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

Article abstract of DOI:10.1016/j.bmc.2006.05.050

Four new side-chain amide (2 and 3) and hydroxamate (4 and 5) analogs of the hormone calcitriol (1) have been prepared. Even though lacking the 25-OH group characteristic of natural calcitriol (1), analogs 2-4 are as antiproliferative in vitro as calcitriol (1) but are 20-40 times less calciuric in vivo than calcitriol (1).

Full text of DOI:10.1016/j.bmc.2006.05.050

Bioorganic & Medicinal Chemistry 14 (2006) 6341–6348
Highly antiproliferative, low-calcemic, side-chain amide and
hydroxamate analogs of the hormone 1a,25-dihydroxyvitamin D3
Sandra Sinishtaj,a Heung Bae Jeon,a Patrick Dolan,b
Thomas W. Kenslerb and Gary H. Posnera,*
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 19 April 2006; revised 22 May 2006; accepted 23 May 2006
Available online 12 June 2006
Abstract—Four new side-chain amide (2 and 3) and hydroxamate (4 and 5) analogs of the hormone calcitriol (1) have been
prepared. Even though lacking the 25-OH group characteristic of natural calcitriol (1), analogs 2–4 are as antiproliferative
in vitro as calcitriol (1) but are 20–40 times less calciuric in vivo than calcitriol (1).
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
biological evaluation of a new series 2–5 of 16,17-saturat-
edside-chainamideandhydroxamateanalogsofcalcitriol
(1), in which the natural 25-OH is absent.
The natural hormone 1a,25-dihydroxyvitamin D3
(1, calcitriol) regulates diverse biochemical functions,
such as immunomodulation, the control of hormonal
systems, inhibition of cell growth, and induction of cell
differentiation.3–5 Maintaining good health requires
low levels of calcitriol, but using this hormone chemo-
therapeutically at supraphysiological levels is not feasi-
ble because of life-threatening hypercalcemia.1,2
Therefore, medicinal organic chemists have developed
a series of structurally modified versions of calcitriol
that are at least as antiproliferative as calcitriol but
substantially less calciuric than calcitriol.4
2. Results
2.1. Chemistry
Syntheses of analogs 2–5 are outlined in Schemes 1
and 2. In Scheme 1, selective N-alkylation allowed con-
version of primary iodide 610 into side-chain amides 7
and 8, and into side-chain hydroxamate 9. Horner–
Wadsworth–Emmons (HWE) coupling of the A-ring
phosphine oxide 1311 occurred as expected exclusively
at the C-8 ketone carbonyl group. In Scheme 2, metal-
promoted Michael addition of iodide 1412 to methyl
acrylate produced 25-carbonyl adduct 15. HWE cou-
pling of NH-hydroxamate 17 with A-ring phosphine
oxide 13 proceeded in modest yield.
The vitamin D3 side chain appears to be conformation-
ally flexible with over 1083 low-energy conformers plau-
sible.6 Knowledge about side-chain flexibility gives the
designer a point of attack, and recently we reported a
series of highly growth-inhibitory, low-calcemic, side-
chain ketone analogs of calcitriol.7 These ketone
analogs, as well as some sulfone8 and sulfoximine9 ana-
logs, represent a group of analogs that lack the traditional
terminal hydroxyl group and still maintain biological
activity. Here, we report the synthesis and preliminary
2.2. Biology
Our standard in vitro murine keratinocyte proliferation
assay13,14 shows that amide analogs 2 and 3 and
hydroxamate analog 4, even though lacking the natural
25-OH group, are as cell growth-inhibitory as calcitriol
(Fig. 1). Noteworthy are analogs 2–4 which show better
activity than calcitriol, specifically at low-nanomolar
concentrations. However, reversing the hydroxamate
Keywords: Vitamin D; Calcitriol; Antiproliferative.
*
Corresponding author. Tel.: +1 410 516 7429; fax: +1 410 516
8420; e-mail: ghp@jhu.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.050

6342
S. Sinishtaj et al. / Bioorg. Med. Chem. 14 (2006) 6341–6348
R
N
H
N
25
25
25
H
H
H
24
OMe
17
16
OH
O
O
H
H
H
2, R=H
3, R=Me
4, R=OMe
5
1
HO
OH
HO
OH
HO
OH
1(1α,25-dihydroxyvitamin D3, calcitriol)
moiety, as in analog 5, is not favorable for biological
activity; analog 5 is less antiproliferative than calcitriol
(Fig. 1).
Our standard in vivo rat urine assay13,14 shows 20–40
times higher doses (10–20 lg/kg) of the amide and
hydroxamate analogs 2–4 are required to cause the same
R
HN
R
N
R
N
I
H
H
H
NaH,
O
1. TBAF
O
O
2. PDC
8
THF
H
OTES
H
OTES
H
O
10, R= H, 91%
11, R= CH3, 94%
12, R= OMe, 75%
7, R= H, 95%
8, R= CH3, 30%
9, R= OMe, 90%
6
R
N
H
P(O)Ph2
OTBS
O
H
1. n-BuLi, then 10,11 or 12
2. TBAF, THF
TBSO
(-)-13
HO
OH
(+)-2, HJK24N(H)25(O)TB, 50%
(+)-3, HJK24N(Me)25(O)TB, 29%
(+)-4, SS24N(OMe)25(O)TB, 40%
Scheme 1.
OMe
H
I
H
H
H
OMe
N
MeONH.HCl
O
OMe
O
O
AlMe3, toluene
Zn, pyridine
.
H
H
55%
H
NiCl2 6H2O
OTES
OTES
OTES
88%
14
15
16
1. TBAF
2. PDC
70%
H
N
H
OMe
H
O
P(O)Ph2
OTBS
H
N
H
1. n-BuLi
2.TBAF
19%
OMe
O
8
H
O
TBSO
17
(-)-13
HO
OH
(-)-5, SS-25(O)26-N(OMe)H
Scheme 2.

