93
Mutation Research, 263 (1991) 93-100 © 1991 Elsevier Science Publishers B.V. 0165-7992/91/$03.50 ADONIS 016579929100045V MUTLET 0495
Mutagenicity of (p-nitrophenyl)adenines in Salmonella typhimurium* A k i r a M a t s u d a I , M a k i k o A k a s h i 2, Y o s h i k o O h a r a 2, Y u s u k e W a t a y a 2, H i k o y a H a y a t s u 2 and Tohru Ueda I 1Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060 and 2Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700 (Japan) (Received 23 October 1990) (Revision received 30 January 1991) (Accepted 4 February 1991)
Keywords: Mutagenicity; Structure-activity relationship; 2-(p-Nitrophenyl)adenine; 8-(p-Nitrophenyl)adenine; 9-(p-Nitrophenyl)adenine; Adenine; Nucleoside
Summary Adenine derivatives having a p-nitrophenyl group at position 2, 8, or 9 were directly mutagenic towards Salmonella typhimurium strains TA98 and TAI00, whereas N~-(p-nitrophenyl)adenine was not mutagenic. 2,9- And 8,9-bis-(p-nitrophenyl)adenines were also mutagenic, but N~,9-bis-(p-nitrophenyl)adenine was not. The study on 13 (p-nitrophenyl)adenine derivatives for their Salmonella mutagenicity indicates that only those having a p-nitrophenyl ring directly linked to the purine ring are mutagenic, implying the importance of the coplanar character of the nitrophenyl and the purine rings. The nitro group seems essential for the mutagenicity, as shown from the results of assays using nitroarene-sensitive and -insensitive Salmonella strains. The mutagenic potency of this class of compounds is high, comparable to that of 2-nitrofluorene.
Recently, we found that 2-(m- and p-nitrophenyl)adenosines are directly mutagenic towards Salmonella typhimurium TA98 and TA100 (Matsuda et al., 1990). It was further noted that 2-(pnitrophenyl)-2'-deoxyadenosine (2) and 2-(p-
Correspondence: Dr. Akira Matsuda, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kitaku, Sapporo 060 (Japan) * This paper is part 99 of Nucleosides and Nucleotides. Part 98: Minakawa, N., A. Matsuda, T. Ueda and T. Sasaki (1990) Nucleosides, Nucleotides, in press.
nitrophenyl)adenine (3) are more potent mutagens than 2-(p-nitrophenyl)adenosine (1). The presence of $9 mix in the assay system diminishes the mutagenicity of these compounds. Compound 2 is the most potent among them, and its mutagenic potency in TA98 (2370 revertants/nmole without $9, 1225 revertants/nmole with $9) is greater than that of 4-nitroquinoline N-oxide. It is noteworthy that 2 exhibits mutagenicity in mouse mammary FM3A cells in culture. The nitro group in the structure of these compounds appears to be essential for the mutagenicity, since the O-acetyltransferasedeficient mutants and the nitroreductase-deficient
94
mutants of TA98 and TA100, i.e., TA98/1,8-DNP6, TAI00/1,8-DNP, TA98NR, and TAI00NR, do not give significant numbers of His + revertants on treatment witti these compounds (Matsuda et al., 1990). However, it is not clear whether the mutagenicity of these compounds arises from their nucleosidic character or from their being aromatic nitro compounds, like 4-nitroquinoline N-oxide and nitrated polyaromatics. We wish to report here the results of a study on the structure-mutagenicity relationship of (p-nitrophenyl)adenines, especially on the effect of changing the position and the number of the p-nitrophenyl group in the adenine ring.
Materials and methods
General Melting points were measured on a Yanagimoto MP-3 micromelting point apparatus and are uncorrected. Electron impact mass spectra (Mass) were measured on a JEOL JMX-DX303 spectrometer. The 1H-NMR spectra were recorded on a JEOL JNM-FX 100 (100 MHz) or JEOL JNM-GX 270 (270 MHz) spectrometer with tetramethylsilane as internal standard. Chemical shifts are reported in parts per million (~), and signals are expressed as s (singlet), d (doublet), t (triplet), or br (broad). All exchangeable protons were detected by addition of D20. Silica gel used for column chromatography was YMC gel 60A (70-230 mesh). Compounds 1-3 were prepared as described previously and were analytically pure (Matsuda et al., 1990).
2,Ng-(Bis-p-nitrophenyl)adenine (7) Sodium hydride (5.6 rag, 0.35 mmole, in 8.4 mg mineral oil) was added to a suspension of 3 (60 mg, 0.23 mmole) in dry N,N-dimethylformamide (DMF) (2 ml). After the mixture was stirred for 30 min at room temperature, p-fluoronitrobenzene (50 mg, 0.35 mmole) was added, and the whole was heated at 70°C for 7 h. The mixture was neutralized with acetic acid and concentrated to dryness in vacuo. The residue was crystallized from ethanol to give 7 (77 mg, 87%); mp > 300°C. Mass m/z: 377 (M+). Anal. Calcd for CI7H11N704: C, 54.12; H,
2.94; N, 25.98. Found: C, 54.18; H, 2.88; N, 25.70.
Ng-(p-Nitrophenyl)adenine (4) Sodium hydride (60 mg, 2.5 mmole, in 40 mg mineral oil) was added to a suspension of adenine (270 mg, 2 mmole) in dry DMF (15 ml). pFluoronitrobenzene (500 mg, 3.55 mmole) was added to the mixture and the reaction mixture was heated at 80°C with stirring. After 14 h, the mixture was neutralized with acetic acid and concentrated to dryness in vacuo. The residue was dissolved in aqueous ethanol, the solution was mixed with silica gel (ca. 5 g), and the resulting suspension was evaporated to dryness. The residue was placed on a silica gel column (3 × 30 cm), which was washed with chloroform containing 4% ethanol and then eluted with 8% ethanol in chloroform. From the latter fractions, 4 was obtained (339 mg, 66%; crystallized from ethanol); mp 270-274°C. Mass m/z: 256 (M+). IH-NMR (DMSO (dimethyl sulfoxide)-d6): 8.81 (s, 1H, H-2 or 8), 8.48 (d, 2H, Ph, J = 9 . 8 Hz), 8.35 (d, 2H, Ph, J = 9 . 8 Hz), 8.27 (s, 1H, H-8 or 2), 8.35 (d, 2H, Ph, J = 8.8 Hz), 7.49 (br s, 2H, 6-NH2). Anal. Calcd for CllHsN602: C, 51.57; H, 3.15; N, 32.80. Found: C, 51.49; H, 3.12; N, 32.71.
