Chem. Res. Toxicol. 1988,1, 204-207
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Articles 5-Alkylresorcinols from Hakea amplexicaulis That Cleave DNA John R. Barr, V. S. Murty, Keiichi Yamaguchi, Shangara Singh, David H. Smith, and Sidney M. Hecht* Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia 22901 Received February 23, 1988
A dichloromethane extract of Hukea amplexicaulis was found to cause strand scission of 9x174 replicative form DNA in the presence of Cu(I1). Bioassay-guided fractionation of this extract afforded five compounds capable of mediating DNA relaxation. Structure determination of the active principles indicated that they were 5-tridecylresorcinol (I),5-pentadec-cis-8-enylresorcinol (2), 5-heptadeca-8,11-dienylresorcinol(3), 5-pentadecylresorcinol (4), and 5-heptadec-cis-8enylresorcinol (5). As noted previously for compounds in this structural series, DNA cleavage was enhanced significantly by incubation under (alkaline) conditions known to promote oxygenation of the parent compounds on the aromatic nucleus.
Plant-derived natural products have long constituted a rich source of compounds useful as probes of biological systems and as medicinal agents (see, e.g., ref 1-8). In addition to their intrinsic activities, the naturally derived principles have provided a clear starting point for structural studies and for structural modification to afford more potent or specific biological and medicinal agents (9-14). In recent years, the potential of natural products and their synthetic congeners to facilitate the dissection of certain biochemical systems at a molecular level has become apparent; one good example is macromolecule-effector interaction (15-18). As part of a program whose goal is the identification of naturally derived principles that bind or cleave DNA by novel mechanisms, we recently reported the isolation from Hukea trifurcata of three 5-alkylresorcinolderivatives that were found to mediate DNA strand scission in the presence of Cu(I1) and O2 (19). Reported herein is the isolation and structure elucidation of five 5-alkylresorcinol derivatives from a plant in the same genus, as well as the ability of the newly derived principles to mediate DNA strand scission in the presence of Cu(I1).
Isolation of Compounds 1-5. A dichloromethane extract prepared from the roots and bark of Hakea amplexicaulis was fractionated initially by succeasive chromatographies on Sephadex LH-20. In a typical experiment, 850 mg of extract was applied to a 165 X 2.5 cm Sephadex LH-20 column. The column was washed with 1:l CHC13-CH30H, and the active fractions were identified by bioassay. The combined extract from three such fractionations (1.1g) was fractionated again on Sephadex LH-20, affording 680mg of material that mediated relaxation of cccDNA.' A portion of this active fraction (76 mg) was purified further by reverse-phase HPLC; elution was with 9 1 CH30H-H20 at a flow rate of 5 mL/min. Compounds 1-5 were isolated in quantities of 20.4, 29.5, 1.9, 1.8, and 4.2 mg, respectively.
OH
1
OH
2 CH=CH~CH=CH-
Experlmental Procedures Materials. Sephadex LH-20 was obtained from Sigma Chemicals; HPLC separations employed a lop Alltech C-18 column (1.0 X 25 cmL ax174 replicative form DNA and agarose were purchased from Bethesda Research Laboratories. Hakea amplexicaulis, collected in Western Australia, was obtained with the assistance of the National Cancer Institute. NMR Experiments. High-field 'H NMR spectra were recorded at 360 MHz on a Nicolet NTC 360 FT N M R spectrometer using Nicolet 1280 software. The NMR spectra were obtained using 16K Fourier transforms and a 6.5-ps pulse (goo)with 12-s delays between pulses. Mass Spectral Analysis. Chemical ionization mass spectra were recorded on a Finnigan MAT 4600 gas chromatograph/mass spectrometer using a direct exposure probe. Methane was employed as a reagent gas with a source pressure of 0.35 Torr and an electron energy of 100 eV. 0893-228x/88/2701-0204$01.50/0
HoQ-
OH
3
OH
4
OH
5
Abbreviations: cccDNA, covalently closed circular DNA; form I DNA, supercoiled cccDNA; form I1 DNA, nicked cccDNA; EDTA, ethylenediaminetetraacetic acid.
