Chem. Res. Toxicol. 1990, 3, 125-132
125
Solvolysis of the Bay-Region Diol Epoxides of ir-Methylbenz[ c ]acridine in Aqueous Solution and Effect of DNA Joan V. Fraser, Colin C. Duke, Gerald M. Holder,* and Douglas E. Moore* Department of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia Received July 10, 1989 T h e hydrolysis of the bay-region syn- and anti-diol epoxides of 7-methylbenz[c]acridine (7MBAC) has been studied in aqueous solution. T h e rates of reaction have been measured a t low ionic strength in the absence and presence of DNA, as a function of p H and temperature, by spectrophotometry and high-pressure liquid chromatography. T h e major products are the corresponding tetrols, but the syn diastereomer also undergoes rearrangement to a keto diol. T h e hydrolysis reaction is catalyzed by both acid and DNA and conforms t o a mechanism in which a physical association complex is formed between diol epoxide and DNA, followed by two parallel pathways in which the diol epoxide either is hydrolyzed or becomes covalently bound to the DNA. The rate constants are significantly lower than the corresponding ones for the diol epoxides of benzo[a]pyrene. However, the extent of covalent binding of 7MBAC diol epoxides to DNA was found to be 12-30%, depending on the p H of reaction, values that are 2-3 times greater than for the benzo[a]pyrene diol epoxides reacted under the same conditions of low ionic strength. The very small changes (2 nm) in spectroscopic characteristics observed for the covalently bound adduct as compared t o the free tetrol suggest that the binding occurs with little intercalation of the compound between the base pairs of DNA.
Introduction Most known chemical carcinogens exert their adverse biological effects after metabolic activation. This occurs with nitrosamines, 2-(acetylamino)fluorene, 2-naphthylamine, and the polycyclic aromatic hydrocarbons (PAH). For many of these compounds, several metabolites are produced, and one or more of these, or their secondary metabolites, behave as alkylating agents and are able to react with cellular nucleophiles. Such covalent binding to DNA is thought to be a critical step in the initiation of cancer ( I ) . Of the many carcinogenic and mutagenic PAH found in environmental pollutants, benzo[a]pyrene (BP) is one of the most widely studied (1, 2). In vivo metabolic activation of BP occurs via the intermediate trans-BP-7,& dihydrodiol (a proximate carcinogen) to anti-BP-7,8diol-9,lO-oxide (an ultimate carcinogen). This ultimate carcinogen (referred to as anti-BPDE, a diol epoxide'), binds to DNA to form a number of covalent adducts. The major one possesses a chemical bond between the 10carbon in the "bay-region" position of the hydrocarbon and the exocyclic amino group of guanine. 5-Methylchrysene (5MeC), another PAH found in cigarette smoke, has also been found to be a potent initiator of skin tumors. It is more tumorigenic than all of the isomeric methylchrysenes, which show very low activities (3, 4). When the major reactive diol epoxide metabolites of BP (5), 5MeC (5), 3-methylcholanthrene (3MC) ( 6 ) ,and 6fluorobenzo[a]pyrene (7) are incubated in aqueous solutions at 25 "C, they undergo solvolysis by acid-catalyzed and spontaneous hydrolytic pathways to yield tetrols; these reactions represent detoxification pathways. In the presence of DNA the overall rate of breakdown of the diol epoxide is accelerated, and covalent binding to DNA occurs as well as hydrolysis to tetrols. The alkylation of DNA is thought to be a reflection of the activation pathways leading to carcinogenesis.
* Correspondence should be addressed to these authors.
Linear dichroism and spectroscopic studies indicate two types of environment for covalent adducts of anti-BPDE with DNA. One BPDE residue is characterized by a 10-nm red shift in the UV absorption spectrum, a negative linear dichroism spectrum which is solvent inaccessible, suggesting an intercalated environment (site I). The other is characterized by a 2-3-nm red shift, a positive linear dichroism spectrum, and solvent accessibility (site 11) (8). In contrast to the large amount of work on diol epoxides of PAH, there has been relatively little dealing with polycyclic azaaromatic compounds. The nitrogenous compounds 7-methylbenz[c]acridine (7MBAC) (1; see Chart I), dibenz [c,h]acridine, dibenz [a,h]acridine, and dibenz[ ajlacridine are carcinogenic azaaromatic hydrocarbons, structurally analogous to PAH, being found as basic components of the particulate fraction of cigarette smoke and as air pollutants. They have different potencies as carcinogens, 7MBAC being most active while dibenz[a,j]acridine is least active (9). It has been established that these nitrogenous hydrocarbons are activated to dihydrodiols which may possibly be further metabolized to vicinal diol epoxides, and these vicinal diol epoxides are most active in mutagenicity tests (10-12). This report compares the hydrolytic reactivities of the isomeric syn- and anti-diol epoxides of 7MBAC (2 and 3, respectively1) and their properties as DNA alkylating agents. The rates of reaction of the two diastereoisomers have been examined in the absence and presence of DNA, and as a function of temperature and pH by means of spectrophotometry and HPLC. Experimental Section General Precautions. Polycyclic azaaromatic hydrocarbons and their derivatives,particularly diol epoxides, should be regarded as probable carcinogens. Precautions should be taken to prevent accidental contact with the skin, inhalation of dust, or ingestion. Syn and anti isomers, also known as diol epoxides 1 and 2, respectively, possess the benzylic hydroxyl group cis and trans to the epoxide oxygen.
0893-228~/90/2703-0125$02.50/0 0 1990 American Chemical Society
Fraser et al.
