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Synthesis, Cytotoxicity, and QSAR Analysis of X-Thiophenols in Rapidly Dividing Cells Rajeshwar P. Verma,† Sanjay Kapur,† Omar Barberena,† Alan Shusterman,‡ Corwin H. Hansch,† and Cynthia D. Selassie*,† Department of Chemistry, Pomona College, Claremont, California 91711, and Department of Chemistry, Reed College, Portland, Oregon 97202 Received November 5, 2002
In this study, the cytotoxicities of a series of X-thiophenols vs rapidly growing mouse leukemia cells in vitro are determined. The resulting ID50 values are then used to formulate a quantitative structure-activity relationship, which is well-correlated by the Brown variation of the Hammett electronic parameter, σ-plus (σ+ ), such that Log 1/ID50 ) -0.93 ((0.18) σ+ + 0.86 ((0.24) IH + 3.99 ((0.13). IH represents an indicator variable that calls attention to the unusual activity of halogens and pseudohalogens. In lieu of σ+, homolytic bond dissociation energies (BDE) are also used successfully to correlate the cellular cytotoxicities of thiophenols. The nature of substituent effects on cellular toxicity is examined, and they reveal that electron-releasing substituted thiophenols such as 4-amino thiophenol and the 4-alkoxy thiophenols are highly cytotoxic and effective at inhibiting cellular proliferation at physiological pH. On the other hand, electron-attracting substituted thiophenols such as the 4-cyano and 4-halogen analogues show a reduced ability to inhibit the cell growth of this cell line. Thus, there is a clear parallel between enhanced biological activity and electron releasing ability as measured by σ+ constants or BDE values. The susceptibility of the cellular interaction to electronic effects as delineated by the coefficient with the σ+ term (also called the Hammett F value) is high (-0.96), suggesting that substantial energetic assistance is provided by the substituents and that a weak initiating radical reactant such as superoxide radical may be involved. Previous cytotoxicity studies of a large diverse data set of X-phenols in this cell line and embryo cells have also revealed a more pronounced dependence on σ+ and ∆BDE. A comparison of reaction constants obtained from thiophenoxy radical formation reactions and phenoxy radical formation reactions in organic media suggests radical-mediated involvement in cell cytotoxicity. Such cells could be more vulnerable to the effects of reactive thiyl species on their metabolism and subsequent proliferation.
Introduction In recent years, the focus in toxicology has gradually shifted toward assessment of the chronic effects of drugs, pesticides, and industrial chemicals, since drugs such as DES1 and thalidomide have proven to be teratogenic. Similar concerns have also been expressed about environmental estrogens (1). DES and thalidomide have provided sobering lessons on the importance of thoroughly testing a drug’s safety before usage. More importantly, they have helped focus attention on certain functional moieties that confer toxic attributes to drug entities. After the toxic side effects experienced by chloramphenicol users, drug design efforts are no longer geared toward the incorporation of the nitro functionality in the modification of lead compounds. Recent work in our laboratory has focused on the phenolic OH group (2-5). An unusual type of phenol toxicity to rat embryos in vitro first attracted our attention. Three different toxic end points were found to * To whom correspondence should be addressed. Tel: (909)621-8446. Fax: (909)607-7726. E-mail:
[email protected]. † Pomona College. ‡ Reed College. 1 Abbreviations: QSAR, quantitative structure-activity relationship; DES, diethylstilbesterol; CHO, Chinese hamster ovary; ∆BDE, relative bond dissociation energy.
correlate as follows: log 1/C ) -0.6σ+ + constant, where C is the molar concentration of phenol producing a standard degree of maldevelopment. The presence of a negative σ+ term was duly noted (6). Around the same time, in CHO cells, the inhibition of DNA synthesis by substituted phenols was found to correlate strongly with σ+ (7). A study of 25 sets of simple phenols revealed that a negative σ+ term appeared in the formation of phenoxyl radicals by various types of initiating radicals (6). These results sparked further study in rapidly dividing cells where reactive oxygen species (ROS) would be expected to be formed in significant amounts. Studies with leukemia cells indicated that cytotoxicities of simple ortho, meta, and para electron-releasing phenols as well as complex phenols such as bisphenol A, diethylstilbestrol, and estradiol were well fit by the following model (8).