Ng-(2, 4-Dinitrophenyl)adenine (10) 2,4-Dinitrofluorobenzene (560 mg, 3 mmole) was added to a suspension of adenine (270 mg, 2 mmole) in dry DMF (15 ml) containing sodium hydride (60 mg, 2.5 mmole). The resulting solution was stirred overnight at room temperature, and then neutralized with acetic acid. The mixture was concentrated to dryness and the residue was taken up in ethanol. The resulting precipitate was crystallized from hot aqueous ethanol with added charcoal to give 10 (266 mg, 44%); mp 288-292°C. Mass m/z: 301 (M +). Anal. Calcd for CIIH7N704: C, 43.86; H, 2.34; N, 32.55. Found: C, 43.78; H, 2.23; N, 32.38.
Ng-(p-Nitrobenzyl)adenine (11) p-Nitrobenzyl bromide (650 mg, 3 mmole) was added to a suspension of adenine (270 mg, 2 mmole) in dry DMF (15 ml) containing sodium
95 hydride (60 mg, 2.5 mmole). The reaction mixture was stirred overnight at room temperature, neutralized with acetic acid, and concentrated to dryness in vacuo. The residue was dissolved in methanol, the solution was mixed with silica gel (ca. 5 g), and the suspension was evaporated to dryness. The resulting material was placed on a silica gel column (3 x 26 cm), and the column was washed with 407o ethanol in chloroform. The column was then eluted with 8070 ethanol in chloroform, and the desired material (11) was obtained (410 mg, 76070; crystallized from hot methanol); mp 250-254°C. Mass re~z: 270 (M+). 1H-NMR (DMSO-d6): 8.29 (s, 1H, H-2 or 8), 8.21 (d, 2H, Ph, J = 8.8 Hz), 8.13 (s, 1H, H-8 or 2), 7.52 (d, 2H, Ph, J = 8.8 Hz), 7.27 (br s, 2H, 6-NH2), 5.54 (s, 2H, CH2Ph). Anal. Calcd for C12H1oN602: C, 53.33; H, 3.73; N, 31.10. Found: C, 53.40; H, 3.72; N, 30.66.
N6-(p-Nitrophenyl)adenine (6) This compound was prepared from 6-chloropurine riboside (Sigma) as shown in Scheme 1. A suspension of 6-chloropurine riboside (2.29 g, 8 mmole) and aniline (2.23 g, 24 mmole) in ethanol (30 ml) was heated under reflux for 17 h. The mixture was evaporated to dryness and coevaporated several times with benzene. The residue was dissolved in anhydrous acetonitrile (50 ml), and to the solution acetic anhydride (5 ml, 50 mmole), triethylamine (7 ml, 50 mmole), and 4-dimethylaminopyridine (10 mg) were added. The reaction mixture was stirred for 1 h at room temperature, and then the solvent was removed under reduced pressure. The residue was mixed in ethyl acetate (100 ml), the organic phase was washed with water (2 × 30 ml), dried with Na2SO4, and concentrated to dryness. The residue was applied to a silica gel column (3 x 25 cm), which was washed with hexane-ethyl acetate (2:1) to elute acetanilide and then eluted with hexane-ethyl acetate (1:2) to give 2',3',5'-tri-O-acetyl-N6-phenyladenosine (2.59 g, 69070; crystallized from ethanol-hexane); mp 113-114°C. Mass m/z: 469 (M÷). 1H-NMR (CDC13): 8.53 (s, 1H, H-2 or 8), 8.00 (s, 1H, H-8 or 2), 7.81-7.84 (m, 3H, Ph and N6H), 7.04-7.74
(m, 3H, Ph), 6.22 (d, 1H, H - I ' , J1'.2, = 5.4 Hz), 5.96 (t, 1H, H-2', J 1 , 2 , = J 2 , 3 , = 5 . 4 Hz), 5.69 (m, 1H, H-3'), 4.43 (m, 3H, H-4', 5'a,b), 2.15, 2.13, 2.09 (each s, 3H, Ac). Anal. Calcd for C22H23NsO7: C, 56.29; H, 4.94; N, 14.92. Found: C, 56.33; H, 4.86; N, 14.82. In a mixture of dichloromethane (20 ml), acetic acid (6 ml) and nitric acid (60%, 9 ml), 2 ' , 3 ' , 5' tri-O-acetyl-N6-phenyladenosine (938 mg, 2 mmole) was dissolved and acetic anhydride (8 ml) was added under cooling in an ice bath. The reaction mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure and the residue was coevaporated several times with aqueous ethanol. The resulting crystalline material was collected by filtration to give 6 (504 mg, 98070; as hemihydrate); mp >300°C. Mass re~z: 256 (M +). IH-NMR (DMSO-d6): 10.57 (brs, 1H, N9H), 8.54 (s, 1H, H-2 or 8), 8.41 (s, 1H, H-8 or 2), 8.26 (m, 5H, Ph and N6H). Anal. Calcd for CI1HsN602"0.5 H20: C, 49.81; H, 3.42; N, 31.69. Found: C, 49.99; H, 3.33; N, 31.32.
N6,N9-(Bis-p-NitrophenyOadenine (9) p-Fluoronitrobenzene (50 mg, 0.35 mmole) was added to a suspension of 6 (60 mg, 0.23 mmole) in dry DMF (5 ml) containing sodium hydride (6 mg, 0.25 mmole). The mixture was heated at 60°C for 2 days with stirring. Water (10 ml) was added to the mixture and the resulting precipitate was collected by filtration. This material was crystallized from hot ethanol to give 9 (71 rag, 8007o); mp >300°C. Mass re~z: 377 (M +). Anal. Calcd for C17HIIN704: C, 54.12; H, 2.94; N, 25.98. Found: C, 53.93; H, 2.84; N, 25.61.
N6-(p-NitrobenzyOadenosine (12) A mixture of adenosine (535 mg, 2 mmole) and p-nitrobenzyl bromide (550 mg, 2.5 mmole) in dry DMF (15 ml) was heated at 80°C for 12 h with stirring. The mixture was concentrated to dryness in vacuo and the residue was dissolved in 0.8 N NaOH in 50070 aqueous ethanol (10 ml). The resulting solution was stirred for 4 h at room temperature, and then neutralized with 1 N HCI. The mixture was evaporated to dryness. The residue was
96 dissolved in methanol and mixed with silica gel (ca. 8 g), and the suspension was concentrated to dryness in vacuo. The residue was placed on a silica gel column (3 × 18 cm), washed with 4070 ethanol in chloroform, and then eluted with 807o ethanol in chloroform. From the latter fractions, 12 was obtained as crystals (134 mg, 17 07o; crystallized from methanol); mp 174-175°C. Mass re~z: 402 (M+). Anal. Calcd for C17H18N606: C, 50.75; H, 4.51; N, 20.89. Found: C, 50.82; H, 4.45; N, 20.91.
ethanolic ethyl acetate (60 ml), the suspension was mixed with silica gel (ca. 10 g), and was concentrated to dryness. The residue was placed on a silica gel column (2.3 × 20 cm), washed with 507o ethanol in chloroform, and then eluted with 10070ethanol in chloroform. From the first portion of the latter fractions, 8 was obtained (31 rag, 35070); mp, >300°C. Mass re~z: 377 (M+). Anal. Calcd for C17HllN704"0.5 H20: C, 52.85; H, 3.13; N, 25.38. Found: C, 52.82; H, 3.01; N, 25.09. From the last portion of the latter fractions, 5 was recovered (35
N6-(p-Nitrobenzyl)adenine (13)
mg).