126 Chem. Res. Toxicol., Vol. 3, No. 2, 1990
R O &OR
CH3
4 5
R
= H
CH3
R = R c
6
R = H
7
R = R c
u
m/z (relative intensity) 294 (M + 1,43), 276 (100); UV spectrum [methanol;, , A nm (e)] 362 (9700), 260 (125000). (*)-3a,4~-Dihydroxy-l~u,2a-epoxy-7-methyl-1,2,3,4-tetrahydrobenz[c]acridine (anti-7MBACDE) (3). trans-3,4-Dihydro-3,4-dihydroxy-7-methylbenz[c]acridine(64.6 mg) was dissolved in freshly distilled anhydrous T H F (25 mL) under N2, and m-chloroperbenzoic acid (110 mg) was added with stirring to give a homogeneous solution. The reaction appeared complete by TLC (silica, ethyl acetate) after 1.5 h. The reaction mixture was poured into ice-cold 2% aqueous NaOH (40 mL), and the product was isolated by using THF/ether (4:l) as solvent and saturated aqueous NaCl for extraction. A yellow solid (3) was obtained (52.9 mg, 77%): mp 182-183 "C dec; 'H NMR [400 MHz, 2 mg/0.3 mL, (CD3)2SO/D20(9:1)] 6 3.13 (s, 7-CH3), 3.83 (bd, H2),3.89 (bd, H3), 4.45 (bd, H4),5.60 (d, Hl), 7.65 (m, Hs), 7.86 (m, Hlo),7.89 (d, Hb), 8.16 (bd, H&, 8.40 (bd, HI'), 8.41 (d, He); J1,2 = 4.5 Hz, J2,3 = -0.5 Hz, 53,4 = 8.8 Hz, J5,6 = 9.3 Hz, J8,s = 8.6 Hz, J,,,,, = 8.6 Hz; CIMS, m / z (relative intensity) 294 (M 1, NO), 276 (70); UV spectrum [methanol;,A, nm (41 361 (9940), 258 (130000). Characterization of the Hydrolysis Products of syn 7MBACDE ( 2 ) . A solution of syn-7MBACDE (2;14.4 mg) in a mixture of T H F ( 5 mL) and 5 mM cacodylate buffer (15 mL, pH 6) was kept a t room temperature for 3 days protected from light. The products isolated with ethyl acetate were fractionated on silica gel H to give, in order of elution, fraction 1 (2.7 mg), fraction 2 (2.0 mg), and fraction 3 (1.7 mg). (~)-2a-Bromo-7-methyl-1,2,3,4-tetrahydro-l8,3a,4~-triFraction 1 was identified as trans -3,4-dihydroxy-7hydroxybenz[ clacridine. T o a stirred solution of trans-3,4methyl-2-0~0-1,2,3,4-tetrahydrobenz[ clacridine (8): 'H NMR dihydro-3,4-dihydroxy-7-methylbenz[c]acridine (86 mg) in freshly [90 MHz, 2.6 mg/0.2 mL, (CD3)2SO]6 3.11 (s, 7-CH3),4.24 (d, distilled tetrahydrofuran (25.5 mL) and water (6.4 mL) a t 0 "C Hl), 4.35 (d, HI,),4.36 (dd, H3), 4.82 (dd, H,), 5.62 (d, OH3), 6.17 under N2 were added N-bromoacetamide (50.3 mg) and 2 M (d, OH,), 7.50-8.60 (m, H5-H11), 53.4 = 8.4 Hz, Jl,l,= 22.5 Hz, J o H , ~ aqueous HCl (2 drops). TLC showed complete reaction after 10 = 4.8 Hz, JOH,, = 5.9 Hz; CIMS NH3, m/z (relative intensity) 294 min. The solid residue obtained on workup was triturated with (M 1,5),276 (loo), CIMS CH,, 276 (100);UV spectrum [MeOH; ethyl acetate to give a yellow crystalline solid (103 mg, 89%): mp , ,A nm (e)] 360 (9400), 308 (16600), 258 (124000). Acetylation 215-225 "C dec; 'H NMR [90 MHz, 3 mg/0.2 mL, (CD3),S0/D2O with acetic anhydride and pyridine at 70 "C gave trans-3,4-di(9:1)] 6 3.11 (s, 7-CH3), 4.22 (dd, H3), 4.64 (d, H4), 4.67 (dd, HZ), hydro-7-methyl-2,3,4-triacetoxybenz[ clacridine (9); see Chart 6.06 (d, HJ, 7.52-7.96 (m, 2 H), 7.72 (d, H5), 8.16 (m, Ha),8.39 11): 'H NMR (400 MHz, 2.5 mg/0.2 mL, CDC1,) 6 2.05 (s, (d, H6), 8.39 (m, HIJ; Jl,z= 4.6 Hz, J2,3 = 2.5 Hz, J3,, = 7.1 Hz; CHSCOO), 2.12 (s, CH,COO), 2.28 (s, CH,COO), 3.12 (s, 7-CH3), 'H NMR [90 MHz, 3 mg/0.2 mL, (CD3),S0, partial spectrum] 5.83 (d, H3), 6.22 (d, Hi), 7.56 (ddd, H3), 7.58 (d, H5), 7.77 (ddd, 6 5.66 (d, OH,), 5.94 (d, OH,), 6.26 (d, OH,); J ~ , o = H 4.4 Hz, J 3 , o ~ = 4.6 Hz, J4,0H = 6.6 Hz; CIMS, m / z (relative intensity) 376 (M HIo), 8.20 (d, Hs), 8.23 (bd, H11), 8.23 (s, HI), 8.26 (bd, Ha); 5 3 , 4 = 2.9 Hz, 55.6 = 9.0 Hz, J8,10 = 1.0 Hz, 58,s = J9,10 = J10,ll = 7.7 + 1, 74), 374 (M + 1,79Br,80), 358 (98), 356 (loo), 294 (95), Hz; CIMS, m / z (relative intensity) 420 (M + 1, 201, 360 (loo), 278 (30), 276 (70). (*)-3a,4@-Dihydroxy-1@,2@-epoxy-7-methy1-1,2,3,4-tetra- 318 (80). A byproduct of the acetylation was identified as 2,3diacetoxy-7-methylbenz[c]acridine:'H NMR (400 MHz, 1mg/0.2 hydrobenz[ clacridine (syn -7MBACDE) ( 2 ) . To a stirred mixture of (*)-2n-bromo-7-methyl-1,2,3,4-tetrahydro-l~,3~,4@- mL, CDCl,) 6 2.39 (s,CH3COO),2.42 (s, CH&OO), 3.12 (s,7-CH3), 7.64 (m, Hg),7.68 (d, H5), 7.73 (s, H,), 7.82 (m, HI,), 8.08 (d, H6), trihydroxybenz[c]acridine(103 mg) and anhydrous T H F (40 mL) 8.29 (d, Ha), 8.33 (d, HI'), 9.34 (5, HI!; J s , = ~ 9.4 Hz, J B ,=~8.7 under N, was added potassium tert-butoxide (47.6 mg). TLC after Hz, Jlo,ll = 8.9 Hz; CIMS, m / z (relative intensity) 360 (M + 1, 25 min showed complete reaction. After working-up using ethyl 67), 318 (100). acetate, a yellow crystalline solid was obtained by precipitation Fraction 2 was identified as (*)-7-methyl-1,2,3,4-tetrafrom a T H F solution with hexane, (53.4 mg, 66%): mp 143-144 hydro-1@,2a,3&4a-tetrahydroxybenz[c]acridine (1 1): 'H "C dec; 'H NMR [400 MHz, 2 mg/0.3 mL, (CD3),SO/D20 (9:1)] NMR [90 MHz, 2.0 mg/0.2 mL, (CD3)2SO/D20(9:1)] 6 3.13 (s, 6 3.14 (s, 7-CH,), 3.87 (m, H2),4.13 (dd, H3), 4.67 (dd, H,), 5.33 7-CH3),3.48 (m, H3),3.74 (m, H2),4.61 (d, H4),5.35 (d,HJ, 7.7-8.5 (d, HJ, 7.65 (m, Hg), 7.66 (d, H5), 7.87 (m, HI,), 8.18 (bd, Ha), = 8.3 H, J3,, = 10.3 Hz; CIMS, (m, H5-Hll); J,,, = 7.7 Hz, 52,3 8.41 (bd, Hi'), 8.41 (d, H6); 51,2 = 4.1 Hz, 52.3 = 2.0 Hz, 5 3 , 4 = 4.3 m / z (relative intensity) 312 (M + 1, loo), 294 (40); UV spectrum Hz, J2,4 = 0.9 Hz, J5,6 = 9.0 Hz, 58.9 = 8.7 Hz, 510,11 = 8.8 Hz; CIMS,
