Nitrite-Induced Nitration Pathways of Retinoic Acid, 5,6-Epoxyretinoic

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Nitrite-Induced Nitration Pathways of Retinoic Acid, 5,6-Epoxyretinoic Acid, and Their Esters under Mildly Acidic Conditions: Toward a Reappraisal of Retinoids as Scavengers of Reactive Nitrogen Species Lucia Panzella,† Paola Manini,† Orlando Crescenzi,‡ Alessandra Napolitano,† and Marco d’Ischia*,† Department of Organic Chemistry and Biochemistry and Department of Chemistry, University of Naples “Federico II”, Via Cinthia 4, I-80126 Naples, Italy Received December 20, 2002

All trans retinoic acid (1), a cancer chemopreventive agent and a pluripotent morphogen, was found to react efficiently with nitrite ions in a biphasic system consisting of CH2Cl2/0.1 M phosphate buffer (pH 3) 1:1 v/v to give a complex mixture of nitration products. Repeated TLC fractionation of the reaction mixtures after methylation allowed isolation of the main products, which could be identified as the 12-nitro derivatives 3a,b and the decarboxylated 12,14-dinitro and 5,6-epoxy-14-nitro derivatives 4 and 5a by spectral analysis. Use of 15NO2- followed by extensive 2D NMR analysis, including 1H,15N heteronuclear multiple bond correlation experiments, allowed identification of nitronitrate derivatives as additional constituents of the mixture. Under similar conditions, 1 methyl ester gave mainly 3a,b. 5,6-Epoxyretinoic acid (2) reacted smoothly with acidic nitrite to give mainly 5a and its isomer 5b whereas its methyl ester afforded 14-nitro derivatives 9a,b as chief products. The observed patterns of reactivity along with mechanistic experiments would suggest that nitrite-induced nitration of 1 proceeds through complex reaction pathways set in motion by attack of NO2 to the 12- and 14-positions. Separate experiments showed that 1 can inhibit nitrite-induced N-nitrosation of 2,3-diaminonaphthalene at pH values of 4 and 5.5, as well as decomposition of caffeic acid under similar conditions. Overall, these results provide the first detailed insight into the reaction behavior of a retinoid toward reactive nitrogen species and shed light on previously overlooked nitrite scavenging properties of 1 of potential relevance to the mechanism of its antiinflammatory, antimutagenic, and cancer chemopreventive action.

Introduction All trans retinoic acid (1)1 is a metabolic product of vitamin A that controls important biological processes, including embryonic development, cellular proliferation and differentiation (1), and tumor growth and invasion (2, 3). The wide-ranging biological and pharmacological properties of 1 (tretinoin), the 9-Z (alitretinoin) and 13-Z (isotretinoin) isomers, and their analogues and derivatives, the retinoids, have warranted their exploitation as chemotherapeutic agents in the treatment of many types of cancer (2, 4), including liver (2), ovarian (5), and breast cancer (6). Orally administered 1 has been employed in the therapy of acute promyelocytic leukemia (7). Topical formulations of 1 are widely used for the treatment of photoaged skin and acne (8, 9), in certain proliferative skin disorders (8), and in the prevention of epithelial carcinogenesis. Retinoids also exhibit antiinflammatory properties (10) and suppress cytokine-induced production of nitric oxide (NO) in several cell types (11). * To whom correspondence should be addressed. Tel: +39-081674132. Fax: +39-081674393. † Department of Organic Chemistry and Biochemistry. ‡ Department of Chemistry. 1 All trans retinoic acid is (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraenoic acid. For the sake of simplicity, throughout this paper, the term retinoic acid (1) was preferably used.

In the past decade, the discovery of nuclear receptors for 1 and other retinoids that are involved in liganddependent transcriptional activation of target genes (12) provided an important breakthrough into the molecular basis of retinoid signaling. Yet, the highly conjugated polyene skeleton of retinoids would raise the possibility that these compounds exert at least in part their potent antiinflammatory, chemopreventive, and protective functions by scavenging reactive oxygen and nitrogen species (13) produced during inflammatory processes. Extensive data indicate that 1 and its isomers are susceptible to oxidation under a variety of conditions, e.g., by the action of prostaglandin H synthase in the presence of hydroperoxides, to give mainly the 5,6-epoxides, e.g., 2, which is also biologically active (14), along with the 4-hydroxy and the 4-oxo derivatives (15-18). The epoxides are labile to acids and undergo a peculiar rearrangement even during chromatography on silica to afford diastereoisomeric dihydrofuran derivatives referred to as 5,8-oxyretinoic acid (15, 19). Isotretinoin was also shown to inhibit lipid peroxidation in rat liver microsomes (20). Under more forcing conditions, oxidation of 1 proceeds through formation of a collection of oxygenation products including epoxides, dioxetanes, an endoperoxide, and doublebond cleavage products (19). A remarkable spin-forbidden

10.1021/tx0256836 CCC: $25.00 © 2003 American Chemical Society Published on Web 03/20/2003

Nitrite-Induced Nitration of Retinoic Acid

addition of triplet oxygen has also been described at high pressures (21).

Much less is known about the reactivity of 1 and its congeners with reactive nitrogen species derived from NO (22). These latter species are abundantly produced following macrophage activation and overactivation/overexpression of NO synthase(s) (13) and are important contributory factors in determining the overall inflammatory response. The major physiologic metabolite of NO is nitrite, which is also an environmental pollutant and an important food additive (23), responsible for mutagenic nitrosamine formation and other genotoxic processes within acidic compartments (24-27). Nitrite concentrations in vivo range from 0.5 to 3.6 µM in plasma (28), 15 µM in respiratory tract lining fluid (29), and 30210 µM in saliva (30), making nitrous acid formation likely in many tissue compartments, especially during periods of excessive nitrite production (e.g., inflammation). Different biological compartments will experience pH conditions low enough to support nitrous acid formation from nitrite in vivo. In the gastric compartment, pH varies from 2.5 to 4.5 during different stages of digestion (31) and the pH of neutrophil phagocytic vesicles has been reported to be as low as pH 3.0 (32). Moreover, tissue acidosis (pH 5.5-6.2) is typically associated with skin inflammation and sensitization during chronic allergy (33-35). Herein, we provide the first detailed study of the reaction behavior of 1, the 5,6-epoxide 2, and their esters with nitrite under mildly acidic conditions. Product analysis was carried out using a combination of 2D 1H,13C, and 15N NMR methodologies. Moreover, the ability of 1 to inhibit nitrosation reactions was also preliminarily evaluated by the DAN2 assay (36-38) and in comparison with caffeic acid, an established nitrite scavenger/antinitrosamine agent (36, 39).