0 1988 American Chemical Society
5-Alkylresorcinols from Hakea amplexicaulis Acetylation of Isolated Compounds. The procedure employed for acetylation of 5-heptadeca-8,11-dienylresorcinol(3) was typical. A 1.3-mg (3.8 pmol) sample of 5-alkylresorcinol3 was dissolved in 0.5 mL of pyridine and cooled to 0 O C . This solution was treated with 100 pL of acetic anhydride, and the combined solution was allowed to stir at 25 "C under argon for 3 h. The reaction was quenched by the addition of ice and was then extracted with CHCl,. The organic extract was dried (MgS04)and concentrated, affording a solid residue: yield, 1.7 mg; silica gel TLC R,0.76 (91 CHCl,-CH30H); chemical ionization mass spectrometry (positive ion), m / z 429 [(M + H)+]. Cleavage of cccDNA. PNA strand scission was carried out in a fashion similar to that described previously (20) and is illustrated here for an alkali-treated sample of 5-tridecylresorcinol (1). To 20 pg of 5-tridecylresorcinol in 34 p L of 8 1 water-dimethoxyethanewas added 43 pL of 0.1 N NaOH. The combined solution was maintained at 25 "C for 5 min, then treated with 51 pL of 270 pM aqueous CuC12.2H20,and aerated with a stream of O2for 3 min. The solution was neutralized with 43 p L of 0.1 N HC1 and placed on ice for 3 min. A 15-pLsample of this solution was transferred to a tube containing 200 ng of ax174 form I DNA in 25 pL of 50 mM sodium cacodylate, pH 7.4. The reaction mixture was maintained at 25 O C for 30 min and then treated with 10 pL of a solution containing 5 mM EDTA, 40% glycerol, and 0.3% bromophenol blue plus 0.4% SDS to quench the reaction. The reaction mixture was maintained on ice for 2 min, centrifuged, and applied to a 1.2% agarose slab gel. Horizontal gel electrophoresis was carried out at 43 V for 12 h in 40 mM Tris buffer, pH 7.8, containing 5 mM NaOAc and 1 mM EDTA.
Results and Discussion The genus Hakea was named for the German botanist Baron von Hake (21). It is comprised of about 100 species of evergreen shrubs and small trees, which are often planted as ornamentals (21). Plants of the genus Hakea are indigenous to Australia; specimens of Hakea amplexicaulis were collected in Western Australia; voucher specimen SPJ-6939 is preserved a t the National Herbarium, Washington, DC. A dichloromethane extract was prepared from the roots and bark. This extract was found to mediate Cu(I1)-dependent DNA strand scission. The principles responsible for mediating DNA cleavage were partially purified by chromatography on Sephadex LH-20. Further fractionation of the active fractions by C18 reverse-phase HPLC afforded five pure compounds capable of relaxing replicative form (PX174 DNA in the presence of Cu(I1) and 02. That portion of the elution profile containing the active principles is illustrated in Figure 1. The ultraviolet spectrum of compound 1 (Amm 280 (t 1900) and 275 nm (2000) was quite similar to that of orcinol,2 suggesting that the isolated compound might be a 5-alkylresorcinol derivative. The chemical ionization mass spectrum of compound 1 indicated a molecular weight of 292, which would be consistent with the empirical formula C19H3202. In fact, based on its 'H NMR spectrum3 and chromatographic properties, this compound was found to be identical with 5-tridecylresorcinol. This compound has been isolated previously (23-28); the assignment of structure was verified by total synthesis (19). Investigation of compound 4 revealed a UV spectrum (A, 280 (t 1600) and 273 nm (1600)) quite similar to that The UV spectrum of 1 had, A 280 and 275 nm, Ami, 278 and 246 nm. Orcinol UV spectrum: see ref 22. Compound I: 'H NMR (CDC13, (CH3)4Si)6 0.95 (t, 3), 1.1-1.8 (m, 22), 2.5 (t, 2, J = 7.5 Hz), 6.10 (t, 1, J = 1.4 Hz), and 6.15 (d, 2, J = 1.5 Hz). Compound 2 'H NMR (CDCl3, (CH3)4Si)6 0.88 (t, 3), 1.30 (m, 16), 1.57 (m, 2), 2.02 (m, 4), 2.48 (t, 2, J = 7.6 Hz), 5.35 (m, 2), 6.17 (br s 1) and 6.24 (br a, 2). Compound 4 'H NMR (CHIOH-& (CHS),Si) 5 6.8i (t, 3), 1.28 (m, 24), 1.55 (q, 21, 2.43 (t, 2, J = 7.2 Hz), 6.07 (d, 1,J = 2 Hz), and 6.12 (d, 2, J = 2 Hz). Compound 5: 'H NMR (CDC13, (CH3)4Si)6 0.88 (t,3), 1.23-1.40 (m, 20), 1.57 (br q, 2), 2.02 (m, 4), 2.49 (t, 2, J = 7.2 Hz), 5.35 (m, 2), 6.16 (br s, l), and 6.24 (br s, 2).
