Stereoselectivity in metabolism of ifosfamide by CYP3A4 and CYP2B6

The aim was to identify the hepatic cytochromes P450 (CYPs) responsible for the enantioselective metabolism of ifosfamide (IFA). The 4-hydroxylation, N2- and N3-dechloroethylation of IFA enantiomers were monitored simultaneously in the same metabolic systems using GC/MS and pseudoracemate techniques. In human and rat liver microsomes, (R)-IFA was preferentially metabolized via 4-hydroxylation, whereas its antipode was biotransformed in favour of N-dechloroethylation. CYP3A4 was the major enzyme responsible for metabolism of IFA enantiomers in human liver. The study also revealed that CYP3A (human CYP3A4/5 and rat CYP3A1/2) and CYP2B (human CYP2B6 and rat CYP2B1/2) enantioselectively mediated the 4-hydroxylation, N2- and N3-dechloroethylation of IFA. CYP3A preferentially supported the formation of (R)-4-hydroxyIFA (HOIF), (R)-N2-dechloroethylIFA (N2D) and (R)-N3-dechloroethylIFA (N3D), whereas CYP2B preferentially mediated the generation of (S)-HOIF, (S)-N2D and (S)-N3D. The enantioselective metabolism of IFA by CYP3A4 and CYP2B1 was confirmed in cDNA transfected V79 cells.     

3'-Intercalation of a N2-dG 1R-trans-anti-benzo[c]phenanthrene DNA adduct in an iterated (CG)3 repeat

The conformation of the 1 R,2 S,3 R,4 S-benzo[ c]phenanthrene- N (2)-dG adduct, arising from trans opening of the (+)-1 S,2 R,3 R,4 S- anti-benzo[ c]phenanthrene diol epoxide, was examined in 5'- d(ATCGC XCGGCATG)-3'.5'-d(CATGCCG CGCGAT)-3', where X = 1 R,2 S,3 R,4 S-B[ c]P- N (2)-dG. This duplex, derived from the hisD3052 frameshift tester strain of Salmonella typhimurium, contains a (CG) 3 iterated repeat, a hotspot for frameshift mutagenesis. NMR experiments showed a disconnection in sequential NOE connectivity between X (4) and C (5), and in the complementary strand, they showed another disconnection between G (18) and C (19). In the imino region of the (1)H NMR spectrum, a resonance was observed at the adducted base pair X (4) x C (19). The X (4) N1H and G (18) N1H resonances shifted upfield as compared to the other guanine imino proton resonances. NOEs were observed between X (4) N1H and C (19) N (4)H and between C (5) N (4)H and G (18) N1H, indicating that base pairs X (4) x C (19) and C (5) x G (18) maintained Watson-Crick hydrogen bonding. No NOE connectivity was observed between X (4) and G (18) in the imino region of the spectrum. Chemical shift perturbations of greater than 0.1 ppm were localized at nucleotides X (4) and C (5) in the modified strand and G (18) and C (19) in the complementary strand. A total of 13 NOEs between the protons of the 1 R-B[ c]Ph moiety and the DNA were observed between B[ c]Ph and major groove aromatic or amine protons at base pairs X (4) x C (19) and 3'-neighbor C (5) x G (18). Structural refinement was achieved using molecular dynamics calculations restrained by interproton distances and torsion angle restraints obtained from NMR data. The B[ c]Ph moiety intercalated on the 3'-face of the X (4) x C (19) base pair such that the terminal ring of 1 R-B[ c]Ph threaded the duplex and faced into the major groove. The torsion angle alpha' [X (4)]-N3-C2-N2-B[ c]Ph]-C1 was calculated to be -177 degrees, maintaining an orientation in which the X (4) exocyclic amine remained in plane with the purine. The torsion angle beta' [X (4)]-C2-N2-[B[ c]Ph]-C1-C2 was calculated to be 75 degrees. This value governed the 3'-orientation of the B[ c]Ph moiety with respect to X (4). The helical rise between base pairs X (4) x C (19) and C (5) x G (18) increased and resulted in unwinding of the right-handed helix. The aromatic rings of the B[ c]Ph moiety were below the Watson-Crick hydrogen-bonding face of the modified base pair X (4) x C (19). The B[c]Ph moiety was stacked above nucleotide G (18), in the complementary strand.     

