Microbial transformation of 17α-ethynyl- and 17α-ethylsteroids, and tyrosinase inhibitory activity of transformed products

Article abstract of DOI:10.1016/j.steroids.2005.05.002

The microbial transformation of the 17α-ethynyl-17β- hydroxyandrost-4-en-3-one (1) (ethisterone) and 17α-ethyl-17β- hydroxyandrost-4-en-3-one (2) by the fungi Cephalosporium aphidicola and Cunninghamella elegans were investigated. Incubation of compound 1 with C. aphidicola afforded oxidized derivative, 17α-ethynyl-17β- hydroxyandrosta-1,4-dien-3-one (3), while with C. elegans afforded a new hydroxy derivative, 17α-ethynyl-11α,17β-dihydroxyandrost-4-en-3-one (4). On the other hand, the incubation of compound 2 with the fungus C. aphidicola afforded 17α-ethyl-17β-hydroxyandrosta-1,4-dien-3-one (5). Two new hydroxylated derivatives, 17α-ethyl-11α,17β- dihydroxyandrost-4-en-3-one (6) and 17α-ethyl-6α,17β-dihydroxy- 5α-androstan-3-one (7) were obtained from the incubation of compound 2 with C. elegans. Compounds 1-6 exhibited tyrosinase inhibitory activity, with compound 6 being the most potent member (IC₅₀ = 1.72 μM).

Full text of DOI:10.1016/j.steroids.2005.05.002

Steroids 70 (2005) 798–802
Microbial transformation of 17␣-ethynyl- and 17␣-ethylsteroids, and
tyrosinase inhibitory activity of transformed products
∗
∗∗
M. Iqbal Choudhary , Sadia Sultan, Mahmud Tareq Hassan Khan, Atta-ur- Rahman
H.E.J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi 75270, Pakistan
Received 29 June 2004; received in revised form 5 May 2005; accepted 10 May 2005
Available online 27 July 2005
Abstract
The microbial transformation of the 17␣-ethynyl-17␤-hydroxyandrost-4-en-3-one (1) (ethisterone) and 17␣-ethyl-17␤-hydroxyandrost-
-en-3-one (2) by the fungi Cephalosporium aphidicola and Cunninghamella elegans were investigated. Incubation of compound 1 with
4
C. aphidicola afforded oxidized derivative, 17␣-ethynyl-17␤-hydroxyandrosta-1,4-dien-3-one (3), while with C. elegans afforded a new
hydroxy derivative, 17␣-ethynyl-11␣,17␤-dihydroxyandrost-4-en-3-one (4). On the other hand, the incubation of compound 2 with the
fungus C. aphidicola afforded 17␣-ethyl-17␤-hydroxyandrosta-1,4-dien-3-one (5). Two new hydroxylated derivatives, 17␣-ethyl-11␣,17␤-
dihydroxyandrost-4-en-3-one (6) and 17␣-ethyl-6␣,17␤-dihydroxy-5␣-androstan-3-one (7) were obtained from the incubation of compound
2
©
with C. elegans. Compounds 1–6 exhibited tyrosinase inhibitory activity, with compound 6 being the most potent member (IC50 = 1.72 ␮M).
2005 Elsevier Inc. All rights reserved.
Keywords: 17␣-Ethynyl-17␤-hydroxyandrost-4-en-3-one; 17␣-Ethyl-17␤-hydroxyandrost-4-en-3-one; Microbial transformation; Cephalosporium aphidicola;
Cunninghamella elegans; Tyrosinase inhibition
1
. Introduction
hydroxyl derivative [8]. It was reported that 17␣-ethynyl
group prevents steroid–enzyme binding in the normal mode
and thus inhibits the formation of 11␣-hydroxylated prod-
uct [9]. However, in this paper we report the formation of
11␣-hydroxylated products of 17␣-ethynyl- and 17␣-ethyl
steroids, along with other oxidized products.
Tyrosinase inhibitors have become increasingly important
for cosmetics and medicines primarily in relation to hyper-
pigmentation and associated conditions. Since the enzyme
plays a key role in melanin biosynthesis, its inhibitors may
help in the prevention of excessive dermal melanin produc-
tion [1]. In addition, tyrosinase is known to be involved in the
molting process of insects and adhesion of marine organisms
2. Experimental
[2].
