Complete 1H and 13C NMR spectral assignment of 17-hydroxy epimeric sterols with planar A or A and B rings.

Article abstract of DOI:10.1002/mrc.1342

Complete 1H and 13C spectral assignments of 17beta- and 17alpha-hydroxy epimers of three biologically active sterols (boldenone, 3-methoxyestradiol and 3-methoxydihydroequilenin) were achieved making use of one- and two-dimensional NMR techniques (1D-HOHAHA, DEPT, COSY, NOESY, TOCSY, HSQC and COLOC). Copyright 2004 John Wiley & Sons, Ltd.

Full text of DOI:10.1002/mrc.1342

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2004; 42: 360–363
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1342
Spectral Assignments and Reference Data
Complete H and 13C NMR spectral
assignment of 17-hydroxy epimeric sterols
with planar A or A and B rings
1
OH
OH
1
1
2∗
P. Ciuffreda, S. Casati and A. Manzocchi
O
1
Dipartimento di Scienze Precliniche LITA Vialba, Universit a` di Milano,
Via G. B. Grassi 74, 20157 Milan, Italy
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina,
1
2
3
2
Universit a` di Milano, Via Saldini 50, 20133 Milan, Italy
Received 1 August 2003; revised 5 September 2003; accepted 9 September 2003
Complete 1H and 13C spectral assignments of
7b- and 17a-hydroxy epimers of three biologically
active sterols (boldenone, 3-methoxyestradiol and 3-
methoxydihydroequilenin) were achieved making use
of one- and two-dimensional NMR techniques (1D-
HOHAHA, DEPT, COSY, NOESY, TOCSY, HSQC and
COLOC). Copyright  2004 John Wiley & Sons, Ltd.
H CO
3
1
OH
KEYWORDS: NMR; H NMR; 13C NMR; 1D-HOHAHA; COSY;
NOESY; TOCSY; HSQC; COLOC; sterols; 17ˇ- and 17˛-hydroxy
epimers
1
H CO
3
a = α
b = β
INTRODUCTION
Figure 1. Structures of compounds 1a–3a and 1b–3b.
The stereochemistry at the 17-position of a steroid plays a key role
in its biological activity. A recent study showed how in rat pituitary
tumor cells vascular endothelium growth factor-A is induced by two
different signaling pathways: through a classical estrogen receptor
necessary in order to use a single solvent instead of mixtures. CDCl3
and not C6D6 was chosen owing to the bad resolution of the 1
H
(ER) interaction by 17ˇ-estradiol and through an ER-independent
signals of ring D in C6D6.
1
pathway by 17˛-estradiol. Studies on new aspects of 17˛-estradiol
2
metabolism in humans showed that it could give health benefits
to aging people, either women or men. Recent studies on 17˛-
RESULTS AND DISCUSSION
dihydroequilenin, an equine estrogen used in menopause therapy
3
All assignments of the 1H and 13C chemical shifts and cou-
pling constants are given in Tables 1 and 2. The resonances of
the compounds were derived from the combined information
since 1942, have shown its positive effects on cardiac synaptosomes
and on brain cells.
4
1H and 13C NMR data, nowadays essential for structural
assignments, can also be useful for the prediction of biological
activity; in fact, the use of 13C spectral data can produce a pre-
14
observed in 1D and 2D (COSY-45, NOESY, TOCSY, HSQC and
15
COLOC ) experiments. Where proton resonances overlapped, 1D
5
dictive model of ER binding activity. In this paper, we report
16
HOHAHA spectra were used to obtain chemical shifts and cou-
1
13
complete H and C NMR data for three pairs of 17ˇ- and 17˛-
hydroxysterols: 17ˇ-hydroxyandrosta-1,4-dien-3-one (boldenone)
1b), 3-methoxyestradiol (2b), 3-methoxydihydroequilenine (3b) and
pling constants.
