Synthesis of novel (N-farnesyl) amino acids and their incorporation into peptides

N-Alkylation, and particularly N-farnesylation, of bioactive peptides might be of wide interest: first, to increase peptide bioavailability by decreasing their elimination and by favouring their transport through biological membranes; second, to target peptides to cellular membranes; and third, to generate therapeutic double-substrate inhibitors of enzymes such as ras-farnesyltransferase. We report the synthesis of novel N-farnesyl amino acids [(N-Frn) amino acids]. We have synthesized (N-Frn)MetOCH₃, (N-Frn)ValOBz and (N-Frn)PheOCH₃, by alkylation of the corresponding natural amino acid esters. In order to demonstrate the feasibility of the introduction of (N-Frn) amino acids into peptides, we have synthesized representative dipeptide analogs: Cys-(N-Frn)ValOBz, Phe-(N-Frn)ValOBz, Lys-(N-Frn)ValOBz, Phe-(N-Frn)MetOCH₃, Glu-(N-Frn)MetOCH₃, Ser-(N-Frn)MetOCH₃, Trp-(N-Frn)PheOCH₃, and Pro-(N-Frn)PheOCH₃, We also describe the synthesis of the model peptide Cys-Val-Phe-(N-Frn)MetOCH₃, which is derived from the tetrapeptide CysValPheMet inhibitor of human p21ras-farnesyl transferase. © Munksgaard 1996.     

N-Hydroxy-N-arylacetamides. II: Molecular aspects of ferrihaemoglobin formation by N-hydroxy-N-arylacetamides and arylhydroxylamines in the rat

1. Ferrihaemoglobin(HbFe³⁺) formation in rats after i.p. injection of 6 N-hydroxy-N-arylacetamides has shown that N-hydroxy-4-chloroacetanilide(N-hydroxy-4CIAA) was the most active and N-hydroxy-2-acetylaminofluorene(N-hydroxy-2AAF) the least active compound tested. As N-hydroxy-N-arylacetamides were thought to produce HbFe³⁺ only after enzymic N-deacetylation, the corresponding arylhydroxylamines were also tested for HbFe³⁺-forming activity and were found to be more active, N-hydroxy-4-chloroaniline(N-hydroxy-4CIA) being one of the most active and N-hydroxy-2-aminofluorene(N-hydroxy-2AF) the least active compound tested.

2. N-Hydroxy-4-chloroacetanilide given i.p. to rats more rapidly invaded the blood and produced larger amounts of ferrihaemoglobin than did N-hydroxy-2-acetylaminofluorene, due to differences in their availability in plasma.

3. Injection of 50mg/kg of N-hydroxy-4-chloroacetanilide gave similar concn of HbFe³⁺ and 4-chloronitrosobenzene(4-CINOB) as injections of 8?mg/kg of N-hydroxy-4-chloroaniline, indicating that the arylhydroxylamine, after N-deacetylation, was the active molecule in vivo.

4. The concn of 4-chloronitrosobenzene declined faster than HbFe³⁺ concn. 4-Chloronitrosobenzene therefore is a further example of a ‘hit-and-run’ chemical.

5. Inhibition by the microsomal carboxylesterase inhibitor, bis(4-nitrophenyl)phosphate(BNPP), indicated that ferrihaemoglobin formation by 4-chloroacetanilide, but not by N-hydroxy-4-chloroacetanilide, depends on the enzymic activity of hepatic microsomal carboxylesterases.     

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 N ³-(R)-(-)-alpha hydroxy-beta-phenethyluridine, possessed hypnotic activity and potentiated pentobarbitalinduced 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.     

Über die Reaktion von Quadratsäure mit Carbodiimiden

Reactions of Squaric Acid with Carbodiimides Reactions of squaric acid ( 5 ) with carbodiimides 2 yield N-(2-hydroxy-3,4-dioxo-1-cyclobutenyl)-N,N'-dialkylureas 6 , or N,N'-diarylsquaric acid diamides 8. Depending on the conditions, squaric acid reacts with N-isopropyl-N'-phenylcarbodiimide ( 2f ) to yield N-(2-hydroxy-3,4-dioxo-1-cyclobutenyl)-N'-isopropyl-N-phenylurea ( 17 ) or 3,4-bis-(3-isopropyl-1-phenylureido)-3-cyclobutene-1,2-dione ( 19 ).     