S. Sinishtaj et al. / Bioorg. Med. Chem. 14 (2006) 6341–6348
6343
4. Experimental
All air and moisture sensitive reactions were carried out
in flame-dried glassware under an inert atmosphere of
argon. All reactive liquid reagents were transferred by
syringe or cannula and were added into the flask
through a rubber septum. Tetrahydrofuran (THF) was
freshly distilled from sodium benzophenone ketyl imme-
diately prior to use. All other solvents and reagents were
used as received unless otherwise stated. n-BuLi was
obtained from commercial sources and was titrated with
N-pivaloyl-O-toluidine prior to use.
The 1H spectra were obtained on a Varian XL 400 spec-
trometer at 400 MHz. The 13C spectra were obtained on
a Varian XL 100 spectrometer at 100 MHz. All NMR
spectra were obtained as a solution in CDCl3 with
TMS as the internal standard unless otherwise stated.
Chemical shifts (d) are reported in parts per million
(ppm) downfield from TMS (0 ppm). Multiplicities of
Figure 1.
1
signals in the H NMR spectra are reported as follows:
p<0.001
*
s (singlet), d (doublet), t (triplet), q (quartet), m (multi-
plet), dd (doublet of doublets), dt (doublet of triplet),
etc.
160
140
160
140
*
Infrared spectra were obtained on a Perkin-Elmer 1600
FT-IR spectrometer as liquid films and thin layer with
NaCl cells.
120
30
120
30
Optical rotations were recorded on JASCO, P-1100
model polarimeter (Japan Spectroscopic Co., Ltd) with
sodium D line at the temperatures as indicated for the
specific compounds.
20
10
0
20
10
0
Analytical thin-layer chromatography (TLC) was
performed on Merck silica gel plates (Merck Kieselgel,
60, 0.25 mm thickness) with F254 indicator. Compounds
were visualized under UV lamp and/or by developing
with iodine, vanillin, p-anisaldehyde or KMnO4. Flash
chromatography was performed on 230–400 mesh silica
gel (E. M. Science) with technical and/or HPLC grade
solvents. Medium pressure liquid chromatography
(MPLC) was performed with a FMI pump and
prepacked silica gel column (Merck, Labor Columns,
LiChroprep Si 60, 40–63 lm). High pressure liquid chro-
matography (HPLC) was performed on a Rainin HPLX
system equipped with two 25 mL pump heads and a
Rainin Dynamax UV-C dual-beam variable wavelength
detector set at 254 or 260 nm using a Phenomenex, Luna
5 l C18 semipreparative (250 · 10 mm) column or
Chiralcel OJ semipreparative (250 · 10 mm) column.
Vehicle
2
Calcitriol
0.5 μg/kg
4
3
10 μg/kg
20 μg/kg
10 μg/kg
Figure 2.
level of urine calcium excretion as the natural hormone
(0.5 lg/kg). The calciuric activities of the three analogs
are similar to one another, with the least calciuric being
the N–(OMe) analog 4, as shown in Figure 2. Also, the
analogs 2–4 do not compromise the weight gain of the
rats even at doses that are more than 40 times higher
than calcitriol (data not shown).
3. Conclusion
The new amide and hydroxamate analogs 2–4 show that
terminating the C,D-ring side chain with a tert-butyl
group instead of the natural tertiary 25-OH group and
introducing a nitrogen atom at the 24 position in the
form of a –N(R)C(O)– group produce analogs that have
a favorable therapeutic window between desirably high
antiproliferative activity and desirably low-calciuric
activity. This desirable biological profile encourages
further evaluation of amide and hydroxamate analogs
2–4 as potential chemotherapeutic agents for various
human illnesses.
HRMS were obtained at the mass spectrometry facility
at the Ohio State University on a Micromass QTOF
Electrospray mass spectrometer, or at The Johns
Hopkins University on a VG70S double focusing
magnetic sector FAB mass spectrometer (VG Analyti-
cal, Manchester, UK, now Micromass/Waters).
4.1. Synthesis of amide 2
4.1.1. Preparation of amide (+)-7. A flame-dried 10 mL
recovery flask equipped with a magnetic stir bar, a

6344
S. Sinishtaj et al. / Bioorg. Med. Chem. 14 (2006) 6341–6348
25
D
septum along with an Ar balloon was charged with 2,2-
dimethylpropionamide (51 mg, 0.50 mmol) and dis-
solved in 5 mL of freshly distilled THF. To this solution,
sodium hydride (12 mg, 0.50 mmol) was added and was
allowed to stir for 30 min before iodide (+)-612 (45 mg,
0.10 mmol) was cannulated into the flask as a solution
in THF (2 mL). The mixture was gradually heated to re-
flux for 20 h. Thin-layer chromatography (TLC) showed
the complete consumption of starting material. The
reaction was quenched by addition of 15 mL of distilled
water and then rinsed into a separatory funnel with ethyl
acetate. The mixture was extracted with ethyl acetate
(3· 30 mL). The combined extracts were washed with
water (1· 5 mL) and brine (1· 5 mL), dried over
Na2SO4, and filtered. The filtrate was concentrated in
vacuo to give the crude product which was purified by
flash chromatography with EtOAc–hexanes (1/4), to af-
10 as an oil. Data for 10: ½aꢀ +12.7 (c 1.00, CHCl3); 1H
NMR (400 MHz, CDCl3) d 5.64 (br s, 1H), 3.29 (m,
1H), 3.13 (m, 1H), 2.41 (dd, J = 11.6, 7.2 Hz, 1H), 2.14–
2.27 (m, 2H), 2.07 (ddd, J = 13.2, 4.0, 2.4 Hz, 1H),
1.79–2.02 (m, 3H), 1.69 (m, 1H), 1.38–1.60 (m, 5H),
1.21–1.31 (m, 2H), 1.15 (s, 9H), 0.97 (d, J = 6.0 Hz,
3H), 0.59 (s, 3H); 13C NMR (100 MHz, CDCl3) d
211.8, 178.2, 61.8, 56.2, 49.8, 40.8, 38.8, 38.5, 36.8, 35.5,
33.6, 27.5, 23.9, 18.9, 18.7, 12.3; IR (neat, cmꢁ1) 3352,
2958, 2873, 1712, 1640, 1532, 1479, 1378, 1308, 1230,
1214, 1056; HRMS: calcd for C19H33NO2Na+
[M+Na+]: 330.2403, found: 330.2415.
