Microbial metabolism. Part 8. The pyranocoumarin, decursin

Article abstract of DOI:10.1248/cpb.55.1512

Microbial transformation of the cancer chemopreventive agent, decursin (1) with Sepedonium chrysospermem (ATCC 13378) yielded two metabolites, (+)-decursinol (2) and (-)-cis-decursidinol (3). The structures were established by spectroscopic data.

Full text of DOI:10.1248/cpb.55.1512

1512
Notes
Chem. Pharm. Bull. 55(10) 1512—1513 (2007)
Vol. 55, No. 10
Microbial Metabolism. Part 8.1) The Pyranocoumarin, Decursin
a
a
,a,b
*
Wimal HERATH, Niranjan REDDY, and Ikhlas Ahmad KHAN
a National Center for Natural Products Research, The University of Mississippi; and b Department of Pharmacognosy,
Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi; University, MS 38677,
U.S.A. Received March 20, 2007; accepted July 13, 2007
Microbial transformation of the cancer chemopreventive agent, decursin (1) with Sepedonium chrysosper-
mem (ATCC 13378) yielded two metabolites, (ꢀ)-decursinol (2) and (ꢁ)-cis-decursidinol (3). The structures were
established by spectroscopic data.
Key words decursin; microbial metabolism; Sepedonium chrysospermem
The genus Angelica of the plant family Apiaceae includes
over sixty species which are medicinally important.2) They
are used to treat many illnesses including, colds, hepatitis,
rheumatism, typhoid and fungal infections and many other
disorders, apart from being utilized them as anti-inflamma-
tory and diuretic agents.2) Angelica gigas is one such species
whose roots have been used to treat anemia. It is also used
as a sedative, under the Korean name Zam Dang Gui.3)
Methanolic extract of the roots of the plant inhibits acetyl-
cholinesterase enzyme (AChE) activity.4) AChE is responsi-
ble for the hydrolysis of acetylcholine (ACh), which is nol (2) and decursidinol (3).
needed to increase the cholinergic functions in the brain.
Decursinol (2) (5 mg, 1.25% yield) was a white solid with
ACh deficiency leads to memory impairments in Alzheimer’s a molecular formula C14H14O4 (HR-ESI-MS data). The pres-
disease.4) In vitro, AChE activity guided isolation yielded ence of –OH, –CH, –CꢁC and –CꢁO groups, in the com-
several coumarins with considerable activity.4) Whilst the pound was suggested by the IR absorption bands at 3440,
coumarin, decursinol (2), exhibited the highest AChE in- 2979, 1626 and 1717 cmꢂ1. Its 1H- and 13C-NMR spectra dif-
hibitory activity, its structural analogue, decursin, showed fered from those of decursin (1) by the absence of signals
relatively poor activity.4) Structure activity data suggested the due to the 3-methylbut-2-enoate side chain. The presence of
need for a free hydroxyl group at C-3ꢀ for AChE activity.4) It a coumarin moiety (d 6.20 and 7.57, a doublet of 1H each
is interesting however, to observe the two compounds ex- due to H-3 and H-4) and a pair of aromatic protons para to
hibiting a reverse effect as anticancer agents on certain each other (d 6.80 and 7.17, a singlet of 1H each), along with
human prostrate carcinoma cells indicating the importance of a geminal dimethyl group (d 1.36 and 1.39, a singlet of 3H
the substituted side chain in decursin for anticancer efficacy.5) each) and a –CH2–CH system (d 2.83 and 3.11, 1H each and
The importance of the senecioic acid moiety of decursin as d 3.86 due to H-3ꢀ proton) indicated its similarity to decursi-
compared to the angelic acid group of decursinol angelate in nol. Comparison of the reported specific rotation and NMR
exhibiting anti-tumor activity has also been demonstrated by data enabled to characterize the metabolite as (ꢃ)-decursinol
in vivo experiments with mice.3) This is the first report of in (2).9)
vivo experiments on anti-tumor activity of decursin.3) How-
Decursidinol (3) (5 mg, 1.25% yield) was isolated as a
ever, there are no reports to-date on the isolation and charac- white solid. The HR-ESI-MS data suggested a molecular for-
terization of mammalian metabolites of this compound. mula C14H14O5 for the compound. Doublets of 1H each due
Since, microbial systems have been successfully used to to H-3 and H-4 at d 6.20 and 7.99 together with two aromatic
mimic mammalian metabolism of drugs,6) we attempted to proton singlets, para to each other at d 7.72 (H-5) and 6.67
1
generate decursin metabolites prospectively7) with the help of (H-8) in the H-NMR spectrum, indicated that the coumarin
such models. Initial screening of decursin with ten fungal architecture remained unchanged during transformation. It
strains showed the formation of two metabolites by seven also showed a small coupling constant (3.6 Hz) of two dou-
cultures. Scale up studies were carried out with Sepedonium blets at d 4.74 and 3.63 due to H-4ꢀ and H-3ꢀ protons indi-
chrysospermem (ATCC 13378) culture to isolate the metabo- cating their cis orientation. All spectroscopic data and spe-
lites in considerable yields. Structure elucidations of the cific rotation were in close agreement with those published
metabolites are discussed.
for (ꢂ)-cis-decursidinol with 3ꢀ(S), 4ꢀ(S) configuration.11)
The compound was thus identified as (ꢂ)-cis-decursidinol
(3).
Results and Discussion
Adopting the standard two stage procedure,8) decursin (1)
was screened using ten fungal cultures. Of the six organisms Conclusion
which showed the ability to transform 1, S. chrysospermem
Decursin (1) is among some of the linear dihydropyra-
(ATCC 13378) was selected for scale up experiments antici- nocoumarins which exhibits a wide range of biological prop-
pating better yields. The metabolites obtained were decursi- erties including cancer chemotherapeutic activity.2,12,13) S.
∗ To whom correspondence should be addressed. e-mail: ikhan@olemiss.edu
© 2007 Pharmaceutical Society of Japan

