Potent, selective and low-calcemic inhibitors of CYP24 Hydroxylase: 24-Sulfoximine analogues of the hormone 1α,25-dihydroxyvitamin D 3

Article abstract of DOI:10.1021/jm040129+

A dozen 24-sulfoximine analogues of the hormone 1α,25- dihydroxyvitamin D₃ were prepared, differing not only at the stereogenic sulfoximine stereocenter but also at the A-ring. Although these sulfoximines were not active transcriptionally and w

Full text of DOI:10.1021/jm040129+

6854
J. Med. Chem. 2004, 47, 6854-6863
Potent, Selective and Low-Calcemic Inhibitors of CYP24 Hydroxylase:
24-Sulfoximine Analogues of the Hormone 1r,25-Dihydroxyvitamin D3
Mehmet Kahraman,† Sandra Sinishtaj,† Patrick M. Dolan,‡ Thomas W. Kensler,‡ Sara Peleg,§ Uttam Saha,|
Samuel S. Chuang,| Galina Bernstein,| Bozena Korczak,| and Gary H. Posner*,†
Department of Chemistry, School of Arts and Sciences, The Johns Hopkins University, Baltimore, Maryland 21218,
Division of Toxicological Sciences, Department of Environmental Health Sciences, Bloomberg School of Hygiene,
The Johns Hopkins University, Baltimore, Maryland 21205, Department of Medical Specialities, The University of Texas,
M. D. Anderson Cancer Center, Houston, Texas 77030, and Cytochroma, Inc., Markham, Ontario, Canada L3R 8E4
Received July 8, 2004
A dozen 24-sulfoximine analogues of the hormone 1R,25-dihydroxyvitamin D3 were prepared,
differing not only at the stereogenic sulfoximine stereocenter but also at the A-ring. Although
these sulfoximines were not active transcriptionally and were only very weakly antiproliferative,
some of them are powerful hydroxylase enzyme inhibitors. Specifically, 24-(S)-NH phenyl
sulfoximine 3a is an extremely potent CYP24 inhibitor (IC50 ) 7.4 nM) having low calcemic
activity. In addition, this compound shows high selectivity toward the CYP24 enzyme in
comparison to CYP27A1 (IC50 > 1000 nM) and CYP27B (IC50 ) 554 nM).
Introduction
racemization but also due to their remarkable biological
activities.8 As part of an ongoing program of research
into design and synthesis of potent and low-calcemic
analogues of 1, we have prepared a range of 24-
sulfoximine analogues of the natural hormone as out-
lined in Schemes 1-4. The 22-iodide (+)-49 underwent
iodide substitution by the required N-tert-butyldimeth-
ylsilyl-protected sulfoximines10 followed by bisdesilyla-
tion to form C,D-ring side-chain NH-sulfoximines 6 as
C-8 secondary alcohols. Oxidation at C-8 produced the
corresponding C-8 ketones 7 (Scheme 1, Table 1).
Horner-Wadsworth-Emmons (HWE) coupling of ke-
tones 7 with A-ring phosphine oxide 8 conjugate base,
followed by HF-promoted desilylation, generated the
target sulfoximine analogues 3a-3g in good yields
(Scheme 2, Table 2). When racemic A-ring 8 (where R3,
R3 ) CH2 and R4 ) OTBS; Table 2, entries 1 and 2)
was used in the HWE coupling reaction, the reaction
afforded a diastereomeric pair of products, 3 and 3′, that
were separated on a semipreparative chiral HPLC
column. Each of these diastereomers was characterized
fully and distinguished from its partner by analogy to
our 24-sulfone analogues.6 When enantiomerically pure
19-nor (where R3, R3 ) H, H and R4 ) OTBS; Table 2,
entries 3 and 4) and 1-deoxy (where R3, R3 ) CH2 and
R4 ) H; Table 2, entry 5) A-rings were used in the
aforementioned coupling reaction, 24-NH-phenyl sul-
foximines were obtained in good yields after desilylation
with HF in acetonitrile at room temperature.
Medicinal chemists are designing analogues to be at
least as potent as, but less calcemic than, the natural
hormone 1R,25-dihydroxyvitamin D3 (calcitriol, 1) for
chemotherapy of diverse human illnesses.1-3 We have
shown that some new vitamin D analogues, even though
lacking the classical side-chain terminal OH group, are
transcriptionally active and low-calcemic.4,5 Among such
new analogues characterized by a 24-sulfone functional
group, 24-sulfone 2 stands out as being a very potent
(IC50 ) 28 nM), low-calcemic, and selective inhibitor of
the human cyctochrome P450C24 (CYP24) hydroxylase
enzyme responsible for the main catabolism of calcitriol
(1).6 Therefore, surrogates for the sulfone functional
group were considered. Now we report on a new series
of 24-sulfoximines 3a-3l in which the traditional side-
chain terminal OH group is lacking but is replaced by
a sulfoximine NH group.
Chemistry
The preparation of 4-fluorophenyl sulfoximine ana-
logues 3h and 3i is outlined in Scheme 3. The first step
is a replacement of the iodide in building block 22-iodide
(+)-911 with racemic sulfoximine (()-10 followed by
desilylation to form para-fluorinated phenyl sulfoximine
analogues 3h and 3i as an approximately 1:1 diaster-
eomeric mixture in 46% overall yield. This mixture was
also separated on a semipreparative chiral HPLC
column, and diastereomers were distinguished from
each other by 1H and 13C NMR spectroscopy correlation
with some of the other sulfoximines described here. The
Sulfoximines,7 the monoimino analogues of sulfones,
have gained considerable attention in synthetic organic
chemistry, not only due to their high stability toward
* Corresponding author: Department of Chemistry, Johns Hopkins
University, 3400 N. Charles St., Baltimore, MD 21218-2685. Phone
410-516-4670; fax 410-516-8420; e-mail ghp@jhu.edu.
† Department of Chemistry, The Johns Hopkins University.
‡ Department of Environmental Health Sciences, The Johns Hopkins
University.
§ The University of Texas.
| Cytochroma, Inc.
10.1021/jm040129+ CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/24/2004

Vitamin D Analogues as Hydroxylase Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6855
Scheme 1
Scheme 2
By use of a standard protocol,15 VDR-mediated in
vitro transcriptional activities of some of the sulfoximine
analogues were tested in CV-1 cells cotransfected with
the recombinant human VDR plasmid and the osteo-
calcin VDRE attached to the thymidine kinase promoter-
growth hormone reporter. Sulfoximines 3a and 3b were
found to be significantly less potent (ED50 ) >1 µM)
than calcitriol (1, ED50 ) 0.4 nM) (data not shown).
As a measure of toxicity and safety in animals,4,15
each of the sulfoximine analogues 3a, 3d, and 3e was
administered orally to rats daily for 1 week at a 20-fold
higher dose (10 µg/kg of body weight) than calcitriol (1,
0.5 µg/kg of body weight). None of the sulfoximines
significantly elevated the levels of calcium in the blood
of rats (Figure 1).
A standard biochemical assay16 showed that, among
the 24-NH sulfoximine analogues, 24-(S)-NH sulfox-
imine analogue 3a is the strongest inhibitor of human
CYP24 hydroxylase enzyme (Table 3). For comparison,
analogue 3a is a more potent CYP24 inhibitor than
commonly used ketoconazole and other azole inhibi-
tors.17
Our learning from the structure-activity relation-
ships (SAR) among these sulfoximines includes the
following: (1) The stereochemical configuration at the
sulfur atom in each pair of diastereomeric sulfoximines
is very important, with sulfoximines of the 24-(S)
configuration having higher CYP24 inhibition potency
than those with the 24-(R) configuration (CYP24 inhibi-
tory activity: 3a > 3b, 3d > 3c, 3f > 3g and 3h > 3i).
(2) Replacement of one of the oxygen atoms of sulfone
analogue 2 by an NH group as in analogue 3a increases
the CYP24 inhibitory activity by about 4-fold. (3) The
nature of the A-ring is of only minor importance, with
CYP24 inhibitory activity being similar for the natural
A-ring and for the 19-nor A-ring (e.g., 3a vs 3d), but
the activity diminished when the 1R-hydroxy group was
replaced by hydrogen atom (e.g., 3e is 10 times less
active than 3a). (4) The electronic nature of the phenyl
group is important, with CYP24 inhibitory activity
decreasing as the terminal aromatic ring acquires lower
electron density (CYP24 inhibitory activity R2 ) phenyl
> 4-fluorophenyl). (5) The 22-ene version 3k designed
to slow side-chain catabolism is inactive. (6) The 22-
Table 1. Synthesis of C,D-Ring Ketones 7
configuration of
entry sulfoximine (5)
R1, R1 R2 6 (yield %)a 7 (yield %)a
1
2
3
(+)-(S)
H, H
H, H
Ph
Ph
6a, 53
6b, 64
6c, 73
7a, 95
7b, 96
7c, 85
(-)-(R)
(()-(R/S)
-(CH2)2- Ph
a Isolated yields.
Table 2. Synthesis of Sulfoximine Analogues 3
configuration of
sulfoximine
3 + 3′
entry
R1, R1
R2
R3
R4 (yield %)a
1
2
3
4
5
6
(+)-(S)
(-)-(R)
(-)-(R)
(+)-(S)
(+)-(S)
(()-(R/S)
H, H
H, H
H, H
H, H
H, H
Ph )CH2 OH
Ph )CH2 OH
47
44
Ph H, H
Ph H, H
Ph )CH2
OH
OH
H
55b
23b
68b
40b
-(CH2)2- Ph )CH2 OH
a Isolated yields for two steps. b Enantiomerically pure A-rings
were used.
MK notation for products 3a-3j refers to the initials of
the researcher who prepared these sulfoximines.