Experimental Section Materials. Retinoic acid (1), caffeic acid, DAN, nitronium tetrafluoroborate (95% containing nitrosonium tetrafluoroborate), and [15N]NaNO2 (99%) were used as obtained. Compound 1 methyl ester was prepared from 1 by treatment with diazomethane. Diazomethane was obtained from N-methyl-N-nitroso-p-toluenesulfonamide by treatment with KOH/ethanol. 2 Abbreviations: DAN, 2,3-diaminonaphthalene; HMQC, heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond correlation; NOESY, nuclear Overhauser effect spectroscopy; PLC, preparative TLC; HOMO, highest occupied molecular orbital; HRMS, high-resolution mass spectrometry.

Chem. Res. Toxicol., Vol. 16, No. 4, 2003 503 Caution: Diazomethane is explosive and must be collected in peroxide-free ether in a dry ice/acetone bath and kept at -20 °C. Note that diazomethane is also toxic and carcinogenic and should be handled in a fume hood. 5,6-Epoxyretinoic acid (2) methyl ester was prepared from 1 methyl ester by treatment with m-chloroperoxybenzoic acid in dry and peroxide-free diethyl ether (40). Compound 2 was obtained by hydrolysis of its methyl ester in KOH/ethanol under an argon atmosphere (40). Methods. UV and IR spectra were performed using a Hewlett-Packard diode array spectrophotometer and a Jasco 430 FT-IR spectrophotometer, respectively. Fluorimetric measurements were performed with a fluorescence spectrophotometer (Jasco). 1H, 13C, and 15N NMR spectra were recorded with a Bruker WM 400 at 400.1, 100.6, and 40.5 MHz. The instrument was equipped with a 5 mm 1H/broadband gradient probe with inverse geometry. Gradient-selected versions of inverse (1H detected) HMQC and HMBC experiments were used. The HMBC experiments used a 100 ms long-range coupling delay. 1H,1H correlation spectroscopy, and NOESY experiments were run using standard pulse programs from the Bruker library. Chemical shifts are reported in δ values (ppm) downfield from TMS (1H and 13C NMR) or relative to NH3 (liquid, 298 K) at 0.0 ppm (15N NMR). CDCl3 was used as solvent. EI-MS spectra were obtained at 70 eV and 230 °C using a Kratos MS 50 spectrometer. Main fragmentation peaks are reported with their relative intensities (percent values are in parentheses). Analytical TLC analyses were performed on F254 0.25 mm silica gel or basic aluminum oxide plates. PLC was performed on F254 0.5 mm silica gel plates. Cyclohexanes-ethyl acetate 90:10 was used as the eluant. Column chromatography was performed using 0.063-0.200 mm silica gel and basic aluminum oxide. Griess reagent (1% sulfanilamide and 0.1% naphthylethylendiamine in 5% phosphoric acid) (41) was used for product detection on TLC plates. HPLC analyses were performed with a Gilson instrument equipped with model 305 pumps and a UV detector model 117 set at 320 nm. A dodecylsilane-coated column, 4.6 mm × 150 mm, 4 µm particle size (Synergi MAX RP 80A) was used at a flow rate of 1.0 mL/min. Acetonitrile-0.05 M formic acid 80:20 (eluant system I) and acetonitrile-0.05 M formic acid 10:90 (eluant system II) were used as the mobile phase. All operations with 1 and its derivatives, including workup of reaction mixtures, were carried out in the dark or under fluorescent light unless otherwise stated. Purity of isolated products was estimated by 1H NMR analysis. Quantum mechanical calculations were carried out using the Gaussian 01 program (42). HOMO atomic coefficients were determined at the HF/STO-3G level on the all transoid conformer of 1, optimized at the PBE0/6-31G* level. Reaction of 1, 2, and Their Methyl Esters with Nitrite. To a solution of 1, 2, or their methyl esters (0.010 mmol) in dichloromethane (2.5 mL), 0.1 M phosphate buffer (pH 3) (1:1 v/v with respect to the organic layer) was added followed by sodium nitrite (0.020 mmol) while the biphasic system was taken under vigorous stirring in a stoppered round bottom flask at room temperature. After 3 h, the organic layer was separated from the aqueous layer, which was extracted with dichloromethane; the combined organic layers were dried over sodium sulfate to give a yellow residue, which was analyzed as described below. When required, Na15NO2 was used in the reaction of 1 and the mixture was worked up as above and directly analyzed by NMR. In other experiments, the reaction of 1 was run (i) as above but with purging of the biphasic system with argon for at least 30 min prior to the addition of sodium nitrite; (ii) using nitrite varying in the range of 0.5-10 molar equivalents with respect to the substrate at 2 mM concentration; (iii) at a 100 µM substrate concentration in the presence of equimolar nitrite; (iv) as in the general procedure but using dichloromethane/0.1 M phosphate buffer (pH 5.5 or 7.4) (1:1 v/v) as the reaction medium taken at 37 °C in thermostated water bath (also in the case of