Synthesis and photobiological activity of new N,N-disubstituted 4-amino-3-chloroangelicins

The synthesis of a series of new N,N-disubstituted 4-amino-3-chloroangelicins (4-amino-3-chloro-2H-furo[2,3-h]-1-benzopyran-2-ones) starting from N,N-disubstituted (E)-5-aminomethylene-6,7-dihydrobenzo[b]furan-4(5H)-ones is described. Their capacity of inhibiting DNA synthesis in Ehrlich ascites tumor cells by U.V.-A light irradiation as well as their phototoxic activity on guinea-pig skin have been studied and the data computed by probit analysis; the results are discussed in the light of the modifications introduced in the molecular structure.     

Design and synthesis of new N1 and C3-substituted 4-fluoroindolic melatoninergics

A series of new C-3 and N1-substituted 4-fluorotryptamides have been prepared and tested for their ability to activate pigment granule aggregation in Xenopus laevis melanophores and bind to the recombinant human MT(1) and MT(2) melatonin receptor subtypes expressed in NIH 3T3 cells. Planar sp(2) geometry at C-3-betaC seems to decrease the population of the preferred conformation as it renders 4-fluoroindoles 4b-d weaker antagonists than their C-3-betaC-unsubstituted congeners 3a-e. This effect is not preclusively linked with the C-3 region, as the same geometry around N1 (compounds 5a-c) similarly leads to weak antagonistic action. Last, the new C-3 substituted 4-fluorotryptamides presented herein are substantially more potent than their respective N-OMe functionalized congeners, previously reported.     

Stereoselective metabolism of ifosfamide by human P-450s 3A4 and 2B6. Favorable metabolic properties of R-enantiomer.

The anticancer prodrug ifosfamide (IFA) contains a chiral phosphorous atom and is administered clinically as a racemic mixture of R and S enantiomers. Animal model studies and clinical data indicate enantioselective differences in cytochrome P-450 (CYP) metabolism, pharmacokinetics, and therapeutic efficacy between the two enantiomers; however, the metabolism of individual IFA enantiomers has not been fully characterized. The role of CYP enzymes in the stereoselective metabolism of R-IFA and S-IFA was investigated by monitoring the formation of both 4-hydroxy (activated) and N-dechloroethyl (DCl) (inactive, neurotoxic) metabolites. In the 4-hydroxylation reaction, cDNA-expressed CYPs 3A4 and 3A5 preferentially metabolized R-IFA, whereas CYP2B6 was more active toward S-IFA. Enantioselective IFA 4-hydroxylation (R > S) was observed with six of eight human liver samples. In the N-dechloroethylation reaction, CYPs 3A4 and 2B6 both catalyzed a significantly higher intrinsic metabolic clearance (V(max)/K(m)) of S-IFA compared with R-IFA. Striking P-450 form specificity in the formation of individual DCl metabolites was evident. CYPs 3A4 and 3A5 preferentially produced (R)N2-DCl-IFA and (R)N3-DCl-IFA (derived from R-IFA and S-IFA, respectively), whereas CYP2B6 correspondingly formed (S)N3-DCl-IFA and (S)N2-DCl-IFA. In human liver microsomes, the CYP3A-specific inhibitor troleandomycin suppressed (R)N2- and (R)N3-DCl-IFA formation by >/=80%, whereas (S)N2- and (S)N3-DCl-IFA formation were selectively inhibited (>/=85%) by a CYP2B6-specific monoclonal antibody. The overall extent of IFA N-dechloroethylation varied with the CYP3A4 and CYP2B6 content of each liver, but was significantly lower for R-IFA (32 +/- 13%) than for S-IFA (62 +/- 17%, n = 8; p <.001) in all livers examined. R-IFA thus has more favorable liver metabolic properties than S-IFA with respect to less extensive N-dechloroethylation and more rapid 4-hydroxylation, indicating that R-IFA may have a distinct clinical advantage over racemic IFA.     