In continuation of our work on microbial transformation
2.1. General experimental procedures
of bioactive compounds [3–7], we describe here the bio-
tranformation of 17␣-ethynyl-17␤-hydroxyandrost-4-en-3-
one (1), 17␣-ethyl-17␤-hydroxyandrost-4-en-3-one (2) and
tyrosinase inhibitory activity of transformed products.
First fungal transformation of compound 1 was reported
by Zakelj-Mavric et al., resulting in the formation of 7␤-
Column chromatography on silica gel, 70–230 mesh. Pre-
coated silica gel plates (Si gel 60 F254, E. Merck) were used
for TLC, UV254 active: detection at 254 nm, and by ceric sul-
phate spray reagent, optical rotations JASCO DIP 360 digital
polarimeter. UV and IR spectra: Hitachi-UV-3200 and Jasco-
1
13
320-A spectrophotometer, respectively. H and C NMR (δ
∗
∗
ppm, J in Hz) COSY, HMQC, HMBC and NOESY spec-
tra, Bruker spectrometers operating at 500 and 400 MHz. EI,
FAB, and HREIMS were recorded on a JEOL JMS-HX110
Corresponding author. Tel.: +92 21 4824924-5; fax: +92 21 4819018-9.
Corresponding author.
E-mail address: hej@cyber.net.pk (M.I. Choudhary).
∗
0
039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2005.05.002

M.I. Choudhary et al. / Steroids 70 (2005) 798–802
799
and JMS-DA-500 mass spectrometers, m/z (rel. int.). PAAR
Hydrogenation apparatus 3916.EA was used for hydrogena-
tion.
on carbon) yielded a compound 2. Recovered yield was
95%.
2.6. Incubation of
2
.2. Microorganisms and chemicals
17α-Ethynyl-17β-hydroxyandrost-4-en-3-one (1)
Cunninghamella elegans (TSY-0865) and Cephalospo-
The substrate 1 (200 mg), distributed in 20 conical flasks,
was incubated as above for C. aphidicola. Chromatography
of the extract on Si gel yielded starting material (90 mg) and
compound 3 (11 mg).
rium aphidicola (IMI 68689) were maintained on Sabouraud-
◦
Glucose-Agar slants and stored in a refrigerator at 4 C. 17␣-
Ethynyl-17␤-hydroxyandrost-4-en-3-one (1) was purchased
from E. Merck, Germany.
2
.6.1. 17α-Ethynyl-17β-hydroxyandrosta-
1,4-dien-3-one (3)
White solid; mp 161–162 C, [α]D +14.2 (c 0.02, CHCl3);
UV (MeOH) λ
2
.3. Preparation of medium
◦
25
Medium (3 L) for C. elegans (TSY-0865) was prepared
(log ∈) 244 nm (3.4); lit value mp
max
26
◦
by mixing glucose (60 g), peptone (15 g), KH2PO4 (15 g)
163–164 C, [α]D 0 (c 1.150, CHCl ); λ 245 nm (15,600)
3
and yeast extract (9 g). The medium for C. aphidicola: (IMI
[10].
6
8689) was prepared by mixing these ingredients in dis-
Again the substrate 1, (300 mg) in 30 conical flasks was
incubated in the culture media of C. elegans as above. Chro-
matography of the extract on Si gel gave; starting material
(160 mg) and 17␣-ethynyl-11␣,17␤-dihydroxyandrost-4-en-
tilled H2O (2 L): glucose 100 g, KH2PO4 (2 g), KCl (2 g),
MgSO4·7H2O (4 g), glycine (4 g) and Gibberella trace ele-
ment solution (4 mL).
3-one (4) (5.5 mg).
2
.4. Culture and fermentation procedure
2.6.2.
The following procedures were used for each fungus:
-day-old spores were aseptically transferred into broth
17α-Ethynyl-11α,17β-dihydroxyandros-4-en-3-one (4)
Gummy [α]2 +4 (c 0.05, CHCl ); UV (MeOH) λ
5
2
D
3
max
medium flask (250 mL) containing 100 mL of freshly pre-
pared and autoclaved media. The seed flasks thus obtained
were incubated on a shake table at 30 C for 2 days. Two-day-
old broth cultures from 100 mL seed flask were inoculated
aseptically into 30 media flasks (250 mL) for C. elegans
and 20 media flasks (250 mL) for C. aphidicola containing
(log ∈) 240 nm (3.4); IR (CHCl ) ν
3407, 1710,
3
max
−
1
1
13
1659 cm ; H NMR (CD OD, 500 MHz), see Table 1;
3
C
◦
3
NMR (CD OD, 100 MHz), see Table 1; EIMS m/z (rel. int.)