To alter C,D-ring side-chain conformation as well as
to slow side-chain catabolism, 22-ene analogues 3j-3l
were synthesized as shown in Scheme 4. The Horner-
Wittig reaction of the building block 22-aldehyde (+)-
1212 with R-fluoro N-silylated sulfoximine (S)-13, fol-
lowed by deprotection resulted in olefinic sulfoximine
3j as a single geometrical isomer (see Scheme 4) about
the C22-C23 double bond in overall 52% yield. Simi-
larly, the reaction of the N-silylated diethylphospho-
nomethyl sulfoximine (S)-14 afforded olefinic analogues
3k and 3l in a 2:1 ratio, in overall 56% yield. Under the
same reaction conditions, analogue 3l also was obtained
as a C-20 epi isomer whose structure was tentatively
assigned by analogy to closely related C-20 epi com-
pounds.13 The SS notation for products 3k-3l refers to
the initials of the researcher who prepared these sul-
foximines.
Biology Results and Discussion
The in vitro antiproliferative potencies of 24-NH
sulfoximine analogues 3a-3l and calcitriol (1) were
determined by our standard murine keratinocyte as-
say.14 However, none of the sulfoximine analogues 3 was
strongly antiproliferative (data not shown).

6856 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Kahraman et al.
Scheme 3
Scheme 4
ene-23-fluoro sulfoximine analogue 3j is also inactive.
(7) The 20-epi analogue 3l is much less active than
parent sulfoximine 3a.
and selective inhibitors of the human cytochrome
P450C24 (CYP24) hydroxylase enzyme. Specifically, 24-
(S) sulfoximine 3a is approximately 40 times more
potent (IC50 ) 7.4 nM) than the commonly used CYP24
inhibitor ketoconazole (IC50 ) 312 nM). Analogue 3a
shows high inhibitory selectivity for CYP24 over the
vitamin D-related cytochromes CYP27A and CYP27B.
Thus, this new sulfoximine entity 3a, having low-
calcemic activity, has the potential to be used chemo-
therapeutically in conjunction with the natural hormone
1R,25-dihydroxyvitamin D3 (1) to extend the hormone’s
lifetime and thus to require less of it for effective
We also analyzed whether sulfoximine 3a had the
ability to inhibit the enzyme activities of CYP27A and
CYP27B, two cytochromes that are critical in the
vitamin D biosynthesis pathway. Analogue 3a had IC50
values of >1000 nM and 554 nM for inhibition of
CYP27A1 and CYP27B, respectively. Thus, sulfoximine
3a showed extraordinary selectivity for CYP24.
In summary, we have shown that NH sulfoximines,
monoimino analogues of sulfones, are in general potent

Vitamin D Analogues as Hydroxylase Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6857
formed 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 with Phenomenex,
Luna 5 m C18 semipreparative (250 × 10 mm) column and
Chiralcel OJ semipreparative (250 × 10 mm) column.
HRMS and LRMS were obtained on a Micromass QTOF
electrospray mass spectroscopy with electronic or chemical
ionization (EI or CI) at The Ohio State University.
(+)-(S)- and (-)-(R)-S-Methyl-S-phenylsulfoximine,18 (()-S-
cyclopropyl-S-phenylsulfoximine,19 compounds 22-iodide (+)-
4,20 N-silylated sulfoximines 5,10 A-ring phosphine oxides 8,14,21
iodide (+)-9,11 and aldehyde (+)-1212 were prepared as re-
ported.
1
The purity of all target compounds was >97% by H NMR
and by analytical HPLC determination.
General Procedure for Preparation of Alcohols 6. A
flame-dried 10-mL recovery flask equipped with a magnetic
stir bar and a septum along with an Ar balloon was charged
with the appropriate N-tert-butyldimethylsilyl sulfoximine 5
(0.32 mmol) dissolved in 3.2 mL of freshly distilled tetrahy-
drofuran (THF) and 0.32 mL of hexamethylphosphoramide
(HMPA). Then the flask was cooled to -78 °C in a 2-propanol/
dry ice bath. To this solution was added n-BuLi (0.33 mmol,
0.23 mL, 0.44 M solution in hexanes) dropwise over several
minutes, during which time a pale yellow color developed. This
mixture was allowed to stir at -78 °C for an additional 30
min. Meanwhile, a flame-dried 10-mL pear-shaped flask
equipped with a septum along with an Ar balloon was charged
with iodide (+)-4 (50 mg, 0.11 mmol) dissolved in 0.5 mL of
freshly distilled THF and cooled to -78 °C in a 2-propanol/
dry ice bath. The solution of iodide (+)-4 was transferred into
the flask containing the lithiated sulfoximine at -78 °C via
cannula over several minutes. After the addition was complete,
the mixture was gradually warmed to room temperature and
stirred at this temperature for about 6 h. Thin-layer chroma-
tography (TLC) showed the complete consumption of starting
material. The reaction was quenched by addition of 2 mL of
pH 7 buffer solution. Then it was 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 solution (1 × 25 mL), dried
over Na2SO4, and filtered. The filtrate was concentrated in
vacuo to give the crude product that was purified by flash
column chromatography.
An argon-purged 5 mL polypropylene vial equipped with a
magnetic stir bar was charged with appropriate alkylated C,D-
ring bis-protected intermediate (0.065 mmol) and dissolved in
1.6 mL of acetonitrile to give ca. 0.04 M solution. To this well-
stirred solution was added 0.26 of HF (6.5 mmol, 0.26 mL, 49%
aqueous solution) via syringe at room temperature, and the
mixture was then allowed to stir at room temperature for 4 h.
TLC showed the completion of the reaction. This reaction
mixture was diluted with ether (25 mL) and a saturated
solution of NaHCO3 was added until no more carbon dioxide
was liberated. The reaction mixture was then rinsed into a
separatory funnel with ethyl acetate and was extracted with
ethyl acetate (4 × 25 mL). The combined extracts were washed
with water (1 × 25 mL) and brine solution (1 × 25 mL), dried
over Na2SO4, and filtered. The filtrate was concentrated in
vacuo to give the crude product.
Figure 1. Effects of vitamin D3 analogues on urinary calcium
excretion in rats. Animals were treated with 0.5-10 µg/kg of
body weight of test compound po for 7 consecutive days, and
urinary excretion of calcium was measured during days 3-7.
Values are mean ( SE from three animals in each group.
Table 3. CYP24 Inhibitory Activities of NH Sulfoximines
NH sulfoximine
CYP24 IC50a (nM)
3a, MK-24(S)-S(O)(NH)Ph
7.4 ( 4.2
28 ( 10
13 ( 11
9.7 ( 3.1
75 ( 40
20.5 ( 3.5
50 ( 38
28 ( 20
44 ( 48
>1000
3b, MK-24(R)-S(O)(NH)Ph
3c, MK-19-nor-24(R)-S(O)(NH)Ph
3d, MK-19-nor-24(S)-S(O)(NH)Ph
3e, MK-1-deoxy-24(S)-S(O)(NH)Ph
3f, MK-23-cyclopropyl-24(S)-S(O)(NH)Ph
3g, MK-23-cyclopropyl-24(R)-S(O)(NH)Ph
3h, MK-24(S)-S(O)(NH)PFP
3i, MK-24(R)-S(O)(NH)PFP
3j, MK-22-ene-23-F-24(S)-S(O)(NH)Ph
3k, SS-22-ene-24(S)-S(O)(NH)Ph
3l, SS-20-epi-22-ene-24(S)-S(O)(NH)Ph
ketoconazole
>1000
549 ( 45
312 ( 1
28
2, KRC-24SO2-Ph-1
a IC50 values represent the means and standard deviations from
at least three independent experiments.
treatment of human diseases. Our progress in this effort
will be reported in due course.
Experimental Section
All air- and moisture-sensitive reactions were carried out
in flame-dried or oven-dried (at 120 °C) 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. Methylene chloride was freshly
distilled from CaH2 prior to use. Tetrahydrofuran and diethyl
ether were freshly distilled from sodium benzophenone ketyl
immediately prior to use. All other solvents and reagents were
used as received unless otherwise stated.
Both 1H and 13C spectra were obtained on a Varian XL 400
spectrometer at 400 and 100 MHz, respectively. All NMR
spectra were obtained as a solution in CDCl3 with tetrameth-
ylsilane (TMS) as the internal standard unless otherwise
stated. Infrared spectra were obtained on a Perkin-Elmer 1600
Fourier transform infrared (FT-IR) spectrometer as liquid films
and thin layers with NaCl cells. Optical rotations were
recorded on Jasco, P-1100 model polarimeter (Japan Spectro-
scopic Co., Ltd.) with sodium D line at the temperatures as
indicated in the experimental details for the specific com-
pounds.
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 an FMI pump and prepacked silica gel
column (Merck, Labor Columns, LiChroprep Si 60, 40-63
mm). High-pressure liquid chromatography (HPLC) was per-
C-8 Alcohol 6a. Flash column chromatography eluted with
100% ethyl acetate afforded 19.4 mg of (+)-6a as a viscous oil
in 86% yield. Data for (+)-6a: [R]D25 ) +30.2 (c 1.45, CHCl3);
1H NMR (CDCl3, 400 MHz) δ 7.97-7.95 (m, 2H), 7.64-7.53
(m, 3H), 4.05 (br s, 1H), 3.20 (ddd, 1H, J ) 4.4, 12.0, and 13.6
Hz), 3.03 (ddd, 1H, J ) 4.4, 12.0, and 13.6 Hz), 2.67 (br s, 1H),
1.93-1.68 (m, 6H), 1.58-1.37 (m, 5H), 1.30-0.95 (m, 5H), 0.87
(s, 3H), 0.84 (d, 3H, J ) 6.4 Hz); 13C NMR (CDCl3, 100 MHz)
δ 141.9, 132.9, 129.1, 128.3, 69.0, 55.8, 54.8, 52.4, 41.8, 40.2,
34.1, 33.5, 28.5, 26.8, 22.3, 18.2, 17.3, 13.4; IR (thin film) 3436
(br, w), 3330 (br, w), 2934 (s), 2871 (s), 1445 (m), 1373 (w),
1219 (br, s), 1161 (w), 1097 (sh, m), 989 (s, m), 753 (s) cm-1
;
HRMS calcd for C20H31NO2SNa+ [M + Na] 372.1967, found
372.1968.

