Cytotoxic flavone analogues of vitexicarpin, a constituent of the leaves of Vitex negundo

Article abstract of DOI:10.1021/np0300784

Bioassay-guided fractionation of the chloroform-soluble extract of the leaves of Vitex negundo led to the isolation of the known flavone vitexicarpin (1), which exhibited broad cytotoxicity in a human cancer cell line panel. In an attempt to increase the

Full text of DOI:10.1021/np0300784

J . Nat. Prod. 2003, 66, 865-867
865
Cytotoxic F la von e An a logu es of Vitexica r p in , a Con stitu en t of th e Lea ves of
Vitex n egu n d o
Fredyc D´ıaz,† Daniel Cha´vez,†,‡ Dongho Lee,†,§ Qiuwen Mi,† Hee-Byung Chai,† Ghee T. Tan,†
Leonardus B. S. Kardono, Soedarsono Riswan,| Craig R. Fairchild,∇ Robert Wild,∇ Norman R. Farnsworth,†
Geoffrey A. Cordell,† J ohn M. Pezzuto,† and A. Douglas Kinghorn*,†
Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, Research Center for
Chemistry, Indonesian Institute of Science, Serpong, 15310 Tangerang, Indonesia, Herbarium Bogoriense,
Research Center for Biology, Indonesian Institute of Science, Bogor, Indonesia, and Bristol-Myers Squibb,
Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New J ersey 08543
Received February 19, 2003
Bioassay-guided fractionation of the chloroform-soluble extract of the leaves of Vitex negundo led to the
isolation of the known flavone vitexicarpin (1), which exhibited broad cytotoxicity in a human cancer cell
line panel. In an attempt to increase the cytotoxic potency of 1, a series of acylation reactions was performed
on this compound to obtain its methylated (2), acetylated (3), and six new acylated (4-9) derivatives.
Compound 9, the previously unreported 5,3′-dihexanoyloxy-3,6,7,4′-tetramethoxyflavone, showed compara-
tive cytotoxic potency to compound 1 and was selected for further evaluation. However, this compound
was found to be inactive when evaluated in the in vivo hollow fiber assay with Lu1, KB, and LNCaP cells
at the highest dose (40 mg/kg/body weight) tested, and in the in vivo P-388 leukemia model (135 mg/kg),
using the ip administration route.
The genus Vitex of the family Verbenaceae is constituted
by 250 species of small trees and shrubs which occur in
tropical to temperate regions.1 Vitex negundo L. is a small
aromatic plant that is widely distributed in South Asia,
the People’s Republic of China, Indonesia, and the Philip-
pines.1 This plant has been used in traditional Indian
medicine due to its anti-inflammatory properties for the
treatment of rheumatoid arthritis.1,2 Previous phytochemi-
cal studies on V. negundo have afforded several types of
compounds, such as flavonoids,2 iridoid glycosides,3 lign-
ans,4 and sesquiterpenes.5 As part of an ongoing collabora-
tive search for novel antineoplastic agents of plant origin,
cytotoxicity-based, bioassay-guided fractionation of the
chloroform-soluble extract of the leaves of Vitex negundo
L. (Verbenaceae), collected in Indonesia, led to the isolation
of the known flavone vitexicarpin (1)6-11 as the only active
principle. In an attempt to increase the resultant cytotoxic
potency of 1, a series of acylation reactions was performed
on compound 1 to afford derivatives 2-9. In this paper,
we describe the isolation of vitexicarpin (1), the synthesis
of several analogues of 1 (2-9), the cytotoxicity determi-
nation of 1-9 in a human cancer cell line panel, and the
in vivo evaluation of 9 in the hollow fiber assay12-14 and
in the P-388 leukemia model.15
F002 (Lu1, 5.9 µg/mL) and F003 (Lu1, 5.1 µg/mL), which
indicated that the mass associated with cytotoxic activity
against Lu1 cells corresponded to the molecular ion at 374
mass units. Compound 1 exhibited significant activity
against the Lu1 (human lung) cancer cell line with an IC50
value of 0.8 µg/mL. In an attempt to increase the cytotoxic
activity, and in order to obtain a preliminary notion of
structure-cytotoxicity relationships, a series of acylation
reactions (Figure 1 in Supporting Information) was per-
formed with compound 1 to yield compounds 2-9.
