Conformationally restrained fentanyl analogues. 2. Synthesis and analgetic evaluation of perhydro-1,6-naphthyridin-2-ones.

Conformational flexibility of the N-acyl portion of fentanyl-type analgetics was restricted through the synthesis of novel perhydro-1,6-naphthyridin-2-one derivatives. Neither the cis-fused derivative (5a), the trans-fused derivative(5b), nor the enamide 8a possessed analgetic activity in the mouse tail-flick assay, reaffirming the sensitivity of this portion of 4-anilidopiperidine analgetics to conformational restraint.

DNA adduct formation and mutation induction by nitropyrenes in Salmonella and Chinese hamster ovary cells: relationships with nitroreduction and acetylation.

Nitrated pyrenes are environmental pollutants and potent mutagens in the Salmonella reversion assay. In this study reversion induction by 1-nitropyrene and 1,8-dinitropyrene in Salmonella typhimurium TA1538 and mutation induction by 1-nitropyrene in Chinese hamster ovary (CHO) cells were related to the extent of metabolism and DNA adduct formation. In suspension cultures of Salmonella typhimurium TA1538, 1,8-dinitropyrene was up to 40-fold more mutagenic than 1-nitropyrene, although both compounds were metabolized at similar rates with nitroreduction being the major pathway. The major metabolite formed from 1-nitropyrene after 2 hr of incubation was 1-nitrosopyrene, while 1-amino-8-nitropyrene was the major metabolite formed from 1,8-dinitropyrene. 1-Nitrosopyrene and 1-nitro-8-nitrosopyrene elicited mutation values consistent with their being intermediates in the activation pathways. However, subsequent to nitroreduction, 1,8-dinitropyrene appeared to be further activated by acetylation, while 1-nitropyrene was not. Each nitrated pyrene produced a major DNA adduct substituted at the C8-position of deoxyguanosine. Although 1,8-dinitropyrene was more mutagenic than 1-nitropyrene, both compounds induced a similar number of revertants per adduct. Incubation of 1-nitrosopyrene with CHO cells produced a rapid concentration- and time-dependent induction of mutations and the conversion of 1-nitrosopyrene to 1-aminopyrene. In contrast, 1-nitropyrene did not induce mutations and was not converted to 1-aminopyrene. Both compounds produced the same major adduct, but adduct formation by 1-nitropyrene was much lower than by 1-nitrosopyrene.(ABSTRACT TRUNCATED AT 250 WORDS)

High-pressure liquid chromatographic identification of drugs used in management of arthritis.

A screening procedure was developed for the identification of drugs used in the clinical treatment of arthritis. Each glucocorticoid, nonsteroidal anti-inflammatory agent, or tranquilizer was characterized by its retention on a reversed-phase high-pressure liquid chromatographic column and by the ratio of the response of dual UV detectors (254 and 280 nm). Although the retention times of all 14 drugs examined were less than 4 min. each drug could be distinguished easily from the other drugs in the series.

Molecular structure of two conformationally restrained fentanyl analogues: cis- and trans-isomers of N-(3-methyl-1-[2-(1,2,3,4-tetrahydro)naphthyl] -4-piperidinyl)-N-phenylpropanamide.

The X-ray crystallographic structures of two analogues of the potent analgetic fentanyl in which the N-phenethyl substituent is restrained through incorporation into a tetrahydronaphthyl ring system are reported. The tetrahydronaphthyl moiety exists in an equatorial conformation with respect to the piperidine ring which exists in the chair conformation. The propanilido group is also equatorial and antiperiplanar. The orientation of the N-phenyl group with respect to the N-acyl moiety is essentially invariant with an approximately 90 degrees dihedral angle. The implications of these conformations in the interaction of fentanyl-type analgetics with opiate receptors are discussed. The following data were obtained: cis-[C25H33N2O+]Cl- . 1/2 C2O4H2, triclinic, P-1; a = 7.005(7), b = 13.189(2), c = 14.312(4)A, alpha = 111.27(2), beta = 99.15(5), gamma = 93.52(4)0, V = 1205.9A, Z = 2, T = 110K, R = 0.0596, Rw = 0.0760; trans-[C25H33N2O+] C2O4H- . 1/2 C2O4H2 . 2 H2O, triclinic, P-1, a = 8.235(6), b = 10.546(8), c = 17.108(9)A, alpha = 107.72(5), beta = 95.73(5), gamma = 90.63(6)0, V = 1406.8A3, Z = 2, T = 110K, R = 0.0737, Rw = 0.0934.