6858 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Kahraman et al.
C-8 Alcohol 6b. Flash column chromatography eluted with
100% ethyl acetate afforded 20.1 mg of (+)-6b as a viscous oil
in 89% yield. Data for (+)-6b: [R]D25 ) +23.7 (c 1.45, CHCl3);
1H NMR (CDCl3, 400 MHz) δ 7.99-7.96 (m, 2H), 7.66-7.54
(m, 3H), 4.07 (br s, 1H), 3.19 (ddd, 1H, J ) 4.8, 12.4, and 13.6
Hz), 3.07 (ddd, 1H, J ) 4.4, 11.6, and 13.6 Hz), 2.68 (br s, 1H),
1.95-1.78 (m, 4H), 1.75-1.62 (m, 2H), 1.58-1.36 (m, 5H),1.32-
0.95 (m, 5H), 0.89 (s, 3H), 0.87 (d, 3H, J ) 6.4 Hz); 13C NMR
(CDCl3, 100 MHz) δ 141.9, 132.9, 129.1, 128.3, 69.0, 55.7, 54.7,
52.4, 42.8, 40.2, 34.1, 33.5, 28.5, 26.8, 22.3, 18.2, 17.3, 13.4;
IR (thin film) 3448 (br, w), 3330 (br, w), 2935 (s), 2871 (s),
1445 (m), 1219 (br, s), 1098 (sh, m), 1078 (br, s), 990 (s), m),
753 (s) cm-1; HRMS calcd for C20H31NO2SNa+ [M + Na]
372.1967, found 372.1981.
C-8 Alcohol 6c. Flash column chromatography eluted with
50% ethyl acetate afforded 50 mg of 6c as a viscous oil in 73%
overall yield. Data for 6c (as a mixture of diastereomers): 1H
NMR (CDCl3, 400 MHz) δ 7.97-7.95 (m, 4H), 7.63-7.58 (m,
2H), 7.55-7.50 (m, 4H), 4.03 (br s, 2H), 2.14 (t, 4H, J ) 11.2
Hz), 1.90 (d, 2H, J ) 13.2 Hz), 1.78-1.20 (m, 22H), 1.08-0.88
(m, 12H), 0.86 (s, 3H), 0.83 (s, 3H), 0.78-0.70 (m, 2H), 0.71
(d, 3H, J ) 6.4 Hz), 0.68 (d, 3H, J ) 6.4 Hz); 13C NMR (CDCl3,
100 MHz) δ 141.2, 140.50, 132.77, 132.70, 128.97, 128.88,
128.74, 69.05, 57.06, 56.97, 52.40, 41.93, 40.26, 40.20, 37.56,
37.13, 33.46, 33.26, 27.18, 27.13, 22.38, 18.78, 17.28, 13.48,
12.41, 12.27, 12.20, 11.67; IR (thin film) 3448 (br, w), 3330
(br, m), 3271 (m), 2931 (s), 2860 (s), 1443 (m), 1219 (br, s),
1067 (sh, m), 984 (s), 967 (m), 755 (s) cm-1; HRMS calcd for
C22H33NO2SNa+ [M + Na] 398.2124, found 398.2121.
1.58 (m, 10H), 1.54-1.35 (m, 6H), 1.28-1.20 (m, 2H), 1.12-
0.91 (m, 6H), 0.81 (d, 3H, J ) 6.4 Hz), 0.77 (d, 3H, J ) 6.4
Hz), 0.79-0.69 (m, 4H), 0.58 (s, 3H), 0.55 (s, 3H); 13C NMR
(CDCl3, 100 MHz) δ 211.65, 139.98, 133.10, 133.02, 129.02,
128.92, 61.75, 57.09, 57.00, 49.85, 40.15, 38.81, 38.78, 37.82,
37.36, 33.62, 27.49, 27.43, 23.89, 19.00, 18.94, 12.71, 12.52,
12.49, 12.44, 11.83; IR (thin film) 3278 (m), 2957 (s), 2874 (m),
1708 (s), 1445 (sh, m), 1378 (w), 1220 (br, s), 1109 (w), 968
(m), 749 (m) cm-1; HRMS calcd for C22H31NO2SNa+ [M + Na]
396.1967, found 396.1955.
General Procedure for Preparation of Sulfoximine
Analogues 3 and 3′. The corresponding A-ring phosphine
oxide 8 and C,D-ring ketone 7 were azeotropically dried with
anhydrous benzene (4 × 4 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 mag-
netic stir bar and a septum along with an Ar balloon was
charged with phosphine oxide 8 (0.11 mmol) dissolved in 1.1
mL of freshly distilled THF to give ca. 0.1 M solution. Then
the flask was cooled to -78 °C in a 2-propanol/dry ice bath.
To this solution was added n-BuLi (68 µL, 0.11 mmol, 1.6 M
solution in hexanes) 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 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 previously charged with CD-ring ketone 7 (12 mg,
0.036 mmol) dissolved in 1 mL of freshly distilled THF and
cooled to -78 °C in a 2-propanol/dry ice bath. The solution of
CD-ring ketone 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 ca. 15 h, during which time it was visually
checked. Upon observation of the light yellow color, the
reaction was quenched at -78 °C by addition of 5 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 (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 eluted with
50% ethyl acetate in hexanes in the presence of 1% triethy-
lamine, affording the coupled product.
This coupled product (0.018 mmol) was charged into a 5 mL
argon-purged polypropylene vial equipped with a magnetic stir
bar and dissolved in 0.9 mL of acetonitrile to give ca. 0.02 M
solution. To this well-stirred solution was added HF (1.8 mmol,
75 mL, 49% aqueous solution) via syringe at room tempera-
ture, and the mixture was then allowed to stir at room
temperature in the dark for 2 h. TLC showed the completion
of the reaction. This reaction mixture was diluted with ether
(25 mL), and a saturated solution of NaHCO3 was added until
no more carbon dioxide was liberated. The reaction mixture
was then rinsed into a separatory funnel with ethyl acetate
and was extracted with ethyl acetate (5 × 25 mL). The
combined extracts were washed with water (1 × 25 mL) and
brine solution (1 × 25 mL), dried over Na2SO4, and filtered.
The filtrate was concentrated in vacuo to give the crude
product that was purified by column chromatography.
General Procedure for Preparation of Ketones 7. A
flame-dried 10-mL recovery flask equipped with a magnetic
stir bar and a septum along with an Ar balloon was charged
with the appropriate alcohol 6 (0.043 mmol) dissolved in 1 mL
of freshly distilled CH2Cl2 to give ca. 0.04 M solution. To this
solution were added PDC (34 mg, 0.09 mmol) and 21 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 column chro-
matography.
Ketone 7a. Flash column chromatography eluted with
100% ethyl acetate afforded 12 mg of ketone (+)-7a in 81%
25
1
yield. Data for (+)-7a: [R]D ) +9.2 (c 0.4, CHCl3); H NMR
(CDCl3, 400 MHz) δ 7.98-7.95 (m, 2H), 7.65-7.54 (m, 3H),
3.21 (ddd, 1H, J ) 4.4, 12.0, and 13.6 Hz), 3.04 (ddd, 1H, J )
4.4, 12.0, and 13.6 Hz), 2.67 (s, 1H), 2.42 (dd, 1H, J ) 8.0 and
11.6 Hz), 2.30-2.16 (m, 2H), 2.05-1.95 (m, 2H), 1.93-1.65 (m,
5H), 1.60-1.35 (m, 4H), 1.27-1.19 (m, 1H), 0.91 (d, 3H, J )
6.4 Hz), 0.58 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 211.6,
141.9, 133.0, 129.2, 128.3, 61.7, 55.9, 54.7, 49.7, 40.8, 38.8, 34.4,
28.6, 27.2, 23.9, 18.9, 18.4, 12.4; IR (thin film) 3271 (w), 2942
(s), 2872 (s), 1701 (s), 1437 (sh, m), 1378 (w), 1219 (br, s), 1102
(w), 978 (m), 755(w) cm-1; HRMS calcd for C20H29NO2SNa+
[M + Na] 370.1811, found 370.1793.
Ketone 7b. Flash column chromatography eluted with
100% ethyl acetate afforded 13 mg of ketone (+)-7b in 87%
25
1
yield. Data for (+)-7b: [R]D ) +8.0 (c 0.4, CHCl3); H NMR
(CDCl3, 400 MHz) δ 7.98-7.95 (m, 2H), 7.65-7.54 (m, 3H),
3.18 (ddd, 1H, J ) 4.8, 12.0, and 13.6 Hz), 3.08 (ddd, 1H, J )
4.8, 12.0, and 13.6 Hz), 2.67 (s, 1H), 2.41 (dd, 1H, J ) 7.6 and
10.8 Hz), 2.30-2.16 (m, 2H), 2.06-1.95 (m, 2H), 1.93-1.80 (m,
2H), 1.78-1.64 (m, 3H), 1.57-1.33 (m, 4H), 1.27-1.19 (m, 1H),
0.92 (d, 3H, J ) 6.8 Hz), 0.59 (s, 3H); 13C NMR (CDCl3, 100
MHz) δ 211.6, 141.9, 133.0, 129.1, 128.3, 61.7, 55.8, 54.6, 49.7,
40.8, 38.8, 34.4, 28.6, 27.1, 23.9, 18.9, 18.4, 12.4; IR (thin film)
3271 (w), 2954 (s), 2872 (s), 1701 (s), 1443 (sh, m), 1219 (br,
s), 1096 (s), 978 (m), 749 (w) cm-1; HRMS calcd for C20H29-
NO2SNa+ [M + Na] 370.1811, found 370.1809.
