Fungal transformation of (1R,2S,5R)-(-)-menthol by Cephalosporium aphidicola

Article abstract of DOI:10.1021/np980057n

Incubation of (1R,2S,5R)-(-)-menthol (1) with Cephalosporium aphidicola for 12 days yielded the six oxidized metabolites: 10-acetoxymenthol (2), 7- hydroxymenthol (3), 4α-hydroxymenthol (4), 3α-hydroxymenthol (5), 9- hydroxymenthol (6), and 10-hydroxymenthol (7). The structures of the novel compounds 2, 4, 5, and 7 were assigned by interpretation of their spectral data.

Full text of DOI:10.1021/np980057n

1
340
J . Nat. Prod. 1998, 61, 1340-1342
F u n ga l Tr a n sfor m a tion of (1R,2S,5R)-(-)-Men th ol by Ceph a lospor iu m
a ph id icola
Atta-ur-Rahman,* Muhammad Yaqoob, Afgan Farooq, Shazia Anjum, Fahim Asif, and M. Iqbal Choudhary
International Center for Chemical Sciences, H. E. J . Research Institute of Chemistry, University of Karachi,
Karachi 75270, Pakistan
Received February 19, 1998
Incubation of (1R,2S,5R)-(-)-menthol (1) with Cephalosporium aphidicola for 12 days yielded the six
oxidized metabolites: 10-acetoxymenthol (2), 7-hydroxymenthol (3), 4R-hydroxymenthol (4), 3R-hydroxy-
menthol (5), 9-hydroxymenthol (6), and 10-hydroxymenthol (7). The structures of the novel compounds
2
, 4, 5, and 7 were assigned by interpretation of their spectral data.
The biotransformation of various classes of terpenoids
Sch em e 1. Microbial oxidation of menthol (1).
by Cephalosporium aphidicola has been studied exten-
sively.1
-6
Our work on the biotransformation of 7R-
hydroxyfrullanolide and sclareolide by Aspergillus species
and Culvularia lunata has resulted in the production of
several interesting metabolites that would have been
7
,8
difficult to obtain synthetically.
The monoterpenoid menthol is the main constituent of
peppermint oil, and it exhibits anesthetic, disinfectant, and
photoprotective effects.9 We now report the biotransfor-
mation of (1R,2S,5R)-(-)-menthol (1) by Cephalosporium
aphidicola (IMI 68981) to yield four new metabolites
characterized as 10-acetoxymenthol (2), 4R-hydroxymen-
thol (4), 3R-hydroxymenthol (5), and 10-hydroxymenthol
(
7) and two known compounds identified as 7-hydroxym-
1
0,11
enthol (3) and 9-hydroxymenthol (6).
The structures
degree of unsaturation. A 6H singlet at δ 1.21 was
observed in the H NMR of the compound, which resonated
due to the two methyls of an isopropyl moiety. This
suggested that hydroxylation took place at the C-7 tertiary
carbon atom in the molecule. The position of the OH group
of these metabolites were established through detailed
physical and spectroscopic studies.
1
Resu lts a n d Discu ssion
The prime investigation aimed at the metabolic trans-
formation of (1R,2S,5R)-(-)-menthol (1) by fermentation
with C. aphidicola has resulted in the production of six
polar compounds as detected by TLC. Column chroma-
tography of the crude material yielded compounds 2-7.
