Microbiological and Chemical Transformations of Argentatin B
Galal T. Maatooq
Department of Pharmacognosy, Faculty of Pharmacy, University of Mansoura,
Mansoura 35516, Egypt. E-mail: galaltm@yahoo.com
Z. Naturforsch. 58 c, 249Ð255 (2003); received October 31/November 14, 2002
Argentatin B is a naturally occurring tetracyclic triterpene isolated from Parthenium argentatum x P. tomentosa. It was microbiologically transformed to 16, 24-epoxycycloartan-3α, 25diol, (isoargentatin D), by Nocardia corallina var. taoka ATCC 31338, Mycobacterium species
NRRL B3683 and Septomyxa affinis ATCC 6737. The later microbe also produced 16, 24epoxycycloartan-3β, 25-diol (argentatin D) and 1, 2-didehydroargentatin B, (isoargentatin
D). Sodium hydroxide converted argentatin B to argentatin D and isoargentatin D. Hydrochloric acid treatment gave cycloartan-25-ol-3, 24-dione. Cerium sulfate/sulfuric acid/aqueous
methanol induced scission of the isopropanol moiety and provided an isomeric mixture of
24-methoxy-25Ð27-trinorargentatin B. Oxidation of this isomeric mixture with pyridinium
chlorochromate, selectively, attacked the isomer with the equatorial proton at position-24 to
give the corresponding lactone, 24-oxo-25Ð27-trinorargentatin B. The produced compounds
were characterized by spectroscopic methods.
Key words: Argentatin B, Biotransformation, Cerium Sulfate
Introduction
Results and Discussion
Incanilin, argentatins A, B, C and D and isoargentatin B are naturally occurring, tetracyclic
triterpenes which were isolated from the rubber
plant Parthenium argentatum Gray (guayule),
Asteraceae (Rodriguez-Hahn et al., 1970; Komoroski et al., 1986 and Romo de Vivar et al., 1990).
These triterpenes were obtained in abundant
quantities, during the isolation of antifungal agents
from the resin of the guayule hybrid P. argentatum
¥ P. tomentosa (Maatooq et al., 1996). Several bioactive tetracyclic triterpenes are structurally related to these compounds (Williams et al., 1992;
Shi et al., 1992). Microorganisms and chemical
reactions can extend types of compounds by
transferring abundant prototypes into new ones.
Microorganisms are mimic of plant metabolism
and thus produce rare compounds from abundant
ones or biologically active and/or less toxic metabolites. In a previous communication, microbial
metabolic products of argentatin A and incanilin
were isolated (Maatooq and Hoffmann, 2002).
The biotransformation and the chemical conversion of argentatin B, isolation and structural elucidation of the produced compound are described
herein.
For the biotransformation of argentatin B, 1, 25
microbes were used for the screening purpose
Scale up the reactions with the three microorganisms, Nocardia corallina var. taoka ATCC 31338,
Mycobacterium species NRRL B3683 and Septomyxa affinis ATCC 6737 gave metabolite 2 as a
common product, while Septomyxa affinis ATCC
6737 produces two more metabolites (3 and 4).
Metabolites 2 and 3 were characterized as isoargentatin D and argentatin D, respectively (Rodriguez-Hahn et al., 1970; Komoroski et al., 1986 and
Romo de Vivar et al., 1990) (Table I and Fig. 1).
The 13C-NMR spectrum of metabolite 4 displayed two new olefinic carbon atoms signals at
δ 154.2 and 127.3, which were correlated to the
proton doublets at 5.94 and 6.77 (J = 11 Hz each),
respectively. This concluded that 4 should have a
new double bond represented by two olefinic
methine groups. The location of this double bond
was concluded to be at position 1, since no significant changes in the chemical shifts values at rings
C and D. However, position 3 carbonyl resonance
is shielded and was found to be at δ 205.1 (ca.