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2 methyl ester). In control experiments, the reaction was carried out under the conditions of the general procedure without added nitrite and with exposure of the reaction mixture to sunlight. The mixture was analyzed by HPLC (eluant system I) after 16 h. Reaction of 1 with NO2BF4. The reaction was carried out as described (23) with modifications. In brief, a solution of 1 (0.010 mmol) in dichloromethane (5 mL) was purged with argon and solid NO2BF4 (0.020 mmol) was added. The reaction was carried out for 3 h under an argon atmosphere at room temperature followed by the addition of water (2 mL). The organic layer was separated and dried over sodium sulfate. The residue was treated with diazomethane and analyzed by TLC. Reaction of 1 with Nitric Oxides. A solution of sodium nitrite in water (1 M) was added to 10% sulfuric acid over 10 min. The red orange gas that developed was conveyed with a flux of argon into a solution of 1 in dry dichloromethane (2 mM). Five minutes after the development of the red fumes had completed, the reaction mixture was washed with water and the organic layer was dried over sodium sulfate. The residue was treated with diazomethane and analyzed by TLC. Isolation of 3a,b, 4, and 5a,b. For preparative purposes, reaction of 1 with nitrite was carried out in dichloromethane/ 0.1 M phosphate buffer (pH 3) as described above using 400 mg of the starting material. After workup of the reaction mixture, the yellow residue (480 mg) was treated with diazomethane and purified by silica gel column chromatography (3.0 cm × 70 cm) using cyclohexanes-ethyl acetate (10:0 to 6:4, gradient mixtures) to give four main fractions. The more polar fraction (150 mg, Rf 0.1-0.2) was not purified. Fraction III (100 mg, Rf 0.2-0.4) was purified on PLC without affording homogeneous fractions. Fraction II (85 mg, Rf 0.4-0.8) was fractionated on PLC to give four main bands, Ia-d. Ia, Ic, and Id were found to consist of 3a (Rf 0.59, 12 mg, 3% yield), 5a (Rf 0.48, 10 mg, 3% yield, 80% purity), and 3b (Rf 0.42, 10 mg, 3% yield) in that order. Fraction Ib was further purified on PLC to give 4 (Rf 0.58, 3 mg, 1% yield). Fraction I (Rf 0.8-0.9) was found to consist of pure 1 methyl ester (Rf 0.84, 42 mg, 10% yield). Compounds 3a,b were also isolated (8 mg, 3% yield, and 15 mg, 5% yield, respectively) from the reaction mixture of 1 methyl ester (300 mg) following fractionation on PLC plates. Residual 1 methyl ester was recovered (30 mg, 10% yield). Each of the isomers 3a,b was exposed to nitrite ions in acid under the standard reaction conditions, and products formed were analyzed by TLC. Reaction of 2 (100 mg) with nitrite was carried out in dichloromethane/0.1 M phosphate buffer (pH 3) as described above. After workup of the reaction mixture, the yellow residue obtained was chromatographed on PLC plates to give 5a (Rf 0.48, 14 mg, 14% yield, 80% purity) and 5b (Rf 0.36, 16 mg, 16% yield, 80% purity) along with 2 methyl ester (Rf 0.68, 21 mg, 20% yield). Methyl (11E,13E)-12-Nitroretinoate (3a). UV λmax (dichloromethane): 394 nm. FT-IR (dichloromethane) νmax: 1712, 1590, 1505, 1310 cm-1. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.06 (6H, s), 1.49 (2H, m), 1.62 (2H, m), 1.74 (3H, s), 2.05 (2H, m), 2.14 (3H, s), 2.33 (3H, d, J ) 1.27 Hz), 3.77 (3H, s), 5.90 (1H, q, J ) 1.27 Hz), 6.10 (1H, d, J ) 12.4 Hz), 6.23 (1H, d, J ) 16.0 Hz), 6.60 (1H, d, J ) 16.0 Hz), 8.07 (1H, d, J ) 12.4 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm): 14.3 (CH3), 19.4 (CH3), 19.8 (CH2), 22.8 (CH3), 29.8 (2 × CH3), 34.1 (CH2), 35.1 (C), 40.4 (CH2), 52.3 (CH3), 122.9 (CH), 125.4 (CH), 131.9 (CH), 133.5 (C), 135.2 (CH), 137.1 (CH), 138.5 (C), 146.5 (C), 150.0 (C), 152.1 (C), 167.0 (C). EI-MS: m/z 359 (M+, 1), 342 (M - OH, 1), 328 (M - OCH3, 1), 313 (M - NO2, 8), 300 (M - COOCH3, 1), 254 (M - COOCH3 NO2, 3). HRMS for C21H29NO4: calcd, 359.2096; found, 359.2114. Methyl (11E,13Z)-12-Nitroretinoate (3b). UV λmax (dichloromethane): 360 nm. FT-IR (dichloromethane) νmax: 1712, 1605, 1505, 1312 cm-1. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.04 (6H, s), 1.47 (2H, m), 1.61 (2H, m), 1.72 (3H, s), 2.05 (2H, m), 2.11 (3H, s), 2.13 (3H, d, J ) 1.60 Hz), 3.64 (3H, s), 5.88 (1H, d, J ) 12.8 Hz), 6.14 (1H, q, J ) 1.60 Hz)), 6.19 (1H, d, J ) 16.0 Hz), 6.54 (1H, d, J ) 16.0 Hz), 8.01 (1H, d, J ) 12.8 Hz). 13C NMR