The EIMS of compound 2 showed a molecular ion at m/z
at C-7 was further confirmed by the appearance of the C-7
quaternary carbon at low field in the 13C NMR spectrum
1
H NMR and 13C NMR spectral assignments
at δ 75.1. The
of 3 in comparison of those of menthol (1) are summarized
in Tables 2 and 3. The synthesis of compound 3 has been
previously reported.10
2
14, which was confirmed by positive ion FABMS, while
its HREIMS displayed a molecular ion at m/z 214.1559
corresponding to the molecular formula C12 . The IR
spectrum displayed absorptions at 3595 (OH) and 1722
Compound 4 showed a molecular ion at m/z 172, which
was confirmed by positive ion FABMS. The exact molec-
ular mass of the compound was determined by HREIMS
and found to be m/z 172.1188, which corresponds to the
22 3
H O
-
1
1
(acetoxy carbonyl) cm . The H NMR spectrum (CDCl
3
,
molecular formula C10
an absorption at 3355 cm (OH). The H NMR spectrum
(CDCl , 500 MHz) exhibited a downfield methine signal at
20 2
H O . The IR spectrum displayed
-
1
1
3
00 MHz) was similar to that of menthol (1) but displayed
an acetyl methyl signal at δ 2.04. These observations
suggested the introduction of an acetoxy group in the
menthol molecule. The presence of the acetoxy group at
3
δ 3.18 (1H, dt, J ) 4.3, 10.6 Hz). This observation
suggested the introduction of an OH group at one of the
C-10 was confirmed by the absence of the CH
in the H NMR spectrum, the absence of the CH
signal in the 13C NMR spectrum, and the appearance of
3
-10 doublet
-10 carbon
2
CH groups, that is, C-3, C-4, or C-6. The assignments of
1
3
all the protons were accomplished from the interpretation
of the HMQC spectrum. The COSY 45° interactions of H-5
(δ 1.41) and H-3 (δ 1.05, 1.83) with the newly oxygenated
methine proton (δ 3.48) suggested the introduction of a
hydroxyl group at C-4. The multiplicity of the H-4 signal
at δ 3.18 (dt, J ) 4.3, 10.6 Hz, H-4) suggested the
â-orientation (equatorial) of H-4 and, hence, of an R (axial)
orientation of the newly introduced OH group at C-4.
The metabolite 5, which was isolated as colorless crys-
tals, displayed a molecular ion peak at m/z 172 in the
EIMS. The positive ion FABMS showed a peak at m/z 173
and confirmed the molecular mass of the compound to be
the carbon signals at δ 171.2 (-OCO) and 22.1 (-OCO-
1
13
3
CH ). The complete H NMR and C NMR assignments
of 2 are presented in Tables 1 and 2, respectively.
The EIMS of 3 showed a molecular ion at m/z 172, which
was confirmed by positive ion FABMS. The exact molec-
ular mass of the compound was found to be 172.1445, which
corresponds to the molecular formula C10
20 2
H O with one
*
To whom correspondence should be addressed. Tel.: (92-21)499007.
Fax: (92-21)4963373. E-mail: hejric@digicom.net.pk.
1
0.1021/np980057n CCC: $15.00
© 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/19/1998

Biotransformation of Cephalosporium
J ournal of Natural Products, 1998, Vol. 61, No. 11 1341
Ta ble 1. 1H NMR Assignments of Menthol (1) and Its Microbial Transformation Products (2-7) (δ ppm, J Hz)
proton (s)
1
2
3
4
5
6
7
1
2
3
R
â
â
3.38 (dt, 4.5, 10.5) 3.42 (m,W1/2 ) 25.2) 3.70 (dt,
3.48 (dt, 4.4, 10.7) 3.75 (dt,
4.4, 10.8)
1.97 (m)
3.44 (dt, 4.3, 10.5)
3.43 (dt, 3.8,
10.6)
1.18 (m)
4
.3, 10.8)
1.08 (ddt, 13.5,