4.1.3. Preparation of HBJ-24N(H)25(O)TB (+)-2. Enan-
tiomerically pure phosphine oxide (ꢁ)-13 and CD-ring
ketone (+)-10 were separately azeotropically dried with
anhydrous benzene (4· 5 mL) on a rotary evaporator
and held under vacuum (ca. 0.1 mmHg) for at least
48 h prior to use.
ford 40 mg (95%) of amide 7 as a colorless oil. Data for
25
D
7: ½aꢀ +46.7 (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3) d 5.53 (br s, 1H), 4.02 (m, 1H), 3.31 (m, 1H),
3.14 (m, 1H), 1.93 (dm, Jd = 12.4 Hz, 1H), 1.74–1.86
(m, 2H), 1.66 (m, 1H), 1.51–1.62 (m, 2H), 1.29–1.45
(m, 4H), 1.16–1.25 (m, 3H), 1.18 (s, 9H), 0.99–1.13 (m,
2H), 0.94 (t, J = 8.0 Hz, 9H), 0.94 (d, J = 6.4 Hz, 3H),
0.89 (s, 3H), 0.54 (q, J = 8.0 Hz, 6H); 13C NMR
(100 MHz, CDCl3) d 178.2, 69.3, 56.5, 53.0, 42.1, 40.7,
38.5, 37.0, 35.5, 34.5, 33.4, 27.6, 27.4, 22.9, 18.7, 17.6,
13.4, 6.9, 4.9; IR (neat, cmꢁ1) 3342, 2952, 2875, 1637,
1537, 1458, 1373, 1230, 1165, 1083, 1025, 725; HRMS:
calcd for C25H49NO2 SiNa+ [M+Na+]: 446.3425, found:
446.3439.
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 (ꢁ)-13 (47 mg,
0.081 mmol) and dissolved in 1.5 mL of freshly distilled
THF to give a ca. 0.07 M solution. The flask was cooled
to ꢁ78 ꢁC in an isopropanol/dry ice bath. To this solu-
tion n-BuLi (50 lL, 0.081 mmol, 1.60 M solution in hex-
anes) was added dropwise over several minutes during
which time a deep red color developed and persisted.
This mixture was allowed to stir at ꢁ78 ꢁC for an addi-
tional 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 CD-ring ketone
(+)-10 (8.0 mg, 0.026 mmol) dissolved in 0.75 mL of
freshly distilled THF and cooled to ꢁ78 ꢁC in an isopro-
panol/dry ice bath. The solution of CD-ring ketone was
transferred dropwise into the flask containing the phos-
phine 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 ca. 5 h, during which time it was checked
visually. Upon observation of a light yellow color, the
reaction was quenched at ꢁ78 ꢁC by the addition of
5 mL of pH 7 buffer and allowed to warm to room
temperature. The mixture was then rinsed into a separ-
atory funnel with ethyl acetate. The mixture was extract-
ed with ethyl acetate (3· 25 mL). The combined extracts
were washed with water (1· 25 mL) and brine
(1· 25 mL), dried over Na2SO4, and filtered. The filtrate
was concentrated in vacuo to give the crude product
which was purified by column chromatography with
EtOAc–hexanes (1/4) to afford 8.0 mg (53.0%) of prod-
uct. The O-silylated analog was transferred into a
flame-dried 5 ml flask, equipped with a stir bar, and
purged with argon. It was then dissolved in 3 mL THF
and cooled to ꢁ78 ꢁC, and TBAF (0.13 mL, 0.13 mmol)
was added dropwise. The mixture was gradually
warmed to room temperature and left to stir overnight.
The next day TLC showed complete consumption of
starting material. The reaction was quenched by the
addition of 15 mL of distilled water and then rinsed into
a separatory funnel with ethyl acetate. The mixture was
extracted with ethyl acetate (3· 30 mL). The combined
4.1.2. Preparation of ketone (+)-10. A flame-dried 10 mL
recovery flask equipped with a magnetic stir bar, a sep-
tum along with an Ar balloon was charged with the TES
protected alcohol 7 (38 mg, 0.090 mmol) dissolved in
3 mL of freshly distilled THF. To this solution,
0.35 mL of tetrabutylammonium fluoride (TBAF)
(0.350 mmol, 1.0 M solution in THF) was added drop-
wise over several minutes and allowed to stir overnight
at room temperature. TLC showed the complete con-
sumption of starting material. The reaction was
quenched by the addition of 5 mL of distilled water
and then rinsed into a separatory funnel with ethyl ace-
tate. The mixture was extracted with ethyl acetate
(3· 25 mL). The combined extracts were washed with
water (1· 25 mL), and brine (1· 25 mL), dried over
Na2SO4, and filtered. The filtrate was concentrated in
vacuo to give the crude product. The residue was sub-
jected to column chromatography with EtOAc–hexanes
(1/1) as eluent to give the desired alcohol as a colorless
oil.
The alcohol was charged into an argon purged 10 mL
recovery flask equipped with a magnetic stir bar, a
septum and dissolved in 4 mL of freshly distilled CH2Cl2.