+
+
328 [M ] (10), 310 (7) [M −18], 261 (17), 187 (15),145
(17), 121 (35), 119 (25), 109 (28), 91 (45), 79 (40), 55 (100);
HREIMS m/z 328.2045 (calcd for C H O , 328.2038).
21
28
3
1
00 mL of medium and fermentation was continued for fur-
ther 2 days.
Compounds 1 and 2 were dissolved in EtOH and solutions
2.7. Incubation of
17α-Ethyl-17β-hydroxyandrost-4-en-3-one (2)
were evenly distributed among 30 flasks (10 mg/0.25 mL in
each flask) for C. elegans, and 20 flasks (10 mg/0.25 mL in
each flasks) for C. aphidicola containing 48-h-old stage II
cultures. Fermentation was carried out for further 12 days on
The substrate 2 (200 mg) in 20 conical flasks was incu-
bated as described above for C. aphidicola. The extract was
chromatographed on Si gel affording; starting material 2
(120 mg) and compound 5 (4.4 mg).
◦
a rotatory shaker (200 rpm) at 29 C. In all experiments, one
control flask without biomass (for checking substrate stabil-
ity) and one flask without exogenous substrate (for checking
endogenous metabolite) were used. The culture media and
mycelium were separated by filtration. The mycelium was
washed with EtOAc (1 L) and the filtrate was extracted with
EtOAc (5 L). The combined organic extract was dried over
anhydrous Na2SO4, evaporated under reduced pressure, and
analyzed by thin layer chromatography. Control flasks were
also harvested and compared with test by TLC to confirm the
bio-transformed products.
2.7.1. 17α-Ethyl-17β-hydroxyandrosta-1,4-dien-3-one
(5)
◦
25
White solid; mp 168–170 C (uncorrected); [α]D +11.1
(c 0.07, CHCl3); UV (MeOH) λmax (log ∈) 242 nm (2.8).
The substrate 2 (300 mg) in 30 conical flasks was also
incubated as described above for C. elegans. The extract
was chromatographed on Si-gel affording starting material
(90 mg); compounds 6 (8.5 mg) and 7 (4.8 mg).
2.7.2. 17α-Ethyl-11α,17β-dihydroxyandrost-4-en-3-one
2
1
.5. Hydrogenation of
7α-Ethynyl-17β-hydroxyandrost-4-en-3-one (1)
(6)
Gummy; [α]25 +45 (c 0.06, CHCl3); UV (MeOH)
D
λmax (log ∈) 241 nm (3.5); IR (CHCl3) νmax 3401, 1708,
−
1
1
13
Sample (1.0 g) dissolved in 50 mL dry MeOH reduced
1658 cm ; H NMR (CD3OD, 400 MHz), see Table 1;
C
in a PAAR hydrogenator with a catalyst (5% palladium
NMR (CD3OD, 125 MHz), see Table 1; EIMS m/z (rel. int.)

8
00
M.I. Choudhary et al. / Steroids 70 (2005) 798–802
Table 1
1
H NMR (500 MHz; CD3OD) and 13C NMR (100 MHz; CD3OD) data for compounds 4, 6 and 7
Carbon no.