Possible existence of a novel receptor for uridine analogues in the central nervous system using two isomers, N3-(S)-(+)- and N3-(R)-(-)-alpha-hydroxy-beta-phenethyluridines.

Uridine analogue binding sites, the so-called uridine receptor, were observed in the experiments on specific [3H]N3-phenacyluridine binding to bovine synaptic membranes using two isomers, N3-(S)-(+)- and N3-(R)-(-)-alpha-hydroxy-beta-phenethyluridine, as ligands. The potent hypnotic, N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine, but not the (R)-isomer, strongly inhibited [3H]N3-phenacyluridine binding. The racemate had inhibitory activity intermediate between that of the two alpha-hydroxy-beta-phenethyluridines ((R)- or (S)-isomers). The inhibitory constants of these compounds were determined. The Ki values of N3-phenacyluridine, alpha-hydroxy-beta-phenethyluridine (racemate), N3-(R)-(-)-, and N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine were 0.65, 397.4, 1908, and 10.2 nM, respectively. The present results indicate the existence of uridine receptors in the central nervous system in relation to their hypnotic activities reported previously.     

Effects of C2-alkylation, N-alkylation, and N,N'-dialkylation on the stability and estrogen receptor interaction of (4R,5S)/(4S,5R)-4,5-bis(4-hydroxyphenyl)-2-imidazolines

(4R,5S)/(4S,5R)-4,5-Bis(4-hydroxyphenyl)-2-imidazolines bearing 2,2'-H (3a), 2,2'-Cl (3b), 2,2',6-Cl (3c), and 2,2'-F (3d) substituents in the aromatic rings were C2-alkylated (5a-i), N-alkylated (7, 7a-c), and N,N'-dialkylated (9a-c). The synthesis started from the diastereomerically pure (1R,2S)/(1S,2R)-1,2-diamino-1,2-bis(4-methoxyphenyl)ethanes 1a-d, which were cyclized to the imidazolines 2a-d and 4a-i with triethylorthoesters or iminoethers. Ether cleavage with BBr(3) yielded the (4R,5S)/(4S,5R)-4,5-bis(4-hydroxyphenyl)-2-imidazolines 3a-d and 5a-i. The N-alkylation and N,N'-dialkylation of 2b, employed for obtaining 7a-c and 9a-c, were performed prior to the ether cleavage with alkyl iodine in dry THF. By use of HPLC, the influence of the substitution patterns in the aromatic rings and alkyl chains at the C2- or N-atoms on the hydrolysis rate of the imidazolines was studied under in vitro conditions. It appeared that only imidazolines with C2- or N-alkyl substituents show sufficient stability to interact as heterocycles with the estrogen receptor (ER). The resulting gene activation was monitored in a luciferase assay using ERalpha-positive MCF-7-2a breast cancer cells stably transfected with the plasmid ERE(wtc)luc. It is interesting to note that C2-alkylation led to a strong reduction or even a complete loss of activity whereas N-alkylation improved the estrogenic profile. The (4R,5S)/(4S,5R)-N-ethyl-4,5-bis(2-chloro-4-hydroxyphenyl)-2-imidazoline 7b has proven to be the most active compound in this structure-activity relationship study (EC(50) = 0.015 microM).     