To this solution were added PDC (35 mg, 0.096 mmol)
and 26 mg of oven-dried Celite in one portion at room
temperature. The resulting mixture was allowed to stir
at room temperature for about 12 h. TLC showed the
complete consumption of starting material. The mixture
was directly purified by flash chromatography with
EtOAc–hexanes (34%) to afford 25 mg (91%) of ketone

S. Sinishtaj et al. / Bioorg. Med. Chem. 14 (2006) 6341–6348
6345
extracts were washed with water (1· 5 mL), and brine
(1· 5 mL), dried over Na2SO4, and filtered. The filtrate
was concentrated in vacuo to give the crude product
which was purified by flash chromatography (100% eth-
The alcohol was charged into an argon purged 10 mL
recovery flask equipped with a magnetic stir bar, a sep-
tum and was dissolved in 3 mL of freshly distilled
CH2Cl2. To this solution were added PDC (37 mg,
0.10 mmol) and 37 mg of oven-dried Celite in one por-
tion at room temperature. The resulting mixture was al-
lowed to stir at room temperature for about 12 h. TLC
showed the complete consumption of starting material.
The mixture was directly purified by flash chromatogra-
yl acetate) to afford 5.0 mg of analog 2 as an oil (50%
25
yield for two steps). Data for 2: ½aꢀ +20 (c 0.25,
D
CHCl3); 1H NMR (400 MHz, CDCl3) d 6.37 (d,
J = 11.2 Hz, 1H), 6.01 (d, J = 11.2 Hz, 1H), 5.54 (br s,
1H), 5.33 (s, 1H), 5.00 (s, 1H), 4.43 (m, 1H), 4.23 (m,
1H), 3.32 (m, 1H), 3.17 (m, 1H), 2.82 (dd, J = 12.0,
4.0 Hz, 1H), 2.60 (dd, J = 13.6, 3.2 Hz, 1H), 2.31 (dd,
J = 13.2, 6.4 Hz, 1H), 1.89–2.04 (m, 5H), 1.42–1.71 (m,
7H), 1.14–1.35 (m, 6H), 1.19 (s, 9H), 0.98 (d,
J = 6.4 Hz, 3H), 0.54 (s, 3H); 13C NMR (100 MHz,
CDCl3) d 178.3, 147.6, 143.0, 132.9, 124.9, 117.1,
111.8, 70.8, 66.8, 56.3, 56.3, 45.9, 45.3, 42.8, 40.4, 38.6,
37.1, 35.7, 34.3, 29.0, 27.8, 27.6, 23.5, 22.2, 18.9, 11.9;
IR (neat, cmꢁ1) 3346, 2949, 2872, 1635, 1539, 1455,
1437, 1349, 1214, 1057, 754; HRMS: calcd for
C28H45NO3 Na+ [M+Na+]: 466.3292, found: 466.3312,
kmax 265 nm (e 10,780).
phy with EtOAc–hexanes (1/2) affording 9.0 mg of ke-
25
D
tone 11 as an oil (94%). Data for 11: ½aꢀ +8.1 (c 0.50,
1
CHCl3); H NMR (400 MHz, CDCl3) d 3.26–3.43 (m,
2H), 3.01 (s, 3H), 2.44 (dd, J = 11.6, 7.6 Hz, 1H),
2.17–2.30 (m, 2H), 2.10 (ddd, J = 12.8, 4.4, 2.4 Hz,
1H), 1.85–2.04 (m, 3H), 1.26–1.78 (m, 8H), 1.26 (s,
9H), 1.02 (d, J = 6.0 Hz, 3H), 0.63 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 211.9, 177.1, 61.9, 56.4, 49.9,
47.9, 40.9, 38.9, 38.7, 36.3, 33.9, 32.9, 28.3, 27.6, 24.0,
19.0, 18.9, 12.4; IR (neat, cmꢁ1) 2957, 2874, 1713,
1626, 1480, 1404, 1378, 1367, 1307, 1116, 1080.
4.2.2. Preparation of HBJ-24N(Me)25(O)TB (+)-3.
Enantiomerically pure phosphine oxide (ꢁ)-13 and
CD-ring ketone (+)-11 were separately azeotropically
dried with anhydrous benzene (4· 5 mL) on a rotary
evaporator and held under vacuum (ca. 0.1 mmHg) for
at least 48 h prior to use.
4.2. Synthesis of amide 3
4.2.1. Preparation of ketone (+)-11. A flame-dried 10 mL
recovery flask equipped with a magnetic stir bar, a sep-
tum along with an Ar balloon was charged with N-meth-
yltrimethylamide (65 mg, 0.56 mmol) and dissolved in
5.6 mL of freshly distilled THF. To this solution sodium
hydride (14 mg, 0.56 mmol) was added and allowed to
stir for 30 min before iodide (+)-6 (50 mg, 0.11 mmol)
was cannulated into the flask as a solution in THF
(2 mL). The mixture was gradually heated to reflux for
20 h. TLC showed the complete consumption of starting
material. The reaction was quenched by the addition of
15 mL of distilled water and then rinsed into a separato-
ry funnel with ethyl acetate. The mixture was extracted
with ethyl acetate (3· 30 mL). The combined extracts
were washed with water (1· 5 mL), and brine
(1· 5 mL), dried over Na2SO4, and filtered. The filtrate
was concentrated in vacuo to give the crude product
which was purified by flash chromatography with
EtOAc–hexanes (1/6) to afford 13 mg (30%) of the amide
8 as a colorless oil.
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 (ꢁ)-13 (35 mg,
0.060 mmol) and dissolved in 1 mL of freshly distilled
THF to give a ca. 0.06 M solution. The flask was cooled
to ꢁ78 ꢁC in an isopropanol/dry ice bath. To this solu-
tion n-BuLi (38 lL, 0.060 mmol, 1.60 M solution in hex-
anes) was added dropwise over several minutes, during
which time a deep red color developed and persisted.