Nonaqueous wastes are disposed of with organic solvent wastes. Instrumentation. Proton NMR spectra were recorded on a JEOL FX90Q or a Bruker WM-400 spectrometer. Chemical ionization mass spectra were obtained on a Finnigan TSQ-46 mass spectrometer with methane or ammonia as reagent gas. UVvisible absorption spectra and kinetic measurements were made on a Perkin-Elmer Lambda 5 spectrophotometer, with kinetic accessory and thermostated multicell sample holder. HPLC was performed with an Altex Model 330 isocratic system RP18 column with 254-nm UV detector using a Brownlee 5 - ~ m (4.6 X 250 mm). For some experiments a Hewlett-Packard diode array spectrophotometric detector was employed. The mobile phase in all cases was methanol/water (60:40) at a flow rate of 1 mL min-'. Short-column vacuum chromatography was performed on silica gel H. Materials. Calf thymus DNA (Sigma Chemical Co., St. Louis, MO) was dissolved in 100 mM cacodylate buffer, dialyzed against 5 mM cacodylate buffer, pH 6, for 48 h. The DNA solution was then centrifuged for 15 min a t 9OOOg. The concentration of the stock DNA was determined from its absorption at 260 nm (c = 6300 M-' cm-', expressed in terms of mononucleotide units) (13), and the appropriate amount was added to buffer solution before the addition of the diol epoxide. trans-3,4-Dihydro-3,4-dihydroxy-7-methylbenz[ clacridine was prepared from 7MBAC by the method previously described (14).
+
+
Solvolysis of 7-Methylbenz[c]acridine
Diol Epoxides
Chart I1 ORC
ORC
I
I
CH3
g
bH3
11
R = H
12
R = Ac
CH3
10
bH3
13 14
R = H R = Ac
[pH 7 phosphate buffer, A,, nm (e)] 359 (9730), 257 (140000). Acetylation gave (*)-7-methyl-1@,2a,3@,4a-tetraacetoxy1,2,3,4-tetrahydrobenz[c]acridine(12): 'H NMR (400 MHz, 1 mg/0.2 mL, CDC13)8 2.00 (s, CH3COO),2.10 (9, CH3COO),2.11 (s, CH&OO), 2.23 (s, CH,COO), 3.12 (s,7-CH3), 5.51 (dd, H3), 5.70 (dd, HZ), 6.50 (d, H4), 7.30 (d, Hi), 7.33 (d, H5), 7.59 (ddd, Hs), 7.77 (ddd, HI&, 8.11 (bd, HE),8.24 (bd, Hi,), 8.31 (d, H6); 52.3 = 5.3 Hz, J 3 , 4 = 7.5 Hz, J5,6 = 9.3 HZ, JB,S= J 9 , l o = J10,ll = 8.7 Hz, JE,lo = Js,ll= 1.0 Hz; CIMS, m/z (relative intensity) 480 (M + 1, 60), 420 (65), 360 (87), 318 (100). Fraction 3 was assigned the structure (*)-7-methyl-1,2,3,4tetrahydro-la,2a,3@,4a-tetrahydroxybenz[c ]acridine (13): 'H NMR [90 MHz, 1.7 mg/0.2 mL, (CD3)2SO/D20(9:1)] 8 3.11 (s, 7-CH3), 3.60 (dd, Hz), 3.99 (dd, H3), 4.47 (d, Hd), 5.91 (d, Hi), 7.5-8.7 (m, H5-HI1); J1,2= 3.6 Hz, J3,4 = 7.2 Hz, J2,3 = 10.3Hz; CIMS, m/z (relative intensity) 312 (M + 1, loo), 294 (60); UV spectrum [pH 7 phosphate buffer; A, nm (e)] 360 (9700), 257 (130000). Acetylation gave (*)-7-methyl-la,2a,3j3,4a-tetraacetoxy-l,2,3,4-tetrahydrobenz[c]acridine(14): 'H NMR (400 MHz, 1.5 mg/0.2 mL, CDCl,) 8 2.03 (s, CH3COO), 2.12 (s, CH,COO), 2.13 (s, CH&OO), 2.23 (s,CH,COO), 3.11 (s, 7-CH3), 5.51 (dd, H2), 5.93 (dd, H3), 6.47 (d, H4), 7.28 (d, HE), 7.59 (ddd, Hs), 7.71 (d, Hi), 7.76 (ddd, Hie), 8.16 (d, Ha), 8.23 (d, H11), 8.31 (d, He); J 1 , 2 = 3.8 Hz, J2,3 = 11.4 Hz, J3,4 = 7.8 Hz, J5,6 = 9.4 Hz, Ja,s = 8.7 Hz, J,o,ll = 8.2 Hz; CIMS, m/z (relative intensity) 480 (M + 1, 95), 420 (85), 360 (80), 318 (100). Characterization of the Hydrolysis Products of anti7MBACDE (3). anti-7MBACDE (3; 17.8 mg) was dissolved in warm T H F (5 mL) and water (1.25 mL), and 0.2 M aqueous HC1 was (100 pL) added. After 8 h at ambient temperature, solvent was removed and the products were chromatographed to give a major polar product (13.8 mg, 73%) and a minor less polar product (1.5 mg, 8%). The major product was recrystallized from methanol/H20 to give pale yellow crystals (5.8 mg, mp 142-145 "C) identified as (&)-7-methyl-1,2,3,4-tetrahydro-la,2@,3@,4a-tetrahydroxybenz[c]acridine (4): 'H NMR [90 MHz, 1.6 mg/0.25 mL, (CD3)2SO/D20 (9:1)] 8 3.13 (s, 7-CH3), 4.01 (dd, H3), 4.14 (dd, H2),4.69 (d, H4),5.65 (d, HJ, 7.51-7.98 (m, Hg and Hlo),7.73 (d, H5), 8.16 (m, Ha), 8.34 (d, Hs), 8.41 (m, HIJ; JI,Z = 4.4 Hz, J2,3 = 2.4 Hz, J3,4 = 7.2 Hz,J 5 , $ = 9.2 Hz; CIMS, m/z (relative intensity) 312 (M + 1, 100), 294 (40); UV spectrum [methanol; A, nm (e)] 357 (9600), 257 (144000). Acetylation gave (*)-71,2,3,4-tetrahydrobenz[c ] methyl- la,2&3@,4a-tetraacetoxyacridine (5): lH NMR (90 MHz, 3 mg/0.25 mL, CDCl,) 8 2.06 (s, 2 X CH,COO), 2.09 (s,CH&OO), 2.25 (s,CH&OO), 3.12 (s, 7-CH3), 5.69 (dd, H3), 5.80 (dd, HZ), 6.55 (d, H4), 7.21 (d, Hi), 7.35 (d, H5), 7.48-7.98 (m, Hs and Hi&, 8.13 (m, Ha), 8.24 (m, Hill, 8.32 (d, He); Ji,2 = 4.4 Hz, J2,3 = 2.4 Hz, J3,4 = 7.2 Hz, 55.6 = 9.2 Hz. The minor product was further purified by preparative TLC [Merck silica gel PF254, solvent CHC13/ethanol (4:1)]to give a yellow solid identified as (*)-7-methy1-1,2,3,4-tetrahydro-
Chem. Res. Toxicol., Vol. 3, No. 2, 1990 127
l@,2j3,3j3,4a-tetrahydroxybenz[c]acridine (6): 'H NMR I90 MHz, 1.6 mg/0.25 mL, (CD3)2SO/D20(lO:l)] 8 3.15 (s, 7-CH3), 3.75 (dd, H3), 4.23 (dd, HZ), 4.85 (d, H4), 5.60 (d, Hi), 7.57-7.80 (m, Hs and Hi,), 7.75 (d, H5), 8.16 (m, Ha),8.37 (d, Hs), 8.44 (m, H11); Ji2 = 3.7 Hz, J2,3 = 2.2 Hz, J3,4 = 6.8 Hz, J5,6= 9.0 Hz; CIMS, m/z (relative intensity) 312 (M + 1, loo), 294 (45). Acetylation as described gave (&)-7-methyl-lj3,2@,3j3,4a-tetraacetoxy1,2,3,4-tetrahydrobenz[c]acridine(7) (1.4 mg, 57%): 'H NMR (90 MHz, 1.3 mg/0.25 mL, CDC13) 6 2.02 (9, CH3COO), 2.08 (s, CH,COO), 2.14 (s, CH,COO), 2.16 (8,CH,COO), 3.12 ( s , ~ - C H ~ ) , 5.55 (ddd, H3), 5.88 (dd, HZ), 6.41 (d, H4), 7.45 (d, H5), 7.46 (dd, Hi), 7.50-7.80 (m, Hg and Hi,), 8.12 (m, HE),8.23 (m, Hi,), 8.32 (d, Hs); Ji,z = 4.9 Hz, J1,3 = 0.5 Hz, J2,3 = 2.5 Hz, J 3 , 4 = 4.1 Hz, J5,6 7 8.8 Hz. Kinetic Studies of t h e Hydrolysis of 7MBACDE. Stock solutions of syn- and anti-7MBACDE in freshly distilled tetrahydrofuran were prepared at approximately 2 mM concentration. For the kinetic studies, a 200-pL volume of the stock solution was added to 10 mL of aqueous sodium cacodylate (5 mM) buffer solution a t the desired pH and temperature and containing the required amount of DNA. The hydrolysis reaction was monitored spectrophotometricallyon a shoulder absorption at 400 nm where the change in absorption was substantially greater than at the maximum a t 360 nm. The reaction was allowed to proceed to completion, and the rate constant for the process was determined by nonlinear regression analysis of the absorbance-time data. A verification of the hydrolysis rates determined by spectrophotometry was obtained independently by reverse-phase HPLC assay of 20-pL aliquots of the reaction mixture taken a t various time intervals. The disappearance of the diol epoxide peak over time was used to determine the rate of reaction. This approach was satisfactory for reactions that were slow in comparison to the HPLC elution time. In order to validate those reactions in the presence of DNA with tljz less than 5 min, the mercaptoethanol quench procedure was used (15). Aliquots (1mL) were removed from the reaction mixture a t fixed time intervals and quenched with 0.1 mL of 2 M aqueous 2-mercaptoethanol (in 0.4 M NaOH). Each sample was then extracted with three (1-mL) volumes of ethyl acetate. The organic solvent was evaporated, the residue was dissolved in methanol (0.3 mL), and 20 pL of this solution was analyzed by reverse-phase HPLC. The reaction rate was determined from the peak due to the mercaptoethanol-diol epoxide adduct. Covalent Binding of 7MBACDE to DNA. An aliquot of the stock 7MBACDE solution (200 pL) was added to 10 mL of 5 mM sodium cacodylate buffer solution containing DNA at 20 times the diol epoxide concentration a t the desired pH. The reaction mixture was incubated for 48 h at 25 "C to allow the reaction to proceed to completion. The reaction mixture was extracted five times with an equal volume of ethyl acetate (saturated with buffer) to remove unbound reaction products. The DNA was then precipitated with 2% potassium acetate in ethanol (20 mL) and washed with cold absolute ethanol (10 mL). The washed pellet was dried under a stream of nitrogen and then redissolved in buffer for spectrophotometric assay at 360 nm. Although the molar absorptivity of the acridine residue covalently bound to DNA is not known, it was clear that relatively little change occurred a t the absorption maximum of 360 nm. The extent of covalent binding was therefore determined by using c = 9600 M-' cm-' as obtained for the major tetrol products, after subtracting the contribution made by the DNA to the total absorption at 360 nm.
