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Conjugation with poly(ethylene glycol) ("PEGylation") is a widely used approach for improving the therapeutic propensities of peptide and protein drugs through prolonging bloodstream circulation, reducing toxicity and immunogenicity, and improving proteolytic stability. In the present study, we investigate how PEGylation affects the interaction of host defense peptides (HDPs) with bacterial lipopolysaccharide (LPS) as well as HDP suppression of LPS-induced cell activation. In particular, we investigate the effects of PEGylation site for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), a peptide displaying potent anti-inflammatory effects, primarily provided by its N-terminal part. PEGylation was performed either in the N-terminus, the C-terminus, or in both termini, keeping the total number of ethylene groups (n = 48) constant. Ellipsometry showed KYE28 to exhibit pronounced affinity to both LPS and its hydrophobic lipid A moiety. The PEGylated peptide variants displayed lower, but comparable, affinity for both LPS and lipid A, irrespective of the PEGylation site. Furthermore, both KYE28 and its PEGylated variants triggered LPS aggregate disruption. To investigate the peptide structure in such LPS complexes, a battery of nuclear magnetic resonance (NMR) methods was employed. From this, it was found that KYE28 formed a well-folded structure after LPS binding, stabilized by hydrophobic domains involving aromatic amino acids as well as by electrostatic interactions. In contrast, the PEGylated peptide variants displayed a less well-defined secondary structure, suggesting weaker LPS interactions in line with the ellipsometry findings. Nevertheless, the N-terminal part of KYE28 retained helix formation after PEGylation, irrespective of the conjugation site. For THP1-Xblue-CD14 reporter cells, KYE28 displayed potent suppression of LPS activation at simultaneously low cell toxicity. Interestingly, the PEGylated KYE28 variants displayed similar or improved suppression of LPS-induced cell activation, implying the underlying key role of the largely retained helical structure close to the N-terminus, irrespective of PEGylation site. Taken together, the results show that PEGylation of HDPs can be done insensitively to the conjugation site without losing anti-inflammatory effects, even for peptides inducing such effects through one of its termini.

Rationale Peptide tandem mass spectra can be analyzed by a number of means. They can be compared against predicted spectra of peptides derived from genome sequences, compared against previously acquired and identified spectra, or - sometimes - sequenced de novo. We recently introduced another method which compares spectra between liquid chromatography/tandem mass spectrometry (LC/MS/MS) datasets to determine the shared spectral content, and demonstrated how this can be applied in a molecular phylogenetic study using sera from human and non-human primates. We will here explore if such a method have other, serendipitous uses. Methods We used the existing compareMS2 algorithm without modification on a diverse set of experiments. Results First we conducted a small phylogenetic study, using (mammalian) bone samples to study old material, and human pathogens aiming to distinguish clinically important strains. Although not as straightforward as primate sera analysis, the method shows significant promise for all these applications. We also used the algorithm to compare 24 different protocols for extraction of proteins from muscle tissue. The results provided useful information in comparing protocols. Finally, we applied compareMS2 aiming for quality control of two traceable protein reference standards (troponin) used in clinical chemistry assays, by analysing the effect of storage conditions. Conclusions The results illustrate a broad applicability of the metric based on shared tandem mass spectra between LC/MS/MS datasets for analysing protein digests in different types of experiments. There is no reason to assume that our instance of this method is optimal in any of these situations, as it makes limited or no use of accurate mass and chromatographic retention time. We propose that with further improvement and refinement, this type of analysis can be applied as a simple but informative first step in many pipelines for bottom-up tandem mass spectrometry data analysis in proteomics and other fields, comparing or analysing large numbers of samples or datasets.

Stationary phases containing pyridine, phenol, aniline and morpholine groups were synthesized using copper (I)-catalyzed azide-alkyne cycloaddition click reactions. The backbone of the stationary phases was aminopropyl silica. The stationary phases were evaluated in packed-column supercritical fluid chromatography (pSFC) with acidic and basic solutes without addition of additives. The analysis of metoclopromide and its impurities by SFC-time-of-flight mass spectrometry (SFC-TOFMS) on the phenol phase is presented. In the click reaction, the 1,2,3-triazole ring is formed and to assess its influence on the polarity/selectivity, the click phases were compared to a commercial available 1,2,4-triazole hydrophilic interaction liquid chromatography (HILIC) phase. The phases were also compared to two extensively used stationary phases in SFC namely 2-ethyl pyridine and bare silica. To allow comparison with other phases used in SFC, linear solvation energy relationships (LSER) of the click phases were determined.