This mixture was allowed to stir at ꢁ78 ꢁC for an addi-
tional 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 CD-ring ketone
(+)-11 (9.0 mg, 0.028 mmol) dissolved in 1 mL of freshly
distilled THF and cooled to ꢁ78 ꢁC in an isopropanol/
dry ice bath. The solution of CD-ring ketone was trans-
ferred 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 ca. 4 h during which time it was checked
visually. Upon observation of a light yellow color, the
reaction was quenched at ꢁ78 ꢁC by the addition of
5 mL of pH 7 buffer and allowed to warm to room tem-
perature. The mixture was then rinsed into a separatory
funnel with ethyl acetate and extracted with ethyl ace-
tate (3· 25 mL). The combined extracts were washed
with water (1· 25 mL) and brine (1· 25 mL), dried over
Na2SO4, and filtered. The filtrate was concentrated in
vacuo to give the crude product which was purified by
column chromatography with EtOAc–hexanes (1/6) to
afford 6.0 mg of product (31%). The O-silylated analog
was transferred into a flame-dried 5 ml flask, equipped
with a stir bar, and purged with argon. Then, the prod-
uct was dissolved in 2 mL THF and cooled to ꢁ78 ꢁC,
A flame-dried 10 mL recovery flask equipped with a
magnetic stir bar, a septum along with an Ar balloon
was charged with amide 8 (38 mg, 0.090 mmol) and dis-
solved in 3 mL of freshly distilled THF. To this solution
0.35 mL of tetrabutylammonium fluoride (TBAF)
(0.35 mmol, 1.0 M solution in THF) was added drop-
wise over several minutes and the contents in the flask
were left to stir overnight at room temperature. TLC
showed the complete consumption of starting material.
The reaction was quenched by the addition of 5 mL of
distilled water and then rinsed into a separatory funnel
with ethyl acetate. The mixture was extracted with ethyl
acetate (3· 25 mL). The combined extracts were washed
with water (1· 25 mL), and brine (1· 25 mL), dried over
Na2SO4, and filtered. The filtrate was concentrated in
vacuo to give the crude product. The residue was puri-
fied by column chromatography with EtOAc–hexanes
(1/1) to give the desired alcohol as a colorless oil.

6346
S. Sinishtaj et al. / Bioorg. Med. Chem. 14 (2006) 6341–6348
and TBAF (0.09 mL, 0.09 mmol) was added dropwise.
The mixture was gradually warmed up to room temper-
ature and left to stir overnight. The next day TLC
showed complete consumption of starting material.
The reaction was quenched by the addition of 15 mL
of distilled water and then rinsed into a separatory fun-
nel with ethyl acetate. The mixture was extracted with
ethyl acetate (3· 30 mL). The combined extracts were
washed with water (1· 5 mL), and brine (1· 5 mL),
dried over Na2SO4, and filtered. The filtrate was concen-
trated in vacuo to afford the crude product which was
purified by flash chromatography with EtOAc (100%)
to afford 4.0 mg of analog 3 as an oil (29% yield for
material. The reaction was quenched by the addition
of 5 mL of distilled water and then rinsed into a separ-
atory funnel with ethyl acetate. The mixture was extract-
ed with ethyl acetate (3· 25 mL). The combined extracts
were washed with water (1· 25 mL), and brine
(1· 25 mL), dried over Na2SO4, and filtered. The filtrate
was concentrated in vacuo to give the crude product
which was purified by flash chromatography with
EtOAc–hexanes (1/10) to give 22 mg (82%) of alcohol
as an oil.
The alcohol was charged into an argon purged 10 mL
recovery flask equipped with a magnetic stir bar, a
septum and dissolved in 3 mL of freshly distilled
CH2Cl2. To this solution were added PDC (27 mg,
0.070 mmol) and 27 mg of oven-dried Celite in one por-
tion at room temperature. The resulting mixture was al-
lowed to stir at room temperature for about 12 h. TLC
showed the complete consumption of starting material.
The mixture was directly purified by flash chromatogra-
25
1
two steps). Data for 3: ½aꢀ +62 (c 0.10, CHCl3); H
D
NMR (400 MHz, CDCl3) d 6.37 (d, J = 11.2 Hz, 1H),
6.01 (d, J = 11.2 Hz, 1H), 5.33 (s, 1H), 5.00 (s, 1H),
4.43 (m, 1H), 4.23 (m, 1H), 3.29–3.42 (m, 2H), 3.01 (s,
3H), 2.82 (dd, J = 12.0, 4.0 Hz, 1H), 2.60 (dd, J = 13.2,
3.2 Hz, 1H), 2.31 (dd, J = 13.2, 6.4 Hz, 1H), 1.89–2.01
(m, 5H), 1.22–1.71 (m, 11H), 1.27 (s, 9H), 0.99 (d,
J = 6.4 Hz, 3H), 0.54 (s, 3H); 13C NMR (100 MHz,
CDCl3) d 177.1, 147.6, 143.0, 133.0, 124.9, 117.1,
111.8, 70.8, 66.8, 56.30, 56.27, 48.0, 45.9, 45.2, 42.8,
40.4, 38.7, 36.3, 34.5, 33.1, 29.0, 28.3, 27.7, 23.5, 22.2,
19.1, 11.9; IR (neat, cmꢁ1) 3388, 2946, 2872, 1608,
1408, 1364, 1261, 1214, 1114, 1056, 753; HRMS: calcd
for C29H48NO3 [M+H+]: 458.3556, found: 458.3634;
kmax 265 nm (e 12,287).