Panzella et al. (100 MHz, CDCl3) δ (ppm): 14.3 (CH3), 19.9 (CH2), 22.7 (CH3), 25.1 (CH3), 29.8 (2 × CH3), 34.1 (CH2), 35.1 (C), 40.4 (CH2), 52.5 (CH3), 122.3 (CH), 124.7 (CH), 130.2 (CH), 133.5 (C), 134.5 (CH), 137.2 (CH), 138.5 (C), 145.0 (C), 147.5 (C), 151.2 (C), 165.8 (C). EI-MS: m/z 359 (M+, 1), 342 (M - OH, 2), 328 (M - OCH3, 6), 313 (M - NO2, 100), 300 (M - COOCH3, 2). HRMS for C21H29NO4: calcd, 359.2096; found, 359.2080. (1E,3E,5E,7E)-2,6-Dimethyl-8-(2,6,6-Trimethyl-1-cyclohexen-1-yl)-1,3-dinitro-1,3,5,7-octatetraene (4). UV λmax (dichloromethane): 410 nm. FT-IR (dichloromethane) νmax: 1605, 1505, 1316 cm-1. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.06 (6H, s), 1.49 (2H, m), 1.63 (2H, m), 1.76 (3H, s), 2.04 (2H, m), 2.19 (3H, s), 2.42 (3H, s), 6.08 (1H, d, J ) 12.4 Hz), 6.28 (1H, d, J ) 16.0 Hz), 6.70 (1H, d, J ) 16.0 Hz), 7.03 (1H, s), 8.21 (1H, d, J ) 12.4 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm): 14.2 (CH3), 19.8 (CH2), 22.7 (CH3), 29.8 (2 × CH3), 34.1 (CH2), 35.1 (C), 40.4 (CH2), 121.3 (CH), 133.5 (C), 136.8 (CH), 138.0 (C), 140.0 (CH), 142.0 (C), 146.0 (C), 152.0 (C). EI-MS: m/z 346 (M+, 3), 329 (M - OH, 1), 316 (M - NO, 4), 300 (M - NO2, 3), 299 (M - HNO2, 4), 254 (M - 2 NO2, 4). HRMS for C19H26N2O4: calcd, 346.1892; found, 346.1875. 2,2,6-Trimethyl-1-[(1E,3E,5E,7E)-2,6-dimethyl-1-nitro1,3,5,7-octatetraen-8-yl]-7-oxabicyclo[4.1.0]heptane (5a). UV λmax (dichloromethane): 380 nm. FT-IR (dichloromethane) νmax: 1588, 1503, 1329 cm-1. 1H NMR (400 MHz, CDCl3) δ (ppm): 0.93 (3H, s), 1.05 (1H, m), 1.11 (3H, s), 1.14 (3H, s), 1.42 (1H, m), 1.45 (2H, m), 1.73 (1H, m), 1.91 (1H, m), 2.00 (3H, s), 2.43 (3H, s), 6.10 (1H, d, J ) 15.6 Hz), 6.23 (1H, d, J ) 14.8 Hz), 6.24 (1H, d, J ) 11.6 Hz), 6.32 (1H, d, J ) 15.6 Hz), 7.12 (1H, s), 7.21 (1H, dd, J ) 14.8, 11.6 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm): 14.1 (CH3), 14.5 (CH3), 17.8 (CH2), 21.8 (CH3), 26.7 (2 × CH3), 30.8 (CH2), 34.6 (C), 36.5 (CH2), 66.3 (C), 72.0 (C), 129.8 (CH), 131.2 (CH), 135.9 (CH), 136.0 (CH), 137.3 (CH), 138.0 (CH), 141.9 (C), 148.0 (C). EI-MS: m/z 317 (M+, 18), 300 (M - OH, 15), 271 (M - NO2, 20). HRMS for C19H27NO3: calcd, 317.1991; found, 317.2008. 2,2,6-Trimethyl-1-[(1Z,3E,5E,7E)-2,6-dimethyl-1-nitro1,3,5,7-octatetraen-8-yl]-7-oxabicyclo[4.1.0]heptane (5b). UV λmax (dichloromethane): 395 nm. FT-IR (dichloromethane) νmax: 1584, 1503, 1328 cm-1. 1H NMR (400 MHz, CDCl3) δ (ppm): 0.94 (3H, s), 1.08 (1H, m), 1.10 (3H, s), 1.14 (3H, s), 1.45 (2H, m), 1.48 (1H, m), 1.75 (1H, m), 1.91 (1H, m), 2.00 (3H, s), 2.08 (3H, s), 6.10 (1H, d, J ) 15.6 Hz), 6.33 (1H, d, J ) 11.2 Hz), 6.35 (1H, d, J ) 15.6 Hz), 6.91 (1H, s), 7.17 (1H, dd, J ) 15.2, 11.2 Hz), 7.73 (1H, d, J ) 15.2 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm): 14.2 (CH3), 17.8 (CH2), 19.1 (CH3), 21.8 (CH3), 26.8 (2 × CH3), 30.8 (CH2), 34.6 (C), 36.5 (CH2), 66.4 (C), 72.0 (C), 127.2 (CH), 129.3 (CH), 131.5 (CH), 134.3 (CH), 137.4 (CH), 137.5 (CH), 142.4 (C), 145.0 (C). EI-MS: m/z 317 (M+, 49), 300 (M - OH, 33), 271 (M - NO2, 26). HRMS for C19H27NO3: calcd, 317.1991; found, 317.2010. Isolation of 9a,b. For preparative purposes, reaction of 2 methyl ester with nitrite was carried out in dichloromethane/ 0.1 M phosphate buffer (pH 3) as described above using 150 mg of the starting material. After workup of the reaction mixture, the yellow residue obtained was fractionated by silica gel column cromatography (1.5 × 50 cm) using cyclohexanesethyl acetate (100:0 to 97:3, gradient mixtures). The fraction eluted with cyclohexanes-ethyl acetate 99:1 was found to consist of 2 methyl ester (45 mg, 30% yield). The fraction eluted with cyclohexanes-ethyl acetate 97:3 was subjected to repeated fractionation steps on PLC plates to give eventually 9a (Rf 0.38, 16 mg, 11% yield) and 9b (Rf 0.34, 18 mg, 12% yield, 90% purity). Methyl (13Z)-5,6-Epoxy-14-nitroretinoate (9a). UV λmax (dichloromethane): 367 nm. FT-IR (dichloromethane) νmax: 1725, 1582, 1535, 1362 cm-1. 1H NMR (400 MHz, CDCl3) δ (ppm): 0.94 (3H, s), 1.10 (1H, m), 1.11 (3H, s), 1.14 (3H, s), 1.45 (2H, m), 1.46 (1H, m), 1.76 (1H, m), 1.89 (1H, m), 2.00 (3H, s), 2.11 (3H, s), 3.83 (3H, s), 6.11 (1H, d, J ) 16.0 Hz), 6.31 (1H, d, J ) 11.2 Hz), 6.34 (1H, d, J ) 16.0 Hz), 7.22 (1H, dd, J ) 15.2, 11.2 Hz), 7.47 (1H, d, J ) 15.2 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm): 14.2 (CH3), 16.2 (CH3), 17.8 (CH2), 21.9 (CH3), 26.8 (2

