High-field NMR studies of 3β-tetrahydropyranyloxy steroids

Article abstract of DOI:10.1016/S0039-128X(00)00103-3

Comprehensive NMR studies were carried out on 3β-hydroxy-pregnene and cholestene analogs, each containing a tetrahydropyranyl ether group at the 3-position. Two-dimensional NMR experiments (COSY, TOCSY, HSQC, and HSQC-TOCSY) permitted the complete assignments of both the ¹H and ¹³C resonances of these derivatives in deuterated benzene or chloroform. The aromatic solvent-induced NMR signal shifts (ASIS) were also investigated. Copyright (C) 2000 Elsevier Science Inc.

Full text of DOI:10.1016/S0039-128X(00)00103-3

Steroids 65 (2000) 415–422
High-field NMR studies of 3␤-tetrahydropyranyloxy steroids૾
a
a,
b
c
Zsuzsanna Szendi , P e´ ter Forg o´ *, Katalin E. K o¨ v e´ r , Frederick Sweet
a
Department of Organic Chemistry, J o´ zsef Attila University, Szeged, Hungary
b
Department of Organic Chemistry, Kossuth Lajos University, Debrecen, Hungary
c
Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO, USA
Received 17 November 1999; received in revised form 20 March 2000; accepted 23 March 2000
Abstract
Comprehensive NMR studies were carried out on 3␤-hydroxy-pregnene and cholestene analogs, each containing a tetrahydropyranyl
ether group at the 3-position. Two-dimensional NMR experiments (COSY, TOCSY, HSQC, and HSQC-TOCSY) permitted the complete
1
13
assignments of both the H and C resonances of these derivatives in deuterated benzene or chloroform. The aromatic solvent-induced
NMR signal shifts (ASIS) were also investigated. © 2000 Elsevier Science Inc. All rights reserved.
Keywords: Steroids; Tetrahydropyranyl ethers; Two-dimensional NMR spectroscopy
1
. Introduction
bling. Chemical shift differences of signals belonging to
different diastereoisomers are only a few Hertz, depending
on the distance from the new chiral center, and exhibit
solvent dependency. Nevertheless, very often, only the
sharp singlets in the proton spectra due to the angular
methyl signals at the C-18 and C-19 positions are used as
sensitive indicators to differentiate the two diastereoiso-
mers. When the 3␤-position contains a tetrahydropyranyl
ether group, the most sensitive signals in the carbon spec-
trum are those due to C-1, C-2, C-3, and C-4 in the steroid
moiety.
The 2-tetrahydropyranyl ether is the most frequently
used blocking group for protecting hydroxyl functions while
modifying the molecular structures of well-known steroids
under strongly alkaline reaction conditions [1–8].
Introducing a tetrahydropyranyloxy (OTHP) group into a
steroid molecule produces an additional asymmetric center
and therefore a new pair of diastereoisomers. Diastereoi-
somerism associated with the 2-tetrahydropyranyl ether
group is well established [1,9–13], and the NMR assign-
ments are available for the simple 2-alkoxy- and 2-phe-
noxytetrahydropyrans from 3,4-dihydro-2H-pyran and the
corresponding alcohols [14–17]. Similarly, the NMR spec-
tra of the tetrahydropyranyl protecting group condensed
with C-3 and C-17␤ in estrone and estradiol serving as
models were comprehensively studied [18].
The tetrahydropyranyl group constituting a 2ЈR and 2ЈS
diastereoisomeric mixture makes the complete configura-
tional determination of new products difficult for validating
stereochemistry by spectroscopic methods. In NMR spec-
troscopy, these diastereoisomeric pairs result in signal dou-
The present report describes complete assignments for
1
13
the H and C NMR spectra from (2ЈR,S) diastereoiso-
meric mixtures of steroid tetrahydropyranyl ethers of the
well-known steroids: 3␤-hydroxypregn-5-en-20-one 3-(2Ј-
tetrahydropyranyl) ether 1; 3␤-hydroxypregna-5,16-dien-
2
0-one 3-(2Ј-tetrahydropyranyl) ether 2; (20R)-pregn-5-
ene-3␤,20-diol 3-(2Ј-tetrahydropyranyl) ether 3; and also
cholesterol analogs: 22,26-epoxy-(20R)-27-norcholesta-
5
3
,22-diene-3␤,20-diol 3-tetrahydropyranyl ether 4 [11];
␤,20-dihydroxy-(20S)-27-norcholest-5-en-25-one ethyl-
ene ketal 3-(2Ј-tetrahydropyranyl) ether 5; 3␤,20,25-trihy-
droxy-(20R)-26,27-bisnorcholest-5-en-22-one 3-(2Ј-tetra-
hydropyranyl) ether 6 [11]. This report also describes the
CDCl and C D solvent influences on the chemical shifts
૾
Financial support was received from the Hungarian National Science
Foundation Grants OTKA T 20938 and FKFP 0728/1997 (to Z.S.), and
National Institutes of Health Grant 5RO1 DK15708-25 (to F.S.).