Vitexicarpin (1) was identified by comparison of its
melting point and spectroscopic characteristics with lit-
erature values.7,17-20 Compounds 4-9 are new derivatives
of 1, while compounds 2 and 3 have been reported previ-
ously in the literature.17,20 The structures of compounds
4-9 were consistent with the spectroscopic values obtained
in each case and were supported with 1H-1H COSY,
HMQC, and HMBC experiments, and by HRMS analysis.
A chloroform extract of the leaves of V. negundo showed
a significant cytotoxic response against the Lu1 cell line
with an IC50 value of 3.4 µg/mL. Bioassay-directed frac-
tionation using this cell line led to the isolation of 1 as the
active component. This result was in agreement with
preliminary results obtained in our laboratory from LC-
MS dereplication experiments16 using the active fractions
1
The H and 13C NMR data for 4-8 are shown in Tables 1
* To whom correspondence should be addressed. Tel: (312) 996-0914.
Fax: (312) 996-7107. E-mail: Kinghorn@uic.edu.
† University of Illinois at Chicago.
and 2, respectively, of the Supporting Information.
The cytotoxic activities of compounds 1-9 were evalu-
ated against a panel of human cancer cell lines (Table 1),
according to established protocols.21,22 Vitexicarpin (1)
exhibited broad cytotoxic activity, except for the Col2 cell
line. Permethylation and benzoylation of this compound to
afford 2 and 4-5, respectively, resulted in total inactivation
‡ Present address: Centro de Graduados
e Investigacion, Instituto
Tecnologico de Tijuana, Apdo. Postal 1166, Tijuana, BC 22000, Mexico.
§ Present address: Chemistry and Life Sciences Group, Research Tri-
angle Institute, P.O. Box 12194, Research Triangle Park, NC 27709.
Research Center for Chemistry, Indonesian Institute of Science.
| Herbarium Bogoriense.
∇ Bristol-Myers Squibb.
10.1021/np0300784 CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/07/2003

866 J ournal of Natural Products, 2003, Vol. 66, No. 6
Ta ble 1. Cytotoxic Activity of Compounds 1-9a,b
Notes
aluminum plates. Fractions were monitored by TLC with
visualization under UV light (254 and 365 nm) and by dipping
the plates into a solution of 10% (v/v) H2SO4 in EtOH.
P la n t Ma ter ia l. Leaves of V. negundo were collected in J uly
1996 at Ujung Genteng, West J ava, Indonesia (07°22′ S,
106°24′ E) by S.R. and J . J . Afriastini. A voucher specimen
(J -050) has been deposited at the Field Museum of Natural
History, Chicago, IL.
Extr a ction a n d Isola tion . The air-dried, powdered leaves
(788 g) were milled and extracted by maceration with MeOH
(6 L × 3) in a percolator at room temperature. The MeOH
solutions were combined, filtered, and evaporated under a
vacuum. The dried MeOH extract (62.5 g) was dissolved in 1
L of a mixture of MeOH-H2O (90:10) and then partitioned
with hexane (500 mL × 6) to afford a dried hexane extract
(20.1 g). The aqueous MeOH solution was then partitioned
with CHCl3 (500 mL × 5). The combined organic layer was
washed using 1% saline solution and concentrated under
vacuum to yield 18.7 g of CHCl3 extract, which showed
significant cytotoxic activity against the Lu1 cell line with an
IC50 value of 3.4 µg/mL.
compound Col2 hTERT-RPE1 HUVEC
KB
LNCaP Lu1
1
2
3
4
5
6
7
8
9
12.7
>20
15.9
>20
>20
>20
11.7
15.0
>20
0.2
>20
0.6
>20
0.1
20
>20
6.5
>20
5.5
11.1
0.2
>20
0.2
0.5 0.8
>20 >20
0.1 0.7
>20
>20
0.4
0.8
0.7
0.5
0.4
3.6
4.3
0.5
0.6
0.6
0.4
>20
>20
16.5
6.5
1.0
1.7
1.4
0.7
0.6
0.5
0.6
0.5
a
b
Results are expressed as ED50 values (µg/mL). Key to cell
lines used: Col2 ) human colon cancer; hTERT-RPE1 ) human
telomerase reverse transcriptase-retinal pigment epithelial;
HUVEC ) human umbilical vein endothelial; KB) human oral
epidermoid carcinoma; LNCaP ) human hormone-dependent
prostate cancer; Lu1 ) human lung cancer.