Sulfoximine Analogues 3a and 3′a. Flash column chro-
matography eluted with 99% ethyl acetate in the presence of
1% triethylamine to afford 8.4 mg of a mixture of diastereo-
mers (+)-3a and (+)-3′a in 92% yield and in a ratio of 2.5:1,
respectively. This diastereomeric mixture was then separated
by HPLC on a Chiralcel OJ column [semipreparative (1 × 25
cm), flow rate ) 2.0 mL/min] eluted with 13% ethanol in
hexanes to afford 1.9 mg of (+)-3a and 1.0 mg of (+)-3′a in
21% and 11% yields, respectively. The retention time for (+)-
3a is 58.1 min, and for (+)-3′a, 45.7 min. Data for (+)-3a:
Ketone 7c. Flash column chromatography eluted with 50%
ethyl acetate afforded 42 mg of ketone 7c as an approximately
1:1 mixture of diastereomers in 85% yield. Data for 7c (as a
mixture): 1H NMR (CDCl3, 400 MHz) δ 7.99-7.94 (m, 4H),
7.65-7.60 (m, 2H), 7.57-7.54 (m, 4H), 2.8 (br s, 2H), 2.39-
2.34 (m, 2H), 2.29-2.14 (m, 4H), 2.04-1.94 (m, 4H), 1.92-
25
1
[R]D ) +80.4 (c 0.13, MeOH); H NMR (CDCl3, 400 MHz) δ
7.98-7.96 (m, 2H), 7.64-7.52 (m, 3H), 6.36 (d, 1H, J ) 11.2
Hz), 5.99 (d, 1H, J ) 11.2 Hz), 5.32 (t, 1H, J ) 1.6 Hz) 4.98

Vitamin D Analogues as Hydroxylase Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6859
(br s, 1H), 4.43-4.40 (m, 1H), 4.23-4.22 (m, 1H), 3.20 (ddd,
1H, J ) 4.4, 12.4, and 13.2 Hz), 3.03 (ddd, 1H, J ) 4.8, 12.4,
and 13.2 Hz), 2.82-2.78 (m, 1H), 2.65 (s, 1H), 2.61-2.58 (m,
1H), 2.33-2.28 (m, 1H) 2.04-1.90 (m, 3H), 1.82-1.75 (m, 2H),
1.69-1.42 (m, 8H), H), 1.28-1.20 (m, 4H), 0.88 (d, 3H, J )
6.4 Hz), 0.49 (s, 3H); 13C NMR (CH3OD-d3, 100 MHz) δ 149.9,
142.3, 140.8, 136.0, 135.2, 130.7, 129.9, 124.9, 119.3, 112.2,
71.6, 67.5, 57.5, 57.2, 55.5, 47.0, 46.2, 43.8, 41.8, 36.4, 30.3,
30.0, 28.4, 24.7, 23.3, 19.1, 12.4; IR 3387 (br, m), 3307 (br, m),
2942 (s), 2872 (m), 1443 (m), 1349 (w), 1213 (s), 1096 (m), 1055
(s), 1008 (m), 984 (sh, s), 749 (s) cm-1; HRMS calcd for C29H41-
NO3SNa+ [M + Na] 506.2699, found 506.2668; UV (MeOH)
and 14.0 Hz), 2.81 (dd, 1H, J ) 3.6 and 12.4 Hz), 2.58 (dd,
1H, J ) 3.6 and 13.6 Hz), 2.40 (dd, 1H, J ) 2.8 and 13.6 Hz),
2.23-2.13 (m, 2H), 2.02-1.94 (m, 2H), 1.87-1.45 (m, 11H),
1.39-1.14 (m, 5H), 0.90 (d, 3H, J ) 6.8 Hz), 0.52 (s, 3H); 13C
NMR (MeOD-d3, 100 MHz) δ 142.11, 141.90, 134.66, 134.19,
130.55, 129.81, 123.50, 117.41, 68.11, 67.83, 57.45, 57.24,
55.70, 46.88, 45.55, 42.80, 41.81, 37.76, 36.41, 30.47, 29.88,
28.44, 24.57, 23.31, 19.12, 12.48; IR 3330 (m, br), 2942 (s), 2872
(s), 1437 (m), 1213 (br, s), 1096 (m), 1049 (m), 978 (m), 749 (s)
cm-1; HRMS calcd for C28H41NO3SNa+ [M + Na] 494.2699,
found 494.2707; UV (MeOH) λmax 251 nm (ꢀ 28 100).
Sulfoximine Analogue 3d. Flash column chromatography
eluted with 99% ethyl acetate in the presence of 1% triethyl-
amine afforded 3.7 mg of (+)-3d in 70% yield. This analogue
was then further purified by HPLC on a Chiralcel OJ column
[semipreparative (1 × 25 cm), flow rate ) 2.5 mL/min] eluted
with 15% ethanol in hexanes to afford 2.3 mg of (+)-3d in 43%
yield. The retention time for (+)-3d is 35.22 min. Data for (+)-
25
λmax 265 nm (ꢀ 16 640). Data for (+)-3′a: [R]D ) +9 (c 0.09,
MeOH); 1H NMR (CDCl3, 400 MHz) δ 7.98-7.96 (m, 2H),
7.64-7.52 (m, 3H), 6.37 (d, 1H, J ) 11.6 Hz), 5.98 (d, 1H, J )
11.6 Hz), 5.31 (m, 1H) 4.98 (br s, 1H), 4.43-4.41 (m, 1H), 4.23-
4.19 (m, 1H), 3.23-317 (m, 1H), 3.07-2.99 (m, 1H), 2.83-2.80
(m, 1H), 2.66 (s, 1H), 2.62-2.60 (m, 1H), 2.32-2.27 (m, 1H)
2.00-1.90 (m, 5H), 1.81-1.64 (m, 7H), 1.25-1.21 (m, 6H), 0.87
(d, 3H, J ) 6.8 Hz), 0.48 (s, 3H); 13C NMR (CD3OD, 100 MHz)
δ 149.8, 142.4, 142.2, 135.9, 130.5, 129.8, 124.9, 124.9, 119.2,
112.4, 71.7, 67.5, 57.2, 55.8, 47.0, 46.4, 43.8, 41.8, 36.4, 30.5,
30.0, 28.4, 24.7, 23.4, 19.1, 12.4; IR: 3320 (br, m), 3307, 2940
(s), 2871 (m), 1445 (m), 1349 (w), 1214 (s), 1093 (m), 1053 (s),
1008 (m), 984 (sh, s), 749 (s) cm-1; HRMS calcd for C29H41-
NO3SNa+ [M + Na] 506.2699, found 506.2690.
25
1
3d: [R]D ) +82.3 (c 0.16, MeOH); H NMR (MeOD-d3, 400
MHz) δ 7.98-7.96 (m, 2H), 7.73-7.61 (m, 3H), 6.20 (d, 1H, J
) 11.2 Hz), 5.86 (d, 1H, J ) 11.2 Hz), 4.05-3.96 (m, 2H), 3.26
(dd, 1H, J ) 4.0 and 11.6 Hz), 3.19-3.13 (m, 1H), 2.81 (dd,
1H, J ) 3.6 and 11.6 Hz), 2.58 (dd, 1H, J ) 4.0 and 13.6 Hz),
2.40 (dd, 1H, J ) 3.6 and 14.0 Hz), 2.23-2.13 (m, 2H), 2.03-
1.93 (m, 2H), 1.84-1.46 (m, 12H), 1.33-1.17 (m, 5H), 0.89 (d,
3H, J ) 6.0 Hz), 0.51 (s, 3H); 13C NMR (MeOD-d3, 100 MHz)
δ 142.17, 141.91, 134.66, 134.20, 130.55, 129.81, 123.51,
117.41, 68.11, 67.84, 57.46, 57.29, 55.81, 46.89, 45.55, 42.81,
41.83, 37.76, 36.43, 30.52, 29.88, 28.46, 24.57, 23.31, 19.10,
12.49; IR 3330 (m, br), 2942 (s), 2872 (s), 1443 (m), 1213 (br,
s), 1096 (m), 1049 (m), 978 (s), 755 (s) cm-1; HRMS calcd for
C28H41NO3SNa+ [M + Na] 494.2699, found 494.2679; UV
(MeOH) λmax 251 nm (ꢀ 32 661).