Compound 4
1H NMR (δ)
Compound 6
1H NMR (δ)
Compound 7
1H NMR (δ)
13C NMR (δ)
13C NMR (δ)
13C NMR (δ)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2.2, m, 2.07, dt (4.3, 14.0)
2.70, dt (4.5, 14.0) 2.27, m
–
5.73, s
–
2.23, m, 2.0, m
1.92, m, 1.88, m
1.65, m
1.12, m
–
3.98, dt (4.9, 10.5)
1.94, m
–
1.67, m
1.68, m, 1.35, m
2.25, m, 1.95, m
–
0.90, s
1.35, s
–
38.5
34.9
202.3
124.4
175.4
34.7
32.6
37.0
60.0
41.3
69.8
45.2
48.3
50.6
23.8
40.0
79.7
14.3
18.7
88.4
74.9
2.24, m, 2.04, dt (4.1, 13.8)
2.68, dt (4.5, 9.0) 2.41, m
–
5.70, s
–
1.60, m, 1.01, m
1.90, m, 1.78, m
1.86, m
1.08, m
–
4.0, dt (4.6, 10.4)
1.82, m, 1.77, m
–
34.8
38.6
202.3
124.0
175.5
33.5
32.8
37.4
60.0
41.5
70.0
44.1
33.6
50.8
24.8
34.9
83.8
29.7
19.8
29.7
9.8
2.02, m, 1.98, m
2.04, dt (13.8, 4.1) 2.41, m
–
2.32, m, 2.14, m
1.92, m
3.60, br s, (W 1/2 = 12.5)
1.71, m, 1.28, m
1.50, m
1.08, m
–
1.50, m, 1.34 m
1.36, m, 1.22, m
–
38.4
36.4
215.0
42.8
51.5
72.6
34.2
41.0
51.2
38.3
20.8
33.5
42.2
50.5
23.5
35.2
84.8
15.2
25.1
29.8
8.2
0
1
2
3
4
5
6
7
8
9
0
1
1.28, m
1.26, m
1.78, m, 1.50, m
1.65, m, 0.97, m
–
0.93, s
1.35, s
1.74, m, 1.47, m
1.64, m, 0.97, m
–
0.90, s
1.21, s
a
a
1.85, m, 1.84, m
0.97, t (7.2)
1.55, m, 1.41, m
0.96, t (7.1)
2.90, s
aThe numbering for C-20 and C-21 denotes ethynyl and ethyl moieties.
+
+
3
(
(
3
32 [M ] (22), 314 [M −18], (11), 191 (3), 149 (17), 124
2000 (Microsoft Corp., USA) for this purpose. All the stud-
ies have been done at least in triplicates and the results here
represents the mean ± S.E.M. (standard error of the mean).
All the reagents, enzyme, substrate and reference compounds
were purchased from Sigma Chem. Co., MO, USA.
27), 123 (23), 111 (86), 109 (82), 83 (23), 79 (23), 57
95), 55 (100); HREIMS m/z 332.2302 (calcd for C21H32O3,
32.2351).
2
.7.3. 17α-Ethyl-6α,17β-dihydroxy-5α-androstan-3-one
(
7)
◦
25
White solid; mp 198–202 C (uncorrected), [α]D 16 (c
3. Results and discussion
−
1 1
0
.03 CHCl3); IR (CHCl3) νmax 3405, 1709, 1657, cm ; H
NMR (CD3OD, 400 MHz) see Table 1; EIMS m/z (rel. int.)
Fermentation of 17␣-ethynyl-17␤-hydroxy-4-androsten-
-one (1) with C. aphidicola for 12 days yielded a known
metabolite, 17␣-ethynyl-17␤-hydroxylandrosta-1,4-dien-3-
one (3) [10,11], (Scheme 1).
+
+
3
1
34 [M ] (11), 316 [M −18] (63), 193 (3), 153 (20), 151 (12),
3
41 (22), 122 (12), 109 (16), 107 (23), 105 (26), 93 (25), 55
(17), 53 (1); HREIMS m/z 334.2140 (calcd for C21H34O3,
3
34.2143).
Further fermentation of compound 1 with C. elegans for
12 days yielded a new hydroxylated metabolite 17␣-ethynyl-
2
.8. Tyrosinase inhibition assay
11␣,17␤-dihydroxyandrost-4-en-3-one (4) (Scheme 1).