Synthesis and bioassay of LHRH-antagonists with N-Ac-d-O-phenyltyrosine and N-Ac-d-3-(2-dibenzofuranyl)alanine in position 1

N-Ac-d -O-phenyltyrosine was synthesized via the corresponding azlactone. Resolution of the dl methyl esters was achieved by Subtilisin Carlsberg. Treatment with palladium(II) acetate in trifluoroacetic acid converted N-Ac-d -O-phenyltyrosine into N-Ac-d -3-(2-dibenzofuranyl)alanine. These two amino acids were incorporated instead of N-Ac-d -2-Nal into position 1 of the LHRH-antagonist (N-Ac-d -2-Nal¹, d -pClPhe², d -3-Pal³, c-PzACAla⁵, d -PiCLyS⁶, ILys⁸,d -Ala¹⁰)-LHRH. The more rigid N-Ac-d -3-(2-dibenzofuranyl)alanine was structurally more effective than N-Ac-d -O-phenyltyrosine; the AOAs for the corresponding analogs were 82 and 38%, respectively, at 0.5 μg. Replacement of c-PzACAla in position 5 by O-phenyltyrosine significantly decreased potency.     

Nitramine, 12. Mitt. Fluormethyl- und Azidomethyl-alkylnitramine

Nitramines, XII: N-Fluoromethyl- and N-Azidomethyl-N-alkylnitramines The N-chloromethyl-N-alkylnitramines 1 react with potassium fluoride and 18-crown-6 to give the unstable N-fluoromethyl-N-alkylnitramines 2 . With sodium azide the stable N-azidomethyl-N-alkylnitramines 3 are formed. With alkynes the latter cyclize to yield 1,2,3-triazoles, the structures of which were studied.     

N-Hydroxy-N-arylacetamides. III: Mechanism of haemoglobin oxidation by N-hydroxy-4-chloroacetanilide in erythrocytes in vitro

1. N-Hydroxy-4-chloroacetanilide(N-hydroxy-4ClAA) was the most active, and N-hydroxy-2-acetylaminofluorene(N-hydroxy-2AAF) the least active compound among six N-hydroxy-N-arylacetamides, in forming ferrihaemoglobin(HbFe³⁺) in bovine erythrocytes in the presence of 11 mM glucose.

2. N-Hydroxy-4ClAA oxidized 25 equiv. of HbFe²⁺, both in the presence and absence of glucose or lactate. Therefore, its catalytic properties did not depend on metabolic regeneration by the NADPH- or NADH-dependent erythrocyte reductases.

3. In contrast, N-hydroxy-4-chloroaniline(N-hydroxy-4ClA) oxidized 760 equiv. of HbFe²⁺ in the presence of glucose, but only 81 equiv. of HbFe²⁺ in the presence of lactate. These results indicate that the catalytic activity depended on the metabolic regeneration from 4-chloronitrosobenzene(4-CINOB) by NADPH-dependent erythrocyte reductases.

4. A relationship was established between HbFe³⁺ concn. and the concn. of N-hydroxy-4ClA and 4-CINOB(determined together), 4-chloroacetanilide(4-ClAA) and 4-chloroaniline(4-CIA), indicating co-oxidation of N-hydroxy-4ClAA and oxyhaemoglobin in erythrocytes and partial reduction of the newly formed 4-CINOB to 4-CIA.

5. In rat blood in vitro incubated with N-hydroxy-4CIAA, 4-CINOB concn. increased with increasing HbFe³⁺ concn., indicating that 4-CINOB was formed by co-oxidation of oxyhaemoglobin and N-hydroxy-4CIAA, and not by enzymic N-deacetylation.     

The Differentiation of N-Oxidation and N-Dealkylation of N-Ethyl-N-methylaniline by Rabbit Liver Microsomes as Distinct Metabolic Routes

Abstract

1. N-Oxidation and N-dealkylation were shown to be separate routes of metabolism for N-ethyl-N-methylaniline.

2. N-Dealkylation, but not N-oxidation, is enhanced when NADH is present in addition to the NADPH-regenerating cofactors.

3. Some inhibitors, e.g. p-chloromercuribenzoate, inhibit N-dealkylation to a very much greater extent than they do N-oxidation.

4. N-Dealkylation is affected more by ‘ageing’ of the microsomal preparation or by the presence of bile salts, than is N-oxidation.

5. N-Oxidation followed by N-O-α-carbon migration of oxygen is only implicated to a very minor extent in the N-dealkylation process.     