or a significant reduction of cytotoxic activity. Compounds
3 and 6-9 showed cytotoxicity profiles similar to that of
vitexicarpin (1) in the cell panel represented, with com-
pounds 3 and 9 exhibiting minor increases in activity
against the LNCaP and/or Lu1 cell lines.
Compounds 1, 3, 4, and 6-9 were further evaluated for
cytotoxicity in an 11-cell-line Oncology Diverse Cell Assay
panel, representing a diverse group of human and murine
tumor cells, according to previously established protocols.23
Since these compounds showed good potency (mean IC50
value of <5.0 µM) and a high cell selectivity against the
K562 human leukemia cell line (maximum/minimum IC50
ratio of >10) in the ODCA assay, they were considered as
high-priority leads. Compound 9 was selected for in vivo
hollow fiber and P-388 antileukemic evaluation rather than
1, as it is a new compound.
Compound 9 inhibited the growth of KB, LNCaP, and
Lu1 cells with ED50 values of 0.5, 0.5, and 0.7 µg/mL,
respectively. Accordingly, compound 9 was evaluated with
the in vivo hollow fiber system12-14 using the KB, LNCaP,
and Lu1 cell lines at doses of 10, 20, and 40 mg/kg. With
LNCaP cells, compound 9 inhibited growth by 0-7.2% at
the ip site and 0-2.4% at the sc site, while with KB cells,
the compound was ineffective at the ip site and inhibited
growth by 0-8.2% at the sc site. Finally, for Lu1 cells, this
compound did not inhibit growth at the ip site, and only a
modest response (0-2.1%) was observed at the sc site. No
significant weight loss was observed in mice during this
test procedure (Figure 2, Supporting Information).
Compound 9 was also evaluated in an in vivo mouse
P-388 leukemia system under a standard protocol.15 How-
ever, when administered intraperitoneally at 135 mg/kg/
injection, this compound was deemed inactive (T/C value
of 100%).
The CHCl3-soluble extract (18.4 g) was subjected to frac-
tionation using an open column with 540 g of silica gel (63-
200 mesh) and eluted with a gradient of increasing polarity
with petroleum ether-CHCl3-MeOH. Ten fractions (F1-F10)
were obtained and monitored using Lu1 cells. An orange,
amorphous solid was precipitated from
a hexane-CHCl3
solution of the active fractions F2 (Lu1, 5.9 µg/mL) and F3
(Lu1, 5.1 µg/mL). This solid was subjected to several purifica-
tion steps by crystallization in hexane and MeOH to produce
orange crystals corresponding to the structure of compound 1
(272 mg), which showed a strong cytotoxic response (ED50: 0.8
µg/mL) against the Lu1 cell line. No other cytotoxic compound
was isolated from the CHCl3-soluble extract in the present
study.
Vitexica r p in (1): orange crystals (CHCl3-MeOH); mp
188-190 °C (lit.7 188-189 °C). Compound 1 exhibited com-
parable spectral (UV, IR, 1H NMR, 13C NMR, and EIMS) data
to published values.17-20
3,5,6,7,3′,4′-Hexa m eth oxyfla von e (2). Permethylation of
5 mg of vitexicarpin (1) was achieved using the Aldrich Diazald
kit to generate CH2N2. Compound 2 (4.9 mg; 90.7% yield) was
obtained as a pale yellow, amorphous solid, mp 179-180 °C.
Compound 2 exhibited comparable spectral (UV, IR, 1H NMR,
13C NMR, and EIMS) data to published values.17
Acyla tion of Vitexica r p in (1). Five milligrams of com-
pound 1 was dissolved with pyridine (0.3 mL) and treated with
the respective anhydride (0.5 mL) to yield compounds 3-9.