+
The HREIMS of 4 displayed the molecular ion (M ) at m/z
Tyrosinase inhibition assays were performed in 96-
328.2045 (C21H28O3, calcd 328.2038) indicating an addi-
tional oxygen atom. The H NMR spectrum of 4 showed
well microplate format using SpectraMax® 340 (Molecu-
lar Devices, CA, USA) microplate reader according to the
method developed by Hearing (1987). Briefly, all the com-
pounds were dissolved in DMSO and finally the solvent mix-
ture (2.5%). 30 Units mushroom tyrosinase (28 nM) was first
preincubated with the compounds in 50 nM Na–phosphate
1
a downfield signal for oxygen-bearing methine proton at
δ 3.98 (dt, J = 10.5 Hz, J = 4.9 Hz) suggesting hydroxyla-
tion of a methylene group. A downfield methine carbon
signal at δ 69.8 (11-CHOH) further supported this infer-
ence. The ␣-stereochemistry of OH-11 as equatorial was
inferred from the multiplicity of the geminal H-11 signal (δ
3.98, dt, J11ax, 9ax = J11ax, 12ax = 10.5 Hz, J11ax, 12eq = 4.9 Hz)
and the NOESY correlations of H-11␤ (δ 3.98) with CH3-
18␤ (δ 0.90) and CH3-19␤ (δ 1.35). The presence of OH
at C-11 was also inferred from HMBC correlations of H-
11 (δ 3.98) with C-9 (δ 60.0), and C-11 (δ 69.8) with H-
◦
buffer (pH 6.8) for 10 min at 25 C. Then the l-DOPA
(
0.5 mM) was added to the reaction mixture and the
enzyme reaction was monitored by measuring the changes
◦
in absorbance at 475 nm (at 37 C) due to the formation of
the DOPA chrome for 10 min. The percent inhibition of the
enzyme and IC50 values of the active compounds were calcu-
®
◦
lated using a program developed with Java and Macro Excel
9 (δ 1.12) and H-12 (δ 1.94), as well as the COSY 45

M.I. Choudhary et al. / Steroids 70 (2005) 798–802
801
Table 2
Tyrosinase inhibitory activity of compounds 1–6
Compounds
IC50 ± S.E.M. (in ␮M)
1
2
3
4
5
6
2.61 ± 0.037328
1.53 ± 0.001088
7.89 ± 0.00128
5.95 ± 0.00078
3.46 ± 0.01046
1.72 ± 0.00089
16.92 ± 0.5268
3.68 ± 0.02234
Kojic acid
l-Mimosine
Metabolite 7 could not be screened because of its insufficient amount, S.E.M.
is the standard error of the mean.
ton was deduced to be ␤ (axial) on the basis of coupling
constants of H-6 signal (W1/2 = 12.5 Hz). The C-6 hydroxyl
group must therefore have an ␣ (equatorial) orientation. The
␣
-stereochemistry of C-6 hydroxyl was further supported by
NOESY correlation of H-6 (δ 3.62) with CH3-19 (δ 0.90).
The position of the newly introduced hydroxyl group (C-
6) was further confirmed by 2D-NMR spectra. The HMBC
spectra of compound 7 showed correlations of H-6 (δ 3.60, br
s, W1/2 = 12.5 Hz) with C-5 (δ 51.1) and C-7 (δ 34.2), and the
COSY 45 correlations of H-6 (δ 3.60) with H-5␣ (δ 1.92)
Scheme 1. Microbial transformation of 17␣-ethynyl-17␤-hydroxyandrost-
-en-3-one (1) and 17␣-ethyl-17␤-hydroxyandrost-4-en-3-one (2).
◦
4
and H-7 (δ 1.73).
Compounds 1–6 exhibited potent inhibition against tyrosi-
nase with some compounds (1–2 and 5–6) more potent than
the standards kojic acid and l-mimosine. Results of tyrosi-
nase inhibition studies on compounds 1–6 have been shown
in the Table 2.
correlations of H-11 (δ 3.98) with H-12 (δ 1.94) and H-9
δ 1.12).
Fermentation of compound 2 with C. aphidicola for
2 days yielded a metabolite which was structurally char-
acterized as 17␣-ethyl-17␤-hydroxyandrosta-1,4-dien-3-one
5) and found to be a known compound [12]. Further
(
1
(
two new metabolites of compound 2 were obtained from
biotransformation by C. elegans which were character-
ized as 17␣-ethyl-11␣,17␤-hydroxyandrost-4-en-3-one (6)
and 17␣-ethyl-6␣,17␤-dihydroxy-5␣-androstan-3-one (7)
Acknowledgements
One of the author (S.S.) gratefully acknowledges the
Glaxo-Smith-Kline (GSK) Private Limited Pakistan for
financial support, while M.T.H.K. is grateful to the Third
World Academy of Sciences (TWAS), Italy, for the award of
South–South Fellowship.
(
Scheme 1).
