1 mixture of diastereomers of the desired
alcohol as a colorless oil.
To a solution of the C,D-ring alcohol (14.1 mg,
0.041 mmol) in CH2Cl2 (5 mL) were added 50 mg of
oven-dried Celite and PDC (46.2 mg, 0.12 mmol) at
room temperature. The reaction mixture was stirred
overnight and then passed through a 2 cm pad of flash
silica gel and washed with EtOAc. The filtrate was con-
centrated and purified by column chromatography (20%
To a solution of ethyl ester (84 mg, 0.18 mmol) and pyr-
idine (0.066 mL, 0.82 mmol) in CH2Cl2 (5 mL), phenyl
chlorothionoformate (0.053 mL, 0.38 mmol) was added.
After being stirred at room temperature for 20 h, the
reaction mixture was diluted with water, extracted with
ether (3· 20 mL), washed with satd NaHCO3 solution
and brine, dried over MgSO4, concentrated in vacuo,
and then purified by column chromatography (5%
EtOAc/hexanes) to give 97.8 mg (90%) of the desired
carbonate as diastereomeric mixtures.
EtOAc/hexanes) to give 13.0 mg (93%) of the desired
25
D
C,D-ring diketone 21 as a colorless oil: ½aꢁ +1.30
1
(c 1.01, CHCl3); H NMR (400 MHz, CDCl3) d 2.45
(dd, J = 11.6, 7.6 Hz, 1H), 2.30–2.21 (m, 2H), 2.11–
2.07 (m, 2H), 2.05–1.97 (m, 1H), 1.96–1.84 (m, 3H),
1.76–1.71 (m, 1H), 1.61–1.42 (m, 5H), 1.33–1.21
(m, 2H), 1.25 (s, 9H), 0.97 (d, J = 6.0 Hz, 3H), 0.64
(s, 3H); 13C NMR (100 MHz, CDCl3) d 211.83, 205.23
(J = 15.5 Hz), 120.74 (J = 253.1 Hz), 61.87, 55.99,
49.81, 43.34, 40.90, 38.89, 34.91, 30.54 (J = 22.7 Hz),
27.30, 27.04 (J = 4.1 Hz), 26.07 (J = 2.3 Hz), 23.99,
19.01, 18.41, 12.47; IR (neat, cmꢀ1) 2954, 2860, 1719,
1460, 1366, 1307, 1231, 1196, 1043, 1008, 967, 943,
914, 843, 773, 737; HRMS m/z [M+Na] calcd 365.2262
for C20H32F2O2Na+, found 365.2252.
To the solution of the resulting phenylthianocarbonate
(97.5 mg, 0.16 mmol) in anhydrous benzene (10 mL),
2,20-azobisisobutyronitrile (AIBN, 5 mg) and tributyltin
hydride (0.066 mL, 0.24 mmol) were added at room
temperature. After being refluxed for 3 h, the mixture
was cooled to 0 ꢁC, diluted with water, extracted with
EtOAc (3· 20 mL), washed with brine, dried over
MgSO4, concentrated in vacuo, and then purified by col-
umn chromatography (3% EtOAc/hexanes) to give
58.2 mg (80%) of the desired difluoro ester 19 as a color-
4.13. 24-F2-25(O)TB analog (ꢀ)-4a
To a solution of 50.0 mg (0.086 mmol) of enantiomeri-
cally pure A-ring phosphine oxide (ꢀ)-11 in 2.0 mL of
anhydrous THF, 53.6 lL (0.086 mmol, 1.6 M in hex-
anes) of n-BuLi was added at ꢀ78 ꢁC, and then the red-
dish solution was stirred for 15 min at ꢀ78 ꢁC. To the
solution, a precooled (ꢀ78 ꢁC) solution of enantiomeri-
cally pure C,D-ring ketone (+)-21 (8.0 mg, 0.023 mmol)
in 1.0 mL of anhydrous THF was added dropwise. The
reaction kept going until the reddish-orange color faded
to yellow (about 6 h). The reaction was quenched by
adding 1.0 mL of pH 7 buffer, and then warmed up to
room temperature, extracted with EtOAc (2· 20 mL),
washed with brine, dried over MgSO4, concentrated in
vacuo, and then purified by column chromatography
(20% EtOAc/hexanes) to afford 13.3 mg (81%) of the
coupled product as a pale yellow oil.