Results Characterization of syn - and anti-7MBACDE. A preliminary report on the synthesis of syn- a n d anti7MBACDE (2 and 3, respectively) has appeared (10). The methods for the syntheses of 2 a n d 3 and t h e yields achieved are similar t o those reported for "bay-region'' synand anti-diol epoxide derivatives of benz[a]anthracene (16) a n d benz[c]acridine ( 17). Acid-catalyzed reaction of trans-7MBAC-3,4-dihydrodiol with N-bromoacetamide followed b y potassium tert-butoxide t r e a t m e n t of t h e bromoacetate gave t h e syn isomer (2),while the anti isomer (3) was produced directly b y oxidation with m-chloro-
128 Chem. Res. Toxicol., Vol. 3, No. 2, 1990 perbenzoic acid. The stereochemistry of the products was confirmed by the value of J3,,of 4.3 Hz and a w coupling of 0.9 Hz between H2 and H, in 2 in which the hydroxyl groups occupy quasiaxial positions, and J3,4of 8.8 Hz (hydroxyl groups quasiequatorial) and the absence of a w coupling in 3. These assignments are consistent with similar results for the diol epoxides of benzo[a]pyrene (281, benz[a]anthracene (16), and benz[c]acridine (17). The hydrolysis of anti-diol epoxide 3 afforded the expected products of trans and cis attack of water at C1 on the epoxide function to give the major la,2&3P,4a-tetrol (4) and the minor 1/3,2&3/3,4a-tetrol (6), respectively. The coupling constants of the methine protons on C1 to C4 in the 'H NMR spectra did not allow structural assignments, but in the tetraacetates the absence of a weak J1,3 in 5 was taken with its clearly quasiaxial H3 and H, to indicate the la,2/3,3&4a configuration in contrast to the tetraacetate 7,which displayed a small J3,4 (4.1 Hz) and a J 1 , 3 of 0.5 Hz. The 1@,2@,3@,4a structure for the latter and its precursor were therefore assigned, the l-acetoxy group adopting a quasiaxial conformation, thereby reducing both steric and electronic repulsion between the ring N atom and the l-acetate. Hydrolysis of the syn-7MBACDE (2) at pH 6 gave a keto diol (8, 50%) with two doublet methylene signals in the 'H NMR at 4.24 and 4.35 ppm (J= 22.5 Hz, slowly exchangeable with D20). Therefore, a 2-keto function rather than a l-keto function is present, analogous to results reported for benzo[a]pyrene, phenanthrene (221, and benzo[c]phenanthrene (23). The formation of the triacetate with a singlet for H1 at 8.23 ppm was accompanied by some 2,3-diacetoxy-7-methylbenz[c]acridine (9). The ratio of hydrolysis products formed under acidic conditions was not investigated. The less polar tetrol was assigned the 1&2a,3/3,4a stereochemistry (11) while the more polar tetrol(l3) possessed the la,2a,3P,4a configuration. These structures were again assigned on the basis of the coupling constants of the methine protons in the spectra of the tetrols and their tetraacetates (12, 14). The coupling constants and calculated van der Waals repulsion energies, molecular mechanics energies, and dihedral angles (29) suggest that the conformation of the tetraacetate of 11 is a half-boat rather than the usual half-chair form, indicating that, as in 14, the l-acetoxy group again prefers the quasiaxial conformation. Kinetic Studies. (A) pH Dependence of the Hydrolysis Rates. The hydrolysis reactions for syn- and anti-7MBACDE followed pseudo-first-order kinetics, under all conditions used. The overall rate constant, kob, was obtained by spectrophotometry at 400 nm. Representative values were also determined by HPLC assay, either by direct analysis or using mercaptoethanol quenching for reactions with a half-life shorter than 5 min, and showed excellent agreement with values obtained by spectrophotometry. The pH dependence of hob in the absence and presence of DNA is shown in Figure 1for syn- and anti-7MBACDE. At pH values below 5.5 the hydrolysis reaction was too fast to be followed by mercaptoethanol quenching and HPLC analysis. A complicating factor was the protonation of the acridine N (pK, 4.1), resulting in a strong bathochromic shift in the ultraviolet absorption spectrum. Under such conditions, it was not possible to detect a significant difference in the spectrum when the diol epoxide was undergoing hydrolysis. Therefore, the study was confined to the physiological pH region 5.5-9. In buffer solution, hobs may contain contributions from specific- and general-acid catalysis. The fact that the rate
Fraser et al. -1 (0)
syn-7MBACDE
i
-2 0
I
n
0
Y
m
0 -
-3.-
\ \ *0 - 4 --
5
-
Buffer
6
7
8
9
lo
PH (b)
anti-7MBACDE
-2 0
n 0
-4
-5 5
6
7
8
9
10
PH Figure 1. pH dependence of the observed rate constant for hydrolysis of (a) syn-7MBACDE and (b) anti-7MBACDE at 25 "C in 0.005 M sodium cacodylate buffer in the absence (0) and presence ( 0 )of 0.8 mM DNA. The curves are calculated from eq 2 by using the constants shown in Table I.