3
6 6
1
13
of H and C in the mixtures of (2ЈR)- and (2ЈS)-tetrahy-
*
Corresponding author. Tel./fax: ϩ36-62-54-42-00.
E-mail address: pforgo@sol.chem.u-szeged.hu (P. Forg o´ ).
dropyranyl ether diastereoisomers.
0
039-128X/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(00)00103-3

4
16
Z. Szendi et al. / Steroids 65 (2000) 415–422
Scheme 1.
2
. Experimental procedures
described in an earlier paper [11]. Melting points (mp) were
measured in a Kofler block and are reported without cor-
rections. The melting points varied with the ratios of the 2ЈR
and 2ЈS diastereoisomers in the tetrahydropyranyl ether
mixtures. Reactions were monitored on thin-layer chroma-
tography plates (TLC) (Merck 5554 layer), eluted with
methanol:benzene (1:9 v/v). The migrating components
were detected under ultraviolet (UV) light (254 and 366
nm), and visualized by spraying the plates with concentrated
2
.1. General
NMR spectra were recorded at room temperature on a
Bruker AM-400 spectrometer using a 5-mm probehead (at
4
6
ton-detected heteronuclear polarization transfer experiments
were carried out on a Bruker AC-400 instrument, using
5
3
00.13 MHz and 100.62 MHz). Samples of compounds 1 to
were dissolved in CDCl or C D . Two-dimensional pro-
3
6 6
H SO and heating them at 120°C for 10 min. Infrared (IR)
2
4
spectra were recorded with KBr pellets in a UNICAM SP
00 instrument.
-mm reverse constructed broad-band probe. An Aspect-
000 workstation was used for data processing. Chemical
2
shift data are presented in ppm values and internal TMS was
used as reference material.
2.2. 3␤-Hydroxypregn-5-en-20-one 3-(2Ј-tetrahydro-
pyranyl) ether (1) [5]
All reagents and solvents were dried and fractionally
distilled prior to their use. In the present study, the conden-
sation of 3,4-[2H]-dihydropyran with 3␤-hydroxysteroids
was complete within 5 min. Compounds 3 and 5 were made
from 3␤-hydroxypregn-5-en-20-one 3-(2Ј-tetrahydropyra-
nyl) ether (1). Preparations of compounds 4 and 6 are
To pregnenolone (3.165 g, 0.01 mol) dissolved in 25 ml
of dry CH Cl :benzene (40:10 v/v) were added 10 ml (0.1
2
2
mol) of 3,4-[2H]-dihydropyran (DHP) and 0.2 g p-toluene-
sulfonic acid hydrate. Within 5 min, all of the starting

Z. Szendi et al. / Steroids 65 (2000) 415–422
417
1
13
Fig. 1. H (bottom) and C (top) spectra of 1.
material was completely converted into 1 in an exothermic
reaction. The mixture was chilled with ice-water, neutral-
ized with triethylamine, and then saturated with cold water.
The product was extracted with benzene, and the organic
(2.93 g, 7.3 mmol); mp: 120–127°C. Concentrating the
mother liquor provided an additional 0.71 g (1.77 mmol,
17.7%) of 1. The overall yield was 91%, R : 0.68.
f
Analysis calculated for C H O : C, 77.95; H, 10.06.
2
6 40 3
layer was dried over MgSO . The combined extracts were
Found: C, 77.82; H, 9.97.
4
filtered, and the solvent was removed under reduced pres-
sure. The light yellow syrup was applied to the surface of a
column packed with neutral, activated alumina and purified
by chromatography. The steroidal tetrahydropyranyl ether
was eluted with benzene, and the solvent was evaporated.
The residue was crystallized by adding small amounts of
benzene and methanol, to give white crystals in 73% yield
2.3. 3␤-Hydroxypregna-5,16-dien-20-one 3-(2Ј-tetrahy-
dropyranyl) ether (2)
Compound 2 was prepared similar to 1 from 9.5 g (0.03
mol) 3␤-hydroxypregna-5,16-dien-20-one. Column chro-
matography with alumina was used for purification. The

4
18
Z. Szendi et al. / Steroids 65 (2000) 415–422
Fig. 2. Two-dimensional heteronuclear single quantum coherence (HSQC) spectrum of 1.
tetrahydropyranyl ether was eluted with acetone:benzene
5:100 v/v). Fractions were collected, and the solvent was
Anal. calculated for C H O : C, 77.56; H, 10.51.