Nitrite-Induced Nitration of Retinoic Acid × CH3), 30.8 (CH2), 34.6 (C), 36.5 (CH2), 53.5 (CH3), 66.5 (C), 72.0 (C), 127.5 (CH), 129.4 (CH), 131.4 (CH), 137.4 (CH), 138.0 (CH), 142.3 (C), 144.2 (C), 150.0 (C), 160.3 (C). EI-MS: m/z 375 (M+, 3), 358 (M - OH, 2), 344 (M - OCH3, 1), 329 (M - NO2, 6), 316 (M - COOCH3, 2), 270 (M - COOCH3 - NO2, 2). HRMS for C21H29NO5: calcd, 375.2046; found, 375.2059. Methyl (13E)-5,6-Epoxy-14-nitroretinoate (9b). UV λmax (dichloromethane): 367 nm. FT-IR (dichloromethane) νmax: 1725, 1582, 1534, 1363 cm-1. 1H NMR (400 MHz, CDCl3) δ (ppm): 0.93 (3H, s), 1.10 (1H, m), 1.10 (3H, s), 1.15 (3H, s), 1.45 (2H, m), 1.46 (1H, m), 1.76 (1H, m), 1.89 (1H, m), 1.99 (3H, s), 2.42 (3H, s), 3.82 (3H, s), 6.12 (1H, d, J ) 15.6 Hz), 6.21 (1H, d, J ) 11.2 Hz), 6.31 (1H, d, J ) 15.6 Hz), 6.33 (1H, d, J ) 14.8 Hz), 7.21 (1H, dd, J ) 14.8, 11.2 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm): 14.2 (CH3), 14.7 (CH3), 17.8 (CH2), 21.9 (CH3), 26.8 (2 × CH3), 30.8 (CH2), 34.6 (C), 36.5 (CH2), 53.5 (CH3), 66.5 (C), 72.0 (C), 126.9 (CH), 129.8 (CH), 131.1 (CH), 137.3 (CH), 137.9 (CH), 143.0 (C), 144.6 (C), 150.0 (C), 161.1 (C). EI-MS: m/z 375 (M+, 5), 358 (M - OH, 4), 344 (M - OCH3, 1), 329 (M - NO2, 10), 316 (M - COOCH3, 3), 270 (M - COOCH3 - NO2, 3). HRMS for C21H29NO5: calcd, 375.2046; found, 375.2031. Inhibition of N-Nitrosation of DAN by 1. The reaction was carried out as described (36) with modifications. In brief, to a solution of 1 (0-0.2 × 10-3 mmol) and DAN (0.04 × 10-3 mmol) in THF (0.100 mL), 0.05 M acetate buffer (pH 4.0) (1:1 v/v with respect to THF) was added followed by sodium nitrite (0.004 mmol). After 30 min, 0.05 M phosphate buffer (pH 7.4, 0.900 mL) was added to stop the reaction, followed by equal volumes of THF. 2,3-Naphthotriazole was quantified as described (36). In other experiments, the reaction was run as above but using THF/0.05 M acetate buffer (pH 5.5) (1:1 v/v) as the reaction medium. Kinetic Experiments. To a solution of 1 or caffeic acid (0.010 mmol) in THF (2.5 mL), 0.05 M acetate buffer (pH 3) (1:1 v/v with respect to THF) was added followed by sodium nitrite (0.040 mmol), and the reaction mixture was taken under vigorous stirring in a stoppered round bottom flask at room temperature. Aliquots of the mixture (0.100 mL) were periodically withdrawn, diluted with 2 mM NaOH (1:50 v/v) to stop the reaction, and analyzed by HPLC (eluant system I or II for 1 or caffeic acid, respectively). In other experiments, caffeic acid was reacted in the presence of 0.5, 1, or 2 molar equivalents of 1 under the above conditions and the mixtures were analyzed by HPLC (eluant system II) at 3 h reaction time.

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reaction and workup steps, with the sole exception of nitrite exposure, and was recovered virtually unchanged with no detectable isomer formation. Repeated fractionation of the reaction mixture on PLC afforded eventually some main products in a chromatographically homogeneous form amenable to spectral characterization. The products at Rf 0.42 and 0.59 (ca. 3% isolated yield each) proved isomers corresponding to the molecular formula C21H29NO4 (EI-MS, m/z 359). Both displayed in the FT-IR spectrum bands at 1505 and 1311 cm-1 due to asymmetrical and symmetrical stretching of N-O bonds of NO2 group, respectively. 1H and 13C NMR analysis indicated lack of the H-12 protons and a markedly downfield shift of the H-11 protons with respect to that of 1 (∆δ ) +0.96 for the more polar product and +1.02 for the less polar isomer). These latter appeared as doublets (J ) 12.4 Hz) correlating in the 1H,13C HMBC spectrum with quaternary carbon signals at δ 147.5/ 150.0, respectively. On these bases, the products were eventually assigned the structures of the methyl esters of (11E,13Z)-12-nitroretinoic acid (3b, more polar isomer) and (11E,13E)-12-nitroretinoic acid (3a, less polar isomer). The E configuration of the nitro-bearing double bond was apparent from the downfield shift of the relevant proton, experiencing the pronounced magnetic anisotropy of the nitro group (43). The relative geometry at the C-13 double bonds was inferred from a NOESY spectrum in which a well-detectable cross-peak was observed between the H-14 and the C-20 CH3 protons in the more polar isomer (3b) but not in the other, in accord with a Z configuration at the C-13 double bond. Furthermore, the allylic coupling constant between the H-14 and the C-20 CH3 protons in that isomer was larger as compared to the other isomer, consistent with literature data for E/Z isomeric alkenes. Additional arguments supporting these assignments came from 1H and 13C chemical shift analysis (44) and inspection of the UV absorption maxima, shifted bathochromically in the case of the E isomer.

Results and Discussion Acid-Promoted Reactions of 1 and 1 Methyl Ester with NO2-. All operations with 1 and its derivatives, including workup of reaction mixtures, were carried out in the dark or under carefully controlled conditions to avoid artifactual isomerizations or other unwanted lightinduced reactions. In a typical experiment, 1 (2 mM) was allowed to react with nitrite (4 mM) in a vigorously stirred biphasic system consisting of CH2Cl2/0.1 M phosphate buffer (pH 3) 1:1 v/v at room temperature. After about 3 h, when the starting material was substantially consumed (>90% HPLC evidence), the organic layer was separated. Unfortunately, the resolution of the chromatographic systems tested (HPLC, TLC) proved inadequate for satisfactory separation of main products. However, after treatment with diazomethane to ensure complete esterification of free carboxyl groups, TLC analysis revealed a very complex mixture of products comprising four main compounds at Rf 0.42, 0.48, 0.58, and 0.59 and an array of relatively polar components positive to the Griess test for nitrosating species (41). Lack of artifactual isomerization and other unwanted light- or workupinduced reactions was confirmed by blank experiments in which pure 1 was subjected to the same sequence of