phy with EtOAc–hexanes (1/10) to afford 12 mg (91%)
25
D
of ketone 12 as an oil. Data for 12: ½aꢀ +11.4 (c 4.0,
1
CHCl3); H NMR (400 MHz, CDCl3) d 4.27–4.24 (dd,
2H, J = 6 Hz, J = 7.6 Hz), 3.72 (s, 3H), 2.48–2.43 (dd,
1H, J = 7.6 Hz, J = 11.6 Hz), 2.31–2.17 (m, 2H), 2.10
(d, 1H, J = 12.8 Hz), 2.05–1.97 (m, 1H), 1.96–1.91 (m,
1H), 1.63–1.49 (m, 3H), 1.11 (s, 9H), 1.00 (d,
J = 6.4 Hz, 3H), 0.65 (s, 3H); 13C NMR (100 MHz,
CDCl3) d 211.9, 162.3, 70.6, 61.9, 61.5, 56.8, 52.9,
49.9, 46.6, 40.9, 38.9, 36.3, 36.1, 32.6, 27.6, 27.5, 24.0,
19.0, 18.9, 12.4; IR (neat, cmꢁ1) 2962, 2873, 1717,
1634, 1462, 1378, 1362, 1300, 1162, 1056, 901; HRMS:
calcd for C20H35NO2 Na+ [M+Na]: 360.2509, found:
360.2513.
4.3. Synthesis of hydroxamate 4
4.3.1. Preparation of ketone (+)-12. A flame-dried 10 mL
recovery flask equipped with a magnetic stir bar, a
septum along with an Ar balloon was charged with
tert-butyl(O-methyl) hydroxamate (88 mg, 0.67 mmol)
and dissolved in 5 mL of freshly distilled DMF, making
a 1.34 M solution. To this solution, sodium hydride
(17 mg, 0.74 mmol) was added and was allowed to stir
for 30 min before iodide (+)-6 (27 mg, 0.67 mmol) was
cannulated into the flask as a solution in DMF
(1.5 mL). The mixture was gradually heated to reflux
for 24 h. TLC showed the complete consumption of
starting material. The reaction was quenched by addi-
tion of 15 mL of distilled water at room temperature
and then rinsed into a separatory funnel with ethyl ace-
tate. The mixture was extracted with ethyl acetate
(3· 30 mL). The combined extracts were washed with
water (1· 5 mL), and brine (1· 5 mL), dried over
Na2SO4, and filtered. The filtrate was concentrated in
vacuo to give the crude product which was purified by
flash chromatography with EtOAc–hexanes (1/4) to
afford 22 mg (89%) of the hydroxamate 9 as a colorless
oil.
4.3.2. Preparation of SS-24N(OMe)25(O)TB (+)-4.
Enantiomerically pure phosphine oxide (ꢁ)-13 and
CD-ring ketone (+)-12 were separately azeotropically
dried with anhydrous benzene (4· 5 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 (ꢁ)-13 (60 mg,
0.10 mmol) and dissolved in 1 mL of freshly distilled
THF to give a ca. 0.1 M solution. The flask was cooled
to ꢁ78 ꢁC in an isopropanol/dry ice bath. To this solu-
tion n-BuLi (60 lL, 0.10 mmol, 1.60 M solution in
hexanes) was added dropwise over several minutes dur-
ing which time a deep red color developed and persisted.
This mixture was allowed to stir at ꢁ78 ꢁC for an addi-
tional 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 CD-ring ketone
(+)-12 (12 mg, 0.030 mmol) dissolved in 1 mL of freshly
distilled THF and cooled to ꢁ78 ꢁC in an isopropanol/
dry ice bath. The solution of CD-ring ketone was trans-
ferred 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 per-
sisted and the mixture was allowed to stir at ꢁ78 ꢁC for
ca. 2 h during which time it was checked visually. Upon
A flame-dried 10 mL recovery flask equipped with a
magnetic stir bar, a septum along with an Ar balloon
was charged with hydroxamate 9 (22 mg, 0.049 mmol)
and dissolved in 2 mL of freshly distilled THF. To this
solution 0.10 mL of tetrabutylammonium fluoride
(TBAF) (0.10 mmol, 1.0 M solution in THF) was added
dropwise over several minutes and the contents of the
flask were left to stir overnight at room temperature.
TLC showed the complete consumption of starting

S. Sinishtaj et al. / Bioorg. Med. Chem. 14 (2006) 6341–6348
6347
observation of a light yellow color, the reaction was
quenched at ꢁ78 ꢁC by the addition of 5 mL of pH 7
buffer and allowed to warm to room temperature. The
mixture was then rinsed into a separatory funnel with
ethyl acetate. The mixture was extracted with ethyl ace-
tate (3· 25 mL). The combined extracts were washed
with water (1· 25 mL) and brine (1· 25 mL), dried over
Na2SO4, and filtered. The filtrate was concentrated in
vacuo to give the crude product which was purified by
flash chromatography EtOAc–hexanes (1/10) to afford
20 mg of product (78%). The O-silylated analog was
transferred into a flame-dried 5 ml flask, equipped with
