Chem. Res. Toxicol. 1990, 3, 268-280
268
Molecular Recognition between Ligands and Nucleic Acids: Novel Pyridine- and Benzoxazole-Containing Agents Related to Hoechst 33258 That Exhibit Altered DNA Sequence Specificity Deduced from Footprinting Analysis and Spectroscopic Studies Yadagiri Bathini, K. Ekambareswara Rao, Regan
G. Shea,? and J. William Lown*
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 Received November 21, 1989
T h e syntheses of certain analogues of the DNA minor groove binding agent Hoechst 33258 designed to exhibit altered sequence recognition are described. T h e structural modifications include the following: substitution of pyridine for the benzene ring of the benzimidazole moiety, replacement of one benzimidazole unit by a benzoxazole in the two possible orientations with respect t o t h e DNA receptor, and a synthesis of 2,2'-m-phenylenebis[6-(4-methyl-lpiperazinyl)benzimidazole]. Sequence recognition of these agents on a HindIIIIEcoRI fragment of pBR322 DNA was determined by MPE footprinting procedures. Some of the analogues exhibited altered DNA sequence preference compared with Hoechst 33258. In particular, a structure bearing a benzoxazole moiety with the oxygen oriented inward to the minor groove together with an inward-directed pyridine nitrogen appears to confer the property of recognition of a GC base pair within the binding sequence. The possible factors, structural, stereochemical, and electrostatic, contributing to the altered DNA sequence recognition properties are discussed.
I ntroductlon As attention is increasingly focused on the problem of control in molecular biology, there is growing interest in attempts to develop DNA sequence specific agents (1-4), ideally to target any predetermined sequence, for the study of gene expression and control. Such agents would have many applications in molecular biology and, quite possibly, in diagnosis and therapy, particularly if one could target a sequence of 15 base pairs which corresponds to a unique sequence in the human genome (5). A number of approaches have been taken to this problem including the use of @-oligonucleotides( I ) , methylphosphonate derivatives of @oligonucleotides,and probes that invoke triplex formation (2). All of these approaches take advantage of the intrinsic sequence recognition imparted by WatsonCrick base pairing. While this property is attractive, the oligonucleotide-based probes suffer from various disadvantages. Thus both a- and @oligonucleotides,since they are charged, cannot easily penetrate the cell membrane. Once introduced into the cell, by transfection or microinjection, @-oligomersare subject to rapid nuclease degradation. Although a-oligomers are more resistant to nuclease action (9) and the (Y-DNA~RNA hybrid is resistant to RNase H ( 5 ) ,nevertheless mRNA translation is not inhibited, presumably as a result of efficient helicase action. The most promising oligonucleotide probes to date are the methylphosphonates (6-8).While they are taken up readily in cells, the existence of S and R forms at each phosphate residue results in a mixture of 2"-l stereoisomers for a sequence of n bases (6-8). Therefore, only a very small fraction anneals to the desired target (IO). An alternative approach is to develop sequence-specific probes based on natural or synthetic DNA binding agents.
* Author to whom correspondence should be addressed. 'Present address: Department of Molecular Biology, Genentech Inc., South San Francisco, CA 94080. 0893-228x/90/2703-0268$02.50/0
Groove binding agents have several advantages over intercalators for this purpose in that, unlike the latter, groove binders should cause minimal structural distortion of the DNA and, correspondingly, less disturbance of the information inherent in the DNA sequence. We have reported a promising approach based on the naturally occurring oligopeptide antibiotics netropsin (11,12)and distamycin (4).Rational structural modification led to the development of lexitropsins, or information-reading oligopeptides. After systematic development and exploration of the several factors influencing molecular recognition between ligands and DNA, later lexitropsins were produced that recognize unique sequences and exhibit no memory for the home site (AT), of the parent antibiotics ( 4 ) . Encouraged by these results, we have begun to explore alternative model vectors. Hoechst dye 33258 is known to bind to double-helical DNA and to have ready access into cells (13-19)and for these reasons has been