The product at Rf 0.58 (ca. 1% isolated yield) displayed a molecular ion at m/z 346 and a proton spin system sharing the basic features with those of 3a,b, with the noticeable exception that the methoxycarbonyl functionality was lacking. Data from extensive shift correlation experiments, and the absorption maximum at 410 nm, eventually allowed us to formulate the product as the decarboxylated dinitro derivative 4. Like 4, the product at Rf 0.48 (ca. 3% isolated yield) lacked the methoxycarbonyl functionality. Diagnostic

506


Figure 1. 1H,15N HMBC spectrum of the reaction mixture of 1 with [15N]NaNO2 at pH 3.0 at 3 h reaction time. Arrows indicate cross-peaks of identified products (see text). Letters denote regions of the spectrum comprising cross-peaks due to nitronitrates.

features of the 1H NMR spectrum were a 1H singlet at δ 7.12 correlating with a carbon signal at δ 138.0 and the presence of five distinct singlets for the methyl protons, indicating a diastereotopic relationship between the methyls at C-1 due to the apparent loss of symmetry of the skeleton of 1. Relevant to this point were two crosspeaks in the 1H, 13C HMBC spectrum between the doublets at δ 6.32 and 6.10 and two telltale resonances for quaternary carbons at δ 72.0 and 66.3. EI-MS analysis indicated a molecular ion peak at m/z 317 and intense fragmentation peaks at m/z 300 and 271, suggesting losses of OH (typical of nitrogroups on unsaturated moieties) (45) and NO2. Taken together, these data allowed straightforward formulation of the product as the decarboxylated nitroepoxide 5a.

All attempts to isolate and characterize the main components of the polar Griess positive fraction were defeated, because of the marked instability during chromatographic purification. To gain an insight into the nature of these elusive species and to provide a broader inventory of the nitr(os)ation products from 1, the reaction was carried out under the same conditions using 15NO2-, and the crude mixture obtained in the organic layer was carefully taken to dryness and subjected to extensive 2D NMR analysis without preliminary methylation. The 1H,15N HMBC spectrum of the whole reaction mixture is shown in Figure 1. Straightforward analysis allowed identification of the resonances due to 3a,b free acids (cross-peaks between the resonances of the H-11 protons at δ 7.98 and 8.08, further split by coupling with 15N (J ) 3.6 Hz) and 15N resonances centered at δ 372);

Panzella et al.

4 (weak cross-peak correlating the H-11 proton resonance at δ 8.20 with a nitrogen signal at δ 371); and 5a (correlation peak between the H-14 proton resonance at δ 7.12 and a 15N resonance centered at δ 376). A complex group of resonances at δ 5.45, 5.55, and 6.25 displayed cross-peaks with a set of nitrogen signals at δ 385-395 (region A′ of Figure 1); that is, in a region typical of nitro groups linked to sp3-hybridized carbons (46). 1H,13C HMQC experiments indicated well-defined one bond connectivities between the protons resonating at δ 5.45, 5.55, and 6.25 and the carbons resonating at δ 91.5-96.5, 96.5, and 92.5, in that order, suggesting mixtures of regioisomeric and diastereoisomeric vicinal nitronitrates (46-48). In particular, the signals at δ 6.25 were ascribed to protons adjacent to nitrato functionalities, whereas those at δ 5.45-5.55 were presumably due to protons adjacent to nitro groups. These structures were possibly responsible for an intense group of cross-peaks correlating nitrogen signals in the range between δ 390410 with groups of proton resonances at δ 1.7-1.9 and δ 2.1-2.5, attributed to allylic methyl groups. The resonances at δ 1.7-1.9 showed, in turn, cross-peaks in the 1H,13C HMBC spectrum with carbon signals at δ 88-93, suggesting sp3-hybridized carbons bearing nitro groups (45, 46). Finally, small cross-peaks correlating the proton resonances at δ 5.45 and 5.55 with nitrogen signals at δ 330-335 (region A of Figure 1) corroborated the presence of nitrato functionalities (46). No definitive conclusion could be drawn about the structural moieties responsible for the intense crosspeaks correlating the nitrogen signals at δ around 406 with the methyl signals at δ 1.69 and 1.84. These latter displayed also multiple bond correlations with groups of carbon resonances at δ 88-92 and 148-152, suggesting nitrogenous groups on positions vicinal to methyl groups, e.g., nitro groups or oximes, but this remained a matter of speculation. Under the above general conditions, 1 methyl ester reacted efficiently with NO2- (ca. 90% substrate consumption after 3 h) to afford a mixture of products. Two of these, deep yellow in color, could eventually be isolated by repeated TLC fractionation and were readily assigned the structures of 3a,b (ca. 3 and 5% yield, respectively) by straightforward spectral analysis. In a separate set of experiments, the effects of various parameters on the kinetic and chemical course of the acid-promoted reaction of 1 with nitrite were investigated. Varying NO2- concentration from 1 to 20 mM, with the concentration of 1 set at 2 mM and all other conditions unchanged, resulted in appreciable effects on the extent of substrate consumption but not on product distribution. When reaction of 1 with NO2- was carried out in the same biphasic system but at pH 5.5 and at 37 °C, product analysis at various intervals of time indicated a lower substrate consumption (ca. 10% after 5 h) while product distribution was apparently unchanged. No appreciable reaction of 1 with NO2- was observed at pH 7.4 (HPLC analysis). Notably, at a concentration as low as 100 µM, 1 was found to react efficiently with 100 µM nitrite ions at pH 3 (ca. 50% substrate consumption after 3 h). The product pattern was substantially similar to that observed when the reaction was run with 1 at 2 mM concentration. When the crude reaction mixture was exposed to the medium for 3 h in the absence of nitrite, product


distribution did not vary to any appreciable extent (TLC evidence). Finally, when the reaction of 1 with nitrite was carried out in an argon atmosphere, to ensure absolute exclusion of oxygen, product analysis after the usual workup showed formation of 3a,b and 4 but not of 5a. Acid-Promoted Reactions of 2 and 2 Methyl Ester with NO2-. Reaction of 2 with acidic NO2- in the usual biphasic system at pH 3 proceeded smoothly (ca. 80% substrate consumption after 3 h) to give an array of products, two of which (molecular ion peaks at m/z 317) could be isolated by TLC fractionation after workup as in the case of 1. The less polar product proved to be 5a (ca. 14% yield), whereas the more polar one (ca. 16% yield) gave a similar 1H NMR spectrum featuring however a markedly downfield 1H doublet (δ 7.73). This latter displayed a cross-peak in the 1H,13C HMBC spectrum with a carbon resonance at δ 134.3, which, in turn, showed one bond correlation with a singlet at δ 6.91. Worthy of note was also a methyl resonance (3H, singlet) at δ 2.08 correlating with the carbon signal at δ 134.3. The close analogies of the EI-MS spectrum with that of 5a eventually led to assign the product the structure of the 13Z isomer of 5a (5b). The proposed Z configuration at the C-13 double bond would well account for the marked downfield shift of the H-12 proton, which would be exposed to the deshielding effect of the nitro group; this configuration is also in agreement with the upfield shift of the C-20 CH3 protons as compared with 5a.