A suspension of 12 (80 mg, 0.2 mmole) in 2 N HC1 (5 ml) was heated at 80°C for 1 day with stirring. The resulting crystalline material was collected by filtration to give 13 (39 rag, 73070); mp > 300°C. Mass m/z: 270 (M ÷). Anal. Caicd for C12HIoN~O2: C, 53.33; H, 3.73; N, 31.10. Found: C, 53.22; H. 3.64; N, 31.00.
8-(p-Nitrophenyi)adenine (5) A mixture of 4,5,6-triaminopyrimidine sulfate hydrate (Aldrich, 1.2 g, 5 mmole), p-nitrobenzonitrile (1.11 g, 7.5 mmole), and triethylamine (1.4 ml, 10 mmole) in methanolic ammonia (saturated at 0°C, 30 ml) was heated in a sealed steel tube at 180°C. After 7 h, the tube was cooled to room temperature and degassed. The solvent was removed under reduced pressure to dryness to leave a reddish solid, which was then suspended in 50°7o aqueous ethanol (ca. 50 ml). The insoluble material was collected by filtration and crystallized from hot ethanol to give 5 (231 mg, 18070); mp > 300°C. Mass m/z: 256 (M+). 1H-NMR (DMSO-d~): 13.65 (br s, IH, N9H), 8.40 (br s, 4H, Ph), 8.17 (s, 1H, H-2), 7.36 (br s, 2H, 6-NH2). Anal. Calcd for C~IHsN602: C, 51.57; H, 3.15; N, 32.80. Found: C, 51.83; H, 3.24; H, 32.67.
8,Ng-Bis-(p-Nitrophenyi)adenine (8) p-Fluoronitrobenzene (50 mg, 0.35 mmole) was added to a mixture of 5 (60 mg, 0.23 mmole) in dry DMF (5 ml) containing sodium hydride (6 mg, 0.25 mmole). The mixture was heated at 70°C for 3 days with stirring, and then evaporated to dryness in vacuo. The residue was suspended in 50070
Mutagenicity assay The Ames assay (Ames et al., 1975) was done with the preincubation technique (Yahagi et al., 1977). The Salmonella typhimurium strains TA98 and TAI00 were obtained from Dr. B.N. Ames of the University of California, Berkeley, strains TA98NR, TA98/1,8-DNP, TA100NR and TAI00/1,8-DNP from Dr. H.S. Rosenkranz of the Case Western Reserve University, and strains YG1020, YG1021, YG1024, YG1025, YG1026 and YG1029 from Dr. M. Ishidate Jr. of the National Institute of Hygienic Sciences, Tokyo. YG1020, YG1021 and YG1024 are derived from TA98; YG1021 and YG1024 are able to overproduce nitroreducase and O-acetyltransferase, respectively, and YG1020 is their non-overproducing control. YG1025, YG1026 and YGI029 are derived from TA100; YG1026 and YG1029 are able to overproduce nitroreductase and O-acetyltransferase, respectively, and YG1025 is their non-overproducing control (Watanabe et al., 1989, 1990). All of the derivative strains of TA98 and TAI00 were checked for their properties according to the instructions given by the source scientists. $9 was prepared from the livers of rats induced with polychlorinated biphenyl (PCB-54, Tokyo Kasai Chemicals; Cl content, ca. 54070). $9 mix containing 50/~l per plate of this $9 and supplementary coenzymes was used. In all mutation assays, each datum represents the mean of 2 plates. For each value, the background revertant score was subtracted. For every compound with every bacterial strain, a dose-response curve was determined, and from the
97
then acetylating the sugar hydroxyls (Scheme 1). N6-6o-Nitrobenzyl)adenosine (12) was obtained through the Dimroth rearrangement of N1-6Onitrobenzyl)adenosine under alkaline conditions (Scheme 2). Acid hydrolysis of the glycosidic linkage of 12 gave N6-6o-nitrobenzyl)adenine (13). 4,5,6-Triaminopyrimidine was condensed with pnitrobenzonitrile in methanolic ammonia giving 8-(p-nitrophenyl)adenine (5) (Scheme 3). All the compounds prepared were pure, as determined by the elemental analysis.