Sulfoximine Analogues 3b and 3′b. Flash column chro-
matography eluted with 99% ethyl acetate in the presence of
1% triethylamine afforded 7.2 mg of a mixture of diastereomers
(+)-3b and (-)-3′b in 82% yield and in a ratio of 2.9:1,
respectively. This diastereomeric mixture was then separated
by HPLC on a Chiralcel OJ column [semipreparative (1 × 25
cm), flow rate ) 2.0 mL/min] eluted with 13% ethanol in
hexanes to afford 2.2 mg of (+)-3b and 1.0 mg of (+)-3′b in
25% and 11% yields, respectively. The retention time for (+)-
3b is 49.2 min, and for (+)-3′b, 40.2 min. Data for (+)-3b:
Sulfoximine Analogue 3e. Flash column chromatography
eluted with 99% ethyl acetate in the presence of 1% triethyl-
amine afforded 10.6 mg of (+)-3e in 88% yield. This analogue
was then further purified by HPLC on a Chiralcel OJ column
[semipreparative (1 × 25 cm), flow rate ) 2.5 mL/min] eluted
with 10% ethanol in hexanes to afford 3.0 mg of (+)-3e in 28%
yield. The retention time for (+)-3e is 31.47 min. Data for (+)-
3e: [R]D25 ) +36.6 (c 0.53, CHCl3); 1H NMR (CDCl3, 400 MHz)
δ 7.98-7.96 (m, 2H), 7.64-7.53 (m, 3H), 6.21 (d, 1H, J ) 11.6
Hz), 6.00 (d, 1H, J ) 11.2 Hz), 5.05-5.04 (m, 1H), 4.80 (d, 1H,
J ) 2.4 Hz), 3.96-3.91 (m, 1H), 3.20 (ddd, 1H, J ) 4.4, 12.4,
and 13.6 Hz), 3.04 (ddd, 1H, J ) 4.4, 11.2, and 13.6 Hz), 2.80
(dd, 1H, J ) 4.4 and 12.4 Hz), 2.56 (dd, 1H, J ) 4.0 and 13.2
Hz), 2.43-2.36 (m, 1H), 2.33 (dd, 1H, J ) 7.6 and 13.2 Hz)
2.20-2.13 (m, 1H), 1.96-190 (m, 4H), 1.82-1.42 (m, 10H),
1.28-1.16 (m, 4H), 0.87 (d, 3H, J ) 6.4 Hz), 0.49 (s, 3H); 13C
NMR (CDCl3, 100 MHz) δ 145.0, 142.0, 141.6, 135.3, 132.9,
129.1, 128.3, 122.2, 117.7, 112.4, 69.2, 56.1, 55.7, 54.8, 45.9,
45.7, 40.3, 35.2, 35.0, 31.9, 28.8, 28.7, 27.3, 23.4, 22.1, 18.5,
11.9; IR 3295 (m, br), 2931 (s), 2860 (m), 1443 (m), 1213 (br,
s), 1096 (m), 1061 (w), 984 (m), 749 (s) cm-1; HRMS calcd for
C29H41NO2SNa+ [M + Na] 490.2750, found 490.2723; UV
(MeOH) λmax 265 nm (ꢀ 15 648).
25
1
[R]D ) +37.3 (c 0.13, MeOH); H NMR (CDCl3, 400 MHz) δ
7.98-7.95 (m, 2H), 7.65-7.52 (m, 3H), 6.36 (d, 1H, J ) 11.6
Hz), 5.99 (d, 1H, J ) 11.2 Hz), 5.31 (m, 1H) 4.98 (br s, 1H),
4.43-4.40 (m, 1H), 4.29-4.22 (m, 1H), 3.18 (ddd, 1H, J ) 4.8,
12.4, and 14.0 Hz), 3.07 (ddd, 1H, J ) 4.8, 12.4, and 14.0 Hz),
2.83-2.80 (m, 1H), 2.66 (s, 1H), 2.61-2.58 (m, 1H), 2.33-2.28
(m, 1H) 2.20-1.80 (m, 6H), 1.74-1.62 (m, 8H), 1.30-1.116 (m,
4H), 0.88 (d, 3H, J ) 6.4 Hz), 0.49 (s, 3H); 13C NMR (CDCl3,
100 MHz) δ 147.6, 142.5, 133.2, 129.6, 128.6, 128.2, 124.8,
117.3, 111.8, 70.8, 66.8, 56.1, 55.7, 54.1, 45.9, 45.2, 42.8, 40.3,
34.9, 28.9, 28.2, 27.2, 23.4, 22.1, 18.5, 11.9; IR 3377 (br, m),
3318 (br, m), 2931 (s), 2872 (m), 1442 (m), 1214 (s), 1096 (m),
1055 (s), 1008 (m), 984 (sh, s), 749 (s) cm-1; HRMS calcd for
C29H41NO3SNa+ [M + Na] 506.2699, found 506.2676; UV
25
(MeOH) λmax 265 nm (ꢀ 15 383). Data for (+)-3′b: [R]D
)
+17.5 (c 0.09, MeOH); 1H NMR (CDCl3, 400 MHz) δ 7.98-
7.95 (m, 2H), 7.64-7.52 (m, 3H), 6.37 (d, 1H, J ) 11.2 Hz),
5.98 (d, 1H, J ) 11.2 Hz), 5.31 (m, 1H), 4.98 (br s, 1H), 4.43-
4.41 (m, 1H), 4.22-4.19 (m, 1H), 3.16 (ddd, 1H, J ) 4.4, 12.0,
and 13.2 Hz), 3.08 (ddd, 1H, J ) 4.4, 12.4, and 13.2 Hz), 2.82-
2.80 (m, 1H), 2.66 (s, 1H), 2.62-2.58 (m, 1H), 2.31-2.27 (m,
1H) 1.91-1.80 (m, 4H), 1.74-1.60 (m, 10H), 1.30-1.19 (m, 4H),
0.88 (d, 3H, J ) 6.4 Hz), 0.50 (s, 3H); due to insufficient
amount, 13C NMR was not obtained; IR 3307 (br, m), 2919 (s),
2860 (m), 1443 (m), 1219 (s), 1090 (m), 1055 (s), 984 (sh, s),
749 (s) cm-1; HRMS calcd for C29H41NO3SNa+ [M + Na]
506.2699, found 506.2673.
Sulfoximine Analogues 3f and 3g. Flash column chro-
matography eluted with 99% ethyl acetate in the presence of
1% triethylamine afforded 8.2 mg of a mixture of diastereomers
(+)-3f and (+)-3g in 79% overall yield and in a ratio of
approximately 1:1. This diastereomeric mixture was then
separated by HPLC on a Chiralcel OJ column [semipreparative
(1 × 25 cm), flow rate ) 2.0 mL/min] eluted with 7% ethanol
in hexanes to afford 1.2 mg of (+)-3g and 1.9 mg of (+)-3f in
30% and 48% yields, respectively. The retention time for (+)-
3g is 123.3 min, and for (+)-3f, 137.5 min. Data for (+)-3f:
Sulfoximine Analogue 3c. Flash column chromatography
eluted with 99% ethyl acetate in the presence of 1% triethyl-
amine afforded 9.1 mg of (+)-3c in 91% yield. This analogue
was then further purified by HPLC on a Chiralcel OJ column
[semipreparative (1 × 25 cm), flow rate ) 2.5 mL/min] eluted
with 15% ethanol in hexanes to afford 7 mg (+)-3c in 70%
yield. The retention time for (+)-3c is 30.25 min. Data for (+)-
25
1
[R]D ) +37.7 (c 0.12, CHCl3); H NMR (CDCl3, 400 MHz) δ
7.98-7.95 (m, 2H), 7.63-7.59 (m, 1H), 7.56-7.52 (m, 2H), 6.36
(d, 1H, J ) 11.2 Hz), 5.99 (d, 1H, J ) 11.2 Hz), 5.33 (m, 1H)
4.99-4.98 (br s, m, 1H), 4.45-4.41 (m, 1H), 4.26-4.17 (m, 1H),
2.79 (dd, 1H, J ) 4.0 and 12.4 Hz), 2.59 (dd, 1H, J ) 3.6 and
13.6 Hz), 2.54 (s, 1H), 2.31 (dd, 1H, J ) 6.4 and 13.6 Hz), 2.13
(d, 1H, J ) 14.8 Hz), 2.05-1.99 (m, 1H), 1.95-1.88 (m, 4H),
1.77-1.59 (m, 5H), 1.48-1.20 (m, 7H), 1.11-0.86 (m, 2H), 0.73
25
1
3c: [R]D ) +101.6 (c 0.46, MeOH); H NMR (MeOD-d3, 400
MHz) δ 7.98-7.96 (m, 2H), 7.73-7.62 (m, 3H), 6.20 (d, 1H, J
) 11.2 Hz), 5.86 (d, 1H, J ) 11.2 Hz), 4.04-3.96 (m, 2H), 3.26
(dd, 1H, J ) 4.8 and 12.0 Hz), 3.15 (ddd, 1H, J ) 4.8, 11.6,

6860 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Kahraman et al.
(d, 3H, J ) 6.4 Hz), 0.76-0.70 (m, 2H), 0.49 (s, 3H); 13C NMR
(CDCl3, 100 MHz) δ 147.59, 142.70, 140.75, 133.08, 129.47,
128.78, 128.55, 124.84, 117.18, 111.79, 70.80, 66.81, 56.94,
56.18, 45.92, 45.22, 42.85, 40.32, 40.29, 37.40, 34.13, 28.95,
27.66, 23.44, 22.22, 19.11, 12.41,12.03,11.74; IR 3334 (br, m),
2936 (s), 2872 (m), 1445 (m), 1284 (s), 1215 (br, m), 1119 (m),
1053 (br, s), 978 (w) 753 (s) cm-1; HRMS calcd for C31H43NO3-
SNa+ [M + Na] 532.2855, found 532.2860; UV (MeOH) λmax
A flame-dried 5-mL recovery flask equipped with a magnetic
stir bar and a septum along with an Ar balloon was charged
with p-fluorophenylmethyl sulfoximine (0.1 g, 0.58 mmol)
dissolved in 1.1 mL of anhydrous pyridine to give ca. 0.5 M
solution. To this solution was added TBSCl (0.1 g, 0.69 mmol)
neat in one portion. This mixture was allowed to stir at room
temperature for 12 h. TLC showed complete consumption of
the starting material. The reaction was quenched by addition
of water and then rinsed into a separatory funnel with 25 mL
of ethyl acetate. The mixture was extracted with ethyl acetate
(3 × 25 mL). The combined extracts were washed with brine
solution (1 × 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
10% ethyl acetate, affording 0.15 g of sulfoximine (()-10 as
an oil in 90% yield: 1H NMR (CDCl3, 400 MHz) δ 7.97-7.93
(m, 2H), 7.20-7.14 (m, 2H), 2.99 (s, 3H), 0.91 (s, 9H), 0.05 (s,
3H), 0.04 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 164.9 (d, J )
252.9 Hz), 141.2 (d, J ) 3.0 Hz), 129.6 (d, J ) 8.1 Hz), 116.0
(d, J ) 22.0 Hz), 49.7, 25.9, 17.9, -2.57; 19F NMR (CDCl3, 375
MHz) δ -107.3; IR 2954 (s), 2958 (s), 2885 (s), 2855 (s), 1589
(m), 1493 (m), 1322 (s), 1302 (s), 1284 (s), 1250 (m), 1163 (s),
1150 (s), 1090 (w), 1006 (w), 953 (w), 834 (s), 814 (m), 774 (s)
cm-1; HRMS calcd for C13H22FNOSSiNa+ [M + Na] 310.1067,
found 310.1043.