Acetyl coenzyme A-dependent binding of carcinogenic and mutagenic dinitropyrenes to DNA.

Dinitropyrenes are mutagenic and carcinogenic environmental pollutants found in diesel emissions and urban air particulates. In Salmonella typhimurium these compounds appear to be activated to mutagens by sequential nitroreduction and acetylation. We have examined whether or not similar activation pathways occur with mammalian nitroreductases and acetylases. When rat liver cytosol, NADPH and calf thymus DNA were incubated with [4,5,9,10(-3)H]1-nitropyrene, [4,5,9,10(-3)H]1,3-, 1,6- or 1,8-dinitropyrene very low levels of nitrated pyrene binding with DNA were detected. Addition of acetyl coenzyme A (AcCoA) to these incubations increased the binding of dinitropyrenes 20- to 40-fold while the binding of 1-nitropyrene was not affected. The extent of AcCoA-dependent binding of dinitropyrenes reflected the amount of nitroreduction, as measured by aminonitropyrene formation. However, the increase in binding of dinitropyrenes to DNA in the presence of AcCoA did not occur with dog liver cytosol which is known to be deficient in N-acetylases. These results suggest that cytosolic nitroreductases catalyze the formation of N-hydroxy arylamine intermediates which in the case of dinitropyrenes are converted to reactive N-acetoxy arylamines by cytosolic AcCoA-dependent acetylases.

Synthesis and mutagenicity of 1-nitro-6-nitrosopyrene and 1-nitro-8-nitrosopyrene, potential intermediates in the metabolic activation of 1,6- and 1,8-dinitropyrene.

1,6-Dinitropyrene and 1,8-dinitropyrene are environmental contaminants which are mutagenic in bacteria and cultured mammalian cells. Since nitroreduction, and possibly O-acetylation, have been implicated in the metabolic activation of these compounds, the reduced intermediates, 1-nitro-6-nitrosopyrene and 1-nitro-8-nitrosopyrene, were synthesized and their mutagenicity examined in Salmonella typhimurium and Chinese hamster ovary (CHO) cells. Nitration of 1-acetylaminopyrene yielded a mixture of 1-acetylamino-6-nitropyrene and 1-acetylamino-8-nitropyrene, which was separated by flash chromatography. Following deacetylation, the amino-nitropyrenes were oxidized to the desired nitronitrosopyrenes with m-chloroperoxybenzoic acid. Both nitronitrosopyrenes showed similar levels of mutagenicity in S. typhimurium strain TA98 and a nitroreductase-deficient analogue, TA98NR, but much lower activity in the esterificase-deficient strain, TA98/1,8-DNP6, which suggested that reduced metabolites require further activation by O-acetylation. In contrast, the analogous compound, 1-nitrosopyrene, was equally mutagenic in all three strains while its parent compound, 1-nitropyrene, demonstrated a much reduced mutagenicity in strain TA98NR. In CHO cells, 1-nitropyrene was not mutagenic and the dinitropyrenes were only weakly active, while all three nitrosopyrene derivatives were highly mutagenic. These data support the hypothesis that nitrated pyrenes are metabolized to mutagens through nitroreduction. In Salmonella the limiting step in the metabolic activation of 1-nitropyrene appears to be the initial reduction to 1-nitrosopyrene, while with the dinitropyrenes subsequent esterification of the reduced intermediates seems critical. With CHO cells, the initial reduction to nitroso derivatives is the limiting step for all nitropyrenes, and esterification does not appear to occur in the activation sequence.