26 42 3
Found: C, 77.48; H, 10.36.
(
evaporated to give 6.6 g (16.55 mmol, 55.2%) of a white
crystalline powder after recrystallizing this material from
2
.5. 3␤,20 Dihydroxy-(20S)-27-norcholest-5-en-25-one
acetone; mp: 165–171°C, R : 0.66. An additional 2.19 g
f
ethylene ketal 3-(2Ј-tetrahydropyranyl) ether (5)
(5.49 mmol, 18.3%) 2 was obtained by evaporating the
mother liquor. Overall yield 73.5% in crystalline form.
Anal. calculated for C H O : C, 78.34; H, 9.61. Found:
C, 78.45; H, 9.59.
Magnesium (0.73 g; 0.03 mol) was activated under N2
2
6 38 3
with a few drops of ether containing one drop of CH I. The
3
reaction flask was slightly warmed to evaporate the ether;
then, 6.23 ml (0.04 mol) 5-chloropentan-2-one-ethylene
ketal was slowly added in 20 ml tetrahydrofuran. A white
solid precipitated, which dissolved after adding all of the
reagent followed by 30 ml of anhydrous tetrahydrofuran.
The reaction mixture was heated under reflux for 1.5 h.
Then 8.0 g (0.02 mol) of 3␤-hydroxypregn-5-en-20-one
3-(2Ј-tetrahydropyranyl) ether (1) in 25 ml tetrahydrofuran
was added dropwise to the solution, and the mixture was
heated under reflux for 4 h until the reaction was complete.
Excess Grignard reagent was decomposed by adding to it a
2
.4. (20R)-Pregn-5-ene-3␤,20-diol 3-(2Ј-tetrahydro-
pyranyl) ether (3)
Lithium-tri-terc-butoxyaluminum-hydride (0.01 mol)
was dissolved in 20 ml of dry tetrahydrofuran and then
heated for 5 min under reflux (N ). The mixture was cooled
2
to 2°C, then 2.0 g (5 mmol) of 3␤-hydroxypregn-5-en-20-
one 3-(2Ј-tetrahydropyranyl) ether (1) in 10 ml tetrahydro-
furan were added. After stirring the resulting mixture at 0°C
for 1.5 h, and at room temperature overnight, it was worked
saturated NH Cl ice water solution, and stirring the result-
4
up by adding 3.0 g NH Cl in 40 ml ice-water and repeatedly
ing mixture at room temperature for 1 h. The product was
extracted with three 50-ml portions of ether. The combined
ethereal fractions were washed twice with 15 ml of satu-
rated aqueous NH Cl, then with water and dried (MgSO ).
The product was purified by chromatography in a column of
alumina, eluted with ether:hexane (1:4 v/v). The yield of the
4
extracting with benzene. The combined benzene extracts
were washed with water, dried (Na SO ), filtered, and then
2
4
concentrated to dryness. Crystallization of the solid from
acetone provided 1.64 g (4.07 mmol, 81.5%) of a white
4
4
crystalline product. R : 0.58; mp: 127–134°C.
f

Z. Szendi et al. / Steroids 65 (2000) 415–422
419
Fig. 3. Two-dimensional HSQC-TOCSY spectrum of 1.
chromatographed product was 9.45 g (17.8 mmol, 89%) and
after its recrystallization from acetone, 7.88 g (14.8 mmol,
C-20 by NMR was complicated by the contribution to its
spectrum of the 3-OTHP group and its C-2Ј diastereoiso-
mers. Many attempts by a variety of methods to separate the
2ЈR and 2ЈS diastereoisomers for resolving their NMR spec-
tra were unsuccessful. However, the NMR spectra of the
3␤-tetrahydropyranyloxy steroids in deuterated benzene
yielded a resolvable doubling of the signals due to diaste-
reoisomeric C-2Ј.
7
4.2%) of the title product was obtained in pure crystalline
form. R : 0.57, mp: 132–134°C.
f
Anal. calculated for C H O : C, 74.67; H, 10.25.
3
3 54 5
Found: C, 74.73; H, 10.39.