N‐heterocyclic carbenes as ligands in palladium‐mediated [11C]radiolabelling of [11C]amides for positron emission tomography

A model palladium‐mediated carbonylation reaction synthesizing N‐benzylbenzamide from iodobenzene and benzylamine was used to investigate the potential of four N‐heterocyclic carbenes (N,N′‐bis(diisopropylphenyl)‐4,5‐dihydroimidazolinium chloride ( I ), N,N′‐bis(1‐mesityl)‐4,5‐dihydroimidazolinium chloride ( II ), N,N′‐bis(1‐mesityl)imidazolium chloride ( III ) and N,N′‐bis(1‐adamantyl)imidazolium chloride ( IV )) to act as supporting ligands in combination with Pd₂(dba)₃. Their activities were compared with other Pd‐diphosphine complexes after reaction times of 10 and 120 min. Pd₂(dba)₃ and III were the best performing after 10 min reaction (20%) and was used to synthesize radiolabelled [¹¹C]N‐benzylbenzamide in good radiochemical yield (55%) and excellent radiochemical purity (99%). A Cu(Tp*) complex was used to trap the typically unreactive and insoluble [¹¹C]CO which was then released and reacted via the Pd‐mediated carbonylation process. Potentially useful side products [¹¹C]N,N′‐dibenzylurea and [¹¹C]benzoic acid were also observed. Increased amounts of [¹¹C]N,N′‐dibenzylurea were yielded when PdCl₂ was the Pd precursor. Reduced yields of [¹¹C]benzoic acid and therefore improved RCP were seen for III /Pd₂(dba)₃ over commonly used dppp/Pd₂(dba)₃ making it more favourable in this case. Copyright © 2010 John Wiley & Sons, Ltd.     

Untersuchungen an 1,3-Thiazinen, 25. Mitt. Transacylierende N-Acyl-tetrahydro- und N-Acyl-dihydro-1,3-thiazin-derivate

1,3-Thiazines, XXV: Transacylating Derivatives of N-Acyltetrahydro- and N-Acyldihydro-1,3-thiazine Novel N-acyl-2-thioxo-3,4-dihydro-1,3-thiazine-4-ones 4 and N-acyl-tetrahydro-1,3-thiazine-2,4-diones 7 were preparee by acylation of the N-unsubstituted 1,3-thiazine derivatives 3 and 6 with acid chlorides. Their characteristics are compared with those of known N-acylthiazine derivatives.     

Microsomal metabolism of N,N-diethyl-m-toluamide (DEET,DET): the extended network of metabolites

1. The aim was to set out to establish the complete network of metabolites arising from the phenobarbital-treated rat liver microsomal oxidation of N,N-diethyl-m-toluamide (DEET). The products formed from DEET and all its subsequent metabolites were identified by HPLC retention times, UV spectroscopy, mass spectrometry and by comparison with authentic standards. 2. DEET (1a) produces three major metabolites, N-ethyl-m-toluamide (1b), N,N-diethyl-m-(hydroxymethyl)benzamide (2a) and N-ethyl-m-(hydroxymethyl)benzamide (2b), and, at low substrate concentrations or extended reaction times, two minor metabolites, toluamide (1c) and N,N-diethyl-m-formylbenzamide (3a). 1b and 2a are primary metabolites and their formation follows Michaelis-Menten-type kinetics. At low DEET concentrations, ring methyl group oxidation is favoured; at saturation concentrations, methyl group oxidation and N-deethylation proceed at similar rates. The rate of formation of 2b decreases with increasing DEET concentration; 2b is therefore a secondary metabolite of DEET and DEET acts as a competitive inhibitor of the metabolism of 1b and 2a. 3. Except for the primary amides, where N-dealkylation is impossible, metabolism of all subsequent compounds, 1b,c, 2a-c, 3a-c and 4a,b, involves an N-deethylation (NEt₂ → NHEt or NHEt → NH₂) competitive with a ring substituent oxidation (CH₃ → CH₂OH, CH₂OH → CHO or CHO → CO₂H). Surprisingly, the aldehydes 3a-c are also reduced to the corresponding alcohols 2a-c (CHO → CH₂OH); CO inhibits the oxidative metabolism of 3a-c, but reduction to 2a-c continues uninhibited. 4. The outcomes of this work are that (1) previously unreported aldehydes 3b and 3c form part of the DEET network of metabolites, (2) the reduction of the aldehydes 3a-c has the potential to inhibit the formation of the more highly oxidized DEET metabolites, (3) amide hydrolysis was not observed for any substrate and (4) no evidence was obtained for N-(1-hydroxyethyl)amide intermediates.     