widely used as a fluorescent cytological stain for DNA (20). Several studies including absorption spectroscopy and the techniques of footprinting have established that, in common with netropsin, Hoechst 33258 binds preferentially a t AT-rich DNA sequences with a minimum binding size of four consecutive A T base pairs (19,21-24). The expected minor groove selectivity at such sites was recently confirmed by X-ray diffraction of a complex of the dye with d(CGCGAATTCGCG1 (25), although the results of such X-ray studies have been controversial (26). Recently, the latter group studied the crystal structure of a complex of Hoechst 33258 and d(CGCGATATCGCG)2and confirmed that the piperazine ring is located in the wider portion of the minor groove near the adjacent GC base pair (27). We report the design, synthesis, and altered sequence specificity, established by methidiumpropyl-EDTA.Fe(I1) (MPE) footprinting, of a series of rationally structurally altered agents related to Hoechst 33258. The structural modifications included substitution of pyridine for the 0 1990 American Chemical Society
Ligand-DNA Molecular Recognition
Chem. Res. Toxicol., Vol. 3, No. 3, 1990 269
Scheme I. Outline of Convergent Synthetic Routes to DNA Binding Ligands"
" Reaction conditions: (a) 4-hydroxybenzaldehyde in ethanol, then Pb(OAc), in acetic acid; (b) LiAlH, in THF, then pyridinium chlorochromate; (c) l-methylpiperazine and K2CO3, DMF; (d) H2, Pd-C 5% in EtOAc; (e) PhN02,heat 24 h; (f) 4-hydroxybenzaldehyde in ethanol, then Pb(OAc)4in acetic acid; (g) LiAIH, in THF, then pyridinium chlorochromate; (h) PhN02, heat 24 h; (i) l-methylpiperazine; fi) Hz, in ethanol, then Pb(OAc), in CH3C0,H; heat 24 h; (1) 1Pd-C, 5 % in EtOAc; (k) 2-(p-hydroxyphenyl)benzoxazole-6-carboxaldehyde methylpiperazine, KzCO3, DMF; (m) H2, Pd-C 5% in EtOAc; (n) 2-(p-hydroxyphenyl)benzoxazole-6-carboxaldehyde, PhN02, heat 24 h. benzene ring of t h e benzimidazole moiety (to permit GC recognition) and replacement of one benzimidazole unit by a benzoxazole moiety in t h e two possible orientations (to introduce two alternative hydrogen bond acceptors). T h e factors contributing t o the resulting altered molecular recognition a n d sequence selectivity are discussed. Synthesis. Hoechst 33258 (compound I), analogues 2-7, and m-phenylenebis[piperazinylbenzimidazole]8 (see C h a r t I) were synthesized as outlined in representative reactions in Scheme I following a convergent approach. T r e a t m e n t of o-hydroxyanilines with phenolic carboxaldehydes followed by oxidative cyclization with Pb(OAc), in benzene or acetic acid afforded t h e substituted benzoxazole moieties in good yield (28). Benzimidazoles were constructed by heating ortho aromatic diamines with t h e appropriate aromatic carboxaldehyde in a 1:l molar ratio in nitrobenzene, a n d t h e progress of t h e reaction was monitored b y TLC. In this reaction the initially formed Schiff base undergoes oxidative cyclization in air t o afford t h e benzimidazole (29). T r e a t m e n t of 5-chloro-2-nitroaniline with l-methylpiperazine in t h e presence of K 2 C 0 3 in DMF afforded t h e required piperazinyl derivative in 64% yield together with a small a m o u n t of 5-(dimethylamino)-2-nitroaniline. T h e latter compound may have resulted from substitution of chlorine by t h e dimethylamino group of DMF. Precedents for this kind of reaction have been reported recently (30a). T h u s t r e a t m e n t of 5-chloro-2-nitrophenol with l-methylpiperazine in t h e absence of K&03 afforded only 5-(dimethylamino)-2nitrophenol (whereas similar treatment in t h e presence of K2C03gave no reaction). T h i s result also s u p p o r t s t h e suggestion t h a t the dimethylamino group of D M F is acting as a nucleophile. T h e required 2-nitr0-5-(4-methyl-lpiperaziny1)phenol was subsequently obtained by heating 5-chloro-2-nitrophenol with l-methylpiperazine in t h e absence of solvent.