High throughput screening and subsequent hit validation identified 4-isopropyl-3-(2-((1-phenylethyl)amino)pyrimidin-4-yl)oxazolidin-2-one as a potent inhibitor of IDH1R132H. Synthesis of the four separate stereoisomers identified the (S,S)-diastereomer (IDH125, 1f) as the most potent isomer. This also showed reasonable cellular activity and excellent selectivity vs IDH1wt. Initial structure–activity relationship exploration identified the key tolerances and potential for optimization. X-ray crystallography identified a functionally relevant allosteric binding site amenable to inhibitors, which can penetrate the blood–brain barrier, and aided rational optimization. Potency improvement and modulation of the physicochemical properties identified (S,S)-oxazolidinone IDH889 (5x) with good exposure and 2-HG inhibitory activity in a mutant IDH1 xenograft mouse model.

It is shown that in radioactive 4-amino-2-phenylpyrimidine, obtained as a minor product in the reaction of 4-chloro-2-phenylpyrimidine-4-14C with potassium amide in liquid ammonia, the 14C label is present in position 4 of the ring. This result proves that the formation of the 4-amino compound cannot be accomodated with a mechanism initiated by the attack of an amide ion on the 6 position, but probably occurs by an addition-elimination mechanism on the C(4) carbon atom.

5-Amino-4-chloro-2-phenylpyrimidine and 4-chloro-5-methyl-2-phenylpyrimidine are converted into 4(5)-cyano-2-phenylimidazole and 4(5)-ethynyl-2-phenyl-imidazole, respectively, by reaction with potassium amide in liquid ammonia at - 33°.

While treatment of 2-chloro-3-phenylpyrazine with potassium amide in liquid ammonia yields only 2-amino-3-phenylpyrazine, from 2-chloro-6-phenylpyrazine on the contrary two ring contraction products, 4(5)-phenylimidazole and 2-cyano-4(5)-phenylimidazole are obtained. From 2-chloro-5-phenylpyrazine a mixture of 2-amino-5-phenylpyrazine, 4(5)-phenylimidazole as well as 2-cyano-4(5)-phenylimidazole is formed. In the diphenyl series it has been established that, whereas 2-chloro-3,5-diphenylpyrazine gives only the corresponding 2-aminopyrazine, 2-chloro- 5,6-diphenylpyrazine gives a mixture of the amino product and two ring contraction products: 4,5-diphenylimidazole and 2-cyano-4,5-diphenylimidazole.2-Chloro-3,6-diphenylpyrazine was found to be relatively inactive towards potassium amide in liquid ammonia. This divergent behaviour is explained by the high susceptibility of the positions 3 and 6 of the 2-chloropyrazine for a nucleophilic attack as established by 14C- and 15N-labelling studies which were made previously.

Starting from D-ribose and 3,4-xylidine, a fifteen-step synthesis is described for the preparation of 5-dFAD (18). The synthesis involves as key intermediates, 5-deazariboflavin (8), its 2′,3′,4′-tris-O-(tetrahydropyranyl) derivative (12) and 2′,3′,4′-tris-O-(tetrahydropyranyl)-5′-O-(morpholinophosphonyl)-5-deazariboflavin (15).

Amination of the 2-X-4,6-diphenylpyrimidines (X=F, Cl, Br) with potassium amide in liquid ammonia yields the corresponding 2-amino compounds, and in addition some dehalogenated product in the case of X = Br. It is proved, using the corresponding [1,3-15N]-labelled pyrimidines, that for the 2-chloro and 2-bromo compounds the amination goes to the extent of about 70% via a ring-opening [SN(ANRORC) mechanism] but that in the amination of the 2-fluoro compound no ring-opening takes place [SN(AE) process]. One of the supposed intermediates during the ring-opening i.e. 3-(cyanoimino)-1,3-diphenyl-1-propenamine could be isolated. Also the amination of 2-chloro-4-phenylquinazoline with potassium amide occurs to the extent of about 70% via the SN(ANRORC) mechanism. If instead of potassium amide in liquid ammonia an ethanolic ammonia solution is used at elevated temperatures, a considerable amount of the corresponding 2-amino compound is also formed via the ring-opening process, i.e. 34-67% (depending on the ammonia concentration). 2-Chloro- and 4-chloro-quinazoline behave similarly on reaction with ethanolic ammonia.