Metabolism of 1-nitropyrene in germ-free and conventional rats.

The distribution, covalent binding and metabolism of radioactive 1-nitropyrene (1-NP) were examined following its oral administration to conventional and germ-free male Wistar rats. With both groups of animals, the liver, kidney, bladder, adipose tissue and gastrointestinal tract had the highest specific radioactivity. However, the maximum concentration of radioactivity occurred at 12 hr in conventional rats as compared to 24 hr in germ-free animals. This difference may be due to the faster transit time of the intestinal contents through conventional rats. At 48 hr after treatment, the covalent binding of 1-NP metabolites was greatest in liver and kidney of conventional rats, while in germ-free rats, substantial binding was also found in the gastrointestinal tract. The mutagenic activity in Salmonella typhimurium TA98 of fecal extracts and urine from conventional rats was greater in the presence of an S9 mix, whereas similar extracts from germ-free animals were more mutagenic in the absence of S9. The major fecal metabolites in germ-free rats were (in order of decreasing concentration): 3-nitropyrenol greater than 1-NP greater than 4,5-dihydroxy-4,5-dihydro-1-NP greater than 6-nitropyrenol greater than 8-nitropyrenol. With the exception of 1-NP, similar metabolites were found in the urine as their glucuronide conjugates. In the feces from conventional rats, substantial nitro reduction and N-acetylation occurred with the major metabolites being: 1-NP greater than 1-aminopyrene greater than 8-acetylaminopyrenol greater than 6-acetylaminopyrenol greater than 3-acetylaminopyrenol. The major metabolites identified in the urine from conventional rats were glucuronide conjugates of 6- and 8-acetylaminopyrenol, while the major biliary conjugates identified were glucuronide conjugates of 4,5-dihydroxy-4,5-dihydro-1-NP and 3-, 6-, and 8-nitropyrenol, although the relative proportion of glucuronide conjugates of 6- and 8-aminopyrenol and 6- and 8-acetylaminopyrenol increased in later stages of the biliary excretion. The polar and beta-glucuronidase-refractory metabolites, which may be sulfate and glutathione conjugates, remain to be identified.

Oxidative microsomal metabolism of 1-nitropyrene and DNA-binding of oxidized metabolites following nitroreduction.

1-Nitropyrene is an environmental mutagen and carcinogen which undergoes both oxidative and reductive metabolism. We have previously shown that nitroreduction to N-hydroxy-1-aminopyrene leads to the formation of arylamine--DNA adducts. In the present study, we have investigated the oxidative metabolism of 1-nitropyrene and the subsequent binding of ring-oxidized metabolites to DNA. In vitro incubations were conducted using hepatic microsomes from uninduced rats or from rats pretreated with phenobarbital, Aroclor 1254, 3-methylcholanthrene, or 3-methylcholanthrene and trans-stilbene oxide. H.p.l.c. analysis of the incubation mixtures indicated the presence of the previously reported metabolites, 1-aminopyrene, 3-, 6-, and 8-hydroxy-1-nitropyrene, and 1-nitropyrene trans-4,5-dihydrodiol. In addition, 1-nitropyrene 4,5-oxide, 1-nitropyrene 9,10-oxide, 1-nitropyrene trans-9,10-dihydrodiol and 1-pyrenol were identified. The formation of both K-region dihydrodiols could be increased by trans-stilbene oxide induction of microsomal epoxide hydrase. Formation of the K-region epoxides was greatest using phenobarbital- and Aroclor-induced microsomes and increased with increasing oxygen tension, while 1-pyrenol formation was highest in 3-methylcholanthrene-induced microsomal incubations and was not affected by the oxygen tension. When calf thymus DNA was added to the microsomal incubations, similar levels of DNA-binding occurred in incubations conducted under oxygen, air, argon or anaerobic conditions. H.p.l.c. analysis of the enzymatically hydrolyzed DNA indicated the presence of multiple DNA adducts with the major product coeluting with N-(deoxyguanosin-8-yl)-1-aminopyrene. The K-region oxides bound directly to DNA to give adducts similar to the minor products detected in the microsomal incubations. Incubation of the K-region oxides with the nitroreductase, xanthine oxidase, increased the DNA-binding and resulted in an additional adduct which coeluted with N-(deoxyguanosin-8-yl)-1-amino pyrene. 3-Hydroxy-1-nitropyrene bound extensively to DNA upon nitroreduction by rat liver cytosol or xanthine oxidase, while 6- and 8-hydroxy-1-nitropyrene bound only slightly. None of these oxidized metabolites was activated to DNA-binding species by cytosolic nitroreduction followed by AcCoA-dependent acetylation. The fact that oxidized metabolites of 1-nitropyrene are reduced to DNA-binding derivatives more easily than 1-nitropyrene itself may be important in vivo where 1-nitropyrene has been shown to be readily oxidized.