Materials and Methods Melting points were determined on an Electrothermal melting point apparatus and are uncorrected. The IR spectra were recorded on a Nicolet 7199 FT spectrophotometer, and only the principal bands are reported. The 'H NMR spectra were recorded on Bruker WH-200 and WH-400 spectrometers. Mass spectra were determined on an Associate Electrical Industries (AEI) MS-9 and MS-50 focusing high-resolution mass spectrometer. Florisil (60-100 mesh) and Kieselgel60 (23Ck400 mesh) of E. Merck was used for chromatography, and precoated silica gel 60F-254 sheets of E. Merck were used for TLC, with the solvent system indicated in the procedure. TLC plates were visualized by using UV light or 2.5% phosphomolybdic acid in methanol with heating. All compounds obtained commercially were used without further purification unless otherwise stated. Ethanol and methanol were freshly distilled from magnesium turnings; tetrahydrofuran was distilled from sodium/benzophenone under an atmosphere of dry argon; ether was dried over sodium; methylene chloride was distilled from phosphorus pentoxide and stored over 3-A molecular sieves; triethylamine was treated with potassium hydroxide, then distilled from barium oxide, and stored over 3-A molecular sieves; dimethyiformamide was distilled from barium oxide and stored over 3-A molecular sieves. Synthesis of Hoechst Analogues 2-7 a n d 2,2'-m Phenylenebis[6-(4-methyl-l-piperazinyl)benzimidazole] (8). 5-(4-Methyl-l-piperazinyl)-2-nitroaniline. A mixture of 860 mg (5 mmol) of 5-chloro-2-nitroaniline, 1 g (10 mmol) of 1methylpiperazine, and 700 mg (5 mmol) of K&03 in 10 mL of dimethylformamidewas stirred at 120 "C for 18 h. The reaction mixture was cooled, poured into 50 mL of cold water, and extracted with ethyl acetate (3 x 30 mL). The combined organic extracts were dried (Na,SO,) and evaporated, and the resulting residue was purified on a column of Florisil (EtOAc/MeOH) to give 764 mg of 5-(4-methyl-l-piperazinyl)-2-nitroaniline as a light yellow solid (64.7% yield): mp 153-154 "C [lit. (306) 155 "C]; IR (Nujol) ,Y, 3440, 3280, 3150, 1620, 1570 cm-'; 'H NMR (DMSO-d,) 6 2.2 (s,3 H, N-CH,), 2.4 (t,4 H, CHZ, J = 5 Hz), 3.3 (t, 4 H, CH2,J = 5 Hz), 6.2 (d, 1 H, Ar-H, J = 2 Hz), 6.38 (dd, 1 H, Ar-H, J = 9, 2 Hz), 7.26 (bs, exch, 2 H, NH2), 7.8 (d, 1 H,
270 Chem. Res. Toxicol., Vol. 3, No. 3, 1990 Chart I. Structures of Minor Groove DNA Binding Ligands Employed in This Study of Molecular Recognition
Ar-H, J = 9 Hz). MS ( m / z ,relative intensity): calcd for CllHIBN4O2236.1273; found 236.1270 (M+,48), 221 (M - CH3, 7), 165 (M - CdHgN, 12), 71 (CIHgN, 32), 70 (C,HSN, 100). 4-(4-Methyl-l-piperazinyl)-l,2-diaminobenzene. A solution of 944 mg (4 mmol) of 5-(4-methyl-l-piperazinyl)-2-nitroaniline in 60 mL of ethyl acetate was hydrogenated over 200 mg of 5% palladium on charcoal a t room temperature. The catalyst was removed by filtration and the filtrate was concentrated in vacuo. Trituration with hexane gave 780 mg of the title product as crystalline solid (95% yield): mp 94 "C; IR (KBr),,u 3400,3280, 3200,2920,2840,2800,1630,1600,1500cm-'; 'H NMR (DMSO-d,) 6 2.18 (s,3 H, N-CH,), 2.38 (t, 4 H, CH2, J = 5 Hz), 2.86 (t, 4 H, CH,, J = 5 Hz), 3.96 (bs, 2 H, NHZ), 4.32 (bs, 2 H, NH,), 6.0 (dd, 1 H, Ar-H, J = 8, 2 Hz), 6.2 (d, 1 H, Ar-H, J = 2 Hz), 6.38 (d, 1H, Ar-H, J = 8 Hz). DzO exchange resulted in the disappearance of the peaks at 6 3.96 and 4.32. MS (m/z,relative intensity): calcd for Cl1HlIN4206.1531; found 206.1533 (M+, loo), 191 (M - CH3, 8), 136 (M - CIHgN, 49), 135 (M - C,HgN, 48),71 (C,HgN, 26), 70 (CdHgN, 20). 5-(4-Methyl-l-piperazinyl)-2-nitrophenol. A solution of 500 mg (3.6 mmol) of 5-chloro-2-nitrophenol in 5 mL of l-methylpiperazine was heated a t 110 "C for 24 h. Excess l-methylpiperazine was removed under reduced pressure, and the residue
Bathini et a1. was treated with saturated ammonium chloride solution. After extraction with three 30-mL portions of an ethyl acetate/tetrahydrofuran (1:l)mixture, the combined organic extracts were dried (Na2S04)and evaporated and the resulting yellow solid was purified on a column of Florisil (EtOAc/MeOH) to give 580 mg of the title compound (85% yield): mp 77 "C; IR (KBr) u- 3440, 1630,1560 cm-'; 'H NMR (DMSO-d,) 6 2.2 (s, 3 H, N-CH,), 2.38 (t, 4 H, CH,, J = 5 Hz), 3.43 (t, 4 H, CH2, J = 5 Hz), 6.4 (d, 1 H, Ar-H, J = 2.5 Hz), 6.63 (dd, 1 H, Ar-H, J = 10, 2.5 Hz), 7.84 (d, 1 H, Ar-H, J = 10 Hz), 11.0 (bs, 1 H, Ar-OH). DzO exchange resulted in the disappearance of the peak a t 6 11.0. MS (m/z, relative intensity): calcd for Cl1Hl5N3O3237.1113; found 237.1118 (M+, 71), 236 (M - 1,8), 166 (M - CdHgN, 16),71 (C4HgN, 47), 70 (C4H8N,100). Anal. Calcd: C, 55.69; H, 6.33; N, 17.72. Found: C, 55.36; H, 6.2; N, 17.8. 