0.5, 3.5)
1.15 (m)
0.90 (m)
1.37 (m)
1.30 (m)
1.34 (m)
1
0.93 (m)
0.95 (m)
1.05 (m)
3.99 (ddd, 6.2, 0.94 (m)
0.8, 13.4)
0.95 (m)
1
3
4
4
5
R
â
R
R
1.39 (m)
0.83 (m)
1.58 (qd, 13.0, 3.5) 1.65 (m)
1.65 (m)
0.85 (m)
1.67 (m)
1.60 (m)
1.00 (m)
1.70 (m)
1.83 (dt, 7.6, 3.9)
3.18 (dt, 4.3, 10.6) 0.95 (m)
1.20 (m)
1.60 (m)
0.82 (m)
1.66 (m)
1.42 (m)
1.10 (m)
0.80 (m)
1.62 (tt, 2.0, 3.9)
1.50 (m)
1.64 (dtd, 9.0,
1.41 (m)
1.41 (m)
1.25 (m)
6
.5, 3.5)
6
6
â
R
0.98 (m)
1.10 (m)
1.95 (m)
1.06 (m)
1.10 (m)
1.10 (m)
0.98 (m)
1.98 (m)
1.00 (m)
2.05 (m)
1.94 (dtd, 12.0,
1.92 (dtd, 12.2, 1.95 (dt, 4.0, 12.7) 1.75 (m)
4.1, 2.1)
3
.5, 2.0)
7
2.15 (dtd, 14.0,
2.13 (m)
2.13 (dtd, 14.0,
7.0, 2.85)
2.08 (m)
2.08 (m)
2.15 (dtd, 14.1,
7.1, 2.8)
7
.0, 3.0)
8
9
0.79 (d, 7.0)
0.90 (d, 7.0)
0.81 (d, 4.2)
0.95 (d, 7.0)
1.21 (s)
1.21 (s)
0.85 (d, 4.2)
0.95 (d, 7)
1.24 (d, 4.2)
0.85 (d, 6.9)
0.855 (d, 7.1)
0.77 (d, 7)
0.90 (d,7)
3.57 (dd, 5.3, 10.5)
3
.50 (dd, 7.5, 10.5)
1
0
0.89 (d, 7.0)
4.00 (dd, 8.5, 9.7)
0.90 (d, 6.5)
1.03 (d, 6.5)
0.93 (d, 7.0)
0.90 (s)
3.38 (m, W1/2
18.5)
)
3
.90 (dd, 4.5, 8.5)
OCOCH
3
2.04 (s)
Ta ble 2. 13C NMR Assignments of Menthol (1) and Its
Microbial Transformation Products (2-7) (δ ppm)
6 are presented in Tables 1 and 2. A synthetic compound
6
has been previously reported.
carbon multiplicities
1
2
3
4
5
6
7
Compound 7 showed a molecular ion at m/z 172, which
was confirmed by positive ion FABMS. The exact molec-
ular mass was found to be m/z 172.1454 by HRMS,
1
2
3
4
5
6
7
8
9
1
CH
CH
71.5 71.0 72.9 70.7 72.3 72.0 71.4
50.2 50.3 53.4 48.6 50.1 46.0 50.6
23.3 22.6 27.1 32.4 68.5 25.6 22.9
34.6 34.3 34.4 76.0 38.4 34.3 29.0
31.7 30.9 31.4 38.3 30.3 31.6 39.5
45.1 39.2 44.6 42.5 48.2 45.1 39.3
25.9 25.9 75.1 25.8 25.9 36.0 26.0
16.2 16.1 23.7 15.9 16.2 13.0 16.2
21.0 20.9 30.1 20.9 20.9 66.7 21.0
22.2 68.7 22.0 18.1 23.8 22.0 68.0
171.2
CH2
CH2
CH
CH2
CH
CH3
CH3
CH3
20 2
corresponding to the molecular formula C10H O . An
-1
absorption at 3415 cm
in the IR spectrum of 7 was
observed showing the presence of hydroxyl groups in the
menthol. A multiplet at δ 3.43(2H) and the disappearance
of the CH
of C-10 methyl to C-10 CH
firmed by the presence of a CH
at low field, that is, at δ 68.6, and the absence of a CH
3
-10 signal provided information of the conversion
OH. This was further con-
-10 oxygen-bearing carbon
-10
signal at δ 22.2 in the C NMR spectrum. The complete
2
0
2
OCOCH3
OCOCH3
3
22.1
13
1
13
H and C NMR assignments of 7 are presented in Tables
1
72. The molecular formula of the compound was found
to be C10 by recording the HREIMS, which showed
an exact mass at m/z 172.1428. A hydroxyl absorption at
2 and 3.
20 2
H O
Exp er im en ta l Section
-
1
1
3
385 cm was observed in the IR spectrum of 5. The H
NMR spectrum (CDCl , 400 MHz) of 5 exhibited a diag-
nostic CH proton signal at δ 3.99 (1H, ddd, J ) 6.2, 10.8,
3.4 Hz), which provided evidence of hydroxylation at C-3,
Gen er a l Exp er im en ta l P r oced u r es. Bruker AMX 500,
3
AM 400, AM 300, and AC 300 NMR spectrometers were used
1
to record H NMR spectra, and the same instruments operat-
1
1
3
ing at 125, 100, and 75 MHz were used to record C NMR
spectra. A Varian MAT 311A mass spectrometer connected
to a Masspec Data system was used to record mass spectra.