11.4 ppm), which implies the likely dehydrogenation of positions-1 and 2 to give the α, β-unsaturated carbonyl group. This is supported by the ap-
0939Ð5075/2003/0300Ð0249 $ 06.00
” 2003 Verlag der Zeitschrift für Naturforschung, Tübingen · www.znaturforsch.com ·
D
250
G. T. Maatooq · Transformations of Argentatin B
C#
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26+
27+
28
29
30
OCH3
33.4
37.3
216.2
50.1
48.5
21.2
26.0
47.4
20.7
25.9
26.2
32.7
45.9
45.8
44.9
74.9
57.5
18.9
29.8
29.0
20.8
35.5
23.4
82.7
73.3
23.9
25.6
19.5
22.3
20.7
Ð
31.9
30.3
78.7
40.4
47.1
21.0
25.7
47.4
19.5
25.8
26.1
32.7
45.8
45.7
44.8
74.8
57.3
18.7
30.0
28.9
20.9
35.4
23.4
82.4
73.2
23.8
25.6
14.0
25.7
19.4
Ð
28.5
27.3
76.9
39.4
40.9
20.8
25.8
47.4
19.5
26.4
25.8
32.7
45.9
45.8
44.7
74.8
57.2
18.6
29.8
28.8
21.1
35.4
23.4
82.4
73.2
237
25.5
20.9
25.7
19.4
Ð
154.2
127.3
203.1
46.3
46.8
21.9
26.1
46.4
25.2
30.2
27.9
32.6
44.9
44.3
43.7
75.1
57.5
18.0
29.8
29.8
19.6
35.9
23.9
83.1
73.7
24.0
24.4
20.0
21.5
19.2
Ð
33.4
37.4
216.5
50.2
48.5
21.3
25.9
47.9
20.9
26.4
26.0
32.6
46.7
46.6
40.9
45.2
57.3
17.7
29.8
29.5
20.1
36.6
29.3
216.8
72.1
18.3
18.4
20.8
22.1
18.9
Ð
33.4
37.4
216.5
50.2
48.4
21.4
26.0
47.5
20.8
26.3
26.1
33.2
46.1
45.2
44.9
80.5
60.8
19.4
29.8
32.5
23.0
36.6
33.8
109.6
Ð
Ð
Ð
20.8
22.1
19.6
55.4
33.4
37.4
216.5
50.2
48.4
21.4
26.0
47.3
20.8
26.3
26.2
32.7
46.2
45.4
44.7
70.4
58.0
18.3
29.8
30.5
22.8
33.8
32.5
101.2
Ð
Ð
Ð
20.8
22.1
18.9
54.5
33.4
37.4
216.4
50.2
48.3
21.3
25.9
47.4
20.7
26.2
26.1
32.4
46.5
45.7
44.1
81.0
56.3
19.1
29.8
29.2
20.2
31.3
29.9
174.2
Ð
Ð
Ð
20.8
22.1
19.7
Ð
pearance of a strong absorption bands at 1610
(double bond) and 1670 cmÐ1 in the IR spectrum
and a UV absorption of 265.0 nm (Silverstein
et al., 1991). The EIMS gave the parent ion peak
at m/z 454 analyzed for C30H46O3 which is consistent with the proposed structure for 4 as 16,
24-epoxycycloart-1-en-25-ol (1, 2-didehydroargentatin B).
Compound 5 was obtained after reflux with hydrochloric acid. The EIMS gave m/z 456 analyzed
for C30H48O3. The 1H-NMR demonstrated the absence of both carbinol methine proton signals of
positions 16 and 24 in the range of δ 3.0Ð5.0. The
13
C-NMR spectrum showed the presence of only
one carbinol methine proton signal at δ 72.1 assigned to position 25 and confirmed the loss of the
epoxide linkage between positions 16 and 24 and
the absence of any other hydroxylations. Two carbonyl signals were observed at δ 216.5 and 216.8,
one of them has to be assigned to position 3, while
the new one at δ 216.8 has to be assigned to position 16 or 24. The location of this carbonyl was
Table I. 13C-NMR spectral
properties of compounds
1Ð8*.
* At 62.5 MHz, using
CDCl3 as a solvent, TMS
is the internal standard
and chemical shifts (δ)
are expressed in ppm.
+
Assignments may be interchangeable.
excluded from position 16 and was confirmed to
be at position 24 based on the appearance of m/z
369 [M-C4H7O2]+ and the strong peak at m/z 313
[M-C8H15O2]+. Thus, HCl reaction demonstrated
an opening of the side chain epoxide ring followed
by oxidation to produce 25-hydroxycycloartan-3,
24-dione.
Oxidation of argentatin B with cerium sulfate/
H2SO4/aq. MeOH, produced an isomeric mixture.