2-Amino-5-(4-methyl-l-piperazinyl)phenol. A solution of 100 mg of 5-(4-methyl-l-piperaziny1)-2-nitrophenol in 20 mL of ethyl acetate was hydrogenated over 25 mg of 5% palladium on charcoal a t room temperature. The catalyst was removed by filtration, and evaporation of the solvent gave 86 mg of the title compound. This aminophenol is very unstable and decomposes rapidly on exposure to atmosphere. IR (KBr) u, 3350, 3280, 1610 cm-'; 'H NMR (DMSO-d,) 6 2.2 (s, 3 H, N-CH,), 2.4 (t, 4 H, CH,, J = 5 Hz), 2.85 (t, 4 H, CH2, J = 5 Hz), 4.0 (bs, 2 H, NHZ), 6.16 (dd, 1 H, Ar-H, J = 8, 2.5 Hz), 6.31 (d, 1 H, Ar-H, J = 2.5 Hz), 6.45 (d, 1 H, Ar-H, J = 8 Hz), 8.9 (bs, 1 H, Ar-OH). DzO exchange resulted in the disappearance of the peaks at 6 4.0 and 8.9 MS ( m / z ,relative intensity): calcd for CllH17N30207.1371; found 207.1374 (M+, loo), 192 (M - CH3, 8), 137 (M - C4H8N, 19), 136 (M - ChHgN, 41), 71 (CdHSN, 18), 70 (C,HSN, 17). 2-Amino-6-chloro-3-nitropyridine. Into a solution of 5 g (26 mmol) of 2,6-dichloro-3-nitropyridine in 100 mL of ethanol was distilled 30 mL of ammonia. The mixture was then stirred for 5 h. The excess of ammonia and ethanol was removed in vacuo. The residue was extracted with THF (3 X 50 mL). The combined organic extract was washed with saturated NaCl solution, dried (Na2S04),and evaporated to give 4.2 g of the title compound (93% yield). 2-Amino-6-(4-methyl-l-piperazinyl)-3-nitropyridine. A mixture of 519 mg (3 mmol) of 2-amino-6-chloro-3-nitropyridine, 600 mg (6 mmol) of 1-methylpiperazine, and 420 mg (3 mmol) of KzCO3 in 5 mL of dimethylformamide was stirred a t room temperature for 3 h. The reaction mixture was diluted with 20 mL of water and extracted with ethyl acetate (3 X 30 mL). The combined organic extract was dried (Na2S04)and evaporated to give 675 mg of the title compound as a pale yellow solid (95% yield): mp 172 "C; IR (Nujol) una. 3420,3240,3110,1605, 1560 cm-'; 'H NMR (DMSO-d,) 6 2.2 (s, 3 H, CH,), 2.36 (t, 4 H, CHz, J = 5 Hz), 3.7 (t, 4 H, CH,, J = 5 Hz), 6.34 (d, 1 H, Ar-H, J = 10 Hz), 7.7 (bs, 2 H, NH,), 8.04 (d, 1 H, Ar-H, J = 10 Hz). D20 exchange resulted in the disappearance of the peak a t 6 7.7. MS ( m / z relative intensity): calcd for CloH15N502237.1226; found 237.1224 (M', 25), 222 (M - CH3, 5 ) , 180 (M - C3H,N, 5), 167 (M - C3H8N,41), 83 (29), 70 (C4H8N,100). Anal. Calcd: C, 50.63; H, 6.37; N, 29.53. Found: C, 50.77; H, 6.57; N, 29.48. 2,3-Diamino-6-(4-methyl-l-piperazinyl)pyridine. A solution of 474 mg (2 mmol) of 2-amino-6-(4-methyl-l-piperazinyl)-3nitropyridine in 40 mL of ethyl acetate was hydrogenated over 100 mg of 5% palladium on charcoal a t room temperature. The catalyst was removed by filtration and the filtrate was concentrated in vacuo. Trituration of the residue with hexane gave 400 mg of the title compound as a crystalline solid (96.6% yield): mp 96-98 "C; IR (KBr) u- 3420,3310,3190,2830,2820,1630,1590 cm-'; 'H NMR (DMSO-de) 6 2.2 (s, 3 H, CHJ, 2.35 (t,4 H, CHz, J = 5 Hz), 3.14 (t,4 H, CH2, J = 5 Hz), 3.4 (bs, 2 H, NHZ), 5.14 (s, 2 H, NH,), 5.76 (d, 1 H, Ar-H, J = 8.5 Hz), 6.64 (d, 1 H, Ar-H, J = 8.5 Hz). D20 exchange resulted in the disappearance of the peaks a t b 3.4 and 5.14. MS ( m / z ,relative intensity): calcd for C10H17N5207.1484; found 207.1487 (M+,47), 192 (M - CH3, 17), 137 (M - C,H,N, loo), 70 (C,HsN, 8). 6-Carbethoxy-2-(4-hydroxyphenyl)benzoxazole.A solution of 905 mg (5 mmol) of 2-amino-5-carbethoxyphenoland 610 mg (5 mmol) of 4-hydroxybenzaldehyde in 30 mL of ethanol was heated under reflux for 3 h. Ethanol was evaporated under reduced pressure, the resulting Schiff base was dissolved in 30 mL of acetic acid, and 2.215 g ( 5 mmol) of lead tetraacetate was
Ligand-DNA Molecular Recognition added and stirred for 30 min at room temperature. The reaction mixture was diluted with 40 mL of water and extracted with three 30-mL portions of ethyl acetate. Solvent was evaporated, and the resulting solid was purified on a column of Florisil (EtOAc/MeOH) to give 1.25 g of the title compound as a crystalline solid (88%yield): mp 220-222 "C; IR (KBr) u,, 3360,3120,1700, 1610,1590,1560,1500 cm-'; 'H NMR (DMSO-d6) 6 1.34 (t, 3 H, OCH2CH3),4.34 (q, 2 H, OCH2CH3),7.0 (d, 2 H, Ar-H, J = 9 Hz), 7.8 (d, 1 H, Ar-H, J = 8.5 Hz), 7.98 (dd, 1 H, Ar-H, J = 8.5, 1.5 Hz), 8.1 (d, 2 H, Ar-H, J = 9 Hz), 8.22 (d, 1H, Ar-H, J = 1.5 Hz), 10.6 (bs exch, 1 H, Ar-OH). MS ( m / z ,relative intensity): calcd for Cl6Hl3NO4283.0844; found 283.0842 (M', 97), 268 (M - CH3, 6), 255 (M - C2H4,28), 238 (M - OCH2CH3,loo), 182 (36). Anal. Calcd: C, 67.84; H, 4.59; N, 4.95. Found C, 67.65; H, 4.53; N, 4.62. 