Metabolism of a novel hypnotic, N3-phenacyluridine, and hypnotic and sedative activities of its enantiomer metabolites in mouse

1. The metabolism of N3-phenacyluridine (3-phenacyl-1-beta-D-ribofuranosyluracil), a potent hypnotic nucleoside derivative, was studied in mouse. 2. Of the radioactivity, 65% was excreted in urine within 48 h after intraperitoneal (i.p.) administration of [3H]N3-phenacyluridine. The urinary metabolites N3-phenacyluracil and N3-alpha-hydroxy-beta-phenethyluridine were extracted, isolated and analyzed by mass spectrometry. 3. Racemates of N3-alpha-hydroxy-beta-phenethyluridine were synthesized and both isomers were separated as N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine and N3-(R)-(-)-alpha-hydroxy-beta-phenethyluridine by hplc (CHIRALCEL-OJ column) with retentions of 13.8 and 17.9 min respectively. The reduction process took place with high stereo-selectivity, which gave an alcohol product in the urine with the same retention (17.9 min) as one of the synthetic isomers separated by hplc. 4. One of urinary metabolites was identified as N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine. N3-phenacyluridine was predominantly converted to an alcoholic metabolite of (S)-(+)-configuration. 5. N3-phenacyluracil and uridine were also identified as minor metabolites. 6. The pharmacological effects of the metabolites and related compounds were also evaluated in mouse. N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine, but not N3-(R)-(-)-alpha-hydroxy-beta-phenethyluridine, possessed hypnotic activity and potentiated pentobarbital-induced sleeping time with a similar potency to the parent compound, N3-phenacyluridine. N3-alpha-hydroxy-beta-phenethyluridine (racemate) had almost two thirds of the hypnotic activity of N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine. No other metabolites exhibited hypnotic activities. 7. The present study indicates that N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine, a major metabolite of N3-phenacyluridine, is an active metabolite and contributes a significant CNS depressant effect.     

Synthesis and H2-antagonistic action of N3, N5-substituted 1,2, 4-oxadiazole-3, 5-diamine. 26. H2-antihistaminics

N5-(3-[3-(1-Piperidinylmethyl)phenoxy) phenoxy]propyl)-1,2, 4-oxadiazole-3, 5-diamines and N5(2-[(5-dimethylaminomethyl-2-furanyl) methylthio] ethyl)-1,2,4-oxadiazole-3,5-diamines with different N3-substituents were prepared and tested for their H2-antihistaminic activity on the isolated guinea-pig atrium. They were synthesized by nucleophilic substitution of the appropriate 5-trichloromethyloxadiazoles with the amines 3 and 11. Of the prepared substances 10a,c in particular showed high H2-antagonistic activity.     

Methotrexate analogues. 33. N delta-acyl-N alpha-(4-amino-4-deoxypteroyl)-L-ornithine derivatives: synthesis and in vitro antitumor activity

N delta-Acyl derivatives of the potent folylpolyglutamate synthetase (FPGS) inhibitor N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-L-Orn) were synthesized from N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-L-ornithine by reaction with an N-(acyloxy)succinimide or acyl anhydride, followed by deformylation with base. The N delta-hemiphthaloyl derivative was also prepared from 4-amino-4-deoxy-N10-formylpteroic acid by reaction with persilylated N delta-phthaloyl-L-ornithine, followed by simultaneous deformylation and ring opening of the N delta-phthaloyl moiety with base. The products were potent inhibitors of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, with IC50's ranging from 0.027 and 0.052 microM as compared with 0.072 microM for APA-L-Orn. Several of the N delta-acyl-N10-formyl intermediates also proved to be good DHFR inhibitors. One of them, N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-N delta-(4-chlorobenzoyl)-L- ornithine, had a 2-fold lower IC50 than its deformylated product, confirming that the N10-formyl group is well tolerated for DHFR binding. While N delta-acylation of APA-L-Orn did not significantly alter anti-DHFR activity, inhibition of FPGS was dramatically diminished, supporting the view that the basic NH2 on the end of the APA-L-Orn side chain is essential for the activity of this compound against FPGS. N delta-Acylation of APA-L-Orn markedly enhanced toxicity to cultured tumor cells. However, N delta-acyl derivatives also containing an N10-formyl substituent were less cytotoxic than the corresponding N10-unsubstituted analogues even though their anti-DHFR activity was the same, suggesting that N10-formylation may be unfavorable for transport. Two compounds, the N delta-benzoyl and N delta-hemiphthaloyl derivatives of APA-L-Orn, with IC50's against L1210 cells of 0.89 and 0.75 nM, respectively, were more potent than either methotrexate (MTX) or aminopterin (AMT) in this system. These compounds were also more potent than MTX against CEM human lymphoblasts and two human head and neck squamous cell carcinoma cell lines (SCC15, SCC25) in culture. Moreover, in assays against SCC15/R1 and SCC25/R1 sublines with 10-20-fold MTX resistance, the N delta-hemiphthaloyl derivative of APA-L-Orn showed potency exceeding that of MTX itself against the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Compared pharmacology of human histamine H3 and H4 receptors: structure-activity relationships of histamine derivatives