dose-response slope the mutagenic potency at 10 /~g was estimated (data in Tables 1 and 2). The 6onitrophenyl)adenines were dissolved in DMSO to prepare stock solutions for the assay. 2-Nitrofluorene used as a reference mutagen was a product of Aldrich. Results and discussion
Synthesis of (p-nitrophenyl)adenines The synthesis of 1, 2 and 3 has been described previously (Matsuda et al., 1990). The synthesis of the Ng-substituted adenines 4, 7-11 was carried out by treating the adenines with p-fluoronitrobenzene, 2,4-dinitrofluorebenzene, or p-nitrobenzyl bromide in the presence of sodium hydride, which serves as a base for promoting the dissociation of the N 9 proton of the adenine ring. N6-(p-Nitro phenyl)adenine (6) was prepared by nitration in acid of 2' ,3' ,5 '-tri-O-acetyl-N6-phenyladenosine, which in turn was obtainable by directly substituting 6-chloropurine riboside with aniline and
Mutagenicity of various (p-nitrophenyl)adenines Fig. 1 lists the structures of compounds that have been assayed for mutagenicity in Salmonella typhimurium. The mutagenicities found are presented in Tables 1 and 2. Potent mutagenicity was observed for compounds 1, 2, 3, 4, 5, 7 and 8 in S. typhimurium TA98 and TA100. The activities were comparable to those of the strong mutagen 2-nitrofluorene. The other compounds showed little or no activity in
TABLE 1 MUTAGENICITY OF (p-NITROPHENYL)ADENINES IN S. typhimurium TA98 AND ITS DERIVATIVE STRAINS Compound
Number of induced revertants/10 #g compounda
number
TA98 - $9
I 2 3 4 5 6 7 8 9 10 11 12 13 2-Nitrofluorene
TA98NR + $9
- $9
2,330 398 33 63,600 35,800 3,980 18,600 1,340 2,570 1,300 709 13 18,600 5,520 1,100 2 1 1 6,840 3,440 1,740 18,100 4,690 470 5 9 0 18 5 4 0 1 4 13 5 0 7 8 0 2,960
1,480
850
TA98/1,8-DNP YG1020
YGI021 + $9
YGI024
+ $9
- $9
+ $9
- $9
- $9
+ $9
- $9
+ $9
22 1,850 134 29 305 1 470 217 0 2 1 0 3
119 2,730 1,630 754 1,180
25 1,450 175 564 158
27,700 5,850 69,900 36,600 69,100 15,500 1,120,000 209,000 4,060,000 2,760,000 1,800,000 1,070,000 21,700 3,430 80,200 12,100 43,000 3,860 788 598 6,910 4,300 4,620 3,190 18,400 1 0 , 8 0 0 349,000 1 2 5 , 0 0 0 47,800 18,800
1
1
0
0
0
3
1
1
885 515 0 0 0 0 0
350 150 5 1 0 1 1
12,100 37,800 6 36 0 57 9
4,850 25,400 6 17 7 15 6
54,400 665,000 75 46 98 1,020 0
43,900 447,000 77 11 32 325 72
120,000 102,000 29 90 9 121 299
28,500 54,200 38 106 28 49 46
1,450
960
380
7,030
2,340
29,100
2,310
30,400
23,500
a The numbers shown are those above the background that was observed for solvent-only control plates. The background colony numbers for individual tester strains were as follows. S t r a i n / - $9 or + S9/number per plate; TA98/-/22--40, +/36-41; T A 9 8 N R / - / 20-22, +/29-92; TA98/1,8-DNP/- /12-15, +/9-118; Y G I 0 2 0 / - / 3 - 51, +/26-49; YG1021/- /18-80, +/5-43; Y G I 0 2 4 / - / 28-38, +/89-96.
98
the assays performed. The positive compounds were all directly mutagenic, and the presence of $9 in the assay mixture almost always diminished the mutagenicity. This fact suggests that when these compounds are metabolically transformed before entering the cells, their mutagenicities decrease. It may also be due to non-enzymatic factors in $9 that are known to be inhibitory for the mutagenicity of certain nitro compounds (Shah et al., 1990). As was reported previously, 2-(p-nitrophenyl)adenine (3) and its nucleosides are mutagenic in both TA98 and TA100, the deoxyribonucleoside (2) being the most potent (Matsuda et al., 1990). Among the mono-p-nitrophenylated adenine derivatives 3-6, only 6 is non-mutagenic, and the mutagenic potencies of the others are not greatly different from each other. It is noteworthy that whereas in compounds 3, 4 and 5 the p-nitrophenyl group is directly linked to the purine ring, in 6 it is linked to the extra-ring nitrogen atom. Thus, in 3, 4 and 5, the phenyl ring is coplanar with the purine ring because of the conjugation between these
rings, while in 6 the phenyl is not coplanar with the purine due to the lack of conjugation. A role of intact 6-NH2 in the mutagenicity may be another explanation for the activity in 3, 4 and 5, although there are no plausible reasons why the 6-NH2 group should be important. Among the bis-p-nitrophenylated adenines, 7 and 8 are mutagenic but 9 is not. The lack of activity in 9 suggests that the presence of a freely rotating p-nitrophenyl ring at the N 6 position abolishes the mutagenicity ascribable to the 9-(p-nitrophenyl)adenine moiety. The absence of mutagenicity in compound 10, in contrast to the potent activity in 4, implies an unfavorable nature of the presence of the o-nitro group for the mutagenicity. Consistent with this observation, 2-(o-nitrophenyl)adenosine is not mutagenic either in TA98 or TA100, irrespective of the presence or absence of $9 (Matsuda et al., 1990). The lack of activity in compounds 11-13 reinforces the view that the coplanarity of the phenyl
TABLE 2 MUTAGENICITY OF (p-NITROPHENYL)ADENINES IN S. typhimurium TAI00 AND ITS DERIVATIVE STRAINS Compound
Number of induced revertants/10/~g compounda
number
TAI00
1 2 3 4 5 6 7 8 9 10 11 12 13 2-Nitrofluorene
TAI00NR
TAI00/I,8-DNP YGI025
YGI026
YGI029
- $9
+ $9
- $9
+ $9
- $9
+ $9
- $9
+ $9
- $9
+ $9
- $9
+ $9
503 14,600 25,000 911 1,140 2 2,280 1,250 0 163 5 15 12
239 4,430 1,890 348 306 0 443 441 0 81 3 0 0
41 808 6,430 34 28 0 252 2 0 0 6 0 0
9 390 1,390 1 51 0 44 7 9 6 0 3 6
56 358 2,120 272 51 0 147 18 0 74 6 0 4
5 83 393 139 26 0 0 16 0 14 3 0 0
630 9,730 184,000 242 721 10 2,310 642 0 84 12 10 11
333 4,860 3,790 269 293 0 836 274 4 67 5 5 4
6,430 23,800 208,000 294 856 3 4,330 5,620 15 21 26 43 117
5,130 12,900 1,010 804 478 0 2,180 2,620 0 78 31 25 33
1,740 25,100 259,000 421 1,866 2 14,300 5,765 21 138 17 22 17
911 13,000 707 912 1,300 3 7,230 2,243 30 87 0 13 31
4,310
1,860
280
900
500
160
1,950
1,750
10,700
7,730
30,400
9,960
a The numbers shown are those above the background that was observed for solvent-only control plates. The background colony numbers for individual tester strains were as follows. S t r a i n s / - S 9 or + S 9 / n u m b e r per plate; T A I 0 0 / - / 7 0 - 1 2 5 , +/78-103; T A I 0 0 N R / - / 8 4 - 1 2 8 , +/86-139; T A I 0 0 / 1 , 8 - D N P / - / 1 5 - 1 1 4 , +/31-100; Y G I 0 2 5 / - / 1 1 5 - 1 2 5 ; +/118-154; Y G I 0 2 6 / - / 1 2 4 - 1 9 7 , +/121-158; YG1029/-/112-180, ÷/61-175.
99
mt•No2 HN~ N O a 4
5
6
t~l a ~
th
-6
¢ Scheme 1. Synthesis of 6 and 9.
HI
o.(
N .m~ x.~j~-o2 _.. HO
2 10
11
IIHO~H
.._..
~~o~,
HO
12
na
~ANd
,H
13
Scheme 2. Synthesis of 12 and 13.
Fig. 1. Structures of (p-nitrophenyl)adenines.