1
The H spectrum arising from the steroid moiety in the
3
. Results and discussion
Earlier, we condensed 3,4-[2H]-dihydropyran or 2,3-di-
3-OTHP derivatives showed double signals for the hydrogens
at the C-2 and C-4 positions. Also, small differences in chem-
ical shifts were observed in the NMR spectra of the angular
hydrofuran with pregnenolone, which provided a novel
starting material for synthesis of a new series of 20-hy-
droxycholesterol analogs [11]. The C-3␤ hydroxyl in preg-
nenolone was protected by a tetrahydropyranyl group
against the strongly alkaline reagents 6-(3,4-[2H]-dihydro-
pyranyl)-lithium and 2-lithiodyhydrofuran. The synthesis
produced excellent yields of the 20R and 20S isomers in
each of the reactions. The major product (20R) was found to
have the same configuration in its side-chain as cholesterol.
The pure 20R isomer was readily isolated by fractional
crystallization. But determination of the configuration at
13
methyl groups. However, the only double signals in the
C
NMR spectra were produced by the carbon atoms in the steroid
moiety; and not from the tetrahydropyranyl group.
1
13
Analyses of the H and C NMR spectra from 4 and 6
in CDCl shows mostly a single line series. However, re-
cording the spectra of the compounds in deuterated benzene
3
1
13
produces line separations. The H and C spectra of the
starting material 1 similarly exhibited doubling of lines in
deuterated benzene. This must be caused by diastereoisom-
erism associated with the OTHP groups.

4
20
Z. Szendi et al. / Steroids 65 (2000) 415–422
Table 1
1
Chemical shift values for H-NMR from compounds 1 through 6 in CDCl or C D
3
6
6
1
2
3
4
5
6
CDCl3
C D
CDCl3
C D
CDCl3
C D
CDCl3
C D
CDCl3
C D
CDCl3
C D
6 6
6
6
6
6
6
6
6
6
6
6
1
2
1.84; 1.05 1.72; 1.01 1.78; 1.00 1.65; 0.96 1.86; 1.08 1.74; 1.05 1.84; 1.03 1.73; 0.98 1.84; 1.05 1.74; 0.97 1.85; 1.07 1.77; 1.03
.74; 1.02
1.88; 1.58 2.13; 1.78 1.84; 1.55 1.86; 1.52 1.89; 1.63 2.10; 1.76 1.88; 1.45 1.91; 1.55 1.87; 1.62 2.05; 1.72 1.89; 1.47 2.12; 1.78
1
1
3.52
2.34
.87; 1.45 1.92; 1.57 1.82; 1.40 2.04; 1.69 1.87; 1.48 1.94; 1.54
1.87; 1.48 1.87; 1.52
3.54 3.69
2.56; 2.39 2.36
1.93; 1.59
3.75
2.69; 2.61
3
4
3.72
3.45
3.67
3.50
3.73
3.54
2.35
3.72
2.71; 2.62 2.36
2.53; 2.40
3.54
2.70; 2.59 2.28; 2.13 2.65; 2.57 2.34; 2.19 2.46
2.48; 2.35
2
.33; 2.18 2.50; 2.38 2.30
2.34; 2.20 2.50; 2.39
6
5.32
5.33
.37
5.28
5.33
5.38
5.35
5.40
5.43
5.35
5.42
5.39
5.35
5.41
5.39
5.33
5.39
5.42
5
7
8
9
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
3
4
5
6
2
3
4
5
1.97; 1.55 1.84; 1.45 1.95; 1.54 1.84; 1.52 1.97; 1.54 1.95; 1.56 1.96; 1.53 1.92; 1.52 1.97; 1.55 1.91; 1.50 1.95; 1.54 1.89; 1.53