a stir bar, and purged with argon. The product was then
dissolved in 2 mL THF and cooled to ꢁ78 ꢁC, and
TBAF (0.30 mL, 0.30 mmol) was added dropwise. The
mixture was gradually warmed to room temperature
and left to stir overnight. The next day TLC showed
complete consumption of starting material. The reaction
was quenched by the addition of 15 mL of distilled
water and then rinsed into a separatory funnel with ethyl
acetate. The mixture was extracted with ethyl acetate
(3· 30 mL). The combined extracts were washed with
water (1· 5 mL), and brine (1· 5 mL), dried over
Na2SO4, and filtered. The filtrate was concentrated in
vacuo to give the crude product which was purified by
flash column chromatography (50% ethyl acetate in hex-
concentrated in vacuo. The crude product was purified
via flash chromatography using EtOAc–hexanes (1/10)
to afford 38 mg (88%) of ester 15, as an oil. For the prep-
aration of the 25(O)26N(OMe)H-CD-ring-8-OTES 16,4
the purified ester 15 was transferred into a 10 mL pear-
shaped flask and placed under vacuum for 24 h. In a
flame-dried 25 mL round-bottomed flask equipped with
a magnetic stir bar, a septum and an argon balloon was
placed well-dried methoxylamine hydrochloride
(0.007 g, 0.08 mmol) and dissolved in 10 mL toluene to
give a ca. 0.08 M solution. This mixture was cooled to
0 ꢁC before trimethylaluminum (0.042 mL, 0.085 mmol)
was added dropwise. This was allowed to stir at 0 ꢁC for
5 min, before the reaction was allowed to warm to room
temperature and left to stir for 1 h before ester 15
(0.030 g, 0.076 mmol) was added via a cannula as a
0.08 M solution in benzene. This was allowed to stir
for 5 h. TLC showed the complete consumption of start-
ing material. The reaction was quenched with water
(1· 10 mL), neutralized with 1 M HCl, and extracted
with ethyl acetate (3· 30 mL). The extracts were com-
bined, dried over NaSO4, filtered, and concentrated in
vacuo. The crude product was purified via flash chroma-
tography using EtOAc–hexanes (1/2) to afford 21 mg
(71%) of ester 16 as a colorless oil. Data for 16:
25
1
½aꢀ +34 (c 0.80, CHCl3); H NMR (400 MHz, CDCl3)
D
anes) to afford 5.0 mg of analog 4 as an oil (40% yield
d 8.26 (s, 1H), 4.02–4.01 (d, 1H, J = 2.4 Hz), 3.76 (s,
3H), 1.95–1.91 (m, 1H), 1.86–1.04 (m, 18H), 0.96–0.89
(m, 15H), 0.57–0.51 (m, 6H); 13C NMR (100 MHz,
CD3OD) d 171.4, 69.5, 62.9, 56.6, 53.0, 41.9, 40.7,
35.0, 34.9, 34.3, 32.7, 26.92, 22.8, 17.6, 17.3, 12.8, 5.93,
4.5; IR (neat, cmꢁ1) 3177, 2944, 2875, 1657, 1455,
1368, 1229, 1166, 1078, 1015, 971; HRMS: calcd for
C23H45NO3 SiNa+ [M+Na]: 434.3061, found: 435.3051.
24
D
for two steps). Data for 4: ½aꢀ +58 (c 0.45, CHCl3);
1H NMR (400 MHz, CDCl3)
d
6.37 (d, 1H,
J = 11.2 Hz), 6.01 (d, 1H, J = 11.2 Hz), 5.33 (s, 1H),
5.00 (s, 1H), 4.44 (m, 1H), 4.27–4.2 (m, 3H), 3.72 (s,
3H), 2.82 (dd, 1H, J = 4.0, 12.4 Hz), 2.60 (dd, 1H,
J = 3.2, 13.2 Hz), 2.31 (dd, 1H, J = 6.4, 13.2 Hz), 2.08–
1.8 (m, 6H), 1.72–1.45 (m, 8H), 1.37–1.22 (m, 4H),
1.15 (s, 9H), 0.96 (d, 3H, J = 6.4 Hz), 0.56 (s, 3H); 13C
NMR (100 MHz, CDCl3) d 162.4, 147.6, 143.0, 133.0,
124.9, 117.1, 111.8, 70.8 (d, J = 6.8 Hz), 66.8, 61.5,
56.6, 56.3, 45.9, 45.2, 42.8, 40.4, 36.3, 36.2, 33.1, 29.7,
29.0, 28.3, 27.7, 27.5, 25.8, 23.5, 22.3, 19.0, 11.9; IR
(neat, cmꢁ1) 3371.4, 2940.9, 2874.6, 1737.5, 1627.1,
1461.5, 1373.2, 1295.9, 1240.7, 1157.9, 1058.6, 893.0;
HRMS: calcd for C29H47NO4 Na+ [M+Na]: 496.3397,
found: 496.3397; kmax 265 nm (e 15,954).
4.4.2. Preparation of ketone (+)-17. A flame-dried 10 mL
recovery flask equipped with a magnetic stir bar, a sep-
tum along with an Ar balloon was charged TES protect-
ed alcohol 16 (0.021 g, 0.049 mmol) and dissolved in
3 mL of freshly distilled THF. Then the flask was cooled
to ꢁ78 ꢁC in an isopropanol/dry ice bath. To this solu-
tion 0.485 mL of TBAF (0.49 mmol, 1.0 M solution in
THF) was added dropwise over several minutes and
the contents of the flask were allowed to stir at ꢁ78 ꢁC
for an additional 30 min. The mixture was gradually
warmed to room temperature and left to stir overnight.