6- (Hydroxymethy1)-2-( 4- hydroxypheny1)benzoxazole. To a slurry of 304 mg (8 mmol) of lithium aluminum hydride in 100 mL of anhydrous ether a t 0 "C was added 1.132 g (4 mmol) of 6-carbethoxy-2-(4-hydroxyphenyl)benzoxazole in 30 mL of dry tetrahydrofuran dropwise with stirring. The reaction mixture was allowed to attain room temperature and stirred for 12 h, then cooled to 0 "C and decomposed with saturated NHICl solution, the organic layer was removed, and the aqueous layer was extracted with two 30-mL portions of ethyl acetate/tetrahydrofuran (1:l)mixture. The combined organic extract was dried (Na2S04) and evaporated to give 870 mg of the title compound as a crystalline solid (90% yield): mp 236-238 "C; IR (KBr) umm 3240, 3120, 1620, 1590, 1550, 1500 cm-'; 'H NMR (DMSO-d6) 6 4.62 (s, 2 H, CH20H),5.34 (bs, 1 H, CH20H), 6.97 (d, 2 H, Ar-H, J = 9 Hz), 7.3 (d, 1 H, Ar-H, J = 8.5 Hz), 7.64 (m, 2 H, Ar-H), 8.03 (d, 2 H, Ar-H, J = 9 Hz), 10.34 (bs, 1 H, Ar-OH). D20 exchange resulted in the disappearance of peaks a t 6 5.34 and 10.34. MS ( m / z ,relative intensity): calcd for C14HllN03241.0739; found 241.0739 (M+, loo), 240 (M - 1, 24), 224 (M - OH, 44), 212 (M - CHO, 49). 2-(4-Hydroxyphenyl)benzoxazole-6-carboxaldehyde.To a suspension of 843 mg (3.5 mmol) of 6-(hydroxymethyl)-2-(4hydroxypheny1)benzoxazolein 30 mL of dichloromethane and 15 mL of tetrahydrofuran was added 2.257 g (10.5 mmol) of pyridinium chlorochromate in portions. The reaction mixture was stirred for 3 h a t room temperature and filtered through a column of Florisil. The solvent was evaporated, and the resulting crude aldehyde was purified on a column of silica gel (EtOAc) (flash chromatography) to give 750 mg of the title compound as a colorless solid (89.6% yield): mp 238-232 "C; IR (KBr) u- 1690, 1600,1560,1500cm-'; 'H NMR (DMSO-d6)6 6.96 (d, 2 H, Ar-H, J = 9 Hz), 7.9 (m, 2 H, Ar-H), 8.08 (d, 2 H, Ar-H, J = 9 Hz), 8.2 (s, 1 H, Ar-H), 10.02 (s, 1 H, CHO), 10.5 (bs, 1 H, Ar-OH). D 2 0 exchange resulted in the disappearance of the peak at 6 10.5. MS ( m / z , relative intensity): calcd for Cl4HBNO3239.0582; found 239.0576 (M+, loo), 238 (M - 1, 71), 182 (28). Anal. Calcd C, 70.29; H, 3.76; N, 5.86. Found: C, 70.15; H, 3.5; N, 5.94. 5-Carbethoxy-2-(4-hydroxyphenyl)benzoxazole.The title compound was prepared following the procedure described above for the isomer. Thus 905 mg of 2-amino-4-carbethoxyhenol and 610 mg of 4-hydroxybenzaldehyde gave 1.2 g of the title compound as a crystalline solid (85% yield): mp 214-216 "C; IR (KBr) u1700, 1620, 1500 cm-'; 'H NMR (DMSO-d6) 6 1.32 (t, 3 H, OCH2CH3),4.34 (q, 2 H, OCH2CH3),6.97 (d, 2 H, Ar-H, J = 9 Hz), 7.83 (d, 1 H, Ar-H, J = 8 Hz), 7.96 (dd, 1 H, Ar-H, J = 8, 2 Hz), 8.05 (d, 2 H, Ar-H, J = 9 Hz), 8.24 (d, 1 H, Ar-H, J = 2 Hz), 10.45 (bs exch, 1 H, Ar-OH). MS ( m / z ,relative intensity): calcd for Cl6Hl3NO4283.0844; found 283.0845 (M+, 100), 268 (M - CH3,7), 255 (M - CzH4,26), 238 (M - OCHZCH3,86), 210 (M - COOEt, 29). Anal. Calcd: C, 67.84; H, 4.59; N, 4.95. Found: C, 67.67; H, 4.60; N, 4.67. 5-(Hydroxymethyl)-2-(4-hydroxyphenyl)benzoxazole. The corresponding ester was reduced to the title hydroxy compound with lithium aluminum hydride following the procedure described above: mp 251-253 "c;IR (KBr) u,, 3240,3120,1620,1600,1500, 1440 cm-'; 'H NMR (DMSO-d6)6 4.62 (s,2 HCH20H), 5.3 (bs, 1 H, CH20H),6.97 (d, 2 H, Ar-H, J = 9 Hz), 7.33 (d, 1 H, Ar-H, J = 8 Hz), 7.67 (m, 2 H, Ar-H), 8.06 (d, 2 H, Ar-H, J = 9 Hz), 10.36 (bs, 1 H, Ar-OH). D 2 0 exchange resulted in the disappearance of the peaks a t 6 5.3 and 10.36. MS ( m / z relative intensity): calcd for C14HllN03241.0739; found 241.0739 (M+,
Chem. Res. Toxicol., Vol. 3, No. 3, 1990 271 100), 240 (M - H, 19), 224 (M - OH, 29), 212 (M - CHO, 42). 2-(4-Hydroxyphenyl)benzoxazole-5-carboxaldehyde.The corresponding hydroxy compound was oxidized to the aldehyde with pyridinium chlorochromate following the procedure described above: mp 252-254 "C; IR (KBr) u- 3260,1680,1610,1500,1440 cm-'; 'H NMR (DMSO-d6) 6 6.98 (d, 2 H, Ar-H, J = 9 Hz), 7.89 (m, 2 H, Ar-H), 8.05 (d, 2 H, Ar-H, J = 9 Hz), 8.22 (s, 1 H, Ar-H), 10.05 (s, 1H, CHO), 10.5 (bs,1 H, Ar-OH). D20 exchange resulted in the disappearance of the peak a t 6 10.5. MS ( m / z ,relative intensity): calcd for CI4H9NO3239.0582; found 239.0578 (M', 1001,238 (M - H, 49), 210 (M - CHO, 29). Anal. Calcd: C, 70.29; H, 3.76; N, 5.86. Found: C, 70.1; H, 3.75; N, 5.9.