1.44
0.96
1.30
0.85
1.61
0.95
1.54
0.97
1.48
1.50
0.97
1.53
1.50
0.95
1.48
1.46
0.92
1.46
1.47
0.91
1.44
1.50
0.90
1.51
1.40
0.87
1.40
1.52
0.95
1.55
1.47
0.94
1.44
1
2
4
5
6
7
8
9
0
1
2
3
4
5
6
Ј
Ј
Ј
Ј
Ј
Љ
Љ
Љ
Љ
1.60; 1.48 1.40; 1.25 1.53
2.03; 1.44 1.81; 1.08 2.34; 1.27 2.71; 1.38 2.08; 1.26 2.19; 1.23 2.06; 1.25 2.12; 1.22 2.10; 1.20 2.02; 1.04 2.17; 1.34 2.04; 1.12
1.13 0.82 1.36 1.26 1.04 0.92 1.03 0.94 0.98 0.84 1.00 0.83
1.67; 1.20 1.50; 1.08 2.23; 1.96 1.93; 1.70 1.68; 1.20 1.46; 0.99 1.59; 1.10 1.57; 1.11 1.60; 1.20 1.54 1.57; 1.16 1.46; 1.09
1.63; 1.14 1.53; 1.01 1.67; 1.57 1.83; 1.66 1.80; 1.62 1.82; 1.63 1.54; 1.17 1.62; 1.12
2.17; 1.60 2.33; 1.48 6.62
6.10
—
0.94*
0.95*
—
2.33
0.63
1.01
—
2.10
0.56*
0.91*
—
—
1.33
0.77
1.01
3.72
1.14
—
1.22
0.74*
1.00*
3.54
1.01
—
1.77
0.83
1.00
—
1.37
—
1.88
0.97*
0.98*
—
1.53
—
1.44
0.85
1.00
—
1.35
0.87*
0.97*
—
1.75
0.89
1.02
—
1.45
—
1.36
1.02
0.97*
—
1.31
—
0.87
0.98
—
2.13
—
1.83
—
2.18
—
1.96*
—
1.31
1.22
1.47; 1.31 1.44; 1.32
—
—
—
—
4.70
1.54
—
—
—
—
4.82
1.65
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
4.84
—
—
—
—
—
—
—
—
1.38
1.58
—
1.26
4.72
1.70
1.68; 1.53 3.66
3.32
1.69
2.38
—
1.67
—
1.33
4.83
1.62
1.87
2.66
—
4.64
1.67; 1.49 1.64
4.82; 4.79 4.70
4.71
1.72; 1.55 1.64
4.73
4.71
4.84
1.73; 1.54 1.65
1.71; 1.55 1.64; 1.44
1.81; 1.30 1.83; 1.33 1.78; 1.48 1.84; 1.53 1.83; 1.53 1.81; 1.32 1.84; 1.52 1.84; 1.34 1.82; 1.53 1.79; 1.62 1.83; 1.53 1.82; 1.32
1.52 1.34 1.48 1.39; 1.31 1.53 1.32 1.52 1.38; 1.30 1.53 1.31 1.54 1.38; 1.30
3.89; 3.48 3.93; 3.44 3.82; 3.40 3.88; 3.39 3.90; 3.48 3.89; 3.42 3.92; 3.49 3.92; 3.42 3.90; 3.48 3.89; 3.43 3.90; 3.49 3.91; 3.41
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3.96
1.76
2.03
4.75
—
3.69
1.44
1.83
4.71
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
(CH O)
3.94
3.61
2
2
*
Chemical shift values labeled with * represent duplicate signals. However, the line separation is so small that the same chemical shifts can be given for
both diastereoisomers.
1
The configurational assignment of (20R)-4 and (20R)-6
at C-20 was readily accomplished after removing the OTHP
group under controlled acidic conditions when single iso-
mers could be obtained. It could then be confirmed that the
main cause of difficulties in establishing the configurations
of the compounds by NMR was diastereoisomerism due to
OTHP in (20R)-4 and (20R)-6. Although each of these
compounds was homogeneous with respect to C-20, they
were diastereoisomeric mixtures due to C-2Ј in the OTHP
group. Subsequently, we isolated both 20R and 20S isomers
in their 3␤-OH and 3␤-acetate forms.