October 2007
1513
Microbial Transformation of Decursin (1) by S. chrysospermem
EtOAc extract of the combined culture filtrates was column chro-
matographed over silica gel (Si gel 230—400 mesh: E. Merck, 30 g, column
diameter: 20 mm.) with CHCl3 gradually enriched with MeOH. Two com-
pounds, 2 (15 mg) and 3 (10 mg) were isolated and identified by means of
chrysospermem and several other fungal strains transform
decursin (1) into (ꢃ)-decursinol (2) and (ꢂ)-cis-decursidinol
(3). Decursinol and few other coumarins including decursin
(1) are constituents of A. gigas.4) Compounds 1 and 2 to-
gether with decursinol angelate show important biological spectroscopic data.
activities.14) trans-Decursidinol is also a natural product
isolated from the roots of Peucedanum decursivum.15) Quan-
tities of decursinol and trans-decursidinol required to in-
vestigate the biochemical and pharmacological effects, in-
cluding the pain relief applications are obtained by organic
synthesis.14) However, except as reaction products there are
no reports on the isolation and bioactivity of cis-decursidi-
nol.
Decursinol (2) was isolated as a white solid (15 mg, 3% yield). Rf 0.32
[hexane–EtOAc (3 : 2)]; [a]D27 ꢃ6.3° (cꢁ0.11, MeOH). UV lmax (MeOH)
nm (log e): 207 (4.71), 220 (4.30), 330 (4.39); IR nmax(CHCl3) cmꢂ1: 3440,
2979, 2334, 1717, 1626, 1563, 1390, 1133, 1067, 821. HR-ESI-MS m/z:
247.1003 [MꢃH]ꢃ (Calcd for C14H15O4, 247.09711).
Decursidinol (3) was purified as a white solid (10 mg, 2% yield). Rf 0.12
[hexane–EtOAc (3 : 2)]; [a]D27 ꢂ42.5 (cꢁ0.11, CH2Cl2). UV lmax (MeOH)
nm (log e): 206 (4.49), 223 (4.15), 328 (4.09); IR nmax(CHCl3) cmꢂ1: 3489,
2922, 2854, 1708, 1623, 1558, 1460, 1384, 1288, 1147, 1957, 826. HR-ESI-
MS m/z: 263.0910 [MꢃH]ꢃ (Calcd for C14H15O5, 263.09202).
Since there are no reports on mammalian metabolites of
decursin, the data on the microbial transformed products,
(ꢃ)-decursinol (2) and (ꢂ)-cis-decursidinol (3) may be used
for further pharmacological evaluation of decursin. They
may also be used as analytical standards for detection in bio-
logical fluids.
The formation of more polar, phase I hydrolyzed (2) and
oxidized (3) products may be viewed as an attempt to reduce
the biological half-life of 1 to prevent its accumulation in the
body.16)
Acknowledgements The authors thank Mr. Frank Wiggers for assis-
tance in obtaining 2D NMR spectra and and Dr. Bharathi Avula for conduct-
ing HR-ESI-MS analysis. This work was supported, in part, by the United
States Department of Agriculture, Agricultural Research Specific Coopera-
tive Agreement No. 58-6408-2-00009.
References and Notes
1) Part 7: Herath W. H. M. W., Ferreira D., Khan I. A., Nat. Prod. Res.,
21, 444—450 (2007).
2) Sarker S. D., Nahar L., Curr. Med. Chem., 11, 1479—1500 (2004).
