J o u m I of Natural Products Vol. 58, No. 4, pp. 475-482, April 1995
475
ANTITUMOR AGENTS, 154.’ CYTOTOXIC AND ANTIMITOTIC FL.AVONOLS FROM POLANISIA DODECANDRA QIAN SHI, KE
CHEN, LEPINGLI,
Natural Products Laboratmy, Division of Medicinal Chemistry and Natural Products, School of Pharmaq, JER-JANG
CHANG, CAN AUTRY,
Laboratmy of Animal Medicine, School of Medicine, University of N m h Carolina, Chapel Hill, North Carolina 27599
Mmsuo KOZUKA,TAKAOKONOSHIMA, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607. Japan
JmR. E m s , Department of Botany, University of Oklahoma, Norman, Oklahoma 73019 CHIIM. LIN, ERNESTHAMEL,
Laboratmy of Molecular Pharmacology, Developmental Tberapeutics Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, BetbesL, Maryland 20892 ANDREWT. McPm,* DONALD R. MCPHAIL,
Department of Chemistry, Paul M. Gross Chemical Laboratmy, Duke University, Durham, Nwth Carolina 27706 and KUO-HSIUNG LEE*
Natural Products Laboratory, Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599 hsTRACT.--Three flavonols, 5,3’-dihydroxy-3,6,7,8,4’-pentamethoxyflavone E l f , 5,4‘dihydroxy-3,6,7,8,3 ’-pentamethoxyflavone [2], and quercetin 3-O-P-~-glucopyranosy1-7-O-aL-rhamnopyranoside 137, were isolated from Polanisia dodecandra. Compound 1 showed remarkable cytotoxicity in vitro against panels of central nervous system cancer (SF-268, SF-539, SNB75, U-25 l), non-small cell lung cancer (HOP-62, NCI-H266, NCI-H460, NCI-H522), small cell lung cancer (DMS-114), ovarian cancer (OVCAR-3, SK-OV-3), colon cancer (HCT-116), renal cancer (U0-31), a melanoma cell line (SK-MEL-S), and two leukemia cell lines (HL-60 ITB], SR), with GI,, values in the low micromolar to nanomolar concentration range. This substance also inhibited tubulin polymerization (IC,,=0.83 k0.2 pM) and the binding of radiolabeled colchicine to tubulin with 59% inhibition when present in equimolar concentrations with colchicine. Compound 2 also showed cytotoxicity against medulloblastoma (TE-67 1) tumor cells with an ED,, value of 0.98 pg/ml. Compound 1 appears to be the first example of a flavonol to exhibit potent inhibition of tubulin polymerization and, therefore, warrants further investigation as an antimitotic agent.
Polanisia dodecandra (L.) DC. (Capparidaceae), Clammy-weed, is native to North America and is abundant from Montana to Wisconsin and from Quebec to Mexico (2). P. dodecandra is a branched glandular-viscid herb with a rank odor and grows in sandy places. Except for our previously published work ( 3 ) ,it is a hitherto phytochemically and biologically uninvestigated species and, therefore, attracted our interest for laboratory investigations. In the course of our continuing search for novel cytotoxic agents that are active against slow-growing solid tumors, especially compounds from previously uninvestigated plants, the MeOH extract of the whole plant of P. dodecandra was found to show significant in vitro cytotoxicity against various tumor cells. Subsequent ‘For part 153, see Wang eta(. (1).
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bioassay-guided fractionation and repeated cc on Si gel of the active extract led to the 117, 5,4’isolation of three flavonols, 5,3’-dihydroxy-3,6,7,8,4’-pentamethoxyflavone dihydroxy-3,6,7,8,3’-pentamethoxyflavone121,and quercetin 3-O-P-~-glucopyranosyl7-O-a-~-rhamnopyranosideC37.These flavonols were identified by their spectral data and the structures of compounds 1 and 2 were confirmed by X-ray diffraction studies. Compound 1 showed remarkable cytotoxicity in vitro against panels of central nervous system cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, colon cancer, renal cancer, a melanoma cell line, and leukemia cell lines with GI,,values in the low micromolar to nanomolar concentration range. Compound 2 exhibited significant cytotoxicity against medulloblastoma tumor cells. Compound 1 also showed significant inhibition of tubulin polymerization and inhibition of radiolabeled colchicine binding to tubulin. In addition, a dammarane-type triterpene, polacandrin, one of the potent cytotoxic principles, was isolated and has been reported recently (3). We report herein on the isolation, identification, and X-ray analyses of these flavonols and on their cytotoxicity.