By treatment with potassium amide in liquid ammonia, 2-chloropyrazine is converted into a mixture of 2-cyanoimidazole, imidazole and 2-aminopyrazine. The mechanism of the formation of the two ring-contracted products is discussed.

Nitriles R–CH2CN where R is CN, SO2C6H5, C6H4-NO2-p, C6H4-CF3-m, are effective reagents for converting 5-nitropyrimidine (1) into 2-amino-5-nitro-3-R-pyridines (7). In reactions of 1 with cyanoacetic acid derivatives (methyl, ethyl, tert-butyl esters), 3-cyano-5-nitro–2(1H)-pyridone is formed in addition to 7. Ketonic nitriles, such as 3-oxobutyronitrile and 3-oxo–3-phenylpropionitrile, react with 1 to form 2-methyl- and 2-phenyl-5-nitro-3-cyanopyridine, respectively. Plausible mechanisms are discussed. The reaction of 1 with N-(cyanoacetyl)carbamate provides a facile synthetic route to 1,2,3,4-tetrahydro-6-nitropyrido[2,3-d]pyrimidine-2,4-dione.

On treatment of 5-bromo-3-methyl-1-phenylpyrazole, 2-bromobenzo[b]thiophene and 2-bromobenzo[b]thiophene-1,1-dioxide with potassium amide in liquid ammonia bromine migration to the adjacent carbon atom was observed. Considerable evidence for the occurrence of an intermolecular transbromination was obtained.

Treatment of 3-nitro-1,6-naphthyridine, its 2-chloro-, 2-ethoxy- and 2-amino derivatives, with liquid ammonia, containing potassium permanganate, gave the corresponding 2-X-4-amino-3-nitro-1,6-naphthyridines (X = H, Cl, OEt, NH2). From 1H NMR spectroscopy sound evidence for the intermediacy of 2-X-4-amino-1,4-dihydro-3-nitro-1,6-naphthyridine is obtained. Similarly, from 5,8-dichloro(dibromo)-1,7-naphthyridine, the corresponding 2-amino-5,8-dichloro-(dibromo)-1,7-naphthyridines were formed on treatment with liquid ammonia/potassium amide/potassium permanganate. 1H NMR evidence for the intermediary C-2 σ-adduct has been obtained.

Treatment of 4-chloroquinazoline with lithium piperidide in piperidine at −75° yields a mixture of a stable open chain compound i.e. o-(piperidinomethylene)iminobenzonitrile and the corresponding 4-piperidino compound. When 4-chloroquinazoline is aminated with potassium amide in liquid ammonia at −75° only 4-aminoquinazoline is formed. By means of 15N-labelling it has been shown that about 50% of the 4-aminoquinazoline is formed via an ANRORC mechanism. The synthesis of 4-chloro-[3-15N]quinazoline is described.

In the reaction of 4-chloro-6-R-pyrimidine-1-oxides (R=CH3 or C6H5) with potassium amide in liquid ammonia at −75°C or with liquid ammonia at −33°C besides the formation of the corresponding 4-aminopyrimidine-N-oxides, a ring contraction into 5-amino-3-R-isoxazoles (R=CH3 or C6H5) has been observed. A mechanism for the ring contraction reaction is postulated.