FABMS and HREIMS spectra were recorded on a J EOL-J MS
HX 110 mass spectromter with a glycerin matrix. The IR
spectra were recorded on a J ASCO IR A-302 IR spectropho-
tometer, and UV spectra were recorded on a Hitachi UV-3200
UV/vis spectrophotometer. Optical rotations were measured
on a J ASCO DIP 360 digital polarimeter. Melting points were
taken on a Buchi 535 melting point apparatus. The purity of
C-4, or C-6. The position of the new hydroxyl group was
established as C-3 on the basis of COSY 45° NMR interac-
tions of H-3 (δ 3.99) with H-2 (δ 1.97) and H-4 (δ 1.20, 1.95).
This was further confirmed by HMBC correlations between
H-3R (δ 3.95) and C-2 (δ 50.1). The new methine proton
exhibited a doublet of double doublets at δ 3.99 (J ) 6.2,
1
0.8, 13.4 Hz), which provided evidence for the 3â (axial)
orientation of H-3, and hence the 3R (equatorial) orienta-
tion of the newly introduced OH was confirmed. The
complete 1H NMR and C NMR assignments of 5 are
summarized in Tables 1 and 3.
13
the samples was checked on precoated plates (Si gel 60 F254
,
0.2 mm, Merck). The metabolites were purified by column
chromatography on flash Si gel and on precoated plates (Si
gel 60 F254, 0.2 mm, Merck). (1R,2S,5R)-(-)-Menthol was
purchased from Aldrich.
The EIMS of compound 6 showed a molecular ion at m/z
1
72, which was confirmed by FABMS. The exact molecular
mass of the compound as determined by HREIMS was
found to be m/z 172.1504, which corresponds to the mo-
P r ep a r a tion of Med iu m . A medium for C. aphidicola
(
IMI 68981) was prepared by mixing glucose (100 g), KH
(2 g), MgSO ‚7H O (4 g), KCl (2 g), glycine (4 g), and Gibberella
trace element solution [Co(NO (0.01% w/v), FeSO ‚7H
0.1%), CuSO ‚5H O (0.1%), ZnSO ‚7H O (0.161%), MnSO
O (0.01%), Mn(NO ‚4H O (0.01%), H O (100 mL)] (4-mL
portion of mixture) into distilled H O (2 L).
2 4
PO
lecular formula C10
20 2
H O . The IR spectrum displayed an
absorption at 3392 cm (OH). The 1H NMR spectrum
-
1
4
2
3
)
2
4
2
O
(
(
CDCl
1) and displayed an AB double doublet at δ 3.57 and 3.50
, while the CH -9 signal of
3
, 400 MHz) was distinctly similar to that of menthol
(
H
4
2
4
2
4
‚
2
3
)
2
2
2
due to an oxygen-bearing CH
2
3
2
menthol did not appear in the spectrum. The position of
the newly introduced OH group at C-9 was indicated by
Cu ltiva tion of C. a ph id icola . Two-day-old spores of C.
aphidicola were aseptically transferred into a broth medium
flask (250 mL) containing 100 mL of freshly prepared and
autoclaved medium. The seed flask thus obtained was incu-
bated on a shake table at 30 °C for 2 days.
the absence of the CH
ance of the CH -9 signal at δ 66.7 in the C NMR
spectrum. The complete H and C NMR assignments of
3
-9 signal at δ 21.0 and the appear-
1
3
2
1
13

1
342 J ournal of Natural Products, 1998, Vol. 61, No. 11
In n ocu la tion of th e Cu ltu r es. A 2-day-old broth culture
Atta-ur-Rahman et al.
Com p ou n d 5. Elution with EtOAc-petroleum ether (7:
13) afforded impure fractions that were mixed and loaded on
precoated plates (Si gel 60 F254 0.2 mm, Merck) using EtOAc-
petroleum ether (1:1) as mobile phase to yield a crystalline
from a 1-mL seed flask was innoculated aseptically into 20
media flasks (250 mL) containing 100 mL of medium and
fermentation was continued for a further 2 days.