The analysis of the NMR spectral data of this mixture indicated that this reagent induced scission of
24Ð25 bond, followed by methoxylation of position-24 to give an isomeric mixture of 24-methoxy25Ð27-trinorargentatin B. The EIMS gave m/z 428
[M]+ analyzed for C28H44O3. The two isomers
were partially separated chromatographically,
where 6 was obtained in a pure form and the remaining mixture was oxidized with pyridinium
chlorochromate. It was noticed that 7 is completely oxidized to the corresponding lactone, 8,
24-oxo-25Ð27-trinorargentatin B, leaving behind
the remains of the unreacted 6.
G. T. Maatooq · Transformations of Argentatin B
251
21
22
18
20
23
12
13
11
17
16
19
1
24
9
14
2
10
26
11
OH
19
O
27
1
8
10
30
3
O
4
HO
29
O
22
18
24
20
26
8
11
19
23
1
OH
11
29
25
12
13
6
28
21
17
16
14
10
O
5
29
21
22
18
20
12
17
16
14
20
12
24
13
11
O
22
18
23
OCH3
8
17
16
14
15
30
4
6
21
8
7
5
4
28
11
8
3
30
13
9
2
27
15
2 = 3α
3 = 3β
7
5
1
6
28
8
3
7
5
4
9
2
15
23
24
O
O
15
6 = 24β
7 = 24α
The 1H-NMR spectral data of 6 displayed five
signals at δ 0.92 (3H, s, H-18), 0.95 (3H, d, J =
7.0 Hz, H-21), 1.05 (3H, s, H-30), 1.11 (3H,s, H-28)
and 1.19 (3H, s, H-29). This indicated the likely
loss of the two skeletal methyl groups at positions26 and 27. HETCOR showed that the proton
multiplet at δ 4.09Ð4.20 (2H) was correlated to
two methine (DEPT) carbon signals at δ 80.5 and
109.6 which were assigned to positions-16 and 24,
respectively. The δ 109.6 chemical shift value is
consistent for a deshielded position-24 as a result
of another oxygenation. The proton singlet at
δ 3.38 (3H) correlated to the carbon signal at
δ 55.4 (HETCOR) was assigned to the methoxyl
group at position-24. The relative stereochemistry
of this methoxyl group at position-24 was determined to be β-oriented which inferred from the
observed relatively high deshielding of the 13CNMR chemical shift of position-24 (δ 109.6) compared to that of 7 (δ 101.2). The overlapping of
the proton signals of positions-16 and 24 made it
difficult to add further confirmations. The 13CNMR data of 6 (Table I) were significantly af-
30
8
Fig. 1. The structure of argentatin B
and its transformation products.
fected with this transformation compared to that
of the substrate (Komoroski et al., 1986). This is
evidenced only at rings-D and E, while rings A, B
and C are not affected. The EIMS of 6 gave m/z
428 [M]+, 413 [M-CH3]+ and 397 [M-OCH3]+,
which are consistent with the proposed structure
for 6. The 1H and 13C-data for 7 was obtained by
subtracting those of 6 from those of the isomeric
mixture. The 1H-NMR spectral data of 7 showed
few differences from those of 6. Positions-16 and
24 protons signals appeared at δ 4.57Ð4.68 as
multiplet and were correlated (HETCOR) to the
carbon signals at δ 70.4 and 101.2, respectively.
The protons singlet at δ 3.31 integrated for three
protons and was correlated to the methyl carbon
signal (DEPT) at δ 54.5 which was assigned to position-24 methoxyl group. The relative stereochemistry of this methoxyl group was assigned to
be α-oriented based on the relative shielding of
position-24 carbon signal (δ 101.2), compared to
that of 6 (δ 109.6). The overlapping of positions16 and 24 anomeric proton signal made it impossible to get more confirmations.
252
The 1H-NMR spectrum of 8 indicated the absence of the methoxyl group, since the proton signal at δ 3.31 was vanished. A downfield shifted
single proton multiplet observed at δ 4.92, which
was correlated to the carbon signal at δ 81.0, was
assigned to position-16, while the anomeric position-24 carbon and proton signals were absent. A
new downfield carbon signal at δ 174.2, in 13CNMR spectrum, was assigned to position-24-oxo
group forming a lactone ring. The location of the
lactone ring was also confirmed by its observed
deshielding effect on H-16 (ca. δ 0.3) and C-16 (ca.
δ 10.0). The IR spectrum showed a strong absorption band at 1680 cmÐ1 (lactone ring). The EIMS
of 8 gave m/z 412 as a parent ion peak analyzed
for C27H40O3, which is consistent with the proposed structure for the oxidation product to be 24oxo-25Ð27-trinorargentatin B.