The carbon type (C, CH, CH2, CH3) was determined using
DEPT experiments. All protonated carbons were assigned without
ambiguity using HSQC experiments. Quaternary carbons are well
correlated with protons in long-range CH correlation experiments
(
their ˛-epimers (1a, 2a and 3a) (Fig. 1).
NMR data collected from the literature are scattered. No 13C
NMR data for 1a are known and partial H NMR data in CDCl3
1
(
COLOC; see Experimental section). For 1a the signal at υ 44.3
6
1
were reported in a study on the synthesis of 17˛-hydroxysterols.
H
was assigned to C-10 owing to its long-range couplings with H-
2, H-4 and H-19, whereas the signal at υ 46.0 was assigned to
C-13 owing to its long-range coupling with H-18 in the COLOC
spectrum. For 1b the same correlations were observed for the
signals at υ 44.3 (assigned to C-10) and υ 43.8 (assigned to C-
13).
1
3
and C NMR data for 1b are known, but whereas the former were
reported in CDCl3 the latter were described only in dioxane. The
only data available for 2a are some characteristic H NMR parameters
in a paper describing its synthesis. For 2b, whereas a C NMR
spectrum in CDCl3 is known, the H NMR spectrum was measured
in C6D6. The proton spectra of estradiol and its 17˛-epimer have
been reported in a mixture of solvents (CDCl3 –DMSO-d6ꢀ and
their C NMR data are known in acetone-d6. Finally no data were
found either for equine estrogens or for methoxy derivatives 3a and
7
8
1
9
13
1
0
1
1
1
Also for 3a the correct chemical shift values for C-5, C-8, C-
9 and C-10 were assigned due to the three-bond correlations,
in particular for C-5 with H-1 and H-7, for C-8 with H-6,
for C-9 with H-1, H-7 and H-12ˇ, and for C-10 with H-2, H-
4 and H-6. For 3b the correct chemical shift values for the
corresponding quaternary carbons were determined in a similar
way. For 3-methoxyestradiol epimers (2a and b) quaternary
carbons assignments were obtained by comparison with literature
1
2
1
3
13
3b.
We report here complete assignments of the 1H and 13C NMR
spectra of 1a–3a and natural epimers 1b–3b in CDCl3 with the aim
of providing mutually comparable data that have not been reported
previously. The methylation of the phenolic hydroxyl group was
1
3
data.
In the 13C NMR spectra, the most important difference was at C-
8, whose signals were 6 ppm downfield in the a series. An opposite
1
Ł
outcome was found for the C-12 signals, lying 5 ppm upfield in
17˛-hydroxy (a) epimers.
The correlations in the NOESY spectra between CH3-18 protons
and 16ˇ, 15ˇ and 11ˇ protons were used to assign the diastereotopic
Correspondence to: A. Manzocchi, Dipartimento di Chimica, Biochimica e
Biotecnologie per la Medicina, Universit a` di Milano, Via Saldini 50, 20133
Milan, Italy. E-mail: ada.manzocchi@unimi.it
Contract/grant sponsor: Universit a` degli Studi di Milano (Fondi FIRST).
Copyright  2004 John Wiley & Sons, Ltd.