Photolysis of 4-R-1,4(or 3,4)-dihydropyrimidines causes ring contraction into imidazoles, provided that the substituent R is capable of sufficiently stabilizing an anionic centre (R = 2-thiazolyl, 2-pyridyl, 4-pyridyl). Chemical evidence is presented to show that the ring contraction of 6-phenyl-4-(2-pyridyl)1,4(or 3,4)-dihydropyrimidine occurs via heterolytic fission of the C(1)-C(6) bond of intermediate 1-phenyl-6-(2-pyridyl)-2,4-diazabicyclo[3.1.0]hex-2(or 3)-ene. The anion-stabilizing effect of R is correlated with the acid strength (pKa) of R–CH3. A pKa value around 30 probably determines the border-line between ring contraction into an imidazole and formation of an isomeric 5-R-1,2(or 2,3)-dihydropyrimidine.

On treatment of 2-bromothiophene, 2,3-, 2,4-, and 2,5-dibromothiophene with potassium amide in liquid ammonia at − 75°, a bromine migration of position α to position β of the thiophene ring takes place. On treatment of the above mentioned compounds with potassium amide in liquid ammonia at − 33°, besides α to β bromine rearrangement, amination to 3-aminothiophene was observed. The mechanism of the transbromination is assumed to be an intermolecular process, involving dibromo- and tribromo-thiophenes. The formation of 3-aminothiophene does not occur via a didehydro intermediate, but it is the result of amination of a polybromothiophene, yielding an amino-bromothiophene, which is debrominated by reaction with amide ion or a thiophene anion.

During a study on the amination of 4-phenylpyrimidine in potassium amide/ liquid ammonia it was found that the extent to which the SN(ANRORC) mechanism operates in the amination largely depends upon whether an ammonium salt is used in quenching the reaction. The composition of the σ-adduct mixture, the structure of the open-chain intermediates, the inhibition of the SN(ANRORC) mechanism and the course of the amination in apolar solvents have been investigated. In addition, it has been found that, in the amination of 4-tert-butylpyrimidine, the SN(ANRORC) mechanism occurs to only a very limited extent.

Evidence is presented that the conversion of 4-bromo-6-phenylpyrimidine into 4-amino-6-phenylpyrimidine by treatment with potassium amide in liquid ammonia at -75° occurs to the extent of about 80% according to a mechanism involving an open-chain intermediate.

Evidence is presented that the amination of 4-chloro-2,6-diphenylpyrimidine with potassium amide in liquid ammonia into the corresponding 4-amino compound occurs to the extent of about 45% by the ANRORC mechanism. In the amination of the 4-iodo- and 4-fluoro derivatives of 2,6-diphenylpyrimidine, however, the ANRORC mechanism is not operative at all. The amination of 5-bromo-4-chloro-2,6-diphenylpyrimidine into 4-amino-5-bromo-2,6-diphenylpyrimidine occurs to the extent of about 18% by the ANRORC mechanism. The synthesis of 5-bromo-4-chloro-2,6-diphenyl-[1(3)-15N]pyrimidine and of the 4-fluoro, 4-chloro and 4-iodo derivatives of 2,6-diphenyl-[1(3)-15N]pyrimidine is described.

On treatment with active methylene compounds in basic media the quaternary pyrimidinium salts, i.e. methyl l-methylpyrimidinium sulfate, 1-methyl-4-phenylpyrimidinium iodide and 1-methyl-5-phenylpyrimidinium iodide are converted into pyridine derivatives. The mechanism of the reaction is discussed.

The mechanism of the conversion of chloropyrazine into imidazole by potassium amide in liquid ammonia has been investigated. It has been proved, using the labelled compounds 2-chloro-[1-15N]pyrazine and 2-chloro-[2-14C]pyrazine, that this ring contraction occurs by a nucleophilic attack of the amide ion on position 3. The synthesis of 2-chloro-[2-14C]pyrazine starting from diethyl malonate labelled in the ethoxycarbonyl group, is described.

It has been found that 2-chloropyrazine, containing an excess of 15N in position 1, by treatment with potassium amide in liquid ammonia at −65° yields 2-aminopyrazine, in which the exocyclic nitrogen contains all the excess of 15N. An A(ddition)-N(ucleophilic)-R(ing)-O(pening)-R(ing)-C(losure) mechanism is proposed. The corresponding animation of 2-chloro[1(4)-15N]quinoxaline at −33° leads to 2-amino[1(4)-15N]quinoxaline. In the last-mentioned conversion an A(ddition)-E(limination) mechanism is operative.