Various histamine derivatives were investigated at the human H3 receptor (H3R) and H4 receptor (H4R) stably expressed in human embryonic kidney (HEK)-293 cells using [125I]iodoproxyfan and [3H]histamine binding, respectively. In Tris buffer, [3H]histamine binding to membranes of HEK(hH4R) cells was monophasic (K(D) of 3.8+/-0.8 nM). In phosphate buffer, the Hill coefficient was decreased (n(H) = 0.5+/-0.1) and a large fraction of the binding was converted into a low-affinity component (K(D) = 67+/-27 nM). The inhibition of [3H]histamine binding by two agonists, a protean agonist and five antagonists/inverse agonists confirms that the potency of many H3R ligands is retained or only slightly reduced at the H4R. Histamine derivatives substituted with methyl groups in alpha, beta or N(alpha) position of the side chain retained a nanomolar potency at the H3R, but their affinity was dramatically decreased at the H4R. With relative potencies to histamine of 282 and 0.13% at the H3R and H4R, respectively, (+/-)-alpha,beta-dimethylhistamine is a potent and selective H3R agonist. Chiral alpha-branched analogues exhibited a marked stereoselectivity at the H3R and H4R, the enantiomers with a configuration equivalent to L-histidine being preferred at both receptors. The methylsubstitution of the imidazole ring was also studied. The relative potency to histamine of 4-methylhistamine (4-MeHA) at the H4R (67%) was similar to that reported at H2 receptors but, owing to its high affinity at the H4R (Ki = 7.0+/-1.2 nM) and very low potency at H1- and H3-receptors, it can be considered as a potent and selective H4R agonist. On inhibition of forskolin-induced cAMP formation, all the compounds tested, including 4-MeHA, behaved as full agonists at both receptors. However, the maximal inhibition achieved at the H4R (approximately -30%) was much lower than at the H3R (approximately -80%). Thioperamide behaved as an inverse agonist at both receptors and increased cAMP formation with the same maximal effect (approximately +25%). In conclusion, although the pharmacological profiles of the human H3R and H4R overlap, the structure-activity relationships of histamine derivatives at both receptors strongly differ and lead to the identification of selective compounds.     

Synthesis and antitubulin activity of N1- and N4-substituted 3,5-dinitro sulfanilamides against African trypanosomes and Leishmania

Thirty analogues of N(1)-phenyl-3,5-dinitro-N(4),N(4)-di-n-propylsulfanilamide (GB-II-5, compound 3), a new antikinetoplastid antimitotic agent, have been synthesized and evaluated. The addition of simple functional groups to the N1 aromatic ring generally decreases antiparasitic and antimitotic potency, but placement of a dibutyl substituent at the N4 nitrogen to give N(1)-phenyl-3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (compound 35) augments antitrypanosomal and antileishmanial activity. Compound 35 possesses IC(50) values of 0.12 and 2.6 microM against cultured T. brucei and L. donovani amastigote-like forms, surpassing the activity of compound 3 against these parasites by 3.4- and 1.9-fold, respectively. Compound 35 inhibits the assembly of leishmanial tubulin with an IC(50) of 6.9 microM and displays antimitotic effects in cultured T. brucei as assessed by flow cytometry, but shows little effect on purified mammalian tubulin, and displays 100-fold selectivity for trypanosomes over two mammalian cell lines. Although 3 and 35 were not effective in initial in vivo antitrypanosomal assays, the in vitro potency and selectivity of these compounds make N(1)-aryl-3,5-dinitro-N(4),N(4)-dialkylsulfanilamides a promising new class of antikinetoplastid agents that act on parasite tubulin.     