Mutation Research, 263 (1991) 93-100 © 1991 Elsevier Science Publishers B.V. 0165-7992/91/$03.50 ADONIS 016579929100045V MUTLET 0495
Mutagenicity of (p-nitrophenyl)adenines in Salmonella typhimurium* A k i r a M a t s u d a I , M a k i k o A k a s h i 2, Y o s h i k o O h a r a 2, Y u s u k e W a t a y a 2, H i k o y a H a y a t s u 2 and Tohru Ueda I 1Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060 and 2Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700 (Japan) (Received 23 October 1990) (Revision received 30 January 1991) (Accepted 4 February 1991)
Keywords: Mutagenicity; Structure-activity relationship; 2-(p-Nitrophenyl)adenine; 8-(p-Nitrophenyl)adenine; 9-(p-Nitrophenyl)adenine; Adenine; Nucleoside
Summary Adenine derivatives having a p-nitrophenyl group at position 2, 8, or 9 were directly mutagenic towards Salmonella typhimurium strains TA98 and TAI00, whereas N~-(p-nitrophenyl)adenine was not mutagenic. 2,9- And 8,9-bis-(p-nitrophenyl)adenines were also mutagenic, but N~,9-bis-(p-nitrophenyl)adenine was not. The study on 13 (p-nitrophenyl)adenine derivatives for their Salmonella mutagenicity indicates that only those having a p-nitrophenyl ring directly linked to the purine ring are mutagenic, implying the importance of the coplanar character of the nitrophenyl and the purine rings. The nitro group seems essential for the mutagenicity, as shown from the results of assays using nitroarene-sensitive and -insensitive Salmonella strains. The mutagenic potency of this class of compounds is high, comparable to that of 2-nitrofluorene.
Recently, we found that 2-(m- and p-nitrophenyl)adenosines are directly mutagenic towards Salmonella typhimurium TA98 and TA100 (Matsuda et al., 1990). It was further noted that 2-(pnitrophenyl)-2'-deoxyadenosine (2) and 2-(p-
Correspondence: Dr. Akira Matsuda, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kitaku, Sapporo 060 (Japan) * This paper is part 99 of Nucleosides and Nucleotides. Part 98: Minakawa, N., A. Matsuda, T. Ueda and T. Sasaki (1990) Nucleosides, Nucleotides, in press.
nitrophenyl)adenine (3) are more potent mutagens than 2-(p-nitrophenyl)adenosine (1). The presence of $9 mix in the assay system diminishes the mutagenicity of these compounds. Compound 2 is the most potent among them, and its mutagenic potency in TA98 (2370 revertants/nmole without $9, 1225 revertants/nmole with $9) is greater than that of 4-nitroquinoline N-oxide. It is noteworthy that 2 exhibits mutagenicity in mouse mammary FM3A cells in culture. The nitro group in the structure of these compounds appears to be essential for the mutagenicity, since the O-acetyltransferasedeficient mutants and the nitroreductase-deficient
94
mutants of TA98 and TA100, i.e., TA98/1,8-DNP6, TAI00/1,8-DNP, TA98NR, and TAI00NR, do not give significant numbers of His + revertants on treatment witti these compounds (Matsuda et al., 1990). However, it is not clear whether the mutagenicity of these compounds arises from their nucleosidic character or from their being aromatic nitro compounds, like 4-nitroquinoline N-oxide and nitrated polyaromatics. We wish to report here the results of a study on the structure-mutagenicity relationship of (p-nitrophenyl)adenines, especially on the effect of changing the position and the number of the p-nitrophenyl group in the adenine ring.
Materials and methods
General Melting points were measured on a Yanagimoto MP-3 micromelting point apparatus and are uncorrected. Electron impact mass spectra (Mass) were measured on a JEOL JMX-DX303 spectrometer. The 1H-NMR spectra were recorded on a JEOL JNM-FX 100 (100 MHz) or JEOL JNM-GX 270 (270 MHz) spectrometer with tetramethylsilane as internal standard. Chemical shifts are reported in parts per million (~), and signals are expressed as s (singlet), d (doublet), t (triplet), or br (broad). All exchangeable protons were detected by addition of D20. Silica gel used for column chromatography was YMC gel 60A (70-230 mesh). Compounds 1-3 were prepared as described previously and were analytically pure (Matsuda et al., 1990).
2,Ng-(Bis-p-nitrophenyl)adenine (7) Sodium hydride (5.6 rag, 0.35 mmole, in 8.4 mg mineral oil) was added to a suspension of 3 (60 mg, 0.23 mmole) in dry N,N-dimethylformamide (DMF) (2 ml). After the mixture was stirred for 30 min at room temperature, p-fluoronitrobenzene (50 mg, 0.35 mmole) was added, and the whole was heated at 70°C for 7 h. The mixture was neutralized with acetic acid and concentrated to dryness in vacuo. The residue was crystallized from ethanol to give 7 (77 mg, 87%); mp > 300°C. Mass m/z: 377 (M+). Anal. Calcd for CI7H11N704: C, 54.12; H,
2.94; N, 25.98. Found: C, 54.18; H, 2.88; N, 25.70.
Ng-(p-Nitrophenyl)adenine (4) Sodium hydride (60 mg, 2.5 mmole, in 40 mg mineral oil) was added to a suspension of adenine (270 mg, 2 mmole) in dry DMF (15 ml). pFluoronitrobenzene (500 mg, 3.55 mmole) was added to the mixture and the reaction mixture was heated at 80°C with stirring. After 14 h, the mixture was neutralized with acetic acid and concentrated to dryness in vacuo. The residue was dissolved in aqueous ethanol, the solution was mixed with silica gel (ca. 5 g), and the resulting suspension was evaporated to dryness. The residue was placed on a silica gel column (3 × 30 cm), which was washed with chloroform containing 4% ethanol and then eluted with 8% ethanol in chloroform. From the latter fractions, 4 was obtained (339 mg, 66%; crystallized from ethanol); mp 270-274°C. Mass m/z: 256 (M+). IH-NMR (DMSO (dimethyl sulfoxide)-d6): 8.81 (s, 1H, H-2 or 8), 8.48 (d, 2H, Ph, J = 9 . 8 Hz), 8.35 (d, 2H, Ph, J = 9 . 8 Hz), 8.27 (s, 1H, H-8 or 2), 8.35 (d, 2H, Ph, J = 8.8 Hz), 7.49 (br s, 2H, 6-NH2). Anal. Calcd for CllHsN602: C, 51.57; H, 3.15; N, 32.80. Found: C, 51.49; H, 3.12; N, 32.71.