25
265 nm (ꢀ 15 684). Data for (+)-3g: [R]D ) +8.5 (c 0.08,
CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.97-7.94 (m, 2H), 7.62-
7.58 (m, 1H), 7.55-7.51 (m, 2H), 6.36 (d, 1H, J ) 11.6 Hz),
5.98 (d, 1H, J ) 11.2 Hz), 5.33 (m, 1H), 4.99 (br s, 1H), 4.45-
4.41 (m, 1H), 4.26-4.20 (m, 1H), 2.80 (dd, 1H, J ) 4.4 and
12.8 Hz), 2.59 (dd, 1H, J ) 3.6 and 13.6 Hz), 2.50 (s, 1H), 2.31
(dd, 1H, J ) 6.8 and 13.6 Hz), 2.15 (d, 1H, J ) 14.4 Hz), 2.05-
1.99 (m, 1H), 1.95-1.88 (m, 4H), 1.77-1.59 (m, 5H), 1.48-
1.20 (m, 7H), 1.11-0.86 (m, 2H), 0.75 (d, 3H, J ) 6.0 Hz),
0.75-0.72 (m, 2H), 0.46 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ
147.62, 142.74, 141.50, 133.06, 129.38, 128.77, 128.46, 124.87,
117.17, 111.76, 70.82, 66.84, 57.05, 56.19, 54.1, 45.93, 45.24,
42.88, 40.33, 37.80, 34.11, 28.96, 27.61, 23.45, 22.21, 19.13,
12.29 (2C), 12.01; IR 3330 (br, m), 2931 (s), 2872 (m), 1443
(m), 1219 (s), 1072 (m), 961 (sh, s), 890 (w) 749 (s) cm-1; HRMS
calcd for C31H43NO3SNa+ [M + Na] 532.2855, found 532.2826;
UV (MeOH) λmax 265 nm (ꢀ 9024).
Sulfoximine Analogues 3h and 3i. A flame-dried 5 mL
recovery flask equipped with a magnetic stir bar and a septum
along with an Ar balloon was charged with (()-10 (8.4 mg,
0.029 mmol) dissolved in 0.5 mL of freshly distilled THF. Then
the flask was cooled to -78 °C in a 2-propanol/dry ice bath.
To this solution was added 19 mL of n-BuLi (0.03 mmol, 1.6
M solution in hexanes) dropwise over several minutes, followed
by addition of 50 mL of HMPA, resulting in a yellow color.
This mixture was allowed to stir at -78 °C for an additional
30 min. Meanwhile, a flame-dried 5 mL pear-shaped flask
equipped with a septum along with an Ar balloon was charged
with iodide (+)-9 (5 mg, 0.0073 mmol), dissolved in 0.5 mL of
freshly distilled THF and cooled to -78 °C in a 2-propanol/
dry ice bath. The solution of iodide (+)-9 was transferred into
the flask containing the lithiated sulfoximine at -78 °C via
cannula over a few minutes. After the addition was complete,
the mixture was stirred at -78 °C for about 4-5 h. TLC
showed almost complete consumption of (+)-9. The reaction
was quenched by addition of 1 mL of pH 7 buffer 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 (1 × 10 mL) and brine
solution (1 × 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 first
with 100 mL of 100% hexanes and then with 10% ethyl acetate
in hexanes, affording 3.4 mg of 11 in 55% yield.
An argon-purged 5 mL polypropylene vial equipped with a
magnetic stir bar along with a cap was charged with 11 (3.4
mg, 0.004 mmol) dissolved in 0.4 mL of acetonitrile to give ca.
0.01 M solution. To this well-stirred solution was added HF
(0.44 mmol, 16 µL, 49% aqueous solution) via syringe at room
temperature, and the mixture was then allowed to stir at room
temperature in the dark for 4 h. TLC showed the completion
of the reaction. This reaction mixture was diluted with ether
(10 mL), and a saturated solution of NaHCO3 was added until
no more carbon dioxide was liberated. The reaction mixture
was then rinsed into a separatory funnel with ethyl acetate
and extracted with ethyl acetate (4 × 10 mL). The combined
extracts were washed with water (1 × 10 mL) and brine
solution (1 × 10 mL), dried over Na2SO4, and filtered. The
filtrate was concentrated in vacuo to give the crude product,
which was purified by flash column chromatography eluted
with 99% ethyl acetate in the presence of 1% triethylamine,
affording 1.7 mg of (+)-3h and (+)-3i in 84% yield. This
mixture was separated by HPLC on a Chiralcel OJ column
[semipreparative (1 × 25 cm), flow rate ) 2.5 mL/min] eluted
with 13% ethanol in hexanes to afford 0.51 mg of (+)-3h and
0.37 mg of (+)-3i in 19% and 26% yield, respectively. The
Preparation of p-Fluorophenylmethyl Sulfoximine 10.
A flame-dried 25-mL recovery flask equipped with a magnetic
stir bar, a septum, and an addition funnel along with an Ar
balloon was charged with p-fluorophenylmethyl sulfide (1 g,
7 mmol) dissolved in 14 mL of freshly distilled CH2Cl2. Then
the flask was cooled to 0 °C in an ice bath. To this solution
was added mCPBA (1.9 g 7.7 mmol, 70%) as a solution in 5
mL of CH2Cl2 dropwise via addition funnel over several
minutes. This mixture was allowed to stir at 0 °C for an
additional 2 h. TLC showed complete consumption of the
starting material. The reaction was quenched by addition of
water and then rinsed into a separatory funnel with 50 mL of
CH2Cl2. The mixture was extracted with CH2Cl2 (3 × 25 mL).
The combined extracts were washed with saturated NaHCO3
solution (1 × 10 mL) and brine solution (1 × 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 100% ethyl acetate.
affording 1 g of sulfoxide as an oil in 90% yield.
This sulfoxide (1 g, 6.3 mmol) was charged into a flame-
dried 25-mL recovery flask equipped with a magnetic stir bar,
a septum, and an addition funnel along with an Ar balloon
and dissolved in 6.3 mL of CHCl3. Then, 0.45 g of NaN3 (6.9
mmol) was added into the flask neat. Meanwhile, 1.53 mL of
concentrated H2SO4 was charged into the addition funnel and
allowed to drip into the reaction flask at 0 °C over several
minutes. The addition funnel was then replaced with a reflux
condenser and the flask was placed into an oil bath and heated
to 45 °C for overnight. TLC showed complete consumption of
the starting material. The reaction flask was cooled to room
temperature and the reaction was quenched by addition of
water, then the product rinsed into a separatory funnel with
50 mL of CHCl3. The mixture was extracted with CHCl3 (3 ×
25 mL). The combined extracts were washed with brine
solution (1 × 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
100% ethyl acetate affording 0.81 g of sulfoximine as a solid
in 74% yield. This was recrystallized from ethyl acetate: mp
1
93-94 °C; H NMR (CDCl3, 400 MHz) δ 8.06-8.02 (m, 2H),
7.26-7.21 (m, 2H), 3.12 (s, 3H), 2.76 (br s, 1H); 13C NMR
(CDCl3, 100 MHz) δ 165.37 (d, J ) 253.7 Hz), 139.4 (d, J )
2.7 Hz), 130.5 (d, J ) 9.1 Hz), 116.3 (d, J ) 22.8 Hz), 46.3; 19
F
NMR (CDCl3, 375 MHz) δ -105.6; IR 3268 (m), 3102 (w), 2928
(w), 1589 (s), 1493 (s), 1404 (w), 1321 (w), 1224 (s), 1094 (s),
1021 (m), 1004 (s), 946 (m), 840 (m), 817 (m), 753 (m) cm-1
;
HRMS calcd for C7H8FNOSNa+ [M + Na] 196.0202, found
196.0201.

Vitamin D Analogues as Hydroxylase Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6861
retention time for (+)-3h is 78.0 min, and for (+)-3i, 61.0 min.
yield. This was further purified by HPLC on a (S,S)-Whelk-
O1 column [semipreparative (1 × 25 cm), flow rate ) 3.0 mL/
min] eluted with 20% ethanol in hexanes to afford 0.8 mg of
3j. The retention time for 3j is 39.7 min. Data for 3j: 1H NMR
(CDCl3, 400 MHz): δ 8.04-8.02 (m, 2H), 7.64-7.62 (m, 1H),
7.57-7.52 (m, 2H), 6.35 (d, 1H, J ) 10.4 Hz), 6.01 (dd, 1H, J
) 11.2 and 32 Hz), 5.98 (d, 1H, J ) 11.6 Hz), 5.31 (br s, 1H)
4.96 (br s, 1H), 4.43-4.40 (m, 1H), 4.23-4.22 (m, 1H), 2.83-
2.80 (m, 1H), 2.29 (s, 1H), 2.66-2.56 (m, 1H), 2.36-2.28 (m,
1H), 2.04-1.90 (m, 3H), 1.82-1.62 (m, 4H), 1.40-1.25 (m, 9H),
1.09 (d, 3H, J ) 6.8 Hz), 0.49 (s, 3H); HRMS calcd for C29H38-
FNO3SNa+ [M + Na] 522.2448, found 522.2439; UV (MeOH)
λmax 266 nm (ꢀ 5447).