less oil: 1H NMR (400 MHz, CDCl3)
d
4.32
(q, J = 7.2 Hz, 2H), 4.03 (s, 1H), 2.14–2.06 (m, 1H),
1.98–1.89 (m, 2H), 1.85–1.75 (m, 2H), 1.69–1.64 (m,
1H), 1.61–1.53 (m, 2H), 1.49–1.43 (m, 1H), 1.38–1.34
(m, 2H), 1.35 (t, J = 7.2 Hz, 3H), 1.26–1.17 (m, 3H),
1.13–0.99 (m, 2H), 0.94 (t, J = 8.0 Hz, 9H), 0.91 (d,
J = 6.0 Hz, 3H), 0.90 (s, 3H), 0.55 (q, J = 8.0 Hz, 6H).
4.12. Difluoro-C,D-ring diketone 21
To a solution of ethyl ester 19 (54 mg, 0.12 mmol) in
THF (5 mL) was added 125.1 lL of tert-BuLi (1.43 M
in pentane, 0.18 mmol) at ꢀ78 ꢁC. After being stirred
for 3 h, the reaction mixture was warmed up to room
temperature, diluted with water, extracted with ether
(3· 20 mL), washed with satd NaHCO3 solution and
brine, dried over MgSO4, concentrated in vacuo, and
then purified by column chromatography (hexanes only)
to give 41.6 mg (75%) of the desired ketone as a colorless
The coupled product (13.2 mg, 0.019 mmol) was dis-
solved in 5 mL of anhydrous THF, and to this solution,
0.075 mL (0.075 mmol) of a 1.0 M solution of TBAF in
THF was added. The reaction was run in darkness over-
night, and then extracted with EtOAc (2· 30 mL),
washed with brine, dried over MgSO4, concentrated in
vacuo, and then purified by column chromatography
1
oil: H NMR (400 MHz, CDCl3) d 4.02 (d, J = 2.4 Hz,
1H), 1.95–1.90 (m, 1H), 1.87–1.74 (m, 2H), 1.69–1.64
(m, 1H), 1.57–1.52 (m, 2H), 1.49–1.43 (m, 2H), 1.39–
1.31 (m, 3H), 1.26 (s, 9H), 1.24–1.20 (m, 2H), 1.12–
0.99 (m, 2H), 0.94 (t, J = 8.0 Hz, 9H), 0.89 (d,
J = 6.0 Hz, 3H), 0.89 (s, 3H), 0.55 (q, J = 8.0 Hz, 6H).
(5% MeOH/CH2Cl2) to give 8.48 mg (95%) of the de-
25
D
sired compound 4a as a colorless oil: ½aꢁ ꢀ23.3
1
(c 0.42, CHCl3); H NMR (400 MHz, CDCl3) d 6.37
(d, J = 11.6 Hz, 1H), 6.01 (d, J = 11.2 Hz, 1H), 5.32

G. H. Posner et al. / Bioorg. Med. Chem. 13 (2005) 5569–5580
5579
(t, J = 1.6 Hz, 1H), 5.00 (t, J = 1.6 Hz, 1H), 4.44–4.41
(m, 1H), 4.24–4.21 (m, 1H), 2.82 (dd, J = 12.8, 4.4 Hz,
1H), 2.60 (dd, J = 13.6, 3.6 Hz, 1H), 3.34–2.28 (m,
1H), 2.07–1.82 (m, 6H), 1.70–1.58 (m, 5H), 1.55–1.42
(m, 5H), 1.31–1.27 (m, 2H), 1.26 (s, 9H) 0.94 (d,
J = 6.4 Hz, 3H), 0.54 (s, 3H); 13C NMR (100 MHz,
CDCl3) d 205.33 (J = 29.1 Hz), 147.58, 142.95, 132.96,
124.93, 120.86 (J = 253.6 Hz), 117.09, 111.81, 70.82,
66.84, 56.26, 55.86, 45.85, 45.24, 43.34, 42.82, 40.39,
35.50, 30.64 (J = 22.7 Hz), 29.03, 27.39, 27.13, 26.08,
23.52, 22.19, 18.51, 11.98; IR (neat, cmꢀ1) 3365, 2942,
2860, 1725, 1642, 1454, 1366, 1225, 1049, 961, 908,
400 MHz) d 4.02–4.01 (m, 1H), 1.93 (dm, J = 12.8 Hz,
1H), 1.83–1.72 (m, 2H), 1.67–1.42 (m, 5H), 1.38–0.99
(m, 7H), 1.37–1.14 (m, 2H), 1.23 (s, 3H), 1.22 (s, 9H),
1.19 (s, 3H), 0.94 (t, J = 8.0 Hz, 9H), 0.88 (s, 3H), 0.87
(d, J = 6.4 Hz, 3H), 0.54 (q, J = 8.0 Hz, 6H); 13C
NMR (CDCl3, 100 MHz) d 218.85, 69.39, 56.49, 53.06,
49.48, 45.66, 42.08, 40.74, 38.08, 35.90, 34.63, 30.99,
28.45, 27.31, 26.63, 26.52, 23.01, 18.55, 17.67, 13.45,
6.93, 4.93; IR (neat, cmꢀ1) 2952, 2876, 1684, 1476,
1367, 1166, 1085, 1019, 725; HRMS m/z [M+Na] calcd
473.3785 for C28H54O2SiNa+, found 473.3873.