Ng-(2, 4-Dinitrophenyl)adenine (10) 2,4-Dinitrofluorobenzene (560 mg, 3 mmole) was added to a suspension of adenine (270 mg, 2 mmole) in dry DMF (15 ml) containing sodium hydride (60 mg, 2.5 mmole). The resulting solution was stirred overnight at room temperature, and then neutralized with acetic acid. The mixture was concentrated to dryness and the residue was taken up in ethanol. The resulting precipitate was crystallized from hot aqueous ethanol with added charcoal to give 10 (266 mg, 44%); mp 288-292°C. Mass m/z: 301 (M +). Anal. Calcd for CIIH7N704: C, 43.86; H, 2.34; N, 32.55. Found: C, 43.78; H, 2.23; N, 32.38.
Ng-(p-Nitrobenzyl)adenine (11) p-Nitrobenzyl bromide (650 mg, 3 mmole) was added to a suspension of adenine (270 mg, 2 mmole) in dry DMF (15 ml) containing sodium
95 hydride (60 mg, 2.5 mmole). The reaction mixture was stirred overnight at room temperature, neutralized with acetic acid, and concentrated to dryness in vacuo. The residue was dissolved in methanol, the solution was mixed with silica gel (ca. 5 g), and the suspension was evaporated to dryness. The resulting material was placed on a silica gel column (3 x 26 cm), and the column was washed with 407o ethanol in chloroform. The column was then eluted with 8070 ethanol in chloroform, and the desired material (11) was obtained (410 mg, 76070; crystallized from hot methanol); mp 250-254°C. Mass re~z: 270 (M+). 1H-NMR (DMSO-d6): 8.29 (s, 1H, H-2 or 8), 8.21 (d, 2H, Ph, J = 8.8 Hz), 8.13 (s, 1H, H-8 or 2), 7.52 (d, 2H, Ph, J = 8.8 Hz), 7.27 (br s, 2H, 6-NH2), 5.54 (s, 2H, CH2Ph). Anal. Calcd for C12H1oN602: C, 53.33; H, 3.73; N, 31.10. Found: C, 53.40; H, 3.72; N, 30.66.
N6-(p-Nitrophenyl)adenine (6) This compound was prepared from 6-chloropurine riboside (Sigma) as shown in Scheme 1. A suspension of 6-chloropurine riboside (2.29 g, 8 mmole) and aniline (2.23 g, 24 mmole) in ethanol (30 ml) was heated under reflux for 17 h. The mixture was evaporated to dryness and coevaporated several times with benzene. The residue was dissolved in anhydrous acetonitrile (50 ml), and to the solution acetic anhydride (5 ml, 50 mmole), triethylamine (7 ml, 50 mmole), and 4-dimethylaminopyridine (10 mg) were added. The reaction mixture was stirred for 1 h at room temperature, and then the solvent was removed under reduced pressure. The residue was mixed in ethyl acetate (100 ml), the organic phase was washed with water (2 × 30 ml), dried with Na2SO4, and concentrated to dryness. The residue was applied to a silica gel column (3 x 25 cm), which was washed with hexane-ethyl acetate (2:1) to elute acetanilide and then eluted with hexane-ethyl acetate (1:2) to give 2',3',5'-tri-O-acetyl-N6-phenyladenosine (2.59 g, 69070; crystallized from ethanol-hexane); mp 113-114°C. Mass m/z: 469 (M÷). 1H-NMR (CDC13): 8.53 (s, 1H, H-2 or 8), 8.00 (s, 1H, H-8 or 2), 7.81-7.84 (m, 3H, Ph and N6H), 7.04-7.74
(m, 3H, Ph), 6.22 (d, 1H, H - I ' , J1'.2, = 5.4 Hz), 5.96 (t, 1H, H-2', J 1 , 2 , = J 2 , 3 , = 5 . 4 Hz), 5.69 (m, 1H, H-3'), 4.43 (m, 3H, H-4', 5'a,b), 2.15, 2.13, 2.09 (each s, 3H, Ac). Anal. Calcd for C22H23NsO7: C, 56.29; H, 4.94; N, 14.92. Found: C, 56.33; H, 4.86; N, 14.82. In a mixture of dichloromethane (20 ml), acetic acid (6 ml) and nitric acid (60%, 9 ml), 2 ' , 3 ' , 5' tri-O-acetyl-N6-phenyladenosine (938 mg, 2 mmole) was dissolved and acetic anhydride (8 ml) was added under cooling in an ice bath. The reaction mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure and the residue was coevaporated several times with aqueous ethanol. The resulting crystalline material was collected by filtration to give 6 (504 mg, 98070; as hemihydrate); mp >300°C. Mass re~z: 256 (M +). IH-NMR (DMSO-d6): 10.57 (brs, 1H, N9H), 8.54 (s, 1H, H-2 or 8), 8.41 (s, 1H, H-8 or 2), 8.26 (m, 5H, Ph and N6H). Anal. Calcd for CI1HsN602"0.5 H20: C, 49.81; H, 3.42; N, 31.69. Found: C, 49.99; H, 3.33; N, 31.32.
N6,N9-(Bis-p-NitrophenyOadenine (9) p-Fluoronitrobenzene (50 mg, 0.35 mmole) was added to a suspension of 6 (60 mg, 0.23 mmole) in dry DMF (5 ml) containing sodium hydride (6 mg, 0.25 mmole). The mixture was heated at 60°C for 2 days with stirring. Water (10 ml) was added to the mixture and the resulting precipitate was collected by filtration. This material was crystallized from hot ethanol to give 9 (71 rag, 8007o); mp >300°C. Mass re~z: 377 (M +). Anal. Calcd for C17HIIN704: C, 54.12; H, 2.94; N, 25.98. Found: C, 53.93; H, 2.84; N, 25.61.
N6-(p-NitrobenzyOadenosine (12) A mixture of adenosine (535 mg, 2 mmole) and p-nitrobenzyl bromide (550 mg, 2.5 mmole) in dry DMF (15 ml) was heated at 80°C for 12 h with stirring. The mixture was concentrated to dryness in vacuo and the residue was dissolved in 0.8 N NaOH in 50070 aqueous ethanol (10 ml). The resulting solution was stirred for 4 h at room temperature, and then neutralized with 1 N HCI. The mixture was evaporated to dryness. The residue was
96 dissolved in methanol and mixed with silica gel (ca. 8 g), and the suspension was concentrated to dryness in vacuo. The residue was placed on a silica gel column (3 × 18 cm), washed with 4070 ethanol in chloroform, and then eluted with 807o ethanol in chloroform. From the latter fractions, 12 was obtained as crystals (134 mg, 17 07o; crystallized from methanol); mp 174-175°C. Mass re~z: 402 (M+). Anal. Calcd for C17H18N606: C, 50.75; H, 4.51; N, 20.89. Found: C, 50.82; H, 4.45; N, 20.91.
ethanolic ethyl acetate (60 ml), the suspension was mixed with silica gel (ca. 10 g), and was concentrated to dryness. The residue was placed on a silica gel column (2.3 × 20 cm), washed with 507o ethanol in chloroform, and then eluted with 10070ethanol in chloroform. From the first portion of the latter fractions, 8 was obtained (31 rag, 35070); mp, >300°C. Mass re~z: 377 (M+). Anal. Calcd for C17HllN704"0.5 H20: C, 52.85; H, 3.13; N, 25.38. Found: C, 52.82; H, 3.01; N, 25.09. From the last portion of the latter fractions, 5 was recovered (35
N6-(p-Nitrobenzyl)adenine (13)
mg).