Reactions of 3-bromo-4-ethoxypyridine and 2-bromo-6-ethoxypyridine with lithium piperidide in piperidine have been investigated. It was found that in these compounds both substituents when occupying positions 2,4 or 6 are replaced by the piperidino group without rearrangements occurring. From 3-bromo-4-ethoxypyridine some 2,4-dipiperidinopyridine is formed as an additional product, probably via 4-piperidino-2,3-pyridyne as an intermediate.

A method is described for the oxidation of some 1-alkyl-3-carbamoylpyridinium chlorides by reaction with immobilized rabbit liver aldehyde oxidase. With the 1-methyl-, 1-ethyl-and 1-n-propyl derivatives, only the 1-alkyl-1,6-dihydro-6-oxo-3-pyridinecarboxamides have been obtained, but with the t-Bu analogue the corresponding 4-oxo compound was found as single product. The i-Pr derivative gave rise to a mixture of the corresponding 4- and 6-oxo compounds. From the kinetic data some information has been acquired with respect to the enzyme active-site environment and the binding of the substrates to the enzyme.

Reactions of 6-substituted derivatives of 2-halogenopyridines with potassium amide in liquid ammonia have been investigated. Together with aminations leading to the formation of 2-amino compounds, two unusual reactions were found to occur, i.e. reactions partially involving meta-rearrangement and conversions involving a ring transformation. The rearrangements took place in aminations of various substances (e.g. of 2-bromo-6-methylpyridine, 2-chloro-6-methylpyridine and 2-bromo-6-ethoxypyridine). Ring transformations occurred in aminations of 2,6-dibromo- and 2,6-dichloro-pyridine, 4-amino-2-methylpyrimidine being formed. The mechanism of the remarkable reaction last-mentioned is discussed.

Treatment of 2-halogenoquinoxalines and 3-substituted 2-halogenoquinoxalines with potassium amide in liquid ammonia yields the corresponding 2-aminoquinoxalines and benzimidazole. It was found - using 2-chloro-[2- 14C]quinoxaline - that the ring contraction does not involve a benzotriazepine derivative as intermediate and that the animation occurs via an AE-mechanism. In the case of 2-fluoroquinoxaline, there are indications that the amination goes partially via an addition-abnormal-elimination (EAa mechanism.

Treatment of 2-(diethylamino)-3-(methylthio)-5-phenyl-3H-azepine (3b, R = SMe) with potassium amide in liquid ammonia, followed by reaction of the anion formed with methyl iodide, gave 2-(diethylamino)-5-methyl-3-(methylthio)-5-phenyl-5H-azepine (5a), the structure of which was confirmed by X-ray analysis. Substitution at position 5 (as well as at C-3) was also observed on reacting the anion of 2-(diethylamino)-5-phenyl-3H-azepine, obtained by deprotonation of its conjugate acid with lithium diisopropylamide in N,N,N′,N′-tetramethyl-1,2-ethanediamine, with different alkyl halides.

The conversion of 1-halogeno-2,7-naphthyridines into the corresponding 1-amino compounds with KNH2/NH3, is shown, by the use of deuterated starting compounds, to proceed via an SN(AE)ipso substitution and not via an SN(AE)tele mechanism, even though convincing evidence for the formation of tele σ-adduct, 8-amino-1-halogeno-dihydro-2,7-naphthyridinide, was obtained. Using the same methods, it is shown that the conversion of 2-bromo-1,8-naphthyridine into 2-amino-1,8-naphthyridine proceeds, to the extent of 40%, via an odd SN(AE)tele pathway.

When pyridine is heated with 100% sulphuric acid in the presence of mercuric sulphate as a catalyst at about 275°, pyridine-3-sulphonic acid is formed as the chief product, together with very small amounts of byproducts. The course of the reaction is more complicated at somewhat higher reaction temperatures. At 330° a mixture is obtained which contains, together with pyridine-3-sulphonic acid and the 4-isomer, 4-hydroxypyridine in considerable amount. The sulphonation of pyridine is a reversible process. When pyridine-3-sulphonic acid is heated with 100% sulphuric acid and mercuric sulphate at ∼ 330°, a mixture is formed containing chiefly pyridine, together with pyridine-4-sulphonic acid, 4-hydroxypyridine and the starting substance.