G. T. Maatooq · Transformations of Argentatin B
cording to the standard two-stages fermentation
protocol (Betts et al., 1974). Preliminary screening
experiments were carried out in 125 ml stainless
steel capped DeLong culture flasks held one fifth
of their volume of the following medium; 2% glucose, 0.5% soybean meal, 0.5% yeast extract, 0.5%
NaCl and 0.5% K2HPO4. The pH of the medium
was adjusted to 7.0 using 6 n HCl before autoclaving for 20 m at 121∞ and 15 psi. After inoculation
with Nocardia corallina var. taoka ATCC 31338, or
Mycobacterium species B3683 NRRL or Septomyxa
affinis ATCC 6737, stage I cultures were incubated
at 27∞ and 250 rpm for 72 h before being used to inoculate stage II culture flasks. Usually, 10% inoculum volumes are recommended. For screening scale
experiments 10 mg of the triterpene in 0.2 ml of
DMF-EtOAc, 1:1 v/v, mixture was added to 24-hold stage II cultures, which were incubated again
and sampled periodically for analysis.
Experimental
Instrumentation
Sampling
Melting points are uncorrected. IR was conducted on Beckman Acculab I IR spectrometer.
UV data were obtained from Beckman Model 26
Spectrophotometer. Optical rotations were measured on Autopole III Automatic Polarimeter
(Rudolph Scientific, Fairfield, New Jersey). 1HNMR and 13C-NMR were measured on a Bruker
WM 250 NMR Spectrometer, at 250 MHz and
62.5 MHz, respectively, using CDCl3 as a solvent
and TMS as the internal standard. The chemical
shifts (δ) are e¥pressed in ppm. DEPT and
HETCOR were measured on a Bruker WM 300
NMR Spectrometer. CIMS (CH4) and EIMS
(70 eV) were conducted on a Hewlett Packard
5988A Spectrometer, equipped with a Hewlett
Packard RTE-6/VM data system.
Samples of 1 ml each were taken after 12, 24, 36
and 48 h and every other day for 2 weeks following
substrate addition. Each sample was extracted by
shaking with 0.5 ml EtOAc and spun at 3000 ¥ g
for 1 min in a desk-top centrifuge. EtOAc extract
of all samples were spotted on Si gel GF254 TLC
plates, and developed in a suitable solvent system.
All the chromatograms were visualized after
spraying with 0.01% vanillin/H2SO4, followed by
heating for 5Ð10 s with a heat gun.
Substrate material
Argentatin B was isolated from Parthenium argentatum ¥ P. tomentosa and was characterized by
1
H-, 13C-NMR and mass spectrometry (Rodriguez-Hahn et al., 1970; Komoroski et al., 1986 and
Romo de Vivar et al., 1990).
Preparative scale conversion of argentatin B with
Nocardia corallina (Reaction-A)
Five 2-liter stage II cultures received 2.0 g of argentatin B in 10 ml of DMF-EtOAc, 1:1 (1 mg substrate per ml of culture medium). After incubation
for 10 days under the usual condition, the cultures
were combined and exhaustively extracted with
3 ¥ 1.5 liter of 10% MeOH/EtOAc. The ethyl acetate extract was concentrated and dried to yield
2.49 g of a dark brown residue.
Fermentation methods
Preparative scale conversion of argentatin B with
Mycobacterium species (Reaction-B)
Microbial transformation studies were carried
out by incubating the cultures, by shaking at
250 rpm, at 25∞. Fermentation was carried out ac-
Seven 2-liter stage II cultures received 2.8 g of
argentatin B in 14 ml of DMF-EtOAc, 1:1 (1 mg
substrate per ml of culture medium). After incuba-
G. T. Maatooq · Transformations of Argentatin B
tion for 18 days under the usual condition, the cultures were combined and exhaustively extracted
with 3 ¥ 2 liter of 10% MeOH/EtOAc. The ethyl
acetate extract was concentrated and dried to yield
3.9 g of a brown residue.
Preparative scale conversion of argentatin B with
Septomyxa affinis (Reaction-C)
Ten 2-liter stage II cultures received 4.0 g of argentatin B in 20 ml of DMF-EtOAc, 1:1 (1 mg substrate per ml of culture medium). After incubation
for 13 days at 250 rpm and 27∞, the cultures were
combined and exhaustively extracted with 3 ¥ 3
liter of 10% MeOH/EtOAc. The extract was concentrated and dried to yield 5.2 g residue.