Chem. Res. Toxicol., Vol. 1, No. 4, 1988 205
I 10
I
I
12
14
16
1s
Elution Time (min)
Figure 1. Purification of active principles that mediate DNA cleavage. The active fractions from Sephadex LH-20 chromatography were fractionated by application to a (250 X 10 mm) C-18 reverse-phaseHPLC column; elution was effected with 9:l CH30H-H20at a flow rate of 5 mL/min. The elution profile was monitored at 254 nm. of 1. The chemical ionization mass spectrum indicated that the molecular weight of 4 was 320, suggesting that it might be a simple homologue of 1. The 'H NMR spectrum3 was consistent with this suggestion and verified the absence of branching within the 5-alkyl substituent; the structure of 4 was, therefore, assigned as 5-pentadecylresorcinol (22, 24, 25, 29). Compound 2 was found to have spectral data quite similar to those of 1 and 4. The W spectrum, for example, had ,A, 280 (c 2300) and 273 nm (2400). The 360-MHz 'H NMR spectrum of Z3 was also similar to those of 1 and 4, with the exception that the spectrum of 2 contained a two-proton resonance at 6 5.35 (br t, J = 7.2 Hz), suggesting the presence of a cis-olefin. The chemical ionization mass spectrum indicated that 2 had M, 318, consistent with the molecular formula C21H,02. Acetylation of 2 afforded the respective di-0-acetate (mlz 403, chemical ionization mass spectrometry) which was analyzed following ozonolysis (03, CS2,-78 "C) (19);the appearance of the pseudomolecular ions a t m / z 321 and 115 were assigned to 8-(1,3-diacetoxypheny1)octanal and heptanal, respectively. On this basis, the structure of 2 was assigned as 5-pentadeca-cis8-enylresorcinol (23-27,29,30). Structure verification was obtained by comparison of the spectra of 2 with those of an authentic synthetic sample (19), as well as by comparative HPLC analysis of the two. The ultraviolet spectrum of compound 5 was essentially identical with those of 1,2, and 4, indicating that it might well be of the same structural type. The 'H NMR spectrum3 was most similar to that of 2, in that it also contained a signal a t 6 5.35 (br t, 2, J = 6 Hz) indicative of a cis-olefin. The 'H NMR spectrum did not reflect any branching within the alkyl substituent; careful analysis of the spectra of 2 and 5 in the region between 1.25 and 1.40 ppm suggested that 5 contained additional aliphatic H's. That 5 was a higher homologue of 2 was also suggested by chemical ionization mass spectrometry of 5 , which indicated a molecular weight of 346, i.e., 28 mass units higher than 2. Following conversion of 5 to the respective di-0-
Barr et at.
206 Chem. Res. Toxicol., Vol. I, No.4, 1988 1
3
5
7
I1
1
Figure 3. DNA strand scission by 5-a1kylresorcinol derivatives 1,2,4, and 5. Cleavage of 9 x 1 7 4 form I DNA was carried out with 30 pM Cu(I1) + alkali-treated 5-alkylresorcinol derivatives 1 (lanes 1 and Z), 2 (lanes 3 and 4), 4 (lanes 5 and 6), or 5 (lanes
Figure 2. DNA strand scission by orcinol and 5-tridecylresorcinol. Cleavage of a x 1 7 4 replicative form (form I) DNA was carried
out with 150 pM orcinol + 30 pM Cu(1I) (lane l), 150 pM orcinol 30 p M Cu(I1) that had been incubated under alkaline conditions prior to DNA cleavage (lane 21, 10 pM Fe(I1) + 0.3% H202 (lane 4), 150 pM 5-tridecylresorcinol + 30 pM Cu(I1) that had been incubated under alkaline conditions prior to DNA cleavage (lane 7), and 150 p M 5-tridecylresorcinol + 30 p M Cu(I1) (lane 8). Control reactions were run by using DNA alone (lane 5), DNA incubated in the reaction solvent (1,2-dimethoxyethane-water) (lane 3), or DNA treated with an aqueous solution incubated under alkaline conditions and then neutralized (lane 6). T h e assay conditions are described under Experimental Procedures.