Log 1/C ) -1.35 ((0.15) σ+ + 0.18 ((0.04) log P + 3.31 ((0.11) (1) where n ) 51, r2 ) 0.895, s ) 0.227, and q2 ) 0.882. In this equation, C represents the concentration of X-phenol that induces 50% inhibition of growth in the murine L1210 cell line. σ+ is the Brown variation of the Hammett electronic parameter that describes a substituent’s effect
10.1021/tx020103q CCC: $25.00 © 2003 American Chemical Society Published on Web 02/19/2003
Synthesis, Cytotoxicity, and QSAR Analysis of Thiophenols
on electron density distribution of an aromatic ring, and log P represents the hydrophobicities of the various phenols. Further work then demonstrated that σ+ values could be replaced by calculated homolytic BDEs and that estriol, equilin, and equilenin were also well fit by eq 1 (8). The carcinogenicity of estrogens (9, 10) and these cytotoxicity results suggest that phenols with low BDE values or large negative σ+ values may be involved in DNA damage. This type of “σ+ toxicity” in phenols has not been detected to date in mature animals or fish. Equation 1 is based on phenols that have electronreleasing substituents only. With electron-attracting substituents, a simpler mechanism is found, one that only involves hydrophobicity (5). Thus, phenols in general, behave in an enigmatic fashion; some of them are harmless, while others appear to be hazardous. For example, phenol itself is not carcinogenic or estrogenic, but para-methoxy phenol is carcinogenic in rats (11). Note that the para-methoxy substituent has a large negative σ+ value. Many polyphenolic compounds that are ubiquitous in fruits, tea, and vegetables appear to act as chemopreventive agents (12-15). It may well be that this type of unusual activity is concentration-dependent; estrogenic hormones are vital to homeostasis, but at high concentration levels, they are carcinogenic. Quercetin, which appears to be one of the best radical scavengers (14), is toxic to leukemia cells at 100 µM concentration. Thus, it is likely that ingestion of high amounts of the natural phytophenols could be as damaging as consuming too little. To better understand the scope of radical toxicity at the molecular level, we have extended our investigations to other functional groups with labile hydrogens. Recent studies by Amrolia et al. of the cytotoxicity of aromatic thiols in human red blood cells have revealed that thiophenols induce the conversion of oxyhemoglobin to methemoglobin (16). Reduction of corresponding disulfides by intracellular glutathione led to cyclic reduction/ oxidation reactions, which culminated in enhanced oxidative stress (16). Sulfur-centered radicals are very similar to their oxygen analogues. There are, however, significant differences, which may be attributed to the reduced electronegativity and greater size of the sulfur atom, which makes it “softer” than oxygen in terms of its electronic properties (17). -e/-H+
(i) RSH 98 RS• f 1/2 RSSR -e
(ii) RSSR 98 (RSSR) + • The strong oxidative character of sulfur-centered radical cations (RSSR+ • ) readily allows for oxidation of thiolates, phenothiazines, phenol derivatives, vitamin E, and vitamin C (18). The reaction constants (F+) for thiophenoxy and phenoxy formation by various radicals (X-C6H4SH + R• f X-C6H4S• + RH) are outlined in Table 1 (6). The coefficients with Hammett’s σ+ electronic term, the F+ values, are all negative albeit some are more negative than others. A negative F+ indicates that electron-releasing substituents increase the rate of homolytic bond cleavage. Example 4 in Table 1 shows that a more electrophilic radical, PhCOO•, leads to a larger F+ value, suggesting that positive charge accumulates on the thiyl moiety in the transition state, i.e., the transition
Chem. Res. Toxicol., Vol. 16, No. 3, 2003 277 Table 1. Hammett Reaction Constants for the Reaction of X-Thiophenols and X-Phenols with Radical Initiatorsa
X-C6H4SH + R• f X-C6H4S• + RH
X-C6H4OH + R• f X-C6H4O• + RH class type 1 2 3 4 5 6 7 8 9 10 a
X-thiophenols X-thiophenols X-thiophenols X-thiophenols X-phenols X-phenols X-phenols X-phenols X-phenols X-phenols
solvent
radical Nb initiator R•
C6H6 6 Me3CO• CCl4 6 Me3CO• CH3COOH 6 Me3CO• C6H5Cl 5 C6H5COO• CCl4 11 Me3CO• benzene 4 Me3CO• acetonitrile 4 Me3CO• chlorobenzene 7 Me3CO• CCl4 5 Me3CO• benzene 12 Me3CO•
(F+)
temp (°C)
-0.29 -0.29 -0.28 -1.18 -1.13 -1.46 -1.02 -0.71 -0.82 -0.82
130 130 130 100 120 130 130 122 25 22
Ref 6. b Number of analogues in each data set.