A suspension of 12 (80 mg, 0.2 mmole) in 2 N HC1 (5 ml) was heated at 80°C for 1 day with stirring. The resulting crystalline material was collected by filtration to give 13 (39 rag, 73070); mp > 300°C. Mass m/z: 270 (M ÷). Anal. Caicd for C12HIoN~O2: C, 53.33; H, 3.73; N, 31.10. Found: C, 53.22; H. 3.64; N, 31.00.
8-(p-Nitrophenyi)adenine (5) A mixture of 4,5,6-triaminopyrimidine sulfate hydrate (Aldrich, 1.2 g, 5 mmole), p-nitrobenzonitrile (1.11 g, 7.5 mmole), and triethylamine (1.4 ml, 10 mmole) in methanolic ammonia (saturated at 0°C, 30 ml) was heated in a sealed steel tube at 180°C. After 7 h, the tube was cooled to room temperature and degassed. The solvent was removed under reduced pressure to dryness to leave a reddish solid, which was then suspended in 50°7o aqueous ethanol (ca. 50 ml). The insoluble material was collected by filtration and crystallized from hot ethanol to give 5 (231 mg, 18070); mp > 300°C. Mass m/z: 256 (M+). 1H-NMR (DMSO-d~): 13.65 (br s, IH, N9H), 8.40 (br s, 4H, Ph), 8.17 (s, 1H, H-2), 7.36 (br s, 2H, 6-NH2). Anal. Calcd for C~IHsN602: C, 51.57; H, 3.15; N, 32.80. Found: C, 51.83; H, 3.24; H, 32.67.
8,Ng-Bis-(p-Nitrophenyi)adenine (8) p-Fluoronitrobenzene (50 mg, 0.35 mmole) was added to a mixture of 5 (60 mg, 0.23 mmole) in dry DMF (5 ml) containing sodium hydride (6 mg, 0.25 mmole). The mixture was heated at 70°C for 3 days with stirring, and then evaporated to dryness in vacuo. The residue was suspended in 50070
Mutagenicity assay The Ames assay (Ames et al., 1975) was done with the preincubation technique (Yahagi et al., 1977). The Salmonella typhimurium strains TA98 and TAI00 were obtained from Dr. B.N. Ames of the University of California, Berkeley, strains TA98NR, TA98/1,8-DNP, TA100NR and TAI00/1,8-DNP from Dr. H.S. Rosenkranz of the Case Western Reserve University, and strains YG1020, YG1021, YG1024, YG1025, YG1026 and YG1029 from Dr. M. Ishidate Jr. of the National Institute of Hygienic Sciences, Tokyo. YG1020, YG1021 and YG1024 are derived from TA98; YG1021 and YG1024 are able to overproduce nitroreducase and O-acetyltransferase, respectively, and YG1020 is their non-overproducing control. YG1025, YG1026 and YGI029 are derived from TA100; YG1026 and YG1029 are able to overproduce nitroreductase and O-acetyltransferase, respectively, and YG1025 is their non-overproducing control (Watanabe et al., 1989, 1990). All of the derivative strains of TA98 and TAI00 were checked for their properties according to the instructions given by the source scientists. $9 was prepared from the livers of rats induced with polychlorinated biphenyl (PCB-54, Tokyo Kasai Chemicals; Cl content, ca. 54070). $9 mix containing 50/~l per plate of this $9 and supplementary coenzymes was used. In all mutation assays, each datum represents the mean of 2 plates. For each value, the background revertant score was subtracted. For every compound with every bacterial strain, a dose-response curve was determined, and from the
97
then acetylating the sugar hydroxyls (Scheme 1). N6-6o-Nitrobenzyl)adenosine (12) was obtained through the Dimroth rearrangement of N1-6Onitrobenzyl)adenosine under alkaline conditions (Scheme 2). Acid hydrolysis of the glycosidic linkage of 12 gave N6-6o-nitrobenzyl)adenine (13). 4,5,6-Triaminopyrimidine was condensed with pnitrobenzonitrile in methanolic ammonia giving 8-(p-nitrophenyl)adenine (5) (Scheme 3). All the compounds prepared were pure, as determined by the elemental analysis.
