6
K. YILDIRIM ET AL.
dC 74.28 ppm, indicating the presence of a 7b-
hydroxyl group (Holland and Thomas 1982). The 13C
NMR spectrum of 11 showed a downfield shift for C-
8 (DdC 7.54 ppm) whereas it showed a c-gauche up-
field shift for C-9 (DdC 3.06 ppm), further indicating
the presence of a 7b-hydroxyl group. NMR data of
11 were consistent with literature (Holland and
Thomas 1982).
addition to this, a minor epimerization at C-17 had
taken place. In the same work, this fungus hydroxy-
lated 2 at C-16b and most of this hydroxyl group was
then oxidized at C-16. This was accompanied by a
minor epimerization and a minor independent oxida-
tion at C-17.
It can also be seen from Table 2, U. chartarum MRC
72584 metabolized 1 different from how C. sphaero-
spermum MRC 70266 did. U. chartarum MRC 72584
hydroxylated 1 at C-6b, C-7a, C-7b and C-14a, accom-
panied by an oxidation at C-6 subsequent to its
hydroxylation, a 5a-reduction and a reduction at C-17.
In a recent work, U. chartarum MRC 72584 hydroxy-
lated 17b-hydroxyandrost-4-en-3-one 2 at C-6b, C-7b,
C-14a and C-12b, accompanied by a 5a-reduction and
oxidations at C-6 and at C-17 (Yildirim, Kuru, and S¸
Yılmaz 2018). These results might have suggested that
U. chartarum MRC 72584 metabolized 1 and 2 in
some different ways due to the presence of a 17b-
hydroxyl group in 2 (Kołek and Swizdor 1998).
Reports of fungal 5a-reduction, oxidations of
hydroxyl groups following hydroxylations, the reduc-
tion of 1 at C-17 and hydroxylations of 1 at C-6b and
C-7a are very common (Mahato and Garai 1997;
Fernandes et al. 2003; Bhatti and Khera 2012; Donova
and Egorova 2012; Nassiri-Koopaei and Faramarzi
2015). However, fungal hydroxylations of 1 at C-7b
and C-15a are rare. For example, Mucor racemosus
hydroxylated 1 at C-6b, C-7a, C-7b and C-11a, accom-
panied by a reduction at C-17 (Faramarzi et al. 2008).
Gongronella butleri (Kollerov et al. 2008) hydroxylated
The fifth metabolite was identified as 7a-hydroxyan-
drost-4-en-3,17-dione 12. NMR spectra of 12 had char-
acteristic resonances at dH 4.15 ppm (1 H, m) and dC
67.10 ppm, indicating the presence of a 7a-hydroxyl
group (Holland and Thomas 1982). The 13C NMR spec-
trum of 12 showed a downfield shift for C-8 (D
5.86 ppm) whereas it showed a c-gauche up-field shift
for C-9 (D 8.42 ppm), further indicating the presence
of a 7a-hydroxyl group. NMR data of 12 were in
agreement with literature (Holland and Thomas 1982).
In this work, as can be seen from Table 2, C. sphaer-
ospermum MRC 70266 hydroxylated 1 mainly at C-6b,
accompanied by a hydroxylation at C-15a, a reduction
at C-17, a 5a-reduction and oxidations at C-6 and C-16
following hydroxylations. In a recent work, however, C.
sphaerospermum MRC 70266 metabolized 17b-hydrox-
yandrost-4-en-3-one 2 in a different way (Yildirim
et al. 2019). The same fungus mainly hydroxylated 2
at C-6b, accompanied by a minor oxidation at C-17, a
minor 5a-reduction and some minor hydroxylations at
C-7b, C-12b, C-15a and C-16b. These results might
have suggested that C. sphaerospermum MRC 70266
metabolized 1 and 2 in some different ways due to
the lack of a 17b-hydroxyl group in 1 as in the bio-
transformations of some 4-ene-3-oxo steroids by
Fusarium culmorum (Kołek and Swizdor 1998). In
another recent work, Cladosporium cladosporioides
MRC 70282 (Yildirim, Kuru, and RF Yılmaz 2018), a dif-
ferent Cladosporium isolate, metabolized 1 and 2 in
different ways from how C. sphaerospermum MRC
70266 did. C. cladosporioides MRC 70282 reduced 1
at C-17, hydroxylated it at C-16b and most of this
hydroxyl group was then oxidized to a ketone. In
1
at C-6b, C-6a, C-7a, C-7b and C-14a whilst
Botryosphaeria obtusa (Smith et al. 1990) hydroxylated
1 at C-6b and C-7b. In another work, Gibberella saubi-
netti hydroxylated 1 at C-6b and C-15a (Urech et al.
1960). In a recent work, Colletotrichum lini hydroxy-
lated 1 at C-11a and C-15a (Wu et al. 2015).
In short, it was shown that C. sphaerospermum MRC
70266 and U. chartarum MRC 72584 metabolized 1 in
different ways and 6b,17b-dihydroxyandrost-4-en-3,16-
dione 8 was determined as a new metabolite. Our
Table 2. Metabolite yields following chromatography.
Fungus
Metabolite
%Yield
C. sphaerospermum MRC 70266
17b-Hydroxyandrost-4-en-3-one 2
5a-Androstane-3,6,17-trione 3
2
3
6b-Hydroxyandrost-4-en-3,17-dione 4
17b-Hydroxyandrost-4-en-3,16-dione 5
6b,17b-Dihydroxyandrost-4-en-3-one 6
15a-Hydroxyandrost-4-en-3,17-dione 7
6b,17b-Dihydroxyandrost-4-en-3,16-dione 8
5a-Androstane-3,6,17-trione 3
17b-Hydroxy-5a-Androstane-3,6-dione 9
14a-Hydroxyandrost-4-en-3,17-dione 10
7b-Hydroxyandrost-4-en-3,17-dione 11
7a-Hydroxyandrost-4-en-3,17-dione 12
37
5
6
4
3
13
3
14
18
10
U. chartarum MRC 72584