chemical shifts for H-NMR signals associated, for exam-
ple, with angular methyl groups or olefinic protons. Yet
more than 70% of the H signals typically exhibit reso-
1
nances in a very narrow region (2.5 ppm) near 1.5 ppm
(bottom spectrum in Fig. 1). The separations between sig-
1
3
1
nals for the steroid C spectra are wider than for H, but too
often they are not wide enough to make unambiguous as-
signments (top spectrum in Fig. 1). Further complicating
this problem, after condensing a tetrahydropyranyl protect-
ing group with a 3␤-hydroxysteroid, five additional carbon
atoms and nine hydrogen atoms are introduced into the
molecule. This renders the high-field NMR region that
much more complex to resolve analytically. Within the
tetrahydropyranyl group, the C-2Ј hydrogen exhibits a
chemical shift at around 4.7 ppm. The 6Ј methylene protons
An important objective of the present study was to derive
1
13
complete assignments for the H and C signals of tetra-
hydropyranyloxy derivatives 1–6 (Scheme 1). Deriving the
molecular structure of steroids from their NMR spectra is
generally based on applying well-established, characteristic
1
resonate at 3.8 and 3.4 ppm. The chemical shifts due to H

Z. Szendi et al. / Steroids 65 (2000) 415–422
421
Table 2
Chemical shifts for C-NMR from compounds 1 through 6 in CDCl or C D
6 6
1
3
3
1
2
3
4
5
6
CDCl3
C D
CDCl3
C D
CDCl3
C D
CDCl3
37.20
C D
CDCl3
C D
CDCl3
C D
6 6
6
6
6
6
6
6
6
6
6
6
1
2
3
4
5
6
37.14
37.58
37.78
28.54
30.33
76.05
37.02
37.27
27.91
29.60
75.83
75.91
38.75
40.23
141.47
141.64
120.77
120.84
31.53
30.15
50.52
38.02
37.83
30.78
29.03
76.33
75.98
41.61
39.93
142.54
142.37
121.53
37.48
37.24
29.69
28.00
75.00
37.81
37.61
30.37
28.59
76.41
76.19
41.15
39.49
141.58
141.41
121.63
37.77
37.57
30.40
28.60
76.28
76.08
41.12
39.44
141.43
141.26
121.73
37.40
37.15
29.64
27.93
75.89
37.87
37.67
30.42
28.62
76.30
76.17
41.11
39.47
141.41
141.25
121.68
121.64
32.24
31.68
50.55
37.40
37.15
29.60
27.89
75.89
37.93
37.72
30.42
28.61
76.31
76.15
41.11
39.45
141.42
141.26
121.63
3
7.39
27.88
9.59
27.98
75.09
2
75.82
7
6.21
38.65
39.36
41.03
141.14
141.31
121.48
40.27
39.87
40.23
40.19
38.72
141.04
140.86
121.41
121.33
31.77
31.27
50.06
50.03
36.76
36.73
20.88
40.15
38.67
4
0.13
140.82
41.00
121.12
141.18
140.88
121.41
121.34
31.91
141.12
121.40
141.07
140.88
121.28
121.20
31.71
1
1
21.20
31.79
31.74
49.95
7
8
9
32.14
31.99
50.39
32.47
30.92
51.42
32.46
32.10
50.69
31.78
31.37
51.16
32.23
31.68
50.59
32.23
31.63
50.68
31.77
50.22
31.24
50.19
4
9.92
36.75
6.71
1
1
0
1
37.02
36.98
21.29
36.90
36.94
20.60
37.71
37.66
21.51
21.47
35.65
46.89
57.10
32.68
143.64
143.57
156.11
16.43
19.84
195.58
27.31
—
36.75
20.93
37.18
37.14
21.36
21.32
40.17
42.64
56.49
25.95
24.90
36.76
20.90
37.15
37.10
21.29
37.02
36.98
21.29
36.75
36.72
20.90
37.12
37.09
21.36
3
21.00
2
1.33
1
1
1
1
1
2
3
4
5
6
38.78
43.95
56.84
24.43
22.75
38.93
43.75
56.87
24.62
23.14
34.61
46.06
56.40
32.19
144.26
38.79
42.51
56.22
25.68
24.58
38.76
42.33
56.51
23.58
22.89
40.35
42.81
57.31
24.03
23.38
40.06
42.63
56.82
23.75
22.34
40.49
42.87
57.11
24.14
22.75
40.39
43.69
56.84
23.94
21.99
40.76
43.96
57.08
24.36
22.45
1
44.31
17
18
19
20
21
22
23
24
25
26
2Ј
63.65
13.16
19.91
209.57
31.49
—
—
—
—
63.58
13.24
19.44
206.91
31.13
—
—
—
—
155.38
15.65
19.23
196.77
27.06
—
—
—
—
58.46
12.36
19.42
70.44
23.71
—
—
—
—
58.63
12.44
19.54
70.19
24.04
—
—
—
—
56.92
13.31
19.36
75.98
25.90
—
—
—
—
56.48
13.67
19.50
75.18
27.13
—
—
—
—
57.14
13.55
18.74
74.99
26.29
43.93
18.74
39.66
109.99
23.72
96.84
96.68
31.21
19.99
19.92
25.47
62.74
62.65
—
58.36
13.72
19.50
75.11
26.66
44.72
19.13
40.45
110.12
24.05
96.66
96.59
31.52
19.74
19.68
26.02
61.91
61.83
—
55.09
13.30
19.33
80.19
24.63
214.24
61.82
26.63
32.02
—
96.90
96.73
31.18
19.99
19.90
25.43
62.82
62.71
—
55.59
13.72
19.53
80.28
25.01
213.96
61.60
27.09
32.06
—
96.76
96.69
31.62
19.91
19.82
26.07
62.04
61.94
—
—
—
—
96.73
96.63
96.69
31.58
19.76
19.85
26.03
61.88
61.98
—
—
—
—
—
96.83
97.21
97.04
32.07
20.29
96.85
96.77
31.29
20.01
96.54
96.95
96.79
31.25
19.99
96.60
9
6.90
3
4
Ј
Ј
31.19
19.91
31.23
20.60
31.59
19.80
31.59
19.85
19.78
26.06
61.82
61.96
66.21
22.64
20.28
92.22
160.33
—
2
0.00
5
6
Ј
Ј
25.42
62.72
25.45
62.73
62.77
—
—
—
—
—
—
26.54
62.38
62.34
—
—
—
—
—
—
25.52
62.76
26.06
61.85
25.49
62.76
6
—
—
—
—
—
—
2.83
2Љ
3Љ
4Љ
5Љ
6Љ
—
—
—
—
—
—
—
—
—
—
—
—
66.21
22.29
19.99
92.64
159.11
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
(
CH O)
—
64.53
64.65
—
—
2
2
in the C-3Ј, C-4Ј, and C-5Ј methylene groups fall within the
those from steroid carbons in the high-field region. Often
these signals cannot readily be identified.