361
Spectral Assignments and Reference Data
Table 1. 13C and 1H chemical shifts, υ (ppm from TMS)
Atom
1b
1a
2b
2a
3b
3a
13C
1
2
3
4
5
6
7
8
9
156.6
128.1
187.7
124.5
169.9
33.4
33.8
36.2
53.2
44.3
23.2
37
156.7
128.1
187.2
124.4
170.0
33.5
34.6
36.8
52.8
44.3
23
127.0
112.0
158.0
114.4
138.6
30.4
28
127.0
112.1
158.1
114.5
138.7
30.6
28.8
39.8
44.3
133.4
26.9
32.2
46.2
48.5
25.0
33.1
80.7
17.8
—
125.5
119.0
157.4
107.3
133.9
125.9
125.8
134.7
131.2
128.1
25.0
125.5
119.0
157.3
107.3
133.8
125.9
126.8
135.7
131.0
128.1
25.2
39.5
44.5
133.3
27.0
37.4
43.9
50.7
23.8
31.2
82.5
11.7
—
10
11
12
13
14
15
16
17
18
19
31.7
46
34.6
29.8
43.8
50.8
24.2
31
43.6
45.7
48.4
25.4
33
47.1
45.4
24.2
25.7
32.0
34.2
82
80.1
17.6
19.4
—
81.7
80
11.8
19.4
—
10.9
16.6
—
—
OCH3
55.8
55.9
56.0
56.0
1H
1
7.03
7.05
7.19
7.20
7.86
7.87
2
6.21
6.05
2.34
2.45
0.98
1.93
1.65a
1.03
1.75
1.66a
1.07
1.85
0.94
1.59a
1.31
2.05
1.44
3.62
—
6.21
6.05
2.34
2.45
1.12a
1.97
1.61a
1.08a
1.80
1.70
1.62
1.55
1.39
1.76
1.22
1.46
2.16
—
6.70
6.61
6.70
6.60
7.14
7.10
7.56
7.14
7.10
7.56
4
6
6
7
7
8
9
1
1
1
1
1
1
1
1
1
1
1
˛
2.83
2.84
ˇ
Overlaps 6˛
1.32a
1.86
Overlaps 6˛
1.42
˛
7.15
7.23
ˇ
1.90
ꢁˇꢀ
ꢁ˛ꢀ
1˛
1ˇ
2˛
2ˇ
4ꢁ˛ꢀ
5˛
5ˇ
6˛
6ˇ
7˛
7ˇ
1.43a
1.39
2.22a
—
—
2.18
—
—
2.30
2.35
3.29
3.18
1.70a
2.19a
2.79
2.17a
1.77
2.34
1.68a
3.94
—
3.36
3.22a
2.13
1.87
3.23a
2.37
1.66
1.75
2.42
—
1.48a
1.28a
1.94
1.52a
1.74
1.60a
1.61a
1.85
1.18
1.70
1.35a
1.26
2.10
1.51a
2.19a
—
1.48a
3.71
3.74
0.72
1.22
—
—
3.80
3.99
0.61
—
1
8-CH3
9-CH3
0.79
1.22
—
0.78
0.68
0.69
—
1
—
—
OCH3
3.76
3.76
3.90
3.90
a
Data from HOHAHA spectra.
protons in 1a, 3a and 3b. NOESY experiments also confirmed that
the 12˛ proton is at higher field than the 12ˇ proton in ˇ compounds
of the ˛ series the H-17ˇ signal (υ 3.74 for 1a, υ 3.80 for 2a and
υ 3.99 for 3a) was a broad doublet showing a coupling constant
of 6.0 Hz with H-16ˇ and a very small coupling constant ꢁ<1 Hzꢀ
(1b, 2b and 3b) whereas in ˛ compounds (1a, 2a and 3a) the chemical
shifts of the same protons showed the opposite trend.
between H-17ˇ and H-16˛, depending on the dihedral angle close
to 90° between them. In contrast, in compounds of the ˇ series, each
1
In the H NMR spectra, the differences between the ˛ and ˇ series
were more evident for ring D signals, as expected. In compounds
H-17˛ signal at υ 3.63 for 1b, υ 3.71 for 2b and υ 3.94 for 3b was
Magn. Reson. Chem. 2004; 42: 360–363
Copyright  2004 John Wiley & Sons, Ltd.