Synthesis and mutagenicity of 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide, microsomal metabolites of 1-nitropyrene.

[4,5,9,10-(3)H]1-Nitropyrene was incubated with liver microsomes prepared from guinea pigs treated with Aroclor 1254 and the products were examined by h.p.l.c. The previously reported metabolites, 1-nitropyrene trans-4,5-dihydrodiol, 1-nitropyrene trans-9,10-dihydrodiol, and 3-, 6-, and 8-hydroxy-1-nitropyrene were detected. In addition, h.p.l.c., nuclear magnetic resonance and mass spectral analyses indicated the presence of 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide. The epoxide hydrase inhibitor, 1,2-epoxy-3,3,3-trichloropropane, decreased the concentration of the 4,5- and 9,10-dihydrodiols in the microsomal incubations and increased the concentration of their corresponding oxides. Reaction of 1-nitropyrene with m-chloroperoxybenzoic acid gave a mixture of 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide, which was separated by chromatography. The mutagenicity of the oxides was determined in Salmonella typhimurium strains TA98, TA98NR, and TA98/1,8-DNP6, both with and without exogenous activation by a rat liver homogenate fraction (S9). In the absence of S9, both oxides showed maximum activity in TA98, slightly decreased mutagenicity in the acetylase-deficient strain TA98/1,8-DNP6, and much reduced activity in the nitroreductase-deficient strain, TA98NR. When assayed in the presence of S9, 1-nitropyrene 4,5-oxide had maximum mutagenicity in TA98, and was 50 and 95% less mutagenic in TA98NR and TA98/1,8-DNP6, respectively. 1-Nitropyrene 9,10-oxide had a similar strain sensitivity, except that its total mutagenicity was lower. Since 1-nitropyrene is metabolized by oxidative pathways in vivo, these K-region oxides may contribute to the toxicities elicited by this compound.

In vivo and in vitro formation of glutathione conjugates from the K-region epoxides of 1-nitropyrene.

4,5-Epoxy-4,5-dihydro-1-nitropyrene (1-nitropyrene 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-nitropyrene 9,10-oxide), which are electrophilic metabolites formed during the metabolism of the environmental pollutant, 1-nitropyrene, reacted slowly with glutathione. The rate of conjugation was greatly enhanced by the addition of purified rat liver glutathione (GSH) transferases, with transferases 3-3 and 4-4 exhibiting higher catalytic activities than transferases 1-1, 2-2 and 7-7. Two GSH conjugates were formed from each of the oxides: 1-nitropyrene 4,5-oxide gave a 1:1 mixture of 4-(glutathion-S-yl)-5-hydroxy-4,5-dihydro-1-nitropyrene and 5-(glutathion-S-yl)-4-hydroxy-4,5-dihydro-1-nitropyrene while 1-nitropyrene 9,10-oxide gave a 2:1 mixture of 9-(glutathion-S-yl)-10-hydroxy-9,10-dihydro-1-nitropyrene and 10-(glutathion-S-yl)-9-hydroxy-9,10-dihydro-1-nitropyrene. Both K-region oxides were converted to trans-dihydrodiols by hepatic microsomal epoxide hydrase, and faster rates were observed with 1-nitropyrene 4,5-oxide. In subsequent experiments [4,5,9,10-3H]1-nitropyrene was administered to Sprague-Dawley rats by intravenous and intraperitoneal injections. HPLC analysis of biliary metabolites indicated the presence of four GSH conjugates that were identical to those obtained from reactions of the K-region oxides with GSH. In addition, glucuronide conjugates were detected from trans-4,5-dihydroxy-4,5-dihydro-1-nitropyrene (1-nitropyrene trans-4,5-dihydrodiol) but not trans-9,10-dihydroxy-9,10-dihydro-1-nitropyrene (1-nitropyrene trans-9,10-dihydrodiol). These data combined with earlier studies indicate that 1-nitropyrene is oxidized preferentially to 1-nitropyrene 4,5-oxide and that, while the main detoxification pathway of 1-nitropyrene 9,10-oxide is GSH conjugation, 1-nitropyrene 4,5-oxide is excreted via both GSH conjugation and dihydrodiol formation followed by O-glucuronidation.