Oxidation of 1-aryl-3-carbamoylpyridinium chlorides with immobilized rabbit liver aldehyde oxidase gave predominantly 1-aryl-1,6-dihydro-6-oxo-3-pyridinecarboxamides, together with the corresponding 1-aryl-1,4-dihydro-4-oxo-3-pyridinecarboxamides. In general, the site of oxidation by this enzyme is determined by steric factors. The rate of oxidation by free aldehyde oxidase is very sensitive to electronic effects. A more electron-withdrawing aryl substituent increases the reaction rate for oxidation to 6-oxo product. Consequently, a positive ρ value of about 3.6 was calculated for free aldehyde oxidase. Oxidation of these compounds by bovine milk xanthine oxidase yielded mainly 4-oxo products. Preparative-scale reactions with both free and immobilized xanthine oxidase gave low product yields. The oxidation rate of free xanthine oxidase is only slightly affected by substituents in the aryl group.

A new synthesis of 9-oxodec-2-enoic acid, using thiophen as one of the starting materials, is described.

Investigations on ring contraction of pyrimidine and some of its homologues into pyrazoles have been extended. When pyrimidine, 4-methyl- and 4,6-dimethylpyrimidine are allowed to react with hydrazine, pyrazole, 3(=5)-methylpyrazole and 3,5-dimethyl-pyrazole, respectively are formed in excellent yields. The same compounds, when treated with methylhydrazine sulfate yield 1 -methylpyrazole, an isomeric mixture of 1,3- and 1,5-dimethylpyrazole and 1,3,5-trimethylpyrazole, respectively. The quaternary salts 1-methylpyrimidinium iodide and 1,4,6-trimethylpyrimidinium methyl sulfate are converted by hydrazine into pyrazole and 3,5-dimethylpyrazole. A mechanism of this ring transformation, involving an open-chain intermediate, is proposed.

A series of human estrogen receptor (hER) mimics were synthesised. On the basis of the knowledge on the structure of the hormone binding domain of the hER, three different peptide chains were constructed onto a tripodal scaffold. By using a fully automated solid-phase synthesis protocol, 120 members with known identity were synthesised in only a week. Affinity towards estrogenic compounds was checked by affinity LC. For this purpose, ethinylestradiol was "clicked" onto a modified-silica phase, and the obtained material was packed into an HPLC column. The results stemming from the affinity LC experiments were confirmed by clicking one library member to silica and by using this solid phase to extract different endocrine-disrupting chemicals (EDCs) from aqueous media.

A convergent synthesis for the preparation of a new orthogonally protected tripodal scaffold has been developed. The scaffold was successfully coupled to a Tentagel solid support and further derivatised at the three attachment points demonstrating its possible use for combinatorial chemistry.

3-Nitro-, 4-nitro-, 7-nitro-, 5,7-dinitro-, and 6,8-dinitroquinoline react with the carbanion of chloromethyl phenyl sulfone to give products of substitution of hydrogen at positions 4, 3, 8, 6,8, and 5,7, respectively. The reactions are found by quantum chemical calculations to be controlled by the interactions of the HOMO of the nucleophile with the LUMO of the substrate. The σ adducts 4-(methylsulfonylphenyl)-3-nitroquinoline and 3-(methylsulfonylphenyl)-4-nitroquinoline are detected by 1H-NMR spectroscopy. In addition, heats of formation of the intermediary σ adducts 2a–d, f, h, and i are calculated.

Monoterpenoid biosynthesis in tobacco was modified by introducing two subsequent enzymatic activities targeted to different cell compartments. A limonene-3-hydroxylase (lim3h) cDNA was isolated from Mentha spicata L. 'Crispa'. This cDNA was used to re-transform a transgenic Nicotiana tabacum'Petit Havana' SR1 (tobacco) line expressing three Citrus limon L. Burm. f. (lemon) monoterpene synthases producing (+)-limonene, gamma-terpinene and (-)-beta-pinene as their main products. The targeting sequences of these synthases indicate that they are probably localized in the plastids, whereas the sequence information of the P450 hydroxylase indicates targeting to the endoplasmatic reticulum. Despite the different location of the enzymes, the introduced P450 hydroxylase proved to be functional in the transgenic plants as it hydroxylated (+)-limonene, resulting in the emission of (+)-trans-isopiperitenol. Some further modifications of the (+)-trans-isopiperitenol were also detected, resulting in the additional emission of 1,3,8-p-menthatriene, 1,5,8-p-menthatriene, p-cymene and isopiperitenone.