The Excretion of [35S]Dapsone and its Metabolites in the Urine,Faeces and Bile of the Rat

Abstract

1. When [³⁵S]dapsone was given orally to rats, 60–70% of the ³⁵S was excreted in 6 days, 45% in the urine and 22% in the faeces. The bulk of the ³⁵S was excreted in the first 24 h. Values were similar but slightly higher when the compound was given intraperitoneally.

2. When [³⁵S]dapsone was given intraperitoneally to bile duct-cannulated rats, 75–80% of the ³⁵S was excreted in 3 days, 32% in the bile and 27% in the urine.

3. The main metabolite in the urine was dapsone N-sulphamate, with smaller amounts of dapsone, acetyldapsone, N-acetyldapsone N'-sulphamate and dapsone N-glucuronide, whereas the main metabolite in the bile was dapsone N-glucuronide with only small amounts of N-sulphamate.

4. The amount of dapsone N-glucuronide detected in the urine increased when the rats were given NaHCO₃ to produce an alkaline urine.

5. The difference in the nature of the major metabolite found in the urine and that found in the bile has been explained in terms of the instability of N-glucuronides at acid pH and the molecular weight values required for significant biliary excretion in the rat, the N-glucuronide having a higher molecular weight than the N-sulphamate.

6. The potassium salts of dapsone N-sulphamate, dapsone N, N'-disulphamate, and N-acetyldapsone N'-sulphamate have been synthesized.     

Derivate des 2-Amino-1,2,3,4-tetrahydronaphthalins,III. Synthese einiger N'-substituierter N-(trans-3-Hydroxy-1,2,3,4-tetrahydro-2-naphthyl)-piperazine

Derivatives of 2-Amino-1,2,3,4-tetrahydronaphthalene, III: Synthesis of Some N'-Substituted N-(trans-3-Hydroxy-1,2,3,4-tetrahydro-2-naphthyl)piperazines The synthesis of the N'-substituted N-(trans-3-hydroxy-1,2,3,4-tetrahydro-2-naphthyl)piperazines 2 – 5 and of trans-2-morpholino-3-hydroxy-1,2,3,4-tetrahydronaphthalene ( 6 ), starting from 2,3-epoxytetralins 1 , is reported. The new Mannich bases 3 show hypotensive and antiarrhythmic activities.     

3-Alkoxyoxazolidin-2,4-dione aus N-Alkoxy-2-hydroxycarbonsäureamiden und 1,1′-Carbonyldiimidazol

3-Alkoxyoxazolidine-2,4-diones from N-Alkoxy-2-hydroxycarboxamides and 1,1′-Carbonyldiimidazole Reaction of N-alkoxy-2-hydroxycarboxamides 3 with 1,1′-carbonyldiimidazole produces N-alkoxy-oxazolidine-2,4-diones 4 . Hydrolysis of N-(tetrahydro-2H-2-pyranyloxy)oxazolidine-2,4-diones affords 3-hydroxyoxazolidine-2,4-diones 5 which are converted by the reaction with phenyl isocyanate into 7 and with benzoyl chloride into 8 . Benzylaminolysis of 4 gave 3-benzyloxazolidine-2,4-diones 10 .     

Synthese von 9-Fluorenalkanaminen, 3. Mitt. Stickstoffalkylierte und -acylierte 2-(9H-Fluoren-9-yl)-2-propanamine

Synthesis of (9-Fluorenylalkan)amines, III: N-Alkylated or N-Acylated 2-(9H-Fluoren-9-yl)-2-propanamines The synthesis of N-alkylated or N-acylated derivatives of the fluorene 3a is described. Compound 3b can be prepared via 1b and 2b , but 3c – 3f have to be synthesised by direct alkylation or acylation of 3a . The reaction of 8 with basic agents yields the stable derivative 9 .     

The Metabolism of N-Ethyl-N-methylaniline by Rabbit Liver Microsomes: The Measurement of Metabolites by Gas-liquid Chromatography

Abstract

1. A method for the determination of N-ethyl-N-methylaniline and its metabolites by g.l.c. is described.

2. Following incubation in N-ethyl-N-methylaniline with rabbit liver microsomes for 60 min, over 95% of the substrate was accounted for as unchanged compound or metabolites.

3. N-Ethyl-N-methylaniline is metabolized in vitro by rabbit tissues mainly by N-oxidation and N-demethylation and to a lesser extent by N-deethylation and di-dealkylation.