Scheme 1. Synthesis of para-Substituted Thiophenols and 4-n-Butylphenyl Disulfide
state for H• transfer may have some polar character as in (PhCO2- ••H•• δ+SC6H4-X). In such a scenario, electronwithdrawing substituents might be expected to hinder thiyl formation. The negative signs and magnitudes of the reaction constants in Table 1 coupled with the unusual results obtained for X-phenol cytotoxicity led to this study on the cytotoxicity of a comprehensive set of 4-X-thiophenols in rapidly proliferating, murine leukemia cells.
Experimental Procedures Chemicals. 4-Fluoro, 4-chloro, 4-bromo, 4-methoxy, 4-hydroxy, 4-methyl, 4-nitro, and 4-amino thiophenols were purchased from Aldrich Chemical Co. (Milwaukee, WI) while 4-tertbutyl and 4-trifluoromethylthiophenols were purchased from Alfa Aesar Chemical Co. (Ward Hill, MA). Phenyl disulfide, p-tolyl disulfide, and 4-nitrophenyl disulfide were purchased from Aldrich Chemical Co. (Milwaukee, WI), while 4-methoxyphenyl disulfide and 4-chlorophenyl disulfide were purchased from Lancaster (Windham, NH). 4-Phenoxy- (19), 4-cyano- (20) and 4-acetamidothiophenol (21) were prepared according to methods reported in the literature. 4-Alkoxythiophenols (4ethoxy, 4-n-propoxy, 4-n-butoxy, 4-isopentoxy, 4-n-pentoxy, 4-nhexyloxy and 4-n-heptyloxy thiophenols), 4-alkylthiophenols (4neopentyl, 4-isopropyl, and 4-n-butylthiophenols), 4-thiomethylthiophenol, 4-iodothiophenol, and 4-n-butylphenyl disulfide were prepared according to Scheme 1. Structures and purities of these compounds were confirmed by 1H NMR, 13C NMR, IR, GC/MS, TLC, and boiling points/melting points. Instrumentation. Melting points were determined on an electrothermal melting point apparatus (MEL-TEMP II with digital thermometer), and boiling points were determined under
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reduced pressure (10 mm) and are uncorrected. 1H NMR and 13C NMR spectra were performed on a Bruker DPX 400 MHz NMR spectrometer with TMS as the internal standard; chemical shifts are given in δ (ppm) scale. IR spectra were recorded on a Perkin-Elmer 1600 series FTIR, and only principal, sharply defined IR peaks are reported. Mass spectra were obtained from GC/MS data from a Hewlett-Packard GC/MS system HP 6890 series with mass selective detector. For purity tests, TLC was performed on silica gel plates (silica gel IB-F Baker). Syntheses. (i) General Procedure for the Syntheses of 4-Alkoxythiophenols. 4-n-Butoxythiophenol. Phenol (14.1 g, 0.15 mol) was dissolved in methanol (100 mL), and to it was added NaOH (6.6 g, 0.165 mol). The reaction mixture was stirred at 50 °C for 20 min to obtain a clear solution. The resulting solution was treated with BuBr (21.92 g, 0.16 mol), and the reaction mixture was heated under reflux for 18 h. After it was refluxed, the reaction mixture separated into two layers. The organic layer was removed and washed with 10% NaOH solution and water in that order and dried over MgSO4 (anhydrous). After it was filtered, it was distilled under reduced pressure to yield phenyl-n-butyl ether (14.5 g, 64.9%): bp/mm 78 °C/10 (lit (22) 82-83.5 °C/10). 