1 R,=H, R,=CH3 2
3
R,=CH,, R,=H
RESULTS AND DISCUSSION Compounds 1 and 2 were both obtained as yellow crystals. The ‘H-nmr spectrum of each compound exhibited the same B-ring proton signals characteristic for H-2’, -5 I, and -6’ of a 3,5,6,7,8,3’,4’-oxygenated flavone with two hydroxy and five methoxy groups. The ‘H-nmr spectra (Table l),nOe studies, and uv behavior ofcompounds 1 and 2 indicated an OH at position 3 ’ in 1 and at position 4’ in 2. Previously, compound 1 has been isolated from Gutierrezia microrephala (4), Polanisia tracbyspwma ( 5 ) , Calycadenia truncata (6), and Calycadenia mollis (6), and compound 2 from Calycoptwis flwibunab (7), Digitalis tbapsi (a), Baccharzs incarum (9), Gutierrezia resinosa (lo), Gutierrezia microrephala (1l), Calycadenia ciliosa (6), and Calycadenia multiglandulosa (6). The reported ‘H-nmr data for 1 ( 4 3 )and 2 (9-1 l), measured in eel,, CDCI,, Me,CO-d6 or DMSO-d, did not effectively distinguish between OMe-3 ’ versus OMe-4’ substitution, since these data for both substitution patterns were very similar. In the present study, the spectral data for 1 and 2 measured in CDCI, were also almost identical (Table 1). However, the spectra measured in C6D6 showed good resolution of the two substitution patterns (Table 1). The previously reported 13C-nmrdata measured in CDC1, did not assign all signals (9). In the present study, HETCOR and long-range HETCOR spectra were used to assign all carbons unequivocally (Table 2). The 13C-nmrdata for compounds 1 and 2 measured in CDC1, are similar to those measured in DMSO-d, ( 5 , l l ) . X-Ray crystallographic analyses confirmed the structures of 1 and 2 and provided details of their solid-state geometries.*Corresponding bond lengths in 1 and 2 agree well 2Fractionalatomic coordinate data and solid-state conformation figuresare deposited in A.T.McPhail’s laboratory.
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Bioactive Flavonols from Polanisia
7 LE 1. 'H-Nmr Spectral Data for 1 a n c ! in Different Solvents.
Compound 1
Compound 2
Proton CDCI,
H-2' . . . . . . . . . . . . H-5' . . . . . . . . . . . . H-6' . . . . . . . . . . . . OH-5 . . . . . . . . . . . . OH-3' . . . . . . . . . . . OH-4' . . . . . . . . . . . OMe-3' . . . . . . . . . . OMe-4' . . . . . . . . . . OMe-3 . . . . . . . . . . . OMe-6 . . . . . . . . . . . OMe-7 . . . . . . . . . . . OMe-8 . . . . . . . . . . .
CDCI,
C6D6
C6D6
7.80, s 7.61, dJ52.0 Hz 7.77, d,J=2.2 Hz 8.03, d,J=2.2 Hz 7.07, d,J=7.5 Hz 7.00, d,J=8.6 Hz 7.00, d,J=7.0 Hz 6.37, d,J=8.7 Hz 7.78, dd,J=2.2 and 7.79, dd,J=2.2 and 7.78, dd, J=1.5 and 7.80, dd,J=2.0 and 7.0 Hz 7.5 Hz 8.6 Hz 8.7 Hz 12.40, s 13.15, s 13.12, s 12.39, s 5.41, s 5.73, s 6.02, s 5.66, s 3.21, S' 3.98, s 4.00, s 3.07, sa 3.88, s 3.84, sb 3.88, s 3.85, sb 3.82, sb 3.81,~~ 3.95, s 3.95, s 4.11, s 4.11, s 3.72, sb 3.69, sb 3.64, Sb 3.65, sb 3.96, s 3.96, s
'In C6D6,only the OMe-3' in compound 1 and OMe-4' in compound 2 were assigned by the NOESY spectra. ?n C6D6, the remainingOMegroupswere not assigned unambiguously and theirassignmentsmaybe incerchangeable.
