E. Sieva¨nen et al.
seems that the amount, position, and orientation of the hydroxyl
groups of the bile acids affect the adduct formation probabil-
ity. The greater amount and the α-orientation of the hydroxyl
group in position 7 seem to decrease the adduct formation
tendency – probably via increasing the dimerization probability
of the bile acid itself. Because bile acids are endogenous com-
pounds – they form as end products of cholesterol metabolism in
the liver[19] – and have an essential role in many of the digestive
processes,[20] they have a huge potential to be used in medici-
nal and pharmaceutical applications. Their large, rigid, and chiral
steroid nucleus, amphiphilicity, a unique disposition of hydroxyl
groups, availability, and inexpensivity have led to the use of bile
acids in pharmacology and in supramolecular applications, as
recently reviewed by us.[21,22]
In conclusion, we have shown how 3-ketosteroids can be
transformed to geminal difluoro derivatives in one step by DAST
treatment. Multinuclear 1H, 13C, and 19F magnetic resonance
techniques have been used to characterize the formed structures
in detail. The couplings induced by fluorine have been determined
to affect mainly on the 13C NMR signals of the steroidal A-ring. ESI
mass spectrometric results prove that 1 has a clear tendency to
form adducts with bile acids. Fluorinated steroids may thus have
potential in acting as agents aimed for binding and removing
steroids from the human system providing new possibilities in
the treatment of cardiovascular diseases, such as atherosclerosis.
These studies are in progress.
a 5 mm dual inverse detection probehead (BBI) and z-gradient
accessory working at 500.13 MHz for proton, 125.76 MHz for
carbon-13, and 470.54 MHz for fluorine-19, respectively. In 1H
NMR experiments, the chemical shifts were referenced to the
trace signal of CHCl3 (δ = 7.26 ppm from internal TMS) and
in 13C NMR experiments to the center peak of CDCl3 septet
(δ = 77.00 ppmfrominternalTMS),respectively.19FNMRchemical
shifts were referenced to the signal of an external neat CFCl3 in
a 1 mm diameter capillary inserted coaxially inside the 5 mm
NMR tube.
In1HNMRexperimentsthespectralwidthwas5000 Hz(10 ppm),
number of data points 65 K giving a 6.6 s acquisition time, and the
relaxation delay was 1 s. The flip angle was 30◦ and the number
of scans was 8. The FID was multiplied by a 0.1 Hz exponential
window function before FT. In proton decoupled (Waltz-16) 13C
NMR experiments the spectral width was 30 300 Hz (230 ppm),
number of data points 65 K giving a 1.1 s acquisition time, and the
relaxation delay was 2 s. The flip angle was 30◦ and the number
of scans was 2300. The FID was multiplied by a 1.0 Hz exponential
window function before FT.
In 19F NMR experiments the spectral width was 19 100 Hz
(40 ppm), number of data points 65 K giving a 1.7 s acquisition
time, and the relaxation delay was 1 s. The flip angle was 30◦ and
the number of scans was 32. The FID was multiplied by a 0.1 Hz
exponential window function before FT.
In PFG 1H,13C HMQC experiments (hmqcgpqf Bruker pulse
program) the matrix size was 1500 Hz (3 ppm)/1024 data points
1
for H-axis ×7560 Hz (60 ppm)/512 data points for 13C-axis. For
every 13C increment, 16 scans have been accumulated using garp
composite pulse decoupling during acquisition. The matrix size
was zerofilled to 2 K × 2 K and multiplied by a sine bell window
function along both axes before FT.
Experimental
5α-Androstan-3,17-dione and bile acids (purity >95%) utilized
were commercial products and used without further purification.
LCA was oxidized to 3-oxo-5β-cholan-24-oic acid at ambient
temperature with chromic acid.[23–25]
In PFG 1H,13C HMBC experiments (hmbcgplpndqf Bruker pulse
program), the matrix size was 1500 Hz (3 ppm)/1024 data points
1
for H-axis ×28 750 Hz (230 ppm)/1024 data points for 13C-axis.
Synthetic procedure for 3,3-difluoro-5β-cholan-24-oic acid (1)
and 3,3-difluoro-5α-androstan-17-one (2)
For every 13C increment 64 scans have been accumulated using
3.45 ms low pass filter to remove direct couplings in the beginning
of the pulse program and 50 ms delay for the evolution of
long-range couplings. The matrix size was zerofilled to 2 K × 2 K
and multiplied by a sine bell window function along both axes
before FT.
To a 25 ml flask equipped with a magnetic bar and starting
compound (50 mg, for 1 0.13 mmol, for 2 0.17 mmol), toluene
(5 ml), and DAST (for 1 645.5 mg, 30 eq., for 2 55 mg, 2 eq.) were
added. The reaction mixture was heated under stirring on an
oil bath for 19 h at 60 ◦C. The mixture was allowed to cool to
ambient temperature, after which 10 ml of water was added. The
formed mixture was extracted with CH2Cl2 (2 × 25 ml) and the
extract was dried with MgSO4. The yield of crude product of 1
was 49 mg. Compound 1 was purified with preparative TLC using
hexane : ethyl acetate 9 : 1 as an eluent giving 21 mg (38% yield)
MS measurements
Electrospray mass spectrometric measurements were performed
using LCT TOF mass spectrometer with ESI (Micromass LCT).
Controlling the LCT as well as acquiring and processing the data
were performed with a MassLynx NT software system. In each
experiment, a flow rate of 40 µl/min was used for the sample
solution and the sample droplets were dried with nitrogen gas.
Compound 1 was best ionized with negative ion mode, whereas
compound 2 gave the desired result with positive ion mode. For
1 the potentials of −65 V and −5 V for the sample and extraction
coneswereapplied.RFlenswassetatapotentialof−800 Vandthe
potential in the capillary at 5000 V. The desolvation temperature
was set at 180 ◦C and the source temperature at 100 ◦C. For 2 the
potentials of 35 V and 4 V for the sample and extraction cones
were applied. RF lens was set at a potential of 900 V and the
potential in the capillary at 4300 V. The desolvation temperature
was set at 120 ◦C and the source temperature at 100 ◦C. In the
adduct formation experiments, the measurement conditions were
identical to those described for 1 above, except that the value of
sample cone was set to −40 V.
1
of white crystalline material. The purity of 1 was checked by H
NMR (500 MHz, CDCl3, 303 K, Table 2) and mass spectrometry. MS
m/z ESI-TOF− found 395.34 [M − H]− (100%), C24H37F2O2 requires
395.28; 791.73 [2M − H]− (4.4%), C48H75F4O4 requires 791.56.
After isolation by preparative TLC, 35 mg of crude product of
2 was obtained. The crude product crystals were washed with
hexane giving 24 mg (45% yield) of white crystalline material.
The purity of 2 was checked by 1H NMR (500 MHz, CDCl3, 303 K,
Table 3) and mass spectrometry. MS m/z ESI-TOF+ found 273.21
(11%); 293.19 [M − F + H]+ (100%), C19H30FO requires 293.23;
311.22 [M + H]+ (25%), C19H29F2O requires 311.22.
NMR measurements
All NMR spectra were run in dilute CDCl3-solutions at 303 K using
Bruker Avance DRX 500 FT NMR spectrometer equipped with
c
Copyright ꢀ 2008 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2008; 46: 392–397