dose-response slope the mutagenic potency at 10 /~g was estimated (data in Tables 1 and 2). The 6onitrophenyl)adenines were dissolved in DMSO to prepare stock solutions for the assay. 2-Nitrofluorene used as a reference mutagen was a product of Aldrich. Results and discussion
Synthesis of (p-nitrophenyl)adenines The synthesis of 1, 2 and 3 has been described previously (Matsuda et al., 1990). The synthesis of the Ng-substituted adenines 4, 7-11 was carried out by treating the adenines with p-fluoronitrobenzene, 2,4-dinitrofluorebenzene, or p-nitrobenzyl bromide in the presence of sodium hydride, which serves as a base for promoting the dissociation of the N 9 proton of the adenine ring. N6-(p-Nitro phenyl)adenine (6) was prepared by nitration in acid of 2' ,3' ,5 '-tri-O-acetyl-N6-phenyladenosine, which in turn was obtainable by directly substituting 6-chloropurine riboside with aniline and
Mutagenicity of various (p-nitrophenyl)adenines Fig. 1 lists the structures of compounds that have been assayed for mutagenicity in Salmonella typhimurium. The mutagenicities found are presented in Tables 1 and 2. Potent mutagenicity was observed for compounds 1, 2, 3, 4, 5, 7 and 8 in S. typhimurium TA98 and TA100. The activities were comparable to those of the strong mutagen 2-nitrofluorene. The other compounds showed little or no activity in
TABLE 1 MUTAGENICITY OF (p-NITROPHENYL)ADENINES IN S. typhimurium TA98 AND ITS DERIVATIVE STRAINS Compound
Number of induced revertants/10 #g compounda
number
TA98 - $9
I 2 3 4 5 6 7 8 9 10 11 12 13 2-Nitrofluorene
TA98NR + $9
- $9
2,330 398 33 63,600 35,800 3,980 18,600 1,340 2,570 1,300 709 13 18,600 5,520 1,100 2 1 1 6,840 3,440 1,740 18,100 4,690 470 5 9 0 18 5 4 0 1 4 13 5 0 7 8 0 2,960
1,480
850
TA98/1,8-DNP YG1020
YGI021 + $9
YGI024
+ $9
- $9
+ $9
- $9
- $9
+ $9
- $9
+ $9
22 1,850 134 29 305 1 470 217 0 2 1 0 3
119 2,730 1,630 754 1,180
25 1,450 175 564 158
27,700 5,850 69,900 36,600 69,100 15,500 1,120,000 209,000 4,060,000 2,760,000 1,800,000 1,070,000 21,700 3,430 80,200 12,100 43,000 3,860 788 598 6,910 4,300 4,620 3,190 18,400 1 0 , 8 0 0 349,000 1 2 5 , 0 0 0 47,800 18,800
1
1
0
0
0
3
1
1
885 515 0 0 0 0 0
350 150 5 1 0 1 1
12,100 37,800 6 36 0 57 9
4,850 25,400 6 17 7 15 6
54,400 665,000 75 46 98 1,020 0
43,900 447,000 77 11 32 325 72
120,000 102,000 29 90 9 121 299
28,500 54,200 38 106 28 49 46
1,450
960
380
7,030
2,340
29,100
2,310
30,400
23,500
a The numbers shown are those above the background that was observed for solvent-only control plates. The background colony numbers for individual tester strains were as follows. S t r a i n / - $9 or + S9/number per plate; TA98/-/22--40, +/36-41; T A 9 8 N R / - / 20-22, +/29-92; TA98/1,8-DNP/- /12-15, +/9-118; Y G I 0 2 0 / - / 3 - 51, +/26-49; YG1021/- /18-80, +/5-43; Y G I 0 2 4 / - / 28-38, +/89-96.
98
the assays performed. The positive compounds were all directly mutagenic, and the presence of $9 in the assay mixture almost always diminished the mutagenicity. This fact suggests that when these compounds are metabolically transformed before entering the cells, their mutagenicities decrease. It may also be due to non-enzymatic factors in $9 that are known to be inhibitory for the mutagenicity of certain nitro compounds (Shah et al., 1990). As was reported previously, 2-(p-nitrophenyl)adenine (3) and its nucleosides are mutagenic in both TA98 and TA100, the deoxyribonucleoside (2) being the most potent (Matsuda et al., 1990). Among the mono-p-nitrophenylated adenine derivatives 3-6, only 6 is non-mutagenic, and the mutagenic potencies of the others are not greatly different from each other. It is noteworthy that whereas in compounds 3, 4 and 5 the p-nitrophenyl group is directly linked to the purine ring, in 6 it is linked to the extra-ring nitrogen atom. Thus, in 3, 4 and 5, the phenyl ring is coplanar with the purine ring because of the conjugation between these
rings, while in 6 the phenyl is not coplanar with the purine due to the lack of conjugation. A role of intact 6-NH2 in the mutagenicity may be another explanation for the activity in 3, 4 and 5, although there are no plausible reasons why the 6-NH2 group should be important. Among the bis-p-nitrophenylated adenines, 7 and 8 are mutagenic but 9 is not. The lack of activity in 9 suggests that the presence of a freely rotating p-nitrophenyl ring at the N 6 position abolishes the mutagenicity ascribable to the 9-(p-nitrophenyl)adenine moiety. The absence of mutagenicity in compound 10, in contrast to the potent activity in 4, implies an unfavorable nature of the presence of the o-nitro group for the mutagenicity. Consistent with this observation, 2-(o-nitrophenyl)adenosine is not mutagenic either in TA98 or TA100, irrespective of the presence or absence of $9 (Matsuda et al., 1990). The lack of activity in compounds 11-13 reinforces the view that the coplanarity of the phenyl
TABLE 2 MUTAGENICITY OF (p-NITROPHENYL)ADENINES IN S. typhimurium TAI00 AND ITS DERIVATIVE STRAINS Compound
Number of induced revertants/10/~g compounda
number
TAI00
1 2 3 4 5 6 7 8 9 10 11 12 13 2-Nitrofluorene
TAI00NR
TAI00/I,8-DNP YGI025
YGI026
YGI029
- $9
+ $9
- $9
+ $9
- $9
+ $9
- $9
+ $9
- $9
+ $9
- $9
+ $9
503 14,600 25,000 911 1,140 2 2,280 1,250 0 163 5 15 12
239 4,430 1,890 348 306 0 443 441 0 81 3 0 0
41 808 6,430 34 28 0 252 2 0 0 6 0 0
9 390 1,390 1 51 0 44 7 9 6 0 3 6
56 358 2,120 272 51 0 147 18 0 74 6 0 4
5 83 393 139 26 0 0 16 0 14 3 0 0
630 9,730 184,000 242 721 10 2,310 642 0 84 12 10 11
333 4,860 3,790 269 293 0 836 274 4 67 5 5 4
6,430 23,800 208,000 294 856 3 4,330 5,620 15 21 26 43 117
5,130 12,900 1,010 804 478 0 2,180 2,620 0 78 31 25 33
1,740 25,100 259,000 421 1,866 2 14,300 5,765 21 138 17 22 17
911 13,000 707 912 1,300 3 7,230 2,243 30 87 0 13 31
4,310
1,860
280
900
500
160
1,950
1,750
10,700
7,730
30,400
9,960
a The numbers shown are those above the background that was observed for solvent-only control plates. The background colony numbers for individual tester strains were as follows. S t r a i n s / - S 9 or + S 9 / n u m b e r per plate; T A I 0 0 / - / 7 0 - 1 2 5 , +/78-103; T A I 0 0 N R / - / 8 4 - 1 2 8 , +/86-139; T A I 0 0 / 1 , 8 - D N P / - / 1 5 - 1 1 4 , +/31-100; Y G I 0 2 5 / - / 1 1 5 - 1 2 5 ; +/118-154; Y G I 0 2 6 / - / 1 2 4 - 1 9 7 , +/121-158; YG1029/-/112-180, ÷/61-175.
99
mt•No2 HN~ N O a 4
5
6
t~l a ~
th
-6
¢ Scheme 1. Synthesis of 6 and 9.
HI
o.(
N .m~ x.~j~-o2 _.. HO
2 10
11
IIHO~H
.._..
~~o~,
HO
12
na
~ANd
,H
13
Scheme 2. Synthesis of 12 and 13.
Fig. 1. Structures of (p-nitrophenyl)adenines.