Each mixture was left to stand at room temperature overnight.
After removal of the reagent under a dried-air stream, the
residue was partitioned between CHCl3 (5 mL × 3) and water
(5 mL). The organic layer was concentrated to afford a pale
yellow, amorphous solid in each case (Figure S1, Supporting
Information).
5,3′-Diacetoxy-3,6,7,4′-tetr am eth oxyflavon e (3): mp 140-
142 °C. Compound 3 exhibited comparable spectral (UV, IR,
1H NMR, 13C NMR, and EIMS) data to published values.20
3′-Be n zoyloxy-5-h yd r oxy-3,6,7,4′-t e t r a m e t h oxyfla -
von e (4): mp 210-211 °C; UV (CHCl3) λmax (log ꢀ) 333 (4.36),
276 (4.33), 241 (4.41) nm; IR (dried film) νmax 2924, 2365, 2333,
1742, 1655, 1458, 1364, 1222, 812, 710 cm-1; 1H and 13C NMR
data, see Tables S1 and S2; LREIMS m/z 478 (47) [M]+, 463
(12), 435 (1) 105 (100); HREIMS m/z 478.1264 (C26H22O9, calcd
478.1264).
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
determined on a Fisher-J ohns melting point apparatus and
are uncorrected. UV spectra were measured on a Beckman
DU-7 spectrometer. IR spectra were obtained with an ATI
Mattson FT-IR spectrometer. NMR spectra were recorded with
TMS as internal standard, using either a Bruker Avance-360
or a Bruker DRX-500 NMR spectrometer (360 or 500 MHz,
respectively). HREIMS and LREIMS were recorded on a
Finnigan MAT 95 mass spectrometer (70 eV), while HR-
FABMS and LRFABMS were recorded on a VG70E-HF mass
spectrometer. Column chromatography was carried out on
silica gel 60 (Merck, Darmstadt, Germany; 63-200 and 230-
400 mesh). Preparative TLC was performed on glass-backed
TLC silica gel plates [2 mm thick (Scientific Adsorbents Inc.,
Atlanta, GA)]. Thin-layer chromatography (TLC) was per-
formed on precoated 0.25 mm thick Merck silica gel F254
5,3′-Diben zoyloxy-3,6,7,4′-tetr a m eth oxyfla von e (5): mp
220-222 °C; UV (CHCl3) λmax (log ꢀ) 322 (4.34), 282 (sh, 3.93),
262 (4.18), 241 (4.53) nm; IR (dried film) νmax 2916, 2848, 1742,
1611, 707 cm-1; 1H and 13C NMR data, see Tables S1 and S2;
LREIMS m/z 582 (25), 477 (28), 105 (100); HREIMS m/z
582.1493 (C33H26O10, calcd 582.1526).
5,3′-Dip r op a n oyloxy-3,6,7,4′-tetr a m eth oxyfla von e (6):
mp 131-132 °C; UV (CHCl3) λmax (log ꢀ) 320 (4.07), 265 (4.15),
241 (4.04) nm; IR (dried film) νmax 2938, 2848, 1764, 1623,
1512, 1464, 1358, 1276 cm-1; 1H and 13C NMR data, see Tables

Notes
J ournal of Natural Products, 2003, Vol. 66, No. 6 867
S1 and S2; LRFABMS m/z 487 [M + H]+; HRFABMS m/z
509.1437 (C25H26O10Na, calcd 509.1423).
Laboratory of the Research Resources Center, University of
Illinois at Chicago, for the provision of NMR facilities used in
this investigation.
5,3′-Dibu tan oyloxy-3,6,7,4′-tetr am eth oxyflavon e (7): mp
108-109 °C; UV (CHCl3) λmax (log ꢀ) 321 (4.25), 264 (4.23), 243
(4.08) nm; IR (dried film) νmax 2961, 2338, 1762, 1623, 1467,
Su p p or tin g In for m a tion Ava ila ble: The 1H and 13C NMR data
for the new analogues 4-8 of vitexicarpin (1) (Tables S1 and S2), a
summary of acylation reactions for compound 1 (Figure S1), and the
results from the in vivo hollow fiber study performed with compound
9 (Figure S2). This information is available free of charge via the
Internet at http://pubs.acs.org.