5(6)-Carbethoxy-2-(4-methoxyphenyl)benzimidazole.A mixture of 900 mg (5 mmol) of 4-carbethoxy-l,2-diaminobenzene and 680 mg (5 mmol) of 4-methoxybenzaldehyde in 20 mL of nitrobenzene was heated a t 140 OC for 24 h. The nitrobenzene was removed under reduced pressure, and purification of the resulting residue on a column of silica gel (hexane/EtOAc) gave the title compound (A), 900 mg, and the bis-Schiff base (B), 300 mg. (A) mp 168-170 "C; IR (Nujol) u,, 3300,1700,1620 cm-'; 'H NMR (DMSO-d6) 6 1.3 (t, 3 H, OCH2CH3),3.85 (s, 3 H, OCH,), 4.34 (q, 2 H, OCH2CH3),7.14 (d, 2 H, Ar-H, J = 9 Hz), 7.72 (m, 2 H, Ar-H), 8.15 (d, 2 H, Ar-H, J = 9 Hz), 8.2 (s, 1H, Ar-H), 13.1 (bs, 1 H, NH). DzO exchange resulted in the disappearance of the peak a t 6 13.1. MS ( m / z ,relative intensity): calcd for C17Hl6N2O3296.1160; found 296.1159. Anal. Calcd: C, 68.91; H, 5.44; N, 8.45. Found: C, 68.73; H, 5.33; N, 8.46. (B) mp 116-118 "C; IR (Nujol) umm 1700,1620,1520 cm-'; 'H NMR (DMSO-d8) 6 1.3 (t, 3 H, OCH2CH3),3.68 ( s , 3 H, OCH,), 3.8 ( s , 3 H,0CH3),4.3 (q, 2 H,0CH2CH,),5.6 (s,2 H,N=CH), 6.9 (m, 4 H, Ar-H), 7.1 (d, 2 H, Ar-H, J = 8.5 Hz), 7.72 (d, 2 H, Ar-H, J = 8.5 Hz), 7.84 (m, 2 H, Ar-H), 8.05 (s, 1 H, Ar-H). MS ( m / z ,relative intensity): calcd for CZ5Hz4N2O4 416.1736; found 416.1740 (M+, 22), 121 (100). Anal. Calcd: C, 72.10; H, 5.81; N, 6.73. Found: C, 72.0; H, 5.76; N, 6.82. 5(6)-(Hydroxymethyl)-2-(4-methoxyphenyl)benzimidazole. To a slurry of 228 mg (6 mmol) of lithium aluminum hydride in 60 mL of ether a t 0 "C was added 888 mg (3 mmol) of 5(6)-carbethoxy-2-(4-methoxyphenyl)benzimidazolein 20 mL of dry tetrahydrofuran dropwise with stirring. The reaction mixture was allowed to attain room temperature and stirred overnight; then it was cooled in an ice bath and decomposed with water. The organic layer was removed, and the aqueous layer was extracted with an ether/THF (1:l)mixture (2 X 30 mL). The combined organic extracts were dried (Na2S04)and evaporated to give 730 mg of the title compound (95.8% yield): mp 218 OC; IR (KBr) v- 3480,3050, 1600,1500 cm-'; 'H NMR (DMSO-d6) 6 3.84 (s, 3 H, OCH,), 4.6 (d, 2 H, CH20H),5.16 (m, 1H, CH20H),7.1 (m, 3 H, Ar-H), 7.4-7.6 (m, 2 H, Ar-H), 8.12 (d, 2 H, Ar-H, J = 9 Hz), 12.67 (bs, 1H, NH). After D20 exchange peaks at 6 5.16 and 12.67 disappeared and the peak at 6 4.6 became a singlet. MS ( m / z , relative intensity): calcd for C15H14N202 254.1055; found 254.1052 (M+, loo), 239 (M - CH3,5), 237 (M - OH, 39), 255 (M - CHO, 42). Anal. Calcd for C15H14N202:C, 70.85; H, 5.55; N, 11.02. Found: C, 70.89; H, 5.56; N, 10.98. 2-(4-Methoxyphenyl)benzimidazole-5(6)-aldehyde.To 860 mg (4 mmol) of pyridinium chlorochromate in 20 mL of dichloromethane was added 635 mg (2.5 mmol) of 5(6)-(hydroxymethyl)-2-(4-methoxyphenyl)benzimidazolein 20 mL of dichloromethane dropwise a t room temperature. The reaction mixture was stirred for 3 h and diluted with 50 mL of ethyl acetate. The solution was filtered through a column of Florisil. The solvent was evaporated to give 530 mg of the title compound as a colorless solid (84% yield): mp 194 "C; IR (CHC13)u,, 3080,1690,1610 cm-'; 'H NMR (DMSO-d6) 6 3.86 (s, 3 H, O W 3 ) , 7.14 (d, 2 H, Ar-H, J = 9 Hz), 7.74 (m, 2 H, Ar-H), 8.1-8.2 (m, 3 H, Ar-H), 10.14 (s, 1 H, CHO), 13.24 (bs, 1 H, NH). D 2 0 exchange resulted in the disappearance of the peak a t 6 13.24. MS ( m / z relative intensity): calcd for C15H12N202 252.0899; found 252.0896 (M', loo), 237 (M - CH3, lo), 223 (M - CHO, 10). Anal. Calcd for C15H12N202: C, 71.41; H, 4.80, N, 11.11. Found C, 71.15; H, 4.88 N, 10.82. Hoechst Analogue 2. A mixture of 85 mg of 4-(4-methyl-lpiperazinyl)-1,2-diaminobenzeneand 100 mg of 2-(4-hydroxyphenyl)benzoxazole-6-carboxaldehydein 5 mL of nitrobenzene
272 Chem. Res. Toxicol., Vol. 3, No. 3, 1990
Bathini e t al.
relative intensity): calcd for CUHnNSO2426.1804; found 426.1806 (M', 36), 411 (M - CH3, 18), 356 (loo),239 (25), 71 (29), 70 (30). Anal. Calcd: C, 67.60; H, 5.16; N, 19.72. Found: C, 67.27; H, 5.58; N, 19.35. Hoechst Analogue 7. A mixture of 125 mg (0.6 mmol) of the pyridinediamine and 152 mg (0.6 mmol) of the appropriate aldehyde in 5 mL of nitrobenzene was heated a t 140 "C for 24 h. The nitrobenzene was removed under reduced pressure, and the resulting residue was chromatographed on Florisil (EtOAc/MeOH) to give 180 mg of 7 as a light yellow solid (68% yield): mp 208-210 "C; IR (KBr) V- 3400,2920,2840,1620,1580,1500 cm-'; 'H NMR (DMSO-de) 6 2.24 (s, 3 H, NCH3), 2.46 (t, 4 H, CH,, J = 4 Hz), 3.5 (t, 4 H, CH,, J = 4 Hz), 3.86 (s, 3 H, OCH,), 6.77 (d, 1 H, Ar-H, J = 8 Hz), 7.13 (d, 2 H, Ar-H, J = 9 Hz), 7.64 (d, 1 H, Ar-H, J = 8.5 Hz), 7.78 (d, 1 H, Ar-H, J = 8 Hz), 8.03 (m, 1 H, Ar-H), 8.17 (d, 2 H, Ar-H, J = 9 Hz), 8.3 (s, 1 H, Ar-H), 13.0 (bs, 2 H, NH). DzO exchange resulted in the disappearance of the peak a t 6 13.0. MS ( m / z , relative intensity): calcd for CZ5Hz6N70 439.2115; found 439.2118 (M+, 44), 424 (M - CH3, 17), 383 (M - CsHaN, %), 369 (M - CdHSN, loo), 70 (CdHgN, 30). And. CdCd C, 68.34; H, 5.69; N, 22.32. Found: C, 68.10; H, 5.75; N, 22.08. 