4. Both major routes of metabolism were observed in homogenates prepared from rabbit liver and lung; in addition N-oxidation occurred in kidney and bladder tissue homogenates.     

Synthese und biologische Wirksamkeit neuer N,N-disubstituierter 5-Alkyliden- bzw. 5-Aralkyliden-3-aminorhodanine

Synthesis and Biological Effects of Novel N,N-Disubstituted 5-Alkyliden- and 5-Aralkyliden-3-aminorhodanines Numerous novel N,N-disubstituted 5-alkyliden- and 5-aralkyliden-3-aminorhodanines 2 have been prepared by condensation of carbonyl compounds with 1 . The effectiveness of some derivatives in an ?akanthose test”? with hairless mice was shown.     

Alkaloid N-Oxides

Abstract

1. Naturally occurring alkaloid N-oxides are reviewed.

2. Examples of indole alkaloid N-oxides from simple indole bases to complex dimeric alkaloids are given.

3. Pyrrolizidine alkaloid N-oxides are listed and their toxicity, metabolism and role in the plant considered.

4. A brief account of quinolizidine, piperidine and miscellaneous alkaloid N-oxides, together with nicotine N-oxide and N-oxides from micro-organisms, is given.     

Biokinetic investigation of the photodynamic activity of new photosensitizers

Results of an in vivo biokinetic investigation of the photodynamic activity of a series of new photosensitizers including a tetraazachlorin derivative (2,3,3α,21α-tetrahydro-2-methyl-3α,8,13,18-tetraphenyl-5,10,15,20-tetraaza-1H,22H,24H-pyrrolo[3, 4-b]porphine), two difluoroboryl-substituted complexes of 3,3′-diphenylazadiisoindolylmethene {N,N-difluoroboryl-N-[3-(4-t-butylphenyl)-2H-isoindol-1-yl)]-N-[3-(4-t-butylphenyl)-1H-isoindol-1-yliden]amine and N,N-difluoroboryl-1-[3-(4-methoxyphenyl)-2H-isoindol-1-yl)]-N-[3-(4-methoxyphenyl)-1H-isoindol-1-yliden]amine}, and a sulfanyl-substituted phthalocyanine [1,8,15,22-tetrakis(t-butylsulfanyl)-29H, 31H-phthalocyanine] are reported. These compounds exhibit pronounced photodynamic activity in mice bearing solid Ehrlich ascites carcinoma and sarcoma S-37 upon intravenous injection as micellar suspensions in aqueous solutions (4%) of Cremophor EL and Proxanol 268. Acomparison of the tumor growth rate and the time of attaining a critical tumor volume in the test and control groups shows evidence for high photodynamic activity of the new photosensitizers.     

Synthesis of 3,7,8‐15N3‐N1‐(β‐D‐erythro‐pentofuranosyl)‐5‐guanidinohydantoin

3,7,8‐¹⁵N₃‐N¹‐(β‐D‐erythro‐pentofuranosyl)‐5‐guanidinohydantoin was synthesized from the oxidation of 1,7,NH₂‐¹⁵N₃‐8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine with 2 equivalents of Ir(IV) in pH 4.5 potassium phosphate buffer. The synthesis of 1,7,NH₂‐¹⁵N₃‐8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine started with bromination of 1,7,NH₂‐¹⁵N₃‐2′‐deoxyguanosine. The resulting 1,7,NH₂‐¹⁵N₃‐8‐bromo‐7,8‐dihydro‐2′‐deoxyguanosine reacted with sodium benzyloxide to afford 1,7,NH₂‐¹⁵N₃‐8‐benzyloxy‐7,8‐dihydro‐2′‐deoxyguanosine. Subsequent catalytic transfer hydrogenation of 1,7,NH₂‐¹⁵N₃‐8‐benzyloxy‐7,8‐dihydro‐2′‐deoxyguanosine with cyclohexene and 10% Pd/C yielded 1,7,NH₂‐¹⁵N₃‐8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine. Purification of 3,7,8‐¹⁵N₃‐N¹‐(β‐D‐erythro‐pentofuranosyl)‐5‐guanidinohydantoin was first carried out on a C18 column and the product was further purified on a graphite column. ESI‐MS was used to confirm the identity and to determine the isotopic purity of all the labeled compounds. The isotopic purity of 3,7,8‐¹⁵N₃‐N¹‐(β‐D‐erythro‐pentofuranosyl)‐5‐guanidinohydantoin was 99.4 atom% based on LC‐MS measurements. Copyright © 2003 John Wiley & Sons, Ltd.