Isolation and purification of reactions A and B
metabolites
The TLC indicated the presence of one major
spot more polar than the substrate, in both reactions, at Rf = 0.50 (Si gel GF254, hexane-EtOAc;
75:25). The crude extract of reactions A (2.49 g)
and B (3.9 g) were, separately flash chromatographed, 200 g silica gel, 63Ð200 µ, 2.5 ¥ 45 cm.
The elution was adopted using EtOAc/hexane
500 ml each of 5%, 10%, 15%, 20%, 25%, 30%
and 100%. Frs 100Ð200 ml each were collected
and TLC investigated. Similar frs were pooled together. Frs eluted with 10Ð15% in both reactions
gave 1.51 g and 1.72 g of recovered substrate,
respectively. Frs eluted with 20% EtOAc/hexane
gave 2 as needles (109 mg from reaction A and
256 mg from reaction B), respectively, after prep
TLC on 1 mm-thick silica gel GF254 plates and
using 30% EtOAc/hexane as a solvent.
Isolation and purification of reaction C metabolites
The TLC displayed two new reddish-brown
spots at Rf = 0.52 and 0.50, respectively (Si gel
GF254, hexane-EtOAc; 75:25). The crude reaction
mixture (5.2 g) was subjected to flash chromatography, silica gel, 400 g, 63Ð200 µ, 3.5 ¥ 45 cm. The
elution profile was EtOAc/hexane, 1000 ml each
of 5%,10%, 15%, 20%, 25%, and 50%. Twenty
four frs were collected, 200Ð300 ml each, and TLC
investigated. Similar frs were pooled together. Frs
eluted with 10Ð15% EtOAc/hexane gave 2.22 gm
253
of recovered substrate. Frs eluted with 15Ð20%
EtOAc/hexane gave 2.01 g residue which displaying 3 spots one of them is the substrate. This 2.01 g
was subjected to MPLC, 140 g silica gel 15Ð25 µ,
2.5 ¥ 45 cm. The elution was adopted using
EtOAc/hexane 500 ml each of 5%, 10%, 15%,
20%, 25% and 50%. Frs 4Ð5 eluted with 10%
EtOAc/hexane gave 59 mg of the substrate. Frs
eluted with 15% EtOAc/hexane gave three different groups. Frs 8Ð10 afforded 96 mg of 2 as
needles. Fr 7 gave 170 mg of 3 as needles. Fr 6
gave 38 mg of 4 which further purified on 1 mmthick prep TLC silica gel GF254 plates, using 30%
EtOAc/hexane as a solvent system. This gave
26 mg of 4 as yellowish gum. Compound 4 is
strongly quenching under Uv light. Compounds 2,
3 and 4 possess Rf = 0.5, 0.52 and .054 (25%
EtOAc/hexane), respectively.
Sodium hydroxide reaction
Argentatin B, 0.50 g was refluxed with 50%
methanolic NaOH, 50 ml, overnight. The reaction
mixture was diluted with H2O (200 ml) and extracted with EtOAc (3 ¥ 150 ml) to give 0.42 g residue. The reaction product was subjected to c. c.,
140 g silica gel 63Ð200 µ, 1.5 ¥ 45 cm. The elution
was adopted using EtOAc/hexane, 250 ml each of
5%, 10%, 15%, 20%, 25% and 50%. Frs eluted
with 15Ð20% displayed 2 spots (Rf = 0.5 and 0.52
in 25% EtOAc/hexane). After prep TLC, on
1 mm-thick silica gel GF254 plates, it gave 93 mg
and 52 mg of 2 and 3, respectively.
Hydrochloric acid reaction
Argentatin B, 1.0 g, was dissolved in 50 ml
MeOH and 30 ml conc. HCl was added. The mixture was refluxed overnight, then diluted with
300 ml H2O and extracted with EtOAc, 3 ¥ 300 ml,
to give 0.82 g residue. The reaction product was
subjected to c. c., 150 g silica gel 63Ð200 µ, 2.5 ¥
45 cm. The elution was adopted using 2.0 l 5%
EtOAc/hexane, 1.0 l 10%, 1.0 l 15% and 1.0 l 20%
v/v. Frs eluted with 10Ð15% gave 240 mg of
recovered substrate. Frs eluted with 15Ð20% gave
503 mg of 5 as needles which was purified on
1 mm-thick prep TLC silica gel GF254 plates, using
30% EtOAc/hexane as a solvent (Rf = 0.53).