is independent of pH above 8 indicates the absence of any base catalysis. Thus the overall rate constant can be represented by (1) + kH[H30+l + kHA[HAl where kH and kHA are the rate constants for specific- and general- (buffer) acid catalysis, respectively; the term ko represents the acid-independent (water-induced) rate constant. kHA was determined by measuring kobs, as a function of cacodylic acid concentration in the range 0-100 mM, maintaining a constant pH of 6. This pH was chosen so that the cacodylic acid was predominantly in the acid form [pK, = 6.1 (19)] and its effect more reliably determined than at more alkaline pH values. The values of 12, and kH were then computed by using kobs a t several pH values. The limited buffer capacity of cacodylic acid above pH 7 was considered, and the variation of rate constant was checked with phosphate buffer. A similar pH dependence was observed, but with slightly greater kob values because of the different general-acid-catalytic effect of phosphate. (B) Effect of DNA. The dependence of the rate constant on the concentration of DNA for both isomers of 7MBACDE is shown in Figure 2 for pH 6. The rate constant hobs increased with increasing DNA concentration and reached a limiting value of 0.0035 s-l at a DNA concentration of 8 X M (pH 6) for syn-7MBACDE and 0.020 s-l for anti-7MBACDE under the same conditions. This concentration of DNA represents a molar ratio of more than 20 DNA bases per initial diol epoxide molecule. The effect of DNA was also pH-dependent as shown in Figure 1. A t pH 6 the rate observed in the presence of 8 X M DNA for both the diol epoxides was increased by about 50-fold compared to buffer solution. As the pH was increased, this difference gradually decreased so that at pH above 8 the catalytic effect of DNA disappeared. The observed rate of hydrolysis was greater for the anti isomer in all systems investigated. kobs
=
k0
Chem. Res. Toxicol., Vol. 3, No. 2, 1990 129
Solvolysis of 7-Methylbent[c]acridine Diol Epoxides
07
/
24
0
I
0
4
9
12
I6
20
24
28
SL
Con0 Ratio [DNA]I[DE]
Table I. Kinetic Constants for Solvolysis of Diol Epoxides of BP and 7MBAC at 25 "C in Aqueous Solutions Containing DNA (Ionic Strenath 0.005) antisyn-7MBACDE anti-7MBACDE BPDEa (1.6 0.2) x 10-4 (0.5 f 0.2) X lo4 4.4 x 10-4 k,, s-' 100f8 kH, M-' s-* 184 f 9 2100 (4.1 f 0.4) X (9.5 f 1.0) x 10-8 0.71 kHA, M-' S-l 64.1 f 4.4 98.6 f 6.3 59.6 E,(uncat), kJ mol-' (1.4 f 0.6) X (0.8 f 0.5) X lo-' 4.4 x 10-4 k4, s-' (5.3 f 1.6) x 103 (2.6 f 0.9)x 104 3.6 X lo6 kH', M-' S-' (1.5 f 0.2) x 103 (1.6 f 0.3) X 10' 1.2 x 104 KD, M-' 34.5 f 2.7 36.5 73.7 f 7.8 &(cat), kJ mol-' -145 -165 f 35 AS*(cat), JK-' -186 f 45 From ref 19 p = 0.005, sodium cacodylate buffer.
ryn-DE
-1
-4
0 0
5
10
15
20
25
30
35
40
-5
Con0 Ratlo [DNA]I[DE]
Figure 2. Effect of DNA on the observed rate constant for hydrolysis of (a) syn-7MBACDE and (b) anti-7MBACDE at 25 "C in 0.005 M sodium cacodylate buffer (pH 6.0). The initial concentration of diol epoxide was 4.0 X lod M in each experiment. The curves are calculated from eq 2 and the kinetic parameters in Table I.