+
The HREIMS of 6 displayed the M at m/z 332.2302
(
C21H32O3 calcd 332.2351) indicating an additional oxygen
1
atom. The H NMR spectrum showed methine proton signal
at δ 4.0 (dt, J = 10.4 Hz, J = 4.6 Hz) suggesting a hydrox-
ylation at C-11 as in compound 4. The ␣-stereochemistry
of C-11 OH was inferred from the multiplicity of the
H-11 signal (δ 4.0, dt, J11ax, 9ax = J11ax, 12ax = 10.4 Hz and
J11ax, 12eq = 4.6 Hz) and the NOESY correlations of H-11 ␤
References
[1] Hearing VJ. Methods in Enzymology, vol. 142. New York: Academic
Press; 1987. p. 154.
(δ 4.0) with H-18␤ (δ 0.93) and H-19␤ (δ 1.35). The methine
[
2] Shiino M, Watanabe Y, Umezawa K. Synthesis of N-substituted N-
nitrosohydroxyl-amines as inhibitors of mushroom tyrosinase. Bioorg
Med Chemdkjdot 2001;9:1233–40.
signal resonated at δ 69.9 was assigned to hydroxyl-bearing
C-11. The HMBC correlation of C-11 (δ 69.9) with H-12␣ (δ
◦
[
3] Choudhary MI, Sultan S, Yaqoob M, Musharraf SG, Yasin A,
Shaheen F, et al. Microbial transformation of cortisol and prolyl
endopeptidase inhibitory activity of its transfomed products. Nat
Prod Res 2003;17:389–95.
1
.82) and COSY 45 correlations of H-11 (δ 4.0) with H-12
(
δ 1.82), H-9 (1.08) and H-19 (1.35) were also observed.
The HREIMS of compound 7 displayed the M at m/z
+
3
34.2140 (C21H34O3 calcd. 334.2143). Only terminal UV
[
[
[
4] Choudhary MI, Musharraf SG, Khan MTH, Adaelrahman D, Pervaz
M, Shaheen F, et al. Microbial transformation of isolongifolen-4-one.
Helv Chim Acta 2003;86:3450–60.
5] Choudhary MI, Musharraf SG, Shaheen F, Atta-ur-Rahman.
Microbial transformation of (+)-androsta-1,4-diene-3, 17-dione by
Cephalosporium aphidicola. Nat Prod Lett 2002;16:377–82.
6] Musharraf SG, Atta-ur-Rahman, Choudhary MI, Sultan S. Microbial
transformation of (+)-adrenosterone. Nat Prod Lett 2002;6:345–9.
absorption indicated the lack of conjugation in the molecule.
1
The H NMR spectrum of 7 was very similar to that of com-
pound 2 with no olefinic proton and an additional hydroxyl-
bearing methine proton signal at δ 3.60. Hydroxylation at
C-6 was further inferred from a downfield hydroxyl-bearing
carbon signal at δ 72.6. The stereochemistry of C-6 pro-

8
02
M.I. Choudhary et al. / Steroids 70 (2005) 798–802
[
[
[
7] Atta-ur-Rahman, Choudhary MI, Asif F, Farooq A, Yaqoob M, Dar
A. Microbial transformation of sarsapogenin by Fusarium lini. Phy-
tochemistry 1998;49:2341–2.
8] Zakelj-Mavric M, Belic I, Gottleib HE. The bioconversions of 17␣-
ethynyl steroids with 11␣-hydroxylase of Rhizopus nigricans. Micro-
biol Lett 1986;33:117–20.
9] Ambrus G, Szarka E, Barta I, Albrecht K, Horvath G. Microbial
hydroxylation of norethisterone. Acta Microbiol Acad Sci Hung
[10] Vischer E, Meystre C, Wettestein A. Further microbiological reac-
tion synthesis of 1-dehydrosteroids. Helv Chim Acta 1955;38.:1502–
7.
[11] Horvath G, Ambrus G. Studies on rearrangements in 17-hydroxy
steroids related to norethisterone. Adv Mass Spectr 1978;7B.:
1280–6.
[12] Albert W, Ehmann, Ludwing. (CIBA Ltd.) Chinese Patent 346538,
Swiss July 15, 1960 [C.A. vol 55, 1961. 627 b].
1975;22:453–61.