Data for (+)-3h: [R]D ) +12.3 (c 0.09, CHCl3); 1H NMR
25
(CDCl3, 300 MHz) δ 8.00-7.95 (m, 2H), 7.25-7.20 (m, 2H),
6.36 (d, 1H, J ) 11.22 Hz), 5.99 (d, 1H, J ) 11.22 Hz), 5.32
(br s, 1H), 4.98 (br s, 1H), 4.44-4.30 (m, 1H), 4.23-4.22 (m,
1H), 3.23-3.15 (m, 1H), 3.07-2.97 (m, 1H), 2.66 (br s, 1H),
2.60 (dd, 1H, J ) 3.39 and 13.26 Hz), 2.30 (dd, 1H, J ) 5.61
and 13.29 Hz), 2.04-1.88 (m, 4H), 1.81-1.44 (m, 11H), 1.30-
1.12 (m, 4H), 0.88 (d, 3H, J ) 6.33 Hz), 0.50 (s, 3H); due to
insufficient amount, 13C NMR was not obtained; IR (neat) 3299
(m), 2926 (s), 2869 (s), 1587 (m), 1491 (w), 1446 (w), 1140 (w),
1350 (m), 1220 (s), 1016 (w), 1057 (w), 994 (m), 836 (w), 752
(s) cm-1; HRMS calcd for C29H40FNO3SNa+ [M + Na] 524.2605,
found 524.2610; UV (MeOH) λmax 263 nm (ꢀ 11 096). Data for
Preparation of Sulfoximine 14. A flame-dried 10 mL
recovery flask equipped with a magnetic stir bar and a septum
along with an Ar balloon was charged with (+)-S-N-tert-
butyldimethylsilyl sulfoximine (43 mg, 0.16 mmol) and dis-
solved in 1.5 mL of freshly distilled THF. Then the flask was
cooled to -78 °C in a 2-propanol/dry ice bath. To this solution
was added 0.16 mL of t-BuLi (0.18 mmol, 1.1 M solution in
pentane) dropwise via syringe over several minutes, resulting
in a yellow color. This mixture was allowed to stir at -78 °C
for an additional 30 min. To this solution was added diethyl-
chlorophosphate (41.3 mg, 35 mL, 0.24 mmol) via syringe.
After the addition was complete, the mixture was stirred at
-78 °C for about 1 h. TLC showed that the starting material
was entirely consumed. The reaction was quenched by addition
of water (5 mL) and then the mixture was rinsed into a
separatory funnel and extracted with ethyl acetate (3 × 10
mL). The combined extracts were washed with water (1 × 10
mL) and brine solution (1 × 10 mL), dried over Na2SO4, and
filtered. The filtrate was concentrated in vacuo to give the
crude product. The crude product was purified by flash
chromatography eluted with a 9:1 mixture of ethyl acetate/
hexanes to give 34 mg of sulfoximine 14 as an oil in 53% yield.
Data for 14: [R]D22 ) +40.3 (c 0.55, CHCl3); 1H NMR (CDCl3,
300 MHz) δ 7.99-7.97 (m, 2H), 7.58-7.48 (m, 3H), 4.13-4.02
(m, 4H), 3.73 (dd, 1H, J ) 3.6 and 15.2 Hz), 3.68 (dd, 1H, J )
3.6 and 15.2 Hz), 1.28-1.21 (m, 6H), 0.91 (s, 9H), 0.05 (s, 6H);
13C NMR (CDCl3, 100 MHz) δ 144.46, 132.47, 128.55, 127.83,
62.74 (d, J ) 6.1 Hz), 62.55 (d, J ) 6.1 Hz), 58.02 (d, J ) 135.8
Hz), 25.85, 17.96, 16.22 (d, J ) 2.1 Hz), 16.16 (d, J ) 2.3 Hz),
-2.63, -2.65; IR (neat) 3066 (w), 2955 (m), 2929 (m), 2855
(m), 1473 (m), 1446 (m), 1391 (m), 1361 (m), 1320 (m), 1300
(s), 1253 (s), 1167 (s), 1052 (s), 1025 (s), 974 (m), 834 (s), 778
(m), 689 (m) cm-1; HRMS calcd for C17H32NO4PSSiNa+ [M +
Na] 428.1451, found 428.1435.
Sulfoximine Analogues 3k and 3l. A flame-dried 5 mL
recovery flask equipped with a magnetic stir bar and a septum
along with an Ar balloon was charged with phosphonate 14
(4.2 mg, 0.01 mmol) dissolved in 0.5 mL of freshly distilled
THF. Then the flask was cooled to -78 °C in a 2-propanol/dry
ice bath. To this solution was added 13 mL of t-BuOK (0.013
mmol, 1.0 M solution in THF) dropwise over several minutes
via syringe, resulting in a yellow color. This mixture was then
allowed to stir at -78 °C for an additional 30 min. Meanwhile,
a flame-dried 5-mL pear-shaped flask equipped with a septum
along with an Ar balloon was charged with 22-aldehyde (+)-
12 (5.0 mg, 0.0087 mmol) dissolved in 1.0 mL of freshly
distilled THF and cooled to -78 °C in a 2-propanol/dry ice bath.
The solution of (+)-12 was transferred into the flask containing
the anion of 14 at -78 °C via cannula over a few minutes.
After the addition was complete, the mixture was stirred at
-78 °C for about 1 h. Then the flask was warmed to room
temperature and allowed to stir for 1.5 h. TLC showed the
consumption of the starting material. Reaction was quenched
by addition of water (5 mL) and then the mixture was rinsed
into a separatory funnel extracted with ethyl acetate (3 × 10
mL). The combined extracts were washed with water (1 × 10
mL) and brine solution (1 × 10 mL), dried over Na2SO4, and
filtered. The filtrate was concentrated in vacuo to give the
crude product as a 2:1 mixture of isomers (5.0 mg, 0.006 mmol).
This mixture was then charged into an argon-purged 5 mL
polypropylene vial, equipped with a magnetic stir bar along
25
1
(+)-3i: [R]D ) +22.6 (c 0.05, CHCl3); H NMR (CDCl3, 300
MHz) δ 8.00-7.95 (m, 2H), 7.25-7.19 (m, 2H), 6.36 (d, 1H, J
) 11.22 Hz), 5.99 (d, 1H, J ) 11.31 Hz), 5.32 (br s, 1H), 4.98
(br s, 1H), 4.45-4.40 (m, 1H), 4.24-4.18 (m, 1H), 3.22-3.00
(m, 2H), 2.80 (dd, 1H, J ) 3.81 and 11.82 Hz), 2.67 (br s, 1H),
2.59 (dd, 1H, J ) 3.48 and 13.38 Hz), 2.31 (dd, 1H, J ) 6.36
and 13.44 Hz), 2.06-1.87 (m, 3H), 1.55-1.41 (m, 13H), 1.29-
1.15 (m, 4H), 0.88 (d, 3H, J ) 6.39 Hz), 0.50 (s, 3H); due to
insufficient amount, 13C NMR was not obtained; IR (neat) 3294
(m), 2921 (s),2866 (m), 1583 (m), 1490 (m), 1348 (m), 1222 (s),
1096 (w), 1052 (m), 997 (m), 838 (w), 750 (s) cm-1; HRMS calcd
for C29H40FNO3SNa+ [M + Na] 524.2605, found 524.2619; UV
(MeOH) λmax 263 nm (ꢀ 10 895).
Sulfoximine Analogue 3j. A flame-dried 5-mL recovery
flask equipped with a magnetic stir bar and a septum along
with an Ar balloon was charged with (S)-13 [7.5 mg, 0.026
mmol; 13 was prepared by treatment of (S)-N-tert-butyldi-
methylsilyl-S-methyl-S-phenylsulfoximine with n-BuLi in THF
at -78 °C followed by N-fluorobenzenesulfonimide (NFSI) in
42% yield] and dissolved in 0.5 mL of freshly distilled THF.
Then the flask was cooled to -78 °C in a 2-propanol/dry ice
bath. To this solution was added 50 mL of LHMDS (0.05 mmol,
1.0 M solution in THF) dropwise over several minutes,
resulting in a yellow color. This mixture was allowed to stir
at -78 °C for an additional 10 min. Meanwhile, 0.5 mL of THF
solution of chlorodiethyl phosphate (4.4 mg, 3.8 µL, 0.026
mmol) was transferred into the flask containing the lithiated
sulfoximine at -78 °C via cannula over a few minutes. After
the addition was complete, the mixture was stirred at -78 °C
for about 1 h. Then, 0.5 mL of a THF solution of aldehyde (+)-
12 (11 mg, 0.002 mmol) was transferred via cannula over a
few minutes at -78 °C. The reaction mixture was allowed to
warm to room temperature over 2 h. TLC showed almost
complete consumption of (+)-12. The reaction was quenched
by addition of 1 mL of pH 7 buffer and then rinsed into a
separatory funnel with ethyl acetate. The mixture was ex-
tracted with ethyl acetate (3 × 5 mL). The combined extracts
were washed with water (1 × 5 mL) and brine solution (1 × 5
mL), dried over Na2SO4, and filtered. The filtrate was concen-
trated in vacuo to give the crude product that was purified by
preparative TLC plate eluted with 10% ethyl acetate in
hexanes to give 7.8 mg of the coupled product in 67% yield.