At a given pH, the catalytic action of DNA observed here follows the saturation kinetics pattern also seen in the hydrolysis of BPDE catalyzed by DNA (19, 20). The catalyzed reaction is postulated to occur following the formation of a complex (with association constant KD) between DNA and the diol epoxide. In principle, two kinetically distinct pathways are possible for the complex; one (kH') is pH-dependent and therefore involves a proton-donating species whose concentration is determined by pH, while the other (kor) is pH-independent and analogous to ko or the "spontaneous" reaction of diol epoxide in aqueous solution. An analysis based on this mechanism yields the overall rate expression: hobs
= (ko+ k ~ [ H 3 0 ++ ] (kd + ~H'[H@+] )KD[DNA]I/ (1 + KD[DNAI) (2)
The rate data of Figures 1 and 2 were analyzed by eq 2 and values computed for each of the individual constants, as summarized in Table I. (C) Temperature Dependence of kobs.For syn- and anti-7MBACDE the temperature dependence of the hydrolysis rate constant in the presence of DNA and in buffer solution is shown in Figure 3. From the slope of each plot, the overall activation energy was calculated and the entropy of activation determined according to the Eyring equation (21).The values are included in Table I. For the anti isomer, which is about 93% physically bound and for which kHris the predominant catalytic pathway, E, and A S are effectively for the acid-catalyzed reaction of the bound diol epoxide. (D) Covalent Binding of syn - a n d anti-7MBACDE to DNA. The fraction of the 7MBACDE molecules that
3
3.1
3.2
3.3
3.4
3.5
3.6
100OlT
(b)
-1O
i
antl-DE
a
-4
-5
-. .I
3
t
3.1
3.2
3.3
3.4
3.5
3.6
lOOOIT
Figure 3. Temperature dependence of the observed rate constant for hydrolysis of (a) syn-7MBACDE and (b) anti-7MBACDE in 0.005 M sodium cacodylate buffer (pH 6.0) in the absence (0) and presence ( 0 )of 0.8 mM DNA. bound covalently to DNA (fmJ in the course of the catalyzed reaction was determined as a function of pH (Figure 4). As the pH was increased from 6 to 8, the fraction of syn-7MBACDE covalently bound to DNA decreased from 0.26 to 0.12, and for anti-7MBACDE it decreased from 0.30 to 0.12. The fraction bound remained constant a t pH above 8 for both compounds. The extent of binding measured is equivalent to one 7MBAC residue for between 30 and 70 DNA base pairs. In a control experiment, a sample of anti-7MBAC tetrol was incubated with DNA, and the same procedure was followed. No binding was observed, verifying the effectiveness of the extraction process.
Discussion Kinetic Studies. The reaction kinetics of solvolysis of the diol epoxides from benzo[a]pyrene have been studied
130 Chem. Res. Toxicol., Vol. 3,No.2, 1990
35
Fraser et al. Scheme I
T
DE
+ DNA I
i
ko
tetrols
3
1.i 0
10
4 5
7
6
a
t
9
PH
Figure 4. pH dependence of the extent of covalent binding to DNA following hydrolysis of (0) syn-7MBACDE and ( 0 )anti7MBACDE at 25 "C in 0.005 M sodium cacodylate buffer in the presence of 0.8 mM DNA. The initial concentration of diol epoxide was 4.0 X M in each experiment.
extensively, together with the effect thereon of DNA, polynucleotides, serum protein, and surfactants (7,13, 19, 20,24). Most of the kinetic studies have concentrated on anti-BPDE, which is highly carcinogenic in contrast to the syn-BPDE (I). The kinetics have generally been interpreted in terms of a mechanism similar to that expressed eqs 1 and 2. Some variations in values of the kinetic constants obtained from different studies are apparent mainly due to differences in the ionic strength used. In particular, all aspects of the catalytic effect of DNA (kH', KD,and f,,,) are markedly affected by an increase of the ionic strength of the medium (13, 19); this is interpreted in terms of the added positive ions reducing the repulsive forces between neighboring negatively charged phosphate groups, leading to a tighter winding of the helix and a decrease in the base pair separation (25). A t low ionic strength the structure of DNA is more open and greater accessibility of the relevant groups for reaction exists. For example, the values of kH', KD, and f,, for anti-BPDE were found to be, respectively, 1200 M-ls-I, 2400 M-l, and 0.05 at p = 0.1 (20) and 3.6 X lo5 M-' s-l, 12000 M-l, and 0.10 at g = 0.005 (19). In the present work, the low ionic strength protocol has been adopted, since it amplifies the effect of DNA, thereby enabling a greater discrimination when comparing the effect of DNA on the rates of solvolysis of the different isomers. The kinetic constants for anti-BPDE measured at low ionic strength (19) are given in Table I, for comparison with those determined here for syn- and anti-7MBACDE. No similar study has been carried out for syn-BPDE. Neither of the 7MBAC diol epoxides reacted as rapidly as BPDE, as indicated by the rate constants, both in the absence and in the presence of DNA. While anti7MBACDE experienced a stronger influence of acid catalysis than did the syn-7MBACDE, its spontaneous hydrolysis rate was less. A similar relative reactivity was observed for syn- and anti-BPDE (20). The relative enhancement of the reaction by DNA can be expressed specifically by the ratio of the individual catalyzed constants in the presence and absence of DNA, namely, kH'/kH and k,'/k,. However, within experimental error, the value of k,' is the same as that of k,, indicating that the dominant role of DNA is the pH-dependent pathway, expressed by the ratio kH/kH'. For anti-BPDE the value for this ratio was found to be about 180 ( p = 0.005) (191, and it was suggested that this enhancement for complexed BPDE may be due to a direct attachment of hydronium ion to the epoxide oxygen forming the Denzylic carbocation in a rate-determining step. At p = 0.1 the ratio was 5.2 for syn-BPDE and 60 for anti-BPDE
+
kn[H+l
KD
[DE*.*DNA]
i [DE-
ko'
DNA]'
kw'[H+I
-
[DE***DNA-H+]'
i
free tetrols or covalently bound products