254
Cerium sulfate reaction
Argentatin B, 0.5 g, was dissolved in MeOH,
40 ml, then 2.0 g cerium sulfate was added, followed by 10 ml 1:1 H2SO4/H2O. The reaction mixture was refluxed for four h where 100% conversion was observed. The reaction mixture was
diluted with H2O, 200 ml, and extracted with
EtOAc, 3 ¥ 300 ml, to give 0.44 g residue. The reaction product, 0.44 g, was subjected to c. c., 150 g
silica gel 63Ð200 µ, 2.5 ¥ 35 cm. The elution was
achieved using 2000 ml 5% EtOAc/hexane and
1000 ml 10% EtOAc/hexane. Twelve frs were obtained. Frs 9Ð11 eluted with 10% EtOAc/hexane
afforded 168 mg needles (Rf = 0.71, 30% EtOAc/
hexane). By NMR this product proved to be an
isomeric mixture of equal proportions of 6 and 7.
Partial chromatographic separation was obtained
by c. c, 140 gm 63Ð200, 1.5 ¥ 45 cm. The eluting
solvent was 500 ml 0.5% iso-Pr-OH/hexane,
3000 ml of 1%, 500 ml 2%, 500 ml 5%, 250 ml
10% and 300 ml Me2CO. Thirty frs were obtained
(100Ð200 ml each). Frs 7Ð10 eluted with 1% isoPr-OH/hexane gave 62 mg of pure 6 as needles
(Rf = 0.31, 2% iso-Pr-OH/hexane). Frs 4Ð6 eluted
with 1% iso-Pr-OH/hexane gave 95 mg unresolved
mixture. This isomeric mixture was dissolved in
5 ml CH2Cl2 and 200 mg of pyridinium chlorochromate was added. The reaction mixture was
left at room temperature for four h where equilibrium was obtained. Water was added (50 ml) and
the reaction mixture was extracted with EtOAc,
3 ¥ 50 ml. The residue left after solvent evaporation was subjected to successive prep TLC on
1 mm-thick silica gel GF254 plates using 4% isoPr-OH/hexane then 20% Me2CO/hexane as a
solvent systems. This gave 20 mg of 6 as needles
and 23 mg of 8 as needles (Rf = 0.32, 20%
Me2CO/hexane).
G. T. Maatooq · Transformations of Argentatin B
H-16), 3.58 (1H, dd, 5, 5, H-24), 3.28 (1H, dd, 3, 9,
βH-3), 1.15 (3H, s, H-28), 1.09 (6H, s, H-26 and
H-27), 0.97 (3H, s, H-29), 0.93 (3H, d, 7, H-21),
0.88 (3H,s, H-30), 0.82 (3H, s, H-18), 0.59 (1H, d,
5, H-19) and 0.34 (1H, d, 5, H-19⬘).
Compound 3, argentatin D, (Ð) 16,
24-epoxycycloartan-3β, 25-diol
Needles, mp 225∞. α[D]25, Ð 13.8∞ (CH2Cl2;
c. 0.5). UV λmax nm; 221.0. EIMS, 70 eV, m/z (rel.
int.); 458 [M]+ (5), 457 [M-H]+ (12), 443 [M-CH3]+
(8), 441 (50), 439 (40), 424 (40), 423 (100), 399
(41), 382 (30), 381 (44), 313 (10), 233 (18), 201
(30), 175 (22), 149 (20) and 127 (42). 1H-NMR
(250 MHz, CDCl3, (δ) ppm, J = Hz); 4.55 (1H, m,
H-16), 3.56 (1H, dd, 5, 5, H-24), 3.44 (1H, br t,
αH-3), 1.11 (3H, s, H-28), 1.07 (3H, s, H-26), 1.06
(3H, s, H-27), 0.92 (3H, s, H-29), 0.87 (3H, d, 7,
H-21), 0.85 (3H,s, H-30), 0.83 (3H, s, H-18), 0.51
(1H, d, 5, H-19) and 0.31 (1H, d, 5, H-19⬘).
Compound 4, (Ð) 16, 24-epoxycycloartan-1-en-25ol-3-one
Yellow gum, α[D]25, Ð 45∞ (CH2Cl2; c. 1.0). IR
υmaxcmÐ1; 3410, 2940, 2910, 2860, 1670, 1610, 1460,
1340, 1270, 1160 , 1100, 1060 and 730. UV λmax
nm; 265.0. EIMS, 70 eV, m/z (rel. int.); 454 [M]+
(2), 439 [M-CH3]+ (2), 436 [M-H2O]+, (40), 421
[M-H2O-CH3]+, (2), 395 [M-C3H7O]+, (2), 396 (3),
381 (4), 337 (2), 297 (5), 233 (6), 203 (11), 159 (23),
137 (25), 109 (29), 93 (33), 59 (100) and 42 (77).