Molecular mechanism of N4-aminocytidine mutagenesis

N4-Aminocytidine is strongly mutagenic towards E. coli, S. typhimurium, B. subtilis and coliphages phi X174 and M13mp2. It also causes mutations in mammalian cell lines and somatic cell mutations in D. melanogaster. The sequence analysis of deoxyribonucleic acid (DNA) from mutated phages revealed that N4-aminocytidine induces both adenine-thymine (AT) to guanine-cytosine (GC) and GC to AT transitions. No transversions are detectable. When E. coli and the mammalian cells were cultured in the presence of [3H]-N4-aminocytidine, [3H]-N4-aminodeoxycytidine was found in their DNA. It is likely that N4-aminocytidine is metabolized within the cells into N4-aminodeoxy-cytidine 5'-triphosphate (dCamTP), which is then incorporated into DNA, thereby causing base-pair transitions. To prove this hypothesis, we studied the incorporation of dCamTP into polynucleotides in the in vitro DNA synthesis catalyzed by E. coli DNA polymerase I large fragment (Klenow enzyme) and DNA polymerase alpha from a mouse cell line. Both polymerases catalyze incorporation of dCamTP into DNA efficiently in place of dCTP opposite guanine, and less efficiently, but to a significant extent, in place of dTTP opposite adenine. These observations prove the erroneous nature of dCamTP as a substrate for DNA synthesis. DNA containing N4-aminocytosine was prepared by the incorporation of dCamTP into single-stranded phage DNA annealed to complementary oligonucleotides. The DNA was transfected to E. coli cells. The analysis of progeny phages indicates that N4-aminocytosine residue in DNA causes A to G or G to A mutation in the position opposite to the site where N4-aminocytosine should be incorporated.     

Identification of (3R)-7-hydroxy-N-((1S)-1-[[(3R,4R)-4-(3-hydroxyphenyl)- 3,4-dimethyl-1-piperidinyl]methyl]-2-methylpropyl)-1,2,3,4-tetrahydro- 3-isoquinolinecarboxamide as a novel potent and selective opioid kappa receptor antagonist

(3R)-7-Hydroxy-N-((1S)-1-[[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl]-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic) was identified as a potent and selective kappa opioid receptor antagonist. Structure-activity relationship (SAR) studies on JDTic analogues revealed that the 3R,4R stereochemistry of the 3,4-dimethyl-4-(3-hydroxyphenyl)piperidine core structure, the 3R attachment of the 7-hydroxy-1,2,3,4-tetrahydroisoquinoline group, and the 1S configuration of the 2-methylpropyl (isopropyl) group were all important to its kappa potency and selectivity. The results suggest that, like other kappa opioid antagonists such as nor-BNI and GNTI, JDTic requires a second basic amino group to express potent and selective kappa antagonist activity in the [(35)S]GTPgammaS functional assay. However, unlike previously reported kappa antagonists, JDTic also requires a second phenol group in rigid proximity to this second basic amino group. The potent and selective kappa antagonist properties of JDTic can be rationalized using the "message-address" concept wherein the (3R,4R)-3,4-dimethyl-4-(hydroxyphenyl)piperidinyl group represents the message, and the basic amino and phenol group in the N substituent constitutes the address. It is interesting to note the structural commonality (an amino and phenol groups) in both the message and address components of JDTic. The unique structural features of JDTic will make this compound highly useful in further characterization of the kappa receptor.     

