The development of reactions in a continuous fashion in plug flow tube reactors (PFR) offers unique advantages to the drug development and scale-up process and can also enable chemistry that would be difficult to perform via batch processing. Herein, we report the development of two different continuous flow approaches to a key 1H-4-substituted imidazole intermediate ( 5). In a first generation approach, rapid optimization and scale-up of a challenging cyclization reaction was demonstrated in a PFR under GMP conditions to afford 29 kg of protected product 2. This material was further processed in batch equipment to deliver di-HCl salt 4. This first generation approach highlights the rapid development of chemistry in research-scale PFRs and speed to material delivery through linear scale up to a pilot-scale PFR under GMP conditions. In a second generation effort, a more efficient synthetic route was developed, and PFRs with automated sampling, dilution, and analytical analysis allowed for rapid and data-rich reaction optimization of both a key cyclization reaction and thermal removal of a Boc protecting group. This work culminated in 1 kg demonstration runs in a 0.22 L PFR for both continuous steps and shows the potential of commercialization from a lab hood footprint (1−2 MT/year).
Several fluorescence properties of two enantiomers, NSC 613862 (S)-(-) and NSC 613863 (R)-(+), have been compared. Even though the two isomers showed the same fluorescence behavior in solution in different solvents, drastic differences were observed after binding to purified calf brain tubulin. Binding measurements for the two compounds were performed both by fluorescence spectroscopy and by column gel permeation, a direct method of measurement. For both isomers, the binding was characterized by the presence of one high-affinity binding site with an apparent association constant of (3.2 f 0.5) X lo6 M-I and (4.1 f 0.9) X lo6 M-' for the Rand S-isomer, respectively, and by several low-affinity sites.Both isomers were also shown to induce GTPase activity in tubulin. The high-affinity binding site seems to be the same for the two isomers. Moreover, fluorescence competition experiments suggest at least a partial overlap of the colchicine and podophyllotoxin site. To explain the differences in fluorescence behavior after binding to tubulin, we hypothesize that the R-isomer is positioned differently in its binding locus as compared with the S-isomer.Initially synthesized by Elliott et al. (1968) to act as analogues of folic acid, a number of compounds that possess an aromatic nucleus with a l-deaza-7,8-dihydropteridine structure have been reported to inhibit mitosis and produce an accumulation of cells in metaphase (Wheeler et al., 1981). Among them, NSC 181928, ethyl (5-amino-1,2-dihydro-3-[ (N-methy1anilino)methyll pyrido [3,4-b]pyrazin-7-yl)carbamate has been studied in particular. Hamel and Lin (1982) demonstrated that this drug was an active antitubulin agent which inhibited both polymerization and binding of colchicine to tubulin and stimulated tubulin-dependent GTP hydrolysis.Bowdon et al. (1987) showed that another member of the same series, NSC 370147, ethyl (5-amino-1,2-dihydro-2methyl-3-phenylpyrido [ 3,4-b] pyrazin-7-yl)carbamate, was able to competitively inhibit the binding of [3H]c~lchicine and slightly enhance the binding of [3H]vincristine to purified tubulin.However, NSC 370147 is a racemic compound. Its two chiral isomers, NSC 613862 (S)-(-) and NSC 613863 (R)- (+) (see Chart I), have displayed significant differences in Abstract published in Advance ACS Abstracts, September 1, 1993. I Abbreviations: BSA, bovine serum albumin; PG buffer, 10 mM sodium phosphate and 0.1 mM GTP, pH 7.0; MDL 27048, trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylam~o)phenyl]-2-methyl-2-pro~n-1 -one; MTC, 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6cyclohep~~en-1-one; SDS, sodium dodecyl sulfate; TME, tropolone methyl ether; AS,,, difference between theapparent sedimentation coefficient of tubulin alone and liganded.
Antitumor activity in mice was observed for the oxime of the previously reported ethyl [6-amino-4-[(1-methyl-2-phenyl-2-oxoethyl)amino]-5-nitropyridin -2-yl] carbamate (8) and several related compounds. These compounds are precursors of the active ethyl pyrido[3,4-b]pyrazin-7-ylcarbamates (e.g., 4), which are potent antimitotic agents. In the 5-nitropyridine series overall biological activity was reduced by replacement of the oxime moiety with a keto or alcohol group and by replacement of the 1-methyl group of the side chain with hydrogen. Reduction of the nitro group of the 5-nitropyridines containing an alcohol in the side chain to the corresponding 5-aminopyridines increased biological activity. Preliminary studies showed that the 5-nitropyridine oximes were considerably less potent than the pyridopyrazines as antimitotic agents and that the former are apparently not converted to the latter in vivo. The inhibition of the incorporation of pyrimidine nucleosides into DNA and RNA was identified as another possible mode of action of the 5-nitropyridine oximes.
The 1,2-dihydropyrido[3,4-b]pyrazines (1) are mitotic inhibitors with significant antitumor activity in mice. Also, the active imidazo[4,5-b]pyridine 3 was shown to cause the accumulation of cells at mitosis. Routes were developed for the synthesis of congeners of 3 by cyclization of 4-(substituted amino)-5,6-diaminopyridines with ethyl orthoformate. Oxidative cyclization of either 4,5- or 5,6-diaminopyridines with aryl aldehydes produced the [4,5-c] and [4,5-b] imidazopyridine ring systems, respectively. The latter reaction with 6-(substituted amino)-4,5-diaminopyridines gave imidazo[4,5-c]pyridine ring analogues of 1. Biological studies indicated that the target compounds were less active than 1 and 3.
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