1
H-NMR (250 MHz, CDCl3, (δ) ppm, J = Hz);
5.94 (1H, d, 11, H-1), 6.77 (1H, d, 11, H-2), 4.59
(1H, m, H-16), 3.60 (1H, dd, 5, 5, H-24), 1.12 (3H,
s, H-28), 1.11 (6H, s, H-26 and H-27), 1.10 (3H, s,
H-29), 0.95 (3H,s, H-30), 0.92 (3H, d, 7, H-21), 0.88
(3H, s, H-18), 0.91 (1H, d, 5, H-19) and 0.74 (1H,
d, 5, H-19⬘).
Compound 2, isoargentatin D, (Ð) 16,
24-epoxycycloartan-3α, 25-diol
Compound 5, (Ð) 25-hydroxycycloartan-3,
24-dione
Needles, mp 148Ð149∞, α[D]25, Ð 31.4∞ (CH2Cl2;
c. 1.5). UV λmax nm; 218.0. EIMS, 70 eV, m/z (rel.
int.); 458 [M]+ (8), 457 [M-H]+ (20), 443 [M-CH3]+
(22), 441 (60), 440 (9), 424 (40), 423 (100), 400
(30), 381 (39), 341 (10), 260 (22), 233 (18), 203
(30), 202 (30), 161 (22) and 127 (52). 1H-NMR
(250 MHz, CDCl3, (δ) ppm, J = Hz); 4.59 (1H, m,
Needles, mp 136Ð138∞, α[D]25, Ð 4.2∞ (CH2Cl2;
c. 1.5). IR υmaxcmÐ1; 3390, 2930, 2850, 1710, 1340,
1100, 1010 and 740. UV λmax nm; 228.0. EIMS,
70 eV, m/z (rel. int.); 456 [M]+ (2), 441 [M-CH3]+
(4), 438 [M-H2O]+, (5), 423 (8), 369 [M-C4H7O2]+,
(4), 313 [M-C8H15O2] (13), 311 (14), 285 (3), 271
(3), 245 (2), 219 (40), 173 (8), 133 (18), 105 (17),
G. T. Maatooq · Transformations of Argentatin B
71 (42), 54 (40), 43 (100) and 42 (33). 1H-NMR
(250 MHz, CDCl3, (δ) ppm, J = Hz); 1.17 (3H, s,
H-28), 1.12 (3H, s, H-29), 1.11 (3H, d, 7, H-21),
1.10 (3H, s, H-26), 1.09 (3H, s, H-27), 1.05 (3H,s,
H-30), 0.91 (3H, s, H-18), 0.82 (1H, d, 5, H-19) and
0.57 (1H, d, 5, H-19⬘).
Compound 6, (+) 24-β methoxy-25Ð27trinorargentatin B
255
Compound 7, 24-α methoxy-25Ð27trinorargentatin B
1
H-NMR (250 MHz, CDCl3, (δ) ppm, J = Hz);
4.57Ð4.68 (2H, m, H-16 and H-24), 3.31 (3H, s,
H-24 methoxy), 1.19 (3H, s, H-28), 1.11 (3H, s,
H-29), 1.05 (3H,s, H-30), 0.95 (3H, d, 7, H-21), 0.92
(3H, s, H-18), 0.83 (1H, d, 5, H-19) and 0.59 (1H,
d, 5, H-19⬘).