DNA binding by 1-nitropyrene and 1,6-dinitropyrene in vitro and in vivo: effects of nitroreductase induction.

1-Nitropyrene, the predominant nitropolycyclic aromatic hydrocarbon found in diesel exhaust, is a mutagen and tumorigen. 1,6-Dinitropyrene is present in diesel exhaust in much smaller quantities than 1-nitropyrene, but is much more mutagenic and carcinogenic. In an attempt to understand this difference in biological potencies, we have compared the extent of DNA binding by these two nitropyrenes in vivo. We have also determined the effect of 1-nitropyrene pretreatment upon the induction of nitroreductases and the subsequent DNA binding by both 1-nitropyrene and 1,6-dinitropyrene. Covalent DNA binding by 1-nitropyrene could not be detected in vivo; however, 1,6-dinitropyrene formed N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene as the major DNA adduct in rat liver, kidney, urinary bladder and mammary gland, with the highest levels being found in the bladder. The capability of liver microsomes to catalyze the oxidative metabolism of 1-nitropyrene was unchanged after treating rats with 8 mg/kg 1-nitropyrene. Cytochrome P-450, NADPH-cytochrome P-450 reductase and cytochrome b5 levels were also unchanged, while slight increases were detected in NADH-cytochrome b5 reductase and epoxide hydrase activities. Liver cytosolic and microsomal nitroreductase activities toward both 1-nitropyrene and 1,6-dinitropyrene were increased 2-fold, and cytosolic nitrosoreductase activity toward 1-nitrosopyrene and 1-nitro-6-nitrosopyrene was elevated by approximately 20%. DNA binding of both 1-nitropyrene and 1,6-dinitropyrene in vitro was 2-fold higher when using cytosol from 1-nitropyrene-pretreated rats. However, pretreatment of rats with 1-nitropyrene only slightly increased the amount of in vivo DNA binding by 1,6-dinitropyrene except in kidney where there was a 60% increase. These results indicate that although nitroreduction is involved in DNA adduct formation by 1,6-dinitropyrene, additional factors (e.g. O-acetylation) limit the extent of DNA binding in vivo.

Potential clinical use of butyric acid derivatives to induce antigen-specific T cell inactivation.

Compounds with the capacity to induce antigen-specific unresponsiveness in CD4(+) T cells can in some clinical situations be more beneficial than general immune suppressants. Newly synthesized ester, ester/amide, and amide derivatives of butyrate with the capacity to induce antigen-specific T cell unresponsiveness in vivo and in vitro were tested here. The ester and ester/amide derivatives of butyrate were shown to block proliferation by interleukin-2-stimulated murine Th1 cells in vitro. A 3-day treatment with these same two derivatives also suppressed a primary antibody response to a thymus-dependent antigen in mice. In addition, even a single injection of the ester derivative of n-butyrate 2-(4-morpholinyl)ethyl butyrate hydrochloride (MEB) on day 2 or 3 after immunization suppressed the generation of memory T cells capable of proliferating to antigen or of promoting a secondary antigen-specific antibody response. MEB also induced antigen-specific unresponsiveness in antigen-activated, but not resting or interleukin-2-activated, T cells in vitro. DNA analysis showed that regardless of when MEB was added to the cultures, it induced the eventual G(1) sequestration of essentially all activated Th1 cells. Because G(1) blockade is associated with Th1 cell anergy, this finding suggests that MEB has the potential to induce anergy in already-activated CD4(+) T cells. Taken together, the results presented here establish MEB as a novel means of inducing anergy in CD4(+) T cells both in vitro and in vivo and underscore the likelihood that MEB and/or other butyrate derivatives can be used as immunotherapeutic reagents.