resonance signals due to analogous ring hydrogens in the
1
3
steroid. The C spectra of these compounds follow similar
patterns. The carbons bonded to oxygen in the tetrahydro-
pyranyl group have large chemical shifts. The signal for
C-2Ј is near 97 ppm and C-6Ј is at 62 ppm. The three ring
methylene carbons C-3Ј, C-4Ј, and C-5Ј exhibit signals near
Knowing that C-2Ј and C-6Ј have nuclei with lower field
chemical shifts, we used these signals as starting points in
polarization transfer experiments for identifying the tetra-
hydropyranyloxy group. Similarly, signals belonging to C-3
and C-6 nuclei can also be used to start polarization trans-

4
22
Z. Szendi et al. / Steroids 65 (2000) 415–422
fers. The HSQC spectrum of 1 is shown in Fig. 2. The
high-field correlations (C-6, C-2Ј, C-3, C-6Ј, and C-17) can
easily be identified from this spectrum. However, low-field
signals are in a crowded region at the upper right of this
two-dimensional map. The HSQC-TOCSY experiment [19]
References
[1] Green TW. Protection for the hydroxyl group. In: Protective groups in
organic synthesis. New York: Wiley, 1981. p. 21.
[
2] Greenhalg CW, Henbest HB, Jones ERH. Studies in the sterol group.
Part LI. Reactions between 17-ketosteroids and organometallic com-
pounds. J Chem Soc 1951;1190–3.
(Fig. 3) provided relayed magnetization transfer inside the
spin-system, thus allowing the complete and unambiguous
assignments to be made for the NMR spectra.
Assignments for the ring methylene carbons in the
OTHP group (C-3Ј, C-4Ј, and C-5Ј) were made by ap-
plying HSQC-TOCSY methods. Also, these types of ex-
periments were useful in making the assignments for the
[
3] Ott AC, Murray MF, Pederson RL. The reaction of dihydropyran with
steroidal alcohols. Utility in the synthesis of testosterone acyl esters.
J Am Chem Soc 1952;74:1239–41.
[4] Van Boom JH, Herschied JDM, Reese CB. p-Toluenesulfonic acid as
a catalyst for the rapid tetrahydropyranylation and methoxy-tetrahy-
dropyranylation of steroidal alcohols. Synthesis 1973;169–70.
[
5] Alper H, Dinkes L. Boron trifluoride etherate-catalized formation of
steroidal O-tetrahydropyran-2-yl derivatives. Synthesis 1972;81.
6] Bongini A, Cardillo G, Orena M, Sandri S. A simple and practical
method for tetrahydropyranylation of alcohols and phenols. Synthesis
1979;618–20.
2
0-substitued side-chain. We found that doubled signals
in both proton and carbon spectra for compounds 1
through 6 are associated with the C-2Ј diastereoisomers
of the OTHP group. The differences between the chem-
ical shifts for the corresponding proton signals in two
different isomers are only a few Hertz for H-2, H-4, H-6,
and for H-18 and H-19 (the angular methyl groups).
Where the line separation was exceedingly small, the
doubled property is marked with an asterisk in Table 1.
Similar differences were found in the carbon spectra at
C-1, C-2, C-3, C-4, C-5, C-6, C-9, C-10, C-11, C-16,
C-2Ј, C-4Ј, and C-6Ј (in 2). The magnitude of these
[
[7] Miyashita N, Yoshikoshi A, Grieco PA. Pyridinium p-toluene-sulfo-
nate. A mild and efficient catalyst for the tetrahydropyranylation of
alcohols. J Org Chem 1977;42:3772–4.