(20). Our values of this ratio for 7MBACDEs ( p = 0.005) are 140 for the anti isomer and 53 for the syn isomer. The KD value calculated for syn-7MBACDE (about 1500 M-l) was an order of magnitude lower than those of anti7MBACDE (16000 M-l) and anti-BPDE a t low ionic strength. A t g = 0.1, KD for the anti-BPDE (2400M-l) was double that of syn-BPDE (20). The fact that there are two parallel pathways contributing to kH' is not distinguishable by kinetic analysis but is evident from product analysis in that a decreasing proportion of the diol epoxide becomes covalently bound to the DNA with increasing pH, while the remainder exists as free tetrols in solution. From these observations, the reaction mechanism for the pH range 6-9 where the nitrogen is unprotonated is adequately described by Scheme I. Although both BP and 7MBAC diol epoxides appear to fit the same mechanism, and there is the same relation between the syn and anti isomers, the significant difference in magnitude of the rate constants indicates some variation in the pathway. On the other hand, the activation energies of the reactions are not significantly different for antiBPDE and anti-7MBACDE. Like anti-BPDE, the activation energy of the kH' step for anti-7MBACDE is substantially lower than the activation energy for hydrolysis of free 7MBACDE in buffer solution. This partially accounts for the acceleration of hydrolysis of 7MBACDE when complexed with DNA. The major difference in activation parameters for the anti isomers lies with the entropy of activation ( A S ) , calculated from kH and kH' on the basis that they are rate determining for the uncatalyzed and catalyzed reactions, respectively. The entropy of activation can be interpreted in terms of the relative change in the structuring of the reacting species in the rate-determining step going from the association complex (DNA-DE) to the activated complex {DNA-DE)*. Different sites of binding for the association complex between DNA and the diol epoxide may exist for the anti isomers of the BP and 7MBAC derivatives, leading to the difference in entropy of activation. One difference between BP and 7MBAC diol epoxides may lie in the nature of their physical association with DNA. anti-BPDE undergoes a rapid initial intercalation into the DNA helix as shown by a bathochromic shift in its absorption maximum (6). Similar observations have been made with benz[a]anthracene-3,4-diol1,2-oxides (26). No such observation was made with 7MBACDEs, which give rise to a small (2-nm) bathochromic shift in their spectra after complexing with DNA (data not shown). Other methods of investigation of the intercalation of 7MBACDEs and related compounds have not yet been investigated. Antibacterial acridine derivatives such as proflavin, anticancer agents such as m-amsacrine, and bis(aminoacridines) are well-studied intercalators. The derivative of benz[c]acridine, 7-aminobenz[c]acridine, is effective as an antibacterial and presumably also intercalates (18). syn-7MBACDE is less affected in the catalysis, and this is reflected by a higher activation energy in both catalyzed and uncatalyzed reactions. The calculated AS* value in Table I cannot be related in a meaningful sense to one
Solvolysis of 7-Methylbenz[c]acridine Diol Epoxides reaction. Although a 20-fold excess of DNA was present, only about 5 5 % of the syn-diol epoxide was physically bound, and therefore no individual reaction can be regarded as rate determining. Consequently, E, and AS* for the syn isomer relate to the temperature effects on both the catalyzed and uncatalyzed reactions. As an alternative compound for comparison, we may consider 5MeCDE, a small molecule that binds physically much more weakly to DNA than BPDE. The values of KDand kH’ have been reported at low ionic strength to be 2800 M-’ and 8.7 X lo4 M-ls-l for anti-5MeCDE. While kH is of the same order of magnitude as recorded for the anti-7MBACDE isomer tested here, KDis substantially lower and f,,, was found to be 0.07 (5). The effect of the nitrogen atom near the bay region has also been studied in a comparison of the hydrolysis of the anti and syn bay-region diol epoxides of dibenz[a,j]anthracene and dibenz[c,h]acridine. A t high ionic strength kH and KO values for the syn and anti homocyclic system were 2-5 times greater than those of the heterocyclic system (Jerina et al., submitted for publication). Covalent Binding to DNA. In this study, the two isomers of 7MBACDE gave rise to a high proportion of covalent binding, almost 3 times as much as in the case of BPDE under similar reaction conditions (19). There was also a pH dependence on the binding, reflecting the pH dependence of kH’. There is a reduced but still significant amount of covalent binding of the 7MBACDEs at higher pH values, and this observation is consistent with Scheme I, which includes an acid-independent component of binding and hydrolysis. It is perhaps surprising that this characteristic was not observed for BPDE. The relatively high level of binding puts 7MBACDE in the same category as anti-3-methylcholanthrenediol epoxide, which binds to DNA at twice the levels of antiBPDE without the intercalative step (alkyl groups are thought to hinder insertion between base pairs of DNA) (6). anti-Benz[a]anthracene-3,4-dioll,Zoxide also binds covalently to a similar extent (26). The physical association equilibrium constant and the reaction rate constants are lower in the case of 3-MCDE (6). It was suggested that, in the transition state of 3-MCDE, the triol carbocation may be better oriented for covalent binding to DNA than is the BPDE triol carbocation, thereby reducing the probability of nucleophilic attack by water. A further postulate was that the proportion of the molecules covalently bound may relate more directly to tumorigenicity than either KDor kH’. However, a comparison of the enantiomers of both bay-region diol epoxides of BP, benz[alanthracene, and benzo[c]phenanthrene showed that the extent of covalent binding did not simply correlate with the tumorigenic properties of these alkylating agents (27). Acknowledgment. Financial assistance for this work was received through the Australian Research Council. We are indebted to B. Tattam for the mass spectral data. Registry No. 2, 101222-81-5; 3, 101135-03-9; 4, 125950-91-6; 5, 125950-92-7; 6, 125994-17-4; 7,125994-18-5; 8,125950-93-8; 9, 125950-94-9; 11, 125994-19-6; 12, 125994-20-9; 13, 125994-21-0; 14, 125994-22-1; 2,3-diacetoxy-7-methylbenz[cJacridine, 12595095-0; trans-3,4-dihydro-3,4-dihydroxy-7MBAC, 125994-23-2; (f)-2n-bromo-1,2,3,4-tetrahydro-lp,3~,4P-trihydroxy-7MBAC, 125950-96-1.
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