A new ursane triterpene from Monochaetum vulcanicum that inhibits DNA polymerase β lyase

Article abstract of DOI:10.1021/np030531b

Bioassay-directed fractionation of a butanone extract of Monochaetum vulcanicum resulted in the isolation of a new triterpene (1) and four known compounds, ursolic acid (2), 2α-hydroxyursolic acid (3), 3-(p-coumaroyl)ursolic acid (4), and β-sitosteryl-β-D-galactoside (5). The structure of the new compound 1 was established as 3β-acetoxy-2α -hydroxyurs-12-en-28-oic acid on the basis of extensive 1D and 2D NMR spectroscopic interpretation and chemical derivatization. Compounds 1-3 and 5 exhibited polymerase β lyase activity.

Full text of DOI:10.1021/np030531b

J . Nat. Prod. 2004, 67, 899-901
899
A New Ur sa n e Tr iter p en e fr om Mon och a etu m vu lca n icu m th a t In h ibits DNA
P olym er a se â Lya se
V. S. Prakash Chaturvedula,† Zhijie Gao,‡ Shannon H. J ones,‡ Xizhi Feng,‡ Sidney M. Hecht,‡ and
David G. I. Kingston*,†
Department of Chemistry, M/ C 0212, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0212,
and Departments of Chemistry and Biology, The University of Virginia, Charlottesville, Virginia 22901
Received December 18, 2003
Bioassay-directed fractionation of a butanone extract of Monochaetum vulcanicum resulted in the isolation
of a new triterpene (1) and four known compounds, ursolic acid (2), 2R-hydroxyursolic acid (3), 3-(p-
coumaroyl)ursolic acid (4), and â-sitosteryl-â-D-galactoside (5). The structure of the new compound 1 was
established as 3â-acetoxy-2R-hydroxyurs-12-en-28-oic acid on the basis of extensive 1D and 2D NMR
spectroscopic interpretation and chemical derivatization. Compounds 1-3 and 5 exhibited polymerase â
lyase activity.
In addition to its polymerization activity, the DNA repair
enzyme DNA polymerase â (pol â) also has an intrinsic
deoxyribose phosphate (dRP) lyase activity, which is im-
portant to its repair function.1,2 This second activity
constitutes a second target for the discovery of potential
anticancer agents, since inhibitors of the lyase activity of
pol â should also potentiate the cytotoxicity of DNA-
damaging agents. It has already been shown by one of our
groups that naturally occurring inhibitors of pol â can be
found in nature,3 and it is thus reasonable to suppose that
specific inhibitors of the lyase activity may also exist in
nature. We thus elected to begin a search for naturally
occurring inhibitors of pol â lyase as a part of our continu-
ing research to identify novel naturally occurring antican-
cer agents,4,5 The assay system used for this purpose has
been described previously.6
A butanone extract of the plant Monochaetum vulcani-
cum Cogn. (Melastomataceae) was selected for bioassay-
guided fractionation on the basis of its strong activity
at 16.2 µg/mL in the pol â lyase assay and the absence
of any reported phytochemical studies of this species.
Earlier phytochemical studies of the genus Monochaetum
resulted in the isolation of ellagitannins and flavonoid
glycosides.7
Initial liquid-liquid partition of the crude extract of M.
vulcanicum indicated that the activity was equally distrib-
uted between the n-hexane and CHCl3 fractions of n-
hexane/aqueous MeOH and CHCl3/aqueous MeOH parti-
tions, respectively. The n-hexane and CHCl3 residues were
combined on the basis of their similar nature as judged by
1H NMR and TLC. The combined residue after separation
by chromatography over MCI gel followed by reversed-
phase PTLC yielded the new triterpenoid 1 in addition to
the four known compounds 2-5. The four known com-
pounds were identified as ursolic acid (2),8 2R-hydroxy-
ursolic acid (3),9 3-(p-coumaroyl)ursolic acid (4),10 and
â-sitosteryl-â-D-galactoside (5)11 by comparison of their
spectroscopic data with literature values.
proton test) spectral data. It gave a positive Liebermann-
Burchard test for triterpenoids. The IR absorption bands
observed at 3450 and 1728 cm-1 indicated the presence of
hydroxyl and carbonyl groups in its structure. The mass
fragments in its EIMS at m/z 469, 454, and 436 were
formed by the successive loss of COOH, AcOH, and H2O
molecules from the molecular ion and, thus, indicated the
presence of carboxylic acid, acetoxy, and hydroxy groups
in its structure. The 1H NMR spectrum showed the
presence of five methyl singlets at δ 0.84, 0.87, 0.89, 0.91,
and 0.98, two methyl doublets at δ 0.82 (J ) 6.8 Hz) and
0.88 (J ) 7.2 Hz), an olefinic proton at δ 5.34 as a triplet
(J ) 2.6 Hz), an oxymethine proton at δ 3.22 (dt, J ) 4.8,
11.5 Hz), a secondary acetate group [δ 4.46 (d, J ) 11.3
Hz), and 2.03 (s, 3H)], eight methylenes, and five methines.
The 13C NMR values for all 32 carbons were assigned on
the basis of APT, HMQC, and HMBC spectral data, which
indicated the presence of eight sp3 methyls, eight sp3
methylenes, seven sp3 methines, five sp3 quaternary car-
bons, one sp2 methine carbon, one sp2 quaternary carbon,
one ester carbonyl group, and one carboxylic acid group. A
literature search suggested that the above spectral data
Compound 1 was obtained as an optically active viscous
liquid and was shown to have the molecular formula
C32H50O5 by HRFABMS, 13C NMR, and APT (attached
* To whom correspondence should be addressed. Tel: (540) 231-6570.
Fax: (540) 231-7702. E-mail: dkingston@vt.edu.
† Virginia Polytechnic Institute and State University.
‡ University of Virginia.
10.1021/np030531b CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/17/2004