An argon-purged 5 mL polypropylene vial equipped with a
magnetic stir bar along with a cap was charged with the
coupled product (7 mg, 0.0083 mmol) dissolved in 0.4 mL of
acetonitrile to give ca. 0.02 M solution. To this well-stirred
solution was added HF (0.83 mmol, 0.34 mL of 49% aqueous
solution) via syringe at room temperature, and the mixture
was then allowed to stir at room temperature in the dark for
2 h. TLC showed the completion of the reaction. This reaction
mixture was diluted with ether (10 mL) and a saturated
solution of NaHCO3 was added until no more carbon dioxide
was liberated. The reaction mixture was then rinsed into a
separatory funnel with ethyl acetate and was extracted with
ethyl acetate (4 × 5 mL). The combined extracts were washed
with water (1 × 10 mL) and brine solution (1 × 10 mL), dried
over Na2SO4, and filtered. The filtrate was concentrated in
vacuo to give the crude product, which was purified on a
preparative TLC plate eluted with 99% ethyl acetate in the
presence of 1% triethylamine to afford 3.2 mg of 3j in 78%

6862 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Kahraman et al.
with a cap, and dissolved in 0.6 mL of anhydrous acetonitrile
to give ca. 0.01 M solution. To this well-stirred solution was
added HF (0.6 mmol, 0.25 mL, 49% aqueous solution) via
syringe at room temperature, and the mixture was then
allowed to stir at room temperature in the dark for 4 h. TLC
showed the completion of the reaction. This reaction mixture
was diluted with ether (10 mL), and a 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
(3 × 10 mL). The combined extracts were washed with water
(1 × 10 mL) and brine solution (1 × 10 mL), dried over Na2-
SO4, and filtered. The filtrate was concentrated in vacuo to
give the crude product, which was passed through a pad of
silica gel to afford 2.3 mg of a mixture of 3k and 3l in 79%
yield. This mixture was separated by HPLC on a Chiralcel OJ
column [semipreparative (1 × 25 cm), flow rate ) 2.5 mL/min]
eluted with 17% ethyl acetate in hexanes to afford 800 mg of
3k and 540 mg of 3l in 28% and 19% yields, respectively. Data
for 3k: [R]D ) +7.7 (c 0.069, CHCl3); H NMR (CDCl3, 300
MHz) δ 7.96-7.93 (m, 2H), 7.61-7.48 (m, 3H), 6.83 (dd, 1H, J
) 8.9 and 15.0 Hz), 6.35 (d, 1H, J ) 12.60 Hz), 6.31 (d, 1H, J
) 15.00 Hz), 5.98 (d, 1H, J ) 10.83 Hz), 5.31 (s, 1H), 4.97 (s,
1H), 4.48-4.38 (m, 1H), 4.28-4.18 (m, 1H), 2.84-2.79 (m, 2H),
2.62-2.57 (m, 1H), 2.34-2.28 (m, 2H), 2.06-1.88 (m, 4H),
1.69-1.33 (m, 7H), 1.33-1.22 (m, 4H), 1.11 (d, 3H, J ) 6.6
Hz), 0.52 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 150.98, 147.59,
142.22, 133.34, 130.10, 129.15, 127.65, 124.74, 117.74, 114.95,
111.80, 70.80, 66.82, 55.91, 55.46, 46.12, 45.22, 42.83, 40.14,
39.24, 28.91, 27.06, 23.38, 22.22, 19.03, 12.31; IR (neat) 3295
(w), 2931 (m), 2872 (w), 1443 (m), 1414 (w), 1349 (m), 1220
(m), 1055 (m), 979 (m), 750 (m), 685 (m) cm-1; HRMS calcd
for C29H39NO3SNa+ [M + Na] 504.2543, found 504.2530; UV
(MeOH) λmax 261 nm (ꢀ 12 799). Data for 3l: 1H NMR (CDCl3,
300 MHz) δ 7.96-7.94 (m, 2H), 7.61-7.48 (m, 3H), 6.83 (dd,
1H, J ) 89.8 and 15.0 Hz), 6.35 (d, 1H, J ) 8.3 Hz), 6.31 (d,
1H, J ) 15.00 Hz), 5.98 (d, 1H, J ) 11.16 Hz), 5.31 (s, 1H)
4.97 (s, 1H), 4.47-4.38 (m, 1H), 4.28-4.18 (m, 1H), 2.81-2.76
(m, 2H), 2.60-2.57 (m, 1H), 2.34-2.25 (m, 2H), 2.06-1.87 (m,
4H), 1.62-1.37 (m, 7H), 1.33-1.25 (m, 4H), 0.99 (d, 3H, J )
6.6 Hz), 0.42 (s, 3H); due to insufficient amount, 13C NMR was
not obtained; HRMS calcd for C29H39NO3SNa+ [M + Na]
504.2543, found 504.2543; UV (MeOH) λmax 265 nm (ꢀ 7718).
Biochemical Studies. A recombinant cell line expressing
human CYP24 enzyme (V79-CYP24) was used to measure the
inhibitory properties of sulfoximine analogues on the enzyme
activity as described before.6,22,23 Briefly, V79-CYP24 cells
(250 000 cells/plate) in 150 µL of medium were incubated with
25 µL of sulfoximine analogue (10-6-10-9 M). After 10 min,
25 µL of substrate [3H-1â]-1R,25(OH)2D3 (20 nM) was added
and the plate was incubated for 2 h at 37 °C in a humidified
atmosphere containing 5% CO2. Both sulfone analogue and
radiolabeled substrate were prepared in Dulbecco’s modified
Eagle medium (DMEM) with 1% bovine serum albumin (BSA)
in the absence and presence of 100 µM 1,2-dianilinoethane,
respectively. The reaction was terminated by the addition of
500 µL of methanol and transferred to a glass tube. The
aqueous phase was extracted by standard Bligh-Dyer extrac-
tion in which we substituted dichloromethane for chloroform.24
Samples were then spun at 4000 rpm for 5 min. Triplicate 100
µL aliquots of aqueous fraction containing water-soluble
CYP24 products were mixed with 600 µL of scintillation fluid
and the radioactivity was measured by use of a scintillation
counter. All values were normalized for background.
Measurement of Enzyme Activity of CYP27A1. CYP27A1
activity was measured as described by Dilworth et al.24 with
the exception of the use of V79-CYP27A cells. Briefly, each
CYP27A assay was performed in 6-well plate format with V79-
CYP27A cells at 500 000 cells/well in a final volume of 600 µL
of DMEM containing 1% BSA. Solutions containing inhibitors
in assay medium were added in a volume of 25 µL to achieve
desired final concentrations (10-9-10-5 M). The final reaction
mixture also contained antioxidant DPPD (Sigma-Aldrich,
Milwaukee, WI) at a concentration of 100 nM and substrate
1R -(OH)D3 (Sigma-Aldrich) at a final concentration of 8 µM.
After 24 h of incubation at 37 °C, reactions were terminated
with the addition of 750 µL of methanol. The reaction mixtures
were transferred individually into disposable glass culture
tubes and subjected to Bligh-Dyer extraction with dichloro-
methane and saturated KCl solution. The lipophilic product
and substrate were recovered in organic phase, dried, and
subjected to HPLC analysis on a SIL 3 µm column in hexane/
2-propanol/methanol (91/7/2) isocratic mobile phase. The area
under the curve for both substrate and product was used to
estimate the conversion rate of 1R-(OH)D3 into calcitriol. The
normalized activity for each concentration point of the inhibi-
tors was calculated to quality control samples without inhibi-
tion with the conversion for noncell controls subtracted.
Measurement of Enzyme Activity of CYP27B. SW900
cells were plated in 24-well plates at 250 000 cells/well. The
cells were washed with 1× PBS followed by addition of 150
µL of actinomycin D (1 µg/mL) in medium per well and
incubation for 1 h to halt the RNA transcription. Solutions
containing inhibitors in assay medium were added in a volume
of 25 µL to achieve desired final concentrations (10-9-10-5 M).
The final reaction mixture also contained antioxidant DPPD
at a concentration of 100 nM and substrate 3H-(23,24)-25-
(OH)D3 (103 Ci/mmol) (Amersham, Piscataway, NJ) at a final
concentration of 10 nM. After incubation at 37 °C for 5 h,
reactions were stopped with the addition of 400 µL of metha-
nol. The reaction mixtures were transferred into Eppendorf
tubes and the solutions were dried in a Speed Vac concentra-
tor. The lipophilic substrate and metabolite were extracted in
80 µL of mobile phase by vortexing and sonication. Aliquots
(50 µL) of the mobile-phase extract were subjected to HPLC
analysis on a SIL 3 µm column in hexane/2-propanol/methanol
(91/7/2) isocratic mobile phase on a fully automated Waters
Alliance 2695 HPLC system (Milford, MA) and a Radioflow
detector LB509 (EG&G Berthold, Bundoora, Australia). The
area under the curve for both substrate and product was used
to estimate the conversion rate of 25-(OH)D3 into calcitriol.
The normalized activity for each concentration point of the
inhibitors was calculated to quality control samples without
inhibition with the conversion for noncell controls subtracted.
25
1
Determination of Urinary Calcium Levels.15 Male F344
rats (150 g) were housed individually in glass metabolism
cages and received food and water ad libitum. After several
days of acclimation, each rat received 1 µg/kg of weight of the
corresponding sulfoximine analogue per os for 7 consecutive
days in 150 µL of propylene glycol/0.05 M Na2HPO4 (80:20).
Urine samples, which were collected on ice, were centrifuged
at 650g for 100 min, adjusted to pH 6 as necessary, and
assayed for calcium content spectrophotometrically at 575 nm
by use of reagents and standards from Sigma calcium kit no.
587.
Acknowledgment. We thank the NIH (Grants CA
52857 and 93547 to G.H.P. and DK 50583 to S.P.) for
financial support. We also thank Kara Ryder and Hui
Wang for their technical assistance.
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(3) Bouillon, R.; Okamura, W. H.; Norman, A. W. Structure-
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Vitamin D Analogues as Hydroxylase Inhibitors
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