in general and are in accordance with expectations (12). The ring N C atoms 0 - 1 4 - 1 0 are approximately coplanar (mean deviation 0.03 1 hi in 1,0.019 hi in 2),whereas several of the directly bonded atoms are displaced significantly from the least-squares planes through the ring atoms [displacements (hi) in 1 , with corresponding values for 2 in parentheses, follow: 0 - 1 1 -0.083 (-0.003), 0-13 -0.091 (0.105), 0 - 1 4 0.004 (-0.029), 0-15 0.197 (0.013), 0-17 0.030 (-0.049), 0-19 -0.099 (0.173), C-1' 0.209 (0.005)]. B-ring atoms are essentially coplanar (mean deviation: 0.005 hi in 1, 0.004 hi in 2)while substituent atoms 0 - 7 ' , 0-8', and C-2 are displaced by small, but significant, amounts from the C - 1 4 - 6 ' least-squares plane (LO-7 -0.044 hi, LO-8 0.038 hi, AC-2 0.061 hi in 1;corresponding values in 2 are -0.017 hi, 0.025 hi, 0.057 hi). The dihedral angle between the 0-1-C-10 and C - 1 ' 4 - 6 ' least-squares planes is 17.5' and 7.2' in 1 and 2, respectively. The OH group at C-5 is intramolecularly hydrogen bonded to 0 - 1 3 in both compounds 10-14...O-13: 2.591(2)hi in 1,2.590(2) A in 21.In the crystals of 1and 2,molecules are associated by 0 - H . ..O hydrogen bonds involving their B-ring hydroxy groups [0-7'. . .O-14 (at - 1+x, y, 1+z) 2.937(3)hi for 1;0-8'. . .O-13 (at x , - 1/2-y, 1/2+z) 2.730(2) hi for 21. ctral Data of 1 and 2.' Compound
Compound Carbon
Carbon 2 ................ 3 ................
4 ................
................ . 6 ................ 7 ................ 8 ................ 9 ................ 10 . . . . . . . . . . . . . . . 1'. . . . . . . . . . . . . . . . 2' . . . . . . . . . . . . . . . . 5
'Measured in CDCI,.
1
2
155.8 138.9 179.4 149.1 136.2 152.9 132.9 144.9 107.5 123.7 114.6
155.9 138.6 179.3 149.2 136.2 152.9 132.8 144.9 107.5 122.5 110.8
3'
..............
4' . . . . . . . . . . . . . .
.............. 6' . . . . . . . . . . . . . . OMe-3 . . . . . . . . . . OMe-6 . . . . . . . . . . OMe-7 . . . . . . . . . . OMe-8 . . . . . . . . . . OMe-3' . . . . . . . . . . OMe-4' . . . . . . . . . .
1
2
145.6 149.0 110.5 121.6 60.1 62.1 61.7 61.2 56.1
146.4 148.6 114.9 122.8 60.1 62.1 61.7 61.2 56.0
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Previously, no cytotoxic activity has been reported for compound 1,and only one cell line (KB) has been examined using compound 2 (10). In the present study, compounds 1-3 were initially evaluated against six tumor cell lines, including human epidermoid carcinoma of the nasopharynx (KB), lung carcinoma (A-549), ileocecal carcinoma (HCT-8), melanoma (PRMI-795 l), and medulloblastoma (TE-671), as well as one murine leukemia cell line (P-388). Compound 1 exhibited strong cytotoxic effects against these cell lines, while compound 2 showed no significant inhibitory effect (ED,,>4.0 pg/ml) for most cell lines tested (Table 3) except for TE-671 (ED,,=0.98
ED,, (kgiml) Compound
1 ............. 2
.............
KB
A459
HCT-8
P-388
PRMI-7591
TE-671
0.045 8.38
0.60
4.40
Ib
Ib
0.055 6.52
0.55 5.93
0.069 0.98
pg/ml). Compound 3 was inactive in all six cell lines. Further testing of the antitumor activity of 1 was performed by the National Cancer Institute (NCI) in their in vitro disease-oriented antitumor screen, which determines a tested agent's effect on growth parameters against a panel of approximately 60 human tumor cell lines (13,14). In this assay, 1 showed strong cytotoxicity against the growth of most of the tumor cell lines, with GI,, values (drug concentration required to cause 50% inhibition) in the low micromolar to nanomolar concentration range. Its cytotoxic effect was especially notable on panels of central nervous system cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, and leukemia cell lines (Table 4). The only structural difference between 1 and 2 is in the substitutions at the 2-aryl moiety, which in 1 has a hydroxyl at position 3' and a methoxy at position 4', while in 2 these substituent locations are interchanged. Compound 1 was highly active, while 2
TABLE 1
Inhibition of in vitro T
lor Cell Growth by Cc
pound 1.' Log,, concentration (M)
Log,, concentration (M) Cell linesb TGI' HL-60 (TB) . . . . . . . . SR . . . . . . . . . . . . . . . HOP-62. . . . . . . . . . . NCI-H226. . . . . . . . . NCI-H460 . . . . . . . . . NCI-HS 2 2 . . . . . . . . . DMS-114. . . . . . . . . . HCT-116, . . . . . . . . .
