6-Substituted-4-(3-bromophenylamino)quinazolines as putative irreversible inhibitors of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER-2) tyrosine kinases with enhanced antitumor activity.

A series of new 6-substituted-4-(3-bromophenylamino)quinazoline derivatives that may function as irreversible inhibitors of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER-2) tyrosine kinases have been prepared. These inhibitors have, at the C-6 position, butynamide, crotonamide, and methacrylamide Michael acceptors bearing water-solublilizing substituents. These compounds were prepared by acylation of 6-amino-4-(3-bromophenylamino)quinazoline with unsaturated acid chlorides or mixed anhydrides. We show that attaching a basic functional group onto the Michael acceptor results in greater reactivity, due to intramolecular catalysis of the Michael addition and/or an inductive effect of the protonated basic group. This, along with improved water solubility, results in compounds with enhanced biological properties. We present molecular modeling and experimental evidence that these inhibitors interact covalently with the target enzymes. One compound, 16a, was shown to have excellent oral activity in a human epidermoid carcinoma (A431) xenograft model in nude mice.

4-Anilino-6,7-dialkoxyquinoline-3-carbonitrile inhibitors of epidermal growth factor receptor kinase and their bioisosteric relationship to the 4-anilino-6,7-dialkoxyquinazoline inhibitors.

The synthesis and SAR of a series of 4-anilino-6, 7-dialkoxyquinoline-3-carbonitrile inhibitors of epidermal growth factor receptor (EGF-R) kinase are described. Condensation of 3, 4-dialkoxyanilines with ethyl (ethoxymethylene)cyanoacetate followed by thermal cyclization gave, regiospecifically, 6,7-dialkoxy-4-oxo-1, 4-dihydroquinoline-3-carbonitriles. Chlorination (POCl(3)) followed by the reaction with substituted anilines furnished the 4-anilino-6, 7-dialkoxyquinoline-3-carbonitrile inhibitors of EGF-R kinase. An alternate synthesis of these compounds starts with a methyl 3, 4-dialkoxybenzoate. Nitration followed by reduction (Fe, NH(4)Cl, MeOH-H(2)O) gave a methyl 2-amino-4,5-dialkoxybenzoate. Amidine formation using DMF-acetal followed by cyclization using LiCH(2)CN furnished a 6,7-dialkoxy-4-oxo-1,4-dihydroquinoline-3-carbonitrile, which was transformed as before. Compounds containing acid, ester, amide, carbinol, and aldehyde groups at the 3-position of the quinoline ring were also prepared for comparison, as were several 1-anilino-6,7-dimethoxyisoquinoline-4-carbonitriles. The compounds were evaluated for their ability to inhibit the autophosphorylation of the catalytic domain of EGF-R. The SAR of these inhibitors with respect to the nature of the 6,7-alkoxy groups, the aniline substituents, and the substituent at the 3-position was studied. The compounds were further evaluated for their ability to inhibit the growth of cell lines that overexpress EGF-R or HER-2. It was found that 4-anilinoquinoline-3-carbonitriles are effective inhibitors of EGF-R kinase with activity comparable to the 4-anilinoquinazoline-based inhibitors. A new homology model of EGF-R kinase was constructed based on the X-ray structures of Hck and FGF receptor-1 kinase. The model suggests that with the quinazoline-based inhibitors, the N3 atom is hydrogen-bonded to a water molecule which, in turn, interacts with Thr 830. It is proposed that the quinoline-3-carbonitriles bind in a similar manner where the water molecule is displaced by the cyano group which interacts with the same Thr residue.

Biosynthetic origin of the carbon skeleton and oxygen atoms of the LL-F28249 alpha, a potent antiparasitic macrolide.

The biosynthesis of LL-F28249 alpha in a culture of Streptomyces cyaneogriseus has been studied using 13C, 14C and 18O labeled precursors. A complete 13C NMR spectrum of F28249 alpha has been assigned. Incorporation studies using 13C labeled precursors indicate that the carbon skeleton of F28249 alpha is derived from seven acetate, six propionate and one 2-methylpropionate units. The origin of the oxygen atoms of F28249 alpha has been examined by feeding [1-13C,18O2]acetate, [1-13C,18O2]propionate, [2-13C]acetate/18O2 and 18O2 separately to the fermentation culture and analyzing the resulting labeled LL-F28249 alpha samples by 13C NMR, electron impact MS and chemical ionization MS. Out of a total of eight oxygen atoms in LL-F28249 alpha, four oxygen atoms are derived from acetate, three from propionate and one from molecular oxygen.

Biosynthesis of the spermidine and guanidino units in the glycocinnamoylspermidine antibiotic cinodine.

The biosynthesis of the spermidine and guanidino groups has been studied with carbon-14 and carbon-13 labeled intermediates. Arginine, citrulline and ornithine are incorporated in good efficiency. The guanidino group of arginine and the ureido group of citrulline both label the guanidino group on the hexose sugar. None of the ureido groups in the antibiotic was enriched. It is likely that citrulline is converted to arginine before use in the biosynthesis. Arginine, citrulline and ornithine are incorporated as the four carbon unit of spermidine. All of the labeled 5 carbon from ornithine or from citrulline appears adjacent to the secondary amine in the four carbon unit of spermidine. This would indicate that unbound putrescine is not an immediate precursor of spermidine.

Studies on the role of protein synthesis and of sodium on the regulation of ornithine decarboxylase activity.

The minimum requirements for eliciting or enhancing ornithine decarboxylase activity (EC. 4.1.1.17); L-ornithine carboxylase) in neuroblastoma cells incubated in salts-glucose solutions have been investigated. These incubation conditions permit the study of changes in ornithine decarboxylase activity independently of the growth-associated reactions that occur in cell culture media (Chen, K.Y. and Canellakis, E.S. (1977) Proc. Natl, Acad. Sci. U.S.A. 74, 3791-3795). Ornithine decarboxylase activity can be elicited by a variety of asparagine and other amino acid analogs, including alpha-aminoisobutyric acid, that cannot participate in protein synthesis. Of the eleven asparagine analogs tested, alpha-N-CH3-DL-asparagine is the most potent in eliciting ornithine decarboxylase activity and is equivalent to asparagine in this regard. Inclusion of polar groups into the asparagine molecule results in the loss of its ability to elicit ornithine decarboxylase activity. With the use of these analogs and of analogs of other amino acids it is shown that the rapid fall in ornithine decarboxylase activity that is noted following cycloheximide treatment may not be a consequence of the inhibition of protein synthesis. The rapid fall in ornithine decarboxylase activity is primarily due to the removal of the agent that elicits and stabilizes its activity. These results, the finding that alpha-aminoisobutyric acid stimulates ornithine decarboxylase activity and that sodium is required for the stimulation of ornithine decarboxylase activity are discussed in relation to the "A" amino acid transport system.