Syntheses and characterisation of N,N'-biscarbazolyl azine and N,N'-carbazolyl hydrazine derivatives and their antimicrobial studies

Reaction of 1-oxo-1,2,3,4-tetrahydrocarbazoles 1a-e with hydrazine hydrate in absolute ethanol afforded N,N'-bis-carbazolylazine derivatives 2a-e. Treatment of compounds 1a-e with hydroxylamine hydrochloride in ethanol with a catalytic amount of pyridine resulted in the formation of 1-hydroxyimino-1,2,3,4-tetrahydrocarbazoles 3a-e. Reduction of 3 with hydrazine hydrate in the presence of palladized carbon afforded N,N'-carbazolyl hydrazine derivatives 4a-e. The newly formed compounds were characterized by IR, 1H NMR, mass spectra and by elemental analysis. All compounds proved to have great potentialities as antibacterial and antifungal agents due to the presence of the azine or the hydrazine group. Particularly, the chloro substituted derivatives 2e and 4e showed excellent antibacterial and antifungal activity.     

Anti-aggregatory and anticoagulant effects of oligoamines. 9. The pharmacokinetics of N,N',N"-tris-4-(3-iodo-4-methoxyphenyl)-butyl-1,3,5-benzene triethanamine

Platelet Aggregation Inhibiting and Anticoagulant Effects of Oligoamines, IX: Pharmakokinetic Investigation of N,N′,N″-tris-4-(3-iodo-4-methoxyphenyl)-butyl-1,3,5-benzene-triethanamine After intraveneous administration to rats the title compound [ 3 *] is accumulated in organs with good vascularization like lung (C_max = 16.6% dose/g) and spleen (C_max = 8.8% dose/g). In whole blood a strong affinity to red blood cells is observed. 70 percent of 3 * bound to the erythrocytes are found in the membranes and could not be washed out with buffer. After oral administration of 3 * to rats only 2% of the applied dose is absorbed from the gastrointestinal tract.     

D2 and D3 dopamine receptor cell surface localization mediated by interaction with protein 4.1N

We identified protein 4.1N as a D2-like dopamine receptor-interacting protein in a yeast two-hybrid screen. Protein 4.1N is a neuronally enriched member of the 4.1 family of cytoskeletal proteins, which also includes protein 4.1R of erythrocytes and the 4.1G and 4.1B isoforms. The interaction of protein 4.1N was specific for the D2 and D3 dopamine receptors and was independently confirmed in pulldown and coimmunoprecipitation assays. Deletion mapping localized the site of dopamine receptor/protein 4.1N interaction to the N-terminal segment of the third intracellular domain of D2 and D3 receptors and the carboxyl-terminal domain of protein 4.1N. D2 and D3 receptors were also found to interact with the highly conserved carboxyl-terminal domain of proteins 4.1R, 4.1G, and 4.1B. Immunofluorescence studies show that protein 4.1N and D2 and D3 dopamine receptors are expressed at the plasma membrane of transfected human embryonic kidney 293 and mouse neuroblastoma Neuro2A cells. However, expression of D2 or D3 receptors with a protein 4.1N truncation fragment reduces the level of D2 and D3 receptor expression at the plasma membrane. These results suggest that protein 4.1N/dopamine receptor interaction is required for localization or stability of dopamine receptors at the neuronal plasma membrane.     

Histamine H3 and H4 receptors as novel drug targets

The identification of histamine H3 (H3R) and H4 (H4R) receptors some years ago revived interest in histamine research and exposed attractive perspectives for the potential therapeutic exploitation of these new drug targets. While the H3R is mainly localised in the CNS, the H4R is primarily expressed in hematopoietic cells, indicating their function in neurotransmission and immunomodulation, respectively. Although structural similarities between H3R and H4R and species differences in their pharmacological profiles are causes of limitations in the evaluation of their biological profile, the development of selective ligands for these receptors facilitates the elucidation of their physiological and pathophysiological functions. The H3R is a recognised drug target for neuronal diseases, such as cognitive impairment, schizophrenia, sleep/wake disorders, epilepsy and neuropathic pain; a small number of selective H3R antagonists have already passed some clinical Phase II trials. The H4R is the newest identified member of the histamine receptor family. Preclinical data strongly suggest its potential therapeutic exploitation in allergy, inflammation, autoimmune disorders and possibly cancer. Considering the topicality of this area of research, this review focuses on the pharmacology of selected promising indications and the rationales for the application of H3R and H4R ligands.     