[
8] Bernadi KF, Floyd MB, Poletto JF, Weis MJ. Prostaglandins, and
congeners. 20. Synthesis of prostaglandins via conjugate addition of
lithium trans-1-alkenyl-trialkylalanate reagents. A novel reagent for
conjugate 1,4-additions. J Org Chem 1979;44:1438–47.
[9] Griffin BE, Jarman M, Reese CB. The synthesis of oligoribonucleo-
tides-IV. Preparation of dinucleoside phosphates from 2Ј,5Ј-protected
ribonucleoside-derivatives. Tetrahedron 1968;24:639–62.
Ϫ2
Ϫ1
differences is in the range from 10 to 10 ppm (Table
).
[10] Beier R, Mundy BP. A facile removal of the tetrahydropyranyl protecting
group from alcohol derivatives. Synth Commun 1979;9:271–3.
2
We used two different solvents to obtain solvent-depen-
[11] Szendi Z, Sweet F. Novel synthesis of cholesterol analogs: conden-
sation of pregnenolone with dihydropyran or dihydrofuran. Steroids
dent structural information. Significant changes were ob-
served in the chemical shifts (up to several tenths of a ppm)
from the skeletal protons by changing the solvent from
chloroform to benzene. Chemical shift changes appeared for
nearly all of the steroid protons. Yet, the directions of these
changes did not appear to follow a pattern. The signal due
to H-16 in 2 exhibited the largest change associated with
changing the solvents. The benzene-induced chemical shift
change for H-16 is Ϫ0.52 ppm. The proton signals in the
side-chain of 6 were fairly sensitive for solvent changes as
were the protons at the steroid C-21 position. However, the
signal from H-6 in the B-ring double bond of the steroids in
the present study did not show any sensitivity to changes in
solvents.
1
991;56:458–63.
[12] Heusler K, Kalvoda J, Wieland P, Wettstein A. Zur dehydrierung von
steroid-allylalkoholen. Helv Chim Acta 1961;44:179–86.
[
13] Tschesche R, Goossens B, Piestert G, T o¨ pfer A. Synthesis of 27-nor-
5-oxocholest-5-en-3␤-yl acetate and 27-nor-25-oxocholestanol from
2
pregnenolone. Tetrahedron 1977;33:735–7.
[
14] Eliel EL, Hargrave KD, Pietrusiewitcz KM, Manoharan M. Confor-
mational analysis. 42. Monosubstituted tetrahydropyrans. J Am Chem
Soc 1982;104:3635–43.
[15] Duggan AJ, Hall SS. The chemistry of 2-alkoxy-3,4-dihydro-2H-pyrans.
III. Synthesis and solvolysis of the dichlorocarbene adducts 3-alkoxy-2-
oxa-7,7-dichloronorcaranes. J Org Chem 1975;40:2234–7.
[16] Hall SS, Weber GF, Duggan AJ. Relative reactivity of substituted
2
-alkoxy-and 2-phenoxy-3,4-dihydro-2H-pyrans with tert-butyl hy-
pochlorite. Effect of substituents on reactivity and products. J Org
Chem 1978;43:667–72.
[
17] De Hoog AJ. Carbon-13-nuclear magnetic resonance spectra of some
2-substituted tetrahydropyrans. Org Magn Reson 1974;6:233–5.
18] Boucheau V, Renaud M, de Ravel MR, Mappus E, Cuilleron CY.
Proton and carbon-13 nuclear magnetic resonance spectroscopy of
diastereomeric 3-and 17␤-tetrahydropyranyl ether derivatives of es-
trone and estradiol. Steroids 1990;55:209–21.
Acknowledgments
[
3
␤-Hydroxypregna-5,16-dien-20-one was generously
donated by Gedeon Richter Chemical Works Ltd. (Budap-
est, Hungary). The authors thank Prof. Dr Ir e´ n Vincze and
Prof. Dr Gy o¨ rgy Dombi for their kind recommendations and
advice, and Dr Antal P e´ ter, Dr Zsolt Szegletes, and Dr J a´ nos
W o¨ lfling provided analytical and preparative HPLC.
[
19] K o¨ v e´ r K, Hruby VJ, Uhrin D. Sensitivity and gradient enhanced
heteronuclear coupled/decoupled HSQC-TOCSY experiments for
measuring long-range heteronuclear coupling constants. J Magn Re-
son 1997;129:125–9.