TLC showed the complete consumption of starting
material. The reaction was quenched by addition of
5 mL of distilled water and then rinsed into a separatory
funnel with ethyl acetate. The mixture was extracted
with ethyl acetate (3· 25 mL). The combined extracts
were washed with water (1· 25 mL), and brine
(1· 25 mL), dried over Na2SO4, and filtered. The filtrate
was concentrated in vacuo to give the crude product
which was purified by flash chromatography EtOAc–
hexanes (7/10) to afford 10 mg (71%) of deprotected
alcohol. The purified alcohol was charged into an argon
purged 10 mL recovery flask equipped with a magnetic
stir bar, a septum and dissolved in 2 mL of freshly
distilled THF to give a ca. 0.02 M solution. To this
solution were added PDC (0.026 g, 0.071 mmol) and
16 mg of oven-dried Celite in one portion at room
temperature. The resulting mixture was allowed to stir
4.4. Synthesis of hydroxamate 5
4.4.1. Preparation of dicarbonyl (+)-16. In a flame-dried
25 mL two-necked round-bottomed flask equipped with
a magnetic stir bar, a septum, a reflux condenser, and an
Ar balloon were placed activated zinc (0.086 g,
1.3 mmol), methyl acrylate 14 (0.14 mL, 1.54 mmol),
and NiCl2Æ6H2O (0.040 g, 0.17 mmol) in pyridine
(3 mL). The mixture was heated at 65 ꢁC for 1 h, during
which time the color of the reaction mixture turned red-
dish brown. The reaction mixture was cooled to 0 ꢁC
and added a solution of pre-cooled (0 ꢁC) 22-iodide
(+)-6 (0.050 g, 0.11 mmol). It was then warmed to room
temperature and allowed to stir for 3 h. The mixture was
then diluted with EtOAc (5 mL) and filtered through a
pad of Celite. The filtrate was washed with 5% HCl
(2· 5 mL) and extracted with EtOAc (3· 5 mL). The
combined extracts were washed with water (1· 5 mL)
and brine (1· 5 mL), dried over NaSO4, and

6348
S. Sinishtaj et al. / Bioorg. Med. Chem. 14 (2006) 6341–6348
at room temperature for about 12 h. TLC showed the
complete consumption of starting material. The mixture
was directly purified by column chromatography
and then rinsed into a separatory funnel with ethyl ace-
tate. The mixture was extracted with ethyl acetate
(3· 30 mL). The combined extracts were washed with
water (1· 5 mL), and brine (1· 5 mL), dried over
Na2SO4, and filtered. The filtrate was concentrated in
vacuo to give the crude product which was purified by
flash chromatography (100% ethyl acetate) to afford
1.4 mg of analog (ꢁ)-5 as an oil (11% yield for two
with EtOAc/Hexanes (1/1) to afford 9.0 mg (89%) of ke-
25
D
tone (+)-17 as an oil. Data for 17: ½aꢀ +10 (c 0.36,
1
CHCl3); H NMR (400 MHz, CDCl3) d 8.22 (s, 1H),
3.77 (s, 3H), 2.46–2.41 (m, 1H), 2.33–2.16 (m, 3H),
2.12–1.97 (m, 3H), 1.95–1.82 (m, 3H), 1.77–1.43 (m,
24
1
10H), 1.34–1.28 (m, 3H), 0.98–0.96 (d, 3H, J = 8Hz),
steps). ½aꢀ ꢁ4.6 (c 0.10, CHCl3); H NMR (400 MHz,
CDCl3) dD8.22 (s, 1H), 6.39–6.36 (d, 1H, J = 11.2 Hz),
6.02–6.00 (d, 1H, J = 10.8 Hz), 5.33–5.32 (m, 1H), 5.00
(s, 1H), 4.44–4.43 (m, 1H), 4.24–4.22 (m, 1H), 3.76 (s,
1H), 2.84–2.81 (m, 1H), 2.62–2.59 (m, 1H), 2.35–2.27
(m, 1H), 2.04–1.85 (m, 7H), 1.68–1.05 (m, 17H), 0.95–
0.94 (d, 3H, J = 6.0 Hz), 0.54 (s, 3H); 13C NMR
(100 MHz, CDCl3) d insufficient material for a carbon
NMR; HRMS: calcd for C26H41NO4 Na+ [M+Na]:
454.2927, found: 454.2913; UV (MeOH) kmax 265 nm
(e 15,958).
0.63 (s, 3H); 13C NMR (100 MHz, CDCl3) d (not enough
material to obtain a clean spectrum); IR (neat, cmꢁ1
3210, 2956, 1711, 1652, 1456, 1378, 1070; HRMS: calcd
)
for C18H33NO3 Na+ [M+Na]: 346.2353, found: 346.2364.
4.4.3. Preparation of SS-23-oxa-24N(Me)25(O)TB-(5).
Enantiomerically pure phosphine oxide (ꢁ)-13 and CD-
ring ketone (+)-17 were separately azeotropically dried
with anhydrous benzene (4· 5 mL) on a rotary evapora-
tor 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 and a septum with an Ar balloon
was charged with phosphine oxide (ꢁ)-13 (59 mg,
0.10 mmol) and dissolved in 0.75 mL of freshly distilled
THF to give a ca. 0.13 M solution. The flask was cooled
to ꢁ78 ꢁC in an isopropanol/dry ice bath. To this solu-
tion, n-BuLi (71 lL, 0.11 mmol, 1.60 M solution in hex-
anes) was added dropwise over several minutes during
which time a deep red color developed and persisted.
This mixture was allowed to stir at ꢁ78 ꢁC for an addi-
tional 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 CD-ring ketone
(+)-17 (10 mg, 0.034 mmol) dissolved in 0.75 mL of
freshly distilled THF and cooled to ꢁ78 ꢁC in an isopro-
panol/dry ice bath. The solution of CD-ring ketone was
transferred dropwise into the flask containing the phos-
phine 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 ca. 2 h during which time it was checked
visually. Upon observation of a light yellow color, the
reaction was quenched at ꢁ78 ꢁC by addition of 5 mL
of pH 7 buffer and allowed to warm to room tempera-
ture. The mixture was then rinsed into a separatory fun-
nel with ethyl acetate and extracted with ethyl acetate
(3· 25 mL). The combined extracts were washed with
water (1· 25 mL) and brine (1· 25 mL), dried over
Na2SO4, and filtered. The filtrate was concentrated in
vacuo to give the crude product which was purified by
column chromatography (50% ethyl acetate in hexanes)
to afford 5.0 mg (24%) of product.
Acknowledgment
We thank the NIH for financial support (CA 93547).
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The O-silylated analog was transferred into a flame-
dried 5 mL flask, equipped with a stir bar and purged
with argon. The product was then dissolved in 1 mL
THF and cooled to ꢁ78 ꢁC, and TBAF (0.040 mL,
0.075 mmol) was added dropwise. The mixture was
gradually warmed to room temperature and left to stir
overnight. The next day TLC showed the complete con-
sumption of starting material. The reaction was
quenched by the addition of 15 mL of distilled water
14. Posner, G. H.; Halford, B. A.; Peleg, S.; Dolan, P. M.;
Kensler, T. W. J. Med. Chem. 2002, 35, 3280–3287.