High-performance liquid chromatography determination of residue levels on chicken carcasses treated with cetylpyridinium chloride.

Cetylpyridinium chloride (CPC) has been found to be effective in reducing contamination of chicken carcasses from a variety of microorganisms, including Escherichia coli O157:H7, Salmonella typhimurium, Campylobacter jejuni, Aeromonas hydrophila, Listeria monocytogenes, and Staphylococcus aureus. A procedure has been developed to determine residue levels on chicken carcasses after CPC treatment. For the analysis, chicken carcasses were extracted with 95% ethanol. The CPC concentration in the extract was measured by high-performance liquid chromatography (HPLC) with ultraviolet detection using dodecylpyridinium chloride (DPC) as an internal standard. The method was validated in the concentration range of 3-200 microg/ml CPC in ethanolic extract. This assay is rapid, precise, and accurate.

Effect of the nitro group conformation on the rat liver microsomal metabolism and bacterial mutagenicity of 2- and 9-nitroanthracene.

The aerobic and hypoxic metabolism of 2-nitroanthracene (2-NA) and 9-nitroanthracene (9-NA), two components of diesel exhaust, was studied and the mutagenicities of the parent compounds and their metabolites were compared. 2-NA was metabolized by 3-methylcholanthrene-induced rat liver microsomes under aerobic conditions to 2-NA trans-5,6-dihydrodiol, 2-NA trans-7,8-dihydrodiol, 2-NA 7-keto-5,6,7,8-tetrahydro-trans-5,6-diol, 2-NA 6-keto-5,6,7,8-tetrahydro-trans-7,8-diol, 2-nitro-9,10-anthraquinone and 2-NA 5,6,7,8-tetrahydrotetrol. When incubations were conducted under hypoxic conditions, 2-aminoanthracene was produced facily. N.m.r. spectral analysis indicated that the nitro-substituent of 2-NA and all of its ring-oxidized metabolites preferentially adopted an orientation in which the nitro group was coplanar or nearly co-planar with the aromatic ring system. 2-NA and its two trans-dihydrodiol metabolites were mutagenic in Salmonella typhimurium strain TA98, both in the presence and in the absence of S9 enzymes while the two tetrahydrodiol-ketones were much less mutagenic. When assayed in strains TA98NR and TA98/1,8-DNP6, the mutagenic activities of 2-NA and the trans-7,8-dihydrodiol were decreased. 2-Aminoanthracene was mutagenic in strain TA98 only in the presence of S9 enzymes. When 2-aminoanthracene was metabolized aerobically, the corresponding trans-5,6- and 7,8-dihydrodiols were not detected. These results suggest that 2-NA can be metabolized to mutagenic products by nitroreduction and ring-oxidation followed by nitroreduction, but not nitroreduction followed by ring-oxidation. Aerobic metabolism of 9-NA produced 9-NA trans-1,2- and 3,4-dihydrodiols, while metabolism was not detected under anaerobic conditions. Previous studies indicated that 9-NA and its two metabolites were not mutagenic in TA98. The differences in the orientation of the nitro substituents in 2-NA and its ring-oxidized metabolites and in 9-NA and its metabolites can be employed to explain the strong mutagenicity of 2-NA and weak mutagenicity of 9-NA when assayed both in the absence and in the presence of S9 activation enzymes.