F er m en t a t ion of (1R,2S,5R)-(-)-Men t h ol. (1R,2S,-
solid with R
mp 59 °C; [R]25
200 (2.5) nm; IR (CHCl
MHz) data, see Table 1; 13C NMR (CDCl
Table 2; positive ion FABMS m/z 173; EIMS (70 eV) m/z 154
(4), 134 (6), 94 (100), 79 (64); HREIMS m/z 172.1428 [M] calcd
f
0.3, identified as 3R-hydroxymenthol (5) (2.3%):
5
R)-(-)-Menthol (1) (1.0 g) was diluted with Me
2
CO (10 mL),
D
34° (c 0.072, MeOH); UV (MeOH) λmax (log ꢀ)
-
1 1
and the resulting solution was evenly distributed among 20
conical flasks having shake cultures, and the fermentation was
continued for 12 days. The mycelium was filtered, washed
with EtOAc (1 L), and the broth thus obtained was extracted
with EtOAc (10 L). The extract was dried over anhydrous
sodium sulfate and concentrated in vacuo to afford a brown
gum (2 g) that was absorbed on flash Si gel (5.0 g), subjected
to column chromatography containing flash Si gel (50 g), and
eluted with various solvent gradients of petroleum ether,
EtOAc, and MeOH.
3
) υmax 3400 cm ; H NMR (CDCl
3
, 500
3
, 125 MHz) data, see
+
for C10
20 2
H O 172.1463.
Com p ou n d 6. Elution with EtOAc-petroleum ether (2:3)
afforded a white crystalline solid identified as 9-hydroxymen-
thol (6) (14.0%); mp 79.5 °C; [R]25
-20° (c 0.2, MeOH); UV
D
-
1
(MeOH) λmax (log ꢀ) 220 (1.6) nm; IR (CHCl
3
) υmax 3400 cm
;
,
1
13
3
H NMR (CDCl , 400 MHz) data, see Table 1; C NMR (CDCl
3
1
25 MHz) data, see Table 2; positive ion FABMS m/z 172.1;
Com p ou n d 1. Elution with EtOAc-petroleum ether (1:4)
+
+
2
5
EIMS (70 eV) m/z 172 (5) [M] 154 (11) [M - 18] , 112 (29),
5.1 (43), 81 (100), 71 (82), 55 (86); HREIMS m/z 172.1504
afforded residual menthol (1) (6.0%); mp 43 °C; [R]
D
507° (c
9
0
3
.06, MeOH); UV (MeOH) λmax (log ꢀ) 215 nm (2.1); IR (CHCl )
+
-
1
1
20 2
[M] , calcd for C10H O , 172.1463.
ν
max 3595 cm ; H NMR (CDCl
3
, 500 MHz) data, see Table 1;
1
3
Com p ou n d 7. Elution with EtOAc-petroleum ether (1:1)
afforded impure fractions that were mixed and loaded on to
precoated plates (Si gel 60 F254 0.2 mm, Merck) and eluted
with EtOAc-petroleum ether (1:1). This afforded a white
C NMR (CDCl
3
, 100 MHz) data, see Table 2; positive ion
+
FABMS m/z 157; EIMS (70 eV) m/z 155 (1.5) [M - 1] , 138
+
(
2
29) [M - H O] , 95 (64), 71 (100)
Com p ou n d 2. Elution with EtOAc-petroleum ether (40-
0) (3:17) afforded impure fractions that were mixed and
purified by TLC on precoated plates (Si gel 60 F254 0.2 mm,
f
crystalline solid with R 0.2, identified as 10-hydroxymenthol
6
(
7) (21.5%): mp 88.4 °C; [R]25
MeOH) λmax (log ꢀ) 230 (2.2) nm; IR (CHCl
D
57.0 (c 0.06, MeOH); UV
-
1
(
3
) υmax 3400 cm
;
,
Merck) using EtOAc-petroleum ether (2:3) as mobile phase
1
13
3
H NMR (CDCl , 300 MHz) data, see Table 1; C NMR (CDCl
3
where two pure compounds (2 and 3) were obtained.
A
7
5 MHz) data, see Table 2; positive ion FABMS m/z 173; EIMS
f
colorless crystalline solid with R 0.4 has been identified as
+
+
0-acetoxymenthol (2) (3.0%): mp 68 °C; [R]2
0
(70 eV) m/z 172 (68) [M] , 154 (12) [M - 18] , 123 (95), 81
1
D
24° (c 0.13,
+
(100), 55 (89); HREIMS m/z 172.1454 [M] , calcd for C10
20 2
H O
MeOH); UV (MeOH) λmax (log ꢀ) 208 (2.20), 273.2 (2.04) nm;
-
1 1
172.1463.