1360, 1277 cm-1 1H and 13C NMR data, see Tables S1 and
;
S2; LRFABMS m/z 515 [M + 1]+; HRFABMS m/z 515.1912
(C27H31O10, calcd 515.1917).
5,3′-Dipen t-4-en oyloxy-3,6,7,4′-tetr am eth oxyflavon e (8):
mp 155-157 °C; UV (MeOH) λmax (log ꢀ) 327 (4.70), 261 (4.60),
234 (sh, 4.60) nm; IR (dried film) νmax 1764, 1623, 1462, 1362,
Refer en ces a n d Notes
1276 cm-1 1H and 13C NMR data, see Tables S1 and S2;
;
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LRFABMS m/z 539 [M + 1]+; HRFABMS m/z 539.1908
(C29H31O10, calcd 539.1917).
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5,3′-Dih exa n oyloxy-3,6,7,4′-t et r a m et h oxyfla von e (9):
mp 100-101 °C; UV (MeOH) λmax (log ꢀ) 324 (4.30), 260 (4.09),
229 (4.22) nm; IR (dried film) νmax 2935, 2355, 2338, 1764,
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;
8.02 (1H, dd, J ) 8.8, 2.2 Hz, H-6′), 7.77 (1H, d, J ) 2 Hz,
H-2′), 7.06 (1H, d, J ) 8.8 Hz, H-5′), 6.85 (1H, s, H-8), 2.82
(2H, t, J ) 7.5 Hz, H-2′′′), 2.63 (2H, t, J ) 7.5 Hz, H-2′′), 1.89
(2H, t, J ) 7.3 Hz, H-3′′), 1.85 (2H, t, J ) 7.3 Hz, H-3′′′), 1.44
(4H, m, H-4′′, H-4′′′), 1.40 (4H, m, H-5′′, H-5′′′), 0.95 (6H, m,
H-6′′, H-6′′′); 13C NMR (CDCl3, 125.78 MHz) δ 172.1 (C, C-1′′,
C-1′′′), 171.8 (C, C-4), 157.6 (C, C-7), 153.0 (C, C-2), 152.9 (C,
C-4′), 142.1 (C, C-3), 140.9 (C, C-3′), 139.8 (C, C-6), 127.5 (CH,
C-6′), 123.3 (C, C-1′), 122.8 (CH, C-2′), 112.1 (CH, C-5′), 111.9
(C, C-10), 97.9 (CH, C-8), 61.5 (CH3, OCH3-6), 59.9 (CH3, OCH3-
3), 56.4 (CH3, OCH3-7), 55.9 (CH3, OCH3-4′), 33.9 (CH2, C-2′′),
34.2 (CH2, C-2′′′), 31.3 (CH2, C-4′′′), 31.2 (CH2, C-4′′), 24.6 (CH2,
C-3′′′), 24.3 (CH2, C-3′′), 22.4 (CH2, C-5′′), 22.3 (CH2, C-5′′′),
14.0 (CH3, C-6′′, 6′′′); LRFABMS m/z 571 [M + 1]+; HRFABMS
m/z 593.2358 (C31H38O10Na, calcd 593.2363).
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a
MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
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assessed for its biological potential in the in vivo hollow
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Ack n ow led gm en t. This investigation was supported by
grant U19-CA-52956, funded by the National Cancer Institute,
NIH, Bethesda, MD. F.D. acknowledges the Colombian Insti-
tute for Science and Technology Development “Francisco J ose´
De Caldas” (COLCIENCIAS) (Bogota´, Colombia), and the
University of Cartagena, Cartagena (Colombia), for a Doctoral
Fellowship. We thank Dr. K. Fagerquist, Mass Spectrometry
Facility, Department of Chemistry, University of Minnesota,
Minneapolis, MN, for the mass spectral data. Dr. Darrick H.
S. L. Kim, Department of Medicinal Chemistry and Pharma-
cognosy, University of Illinois at Chicago, is acknowledged for
helpful suggestions in the chemical synthesis aspects of the
work. We are grateful to the Nuclear Magnetic Resonance
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NP0300784