+
acetate, ozonolysis afforded peaks a t m/z 321 and 143, assigned as the pseudomolecular ions of 8-(1,3-diacetoxypheny1)octanal and nonanal, respectively. The structure of 5 was, therefore, concluded to be 5-heptadeca-cis-8enylresorcinol. This compound has been identified previously as a natural product (25). Analysis of the structure of 3 suggested that it was also a resorcinol derivative (Ama 280 (e 2300) and 274 nm (2800)). Consistent with this suggestion, the 'H NMR spectrum (CDC13)indicated the presence of three aromatic protons a t 6 6.25 (br s, 2) and 6.18 (br s, 1). Also present was a four-proton multiplet a t 6 5.36, suggesting the presence of two olefins within the alkyl substituent. That these olefins were separated by single methylene group was suggested by the presence of a two-proton triplet a t 6 2.78 ( J = 7 Hz) and a four-proton signal corresponding to the allylic H's a t 6 2.06 (m). Further support for these assignments was obtained by irradiation of the presumed vinylic protons a t 6 5.36, which caused the two-proton triplet a t 6 2.78 to collapse to a singlet and the four-proton multiplet a t 6 2.06 to collapse to a broad singlet. Irradiation of the signal a t 6 2.78 caused sharpening of the multiplet a t 6 5.36. Also present in the spectrum were signals for the benzylic (6 2.49 (t, 2, J = 7.2 Hz)), homobenzylic (6 1.58 (br, 2)), methylene (6 1.32 (br, 14)), and methyl (6 0.90 (br t, 3)) protons. The chemical ionization mass spectrum of 3 gave a strong pseudomolecular ion a t m / z 345 (positive ion spectrum); acetylation provided a di-0-acetate (M, 428), which was analyzed for position of unsaturation by chemical ionization mass spectrometry following ozonolysis. On the basis of the accumulated data, the structure of 3 was assigned as 5-heptadeca-8,l l-dienylresorcinol. The isolation of a number of structurally related 5-alkenylresorcinol derivatives, all of which have been identified as cis-olefins (25,26,28-30), suggests on biogenetic grounds that the structure of 3 may be 5-heptadeca-cis,cis-8,1l-dienylresorcinol.However, the 360MHz 'H NMR spectrum lacked sufficient resolution to permit the J values for the vinylic protons to be determined, so the assignment of geometry to 3 cannot be made with certainty. Also characterized was the ability of each of the purified 5-a1kylresorcinol derivatives to mediate DNA strand
7 and 8) a t 400 p M (lanes 1,3,5, and 7) or 80 p M (lanes 2,4,6, and 8) concentrations. Control reactions lacking resorcinol derivatives gave no significant DNA strand scission. The assay conditions are described under Experimental Procedures.
scission in the presence of Cu(I1) and oxygen. This was measured by conversion of supercoiled cccDNA (form I DNA) to nicked circular (form 11) DNA having altered mobility on an agarose gel. As noted previously (19),the purified principles were only weakly active, but activity increased when the compounds were first incubated in aerated aqueous solutions a t alkaline pH, which presumably resulted in initial oxygenation of the benzene nucleus (31-36) (Figure 2). Also illustrated in Figure 2 is the greater activity of 5-alkylresorcinol derivatives having longer alkyl substituents (19). Comparative testing of four of the five isolated 5-alkylresorcinols is shown in Figure 3. The derivatives were base-treated prior to admixture of DNA and then tested a t 400 and 80 pM concentrations in the presence of 30 pM Cu(I1). As shown in the figure, none of the tested compounds differed dramatically in activity from the others, but activity generally did seem to increase with increasing alkyl chain length and increasing unsaturation. When tested a t 80 p M concentration under comparable conditions 5-a1kylresorcinol3 gave similar results (77% form I; 23% form 11; densitometry data not shown).
Acknowledgment. This work was supported by PHS Research Grant CA 40291, awarded by the National Cancer Institute, DHHS. The plant material used in this study was obtained from the National Cancer Institute through the courtesy of Dr. Matthew Suffness. Registry No. 1, 5259-01-8; 2, 22910-86-7; 3, 114679-03-7; 4, 3158-56-3; 5, 52483-19-9; CU, 7440-50-8.
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