796, 749, 737, 596; UV (MeOH)
(e = 3166).
k
max = 252 nm
To
a
solution of tert-butyl ketone (40 mg,
0.0887 mmol) in THF (3 mL), a solution of tetrabutyl-
ammonium fluoride (TBAF, 1.0 M in THF, 0.89 mL,
0.89 mmol) was added. After being stirred for 4 h at
room temperature, the reaction mixture was concen-
trated. Purification of the residue by flash column chro-
matography (20% EtOAc/hexanes) provided 29.6 mg
(0.088 mmol, 99% yield) of the corresponding alcohol.
A solution of this alcohol (29.6 mg) in CH2Cl2
(3 mL) was added to pyridinium dichromate (PDC,
66.7 mg, 0.177 mmol, 2 equiv) and Celite (70 mg) at
room temperature under argon atmosphere. After
being stirred overnight, the reaction mixture was dilut-
ed with EtOAc and filtered through a silica gel plug.
The filtrate was concentrated in vacuo and then puri-
fied by column chromatography (20% EtOAc/petro-
4.14. Ester 20
Lithium diisopropylamide (LDA) solution was prepared
by treating diisopropylamine (213.5 mg, 2.11 mmol) in
THF (5 mL) at ꢀ78 ꢁC with a 1.6 M solution of n-BuLi
in hexanes (1.3 mL, 2.08 mml). The LDA solution was
stirred at ꢀ78 ꢁC for 30 min, and then a solution of ethyl
isobutyrate (245 mg, 2.11 mmol) in THF (2 mL) was
added. After being stirred for 1 h at ꢀ78 ꢁC, a solution
of iodide (+)-6 (95 mg, 0.211 mmol) in THF (2 mL) was
added. The reaction mixture was stirred at ꢀ78 ꢁC for
2 h, then warmed up to room temperature and quenched
with the addition of water (5 mL). The reaction mixture
was extracted with diethyl ether (4· 15 mL). The organic
layer was washed with water and brine, dried over sodi-
um sulfate, concentrated in vacuo, and then purified by
column chromatography (5% EtOAc/petroleum ether)
to give 91.8 mg of ester 20 (0.209 mmol, 99% yield) as
leum ether) to give 27.6 mg of the diketone 22
(0.0825 mmol, 94% yield) as colorless oil: ½aꢁ +8.7
24
D
1
(c 1.33, CHCl3); H NMR (CDCl3, 400 MHz) d 2.42
(dd, J = 11.4, 7.4 Hz, 1H), 2.29–2.16 (m, 2H), 2.09
(dm, J = 13.2 Hz, 1H), 2.03–1.81 (m, 3H), 1.76–1.39
(m, 8H), 1.37–1.14 (m, 2H), 1.23 (s, 3H), 1.21
(s, 9H), 1.20 (s, 3H), 0.94 (d, J = 6.4 Hz, 3H), 0.61
(s, 3H); 13C NMR (CDCl3, 100 MHz) d 218.68,
212.04, 61.93, 56.27, 49.84, 49.34, 45.70, 40.93, 38.91,
38.12, 36.01, 30.96, 28.36, 27.46, 26.59, 26.55, 24.02,
19.05, 18.66, 12.41; IR (neat, cmꢀ1) 2957, 2873, 1715,
1682, 1477, 1385, 1366, 1220, 1044, 982; HRMS m/z
[M+Na] calcd 357.2764 for C22H38O2Na+, found
357.2750.