Chem. Res. Toxicol., Vol. 16, No. 4, 2003 507 Table 1. Substrate Consumption by Reaction with Nitrite Ions at pH 3 and Product Yieldsa unreacted (%)

substrate 1

10

1 methyl ester

10

2

20

2 methyl ester

30

product

yield (%)

3a 3b 4 5a 3a 3b 5a 5b 9a 9b

3 3 1 3 3 5 14 16 11 12

a Determined on isolated products at 3 h reaction time after methylation when required.

Mechanistic Issues. The acidic pH conditions adopted in the present study are compatible with a mechanism involving generation and decomposition of nitrous acid (pKa ) 3.25) (49) as the event triggering nitrite-induced nitr(os)ation pathways. These reactions produce a collection of ionic, free radical, and neutral nitrogenous species (see equations below), which, once formed, can rapidly equilibrate (45, 50) and can bring attack to the polyene reactive moiety of 1.

NO2- + H+ a HNO2 HNO2 + H+ a NO+ + H2O 2HNO2 a N2O3 + H2O N2O3 a NO + NO2 2NO + O2 a 2NO2

Under similar conditions, 2 methyl ester reacted efficiently (70% consumption after 3 h) to give two main related products (molecular ion peaks at m/z 375), which were readily identified as the 14-nitro derivatives 9a,b (11 and 12% yields, respectively) differing in the configuration at the C-13 double bond. Assignment of relative configurations to each isomer was made possible by chemical shift arguments similar to those invoked for products 5a,b.

No evidence was obtained for the generation of 5,8oxyretinoic acid derivatives (15, 19) from 2. At pH 5.5 and at 37 °C, 2 methyl ester (2 mM) reacted slowly with nitrite ions (about 20% consumption after 5 h), to give the same pattern of products. Product yields and substrate consumption in the reactions of 1 and 2 and their methyl esters with nitrite ions at pH 3 are summarized in Table 1.

To assess what species is/are actually responsible for nitration of 1, nitrite was allowed to decompose in a separate solution containing 1% sulfuric acid, and the gaseous species that evolved were conveyed by a stream of argon into a flask containing a solution of 1 in anhydrous CH2Cl2. Product analysis after the usual workup and methylation indicated again formation of 3a,b and 4 but little or no 5a. Allowing air into the reaction mixture after the bubbling of gas had ceased did not affect product distribution, ruling out any involvement of oxygen in the later stages of the reaction after exposure to nitr(os)ating agent(s) or during workup. On the other hand, reaction of 1 in CH2Cl2 with nitronium tetrafluoborate (containing trace of nitrosonium salt) (23) resulted in a rapid degradation of the substrate to a complex pattern of products, none of which displayed chromatographic properties matching with those of the products formed by reaction with acidic nitrite. Overall, these experiments, although not conclusive, supported the view that volatile nitrogen oxides were the main, although possibly not the sole, nitr(os)ating species responsible for the conversion of 1. These arguments concurred to delineate a possible mechanistic scenario in which the main sequence of reactions was set in motion by free radical attack of NO2, produced by acid-induced decomposition of nitrite, onto the C-12 or C-14 positions of the polyene system of 1 (Scheme 1). Although the incomplete mass balance did not allow us to draw a detailed picture of the possible events

508


Panzella et al. Scheme 1

Table 2. π HOMO Coefficients of 1a atom C-5 C-6 C-7 C-8 C-9 C-10 C-11

atom 0.322 0.220 -0.352 -0.307 0.337 0.383 -0.240

C-12 C-13 C-14 C-15 dO -O(H)

-0.361 0.121 0.291 -0.010 -0.144 -0.012

a HF/STO-3G//PBE0/6-31G* determined on the all transoid conformer of 1.

ensued by exposure of 1 to NO2, it was apparent that positions 14 and 12 were the primary reactive sites. The preferential radical attack of NO2 to the 14-position (path b) was predictable on theoretical grounds, in view of the obvious requirement of maximum conjugative delocalization over four double bonds of the resulting β-nitroalkyl radical I. The observed involvement of the 12-position was somewhat unanticipated and raised intriguing mechanistic issues. Although this position is vinylogous to the R-position, it gives rise to a radical intermediate (II, path a) in which the unpaired electron can be delocalized over only three double bonds. This would entail that the radical stabilization due to maximum electron delocalization is not sufficient to tip the balance in favor of the 14-position and that other factors control the regiochemistry of nitration. It is possible, for instance, that sterical effects opposed by the carboxyl and methyl groups somewhat hinder access of NO2 to the R-position. Interestingly, inspection of the HOMO coefficients of 1 indicated a slightly greater HOMO coefficient on the 12position as compared to the 14-position (Table 2). This makes it possible that frontier orbitals direct at least in part the approach of soft electrophiles such as NO2 or related nitrating species to the polyene chain. However, partial charges may also be a contributory factor, in accord with the relatively high value reported for the 12position (51). The next issue, then, was the fate of the primary nitro radicals I and II. Careful HPLC analysis of the reaction