362
Spectral Assignments and Reference Data
Table 2. J(H,H) values (Hz)
Atoms
1b
1a
2b
2a
3b
3a
1
2
4
6
6
6
6
6
7
7
7
8
8
9
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
, 2
10.2
1.7
10.2
1.9
8.5
2.8
8.6
2.8
9.1
2.8
9.1
2.8
, 4
, 6ˇ
1.7
1.7
—
—
˛, 6ˇ
ꢀ13.2
4.5
ꢀ13.2
4.4
Not detectable
Not detectable
6.6
—
—
˛, 7˛
6.7a
8.4
8.4
˛, 7ˇ
2.5
2.5
2.4
2.2
ˇ, 7˛
13.3
4.9
13.4
4.9
11.9a
6.1
11.4
6.1
ˇ, 7ˇ
˛, 7ˇ
ꢀ12.6
11.2
ꢀ13.5
11.2
ꢀ12.7
ꢀ11.4
11.6
2.6
9.9a
10.8a
—
—
a
a
a
˛, 8ˇ
11.6
—
—
—
—
ˇ, 8ˇ
3.8a
12.0
11.0
4.2
10.5a
ꢀ13.2
4.5
3.8a
12.2a
11.1
4.2a
10.8a
ꢀ13.2
4.0
2.8
11.2
10.7
4.3
ˇ, 9˛
—
—
ˇ, 14˛
, 11˛
—
—
4.2
—
—
, 11ˇ
11.2
ꢀ13.2
4.2
12.6a
ꢀ13.2
4.3
—
—
1˛, 11ˇ
1˛, 12˛
1˛, 12ˇ
1ˇ, 12˛
1ˇ, 12ˇ
2˛, 12ˇ
4˛, 15˛
4˛, 15ˇ
5˛, 15ˇ
5˛, 16˛
5˛, 16ˇ
5ˇ, 16˛
5ˇ, 16ˇ
6˛, 16ˇ
7˛, 16˛
7˛, 16ˇ
7ˇ, 16˛
7ˇ, 16ˇ
8-CH3, 12˛
1ˇ, 14˛
ꢀ18.0
7.8
1
11a
7.8a
ꢀ17.7
7.8
2.8
3.4
2.9
13.0a
2.8
1
12.6
4.2
12.3
12.9
4.0a
11.7
7.8
4.7
3.8
a
a
ꢀ12.7
7.4
ꢀ12.7
ꢀ12.7
7.2
ꢀ12.7
7.2
ꢀ12.7
ꢀ12.7
7.4
a
7.2
7.7
11.9
ꢀ12.2
9.6
12.2
ꢀ12.2
9.6
12.2
12.2
ꢀ12.0
9.4
12.2
ꢀ12.0
9.4
12.0a
ꢀ12.0
9.1
a
ꢀ12.0
9.3
2.8
3.5
3.3
2.8
2.8a
2.8
5.7
6.8
5.8
6.7
5.8
6.8
11.7
ꢀ13.3
8.4
11.0
ꢀ15.0
—
12.0
ꢀ13.1
8.4
11.0
11.5
ꢀ13.2
8.4
11.2
ꢀ15.0
a
ꢀ15.0
—
—
8.4
—
8.4
8.4
<1
<1
6.0
<1
<1
6.0
<1
1.5a
—
6.1
—
<1
1
<1
<1
<1
a
Data from HOHAHA spectra.
a double doublet of the same coupling constant with H-16˛ and
H-16ˇ.
EXPERIMENTAL
General procedures
Melting-points were recorded on a Stuart Scientific SMP3 instrument
and are uncorrected. All reagents were obtained from commercial
sources and used without further purification. The starting sterols
In both the ˛ and ˇ series, the signals of H2-16 showed a
positional difference of 0.6–0.7 ppm: the 16˛ proton was downfield
in 17ˇ-epimers whereas in the 17˛-epimers the downfield signal
was that of H-16ˇ. For H-14, a difference of 0.45 ppm downfield
was observed between the position of the proton in 17˛-epimers
with respect to the 17ˇ-epimers. The shift of the H3-18 singlet is
upfield in 17˛-epimers and for a long time it has been diagnostic of
epimerization of the 17-hydroxy functionality. In ring C, the position
of the H2-12 signals was significantly affected by the stereochemistry
of 17-hydroxy functionality. The H-12˛ signal of the 17ˇ-epimers
was upfield of that of the ˇ proton (0.5–0.8 ppm) whereas in the 17˛-
epimers the positions were reversed and much closer. The greatest
difference was in 3a where H-12˛ was at υ 2.13 and H-12ˇ at
υ 1.87 ppm.