1H NMR (acetone-d6): δ 0.95 (t, 3H, -CH3), 1.48 (q, 2H, -CH2-), 1.73 (q, 2H, -CH2-), 3.95 (t, 2H, -CH2), 6.90 (m, 3H, Ar), 7.25 (m, 2H, Ar). Phenyl-n-butyl ether (12 g, 0.08 mol) was dissolved in chloroform (40 mL) and cooled to -8 °C. Over the period of an hour, the cooled solution was treated with chlorosulfonic acid (12 mL, 0.18 mol) at or below -5 °C. The reaction mixture was stirred for 10 min with cooling and then without cooling until it reached room temperature. Then, it was poured into 100 g of crushed ice and extracted with chloroform. The chloroform extract was washed with water and dried over anhydrous MgSO4. After it was filtered, the chloroform was evaporated and the residue was distilled under reduced pressure to give 4-n-butoxybenzene sulfonyl chloride (8.8 g, 44.5%): bp/mm 100 °C/10. 1H NMR (acetone-d6): δ 0.98 (t, 3H, -CH3), 1.54 (q, 2H, -CH2-), 1.82 (q, 2H, -CH2-), 4.19 (t, 2H, -CH2-), 7.24 (d, J 7.5 Hz, 2H, Ar), 8.04 (d, J 7.5 Hz, 2H, Ar). A mixture of 4-n-butoxybenzene sulfonyl chloride (4.8 g, 0.02 mol), crushed ice (50 g), and concentrated sulfuric acid (10 mL) was cooled to -7 °C. The reaction mixture was treated with 10 g of Zn powder at 0 °C over a period of 30 min. It was stirred for 1.5 h at 0° and 3 h at room temperature, kept overnight, and steam-distilled. The distillate was extracted with Et2O and dried over anhydrous magnesium sulfate. After it was filtered, the solvent was evaporated and the residue was distilled under reduced pressure to give 4-n-butoxythiophenol (1.8 g, 51.28%): bp/mm 138 °C/10 (lit (22) 133-135 °C/12). 1H NMR (acetone-d6): δ 0.95 (t, J 7.2 Hz, 3H, -CH3), 1.48 (q, 2H, -CH2), 1.73 (q, 2H, -CH2-), 3.94 (t, J 6.5 Hz, 2H, -CH2-), 3.98 (s, 1H, -SH), 6.84 (d, J 8 Hz, 2H, Ar), 7.25 (d, J 8 Hz, 2H, Ar). 13C NMR (acetone-d6): δ 14.63 (CH3), 20.34 (CH2), 32.57 (CH2), 68.76 (CH2), 116.73 (C3 and C5), 121.780 (C1), 132.71 (C2 and C6), 159.18 (C4). IR: 2560.7 cm-1 (SH). GC/MS (m/z, %): 182 (M+, 28), 126 (100), 97 (20). The other alkoxythiophenols were prepared in a similar fashion from their respective phenyl-alkyl ethers. Yields, mp/ bp, and spectral data are given below. 4-Ethoxythiophenol. Yield, ∼36%; bp/mm 108-110 °C/10 (lit (22) 110-112 °C/10). 1H NMR (CDCl3): δ 1.41 (t, J 7.5 Hz, 3H, CH3), 3.38 (s, 1H, -SH), 3.97 (q, 2H, -CH2-), 6.78 (d, J 8.2 Hz, 2H, Ar), 7.24 (d, J 8.2 Hz, 2H, Ar). 13C NMR (CDCl3): δ 15.04 (CH3), 63.79 (CH2), 115.37 (C3 and C5), 128.45 (C1), 132.68 (C2 and C6), 159.30 (C4). IR: 2564 cm-1 (SH). GC/MS (m/z, %): 154 (M+, 100), 126 (91), 97 (67). 4-n-Propoxythiophenol. Yield, ∼14.3%; bp/mm 112-115 °C/10 (lit (22) 117-119 °C/10). 1H NMR (CDCl3): δ 1.07 (t, J 7.5 Hz, 3H, -CH3), 1.84 (m, 2H, -CH2-), 3.34 (s, 1H, -SH), 3.91 (t, J 6.5 Hz, 2H, -CH2-), 6.83 (d, J 8 Hz, 2H, Ar), 7.27 (d, J 8 Hz, 2H, Ar). 13C NMR (CDCl3): δ 11.00 (CH3), 23.45 (CH2), 70.20 (CH2), 116.28 (C3 and C5), 128.71 (C1), 133.50 (C2 and C6), 160.92 (C4). IR: 2575.9 cm-1 (SH). GC/MS (m/z, %): 168 (M+, 45), 126 (100), 97 (22).