2,2'-m -Phenylenebis[6-(4-methyl- I-piperaziny1)benzJ=gHz),8.14(dd,lH,Ar-H,J=8,2Hz),8.38(d,lH,Ar-H, imidazole] (8). A mixture of 100 mg of 4-(4-methyl-lJ = 2 Hz). MS ( m / z relative intensity): calcd for CZ5Hz3N5O2 piperazinyl)-1,2-diaminobenzeneand 33 mg of isophthalaldehyde 425.1851; found 425.1843 (M', loo), 410 (M - CH3, 5), 355 (M in 5 mL of nitrobenzene was heated at 140 "C for 30 h. The - CdHsN, 34), 354 (M - CdHgN, 42), 71 (CdHgN, 39), 70 (CdHgN, nitrobenzene was removed under reduced pressure, and the re38). Anal. Calcd: C, 70.59; H, 5.41; N, 16.47. Found: C, 70.19; sulting residue was purified on a column of Florisil to give 105 mg of 8 as a pale yellow solid (84% yield): mp 245-247 "C; IR H, 5.66; N, 16.19. (KJ3r),Y 3400,3060,2930,2800,1630,1600,1580 cm-'; 'H NMR Hoechst Analogue 4. A solution of 100 mg of the diamine (DMSO-de) 6 1.6 (s, 6 H, NCHJ, 1.76 (bs, 8 H, CHJ, 2.48 (bs, and 86 mg of the aldehyde in 15 mL of ethanol was heated under reflux for 1 2 h. Ethanol was removed under reduced pressure, 8 H, CHJ, 6.24 (s, 1H, Ar-H), 6.3-6.4 (singlet merged with doublet, the resulting Schiff base was dissolved in 5 mL of acetic acid, 184 2 H, Ar-H), 6.85 (d, 2 H, Ar-H, J = 9 Hz), 7.04 (t, 1 H, Ar-H, J mg of lead tetraacetate was added, and the mixture was stirred = 8 Hz), 7.54 (d, 2 H, Ar-H, J = 8 Hz), 8.36 (s, 1 H, Ar-H). MS for 5 min at room temperature. The reaction mixture was diluted ( m / z , relative intensity): calcd for C30H34N8506.2906; found 506.2914 (loo), 491 (6), 436 (17), 435 (6), 205 (30), 71 (39), 70 (57). with 15 mL of ethyl acetate and 10 mL of water. The organic Anal. Calcd: C, 71.15; H, 6.72; N, 22.13. Found: C, 70.84; H, layer was removed, and the aqueous layer was extracted with two 20-mL portions of tetrahydrofuran/ethyl acetate (1:l) mixture. 6.38; N, 21.89. The combined organic extract was concentrated and chromatoBiochemicals. Sonicated calf thymus and pBR322 DNAs, poly(dA-dT),poly(dG-dC), and restriction enzymes Hind111 and graphed on Florisil to give 40 mg of recovered aldehyde and 90 mg of 4 (84% yield): mp 260 "C dec; IR (KBr) vnBx 3440, 2920, EcoRI were purchased from Pharmacia P-L Biochemicals. The 1610,1580, 1490,1430 cm-'; 'H NMR (DMSO-d,) 6 2.22 (s, 3 H, polynucleotides were used without further purification. The calf NCH3), 2.5 (m, 4 H, CH,), 3.2 (m, 4 H, CH,), 6.8 (d, 2 H, Ar-H, thymus (ct) and T4 coliphage DNAs were from Sigma. Ct DNA J = 9 Hz), 7.08 (dd, 1 H, Ar-H, J = 9, 2 Hz), 7.3 (d, 1 H, Ar-H, was deproteinated by the phenol extraction method and extenJ = 2 Hz), 7.62 (d, 1 H, Ar-H, J = 9 Hz), 7.85 (d, 1 H, Ar-H, J sively dialyzed against 20 mM NaCl solution, pH 7.1, before use. = 8 Hz), 8.0 (d, 2 H, Ar-H, J = 9 Hz), 8.13 (dd, 1 H, Ar-H, J = T4 polynucleotide kinase and AMV reverse transcriptase and urea 8, 2 Hz), 8.33 (d, 1 H, Ar-H, J = 2 Hz); MS ( m / z ,relative inwere from Bethesda Research Labs. Dithiothreitol was from tensity): calcd for C25HnN403426.1691; found 426.1687 (M', 1001, Calbiochem. Acrylamide, bromophenol blue, and xylene cyano1 411 (M - CH3, 5), 356 (M - CdH,N, 16), 355 (M - CdHgN, 32), were from Serva. Methidiumpropyl-EDTA (MPE) was a generous 71 (C4HgN,29), 70 (C4H8N,32). Anal. Calcd: C, 70.42; H, 5.16; gift from Professor Dervan. Ferrous ammonium sulfate was from N, 13.14. Found: C, 70.11; H, 4.87; N, 13.0. BDH. [y-32P]ATP,[ ( u - ~ ~ P I ~ A[a-3?]cordycepin TP, triphosphate, Hoechst Analogue 5. A mixture of 210 mg of the appropriate and terminal transferase were purchased from New England diamine and 240 mg of the aldehyde in 10 mL of nitrobenzene Nuclear. Hoechst 33258 was obtained from Aldrich. All other reagents were of analytical grade. was heated at 140 "C for 24 h. The nitrobenzene was removed under reduced pressure, and the resulting residue was chromaFootprinting. MPE.Fe(I1) Footprinting. HindIII-digested pBR322 DNA was either 5'-32P-labeled ( [ Y - ~ ~ P I A T and P T4 tographed on Florisil (EtOAc/MeOH) to give 300 mg of 5 as a light yellow solid (70% yield): mp 250 "C dec; IR (KBr) v- 3400, polynucleotide kinase) or 3'-32P-labeled ([( U - ~ ~ P ] ~and A T AMV P 3100,2930,2840,2800,1620,1590 cm-'; 'H NMR (DMSO-d6)6 reverse transcriptase or [(~-~~P]cordycepin triphosphate and terminal transferase) and then digested with EcoRI. The resulting 2.22 (s,3 H, NCH,), 2.44 (t, 4 H, CHJ, 