IR (CHCl
see Table 1; C NMR (CDCl
positive ion FABMS m/z 215; EIMS (70 eV) m/z 213 (3) [M -
3
) λmax 3600 cm ; H NMR (CDCl
3
, 300 MHz) data,
, 125 MHz) data, see Table 2;
1
3
3
Ack n ow led gm en t. We are thankful to the Pakistan
Science Foundation for a grant no. PSF/S-Ku/CHEM(218) to
A.R. We are also thankful to the Bismillah Bibi Trust for
financial assistance to M.Y.
+
1
] , 165 (5), 154 (70), 136 (81), 112 (74), 55 (100); HREIMS
+
m/z 214.1559 [M] , calcd for C12
Com p ou n d 3. The second compound obtained by TLC (see
compound 2) was a colorless crystalline solid with R 0.2, and
identified as 7-hydroxymenthol (3) (4.8%); mp 58.9 °C; [R]
0° (c 0.06, MeOH); UV (MeOH) λmax (log ꢀ) 220 (2.7) nm; IR
22 3
H O 214.1569.
Refer en ces a n d Notes
f
2
5
D
(1) Farooq, A.; Hanson, J . R. Phytochemistry 1995, 40, 815-817.
(2) Gand, E.; Hanson, J . R.; Nasir, H. Phytochemistry 1995, 39, 1081-
1084.
5
(
-
1 1
CHCl
Table 1; C NMR (CDCl
ion FABMS m/z 173; EIMS (70 eV) m/z 172 (0.08) [M] 154
3
) λmax 3440 cm ; H NMR (CDCl
3
, 300 MHz) data, see
(
3) Ackland, M. J .; Gordon, J . F.; Hanson, J . R.; Yeoh, B. L.; Ratcliffe, A.
1
3
3
, 125 MHz) data, see Table 2; positive
H. Phytochemistry 1988, 27, 1031-1033.
+
(4) Hanson, J . R.; Nasir, H. Phytochemistry 1993, 33, 831-834.
(5) Hanson, J . R.; Hitchcock, P. B.; Manickavasagar, R. Phytochemistry
1994, 37, 1023-1026.
+
(
2) [M - 18] , 139 (12), 121 (9), 96 (65), 81 (100), 59 (92);
+
HREIMS m/z 172.1445 [M] , calcd for C10
20 2
H O , 172.1463.
(6) Hanson, J . R.; J arvis, A. G.; Laboret, F.; Takahashi, J . Phytochemistry
Com p ou n d 4. Elution with EtOAc-petroleum ether (3:7)
afforded impure fractions that were loaded on TLC precoated
plates (Si gel 60 F254 0.2 mm, Merck) and eluted with EtOAc-
1995, 38, 73-76.
(7) Atta-ur-Rahman; Choudhary, M. I.; Ata, A.; Alam, M.; Farooq, A.;
Perveen, S.; Shekhani, M. S.; Ahmed, N. J . Nat. Prod. 1994, 57,
1
251-1255.
petroleum ether (1:1) to yield a colorless crystalline solid, R
f
(
8) Atta-ur-Rahman; Farooq, A.; Choudhary, M. I. J . Nat. Prod. 1997,
0
.7, identified as 4â-hydroxymenthol (4) (7.5%): mp 55 °C;
60, 1038-1040.
25
(9) Belukha, U.K. Med. Zh. Uzb. 1969, 10, 23-25.
[R]
D
20° (c 0.108, MeOH); UV (MeOH) λmax (log ꢀ) 220 (2.5)
-
1
1
(10) Baron, C.; Maume, B.; Lhuguenot, L. C. Fr. Sec. Parfums 1970, 13(67),
nm; IR (CHCl
3
) υmax 3400 cm ; H NMR (CDCl
3
, 500 MHz)
3
1-35.
1
3
data, see Table 1; C NMR (CDCl
3
, 125 MHz) data, see Table
(
11) Schulte-Elte, K. H.; Ohloff, G. Helv. Chim. Acta, 1967, 50, 153-165.
2
1
1
; positive ion FABMS m/z 173; EIMS (70 eV) m/z 155 (11),
+
54 (94) [M - 18] , 139 (100), 97 (81), 55 (79); HREIMS m/z
+
72.1188 [M] , calcd for C10
H
20
O
2
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