900 J ournal of Natural Products, 2004, Vol. 67, No. 5
Notes
(B603644, leaves) and CR-4 (B603645, stems). Voucher speci-
mens are located at the National Herbarium, Washington, DC.
P olym er a se Beta Lya se Bioa ssa y. The assay was per-
formed at the University of Virginia as previously reported.6
Extr a ct P r ep a r a tion . The plant samples were dried,
ground, and soaked sequentially in hexane, butanone, and
MeOH. Concentrations of the individual solutions provided the
dried extract. The butanone extract designated PC-9-145 was
used in the present study.
Extr a ction a n d Isola tion . The crude extract (0.45 g) was
suspended in aqueous MeOH (MeOH-H2O, 9:1, 50 mL) and
extracted with hexane (3 × 50 mL). The aqueous layer was
then diluted to 70% MeOH (v/v) with H2O and extracted with
CHCl3 (3 × 50 mL). The aqueous layer was concentrated, and
the residue obtained was suspended in H2O (25 mL) and
extracted with BuOH (2 × 25 mL). The hexane and CHCl3
extracts were found to be equally active and were combined
on the basis of their similar nature on TLC and their similar
1H NMR spectra. The combined residue (0.35 g) was fraction-
ated over MCI gel using MeOH-H2O (75:25 to 100:0) to
furnish 11 fractions (A-K), of which fractions C, E, and H-K
were selected for further fractionation on the basis of their
activity and their 1H NMR spectra. Fraction C on reversed-
phase preparative TLC (MeOH-H2O, 80:20) yielded ursolic
acid (2, 1.8 mg). Similarly, fraction E on reversed-phase
preparative TLC (MeOH-H2O, 85:15) afforded 2R-hydroxyur-
solic acid (3, 2.6 mg). Fractions I and J on reversed-phase
preparative TLC with mobile phase MeOH-H2O (90:10)
afforded 3-(p-coumaroyl)ursolic acid (4, 2.2 mg) and â-sito-
steryl-â-D-galactoside (5, 2.8 mg), respectively. Fraction K on
reversed-phase preparative TLC (MeOH-H2O, 90:10) yielded
the new triterpene 1 (1.5 mg). The four known compounds 2-5
were identified by comparison of their spectral data with
literature values.8-11
F igu r e 1. Selected HMBC correlations of 1.
Ta ble 1. IC50 of Polymerase â Lyase Inhibiition of Compounds
Isolated from Monochaetum vulcanicuma
compound
IC50 (µM)
1
2
3
4
5
21.5
18.6
12.6
>50
26.5
a
Data are the meam of three determinations.
were consistent with the presence of an urs-12-ene type of
pentacyclic triterpenoid skeleton in 1. The appearance of
the two oxymethine protons indicated their presence in the
A-ring of an urs-12-en-28-oic acid skeleton.12 This was
further supported by the mass fragment observed at m/z
198, formed by the loss of C11H18O3 from the molecular ion,
corresponding to the A-ring of 1. The placement of the
secondary hydroxy and acetoxy groups in the A-ring of 1
at the C-2 and C-3 positions was made on the basis of the
key HMBC correlations: H-2/C-1, C-3, C-4, C-25 and H-3/
C-1, C-2, C-4, C-23, C-24 (Figure 1). The stereochemistry
of the hydroxy and acetoxy groups was assigned as rel R
and â on the basis of their almost identical coupling
constants with reported 2R,3â-dihydroxy- and diacetoxyurs-
12-en-28-oic acid derivatives.8,13 This was supported by the
NOESY correlations of 1, in which the oxymethine at the
C-2 carbon showed correlations to the methyl protons of
C-24 and C-25, whereas the acetoxymethine proton at C-3
showed a correlation to the protons of the C-23 methyl
group. In addition, acetylation of compound 1 with Ac2O-
pyr afforded a product whose mp, rotation, and 1H NMR
data were identical with those of 2R,3â-diacetoxyurs-12-
en-28-oic acid (6),13 thus confirming the structure. The