Compound 8, (Ð) 24-oxo-25Ð27-trinorargentatin B
Needles, mp 162Ð164∞, α[D] , + 6.1∞ (CH2Cl2;
c. 2.5). IR υmaxcmÐ1; 3090, 2930, 2850, 1710, 1340,
1100, 1010 and 740. UV λmax nm; 226.0. EIMS,
70 eV, m/z (rel. int.); 428 [M]+ (2), 413 [M-CH3]+
(2), 397 [M-CH3O]+ (3), 381 (3), 363 (2), 311 (6),
290 (12), 258 (4), 219 (10), 159 (32), 133 (40), 121
(55), 107 (62), 71 (100), 55 (90) and 43 (75). CIMS
(CH4), m/z (rel. int.); 429 [M+1]+ (7), 428 [M]+
(18), 427 (14), 411 (20), 397 [M-CH3O]+ (100), 379
(85), 311 (11), 269 (7), 231 (12), 219 (14), 193 (27),
177 (26), 175 (32), 163 (12), 219 (14), 193 (27), 177
(26), 175 (35), 163 (12), 133 (10), and 85 (17). 1HNMR (250 MHz, CDCl3, (δ) ppm, J = Hz); 4.09Ð
4.20 (2H, m, H-16 and H-24), 3.38 (3H, s, H-24
methoxy), 1.19 (3H, s, H-28), 1.11 (3H, s, H-29),
1.05 (3H,s, H-30), 0.95 (3H, d, 7, H-21), 0.92 (3H,
s, H-18), 0.83 (1H, d, 5, H-19) and 0.59 (1H, d, 5,
H-19⬘).
Needles, mp 191Ð192∞, α[D]25, Ð 35.8∞ (CH2Cl2;
c. 1.0). IR υmaxcmÐ1; 2910, 2860, 1730, 1680, 1350,
1090, 1030 and 790. UV λmax nm; 232.0. EIMS,
70 eV, m/z (rel. int.); 412 [M]+ (10), 397 [M-CH3]+
(10), 311 (11), 275 (11), 274 (32), 259 (20), 219
(10), 193 (20), 173 (28), 133 (40), 91 (45), 67 (43),
55 (100), 43 (60) and 42 (80). 1H-NMR (250 MHz,
CDCl3, (δ) ppm, J = Hz); 4.92 (H, m, H-16), 1.18
(3H, s, H-28), 1.14 (3H, s, H-29), 1.06 (3H,s, H-30),
1.00 (3H, d, 7, H-21), 0.95 (3H, s, H-18), 0.85 (1H,
d, 5, H-19) and 0.61 (1H, d, 5, H-19⬘).
Betts R. E., Walters D. E., and Rosazza J. P. N. (1974), Microbial transformation of antitumor compounds. 1.
Conversion of acronycine to 9-hydroxyacronycine by
Cunninghamella echinulata. J. Med. Chem. 17, 599Ð602.
Komoroski R. A., Gregg E. C., Shockcer J. P., and
Geckle J. M. (1986), Identification of guayule triterpenes by two-dimentional and multiple NMR techniques. Magn. Res. Chem. 24, 534Ð543.
Maatooq G. T., Stumpf D. K., Hoffmann J. J., Hutter
L. K., and Timmermann B. N. (1996), Antifungal
eudesmanoids from Parthenium argentatum ¥ P. tomentosa. Phytochemistry 41, 519Ð523.
Maatooq G. T., and Hoffmann J. J. (2002), Microbial
transformation of a mixture of argentatin A and Incanilin. Z. Naturforsch. 57 c, 498Ð495.
Rodriguez-Hahn L., Romo de Vivitar A., Ortega A.,
Aguilar M., and Romo J. (1970), Determinacion de
las estracturas de las argentatinas A, B y C de las
guayule. Rev. Latinoam. Quim 1, 24Ð38.
Romo de Vivar A., Martinez-Vazquez M., Matsubara C.,
Prez-Sanchez and Joseph-Nathan P. (1990), Triterpenes in Parthenium argentatum, structures of argentatins C and D. Phytochemistry, 29, 915Ð918.
Shi Q., Chen K., Fujiboka T. I., Kashiwada Y., Chang J.,
Kozoka M., Estes J. R., McPhail A. T., McPhail D. R.,
and Lee K. (1992), Antitumor agents, 135. Structure
and stereochemistry of polacandrin, A new cytotoxic
triterpene from Polanisia dodecandra. J. Nat. Prod. 55,
1488Ð1497.
Silverstein R. M., Bassler G. C., and Morrill T. C. (1991),
Spectrometric Identification of Organic Compounds,
5th ed. John Wiley & Sons, New York, pp. 115 &
303.
Williams D. E., Sinclair A. R. E., and Andersen R. J.
(1992), Triterpene constituents of the dwarf birch, Betula glandulosa. Phytochemistry 31, 2321Ð2324.
25
Dedication
This work is dedicated to the spirit of my late
colleague Prof. Dr. Joseph J. Hoffmann, Southwest
Center for Natural Products Research and Commercialization, Office of Arid Lands Studies, College of Agriculture, University of Arizona, USA.