1
7ˇ-hydroxyandrosta-1,4-dien-3-one (1b), estradiol and dihydroe-
quilenine were purchased from Sigma.
Representative general synthetic procedure
3-Methoxyestradiol (2b) and 3-methoxydihydroequilenine (3b) were
prepared from equilenin and estradiol, respectively, by selective
methylation (K2CO3, MeI). 17ˇ Alcohol inversion was obtained using
1
7
a modified Mitsunobu protocol as follows. Diethyl azodicarboxy-
late (0.68 ml, 4.4 mmol) was added dropwise to an ice-cold, mag-
netically stirred suspension of 1b (0.50 g, 1.75 mmol), 4-nitrobenzoic
acid (0.74 g, 4.4 mmol) and triphenylphosphine (1.15 g, 4.4 mmol) in
In conclusion, the H and 13C chemical shifts and H,H coupling
constant assignments for 1a–3a and 1b–3b have been achieved by
the combined use of one- and two-dimensional NMR techniques and
have been given as mutually comparable data.
1
dry toluene under nitrogen. After complete addition, the reaction
mixture was heated to 80 °C for 2 h. Evaporation of the solvent and
flash chromatography [light petroleum–ethyl acetate (1 : 1) as eluant]
Copyright  2004 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2004; 42: 360–363

363
Spectral Assignments and Reference Data
Table 3. Melting-points and yields of
7˛-alcohols
Typical acquisition conditions for HSQC experiments were as
follows: relaxation delay 2.0 s, 512 t1 increments, 2048 t2 points,
1
1
13
1
sweep width 8.0 ppm for H and 150 ppm for C, JꢁC,Hꢀ 140 Hz.
Zero filling in F1, sine-bell squared and shifted (ꢂ/2 in both
dimensions) apodization functions were used for processing.
For COLOC experiments the relaxation delay was 2.0 s. The
Melting-point
Yield
(%)
Compound
( °C)

1 and 2 delays, optimized via refocused INEPT experiments,
1a
2a
3a
176–177
114–115
108–109
56
58
62
were slightly different for 1a and b and 3a and b: in the former
3
case they were 72 and 36 ms, corresponding to JꢁC,Hꢀ D 6.94 Hz,
whereas in the latter they were 74 and 37 ms, corresponding to
3
JꢁC,Hꢀ D 6.76 Hz. The proton sweep width was 7.5 ppm for all
1
3
compounds, whereas for C the sweep widths were different, i.e.
5
.2 ppm for 1a, 3 ppm for 1b, 14 ppm for 3a and 18 ppm for 3b.
afforded the product as a white solid (0.30 g, 0.7 mmol). The com-
pound was dissolved in a 1% solution of NaOH in MeOH (10 ml) with
stirring at room temperature. After 2 h, the solvent was evaporated
under vacuum. The residue was extracted with CH2Cl2 ꢁ3 ð 10 mlꢀ
and the residue was purified on a chromatographic column [light
petroleum –ethyl acetate (1 : 1) as eluant] to give 1a (0.28 g, 56%),
which crystallized from MeOH as a white solid. The yields and
melting-points of 1a, 2a and 3a are reported in Table 3.
All experimental data processed using the Bruker software
package.
Acknowledgement
This work was financially supported by the Universit a` degli Studi
di Milano (Fondi FIRST).
NMR spectroscopy
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1
13
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4
5
1
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1
3
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1
2
048 t2 points; sweep width 7.5 ppm. Zero filling in F1, sine-bell
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1
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1
pulse for H; 90° pulse for MLEV17; TRIM pulse 2.5 ms, mixing
1
time 80 ms; 512 t1 increments; 2048 t2 points; sweep width 7.5 ppm
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Copyright  2004 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2004; 42: 360–363