24
1
a colorless oil: ½aꢁ +41.8 (c 1.50, CHCl3); H NMR
D
(CDCl3, 400 MHz) d 4.104 and 4.102 (q, J = 7.2 Hz,
2H), 4.03–4.02 (m, 1H), 1.93 (dm, J = 12.4 Hz, 1H),
1.83–1.72 (m, 2H), 1.67–1.65 (m, 1H), 1.61–1.50 (m,
5H), 1.43–1.27 (m, 6H), 1.24 (t, J = 7.2 Hz, 3H), 1.19–
0.97 (m, 2H), 1.14 (s, 3H), 1.137 (s, 3H), 0.94 (t,
J = 8.0 Hz, 9H), 0.88 (s, 3H), 0.87 (d, J = 6.4 Hz, 3H),
0.55 (q, J = 8.0 Hz, 6H); 13C NMR (CDCl3, 100 MHz)
d 178.12, 69.38, 60.10, 56.28, 53.07, 42.15, 42.07,
40.74, 36.97, 35.40, 34.65, 30.61, 27.11, 25.25, 25.11,
22.99, 18.63, 17.67, 14.28, 13.46, 6.93, 4.93; IR (neat,
cmꢀ1) 2950, 2875, 1731, 1472, 1144, 1025, 742; HRMS
m/z [M+Na] calcd 461.3421 for C26H50O3SiNa+, found
461.3398.
4.16. 24-(CH3)2-25(O)TB analog (+)-4b
Phosphine oxide (ꢀ)-11 and enantiomerically pure C,D-
ring diketone 22 were separately azeotropically dried
four times with benzene and held under vacuum for
60 h immediately prior to use. To a solution of phos-
phine oxide (ꢀ)-11 (62.0 mg, 0.106 mmol) in THF
(2 mL), a 1.6 M solution of n-BuLi in hexanes (66 lL,
0.106 mmol) was added dropwise at ꢀ78 ꢁC under argon
atmosphere. The resulting deep reddish-orange solution
was allowed to stir for 20 min, at which time a precooled
(ꢀ78 ꢁC) solution of enantiomerically pure C,D-ring
diketone (+)-22 (15.8 mg, 0.0472 mmol) in THF
(2 mL) was transferred dropwise via cannula. The deep
reddish-orange solution was stirred in the dark for 5 h,
during which time the color was faded. On observation
of a light yellow color, the reaction mixture was
quenched at ꢀ78 ꢁC with 3 mL of buffer water (pH 7).
The mixture was allowed to come up to rt, extracted
with EtOAc (4· 10 mL), dried over Na2SO4, filtered,
4.15. Diketone 22
To a solution of ester 20 (90 mg, 0.205 mmol) in diethyl
ether (4 mL), a solution of tert-butyllithium (1.43 M in
pentane, 0.15 mL, 0.215 mmol) was added dropwise at
ꢀ78 ꢁC. After being stirred for 1 h at ꢀ78 ꢁC, the reac-
tion mixture was warmed up to room temperature,
and then quenched with the addition of water (4 mL).
The reaction mixture was extracted with diethyl ether
(4· 15 mL). The organic layer was washed with water
and brine, dried over sodium sulfate, concentrated in
vacuo, and then purified by column chromatography
(5% EtOAc/petroleum ether) to give 83.1 mg of tert-
butyl ketone (0.184 mmol, 90% yield) as a colorless oil:
24
D
½aꢁ +41.1 (c 0.71, CHCl3); 1H NMR (CDCl3,

5580
G. H. Posner et al. / Bioorg. Med. Chem. 13 (2005) 5569–5580
concentrated, and purified by silica gel column chroma-
tography (5% EtOAc/petroleum ether) to give 17 mg of
the coupled product (0.0243 mmol, 52% yield).
References and notes
1. Holick,M.F.VitaminD:MolecularBiology,Physiology,and
Clinical Applications; Humana Press: Totowa, NJ, 1999.
2. Feldman, D.; Glorieux, F.; Pike, J. W. Vitamin D;
Academic Press: San Diego, CA, 1997.
3. Bouillon, R.; Okamura, W. H.; Norma, A. W. Endocr.
Rev. 1995, 16, 200–257.
This coupled product was dissolved in THF (2 mL) and
treated with a solution of tetrabutylammonium fluoride
(TBAF, 1.0 M in THF, 0.24 mL, 0.24 mmol). After
being stirred overnight at room temperature, the reac-
tion mixture was concentrated. Purification of the resi-
due by flash column chromatography (100% EtOAc)
4. Posner, G. H.; Kahraman, M. Eur. J. Org. Chem. 2003,
20, 3889–3895.