mixture during the early stages indicated formation of small amounts of isomers of 1 as those produced by light or UV exposure (52). This is likely to reflect reversion of the nitro radicals back to the reagents, whereby the polyene skeleton of 1 would be restored with partial double bond isomerization (53). Formation of 3a free acid can be rationalized in terms of coupling of the 12-nitro radical II with another molecule of NO2 to give unstable nitronitrite(s) or related species, which would lose HNO2 in the acidic reaction medium. This mechanism would be akin to that commonly accepted for nitrite-induced nitration of alkenes (54, 55). Isomerization at the C-13 double bond might then be induced by reversible addition of NO2 at the 14position of 3a free acid. Indeed, in a separate experiment, purified 3a was exposed to nitrite under the usual conditions and was found to give some 3b along with other uncharacterized products. Surprisingly enough, however, no 3a was detected among the products formed by a similar reaction run on 3b. On the other hand, 3b free acid might derive from nitration at the 12-position of the 13-Z isomer of 1. The origin of the decarboxylated products 4 and 5a is less straightforward. In principle, 4 could arise by nitration-decarboxylation of 3a free acid or by nitrationdecarboxylation of 1 via the putative 14-nitro derivative III. In both cases, decarboxylation may occur either by a heterolytic mechanism driven, for example, by loss of nitrite ion from a 1,2-nitronitrite intermediate (path b), or by homolytic fragmentation of a mixed carboxylicnitrous anhydride arising from reaction of 1 with N2O3 or a related species (path c). Formation of 5a from both 1 and 2 would suggest that 2 is an intermediate in the route from 1. However, no evidence for the formation of 2 was observed throughout the reaction course (HPLC). Alternatively, product 5a could arise by oxidation of III by a mechanism akin to that commonly accepted for the oxidative conversion of 1 to 2 (15). Although the chemistry depicted in Scheme 1 hinges on reversible homolytic attack of NO2 to the


Figure 2. Inhibition of N-nitrosation of DAN by 1 measured as fluorescence emission at 450 nm of 2,3-naphthotriazole. Shown are the mean ( SD values for two separate experiments. Compound 1 was incubated at varying concentrations in 1:1 THF/50 mM acetate buffer at pH 4.0 ([) or 5.5 (9) in the presence of DAN (0.2 mM) and NO2- (20 mM) as detailed in the Experimental Section. Relative fluorescence represents the ratio of fluorescence values measured in the presence vs those determined in the absence of the inhibitor.

polyene moiety of 1, heterolytic pathways cannot be ruled out. Nitrite-Scavenging Properties of 1. The observed reactivity of 1 toward nitrite at acidic pH values provided a basis to postulate that 1 and related retinoids could act as nitrite scavengers or antinitrosamine agents. Accordingly, the ability of 1 to inhibit nitrosation reactions was assessed by the fluorimetric DAN assay (3638) at pH 4.0, according to a reported procedure (36). Data in Figure 2 showed that 1 was quite effective at inhibiting nitrosation of DAN, causing ca. 40% inhibition at equimolar concentration. From fluorescence data, the ratio of the kinetic constants (k1/kDAN) for the reactions of 1 and DAN with nitrite could be calculated as 0.31 ( 0.05 using the equation below (36):

f/F ) 1 - k1[1]/kDAN[DAN] where f and F are the fluorescence intensities determined in the presence and in the absence of 1, respectively. A value of 8.6 × 109 M-1 s-1 (36) has been reported for the reaction of DAN with nitrite under the same experimental conditions adopted in the present study. The inhibitory effect of 1 on nitrosation of DAN was also determined at pH 5.5 (Figure 2). In the latter case, inhibition did not exceed a value of ca. 60%, with 1 at a 600 µM concentration. This is probably due to the lower generation of nitrosating species with increasing pH. Figure 3 shows the decay of 1 (2 mM) induced by 4 molar equivalents of nitrite in a homogeneous system consisting of THF/acetate buffer (pH 3) 1:1 v/v, in comparison with that of an established antinitrosamine agent, caffeic acid (36, 39). The results indicated similar rates of consumption, with 50% decay of both substrates at 90 min reaction time. A 50% inhibition of caffeic acid decay was consistently observed in the presence of equimolar amounts of 1 (data not shown). Conclusions. This study provides the first systematic inventory of products formed by reaction of 1 with reactive nitrogen species. Chemical highlights included

Chem. Res. Toxicol., Vol. 16, No. 4, 2003 509

Figure 3. Decay of 1 (O) and caffeic acid (b) in the presence of acidic nitrite. Substrates (2 mM) were exposed separately to NO2- (8 mM) in 1:1 THF/50 mM acetate buffer (pH 3.0); all other reaction conditions are as detailed in the Experimental Section. Shown are the mean ( SD values for three separate experiments.

the unexpected reactivity of 1 at the 12-position and the marked facility to decarboxylation following nitration at C-14. Besides the chemical interest, these results may offer a novel basis for biologically and pharmacologically oriented research aimed at inquiring into the mechanism of action of retinoids. The concentrations used in this work were much higher than those encountered under normal physiological or pharmacological conditions, to ensure sufficient product formation for isolation and structural characterization. Yet, experiments with lower substrate concentrations (e.g., 0.1 mM) support the same pattern of reactivity. Moreover, nitrite-induced conversions of 1 and 2 occur over a relatively broad pH range, up to 5.5, which may warrant investigation of the underlying chemistry in suitable biological models. The recent report of the chemopreventive potential of 1 and β-carotene against a diethylnitrosamine-initiated and phenobarbital-promoted experimental model of hepatocarcinogenesis (56) seems relevant in this perspective.

Acknowledgment. This work was carried out in the frame of a research program on antitumor agents sponsored by MURST (PRIN 2001). We thank the “Centro Interdipartimentale di Metodologie Chimico-Fisiche of Naples University” for NMR facilities and the “Servizio di Spettrometria di Massa del CNR e dell’Universita` di Napoli” for mass spectra. Supporting Information Available: 1H NMR, 1H,13C HMQC, 1H,13C HMBC, and NOESY spectra of compounds 3a,b. 1H NMR spectrum and 1H,13C HMBC spectrum of compound 4. 1H NMR, 1H,13C HMQC and 1H,13C HMBC spectra of compounds 5a,b and 9a,b. 1H NMR, 1H,13C HMQC, 1H,13C HMBC and 1H,15N HMBC spectra of 15N-labeled reaction mixture of 1. This material is available free of charge via the Internet at http://pubs.acs.org.

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Nitrite-Induced Nitration Pathways of Retinoic Acid, 5,6-Epoxyretinoic

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