4-Hydroxy-2-nonenal and ethyl linoleate form N(2),3-ethenoguanine under peroxidizing conditions

In these studies, we demonstrate that N(2),3-ethenoguanine (N(2), 3-epsilonGua) is formed from lipid peroxidation as well as other oxidative reactions. Ethyl linoleate (EtLA) or 4-hydroxy-2-nonenal (HNE) was reacted with dGuo in the presence of tert-butyl hydroperoxide (t-BuOOH) for 72 h at 50 degrees C. The resulting N(2), 3-epsilonGua was characterized by liquid chromatography/electrospray mass spectroscopy and by gas chromatography/high-resolution mass spectral (GC/HRMS) analysis of its pentafluorobenzyl derivative following immunoaffinity chromatography purification. The amounts of N(2),3-epsilonGua formed were 825 +/- 20 and 1720 +/- 50 N(2), 3-epsilonGua adducts/10(6) normal dGuo bases for EtLA and HNE, respectively, corresponding to 38- and 82-fold increases in the amount of N(2),3-epsilonGua compared to controls containing only t-BuOOH. Controls containing t-BuOOH but no lipid resulted in a >1000-fold increase in the level of N(2),3-epsilonGua over dGuo that was not subjected to incubation. EtLA and HNE, in the presence of t-BuOOH, were reacted with calf thymus DNA at 37 degrees C for 89 h. The amounts of N(2),3-epsilonGua formed in intact ctDNA were 114 +/- 32 and 52.9 +/- 16.7 N(2),3-epsilonGua adducts/10(6) normal dGuo bases for EtLA and HNE, respectively. These compared to 2.02 +/- 0. 17 and 2.05 +/- 0.47 N(2),3-epsilonGua adducts/10(6) normal dGuo bases in control DNA incubated with t-BuOOH, but no lipid. [(13)C(18)]EtLA was reacted with dGuo to determine the extent of direct alkylation by lipid peroxidation byproducts. These reactions resulted in a 89-93% level of incorporation of the (13)C label into N(2),3-epsilonGua when EtLA and dGuo were in equimolar concentrations, when EtLA was in 10-fold molar excess, and when deoxyribose (thymidine) was in 10-fold molar excess. Similar reactions with ctDNA resulted in an 86% level of incorporation of the (13)C label. These data demonstrate that N(2),3-epsilonGua is formed from EtLA and HNE under peroxidizing conditions by direct alkylation. The data also suggest, however, that N(2),3-epsilonGua is also formed by an alternative mechanism that involves some other oxidative reaction which remains unclear.     

Selective inhibition of gamma-aminobutyric acid aminotransferase by (3R,4R),(3S,4S)- and (3R,4S),(3S,4R)-4-amino-5-fluoro-3-phenylpentanoic acids

(3R,4R),(3S,4S)- and (3R,4S),(3S,4R)-4-amino-5-fluoro-3-phenylpentanoic acid (1a and 1b) were synthesized and studied as selective inactivators of gamma-aminobutyric acid (GABA) aminotransferase. Neither compound caused time-dependent inactivation of the enzyme. Neither compound underwent enzyme-catalyzed transamination nor was fluoride ion eliminated from either compound by the enzyme. No 3-phenyllevulinic acid, the product of elimination of HF followed by enamine hydrolysis, was detected. However, both 1a and 1b were competitive reversible inhibitors of GABA aminotransferase; the Ki for 1a was smaller than the Km for GABA. These results suggest that 1a and 1b bind to the active site of GABA aminotransferase, but gamma-proton removal does not occur. Whereas (S)-4-amino-5-fluoropentanoic acid (AFPA) is a potent inhibitor of L-glutamic acid decarboxylase (GAD), neither 1a nor 1b at concentrations 40 times the Ki of AFPA caused any detectable competitive inhibition of GAD. Therefore, the incorporation of a phenyl substituent at the 3-position of AFPA confirms selective inhibition of GABA aminotransferase over GAD.