3.54 (t, 4 H, CHZ), 6.8 (d, 1 H, Ar-H, J = 9 Hz), 7.0 (d, 2 H, Ar-H, J = 9 Hz), 7.8-7.9 (two 4332 bp and 31 bp DNA fragments (31,32)were not separated doublets merged, 2 H, Ar-H), 8.08 (d, 2 H, Ar-H, J = 9 Hz), 8.2 prior to the cleavage reactions. Reaction mixtures included so(dd, 1 H, Ar-H, J = 8 , l Hz), 8.4 (d, H, AT-H,J = 1Hz). MS (m/z, nicated calf thymus DNA, radiolabeled DNA, and ligand (omitted relative intensity): calcd for CUHz2N6O2426.1804; found 426.1800 in control reactions) in buffer (10 mM Tris, 20 mM NaCl, pH 7.4). (M', 14), 411 (M - CH3,6), 356 (34), 309 (37), 294 (16), 240 (15), After equilibration of the ligand-DNA mixtures for 20 min at room 239 (1001, 205 (14), 71 (27), 70 (27). Anal. Calcd: C, 67.60; H, temperature, MPE.Fe(II) [from a freshly prepared mixture of 5.16; N, 19.72. Found: C, 67.23; H, 5.45; N, 19.35. Fe(NH,)2(S0,)2 and MPE], followed by DTT, was added to each. The final solutions contained 100 pM DNA base pairs, 10 mM Hoechst Analogue 6. A mixture of 113 mg of the appropriate diamine and 130 mg of the aldehyde in 5 mL of nitrobenzene was Tris, 20 mM NaC1, 10 pM MPE.Fe(II), 2.5 mM DTT, and ligand heated a t 140 "C for 24 h. Nitrobenzene was removed under ( 8 , 16, or 78 pM). Reactions were run at room temperature (7 min) and were then stopped by freezing at -78 "C. The solutions reduced pressure, and the resulting residue was chromatographed were then lyophilized and resuspended in formamide loading on Florisil (EtOAc/MeOH) to give 180 mg of 6 as a light yellow buffer (33,34) for gel electrophoresis. After electrophoresis (0.4 solid (77% yield): mp 223-225 "C; IR (KBr) ,Y 3400,3100,2930, mm thick, 55 cm long, 6% polyacrylamide, 7 M urea, 2200 V, 55 2840,2800,1620,1590 cm-'; 'H NMR (DMSO-&,) 6 2.22 (s, 3 H, "C), the polyacrylamide sequencing gels were dried (Bio-Rad NCHJ, 2.43 (t, 4 H, CH,), 3.52 (t, 4 H, CH,), 6.8 (d, 1 H, Ar-H, Model 483 slab dryer) and autoradiographed a t -70 "C by using J = 9 Hz), 6.96 (d, 2 H, Ar-H, J = 9 Hz), 7.8-7.9 (two doublets Kodak X-Omat AR film. The resulting autoradiographs were merged, 2 H, Ar-H), 8.05 (d, 2 H, Ar-H, J = 9 Hz), 8.18 (dd, 1 scanned by using an LKB Ultroscan XL laser densitometer. H, Ar-H, J = 8, 1 Hz), 8.42 (d, 1 H,Ar-H, J = 1 Hz). MS ( m / z , was heated a t 140 "C for 24 h. The nitrobenzene was removed under reduced pressure, and the resulting residue was chromatographed on Florisil (EtOAc/MeOH) to give 155 mg of 2 as a yellow solid (88% yield): mp 270-272 "C; IR (KBr) ,Y, 3400, 3040,2920,2800,1610,1550,1500,1440 cm-'; 'H NMR (DMSO-d6) 6 2.24 (s,3 H, NCH3), 2.5 (bs, 4 H, CHZ), 3.14 (bs, 4 H, CH,), 6.9-7.0 (d, 4 H, Ar-H), 7.46 (d, 1 H, Ar-H, J = 8 Hz), 7.83 (d, 1 H, Ar-H, J = 8 Hz), 8.07 (d, 2 H, Ar-H, J = 9 Hz), 8.17 (d, 1 H, Ar-H, J = 8 Hz), 8.4 (s, 1 H, Ar-H). MS ( m / z ,relative intensity): calcd for CZ5Hz3N5O2 425.1851; found 425.1852 (M+, 87), 355 (M CdHBN, 27), 354 (M - CdHgN, 31), 308 (loo), 238 (34), 237 (43), 183 (41),71 (C4HgN,44), 70 (C4H8N,48). Anal. Calcd: C, 70.59; H, 5.41; N, 16.47. Found: C, 70.27; H, 5.74; N, 16.18. Hoechst Analogue 3. A mixture of 104 mg of the diamine and 120 mg of the aldehyde in 5 mL of nitrobenzene was heated at 140 "C for 24 h. The nitrobenzene was removed under reduced pressure, and the resulting residue was chromatographed on Florisil (EtOAc/MeOH) to give 170 mg of 3 as a crystalline solid (79% yield): mp 180 "C dec; IR (KBr) Y- 3420,2880,1610,1500 cm-'; 'H NMR (DMSO-d,) b 2.2 (s, 3 H, NCH3), 2.46 (m, 4 H, CH,), 3.06 (m, 4 H, CHJ, 6.8-7.0 (m, 4 H, Ar-H), 7.43 (d, 1 H, Ar-H, J = 9 Hz), 7.83 (d, 1 H, Ar-H, J = 9 Hz),8.04 (d, 2 H, Ar-H,
Chem. Res. Toxicol., Vol. 3, No. 3, 1990 273
Ligand-DNA Molecular Recognition 1
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-78
Figure 1. A portion of a footprinting autoradiogram from partial MPEoFe(I1) digestion of a 32P-end-labeled4332 base pair DNA fragment. Lanes 1-9 are 5’-labeled; lanes 10-18 are 3’-labeled. Lanes 1 and 10 contain intact DNA; lanes 9 and 18 contain the products of the Maxam-Gilbert “G” reaction (34);lanes 2 and 11resulted from cleavage in the absence of ligand; lanes 3,5,7, 12,14, and 16 contain added ligands a t 0.78/base pair; and lanes 4,6,8,13, 15, and 17 contain 0.08/base pair added ligands. The added ligands are 5 (lanes 3,4,12, and 13), 6 (lanes 5,6,14, and E ) , and Hoechst 33258 (1) (lanes 7, 8, 16, and 17). Footprint locations at rt 0.78 (bases 78-163) are given in Figure 4. The densitometric data were corrected for the background absorbance of the film (