Verma et al. 4-Isopentoxythiophenol. Yield, ∼26.9%; mp 44-46 °C. 1H NMR (CDCl3): δ 1.04 (d, J 7.5 Hz, 6H, 2× -CH3), 1.69 (q, 2H, -CH2-), 1.87 (m, 1H, -CHCH-), 38.87 (CH2), 67.30 (CH2), 116.37 (C3 and C5), 128.36 (C1), 133.52 (C2 and C6), 160.645 (C4). IR: 2562.9 cm-1 (SH). GC/ MS (m/z, %): 196 (M+, 27), 126 (100), 97 (18). 4-n-Pentoxythiophenol. Yield, ∼22.5%; bp/mm 140-141 °C/10. 1H NMR (CDCl3): δ 0.97 (t, J 7.0 Hz, 3H, -CH3), 1.45 (m, 4H, 2× -CH2-), 1.77 (m, 2H, -CH2-), 3.37 (s, 1H, -SH), 3.93 (t, J 6.5 Hz, 2H, -CH2-), 6.82 (d, J 8.0 Hz, 2H, Ar), 7.26 (d, J 8.0 Hz, 2H, Ar). 13C NMR (CDCl3): δ 14.54 (CH3), 23.24 (CH2), 29.11 (CH2), 31.07 (CH2), 68.95 (CH2), 116.14 (C3 and C5), 128.63 (C1), 133.49 (C2 and C6), 160.87 (C4). IR: 2563.5 cm-1 (SH). GC/MS (m/z, %): 196 (M+, 22), 126 (100), 97 (10). 4-n-Hexyloxythiophenol. Yield, ∼21.27%; bp/mm 170 °C/ 10 (lit (22) 160 °C/12). 1H NMR (acetone-d6): δ 0.88 (t, J 7.5 Hz, 3H, -CH3), 1.32 (m, 4H, -CH2-CH2-), 1.45 (m, 2H, -CH2), 1.75 (m, 2H, -CH2-), 3.93 (t, J 6.5 Hz, 2H, -CH2-), 3.99 (s, 1H, -SH), 6.84 (d, J 7.8 Hz, 2H, Ar), 7.24 (d, J 7.8 Hz, 2H, Ar). 13C NMR (acetone-d ): δ 14.54 (CH ), 23.40 (CH ), 26.57 (CH ), 6 3 2 2 31.26 (CH2), 32.38 (CH2), 68.72 (CH2), 115.94 (C3 and C5), 121.24 (C1), 132.31 (C2 and C6), 158.92 (C4). IR: 2561.2 cm-1 (SH). GC/MS (m/z, %): 210 (M+, 24), 126 (100), 97 (13). 4-n-Heptyloxythiophenols. Yield, ∼31.7%; bp/mm 150-152 °C/10. 1H NMR (CDCl3): δ 0.95 (t, J 6.5 Hz, 3H, -CH3), 1.381.91 (m, 10H, 5× -CH2-), 3.38 (s, 1H, -SH), 3.93 (t, J 6.5 Hz, 2H, -CH2-), 6.82 (d, J 8.0 Hz, 2H, Ar), 7.27 (d, J 8.0 Hz, 2H, Ar). 13C NMR (CDCl3): δ 14.71 (CH3), 23.55 (CH2), 26.97 (CH2), 32.72 (CH2), 33.58 (CH2), 34.62 (CH2), 69.02 (CH2), 116.30 (C3 and C5), 128.77 (C1), 133.48 (C2 and C6), 160.57 (C4). IR: 2562.5 cm-1 (SH). GC/MS (m/z, %): 224 (M+, 22), 126 (100), 97 (12.5). (ii) General Procedure for the Synthesis of 4-Alkyl-, 4-Thiomethyl-, and 4-Iodothiophenol. 4-Neopentylthiophenol. 4-Neopentylbenzene sulfonyl chloride was prepared by the chlorosulfonation of neopentylbenzene (yield, ∼83.42%); mp 60 °C. 1H NMR (acetone-d6): δ 0.93 (s, 9H, 3× -CH3), 2.72 (s, 2H, -CH2), 7.59 (d, J 7.5 Hz, 2H, Ar), 8.04 (d, J 7.5 Hz, 2H, Ar). GC/MS (m/z, %): 246 (M+, 11), 211 (5), 90 (10), 57 (100). Concentrated sulfuric acid, 22 g (0.225 mol), was added to 70 g of ice in a 250 mL, three-necked flask, and the mixture was kept at -10° to -15 °C with an ice-salt bath while 7.9 g (0.032 mol) of 4-neopentylbenzene sulfonyl chloride was added with vigorous stirring. Then, 11 g (0.168 atom) of zinc dust (95%) was added rapidly, always maintaining the temperature at -5° to -10 °C (15 min). After it was stirred for 1.5 h at 0 °C, the mixture was allowed to warm to room temperature and finally refluxed for 5 h, after which it became a clear solution. After it was steamdistilled, the distillate was extracted with ether and dried over anhydrous magnesium sulfate. After the ether was evaporated, the residue was distilled at 113 °C (10 mm) to give 4-neopentylthiophenol (3.2 g, 56.14%); bp/mm 113 °C/10. 1H NMR (acetone-d6): δ 0.86 (s, 9H, 3× -CH3), 2.43 (s, 2H, -CH2-), 4.11 (s, 1H, -SH), 7.03 (d, J 7.8 Hz, 2H, Ar), 7.22 (d, J 7.8 Hz, 2H, Ar). 13C NMR (acetone-d6): δ 29.93 (3× -CH3), 32.68 (>C