3) Lee S., Lee Y. S., Jung S. H., Shin K. H., Kim B.-K., Kang S. S., Arch.
Pharm. Res., 26, 727—730 (2003).
4) Kang Y. K., Lee K. Y., Sung S. H., Park M. J., Kim Y. C., J. Nat.
Prod., 64, 683—685 (2001).
Experimental
General Experimental Procedures IR spectra were measured in
CHCl3 on an ATI Mattson Genesis series FTIR spectrophotometer. UV spec-
tra were run on a Hewlett Packard 8452A diode array spectrometer. Specific
5) Yim D., Singh R. P., Agarwal C., Lee S., Chi H., Cancer Res., 65,
1035—1044 (2005).
1
rotations were measured with a Jasco DIP-370 digital polarimeter. H- and
6) Clark A. M., Hufford C. D., Med. Res. Rev., 11, 473—501 (1991).
7) Davis P. J., “Antibiotics and Microbial Transformations,” Chap. 3, ed.
by Lamber S. S., Walker C. A., CRC Press, Florida, 1987, pp. 47—68.
8) Abourashed E. A., Khan I. A., Chem. Pharm. Bull., 48, 1996—1998
(2000).
13C-NMR spectra were obtained in CDCl3 on a Varian Unity Inova 600 spec-
trometer unless otherwise stated. HR-ESI-MS data were acquired using a
Bruker GioApex 3.0.
Substrate Decursin (1) was isolated as a white gummy solid from the
MeOH extract of the roots of A. gigas. Its authenticity was confirmed by
physical NMR data.
9) Nemoto T., Ohshima T., Shibasaki M., Tetrahedron, 59, 6889—6897
(2003).
Organisms and Metabolism Initial screening of decursin (1) was car-
ried out with ten culture samples from the microbial collection of The Na-
tional Center for Natural Products Research of The University of Missis-
sippi. A two-stage screening procedure was followed using 25 ml medium a
in 125 ml Erlenmeyer flasks.8) Compound 1 was added in dimethylfor-
mamide (0.5 mg/ml) to 24 h old stage II cultures and incubated for 14 d on a
rotary shaker (New Brunswick Model G10-21) at 100 rpm. Precoated Si gel
60 F254 TLC plates (E. Merck) with p-anisaldehyde as the spray reagent were
used to monitor the reaction. Preparative scale fermentations were carried
out in five 2 l flasks, each containing 100 mg of substrate in 500 ml medium
a. EtOAc was used to extract the combined culture filtrates. Metabolites
were isolated by column chromatography over silica gel. Culture and sub-
strate controls were run along with the above experiments.8)
10) Sano K., Yosioka I., Kitagawa I., Chem. Pharm. Bull., 23, 20—28
(1975).
11) Kong L., Yao N., Niwa M., Heterocycles, 53, 2019—2025 (2000).
12) Ahn K.-S., Sim W.-S., Kim I.-H., Planta Med., 62, 7—9 (1996).
13) Ahn K.-S., Sim W.-S., Lee I.-K., Seu Y.-B., Kim I.-H., Planta Med.,
63, 360—361 (1997).
14) Kim S., Ko H., Son S., Shin K. J., Kim D. J., Tetrhedron Lett., 42,
7641—7643 (2001).
15) Yao N. H., Kong L. Y., Acta Pharmac. Sin., 36, 351—355 (2001).
16) Vermeulen N. P. E., “Cytochromes P450: Metabolic and Toxicological
Aspects,” Chap. 2, ed. by Ioannides C., Parke D. V., CRC Press,
Florida, 1996, pp. 29—53.