5. Posner, G. H.; Crawford, K. R.; Yang, H. W.; Kahraman,
M.; Jeon, H. B.; Li, H.; Lee, J. K.; Suh, B. C.; Hatcher, M.
A.; Mirza, T.; Usera, A.; Dolan, P.; Kensler, T. W.; Peleg,
S.; Jones, G.; Zhang, A.; Korczak, B.; Saha, U.; Chuang,
S. S. J. Steroid Biochem. Mol. Biol. 2004, 89–90, 5–12.
6. Kahraman, M.; Sinishtaj, S.; Dolan, P. M.; Kensler, T. W.;
Peleg, S.; Saha, U.; Chuang, S. S.; Bernstein, G.; Korczak,
B.; Posner, G. H. J. Med. Chem. 2004, 47, 6854–6863.
7. Uskokovic, M. R.; Studzinski, G. P.; Gardner, J. P.;
Reddy, S. G.; Campbell, M. J.; Koeffler, H. P. Curr.
Pharm. Des. 1997, 3, 99–123.
8. Siu-Caldera, M. L.; Clark, J. W.; Santos-Moore, A.; Peleg,
S.; Liu, Y. Y.; Uskokovic, M. R.; Sharma, S.; Reddy, G.
S. J. Steroid Biochem. Mol. Biol. 1996, 59, 405–412.
9. Posner, G. H.; Halford, B. A.; Peleg, S.; Dolan, P.;
Kensler, T. W. J. Med. Chem. 2002, 45, 1723–1730.
10. Plum, L. A.; Pradl, J. M.; Ma, X.; Sicinski, R. R.;
Gowlugari, S.; Clagett-Dame, M.; DeLuca, H. F. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 6900–6904.
provided 9.9 mg of 24-(CH3)2-25(O)TB 4b (0.021 mmol,
24
86% yield) as a white solid: mp (ꢁC) 109–112; ½aꢁ +40.7
(c 0.48, CHCl3); 1H NMR (CDCl3, 400 MHz) dD6.37 (d,
J = 11.2 Hz, 1H), 6.01 (d, J = 11.2 Hz, 1H), 6.07 (d,
J = 16.0 Hz, 1H), 5.32 (m, 1H), 5.00 (m, 1H), 4.43 (m,
1H), 4.24–4.22 (m, 1H), 2.81 (dd, J = 12.0, 4.0 Hz,
1H), 2.59 (dd, J = 13.2, 3.2 Hz, 1H), 2.31 (dd, J = 13.4,
6.6 Hz, 1H), 2.05–1.81 (m, 5H), 1.72–1.43 (m, 10H),
1.32–1.19 (m, 4H), 1.23 (s, 3H), 1.22 (s, 9H), 1.21 (s,
3H), 0.91 (d, J = 6.0 Hz, 3H), 0.52 (s, 3H); 13C NMR
(CDCl3, 100 MHz) d 218.85, 147.60, 143.18, 132.81,
124.99, 116.99, 111.77, 70.82, 66.85, 56.30, 56.17,
49.45, 45.86, 45.25, 42.83, 40.41, 38.10, 36.64, 31.06,
29.06, 28.43, 27.58, 26.66, 26.51, 23.57, 22.25, 20.70,
18.78, 11.95; IR (neat, cmꢀ1) 3350, 2949, 2872, 1682,
1475, 1054, 755; HRMS m/z [M+Na] calcd 493.3652
for C31H50O3Na+, found 493.3675; UV (MeOH)
11. Dai, H.; Posner, G. H. Synthesis 1994, 1383–1398.
12. Posner, G. H.; Lee, J. K.; Wang, Q.; Peleg, S.; Burke, M.;
Brem, H.; Dolan, P.; Kensler, T. W. J. Med. Chem. 1998,
41, 3008–3014.
k
max = 265 nm (e = 14,822).
Acknowledgment
We thank the NIH for financial support (CA 93547).
13. For a high-calcemic 19-nor analog, see: Sicinski, R. R.;
Rutkiewicz, P.; Kolinski, A.; Sicinska, W.; Prahl, J. M.;
Smith, C. M.; Luca, H. F. J. Med. Chem. 2002, 45, 3366.