same product was also obtained by acetylation of 3. On the
basis of the above spectroscopic and chemical evidence, 1
was assigned as 3â-acetoxy-2R-hydroxyurs-12-en-28-oic
acid (3-acetyl-2R-hydroxyursolic acid).
3â-Acetoxy-2r-h yd r oxyu r s-12-en -28-oic a cid (1): color-
less oil; [R]D +56.2° (c 0.64, CHCl3); UV (MeOH) λmax 216 nm
(ꢀ 14 200); IR νmax 3450, 2955, 1728, 1435, 1110, 1050 cm-1
;
1H NMR, δ 0.82 (d, J ) 6.8, H-29), 0.84 (s, 3H, H-26), 0.87 (s,
3H, H-25), 0.88 (d, J ) 7.2, H-30), 0.89 (s, 3H, H-23), 0.91 (s,
3H, H-24), 0.98 (s, 3H, H-27), 1.04 (1H, m, H-5), 1.22 (1H, m,
H-7), 1.30 (2H, m, H-6 and H-16), 1.32 (1H, m, H-15), 1.36
(1H, m, H-21), 1.38 (1H, m, H-19), 1.40 (1H, m, H-11), 1.51
(1H, m, H-9), 1.52 (1H, m, H-6), 1.54 (1H, m, H-22), 1.60 (1H,
m, H-21), 1.62 (1H, m, H-16), 1.64 (2H, m, H-7 and H-1), 1.98
(1H, m, H-15), 2.02 (1H, m, H-22), 2.03 (3H, 3-OAc), s 2.04
(1H, m, H-19), 2.10 (1H, m, H-11), 2.16 (1H, m, H-1), 2.27 (1H,
d J ) 11.2, H-18), 3.22 (1H, dt, J ) 4.8, 11.5, H-2), 3.63 (1H,
m, 2-OH), 4.46 (1H, d, J ) 11.3, H-3), and 5.34 (1H, t, J )
2.6); 13C NMR, δ 50.3, 68.3, 80.4, 38.4, 55.5, 18.3, 33.3, 40.2,
47.8, 39.2, 24.8, 125.6, 138.2, 42.4, 28.2, 24.6, 49.8, 53.2, 39.6a,
39.4a, 31.8, 37.9, 28.3, 17.5b, 16.9b, 17.0b, 23.6, 179.1, 21.3, 17.3b
for carbons 1-30, respectively (values having the same
superscript are interchangeable), 21.3 (3-Ac), and 170.8 (3-
Ac); EIMS m/z (rel int) 514 [M+] (18), 469 (32), 454 (24), 436
(12), 376 (13), 335 (15), 334 (23), 323 (15), 248 (100), 235 (10),
226 (32), 198 (21), 181 (21), 180 (16), 121 (14), 61 (24);
HRFABMS m/z 437.3431 [M - AcOH - H2O]+ (calcd for
All the isolated compounds were tested for inhibition of
DNA polymerase â lyase activity, and the results are shown
in Table 1. Compounds 1-3 and 5 were weakly active, with
IC50 values ranging from 12.6 to 26.5, with 2R-hydroxyur-
solic acid (3) having the greatest activity.
C
30H45O2, 437.3420).
Acetyla tion of 3â-Acetoxy-2r-h yd r oxyu r s-12-en -28-oic
Acid (1). Compound 1 (0.8 mg) was treated with Ac2O-pyr
(1:1, 0.5 mL) at room temperature for 10 h. Concentration of
the mixture under vacuum followed by reversed-phase pre-
parative TLC (MeOH-H2O, 98:2) gave 6 (0.6 mg), which had
1H NMR and MS data identical with those of 2R,3â-diacetyl-
oxyurs-12-en-28-oic acid.13
Acetyla tion of 2r-Hyd r oxyu r solic Acid (3). Acetylation
of 2R-hydroxyursolic acid (3, 1.5 mg) as described above gave
a product (1.2 mg) that was identical with 6.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
recorded with an Electrothermal digital apparatus and are
uncorrected. Optical rotations were recorded on a Perkin-
Elmer 241 polarimeter. IR (KBr) and UV (MeOH) spectra were
measured on MIDAC M-series FTIR and Shimadzu UV-1201
spectrophotometers, respectively. NMR spectra were obtained
on a J EOL Eclipse 500 spectrometer, and chemical shifts are
given in ppm (δ) with TMS (tetramethylsilane) as an internal
reference; coupling constants (J ) are in Hz. The HRFAB mass
spectra were obtained on a J EOL HX-110 instrument.
Ack n ow led gm en t. This work was supported by a Na-
tional Cooperative Drug Discovery Group award to the Uni-
versity of Virginia by the National Cancer Institute (U19 CA
50771, S.M.H., Principal Investigator); this support is grate-
fully acknowledged.
P la n t Ma ter ia l. Monochaetum vulcanicum Cogn. (Melas-
tomataceae) was collected in J une 1964 in Costa Rica as CR-3

Notes
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NP030531B