A novel route to pyrazole derivatives bearing sulfonyl, aminosulfonyl, alkoxy- and aryloxysulfonyl groups at C-3 by ring transformation of 3-chloro-6-aryl-1,2,4-triazines 1a-c with alpha-halocarbanions 2a-h is described.

(2001). A NOVEL SYNTHESIS OF 2-ACYLPYRIDINES via INVERSE ELECTRON DEMAND DIELS-ALDER REACTION OF 5-ACYL-1,2,4-TRIAZLNES. Organic Preparations and Procedures International: Vol. 33, No. 5, pp. 501-505.

The reactions of 3-X-6-phenyl-1,2,4-triazines (X = SMe, SPh, SO2Ph) with phenylacetonitrile anion in DMF were studied. In these reactions the ring transformation product 3-amino-4,6-diphenylpyridazine, the covalent addition product 3-X-5-(α-cyanobenzyl)-6-phenyl-2,5-dihydro-1,2,4-triazine, and the ipso-substitution product 3-(1-cyano-1-phenylmethyl)-6-phenyl-1,2,4-triazine were obtained. Analogous reactions carried out in DMA gave only the addition products in excellent yields as diastereomeric mixtures of the corresponding 2,5-dihydro-1,2,4-triazines.

When pyrimidine, 1-methylpyrimidinium methosulfate, 1,3-diethylpyrimidinium tetrafluoroborate or pyrimidine N-oxide is reacted with hydroxylamine hydrochloride, isoxazole is formed in a good yield. On reaction of 4,6-dimethylpyrimidine and its corresponding N-oxide with the same reagent 3,5-dimethylisoxazole is obtained. Similarly, from 6-methyl-4-phenylpyrimidine 1-oxide 3-methyl-5-phenylisoxazole is formed. It has been found by 15N-labelling experiments that the NO moiety of the isoxazole ring originates from the hydroxylamine and not from the function of the pyrimidine N-oxide.

Reaction of 4-chloro-3,6-diphenylpyridazine with potassium amide in liquid ammonia gives 4-amino-3,6-diphenylpyridazine and imino-4,4′-bis(3,6-diphenylpyridazine). Starting with 4-chloro-3,6-diphenylpyridazine-[5-13C], it was proved by quantitative 13CNMR measurements that both products are formed via the intermediacy of 4,5-didehydro-3,6-diphenylpyridazine.

Treatment of 3-(methylthio)-1,2,4-triazine with potassium amide in liquid ammonia gives 3-amino-1,2,4-triazine, 3,3′-bis(methylthio)-5,5′-bi-1,2,4-triazine and 3-amino-3′-(methylthio)-5,5′-bi-1,2,4-triazine. In the 3-amino compound formed from 3-(methylthio)-[4-15N]-1,2,4-triazine, 93% of the 15N is present in the exocyclic nitrogen of the amino group. A SN(ANRORC) mechanism is proposed for this amino-de(methylthio)lation reaction.

Pyrimidine is converted by heating with aqueous methylamine or ammonia at 190° into 5-ethyl-2-methylpyridine (5-10%).

The reaction of 3-chloro-6-phenyl-1,2,4-triazine 1a with carbon nucleophiles 2ad bearing a cyano substituent at a carbanionic center has been studied. In all reactions the formation of the corresponding 3-aminopyridazines 3ad takes place via ANRORC mechanism involving addition of the nucleophile at position 5 in compound 1a, ring opening with breaking of the N4C5 bond and intramolecular ring closure of the resulting open-chain intermediate. A 15N study with labeled phenylacetonitrile 2a* has shown that the nitrogen atom of the exocyclic amino group of 3-amino-4,6-diphenylpyridazine 3a was originally present in phenylacetonitrile.